Multiple Myeloma Cells In Bone Marrow Research Articles

In Vitro Low-Bortezomib Doses Induce Apoptosis and Independently Decrease the Activities of Glutathione S-Transferase and Glutathione Peroxidase in Multiple Myeloma, Taking into Account the GSTT1 and GSTM1 Gene Variants.

Multiple myeloma (MM) is a malignancy derived from plasma cells. Bortezomib affects the concentration of reduced glutathione (GSH) and the activity of glutathione enzymes. The aim of our study was to analyze deletion (null/present) variants of GSTT1 and GSTM1 genes and their association with the levels of glutathione and its enzymes in bortezomib-treated cell cultures derived from MM patients. This study included 180 individuals (80 MM patients and 100 healthy blood donors) who were genotyped via multiplex PCR (for the GSTT1/GSTM1 genes). Under in vitro conditions, MM bone marrow cells were treated with bortezomib (1-4 nM) to determine apoptosis (via fluorescence microscopy), GSH concentration, and activity of glutathione enzymes (via ELISA). Bortezomib increased the number of apoptotic cells and decreased the activity of S-glutathione transferase (GST) and glutathione peroxidase (GPx). We found significant differences in GST activity between 1 nM (GSTT1-null vs. GSTT1-present), 2 nM (GSTT1-null vs. GSTT1-present), and 4 nM (GSTM1-null vs. GSTM1-present) bortezomib: 0.07 vs. 0.12, p = 0.02; 0.06 vs. 0.10, p = 0.02; and 0.03 vs. 0.08, p = 0.01, respectively. Bortezomib affects the activities of GST and GPx. GST activity was associated with GSTT1 and GSTM1 variants but only at some bortezomib doses.

Clarithromycin overcomes stromal cell-mediated drug resistance against proteasome inhibitors in myeloma cells via autophagy flux blockage leading to high NOXA expression.

We previously reported that macrolide antibiotics, such as clarithromycin (CAM), blocked autophagy flux, and simultaneous proteasome and autophagy inhibition by bortezomib (BTZ) plus CAM resulted in enhanced apoptosis induction in multiple myeloma (MM) cells via increased endoplasmic reticulum (ER) stress loading. However, in actual therapeutic settings, cell adhesion-mediated drug resistance between bone marrow stromal cells (BMSC) and MM cells has been known to be a barrier to treatment. To investigate whether CAM could enhance BTZ-induced cytotoxicity in MM cells under direct cell adhesion with BMSC, we established a co-culture system of EGFP-labeled MM cells with BMSC. The cytotoxic effect of BTZ on MM cells was diminished by its interaction with BMSC; however, the attenuated cytotoxicity was recovered by the co-administration of CAM, which upregulates ER stress loading and NOXA expression. Knockout of NOXA in MM cells canceled the enhanced cell death by CAM, indicating that NOXA is a key molecule for cell death induction by the co-administration of CAM. Since NOXA is degraded by autophagy as well as proteasomes, blocking autophagy with CAM resulted in the sustained upregulation of NOXA in MM cells co-cultured with BMSC in the presence of BTZ. Our data suggest that BMSC-associated BTZ resistance is mediated by the attenuation of ER stress loading. However, the addition of CAM overcomes BMSC-associated resistance via upregulation of NOXA by concomitantly blocking autophagy-mediated NOXA degradation and transcriptional activation of NOXA by ER stress loading.

The Role of Macrophage Migration Inhibitory Factor (MIF) in Regulation of T-Cell Activity in Multiple Myeloma

