Mouse Model Of Multiple Myeloma Research Articles

Characterization of the Role of Regulatory T Cells (Tregs) in Inducing Progression of Multiple Myeloma

Introduction. Growing evidence suggests that immune cells that reside within the tumor microenvironment are dysregulated and functionally impaired, leading to defective anti-tumor immunity of the host. One of the major immunosuppressive mechanisms during tumor progression is expansion of regulatory immune cells. Here, we analyzed the immune cells within the bone marrow (BM) and the peripheral blood (PB) of 2 immunocompetent multiple myeloma (MM) mouse models. We next studied the role of regulatory T cells (Tregs) in MM pathogenesis.Materials and methods. To study the immune cell populations of the BM and PB, we used two immuncompetent mouse models and transplanted VK*MYC cells or 5TGM1 cells into C57BL/6 and C57BL/Kalwrij mice respectively. The immune cell populations and checkpoint receptor expressions were analyzed by CyTOF mass cytometer or flow-cytometry. Treg induction assay was performed in vitro to study the mechanism of Treg increase in the BM of myeloma injected mice. CD4+ CD25- cells were obtained from C57BL/Kalwrij mice and were co-cultured with 5TGM1 cells or B cells from C57BL/Kalwrij mice in vitro. Treg induction was compared by flow-cytometry.Transplantable VK*MYC cells were injected into "depletion of regulatory T cell" (DEREG) mice, which expresses a simian diphtheria toxin (DT) receptor-enhanced GFP fusion protein under the control of the FOXP3 gene locus, or their wild type littermates. DT injection into these mice leads to depletion of Tregs as previously described (J Exp Med. 2007; 204: 57-63). DT was given once every week for a total 3 times i.p to the DEREG mice or the littermate controls to specifically deplete Tregs and to study the role of Tregs during MM progression. Tregs (CD4+ FOXP3-GFP+ cells) were sorted from VK*MYC injected mice or non-injected DEREG mice BM using FACSAria cell sorter. Cells isolated were subjected to RNA sequencing. Gene Set Enrichment Analysis (GSEA) was performed to define differences in molecular signatures between MM-associated and normal Tregs.Results. The Treg proportion was significantly increased within the CD4+ T cells in the BM of myeloma cell injected mice from the early stage of disease compared to control mice, while in the PB, the increase was observed only at the late stages of disease progression. The effector T cell (Teff)/Treg ratio was significantly decreased in the BM at the end-stage myeloma bearing mice (P<0.01). Checkpoint related molecules (PD-1, LAG-3 and Tim-3) on Tregs and Teffs were up-regulated at the protein level in the BM of myeloma injected mice compared to control (P<0.01). These data indicate the activation of Tregs and decrease of Teff activity, leading to suppression of anti-myeloma T cell activity in the MM BM. Under in vitro co-culture conditions, 5TGM1 cells induced a significant increase in the number of Tregs from non-Treg CD4+ T cells compared to controls (P<0.01). 5TGM1 cell-induced Tregs presented with enhanced ki-67 expression, thus suggesting the ability of MM cells to induce Treg proliferation. Additionally, the trans-well co-culture experiment showed that the major mechanism for Treg induction by 5TGM1 was by direct contact with T cells rather than secreting factors. Significant increase in survival was observed in the VK*MYC injected DEREG mice under Treg depletion compared to the DEREG mice without Treg depletion and wild type littermates under DT treatment (P<0.001). The Treg depleted DEREG mice were accompanied with an increase of Teffs (P<0.01), indicating recovery of anti-myeloma T cell activity. RNA sequencing of BM Tregs from VK*MYC injected mice showed increased expression of immune checkpoint related molecules and increased Treg effector molecules (IL-10, Granzyme B) compared to BM Tregs from control mice, indicating a more functionally active phenotype of VK*MYC associated Tregs. GSEA showed an enrichment of genes involved in type-1 interferon signaling in VK*MYC associated Tregs.Conclusions. We used 2 immunocompetent MM mouse models to study the characterization and significance of Tregs in MM progression. CyTOF analysis and RNA sequencing data indicated Treg activation in the MM BM microenvironment. The Treg in vivo depletion experiment showed that Tregs have a significant role in MM progression. These data indicate that immunotherapy targeting Tregs may represent a novel therapeutic strategy for MM. Studies are ongoing to understand the roles of Tregs in human MM patients. DisclosuresNo relevant conflicts of interest to declare.

Targeting Redox Overcomes Proteasome Inhibitor Resistance in Multiple Myeloma

Proteasome inhibitors (PIs) like bortezomib (Btz) and carfilzomib (Crflz) induce an oxidative stress response in Multiple Myeloma (MM) cells. Oxidative stress is a key effector pathway in PI-induced cell death, and altered redox signaling has been implicated in the acquisition of PI resistance. The potential of redox as a therapeutic target/pathway for PI resistant MM has not been realized due to the absence of a precise molecular targeted strategy that exploits redox signaling in a way that attacks PI resistant cells while sparing normal cells. Therefore, we set out in this study to characterize redox adaptations that contribute to PI resistance in MM, and to use drug screening platforms to identify specific redox-targeted small molecules that restore PI sensitivity. Using multiple isogenic pairs of PI sensitive and resistant MM cell lines, we found that resistant cells exist under high basal levels of reactive oxygen species (ROS) and oxidation of protein thiols (i.e., oxidative damage). Resistant cells induce significantly higher relative levels of ROS following PI treatment, but exhibit no further increase in oxidative damage. By comparison, their PI sensitive counterparts have relatively low levels of basal and PI-induced ROS levels, but undergo significantly higher levels of oxidative damage following PI treatment. These findings demonstrate that PI resistance is associated with alterations in redox balance; they further suggest that PI resistant cells have acquired adaptations that allow them to survive under high basal levels of oxidative stress, and that provide protection from PI-induced oxidative damage. We also identified significant changes in cellular bioenergetics that are typical of PI resistant cells. Generally, PI resistant cells appear to be more metabolically efficient, relying on mitochondrial respiration as their primary source of ATP production. Specifically, PI resistant cells have higher basal oxygen consumption rates (OCR), expanded respiratory capacity, increased NAD(P)H levels and pyruvate dehydrogenase (PDH) activity, and nearly absent activation of the AMP kinase energy stress signaling pathway. Thus, the acquisition of PI resistance is associated with significant changes in redox balance as well as in cellular bioenergetics. Given these findings, we next used a cell-based drug screening method to screen for redox-targeted small molecules capable of restoring PI sensitivity to resistant cells. We screened a compound collection of known pro- and anti-oxidant small molecules with wide-ranging mechanisms of action. From this screen we identified compound E61, which demonstrated strong synergy with multiple PIs, including Btz, Crflz, ixazomib, and oprozomib. E61 induced an oxidative stress response characterized by a burst of ROS generation and oxidation of protein thiols, and synergistically enhanced the PI-induced oxidative stress response in resistant cells. The synergistic cytotoxic response to E61 and PI co-treatment was dependent on ROS, and was evident across several models of PI resistance, representing cells of diverse genetic backgrounds. While E61 enhanced PI-induced cell death in resistant MM cells, its effects were protective in normal cell types, including peripheral blood mononuclear cells (PMBCs) and lymphocytes from normal human donors. These findings suggest that compound E61 will have a wide therapeutic index in combination with PI therapy in preclinical mouse models of MM, a hypothesis that we are currently testing. All together, our findings identify specific redox and bioenergetics changes that are acquired by PI resistant MM cells. Furthermore, our work offers a novel redox-targeted small molecule, E61, to be used in combination with PI-based therapeutic regimens in refractory MM. DisclosuresNo relevant conflicts of interest to declare.

