Preclinical studies of the pan-Bcl inhibitor obatoclax (GX015-070) in multiple myeloma

Blood ◽  
2007 ◽  
Vol 109 (12) ◽  
pp. 5430-5438 ◽  
Author(s):  
Suzanne Trudel ◽  
Zhi Hua Li ◽  
Jennifer Rauw ◽  
Rodger E. Tiedemann ◽  
Xiao Yan Wen ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Single Agent ◽  
Myeloma Cell ◽  
Bone Marrow Stroma ◽  
Cytochrome C Release ◽  
Marrow Stroma ◽  
Combination Studies ◽  
Binding Groove ◽  
Cytotoxic Responses

Abstract Bcl family members Bcl-2, Bcl-xL, and Mcl-1, are frequently expressed and implicated in the survival of myeloma cells. Obatoclax (GX015-070) is a novel, small-molecule antagonist of the BH3-binding groove of the Bcl family of proteins. We show that GX015-070 inhibits the binding of Bak to Mcl-1, up-regulates Bim, induces cytochrome c release, and activates capase-3 in human myeloma cell lines (HMCLs), confirming the predicted mechanism of action. Consequently, GX015-070 potently inhibited the viability of 15 of 16 HMCLs (mean IC50 of 246 nM), including those resistant to melphalan and dexamethasone. In combination studies, GX015-070 enhanced the antimyeloma activity induced by melphalan, dexamethasone, or bortezomib. Sensitivity to GX015-070 correlated with the absence or near absence of Bcl-xL. Coculture with interleukin-6 or adherence to bone marrow stroma conferred modest resistance; however, it did not overcome GX015-070–induced cytotoxicity. Of importance, GX015-070 as a single agent induced potent cytotoxic responses against patient-derived tumor cells. GX015-070 inhibited normal bone marrow–derived colony formation; however, cytotoxicity to human blood lymphocytes was not observed. Taken together, these studies describe a novel BH3 mimic with selectivity for Mcl-1, and support the therapeutic application of GX015-070 for diverse neoplasias including multiple myeloma.

Discovery and Characterization of Macrocyclic Thiopeptide Proteasome Inhibitors for Hematologic Malignancies

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3669-3669
Author(s):  
Sridevi Ponduru ◽  
Raymond Moellering ◽  
Edward Greenberg ◽  
John Paul Ying-Ching Shen ◽  
Benjamin Z Stanton ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Protein Degradation ◽  
High Throughput ◽  
Active Sites ◽  
Proteasome Inhibitors ◽  
Hematologic Malignancies ◽  
Bone Marrow Stroma ◽  
Shh Signaling ◽  
Marrow Stroma

Abstract The ubiquitin proteasome pathway comprises a coordinated, dynamic cellular system critical to cellular metabolism, signaling and proliferation. The expanding clinical utility of the peptide boronate, bortezomib, in the treatment of patients with multiple myeloma and other hematologic malignancies has established the human 26S proteasome as a validated target in cancer. Still, only one FDA-approved proteasome inhibitor presently exists. Restricted activity against one enzymatic function of the proteasome and dose-limiting toxicities associated with bortezomib warrant further discovery efforts aimed at the identification of structurally and functionally distinct protein degradation inhibitors (PDIs). Here, we report a novel family of natural product proteasome inhibitors discovered by high-throughput, high-content screening at the National Cancer Institute Initiative for Chemical Genetics. A primary screen of 14,000 small molecules was performed in 384-well plate format using a cell line stably transfected with a destabilized fluorescent protein chimera. Assay positives were retested in the primary screen in dose-response format. Thiostrepton was selected for further characterization due to its unique macrocyclic chemical structure, the recent publication of its total synthesis, reports of anticancer properties and the lack of prior annotation as a PDI. First, thiostrepton was linked to previously characterized molecules acting on the protein degradation pathways by transcriptional small molecule connectivity mapping (CMAP). Subsequent cell-state analyses confirmed strong induction of functional and annotated gene sets associated with misfolded protein stress and proteasome inhibition. Mechanism of action was confirmed by biochemical profiling of human 20S proteasome active site inhibition and specificity using homogeneous assays and selective substrates for each of three catalytic active sites. Importantly, inhibitory activity of thiostrepton differs from bortezomib by blocking both the chymotryptic-like and PGPH active sites with sub-micromolar potencies. Dose-dependent inhibition of multiple myeloma cell growth was observed, with a concomitant increase in polyubiquitinated protein stress and induction of apoptosis. Inhibition of conferred proliferation by bone marrow stroma was confirmed using a novel miniaturized high-content assay modeling the bone marrow stroma-multiple myeloma microenvironment. Structurally related compounds to thiostrepton, nosiheptide and siomycin, were confirmed also as proteasome inhibitors as above. Our discovery of this class of natural products as proteasome inhibitors and a recent report of siomycin inhibition of Sonic Hedgehog (Shh) signaling begged the question whether established proteasome inhibitors would inhibit Shh signaling in human cancer. This hypothesis was confirmed in a set of reporter-gene assays. In sum, these studies identify thiopeptide macrocycles as a class of naturally-occurring proteasome inhibitors poised for clinical development in hematologic malignancies, establish novel high-throughput assays for modeling MM-stroma microenvironment interactions and pave the way for the development of proteasome inhibitors in disease states where Shh signaling is central to pathogenesis.

