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Winter 2002/2003 Volume 5, Issue 4:
YOUR GIFTS AT WORK: 2003 Brian D. Novis Research Grant Awards
(l-r) Dr. Gareth Morgan, Ashley Barit, Dr. Philip Greipp, Dr. Federico Caligaris-Cappio,
Benson Klein, Dr. Kewal Asosingh, Carol Klein, Dr. Takashi Sonoki,
Dr. Jesus San Miguel. Dr. Sun J. Choi, Dr. Brian Van Ness

The IMF Brian D. Novis Reseach Grant Award presentations took place on Saturday, December 7th, 2002 in Philadelphia, PA during the 44th annual meeting and exposition of the American Society of Hematology. In attendance were many members of the IMF Scientific Advisory Board, IMF Directors, staffers, and supporters whose efforts helped make the grants possible.

Susie Novis with Senior Grant Recipients
Drs. Brian Van Ness and Gareth Morgan

IMF President Susie Novis opened the reception with a warm welcome, and provided background information on the IMF research program that dates back to 1995. Dr. Robert A. Kyle, Chairman of the IMF Scientific Advisory Board, and Dr. Brian G.M. Durie, Chairman of the Board, were on hand to present the awards. Individual donors were introduced to the researchers whose projects they are funding. The grant recipients spoke of the objectives of their research projects.

This issue of Myeloma Today features research abstracts of several Junior Grant recipients. Others will be profiled in the upcoming issue of the newsletter.

Senior Grant Recipients Drs. Jesus San Miguel and Philip Greipp
with IMF Chairman of the Board Dr. Brian Durie

Dr. Kewal Asosingh
Tumor-host interactions during MM disease progression; functional roles of CD45 subsets
Vrije Universiteit Brussel (VUB)
Brussels, Belgium

For the development of new, more effective strategies in the treatment of cancer, understanding of the biological heterogeneity in the tumor population is an important goal. Only specific subsets within the parental tumor have the necessary receptors for motility, invasiveness, and tumor spread. In different tumor stages remodeling of the extracellular matrix occurs, by exchange of proteinases and cytokines between stromal cells and cancer cells. This modified microenvironment stimulates invasion and promotes survival and proliferation of the cancer cells. Only specific cancer cell populations are able to induce the production of survival/growth factors and proteolytic enzymes by local stromal cells and have the capacity to induce angiogenesis, which is pivotal for progressive tumor growth. In multiple myeloma (MM) research, understanding of the heterogeneity, in general, and understanding of the specific roles of CD45 subsets, in particular, is still in its infancy. CD45 is a transmembrane tyrosin phosphatase. While immature plasma cells are CD45 positive, fully matured plasma cells lose all CD45 expression. Myeloma patients have a heterogeneous CD45 expression pattern and very recently CD45 appeared to be a predictor of therapeutical response. In our previous work the in vivo bone marrow homing, differentiation and proliferation of CD45 subsets in the 5T experimental mouse model (11, 12) were investigated. In the current project the roles of CD45+ and CD45- 5TMM cells during myeloma disease progression will be analyzed. Experiments with mice injected with the heterogeneous 5T2MM cell population indicated major alteration in the CD45 subset composition during disease progression. In the early stage of the disease (few weeks after intravenous injection) the majority of the MM cells had an immature, invasive and apoptosis resistant phenotype (CD45+CD138-IL6-Ra+). In contrast, end stage MM cells had a mature, less-invasive and apoptosis sensitive phenotype (CD45-CD138+IL6-Ra-). In between there was a gradual progression in the subset composition towards the endstage phenotype. The early-, intermediate- and endstage myeloma cells will be investigated for functional differences. Invasion, apoptosis and proliferation will be analyzed. Bone marrow samples collected at different time points (with one-week time interval) will be examined for the presence of angiogenesis and osteolysis by microvessel density quantification and micro CT, respectively. Correlations with the presence of CD45 subsets will be analyzed. The observed alterations in the subset composition suggest alterations in tumor-host interactions during the disease progression. Alterations in gene expression profile of BM stromal cells and MM cells during disease progression will be identified by differential gene expression analysis (cDNA micro array) at different time points (early tumor stage, middle and endstage). In particular, differential expression of genes involved in apoptosis, invasion, proliferation, angiogenesis and osteolytic bone disease and in the signaling cascades of these processes will be analyzed. The differential expression will be confirmed by Real Time-PCR and at protein level by flow cytometry, Western blot or immunohistochemistry. The overexpressed/ down-regulated proteins will be tested for their functional involvement.

