Mouse Model Of Myeloma Research Articles

Myeloma Derived Mitochondrial Damage Associated Molecular Patterns Promote Pro-Tumoral Expansion By Inducing a Pro-Inflammatory Signature in the Bone Marrow Microenvironment

Multiple Myeloma (MM) is a malignancy of antibody producing B cells (plasma cells), leading to the accumulation of tumour cells within the bone marrow (BM) microenvironment. Mitochondrial DNA (mtDNA) is a damage associated molecular pattern (DAMP), the mitochondrial genome contains islands of unmethylated CpG nucleotide motifs that have been shown to activate and promote memory B cell proliferation and antibody secretion. Recent studies have indicated that mtDNA is elevated in the circulation of trauma and cancer patients and have highlighted the need to determine the functional purpose of elevated mtDNA within the BM microenvironment. Here we investigate the hypothesis that multiple myeloma cells secrete mtDNA and other mtDAMPs to induce a pro-inflammatory bone marrow microenvironment, supportive of the progression and survival of MM. As well as investigating how MM affects the haematopoietic stem cell niche through establishing a syngeneic myeloma mouse model. We show that immunocompromised NSG mice transplanted with human MM cell lines released higher levels of MM derived mtDNA detected in the plasma by real-time PCR. Furthermore, in vitro experiments showed that pro-inflammatory cytokines IL-1B, IL-6, and IL-8 were elevated in both primary and cell line myeloma samples upon treatment with CpG oligonucleotides. Murine bone marrow derived macrophages (BMDMs) treated with mtDNA or mtDAMPs showed increased expression of pro-inflammatory cytokines. To understand the significance of the mtDAMP proinflammatory induction, C57BL/6 mice were successfully engrafted with murine 5TGM1 myeloma cell line to establish a syngeneic mouse model. Analysis of the haematopoietic stem and progenitor cell populations showed that 5TGM1 induced the expansion of these cell populations when compared to control WT animals. To understand the significance of mtDAMPs in regulating this HSPC expansion we used blocking antibodies to TLR9 (receptor for mtDNA) and FPR1 (receptor for formylated mitochondrial proteins) in 5TGM1 engrafted animals. Results show that myeloma tumour burden was reduced when FPR1 and TLR9 are blocked compared to control animals. Here we highlight the involvement of mtDAMPs in MM to create a favourable proinflammatory microenvironment which provides the conditions for MM survival and proliferation. This data highlights the importance of mtDAMPs in MM disease progression and suggests targeting these activating receptor pathways may provide a novel therapeutic intervention. Disclosures Bowles: Janssen: Research Funding; AbbVie: Research Funding. Rushworth:Janssen: Research Funding; AbbVie: Research Funding.

Multiple Myeloma Cell Defects Erythropoiesis and Results Anemia Via High Level of CCL3 in Bone Marrow Microenvironment

Background: Anemia is the most common complication of myeloma, and is associated with worse clinical outcomes including diminished quality of life, and increased risk of morbidity and mortality. Bone marrow (BM) is the main location of the growth and differentiation of HSPCs. Although marrow replacement by myeloma cell is widely considered a mechanistic rationale for myeloma-related anemia, however the molecular mechanism has not been fully understood. Methods: The clinical characteristics and the prognostic impact of myeloma-related anemia were investigated in patients with multiple myeloma. The erythroid differentiation defects of HSPCs were examined by flow cytometry and colony-formation assay. RNA-seq were conducted to clarify the different gene expression and the signaling pathway in HSPCs. Relative protein levels were determined by immunoblotting and relative gene levels were determined by quantitative real-time PCR. 5TGM1 murine myeloma mouse model was utilized. Results: The data of our large cohort of 1,363 myeloma patients demonstrated that 84.4% of patients had anemia (Hgb<120 g/L), more than 50% of patients had moderate (Hgb=90-120 g/L) or severe anemia (Hgb=60-90 g/L) at the time of diagnosis. Anemia positively correlated with myeloma cell infiltration in the BM and worse outcome of patient. Patient sample and mouse model indicated that the quantity and the erythroid differentiation of HSPCs were affected by myeloma cell infiltration. The number of HPCs was significantly declined in the BM of myeloma, and negatively correlated with the quantity of tumor cells. The master regulator of erythropoiesis GATA1 and KLF1, was obviously down-regulated in myeloma HSPCs cells. However, the gene of chemokine CCL3 was significantly upregulated. Elevated CCL3 in the BM plasma of myeloma further inhibited erythropoiesis of HSPCs through activation of CCL3/CCR1/p38 signaling, and suppressed the expression of GATA1. CCR1 antagonist treatment effectively inhibits CCL3 activity and recovered the expression of GATA1 and rescued the erythropoiesis of HSPCs. Conclusions: myeloma cell infiltration causes the elevation of CCL3 in microenvironment that suppresses the erythropoiesis of HSPCs and results in anemia by downregulating the level of GATA1 of HSPCs. Thus, our study indicates targeting CCL3 would be a potential strategy against anemia and improvement the survival of myeloma patient. Disclosures No relevant conflicts of interest to declare.