Aberrant Expression Of miRNA and mRNA Of Cell Cycle and Adhesion-Related Genes In Bone Marrow Stroma Cells Derived From Patients With Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3149-3149 ◽  
Author(s):  
Rimma Berenstein ◽  
Blau Igor Wolfgang ◽  
Axel Nogai ◽  
Marlies Wächter ◽  
Antonio Pezzutto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Genetic Alterations ◽  
Bone Marrow Stroma ◽  
Aberrant Expression ◽  
Healthy Donors ◽  
Marrow Stroma ◽  
Bone Marrow Stroma Cells

Abstract Multiple myeloma (MM) is a B-cell malignancy characterized by accumulation of malignant plasma cells (PC) within the bone marrow. The bone marrow microenvironment such as bone marrow stroma cells (BMSC) supports MM disease progression, resistance to chemotherapy, protects the tumor cells against apoptosis and causes osteolytic bone disease and angiogenesis. The aim of this study was to identify constitutive genetic alterations in BMSC derived from patients with MM (MM-BMSC) in comparison to BMSC from healthy donors. For BMSC selection, mononuclear cells were isolated from fresh bone marrow aspirates and were further expanded in cell culture. We studied 25 MM patients and 5 healthy donors. Senescence status was determined in passage 1 of cell cultures. MM-BSMC displayed a considerably higher percentage of senescence cells (p<0,05). We investigated the expression of cell cycle and adhesion-associated genes (CCNE1, CCND1, CDKN1B, VCAM, ICAM, IKK-alpha) in BMSC (passage 4) using SYBR-Green Real-Time PCR and relative quantification by linear regression. A downregulation of CCNE1 (p=0,05), CDKN1B (p=0,29), and an upregulation of CCND1 (p=0,05), VCAM-1 (p=0,33), ICAM-1 (p=0,33), and IKK-alpha (p=0,05) was demonstrated. Furthermore, the expression profile of miRNAs, targeting the analyzed mRNA genes or correlating with senescence, was studied (miR-16, miR-221, miR-126, miR-223, miR-485-5p and miR-519d). For miRNA detection treatment with Poly(A)-Polymerase and cDNA-Synthesis with a Poly(T)VN-Adaptor primer were carried out following an amplification with an universal reverse primer corresponding to the adaptor sequence and a miRNA-specific primer. miR-16, miR-223, miR-485-5p and miR-519d were significantly upregulated, (p=0,02; p=0,004; p=0,02; and p=0,002, respectively), whereas miR-221 and miR-126 showed no considerable differences to BMSC obtained from healthy donors. Next we investigated incubation of immunmodulatory drug Lenalidomid in BMSC cultures. Cells were treated with 10 µM Lenalidomid over 72 hours and expression was normalized to a 0,01 % DMSO control. Significant difference in gene expression were visible for ICAM-1 (p=0,01). For CDKN1B (p=0,16) and VCAM-1 (p=0,12) we demonstrated changes in gene expression. Our results indicate aberrant expression of cell cycle and adhesion-related genes, such as CCNE1, CCND1 and CDKN1B VCAM-1, ICAM-1 and IKK-alpha in MM-BMSC as compared with healthy donors. Furthermore, we found significant upregulation of miR-16, miR-223, miR-485-5p and miR-519d. Further investigation are needed to determine molecular mechanisms in MM-BMSC and PC interaction that lead to constitutive changes in BMSC characteristics and gene expression patterns. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Overexpression of c-maf is a frequent oncogenic event in multiple myeloma that promotes proliferation and pathological interactions with bone marrow stroma

