Chronic lymphocytic leukemia (CLL) is characterized by the relentless accumulation of neoplastic B lymphocytes (CLL cells) in the blood, lymphatic tissues, and the bone marrow. Despite major advances in treatment, CLL currently is considered incurable with standard treatments and new therapeutic approaches therefore are urgently needed. In patients, CLL cells accumulate not because they grow faster, but because they survive longer than normal cells. However, when CLL cells are removed from the patient and placed into cell culture, they rapidly undergo cell death unless they are cultured in contact with “feeder” cells. Therefore, it is hypothesized that contact with “feeder” cells such as stromal cells or nurse-like cells is essential for growth and survival of CLL cells.

CLL cells are tethered to “feeder” cells in the bone marrow or the lymphatic tissues by a factor called “CXCL12” which is secreted by “feeder” cells and binds to CXCR4 receptors on CLL cells. We previously demonstrated that CXCR4 blocking molecules inhibit the contact between CLL cells and their nurturing counterparts and thereby make them more sensible to spontaneous or chemotherapy-induced cell death. However, the protective effect of “feeder” cells was only partially antagonized by CXCR4 blockers. This research proposal will further dissect which other molecules participate in supporting CLL cells in their microenvironment. Different factors that may be involved in this process will be placed alone or in combination in culture with CLL cells and we will assess whether they can substitute the “feeder” cell effect.

In parallel, studies will be performed to demonstrate the presence of these factors in tissue sections from CLL patients. Such a dissection of the interactions between CLL cells and their supportive microenvironment will allow us to identify new potential targets for improvement of current treatments for CLL patients.

Malignant leukemia cells from patients with CLL rapidly die once they are removed from the patient’s blood, bone marrow, or lymph nodes. This suggests that interactions between CLL cells and neighbor cells, called stromal cells and “nurse-like cells”, that are part of the “microenvironment” in which leukemia cells live in the patients’ tissues, are critical for maintenance of the leukemia cells.

The aim of the research supported by the CLL Global Research Foundation was to study which cells and molecules are important for interactions between CLL cells and their microenvironment. Initial results were submitted for publication, and also summarized in a review article published in the British Journal of Haematology.

During this funding period, we found that a molecule on CLL cells called “CXCR5” is over expressed in CLL and mediates contact between CLL cells and “nurse-like cells”. Because of the high-level expression of CXCR5, and its function in migration and adhesion to “nurse-like cells,” CXCR5 is an attractive therapeutic target in this disease. These results have been submitted for publication.

In order to systematically dissect the interactions between CLL cells and the microenvironment, we performed experiments to analyze which genes in CLL cells are turned on, and which genes are turned off when CLL cells are grown with stroma or “nurse-like cells”. The experiments, in collaboration with Dr. Andreas Rosenwald from the Department of Pathology at Würzburg University, Germany, revealed strong up-regulation of 2 molecules that normally regulate interactions of immune cells (B-cells and T-cells). These studies are currently ongoing and are a central part of the project funded by the CLL Global.

To further evaluate if the findings in cell cultures can be reproduced with tissues from CLL patients, biopsy specimen from CLL patients were stained in collaboration with Professor A. Schmitt-Gräff (Hematopathology, Freiburg University). We found that “nurse-like cells” secrete a protein called CXCL13 that binds to CXCR5.

Collectively, the CLL Global funding allowed us to initiate studies with international collaborators to determine how CLL cells interact with the microenvironment. These studies provide novel insight into the mechanism of this disease and help to identify new therapeutic targets for CLL patients.

Clonal proliferation and growth are key elements in the development and progression of cancer. Despite the apparent lack of an easily detectable proliferative compartment, B-cell type chronic lymphocytic leukemia (B-CLL) is not an exception to this rule. Indeed, recent data highlight this point. Using the non-radioactive stable isotope deuterium, administered to patients in the form of heavy water (2H2O), we recently determined that B-CLL cell birth and death occur at a substantially higher level than previously realized.

Several unanswered questions should now be addressed: What are the proliferating cells in B-CLL? Is there a normal circulating B-cell subset that has kinetic profiles similar to B-CLL cells? What is the scope of genetic changes that occur in a normal B-cell to transform them into B-CLL cells and to transform B-CLL cells into more dangerous clones? Finally, when a larger number of patients are studied, will there be a correlation between B-CLL cell kinetics and the serum, cellular, and molecular markers of outcome?

Having demonstrated the success of 2H2O labeling in defining the proliferative history of B-CLL cells, we expect that, by combining this approach with established techniques of immunofluorescence and cell isolation, we will identify and characterize the proliferating cells in this leukemia. We will then compare the kinetics of these cells with potential normal counterparts that are available in the blood. In these studies, we will be looking to define the development of new cytogenetic lesions that could presage changes in the clinical behavior of the leukemic clone. Furthermore, we intend to characterize those normal B-lymphocytes that eventually become B-CLL cells, especially those which are clonally expanded in normal individuals and, more so, in apparently healthy relatives of patients having B-CLL.

Cell growth is a dominant feature of all cancers. Using a safe, non-radioactive technique (heavy water ingestion) that labels the DNA of doubling cells, we previously determined that the birth and death of B-CLL cells occur at a much higher level than once realized.

Utilizing B-CLL cells from 25 patients who have consumed heavy water, we have confirmed that the birth of the leukemic cells from patients with B-CLL can be measured safely and conveniently, and that these rates vary among patients.

We have now also defined the specific cells in the blood of B-CLL patients that have incorporated the most heavy water, indicating that these are the cells that have divided during the study period. These cells are marked by the simultaneous expression on their surface membranes of a high density of the molecule CD5, along with the expression of the molecules CD38.

These cells may be important targets for therapy, since they likely give rise to the bulk of the leukemia cells in B-CLL

Several decades of experience in the management of CLL patients clearly indicate that the clinical behavior of this chronic leukemia is highly heterogeneous, with some patients requiring treatment from the start while others do not. One of today’s most important challenges in CLL management is to rapidly and correctly identify high-risk and low-risk patients and consequently to choose the best therapy for the individual’s particular variant of the disease, while minimizing the adverse effects of drugs.

Today, patients with aggressive CLL can be identified by molecular markers such as the status of the immunoglobulin genes, the presence of CD38 (a surface receptor which may be expressed by the leukemic cells) and of ZAP-70 (a signaling element which may be present inside the cells).

In previous studies, our lab analyzed the role of CD38 expression in CLL and how its interactions with the microenvironment can lead to tumor proliferation. We now propose to show that CD38 and ZAP-70 are part of the chain of events that leads to the complex mechanisms which maintain leukemic cell growth, defeating the normal control mechanisms. This goal will be achieved by means of biochemical, morphologic and functional analyses, which will show how CD38 and ZAP-70 function, where they are localized in the cell and how they interact with one another.

A third part of the project is centered on the CD38 gene. Two forms of the gene have been described in healthy individuals and we wish to see if there is a privileged association between one of the CD38 variants and a CLL subtype.

We hope to apply what we learn in the laboratory about modulating CD38 and ZAP-70 to our clinical treatment decisions.

CD38 and ZAP-70 are two molecules acting as negative prognostic markers for CLL patients. Clinical trials have shown that a combined analysis of CD38 and ZAP-70 expression results in more efficient identification of patients with high risk CLL. The molecular basis for this observation is that CD38 and ZAP-70 are functionally linked and control growth of CLL cells. We also showed that CD38 and ZAP-70 favor migration of CLL cells from the blood to peripheral lymphoid organs (such as spleen and lymph nodes) where tumor cells are met with a microenvironment that favors growth. We have now shown that this happens because CD38 facilitates molecular responses to chemokines (such as CXCL12) which control movement of CLL cells. As a consequence, administration of anti-CD38 antibodies potently blocks migration of CLL cells, trapping them in the blood, where they are more accessible to drug attack.

This information provides the rationale for devising a CLL therapy that targets CD38. The use of reagents specifically blocking this molecule might provide a new approach for interfering with harmful growth circuits, therefore increasing the susceptibility of leukemic cells to conventional chemotherapy

In chronic lymphocytic leukemia (CLL), some of the leukemic cells proliferate whereas others are long-lived and accumulate. CLL cells from various patients differ in that they may have aberrations in certain genes named V genes, and they may express either high or low levels of proteins called CD38 and ZAP-70. However, despite this patient-to-patient heterogeneity, a protein called STAT-3 is activated in almost all CLL patients.

Hormone-like substances, termed cytokines and growth factors, circulate in the blood, attach to cells, and induce cellular multiplication. They accomplish this by activating a pathway that exists in cells, called JAK-STAT. When cytokines or growth factors attach to a cell, the JAK component of this pathway is stimulated, and JAK proteins activate STAT proteins. Activated STATs move to the cell’s nucleus and induce the cells to proliferate and to better protect themselves from insults that might cause cell death. We found that in 31 of 32 patients with CLL, one of the STAT proteins, STAT-3, is activated in an unusual manner. The phosphate that induces STAT-3 activation is attached to a serine rather than a tyrosine (as usually found in other leukemias). Nevertheless, our preliminary data suggest that serine-phosphorylated STAT-3 is biologically active in CLL.

In the proposed study we intend to: 1) Find out what causes STAT-3 to be serine-phosphorylated and activated in CLL; 2) Determine whether serine-phosphorylation of STAT-3 induces the proliferation of CLL cells and whether it improves the capability of CLL cells to protect themselves from insults that might cause cell death; and 3) Identify drugs that inhibit the activity of STAT-3 and have the potential to be used in therapies for CLL.

In chronic lymphocytic leukemia (CLL), some of the leukemic cells proliferate whereas others are long-lived and accumulate. Hormone-like proteins, termed cytokines and growth factors, circulate in the blood, attach to cells’ membrane and activate STAT proteins that bind to DNA and enhance protection from insults that cause cell death and cellular multiplication. We studied CLL cells from about 100 patients, and found in all of the patients, that one of the STAT proteins, STAT-3, is activated spontaneously in an unusual manner. The phosphate that induces STAT-3 activation is attached to a different location on the DNA than other malignant disorders.

The unusual spontaneous activation of STAT-3 remains unchanged for days when CLL cells are maintained in culture, and the inhibition of STAT-3 results in CLL cell death. Because Azacytidine, a drug recently approved for treatment of myelodysplastic syndrome, inhibits the activation of STAT-3, we initiated a phase II trial of Azacytidine in CLL. Thus far, 1 of the 7 patients with CLL enrolled onto the study has benefited clinically.

We have been concentrating our efforts on identifying the mechanism(s) that induces spontaneous activation of STAT-3 which, in our view, is crucial for unraveling the molecular event(s) that contribute to the evolution of CLL

Programmed cell death, also known as apoptosis, maintains the optimal level of normal cells in the body. This process is regulated by proteins that are either prosurvival or proapoptotic. For chronic lymphocytic leukemia (CLL) cells, there is a survival advantage due to the presence of anti-apoptotic proteins. The first member of this family was identified as Bcl-2 protein. Another member is known as Mcl-1.

Several investigations have demonstrated that CLL cells express high levels of Mcl-1 protein which provides a survival advantage by blocking apoptosis and consequently increasing CLL lymphocytes in the body. In addition, microenvironment factors present in bone marrow stromal cells or in the lymph nodes, further increase levels of Mcl-1 in CLL lymphocytes. We hypothesize that the increase in Mcl-1 in CLL cells by the microenvironment happens through several pathways. Identification of these mechanisms will provide therapeutic options to disrupt them.

Based on our preliminary data, we propose to identify the mechanism by which Mcl-1 is increased in CLL cells when they are co-cultured with stroma cells. This will be achieved by determining transcription of Mcl-1 gene, synthesis of Mcl-1 protein, and changes on the protein to make it more stable.

No update is available at this time. Please check back soon.

We have detected that CLL plasma contains elevated levels of extracellular vesicles termed “microvesicles”. These microvesicles play a critical role in CLL by modulating, or altering, stromal cell function. Aberrant modulation of stromal cell function is likely to enhance the progression of CLL. Our recent findings demonstrate that with therapy, microvesicle levels in CLL plasma fall in some patients. In other patients there is no change or an increase. We predict that these latter patients will relapse more quickly given the microvesicles’ ability to modify host stromal function.