Carol and Benson Klein with Dr. Sun J. Choi

Dr. Chao-Lan Yu
Suppressor of Cytokine Signaling (SOCS): Its Regulation and Potential Implication as a Therapeutic Agent in Multiple Myeloma
Vanderbilt University School of Medicine
Nashville, Tennessee

Interleukin-6 (IL-6) is the major growth and survival factor in human multiple myeloma (MM) cells. Through the signaling receptor subunit gp130, IL-6 activates the receptor-associated JAK family protein tyrosine kinases and, subsequently, the downstream signal transducer and activator of transcription 3 (STAT3). STAT proteins are latent cytoplasmic transcription factors that regulate the expression of specific cellular genes. Consistent with the critical role of STAT3 in IL-6 signaling, constitutive STAT3 activation has been reported both in human myeloma cell lines and in bone marrow cells from MM patients. Aberrant STAT3 activation may contribute to the malignant progression of MM by the induction of antiapoptotic proteins Bcl-2 and Bcl-xL. Furthermore, STAT3 activation is also linked to cell cycle progression by the induction of cyclinD1. However, the molecular mechanisms of constitutive STAT3 activation in MM cells still remain largely unclear.

STAT3 can be down-regulated through negative feedback control following cytokine-induced activation. The suppressor of cytokine signaling (SOCS) family members are key players in this process. SOCS proteins are induced by cytokines to prevent further activation of the JAK-STAT pathway. Among eight SOCS family members, both SOCS-1 and SOCS-3 have been shown to be induced by IL-6 and to inhibit subsequent IL-6 signaling. SOCS-1 and SOCS-3 inhibit JAK kinase activity either by direct association with JAK kinases or by simultaneous interactions with JAK and the receptor. We have preliminary data indicating that the absence of SOCS gene expression correlates with constitutive STAT activation in leukemic T cells. Therefore, we would like to hypothesize that defect of negative feedback control through SOCS-1 and SOCS-3 proteins may contribute to constitutive STAT3 activation and resistance to apoptosis in MM cells. This hypothesis will be tested in a well-characterized human myeloma cell line, U266, through the following specific aims:

1. To determine if the negative feedback control involving SOCS-1 and SOCS-3 proteins is intact in MM cells. The observation that exogenous IL-6 could induce further STAT3 activation in U266 myeloma cells suggested that JAK kinases are not effectively inhibited by SOCS-1 and SOCS-3. It is possible that, in U266 myeloma cells, IL-6 autocrine loop cannot induce SOCS-1 and SOCS-3 expression to a level high enough to inhibit JAK kinases. We will first compare the expression levels of SOCS-1 and SOCS-3 in U266 myeloma cells with varying degrees of STAT3 activity. This can be achieved by treating U266 cells with a high dose of exogenous IL-6 or AG490, a JAK-specific inhibitor. A constant low or undetectable SOCS expression regardless of endogenous STAT3 activity will suggest an intrinsic defect in SOCS expression in U266 myeloma cells. On the other hand, if SOCS expression correlates with STAT3 activity in U266 myeloma cells, we will determine its ability to associate with and inhibit JAK kinases. Our expectation is that functional expression of SOCS-1 and/or SOCS-3 will be defective in U266 myeloma cells.

