Mouse Model Of Multiple Myeloma Research Articles

Potent anti-tumor response by targeting B cell maturation antigen (BCMA) in a mouse model of multiple myeloma

Multiple myeloma (MM) is an aggressive incurable plasma cell malignancy with a median life expectancy of less than seven years. Antibody-based therapies have demonstrated substantial clinical benefit for patients with hematological malignancies, particular in B cell Non-Hodgkin's lymphoma. The lack of immunotherapies specifically targeting MM cells led us to develop a human-mouse chimeric antibody directed against the B cell maturation antigen (BCMA), which is almost exclusively expressed on plasma cells and multiple myeloma cells. The high affinity antibody blocks the binding of the native ligands APRIL and BAFF to BCMA. This finding is rationalized by the high resolution crystal structure of the Fab fragment in complex with the extracellular domain of BCMA. Most importantly, the antibody effectively depletes MM cells in vitro and in vivo and substantially prolongs tumor-free survival under therapeutic conditions in a xenograft mouse model. A BCMA-antibody-based therapy is therefore a promising option for the effective treatment of multiple myeloma and autoimmune diseases.

Anti-β₂-microglobulin monoclonal antibodies overcome bortezomib resistance in multiple myeloma by inhibiting autophagy.

Our previous studies showed that anti-β2M monoclonal antibodies (mAbs) have strong and direct apoptotic effects on multiple myeloma (MM) cells, suggesting that anti-β2M mAbs might be developed as a novel therapeutic agent. In this study, we investigated the anti-MM effects of combination treatment with anti-β2M mAbs and bortezomib (BTZ). Our results showed that anti-β2M mAbs enhanced BTZ-induced apoptosis of MM cell lines and primary MM cells. Combination treatment could also induce apoptosis of BTZ-resistant MM cells, and the enhanced effect depended on the surface expression of β2M on MM cells. BTZ up-regulated the expression of autophagy proteins, whereas combination with anti-β2M mAbs inhibited autophagy. Sequence analysis of the promoter region of beclin 1 identified 3 putative NF-κB-binding sites from -615 to -789 bp. BTZ treatment increased, whereas combination with anti-β2M mAbs reduced, NF-κB transcription activities in MM cells, and combination treatment inhibited NF-κB p65 binding to the beclin 1 promoter. Furthermore, anti-β2M mAbs and BTZ combination treatment had anti-MM activities in an established MM mouse model. Thus, our studies provide new insight and support for the clinical development of an anti-β2M mAb and BTZ combination treatment to overcome BTZ drug resistance and improve MM patient survival.

CDKN1A and FANCD2 are potential oncotargets in Burkitt lymphoma and multiple myeloma.

BackgroundComparative genetic and biological studies on malignant tumor counterparts in human beings and laboratory mice may be powerful gene discovery tools for blood cancers, including neoplasms of mature B-lymphocytes and plasma cells such as Burkitt lymphoma (BL) and multiple myeloma (MM).MethodsWe used EMSA to detect constitutive NF-κB/STAT3 activity in BL- and MM-like neoplasms that spontaneously developed in single-transgenic IL6 (interleukin-6) or MYC (c-Myc) mice, or in double-transgenic IL6MYC mice. qPCR measurements and analysis of clinical BL and MM datasets were employed to validate candidate NF-κB/STAT3 target genes.ResultsqPCR demonstrated that IL6- and/or MYC-dependent neoplasms in mice invariably contain elevated mRNA levels of the NF-κB target genes, Cdkn1a and Fancd2. Clinical studies on human CDKN1A, which encodes the cell cycle inhibitor and tumor suppressor p21, revealed that high p21 message predicts poor therapy response and survival in BL patients. Similarly, up-regulation of FANCD2, which encodes a key member of the Fanconi anemia and breast cancer pathway of DNA repair, was associated with poor outcome of patients with MM, particularly those with high-risk disease.ConclusionsOur findings suggest that CDKN1A and FANCD2 are potential oncotargets in BL and MM, respectively. Additionally, the IL-6- and/or MYC-driven mouse models of human BL and MM used in this study may lend themselves to the biological validation of CDKN1A and FANCD2 as molecular targets for new approaches to cancer therapy and prevention.Electronic supplementary materialThe online version of this article (doi:10.1186/s40164-015-0005-2) contains supplementary material, which is available to authorized users.

The effect of the PI3K inhibitor BKM120 on tumour growth and osteolytic bone disease in multiple myeloma

The plasma cell malignancy multiple myeloma (MM) is unique amongst haematological malignancies in its capacity to cause osteoclast-mediated skeletal destruction. The PI3K/Akt pathway mediates proliferation, survival and drug resistance in MM plasma cells and is also involved in regulating the formation and activity of bone-forming osteoblasts and bone-resorbing osteoclasts.NVP-BKM120 (Buparlisib, Novartis) is a PI3K inhibitor that is currently undergoing clinical evaluation in several tumour settings. In this study, we have examined the anti-tumorigenic effects of BKM120 in an immunocompetent mouse model of MM and its effects on osteoblast and osteoclast formation and function. BKM120 treatment (40mg/kg) resulted in a significant decrease in serum paraprotein and tumour burden, and μCT analysis of the proximal tibia revealed a significant reduction in the number of osteolytic bone lesions in BKM120-treated animals. BKM120 also mediated a significant increase in serum levels of the osteoblast marker P1NP, and a significant decrease in serum levels of the osteoclast marker TRAcP5. In vitro, BKM120 decreased MM plasma cell proliferation, osteoclast formation and function, and promoted osteoblast formation and function. These findings suggest that, in addition to its anti-tumour properties, BKM120 could be used to treat osteolytic bone disease in MM patients.

α-Radioimmunotherapy with ²¹³Bi-anti-CD38 immunoconjugates is effective in a mouse model of human multiple myeloma.

In spite of development of molecular therapeutics, multiple myeloma (MM) is fatal in most cases. CD38 is a promising target for selective treatment of MM. We tested radioimmunoconjugates consisting of the α-emitter ²¹³Bi coupled to an anti-CD38 MAb in preclinical treatment of MM. Efficacy of ²¹³Bi-anti-CD38-MAb was assayed towards different MM cell lines with regard to induction of DNA double-strand breaks, induction of apoptosis and initiation of cell cycle arrest. Moreover, mice bearing luciferase-expressing MM xenografts were treated with ²¹³Bi-anti-CD38-MAb. Therapeutic efficacy was monitored by bioluminescence imaging, overall survival and histology. ²¹³Bi-anti-CD38-MAb treatment induced DNA damage which did not result in activation of the G2 DNA-damage-response checkpoint, but instead in mitotic arrest and subsequent mitotic catastrophe. The anti-tumor effect of ²¹³Bi-anti-CD38-MAb correlated with the expression level of CD38 in each MM cell line. In myeloma xenografts, treatment with ²¹³Bi-anti-CD38-MAb suppressed tumor growth via induction of apoptosis in tumor tissue and significantly prolonged survival compared to controls. The major organ systems did not show any signs of ²¹³Bi-induced toxicity. Preclinical treatment of MM with ²¹³Bi-anti-CD38-MAb turned out as an effective therapeutic option.