Thus we hypothesize that 1) microvesicle levels in CLL are dynamic, 2) increase with progression of CLL and 3) that while levels may decrease with therapy they will persist with residual disease and rise with relapse. CLL microvesicles contain, and can transfer, specific non-coding RNAs (small genes that regulate the expression of proteins.) These particular non-coding RNAs are linked to CLL progression and may explain how microvesicles are able to modify stromal cell function. During our study, we will examine microvesicle parameters including levels, phenotypes, and contents in CLL plasma as patients progress through their clinical course and treatments. This study can help direct and/or modify approaches for understanding and preventing disease progression and better predict clinical/therapeutic outcomes for CLL patients.

No update is available at this time. Please check back soon.

Up to 10% of patients with CLL/SLL may develop a type of lymphoma which is known as Richter transformation. This may be due to either changes in the original CLL cells or development of a completely new lymphoma. Epstein-Barr virus (EBV), which also causes infectious mononucleosis, has been reported as a possible initiating factor but this has yet to be conclusively shown.

In this grant we will determine whether EBV is involved in Richter transformation by examining tumor samples to determine whether they express different proteins made by EBV. Once we have defined which combination of EBV proteins are expressed, we will develop a therapy to specifically target these proteins using killer T-cells. We have shown that it is possible to grow killer T-cells specific for EBV from normal individuals and patients with some types of lymphoma. In this proposal we aim to develop a similar customized therapy for CLL patients with Richter transformation that will target the particular EBV proteins expressed on tumor cells.

No update is available at this time. Please check back soon.

Emerging evidence in solid tumors and acute leukemias suggests that the ability of a tumor to grow and propagate depends on a small subset of cells, termed “tumor initiating cells” or “cancer stem cells”. The cancer stem cell model postulates that tumors are maintained by a fraction of cancer cells with stem cell properties (self-renewal) and high resistance to apoptosis. There is also evidence suggesting that a number of embryonic signaling pathways, involved in adult stem cell maintenance (i.e., BMI-1, NOTCH, WNT, and SHH), are aberrantly re-activated in tumors, supporting the hypothesis that deregulated self-renewal and proliferation of “cancer stem cells” may be critical for tumor growth.

Using flow cytometry and the DNA-binding dye, Hoechst 33342, we identified a distinct small subset of neoplastic cells, termed the side population (SP), in several human and murine lymphoma cell lines and in CLL samples. Our experimental data in lymphoma cell lines indicates that the SP-cell fraction, in comparison with the non-SP fraction, is enriched with cells with stem cell-like properties including longer telomere length, higher levels of BCL-2 and ABCG-2 proteins, higher clonogenicity capacity and higher capacity to reconstitute tumors in xenograft transplant experiments.

We are also investigating the activation status and the biologic role of the Sonic Hedgehog (SHH) pathway in CLL. Our preliminary data suggest that SHH pathway may be a survival factor for CLL cells, either in an autocrine and/or paracrine manner, and could represent a new target for therapy in CLL.

Based on our preliminary data, this proposal has the following specific aims:
1) To isolate and characterize putative tumor initiating cells in CLL.
2) To elucidate the biologic role of SHH signaling pathway in CLL cell biology.

No update is available at this time. Please check back soon.

The way in which CLL cells are initiated and develop into a life-threatening disease makes B-cell chronic lymphocytic leukemia (B-CLL) an enigma. Other family members of CLL patients also develop CLL or other types of cancer much more frequently than expected by chance. We propose that the responsible genes, at least for a portion of the patients, are genes recently identified and named microRNAs (miRNAs). These are very small genes (about 20 to 22 nucleotides) that do not codify for proteins but interact and degrade the messenger of the protein coding genes. Previously, we reported that mir-15a and mir-16-1 are located at chromosome 13q14, a region deleted in >50% of sporadic and familial B-CLLs and that a germline mutation in mir-16-1 gene is influencing the transcription levels of this gene.

To achieve our goal we will use a panel of 50 to 100 familial CLLs and the available family members from the CLL consortium registry. We will analyze the expression of miRNAs in familial cases versus non-familial cancers and versus normal hematopoietic cells using a microarray technology that we developed. We will also screen for the presence of alterations in the DNA sequence of microRNAs in malignant and normal cells from CLL patients. The endpoint of the first year will be to be able to identify microRNAs that, when altered, can cause the familial form of CLL. In the next step we will use computer-assisted research and various experimental approaches to determine the way in which these miRNAs act on different targets and the cellular mechanisms that are altered. We will create a list of possible molecules with important therapeutic implications.

As a starting point, we used the well-described peculiarity that family members of CLL patients also develop CLL or other types of cancer much more frequently than to be expected by chance. We propose that the responsible genes, at least for a portion of the patients, are genes recently identified and named microRNAs (miRNAs).

Research performed during the first 18 months of the grant maintains the view that these very small genes could play an important role in the initiation of familial CLL. We identified that in familial cases the malignant cells with miRNAs were deleted or mutated. We have also shown that inserting the missing genes into the malignant cells decreases the potential of formation of leukemic cells. We have also pioneered the idea that miRNAs, and other larger non-codingRNAs, named ultraconserved genes, are involved in the formation and production of tumors, particularly in leukemias and lymphomas. These results offered the genetic basis for performing laboratory studies aimed at understanding the therapeutic potential of these miRNAs in patients with familial form of CLL.

The broad, long-term purpose of my research is to decipher the roles of non-codingRNAs, including miRNAs, in the initiation and progression of blood cancers. The final results will reveal new markers for molecular diagnosis and prognosis of blood cancers, such as CLL, and new targets for drug therapy.

Chronic lymphocytic leukemia (CLL) is caused by the interplay between large genes that help make proteins and small genes which regulate the expression of proteins. The small genes are known as non-coding RNAs (ncRNAs). The mechanism(s) through which genes affect the development and prognosis of CLL is still largely unknown. We do know that there are chromosomal abnormalities (13q, 17p, 11q and 6q deletions and trisomy 12) in CLL cells that correlate with survival. By studying the genes, proteins and other molecules associated with these abnormalities, we may better understand the mechanisms for disease development and prognosis.

We previously discovered microRNAs (miRNAs), a type of ncRNA, associated with the loss of genetic information on chromosome 13q (miRNA-15a/16 cluster). More recently, miRNAs have also been found on chromosome 11q.  The purpose of this current CLL Global research project is to discover ncRNAs involved in trisomy 12 and 6q deletion. These abnormalities are currently less understood than other chromosomal abnormalities involved in CLL. Also, trisomy 12 generally follows an indolent course whereas 6q deletion (or 6q-) tends to be related to a more aggressive evolution. By understanding two abnormalities at opposite spectrums we feel we can better grasp the disease as a whole.
At the end of this project we will be able to identify the first miRNAs and other small genes associated with 6q- and trisomy 12 subtypes of CLL. We also intend to define the interactions of these small genes with other genes and molecules that lead to chromosomal abnormalities. This project is innovative as it focuses on a large spectrum of ncRNAs in poorly deciphered categories of CLL. This approach can generate new diagnostic tools and new therapeutic targets for future development.

No update is available at this time. Please check back soon.

It is well known that CLL patients who have certain abnormalities in the genes of their leukemic cells respond to treatment less well and with a shorter survival than others. Frozen cells have been stored in the Royal Marsden Cell Bank from the blood or bone marrow of over 700 patients enrolled in the recently closed UK CLL4 trial. This trial randomly assigned previously untreated CLL patients to treatment with either chlorambucil or fludarabine (with or without cyclophosphamide). The genes in these stored cells have been analysed and abnormalities of the “11q” and “17p” gene regions have been found to be linked with poorer outcomes. Only 58% of the 79 patients with deleted 11q genes (and 18% of the 44 with deleted 17p genes) survive for four years, compared to 72% of patients without these abnormalities. In addition, 79% of those with 17p deletions failed to respond to treatment, compared to only 12% of those with “normal” genes. But the course of the disease will not be the same for all patients within these groups.

We propose to use a variety of new, highly sensitive laboratory techniques to:
1. Map the deletions at 11q and 17p more precisely
2. Define exactly which genes within these regions are associated with poor outcome
3. Identify new altered regions, and define which particular malfunctioning genes within them affect outcome
4. Look for gene abnormalities which affect how CLL cells arise, behave and proliferate, and which may be targets for new treatments

Prognostic markers in CLL are becoming increasingly important for patients and clinicians alike. Cytogenetic markers, such as 17p deletion, are associated with a very poor outcome in CLL, and, over the grant period, we have examined, in detail, samples from patients in the LRF CLL4 clinical trial in the U.K. In samples with a known 17p abnormality, we used a technique to look for changes in other genes.
We identified highly significant changes in other chromosomes which are associated with the 17p abnormality and may therefore be involved in the development of high-risk disease. In addition, we have also examined what happens to the copy of the gene remaining on the non-deleted chromosome. We have shown that, in the majority of cases, there is a mutation in this remaining copy.

This explains why the TP53 gene is not active in cases with the 17p abnormality and why there is a strong association with resistance to chemotherapy which is dependent on a functional TP53 pathway for activity. These results have helped further our understanding of the reasons behind the poor outcome for patients with these specific genetic changes.

Gene silencing, or the inactivation of genes, is often associated with cancer. Often, the addition of methyl groups to specific regions on the gene results in gene silencing. Gene silencing inactivates genes that are important for cell function. We are working in the laboratory to identify subsets of patients with specific DNA methylation (alterations) which may predict specific prognoses. DNA methylation can be reversed and we are studying drugs that have proven reversal activity in myeloid leukemias. Understanding methylation alterations in CLL may allow the selective use of these relatively non-toxic therapies.

Based on these concepts, we propose to:
1-Develop an epigenetic/genetic profile of patients with chronic lymphocytic leukemia (CLL).
2-Develop systems to test the molecular effects of hypomethylating agents on CLL cells.
3-Develop clinical trials based on information derived from #1 & #2 using hypomethylating agents in patients with CLL.

We will analyze a set of untreated CLL samples for both gene specific and global methylation. The data generated will be compared to known prognostic alterations in CLL such as IgVH mutational status, FISH analysis, and expression of ZAP-70 and CD38.

CLL cells will be epigenetically profiled prior to treatment. Two different drugs with hypomethylating activity, 5-aza-2′-deoxycytidine and 5-azacytidine, will be given and the cells will be analyzed for methylation changes and cell kill dynamics. This data will then be used to develop a phase I clinical trial of one of these two agents. The data generated by aims #1-3 will be used to develop a final methylation classification of CLL and to introduce the use of hypomethylating agents for the treatment of CLL patients.

Our project focuses on the study of epigenetic alterations in CLL. In particular, we analyzed aberrant DNA methylation patterns. DNA methylation is a biochemical modification of DNA that results in gene silencing. If this occurs in tumor suppressor genes, it may contribute to the development of CLL.

We have developed a new strategy to detect previously unknown genes methylated in CLL. This strategy consists of the use of a new promoter microarray completed with a technique known as MCA. We have identified in excess of 200 new potential tumor suppressor genes in CLL, and we have validated approximately 30 of them (both at the DNA and gene expression levels). These genes clustered more frequently on chromosome 17 (a poor prognostic region in CLL), and can be grouped into 20 molecular networks. Some of these genes also have prognostic value. Next, we plan to study these genes in the setting of FCR (fludarabine, cyclophosphamide & rituximab) therapy in collaboration with Dr. Wierda.

CLL researchers are working to define which patients should be treated, when and how. It is important to look at the molecular differences of CLL which translates into remarkable clinical differences. The presence of somatic mutations in the immunoglobulin variable region (IGHV) genes and the expression of CD38 and ZAP-70 are examples that link biology to prognosis. We have used a proteomic approach to establish the protein expression profile of CLL cells from different subsets of patients.
We focused on the proteins involved in the transduction of signals delivered by the stimulation of surface antigen receptors, as these proteins might be responsible for a more persistent cell proliferation and/or prevention of apoptosis. We have discovered that a discriminating molecule is hematopoietic-lineage-cell-specific protein 1 (HS1), a protein pivotal in the signal cascade triggered by the B-cell-receptor stimulation. In 40 CLL cases we found that patients with aggressive disease expressed most HS1 protein in the phosphorylated form, while patients with stable disease had most HS1 in the unphosphorylated form. These observations have led us to ask three questions: 1) Can HS1 be used to build an integrated prognostic card for individual CLL patients? 2) Can HS1 be targeted for therapeutic purposes? 3) Can we detect other proteins that might become selective therapeutic targets?