Anti-BCMA Immunotoxins: Design, Production, and Preclinical Evaluation.

Multiple myeloma (MM) is a B-cell malignancy that is incurable for a majority of patients. B-cell maturation antigen (BCMA) is a lineage-restricted differentiation protein highly expressed in multiple myeloma cells but not in other normal tissues except normal plasma B cells. Due to the restricted expression and being a cell surface membrane protein, BCMA is an ideal target for immunotherapy approaches in MM. Recombinant immunotoxins (RITs) are a novel class of protein therapeutics that are composed of the Fv or Fab portion of an antibody fused to a cytotoxic agent. RITs were produced by expressing plasmids encoding the components of the anti-BCMA RITs in E. coli followed by inclusion body preparation, solubilization, renaturation, and purification by column chromatography. The cytotoxic activity of RITs was tested in vitro by WST-8 assays using BCMA expressing cell lines and on cells isolated from MM patients. The in vivo efficacy of RITs was tested in a xenograft mouse model using BCMA expressing multiple myeloma cell lines. Anti-BCMA recombinant immunotoxins are very effective in killing myeloma cell lines and cells isolated from myeloma patients expressing BCMA. Two mouse models of myeloma showed that the anti-BCMA immunotoxins can produce a long-term complete response and warrant further preclinical development.

Complete Tumor Regression by Liposomal Bortezomib in a Humanized Mouse Model of Multiple Myeloma

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Abstract 5655: Efficacy of antibodies targeting TfR1 in xenograft mouse models of AIDS-related non-Hodgkin lymphoma

Abstract Infection with the human immunodeficiency virus (HIV) and the development of acquired immunodeficiency syndrome (AIDS) increases the risk of developing B-cell lymphomas. AIDS-related B-cell non-Hodgkin lymphoma (AIDS-NHL) remains a significant clinical problem in the era of effective combination anti-retroviral therapy. In fact, AIDS-NHL is currently the most common AIDS-related malignancy in developed countries, where NHL accounts for 23-30% of all AIDS-related deaths. Transferrin receptor 1 (TfR1), also known as CD71, is a type II transmembrane homodimeric protein involved in the cellular uptake of iron and the regulation of cell growth. It is expressed on normal B cells at low levels and is upregulated upon activation. HIV infection leads to the chronic activation of B cells, which results in high expression of TfR1 on these cells, B-cell dysfunction, and ultimately the development of AIDS-NHL. Importantly, TfR1 expression is correlated with stage and prognosis of NHL including AIDS-NHL. Thus, it is a meaningful target for antibody-based NHL therapy. We previously developed a mouse/human chimeric IgG3 specific for human TfR1 (ch128.1/IgG3) and have shown that this antibody administered intraperitoneally exhibits antitumor activity in an in vivo model of AIDS-NHL where NOD-SCID mice were challenged intraperitoneally with 2F7 human Burkitt lymphoma (BL) cells that are positive for the Epstein-Barr Virus (EBV). More recently, we have developed a mouse/human chimeric IgG1 version of ch128.1 that shows significant antitumor activity in various disseminated mouse models of human multiple myeloma (MM), another B-cell malignancy. In the present studies, we sought to further explore in two novel mouse models of human AIDS-NHL the utility of targeting the TfR1 with ch128.1/IgG1 and its recently developed humanized version (hu128.1). To accomplish this goal, we used the 2F7 cell line variant 2F7-BR44, which forms metastases in the brain of mice upon intravenous (i.v.) injection. We also used human JB cells, which are of particular relevance since they are human BL cells that are EBV negative. We found that systemic (i.v.) treatment with ch128.1/IgG1 or hu128.1 of SCID-Beige mice challenged locally (subcutaneously) with 2F7-BR44 or JB tumor cells results in significant antitumor activity against different stages of the disease. Treatment of mice challenged systemically (i.v.) with 2F7-BR44 or JB tumor cells also showed antitumor activity, including long-term survival in some cases. Taken together, our results suggest that targeting TfR1 with antibodies such as ch128.1/IgG1 and hu128.1 has potential as an effective therapy for B-cell malignancies, including MM and AIDS-NHL. Citation Format: Tracy R. Daniels-Wells, Pierre V. Candelaria, Emiko Kranz, Jing Wen, Lan Weng, Masakazu Kamata, Juan Carlos Almagro, Otoniel Martinez-Maza, Manuel L. Penichet. Efficacy of antibodies targeting TfR1 in xenograft mouse models of AIDS-related non-Hodgkin lymphoma [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 5655.