Cancer Cell ◽  
2004 ◽  
Vol 5 (2) ◽  
pp. 191-199 ◽  
Author(s):  
Elaine M Hurt ◽  
Adrian Wiestner ◽  
Andreas Rosenwald ◽  
A.L Shaffer ◽  
Elias Campo ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Bone Marrow Stroma ◽  
Marrow Stroma

scholarly journals The sumoylation pathway is dysregulated in multiple myeloma and is associated with adverse patient outcome

Blood ◽  
2010 ◽  
Vol 115 (14) ◽  
pp. 2827-2834 ◽  
Author(s):  
James J. Driscoll ◽  
Dheeraj Pelluru ◽  
Konstantinos Lefkimmiatis ◽  
Mariateresa Fulciniti ◽  
Rao H. Prabhala ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Mammalian Cells ◽  
Treatment Strategies ◽  
Dominant Negative ◽  
Bone Marrow Stroma ◽  
Stroma Cell ◽  
Bone Marrow Stroma Cell ◽  
Marrow Stroma ◽  
Conjugating Enzyme

Abstract Multiple myeloma (MM) is a plasma cell neoplasm that proceeds through a premalignant state of monoclonal gammopathy of unknown significance; however, the molecular events responsible for myelomagenesis remain uncharacterized. To identify cellular pathways deregulated in MM, we addressed that sumoylation is homologous to ubiquitination and results in the attachment of the ubiquitin-like protein Sumo onto target proteins. Sumoylation was markedly enhanced in MM patient lysates compared with normal plasma cells and expression profiling indicated a relative induction of sumoylation pathway genes. The Sumo-conjugating enzyme Ube2I, the Sumo-ligase PIAS1, and the Sumo-inducer ARF were elevated in MM patient samples and cell lines. Survival correlated with expression because 80% of patients with low UBE2I and PIAS1 were living 6 years after transplantation, whereas only 45% of patients with high expression survived 6 years. UBE2I encodes the sole Sumo-conjugating enzyme in mammalian cells and cells transfected with a dominant-negative sumoylation-deficient UBE2I mutant exhibited decreased survival after radiation exposure, impaired adhesion to bone marrow stroma cell and decreased bone marrow stroma cell–induced proliferation. UBE2I confers cells with multiple advantages to promote tumorigenesis and predicts decreased survival when combined with PIAS1. The sumoylation pathway is a novel therapeutic target with implications for existing proteasomal-based treatment strategies.

Targeting the Wnt/Beta-Catenin Pathway in Multiple Myeloma with a Novel Orally Bioavailable Beta-Catenin Inhibitor

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1842-1842
Author(s):  
Ioanna Savvidou ◽  
Tiffany T. Khong ◽  
Stephen K. Horrigan ◽  
Andrew Spencer
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Death ◽  
Stromal Cell ◽  
Single Agent ◽  
Autologous Bone Marrow ◽  
Myeloma Cell ◽  
Tumour Cells ◽  
Beta Catenin ◽  
Orally Bioavailable

