Model Of Human Myeloma Research Articles

Hdp-101, an Anti-BCMA Antibody-Drug Conjugate with a Novel Payload Amanitin in Patients with Relapsed Multiple Myeloma, Initial Findings of the First in Human Study

Introduction Several antibody-drug conjugates (ADCs) are currently being evaluated in clinical trials in a variety of malignancies. The vast majority of these are based on a few toxic compounds, largely limited to microtubule- or DNA-targeting toxins which impact proliferating cells and have limited efficacy in diseases with a low proliferative fraction such as multiple myeloma. Thus, new compounds with alternative modes of action and the ability to actively induce cell death in non-proliferating tumor cells could enhance the therapeutic potential of ADCs. We are currently developing amanitin based ADCs. Amanitin specifically inhibits RNA polymerase II thereby inhibiting the cellular transcription process at very low concentrations irrespective of the proliferation status of the target cell. Subsequently tumor cells enter apoptosis and are eliminated. HDP-101 an Amanitin based antibody drug conjugate HDP-101 is a new ADC targeting BCMA carrying a synthetic version of amanitin as a payload. In vitro cytotoxic potency of HDP-101 was demonstrated on BCMA-positive myeloma cell lines, as well as on non-proliferating primary CD138+ cells isolated from patients with refractory myeloma The cytotoxic effects of HDP-101 were seen even in non-proliferating myeloma cells with low BCMA density. Toxicity was observed neither in non-BCMA expressing control cells nor in myeloma cells exposed to an amanitin-loaded non-target control antibody. In murine xenograft models of human myeloma, HDP-101 caused dose-dependent tumor regression including complete remissions after a single dose in subcutaneous and as well as in disseminated models. Safety profiling in Cynomolgus monkeys revealed a good therapeutic index after repeated dosing. Our non-clinical studies concluded that this amanitin-based ADC is a novel promising approach in the therapy of multiple myeloma to overcome drug resistance and improve patient outcome. HDP-101-01 Clinical study HDP-101-01 is a first-in-human, open label, non-randomized, multicenter, phase 1/2a trial with HDP-101 in patients with myeloma whose disease has progressed. The aim of the Phase 1 dose escalation part is to determine the Maximum Tolerated Dose and/or establish the Recommended Phase 2 Dose. The primary objective of the phase 2 dose expansion phase is to assess the preliminary anti-tumor activity of HDP-101. An adaptive Bayesian logistic regression model with overdose control principle is used to guide the dose escalation steps. An Interim Analysis is planned after each cohort is completed. The design of the study ensures a safe and adaptive dose escalation to reach a potential clinical benefit in a patient who have limited or no therapeutic options. HDP-101-01 Study progress The study started enrollment in February, 2022. As of 10th of July, 2023, eight (2 female and 6 male) patients were dosed in 3 consecutive dose cohorts. The median age of the patients was 70 years, ranging between 50 and 80. All 8 patients were heavily pre-treated and multidrug-resistant. The median previous lines of treatment were 7 (5 to 15). Table 1 HDP-101-01 Results Seven of 8 patients were evaluable for dose limiting toxicities (DLTs) in the first 3 treatment cohorts. The initial 3 cohorts were well tolerated, without any DLTs, there were no signs of liver and kidney toxicity, no infusion reaction was detected. No reports of keratopathy or visual acuity loss were observed. Free payload was not detected in any of the available pharmacokinetic samples. Based on the limited data, the PK of HDP-101 was in line with our expectation based on the preclinical observations, exposure to HDP-101 is dose proportional. Anti-drug antibody (ADA) was not detected and there's no sign of immunogenicity. Objective responses were not reported in these initial cohorts however in Cohort #3 (60μg/Kg) there was one patient ongoing after 8 cycles of treatment with SD. This patient had a slow decrease in M-protein levels. Image 1 The initial dose cohorts showed good tolerability in late stage relapsed and/or refractory multiple myeloma patients. The study continues to enroll patients to higher dose cohorts at selected sites in the US, Germany, Poland and Hungary. Currently the study is enrolling patients in Cohort #4 at the dose of 80μg/Kg. Additional updates will be provided at the 2023 ASH Annual Meeting.

Hdp-101, an Anti-BCMA Antibody-Drug Conjugate with a Novel Payload Amanitin in Patients with Relapsed Multiple Myeloma, Initial Findings of the First in Human Study

