Mouse Model Of Multiple Myeloma Research Articles

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.

Identification and Characterization of Tumor-Initiating Cells in Multiple Myeloma.

Treatment failures in cancers, including multiple myeloma (MM), are most likely due to the persistence of a minor population of tumor-initiating cells (TICs), which are noncycling or slowly cycling and very drug resistant. Gene expression profiling and real-time quantitative reverse transcription polymerase chain reaction were employed to define genes differentially expressed between the side-population cells, which contain the TICs, and the main population of MM cells derived from 11 MM patient samples. Self-renewal potential was analyzed by clonogenicity and drug resistance of CD24+ MM cells. Flow cytometry (n = 60) and immunofluorescence (n = 66) were applied on MM patient samples to determine CD24 expression. Therapeutic effects of CD24 antibodies were tested in xenograft MM mouse models containing three to six mice per group. CD24 was highly expressed in the side-population cells, and CD24+ MM cells exhibited high expression of induced pluripotent or embryonic stem cell genes. CD24+ MM cells showed increased clonogenicity, drug resistance, and tumorigenicity. Only 10 CD24+ MM cells were required to develop plasmacytomas in mice (n = three of five mice after 27 days). The frequency of CD24+ MM cells was highly variable in primary MM samples, but the average of CD24+ MM cells was 8.3% after chemotherapy and in complete-remission MM samples with persistent minimal residual disease compared with 1.0% CD24+ MM cells in newly diagnosed MM samples (n = 26). MM patients with a high initial percentage of CD24+ MM cells had inferior progression-free survival (hazard ratio [HR] = 3.81, 95% confidence interval [CI] = 5.66 to 18.34, P < .001) and overall survival (HR = 3.87, 95% CI = 16.61 to 34.39, P = .002). A CD24 antibody inhibited MM cell growth and prevented tumor progression in vivo. Our studies demonstrate that CD24+ MM cells maintain the TIC features of self-renewal and drug resistance and provide a target for myeloma therapy.

Abstract 3863: Specifically targeting PDIA1 with indene inhibitors leads to bortezomib-potentiation in multiple myeloma

Abstract Protein disulfide isomerase (PDI) is an emerging therapeutic target in oncology. PDI inhibitors have demonstrated a unique propensity to overcome resistance to existing cancer 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 (PIs) in panels of Multiple Myeloma (MM) cells and MM mouse models [1]. An extensive medicinal chemistry program has led to the generation of a diverse library of indene-containing molecules with varying degrees of PI-potentiating activity. The results show detailed structure activity relationships (SAR) with a high degree of correlation between potency of PDIA1 inhibition and bortezomib (Btz) potentiation in MM cells. Inhibition of PDIA1 in MM 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. Furthermore, an advanced derivative, E64FC65, was identified as a PDIA1 selective inhibitor as it was significantly weaker against PDIA3, PDIA4, TXNDC5, PDIA6, and thioredoxin when compared to E64FC26 (Table 1). We purified the redox-active a and a’ thioredoxin-like domains of PDI and found that E64FC65 only inhibited the a domain while E64FC26 inhibited both a and a’ domains. The PDI isoform and domain selectivity of E64FC65 imparted increased anti-cancer activity while reducing toxicity against primary activated human T-cells. Additionally, E64FC65 exhibited improved pharmaceutical properties in ADME assays. This work shows the generation of a PDIA1 a domain specific indene inhibitor that potentiates Btz cytotoxicity in MM cells while abrogating toxicity in normal cells. E64FC65 is thus a favorable PDI inhibitor candidate for further development. Citation Format: Reeder M. Robinson, Leticia Reyes, Ravyn Duncan, Nathan Dolloff. Specifically targeting PDIA1 with indene inhibitors leads to bortezomib-potentiation in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3863.