Anti-Tumor Phagocytic Cell Activation in Multiple Myeloma By the IAP Antagonist LCL161: Results of a Phase II Clinical Trial

Dexamethasone is a powerful anti-inflammatory, immunosuppressive agent widely used in the treatment of MM with pleiotropic effects on both the tumor, and host immune cells. We report on a completely novel approach to immune modulation using an agent with almost directly opposite effects to glucocorticoids on innate immune cells. LCL161 is an IAP antagonist targeting the cellular inhibitor of apoptosis proteins cIAP1 and -2 (cIAP1/2) that in a phase I study in solid tumors was well tolerated with a cytokine release syndrome as the dose limiting toxicity. We previously reported that loss of cIAP1/2 in MM cells results in the stabilization of NIK and activation of the non-canonical NFkB pathway, a pathway we found frequently activated by a promiscuous array of mutations in multiple myeloma (MM). As expected, we found that LCL161 has no direct anti-tumor activity against MM cells in vitro and ex vivo. However, the non-canonical NFkB pathway is also a key activator of the innate immune response. Remarkably, we found that in vivo LCL161 has dramatic, apoptosis-independent activity associated with marked phagocytosisagainst MM that develops spontaneously in an immunocompetent genetically engineered mouse model of MM (Vk*MYC). Cyclophosphamide (Cy), which has been shown to induce an acute secretory activating phenotype in tumor-associated macrophages, markedly potentiated the anti-tumor effects of LCL161. We show that phagocytes (depleted by liposomal clodronate), but not adaptive immune cells, are required for the anti-tumor effect. Consistently we show that LCL161 activates dendritic cells and macrophages in vitro, and in vivo, and upregulates production of inflammatory cytokines.A phase 2 clinical trial of LCL161 was conducted in 24 patients with relapsed MM previously exposed to both IMiDs and PIs, but less than 5 prior lines of therapy. Patients received LCL161 1200mg po weekly, with Cy 500mg po weekly added for progressive disease or lack of response. Single agent LCL161 was well-tolerated, with grade 3 or 4 non-hematologic toxicity in four patients, and following the addition of cyclophosphamide in 9 patients. No patients responded to single agent LCL161, and the best response was stable disease in 6/24. In 21 patients, Cy was added after a median of 2 cycles, in whom the best response was stable disease or better in 18/21 (p<0.001 compared to LCL161 alone), and a PR or better (including 1 VGPR and 1 CR) in 5/21 (p<0.001 compared to LCL161 alone). Six months following the initiation of Cy, 35% of patients remain event-free. Two patients had stable disease for more than nine months after stopping treatment. One, relapsing after VDT-PACE, HDM200, RVd x 3 years with t(11;14) and del17p, received only three months of LCL161, the last month in combination with Cy. He then achieved a PR, reconstitution of his uninvolved IgG to normal levels for the first time in more than 3 years and still has not progressed one year after stopping therapy. Similar changes in serum and bone marrow inflammatory cytokines and gene expression were observed in patients following treatment with LCL161 to those observed in the mice. We conclude that LCL161 is able to modulate the tumor microenvironment to exert long-term disease control in combination with Cy, and deserves further study in combination with agents that more directly depend on phagocytic cell activity (e.g., monoclonal antibodies, inhibitors of "Don't eat me" signals). DisclosuresBergsagel:Novartis: Research Funding; Constellation Pharmaceuticals: Research Funding; Incyte: Consultancy; Jannsen: Consultancy; Mundipharma: Consultancy. Off Label Use: Use of LCL161 to treat multiple myeloma. Kumar:Celgene: Research Funding; Millenium/Takeda: Research Funding; Onyx: Research Funding; AbbVie: Research Funding; Janssen: Research Funding; Sanofi: Research Funding; Celgene, Millenium, Sanofi, Skyline, BMS, Onyx, Noxxon,: Other: Consultant, no compensation,; Skyline, Noxxon: Honoraria. Stewart:Oncospire Genomics: Research Funding.

The Myeloma Cells Escape from Bone Marrow to Skin Extramedullary Localization upon Bortezomib Resistance: Role of CXCR4