2. To evaluate the effects of ectopic expression of SOCS-1 and SOCS-3 on survival and growth of MM cells. We will transiently transfect U266 cells with an enhanced green fluorescent protein (EGFP) expression construct encoding SOCS-1, SOCS-3, or the empty vector. EGFP-expressing cells will be isolated by FACS sorting and then subjected to biochemical assays to confirm the expression of SOCS proteins and inhibition of STAT3 activity. Northern blot analysis will follow determine if SOCS proteins reduce the expression of bcl-2 and bcl-xL, two known antiapoptotic genes. To determine if SOCS proteins promote Fas-mediated apoptosis, transfected U266 cells will be stimulated with Fas ligand or Fas agonistic antibody CH-11, and then examined by Annexin V-PE staining and FACS analysis. We predict that exogenous expression of SOCS-1 and/or SOCS-3 in U266 myeloma cells will result in lower STAT3 activity, reduced levels of bcl-2 and bcl-xL transcripts, and increase in Fas-mediated apoptosis. In addition, we will perform cell cycle analysis and examine cyclinD1 expression on transfected U266 myeloma cells. We hypothesize that forced SOCS-1 and/or SOCS-3 expression may also induce cell cycle arrest by inhibiting cyclinD1 expression.

SOCS proteins have been shown to inhibit proliferation signals induced by a number of oncoproteins. Consistent with the role of SOCS as tumor suppressor, higher incidence of malignancies have been reported both in humans and in mice with defective SOCS expression. Furthermore, SOCS proteins have also been shown to be more effective than dominant-negative STAT proteins in the treatment of certain immune disorders. Results from this proposed research project, therefore, should provide additional insights into the molecular mechanisms that contribute to the pathogenesis of MM and may suggest novel strategies for potential treatment of MM.

Dr. Lee Ann Garrett-Sinha
Identification of the Role of Ets-1 in Regulating Plasma Cell Numbers
University of Buffalo
State University of New York
Buffalo, New York

Plasma cells normally function to produce antigen-specific antibodies and thus help control infections. As such they are a critical part of the immune system. Indeed, people who lack plasma cells due to the genetic disease, X-linked agammaglobulinemia (XLA) are subject to severe recurrent bacterial infections. On the other hand, unregulated plasma cell growth can lead to the development of certain tumors (plasmacytomas and multiple myelomas). Thus, it is critically important for an organism to strictly control the production and the loss of plasma cells to ensure that sufficient cells are available to fight infection, but that excess cells are eliminated.

The processes of cellular differentiation, proliferation, and apoptosis require specific proteins that promote each cell’s fate. Transcription factors regulate the expression of the various proteins necessary for each of these different cellular outcomes. Ample evidence implicates members of the Ets family of transcription factors in the regulation of B cell development, expansion, maturation, and death. Ets proteins regulate the expression of membrane receptors that promote cellular differentiation and proliferation such as the interleukin (IL)-7 receptor, the granulocyte/macrophage colony stimulating factor (GM-CSF) and the B cell receptor (BCR) (11,13-18). Ets proteins also regulate the expression of anti-apoptotic proteins such as Bcl-XL and c-rel as well as pro-apoptotic proteins such as Fas and caspase-3. Therefore, Ets proteins regulate diverse pathways that can lead to cell survival, cell proliferation, or cell death.

Interestingly, up-regulation of Ets-1 protein is detected in a sub-set of tumors. In addition to the receptor genes, pro- and anti-apoptotic genes discussed above, Ets-1 is also known to regulate the expression of certain metalloproteinase genes. Expression of proteinases is required for tumor cells to degrade surrounding extracellular matrix, invade nearby tissues, and metastasize to distant sites. Over-expression of Ets-1 in tumors generally correlates with their increased invasiveness and metastatic potential and with a poorer prognosis for the patient. In addition, expression of Ets-1 in vascular endothelial cells is important during the process of angiogenesis. Since tumor growth requires new blood vessels, the role of Ets-1 in angiogenesis has important implications for cancer growth.

Gene-targeted mice lacking Ets-1 exhibit a ten-fold increase in the numbers of plasma cells in the spleen. The deregulated production of plasma cells found in these animals is reminiscent of unregulated plasma cell growth found in plasmacytomas and multiple myelomas. Thus, Ets-1-/- animals provide an animal model to study factors affecting the equilibrium between plasma cell formation and deletion. The investigations we propose in this application will address how Ets-1 regulates the differentiation of plasma cells, their proliferation and/or their survival. We believe that our studies will have direct bearing on understanding, and perhaps treating, diseases caused by uncontrolled plasma cell growth.

Dr. Brian Durie with the Barit family and supporters

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