Abstract 4441: CS3003, an HDAC6-selective inhibitor, improves anti-PD-1 immune checkpoint blockade therapy efficacy

Abstract Background: Histone deacetylases (HDACs) are involved in diverse cellular regulatory function including histone modification and non-histone modification. Several publications have demonstrated that selective HDAC inhibitors (HDACi) can influence tumor growth and immunogenicity. In particular, the selective inhibition of HDAC6 has function of inhibiting tumor growth in several malignancies. However, the mechanism of this effect is still not clear. Methods: In this study, we evaluated CS3003, a selective HDAC6 inhibitor, inhibition profile using cell-based free assays. In vivo anti-tumor efficacy was evaluated in MM.1S xenograft mouse model. CS3003 and PD-1 blockade combination effect was evaluated in CT26 syngeneic mouse model. RNA-seq method was used to explore the transcription regulation by CS3003 compared to other pan-HDAC, Class I HDAC inhibitor and selective HDAC6 inhibitors. Results: CS3003 has distinct inhibition profile in HDAC family members. Combination of CS3003 and proteasome inhibitor synergistically enhanced anti-tumor function in human multiple myeloma mouse model (coefficient of drug interaction, CDI=0.69). In addition, CS3003 improves the anti-tumor activity of anti-PD-1 immune checkpoint blockade in solid tumor model (CDI=0.62 and 0.88 when the dosage of CS3003 was 30 and 70mg/kg, respectively). Transcriptome analysis demonstrated a unique gene expression pattern of CS3003 from other pan-HDAC and selective HDAC6 inhibitors. Conclusions: These data provide a comprehensive pre-clinical characterization of CS3003 that supports its unique HDAC inhibition profile and its capability of improving anti-PD-1 immune checkpoint blockade therapy efficacy. Citation Format: Zhenhu Li, Zhaoxiang Ren, Jingshu Ma, Liang Tang, Liang Lu, Ying Zhu, Yunfei Wu, Tiancheng Liu, Juan Zhang, Yuanwu Bao, Kangyu Zhang, Changqing Wei, Shuhui Chen, Xinzhong Jon Wang. CS3003, an HDAC6-selective inhibitor, improves anti-PD-1 immune checkpoint blockade therapy efficacy [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 4441.

Preclinical Characterization of an Antibody-Drug Conjugate Targeting CS-1 and the Identification of Uncharacterized Populations of CS-1-Positive Cells.

Multiple myeloma is a hematologic cancer that disrupts normal bone marrow function and has multiple lines of therapeutic options, but is incurable as patients ultimately relapse. We developed a novel antibody-drug conjugate (ADC) targeting CS-1, a protein that is highly expressed on multiple myeloma tumor cells. The anti-CS-1 mAb specifically bound to cells expressing CS-1 and, when conjugated to a cytotoxic pyrrolobenzodiazepine payload, reduced the viability of multiple myeloma cell lines in vitro In mouse models of multiple myeloma, a single administration of the CS-1 ADC caused durable regressions in disseminated models and complete regression in a subcutaneous model. In an exploratory study in cynomolgus monkeys, the CS-1 ADC demonstrated a half-life of 3 to 6 days; however, no highest nonseverely toxic dose was achieved, as bone marrow toxicity was dose limiting. Bone marrow from dosed monkeys showed reductions in progenitor cells as compared with normal marrow. In vitro cell killing assays demonstrated that the CS-1 ADC substantially reduced the number of progenitor cells in healthy bone marrow, leading us to identify previously unreported CS-1 expression on a small population of progenitor cells in the myeloid-erythroid lineage. This finding suggests that bone marrow toxicity is the result of both on-target and off-target killing by the ADC.

Bovine pestivirus is a new alternative virus for multiple myeloma oncolytic virotherapy

BackgroundThe oncolytic viruses have shown promising results for the treatment of multiple myeloma. However, the use of human viruses is limited by the patients’ antiviral immune response. In this study, we investigated an alternative oncolytic strategy using non-human pathogen viruses as the bovine viral diarrhea virus (BVDV) that were able to interact with CD46.MethodsWe treated several human myeloma cell lines and non-myeloma cell lines with BVDV to evaluate the expression of CD46 and to study the effect on cell viability by flow cytometry. The possible synergistic effect of bortezomib in combination with BVDV was also tested. Moreover, we infected the bone marrow mononuclear cells obtained from myeloma patients and we checked the BVDV effect on different cell populations, defined by CD138, CD14, CD3, CD19, and CD56 expression evaluated by flow cytometry. Finally, the in vivo BVDV effect was tested in NOD-SCID mice injected subcutaneously with myeloma cell lines.ResultsHuman myeloma cells were selectively sensitive to BVDV treatment with an increase of cell death and, consequently, of apoptotic markers. Consistently, bone marrow mononuclear cells isolated from myeloma patients treated with BVDV, showed a significant selective decrease of the percentage of viable CD138+ cells. Interestingly, bortezomib pre-treatment significantly increased the cytotoxic effect of BVDV in myeloma cell lines with a synergistic effect. Finally, the in vitro data were confirmed in an in vivo myeloma mouse model showing that BVDV treatment significantly reduced the tumoral burden compared to the vehicle.ConclusionsOverall, our data indicate, for the first time, a direct oncolytic effect of the BVDV in human myeloma cells suggesting its possible use as novel alternative anti-myeloma virotherapy strategy.