Macrophage Migration Inhibitory Factor Regulates Multiple Myeloma Bone Marrow Homing

Multiple myeloma (MM) is the most frequently occurring bone cancer and is characterized by malignant antibody-producing cell accumulation in bone marrow (BM). MM is still incurable, and the disease management is complicated by crosstalk between MM cells and the BM microenvironment. Why MM cells predominantly locate in bone is not well understood. The process by which MM cells are recruited into BM and reside in bone for colonization is called MM BM homing. BM homing is also critical for MM metastasis to distal BM sites. Therefore, MM BM homing is an active process throughout the disease pathogenesis. Here for the first time, we report that macrophage migration inhibitory factor (MIF) regulates MM BM homing via a mechanism that involves MM adhesion in the BM microenvironment. More importantly, we also show that MIF might be a promising target for MM treatment.MIF is an inflammatory cytokine, secreted by various human cell types. Our results here showed that BM biopsies from MM patients had significantly higher MIF expression, compared with that from healthy donors as determined by both immunohistochemistry of BM biopsies and ELISA of BM aspirates (P<0.05). Animal studies using a human MM SCID mouse model (ARP-1 in the SCID mouse) and a murine MM mouse model (5-TGM1 in the C57BL/KawRij mouse) showed that intravenously inoculated (IV) MIF knocked-down (MIF-KD) MM cells did NOT form tumors in bone, whereas control knocked-down cells did form tumors in bone, as determined by both in vivo bioluminescent assay and FACS analysis of BM cells. Unlike the previously identified SDF-1/CXCR4 axis, which regulates MM chemotaxis to BM, MIF knock-down did NOT affect MM cell migration to BM. However, MIF-KD MM cells showed decreased adhesion to BM stromal cells. A mechanistic study suggested that MIF regulation of MM BM homing was mediated by MIF and its receptor CXCR4, but was independent of its primary receptor CD74. The MIF/CXCR4 axis regulated a panel of adhesion molecules via NFκB and JNK intracellular signaling, and MIF-KD and CXCR4-KD MM cells had similar downstream cell signaling and adhesion molecule expression. In addition, exposing MM cells to a MIF inhibitor (4-IPP) down-regulated expression of those adhesion molecules. Further, in the murine MM model with established MM tumors in bone, administering 4-IPP resulted in extramedullary metastasis. Overall, the findings above suggest that MIF regulates MM cell homing to the BM.We also explored MIF-targeting therapy for MM treatment. In the human MM SCID model, IV-inoculated ctrl-KD MM cells formed tumors both in BM and outside of the bone, such as in the abdomen and lung. When tumor-bearing mice were treated with the chemotherapeutic melphalan, the extramedullary tumors regressed significantly (P<0.05), whereas the BM tumors were not sensitive to treatment, probably because the BM microenvironment conferred MM chemoresistance. In the same mouse model inoculated with MIF-KD MM cells, because the MIF-KD MM cells had impaired attachment and could NOT form tumors in bone, the tumor-bearing mice had a complete response to the drug treatment as determined by tumor burden and mouse survival (P<0.05). These encouraging results suggested that MIF might be a valid target for MM treatment. To test this hypothesis, we used the murine MM mouse model and treated the tumor-bearing mice with both melphalan and MIF inhibitor. This combined therapy significantly repressed the MM tumor growth in vivo and lengthened survival (P<0.05). Thus, MIF might be a promising target for MM treatment, in combination with conventional chemotherapy.To summarize, our findings suggest a novel function for MIF in MM BM homing regulation. MIF regulates a panel of adhesion molecules and promotes MM cell attachment within the BM. MIF inhibition disrupts the boundary between MM cells and the BM microenvironment and may raise MM cell drug sensitivity. DisclosuresNo relevant conflicts of interest to declare.

Constitutive gp130-JAK-STAT3 Signaling Induces a Plasma Cell Disorder Representing Genetic and Clinical Features of Human Myeloma – a Novel in Vivo Model for the Patient Subgroup Characterized By a STAT3 Target Gene Signature

Multiple myeloma (MM) is a plasma cell neoplasm characterized by the clonal expansion of an immunoglobulin-secreting terminally differentiated B cell. MM patients classically suffer from the triade consisting of plasma cell infiltration of the bone marrow, lytic bone lesions and monoclonal gammopathy in plasma and/or urine. During disease progress patients develop end organ damage such as nephropathy, neuropathy and bone marrow insufficiency. New treatment options including immunomodulatory drugs and proteasome inhibitors have prolonged overall survival and quality of life. However MM is still considered an incurable disease by conventional treatment approaches.Deregulated signal transduction pathways, in particular the Interleukin-6-JAK-STAT3 pathway, play an important role in the growth regulation and survival of MM cells. IL-6 binds to the specific IL-6 receptor and this complex associates with two molecules of the ubiquitously expressed common signal transducer gp130, which leads to JAK-STAT3 activation and STAT3 target expression. Using immunohistochemistry for STAT3 phosphorylation and by assessing gene expression data we have identified a recurrent STAT3 pathway activation pattern in approximately 40% of all MM patients, strongly indicating that deregulated gp130 downstream activation constitutes an attractive therapeutic target in MM. Based on these findings we generated a novel retroviral bone marrow transduction-transplantation MM mouse model using a constitutively active form of gp130, L-gp130. This L-gp130 mouse model shows all characteristics of the human disease, including monoclonal gammopathy, bone marrow infiltration with lytic bone lesions, and protein deposition in the kidney. Essentially, as compared to previous MM models, MM penetrance is very high and tumor latency with a median of approximately 200 days short. The disease is easily transplantable into secondary recipient mice. To assess clonality we subcloned and sequenced IgH rearrangements from individual tumors. Our analyses revealed that diseased mice contained monoclonal and oligoclonal B cell expansions, and also demonstrated a germinal center passage. Fluorescence in situ hybridization revealed the occurrence of Myc aberrations, a genetic feature that is associated with aggressive disease and short survival in human MM. A genetic in vivo approach revealed that gp130 signaling collaborates with Myc to induce MM, and that gp130 signaling is responsible and sufficient for directing the plasma cell phenotype and to dominantly regulate the expression of antiapoptotic proteins. Finally, testing of murine primary MM cells isolated from diseased L-gp130 animals with currently used anti-myeloma drugs such as bortezomib, carfilzomib, melphalan and doxorubicin revealed significant anti-tumor activity.In summary, the L-gp130 MM mouse model provides a novel tool with clinical and genetic features of human MM. It offers several clear advantages over existing murine MM models. Regarding the patient subgroup with a recurrent STAT3 pathway activation pattern it may serve as a genetic and preclinical tool for delineating MM pathogenesis, for evaluation of targeted therapies, and for assessment of in vivo treatment resistance. DisclosuresKeller:Cellgene: Research Funding.

