Mouse Model Of Multiple Myeloma Research Articles

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.

Abstract PR02: Glyphosate as a potential carcinogen for multiple myeloma

Abstract Glyphosate is the most commercially successful and the most widely used herbicide in the United States and worldwide. It represents about 50% of United States herbicide usage at approximately 250 million pounds annually; the annual use of glyphosate worldwide is about 1.8 billion pounds. There has been considerable debate around the carcinogenicity of glyphosate. IARC labeled glyphosate as “probably carcinogenic to humans.” However, other agencies like the EPA have maintained that glyphosate is unlikely to pose a carcinogenic risk. In epidemiologic studies on the association of cancer and glyphosate, only multiple myeloma (MM) and non-Hodgkin lymphoma (NHL) have a positive and statistically significant association with glyphosate. MM is a plasma cell neoplasm and the second most common hematologic malignancy in the United States. Virtually all MM cases are preceded by a precursor condition, monoclonal gammopathy of undetermined significance (MGUS). MM is regarded as a disease with environmental causes, yet its etiology and what prompts progression remain largely unclear. We employed the Vk*Myc mouse model of MM to assess the impact of glyphosate exposure on MM pathogenesis. In both wild-type and Vk*Myc mice, glyphosate induced splenomegaly and multiple peripheral blood abnormalities, including anemia and high serum IgG levels, hallmarks of MGUS and MM in humans. In Vk*Myc mice, glyphosate triggered bone lytic lesions, as well as renal damage, a phenomenon associated with human MM kidney disease. Most importantly, glyphosate upregulated Aicda, which encodes the activation-induced cytidine deaminase (AID) in the spleen and bone marrow of both wild-type and Vk*MYC mice. AID mediates somatic hypermutation and class-switch recombination of immunoglobulin genes in B cells to produce diverse immunoglobins in secretive forms. Our data indicate that glyphosate accelerates MGUS development and promotes progression to MM in genetically predisposed mice. Given that AID dysregulation is a key pathogenic driver in both MM and NHL, this work offers the first direct experimental evidence establishing glyphosate as an environmental risk factor for MM and NHL. This abstract is also being presented as Poster A50. Citation Format: Yong Li. Glyphosate as a potential carcinogen for multiple myeloma [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr PR02.

Osteotropic Radiolabeled Nanophotosensitizer for Imaging and Treating Multiple Myeloma.

Rapid liver and spleen opsonization of systemically administered nanoparticles (NPs) for in vivo applications remains the Achilles' heel of nanomedicine, allowing only a small fraction of the materials to reach the intended target tissue. Although focusing on diseases that reside in the natural disposal organs for nanoparticles is a viable option, it limits the plurality of lesions that could benefit from nanomedical interventions. Here we designed a theranostic nanoplatform consisting of reactive oxygen (ROS)-generating titanium dioxide (TiO2) NPs, coated with a tumor-targeting agent, transferrin (Tf), and radiolabeled with a radionuclide (89Zr) for targeting bone marrow, imaging the distribution of the NPs, and stimulating ROS generation for cell killing. Radiolabeling of TiO2 NPs with 89Zr afforded thermodynamically and kinetically stable chelate-free 89Zr-TiO2-Tf NPs without altering the NP morphology. Treatment of multiple myeloma (MM) cells, a disease of plasma cells originating in the bone marrow, with 89Zr-TiO2-Tf generated cytotoxic ROS to induce cancer cell killing via the apoptosis pathway. Positron emission tomography/X-ray computed tomography (PET/CT) imaging and tissue biodistribution studies revealed that in vivo administration of 89Zr-TiO2-Tf in mice leveraged the osteotropic effect of 89Zr to selectively localize about 70% of the injected radioactivity in mouse bone tissue. A combination of small-animal PET/CT imaging of NP distribution and bioluminescence imaging of cancer progression showed that a single-dose 89Zr-TiO2-Tf treatment in a disseminated MM mouse model completely inhibited cancer growth at euthanasia of untreated mice and at least doubled the survival of treated mice. Treatment of the mice with cold Zr-TiO2-Tf, 89Zr-oxalate, or 89Zr-Tf had no therapeutic benefit compared to untreated controls. This study reveals an effective radionuclide sensitizing nanophototherapy paradigm for the treatment of MM and possibly other bone-associated malignancies.

