CLL Stromal Interaction Funded Projects

In CLL, there is a fatal attraction between the leukemia cells and “feeder cells” that are present in lymph nodes and the bone marrow. These “feeders” (which also are called nurselike cells or stromal cells) provide CLL cells with nutrients and drug-resistance signals. Therefore, the main focus of our research program is to find out which molecules are important for this interaction between CLL and the “feeder cells,” and to find novel drugs to interrupt this “food chain”.

Our research has already identified two factors called CXCR4 and CXCR5 that make CLL cells move and stick to “feeder cells”. We now are testing new drugs that blocks CXCR4, and are planning to bring these drugs into clinical trials in CLL patients in the near future.

However, once CLL cells stick to “feeder cells,” CXCR4 is clearly not the only survival factor. Therefore, we will conduct studies to systematically analyze how CLL cells “talk” with “feeder cells”. This will be done by a new technique called gene expression profiling. This technique allows us to determine which genes get turned on once CLL cells stick to the “feeder cells”. Using this approach, we should be able to define new targets for therapy. These experiments will be conducted in collaboration with Dr. Rosenwald from Würzburg University in Germany, who is a pioneer in this field.

Moreover, we have established a standardized method to culture CLL cells with different types of “feeder cells”. Previously, drugs for treatment of CLL were tested in the laboratory without any “feeder cells,” not taking into account the drug resistance signals that CLL cells receive from the “feeder cells” in CLL patients. Establishing this new method will allow us to systematically analyze how well new drugs work, and to test drugs that interfere with the fatal attraction between CLL cells and “feeder cells”.

Ultimately, this research will lead to innovative, targeted therapies for CLL patients, but with a paradigm shift: from targeting only the CLL cells towards targeting the CLL cells in the context of their complex social network.

The funding through the CLL Global Research Foundation has allowed us to establish several very productive national and international collaborations. These research collaborations are leading to discoveries that were published in 4 articles in Blood over the last year. Moreover, these findings have identified several new therapeutic targets that now will be pursued in pre-clinical development and then in clinical trials.

Our research is based upon the concept that current therapies for CLL patients are not curative because CLL cells survive in the tissues where they are protected from “conventional” drugs by neighbor cells called “stromal” or “nurselike”cells. When CLL cells are placed in cultures with these stromal cells, CLL cells become attracted to the stromal cells, and attach to them. This “fatal attraction” protects CLL cells from drugs, leading to minimal residual disease, and paving the way to relapses. Therefore, the aim of our research is to find out which signals between the stroma and the CLL cells are important, and which of these signals could be targeted therapeutically.

In our first study, we screened over 20,000 genes for their regulation by stromal cells. We found that several genes become activated by the stromal cells. Then, we focused on 2 genes that are called CCL3 and CCL4. These genes are activated by stromal cells, and make CLL cells produce and release high amounts of CCL3 and CCL4 protein. These proteins function as attraction factors for normal immune cells called T cells that help CLL cells survive and proliferate in lymph tissues. This study was done in collaboration with Dr. Rosenwald’s group in Wuerzburg,Germany, and was published in Blood. These findings now are further pursued by studying the blood levels of CCL3 and CCL4 in collaboration with Dr. Wierda’s group, and by testing a compound from Pfizer that targets this pathway. This may lead to a clinical trial of this Pfizer drug in CLL patients.

In our second study, we tested different stromal cells for their capacity to protect CLL cells from drugs that we commonly use for treatment of CLL patients, such as fludarabine, cyclophosphamide, or steroids. We found that all stromal cells tested were highly effective in protecting CLL cells from fludarabine-, dexamethasone-, and cyclophosphamide-induced cell death, and we established standardized conditions for drug testing with stromal cells in collaboration with our colleagues from the Mayo clinic (Dr. Kay’s group), MD Anderson’s Department of Experimental Therapeutics (Dr. Plunkett’s and Gandhi’s groups), and Vienna University, Austria (Dr. Jaeger’s group). These findings were presented at the 2008 ASH meeting and are currently in preparation for publication. These findings allow us to standardize in vitro testing of novel and established drugs, and determine the effects of MSC on drug resistance. Collectively, in this project we have developed a novel tool for drug testing that takes into account the effect of the microenvironment. This will have an impact on drug development, and hopefully lead to development of novel therapy strategies that target the CLL stroma.

In our third study in collaboration with Dr. Gandhi’s group, we have explored the activity of AT-101, a novel drug that targets the Bcl-2 family of survival proteins in CLL. This work was recently published in Blood.

In our fourth study, in collaboration with Dr. Shokat’s group (Pharmacology, UCSF), we have explored the activity of a novel class of drugs called phosphoinoside 3′-kinase inhibitors (PI3K inhibitors) to overcome stromal cell-mediated drug resistance in CLL. These drugs block a signaling pathway within the CLL cells. This pathway becomes activated in CLL cells by stromal cells. These findings will lead to clinical trials in CLL patients with these promising agents. The study has recently been published in Blood.