PF575 OVEREXPRESSION DNP73 PROMOTES THE PROLIFERATION AND DRUG RESISTANCE OF MULTIPLE MYELOMA

Background:Genomic instability and chromosomal abnormalities induced by DNA damage are important biological characteristics of multiple myeloma (MM). DNp73 is one of the main isomers of TP73, a member of the TP53 protein family. We found that DNp73 was highly expressed in MM which could resist the apoptosis induced by DNA damage in MM cells, leading to clinical drug resistance.Aims:In order to further explore the mechanism of DNp73 mediating drug resistance to DNA damage in MM, this study verified and explored from multiple levels including cell phenotype changes in vivo and in vitro.Methods:MM cell lines OCI‐MY5 and ARP1 were used to constructed DNp73 overexpression (OE) cell lines, respectively. Cell proliferation activity of DNp73 OE and EV (empty vector) cell lines was compared by absolute cell count method and CCK8 method, and cell proliferation was determined under the action of doxorubicin (5nM &amp;50 nM) or epirubicin (5nM &amp; 10 nM) at different concentrations. The cloning formation and drug sensitivity of ARP1 DNp73 OE/EV cells were determined by soft agar cloning formation assay. Flow cytometry was used to detect the apoptosis and cell cycle arrest of cells in DNp73 OE and EV cells under UV and at different concentrations of reagents treatment. Xenografted model and 5TGM1 spontaneous mouse MM model were used to detect the effect of DNp73 on the proliferation of MM cells and the treatment of DNA damage reagents in vivo, as well as the survival period of tumor‐bearing mice were analyzed. RNA‐seq and gene enrichment analysis (GSEA) were performed to explore the potential signaling pathway of DNp73 against apoptosis. Further western blotting and quantity‐real time PCR were performed for validation. ChIP and CoIP were used to search for the potential downstream key target genes and target proteins of DNp73.Results:DNp73‐OE ARP1 &amp; OCI‐MY5 cell lines showed stronger proliferation activity than EV MM cell lines, and showed significant resistance to DNA damage reagents, doxorubicin and epirubicin treatment. The cloning formation experiment of soft agar confirmed that DNp73 induced the resistance of MM cells to those drugs. Flow cytometry analysis showed that DNp73 OE suppressed the MM cells apoptosis and suppressed G1 phase arrest induced by those drugs treatment (P &lt; 0.05). In vivo experiments showed that the tumor burden of mice was significantly more severe compared with the mice of control group. The tumor‐bearing mice in DNp73 OE group were resistant to the treatment of DNA damage reagents. Moreover, the survival of DNp73 OE tumor‐bearing mice was significantly shortened compared with the control group. RNA‐seq, GSEA analysis and western blot assay revealed that DNp73 overexpression induce drug resistance and proliferation through the activation of c‐myc and AKT‐related pathways and inhibition of p21/p27 signal pathway which caused cell cycle arrested in MM.Summary/Conclusion:DNp73 promote MM cell proliferation, anti‐apoptosis and inhibit cell cycle arrest through activating c‐myc and Akt‐related pathways. DNp73 overexpression is related to the occurrence of drug resistance in MM.image

PF581 FUNCTIONAL HIGH‐THROUGHPUT SCREENING FOR IDENTIFICATION OF NOTCH1 DOWNSTREAM EFFECTORS AS NOVEL THERAPEUTIC TARGETS IN MULTIPLE MYELOMA

Background:Intrinsic and acquired drug resistance of multiple myeloma (MM) cells often cause disease progression in MM patients. Therefore, the identification of novel drug targets is needed to further improve treatment strategies. The deregulated Notch1 pathway is a master regulator of MM onset, progression and drug resistance. Inhibition of Notch through use of γ‐secretase inhibitors causes serious side effects and is not used in the clinic for treatment of MM patients.Aims:In this study, we identify novel druggable downstream effectors of the Notch1 pathway to overcome drug resistance of MM cells.Methods:We employ high‐throughput (HT) functional screening based on tailored Notch1 shRNA libraries under selective pressure of chemotherapeutic agents. First, we performed genome‐wide expression profiling through RNA sequencing (RNAseq) in Notch‐positive MM.1S cells after inhibition of the pathway. Strongly and consistently regulated genes (log2FC; padj &lt;0.05) were chosen as targets for two tailored shRNA libraries. Functional screens are conducted using native U266 cells (positive selection screens) or U266 cells engineered to express the Notch1 variants NIC or NΔE (negative selection screens). NIC is the active and γ‐secretase‐independent form of Notch1. By using the membrane‐bound and γ‐secretase‐dependent NΔE variant, screening additionally comprises investigation of cooperation between Notch1 and cytoplasmic signaling complexes. Following transduction of U266 cells with the corresponding shRNA library, cells are cultured under selective pressure of Melphalan, Bortezomib or Lenalidomide. HT sequencing and bioinformatics analysis of changes in the representation of shRNAs will indicate critical Notch1 effectors. For in vitro validation of candidate genes, MM cell lines will be treated with target gene‐specific shRNAs or chemical inhibitors in combination with chemotherapeutic agents. Analyses of proliferation, cell cycle progression and apoptosis will reveal candidate genes, which contribute to growth and survival advantages. Most promising genes will be further tested in vivo using the MM mouse models BALB/c‐MOPC315.BM and VK∗MYC.Results:RNAseq analysis revealed 842 Notch1‐regulated genes. 40 downregulated and 28 upregulated genes were chosen as targets for the shRNA libraries. Specificity of the RNAseq approach was verified by quantitative PCR for known Notch1 target genes such as HEY1 and HES6. Interestingly, the group of Notch1‐upregulated genes further includes bone‐related genes supporting MM growth and the aggressive MM bone phenotype such as RUNX2, osteocalcin or cathepsin K (Trotter et. al., Blood, 2015).Expression of NIC and NΔE in engineered U266 cells was validated by flow cytometric and western blot analysis. Cell viability analysis by Annexin V/PI staining and CellTiter‐Glo® luminescent assay showed reduced drug sensitivity of Notch1‐expressing U266 cells compared to Notch1‐negative U266 cells, confirming the role of Notch1 as survival factor.Based on these results, the first functional screen was recently performed. Intermediate analysis of cell viability clearly showed a sensitizing effect of shRNAs, indicating promising screen hits. First outcomes of the screen will be presented at the meeting.Summary/Conclusion:ShRNA‐based functional HT screening is a potent tool for unveiling Notch1 downstream effectors as specific therapeutic targets in MM to improve treatment efficacy and reduce side effects.