Several antibody-drug conjugates (ADCs) are currently being evaluated in clinical trials in a variety of malignancies. Vast majority of these ADCs are based on a few toxic compounds, largely limited to microtubule- or DNA-targeting toxins targets proliferating cells and have limited efficacy in diseases with a low proliferative fraction such as multiple myeloma. Thus, new compounds with alternative mode of actions and the ability to actively induce cell death in non-proliferating tumor cells could enhance the therapeutic potential of ADCs. We are currently developing amanitin based ADCs. Amanitin specifically inhibits RNA polymerase II thereby inhibiting the cellular transcription process at very low concentrations irrespective of the proliferation status of the target cell. Subsequently tumor cells enter apoptosis and are eliminated. HDP-101 is a new ADC targeting BCMA (B cell maturation antigen) carrying a synthetic version of amanitin as a payload. In vitro cytotoxic potency of HDP-101 was demonstrated on BCMA-positive myeloma cell lines, as well as on non-proliferating primary CD138+ cells isolated from patients with refractory myeloma. Furthermore, the cytotoxic effects of HDP-101 were seen even in non-proliferating myeloma cells with low BCMA density. Toxicity was observed neither in non-BCMA expressing control cells nor in myeloma cells exposed to an amanitin-loaded non-target control antibody. In murine xenograft models of human myeloma, HDP-101 caused dose-dependent tumor regression including complete remissions after a single dose in subcutaneous and as well as in disseminated models. Safety profiling in Cynomolgus monkeys revealed a good therapeutic index after repeated dosing. Our non-clinical studies concluded that this amanitin-based ADC is a novel promising approach in the therapy of multiple myeloma to overcome drug resistance and improve patient outcome. HDP-101-01 is a first-in-human, open label, non-randomized, multicenter, phase 1/2a trial with HDP-101 in patients with multiple myeloma whose disease has progressed. The aim of the Phase 1 dose escalation part is to determine the Maximum Tolerated Dose and/or establish the Recommended phase 2 Dose. The primary objective of the phase 2 dose expansion phase is to assess the preliminary anti-tumor activity of HDP-101. An adaptive Bayesian logistic regression model with overdose control principle is used to guide the dose escalation steps. An Interim Analysis is planned after each cohort is completed. The design of the study ensures a safe and adaptive dose escalation to reach a potential clinical benefit in a patient who have limited or no therapeutic options. The study started enrollment in February 2022. As of 12th of July 2022 four (1 female and 3 male) patients were dosed in 2 consecutive dose cohorts. The median age of the patients was 63.5 years, ranging between 51 and 80. All 4 patients were heavily pre-treated and multidrug-resistant. The median previous lines of treatment were 11 (5 to 16). Three of 4 patients were evaluable for dose limiting toxicities (DLT). The initial 2 cohorts were well tolerated, without any reports on DLTs. No reports of keratopathy or visual acuity loss were observed. Free payload was not detected in any of the available pharmacokinetic samples. In line with our expectation objective responses also were not reported in these initial cohorts. The initial dose cohorts showed good tolerability in late stage relapsed and/or refractory multiple myeloma patients. The study continues to enroll patient to higher dose cohorts. An updated dataset will be presented at the ASH2022 meeting.

CD8+ Anti-BCMA mRNA CAR T-Cells Effectively Kill Human Multiple Myeloma Cells In Vitro and In Vivo

Potent anti-multiple myeloma (MM) responses have been observed in early-stage clinical trials with virally-transduced chimeric antigen receptor (CAR) T-cells redirected to B-cell maturation antigen (BCMA), a membrane protein with extremely high sensitivity and specificity for MM and plasma cells. Despite significant potency, CAR T-cells engineered by virally-mediated gene transfer present a poor risk profile. Permanent modification enhances CAR persistence but also increases the risk of severe cytokine release syndrome (CRS), neurotoxicity, and prolonged plasma cell aplasia due to uncontrolled in vivo expansion and long-term persistence of the permanently-modified CAR T-cells. In an effort to address these shortcomings while preserving efficacy, we have developed CD8+ anti-BCMA CAR T-cells modified transiently by mRNA transfection (Descartes-08). Descartes-08 manufactured at clinical scale showed high (>90%) purity, post-cryopreservation viability, and transfection efficiency. Descartes-08 demonstrated robust dose- and time- dependent killing of BCMA+ MM cell lines, including those with acquired resistance to immunomodulatory drugs (IMiDs), as well as primary MM cells co-cultured with autologous bone marrow stromal cells. Compared with transfected pan- (CD3+) anti-BCMA CAR T-cells, Descartes-08 (CD8+) demonstrated enhanced transfection and killing but reduced secretion of IFN-g, an inflammatory cytokine highly correlated with CRS. Descartes-08 also demonstrated robust dose-dependent efficacy in a disseminated human MM model (i.v. MM1SLuc cells in NSG mice), and could be safely administered repetitively to deepen responses. These results advocate for rapid clinical translation of Descartes-08, which may improve the benefit: risk profile of CAR T-cells in MM and enable their use in patients with earlier-stage disease. DisclosuresKurtoglu:2Cartesian Therapeutics, Inc: Employment, Equity Ownership. Holderness:Cartesian Therapeutics, Inc: Employment, Equity Ownership. Kalayoglu:Cartesian Therapeutics, Inc: Employment, Equity Ownership. Richardson:Celgene: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides AB: Membership on an entity's Board of Directors or advisory committees. Anderson:Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; C4 Therapeutics: Other: scientific founder.

Preclinical Evaluation of Hdp-101, a Novel Anti-BCMA Antibody-Drug Conjugate, in Multiple Myeloma