Potent anti-myeloma efficacy of dendritic cell therapy in combination with pomalidomide and programmed death-ligand 1 blockade in a preclinical model of multiple myeloma.

Dendritic cell (DC)-based vaccines are recognized as a promising immunotherapeutic strategy against cancer; however, the efficacy of immunotherapy with DCs is controlled via immune checkpoints, such as programmed death-ligand 1 (PD-L1). PD-L1 expressed on DC and tumor cells binds to programmed death-1 (PD-1) receptors on the activated T cells, which leads to the inhibition of cytotoxic T cells. Blocking of PD-L1 on DC may lead to improve the efficacy of DC therapy for cancer. Here we demonstrated that DC vaccination in combination with pomalidomide and programmed death-ligand 1 (PD-L1) blockade inhibited tumor growth of a multiple myeloma (MM) mouse model. DCs + pomalidomide with dexamethasone + PD-L1 blockade significantly inhibited immune immunosuppressive factors and promoted proportions of immune effector cells in the spleen and tumor microenvironment. Additionally, functional activities of cytotoxic T lymphocytes and NK cells in spleen were enhanced by DCs + pomalidomide with dexamethasone + PD-L1 blockade. Taken together, this study identifies a potential new therapeutic approach for the treatment of MM. These results also provide a foundation for the future development of immunotherapeutic modalities to inhibit tumor growth and restore immune function in MM.

SR18292 exerts potent antitumor effects in multiple myeloma via inhibition of oxidative phosphorylation

AimsMultiple myeloma (MM) was recently reported to rely on increased oxidative phosphorylation (OXPHOS) for survival, providing a potential opportunity for MM therapy. Herein, we aimed to propose a novel targeted drug for MM treatment, followed by the exploration of reason for OXPHOS enhancement in MM cells. Materials and methodsThe expression of OXPHOS genes and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) was analyzed using bioinformatics analyses, followed by verification in MM cell lines. The effects of SR18292 on OXPHOS were measured by qRT-PCR, Western blot, transmission electron microscopy, oxygen consumption rate and so on. The proliferation and apoptosis were evaluated by CCK-8, flow cytometry and Western blot. The efficiency and safety of SR18292 were assessed in a mouse model of MM. Key findingsThe OXPHOS genes were generally overexpressed in MM cells, which was associated with poorer prognosis of MM patients. PGC-1α, a transcriptional coactivator, was upregulated in MM cells, and MM patients with higher PGC-1α expression exhibited increased enrichment of the OXPHOS gene set. Treatment with SR18292 (an inhibitor of PGC-1α) significantly impaired the proliferation and survival of MM cells due to OXPHOS metabolism dysfunction, which leads to energy exhaustion and oxidative damage. Besides, SR18292 potently inhibited tumor growth at a well-tolerated dose in MM model mice. SignificanceThe overexpression of OXPHOS gene set mediated by upregulated PGC-1α provides a structural basis for enhanced OXPHOS in MM cells, and SR18292 (a PGC-1α inhibitor) exerts potent antimyeloma effects, offering a potential tangible avenue for MM therapy.

Runx2 Deficiency in Osteoblasts Promotes Myeloma Progression by Altering the Bone Microenvironment at New Bone Sites.

Multiple myeloma is a plasma cell malignancy that thrives in the bone marrow (BM), with frequent progression to new local and distant bone sites. Our previous studies demonstrated that multiple myeloma cells at primary sites secrete soluble factors and suppress osteoblastogenesis via the inhibition of Runt-related transcription factor 2 (Runx2) in pre- and immature osteoblasts (OB) in new bone sites, prior to the arrival of metastatic tumor cells. However, it is unknown whether OB-Runx2 suppression in new bone sites feeds back to promote multiple myeloma dissemination to and progression in these areas. Hence, we developed a syngeneic mouse model of multiple myeloma in which Runx2 is specifically deleted in the immature OBs of C57BL6/KaLwRij mice (OB-Runx2-/- mice) to study the effect of OB-Runx2 deficiency on multiple myeloma progression in new bone sites. In vivo studies with this model demonstrated that OB-Runx2 deficiency attracts multiple myeloma cells and promotes multiple myeloma tumor growth in bone. Mechanistic studies further revealed that OB-Runx2 deficiency induces an immunosuppressive microenvironment in BM that is marked by an increase in the concentration and activation of myeloid-derived suppressor cells (MDSC) and the suppression and exhaustion of cytotoxic CD8+ T cells. In contrast, MDSC depletion by either gemcitabine or 5-fluorouracil treatment in OB-Runx2-/- mice prevented these effects and inhibited multiple myeloma tumor growth in BM. These novel discoveries demonstrate that OB-Runx2 deficiency in new bone sites promotes multiple myeloma dissemination and progression by increasing metastatic cytokines and MDSCs in BM and inhibiting BM immunity. Importantly, MDSC depletion can block multiple myeloma progression promoted by OB-Runx2 deficiency.Significance: This study demonstrates that Runx2 deficiency in immature osteoblasts at distant bone sites attracts myeloma cells and allows myeloma progression in new bone sites via OB-secreted metastatic cytokines and MDSC-mediated suppression of bone marrow immunity.