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.

Monosomic loss of MIR15A/MIR16-1 is a driver of multiple myeloma proliferation and disease progression.

The most common genetic abnormality in multiple myeloma (MM) is the deletion of chromosome 13, seen in almost half of newly diagnosed patients. Unlike chronic lymphocytic leukemia, where a recurrent minimally deleted region including MIR15A/MIR16-1 has been mapped, the deletions in MM predominantly involve the entire chromosome and no specific driver gene has been identified. Additional candidate loci include RB1 and DIS3, but while biallelic deletion of RB1 is associated with disease progression, DIS3 is a common essential gene and complete inactivation is not observed. The Vk*MYC transgenic mouse model of MM spontaneously acquires del(14), syntenic to human chromosome 13, and Rb1 complete inactivation, but not Dis3 mutations. Taking advantage of this model, we explored the role in MM initiation and progression of two candidate loci on chromosome 13: RB1 and MIR15A/MIR16-1. Monoallelic deletion of Mir15a/Mir16-1 but not Rb1 was sufficient to accelerate the development of monoclonal gammopathy in wildtype mice, and the progression of MM in Vk*MYC mice, resulting in increased expression of Mir15a/Mir16-1 target genes and plasma cell proliferation, which was similarly observed in patients with MM.

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.

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.

Tuning isoform selectivity and bortezomib sensitivity with a new class of alkenyl indene PDI inhibitor

Protein disulfide isomerase (PDI, PDIA1) is an emerging therapeutic target in oncology. PDI inhibitors have demonstrated a unique propensity to selectively induce apoptosis in cancer cells and overcome resistance to existing therapies, although drug candidates have not yet progressed to the stage of clinical development. We recently reported the discovery of lead indene compound E64FC26 as a potent pan-PDI inhibitor that enhances the cytotoxic effects of proteasome inhibitors in panels of Multiple Myeloma (MM) cells and MM mouse models. An extensive medicinal chemistry program has led to the generation of a diverse library of indene-containing molecules with varying degrees of proteasome inhibitor potentiating activity. These compounds were generated by a novel nucleophilic aromatic ring cyclization and dehydration reaction from the precursor ketones. The results provide detailed structure activity relationships (SAR) around this indene pharmacophore and show a high degree of correlation between potency of PDI inhibition and bortezomib (Btz) potentiation in MM cells. Inhibition of PDI leads to ER and oxidative stress characterized by the accumulation of misfolded poly-ubiquitinated proteins and the induction of UPR biomarkers ATF4, CHOP, and Nrf2. This work characterizes the synthesis and SAR of a new chemical class and further validates PDI as a therapeutic target in MM as a single agent and in combination with proteasome inhibitors.

Long-term survival without graft-versus-host-disease following infusion of allogeneic myeloma-specific V\u03b2 T cell families

BackgroundDespite chemo-induction therapy and autologous stem cell transplantation (ASCT), the vast majority of patients with Multiple Myeloma (MM) relapse within 7 years and the disease remains incurable. Adoptive Allogeneic T-cell therapy (ATCT) might be curative for MM, however current ATCT protocols often lead to graft versus host disease (GvHD). Transplanting only tumor reactive donor T cells that mediate a graft-versus-myeloma (GvM) but not GvHD may overcome this problem.MethodsWe used an MHC-matched/miHA-disparate B10.D2 → Balb/c bone marrow transplantation (BMT) murine model and MOPC315.BM MM cells to develop an ATCT protocol consisting of total body irradiation, autologous-BMT and infusion of selective, myeloma-reactive lymphocytes of T cell receptor (TCR) Vβ 2, 3 and 8.3 families (MM-auto BMT ATCT).ResultsPre-stimulation ex vivo of allogeneic T cells by exposure to MOPC315.BM MM cells in the presence of IL-2, anti-CD3 and anti-CD28 resulted in expansion of the myeloma-reactive T cell TCRVβ 2, 3 and 8.3 subfamilies. Their isolation and infusion into MM-bearing mice resulted in a vigorous GvM response without induction GvHD and long-term survival. Repeated infusion of naïve myeloma-reactive T cell TCRVβ 2, 3 and 8.3 subfamilies was also effective.ConclusionsThese data demonstrate that a transplantation protocol involving only selective tumor-reactive donor T cell families is an effective immunotherapy and results in long-term survival in a mouse model of human MM. The results highlight the need to develop similar ATCT strategies for MM patients that result in enhanced survival without symptoms of GvHD.

