New Drugs Funded Projects

Important characteristics of CLL are that they lack the ability to conduct the immune surveillance function of their normal counterparts, and they are able to outlive their normal counter parts. The viability of CLL cells is governed by a family ofstructurally related proteins called the Bcl-2 family proteins. A major means that the Bcl-2 survival proteins extend the life of the CLL is by binding to other Bcl-2 family proteins that normally initiate the processes of cell death. Thus, the balance between pro-life and pro-death proteins determines whether the CLL cell will live or die. This has been the basis for the development of therapies, a few of which are now in the clinic. A missing portion of our understanding of how this balance is regulated is knowledge of how many of each of these proteins the CLL cell contains, and how this affects different responses to therapies. This application proposes to use a new assay that quantifies the amount of proteins in CLL cells to answer these questions It is known that genetic changes (mutations) cause the increased levels of the pro-life proteins in CLL. Thus a first approach we will take is to determine how each mutation affects the presence of the pro-life and pro-death proteins. We will then determine howthe ratios of pro-life to pro-death protein change in response to therapies directed at the ability of CLL cells to produce these proteins. It will also be important to learn how therapies directed to Bcl-2 family proteins interact with established CLL therapies. To translate these findings to the clinic, we will investigate responses of CLL cells to targeting the two the pro-life proteins in combinations. The results of these investigations will used to guide the design of clinical trials of combination..

Chronic lymphocytic leukemia (CLL) is a B-cell disease where patients show relentless accumulation of leukemic B-lymphocytes in bone marrow, blood, and lymph nodes. Unlike normal lymphocytes that have an 8-10 day life span, CLL cells survive for long time in human body. Reason for this prolonged life of these cells is the presence of Bcl-2 family anti-apoptotic proteins. Among the six members of Bcl-2 pro-survival proteins, Bcl-2 and Mcl-1 are most abundant proteins and have been shown to help in increased lifespan of these leukemia cells. One could postulate that if these proteins are targeted, the CLL cells will succumb to death. Bcl-2-selective antagonist, venetoclax, clearly demonstrated that when Bcl-2 is targeted, CLL cells undergo programmed cell death in preclinical setting. Neutralization of Bcl-2 function by venetoclax was also successful during early clinical trials resulting in approval of this agent. With these encouraging data, three new clinically testable Mcl-1 antagonists have been designed and developed. We hypothesize that direct neutralization of Mcl-1 protein using these drugs will induce apoptosis in CLL lymphocytes and will render these cells susceptible to other agents. Such knowledge will provide new avenues for therapeutic options. The three agents are S64315 from Novartis; AMG176 from Amgen, and AZD5991 from AstraZeneca. We have purchased AMG176 and AZD5991 and are negotiating with Novartis to get S64315. We have tested AMG176 and AZD5991 using in vitro investigations in primary CLL cells. Based on our preliminary data we propose to evaluate in vitro biological activity and mechanism of action of these agents in CLL cells. Next, we will focus on understanding mechanism by which these agents induce cell death. Finally, we will test combination of Mcl-1 antagonists with BCR pathway inhibitors such as ibrutinib and Bcl-2 survival protein antagonist venetoclax. We believe that these investigations may provide several new options to neutralize Mcl-1 protein and a novel treatment options for patients with CLL.

We propose to develop a comprehensive understanding of new CLL agents at an early stage of clinical development as CLL therapies. The underlying hypothesis is that the understanding of their mechanisms of action and also their interaction with other agents in the context of primary CLL cell biology will identify new targets, indicate informative biomarkers, and guide the optimal protocol design for CLL therapy.
Investigations will be conducted in primary CLL cells to maintain the metabolism, survival mechanisms, and stress responses that are unique to the tumor. Knowledge generated in this biological context will guide protocol design with respect to dose, rate, and schedule to maximize the killing of CLL cells. An understanding of the actions of these agents in CLL cells will form the rationale for the design of combinations that could have synergistic effect in killing CLL cells and overcoming resistance mechanisms.