Background: Multiple myeloma (MM) is the second most common hematological malignancy associated with the overproduction of light chain or monoclonal immunoglobulin. MM remains incurable, with high relapses and refractory disease rates after first-line treatment. Hence, novel therapeutic approaches are needed to improve immune responses to prevent relapses or drug resistance after initial therapies. Immune responses are shaped by several factors, including mutations, pro- and anti-inflammatory cytokines and chemokines, and activation of immune checkpoints in the tumor microenvironment (TME) (Gonzalez et al., 2018). Macrophage migration inhibitory factor (MIF) as a soluble pro-inflammatory cytokine has become one of the most puzzling regulators of innate and adaptive immune responses. In myeloma, MIF was found to be significantly higher in MM cells from relapsed patients than those responding to the treatment. MIF has been implicated in drug resistance and suggested as a biomarker for predicting MM patient responses to proteasome inhibitors (Wang et al., 2020). We previously reported that MIF is highly expressed in MM and regulates CD84 (SLAMF5) expression in MM TME. We showed that MIF secreted from MM cells regulates PD-L1 expression through CD84 on CD14 + monocytic cells in the TME. Inhibition of MIF significantly reduced CD84 and PD-L1 expressions on human CD14 + cells and on myeloid-derived suppressor cells of the mice bearing MM (Lewinsky and Gunes et al., 2021). However, whether MIF directly regulates T-cell activity in MM TME has yet to be explored. Methods and Results: The current study reports that MIF may play a pivotal role in T-cell exhaustion in MM. Ouranalyses in published MM transcriptomic databases show that MM patients with low MIF had significantly longer overall and post-progression survival, indicating the therapeutic potential of MIF in MM. We also noted upregulated MIF gene expression in relapsed or refractory MM (R/RMM) patients (n=2) compared to that in newly diagnosed MM patients (n=7) and healthy individuals (n=9) in our and publicly available MM single-cell RNA sequencing datasets. We detected a profound MIF expression in MM cells and myeloid cell fractions, including monocytes, macrophages, and dendritic cells from the patients' bone marrow (BM) specimens. We previously reported that human MM cell lines highly express MIF. In this study, we assessed the impact of MIF secreted by MM cells on the immune cells, specifically CD8 + T cells. Our results showed that inhibition of MIF reduced the elevated T-cell exhaustion markers, PD1 and CTLA-4, on CD8 + T cells in healthy donors (HDs) peripheral blood mononuclear cells (PBMCs) following the coculturing with MM1.S cells (n=3 *P < 0.05 ). We also found that MIF inhibition induced the T-cell activation marker, LAMP1, on CD8 + T cells in this coculturing setting (n=3, **P < 0.01). Furthermore, we noted MIF inhibition reduced PD1 expression on CD8 + T cells in HDs PBMCs (n=3, **P < 0.01), suggesting MIF may regulate the exhaustion profile of CD8 + T cells. MIF can conduct paracrine or autocrine signalings by binding to its receptors, including CD74 and CD44, and lead to intracellular signaling cascades. Hence, we assessed CD74 gene expression as the counterpart of MIF in R/RMM, MM, and healthy patients. We noted significantly upregulated CD74 expression in R/RMM patients compared to newly diagnosed MM patients and healthy individuals. We also noted higher CD74 expression in CD8 + T cells than in CD4 + T cells in R/RMM patients, suggesting that MIF/CD74 axis may be a potential target to enhance CD8 + T cell-mediated immunity in R/RMM. To further explore the impact of MIF inhibition on MM progression, we performed a pilot in vivo study by inoculating murine 5TGM1 cells into two groups of syngeneic C57BL/KaLwRijHsd mice. After two weeks, the mice were injected with either sterile PBS as a control or a MIF inhibitor. The results of this pilot study showed that MIF inhibition significantly decreased MM cells in BM and PB of myeloma-bearing mice and reduced T-cell exhaustion markers on CD8 + T cells compared to that in the control mice (n=3-4, *P < 0.05, **P < 0.01, ***P < 0.001). Conclusion: Overall, our preliminary data indicate that MIF may have a crucial role in T-cell activity by regulating T-cell exhaustion markers in MM. Further studies are needed to explore the therapeutic potential of MIF to reduce T-cell exhaustion to improve anti-tumor immunity in MM.

The Impact of Soluble BCMA and BCMA Gain on Anti-BCMA Immunotherapies in Multiple Myeloma