Sialyltransferase inhibition leads to inhibition of tumor cell interactions with E-selectin, VCAM1, and MADCAM1, and improves survival in a human multiple myeloma mouse model.

Aberrant glycosylation resulting from altered expression of sialyltransferases, such as ST3 β-galactoside α2-3-sialyltransferase 6, plays an important role in disease progression in multiple myeloma (MM). Hypersialylation can lead to increased immune evasion, drug resistance, tumor invasiveness, and disseminated disease. In this study, we explore the in vitro and in vivo effects of global sialyltransferase inhibition on myeloma cells using the pan-sialyltransferase inhibitor 3Fax-Neu5Ac delivered as a per-acetylated methyl ester pro-drug. Specifically, we show in vivo that 3Fax-Neu5Ac improves survival by enhancing bortezomib sensitivity in an aggressive mouse model of MM. However, 3Fax-Neu5Ac treatment of MM cells in vitro did not reverse bortezomib resistance conferred by bone marrow (BM) stromal cells. Instead, 3Fax-Neu5Ac significantly reduced interactions of myeloma cells with E-selectin, MADCAM1 and VCAM1, suggesting that reduced sialylation impairs extravasation and retention of myeloma cells in the BM. Finally, we showed that 3Fax-Neu5Ac alters the post-translational modification of the α4 integrin, which may explain the reduced affinity of α4β1/α4β7 integrins for their counter-receptors. We propose that inhibiting sialylation may represent a valuable strategy to restrict myeloma cells from entering the protective BM microenvironment, a niche in which they are normally protected from chemotherapeutic agents such as bortezomib. Thus, our work demonstrates that targeting sialylation to increase the ratio of circulating to BM-resident MM cells represents a new avenue that could increase the efficacy of other anti-myeloma therapies and holds great promise for future clinical applications.

Estrogen promotes multiple myeloma through enhancing the immunosuppressive activity of MDSC

Although the role of estrogen in solid cancers has been widely investigated, its effect in hematologic malignancies including multiple myeloma (MM) is not known. Here, we utilized a syngeneic mouse model of MM to address this question. In this model, treatment with 17β-estradiol significantly promoted progression of the disease. This effect has not been attributed to the direct effect of estrogen on MM cells but rather was mediated through estrogen-induced alterations in tumor microenvironment. In MM bone marrow, myeloid-derived suppressor cells (MDSCs) represent one of the major cellular populations. 17β-estradiol did not promote expansion and accumulation of MDSCs. However, it significantly increased their ability to suppress T cells proliferation. Thus, these data demonstrated that estrogen promotes progression of MM by enhancing an immunosuppressive function of the bone marrow MDSCs.

Repression of Multiple Myeloma Cell Growth In Vivo by Single-wall Carbon Nanotube (SWCNT)-delivered MALAT1 Antisense Oligos.

The single-wall carbon nanotube (SWCNT) is a new type of nanoparticle, which has been used to deliver multiple kinds of drugs into cells, such as proteins, oligonucleotides, and synthetic small-molecule drugs. The SWCNT has customizable dimensions, a large superficial area, and can flexibly bind with drugs through different modifications on its surface; therefore, it is an ideal system to transport drugs into cells. Long noncoding RNAs (lncRNAs) are a cluster of noncoding RNA longer than 200 nt, which cannot be translated to protein but play an important role in biological and pathophysiological processes. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a highly conserved lncRNA. It was demonstrated that higher MALAT1 levels are related to the poor prognosis of various cancers, including multiple myeloma (MM). We have revealed that MALAT1 regulates DNA repair and cell death in MM; thus, MALAT1 can be considered as a therapeutic target for MM. However, the efficient delivery of the antisense oligo to inhibit/knockdown MALAT1 in vivo is still a problem. In this study, we modify the SWCNT with PEG-2000 and conjugate an anti-MALAT1 oligo to it, test the delivery of this compound in vitro, inject it intravenously into a disseminated MM mouse model, and observe a significant inhibition of MM progression, which indicates that SWCNT is an ideal delivery shuttle for anti-MALAT1 gapmer DNA.