Major breakthroughs in the treatment of CLL have come after the introduction of new drugs into clinical trials. The objective of this project is to understand the mechanism by which several new agents work and provide information that will guide their use and evaluation in clinical trials. Three new agents have been studied. All act by different mechanisms, and all are now in clinical trials.

The first drug causes the premature destruction of proteins that are necessary for CLL cells to live. The second drug is incorporated into the genetic material, DNA. This causes reactions that lead to CLL death in much of the tumor population, and prevents the survival of cells that may survive as they try to reproduce. The third agent directly damages DNA and prevents generation of information for viability. Furthermore, attempts by the CLL cells to remove this drug from DNA leads to still greater damage to the DNA and cell death. The different mechanisms that we have identified give rise to the promise that CLL cells will be less likely to become resistant to these drugs.

CLL is an incurable disease representing the most common form of leukemia in North America. This disorder is characterized by a disrupted cell death pathway rather than increased rate of cell production. The CLL cell death pathway is disrupted because of a prominence of anti-apoptotic (anti-cell death) proteins belonging to Bcl-2 family proteins.

Recently, agents have been identified that target these anti-apoptotic proteins directly by binding to them. Such an action results in inhibition of these proteins leading to removal of survival advantage from the CLL cells. We plan to use three agents that target Bcl-2 family of anti-apoptotic proteins.
Our plan is to first identify the characteristics and functional role of anti-apoptotic family proteins in primary CLL cells and to investigate the mechanism by which anti-apoptotic protein antagonist(s) induce cell death. Second, we will test if microenvironment induces expression of Bcl-2 family proteins in CLL lymphocytes and protects these primary cells from Bcl-2 antagonist-induced cell death. Finally, we plan to evaluate mechanism-based combinations of established and experimental chemotherapeutic agents with Bcl-2 antagonist(s) in CLL lymphocytes.

Chronic lymphocytic leukemia (CLL) is an incurable disease representing the most common type of adult leukemia in the western world. This disorder is characterized by disrupted cell death pathway rather than increased rate of proliferation. The CLL cell death pathway is disrupted because of an overexpression of anti-apoptotic proteins belonging to Bcl-2 family proteins. Recently, agents have been identified that target these anti-apoptotic proteins directly by binding to them. Such an action results in inhibition of these proteins leading to removal of survival advantage from the CLL cells. We used two different agents (gossypol and AT-101) to target Bcl-2 family of anti-apoptotic proteins. We demonstrated mechanism of action of these agents and cytotoxicity in CLL cells. We published three papers in the journal Blood.

Combinations of drugs acting by different mechanisms will be tested on CLL cells freshly isolated from patients. For treatment with drug combinations, the CLL cells will be grown in medium alone or in co-cultures with bone marrow stromal cells, which should mimic the microenvironment of CLL cells. Drug induced changes in cell survival and intracellular signaling will be monitored.

Due to previous work in our laboratory, as a starting compound, we chose the orally available protein tyrosine kinase enzyme inhibitor, dasatinib, which is successfully used for treating leukemias which have a unique BCR-ABL fusion gene. Since dasatinib also targets the activity of Src-family kinases, these enzymes are involved in microenvironment CLL cell interactions and cellular survival functions. We found that dasatinib induces programmed cell death preferentially in patient cells with unfavorable prognostic predictors.

Fludarabine, a key drug in CLL therapy, is the first candidate for combination with dasatinib. The combination of dasatinib and fludarabine increased CLL cell death effects in the laboratory and we will explore these results in a quantitative manner, taking into account patient characteristics.

The next group of combination partners planned for the lab assays are anti-CD20 antibodies, rituximab and GA101, which appear to induce cell death by similar pathways observed with dasatinib. Further agents to be included in the combination schemes are Bcl-2 antagonists and lipopeptides which modify cellular messages. In the long run, the preclinical testing of drug combinations will be extended to a mouse model of CLL.