CAR T Cells Redirected to CD44v6 Control Tumor Growth in Lung and Ovary Adenocarcinoma Bearing Mice.

The main challenge of adoptive therapy with Chimeric Antigen Receptor modified T cells (CAR T) is the application to the field of solid tumors, where the identification of a proper antigen has emerged as one of the major drawbacks to CAR T cell treatment success. CD44 is a glycoprotein involved in cell-cell and cell-matrix interactions. The isoform containing the variant domain 6 of CD44 gene (CD44v6) has been implicated in tumorigenesis, tumor cell invasion and metastasis and represents an attractive target for CAR T cell therapies. Targeting CD44v6 antigen has been shown to control tumor growth in acute myeloid leukemia and multiple myeloma mouse models. While CAR T approach for the treatment of B cell malignancies has shown great success, response rates among patients with solid cancer are less favorable. The purpose of our study was to test the efficacy of CD44v6.CAR T cells, produced in compliance with Good Manufacturing Practice (GMP), in adenocarcinoma tumor models. We generated a bicistronic retroviral vector containing the CD44v6 CAR and the HSV-TK Mut2 suicide gene to enhance the safety of the proposed CAR T cell therapy. CD44v6 transduced CAR T cells were homogeneously positive for ΔLNGFR selection marker, were enriched in T central memory (TCM) and T memory stem cells (TSCM) and displayed a highly activated phenotype. In vitro assays revealed antigen-specific activation and cytotoxicity of human CD44v6.CAR T cells against CD44v6 expressing tumor cell lines. When infused in immunodeficient tumor bearing mice, human CD44v6.CAR T cells were able to reach, infiltrate and proliferate at tumor sites, finally resulting in tumor growth control. Next, we checked if cells produced in compliance with GMP grade standards retained the same antitumor activity of those produced with research grade materials and protocols. Noteworthy, no differences in the potency of the CAR T obtained with the two manufacturing processes were observed. In conclusion, our preclinical results suggest that CD44v6.CAR T based adoptive therapy could be a promising strategy in solid cancer treatment.

PINK1-Dependent Mitophagy Regulates the Migration and Homing of Multiple Myeloma Cells via the MOB1B-Mediated Hippo-YAP/TAZ Pathway.

The roles of mitochondrial dysfunction in carcinogenesis remain largely unknown. The effects of PTEN‐induced putative kinase 1 (PINK1)‐dependent mitophagy on the pathogenesis of multiple myeloma (MM) are determined. The levels of the PINK1‐dependent mitophagy markers PINK1 and parkin RBR E3 ubiquitin protein ligase (PARK2) in CD138+ plasma cells are reduced in patients with MM and correlate with clinical outcomes in myeloma patients. Moreover, the induction of PINK1‐dependent mitophagy with carbonylcyanide‐m‐chlorophenylhydrazone (CCCP) or salinomycin, or overexpression of PINK1 leads to inhibition of transwell migration, suppression of myeloma cell homing to calvarium, and decreased osteolytic bone lesions. Furthermore, genetic deletion of pink1 accelerates myeloma development in a spontaneous X‐box binding protein‐1 spliced isoform (XBP‐1s) transgenic myeloma mouse model and in VK*MYC transplantable myeloma recipient mice. Additionally, treatment with salinomycin shows significant antimyeloma activities in vivo in murine myeloma xenograft models. Finally, the effects of PINK1‐dependent mitophagy on myeloma pathogenesis are driven by the activation of the Mps one binder kinase activator (MOB1B)‐mediated Hippo pathway and the subsequent downregulation of Yes‐associated protein (YAP)/transcriptional co‐activator with PDZ‐binding motif (TAZ) expression. These data provide direct evidence that PINK1‐dependent mitophagy plays a critical role in the pathogenesis of MM and is a potential therapeutic target.

Selective Antisense Oligonucleotide Inhibition of Human IRF4 Prevents Malignant Myeloma Regeneration Via Cell Cycle Disruption