B cell maturation antigen (BCMA) targeting chimeric antigen receptor T cells (CAR T) and bispecific T cell engagers (TCE) have remarkable efficacy in multiple myeloma (MM). While BCMA antigen escape, driven by TNFRSF17 deletions or mutations, is a mediator of anti-BCMA CAR T/ TCE resistance, the majority of patients retain BCMA surface expression at progression. Furthermore, patients with high disease burden and extramedullary disease associated with poorer response to anti-BCMA therapies, have high levels of soluble BCMA (sBCMA). The mechanisms mediating this resistance, and in particular the contribution of sBCMA, have not been fully elucidated. In order to assess the effect of sBCMA on anti-BCMA CAR T function, we measured CAR T cells activation with an in-house manufactured anti-BCMA CAR transduced with an NFAT-GFP reporter gene after co-culture with increasing concentrations of sBCMA (0-2500 ng/mL). sBCMA bound to CAR scFv in a dose dependent manner, effectively competing with the fluorophore tagged BCMA peptide binding. Despite their stable engagement, sBCMA failed to induce the expression of GFP, CD69, CD25, or 41BB on CAR T cells. To further examine the functional effect of sBCMA on CAR T cytotoxicity, we generated isogenic OPM2 cell lines with distinct BCMA expression profiles: OPM2 over-expressing BCMA (OPM2_BCMA high) generated by lentiviral transduction of TNFRSF17, and Cas9 BCMA knockout OPM2 (OPM2_BCMA -/-). OPM2_BCMA high cells exhibited one-log-fold increase in surface BCMA expression and 10-fold increase in sBCMA and were more resistant to CAR T mediated lysis. In addition, in co-culture assays, elevated sBCMA impaired OPM2 target cell lysis by anti-BCMA CAR T in a dose dependent-manner. To gain insight into the effect of chronic sBCMA exposure on CAR T, we exposed anti-BCMA CAR T to OPM2 cells secretome in the absence of direct cell to cell contact in a trans-well co-culture system. On day 24, CAR T in co-culture with OPM2_BCMA high (sBCMA levels of 2343 ng/mL) significantly contracted with no viable cells, while CAR T in culture with parental OPM2 (sBCMA level of 267 ng/mL) did persist with up-regulated TIM3 and TIGIT. In contrast CAR T cultured with OPM2_BCMA -/- remained viable with no expression of exhaustion markers. In examining the impact of sBCMA on TCE activity, sBCMA level as low as 50 ng/mL in vitro impaired anti-BCMA TCE binding to K562 cells transduced to stably express BCMA (K562_wtBCMA) . Consistent results were found in cytotoxicity assays wherein the addition of 2 ng/mL of sBCMA was sufficient to induce a reduction in anti-BCMA TCE mediated cytotoxicity of K562_wtBCMA cells by healthy donor peripheral blood mononuclear cells. To identify genomic events that lead to elevated sBCMA expression and subsequent resistance to CAR T/TCE, we conducted bulk whole genome sequencing (100X) on CD138+ bone marrow MM cells obtained from 40 MM patients treated with anti-BCMA CAR T/ TCE. Structural variants (SVs) at or adjacent to TNFRSF17 locus were identified in four patients. These included i) focal copy number gain of TNFRSF17 (3 copies) in a patient who progressed within 3 months post anti-BCMA TCE, ii) subclonal duplication of TNFRSF17 locus in another patient (n=1), as well as iii) focal copy number losses 5‘ or 3‘ to the TNFRSF17 gene locus (n=2). In addition to SVs affecting TNFRSF17 locus, we also identified mutations involving TNFRSF17 transcriptional regulators. Notably, MM clones at progression 4 months post anti-BCMA TCE (n=1) harboured copy number gains (3 copies) of POU2AF1 gene on chr11q23. POU2AF1 encodes a key transcriptional regulator of TNFRSF17 expression. Indeed, sBCMA level from this patient was significantly elevated at relapse at a level (638 ng/mL) correlated with resistance to anti-BCMA TCE. In addition, we identified translocation involving MAP3K14 encoding the NF-kB regulator NIK in a patient with short remission post anti-BCMA CAR T. Of interest, analysis of the CoMMpass dataset revealed significantly higher BCMA transcripts in patients with translocations involving MAP3K14 locus. Our findings highlight the impact of sBCMA levels and its chronicity of exposure on anti-BCMA TCE and CAR T activities in MM. We further identified structural genomic events driving TNFRSF17 over-expression and their correlation to sBCMA levels facilitating tumoral immunotherapeutic escape.

