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B206 Characterization of Cancer Stem Cells in Multiple Myeloma
Basic Biology and Preclinical Studies B205 Efficacy of CEP-18770 on Myeloma Growth H Chen,1 JR Berenson,1 RA Campbell,1 J Steinberg,1 M Li,1 E Sanchez,1 B Bonavida2 1
Institute for Myeloma & Bone Cancer Research; 2Geffen School of
Medicine at the University of California at Los Angeles
Introduction and Aim: Proteasome inhibitors (PI) are effective agents for the treatment of multiple myeloma (MM) and enhance the effects of chemotherapeutic agents. Determination of the antiproliferative effects of treating MM cell with CEP-18770, a PI, alone and in combination with anti-MM agents was examined. Materials and Methods: MM cell lines were incubated in the presence of CEP-18770 alone and in combination with anti-MM agents for 48 hours. Cell growth was measured using an MTS assay. Results: Although single-agent treatment showed antiproliferative effects, combination indices as calculated by the Chou-Talalay method showed that CEP-18770 had synergistic effects on RPMI8226 and U266 cells with either doxorubicin or melphalan. Similarly, synergism was observed using CEP-18770 plus ATO on RPMI8226. In vivo studies were conducted using our SCID-hu models of MM. Mice received no treatment or CEP-18770 twice weekly at 0.1, 0.3, 1, or 3 mg/kg injected intravenously or at 10 mg/kg via oral gavage. LAGL-1-bearing mice treated with CEP-18770 showed inhibition of tumor growth as determined by human immunoglobulin (hIg) G levels and tumor volume (P = .0008) compared with mice receiving vehicle. Similarly, inhibition of tumor growth was also observed in LAGK-1A–bearing mice treated with CEP-18770 at 1, 3 and 10 mg/kg (hIgG: P = .0001, P = 0.0002, and P = .0001, respectively; tumor volume: P = .0001, P = .0001, and P = .0001, respectively) and LAGK-1B–bearing mice treated with CEP-18770 at 3 and 10 mg/kg (hIgG: P = .0008 and P = .0034, respectively; tumor volume: P = .0008 and P = .0028, respectively) compared with mice receiving vehicle alone. Combination of CEP-18770 (1 mg/kg) plus melphalan (3 mg/kg administered once weekly via intraperitoneal injection), tested on LAGK-1B–bearing mice, showed markedly smaller tumors compared with vehicle (P = .0008) or melphalan alone (P = .0204). Conclusion: These studies provide promising preclinical data to suggest the potent anti-MM effects of CEP-18770 both in vitro and in vivo and that this new PI might enhance the effects of anti-MM agents.
B206 Characterization of Cancer Stem Cells in Multiple Myeloma E Sanchez,1 JR Berenson,1 H Chen,1 M Li,1 CS Wang,1 AM Berenson,1 J Li,1 J Steinberg,1 J Wang,1 J Shen,1 Z Li,1 B Bonavida2 1
Institute for Myeloma & Bone Cancer Research; 2Geffen School of
Medicine at the University of California at Los Angeles
Introduction: Development of therapies targeted at cancer stem cells should improve survival for cancer patients. Multiple myeloma (MM) is characterized by clonal expansion of terminally differentiated B cells. Materials and Methods: To characterize
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whether cancer stem cells can be identified in MM, fresh MM bone marrow biopsies were implanted into SCID mice. The tumors were excised from donor mice 2 months following implantation and digested with proteinase E to produce a single-cell suspension. The cells were applied to a magnetic column and unbound cells were passed through the column with washing followed by centrifugation and finally resuspended. Total RNA was purified from cells, and gene expression of each population was examined using RT-PCR analysis of specific previously identified stem cell–related transcription factors. Results: We found that the CD20 /CD138- and CD133 /CD138- subpopulations both expressed high levels of B-catenin, KLT-4, Oct-4, SOX2, and c-Myc. These small tumor populations are likely to represent MM stem cells because they express genes consistently identified in cancer stem cells found in other cancer types. We unexpectedly found that CD138 cells also expressed B-catenin, KLT-4, Oct-4, SOX2, and c-Myc. This population of MM cells might consist of “premature” tumor cells at a middle stage of tumor cell differentiation, which ultimately develops into mature MM cells. Only CD20-/CD138cells showed no expression of B-catenin, KLT-4, and SOX2 and markedly reduced Oct-4 gene expression, whereas the amount of cMyc gene expression was similar to the levels in the other tumor cell subtypes. Only CD133-/CD138- cells lost B-catenin and showed a reduction in Oct-4 gene expression but still expressed the KLT-4, SOX2, and c-Myc genes. Conclusion: To further examine these cancer stem cell and mature tumor cell populations in terms of growth in vivo, we have injected subcutaneously CD20 /CD138-, CD133 /CD138-, CD20-/CD138-, and CD133-/CD138- tumor cell subpopulations back into SCID mice. These studies have uncovered specific subpopulations within the tumor clone of MM and identified differences in expression of genes known to be involved in stem cell function.