Abstract Abstract 1842 The introduction of novel chemotherapeutics and stem cell transplantation has resulted in an improved outlook for patients with multiple myeloma (MM) but the disease remains incurable emphasising the need for alternative therapeutic approaches. Beta-catenin, the central molecule of the Wnt canonical pathway is dysregulated in multiple solid tumours and haematological malignancies, including MM, providing a rationale to evaluate the potential of beta-catenin inhibitors as a therapeutic paradigm for MM. We have demonstrated that the orally bioavailable beta-catenin inhibitor BC2059 (BetaCat Pharmaceuticals, Gaithersburg, USA) induced apoptosis in a range of genetically heterogenous human myeloma cell lines (HMCL) (n=10). Dose responsiveness to BC2059 was initially determined by MTS assays with concentrations ranging from 50nM to 10000nM showing a dose and time dependent BC2059-induced inhibition of HMCL viability. Further analyses demonstrated the accumulation of cells in the G0/G1 phase suggesting G1 cell cycle arrest with IC50 concentrations for the 10 HMCL ranging from 53nM (KMS-18) to 247nM (NCI H929). The expression of the active nuclear non-phosphorylated form of beta-catenin was confirmed by immunoblotting and intranuclear flow cytometry in all 10 HMCL but the levels did not correlate with BC2059 IC50. Mimicking the bone marrow microenvironment utilising HS5, an immortalised human stromal cell line, by co-culturing with HMCL interestingly showed the capacity of BC2059 to overcome the protective effect of the stromal layer. At the IC80 of BC2059 for 5 HMCL, the level of apoptosis in the absence or presence of HS5 was comparable. For example, forANBL-6 (IC50 =115nM) BC2059-induced cell death at 100nM was 65% when cultured alone and 35% in the context of co-culture, whereas at 220nM cell death was 83% and 70% respectively. Beta-catenin undergoes proteasome mediated destruction and has been demonstrated to be elevated subsequent to exposure to bortezomib, we therefore evaluated whether BC2059 could mitigate this pro-survival stress response. 5 HMCL were tested and the combination of BC2059 and bortezomib was found to be synergistic in all instances with synergy quotients [SQ] ranging from 1.5 to 2.25 (where a SQ>1 confirms synergy). Furthermore, optimal killing was induced with either concomitant exposure to both drugs or by drug sequencing with bortezomib exposure 4 hours prior to BC2059 but not the reverse sequence. Finally, as a single agent BC2059 effectively killed primary MM tumour cells from relapsed and/or refractory MM patients (n=11) in an autologous bone marrow (BM) co-culture assay with a median cell death of 46±7,5% and 59±5.6% at 1μM and 5μM, respectively. In conclusion BC2059 effectively kills both HMCL and primary MM tumour cells at therapeutically feasible concentrations and can overcome the pro-survival effect of stromal cell and BM co-culture. BC2059 warrants further evaluation as a potential anti-MM therapeutic. Disclosures: Horrigan: BetaCat Pharmaceuticals: Employment.

VLA4 Blockade In Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3944-3944 ◽  
Author(s):  
Yao-Te Hsieh ◽  
Enzi Jiang ◽  
Jennifer Pham ◽  
Hye-Na Kim ◽  
Hisham Abdel-Azim ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Single Agent ◽  
Leukemia Cell ◽  
Chemotherapeutic Agents ◽  
Bone Marrow Stroma ◽  
Nsg Mice ◽  
Marrow Stroma ◽  
Acute Myeloid