Identification of Novel Therapeutic Targets in the Clinically Predictive Vk*MYC Mouse Model of Multiple Myeloma

A genetic rearrangement of the MYC locus, resulting in dysregulated expression of MYC, is the most common mutation in human multiple myeloma (MM). The genetically engineered Vk*MYC mouse model is based on dysregulation of MYC, and has been extensively validated as a clinically and biologically faithful model of untreated MM. We previously reported 9 drugs or classes of drugs (DNA alkylators, glucocorticoids, proteasome inhibitors, IMiDs, nab-paclitaxel, histone deacetylase inhibitors, TACI-Ig, perifosine and SNS-032, a CDK2,7,9 inhibitor) with more than 20% partial response (PR) rate in Vk*MYC MM. Among those, the first 5 also have more than 20% PR in patients with MM for a positive predictive value of 56%. Although the HDACi did not show single agent activity in relapsed/refractory MM patients, the PANORAMA phase 3 study of panobinostat in combination with bor+dex has shown superior PFS compared to bor+dex, suggesting the possibility that HDACi may have shown single agent activity if tested in a less advanced clinical setting. In contrast, 11/12 drugs that have less than 20% PR in Vk*MYC MM also have less than 20% PR in patients with MM for a NPV of 92%. Confident that drugs with activity in Vk*MYC mice will likely be effective in the treatment of MM, we have used this model to study novel drugs for prioritization in clinical trials.Pim kinases are constitutively active serine/threonine kinases identified using retroviral mutagenesis as potent suppressors of MYC induced apoptosis and can be inhibited by the pan-PIM kinase inhibitor LGH447. KPT-276 is a selective inhibitor of nuclear export CRM1/XPO1 inhibitor. Both drugs are active in Vk*MYC MM and are showing early evidence of promising clinical activity, increasing the PPV of the Vk*MYC model of MM to 64% (or 73% if HDACi are considered clinically active).Also active in the Vk*MYC model were the bromodomain inhibitors GSK I-BET151, CPI-203, CPI-456, OTX-015, which, like JQ1, compete with acetylated histones for the binding to BRD4, inhibiting super-enhancer activity and MYC transcription. The histone methyltransferase enhancer of Zeste homolog 2 (EZH2) induces transcriptional repression through histone H3 lysine 27 trimethylation (H3K27me3) at the promoter of silenced genes. Activating mutations in the SET domain of EZH2 are prevalent in germinal center DLBCL and follicular lymphoma but have not been identified in MM, where the frequent dysregulation of MMSET by t(4;14) translocation or biallelic deletion of UTX are thought to play a tumorigenic role equivalent to EZH2 activation. EZH2 inhibitors have demonstrated activity against lymphoma with EZH2 activating mutations. Interestingly, we found the EZH2 inhibitor CPI-169 active against Vk*MYC MM, identifying EZH2 as a promising new epigenetic target in MM.EDO-S101 is a novel drug resulting from the fusion of a molecule of bendamustine with a molecule of vorinostat, with the aim of increasing the efficacy of the alkylator through the HDACi-mediated chromatin relaxation that would make DNA more accessible to the damaging effect of bendamustine. It induced a high rate of response in Vk*MYC MM that was sustained for more than 3 months in mice receiving only 2 doses, one week apart. Remarkably EDO-S101 is the only drug we have identified with single agent activity in the very aggressive, multi-drug resistant Vk12653 transplant model of relapsed/refractory MM.Finally, 2 agents (anti-murine PD-L1 and LCL161) that primarily modulate the immune microenvironment, with little to no direct anti-MM activity, also induced responses. Anti-PD-L1 blocks immunosuppressive signaling from MM cells to PD1 on T cells. LCL161 is an IAP antagonist that leads to constitutive activation of the alternative NFkB pathway with direct stimulation of the innate and adaptive immune systems. Although antibodies to CD38 are among the most exciting new agents for the treatment of MM, they cannot be evaluated in Vk*MYC MM since murine normal or malignant plasma cells do not express CD38. In contrast while SLAMF7 is expressed in Vk*MYC MM, there is no murine equivalent of elotuzumab.In summary, we report 7 new drugs with single agent activity in Vk*MYC MM, nearly doubling the number of drugs or classes of drugs with promising anti-MM activity. Two (LGH447 and KPT-330) are already beginning to demonstrate clinical efficacy in MM. All of these agents should be prioritized for rapid evaluation in clinical trials of patients with MM. DisclosuresBergsagel:Novartis: Research Funding; Constellation Pharmaceutical: Research Funding; OncoEthix: Research Funding; MundiPharma: Research Funding.

Identification of an Epithelial-to-Mesenchymal Transition (EMT)-like Programme in t(4;14)-Positive Multiple Myeloma Reveals Novel Targets for Therapeutic Intervention