Several antibody-drug conjugates (ADCs) are currently being evaluated in clinical trials in a variety of hematologic malignancies. Surprisingly, most ADCs are based on only few toxic compounds, largely limited to microtubuli- or DNA-targeting toxins (auristatins and maytansines or duocarmycins and pyrrolobenzodiazepines, PBDs). These payloads are mainly targeting proliferating cells potentially leading to limited efficacy in diseases with a low proliferation rates such as indolent lymphomas or multiple myeloma. Thus, new compounds with alternative toxicity mechanisms could enhance the therapeutic potential of ADCs. We therefore focus on amanitin based ADCs, so called ATACs (antibody-targeted Amanitin conjugates) comprising a new class of antibody-drug conjugates with amanitin as toxic payload. Amanitin is the most well-known toxin of the amatoxin family and binds to the eukaryotic RNA polymerase II thereby inhibiting the cellular transcription process at very low concentrations irrespective of the proliferation status of the target cell.In the current study, we have assessed in vitro and in vivo specificity andefficacy of HDP-101, an ATAC targeting BCMA (B cell maturation antigen). BCMA (also known as CD269) is expressed on cells of the B cell lineage, predominantly on plasma blasts and plasma cells. It is not expressed on naïve, germinal center, and memory B cells. BCMA is highly expressed on malignant plasma cells and therefore considered an ideal target in multiple myeloma.In vitro cytotoxic potency of HDP-101 was tested on several BCMA-positive myeloma cell lines, as well as on non-proliferating primary CD138+ cells isolated from triple and quadruple refractory myeloma patients. Profound cytotoxic effects were seen at pico- to nanomolar concentrations of HDP-101 even in myeloma cells with low epitope density. Importantly, toxicity was observed neither in non-BCMA expressing control cells nor in myeloma cells exposed to an amanitin-loaded non-target control antibody. In mouse xenograft models of human myeloma, HDP-101 caused dose-dependent tumor regression including complete remissions after a single i.v. dose of 2.0 mg/kg and 4.0 mg/kg in subcutaneous xenografts and after single i.v. doses from 0.1 mg/kg to 2.0 mg/kg in disseminating xenografts.Safety profiling in Cynomolgus monkeys revealed a good tolerability and therapeutic index after sequentially applied doses of 0.3 mg/kg, 1.0 mg/kg, and multiple dose applications of 4 x 3.0 mg/kg. Hematology and clinical chemistry parameters were unaffected except for a mild to moderate and transient increase of liver enzymes and lactate dehydrogenase. The half-life of the HDP-101 in serum was ~12 days; the free toxin was detectable at levels close to the lower limit of quantification only.In conclusion, targeted cytotoxic drug delivery to BCMA positive myeloma cell lines was achieved by using HDP-101, an anti-BCMA-ATAC. The mode of action of the payload amanitin led to an efficient anti-tumor response in vitro and in vivo with good tolerability in non-human primate studies yielding a very favorable therapeutic index. Amanitin-based ADCs in the therapy of multiple myeloma is a novel promising approach with a distinct mode of action to overcome drug resistance and improve patient outcome. A First-in-human trial with HDP-101 is expected to start in 2018. DisclosuresBreunig:Heidelberg Pharma GMBH: Employment, Other: Employee Stock Options. Pálfi:Heidelberg Pharma GMBH: Employment, Other: Employee Stock Options. Müller:Heidelberg Pharma GMBH: Employment, Other: Employee Stock Options. Lutz:Heidelberg Pharma GMBH: Employment, Other: Employee Stock Options. Hechler:Heidelberg Pharma GMBH: Employment, Other: Employee Stock Options. Kulke:Heidelberg Pharma GMBH: Employment, Other: Employee Stock Options. Goldschmidt:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Chugai: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Morphosys: Research Funding; Millenium: Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Onyx: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Pahl:WILEX AG: Employment, Membership on an entity's Board of Directors or advisory committees, Other: Employee Stock Options; Heidelberg Pharma GMBH: Employment, Membership on an entity's Board of Directors or advisory committees, Other: Employee Stock Options. Raab:Heidelberg Pharma GMBH: Research Funding.

A First in Human Study Planned to Evaluate Hdp-101, an Anti-BCMA Amanitin Antibody-Drug Conjugate with a New Payload and a New Mode of Action, in Multiple Myeloma

A First in Human Study Planned to Evaluate Hdp-101, an Anti-BCMA Amanitin Antibody-Drug Conjugate with a New Payload and a New Mode of Action, in Multiple Myeloma

Abstract 4557: BCMA-CD3 bispecific antibody PF-06863135: Preclinical rationale for therapeutic combinations

Abstract BCMA-CD3 bispecific antibody PF-06863135 is currently under evaluation in an ongoing Phase 1 clinical trial in relapsed/refractory (RR) multiple myeloma (MM) patients (NCT03269136). We showed previously that BCMA-CD3 bispecific has potent single agent activity in RR MM human bone marrow aspirates in vitro and in preclinical models of human myeloma (Panowski et al, MCT 2019). Herein we used in vitro assays, ex vivo MM patient 3D cultures, and in vivo efficacy models to study therapeutic combinations of PF-06863135 with immune checkpoint inhibitors, immunomodulatory drugs (IMiD), and gamma secretase inhibitors (GSI). Upregulation of immune checkpoints, such as programmed cell death protein-1 (PD-1) or programmed death ligand 1 (PD-L1) has been demonstrated downstream of response to CD3 bispecifics. While PF-06863135 is effective as a single agent in ex vivo 3D primary MM cultures (EC50 0.2 nM), PD-L1 was upregulated on myeloma cells. We combined PF-06863135 with anti-PD-1 in subcutaneous and orthotopic myeloma models MM.1S and MM.1S-PD-L1 in NSG mice engrafted with human T cells. PF-06863135 demonstrated single agent anti-tumor activity in the MM.1S models; however, in the MM.1S-PD-L1 model, PF-06863135 activity was blunted. When given in combination with an anti-PD-1 blocking antibody, the full thrust of the single agent anti-tumor activity of PF-06863135 was restored. Dysfunctional, senescent like T cells may emerge downstream of T cell immunotherapies. IMiDs, such as lenalidomide, are a standard of care therapy in MM and result in immunomodulatory effects on T cells leading us to hypothesize PF-06863135 mediated tumor growth inhibition could be enhanced by combining with lenalidomide. When PF-06863135 and lenalidomide were given in combination in the human T cell engrafted established orthotopic MM.1S and MOLP8 models, an even greater anti-tumor activity was observed as compared to either agent alone. These studies suggest that PF-06863135 activity can be augmented by combinations with immunomodulatory agents such as anti-PD-1 or lenalidomide. BCMA is shed from the surface of myeloma cells by gamma secretases, not only reducing cell surface target density but contributing to a soluble sink. When treated with GSI in vitro, myeloma cell surface BCMA expression increased concurrent with reduction in soluble/shed BCMA across a panel of myeloma and lymphoma cell lines expressing a range of BCMA. In cytotoxic T lymphocyte co-culture assays, the activity of PF-06863135 was potentiated by combining with GSI. Studies of the combination of PF-06863135 and GSI are ongoing in preclinical in vivo models of myeloma. Taken together, our preclinical studies provide insights into mechanisms of action and resistance for PF-06863135, which has potential for profound single agent activity that can be enhanced with therapeutic combinations. Citation Format: Katarzyna Karwacz, Andrea T. Hooper, Chao-Pei Betty Chang, Heike Krupka, Jeffrey Chou, Viola Lam, Ivana Djuretic, Javier Chaparro-Riggers, Puja Sapra. BCMA-CD3 bispecific antibody PF-06863135: Preclinical rationale for therapeutic combinations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4557.