The malignant plasma cells (PC) dependence on the bone marrow (BM) microenvironment is a main feature of multiple myeloma (MM), mainly due to high expression of cell adhesion molecules including CD44, CD54 (ICAM1), CD56, CD49d and chemokines receptors such as CXCR4. At the end stage of the disease, a rare MM complication, that may occurs, is a secondary extramedullary involvement without a leukemic phase and, among the extramedullary localizations, the skin is one of the possible sites, due to the physiological homing of PCs. However, the mechanisms behind the extramedullary spread are not completely understood.In a patient with refractory resistant MM, who has developed a cutaneous localization after 16 months from the diagnosis under Bortezomib (BOR) treatment, we analyzed the gene expression profiles (GEPs) by GeneChip U133 Plus 2.0 (Affymetrix), the immunophenotypic and immunohistochemistry (IHC) profiles of MM cells, across the course of the disease. To confirm the results, IHC profiles on selected molecules were then analyzed in other six MM patients with secondary skin involvement without PC leukemia.On BM CD138+ PCs at diagnosis and BM relapse, by GEP analysis, 742 genes resulted differentially expressed. Moreover, a more stringent analysis identified 19 down-regulated and 42 up-regulated genes; data were confirmed by Real Time PCR on selected genes mainly involved in PC homing. At the BM relapse, we found that MM cells expressed CXCR4 and were negative for CD56; moreover, a significant down-regulation of ICAM1 (CD54) and ALCAM was observed together with the up-regulation of MAGE family genes, DKK1 and SEMA3A in the BM relapse sample compared to diagnosis one. At the cutaneous involvement development, 4 months after BM relapse, by IHC analysis, the MM cells localized in the skin showed the expression of both CD56 and CD54. On the other hand, the BM MM cells immunophenotype confirmed the presence of CD56 but showed the lack of CXCR4 expression. IHC for CD54, CD56 and CXCR4 expression was then performed on both skin and bone biopsies of the other patients analyzed. To go further inside on the role of CXCR4 we treated with BOR (5-10nM) for 24-48h human myeloma cell lines (HMCLs) RPMI-8226 and U266, known to be resistant to BOR, evaluating the CXCR4 expression at both mRNA and protein level, by real-time PCR and by cytofluorimetry, respectively. CXCR4 resulted downregulated after BOR treatment in BOR resistant HMCLs. Consistently, the loss of CXCR4 expression was recently associated to BOR resistance and extramedullary disease in a mouse model of MM suggesting that CXCR4 loss can be correlated with BOR resistance and PCs mobilization from BM, leading to an extramedullary disease.In conclusion, our data indicate that the loss of CXCR4 expression by MM cells across the disease progression, together with CD54 and CD56 down-regulation and their re-acquisition at the extramedullary site, are involved driving the escape of PCs from BM to the extramedullary localization in the skin in the context of Bortezomib resistance. DisclosuresNo relevant conflicts of interest to declare.

MOR202, a Human Anti-CD38 Monoclonal Antibody, Mediates Potent Tumoricidal Activity In Vivo and Shows Synergistic Efficacy in Combination with Different Antineoplastic Compounds

Background: MOR202, a human anti-CD38 monoclonal antibody, is currently being evaluated in a phase I/IIa clinical trial for the treatment of multiple myeloma (MM). Pharmacodynamically, MOR202 exerts its tumoricidal effect by eliciting antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Aside from a promising single-agent activity profile, experiments in vitro indicate additive or synergistic effects of MOR202 in combination with different types of standard of care compounds in MM such as lenalidomide (LEN) or bortezomib (BOR).Aims: To compare in vitro the ADCC profile of MOR202 on MM cells and normal hematopoietic cellswith the respective profiles of two other anti-CD38 antibodies in clinical development: daratumumab and isatuximab. To determine the tumoricidal efficacy of MOR202 in vivo in combination with either LEN, BOR or melphalan (MEL), representing immunomodulatory drugs (IMiDs), proteasome inhibitors, and alkylating agents, respectively.Method: The in vitro ADCC profile of the antibodies was assessed by fluorescence activated cell sorting using a panel of CD38+ MM cell lines in the presence of peripheral blood mononuclear cells. The potential for cytotoxicity towards normal hematopoietic cells was tested on purified natural killer (NK) cells by measuring NK-NK killing. In the in vivo studies, two independent mouse models were employed to assess combinatorial effects. Firstly, as a model for MM, NCI-H929 cells were inoculated intratibially into the bone marrow to establish an orthotopic model characterized by prototypical hallmarks of the disease, such as bone lysis, and the emergence of serum M protein. Bone density, evaluated by microcomputed tomography (μCT), and M protein serum levels were used as endpoint analyses for treatment efficacy. Secondly, as a model for disseminated lymphoma, Ramos non-Hodgkin lymphoma cells were intravenously injected into immunodeficient mice. Survival served as the primary endpoint, reflecting the success of medication. Combinatorial drug interactions were evaluated according to Clarke, 1997 (Breast Cancer Res Treat; 46:255-78) and Chou, 2006 (Pharmacol Rev; 58:621-81).Results: In the in vitro ADCC studies on MM cell lines, the maximum killing of MOR202 was equivalent to the tested comparator antibodies. In contrast, the potential to induce killing of normal NK cells expressing low levels of CD38 was significantly reduced for MOR202 compared with daratumumab and isatuximab (n=6 donors; median of 7% specific killing vs 30% and 37%, respectively). This appeared to be independent of FcγRIIIa receptor polymorphism status. In the mouse MM model, MOR202 administration at 3 mg/kg significantly reduced bone lysis by up to 72.5%, relative to vehicle control. Treatment with LEN, BOR, or MEL alone also decreased bone lysis significantly, as compared with vehicle control. However, coadministration of MOR202 with LEN, BOR, or MEL completely abolished or dramatically reduced bone lysis (Figure). Drug interaction analyses indicated synergistic efficacy. The markedly reduced bone lysis seen with combination therapy was accompanied by a reduction (>90%) of serum M protein levels indicating a significant decrease in tumor load (Figure). In the disseminated lymphoma model, LEN or BOR administration had no impact on survival time as compared with vehicle control. In contrast, MOR202 treatment resulted in significantly improved survival compared with vehicle control (median 20 and 20.5 days vs 42 and 43.5 days, respectively, in two independent experiments). Notably, coadministration of MOR202/LEN, or MOR202/BOR further improved survival, indicating potentiation of MOR202 activity by agents which by themselves had little effect (i.e., a subtype of synergy). Of note, all mice receiving monotherapy finally succumbed to the tumor. In contrast, 37.5% of mice treated with MOR202/LEN and 40% of mice treated with MOR202/BOR were completely free of tumor until study termination at day 98.Conclusions: MOR202 shows equivalent ADCC to CD38 high expressing MM cells as compared with daratumumab and isatuximab, while provoking significantly reduced off-tumor killing of CD38 low expressing normal NK cells. In addition, MOR202 acts synergistically in vivo in combination with different compounds representative of classes of agents commonly used in the treatment of hematologic malignancies. [Display omitted] DisclosuresBoxhammer:MorphoSys AG: Employment. Weirather:MorphoSys AG: Employment. Steidl:MorphoSys AG: Employment. Endell:MorphoSys AG: Employment.