Introduction: Prognosis for multiple myeloma (MM) patients has improved dramatically since the introduction of novel therapies. However, prognosis in the t(4;14) subgroup, characterized by expression of the histone methyltransferase MMSET, remains relatively poor due to the acquisition of a highly aggressive, motile and invasive phenotype and a generally modest response to therapy. We have previously demonstrated that MMSET promotes an epithelial-to-mesenchymal transition (EMT) in prostate cancer1. While the term EMT is not commonly used to describe MM, we hypothesise that an EMT-like process plays a critical role in t(4;14)­-positive MM disease pathogenesis. In this study, we conducted a comprehensive evaluation of the association between t(4;14) and an EMT-related gene expression signature in MM and identify N-cadherin as a therapeutically-targetable EMT-related gene in t(4;14)-positive MM.Methods and results: Expression of a core EMT-related signature, comprising 169 mesenchymal genes and 49 epithelial genes, was assessed in CD138-selected MM plasma cells from newly-diagnosed MM patients in four independent microarray datasets (E-GEOD-19784 [n = 327 MM patients], E-GEOD-26863 [n = 304], E-MTAB-317 [n = 226] and E-MTAB-363 [n = 155]), accessed through ArrayExpress (EMBL-EBI). In each dataset, gene expression was compared in t(4;14)-positive and t(4;14)-negative patients using linear models for microarray data (LIMMA). Twenty six mesenchymal genes were found to be upregulated in t(4;14)-positive patients across the 4 datasets (p < 0.05, Fisher’s method). A general loss of epithelial genes was not observed, likely due to the haematopoietic origins of MM. Upregulated genes included key EMT drivers (TWIST1, SOX9, TCF4) and genes associated with the cytoskeleton (VIM), adhesion and migration (CDH2, ITGB1, NCAM1) and signalling pathways involved in EMT (BMPR1A, IL6R, TGFB2). The t(4;14)-mediated regulation of EMT-related genes including TWIST1, CDH2, BMPR1A and ITGB1 was confirmed by assessing the effects of MMSET knockdown, knockout and add-back in the t(4;14)-positive human myeloma cell line KMS-11.We have previously demonstrated that N-cadherin (CDH2) expression is elevated in plasma cells from approximately 50% of newly diagnosed multiple myeloma (MM) patients and that elevated serum N-cadherin is associated with poor prognosis2. In order to identify whether N-cadherin is a potential therapeutic target in t(4;14)-positive myeloma, the effects of an N-cadherin peptide inhibitor ADH-1 (Exherin™) or shRNA-mediated N-cadherin knock-down were assessed on MM PC adhesion and proliferation in vivo and in vitro. The role of N-cadherin in MM tumour establishment and intramedullary growth was investigated using the C57BL/KalwRijHsd mouse model of MM. In this model, intravenously injected luciferase-expressing mouse MM PC cells (5TGM1-SFG) home to the bone marrow and initiate systemic MM disease. C57BL/KalwRijHsd mice bearing 5TGM1-SFG N-cadherin knock-down cells had significantly reduced tumour burden, as assessed by bioluminescent imaging and serum paraprotein levels, after 4 weeks compared with mice bearing control 5TGM1-SFG cells. Furthermore, daily intraperitoneal ADH-1 administration (100 mg/kg/day) to 5TGM1-SFG cell-bearing mice significantly significantly decreased tumour burden when administered at the time of tumour inoculation, but had no effect on established tumours. These results suggest that N-cadherin plays a role in extravasation, homing and/or tumour establishment in vivo.Conclusion: This study identified an extensive EMT-like gene expression signature driven by MMSET in t(4;14)-positive MM patients. This provides insight as to how the t(4;14) translocation leads poor prognostic outcomes in up to 20% of MM patients. Furthermore, these studies demonstrate a potential role for N-cadherin in MM tumour dissemination in t(4;14)-positive MM and suggest that N-cadherin represents a novel candidate for therapeutic targeting in these patients.1Ezponda et al. Oncogene, 20132Vandyke et al. Br J Haematol, 2013 DisclosuresNo relevant conflicts of interest to declare.

Phase 1 Study Update of the Novel Pan-Pim Kinase Inhibitor LGH447 in Patients with Relapsed/ Refractory Multiple Myeloma

Phase 1 Study Update of the Novel Pan-Pim Kinase Inhibitor LGH447 in Patients with Relapsed/ Refractory Multiple Myeloma

Pre-Clinical Activity of the Novel, First-in-Class p97 Inhibitor, CB-5083, in Multiple Myeloma

Hematological malignancies such as multiple myeloma (MM) have an increased reliance on the ubiquitin proteasome system (UPS) presumably as a consequence of their high protein synthetic and secretory burden. Chemical agents that target the proteasome, such as bortezomib and carfilzomib, have been successful in treating multiple myeloma; however patients treated with these drugs ultimately relapse. The AAA-ATPase p97/VCP (p97) facilitates ATP-dependent extraction and degradation of ubiquitinated proteins destined for proteasomal elimination. In addition to ubiquitin-dependent protein degradation, p97 is also closely involved in other aspects of protein homeostasis, including endoplasmic reticulum-associated degradation (ERAD) and autophagy. Pharmacologic inhibition of p97 provides a compelling therapeutic approach for hematological malignancies that rely on tight regulation of protein homeostasis as a component of their survival.CB-5083 is a novel small molecule inhibitor of p97 ATPase activity with nanomolar enzymatic and cellular potency. Treatment of cancer cells with CB-5083 causes a dramatic increase in poly-ubiquitinated proteins as well as an accumulation of substrates of the UPS and ERAD. CB-5083 causes a profound induction of the unfolded protein response (UPR) with consequent activation of the DR5 death receptor, caspase 8, caspase 3/7 and ultimately cell death. Induction of the UPR occurs to a greater magnitude with CB-5083 when compared to the proteasome inhibitor, bortezomib, suggesting the potential for increased efficacy in cancers with sensitivity to UPR-mediated cell death. In addition, activation of apoptosis and cell death occur more rapidly with CB-5083 than with bortezomib. Sequencing of cell lines made resistant to CB-5083 reveals missense mutations mapping to the D2 ATPase site in p97, supporting on-target association with cytotoxicity.In an expanded panel of MM cell lines there is no correlation between the cytotoxic sensitivity to CB-5083 and the cytotoxic sensitivity to proteasome inhibitors, suggesting differential mechanisms of cytotoxicity and potential activity of CB-5083 in proteasome inhibitor resistant settings. Compared to myeloma cell lines, CB-5083 has reduced cytotoxic potency in immortalized stromal cell lines and in patient-derived CD138-negative bone marrow mononuclear cells. Furthermore, unlike the reduced potency demonstrated by carfilzomib in the context of MM cell-bone marrow stromal cell (BMSC) interactions, the cyto-reductive potential of CB-5083 is unaffected in co-cultures of MM cells with patient-derived BMSCs or immortalized BMSCs from healthy donors.In vivo, CB-5083 is orally bioavailable, shows a pharmacodynamic effect in tumor tissue (as measured by poly-ubiquitin accumulation) and demonstrates robust anti-tumor activity across several MM models. CB-5083 treatment of mice bearing subcutaneous xenografts leads to tumor stasis and regression in RPMI8226 and AMO1 MM models, respectively. In advanced models of disseminated, ortho-metastatic disease, intermittent oral administration of CB-5083 demonstrates significant inhibition of myeloma burden and improves survival, with an overall efficacy profile that compares favorably to that of clinically approved proteasome inhibitors. Furthermore, in the Vk*Myc genetically engineered mouse model of MM, treatment with CB-5083 results in a significant reduction in M-spike by 55%. Combination treatment of mice bearing the RPMI8226 subcutaneous xenograft model with CB-5083, dexamethasone and lenalidomide results in tumor regression. Taken together, these data demonstrate that CB-5083 is a potent and selective inhibitor of the p97 ATPase with robust activity in vitro and in vivo in numerous MM models and strongly support clinical evaluation. Based on these observations, a phase 1 dose-escalation trial has recently been initiated and is currently underway in patients with relapsed/refractory multiple myeloma. DisclosuresAnderson:Cleave Biosciences: Employment. Le Moigne:Cleave Biosciences: Employment. Djakovic:Cleave Biosciences: Employment. Rice:Cleave Biosciences: Employment. Wong:Cleave Biosciences: Employment. Kumar:Cleave Biosciences: Employment. Valle:Cleave Biosciences: Employment. Menon:Cleave Biosciences: Employment. Kiss von Soly:Cleave Biosciences: Employment. Wang:Cleave Biosciences: Employment. Yao:Cleave Biosciences: Employment. Soriano:Cleave Biosciences: Employment. Bergsagel:ONYX: Consultancy; Janssen: Consultancy; BMS: Consultancy; Novartis: Research Funding. Yakes:Cleave Biosciences: Employment. Zhou:Cleave Biosciences: Employment. Wustrow:Cleave Biosciences: Employment. Rolfe:Cleave Biosciences: Employment.