B207 In the Crosshairs: Targeting Myeloma CD28-B7 Interactions JR Nair,1 LM Carlson,1 LH Boise,2 AA Chanan-Khan,1 KP Lee1 1
Roswell Park Cancer Institute; 2Miller School of Medicine,
University of Miami
Introduction: Multiple myeloma (MM) cells are critically dependent on their interactions with bone marrow stromal cells for essential survival signals/growth/immunosuppressive factors. However, very little is known about the cellular and molecular elements in these interactions. Clinical studies show a strong correlation (P = .006) between CD28 expression on MM and disease progression. MM cells that express CD28 invariably also express its ligand CD86 (B7-2). Also, the close association of B7 (CD80/CD86 — ligands for CD28) dendritic cells (DC) with MM cells in patient BM biopsies suggest their potential role as partners in a pro-survival CD28-B7 interaction. Aims: The purpose of this study was to characterize the pro-survival relevance of cellular interactions between myeloma-CD28 and other B7 partners such as MM cells or DCs, identify the induced pro-survival factors, and
Clinical Lymphoma & Myeloma Supplement February 2009
Institute for Myeloma & Bone Cancer Research; 2Geffen School of
Medicine at the University of California at Los Angeles
Introduction and Aim: Proteasome inhibitors (PI) are effective agents for the treatment of multiple myeloma (MM) and enhance the effects of chemotherapeutic agents. Determination of the antiproliferative effects of treating MM cell with CEP-18770, a PI, alone and in combination with anti-MM agents was examined. Materials and Methods: MM cell lines were incubated in the presence of CEP-18770 alone and in combination with anti-MM agents for 48 hours. Cell growth was measured using an MTS assay. Results: Although single-agent treatment showed antiproliferative effects, combination indices as calculated by the Chou-Talalay method showed that CEP-18770 had synergistic effects on RPMI8226 and U266 cells with either doxorubicin or melphalan. Similarly, synergism was observed using CEP-18770 plus ATO on RPMI8226. In vivo studies were conducted using our SCID-hu models of MM. Mice received no treatment or CEP-18770 twice weekly at 0.1, 0.3, 1, or 3 mg/kg injected intravenously or at 10 mg/kg via oral gavage. LAGL-1-bearing mice treated with CEP-18770 showed inhibition of tumor growth as determined by human immunoglobulin (hIg) G levels and tumor volume (P = .0008) compared with mice receiving vehicle. Similarly, inhibition of tumor growth was also observed in LAGK-1A–bearing mice treated with CEP-18770 at 1, 3 and 10 mg/kg (hIgG: P = .0001, P = 0.0002, and P = .0001, respectively; tumor volume: P = .0001, P = .0001, and P = .0001, respectively) and LAGK-1B–bearing mice treated with CEP-18770 at 3 and 10 mg/kg (hIgG: P = .0008 and P = .0034, respectively; tumor volume: P = .0008 and P = .0028, respectively) compared with mice receiving vehicle alone. Combination of CEP-18770 (1 mg/kg) plus melphalan (3 mg/kg administered once weekly via intraperitoneal injection), tested on LAGK-1B–bearing mice, showed markedly smaller tumors compared with vehicle (P = .0008) or melphalan alone (P = .0204). Conclusion: These studies provide promising preclinical data to suggest the potent anti-MM effects of CEP-18770 both in vitro and in vivo and that this new PI might enhance the effects of anti-MM agents.