The Transmembrane Receptor Roundabout 1 (ROBO1) Is Necessary for Multiple Myeloma Proliferation and Homing to the Bone Marrow Niche

Introduction Multiple myeloma (MM) is characterized by dissemination and accumulation of plasma cells in the bone marrow (BM), which promotes tumor cell growth and therapy resistance. ROBO1 is a conserved transmembrane receptor of the Ig superfamily with no intrinsic catalytic activity, and its role in MM pathogenesis is unknown. Material and Methods We first analyzed ROBO1 expression via western blot and/or immunohistochemistry (IHC). Gene expression profiling in a cohort of 170 newly diagnosed MM patients (IFM170) was used to compare ROBO1 expression across primary MM and BM stroma cells (BMSC), and normal BM plasma cells (PC). We used short hairpin RNA (shRNA) for stable ROBO1 knock down (KD) and CRISPR-Cas9 for ROBO1 knock out (KO). For protein structure-function and rescue studies, ROBO1 KO MM cells were transduced with a lentiviral vector expressing either full-length (FL) or truncated ROBO1 mutants devoid of extracellular (Cyt) or intracellular domain (DeltaCyt), including patient-derived truncating mutations, with a C-terminus triple FLAG tag. FLAG immunoprecipitation (IP) followed by mass spectrometry or western blotting and immunofluorescence (IF) were used to identify ROBO1 interacting partners and ROBO1 cellular localization. We used a hydrogel encapsulation system to study proliferation in a 3D system. To study extramedullary and intramedullary MM growth in vivo, WT and ROBO1 KO OPM2 were injected either subcutaneously (plasmacytoma model) or intra-medullary in femoral bones of donor mice which were then implanted subcutaneously in recipient SCID mice (µ-SCID model). PET-CT was used to assess tumor volume. Mouse tumors were retrieved for IHC and RNA extraction followed by RNA sequencing. To study dissemination and homing, KO and FL addback OPM2 cells were injected intravenously in SCID mice. Femurs and plasmacytoma were retrieved at endpoint for IHC. Results ROBO1 is highly expressed in human MM cell lines and primary MM cells with highest expression in cells carrying the high risk t(4;14) cytogenetic and low/absent expression in normal PC. Of human cancer cell lines, ROBO1 expression was limited to late B cell lineage; and ROBO1 KD was selectively cytotoxic against MM, but not other hematologic cancers. ROBO1 KO significantly decreases proliferation in a 3D culture system and tumor growth in extramedullary (mean tumor volume KO versus WT plasmacytoma: 457 versus 1323 mm3, p value= 0.02) and intramedullary (mean tumor volume KO versus WT: 823 versus 2684 mm3, p value= 0.001) murine models of human MM. ROBO1 KO MM cells show decreased adhesion to BM endothelial and BMSC, which is fully rescued by FL ROBO1 addback. To address whether ROBO1 loss alters dissemination/homing of MM cells in vivo, we injected mice intravenously with ROBO KO or FL addback OPM2 cells. While ROBO1 KO resulted in a modest, non-statistically significant prolongation in mouse OS (90 versus 75 days, respectively, p value 0.2), the pattern of disease was strikingly different. As expected, ROBO1 FL mice developed hindlimb paralysis with extensive BM infiltration with MM. Importantly, ROBO1 KO mice demonstrated reduced BM infiltration and developed solitary plasmacytoma. We next showed that ROBO1 C-terminus domain is necessary and sufficient to rescue ROBO1 KO proliferative defect while expression of ROBO1 truncations, including patient-derived frameshift mutations, acted as dominant negative. IP showed avid interaction of ROBO1 with ABL1. Interestingly, we showed that the cytosolic domain of ROBO1 undergoes cleavage and translocates to the nucleus, where its function is now being studied. Conclusions We show that ROBO1 is necessary for MM homing to the BM niche and for MM growth within and outside the BM space. ROBO1 cytosolic domain undergoes proteolytic cleavage and translocates to the nucleus and is necessary and sufficient to rescue ROBO1 KO defective proliferation. Based on our data, we propose a dual model for ROBO1 in MM: the full transmembrane receptor is involved in regulating adhesion, dissemination and homing of MM cells within the BM niche; the cleaved intracellular C-terminus domain participates in transcriptional regulation, promoting MM proliferation. These data suggest that ROBO1 C-terminus may be a novel molecular target in MM. Disclosures Roccaro: Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; European Hematology Association: Research Funding; Transcan2-ERANET: Research Funding; European Hematology Association: Research Funding; AstraZeneca: Research Funding; Transcan2-ERANET: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding. Ghobrial:Takeda: Consultancy; Sanofi: Consultancy; Amgen: Consultancy; BMS: Consultancy; Celgene: Consultancy; Janssen: Consultancy. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

Osteolytic disease in IL-6 and Myc dependent mouse model of human myeloma

Myeloma bone disease (MBD) is an important unmet medical need characterized by focal and generalized bone loss causing severe pain, pathological fractures, instability of the vertebral column, and medullary cord / spinal nerve root compression. MBD is a disease-defining feature of multiple myeloma, the second most common blood cancer in the United States. MBD is poorly recapitulated in genetically engineered mouse models (GEMMs) of human myeloma developed in the past. Objective: To address this shortcoming, we determined onset, incidence and severity of osteolytic disease in a new GEMM of human myeloma developed in our laboratory.