To answer these questions we have devised the following experimental plan:
1) The prognostic significance of phosphorylated HS1 and its relationship with other prognostic markers will be evaluated in a large cohort of patients
2) The possibility of therapeutically targeting the phosphorylated form of HS1 will be investigated to provide the basis for designing specific inhibitors.
3) As preliminary data show that other proteins are differentially expressed in different CLL subsets, their expression pattern and functional role will be studied.

In the past two years, we have studied the protein expression profile of CLL cells from different subsets of patients. We were hoping to identify therapeutic targets and/or prognostic markers, differentially expressed in CLL patients, which could predict patients with slowly progressing or aggressive disease.

First, we focused our attention on HS1, a protein that we have previously described to be differentially activated in CLL patients. Patients with aggressive disease expressed the protein in a modified (phosphorylated) form. We have now reached several novel conclusions that lay the ground for future intensive research. In particular, we have shown that HS1 is involved in the framework of the leukemic cells. When we completely, or partially, blocked HS1’s function in leukemic B-cells, we observed a severe impairment of the CLL cell’s ability to migrate. This might indicate that HS1 plays a relevant role in directing cells toward the marrow, as occurs in the late stages of the disease. Interfering with the HS1 protein may produce a potential therapeutic effect. This deserves further studies, which we plan to analyze in our future research activities.

Secondly, we have identified a novel activation site of HS1 that could be used as a target for the production of specific monoclonal antibodies. Specific reagents could be designed to discriminate between the two forms of the protein, thereby predicting patients with a different prognosis. This could be incorporated into a routine diagnostic work-up, using standard methodologies.

Finally, we produced and compared proteomic maps obtained from CLL patients and normal B-cell subpopulations in order to find molecular level differences that could explain the cell’s behavior. In particular, we have discovered that 1) a protein (Glo I), involved in cell detoxification, appears to discriminate between patients with a different response to therapy. 2) ZAP-70, a molecule thought to be aberrantly expressed in CLL cells, is actually expressed in all normal B-cell subsets analyzed, and it depends on the activation status of the cells. 3) Cortactin, a protein in epithelial cells, is actually expressed in normal and leukemic B-cells at different levels. All these molecules need further studies to elucidate their importance in CLL.

This project is a collaboration between MD Anderson Cancer Center and the University of Cologne in Germany and focuses on the role of the leukemia-causing gene TCL1 in CLL. Abnormal activation of the gene TCL1 leads to a T-cell variant of human CLL, produces CLL-like tumors in mice and has been implicated as a cancer gene in the more common human B-cell CLL. We have shown that TCL1 is found at high levels in a genetic subset of CLL patients with poor outcome. Consequently, TCL1 represents a promising therapeutic target for aggressive variants of this leukemia.

The exact function of TCL1 is still not completely understood. We are using a range of analysis techniques to find which proteins TCL1 interacts with and how its presence alters the growth response of CLL cells to external stimulatory factors. In particular, we have shown in CLL cells that TCL1 interacts with and regulates a key cellular protein, called Akt, which controls tumor cell growth and death. We have shown that inhibitors of Akt alter its association with TCL1 and shift TCL1-containing protein complexes to different locations within the tumor cell.

We are utilizing our range of antibodies developed against TCL1 to isolate novel TCL1-interacting proteins and to track changes in TCL1-containing complexes within CLL cells as the tumor grows. The project will use small molecule inhibitors, TCL1 gene silencing, and TCL1 mimetic peptides to test the effects of interfering with TCL1-complex formation / localization as a therapeutic approach in CLL.

Useful Definitions:
TCL1: a human gene that is associated with the development of T-cell and B-cell leukemias and lymphomas
Akt: a cellular protein that functions in growth signaling and prevention of tumor cell death

Our group is studying the role of growth signaling pathways in CLL, especially TCL1.

We have shown that the levels of TCL1, a small molecule which functions as an oncogene in CLL, raises and falls when leukemia cells divide, differentially regulating the activity of cellular enzymes (specifically serine/threonine kinases, including Akt), through the cell cycle. This effect shifts the balance between growth and death in CLL cells.

We have discovered that TCL1 interacts with the signals from the B-cell receptor (antibody) complex in B-cell CLL and the T-cell receptor in T-cell CLL types. We are currently assessing whether alteration of TCL1 levels, by disrupting its protein complexes with small molecule mimics, can reduce CLL growth.

Cancers such as chronic lymphocytic leukemia (CLL) are characterized by a slow accumulation of cells that display enhanced survival. These cells do not divide or synthesize DNA. Therefore, agents that require active DNA replication or cell division are ineffective against this disease. My project focuses on the development a new class of agents, the histone deacetylase inhibitors (HDACI), for the treatment of leukemias.

HDACIs work by acetylating proteins so that genes synthesize (or transcribe) new messenger RNA’s. These new messenger RNA’s produce proteins which activate cell death in CLL lymphocytes. The exposure of CLL cells to LBH589 (an HDACI) increases the level of protein modification and the activation of cell death. LBH589 also inhibits the transcription of genes needed for the survival of CLL cells. These features make LBH589 an ideal drug to target CLL cells.

Our hypothesis is that because of these DNA independent actions of LBH589, quiescent (non-dividing) CLL lymphocytes will undergo death. We hope to understand the mechanism of action of this agent in leukemia cells that are freshly obtained from blood of patients with CLL. These data will help in the development of HDACIs as a treatment of CLL. We will compare all our data in CLL lymphocytes with normal lymphocytes. We will obtain blood from CLL patients and healthy donors, as permitted under a protocol approved by the Institutional Review Board (IRB). We also have an IRB approved phase I protocol to use LBH589 for patients with leukemias including CLL.

Useful definitions:
Acetylation: Protein modification that allows for the synthesis of new gene products.

Proteins are produced when genes make messenger RNA (mRNA) that is then translated into proteins. MicroRNAs (miRs) are small molecules that bind to specific mRNA in order to reduce the levels of protein produced by that mRNA. I have identified that specific miRs are not produced in CLL cells. Absence of these miRs causes their target proteins to accumulate. For instance, absence of miRI06b causes its target protein, Itch, to accumulate. Itch causes the destruction of another protein, p73. Reduction in the levels of p73 provides a survival advantage to CLL cells.

I have also identified that the reason these miRs are silenced in CLL is due to the action of a class of proteins, the histone deacetylases, that cause the transcription of these genes to shut off. Chemotherapeutic drugs that inhibit deacetylases cause the re-expression of miRI06b. This causes a reduction in the levels of Itch leading to a reciprocal increase in the levels ofp73. Increases in p73 are linked to activation of proteins that cause cell death in CLL. Thus, these drugs may offer a new therapeutic strategy for the treatment of CLL.

Chronic lymphocytic leukemia (CLL) is a disease with high inheritance, meaning that blood relatives face an increased risk to develop the disease, yet genetic analyses have revealed no responsible disease gene so far. Partial losses of chromosomes are common events in the abnormal B-cells that cause CLL. Loss of a region in chromosome 13 occurs in >50% of all CLL cases and less frequent losses are reported in other chromosome such as 11, 17, and 6.

Our genome-wide search pointed to chromosome 9, when a segment of this chromosome co-segregated with the disease in a large family with CLL. The DAPK1 gene located in this region was downregulated due to a mutation that facilitates binding of the HOXB7 inhibitor. Moreover, in many CLL cases DAPK1 expression is downregulated due to chemical (epigenetic) modification of the DNA. Thus, we showed that downregulation of the cell death-promoting gene DAPK1 is an almost universal finding in CLL, which may explain why the CLL causing cells do not die as programmed. However, we do not yet fully understand what leads to the silencing of this gene. Thus, our research aims to determine the mechanisms involved in downregulation of DAPK1 in familial and sporadic CLL.

Chronic lymphocytic leukemia (CLL) is a disease with high inheritance, meaning that blood relatives face an increased risk to develop the disease, yet the responsible gene has not been identified. Our genome-wide search pointed to the DAPK1 gene that was downregulated in many CLL cases, which is found almost universally in CLL. This may explain why the CLL causing cells do not die as programmed. However, our first hypothesis of how this downregulation was caused turned out to apply to very few cases and we quickly decided to test an alternative hypothesis that could explain the silencing of this gene more generally. In fact, we have successfully used this idea in colorectal cancer (CRC), showing that lower expressed variants of the TGFBR1 gene makes people almost 9 times more susceptible to CRC (Valle et al 2008 SCIENCE).

We now have preliminary evidence that lower expressed variants of DAPK1 are found at greater frequency in CLL and we therefore refocused our project to 1) investigate to what extent this mechanism could explain the incidence of CLL and 2) what causes these variants to be expressed at lower levels. We remain determined to elucidate the mechanisms involved in downregulation of DAPK1 in familial and sporadic CLL.

This CLL Global award will fund a new clinical trial to study the therapeutic benefit and safety of infusing patient immune cells (T-cells), genetically modified to recognize and kill CLL tumor cells. These T-cells will be given as an additional therapy following initial standard chemotherapy in previously untreated CLL patients.  In currently ongoing clinical trials, gene modified T-cell infusions have demonstrated therapeutic benefit in patients with relapsed and markedly advanced CLL. However, this has not resulted in complete remissions of the disease.  This suboptimal therapeutic outcome is likely related to the advanced nature of the disease in these already treated patients.

In this project, we will assess whether this approach may demonstrate more significant clinical benefit in previously untreated CLL patients. Patients will receive standard initial chemotherapy to markedly reduce the tumor burden.  In most cases this therapy will not completely eradicate the disease. With low levels of residual tumor cells, we will further treat these patients with an infusion of their own genetically modified T-cells in order to eradicate residual CLL cells. Our intention is for patients to achieve a sustained complete remission of disease.

No update is available at this time. Please check back soon.

Recurrent CLL is unfortunately resistant to conventional therapies and new treatments are urgently needed. A promising new therapy is based on manipulating cells from the immune system to directly fight the CLL tumor cells. One major barrier to achieving therapeutic success is that the immune cells, while able to kill tumor cells, lack the necessary receptors to enable them to distinguish “good” (normal) cells from “bad” (cancerous) CLL cells.

This can now be solved using gene therapy to introduce a desired immunoreceptor into T-cells, a type of immune cell, to redirect specificity. In collaboration with Drs. Bill Wierda at MDACC and Tom Kipps at UCSD, we will develop a receptor that recognizes a molecule called ROR1 which is exclusively expressed on CLL cells. Once this novel immunoreceptor binds to ROR1 it should activate the T-cells to proliferate (make more ROR1-specific T-cells) and to lyse, or destroy, the CLL cell that it is bound to. In addition to work in the laboratory, we will also investigate the ability of the ROR1-specific T-cells to target a tumor in a mouse model. These preclinical data will lay the foundation for a clinical trial infusing ROR1-specific T-cells into patients with CLL.

No update is available at this time. Please check back soon.

Chronic B-lymphocytic leukemia (B-CLL) is a common blood cancer of B-lymphocytes, which are a cellular component of the immune system. B-CLL cells can occasionally be recognized and eliminated by T-lymphocytes, another type of cell of the immune system. We have generated an artificial receptor to attach to T-lymphocytes that specifically targets a molecule (CD19) expressed on the surface of the cancerous B-cells. When the T-lymphocytes contact the tumor cells, they become activated and release substances that kill the cancer. This effect should not significantly damage the remaining normal B-lymphocytes since only some of these lymphocytes express CD19, and the normal B- lymphocytes that lack CD19 should be able to expand and compensate for any losses. We are now proposing to further potentiate the process by the inclusion of a growth factor for the T-lymphocytes and a molecule to eliminate these cells when the anti-tumor effects are obtained. This approach will be tried in cultures and in animals.

No update is available at this time. Please check back soon.

The working hypothesis being examined here is that cancer cells, including CLL cells, express proteins that can be seen as foreign by the immune system. These proteins are the target of donor immune cells following stem cell transplants, an effect known as the “graft versus leukemia” (GVL) effect. Although this effect is well characterized in CLL, it is not known what the target proteins are in CLL cells.