Bypassing the Constraint for Chimeric Antigen Receptor (CAR) Development in T-Cells Expressing the Targeted Antigen: Improvement of Anti-CS1 CAR Activity in Allogenic TCRa/CS1 Double Knockout T-Cells for the Treatment of Multiple Myeloma (MM)

Adoptive immunotherapy with autologous T-cells expressing chimeric antigen receptors (CARs) targeting CD19 has achieved long-term remissions in patients with B cell leukemia, pointing out that CAR technology may become a new alternative in cancer treatment. In this work we assessed the feasibility of targeting the CS1 antigen (SLAMF7) for the treatment of Multiple Myeloma (MM).MM is a B-cell neoplasia characterized by clonal expansion of malignant plasma cells in the bone marrow. Even if currently available therapies can improve overall survival, MM still remains incurable in most patients. Immunotherapy against MM is therefore an area in which extensive research is being made, with novel antigenic targets being considered. Among these is the CS1 glycoprotein, which is highly expressed on tumor cells from most patients with MM. However, CS1 is also expressed on normal CD8+ T-cells, which may be problematic for a CAR-based approach as antigen-expressing T cells will be targeted, impacting both the number and the phenotype of the final CAR T cell population.To circumvent this issue we have used our highly-efficient transcription activator-like effector nuclease (TALEN) gene-editing technology to inactivate CS1 in T-cells prior to transduction with a viral vector encoding an anti-CS1 CAR. Our results demonstrate that while non-gene-edited T-cells expressing an anti-CS1 CAR display limited cytolytic activity against MM cell lines, and resulted in a progressive loss of CD8+ T-cells. CS1-gene-edited CAR cells display significantly increased cytotoxic activity, with the percentage of CD8+ T-cells remaining unaffected. In addition, experiments in an orthotopic MM mouse model showed that CS1 disrupted T-cells were able to mediate an in vivo anti-tumoral activity.Subsequently, we have utilized this strategy for CS1 in the context of our allogeneic "off-the-shelf" engineered CAR+ T-cell platform. This allogenic platform utilizes TALEN gene editing technology to inactivate the TCRα constant (TRAC) gene, eliminating their potential to mediate Graft versus Host Disease (GvHD). We have previously shown that editing of the TRAC gene can be achieved at high frequencies, allowing efficient production of TCR-deficient T-cells that no longer mediate alloreactivity in a xeno-GvHD mouse model. Our results also show that multiplex genome editing is possible and can lead to the production of double KO (TRAC and CS1) T-cells, allowing large scale manufacturing of allogeneic, non alloreactive CS1 specific T-cells with enhanced antitumor activity. Moreover, these allogenic T-cells could be easily available for administration to a large number of MM patients. DisclosuresGaletto:Cellectis SA: Employment. Chion-Sotinel:Cellectis SA: Employment. Gouble:Cellectis SA: Employment. Smith:Cellectis: Employment, Patents & Royalties.

Drug Resistant Myeloma Cells Cause More Severe Bone Destruction in Myeloma

Background: Multiple myeloma (MM) is the second most common hematologic malignancy in the US and is characterized by osteolytic disease. More than 80% of MM patients have osteolysis, leading to in fractures, severe bone pain, spinal cord compression, and hypercalcemia, greatly compromising patient quality of life and resulting in mortality. While bone exists in a dynamic state between resorption and deposition, the molecular mechanisms underlying bone destruction in MM remain elusive. One contributing factor is heparanase (HPSE). In our work, we have previously shown that the expression of NEK2 and HPSE are closely correlated. We have also shown that high expression of chromosomal instability-related genes (e.g. NEK2) induces drug resistance and poor prognosis in MM. Though tumor burden is dramatically decreased in remission, bone destruction is poorly repaired, suggesting drug resistant MM cells may play a critical role in inducing bone destruction or in preventing the repair of bone disease in remission.Materials and Methods: Gene expression profiling (GEP) data and bone lytic lesions detected by MRI were collected from 333 newly diagnosed MM patients from the Total Therapy 2 (TT2) clinical trial. MM cell lines with stable overexpression or knockdown of NEK2 and/or HPSE were created using lentiviral delivery. Protein and mRNA expression was assessed by immunoblotting and real-time PCR, respectively. TRAP staining was used to assess osteoclast differentiation. In vivo studies were performed in the KaLwRij 5TGM1 MM mouse model in 3 different groups: empty vector (EV), NEK2 overexpression (NEK2 OE), or NEK2 OE with HPSE shRNA. Bone density and imaging assessment was performed via X-ray and microCT scanning. HPSE, NFκB, and USP7 contributions were examined using the specific small molecule inhibitors SST0001, BSM-345541, and P5091, respectively.Results: In correlating NEK2 expression with osteolytic lesion from 333 newly diagnosed MMs, we found the expression of NEK2 was significantly higher in MM patients with bone lytic lesion than those without lesions. NEK2 expression was also higher in patients with severe bone disease (7 or more lesions) than those fewer bone lytic lesions (< 7).To determine if NEK2 causes bone lytic lesions in MM, we performed in vivo studies injecting NEK2 OE 5TGM1 cells into KaLwRij mice. MicroCT showed clearly that bone volume and thickness of trabecular bone were decreased significantly in mice injected with NEK2 OE cells compared to EV cells. Strikingly, treatment with the HPSE inhibitor SST0001 dramatically rescued bone mass loss induced by NEK2 overexpression. Histological TRAP staining of decalcified bone sections revealed a significant increase in the number of osteoclasts in the bone surface area in mice injected with NEK2 OE cells compared to EV-treated mice, while addition of the SST0001 significantly reduced the number of osteoclast.Because osteoclast causes bone resorption, we examined the effect of applying conditioned media from MM cells with or without NEK2 overexpression to pre-osteoclast cells derived from MM patients, C57BL6 mice, and the pre-osteoclast RAW264.7 cell line. We found significantly increased mature osteoclasts (>3 nuclei) after culture with media collected from NEK2 MM cells compared to conditioned media collected from EV cells. Consistently, osteoclast differentiation-associated genes (i.e. CTSK, NFATc1, RANK, and TRAP) were upregulated in pre-osteoclasts cultured with NEK2 OE conditioned media. SST0001 suppressed osteoclast differentiation induced by NEK2, strongly suggesting that HPSE mediates NEK2 induced osteoclastogensis and bone destruction in MM. Mechanistic studies revealed that NEK2 directly binds to and activates USP7 resulting in deubiquitination of NFκB. Inhibition of NFκB decreased NEK2-induced HPSE expression induced by NEK2 at both the mRNA and protein level.Conclusions: Our findings suggest that high NEK2 expression may be a biomarker for bone disease in MM. We demonstrate that a subpopulation of drug-resistant, NEK2-overexpressing MM cells leads to increased production and activity of HPSE, and increased osteoclast differentiation contributing to severe bone disease. Mechanistically, we show that NEK2 interacts with USP7, stabilizing NFκB and upregulating HPSE. Our results demonstrate drug resistant MM cells may not only promote tumor cell survival but also contribute to bone destruction in MM. DisclosuresNo relevant conflicts of interest to declare.