EXOSOME MIRNAS PROFILING IN SERUM AND PROGNOSTIC EVALUATION IN PATIENTS WITH MULTIPLE MYELOMA

Introduction: Multiple Myeloma (MM) is an incurable hematological malignancy. Tightly crosstalk through microRNAs (miRNAs) carried by exosomes between the bone marrow microenvironment and MM cells plays pivotal roles in MM. Our study aimed to identify the expression profile of exosomal miRNAs (exo-miRNAs) in the serum of MM patients and investigate the regulation network and their potential functions. Methods: Exosomes in serum from 19 newly diagnosed multiple myeloma patients and 9 healthy donors were isolated and the miRNA profile was investigated by small RNA sequencing. Differential expression of exo-miRNAs was calculated and target genes of miRNAs were predicted. CytoHubba was applied to identify the hub miRNAs and core target genes. The LASSO Cox regression model was utilized to develop the prognostic model, and the ESTIMATE immune score was calculated to investigate the correlation between the model and immune status in MM patients. Results: Our study clarified 313 differentially expressed serum exo-miRNAs between MM patients and HD. GO analysis of target genes of differential miRNAs showed that these target genes are mainly involved in the critical biological processes related to MM pathogenesis such as proteasome-mediated ubiquitin-dependent protein catabolic process, indicating the indispensable roles of these miRNAs in MM pathogenesis. Top six hub differentially expressed serum exo-miRNAs (hsa-miR-4728-5p, hsa-miR-455-3p, hsa-miR-6779-5p, hsa-miR-124-3p, hsa-miR-615-3p, and hsa-miR-7106-5p) were identified. 513 target genes of six hub exo-miRNAs were confirmed to be differentially expressed in MM cells in Zhan Myeloma microarray dataset. Functional enrichment analysis indicated that these target genes mainly involved mRNA splicing, cellular response to stress, and deubiquitination. Thirteen core exo-miRNA target genes were applied to create a novel prognostic signature to facilitate the risk stratification of MM patients, and two groups of MM patients with diverse outcomes were recognized (Figure 1A). The high-score patients identified by thirteen core genes signature displayed a higher 70 high-risk gene set (UAMS-70) score and the 56 drug-resistance genes set score, which might partly explain their inferior outcomes (Figure 1B–C). Additionally, our results suggest that the thirteen core genes signature model was highly correlated with the status of the immune microenvironment, and patients in the low-score group had higher immune cell infiltration levels (Figure 1D). Besides, patients in the high-risk group displayed lower HLA family gene expression (Figure 1E). Encore Abstract - previously submitted to EHA 2023 Keywords: Bioinformatics, Computational and Systems Biology, Diagnostic and Prognostic Biomarkers, Risk Models No conflicts of interests pertinent to the abstract.

Bispecific CS1-BCMA CAR-T Cells Are Clinically Active in Relapsed or Refractory Multiple Myeloma: An Updated Clinical Study