Collectively, our research on the microenvironment in CLL has produced several discoveries of new pathways that promote CLL cell survival. These can be targeted by new drugs, such as CCL3/4 antagonists, PI3K inhibitors, AT-101, and others. The first microenvironment targeting treatment program, based upon our research, and funded through the CLL Global, opened in May, 2009. In this study, we will co-administer Rituximab with a drug called Plerixafor (Mozobil) that mobilizes CLL cells from the tissues to the blood and thereby makes CLL cells better accessible to Rituximab. Given the rapid progress and the variety of new drugs that can target the microenvironment in CLL, we expect to have several new, targeted treatments available in the near future.

Signals coming from the microenvironment or stromal supporting cells work with genetic defects to establish the natural history of CLL and contribute structural growth of the malignant clone. CLL B-cells retain the capacity to migrate into and out of secondary lymphoid organs, such as nodes, to interact with normal bystander cells in tissue microenvironments and to respond to microenvironment-derived signals.

These interactions keep the CLL cells alive and growing. Stromal cells, helper cells and T-lymphocytes appear to be the key microenvironmental elements involved in the onset and progression of the disease. A number of molecules such as Hematopoietic Cell Lineage Specific 1 (HS1) appear to be key players in microenvironmental stimuli, triggering a response in the CLL cells.

The aim of this project is to dissect the molecular pathways involved in the interactions between microenvironment and CLL cells. The project will be have four integrated tasks: 1) Definition of the role of HS1 in cell structure regulation and migration of normal and CLL lymphocytes; 2) Molecular and functional characterization of bystander cells tissue growth centers; 3) Creation of protein maps of CLL cells from different microenvironmental compartments; 4) Analysis in animal models of the role of stroma in the accumulation and spread of CLL cells

The aim of the project is to understand the elements responsible for the clinical presentation and the natural history of CLL. In addition to “causal genes”, there are a number of other “influential genes” involved in the presentation of CLL. All of these genes interact with the microenvironment to induce proliferation, survival and clonal expansion of CLL cells within the tissues. We hope to identify molecules and mechanisms that can be exploited as potential therapeutic targets.

Under this grant, we have started defining the role of the HS1 molecule in the regulation of leukemic B-cell framework. The HS1 molecule controls the leukemic cell traffic, migration and homing to specific tissues. We have also established two new mouse models. The first model is a cross of two models and suggests that HS1 influences the movement and replication of leukemic cells. The second model is created by injecting the CLL cell line, MEC-1, into adult immunodeficient mice. Both of these models may prove to be useful tools to explore the leukemic tissue microenvironment and its interactions with CLL cells.

CLL growth and survival is impacted by signals within the CLL cell. In the bone marrow, certain signals cause the CLL cells to undergo “spontaneous” death. Recently, we identified an intracellular signaling protein, STAT-3, that stimulates CLL cell growth and survival. (STAT-3 has a similar activation effect in other leukemias).

Specific inhibitors of STAT-3 suppress CLL cell proliferation and kill CLL cells. Interleukin-6, a cytokine or regulatory protein derived from bone marrow cells, appears to counteract the inhibitor effect thereby keeping the CLL cells alive. Therefore, we asked whether external signals such as cytokines would hamper the effects of STAT-3 inhibitors.

To answer this question, we will conduct experiments with CLL bone marrow cells, normal cells and outside growth factors. We will explore how bone marrow-supportive cells “fight” CLL cells. Our hypothesis is that bone marrow supportive cells recognize an expanding population of CLL cells and generate factors to stop cell growth. While cytokines may be capable of killing CLL cells, they might also suppress normal cells thereby inducing low blood count in patients with CLL.

We will investigate how the bone marrow responds to a growing clone of CLL cells, and whether CLL marrow supports and stimulates the development of precursor cells. Understanding the roles of these factors in maintaining CLL cells will allow us to development future CLL treatments.

We studied the interaction between chronic lymphocytic leukemia (CLL) cells and their surrounding bone marrow. We found that CLL cells produce a hormone-like factor called vascular endothelial growth factor (VEGF). Previous studies showed that VEGF induces the growth of small blood vessels. Indeed, we found that the bone marrow of patients with CLL contains more small blood vessels than the bone marrow of healthy individuals. These findings suggest that by producing VEGF, CLL cells change the composition of their surrounding bone marrow.

We also found that CLL cells stick to bone marrow blood vessels including the small blood vessels whose growth was induced by VEGF. CLL cells bind to the cells that form the lining of the inner surface of blood vessels, termed vascular endothelial cells. Our experiments show that VEGF, as well as endothelaial cells, provide CLL cells with protection against cell death. In addition, we discovered that CLL cells express the receptor for VEGF on their surface and that when VEGF (including the VEGF that is produced by CLL cells) binds to this receptor, CLL cells are better protected from cell death. These data suggest that inhibitors of VEGF should be used as anti-CLL agents.