Hypoxia-Inducible Factor (HIF)-1alpha Inhibitionin Myeloma Cells Significantly Increases the Anti-Myeloma Effect of Lenalidomide in Vivo

The immunomodulatory drugs (IMiDs®) exert an anti-myeloma effect by cereblon-dependent destruction of IKZF proteinseither through a direct action on multiple myeloma (MM) cells or through indirect immunomodulatory and anti-angiogenic effects.Previous data indicated that MM cells overexpress hypoxia inducible factor (HIF)-1α and that HIF-1α suppression significantly blocks MM-induced angiogenesis and reduces in vivo tumoral burden in MM mouse models. Interestingly, it has been recently reported that HIF-1α knock-down in MM cells potentiates the in vitro effect of Lenalidomide (LEN) on cell proliferation without changing cell viability and that LEN is not able to suppress HIF-1α expression in MM cells. These evidences give the rationale design to investigate the in vivo effect of HIF-1α stable suppression in MM cells on LEN sensitivity. Thus, in this study, we assessed the effect of LEN in vivo in combination with HIF-1α inhibition in a non-obese diabetic/severe combined immunodeficiency (NOD/SCID) subcutaneous mouse model using JJN3, a cell line known to be resistant to the cytotoxic effect of LEN.Different groups of animals were injected with JJN3-pLKO.1 (empty vector) or JJN3-anti-HIF1α, obtained by anti-HIF1α lentiviral shRNA pool. When tumors became palpable, mice were treated with LEN (5mg/kg), using the intraperitoneal route. After three weeks, we evaluated tumor volume and weight. Moreover, by immunohistochemistry on ex vivo plasmacytomas, we evaluated the expression of p27 and the microvascular density (MVD), checked by CD34 immunostaining. In addition, the expression of a p27 inhibitor, the S-phase kinase-associated protein 2 (SKP2), the expression of the HIF-1α target key mediator of glycolysis and tumoral growth, Hexokinase II (HK2), and the levels of pERK 1/2, and total Caspase-3 (Casp-3) were evaluated in the ex vivo plasmacytomas lysates by western blot. We found that LEN treatment induced a significant weight and volume reduction of the tumor burden in mice injected with JJN3 anti-HIF1α as compared to JJN3-pLKO.1. The p27 nuclear expression was significantly increased by LEN treatment in JJN3-anti-HIF1α as compared to JJN3 pLKO.1 mice and compared to JJN3-anti-HIF1α mice treated with vehicle. In addition, we demonstrated that LEN in combination with HIF-1α inhibition significantly reduced in vivo the expression of p-ERK1/2, total Casp-3, HK2 and the p27 inhibitor, SKP2.Because it is known that LEN exerts its anti-MM effect targeting the IKZF proteins, we further checked in vitro whether the effect of HIF-1α suppression and LEN treatment combination could be mediated by IKZF proteins modulation. Interestingly, after LEN (2-10µM) treatment we found that both IKZF1 and IKZF3 were not differentially expressed, whereas IRF4 was down regulated, in JJN3-anti-HIF1α as compared to JJN3 pLKO.1. Finally, regarding a possible combinatory effect on the in vivo angiogenesis, we found that both the number of CD34 positive vessels and the MVD were reduced in mice colonized by JJN3-anti-HIF1α as compared to JJN3-pLKO.1. On the other hand, LEN treatment did not further significantly reduce the number of CD34 positive vessels and the MVD. Accordingly, we did not find any significant inhibitory effect by LEN treatment on the main pro-angiogenic molecules in JJN3 anti-HIF1α as compared to JJN3 pLKO.1 even after 72 hours.Overall, our data indicate that HIF-1α suppression in MM cells significantly increases the anti-MM effect of LEN in vivo, mainly through the inhibition of proliferation signaling including the modulation of p27 pathway and the IKZF target protein IRF4, rather than to an anti-angiogenic effect. These data suggest that the combination of LEN and HIF-1α inhibition has a therapeutic rationale in MM. DisclosuresNo relevant conflicts of interest to declare.

A Novel and Highly Potent CAR T Cell Drug Product for Treatment of BCMA-Expressing Hematological Malignances