Background Bispecific CAR-T cell therapy has been proposed to mitigate some limitations of single-targeted CAR-T cells. CS1 plays a vital role in myeloma pathogenesis, and is also highly expressed on tumor cells in almost all patients (pts) with multiple myeloma (MM). Therefore, we aim to augment BCMA targeting with CS1. Bispecific CS1-BCMA CAR-T cells are effective in targeting MM cells in preclinical studies (Biomedicines 2021). Here we report the outcomes of 16 pts with refractory or relapsed (RR) MM in our phase I clinical trial (NCT04662099). Methods CS1-BCMA bispecific CAR contains a murine anti-CS1 scFv (clone 7A8D5) and a murine anti-BCMA scFv (clone 4C8) in tandem, and a 4-1BB costimulatory domain. The enrolled pts must have received at least 2 prior lines of therapy, and previous BCMA- or CS1-targeted immunotherapies were allowed. Pts were subjected to lymphodepleting regimens with cyclophosphamide (250 mg/m2, d-5 to d-3) and fludarabine (30 mg/m2, d-5 to d-3) daily prior to the CAR-T infusion (d0). Planned dose levels were 0.75x106, 1.5x106, and 3.0x106 CAR+T cells/kg, and repeated infusions were allowed. Primary objectives were incidence of adverse events (AEs). Cytokine release syndrome (CRS) and neurotoxicity were graded using the ASTCT criteria, and other AEs using CTCAE v5.0. Secondary objectives were overall response rate (ORR), overall survival (OS), duration of response (DOR), and progress-free survival (PFS). Response was assessed per the IMWG criteria 2016. Other objectives included in vivo kinetics of CAR-T cells, changes of soluble BCMA, immune assessment of infused CAR-T cells and patients’ lymphoid and immunosuppressive phenotype before and after infusion. Results As of July 31, 2022, 16 patients received the infusion of CS1-BCMA CAR-T cells and were included in the final analysis. Nine pts (56%) carried cytogenetic abnormalities, and 3 pts had extramedullary diseases (EMD) alone without detectable MM cells in bone marrow (BM). BCMA and CS1 were highly expressed on MM cells in BM by flow cytometry (mean BCMA+76.7% and CS1+ 96.5%) and EMD by trichromatic immunofluorescence. Nine pts (56%) were refractory, and 7 pts (44%) were relapsed and refractory. The median prior lines of treatment were 5.4 (range 2-10). All pts (100%) were exposed to bortezomib, 6 (38%) to ixazomib and 1 (6%) to cafilzomib. 13 pts (81%) received lenalidomide, 6 (38%) received thalidomide and 5 (31%) received pomadomide. 7 pts (44%) received autologous transplantation, 6 (38%) were exposed to daratumumab, 2 (13%) received selinexor and 2 (13%) had previous BCMA-targeted CAR-T therapy. Six pts (38%) experienced CRS, with median duration of 4 days (range 4-7). Grade 3 CRS occurred once and lasted for 5 days. Neurotoxicity was not observed. The ORR was 81% (13/16) for all patients, including 6 stringent complete, 3 very good partial, and 4 partial responses (Fig.1A). The ORR was 100% (13/13) for patients with MM cells in BM. Among them, 7 pts (54%) maintained ongoing response, 2 pts progressed after response, 1 withdraw from the study and 3 were lost due to poor compliance. Pt 16 had received prior anti-BCMA CAR-T treatment and achieved a partial response after CS1-BCMA CAR-T infusion. Although CAR-T cells infiltrated into the tumor tissue, response was not observed in the 3 pts with EMD alone (Fig.1B). Soluble BCMA decreased remarkably in peripheral blood (PB) and BM after infusion. At a median follow-up of 290 days, the median OS, PFS and DOR weren't reached, and OS, PFS and DOR at 12-month were 83.9%, 55.2% and 68.8%, respectively. On day 14 after infusion, CAR-T cells peaked at 138245 copies/ug DNA in PB (n=16) and 86654 copies/ug DNA in BM (n=13) by droplet digital PCR; CAR-T cells peaked at 493 CAR+T cells/ul PB (n=16) and 312 CAR+T cells/ul BM (n=13) by flow cytometry. CAR was detectable in vivo at a median of 6 months. Because CS1 is expressed on T and NK cells, we systematically evaluated the immune characteristics (differentiation, Fas, LAG3, PD1, TIM3, CS1) of infused products, absolute quantification and CS1 expression on T and NK cells in PB as well as myeloid derived suppressor cells, regulatory B and T cells in BM and PB. Further data will be released in the meeting. Conclusions Our study demonstrates bispecific CS1-BCMA CAR-T cells are clinical active with a good safety profile in heavily pretreated pts with MM, even after BCMA-targeted CAR-T cell therapy. Further studies are needed for pts with EMD alone. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Intravenous and Subcutaneous Administration of RG6234, a Novel GPRC5DxCD3 T-Cell Engaging Bispecific Antibody, Is Highly Active in Patients with Relapsed/Refractory Multiple Myeloma (RRMM): Biomarker Results from a Phase I Study