B206 Characterization of Cancer Stem Cells in Multiple Myeloma E Sanchez,1 JR Berenson,1 H Chen,1 M Li,1 CS Wang,1 AM Berenson,1 J Li,1 J Steinberg,1 J Wang,1 J Shen,1 Z Li,1 B Bonavida2 1
Institute for Myeloma & Bone Cancer Research; 2Geffen School of
Medicine at the University of California at Los Angeles
Introduction: Development of therapies targeted at cancer stem cells should improve survival for cancer patients. Multiple myeloma (MM) is characterized by clonal expansion of terminally differentiated B cells. Materials and Methods: To characterize
S112
•
whether cancer stem cells can be identified in MM, fresh MM bone marrow biopsies were implanted into SCID mice. The tumors were excised from donor mice 2 months following implantation and digested with proteinase E to produce a single-cell suspension. The cells were applied to a magnetic column and unbound cells were passed through the column with washing followed by centrifugation and finally resuspended. Total RNA was purified from cells, and gene expression of each population was examined using RT-PCR analysis of specific previously identified stem cell–related transcription factors. Results: We found that the CD20 /CD138- and CD133 /CD138- subpopulations both expressed high levels of B-catenin, KLT-4, Oct-4, SOX2, and c-Myc. These small tumor populations are likely to represent MM stem cells because they express genes consistently identified in cancer stem cells found in other cancer types. We unexpectedly found that CD138 cells also expressed B-catenin, KLT-4, Oct-4, SOX2, and c-Myc. This population of MM cells might consist of “premature” tumor cells at a middle stage of tumor cell differentiation, which ultimately develops into mature MM cells. Only CD20-/CD138cells showed no expression of B-catenin, KLT-4, and SOX2 and markedly reduced Oct-4 gene expression, whereas the amount of cMyc gene expression was similar to the levels in the other tumor cell subtypes. Only CD133-/CD138- cells lost B-catenin and showed a reduction in Oct-4 gene expression but still expressed the KLT-4, SOX2, and c-Myc genes. Conclusion: To further examine these cancer stem cell and mature tumor cell populations in terms of growth in vivo, we have injected subcutaneously CD20 /CD138-, CD133 /CD138-, CD20-/CD138-, and CD133-/CD138- tumor cell subpopulations back into SCID mice. These studies have uncovered specific subpopulations within the tumor clone of MM and identified differences in expression of genes known to be involved in stem cell function.
B207 In the Crosshairs: Targeting Myeloma CD28-B7 Interactions JR Nair,1 LM Carlson,1 LH Boise,2 AA Chanan-Khan,1 KP Lee1 1
Roswell Park Cancer Institute; 2Miller School of Medicine,
University of Miami
Introduction: Multiple myeloma (MM) cells are critically dependent on their interactions with bone marrow stromal cells for essential survival signals/growth/immunosuppressive factors. However, very little is known about the cellular and molecular elements in these interactions. Clinical studies show a strong correlation (P = .006) between CD28 expression on MM and disease progression. MM cells that express CD28 invariably also express its ligand CD86 (B7-2). Also, the close association of B7 (CD80/CD86 — ligands for CD28) dendritic cells (DC) with MM cells in patient BM biopsies suggest their potential role as partners in a pro-survival CD28-B7 interaction. Aims: The purpose of this study was to characterize the pro-survival relevance of cellular interactions between myeloma-CD28 and other B7 partners such as MM cells or DCs, identify the induced pro-survival factors, and
Clinical Lymphoma & Myeloma Supplement February 2009