Osteolytic disease in IL-6 and Myc dependent mouse model of human myeloma

Focal and generalized bone loss is a hallmark of multiple myeloma (MM) that is poorly recapitulated in genetically engineered mouse models (GEMM) of myeloma development. To address this shortcoming, we used integrated whole-body ex vivo X-ray microscopy and micro-computed tomography (μCT) to

CircRNA circ_0000190 inhibits the progression of multiple myeloma through modulating miR-767-5p/MAPK4 pathway

BackgroundMultiple myeloma (MM) accounts for 10% of all hematological malignancies. Dysregulation of microRNAs (miRNAs) or long non-coding RNAs (lncRNAs) has important impacts on progression of MM. Circular RNAs (circRNAs) are correlated with malignancy in the modulation of tumor progression. This study aims to investigate the effect of circ_0000190 on regulating the progression of MM.MethodMicroscopic examination via single molecule fluorescent in situ hybridization indicates the location of circ_0000190. qRT-PCR and Western blot were used to evaluate the expression of RNAs and proteins. Potential target of circ_0000190 was searched as miRNA, and examined by luciferase reporter assay. A computational screen was also conducted to search the potential target of miRNA. In vitro cell viability, proliferation, apoptosis assays and flow cytometric were performed to assess the effects of circ_0000190 and its target on MM. Mice model of human MM was established with subcutaneous xenograft tumor, qRT-PCR and western blot were performed to detect the underlying mechanisms of circ_0000190 on MM.ResultsCirc_0000190 was located in the cytoplasm, and down-regulated in both bone marrow tissue and peripheral blood, while the target of circ_0000190, miR-767-5p, was up-regulated, suggesting a negative correlation between them. The binding ability between circ_0000190 and miR-767-5p was confirmed by luciferase reporter assay. Moreover, circ_0000190 inhibited cell viability, proliferation and induced apoptosis of MM thus inhibiting cell progression, which is partially through the negative regulation of miR-767-5p. Mitogen-activated protein kinase 4 (MAPK4) is a direct target of miR-767-5p. In addition, over-expression of miR-767-5p promoted cell progression by directly targeting and regulating MAPK4. The MM model mice with administration of circ_0000190 suppressed tumor growth and progression.ConclusionOur results revealed that the ability of circ_0000190 to protect against MM was inherited through repression of miR-767-5p, and miR-767-5p might be a tumor drive through targeting MAPK4. Therefore, a novel role of circ_0000190 on regulating the progression of MM was found, and the clinical application of circRNAs might represent a strategy in MM.

The Novel Pan-PIM Kinase Inhibitor, PIM447, Displays Dual Antimyeloma and Bone-Protective Effects, and Potently Synergizes with Current Standards of Care.

PIM kinases are a family of serine/threonine kinases recently proposed as therapeutic targets in oncology. In the present work, we have investigated the effects of the novel pan-PIM kinase inhibitor, PIM447, on myeloma cells and myeloma-associated bone disease using different preclinical models. In vitro/ex vivo cytotoxicity of PIM447 was evaluated on myeloma cell lines and patient samples. Synergistic combinations with standard treatments were analyzed with Calcusyn Software. PIM447 effects on bone cells were assessed on osteogenic and osteoclastogenic cultures. The mechanisms of PIM447 were explored by immunoblotting, qPCR, and immunofluorescence. A murine model of disseminated multiple myeloma was employed for in vivo studies. PIM447 is cytotoxic for myeloma cells due to cell-cycle disruption and induction of apoptosis mediated by a decrease in phospho-Bad (Ser112) and c-Myc levels and the inhibition of mTORC1 pathway. Importantly, PIM447 demonstrates a very strong synergy with different standard treatments such as bortezomib + dexamethasone (combination index, CI = 0.002), lenalidomide + dexamethasone (CI = 0.065), and pomalidomide + dexamethasone (CI = 0.077). PIM447 also inhibits in vitro osteoclast formation and resorption, downregulates key molecules involved in these processes, and partially disrupts the F-actin ring, while increasing osteoblast activity and mineralization. Finally, PIM447 significantly reduced the tumor burden and prevented tumor-associated bone loss in a disseminated murine model of human myeloma. Our results demonstrate dual antitumoral and bone-protective effects of PIM447. This fact, together with the very strong synergy exhibited with standard-of-care treatments, supports the future clinical development of this drug in multiple myeloma. Clin Cancer Res; 23(1); 225-38. ©2016 AACR.

P21-Associated Kinase 4 (PAK4) Addiction in Multiple Myeloma: A Potential Therapeutic Application with Enhanced Activity in FGFR3 Expressing Myeloma

Dysregulated oncogenic serine/threonine kinases (STKs) play a pathological role in diverse forms of malignancies, including multiple myeloma (MM), and thus represent potential therapeutic targets. Here, we evaluated the biological and functional role of p21-activated kinase 4 (PAK4), and its potential as a new therapeutic target in MM. The PAK4 gene lies within chromosome region 19q13.2 commonly amplified in 30% of MM patients. We confirmed a correlation between copy number amplification and increased expression of PAK4 in two large myeloma patient datasets. High expression of total and phosphorylated PAK4 was also observed in the premalignant MGUS stage, suggesting that its overexpression may be an early event in the pathogenesis of myeloma. Myeloma cells with high PAK4 expression display sensitivity to conditional PAK4 knockdown, implying that an oncogene-addicted state exists in such cells. Moreover, we show that PAK4 promotes myeloma cell proliferation in vitro as well as in vivo in murine models of human myeloma through activation of the MEK/ERK pathway. This is further highlighted by the correlation between the basal levels of ERK activity and sensitivity to a novel small molecule inhibitor of PAK4, KPT-9274, a lead clinical agent. KPT-9274 effectively inhibits MM cell growth and survival via ERK inhibition in a large panel of MM cell lines and primary MM cells, also in the presence of bone marrow microenvironment, with no significant effect on normal PBMCs suggesting a potentially favorable therapeutic index. Inhibition of ERK by KPT-9274 also correlates with decreased DNA binding activity of ERK-dependent transcription factors AP1, ETS, CREB and EGR and decreased expression of ERK target genes such as CCND2, CCR1 and MYC. Finally, using a protein binding array, we have identified FGFR3, a commonly disrupted tyrosine kinase receptor, and Grb2, an adaptor protein involved in RAS activation, as novel PAK4 binding partners in myeloma and report disruption of this binding by KPT-9274. As a results, t(4;14)-positive MM cells expressing FGFR3 show greater sensitivity to PAK4 inhibition compared to MM cell lines without FGFR3 aberrations (e.g. U266 and ANBL6). FGFR3 may therefore serve as a predictive marker of KPT-9274 sensitivity in MM patients, with the potential for broader application to other malignancies associated with dysregulation of FGFR3 such as human bladder and cervical carcinomas.In conclusions, our study sheds light on the oncogenic role of the serine/threonine kinase PAK4 as survival and anti-apoptotic factor in myeloma and its inhibition with a new allosteric modulator, KPT-9274, as a potential novel therapeutic intervention in MM especially in high risk t(4:14)-positive patients. DisclosuresSenapedis:Karyopharm Therapeutics Inc: Employment. Oliva:Celgene: Honoraria; Takeda: Honoraria; Amgen: Honoraria. Anderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Oncoprep: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