An Open-Label, Dose Escalation, Multi-Center Phase 1 Study of PRLX 93936, an Agent Synthetically Active Against the Activated Ras Pathway, in the Treatment of Relapsed or Relapsed and Refractory Multiple Myeloma

An Open-Label, Dose Escalation, Multi-Center Phase 1 Study of PRLX 93936, an Agent Synthetically Active Against the Activated Ras Pathway, in the Treatment of Relapsed or Relapsed and Refractory Multiple Myeloma

Effects of Cabozantinib Alone and in Combination with Bortezomib in the 5TGM1 Murine Multiple Myeloma Model

Introduction: Cabozantinib (cabo) is an inhibitor of tyrosine kinases including MET, VEGFR2, RET, and the TAM family kinases TYRO3, AXL, and MER. Cabo has shown clinical activity in patients with castration-resistant prostate cancer and other solid tumors with bone metastases. Multiple myeloma (MM) is the second most common hematologic malignancy, and represents ~2% of all cancer deaths. MM is a monoclonal B-cell (plasma cell) neoplasia with clinical hallmarks of multiple osteolytic lesions causing bone pain, pathologic fractures, and hypercalcemia. Circulating levels of HGF and VEGF are upregulated in MM patients, and regulation of plasma cell-osteoblast communication by the HGF-MET signaling pathway has been implicated in the development of lytic bone disease in these patients. Thus, the primary objectives of this research were to A): determine the activity of cabo on bone lesions and tumor burden in the syngeneic 5TGM1 mouse MM model (study 1), and B): investigate the impact of cabo on overall survival of these mice when dosed alone or in combination with bortezomib (btz) (study 2).Methods: Female C57BL/KaLwRij mice were allocated to treatment groups (n=15-16 per group) with equivalent average body weights. Four experimental groups were utilized in each of 2 studies: a vehicle control group, and groups receiving single agent btz (0.5 mg/kg ip twice a week) or cabo (10 mg/kg, PO QD). Study 1 also included a higher dose cabo group (30 mg/kg, PO QD), and study 2 included a combination group: btz (0.5 mg/kg ip twice a week) plus cabo (10 mg/kg, PO QD). In study 2, each single agent group also received the vehicle from the alternate single-agent group via the appropriate route and schedule.On day 0, animals were inoculated with 5TGM1 mouse myeloma cells by IV administration. Dosing began on day 1 and continued daily until euthanasia at day 35 (study 1) or day 70 (study 2). Body weights were determined twice a week and blood samples were collected on days -1, 15, 22, and 34 for analysis of paraprotein (IgG2b) and TRACP 5b. In study 1 the development of osteolytic lesions was detected by radiography at the end of the study. Some animals were euthanized before the end of the experiment due to paraplegia. Animals euthanized within four days of the end of the experiment in study 1 were included in the analyses.Results: In study 1, btz reduced serum IgG2b levels, and decreased the frequency of soft tissue lesions, but did not show bone protective properties. Cabo exhibited bone protective effects: mean and total area of osteolytic lesions were reduced at the 30 mg/kg dose, and serum TRACP 5b values and osteoclast counts at the tumor-bone interface were reduced at both the 10 and 30 mg/kg doses. Relative bone area did not differ from control according to histomorphometry. The rise in serum IgG2b started earlier than vehicle control in both cabo-treated groups, but a significant difference was not observed in relative IgG2b at sacrifice. Cabo dose dependently increased the necrotic tumor area in bone, indicating the possibility that the rise in IgG2b may have been due to lysis of plasma cells. Both doses of cabo decreased the frequency of soft tissue lesions.In study 2, the median survival times were 36 d (vehicle), 43 d (btz), 48 d (cabo), and 55 d (cabo+btz). The prolongation of OS compared to vehicle was statistically significant for the cabo group but not for the btz group. Prolongation of OS in the combination group was significant compared to btz alone, but not when compared to cabo alone.Conclusions: Cabo showed both bone-protective and anti-tumor effects in this murine model of MM. In addition, statistically-significant prolongation of overall survival was observed with the combination of cabo+btz compared to btz alone, and with single-agent cabo compared to vehicle. Based on these results, further investigation of cabozantinib alone or in combination with other agents in multiple myeloma is warranted. DisclosuresAftab:Exelixis: Employment, Equity Ownership. Suominen:Pharmatest: Employment; Exelixis: Research Funding. Clary:Exelixis: Employment, Equity Ownership. Käkönen:Pharmatest: Employment; Exelixis: Research Funding. Fagerlund:Pharmatest: Employment; Exelixis: Research Funding. Alhoniemi:Pharmatest: Employment; Exelixis: Research Funding. Rissanen:Pharmatest: Employment; Exelixis: Research Funding. Halleen:Pharmatest: Employment; Exelixis: Research Funding.

Chemokines CCL14 and CCL3 Facilitate Monocytes/Macrophage Infiltration in Multiple Myeloma Bone Marrow

