IMPACT OF FUNDED RESEARCH:
Previously Funded Individual Grants
BIOLOGY
Grant Awarded in 2005
Jan. A. Burger, M.D., Ph.D.
University of Texas MD Anderson Cancer Center
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.
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.
Grant awarded in 2006
Nicholas Chiorazzi, M.D.
The Feinstein Institute for Medical Research
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.
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
Grant awarded in 2006
Silvia Deaglio, M.D., Ph.D.
University of Torino, Medical School (Italy)
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.
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
Grant awarded in 2006
Zeev Estrov, M.D.
University of Texas MD Anderson Cancer Center
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.
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
Grant awarded in 2009
Varsha Gandhi, Ph.D.
University of Texas MD Anderson Cancer Center
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.
Grant awarded in 2013
Asish Ghosh, BS, MS, Ph.D.
Mayo Clinic
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.
Grant awarded in 2009
Helen Heslop, M.D.
Baylor College of Medicine
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.
Grant awarded in 2007
Francisco Vega, M.D., Ph.D.
University of Texas MD Anderson Cancer Center
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.
GENETICS
Grant awarded in 2006
George Adrian Calin. M.D., Ph.D.
University of Texas MD Anderson Cancer Center
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.
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.
Grant awarded in 2011
George Adrian Calin. M.D., Ph.D.
University of Texas MD Anderson Cancer Center
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.
Grant awarded 2005
Claire Dearden BSc, M.D., FRCP, FPCPath
Royal Marsden Hospital/
Institute of Cancer Research (United Kingdom)
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
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.
Grant awarded in 2005
Guillermo Garcia-Manero, M.D.
University of Texas MD Anderson Cancer Center
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.
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.
Grant awarded in 2005
Paolo Ghia, M.D., Ph.D.
Instituto Scientifico San Raffaele (Italy)
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.
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.
Grant awarded in 2006
Dan Jones, M.D., Ph.D.
University of Texas MD Anderson Cancer Center
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
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.
Grant awarded in 2006 while Dr. Sampath was at MD Anderson Cancer Center
Deepa Sampath, Ph.D.
Ohio State University
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.
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.
Grant awarded in 2008
Stephan M. Tanner, Ph.D.
The Ohio State University
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.
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.
IMMUNOLOGY
Grant awarded in 2011
Renier Brentjens, M.D., Ph.D.
Memorial Sloan-Kettering Cancer Center
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.
Grant awarded in 2009
Laurence Cooper, M.D., Ph.D.
University of Texas MD Anderson Cancer Center
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.
Grant awarded in 2009
Gianpietro Dotti, M.D.
Baylor College of Medicine
Grant awarded in 2006
John Gribben, M.D., DSc
Barts Cancer Center of Excellence/
The London School of Medicine (United Kingdom)
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.
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.
Grant awarded in 2006
Stephen P. Mulligan, M.B., B.S., Ph.D.
University of Sydney (Australia)
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.
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.
Grant awarded in 2004
William G. Wierda, M.D., Ph.D.
University of Texas MD Anderson Cancer Center
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.
THERAPY/PROGNOSTIC
Grant Awarded in 2004 while Dr. Abruzzo was at MD Anderson Cancer Center
Lynne V. Abruzzo, M.D., Ph.D.
Ohio State University
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.
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.
Grant Awarded in 2009 while Dr. Abruzzo was at MD Anderson Cancer Center
Lynne V. Abruzzo, M.D., Ph.D.
Ohio State University
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.
Grant awarded in 2012
Xavier Badoux, MBBS, FRACP, FRCPA
University of Sydney, Royal North Shore Campus
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.
Grant awarded in 2012
Kumudha Balikrishnan, Ph.D.
University of Texas MD Anderson Cancer Center
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.
Grant awarded in 2013
Rosa Bernardi, Ph.D.
San Raffaele Scientific Institute
Ospedale San Raffaele, Milan, Italy
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.
Grant awarded in 2005
Januario E. Castro, M.D.
University of California, San Diego
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 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.
Grant awarded in 2008
Randall S. Davis, M.D.
University of Alabama at Birmingham
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.
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.
Grant awarded 2006
Alexander Dobrovic, Ph.D.
Peter MacCallum Cancer Centre (Australia)
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.
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
Grant Awarded in 2005
Varsha Gandhi, Ph.D.
University of Texas MD Anderson Cancer Center
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
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.
Grant awarded in 2011
Spencer Gibson, M.D.
University of Manitoba
Grant awarded in 2006
Michael Hallek. M.D.
University of Cologne (Germany)
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.
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.
Grant awarded in 2011
Marco Herling, M.D.
University of Cologne
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
Grant awarded in 2006
Richard S. Houlston, M.D., Ph.D.
Institute of Cancer Research (United Kindom)
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
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.
Grant awarded in 2005
Peng Huang, M.D., Ph.D.
University of Texas MD Anderson Cancer Center
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.
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.
Grant awarded in 2008
Peng Huang, M.D., Ph.D.
University of Texas MD Anderson Cancer Center
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
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.
Grant awarded in 2009
Peng Huang, M.D., Ph.D.
University of Texas MD Anderson Cancer Center
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.
Grant awarded in 2004
Neil E. Kay, M.D.
Mayo Clinic
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.
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.
Grant awarded in 2006
Priyabrata Mukherjee, Ph.D.
Mayo Clinic
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
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.
Grant awarded in 2011
Satoshi Nagata, Ph.D.
Sanford Research/University of South Dakota
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.
Grant awarded in 2006
David Oscier MA, MB, FRCP, FRCPath
Royal Bournemouth Hospital (United Kingdom)
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.
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.
Grant awarded in 2013
Graham Packham, Ph.D.
University of Southampton, United Kingdom
Grant awarded in 2006
Yuri Pekarsky, Ph.D.
Ohio State University
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.
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.
Grant awarded in 2012
Yuri Pekarsky, Ph.D.
Ohio State University
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.
Alfonso Quintás-Cardama, M.D.
University of Texas MD Anderson Cancer Center
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.
Grant awarded in 2008
John C. Reed, M.D., Ph.D.
Burnham Institute for Medical Research
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.
Grant awarded in 2012
Carmen Schweighofer, MD
University of Cologne (Germany)
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.
Grant awarded in 2006
Stephan Stilgenbauer, M.D.
University of Ulm (Germany)
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.
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.
SUPPORTIVE CARE
Grant awarded in 2005
Dimitrios P. Kontoyiannis, M.D., Sc.D., FACP, FIDSA
University of Texas MD Anderson Cancer Center
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.
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.