Recently, B cell maturation antigen (BCMA) expression has been proposed as a marker for identification of malignant plasma cells in patients with multiple myeloma (MM). Nearly all MM and some lymphoma tumor cells express BCMA, while normal tissue expression is restricted to plasma cells and a subset of mature B cells. Targeting BCMA maybe a therapeutic option for treatment of patients with MM and some lymphomas.We are developing a chimeric antigen receptor (CAR)-based therapy for the treatment of BCMA-expressing MM. Our anti-BCMA CAR consists of an extracellular single chain variable fragment (scFv) antigen recognition domain derived from an antibody specific to BCMA, fused to CD137 (4-1BB) co-stimulatory and CD3zeta chain signaling domains. Selection of our development candidate was based on the screening of four distinct anti-BCMA CARs (BCMA01-04) each comprised of unique single chain variable fragments. One candidate, BCMA02 (drug product name bb2121) was selected for further studies based on the robust frequency of CAR-positive cells, increased surface expression of the CAR molecule, and superior in vitro cytokine release and cytolytic activity against the MM cell lines. In addition to displaying specific activity against MM (U226-B1, RPMI-8226 and H929) and plasmacytoma (H929) cell lines, bb2121 was demonstrated to react to lymphoma cell lines, including Burkitt's (Raji, Daudi, Ramos), chronic lymphocytic leukemia (Mec-1), diffuse large B cell (Toledo), and a Mantle cell lymphoma (JeKo-1). Based on receptor density quantification, bb2121 can recognize tumor cells expressing less than 1000 BCMA molecules per cell. The in vivo pharmacology of bb2121 was studied in NSG mouse models of human MM and Burkitt's lymphoma. NSG mice were injected subcutaneously (SC) with 107 RPMI-8226 MM cells. After 18 days, mice received a single intravenous (IV) administration of vehicle or anti-CD19Δ (negative control, anti-CD19 CAR lacking signaling domain) or anti-BCMA CAR T cells, or repeated IV administration of bortezomib (Velcade®; 1 mg/kg twice weekly for 4 weeks). Bortezomib, which is a standard of care for MM, induced only transient reductions in tumor size and was associated with toxicity, as indicated by substantial weight loss during dosing. The vehicle and anti-CD19Δ CAR T cells failed to inhibit tumor growth. In contrast, treatment with bb2121 resulted in rapid and sustained elimination of the tumors, increased body weights, and 100% survival. Flow cytometry and immunohistochemical analysis of bb2121 T cells demonstrated trafficking of CAR+ T cells to the tumors (by Day 5) followed by significant expansion of anti-BCMA CAR+ T cells within the tumor and peripheral blood (Days 8-10), accompanied by tumor clearance and subsequent reductions in circulating CAR+ T cell numbers (Days 22-29). To further test the potency of bb2121, we used the CD19+ Daudi cell line, which has a low level of BCMA expression detectable by flow cytometry and receptor quantification analysis, but is negative by immunohistochemistry. NSG mice were injected IV with Daudi cells and allowed to accumulate a large systemic tumor burden before being treated with CAR+ T cells. Treatment with vehicle or anti-CD19Δ CAR T cells failed to prevent tumor growth. In contrast, anti-CD19 CAR T cells and anti-BCMA bb2121 demonstrated tumor clearance.Adoptive T cell immunotherapy approaches designed to modify a patient's own lymphocytes to target the BCMA antigen have clear indications as a possible therapy for MM and could be an alternative method for treatment of other chemotherapy-refractory B-cell malignancies. Based on these results, we will be initiating a phase I clinical trial of bb2121 for the treatment of patients with MM. DisclosuresChekmasova:bluebird bio, Inc: Employment, Equity Ownership. Horton:bluebird bio: Employment, Equity Ownership. Garrett:bluebird bio: Employment, Equity Ownership. Evans:bluebird bio, Inc: Employment, Equity Ownership. Griecci:bluebird bio, Inc: Employment, Equity Ownership. Hamel:bluebird bio: Employment, Equity Ownership. Latimer:bluebird bio: Employment, Equity Ownership. Seidel:bluebird bio, Inc: Employment, Equity Ownership. Ryu:bluebird bio, Inc: Employment, Equity Ownership. Kuczewski:bluebird bio: Employment, Equity Ownership. Horvath:bluebird bio: Employment, Equity Ownership. Friedman:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership.

HDAC inhibitor AR-42 decreases CD44 expression and sensitizes myeloma cells to lenalidomide.

Multiple myeloma (MM) is a hematological malignancy of plasma cells in the bone marrow. Despite multiple treatment options, MM is inevitably associated with drug resistance and poor outcomes. Histone deacetylase inhibitors (HDACi's) are promising novel chemotherapeutics undergoing evaluation in clinical trials for the potential treatment of patients with MM. Although in preclinical studies HDACi's have proven anti-myeloma activity, but in the clinic single-agent HDACi treatments have been limited due to low tolerability. Improved clinical outcomes were reported only when HDACi's were combined with other drugs. Here, we show that a novel pan-HDACi AR-42 downregulates CD44, a glycoprotein that has been associated with lenalidomide and dexamethasone resistance in myeloma both in vitro and in vivo. We also show that this CD44 downregulation is in part mediated by miR-9-5p, targeting insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), which directly binds to CD44 mRNA and increases its stability. Importantly, we also demonstrate that AR-42 enhances anti-myeloma activity of lenalidomide in primary MM cells isolated from lenalidomide resistant patients and in in vivo MM mouse model. Thus, our findings shed light on potential novel combinatorial therapeutic approaches modulating CD44 expression, which may help overcome lenalidomide resistance in myeloma patients.

Abstract B46: VLA-4 targeted nanoparticles carrying a novel anti-Myc prodrug prolongs survival in a mouse model of multiple myeloma.

Abstract The MYC oncoprotein drives multiple myeloma (MM) pathogenesis, but the utility of small molecule inhibitors of MYC-MAX dimerization is limited by poor efficacy. We determined the efficacy of lipase labile MYC-MAX inhibitor prodrug (MI1-PD) loaded nanoparticles targeted via integrin {alpha 4 beta1 α4β1) or Very Late Antigen-1 (VLA-4)} in myeloma mice. We hypothesized that NPs containing specific targeting to myeloma cells, would enhance the therapeutic efficacy of MI1-PD containing NPs. The antiproliferative activity of intergin VLA-4-Targeted MI-1PD NPs (iTM-NPs) was determined in mouse cell line (5TGM1) in vitro and its effect on the survival in C57Bl/KaLwRij-5TGM1 murine model of myeloma in vivo. iTM-NPs (approximately 20 nm in diameter, 5% w/w MI1-PD loading) under in vitro conditions exhibited inhibition of cell growth compared to the controls. Also, C57Bl/KaLwRij-5TGM1 mice that received intravenous injection of iTM-NPs demonstrated greater survival rate over the controls. These results demonstrate the feasibility of using VLA-4 targeted nanotherapy approach to improve the availability of anti-Myc small molecule inhibitors for multiple myeloma and other cancers. Citation Format: Deepti Sood Gupta, Dipanjan Pan, Grace Hu, Anagana Senpan, Xiaoxiao Yang, Edward V. Prochownik, Gregory M. Lanza, Michael H. Tomasson. VLA-4 targeted nanoparticles carrying a novel anti-Myc prodrug prolongs survival in a mouse model of multiple myeloma. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B46.

Abstract B15: Targeting SQSTM1/p62 induces cargo-loading failure and converts autophagy to apoptosis via NBK/Bik in human multiple myeloma cells.