We aim to identify these proteins using molecular techniques and demonstrate that donor immune cells targeting these proteins can be expanded in the test tube and are capable of killing CLL cells. Once we have established this, we shall use the mouse model of CLL to determine if these cells can be given back safely. This will provide the pre-clinical studies that will be required to be able to move on to clinical trials in patients with CLL. In the proposed clinical trial, we would obtain blood samples from matched sibling donors and expand the CLL specific immune cells outside the body that target the proteins expressed by the CLL cells. These cells would then be infused back to CLL patients who have undergone stem cell transplants and who still have evidence of disease.

We believe that this approach will increase the cure rate after stem cell transplantation for CLL, as well as decreasing the risk of the procedure. If successful, we would aim also to induce immune responses against these proteins using the patients own cells.

Our studies have focused on trying to determine why the immune system does not recognize CLL cells and lead to regression of the disease. We have used three approaches to address this question: 1) Study the CLL cells to try to find the proteins expressed on the cells that might be a target for immune cells. 2) Identify why CLL cells are poor targets for immune cells and why they are resistant to killing. 3) What is wrong with the cells of the immune system in patients with CLL?

We have made progress in our understanding of each of these. Using a serial analysis of gene expression (SAGE) array, we identified a novel protein called TOSO in CLL cells that makes these cells more resistant to killing. We have also extended our previous studies and have demonstrated why the T-cells in patients with CLL are so defective. These cells have problems in their internal machinery and cannot bring together the proteins that are essential to switch on T-cells. These defects are induced when immune cells are in contact with CLL cells, so it looks as if CLL cells have developed ways to “switch off” the immune system. We now are looking at ways to reverse this and have ongoing collaborations with investigators at MD Anderson to study this together.

We have developed a new method called ‘DotScan’ that enables 82 markers to be identified on leukemia cells rather than the usual 15-20 using standard techniques. This method is simple and uses a glass slide coated with a specialized membrane (nitrocellulose) on which tiny amounts – about 10 nanolitres (10-8 litres) – of 82 different antibodies are applied. Cells from leukemia patients are then incubated on the slide, and particular markers on the cells are recognized by their corresponding antibodies; the cell is ‘captured’ on the array and does not wash off. The results are read by a simple scanner and displayed on a computer screen. By comparison, flow cytometry is the standard method for leukemia typing with relatively high capital cost, requiring a dedicated operator and relatively high volumes of expensive monoclonal antibodies – typically 5-20 µl (10-5 litres) per test. This technique becomes cost-limiting when more than 20 antigens are studied.

The DotScan microarray enhances our capacity to diagnose and classify different types of leukemia. In a study involving 796 patients, DotScan provided an unequivocal diagnosis when a clear leukemia clone or population is present. We have also obtained preliminary data using DotScan showing different antigen expression between patients with Chronic Lymphocytic Leukemia (B-CLL) with adverse prognostic factors (unmutated immunoglobulin variable genes and the intracellular signaling protein, ZAP-70) compared to patients with favorable prognostic factors.

We therefore propose to expand the array capacity to study CLL specifically by examining all surface antigens that are important in the biology of CLL cells. These include receptors involved in homing of the leukemia cell (adhesion receptors), receptors for survival and proliferation signaling, and receptors that induce the cell death pathway (apoptotic receptors). We believe this “Global CLL Phenotype Profiling” will lead to new insights on the biology of CLL and will be very valuable to both researchers and patients.

This project headed by myself and colleagues at the University of Sydney attempts to give additional information on the proteins which are expressed on CLL cells. The proteins on the surface of the cell identify whether they are T-lymphocytes or B-lymphocytes and also whether they are in an activated or an inactive form. The traditional methodology of trying to establish the protein profile on the cells is by conventional flow cytometry. We developed a machine that characterizes the proteins on the cells’ surface. The cells are incubated with fluorescent antibodies which after going through this machine will send a signal if the protein is on the surface.

Antibodies used in this flow cytometry technique are expensive and there is a limit to the number of samples that can be done at any one time. This project is analyzing 147 proteins. These proteins were selected based on some exploratory work which was conducted by our group. 50 CLL patients have been explored and we have begun a large scale study of patients with known treatment and survival outcomes.

ZAP-70 is an important protein which switches on CLL cells. The methodology to accurately access the amount of ZAP-70 and its activity in CLL cells is complicated and there is a lack of standardization. Our group has developed a method for identifying ZAP-70 by measuring the amount in cell extracts. Linking ZAP-70 with all the other surface proteins on the cells will give more accurate information regarding the level of activity, whether the cells are likely to be susceptible to different treatments and overall outcome. This method is reproducible and considerably cheaper than any other methods which have been developed to identify the protein profile of CLL cells.

Chronic lymphocytic leukemia (CLL) is currently considered incurable with standard treatments. We can now achieve complete remission in the majority of chemotherapy naïve patients; however, their leukemia will likely eventually return. In order to advance therapy for patients with CLL, new treatments that work through complimentary mechanism to chemotherapy are needed.

Recruiting the immune system to react against leukemia cells is one such treatment modality. A strategy has been developed whereby patients’ leukemia cells are treated with a virus that causes them to produce a protein called ISF35. Production of ISF35 by the leukemia cells stimulates the patients’ immune system to recognize and react against the leukemia. The treated cells are administered to patients as a vaccine. The virus does not reproduce and therefore is of little risk for infection to patients. Preliminary work aimed at assessing the tolerability of this treatment in which patients received a single dose of cells treated with ISF35 showed not only that this treatment can safely be given and was well tolerated, but also showed indications of therapeutic benefit such as reduction in leukemia cell counts, lymph node size, and spleen size.

The next step in developing this therapy into treatment for patients with CLL, and the goal of this proposal, is to administer repeated doses and evaluate patients for therapeutic response. This proposal will also evaluate immune function of treated patients to confirm that the treatment is working by the proposed mechanism and in order to optimize effectiveness of the therapy. This will provide an important and novel therapeutic modality to advance treatment of patients with CLL and potentially other malignancies.

CLL is currently considered incurable with standard treatments. We are exploring a treatment modality where we recruit the immune system to react against and eliminate the leukemia cells.

A strategy has been developed whereby patients’ leukemia cells are infected with a virus in the lab that causes them to produce a protein called ISF35. Production of ISF35 causes the immune system to recognize and react against the leukemia cells. The infected cells producing ISF35 are given as a vaccine to patients. We completed enrollment in the Phase I trial of this treatment; patients received a single dose of infected leukemic cells (vaccine). The reaction was not only directed against the infected leukemia cells, but against all of the leukemia cells in general. It was demonstrated that a single infusion was well tolerated and was not associated with any unacceptable side-effects. Also, the Phase I trial indicated therapeutic benefits, such as reduction in leukemia cell counts, lymph node size, and spleen size. We continue to follow the patients on this study; some have moved on to chemotherapy treatments, and others have received a second dose of the vaccine.

A Phase Ib extension trial was done for some of the patients who participated in the Phase I trial. This trial allowed for participating patients to receive re-treatment with repeated doses of the vaccine. This provided us with additional information, as we intend to give the vaccine as multiple-dose treatment. This also enabled us to have more information on the side effect profile and toxicities associated with repeated doses, and I am happy to report that the repeated doses were very well tolerated and also associated with a drop in leukemia counts and reduction in lymph node size.
A collaboration with Dr. Deepa Sampath at MD Anderson Cancer Center has also developed as a result of this clinical trial. The foundation for this collaboration is the observation that the leukemia cells become more sensitive to chemotherapy after patients received their modified leukemia cells. The basis for this sensitivity is being studied.

The Phase II clinical trial has been approved by the MD Anderson Cancer Center’s Institutional Review Board. The Phase II trial will be 5 repeated doses of the patients’ own leukemia cells that are modified to express ISF35. The focus of our work will be to study both the immune reaction that develops against the leukemia cells as a result of this treatment and at the sensitivity the leukemia cells develop to chemotherapy as a result of the treatment.

Although there are promising new treatments for CLL, it is generally incurable. In some patients, the malignant CLL cells accumulate slowly. These patients live for many years and may never require treatment. In other patients, the malignant CLL cells accumulate rapidly. Despite treatment, many of these patients succumb to the disease within a few years. Hematologists have searched for markers that will allow them to predict which patients will have slowly progressing disease and which patients will have aggressive disease that requires treatment in the near future. Traditional markers, such as the number of CLL cells circulating in the blood and how rapidly the CLL cells divide, have been helpful, but are not always accurate.

Recent advances in the field of molecular biology have allowed researchers to identify and measure many genes that distinguish different types of cancer cells (for example, colon cancer and breast cancer) from their normal counterparts. In studies of CLL, researchers have identified hundreds of genes that are expressed at different levels in patients with slowly progressing CLL compared to those with aggressive CLL. We have re-analyzed the data from several studies of CLL using new statistical tests.

Our results suggest that it may be possible to predict whether a patient will have slowly progressing or aggressive CLL by measuring the levels of only a few of these genes. If we are correct, then it would be possible to develop a rapid and reliable blood test to predict whether a patient will have slowly progressing or aggressive CLL. We will use a relatively new laboratory technique, quantitative real-time polymerase chain reaction (QRT-PCR) assay, which measures gene expression levels rapidly and accurately. We will also use new microfluidics technology to perform the QRT-PCR assays. This technology miniaturizes the QRT-PCR assay, so that we can measure many genes simultaneously using only a small amount of blood. Our goal is to develop a rapid and reliable blood test to predict which patients will require treatment soon after they learn that they have CLL, and which patients may never require treatment.

Recent advances in molecular biology have allowed researchers to identify and measure many genes that distinguish different types of cancer cells from their normal counterparts. In studies of CLL, researchers have identified hundreds of genes that are expressed at different levels in patients with slowly progressing CLL compared to those with aggressive CLL. We have re-analyzed the data from several of these studies using new statistical tests to identify genes that may help predict whether a patient will have slowly progressing or aggressive CLL.

Our preliminary results suggest that it is possible to predict a patient’s prognosis by measuring the levels of only a few of these genes. We have used a relatively new laboratory technique, quantitative real-time polymerase chain reaction (QRT-PCR) assay, which measures gene expression levels rapidly and accurately. We also use new microfluidics technology to perform the QRT-PCR assays. This technology miniaturizes the QRT-PCR assay, so that we can measure many genes simultaneously using only a small amount of blood. Our goal has been to evaluate the performance of our test on a much larger set of samples; we are in the process of evaluating these data. If we are able to validate our preliminary results, then we will have developed a rapid and reliable blood test to predict which patients will require treatment soon after they learn that they have CLL, and which patients may never require treatment.

Deletion of chromosomal material in the long arm of chromosome 11, i.e., del(11q), is identified in about 10-20% of CLL patients and is one of the most important predictors of poor prognosis. This region contains the ATM gene (as well as many other genes), which normally suppresses the growth of tumors. Thus, the poor prognosis of patients with del(11q) has been attributed to deletion of this gene. Recently, we have identified CLL cases with del(11q) in which the ATM gene appears intact. This suggests that genes in this region other than ATM may account for the poor prognosis of these patients.

In our experiments, we will look for differences in messenger RNA and protein expression in CLL samples with and without del(11q). We will look for genes within this region whose known function suggests that they contribute to the aggressive behavior of CLL cases with del(11q). We will then explore the functional outcome of either restoring or knocking-down the expression of these quantitatively or structurally aberrant proteins in primary CLL cells or cell lines. In summary, our goal is to identify dysregulated genes in del(11q) CLL patients, other than ATM, that may promote disease progression and relapse, and require a distinct therapeutic approach.

No update is available at this time. Please check back soon.

Many CLL patients have benefited from chemoimmunotherapy including the fludarabine, cyclophosphamide, rituximab (FCR) regimen. Patients with TP53 abnormalities are an exception. There is also suggestion that patients with abnormalities of the ataxic telangiectasia (ATM) gene on chromosome 11q may relapse early after FCR treatment. Most data regarding FCR and the TP53 and ATM abnormalities are derived from clinical trials where patients are younger than the average age of CLL patients (72 years). The relationship is not fully understood between the poor outcomes in older CLL patients and the defects in ATM/TP53.