Quercetin induces cell apoptosis of myeloma and displays a synergistic effect with dexamethasone in vitro and in vivo xenograft models.

Quercetin, a kind of dietary flavonoid, has shown its anticancer activity in many kinds of cancers including hematological malignancies (acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, and MM) in vitro and in vivo. However, its effects on MM need further investigation. In this study, MM cell lines were treated with quercetin alone or in combination with dexamethasone. In order to observe the effects in vivo, a xenograft model of human myeloma was established. Quercetin inhibited proliferation of MM cells (RPMI8226, ARP-1, and MM.1R) by inducing cell cycle arrest in the G2/M phase and apoptosis. Western blot showed that quercetin downregulated c-myc expression and upregulated p21 expression. Quercetin also activated caspase-3, caspase-9, and poly(ADP-ribose)polymerase 1. Caspase inhibitors partially blocked apoptosis induced by quercetin. Furthermore, quercetin combined with dexamethasone significantly increased MM cell apoptosis. In vivo xenograft models, quercetin obviously inhibited tumor growth. Caspase-3 was activated to a greater extent when quercetin was combined with dexamethasone. In conclusion, quercetin alone or in combination with dexamethasone may be an effective therapy for MM.

Impaired Lysophosphatidic Acid Receptor 3 Signaling in Mesenchymal Stromal Cells Promotes Multiple Myeloma Progression through Cellular Senescence and Transdifferentiation into Tumor-Associated Fibroblasts

IntroductionMultiple myeloma (MM) is one of the hematologic malignancies characterized as accumulation of monoclonal tumor of plasma cells. Although novel therapeutic agents have significantly improved the survival of MM patients, MM is still a mostly incurable disease. The most common reason of relapsed and refractory MM is that myeloma cells intimately communicate with bone marrow stroma and acquire drug resistance.Stroma is known to secrete soluble factors and express some adhesion molecules advantageous for progression of MM. Recently, there are accumulating evidences that bone marrow-derived mesenchymal stem cells (MSC) serve as a component of stroma and support growth and drug resistance of myeloma cells. Although the exact etiology has not been clearly defined, it has been suggested that the incidence of MM increases with aging.In this study, we tried to explore the relationship between MM progression and cellular senescence in MSC.MethodsHuman myeloma cell lines used in this study were IM-9, OPM-2, and RPMI-8226. Human MSC used in this study were obtained from Texas A&M Health Science Center for the Preparation and Distribution of Adult Stem Cells. To establish a mouse xenograft model of human MM, 1.0 x 106 of IM-9 cells were co-injected with 4.0 x 105 of human MSC subcutaneously into the flank of BALB/c nude mice. Visible and palpable tumors were formed in all animals after 20 days. Tumor specimens were resected surgically and diagnosed pathologically as MM based on marker expression. For in vivo bioimaging, luciferase-expressing IM-9 (IM-9-luc+) cells were co-injected with human MSC subcutaneously into BALB/c-nude mice, and photon emission was detected with a sensitive CCD camera 10, 15 and 20 days later.ResultsIn our earlier research, we have demonstrated that the signaling of lysophosphatidic acid (LPA), a bioactive lipid mediator, modulates cellular senescence in MSC (Kanehira et al. PLoS One. 2012). MSC produced autotaxin (ATX), a key enzyme in LPA synthesis, in response to myeloma cells via Toll-like receptor 4 (TLR4)/NF-kappaB-dependent pathway. To determine the LPA receptor responsible for cellular senescence in MSC, six LPA receptors (LPAR1-6) were individually knocked down using siRNA for each receptor. In BrdU incorpotaion assay, LPAR3 gene-silenced MSC (siLPAR3-MSC) were less proliferative than control MSC, and in cell cycle analysis with 7-AAD and Pyronin Y, siLPAR3-MSC was arrested in G1 phase. Interestingly, siLPAR3-MSC exhibited some characteristics of cellular senescence, such as up-regulation of senescence-associated beta-galactosidase activity, increased cell size, and flattened morphology. In a mouse xenograft model of MM, siLPAR3-MSC promoted progression of MM and also tumor-associated angiogenesis. In in vitro study, we confirmed that siLPAR3-MSC easily transdifferentiated into alpha-SMA+ tumor-associated fibroblast and secreted FGF2 in response to myeloma cells. The MM promoting effect and elevated tumor-associated angiogenesis observed in siLPAR3-MSC co-injection were both completely cancelled by FGF2 gene-silencing in siLPAR3-MSC (Figure 1 and 2).Conclusion and discussionIn this study, we verified the impairment of LPAR3 signaling may accelerate cellular senescence in MSC. And senesced MSC can provide an advantageous microenvironment for MM progression by FGF2-dependent formation of tumor-stroma milieu. Here, we provide the possibility that LPAR3 signaling could be promising as a therapeutic target in MM. [Display omitted] [Display omitted] DisclosuresFujiwara:Chugai Pharmaceutical CO., LTD: Research Funding. Fukuhara:Gilead Sciences: Research Funding. Harigae:Chugai Pharmaceutical Co., Ltd.: Research Funding.