Abstract Purpose: The purpose of this study was to determine whether transcriptional repression of the adaptor protein SQSTM1/p62, which plays a critical role in recognizing/loading cargo (e.g., malfolded proteins) into autophagosomes for lysosomal degradation, could potentiate multiple myeloma (MM) cell death in response to agents (e.g., BH3-mimetics) that elicit a cytoprotective autophagic response. Methods: To this end, various multiple myeloma cell lines (e.g., U266, RPMI8226, and bortezomib-resistant PS-R U266 cells) were exposed to the pan BH3-mimetic obatoclax (Teva) in the presence or absence of pan-CDK inhibitors e.g., flavopiridol (Telero), dinaciclib (Merck), after which the induction of autophagy (LC3-II expression, GFP-LC3 puncta by confocal microscopy) and apoptosis (Annexin V staining by flow cytometry) were monitored. Autophagic flux was determined using cells transfected with a pBABE mCherry-EGFP-LC3B plasmid, followed by observation of autophagosomes and autolysosomes. Accumulation of ubiquitinated protein aggregates was assessed by filter trap (retardation) assays and immunofluorescent staining using an anti-ubiquitin antibody. The functional significance of perturbations in protein expression (e.g., p62, CDK9, cyclin T, Bik) was determined using cells in which target gene expression was down-regulated by shRNA. Alterations in the appearance of autophagic vacuoles (AV) were observed using electron microscopy. Lysosome biogenesis was analyzed by LysoTracker Probe. Gene expression was determined by quantitative real-time PCR (qPCR), and protein-protein interactions characterized by co-immunoprecipitation. The in vivo effects of combined treatment were assessed in mouse MM flank xenograft models. Results: Here we report that whereas p62 levels fluctuated in a time-dependent manner during autophagy induced by obatoclax, inhibition (e.g., by CDK inhibitors administered at sub-micromolar concentrations) or knockdown of CDK9 or cyclin T1 transcriptionally suppressed p62 expression, but did not affect autophagic flux or modify lysosome biogenesis. These interventions, or shRNA directly targeting SQSTM1/p62, resulted in cargo-loading failure and inefficient autophagy, phenomena recently described in Huntington's disease neurons. Inefficient autophagy was characterized by the formation of “empty” AVs accompanied by the accumulation of ubiquitinated proteins, presumably due to failure of loading into autophagosomes for degradation. These events led to accumulation of the BH3-only protein NBK/Bik on ER, most likely by blocking loading and autophagic degradation of Bik. Whereas Bik up-regulation was further enhanced by disruption of distal autophagic events (e.g., autophagosome maturation) by chloroquine or Lamp2 shRNA, it was substantially diminished by inhibition of autophagy initiation (e.g., by shRNA targeting Ulk1, Beclin-1, or Atg5, or pharmacologically by 3-MA or spautin-1), arguing that Bik accumulation primarily stems from inefficient autophagy. Moreover, NBK/Bik knock-down markedly attenuated apoptosis in vitro and in in vivo. Finally, similar interactions between a CDK inhibitor and obatoclax were observed in vivo in MM mouse models. Together, these findings identify a novel cross-link between autophagy and apoptosis, wherein targeting p62 converts cytoprotective autophagy to an inefficient form due to cargo loading failure, leading to NBK/Bik accumulation which triggers apoptosis. Conclusions: Collectively, these findings highlight a novel approach to target the cargo-loading process during cytoprotective autophagy by the transcriptional down-regulation of the adaptor protein p62, rather than direct inhibition of autophagy at either early or late stages (e.g., by chloroquine). Together, these findings provide new mechanistic insights into and a theoretical foundation for developing a autophagy-targeting therapeutic strategy based on induction of inefficient autophagy in MM. Citation Format: Yun Dai, Shuang Chen, Liang Zhou, Yu Zhang, Yun Leng, Xin-Yan Pei, Hui Lin, Richard Jones, Orlowski Robert, Steven Grant. Targeting SQSTM1/p62 induces cargo-loading failure and converts autophagy to apoptosis via NBK/Bik in human multiple myeloma cells. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B15.

Abstract 786: Effects of combination treatment with cabozantinib and bortezomib in the 5TGM1 murine multiple myeloma model

Abstract Cabozantinib (cabo) is an inhibitor of tyrosine kinases including MET, VEGFR2, RET, and the TAM family kinases TYRO3, AXL, and MER, and has shown clinical activity in patients with castration-resistant prostate cancer and other solid tumors with bone metastases. Multiple myeloma (MM) is a monoclonal B-cell (plasma cell) neoplasia representing ∼2% of all cancer deaths. The clinical hallmark is presence 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. We have previously shown that cabo is active in the syngeneic 5TGM1 mouse MM model. This study aimed to determine whether combination with bortezomib would yield additional benefit. Four experimental groups were included: 1) Control group receiving vehicle, 2) Bortezomib (0.5 mg/kg ip twice a week), 3) Cabo (10 mg/kg, PO QD) and 4) Combination (bortezomib + cabo, same doses). Female C57BL/KaLwRij mice were allocated to treatment groups (n = 15 per group) with equivalent average body weights. At day 0, animals were inoculated with 5TGM1 cells by IV administration. Dosing began at day 1 and continued daily until euthanasia. Body weights were determined twice a week and blood samples were collected at days -1, 16, 23, 35 and at sacrifice for analysis of paraprotein (IgG2b), PINP and TRACP 5b. Development of osteolytic lesions was detected by radiography at day 35 and at sacrifice. Mice were sacrificed individually when they became paraplegic, lost over 20% of body weight, or had severe breathing problems. The maximum length of the study was 70 days. By study day 35, the osteolytic lesions were not affected by bortezomib, were reduced by cabo alone, and further reduced by the combination treatment. Bortezomib had inhibited the rise in serum IgG2b levels, but cabo and the combination treatment had not. Despite the effects on serum IgG2b, bortezomib did not significantly increase survival, whereas cabo and the combination treatment did. Increased survival with the combination was significant when compared to bortezomib monotherapy, but not when compared to cabo monotherapy. Earlier we have shown that cabo dose-dependently increases the necrotic tumor area in bone, and proposed that the rise in IgG2b was due to lysis of plasma cells and not tumor growth. Consistent with this hypothesis, the IgG2b levels of cabo treated mice were lower at sacrifice than at day 35 in this study. In summary, cabo increased survival and exhibited bone-protective and anti-tumor effects in this murine model of MM. Combination with bortezomib showed additive effects on survival. Based on these results, further investigation of cabozantinib in multiple myeloma is warranted. Citation Format: Mari I. Suominen, Katja M. Fagerlund, Esa Alhoniemi, Jukka P. Rissanen, Jussi M. Halleen, Dana T. Aftab. Effects of combination treatment with cabozantinib and bortezomib in the 5TGM1 murine multiple myeloma model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 786. doi:10.1158/1538-7445.AM2015-786

Epigenetic Activity of Peroxisome Proliferator-Activated Receptor Gamma Agonists Increases the Anticancer Effect of Histone Deacetylase Inhibitors on Multiple Myeloma Cells.