In multiple myeloma, pro-inflammatory factors such as interleukin-6 signaling and downstream anti-viral response pathway activation have been correlated with disease progression, symptom severity, and cancer stem cell generation. However, the role of interferon-response factors in the maintenance of malignant progenitors has not been explored. We hypothesized that the key B-cell progenitor fate determinant, interferon-response factor-4 (IRF4), governs regeneration of drug-resistant malignant progenitors in multiple myeloma. Using diverse pre-clinical models, including a humanized in vivo model that recapitulates IRF4 pathway activation detected by RNA-sequencing in primary high-risk samples, we tested whether direct targeting of human IRF4 using state-of-the-art antisense oligonucleotide (ASO) agents could impair myeloma regeneration. While human IRF4 overexpression expanded a putative progenitor population in vitro, IRF4 ASO treatment impaired myeloma cell survival commensurate with reduced IRF4 and MYC gene and protein expression. In addition to impeding subcutaneous tumor formation and myeloma dissemination in xenograft immunocompromised mouse models, human IRF4-targeted ASO monotherapy significantly improved overall animal survival. Moreover, in a patient-derived mouse model of high-risk myeloma, treatment with a lead IRF4 ASO that will advance to a Phase 1 trial (NCT04398485) eradicated CD138-negative MM progenitors and their malignant plasma cell progeny at doses that spared normal human hematopoietic stem cells and B cell development. Complementary mechanistic studies uncovered a role for IRF4 inhibition in disrupting microenvironment-responsive cell cycle progression, down-regulating stem cell regulatory and cell adhesion transcript expression including MYC and CXCR4, and promoting sensitivity to standard-of-care therapeutic agents. These comprehensive therapeutic index and proof-of-concept studies will enable rapid clinical development of IRF4 inhibition to prevent myeloma relapse driven by retention of dormant, drug-resistant progenitors in inflammatory microenvironments.

Differential Pain-Related Behaviors and Bone Disease in Immunocompetent Mouse Models of Myeloma.

ABSTRACTBone pain is a serious and debilitating symptom of multiple myeloma (MM) that impairs the quality of life of patients. The underlying mechanisms of the pain are unknown and understudied, and there is a need for immunocompetent preclinical models of myeloma‐induced bone pain. The aim of this study was to provide the first in‐depth behavioral characterization of an immunocompetent mouse model of MM presenting the clinical disease features: osteolytic bone disease and bone pain. We hypothesized that a widely used syngeneic model of MM, established by systemic inoculation of green fluorescent protein‐tagged myeloma cells (5TGM1‐GFP) in immunocompetent C57Bl/KaLwRijHsd (BKAL) mice, would present pain‐related behaviors. Disease phenotype was confirmed by splenomegaly, high serum paraprotein, and tumor infiltration in the bone marrow of the hind limbs; however, myeloma‐bearing mice did not present pain‐related behaviors or substantial bone disease. Thus, we investigated an alternative model in which 5TGM1‐GFP cells were directly inoculated into the intrafemoral medullary cavity. This localized myeloma model presented the hallmarks of the disease, including high serum paraprotein, tumor growth, and osteolytic bone lesions. Compared with control mice, myeloma‐bearing mice presented myeloma‐induced pain‐related behaviors, a phenotype that was reversed by systemic morphine treatment. Micro‐computed tomography analyses of the myeloma‐inoculated femurs showed bone disease in cortical and trabecular bone. Repeated systemic bisphosphonate treatment induced an amelioration of the nociceptive phenotype, but did not completely reverse it. Furthermore, intrafemorally injected mice presented a profound denervation of the myeloma‐bearing bones, a previously unknown feature of the disease. This study reports the intrafemoral inoculation of 5TGM1‐GFP cells as a robust immunocompetent model of myeloma‐induced bone pain, with consistent bone loss. Moreover, the data suggest that myeloma‐induced bone pain is caused by a combinatorial mechanism including osteolysis and bone marrow denervation. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

A Combination Therapy with Dendritic Cells, Pomalidomide and Programmed Death-Ligand 1 Blockade Exerts a Potent Antitumor Immunity in a Murine Model of Multiple Myeloma