Drug resistance is the most common reason for treatment failure in multiple myeloma (MM), a plasma cell cancer of the bone marrow (BM). Our previous studies have shown that macrophage (MΦ) infiltration is increased in MM BM, and these MM-associated MΦs induce MM drug resistance by protecting MM cells from chemotherapeutic-induced cell death. A recently published clinical study also suggests that the number of BM MΦs negatively correlates with MM drug response and patient survival. Why MM BM harbors more MΦ is not well understood. In general, MΦs are derived from circulating monocytes, and monocytes are recruited to tissues by chemokines and differentiate into MΦs. In this study, we examined monocyte/MΦ chemokine expression in MM BM and determined that CCL14 and CCL3 were functional chemokines that regulated monocyte/MΦ infiltration in MM BM.To identify chemokines that regulate monocyte/MΦ infiltration in the MM tumor bed, we examined a panel of chemokines expressed in MM patient BM cells. Specifically, total BM cells (both CD138+ and CD138- cells) from MM patients were assayed by qPCR for target gene expression analysis. MM BM cells highly expressed CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), and CCL14 (HCC-1), but not CCL8 (MCP-2), CCL7 (MCP-3) or CCL13 (MCP-4). Next, we compared expression of those chemokines in MM BM vs. healthy BM aspirates by ELISA. MIP-1 α and HCC-1 were highly expressed in MM BM aspirates (n=11), but not in BM from healthy donors (n=7; P<0.05). Immunohistochemistry staining also confirmed that MM BM (n=5 patients) highly expressed MIP-1 α and HCC-1. Based on our findings, we hypothesized that elevated MΦ infiltration in MM BM might be due to MM BM overexpression of MIP-1α and HCC-1. To test this hypothesis, we first examined MIP-1α and HCC-1 function in monocyte migration. In vitro chemotactic assay showed that adding MIP-1α or HCC-1 neutralizing antibody inhibited monocyte migration to cocultured BM stromal cells (BMSCs) and MM cells (P<0.05). The antibodies also inhibited monocyte migration to ex vivo cultured BM cells from MM patients (P<0.05). In a murine MM mouse model, C57BL/KawRij mice inoculated with 5TGM1, a murine MM cell line, developed tumors in hind leg bones. Both flow cytometry analysis and immunohistochemistry staining suggested that the tumor-bearing mice had an increased number of MΦs in the BM, compared with healthy mice (P<0.05). Because HCC-1 does not have murine homology but it has the highest amino acid sequence similarity to MIP-1α, we treated the mice with MIP-1α neutralization antibody. Intra-peritoneal injection of mouse MIP-1α antibody decreased the MΦ number in BM (P<0.05). Such in vivo findings strongly suggest that MIP-1α regulates MΦ infiltration in MM BM. We also examined the source cells that contributed to high MIP-1 α and HCC-1 expression in MM BM. Primary MM cells (CD138+) and non-malignant BM cells (CD138-) were isolated from patient (n=5) BM aspirates. qPCR analysis showed that both CD138+ and CD138- cells had high MIP-1α and HCC-1 expression. Further, BMSC/MM coculture stimulated MIP-1α overexpression in BMSCs. Finally, we examined the association between MIP-1α or HCC-1 expression and the number of BM MΦ in MM patients. The chemokine expression in BM aspirates was determined by ELISA, and the number of MΦ was measured by flow cytometry analysis for CD14+/CD68+ cells. We found that MIP-1α expression was positively associated with the number of BM MΦs (P<0.05).To summarize, our findings suggest that CCL14 and CCL3 facilitate monocyte/MΦ infiltration in MM BM. Inhibiting these 2 chemokines may decrease the number of MM-associated MΦs, therefore increasing MM cell vulnerability to chemotherapy. DisclosuresNo relevant conflicts of interest to declare.

Initial Evaluation of Novel Dual PIM/PI3K and Triple PIM/PI3K/mTOR Inhibitors in Multiple Myeloma

Background:Despite advances in treatment, Multiple Myeloma (MM) remains incurable. The PI3K/AKT pathway is activated in MM cells in > 50% of cases due to factors such as bone marrow (BM) microenvironmental signaling and hyperactivation following treatment with proteasome inhibitors (PI). Multiple small-molecule inhibitors have been developed to target PI3K/AKT or mTOR kinases, but the efficacy of these drugs is likely to be compromised by the stimulation of compensatory signaling pathways. The redundancy of signaling pathways provides back-up mechanisms allowing escape from targeted inhibition. One such compensatory pathway is that driven by PIM kinases, which produce parallel oncogenic signals to AKT and mTOR and share several downstream molecular targets. As with PI3K/AKT, the BM microenvironment plays a major role in PIM activation and other factors increasing PIM levels include hypoxia and PI treatment. PIM1 and particularly PIM2 are known to be highly expressed in MM and play important roles in regulating MYC-driven transcription, apoptosis, cytokine signaling, cell proliferation and protein translation. Combinations of separate PI3K and PIM inhibitors have shown evidence of synergy in MM cell lines and animal models and a PIM kinase inhibitor has recently shown activity in relapsed/refractory MM. Given this background we wished to evaluate the activity of a novel family of kinase inhibitors capable of inhibiting not only PIM kinases but also PI3K/AKT (dual inhibitors) and PI3K/AKT/mTOR (triple inhibitors).Methods:We evaluated the in-vitro activities of a single pan-PIM (pPIMi), dual PIM/PI3K (IBL-202) and triple PIM/PI3K/mTOR (IBL-301) inhibitor in MM cell lines: MM1.S, NCI-H929, RPMI8226 and KMS11, which is known to be PIM2 dependent, alongside the pan-PI3K inhibitor GDC-0941 and the pan-PIM inhibitor AZD1208. IBL-202 and IBL-301 are optimized lead compounds and are low nanomolar pan-PIM/PI3K and pan-PIM/PI3K/mTOR inhibitors respectively. These dual and triple inhibitors show excellent kinase selectivity profile against a panel of 456 kinases. Cell viability was assessed using the Cell-Titre Glo assay and apoptosis determined by Annexin-V/PI staining. Co-culture experiments were performed with HS-5 stromal cells. Combination treatment was performed with bortezomib and IBL-202 to assess synergy.Results and discussion:IBL-202 and IBL-301 were significantly more potent than pPIMi in all MM cell lines tested (figure 1). IBL-202 and IBL-301 caused a loss in cell viability 50% and 70%, respectively, greater than pPIMi alone. IBL-202 and IBL-301 induced 50-80% and 80-100% cell death, respectively .v. 10% for pPIMi after 48 hrs, p<0.001. The Pim2 dependent MM cell line KMS11 showed a loss in cell viability following treatment with IBL-202 and IBL-301 up to three times greater than either of the PIM kinase inhibitors or GDC-0941. IBL-202 treatment caused a 90% reduction in cell viability at a dose of 5µM and IBL-301 was equally effective at a concentration of just 1µM. GDC-0941(5µM) caused a loss of approximately 30% in cell viability whereas cells remained entirely resistant to pPIMi and AZD1208 at concentrations up to 10µM (p< 0.001). IBL-202 in combination with bortezomib was synergistic in MM cell lines (CI<1).While co-culture with HS-5 cells protected MM cell lines against bortezomib-induced cell death, it promoted the apoptotic effect of both IBL-202 and IBL-301 with an increase in Annexin V positive cells from 15% to 40%. This suggests that micro-environmental stimulation could potentially induce synthetic lethality in the presence of these inhibitors. We observed strong induction of PIM2 in MM1.S cells following co-culture. Mechanistically, cells respond to dual and triple inhibitors with cell cycle arrest, marked apoptosis and strong down-regulation of biomarkers. The dual and triple inhibitors are optimized with respect to their in vitro ADME properties and have excellent oral bioavailability. In-vivo IBL-301 has been well tolerated, with no signs of toxicity even 20 times above the efficacious dose in a transgenic (KRASV12NSCLC) mouse model. Testing of IBL-202 in a relevant MM mouse model is planned in the near future.Conclusions:IBL-201 and IBL-301 show promising activity in MM cellular models with increased potency compared to inhibitors targeting PIM or PI3K alone and warrant further evaluation in this disease. [Display omitted] DisclosuresO’Neill:Inflection Biosciences: Employment, Equity Ownership. O’Dwyer:Inflection Biosciences: Membership on an entity’s Board of Directors or advisory committees.