Epigenetic modifications play a major role in the development of multiple myeloma. We have previously reported that the PPARγ agonist pioglitazone (PIO) enhances, in-vitro, the cytotoxic effect of the Histone deacetylase inhibitor (HDACi), valproic acid (VPA), on multiple myeloma cells. Here, we described the development of a new multiple myeloma mouse model using MOLP8 cells, in order to evaluate the effect of VPA/PIO combination on the progression of myeloma cells, by analyzing the proliferation of bone marrow plasma cells. We showed that VPA/PIO delays the progression of the disease and the invasion of myeloma cells in the bone marrow. Mechanistically, we demonstrated that VPA/PIO increases the cleavage of caspase 3 and PARP, and induces the acetylation of Histone 3 (H3). Furthermore, we provided evidence that PPARγ agonist is able to enhance the action of other HDACi such as Vorinostat or Mocetinostat. Using PPARγ antagonist or siPPARγ, we strongly suggest that, as described during adipogenesis, PIO behaves as an epigenetic regulator by improving the activity of HDACi. This study highlights the therapeutic benefit of PIO/VPA combination, compared to VPA treatment as a single-arm therapy on multiple myeloma and further highlights that such combination may constitute a new promising treatment strategy which should be supported by clinical trials.

Small Molecule MYC Inhibitor Conjugated to Integrin-Targeted Nanoparticles Extends Survival in a Mouse Model of Disseminated Multiple Myeloma.

28 days, P < 0.05 and T/D 20: 52 days vs. 29 days, P = 0.001). The smaller particles performed better of the two sizes. Neither MI1 nor MI1-PD provided survival benefit when administered systemically as free compounds. These results demonstrate for the first time that a small molecule inhibitor of the MYC transcription factor can be an effective anticancer agent when delivered using a targeted nanotherapy approach.

Upregulation of CD38 expression on multiple myeloma cells by all-trans retinoic acid improves the efficacy of daratumumab.

Daratumumab is an anti-CD38 monoclonal antibody with lytic activity against multiple myeloma (MM) cells, including ADCC (antibody-dependent cellular cytotoxicity) and CDC (complement-dependent cytotoxicity). Owing to a marked heterogeneity of response to daratumumab therapy in MM, we investigated determinants of the sensitivity of MM cells toward daratumumab-mediated ADCC and CDC. In bone marrow samples from 144 MM patients, we observed no difference in daratumumab-mediated lysis between newly diagnosed or relapsed/refractory patients. However, we discovered, next to an expected effect of effector (natural killer cells/monocytes) to target (MM cells) ratio on ADCC, a significant association between CD38 expression and daratumumab-mediated ADCC (127 patients), as well as CDC (56 patients). Similarly, experiments with isogenic MM cell lines expressing different levels of CD38 revealed that the level of CD38 expression is an important determinant of daratumumab-mediated ADCC and CDC. Importantly, all-trans retinoic acid (ATRA) increased CD38 expression levels but also reduced expression of the complement-inhibitory proteins CD55 and CD59 in both cell lines and primary MM samples. This resulted in a significant enhancement of the activity of daratumumab in vitro and in a humanized MM mouse model as well. Our results provide the preclinical rationale for further evaluation of daratumumab combined with ATRA in MM patients.

281. Engineering Hematopoiesis for Tumor-Targeted Interferon-alpha Delivery Inhibits Multuple Myeloma and B Cell Malignancies

A protective and immunosuppressive tumor microenvironment is considered a key factor for the failure of anti-cancer treatments. We developed a strategy to turn a pro-tumoral into an anti-tumoral microenvironment by transplanting genetically engineered hematopoietic stem/progenitor cells (HSPC). We exploit a transcriptionally and post-transcriptionally regulated Interferon-alpha (IFNa) cassette to drive specific expression of this pleiotrophic antitumor cytokine in tumor-infiltrating monocytes/macrophages (IFNa gene therapy). When applied to spontaneous mouse or orthotopic human breast cancer models, IFNa gene therapy with mouse or human HSPC, respectively, inhibited tumor and metastases growth by activating innate and adaptive immune cells in the tumor (Escobar et al, Sci Transl Med 2014). To test this approach in clinics, we aim for a first-in-human trial of IFNa gene therapy in patients undergoing autologous transplantation, a procedure widely practiced for Multiple Myeloma (MM) and Lymphoma. To detect potential untoward effects of chronic IFNa exposure on human hematopoiesis, we transduced mobilized peripheral blood CD34+ cells with the human IFNa lentiviral vector (LV). A single round of transduction yielded vector copy numbers (VCN) of 0.4 to 4 according to the protocol used, with 30-90% transduction efficiency and 1-4 VCN per cell. This IFNa-engineered graft was mixed with mock-transduced CD34+ cells in various proportions and transplanted into NSG mice. All mice engrafted and showed long-term, multilineage hematopoietic output, with a dose-dependent decrease in the human graft at high, supratherapeutic VCN. To explore the efficacy of IFNa gene therapy on MM, we intravenously injected NSG mice reconstituted with IFNa LV-transduced CD34+ cells (VCN:1-3; chimerism 20-50%) with the human MM.1S cell line and followed MM growth by bioluminescence imaging and serial blood and BM exams. IFNa gene therapy strongly delayed myeloma bone disease and improved survival (9 wks vs. 13 wks median survival, p=0.03). Moreover, we tested the efficacy of IFNa gene therapy on human primary Ph+ B-ALL blasts as a model of high-grade B cell malignancies. Strikingly, IFNa gene therapy substantially reduced B-ALL growth in NSG and NSG3GS mice (p=0.002 and p=0.015), and Imatinib treatment, the standard of care for Ph+ALL, gave an additive effect when combined with IFNa gene therapy. These encouraging results pave the way for a phase I/II trial in patients with MM or B cell malignancies. To better understand the mechanism of IFNa gene therapy, we are setting up immuno-competent mouse models of MM and lymphoid malignancies. We developed a spontaneous model of high grade B cell leukemia/lymphoma based on ectopic expression of miR-126. IFNa gene therapy significantly reduced leukemic burden. Moreover, we derived more immunogenic leukemic subclones engineered with the ovalbumin or huCD20 antigen. These lines will help defining the immunomodulatory effect of IFNa gene therapy and allow testing combination with monoclonal antibodies or adoptive T cell transfer.

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.