Quercetin Displays Anti-Myeloma Activity and Synergistic Effect with Dexamethasone in Vitro and In Vivo Xenograft Models

Background: Multiple myeloma (MM) is a hematological malignancy with clonal plasma cell hyperplasia, which is still an incrurable disease since chemoresistance remains the major problem in MM management. Quercetin, a kind of dietary flavonoids, has shown its anticancer activity in many kinds of cancer cell lines and we tried to explore the effect of quercetin in MM.Methods: In vitro, we examined the proliferation of MM cell lines(RPMI8226,ARP-1,MM1R) after treatment with quercetin combined with or without dexamethasone by MTT.Flow cytometry was used to detect apoptosis and cell cycle of MM cells induced by quercetin with or without dexamethasone.Then we detected mRNA and protein expression associated with apoptosis and cell cycle arrest by semiquantitative real time-polymerase chain (qRT-PCR)and western blot analysis. In vivo,a xenograft mice model of human myeloma was established and the mice received vehicle or quercetin alone or dexamethasone alone or quercetin combinded with dexamethasone, and the tumorburdern and the tumor tissue samples were analyzed by tumor volume and immunohistochemistry.Results: Quercetin inhibited proliferation of MM cells by inducing apoptosis and cell cycle arrest in the G0/G1 or G2 phase(quercetin group vs control,p<0.05).Western blot showed that quercetin activated caspase3,caspase9,PARP-1 and increased cytochrome C release. C-myc and cyclinD1 expression were down-regulated and p21 were upregulated. Quercetinalso displays synergistic inhibition effect with dexamethasone in vitro (quercetin with dexamethasone vs quercetin only or dexamethasone only,p<0.05) and western bolt confirmed these results.In vivo,tumor burdern of xenograft mice modeltreated by quercetin was significantly lower than those of control(quercetin group vs control,p<0.05).Conclusions: Quercetin inhibits proliferation of MM cells by inducing apoptosis and cell cycle arrest in the G0/G1 or G2 phase through downregulating c-myc and cyclinD1 and upregulating p21 .Quercetinalso displays synergistic inhibition effect with dexamethasone.Thus,quercetin combination with dexamethasone therapy may be an effective option for MM patients. DisclosuresNo relevant conflicts of interest to declare.

NAD⁺-Metabolizing Ectoenzymes in Remodeling Tumor-Host Interactions: The Human Myeloma Model.

Nicotinamide adenine dinucleotide (NAD+) is an essential co-enzyme reported to operate both intra- and extracellularly. In the extracellular space, NAD+ can elicit signals by binding purinergic P2 receptors or it can serve as the substrate for a chain of ectoenzymes. As a substrate, it is converted to adenosine (ADO) and then taken up by the cells, where it is transformed and reincorporated into the intracellular nucleotide pool. Nucleotide-nucleoside conversion is regulated by membrane-bound ectoenzymes. CD38, the main mammalian enzyme that hydrolyzes NAD+, belongs to the ectoenzymatic network generating intracellular Ca2+-active metabolites. Within this general framework, the extracellular conversion of NAD+ can vary significantly according to the tissue environment or pathological conditions. Accumulating evidence suggests that tumor cells exploit such a network for migrating and homing to protected areas and, even more importantly, for evading the immune response. We report on the experience of this lab to exploit human multiple myeloma (MM), a neoplastic expansion of plasma cells, as a model to investigate these issues. MM cells express high levels of surface CD38 and grow in an environment prevalently represented by closed niches hosted in the bone marrow (BM). An original approach of this study derives from the recent use of the clinical availability of therapeutic anti-CD38 monoclonal antibodies (mAbs) in perturbing tumor viability and enzymatic functions in conditions mimicking what happens in vivo.

Adoptive B-cell transfer mouse model of human myeloma

Adoptive B-cell transfer mouse model of human myeloma

Abstract 1364: Blocking of S100A9 with tasquinimod demonstrates a potent anti-myeloma activity

Abstract Multiple myeloma (MM) is an incurable hematological malignancy characterized by clonal proliferation of plasma cells localized preferentially in the bone marrow (BM). Using an in vivo model of MM, we have recently demonstrated that a population of myeloid-derived suppressor cells in the BM microenvironment is involved in the regulation of MM progression. These cells abundantly produce the pro-inflammatory protein S100A9. Function of S100A9 could be blocked with tasquinimod (ABR-215050, Active Biotech/IPSEN), a quinoline-3-carboxamide derivative that binds to S100A9 and inhibits interaction with its receptors. Here, using pre-clinical models of MM, we investigated the anti-tumor effect of tasquinimod. The anti-tumor effect of tasquinimod was initially evaluated in vivo in a syngeneic MM model. In this model, mouse MM cells, injected intravenously, home to the BM and grow as MM that closely resembles the human disease. Tumor-bearing mice were randomly assigned to treatment or control groups. The treatment group received tasquinimod at a dose of 30 mg/kg/day in drinking water for 28 days. Administration of tasquinimod significantly improved survival of MM-bearing mice (p&amp;lt;0.005). Xenograft models of human MM established in immunodeficient SCID-beige mice were utilized to exclude the involvement of B-, T- and NK cells in the pro-survival effect of tasquinimod. Treatment with tasquinimod significantly (p&amp;lt;0.0001) reduced growth of H929 and RPMI-8226 tumors in these mice. We next utilized S100A9 knockout (KO) mice to address the question of whether the anti-tumor effect of tasquinimod is mediated through inhibition of S100A9. Administration of tasquinimod did not improve survival of MM-bearing S100A9 KO mice. These data suggest that the anti-MM effect of tasqunimod is indeed mediated through inhibition of S100A9. BM angiogenesis plays a critical role in MM progression. We evaluated whether inhibition of S100A9 with tasquinimod would block angiogenesis in MM. Immunohistochemical staining with anti-CD31 antibody demonstrated increased angiogenesis in the BM of MM-bearing mice compared with control tumor-free mice, and treatment with tasquinimod significantly reduced angiogenesis (p&amp;lt;0.005). Serum collected from tasquinimod or vehicle control treated MM- bearing mice two weeks after tumor cell inoculation, was subjected to a Mouse Angiogenesis Proteome Profiler Antibody Array (R&amp;D). A significant decrease in serum levels of pro-angiogenic factors including VEGF, FGF2, tissue factor, and endoglin was detected in tasquinimod-treated mice. Taken together, our data suggest that targeting S100A9 with tasquinimod results in a strong anti-tumor effect in pre-clinical models of MM. This effect is associated with reduced angiogenesis in the BM. Therefore, tasquinimod could potentially be therapeutically beneficial for patients with MM. Citation Format: Indu Ramachandran, Cindy Lin, Dmitry I. Gabrilovich, Yulia Nefedova. Blocking of S100A9 with tasquinimod demonstrates a potent anti-myeloma activity. [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 1364. doi:10.1158/1538-7445.AM2015-1364