Multiple myeloma (MM) is the second-most-common hematologic malignancy, and develops from clonal malignant plasma cells within bone marrow. Despite tremendous improvements in therapeutic strategies (e.g. stem cell transplantation, immune-modulatory drugs (IMiDs), proteasome inhibitors, and, more recently, immunotherapy), which have led to improved responses to treatment and overall survival, most patients eventually relapse. We have previously shown that the immunization with tumor antigen-loaded dendritic cells (DCs) and pomalidomide/dexamethasone synergistically potentiates the enhancing the antitumor immunity in a myeloma mouse model. In the present study, we investigated whether a DC-based vaccine combined with pomalidomide and PD-L1 blockade has a synergistic effect in a murine MM model. MOPC-315 cell lines were injected subcutaneously to establish MM-bearing mice. Four test groups were used to mimic the clinical protocol: (1) PBS control, (2) DCs + pomalidomide/dexamethasone, (3) pomalidomide/dexamethasone + PD-L1 blockade, and (4) DCs + pomalidomide/dexamethasone + PD-L1 blockade. After treatment, preclinical response and in vitro immunological responses were evaluated. The study was designed to closely mimic the clinical MM treatment protocol and clearly demonstrated that combination treatment with DCs + pomalidomide with dexamethasone + PD-L1 blockade more strongly inhibited MM tumor growth. Consequently, the mice treated with DCs + pomalidomide with dexamethasone + PD-L1 blockade displayed markedly induced tumor regression and significantly prolonged survival, as well as very strong anti-myeloma CTL responses and increased numbers of effector cells (such as CD4+ T cells, CD8+ T cells, memory T cells, NK cells and M1 macrophages) associated with antitumor effects. This treatment also effectively decreased the proportions of suppressor cells, including MDSCs, Tregs and M2 macrophages, in the spleen and tumor microenvironment of treated mice. Tregs, MDSCs and M2 macrophages play crucial roles in immunosuppression and tolerance, which are mediated by tumor-secreted cytokines. The inhibition of Tregs, MDSC and M2 macrophage accumulation may enhance systemic cell-mediated immunity through the activation of DCs or CTLs. Importantly, treatment with pomalidomide with dexamethasone + PD-L1 blockade led to decreased expression of PD-L1 and CTLA-4 in treated mice, which further induced effector cell infiltration of the tumor microenvironment. Moreover, DCs + pomalidomide with dexamethasone + PD-L1 blockade induced the activation of cell-mediated immunity by increasing Th1-specific immune responses, as evidenced by the increased production of IFN-γ and a decrease in the regulatory-specific immune response, as evidenced by the decreased production of TGF-β, IL-10 and VEGF in the spleen and tumor microenvironment. These findings show that inducing the systemic immune response represent a means of treating myeloma. Immunotherapy clearly represents a revolution in cancer care, and promising responses have been shown to various treatments, particularly immune checkpoint inhibitors, IMiDs, DCs and CAR T cells. However, not all patients are responsive to current immunotherapies, and among those patients who do respond, the effects are not always long-lasting. Thus, combination approaches are a cornerstone of cancer therapy for improving patient outcomes in MM. This study demonstrated that the combination of DC vaccination + pomalidomide with dexamethasone + PD-L1 blockade synergistically enhances myeloma immune responses to inhibit tumor growth, restores and enhances host immune effector cells, and reduces the generation of immune suppressor cells in MM. This study provides a framework for developing and understanding the role of immunotherapeutic modalities employing DCs, pomalidomide and PD-L1 blockade to inhibit tumor growth and restore immune function in myeloma-bearing mice. Figure Disclosures No relevant conflicts of interest to declare.

High Level of CCL3 in the Bone Marrow Microenvironment Promotes Anemia By Suppressing the Erythropoiesis Differentiation of Hematopoietic Stem Cells in Myeloma

Background and aims: Multiple myeloma (MM) is a hematologic malignancy characterized by monoclonal plasma cells infiltrating the bone marrow. Anemia is the most common symptom and notably characteristic of myeloma patient. The mechanism of anemia of chronic disease (ACD) is impaired underlies that of MM-related anemia. However, the mechanisms underlying myeloma related anemia have not been fully elucidated. Clarify the pathogenesis of myeloma related anemia and identify the key molecular foundation will be helpful to develop the target treatment and reverse the inferior outcome of those patients. Methods: The correlation of tumor burden and the level of HGB was analyzed in the cohort of 776 newly diagnosed myeloma patients. The number and the differentiation activity of HSPCs under the MM microenvironment were analyzed using both primary myeloma patient samples and NSG myeloma mouse model. The HSPC of bone marrow was isolated by the sorting of CD34 positive cells. Colony- Forming Cell (CFC) assay was performed to identify the differentiation activity of HSPC derived from myeloma patient and healthy donor. Furthermore, 5TGM1 myeloma mouse model was utilized to verify the effects of myeloma cells on the differentiation activity of HSPCs during MM progression in vivo. Results: Our clinical analysis showed that more than 80% (620/776) myeloma patients had anemia (HGB< 120g/L) and more than 60% patients had moderate or severe anemia (HGB< 90g/L) at newly diagnosis . The degree of anemia was positively correlated with the tumor burden of CD138+ cells and also highly positive correlated with bone lesions in myeloma patients (p<0.001). Moreover, the higher level of CD138+ cells was significantly negative correlated with the number of CD34+ HSPCs in the bone marrow (r=-0.526, p=0.0082). The flow cytometry data indicated that the percentage of HSPCs population in NDMM patients was significantly lower than that of normal controls (P<0.001). We did not find the decrease of HSC (Lin-CD34+CD38-) in MM bone marrow compared with healthy control. However, the population of HPC (Lin-CD34+CD38+) significantly decreased (P<0.001). The proportion of megakaryocyte-erythrocyte progenitors (MEP) was notably decreased (p<0.0001) in MM patient samples. On the contrary, the proportion of granulocyte-macrophage progenitors (GMP) was increased in MM patients compared to healthy donors (p<0.01). These data suggested that the myeloma bone marrow microenvironment suppressed the CD34+ HPSC proliferation and differentiation to MEP which may contribute the myeloma related anemia. 5TGM1 mouse model of myeloma was used to verify these clinic findings. The number of HSCs (LKS+) and HPCs (LKS-) during MM cell progression was monitored at different time point by flow cytometry after 5TGM1 MM cells injection. The results indicated clearly that the HSCs (LKS+) and HPCs (LKS-) were suppressed in the myeloma bone marrow microenvironment. Similar to MM patient, the proportion of MEP was the most significantly suppressed population among all of the hematopoietic subsets in this MM mouse model. CFC assays indicated that the BFU-E colony formation of CD34+ cells from MM patients was significantly suppressed (p<0.01). RNA sequencing analysis indicated that the transcription factor in erythropoiesis differentiation, GATA1 and KLF1, was obviously decreased in myeloma CD34+ cells compared with that in healthy donor. Further investigated showed that high level of CCL3 in the bone marrow microenvironment suppressed the differentiation of erythropoiesis which played a pivotal role in myeloma related anemia. Administration of the CCL3 receptor (CCR1) antagonist partially recovered the yield of erythroid colonies in the presence of myeloma plasma. Conclusion: Bone marrow microenvironment of multiple myeloma suppresses the erythropoiesis differentiation of hematopoietic stem cells and promotes the myeloma associated anemia. High level of CCL3 may down regulate GATA1 and KLF1 and be one of the important reasons for myeloma-related anemia. Disclosures No relevant conflicts of interest to declare.