Abstract Despite aggressive chemotherapy and early allogeneic transplantation, acute myeloid leukemia (AML) frequently relapses, so that over-all disease-free survival remains below 50%. Strategies to overcome the chemoresistance of relapse-initiating residual AML blasts are, therefore, warranted. Evidence has been provided that AML cells are sheltered from the insult of chemotherapeutic agents by interacting with bone marrow stroma. Integrin alpha4beta1 (VLA4) mediates adhesion of hematopoietic cells to bone marrow stroma cells and extracellular matrix and has been implicated in cell adhesion-mediated chemotherapy resistance. Based on the evidence thereof provided for ALL blasts, VLA4 is here proposed as a therapeutic target for refractory AML. For this purpose, VLA4 was functionally blocked in vitro and in vivo on patient-derived AML cells using an anti-functional humanized VLA4 antibody, Natalizumab (NZM). VLA4-positive (>90%) patient-derived (primary) AML cells were plated on immobilized human VCAM1 or human stromal cell line HS-5 and treated with control (IgG4) or Natalizumab (NZM) for 2 days. NZM de-adhered 94.0%±7.6 AML cells from its counter receptor VCAM-1, yet only 31.3%±13.8 from HS-5, indicating that stroma cells offer ligands for a wider panel of adhesion receptors besides VLA4. We tested also whether VLA4 blockade is beneficial against AML when combined with chemotherapy. For this purpose, primary AML cells were incubated with NZM and incubated on uncoated tissue culture plates or HS-5 stromal layers in the presence or absence of Ara-C (1µM) for two days. AML cells showed higher viability under Ara-C therapy when incubated with HS-5 cells compared to controls, indicating the chemoprotective effect of the stromal layer. The viability of the AML cells treated with combined Ara-C and NZM was similar to the controls, indicating that HS-5-mediated chemo-protection was completely abrogated by NZM. Significantly more AML cells treated with Ara-C+NZM stained AnnexinV+/7AAD- than after Ara-C+control Ig4 treatment (44.4%±5.6 vs. 29.8%±4.8, p=0.03) indicating increased apoptosis of AML cells. On its own, NZM did not induce apoptosis. Next, we tested NZM as a single agent in our NOD/SCIDIL2Rγ deficient (NSG) xenograft model of primary AML. Luciferase-labeled AML cells were intrafemorally injected into NSG mice (1x105 cells / mouse). NZM (5mg/kg) was given intraperitoneally once per week for 4 weeks. NZM-treated animals survived significantly longer than control Ig-treated animals (Median Survival Time, MST=107 days vs. MST=76 days; *p=0.008 by Log-rank Test.To determine effects of NZM on leukemia cell burden/distribution in different organs, primary AML cells were injected into NSG mice and allowed to engraft for 3 days, subsequently treated with a single dose of NZM or Ig control. 72 hours later, AML cell burden in femurs and spleens of NZM-treated animals was significantly decreased compared to control treated mice, however AML cells were not increased into the peripheral blood, so that whether leukemia cells were selectively killed ormobilized and then retained in non-hematopoietic organs remains to be determined. Further studies addressing molecular mechanisms of increased apoptosis after combined VLA4 blockade and chemotherapy are ongoing. Our data suggest that the paradigm of leukemia cell targeting by VLA4 blockade, previously demonstrated by us for ALL, can also be applied to AML. Disclosures: No relevant conflicts of interest to declare.

CD28 and CD86 Regulate Integrin Surface Expression In Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4450-4450
Author(s):  
Catherine M Gavile ◽  
David Egas ◽  
Gregory H Doho ◽  
Sagar Lonial ◽  
Kelvin P Lee ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Cell Surface ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Bone Marrow Stroma ◽  
Rna Seq ◽  
Myeloma Cells ◽  
Surface Molecules ◽  
Marrow Stroma