NEK2 Promotes Bortezomib Resistance through Enhancing Autophagy By Upregulation of Beclin1 in Multiple Myeloma Cells

Background: Multiple Myeloma(MM) is a neoplastic plasma-cell disorder that is characterized by clonal proliferation of malignant of plasma cells in the bone microenvironment, and monoclonal protein in the blood or urine. Drug resistance is one of the main causes of cancer relapse, thus exploring the molecular mechanisms of drug resistance is important for improving therapeutic effect in MM. Our previous publications have demonstrated NEK2 mediated drug resistance via up-regulation of multidrug resistance proteins. In this study, we found autophagy was involved in NEK2 induced Bortezomib resistance in MM cells. Materials and Methods: Primary CD138 positive cells derived from healthy donors(n=6), newly diagnosed MM patients(n=9) and relapsed MM patients(n=7) were isolated by using beads conjugated with human CD138 antibody. Immunofluorescence was performed to detect the expression of NEK2 and LC3B(LC3B-Ⅰand LC3B-Ⅱ) in primary CD138 positive cells. DALGreen was used to detect autolysosome in MM cells. TAP-MS and CO-IP were used to analyze NEK2 interacting proteins. Cell proliferation were examined with soft agar colony formation and cell count. Cell apoptosis was tested through detecting cleaved Caspase3 and cleaved PARP by western blot. Xenograft mouse model of MM were prepared by subcutaneous injection of MM cells(KMS11 NEK2 OE+Scramble, KMS11 NEK2 OE+Beclin1 sh) into immunodeficient B-NDG mouse(1×106 cell/mice). Results: Firstly, immunofluorescence results showed NEK2 expression and LC3B-Ⅱ labeled autophagosome were significantly increased in CD138 positive cells derived from relapsed MM patients as compared with newly diagnosed MM patients and HD. Moreover, over-expression of NEK2 enhanced antophagy, while knockdown of NEK2 suppressed autophagy in MM cells. To explore the underling mechanisms of NEK2 induces autophagy in MM cells, TAP-MS was performed. As a result, Beclin1, an important regulatory protein of autophagy, was identified as NEK2 interacting protein, which was further confirmed by CO-IP in MM cell lines KMS11 and RPMI 8226. Additionally, we found that NEK2 regulated the expression of Beclin1 at protein level, but not at transcriptional level. Subsequent mechanism study indicated NEK2 increased the stability of Beclin1 protein through USP7 mediated deubiquitination, thereby promoting the formation of the Beclin1-Vps15-Vps34 complex, and finally, enhancing autophagy in MM cells. Furthermore, knockdown of Beclin1 significantly suppressed NEK2 induced autophagy in MM cells. Alternatively, down-regulation of Beclin1 overcame NEK2 mediated Bortezomib resistance in xenograft mouse model of MM , suggesting that targeting Beclin1 is a promising approach to therapy MM patients with high NEK2 expression. Conclusion: Our findings revealed NEK2 induced autophagy through up-regulation of Beclin1 at protein level, and confirmed autophagy was involved in NEK2 mediated Bortezomib resistance in MM. Taken together, this study provided novel insight into treating relapsed MM patients. Disclosures No relevant conflicts of interest to declare.