Cytoskeleton Regulator PAK4 Plays a Role in Growth and Survival of Myeloma with a Potential Therapeutic Intervention Using PAK4 Allosteric Modulators (PAMs)

P21-activated serine/threonine kinase 4 (PAK4) is a major effector of Cdc42 and is essential for cytoskeleton reorganization. PAK4 is activated in cancer cells, promotes cell migration and anchorage-independent cell growth, and protects against apoptosis induction. With cellular migration playing a significant role in multiple myeloma (MM) cell growth and survival, we investigated the expression and subcellular localization of PAK4 in MM cells. We observed a high level of un-phosphorylated PAK4 in the cytoplasm and high levels of phosphorylated PAK4 in the nucleus. In a gain-of-function study, over-expression of PAK4-eGFP in PAK4-deficient MM cells (RPMI8226) significantly increased cell proliferation and survival. Conversely, in a loss-of-function study, conditional knock-down of PAK4 expression with TRIPZ-lentiviral vectors decreased MM cell proliferation and survival proportionally to the reduction in PAK4. With a significant impact of PAK4 on MM cell growth, we identified a class of orally bioavailable PAK4 allosteric modulators (PAMs; e.g. KPT-6604, -7189, -7657, -8752). We observed inhibition of MM cell growth and survival after treatment with PAMs even in the presence of bone marrow microenvironment. In addition, there is a significant correlation between PAK4 expression and the inhibition concentration (IC50s) of PAMs in proliferation assays. Moreover, inhibition of PAK4 induced receptor and mitochondrial-mediated apoptotic pathways via Caspase-3, -8, and -9 activation. PAMs had no significant effect on normal PBMCs, suggesting a favorable therapeutic index in MM treatment. Finally, in two murine models of human myeloma, orally bioavailable KPT-8752 given daily was able to inhibit tumor growth in vivo and prolong overall survival. In summary, PAK4 plays an important cellular and molecular function in myeloma and its inhibition with a new class of PAK4 allosteric modulators provides a novel therapeutic approach for the treatment of MM. DisclosuresSenapedis:Karyopharm: Employment. Baloglu:Karyopharm: Employment. Anderson:Celgene: Consultancy; Sanofi-Aventis: Consultancy; Onyx: Consultancy; Acetylon: Scientific Founder, Scientific Founder Other; Oncoprep: Scientific Founder Other; Gilead Sciences: Consultancy.

Multiple Myeloma Cells Express IL-17A Creating An Autocrine Loop: An Attractive Therapeutic Target

We have previously demonstrated that Th17 cells, which produce IL-17A, are significantly elevated in peripheral blood and bone marrow (BM) of patients with Multiple Myeloma (MM) and IL-17A promotes MM cell growth and survival, both in vitro and in vivo via IL-17A receptor. We have recently evaluated and observed that anti-IL-17A monoclonal antibody (mAb) significantly inhibited MM cell growth in vitro, while IL-17A induced proliferation of MM cells compared to control. We have also observed significant down-regulation of IL-6 production by anti-IL-17A mAb in MM-BMSC co-culture. Importantly, the administration of anti-IL-17A mAb weekly for 4 weeks in the SCIDhu model of human myeloma, where MM cells grow within the human microenvironment in mice led to a significant inhibition of tumor growth compared to the control mice. This remarkable activity of anti-IL17 mAb raised the question of whether the myeloma cells themselves are a possible source of IL-17. In this study, we used transcriptome sequencing (RNA-Seq) data to evaluate the expression of IL-17A in primary CD138+ myeloma cells (N=17) compared to normal plasma cells (NPC) (N=5). Whereas none of the NPCs expressed IL-17A, it was significantly over-expressed in majority of MM cells. In addition, these data also showed that the expression of other IL-17 family members (IL-7B, C, D, E & F) and Th17-associated pro-inflammatory cytokines (IL-21, IL-22 & IL-23) were not significantly elevated in primary myeloma cells compared to normal donor plasma cells. We further validated these observations by IL-17 immunoblot showing IL17 expression in all MM cell lines and 10 out of 14 primary patient MM cells; confirmed IL-17 expression in MM cells by quantitative RT-PCR, and flow cytometry and by immuno-histochemistry and confocal microscopy. We observed that IL-17 knock down by IL-17-specific siRNA inhibited MM cell growth as well as their ability to induce IL-6 production in co-cultures with BMSC. Finally, expression profile data from 172 uniformly treated patients showed that patients with lower IL-17A expression had superior overall survival compared to those with higher expression. These data confirms that MM cells express IL-17 and targeting it with a mAb will abrogate the autocrine loop making it an attractive therapeutic target. Disclosures:No relevant conflicts of interest to declare.