Multiple myeloma is a malignancy of long lived plasma cells. Like normal plasma cells, myeloma cells are dependent on the bone marrow microenvironment for survival. While the specific interactions and downstream signals mediated by the bone marrow stroma have yet to be fully characterized, drug resistance is linked to these pathways, and further understanding will uncover new therapeutic avenues for myeloma. CD28 and CD86 are best known for their role in T-cell activation; however they have recently been shown to play important roles in the generation and survival of normal long lived plasma cells. CD28 is the canonical costimulatory receptor known to activate the PI3K-Akt pathway in T-cells upon binding to CD80 or CD86 from an antigen-presenting cell. CD28 and CD86 are also expressed by normal plasma and myeloma cells, and we have previously shown that both CD28 and CD86 are necessary for myeloma cell survival. Silencing of either CD28 or CD86 results in cell death in 3 human myeloma cell lines (RPMI8226, MM.1s, and KMS18). Interestingly, in 2 cell lines, knockdown of CD86 results in higher levels of cell death than CD28. In these lines, silencing CD28 or addition of a soluble inhibitor CTLA4-Ig (Abatacept), results in an increase in CD86 expression. Taken together, these data suggest that CD28 and CD86 regulate the survival of myeloma cells through either cis or trans signals, and feedback signals from CD28 control CD86 expression. The data also suggest that signaling events result from ligation of either CD28 or CD86. To better define the nature of the survival signals emanating from CD28 and CD86, we performed RNA-Seq on myeloma cells where CD28 or CD86 expression had been silenced. For silencing of CD28 we found 1292, 1195, and 1697 transcripts that were significantly changed compared to vector control in KMS18, MM.1s and RPMI8226, and silencing CD86 results in a similar number of changes (1405, 1200 and 1866 transcripts respectively). Since CD28 and CD86 form a receptor-ligand pair, we focused on genes that were common to silencing of both. Of genes that had significant expression changes compared to vector control, we found 229, 221 and 399 transcripts that were commonly regulated by CD28 and CD86 in KMS18, MM.1s and RPMI8226 respectively. Most transcripts were either upregulated (45.4 to 57.5%) or downregulated (28.8 to 41.5%) by silencing of either CD28 or CD86. A subset of transcripts (13.1 to 14%) showed a pattern of expression similar to CD86 - silencing of CD28 and CD86 had opposite effects on expression. This subset of transcripts may represent genes that are regulated by CD86 signaling, and may explain the difference in sensitivity to CD28 vs. CD86 silencing. Curiously, in KMS18 where this difference was not observed, RNA-Seq indicates that these cells are homozygous for a CD86 SNP that is associated with increased cancer susceptibility and lower transplant rejection, and may represent a hypomorphic allele. Surprisingly, we did not observe any significant changes in either pro- or anti-apoptotic Bcl-2 genes in any cell line except for upregulation in minor transcripts of Bcl2L11 (Bim) in KMS18 cells. This change did not affect overall expression as confirmed by qRT-PCR. However, expression of several cell surface proteins associated with myeloma cell survival did change. Integrin-ß1 (ITGB1) and -ß7 (ITGB7) are surface molecules that facilitate both cell-matrix and cell-cell interactions, and have been implicated in myeloma growth, survival, and drug-resistance. Knockdown of CD28 or CD86 resulted in downregulation of ITGB7 that was confirmed by qRT-PCR. We also saw a reduction of ITGB7 at the cell surface with CD86 knockdown, but not with CD28. ITGB1 expression was reduced at the mRNA and cell surface levels with knockdown of CD86, but was induced with CD28 knockdown. Based on their patterns of expression, ITGB7 may be regulated by CD28 signaling, while ITGB1 may be downstream of CD86 signaling. These data indicate that CD28-86 signaling regulates the expression of integrins on the surface of myeloma cells. Because drug resistance has been linked to the myeloma cells’ interaction with the bone marrow stroma and its resident cells (CAM-DR), these surface molecules could be important mediators of CD28 and CD86 survival signaling. Taken together, our data indicate that targeting CD28-86 signaling is a promising therapeutic approach to CAM-DR, and may be a useful addition to current regimens against myeloma. Disclosures: Lonial: Onyx: Consultancy; Celgene: Consultancy; Millennium: Consultancy; Novartis: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Onyx: Consultancy. Boise:Onyx Pharmaceuticals: Consultancy.

Studies of the Bcl Inhibitor GX15-070 in Multiple Myeloma Demonstrate Substantial Pre-Clinical Activity.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1572-1572 ◽  
Author(s):  
A. Keith Stewart ◽  
Zhi Hua Li ◽  
Xiao Yan Wen ◽  
Jennifer Rauw ◽  
Latha Pathangey ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Time Course ◽  
Myeloma Cell ◽  
Clinical Activity ◽  
Western Blots ◽  
Cell Line Panel ◽  
Normal Human ◽  
Binding Groove

Abstract GX15-070 is a small-molecule antagonist of the BH3-binding groove of the Bcl-2 family of proteins and can thus induce apoptosis by inhibiting the interaction between pro- and anti-apoptotic proteins. In addition, GX15-070 activates a cellular pathway, which leads to the activation of the pro-apoptotic BH3-only protein Bim, a natural cellular inhibitor of anti-apoptotic members. Together, the direct inhibition of anti-apoptotic Bcl-2 family proteins and the activation of pro-apoptotic Bim by GX15-070 can sensitize tumor cells to apoptosis. Bcl-2 family members, particularly Bcl-XL and Mcl-1 have been implicated in the survival of myeloma cells and may be responsible, in part, for drug resistance. Consequently, GX15-070 was tested against the MMRC 12 validation cell line panel, a panel of 12 standardized and annotated human myeloma cell lines (HMCLs). Western blots preformed on 8 HMCLs confirmed expression of Bcl-2, Mcl-1 and/or Bcl-XL. Basal levels of Bcl-2 and MCl-1 were relatively similar across all HMCLs studied whereas levels of Bcl-XL were variable GX15-070 inhibited the viability of all 12 myeloma cell lines as determined by MTT assay. The mean IC50 was 215 nM; range (6.4-678nM) and sensitivity did not correlate with level of Bcl-2, MCL-1 or Bcl-XL expression. On the other hand cytotoxicity to normal human blood lymphocytes, stroma and bone marrow CFUs was not observed at concentrations which were effectively cytotoxic to HMCL. Time course experiments demonstrate that apoptosis and cleavage of caspase 3 began by 12 hours and continued to increase over a 96 hour period. Co-culture with human bone marrow stroma cells (BMSCs) failed to protect HMCL from GX15-070 induced cytotoxicity. Similarly, potent MM growth factors, Interleukin 6 (IL-6) and Insulin like growth factor (IGF)-1 did not confer resistance to GX15-070. Of interest, overexpression of Bcl-2 has been shown to attenuate PS-341-induced cell death (Mitisades); however, the combination of PS-341 and GX15-070 did not reveal synergistic interaction in preliminary results. Studies looking at other drug combinations are planned. Finally, GX15-070 produced cytotoxic responses in the first primary myeloma sample tested with further testing ongoing. Mouse xenograft studies are also ongoing and will be presented. These studies indicate extensive pre-clinical activity of GX15-070 in multiple myeloma and set the stage for planned Phase II clinical trials.