A CK1δ/CK1ε Regulated Metabolic Circuit Is a Therapeutic Vulnerability for Multiple Myeloma

Multiple Myeloma (MM) remains an incurable malignancy, despite the advent of several new therapeutic agents, including immunomodulatory drugs (IMiDs, e.g., Lenalidomide (Len)) and proteasome inhibitors (PIs, e.g., Bortezomib (Btz)). Accordingly, there is an urgent need to identify new targetable vulnerabilities for MM patients. We developed an ex vivo 384-well platform that allows one to define drug sensitivities of primary patient CD138+ MM cells in the context of a reconstructed tumor microenvironment (TME), including allogeneic bone marrow stromal cells, extracellular matrix and MM patient serum. Using this platform and activity-based proteomic profiling (ABPP), we identified shared signaling pathways induced by the interactions of MM with stromal cells and integrated these data with screens performed using a bank of protein kinase inhibitors (PKI) and current anti-MM therapeutics. These analyses revealed that the serine/threonine kinases casein kinase-1δ (CK1δ) and CK1ε as high priority targets for MM. Indeed, a highly selective and potent dual inhibitor of CK1δ/CK1ε coined SR-3029 is the most potent PKI versus MM. Further, our studies revealed SR-3029 has potent activity in 138/153 primary patient MM specimens tested thus far, including quad and penta-refractory MM. Analysis of RNAseq data of over 600 Moffitt Cancer Center (MCC) MM patients revealed that patients with high expression of CK1ε had worse survival outcomes while no survival difference was seen with CK1δ expression. Importantly, using the established 5TGM1/Kal-Ridge (C57B6/KaLwRijHsd) syngeneic mouse model of multiple myeloma, we show that tumors derived from 5TGM1 MM cells, which rapidly die following exposure to SR-3029 ex vivo, are also sensitive to CK1δ/CK1ε inhibition in vivo, where SR-3029 treatment reduced tumor burden and significantly improved survival. Similar results were observed using NSG immune compromised animals inoculated with human MM1.S multiple myeloma cells (both flank and tail vein models), where SR-3029 treated animals had reduced tumor burden and extended survival. Analysis of RNAseq on patients' samples (on stroma) treated ex vivo with SR-3029 revealed CK1δ/CK1ε inhibition suppressed multiple metabolic pathways (oxidative phosphorylation, glycolysis, xenobiotic metabolism). Interestingly, analyses of MCC MM patient RNAseq data revealed upregulation of the genes identified in these metabolic pathways as patients progress from pre-treatment to relapse, and that patient MM samples that were resistant to CK1δ/CK1ε inhibition had an upregulation of some of these metabolic genes. Functional studies are being performed to define the mechanism(s) by which CK1δ/CK1ε inhibition disables MM metabolism. Collectively, these findings establish CK1ε and/or CK1δ as attractive targets for anti-myeloma therapy that are required to sustain MM metabolism. Disclosures Dai: M2Gen: Employment. Shain:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Sanofi Genzyme: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

γ-Secretase Inhibitors Improve Multiple Myeloma BCMA CAR-T Therapy

γ-Secretase inhibitors with BCMA CAR-T therapy improved survival in a multiple myeloma mouse model.

The bone marrow microenvironment of multiple myeloma promotes myeloma related anemia by suppressing the differentiation of hematopoietic stem cells

Multiple myeloma (MM) is a hematologic malignancy characterized by monoclonal plasma cells infiltrating the bone marrow thereby causing anemia and lytic bone lesions. MM patients always accompanied with severe anemia, which may significantly contribute to patient mortality. However, the mechanisms underlying myeloma related anemia have not been fully elucidated. We hypothesized that the MM microenvironment plays a pivotal role on their hematopoiesis differentiation. In the present study, the number and the function of HSPCs in the MM microenvironment were analyzed by MM patient primary samples and myeloma mouse model.