Abstract 1814: Harnessing the potential of idiotypic peptide therapy for the treatment of multiple myeloma

Abstract BACKGROUND: The idiotype determinant of surface Immunoglobulin B-Cell Receptor (IgBCR) is the most reliable clonal marker of tumor cell population and can be exploited for novel targeting approaches. We previously developed a novel experimental approach to identify peptide ligands of the IgBCR idiotype determinant of neoplastic B cells, named “Id-peptides” (Palmieri C., Blood 2010). These Id-peptides were validated as a tool for specific targeting of B-cell malignancies with a potential role as modulators of tumor microenvironment via sIgBCR signalling. In Multiple Myeloma (MM), both mature and clonogenic tumor stem cells (clonotype B-lymphocytes, CBLs) share the same antigenic specificity, making these cells a suitable target for Id-peptides. In this regard, we have recently developed Id-peptides for tumor targeting in primary neoplastic plasma cells as well as in the 5T33MM mouse model of MM. RESULTS: Tumor-specific Id-peptides of MM (n=8), or MGUS (n=2) patients were selected by screening phage-displayed random peptide libraries using the cognate serum IgG paraprotein as bait. The selected Id-peptides specifically bound to the cognate primary neoplastic plasma cells, as shown by FACS analysis. By confocal microscopy, we observed that each patient-specific Id-peptide co-localized with the sIgG-BCR at the cell surface of the cognate neoplastic plasma cell. Further, the Id-peptides were able to target CD138pos differentiated plasma cells as well as a number of less differentiated CD138neg, IgGpos and CD19pos bone marrow B-cells, suggesting that they tracked the clonogenic precursors of MM cells. Using the same screening approach in the 5T33MM mouse model of MM, Id-peptides were able to specifically detect a significant proportion of target cancer cells in the peripheral blood of diseased animal. Interestingly, the in vivo exposure of 5T33 murine MM cells to Id-peptides resulted in a reduced expression of immunosuppressive cytokines genes (Tgfb1 and Il10) and pro-angiogenic genes (Vegf, Tbxas, SDF, Il8, CXCr4). CONCLUSION: This study demonstrates that Id-peptides can be used to identify and purify the clonotypic precursors of MM cells, thus providing a unique tool for assessing both the phenotypic and functional features of the still poorly characterized MM “cancer stem cell” compartment. Moreover, the Id-peptides can inhibit the expression of genes that are crucial to promote a functional MM microenvironment and angiogenesis, thus representing a potential tool for MM therapy. Note: This abstract was not presented at the meeting. Citation Format: Enrico Iaccino, Selena Mimmi, Cristina Falcone, Eleonora Vecchio, Roberta Crescenzo, Giuseppina Maggisano, Cesare Carvelli, Samuela Russo, Annamaria de Laurentiis, Marilena Pontoriero, Antonio Pisano, Simona Ceglia, Francesca Fasanella Masci, Annarita Scialdone, Annalisa Rossi, Giuseppe Fiume, Camillo Palmieri, Ileana Quinto, Giuseppe Scala. Harnessing the potential of idiotypic peptide therapy for the treatment of multiple myeloma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1814. doi:10.1158/1538-7445.AM2014-1814

Abstract 951: CB-5083 is a novel first in class p97 inhibitor that disrupts cellular protein homeostasis and demonstrates anti-tumor activity in solid and hematological models

Abstract Background: The AAA-ATPase p97/VCP facilitates the extraction and degradation of ubiquitinated proteins by converting chemical energy into mechanical force. p97 is closely involved in several facets of protein homeostasis, including ubiquitin-dependent protein degradation, endoplasmic reticulum-associated degradation (ERAD) and autophagy. p97 has been increasingly linked to cancer: it showed elevated protein expression in tumors, it can mediate the degradation of proteins in cancer-relevant pathways and is required for orchestrating the ubiquitin-governed DNA-damage response. In this context, p97 inhibitors may have an advantage versus other protein homeostasis inhibitors and may be active in solid tumors where 26S proteasome inhibitors, bortezomib and carfilzomib, have shown poor efficacy. We report here p97 inhibition as a novel approach to exploit cancer cell addiction to protein homeostatic mechanisms. Results: We have discovered novel small molecule inhibitors of p97 ATPase activity with nanomolar enzymatic and cellular potency. In cellular models, treatment of cancer cells with our lead compound CB-5083 causes disruptions in specific p97 functions, including ubiquitin-dependent protein degradation, ERAD, endocytosis and autophagy. In mouse models, CB-5083 is orally bio-available and causes rapid and sustained accumulation of poly-ubiquitin in tumor xenografts after a single administration. Concurrent with increases in polyubiquitin levels, activation of ER stress response pathways and induction of apoptosis markers are also observed. Sub-chronic oral administration of CB-5083 is generally well-tolerated with &amp;lt;10% body weight loss and results in potent tumor growth inhibition in several solid tumor xenograft models. This result is in marked contrast to proteasome inhibitors that are inactive in the same solid tumor models. In the Vk*MYC transgenic mouse model of multiple myeloma, CB-5083 treatment resulted in &amp;gt;50% reduction in M-spike. Additional efforts are focused on the development of translational assays to monitor p97 target engagement and antitumor efficacy in upcoming clinical trials of CB-5083. Conclusion: These data demonstrate that CB-5083 is a potent inhibitor of p97 that translates to tumor growth inhibition in multiple rodent models of human cancer. Furthermore, CB-5083 appears to exhibit greater potency over current proteasome inhibitors that further validate targeting p97 and protein homeostasis in the treatment of cancer. Citation Format: Ronan Le Moigne, Steve Wong, Ferdie Soriano, Eduardo Valle, Daniel J. Anderson, Stevan Djakovic, Mary-Kamala Menon, Bing Yao, Julie Rice, Jinhai Wang, Szerenke Kiss Von Soly, Brajesh Kumar, Marta Chesi, P. Leif Bergsagel, Han-Jie Zhou, David Wustrow, Mark Rolfe, F. Michael Yakes. CB-5083 is a novel first in class p97 inhibitor that disrupts cellular protein homeostasis and demonstrates anti-tumor activity in solid and hematological models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 951. doi:10.1158/1538-7445.AM2014-951