Background: RG6234 is a GPRC5DxCD3 T-cell engaging bispecific antibody with a novel 2:1 format. GPRC5D is overexpressed on multiple myeloma (MM) cells and concurrent binding of RG6234 to GPRC5D on tumor cells and CD3 on T cells results in immunological synapse formation and potent T-cell directed tumor cell killing (Eckmann et al. ASH 2022). An ongoing Phase I study (NCT04557150) is investigating the safety, clinical activity, pharmacodynamics (PD), and pharmacokinetics of intravenous (IV) and subcutaneous (SC) administration of RG6234 in patients with RRMM. Clinical activity was observed in both dose escalations (overall response rate [ORR] IV: 71.4%; ORR SC: 60.4%) (Carlo-Stella et al. ASH 2022). Here, we report clinical biomarker data comparing the PD effects of IV and SC administration and confirming the mechanism of action and potency of RG6234 when given by both administration routes. Methods: Exploratory biomarker analyses included dose-escalation data from 50 patients who received IV doses ranging from 6-10000μg and 46 patients who received SC doses ranging from 30-7200μg. In both cohorts, RG6234 administration was initiated with step-up dosing, reaching the target dose 2 weeks after the initial step dose. Peripheral biomarkers were evaluated using whole blood flow cytometry (IV, n=33; SC, n=40), plasma cytokines (IV, n=50; SC, n=46) and sBCMA (IV, n=36; SC, n=27) Protein Simple ELLA. MM cells were assessed at baseline and on-treatment by bone marrow (BM) aspirate flow cytometry and by BM biopsy CD138/CD8 immunohistochemistry. The data cut-off for the current analysis was June 8, 2022. Updated data will be presented. Results: RG6234 induced rapid PD changes at all tested doses. With IV administration, cytokines (IFNγ, TNFa, CXCL10, IL6, IL10, IL2, IL8) peaked at 4 to 24 hours after the first infusion and cytokine peaks diminished significantly with subsequent administrations. SC administration resulted in a delayed cytokine release that peaked at 24 to 72 hours after the first injection and was 2.2-24.2-fold lower than with IV dosing. Cytokine peaks were comparably low after each SC step-up administration. After SC dosing, T-cell margination was delayed by 20 hours and recovery to 50% of baseline levels took on average 56 hours longer than with IV dosing. Analysis of paired baseline and on-treatment BM biopsies (IV, n=19; SC, n=8) revealed a comparable degree of T-cell recruitment towards the tumor with IV and SC dosing (2.3- and 1.6-fold increase, respectively, in CD8 T-cell density in responders at the end of Cycle 1). Both IV and SC administration induced rapid depletion of MM cells in responding patients, as demonstrated by a decrease in plasma sBCMA 8 days after the initial administration (median 24.9% [n=27] and 26.2% [n=18] reduction from baseline, respectively). By Cycle 3 Day 1, median decreases from baseline in plasma sBCMA among responders were 91.8% (n=24) after IV administration and 95.6% (n=12) after SC administration. Moreover, at the end of Cycle 1, the majority of patients (15/16 [93.8%] in the IV group and 14/15 [93.3%] in the SC group) had <1% MM cells in BM based on flow cytometry. GPRC5D expression was detected at baseline in almost all patients with evaluable BM aspirate (44/45, median of 94.7% GPRC5D+ MM cells). Conclusions: Biomarker analysis indicates that RG6234 leads to T-cell engagement in the BM of patients with RRMM and demonstrates rapid and effective T-cell mediated anti-myeloma activity irrespective of the route of administration. Cytokine release, T-cell activation followed by BM infiltration, and MM cell depletion are early PD changes after IV and SC administration and precede clinical responses. Compared with IV dosing, SC administration reduces peak cytokine levels in Cycle 1, suggesting a more subtle immune activation and a potential safety benefit for patients.

S173: T-CELL ACTIVATION AND MYELOMA CELL KILLING CONFIRM THE MODE OF ACTION OF RG6234, A NOVEL GPRC5D T-CELL ENGAGING BISPECIFIC ANTIBODY, IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA

Background: RG6234 is a novel T-cell engaging bispecific antibody targeting G protein-coupled receptor 5D (GPRC5D) with a unique 2:1 format. GPRC5D is highly expressed on multiple myeloma (MM) cells and concurrent binding of RG6234 to GPRC5D and CD3 on T cells results in immunological synapse formation and potent T-cell directed tumor cell killing. An ongoing Phase 1 dose-escalation study (NCT04557150) is investigating the safety, clinical activity, pharmacodynamics (PD), and pharmacokinetics of RG6234 monotherapy in patients (pts) with relapsed/refractory MM (RRMM). Clinical activity was observed during dose escalation, and the safety profile was manageable (Riley et al. EHA 2022). Aims: Here, we present preliminary clinical biomarker data highlighting PD effects after intravenous (IV) administration that confirm the mechanism of action and high potency of RG6234. Methods: Exploratory biomarker analyses included data from pts treated with RG6234 doses ranging from 0.006mg to 4.8mg in dose escalation. RG6234 was administered as an IV infusion under a step-up dosing regimen, reaching the target dose not later than 2 weeks after the priming dose. Peripheral biomarkers were evaluated using whole blood flow cytometry (n=28), plasma cytokine Protein Simple ELLA (n=33), and plasma sBCMA Protein Simple ELLA (n=26). MM cells were assessed at baseline and on-treatment by bone marrow (BM) aspirate flow cytometry and by BM biopsy CD138/CD8 immunohistochemistry. Informed consent was obtained from participating pts. The clinical cut-off date for the current analysis was January 31, 2022. Results: PD changes were observed in peripheral blood at all tested doses. Cytokines (IFNg, TNFa, CXCL10, IL6, IL10, IL2, IL8) and sCD25 peaked at 4 to 24 hours (h) following the first administration, while cytokine peak magnitudes decreased at subsequent administrations. Cytokine release was followed by transient reduction of circulating T cells at 4 h after infusion, with partial recovery of peripheral T-cell counts by Day 8 after first administration. Elevation of sCD25 and IFNg in plasma (~3.2 and 33 median fold change from baseline, respectively) together with increase in T-cell proliferation (~4 median fold increase of Ki67+CD8+ T-cells) within 72 h post first infusion indicated T-cell activation. Analysis of a limited number of paired baseline and on-treatment BM biopsies (n=13) revealed that the density of CD8+ tumor-infiltrating T cells increased upon treatment in responders, indicating T-cell recruitment towards the tumor. RG6234 induced rapid depletion of MM cells, as demonstrated by a decrease of sBCMA in the plasma of responding pts already 8 days after the first administration (median 33.5% reduction from baseline in responding pts; n=15). Moreover, at the end of Cycle 1, the majority of pts (14/15) had <1% of MM cells in BM based on flow cytometry readout. GPRC5D expression was detected at baseline in all pts with evaluable bone marrow aspirate and >20 detectable MM cells (n=16). Updated exploratory biomarker data will be presented. Summary/Conclusion: Cytokine release, T-cell activation, BM infiltration, and MM cell depletion are early PD changes seen after treatment with RG6234 and precede clinical responses. These PD changes indicate that RG6234 leads to T-cell engagement in the BM of pts with RRMM and clearly demonstrate rapid and effective T-cell mediated anti-MM activity.