Development of an Easily Accessible Patient-Derived Xenograft (PDX) Mouse Model in Multiple Myeloma

Preclinical mouse models are important tools to recapitulate human multiple myeloma (MM) disease. Different preclinical models allow for specific hypothesis-driven research and enables researchers to address multiple questions. Though the SCID-Hu and SCID-synth-hu mice, and a recently established humanized mouse model containing the knock-in of human cytokine genes permit the growth of primary pre-neoplastic and malignant plasma cells, the high-cost, long-term workflow, lack of access to genetically engineered mice are overwhelming disadvantages of these current humanized MM mouse models. Our objective is to establish a unique patient-derived-xenograft (PDX) MM mouse model as an easily accessible approach for prevention and therapy of human MM disease. Bone marrow aspirates from MM patients upon diagnosis were obtained from the Multiple Myeloma Molecular Epidemiology Resource (University of Iowa) and mononuclear cells were isolated. Groups of 7-8 weeks old NOD/SCID/IL2RΥgnull (NSG) mice were administrated with sub-lethal irradiation. 3-5×106 unsorted MM patient-derived bone marrow mononuclear cells were intravenously injected into each recipient NSG mouse after irradiation. In order to monitor engraftment, recipient mice were bled weekly from week 2 after inoculation and serial Serum Protein Electrophoresis (SPEP) tests of recipient mice were performed. Detection of distinct M-protein band by the SPEP test with weight loss and/or limited mobility of injected recipient mice were indicative of successful MM engraftment and the endpoint of this study. M protein was found in all 30 mice after 3 ~ 5 weeks of injection human MM mononuclear cells. To further confirm that the M protein was secreted from human MM cells, we performed flow cytometry to determine human MM cells using anti-human CD138 antibody from mouse tissues. About 10% human CD138+ MM cells were detected in spleen and bone marrow from these PDX-NSG mice by flow cytometry, whereas human CD138+ cells were absent in irradiated control mice without injection of human MM cells. We also performed immunohistochemistry on bone marrow sections of PDX-NSG mice. Human CD138 protein and human light chain protein were positively stained on these samples. We next examined MM related organ damage, which is part of the defining criteria of human MM disease. Elevated blood urea nitrogen (BUN) was detected in xenograft mouse serum compared to control mice, suggesting renal insufficiency rendered by MM engraftment. Meanwhile, xenograft mouse kidney sections were stained with PAS (Periodic acid-Schiff), which demonstrated protein and cellular cast nephropathy and inflammatory infiltration. We also performed TRAP staining on representative xenograft mouse bone sections. TRAP positive osteoclasts were increased in the distal portions of the femur bones derived from these PDX-NSG mice. We present robust data that a newly developed PDX-NSG model can grow primary human MM cells. Our hypothesis holds that cells from the same patient bone marrow microenvironment support tumor plasma cells survival and growth. These factors enables this new model to recapitulate more accurately the features of human MM. We will further investigate whether this new humanized PDX-NSG model provides a better tool for understanding MM development and for a personalized medicine. Disclosures Zhan: BIPHARM LLC: Consultancy, Other: % Allocation of Profit.

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.

Analysis of Intestinal Microbiome in Multiple Myeloma Reveals Progressive Dysbiosis Compared to MGUS and Healthy Individuals