Current techniques available for detecting ATM/TP53 abnormalities require experienced operators and are difficult to standardize. Massively parallel sequencing (MPS), or next generation sequencing, is a new technology that allows multiple large genes to be interrogated at a reasonable cost. We plan to perform MPS on available samples at our institution in addition to patients enrolled on the CLL5 trial (CLL patients age 65 or older receiving first-line treatment with FCR). The results will be correlated with other available data from the samples and patients including cytogenetics, FISH, and P53 function assay (Best et al., 2008). Sequential sampling of patients through the course of their disease and during therapy will enable clinical monitoring of patients and will identify those at risk of treatment failure.

There are two main aspects of our CLL Global project. First, the integrity of the TP53 and ATM genes will be assessed. Given the overwhelming evidence linking mutations in these genes with clinical outcomes, we believe that MPS of these genes will provide clinically useful information. Second, each assay (MPS test) will contain a panel of additional targets that we hypothesize may also be involved either in the response of CLL cells to treatment or in CLL cell survival. These targets have been selected on the basis of literature that documents their mutation in CLL or in a variety of other tumors.

No update is available at this time. Please check back soon.

Normal cells go through a process of programmed cell death, or apoptosis. Defective apoptosis is a hallmark feature of chronic lymphocytic leukemia (CLL) and is a major target of cancer therapy development.  Several chemotherapeutic agents that are known to induce apoptosis are non-selective to tumor cells, leading to lack of specificity and global toxicity. Therefore, reactivating apoptosis in CLL cells by manipulating specific proteins in the cell should be a novel strategy to kill cancer cells with minimal toxicity.

Inhibitors of apoptosis proteins (IAPs) are proteins that inhibit programmed cell death by deactivating other proteins in the cell needed to execute apoptosis. IAP proteins are expressed at high levels in CLL compared to normal B-cells. In the normal process of apoptosis, a specific protein SMAC/DIABLO is released in response to apoptotic stimuli and activates apoptosis by antagonizing the IAPs.

With this background, we hypothesize that mimicking SMAC and the pro-apoptotic sequence with smac-mimetics should sensitize CLL cells to apoptosis by neutralizing the anti-apoptotic properties of IAPs.  The specific aims associated with the project should unveil the mechanisms that fine-tune the balance between the pro-survival and pro-death pathways.  In addition, studies involving the microenvironments that enhance the stabilization of CLL cells should provide knowledge critical for optimizing therapies.

No update is available at this time. Please check back soon.

Our project will study a group of specific proteins called Hypoxia-inducible transcription factors (HIFs).  These proteins play a role in the regulation of gene expression in response to low oxygen levels in the cellular environment.  When oxygen decreases occur, these proteins induce a wide array of responses, ranging from new vessel formation to metabolic adaptation and cell migration. This group of proteins is increasingly associated with the development of solid tumors and some hematological malignancies. Accordingly, we are interested in inhibiting these factors as a potential cancer treatment.

The specific protein HIF-1a is highly expressed in CLL cells compared to normal B cells. This protein has been suggested to stimulate new vessel formation and resistance to cell death. We found that in CLL B cells HIF-1a also regulates the expression of a number of receptors and molecules known to promote the interaction of leukemic cells with protective stromal microenvironments. Based on these data, we now hypothesize that in addition to preventing new vessel formation, compounds with HIF-inhibitory activity may also work as chemosensitizers in CLL, by promoting the release of CLL B cells from protective niches and exposing them to the toxic effects of chemotherapy.

No update is available at this time. Please check back soon.

This proposal will study signaling pathways and protein interaction of the zeta associated protein kinase of 70 kDa (ZAP-70) in chronic lymphocytic leukemia (CLL), with the goal to develop innovative treatments for patients with high-risk disease.

Leukemia cells of patients with aggressive CLL aberrantly express ZAP-70. The abnormal expression of ZAP-70 provides a potential growth factor stimulus to CLL cells that may drive disease progression.

The specific aims of our proposal are the following:
1. Examine whether the presence of a protein (active Hsp90) is a prognostic factor in CLL-B cells either independently or in association with other factors.
2. Examine the molecular and biochemical basis that determines protein expression in CLL-B cells but not in normal T-cells.
3. Characterize the process of ZAP-70 associated survival in CLL-B cells and the mechanisms involved in CLL-B cell death after Hsp90 inhibition alone or in combination with other agents such as Fludarabine and Rituximab.
4. Develop a phase I/II clinical trial in patients with CLL using a novel lipid formulation of 17-AAG and assess: 1) safety and toxicity profile, 2) pharmacokinetic, pharmacodynamic and biomarker parameters, and 3) evaluate response to treatment.

We have identified and validated a new independent prognostic marker for patients with CLL based on an assay that measures activation of the protein Hsp90. This test can potentially identify, at the time of diagnosis, patients that may have a more aggressive clinical course in spite of having good prognostic markers such as low expression of ZAP-70.

We have completed testing on 60 additional samples out of 300 patients that constitute a validation cohort of this test. With this information, we are proposing to design a clinical test that can be used as a biomarker for disease activity and prognosis in CLL and other forms of cancer.
We have found a panel of genes that are differentially regulated in CLL cells that are undergoing cell death induced by Hsp90 inhibitors. These genes can be used to better understand the biological pathways used by Hsp90 inhibitors and also to evaluate the activity of Hsp90 inhibitors as treatment of patients with cancer. We believe that this set of genes will provide a valuable tool for the development of Hsp90 inhibitors in cancer.

More importantly, we have initiated a trial in which patients receive treatment with an Hsp90 inhibitor (CNF2024). This clinical trial is an early phase I dose escalation study in which six patients have received treatment. So far no serious adverse events have been observed, and dose escalation continues.

This project explores the basic biology of a newly identified family of molecules that are expressed on antibody-producing B-cells of the immune system. The molecules (FC receptor-like molecules, FCRL 1-5) are expressed on the surface of different types of B-cells and may modulate signaling based on components in their intracellular tails. Importantly, B-cells are responsible for greater than 75% of non-Hodgkin’s lymphomas including B-cell chronic lymphocytic leukemia (B-CLL). We have found that four FCRL molecules are expressed by B-CLL cells and at significantly higher levels by indolent CLL cells with mutated antibody genes.

An analysis of 107 samples indicates that the expression pattern of the FCRL2 representative has ~94% concordance with the mutated subtype and can predict time from diagnosis to initial treatment. Because antibody gene sequencing is not routinely performed in hospital laboratories, FCRL2 may serve as a novel prognostic marker for CLL and could help determine which patients have aggressive or indolent disease.

This study will optimize FCRL2 detection on CLL cells and explore the biological basis for its expression in CLL. It is anticipated that this work will contribute to better therapeutic strategies for some of the patients afflicted with B-cell malignancies and other B-cell related disorders, and could help elucidate basic pathogenic defects underlying their disease.

Over the last 9 months we have made substantial progress in our effort to define the biological importance of FCRL2 in CLL. We were fortunate to publish our initial results concerning this receptor last February in the journal Blood which was highlighted by an accompanying commentary. This publication was the first to identify the FCRL2 protein as a useful prognostic marker in CLL. Since CLL Global funding commenced, we have been continually recruiting blood samples from individuals with CLL and testing additional reagents to optimize staining for the FCRL2 molecule on CLL B cells. So far our initial anti-FCRL2 reagent remains the best for staining purposes. Recruitment of additional CLL patients into the study has revealed consistency with our original observations that a higher level of FCRL2 staining on the surface of CLL cells is predictive of a more favorable clinical course.

We have also been extensively surveying different human tissues to define the normal expression pattern of the FCRL2 protein. We have made a very interesting observation concerning FCRL2 expression by a distinct population of blood cells involved in immune system memory. FCRL2 appears to mark a unique type of memory B cell with features similar to mutated CLL subtype B cells. Thus, in addition to its promise as a useful prognostic marker, FCRL2 could also be helpful for defining a pre-transformed CLL counterpart.

DNA repair genes are involved in the detection, response to and repair of DNA damage. The knowledge of which DNA repair genes are abnormal in a patient’s leukemic cell population can be used to guide the appropriate choice of therapy for CLL patients.

We will use a new high throughput methodology that will allow an unprecedented view of the repair status of the leukemic cells. We further seek to use this information to choose appropriate DNA damaging therapy to target CLL.

Our underlying hypotheses are:
” defects in DNA repair occur in many cases of CLL
” identifying specific DNA repair defects may guide the choice of appropriate therapy.

SPECIFIC AIMS:
1. To identify alterations of DNA repair genes in CLL.
2. To identify DNA repair genes which are switched off in CLL.
3. To relate specific repair deficiencies with response to therapy.

EXPERIMENTAL DESIGN:
This proposal uses a new methodology based on accurately measuring the level of messages for all known DNA repair genes at one time to profile DNA repair in tumors. Model systems will be used to evaluate the therapeutic implications of any repair deficiencies identified.

POTENTIAL OUTCOMES AND BENEFITS OF THE RESEARCH:
This is the first systematic study of DNA repair gene deficiencies in any cancer. These studies are targeted towards discoveries that will allow us to improve the appropriateness and effectiveness of DNA damaging therapies in CLL. Knowing which repair pathways are altered specifically in the CLL cells can allow us to target the CLL while leaving normal cells comparatively unscathed.

Personalized medicine involves treating each patient with CLL in an individualized fashion taking into account the specific alterations in the leukemia cells. This project involves identifying those individual differences that occur in CLL, particularly those in DNA repair and cell death that might make the CLL cancer cells sensitive to particular therapies.

For this project, we required purified leukemic cells and developed a new way to do this by removing the normal cells in the blood (Essakali et al, 2008). We then used a new all-exon array methodology that surveys the expression of every known gene in humans. Our plan is to identify differences that occur in gene expression in the abnormal cells which might be targeted by existing or future therapies which can selectively kill the cancer cells while leaving the normal cells intact. We identified a new mechanism that makes CLL resistant to some therapies as well as deficiencies of DNA repair in individual patients which may be useful as potential targets (Essakali et al, in preparation).

Essakali S, Carney D, Westerman D, Gambell P, Seymour JF, Dobrovic A. Negative selection of chronic lymphocytic leukaemia cells using a bifunctional rosette-based antibody cocktail. BMC Biotechnol. 2008 Jan 29;8:6

Chronic lymphocytic leukemia (CLL) is characterized by disrupted cell death pathway rather than increased rate of proliferation. Because these cells do not divide they do not synthesize DNA or go through cell replication. Hence agents that are DNA replication- or cell division-directed do not work well for this disease. We are focusing on developing chemotherapy that is not directed to DNA. Studies from my group have reported that chlorinated adenosine (8-Chloro-adenosine, 8-Cl-Ado) kills human myeloma and leukemia cell lines. When these cells are treated with 8-Cl-Ado, the cellular energy, i.e. ATP, declines. Also, by incorporating it into RNA (a transmitter of genetic information from gene to protein), this agent inhibits transcription of genes needed for survival of CLL cells. These features make 8-Cl-Ado an ideal drug to target CLL cells.

Our hypothesis is that because of these actions, which are DNA independent, quiescent CLL lymphocytes will undergo death. We plan to understand metabolism and mechanism of action of this agent in leukemia cells that are freshly obtained from peripheral blood of CLL patients. We will compare all our data in CLL lymphocytes with normal lymphocytes. We have IRB-approved LAB protocols to get blood from CLL patients and healthy donors. Eventually, we will use this knowledge and test this compound in the clinic for patients with CLL. With that respect, we have finished all the toxicology and pharmacology studies with this agent and are currently doing IND-directed toxicology to seek an FDA approval to use this agent as investigational new drug in the clinic for patients with CLL. We already have an IRB approved phase I protocol to use this agent for CLL patients.

T

CLL is characterized by a disrupted cell death pathway rather than increased rate of proliferation. Because these cells do not divide, they do not synthesize DNA, or go through cell replication. Hence, agents that are DNA replication-directed do not work well for this disease.

We focused on developing chemotherapy that is not directed to DNA. We were able to determine metabolism and mechanism of action of a new agent, 8-chloro-adenosine, in CLL cells. We used cell samples freshly obtained from peripheral blood of patients with CLL. We compared this drug in CLL lymphocytes and in normal lymphocytes. Our data demonstrated that the drug works in a DNA independent way in CLL cells, and in laboratory experiments, the drug was effective in killing CLL cells. We have published two papers from this work during the first 18 months.