Recent evidence suggests that the microenvironment (the physical space in the bone marrow where CLL cells reside), with its complex network of physical and chemical signals, affects the proliferation rate and survival of CLL cells. We, therefore, plan to investigate several characteristics of one of the cells in the microenvironment called mesenchymal stromal cell (MSC). The MSCs will be obtained from bone marrow biopsies of CLL patients. We believe that these cells will provide crucial information to better understand the factors that are involved in disease progression in CLL.

In the first aim, we want to determine if MSCs are “true” stem cells. We will do this by studying the following characteristics: protein expression on the surface of the cell, the ability to mature into more differentiated cells and the ability to secrete chemical products. We know that MSCs from patients with CLL are able to support the CLL cells and prolong their survival. We plan to compare the ability of MSCs obtained from normal individuals to provide the same enhanced survival to CLL cells. Furthermore, we plan to compare the protective effect of MSCs from CLL patients with low-risk disease (slow or no clinical progression) with the MSCs of patients with high-risk CLL (rapid progression).

The second aim is to study the reciprocal (two-way) interaction between the CLL cells and the MSCs. We are particularly interested in knowing what specific messages are activated in MSCs of patients with low-risk CLL versus high risk disease. We will evaluate the factors involved in making the MSCs more effective in favoring CLL cell survival, activation and proliferation. Is an MSC more effective in induction of CLL B-cell survival if it has been exposed to a CLL B-cell population?

We will also evaluate the so-called “angiogenic switch” phenomenon. This is a process during which the MSC influences the CLL cells in their ability to release substances involved in new blood vessel formation. The possible subsequent increase in blood-vessel production is known to be associated to disease progression. These substances can also directly increase the CLL B-cell survival capacity.

Finally, in our third aim, we will use the information obtained in aims one and two to design a therapeutic strategy that can block the crucial point of interaction between the MSC and CLL cells, and therefore diminish the MSCs protective effect. We hope this will allow us to develop novel therapeutic strategies for our patients.

To more completely understand the nature and extent of the influence of leukemic cells we have developed a culture system from the bone tissue of CLL patients. We are able to consistently grow out from the bone tissue a specific type of stromal cell (cells known to make up the structure of the microenvironment of CLL) from both CLL patients and normal volunteers. These cells are called “mesenchymal stem cells”, or MSC, and are a known constituent of the stroma in the bone marrow of humans.

Using this culture system, we have found that the leukemic cells and these MSC are able to “talk to each other” and influence each other’s behavior. For example, the MSC are able to induce a longer life span for the leukemic B cell and also confer on it more of an activation status which could be related to disease progression.

In contrast, we have found that the leukemic B cells from the CLL patient can activate the MSC through specific types of secreted factors. This activation, we believe, leads to the secretion of more molecules into the blood of CLL patients that leads to what is called an “angiogenic phenotype” which is also related to progression of the disease. For example, these angiogenic factors can also help leukemic CLL cells survive, and in the tissue sites where the CLL cells reside, generate more blood vessels that sustain their growth. Since these angiogenic factors can circulate in the blood of these patients, they can influence distant tissue sites that may help in leukemia cell growth or survival.

Thus, we have identified ways that CLL patients may experience disease progression by the cross-talk between malignant cells and their environment. Because we have identified specific factors, signaling pathways and the cells that are affected by this type of interaction, we hope to intervene to block this dialogue between the two cells and in this aspect treat patients more effectively.

Although major advances in the treatment of CLL in the last two decades have led to a considerable increase in the number of remissions, there is still no curative strategy for this disease. CLL is characterized by the accumulation of mature-looking, but functionally incompetent lymphocytes in the blood, bone marrow, lymph nodes and other organs, leading to a replacement of the normal inhabitants of these compartments.

There is growing evidence that survival signals provided from the cellular environment (e.g. T-cells etc.) towards the malignant CLL cells contribute substantially to the accumulation of the malignant cells. Whereas current therapeutic approaches are highly efficient in clearing circulating leukemic cells, residual CLL cells in secondary lymphatic tissues or bone marrow are likely the source of minimal residual disease and relapses. In these compartments, the close contact between CLL cells and accessory cells likely protects CLL cells from spontaneous and drug-induced cell death and thereby governs tumor progression.

The interaction between the CLL cells and accessory cells is currently being investigated in many experimental ‘ex vivo’ (or outside the body) approaches (laboratory experiments of CLL and accessory cells), but the ‘in vivo’ situation remains foggy. This research proposal aims at the characterization of the microenvironment in CLL lymph node and bone marrow specimens by in situ (localized) techniques (immunohistochemistry and in situ hybridization). The identification of ‘key players’ in the microenvironment that prevent cell death of CLL cells and promote their growth will help to develop therapeutic molecules that target the pivotal interaction of CLL cells and their microenvironment.

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