Abstract 734: Effects of Cabozantinib in the 5TGM1 murine multiple myeloma model

Abstract Cabozantinib is an inhibitor of tyrosine kinases including MET, VEGFR2, and RET, and has shown clinical activity in patients with castration-resistant prostate cancer and other solid tumors with bone metastases. Multiple myeloma (MM) is the second most common hematologic malignancy, and represents ∼2% of all cancer deaths. MM is a monoclonal B-cell (plasma cell) neoplasia with clinical hallmarks of multiple osteolytic lesions causing bone pain, pathologic fractures, and hypercalcemia. Circulating levels of HGF and VEGF are upregulated in MM patients, and regulation of plasma cell-osteoblast communication by the HGF-MET signaling pathway has been implicated in the development of lytic bone disease in these patients. Thus, our aim was to determine the activity of cabozantinib on bone lesions and tumor burden in the syngeneic 5TGM1 mouse MM model. Four experimental groups were included: negative control group receiving vehicle, positive control group receiving bortezomib (0.5 mg/kg ip twice a week), low dose cabozantinib group (10 mg/kg, PO QD) and high dose cabozantinib group (30 mg/kg, PO QD). Female C57BL/KaLwRij mice were allocated to treatment groups (n=15 per group) with equivalent average body weights. On day 0, animals were inoculated with 5TGM1 mouse myeloma cells by IV administration. Dosing began on day 1 and continued daily until euthanasia at day 35. Body weights were determined twice a week and blood samples were collected on days -1, 15, 22, and 34 for analysis of paraprotein (IgG2b) and TRACP 5b. The development of osteolytic lesions was detected by radiography at the end of the study. Some animals (4/15) were euthanized before the end of the experiment due to paraplegia in control and bortezomib groups, but none in cabozantinib groups. Animals euthanized within four days of the end of the experiment were included in the analysis. Bortezomib reduced serum IgG2b levels and decreased the frequency of soft tissue lesions, but did not show bone protective properties. Cabozantinib exhibited bone protective effects: mean and total area of osteolytic lesions were reduced at the 30 mg/kg dose, and serum TRACP 5b values and osteoclast counts at the tumor-bone interface were reduced at both the 10 and 30 mg/kg doses. Relative bone area did not differ from control according to histomorphometry. The rise in serum IgG2b started earlier than vehicle control in both cabo-treated groups, but a significant difference was not observed in relative IgG2b at sacrifice. Cabozantinib dose dependently increased the necrotic tumor area in bone, indicating the possibility that the rise in IgG2b may have been due to lysis of plasma cells. Both cabozantinib doses decreased the frequency of soft tissue lesions. In summary, cabozantinib showed both bone-protective and anti-tumor effects in this murine model of MM. Based on these promising results, further investigation of cabozantinib in multiple myeloma is warranted. Citation Format: Mari I. Suominen, Douglas O. Clary, Rami Käkönen, Katja M. Fagerlund, Esa Alhoniemi, Jukka P. Rissanen, Jussi M. Halleen, Dana T. Aftab. Effects of Cabozantinib in the 5TGM1 murine multiple myeloma model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 734. doi:10.1158/1538-7445.AM2014-734

Abstract 1803: A new spectrum-selective cathepsin inhibitor, VBY-825, inhibits bone destruction in a syngeneic 5TGM1 multiple myeloma mouse model

Abstract Multiple myeloma (MM) is the second most common blood cancer after non Hodgkin lymphoma. It is a monoclonal B-cell neoplasia with clinical hallmarks of multiple osteolytic lesions causing bone pain, fractures and hypercalcemia. Chemo- or radiotherapy may induce remissions, but MM is generally thought to be incurable. Our aim was to observe the effects of a cathepsin inhibitor VBY-825 on bone lesions and tumor burden in the syngeneic 5TGM1 mouse MM model using immunocompetent C57BL/KaLwRij mice. VBY-825 is a potent inhibitor of cathepsins K, L, B, V, and S. 5TGM1 cells were inoculated via tail vein in 7 weeks old female C57BL/KaLwRij mice, which were divided to 4 groups: Control group received vehicle of VBY-825 (5% dextrose 10 ml/kg daily), Control group received bortezomib vehicle (3 ml/kg twice a week), Reference group received bortezomib (0.5 mg/kg twice a week) and Study group received VBY-825 (100 mg/kg daily). Administration of all compounds began one day before tumor cell inoculation and continued until day 34. Disease progression was followed by measuring the serum levels of paraprotein (IgG2b) and TRACP 5b, radiography, and body weight. The animals were sacrificed 5 weeks after inoculation, examined macroscopically, and their bones were collected for histomorphometric analysis. The reference compound bortezomib had no effects on body weight but it delayed the disease progression based on IgG2b measurements. It also decreased the number and total area of osteolytic lesions, but not mean osteolytic lesion area (MOLA). VBY-825 had no effect on body weight or IgG2b level, frequency of soft tissue tumors or intraosseous tumor area. VBY-825 decreased total and MOLA, consistent with inhibited resorption. There was also a trend of increased relative trabecular bone area. Serum TRACP 5b activity in the VBY-825 treated group did not differ from the respective vehicle group, whereas the number of osteoclasts at tumor-bone interface was increased in VBY-825 treated animals. These findings suggest that VBY-825 decreased osteoclast function and resorption activity without decreasing the number of osteoclasts In conclusion, VBY-825 had no effects on tumor growth but it inhibited bone destruction in this mouse model of MM, which is consistent with its potency on cathepsin S and K, which are known to be important in osteoclast-mediated bone resorption. VBY-825 is a promising candidate for the treatment of tumor-associated bone disease. Citation Format: Mari I. Suominen, Johanna Tuomela, Esa Alhoniemi, Katja M. Fagerlund, Jukka P. Rissanen, Jussi M. Halleen, Leslie J. Holsinger. A new spectrum-selective cathepsin inhibitor, VBY-825, inhibits bone destruction in a syngeneic 5TGM1 multiple myeloma mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1803. doi:10.1158/1538-7445.AM2014-1803

The effect of the dual PI3K and mTOR inhibitor BEZ235 on tumour growth and osteolytic bone disease in multiple myeloma.

The plasma cell malignancy multiple myeloma (MM) is unique among haematological malignancies in its capacity to cause osteoclast-mediated skeletal destruction. The PI3K/Akt/mTOR pathway mediates proliferation, survival and drug resistance in MM plasma cells and is also involved in regulating the formation and activity of bone-forming osteoblasts and bone-resorbing osteoclasts. NVP-BEZ235 is a dual pan class I PI3K and mTOR inhibitor that is currently undergoing clinical evaluation in several tumour settings. In this study, we examined the anti-tumorigenic effects of BEZ235 in an immunocompetent mouse model of MM and assessed the effects of BEZ235 on osteoblast and osteoclast formation and function. BEZ235 treatment (50mg/kg) resulted in a significant decrease in serum paraprotein and tumour burden, and μCT analysis of the proximal tibia revealed a significant reduction in the number of osteolytic bone lesions in BEZ235-treated animals. Levels of the serum osteoblast marker P1NP were significantly higher in BEZ235-treated animals, while levels of the osteoclast marker TRAcP5 were reduced. In vitro, BEZ235 decreased MM plasma cell proliferation, osteoclast formation and function and promoted osteoblast formation and function. These findings suggest that, in addition to its anti-tumour properties, BEZ235 could be useful in treating osteolytic bone disease in MM patients.