Because multiple myeloma is also a B-cell neoplasm and the plasma cells are not actively dividing, we hypothesized that 8-Cl-Ado would work in this disease also. Our data demonstrated that multiple myeloma cell lines treated with 8-Clo-Ado results in a decrease in Met transcript and protein levels. We further established that Met tyrosine kinase is a survival factor for these cells and once there is a decrease in the protein, cells undergo cell death. This was recently published in Cancer Research.

Using separate funding sources, we conducted Investigational New Drug (IND) directed toxicology studies and have now received IND approval from the FDA to move 8-Cl-Adenosine in the clinic for patients with CLL.

Chronic lymphocytic leukemia (CLL) patients can be divided into stable and aggressive disease categories. Patients with aggressive CLL often become drug resistant and have short overall survival times. CLL cells that contain the ZAP-70 protein tend to be closely associated with aggressive disease. Currently there are no targeted treatments designed for this aggressive form of CLL. In this study, we shall assess whether the drug gefitinib can selectively kill ZAP-70 expressing CLL cells. Gefitinib is currently used to treat lung cancer, but has potential to be effective against CLL. We will also evaluate the ability of gefitinib to work in combination with standard chemotherapy to kill these cells by inhibiting ZAP-70 function.

No update is available at this time. Please check back soon.

The malignant cells in CLL accumulate mostly due to deficiencies in the regulation of cell death. Phosphorylation of tyrosine residues in proteins turns many proteins on and off. It was the first discovered mechanism of signal transduction inside the cell. A family of enzymes, Src-family kinases (SFKs) catalyzes this protein modification step. Since they carry a fatty acid residue, SFKs are usually located close to the inside of the cell membrane and transduce the signals resulting from the stimulation of cell surface receptors that sometimes lack own enzyme activity.

We are going to investigate the influence of B-cell receptor (BCR) stimulation on signaling processes that might determine whether tumor cells survive or die. Another transmembrane receptor CD5, which is always present on CLL cells, modulates BCR signaling. Both BCR and CD5 are known to interact with at least two SFKs, namely Lyn and Lck, respectively. Our goal is to understand and to correct aberrant signaling processes by interrupting protein-protein associations between cell surface receptors and SFKs by means of cell-permeant peptides.

Chains of less than 20 amino acids are chemically synthesized and contain the fatty acid, myristic acid, which enables the molecules to cross membranes. In cell cultures treated with such membrane-permeant lipopeptides we will examine their effects on the phosphorylation status of certain signaling proteins, on the ability of SFKs and receptors to associate with each other and finally on biological phenomena, e.g. cytokine production and frequency of cell death. The sequence and structure of peptides blocking survival pathways is expected to yield valuable information for developing small molecule drugs.

Src family kinases (SFKs) appear to be involved in the transduction of signals from the microenvironment of CLL cells to the inside. Therefore, we proposed to study the role of SFKs in CLL cells and tested the effects of small molecule kinase inhibitors on CLL cells freshly isolated from patients´ blood samples.

With dasatinib, a Src/Abl inhibitor that is already successfully used for treating chronic myeloid leukemia, we observed surprisingly clear-cut inhibition of SFK activity in CLL cells. Treatment with dasatinib killed freshly isolated CLL cells and model cell lines. Cell death induction was strongest in CLL cells belonging to unfavorable prognostic subgroups. In summary, our findings in cell culture systems suggest the study of dasatinib for treating CLL in combination with other drugs.

Cancer (including its resistance to therapy) is to a large degree associated with the abnormal cellular accumulation of reactive oxygen species (ROS). Although these ROS are by-products of normal cellular processes, they can also act as harmful mediators of genome damage, which in turn can lead to the development of tumors. A normal cell protects itself by buffering ROS or by inducing apoptosis (cell suicide) if ROS levels increase over a certain toxic threshold. This is done to prevent DNA damage from being inherited to a thereby potentially cancerous daughter cell. In CLL, these sensitive regulatory mechanisms seem to be out of order.
We have demonstrated in CLL that ROS are elevated and that ROS buffers are decreased as compared to normal blood cells. Therefore, we linked ROS to be important for CLL development, but postulated that even in these leukemic cells, a certain toxic threshold exists, above which ROS levels become intolerable. In CLL, both the basal ROS levels and this toxic threshold are elevated. This prompted us to test whether this feature could be turned into CLL’s “Achilles’ heel” via further increase of ROS selectively in the leukemic cell by exploiting its already high ROS burden. Consequently, we designed, synthesized, and pre-screened a panel of ROS-producing catalysts, which have enhanced activity when more basal ROS are present in a cell.* By inducing ROS levels that exceed the toxic threshold, these catalysts should mediate preferential tumor cell toxicity as compared to normal cells.

We have already generated very promising data on the killing efficiency and selectivity of our ROS catalysts. Here, we refine this targeting strategy of abnormally high ROS levels for selective anti-CLL activity. Our mid-term goal 1) is to finish the pre-clinical efficacy testing of the best performing chemically optimized ROS catalysts. We further attempt to gain better knowledge on 2) the molecular modes of ROS accumulation and catalyst mediated killing; 3) which CLL subsets are the most sensitive to our novel reagents; and 4) the synergistic cooperation with established conventional drugs including their combined testing in CLL animal models.

* concept, compound design and synthesis originated from the laboratory of collaborator C. Jacob at Saarland University, Germany

No update is available at this time. Please check back soon.

The clinical behavior of chronic lymphocytic leukemia varies among patients. It is likely that part of the clinical difference is influenced by inherited genetic factors common in the population. We shall conduct a detailed analysis looking at variation in the human genome to tease out clinically useful markers of outcome. The identification of prognostic markers should allow for more accurate assessments and be helpful in the choice of therapeutic options. Specifically, these markers should allow for the prediction of likelihood of response and the potential of adverse reactions to chemotherapeutic treatments. This will allow patient-tailored decisions on drug selection and ultimately improvements in outcome.

We propose to conduct a genome-wide scan of single nucleotide polymorphisms (SNPs) to identify variants influencing the clinical behavior of CLL. The study will be based on an analysis of 10,000 non-synonymous SNPs (nsSNPs). SNPs alter the encoded amino acid sequence and have the potential to directly affect the function of expressed proteins, thereby providing a powerful strategy for identifying influential factors.

To generate unbiased findings we will analyse DNA from 400 patients entered into a large phase III trial (CLL4). The trial is comparing the purine analogue flurdarabine used alone or in combination with cyclophosphamide as a first-line treatment. Our analysis will examine the relationship between genotype and overall survival as well as the relationship between genotype and progression free survival, time to progression and toxicity.

Useful Definition:
single nucleotide polymorphisms (SNPs): a common DNA sequence variation among individuals that can help determine the likelihood that someone will develop a particular disease

We are conducting a genome-wide scan of single nucleotide polymorphisms (SNPs) to identify variants influencing the clinical behaviour of CLL. To generate unbiased findings, we are evaluating the genetic makeup of 10,000 SNPs DNAs from patients entered into a large phase three trial, CLL4. CLL4 is the most comprehensive trial thus far conducted in the UK and incorporates several important elements in addition to establishing the possible benefits of the purine analogue flurdarabine used alone or in combination with cyclophosphamide as a first-line treatment for patients. In addition to examining the relationship between genotype and overall survival the analysis will allow us to examine the relationship between genotype and progression free survival, time to progression, and toxicity.

Progress has been in line with that anticipated. To date we have completed all genotyping and are engaged in relevant statistical analyses. Any associates between genotype and clinical outcome will be validated through collaborations with other researchers engaged in similar work. To this end we have already established collaborations with two research groups who have access to large datasets. Ultimately, it will be highly desirable to establish the biological basis of any significant associations.

Significant progress has been made in recent years in our understanding of the biology and clinical treatment of chronic lymphocytic leukemia (CLL). However, many challenges still remain. Development of drug resistance to current anti-CLL drugs remains a significant problem in the treatment of disease. Thus, identification and development of new compounds that preferentially kill CLL cells with relatively low toxic effect on normal cells are important research tasks. Our recent studies showed that CLL cells exhibit certain abnormalities including high frequency of mutations in mitochondrial DNA, aberrant mitochondrial biogenesis, and increased generation of reactive oxygen species (ROS). These abnormalities appear to be associated with less sensitivity to fludarabine, a drug of choice from frontline treatment of CLL.

Based on our study and understanding of energy metabolism in cancer cells, we speculate that such molecular and biochemical alterations in CLL cells may be exploited for therapeutic benefits, and new drugs can be designed to preferentially target these abnormalities. We hypothesize that malfunction of mitochondria (the cellular organelle that normally produce ATP as the energy source for the cells) may render CLL cells highly dependent on the alternative ATP generation pathway known as glycolysis, and that inhibition of this alternative pathway would cause a severe depletion of ATP in CLL cells, leading to the death of these cancer cells. On the other hand, normal cells may be able to better tolerate glycolytic inhibition due to normal mitochondrial functions.

Our laboratory has synthesized a novel compound named glycolycin that effectively inhibits glycolysis. Preliminary lab studies showed that this compound effectively depletes cellular ATP and kills CLL cells. Importantly, glycolycin remains active in CLL cells that have become resistant to fludarabine. In this research project, we propose to evaluate the anti-CLL activity of glycolycin and validate the drug target in CLL cells, to test the in vivo therapeutic activity of glycolycin in CLL animal model, and to conduct animal toxicology study to determine the toxicity/safety profiles of this compound. We anticipate that this research project will generate important data leading to the development of a novel anticancer agent for the treatment of CLL and other cancers, and provide important information necessary for the design of future clinical trials.

There are three major objectives in this research project. First, we proposed to test the anti-CLL activity of a novel compound known as glycolycin in cell culture and to validate the drug target. Second, we planned to test the potential therapeutic activity of glycolycin in animal models. Third, we proposed to evaluate the potential toxic effects of this compound in animals. The information from this research project is important for future development of glycolycin as a new agent for cancer treatment.

Overall, during the grant funding, we have achieved most of our research objectives, as proposed in the original specific aims, and in some areas exceeded the original goals:
(1) We found that glycolycin is effective in killing CLL cells in culture, and can overcome multi-drug resistance in leukemia cells.
(2) The combination of glycolycin with another drug, rapamycin, had synergistic effect against leukemia cells.
(3) We verified hexokinase II as a key drug target in CLL. Hexokinase II is an enzyme important for glycolytic metabolism.
(4) Using another cancer cell model, we discovered that glycolycin was effective in elimination of residual cancer cells resistance to traditional anticancer agents. Because this subset of malignant cells may play an important role in the persistence of residual disease and drug resistance, the ability of glycolycin to kill this subpopulation of cancer cells may have significant clinical implications.
(5) We have developed a new formulation of P-glycolycin, which is important for drug administration in animals, to test the in vivo therapeutic activity of this compound.
(6) Preliminary dose-finding and toxicology studies in mice have been performed, and the results show that P-glycolycin is tolerable in mice without significant toxicity, at the dosage that has anticancer activity. However, comprehensive animal toxicology studies still remain to be completed.

Although significant progress has been made in clinical treatment of CLL, many CLL cells, especially those with certain genetic abnormalities, are resistant to currently available chemotherapeutic agents such as fludarabine and cyclophosphamide. This imposes a major challenge in clinical treatment of CLL, and underscores the urgent need to identify and develop new agents for effective treatment of refractory CLL. The main goal of this study is to test the therapeutic potential of a natural compound known as ?-phenethyl isothiocyanate (PEITC) for treatment of CLL. Our previous studies showed that CLL cells isolated from patients, especially those in advanced disease stages, exhibit increased stress of reactive oxygen species (ROS) and are vulnerable to compounds that cause further ROS accumulation in the cells.

More recent work further showed that PEITC preferentially kills malignant cells with ROS stress through a ROS-mediated mechanism. Based on these observations, we propose to test the hypothesis that the increase in ROS generation in CLL cells may render them highly sensitive to PEITC, whereas normal lymphocytes with low ROS output are less vulnerable to this compound. We will also compare the cytotoxic activity of PEITC in CLL cells that are either sensitive or resistant to fludarabine, and test the possibility that primary CLL cells from patients in advanced stages refractory to fludarabine-based therapy still remain highly sensitive to PEITC due to their increased ROS generation.