Study of Bone Marrow Mesenchymal Stem Cells Regulating NK Cells Function through Tigit/ CD226 in Patients with Multiple Myeloma

Introduction：Bone marrow microenvironment plays a significant supporting role in the proliferation, differentiation, migration, survival and drug resistance of myeloma cells. To detect the expression of the co-inhibitory receptor TIGIT and the co-stimulatory receptor CD226 on the surface of natural killer cells (NK cells) in multiple myeloma (MM) patients, and changes in the immune phenotypes and killing function of NK cells. Furthermore, to explore the underlying mechanism of bone marrow mesenchymal stem cells (BMSCs) regulating NK cells function through TIGIT/CD226 in patients with MM. The expressions of TIGIT, CD226, activated molecules NKG2D and CD107a and functional molecules IFN-γ and Perforin on the NK cells in bone marrow or peripheral blood were detedted by FCM. BMSCs and NK cells were co-cultured at a ratio of 1:4 in vitro, which was grouped according to different monoclonal antibodies (mAbs) added. After co-cultured, the changes in the expression of NKG2D, NKp30, NKp44 and CD69 of NK cells and IFN-γ concentration were examined by FCM. NK cells and U266 were co-cultured with different E:T ratio and the apoptosis of U266 was detected to evaluate the killing function of NK cells by FCM. The expressions of CD155 on BMSCs in the bone marrow were detected by FCM. The correlations between CD155 expression on BMSCs and the production of IFN-γ and Perforin in bone marrow NK cells, as well as clinical characteristics were analyzed.Results: 1. In bone marrow, expression of TIGIT was significantly higher in patients with NDMM than those in patients with CR and HC (both P values <0.01). CD226 was significantly lower in NDMM patients than those in patients with CR and HC (both P values <0.01). NK cell surface activated molecules NKG2D and CD107a, and functional molecules IFN-γ and Perforin were significantly decreased in NDMM (Fig. A). In peripheral blood, TIGIT, CD226 and above immune phenotype expression levels were basically consistent with the trend in bone marrow. 2. In co-culture experiments of BMSCs and NK cells, only added anti-TIGIT mAbs, compared to both added anti-TIGIT and CD226 mAbs, the immune phenotypes of NK cells significantly increased (Fig. B). And the concentration of IFN-γ in the co-culture supernatants also increased (Fig. C). 3. After co-cultured with BMSCs, NK cells were co-cultured again with U266 in vitro, the apoptosis level of U266 in TIGIT mAbs group increased than those in TIGIT+ CD226 mAbs group at different E: T ratio (Fig. D). 4. The expression of CD155, the ligand of TIGIT and CD226, increased notably on the surface of BMSCs in MM patients(P<0.01) (Fig. E). 5. The expression of CD155 on BMSCs was negatively correlated with the production of IFN-γ and perforin in bone marrow NK cells with NDMM patients. And it has correlation with clinical characteristics including β2-MG, LDH, ALB, Hb, ISS stage and R-ISS stage in patients with MM.Summary: BMSCs may regulate NK cells through TIGIT/CD226. High expression of TIGIT on NK cells may mediate the inhibitory effect of BMSCs in MM patients. Blocking TIGIT could restore NK cells activation and killing function. CD155, a common ligand of TIGIT and CD226, was overexpressed on BMSCs in NDMM patients, but was low expressed on bone marrow MM cells. And it has correlation with NK cells function and clinical characteristics, indicating that CD155 may be involved in the regulation of NK cells function by BMSCs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Genomic Discovery and Clonal Tracking in Multiple Myeloma By Cell Free DNA Sequencing