Bortezomib Reverts Stroma-Mediated Resistant to APO2L/TRAIL in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2806-2806
Author(s):  
Nancy Parquet ◽  
Mark Mead ◽  
Claudio Anasetti ◽  
William S. Dalton ◽  
Lia Perez
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cancer Cells ◽  
Induced Resistance ◽  
Soluble Factor ◽  
Bone Marrow Stroma ◽  
Myeloma Cells ◽  
Bortezomib Treatment ◽  
Trail Receptors ◽  
Marrow Stroma

Abstract Abstract 2806 Poster Board II-782 Hematopoietic stroma provides a sanctuary for multiple myeloma (MM) cells and promotes resistance to immune control mediated by APO2L/TRAIL in part by increasing synthesis of the anti-apoptotic protein c-FLIP (J Immunol, 180: 3; 2008). Bortezomib, a reversible 26S proteosome inhibitor, sensitizes cancer cells to APO2L/TRAIL induced apoptosis in a variety of malignancies including MM. In addition, it has been well established that bortezomib interferes with the protective interaction between cancer cells and the bone marrow stroma. In this study we tested whether bortezomib can reverse the APO2L/TRAIL environmental mediated-immune resistant (EM-IR). Using MM cell lines (RPMI 8226 and U266) or CD138+ positive selected cells from MM patient's bone marrow, we found that exposure to HS5 stroma cells by direct adhesion or in a transwell system induced resistance to bortezomib (10nM for 24 hours) and that pretreatment with bortezomib (10nM for 20 hours) effectively overcomes APO2L/TRAIL resistance (10ng/mL for 4 additional hours). Conditioned medium made from a 14-day culture of MM patient's bone marrow stroma induced resistance to bortezomib and pretreatment with bortezomib sensitized cells to APO2L/TRAIL induced cell death as shown with HS5 cells. Bortezomib showed not to be cytotoxic to HS5 stroma cells and only MCP-2-3 and IL-10 levels were altered in the stroma-MM milieu within cytokines measured by ELISA array. IL-6, a cytokine shown to induce APO2L/TRAIL mediated resistance, remained unchanged with bortezomib treatment. We found that bortezomib increased expression of TRAIL receptors (DR5, DCR1 and DCR2), but such expression did not predict for sensitivity to apoptosis as DR4 demonstrated to be the receptor responsible for activation of APO2L/TRAIL. Soluble factor(s) released by HS5 stroma increased expression of c-FLIP and induced STAT-3 and ERK phosphorylation in myeloma cells. However, only c-FLIP protein expression was effectively reduced by bortezomib. c-FLIP quantitative-PCR found that HS5 increased transcription only after 20 hours bortezomib treatment and significantly reduces soluble factor(s) induced c-FLIP transcription. In a transwell assay, HS5 stroma cells induced NF-κB activation and the addition of bortezomib diminished its activation at 20 hours. These findings provide the rationale to combine bortezomib and APO2L/TRAIL to disrupt the influence of the stroma microenvironment on myeloma cells. Disclosures: Off Label Use: sirolimus for graft-versus-host disease.

Sign in / Sign up

Export Citation Format

Share Document