The cause-effect relationship between ROS stress and drug sensitivity will be examined. We will also develop mechanism-based drug combination strategies to enhance the therapeutic activity of PEITC against CLL cells. The main purpose is to identify proper agents for combination with PEITC to obtain optimal activity. The therapeutic activity of PEITC, alone or in combination with other drugs, will be further evaluated in an animal model that mimics human CLL disease. We anticipate that this study will provide important information on the in vitro and in vivo activity of PEITC against CLL, and serve as a basis for the future design of clinical trials to use PEITC for treatment of CLL patients.T

During the first 9 months of this grant period, we have largely accomplished the objectives of the research project as originally proposed. Specifically, the proposed studies using primary CLL cells from pateint blood samples to test the therapeutic activity of PEITC alone or in combination with other drugs in cell culture have been accomplished while the animal study is still ongoing and is expected to be completed on schedule as originally planned. Our studies demonstrate the promising therapeutic activity of PEITC against CLL cells, especially the CLL cells resistant to standard anit-CLL drugs.

Our research on PEITC resulted in two new publications in 2008. In addtion, we have expanded our research beyond the original specific aims and showed that PEITC was also effective in killing drug-resistant chronic myoliod leukemia (CML) with gene mutations. Overall, our research project is moving forward as planned, and in some areas exceeded the original goals. Our research should have significant application in clinical treatment of CLL.

In CLL patients, the normal tissue stromal cells may interact with the leukemia cells and promote the ability of CLL cells to survive and resist drug treatment. This poses a major challenge in CLL treatment due to the difficulty in the body to eliminate the leukemia cells protected by the tissue stromal cells. Thus, it is extremely important to develop new drugs that can effectively kill CLL cells in the tissue environment.

We recently discovered a compound called selecticine which is capable of preferentially killing CLL cells in the presence of stromal cells. This compound exhibits only moderate toxicity to CLL cells when cultured without stromal cells. Importantly, selecticine shows minimum toxicity in normal cells.

Based on these observations, we propose to investigate why selecticine is highly potent against CLL cells when stromal cells are present, and how stromal cells may sensitize CLL cells to this compound. In particular, we will test if stromal cells might convert selecticine to a compound that is selectively toxic to CLL cells, and will also test if selecticine can be combined with other anti-CLL drugs to improve their anticancer activity. It is expected that this study will have significant implications in clinical treatment of CLL.

No update is available at this time. Please check back soon.

Currently there is no cure for this very common leukemia, and despite the word “chronic” in its title, the disease is very frequently progressive. When this happens, traditional therapies are effective for inducing responses, but this has yet to translate to cures in the majority of patients. In addition, there are frequent complications of traditional therapy in this cohort of patients including further suppression of immunity.

We have defined a biochemical pathway mediated by a circulating protein designated as vascular endothelial growth factor (VEGF). VEGF is secreted by CLL B-cells and not only increases the leukemic B-cell resistance to death but may play a role in a phenomenon known as an “angiogenic switch.” This latter feature has been strongly associated with disease progression and metastases in human solid tumors.

Happily, there are newer agents available that can interrupt this pathway and delay disease progression. We have tested several of these drugs and found one, epigallocatechin (EGCG), a major component of green tea, which can kill significant numbers of leukemic B-cells in at least 80 percent of CLL patients. Even more importantly, this drug has been tested in healthy volunteers and is found to be safe. The formulation is designated as polyphenon E and contains EGCG. This is now being tested at the National Cancer Institute and will be made available to us for testing.

Based on our preclinical work and the recent testing and availability of polyphenol E, we have designed a clinical trial for early stage CLL. This trial will be accompanied by correlative laboratory work designed to test two primary aspects: 1) to stratify CLL patients by risk parameters to see if responses to EGCG relate to risk and 2) to assess critical components of the VEGF pathway to see if responses induced by EGCG are associated with interruption of the pathway.

The Mayo Clinic Trial of Polyphenol E for patients with asymptomatic, early stage CLL opened to accrual at the end of August 2005. 36 patients have been accrued to the trial to date. Of the 33 evaluable patients, few have experienced significant side effects. After additional follow-up is available for the 3 remaining patients, we will proceed with the second phase of the study during which 34 additional patients will be treated at the optimal dose level derived from the first phase of the study.

Only preliminary data on the effectiveness of this treatment is available to date. Among the first 33 patients treated, 25 (76%) have had at least a small decline in their circulating leukemia cell count with 10-40% of those patients experiencing a more substantial decline in circulating leukemia cell count. Although the durability of these benefits has varied between patients, some have experienced significant reduction in the size of enlarged lymph nodes. In some patients, the nodes persist at the end of 6 months of treatment, while in others they have been transient. An abstract detailing the results of the Phase I portion of the trial was submitted for presentation at the ASH 2007 Annual Meeting. Finally we have prepared a full manuscript completely detailing the clinical and toxicity profiles for this initial phase of testing the compound Polyphenol E.

Background and Rationale: Angiogenesis (or the formation of new blood cells) plays a crucial role in the survival of CLL B -cells. Abnormal angiogenesis was found in marrow sections of B-CLL patients and significantly higher levels of bFGF and VEGF in the urine of B-CLL patients. In support, it was found that abnormal marrow angiogenesis was positively associated with advancing stage of CLL. In addition, it is also found that a VEGF based pathway can generate signals that lead to increased resistance to both spontaneous and drug induced apoptosis in CLL. Preliminary work has indeed shown that we can induce significant apoptosis with a variety of anti-angiogenic agents, but we wish to increase the ability to do this with use of more advanced and effective delivery systems. To do this we have now focused on the new technology of nanoscience.

Advantage of Nanotechnology: Nanotechnology has the potential to change the very foundation of cancer treatment, detection and diagnosis. The primary rational for selecting gold nanoparticles is their biocompatibility, very high surface area so that multiple drugs can be loaded, and ease of characterization and surface functionalization (easy to tether molecules of choice on the surface). When the drugs/antibodies will be delivered as nanoconjugates, they will have better efficacy with reduced toxicity.

Preliminary Results: In our preliminary studies we have seen significant enhancement (2-10 fold) in apoptosis induced by gold v-EGF antibody conjugates compared to VEGF antibody alone (N=5).

Specific Aims: In conjunction with our preliminary results, our specific aims include to test:
1. The effect of gold nanoparticles of different sizes to kill CLL B-cells.
2. The effect of gold-AVF nanoconjugates to kill the CLL B-cells compared to only AVF and finding out the mechanism of such activity.
3. The effect of gold-AVF bioconjugates in combination with chemotherapeutic agents to kill the CLL B-cells and elucidate the mechanism of action.

Useful definitions
Angiogenesis: The formation of new blood cells

In an effort to develop both greater insight into the disease, and come up with alternative treatment strategies, we began to assess the role of angiogenesis (the formation of new blood vessels) in CLL. Preliminary work has indeed shown that we can induce significant apoptosis with a variety of anti-angiogenic agents (i.e. avastin). However, a very high dose of anti-angiogenic agents was required, and unfortunately, they are associated with serious toxicities. We hypothesized that, when delivered in a targeted fashion with gold nanoparticles, these anti-angiogenic agents will show better therapeutic efficacy with reduced systematic toxicity.

We have shown that the anti-VEGF antibody, discussed in the original abstract, when conjugated to gold nanoparticles is far more effective in inducing apoptosis in primary CLL B-cells, as compared to anti-VEGF or gold nanoparticles alone. These findings reinforce the advantages of using gold-nanoparticle-based drug delivery systems in human malignancies. We continue to develop this system to combine other known chemotherapeutic or monoclonal agents to these nanoparticles as well.

In one year, we published three papers and submitted two invited reviews (in press). We also received extramural funding from the state of Minnesota to advance cancer nanotechnology.

Antibodies that bind to cancer cells show promise as new agents to treat the cancer.  The overall goal of the proposed research is to develop new therapeutic monoclonal antibodies specific to FCRL5, a protein highly expressed on the surface of CLL cells.

A therapeutic antibody can directly block the growth of cancer cells through initiation of intracellular signaling.  Alternatively, a therapeutic antibody can cause host proteins or white blood cells to the attack and kill the cancer cells.  Most therapeutic antibodies exhibit multiple modes of action.
Monoclonal antibodies are most efficiently made in rodents.  For therapeutic use of a rodent antibody in humans, the antibody must undergo “humanization” through a process called antibody engineering.  This is necessary to reduce unwanted immune reactions to the rodent antibody.  Antibody engineering is also used for the enhancement of antibody effector functions to produce stronger indirect therapeutic effects.

We have produced anti-FCRL5 mouse monoclonal antibodies and demonstrated frequent and high level expression of FCRL5 proteins on patients’ CLL cells.  Recently, we also discovered that treatment with our anti-FCRL5 antibodies induce intracellular signaling in human peripheral B-cells and B-cell lines.

In this project we will (1) analyze intracellular molecular events induced by anti-FCRL5 stimulation and (2) use antibody engineering to humanize our anti-FCRL5 monoclonal antibody and to enhance its indirect effector functions.

No update is available at this time. Please check back soon.

Most patients with chronic lymphocytic leukemia (CLL) are asymptomatic at diagnosis and wish to know the likely natural history of their disorder. ZAP-70 is signaling molecule essential for the function of a subset of normal white blood cells (T-lymphocytes). It is also expressed in the leukemic cells of a subset of patients with CLL and correlates with a shorter time from diagnosis to first treatment and with shorter survival. There is increasing evidence that ZAP-70 expression contributes to the poorer prognosis of ZAP-70 positive cases and is not simply a marker of aggressive disease. Recent data suggest that ZAP-70 may also be a target for therapy.
Most laboratories measuring ZAP-70 expression use a flow cytometric assay, which is widely available, rapid and inexpensive. Unfortunately this assay is not standardized such that a sample may be considered positive for ZAP-70 in one laboratory and negative in another.

We have shown that a modification of the ZAP-70 gene (DNA methylation) correlates closely with ZAP-70 expression measured by flow cytometry. The methylation assay is not subject to many of the factors such as sample age, which can affect the flow cytometric assay. However, the methylation assay we have used so far is only semi quantitative and gives an equivocal result in about 6% of cases.

We propose to use a rapid, sensitive and fully quantitative method (Pyrosequencing) for detecting ZAP-70 methylation. The prognostic value of this assay will then be tested retrospectively in a cohort of 300 patients with prolonged follow up, managed at a single center, and prospectively in 300 patients entered into the UK CLL4 randomized trial for previously untreated patients. Multivariate analysis will be employed to determine whether ZAP-70 methylation provides additional information to known prognostic factors.

We have tested DNA for a chemical change (methylation) in the ZAP-70 gene from the leukaemic cells of 630 patients with either early disease or more advanced CLL requiring treatment. The presence of methylation correlates closely with stable disease and with good response to treatment. This assay is comparable in prognostic value to more widely used assays such as measuring ZAP-70 expression, by flow cytometry, and the mutational status of immunoglobulin VH genes.

By assessing the methylation status of two regions of the ZAP-70 gene, we can obtain clear-cut results in 90% of patients. Further studies are in progress to identify the reason why equivocal results are found in the remaining 10% of patients and to modify the assay so that all patients can be classified. Once achieved, this assay will be particularly suited for testing large numbers of samples, such as those from multi-center trials. The methylation status of other genes is also being evaluated and their prognostic significance determined.

The B-cell receptor (BCR) is a specialized protein that sits on the surface of chronic lymphocytic leukemia (CLL) cells. It can “sense” cues from outside the cell and then trigger responses within the cell that promote the survival and growth of leukemic cells. The BCR also communicates with other proteins on the surface of CLL cells, including CXCR4, which helps to control how CLL cells move around the body. The project will investigate the detailed mechanisms that CLL cells use to allow communication between the BCR and CXCR4. By studying these pathways, we hope to identify markers that can be used to select optimal treatment regimens for patients. It may also be possible to develop new treatment strategies to interfere with BCR/CXCR4 communication.

No update is available at this time. Please check back soon.

B-cell chronic lymphocytic leukemia (B-CLL) results from an expansion of a rare population of mature B-lymphocytes. In human B-CLL, expression of the TCL1 gene is abnormally increased in the aggressive form of B-CLL. This increased TCL1 expression in mice results in a disease similar to the aggressive form of human B-CLL. Our investigation of the function of TCL1 led to the discovery that TCL1 acts in the same pathway as Akt, a molecule that regulates several targets critical for malignant transformation of normal cells.