Background: In addition to acquired genetic changes in neoplastic plasma cells, progression of MGUS to multiple myeloma (MM) is mediated by changes in the proportion of dominant subclones whose expansion is driven by their interaction with the immune microenvironment. Experiments in mouse models of MM have identified a significant contribution to the microenvironment mediated via the gut microbiome and IL-17 responses (Calcinotto et al. Nat Commun 2018;(9)4832:1-7). Despite these preclinical studies, little is known about the composition of the human microbiome in MM at diagnosis, and we hypothesize that it is abnormal and can serve as an axis for immune dysregulation and selection of clonal B cells. In this study we analyzed gut microbial diversity in patients with MGUS and MM and compared these to healthy individuals. Methods: Stool samples were obtained from 10 untreated MGUS and 10 MM patients with ECOG performance status 0-2. Exclusion criteria included those with prior anti-myeloma therapy, primary gastrointestinal disorders, or transient gastrointestinal processes or antibiotic use within the past 3 months. Healthy volunteers from the same geographic region served as controls. 16s rRNA gene sequencing was performed on fecal samples obtained with uBiome Explorer kits processed using Illumina NextSeq500. Qualitative analyses included determination of genera-level operational taxonomic units compared to an established healthy reference range and quantitative assessments of microbial diversity. Cluster analysis was performed using ClustVis (http://biit.cs.ut.ee/clustvis). Results: The mean age at sampling was 74 years for MGUS and 71 for MM. Revised ISS stages I, II, and III for MM patients were 14%, 44%, and 42%, respectively. Median microbial diversity indices showed 84% in MM (range 38-96%), 85% in MGUS (range 42-93%), and 50% in healthy controls (range 23-89%). Supervised clustering of genus-level microbial sequences distinguished specific microbiota separating MM patients from MGUS and healthy controls (Figure). Odoribacter and Lactobacillus were the most enriched genera in MM compared to healthy, conversely Blautia and Faecalibacterium were most reduced. When comparing MM to MGUS, Kluyvera and Bacteroides were found to be most highly represented in MM, whereas Blautia and Parabacteroides showed the greatest decrease compared to MGUS. Data from a MM patient initially enrolled at diagnosis and resampled 4 months after achieving a partial response to induction with VCD showed a decrease in enrichment of Odoribacter from 12.2 to 2.1% as well as Bacteroides from 21.8 to 10.3%. Conclusions: This study identifies distinct changes in the intestinal microbiota that distinguish individuals with MM from its precursor and healthy controls. This is the first time such changes have been identified in untreated MM patients, and some of the genera identified here, such as Faecalibacteria, have been shown to be similarly altered in recent reports of MM patients with persistently active disease despite treatment (Pianko et al. Blood Advances 2019;(3)13:2040-2044). These preliminary studies reveal distinct genus-level differences that exist between MM and MGUS, suggesting increasing dysbiosis with progression to MGUS and MM. We are currently extending these studies to include sequential sampling of additional patients following response to induction chemotherapy. These results provide a rationale for further studying the pathogenic impact of the intestinal microbiome on dysregulation of the MM microenvironment. Figure Disclosures Morgan: Amgen, Roche, Abbvie, Takeda, Celgene, Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Other: research grant, Research Funding. Braunstein:Celgene: Consultancy, Other: Advisory Boards; Amgen: Consultancy, Other: Advisory Board; AstraZeneca: Consultancy, Other: Advisory Board; Janssen: Consultancy, Other: Advisory Board, Research Funding; Verastem: Consultancy, Other: Advisory Board; Takeda: Consultancy, Other: Advisory Boards.

High Glycine Promotes Proliferation and Progression Though Increase of Glutathione Synthesis in Multiple Myeloma