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Development of drug resistance in CLL is a major challenge in clinical treatment of CLL patients. There are multiple mechanisms by which the leukemia cells may acquire the ability to survive drug treatment, and stromal (the supporting framework of cells and tissue) influence on CLL cells has recently been recognized as an important factor. The observations that CLL cells have long surviving time in vivo (inside the body) but are prone to undergo cell death when the leukemia cells are isolated from the patient blood and cultured in vitro (outside the body) suggest that the tissue microenvironment may play an important role in the survival of CLL cells.

Recent studies also suggest that the tissue microenvironment may also significantly affect cellular sensitivity to anticancer agents and lead to drug resistance. However, the biochemical and molecular mechanisms underlying the CLL-stromal interactions that present drug resistance remain unclear, and therapeutic strategies to overcome this type of drug resistance remain to be developed.

Our recent study suggests that certain soluble factors from bone marrow stromal cells seem to stimulate CLL cells to produce molecules that are important for the cells to survive under stress and to maintain a stable metabolic state. Removal of such a protective mechanism renders the CLL cells more vulnerable to drug treatment. Based on these observations, we now propose to use biochemical and molecular methods and the CLL-stromal co-culture system to identify the key molecules responsible for promoting the protective mechanisms in CLL cells leading to increased cell survival and drug resistance.

We also plan to test novel compounds and drug combination strategies to effectively disable the protective mechanism caused by stromal-CLL interaction. Because stromal-CLL interactions play a very important role in protecting CLL cells from the cytotoxic action of anticancer drugs, a successful identification of novel agents and drug combination protocols to overcome this type of drug resistance would have significant therapeutic implications.

We anticipate that the results of this research project will further our understanding of drug resistance mechanisms in CLL, and provide important information for the design of new therapeutic protocols to improve the outcomes of clinical treatment of CLL.

The main purpose of this research project is to use a special cell culture system in which leukemia cells isolated from CLL patients are co-cultured with stromal cells of the bone marrow. This has allowed us to investigate the interactions between leukemia cells and the tissue microenvironment (stromal). The specific goals are to understand how stromal cells may protect leukemia cells and make them more resistant to chemotherapy and to test new drugs that can overcome such drug resistance.

During the first year of this research project, we have successfully accomplished the proposed studies in accordance with the original timetable and milestones. All proposed studies for the first year have been completed with significant new findings on the biochemical processes by which stromal cells promote CLL cell survival and drug resistance through an increased synthesis of an important molecule known as glutathione. Furthermore, we found an effective way to abolish this glutathione protection so that the leukemia cells become sensitive to drug treatment with standard agents such as fludarabine.

In addition, our study also led to an invention of novel drug-containing nanomolecules for potential use in CLL treatment, and a discovery of a small molecular weight compound that can selectively kill CLL cells in the presence of stromal cells. These new agents have high potential to improve the CLL treatment outcome, since they are effective in eliminating CLL cells in the stromal environment of the CLL patients.

Certain cancers such as CLL are characterized by a slow accumulation of cells that display enhanced survival. These cells do not divide. Therefore, agents that require active DNA replication or cell division are ineffective against this disease. My project focuses on the development of 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 RNAs. Generally speaking, the production of these RNAs is suppressed in CLL. When CLL cells are exposed to LBH589, an HDACI, there is an increased level of protein modification on genes and these RNAs are now re-expressed. They in turn control the activity of other proteins which induce 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 drug for the 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.

This project was aimed at developing histone deacetylase inhibitors for treatment of CLL. Histone deacetylase inhibitors allow tumor suppressor genes that are silenced in CLL cells to be re-expressed. Through our research, we identified that the re-expression of microRNA genes, a newly discovered class of genes, causes certain proteins to be produced which in turn causes the death of CLL cells. In other experiments, re-expression of microRNA caused a decrease in Mcl-1, a protein that normally protects CLL cells from death. These two actions make the histone deacetylase inhibitors promising drugs to target CLL.