In preliminary studies we determined that we can pass mouse B-CLL to other mice. Therefore we can treat the same B-CLL in a number of mice. We further investigated the regulation of TCL1 expression by microRNAs (miR), small molecules that inhibit TCL1. We found that TCL1 is inhibited by two such molecules: miR-181 and miR-29. These molecules may be excellent candidates to use in the treatment of TCL1-driven B-CLL. Our goal is to establish microRNAs as novel therapeutic agents for B-CLL.

We propose to verify that miR-181 and miR-29 inhibit TCL1 protein expression and determine whether this effect is cumulative. We intend to produce a TCL1 transgenic mouse line specifically designed to treat B-CLL with miR-181 and miR-29. We will further determine if treatment with miR-181 and miR-29 prevents or cures transplanted B-CLL in these mice.

Useful Definitions:
TCL1: a human gene that is associated with the development of T-cell and B-cell leukemias and lymphomas
MicroRNAs: small molecules that interfere with the ability of a gene to manufacture a protein. These molecules can regulate the expression of many genes and may play a role in establishing a treatment for B-CLL.

As we stated in our original application, our goal is to establish microRNAs targeting TCL1 as therapeutic agents for B-CLL. MicroRNAs are small molecules that interfere with the ability of a gene to manufacture a protein. TCL1 is a human gene associated with the development of T-cell and B-cell leukemias and lymphomas. During this grant, we are hoping to verify that two specific microRNAs can inhibit TCL1 protein expression, and to determine if these molecules could prevent or cure transplanted B-CLL in mice.

During the first year of this grant, we proposed to establish a mouse model suitable to test if microRNAs could be used as drugs in B-CLL, and to verify that the two small molecules, miR-181 and miR-29, regulate Tcl1 protein expression. Since funding started, we successfully achieved these two goals. We produced a gene-modified mouse line. We also investigated, and proved, that microRNAs regulate the Tcl1 protein. We found that Tcl1 is inhibited by the two molecules, miR-181 and miR-29. These findings were published in Cancer Research. Since miR-181 and miR-29 are natural TCL1 inhibitors, these two miRNAs are excellent candidates to evaluate in the treatment of Tcl1 driven B-CLL.

Deletions of chromosome 13, specifically the 13q14 region, are the most common genomic alterations in CLL. It was previously reported that two small molecules miR-15 and miR-16 are located in this deleted region. Furthermore, miR-15/16are reduced in expression in most CLL cases. The loss of these two small molecules plays an important role in the initiation of CLL.

We recently investigated another molecule, DLEU7, which is located in the same deleted region as miR-15/16 and may also play an important role in the development of CLL. In our recent publication in Blood we demonstrated that DLEU7 expression is decreased in CLL when compared to normal cells. We also found that DLEU7 normally functions as a potent inhibitor of pathways previously shown to be important in CLL (NF-kB and NFAT pathways). In addition, overexpression of DLEU7resulted in the death of tumor cells. Our results are consistent with previously reported observations that DLEU7 essentially is not expressed in CLL. Thus, we hypothesize that DLEU7 may cooperate with miR-15/16 to function as a tumor suppressor.
To test this hypothesis we propose three specific aims for our CLL Global project. Aim 1. We will delete DLEU7 in mice and determine whether these mice will develop CLL or other hematological malignancies. Aim 2. We will combine DLEU7deletion with miR-15/16 deletion in mice and determine whether they cooperate in the initiation of CLL. Aim 3. We will clarify on the molecular level how inactivation of DLEU7 contributes to CLL. Our experiments will establish DLEU7 as a target for developing therapeutic approaches for CLL.

No update is available at this time. Please check back soon.

Patients with CLL that have a loss of the short arm of chromosome 17 (17p deletions) fail to respond to standard treatment and generally have very poor outcomes. This is due to the loss of the tumor suppressor p53, which maps to 17p-. In patients that lose the short arm of chromosome 17 (and therefore lose one copy of p53), the remaining p53 copy is mutated. p53 mutations are detected in 7–9% of newly diagnosed patients with CLL and in 30-40% of patients who have failed frontline chemoimmunotherapy. Recently, ibrutinib, an inhibitor of the Bruton’s tyrosine kinase (Btk), has shown preliminary activity in CLL with 17p-.

In our project, we will take advantage of the power of a genetically engineered mouse. We will use a mouse model of CLL (TCL1) with or without p53 mutations to study the impact of the mutation on response and survival to conventional (fludarabine) or Btk inhibitor (ibrutinib) therapy. In addition to the mouse model, we will study the signaling and activation response of B-CLL cells obtained from 17p- patients receiving ibrutinib in a clinical trial at our facility. The results from this study could be extended to other high-risk B-CLL patient populations and may facilitate combination approaches using ibrutinib.

No update is available at this time. Please check back soon.

The genome of every cell in the human body contains the same DNA and same genes, with differences in the repertoire of genes active versus those silenced constituting the basis for the hundreds of different types of cells that make up the human body. During development of cancers and leukemias, the regulation of the genome becomes altered. DNA-binding proteins are responsible for dictating which genes are active versus silence. NF- B proteins regulate the activity of genes involved in immune system function. These gene regulators become dysfunctional in aggressive Chronic Lymphocytic Leukemias (CLLs), driving leukemic cell growth and protecting leukemic cells against chemotherapy and immunotherapy.

Our proposal seeks to identify, characterize, and optimize chemicals that inhibit NF- B activity, but which do so in a way that preserves many of the normal functions of NF- B required for immune defense against bacteria and viruses, while thwarting the adverse effects of NF- B that contribute to aggressive behaviors of CLL. By performing screens of large collections of chemicals using robotic instrumentation and high-throughput technologies, we have identified candidate chemical inhibitors of NF- B.

The aims of this project are to: (1) complete the characterization of these chemical inhibitors of NF- B; (2) optimize the potency of the chemicals; (3) explore the activity of these inhibitors against CLL cells removed from the blood of patients; (4) perform experiments to understand how the chemical inhibitors of NF-kB behave in the body and adjust their properties to achieve desirable properties for eventual clinical use; and (5) test our best chemical inhibitors in mouse models of CLL. Altogether, these studies will lay a foundation for eventual human clinical testing.

No update is available at this time. Please check back soon.

Patients with chronic lymphocytic leukemia (CLL) present with a variable clinical course that is hard to predict for physicians, particularly at diagnosis or early stages of the disease. In studies performed at the UT MD Anderson Cancer Center we have recently discovered gene-signatures which can be tested in peripheral blood leukemic cells of patients and which  enabled us to predict time-to-treatment  and overall survival in previously untreated CLL patients. Specifically, expression levels of only 2 and up to 13 genes allowed us to identify subgroups of patients with an unfavorable prognosis.

In the present study, we will validate these gene signatures in an independent study cohort of patients treated and followed outside of the MD Anderson Cancer Center, specifically at the University of Cologne (Germany) and other collaborating German centers.  Particularly, we will explore the practical and clinical utility of such gene signatures for diagnostic testing purposes.  Upon validation, we will further investigate those genes which performed as the strongest predictors of outcome on a (sub-)cellular level. We will study functional consequences of their dysregulation in leukemic cells, for example with respect to cellular survival, growth and migratory behavior. Ultimately, the gained knowledge of this project will allow us 1) to establish new gene-based methods to perform diagnostic outcome prediction from peripheral blood and 2) to biologically characterize new molecular key and target molecules in CLL.

No update is available at this time. Please check back soon.

CLL is a disease with a highly variable clinical course; some patients die from the disease, whereas others live for many years without problems. CLL is characterized by an increased white blood cell count with the expansion of lymphocytes in the peripheral blood, bone marrow and lymphoid organs. Genomic aberrations (chromosome abnormalities) are found in the CLL cells in almost all cases and define clinical subgroups. The response and survival time after chemotherapy and the antibody rituximab are poor in some CLL patients, while the antibody alemtuzumab is effective in these cases. The differential mechanisms of the response to the diverse agents are not well understood but there is an association with specific genetic defects of the CLL cells (17p deletion, p53 mutation).

To gain better insight into the different mechanisms of cellular response to therapy, CLL cells were collected and individually treated with chemotherapeutic agents or the antibody alemtuzumab. The treatment with chemotherapy led to induction of cell death, with significantly lower rates of dead cells in some of the cases with specific genomic aberrations. Treatment with the antibody alemtuzumab induced cell death in all CLL cases independently of the genomic aberrations.

We plan to extend these preliminary analyses to approximately 50 cases to gain more reliable results. The samples will be studied for different prognostic markers (genomic aberrations, VH mutations status and ZAP-70 expression) and will be related to the results after different treatments. The response to treatment and mechanisms of induction of cell death will be examined in the different risk-groups. Protein expression levels of candidate genes involved in cell growth and cell death (apoptosis) control will be studied to identify different pathways leading to cellular response or resistance after treatment with chemotherapeutic agents or antibody therapy.

In addition, we plan to analyze blood samples of CLL patients during therapy to compare the cell culture results with patient responses and to gain detailed information on the mechanism of cell death in different biological CLL risk groups. Overall, the results of this study should allow a detailed insight into the mechanisms of action of different therapeutic agents and form the basis for the development of novel treatment strategies in CLL.

Genomic aberrations (chromosome abnormalities) are found in the CLL cells in almost all cases. The presence of certain genomic aberrations defines clinical subgroups. To gain better insight into the different mechanisms of cellular response to therapy, CLL cells were collected from patients and individually treated with chemotherapeutic agents or the antibody alemtuzumab. Protein expression levels of candidate genes, involved in cell growth and cell death control, were studied to identify different pathways leading to cellular response or resistance after treatment.

Specific changes in protein regulation could be detected in response to treatment with the chemotherapy agents, fludarabine and etoposide, respectively. No changes in protein expression were found upon treatment with alemtuzumab; the mechanism of action of alemtuzumab seems to be a mixture of different ways of cell death and complementary, dependent toxic effects on cells. T-cells seem to play a minor role in this setting.

Infections are a major cause of illness and death in patients with CLL. These patients are predisposed to infectious complications due to immune compromise inherent to the leukemia itself. In addition, chemotherapy, corticosteroids and/or monoclonal antibodies that are commonly used as treatment of CLL further predispose these patients to severe, often fatal infections. Despite advances in supportive care and the widespread use of antibacterial and antiviral prophylaxis, infection still accounts for over 60% of deaths in patients with CLL. Low levels of natural antibodies that fight infections in the serum (immunoglobulins) and low numbers of some important immune cells (e.g., white cell count) have long been considered to be major predisposing factors for severe, life-threatening infections in this patient population. However, persistent qualitative deficiencies in cellular immune function might equally be as important, since these immune cells are the initial critical line of defense protecting patients from catastrophic infection from a broad spectrum of pathogens.

Currently available laboratory tests can detect only quantitative deficiencies in immune cells and immunoglobulins through blood tests. However, these tests do not provide information on qualitative defects of cellular immunity critical for identifying patients who are at high risk of for serious infectious despite near normal cell counts. This pilot study, first of its kind, attempts to characterize the qualitative immunodeficiency in CLL patients, and focuses on differences in their phagocytic killing against key bacterial pathogens and fungi that frequently lead to death in CLL patients. Our long-term goal is to use data from this study to develop unique diagnostic tools that will enable clinicians to effectively identify patients who are at the highest risk for catastrophic infection. These patients could then be treated more aggressively with antibacterial, antifungal and antiviral prophylaxis to prevent death from an undiagnosed infection.

Infections are a major cause of illness and death in patients with CLL. This pilot study, first of its kind, aims at evaluating the qualitative deficiencies in the white blood cells of patients with CLL. White blood cells are the most important line of defense against a broad spectrum of bacterial and fungal pathogens.

Currently, there are no laboratory tests to detect functional deficiencies in the white blood cells. In the first 20 months of the study, after obtaining regulatory approval from our institution, we established reliable and reproducible functional neutrophil assays in the laboratory against the key bacterial and fungal pathogens afflicting patients with CLL. Our preliminary laboratory data is quite promising and suggests that CLL patients have neutrophils that have suboptimal responses to common pathogens that afflict the patient population, such as Pseudomonas aeruginosa and Staphylococcus aureus. We believe our pilot study will provide important data for a larger prospective study of monitoring functional immune status in CLL patients in an effort to reduce early deaths due to infections. We have already enrolled 24 patients and we anticipate completion of the study within 12-15 months.