Background: Metabolites in tumor microenvironment have been confirmed to contribute to cancer progression. Our previous untargeted metabolomics study has indicated that glycine was significantly increased in bone marrow and peripheral blood derived from Multiple Myeloma(MM) patients compared with health donors(HD). However, the role of glycine in MM progression and its underling mechanisms remain unclear. Materials and Methods: Liquid chromatography-mass spectrometry (LC-MS) was used to detect the concentration of glycine in peripheral blood derived from (25) MM patients and (21) HD. Metabolic flux experiment was performed to explore the distribution of exogenous glycine in MM cell lines ARP1 and 5TGM1. Soft agar colony formation and cell cycle assay were performed to detect MM cells proliferation. 5TGM1 MM mouse models were prepared to examined the effect of glycine on MM in vivo. The unpaired t test was used to evaluate the difference between two different groups. Two-sided Fisher's exact tests were used to assess the associations between glycine abundance and clinical characteristics in MM patients, with a confidence coefficient (confidence interval, CI) of 95%. Results: Targeted metabolic assay of glycine in peripheral blood confirmed that glycine was significantly higher in MM patients than HD(HD vs. MM patients, 14000 vs. 15200, p=0.047). To explore the role of high glycine in MM progression, the associations between glycine abundance and clinical characteristics were investigated. We found that MM patients with high glycine had significantly higher plasma cells percentage(High glycine vs. Low glycine, 11.00% vs. 27.95, p=0.039) and lower hemoglobin concentration(High glycine vs. Low glycine, 96g/l vs. 77g/l, p=0.016). Moreover, high glycine was found to associate with bone damage(p=0.031). Additionally, colony formation and cell cycle assay results showed Glycine-free RPMI 1640 media inhibited MM cells proliferation. Furthermore, 5TMG1 MM mouse fed with glycine-deficiency fodder had slower progression as compared with 5TMG1 MM mouse fed with normal fodder(p=0.0007). These data suggested that exogenous glycine contributes to MM progression. To characterize how exogenous glycine is metabolized in MM cells, MM cell lines ARP1 and 5TGM1 were cultured in the presence of uniformly labeled 13C-glycine for 2, 4, and 6 hours, then the concentration of glycine metabolism related metabolites in conditional media and MM cells were tested by using LC-MS. As a result, 13C-glycine derived GSH was observed in ARP1 as well as 5TGM1, accounting for 37.2% and 52.7% of total GSH after 6 hours of culture, respectively, alternatively, the levels of 13C-GSH in both cell lines were up-regulated with the extension of culture time, indicating that exogenous glycine was involved in GSH synthesis in MM cells. Furthermore, addition of GSH(10 uM) to glycine-free RPMI 1640 media recover the proliferation ability of ARP1 and 5TGM1. Interestingly, betaine, a competitive similar of glycine, was found to suppress MM cell proliferation, and addition of GSH partially counteracted the effect of betaine on MM cells. Conclusion: These findings thus indicate that glycine promotes MM proliferation in vivo and in vitro, and GSH synthesis is the main metabolic pathway contributing to proliferation. Pharmacological blockage of glycine uptake and utilization shows therapeutic potential in MM treatment. 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.

Adhesion-Mediated Multiple Myeloma (MM) Disease Progression: Junctional Adhesion Molecule a Enhances Angiogenesis and Multiple Myeloma Dissemination and Predicts Poor Survival

Multiple myeloma (MM) plasma cell (MMPC) interactions with the microenvironment control MMPC growth, survival, drug-resistance and intra- and extramedullary dissemination. Dissemination of MMPCs through bone marrow niches and in extra-medullary sites is an active process of invasion involving bone marrow endothelial cells, multiple adhesion molecules and chemokine receptors. Since enhanced angiogenesis characterizes MM, we investigated whether junctional adhesion molecule-A (JAM-A) mediated interactions between MM bone marrow endothelial cells (MMECs) and MMPCs impact disease progression. To this end, we analyzed JAM-A expression levels in MMECs of 312 MM patients in two independent cohorts with flow cytometry, namely 111 newly diagnosed (NDMM) and 201 relapsed/refractory (RRMM) and compared them to 36 monoclonal gammopathy of undetermined significance (MGUS) and healthy subjects. To corroborate our data and investigate at a gene-expression level the prognostic value of deregulated genes (FDR<0.1 & P<0.05) we used a Cox-regression model in the CoMMpass dataset (n=326, IA13 release). The role of JAM-A was evaluated by shRNA knockdown and an anti-JAM-A blocking monoclonal antibody. Subsequently, we functionally validated the JAM-A downstream pathways related to cytoskeleton rearrangement, cell proliferation, epithelial mesenchymal transition, invasion and MM dissemination in vitro and in vivo. Surface protein expression of JAM-A on MMECs predicted poor overall survival (OS) in NDMM (not reached (NR) vs. 78 months univariate hazards ratio-HR=9.14, 95% CI 2.8-29.76, P<0.0001) and RRMM patients (NR vs. 130 months, HR=2.96, 95% CI 1.37-6.37, P=0.006) with significant impact also in the progression free survival (PFS) in the advanced stage cohort (8.3 vs. 27 months, HR=1.41, 95% CI 1.05-1.88; P=0.019). A sub-analysis on our cohort with extramedullary disease (EMD) MM revealed that the median OS decreased significantly in patients with JAM-Ahighvs. those with JAM-Alow MMEC expression: 84.1 months vs. not reached, irrespective from the EMD status (log-rank=4.19, P=0.04). Strikingly, among NDMM, these results maintained their significance also in the multivariate analysis (HR=9.11, 95% CI 2.79-29.76, P<0.001); within the RRMM cohort the multivariate analyses confirmed JAM-Ahigh MMECs as a statistically significant independent risk factor for short OS (HR=2.39, 95% CI 1.09-5.28, P=0.03) in much the same way were the Revised international staging system (R-ISS) stages such as stage II (HR=5.34, 95% CI 1.24-22.97, P=0.024), stage III (HR=6.57, 95% CI 1.25-34.54, P=0.026) and chronic kidney disease (HR=2.21, 95% CI 1.06-4.62, P=0.034). Cox stratified model implemented for PFS confirmed only JAM-Ahigh MMECs as a statistically significant risk factor (HR=1.35, 95% CI 1.00-1.81, P=0.044) stratified by chronic kidney disease. Notably, also transcriptional upregulation of JAM-A (RNA-Seq data, CoMMpass) corroborated the prognostic impact (log-rank=13, P=0.0003) and revealed a unique gene-expression signature of activated epithelial-mesenchymal-transition, mTOR/PI3K and focal adhesion pathways in high-risk MM patients with EMD and JAM-Ahigh. Ensuing functional shRNA knockdown of JAM-A reduced MM invasion (P<0.002), angiogenesis (P<0.0001), cell dissemination and migration (P<0.002), cell survival (P<0.001) and expression of cellular-adhesion system molecules such as integrin-beta-1, fibronectin, RAC1 and RHOA (P<0.001). Notably, adding recombinant JAM-A to MMECs enhanced angiogenesis while this was impaired by blocking JAM-A with a specific monoclonal antibody in functional 2D and 3D chorioallantoic membrane-assay and two in vivo MM mouse models. Conclusively, in vivo experiments corroborated our findings that JAM-A blocking halted angiogenesis and reduced MM progression. Collectively, our findings pinpoint JAM-A as a key player propagating a vicious cycle of MMECs and MMPCs interaction; the expression of JAM-A and the related adhesion pathways can prognostically stratify patients in the late disease stages impacting two main MM progression processes: angiogenesis and extra-medullary dissemination. Therefore, we propose JAM-A as a promising MM biomarker and novel therapeutic target in advanced disease. Disclosures Klapper: Roche, Takeda, Amgen, Regeneron: Honoraria, Research Funding. Rosenwald:MorphoSys: Consultancy. Cavo:celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; novartis: Honoraria; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Excess Serine from the Microenvironment Caused the Impaired Megakaryopoiesis and Thrombocytopia in Multiple Myeloma

Background: Thrombocytopenia is major complication in a subset of patients with multiple myeloma (MM). However, a lack of detailed studies about the megakaryopoiesis, thrombopoiesis as well as their connections with the survival of patients limits us to explore whether thrombocytopenia could be used as a reliable prognostic factor for MM. Materials and Methods: In this study, 1393 newly diagnosed MM patients were selected for investigating the potential connection between PLT counts and clinical characteristics including ISS stage, overall survival as well as progression free survival. Besides, 5T33MMvt-KaLwRij mouse model were also used to examine the megakaryopoiesis and thrombopoiesis during disease progression. The proportion and function of different subpopulations of cells including megakaryocytes, megakaryocytic-erythroid progenitors (MEPs), common myeloid progenitors (CMPs) and Lin-Sca-1+c-kit+ (LSK) cells were measured both in MM patients and mouse models. Gas chromatography-time-of-flight mass spectrometry (GC-TOFMS)-based metabolomics was used to analyze the metabolites. Results: Of the 1393 studied patients, 298 cases of MM patients are found with thrombocytopenia at the time of diagnosis. PLT counts were lower both in stage Ⅱ (P<0.01) and Ⅲ patients (P<0.001) than stageⅠpatients. Interesting, we found MM patients with thrombocytopenia had a significantly lower OS (P<0.001) and PFS (P<0.001). In mouse model, we also found PLT counts gradually decreased in peripheral blood during the disease progression (P<0.001). Further analysis demonstrated the proportion and the absolute numbers of megakaryocytes and MEPs were diminished both in mouse and MM patients with thrombocytopenia. PLT counts were negative correlated with the percentage of plasma cells or IgG2b levels (P<0.001), suggesting a potential connection of malignant cells infiltration and thrombocytopenia. In mechanism, metabolomics analysis with BM plasma identified 16 differential metabolites in MM patients with thrombopoiesis (VIP > 1.2, P < 0.05). Among them, serine was observed significantly be elevated in MM mouse and suffice to inhibit the megakaryopoieis and thrombopoiesis in vitro. Conclusion: PLT counts might be used as a reliable prognostic factor for MM patients since the thrombocytopenia was associated with poor survival in MM patients. The thrombocytopenia in MM might attribute to, at least partially, the inhibition by serine from the microenvironment. Our findings revealed novel mechanism of MM and might eventually shed light on the treatment of MM patients. Disclosures No relevant conflicts of interest to declare.

Syrbactin proteasome inhibitor TIR-199 overcomes bortezomib chemoresistance and inhibits multiple myeloma tumor growth in vivo

Multiple myeloma (MM) and mantle cell lymphoma (MCL) are blood cancers that respond to proteasome inhibitors. Three FDA-approved drugs that block the proteasome are currently on the market, bortezomib, carfilzomib, and ixazomib. While these proteasome inhibitors have demonstrated clinical efficacy against refractory and relapsed MM and MCL, they are also associated with considerable adverse effects including peripheral neuropathy and cardiotoxicity, and tumor cells often acquire drug resistance. TIR-199 belongs to the syrbactin class, which constitutes a novel family of irreversible proteasome inhibitors. In this study, we compare TIR-199 head-to-head with three FDA-approved proteasome inhibitors. We demonstrate that TIR-199 selectively inhibits to varying degrees the sub-catalytic proteasomal activities (C-L/β1, T-L/β2, and CT-L/β5) in three actively dividing MM cell lines, with Ki50 (CT-L/β5) values of 14.61 ± 2.68 nM (ARD), 54.59 ± 10.4 nM (U266), and 26.8 ± 5.2 nM (MM.1R). In most instances, this range was comparable with the activity of ixazomib. However, TIR-199 was more effective than bortezomib, carfilzomib, and ixazomib in killing bortezomib-resistant MM and MCL cell lines, as judged by a low resistance index (RI) between 1.7 and 2.2, which implies that TIR-199 indiscriminately inhibits both bortezomib-sensitive and bortezomib-resistant MM and MCL cells at similar concentrations. Importantly, TIR-199 reduced the tumor burden in a MM mouse model (p < 0.01) confirming its potency in vivo. Given the fact that there is still no cure for MM, the further development of TIR-199 or similar molecules that belong to the syrbactin class of proteasome inhibitors is warranted.

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.

Neoantigen-specific CD4+ T cells induce protective, tumor-specific CD8+ T cells in a mouse model of multiple myeloma.

Cancer immunotherapies have primarily focused on generating tumoricidal CD8 T cells. However, recent data demonstrate a critical role for CD4 T cells in tumor immunity. CD4 T cells against epitopes derived from mutated tumor-associated neo-antigens (neoAg) conferred protection against tumor growth in animal models of neoAg vaccine therapy. In clinical studies, immunity elicited by neoAg vaccines was associated with improved survival, even though the majority of immune responses were CD4 T cells that did not have cytolytic activity.