Summary: Development of drug resistance and clonal evolution is a major problem in multiple myeloma (MM) and may be due to genetic heterogeneity and clonal evolution but its longitudinal genetic characterization has been impractical, thus far. We have developed a genomic discovery method to monitor genetic evolution of the disease through whole exome sequencing and low-pass whole genome sequencing of myeloma-derived circulating cell-free DNA (cfDNA). Low-pass whole genome sequencing of 110 cfDNA samples from 75 randomly selected MM patients was used to develop an approach to predict the utility of further whole exome sequencing based on cfDNA concentration as a quick and cost-effective marker. Whole exome sequencing of cfDNA, matched normal blood cells, and bone marrow MM cells obtained from five myeloma patients at the same time point demonstrated that identification of clonal somatic mutations was robust and highly concordant between cfDNA and bone marrow, and provided sufficient resolution to define the subclonal composition by cfDNA. cfDNA allowed longitudinal tracking of disease burden and captured clonal evolution of MM. We provide a framework for effectively utilizing comprehensive discovery-oriented cfDNA sequencing in MM.Methods and Results: In order to estimate the subclonal composition of MM from “blood biopsy”,we collected 110 plasma samples from 75 randomly selected patients with multiple myeloma, who were undergoing treatment, and isolated cfDNA. As a control, we also isolated cfDNA from the plasma of 12 healthy blood donors. We prepared next-generation sequencing libraries and performed low-pass whole genome sequencing to a sequencing depth of 0.1-1 fold coverage across the entire genome for all of these samples. We deployed a computational approach that enables assessment of the fraction of myeloma-derived DNA in cfDNA by utilizing the relative ratio of somatic copy number alterations (SCNAs) from low pass whole genome sequencing data, after correcting for GC bias. Using a detection threshold cutoff of ~5%, we detected myeloma-derived cfDNA in 22% of total MM samples. In two patients from whom multiple serial samples were available, we found strong concordance between the myeloma DNA amount and serum free light chains, suggesting that low pass whole genome sequencing of cfDNA allows tracking of progression of MM longitudinally in patients.We then asked if whole exome sequencing (WES) and robust calling of somatic mutations is possible from cfDNA in myeloma patients. We performed deep whole-exome sequencing of cfDNA from five patients with multiple myeloma in whom we detected the presence of myeloma-derived circulating DNA by low-pass whole genome sequencing of cfDNA. We also performed whole exome sequencing of CD138+ cells that were flow-sorted from the bone marrow of the same patients, as well as CD45- cells as a matched normal sequencing control. cfDNA was obtained on the same day as the bone marrow aspirate, to rule out ongoing clonal evolution between the time of blood and bone marrow harvest. We first determined if canonical mutations that are typically seen in MM can be detected in cfDNA. We identified mutations in NRAS, KRAS, DIS3, TP53, and RB1, all known to be recurrently mutated in MM. These somatic mutations were identified in both the cfDNA as well as in the CD138+ sorted MM cells from the bone marrow aspirate.We then determined that somatic mutations in the BM are well represented in the cfDNA in general, and that the allelic fractions of somatic mutations correlated well between cfDNA and matched bone marrow biopsies (Pearson's r=0.704; 5 patients with a tumor fraction >16%). We found overall that 87% of 840 mutations in the bone marrow were detected in cfDNA and 92% of clonal mutations were also identified in cfDNA. Some distinct subclones could be identified either only in the cfDNA or only in the bone marrow. In one case, extensive clonal evolution occurred during treatment and individual subclones likely accounting for the development of drug resistant disease could be distinguished enabling the reconstruction of evolutionary trees.These results show that low-pass whole genome as well as whole-exome sequencing of cfDNA is feasible in patients with MM, can recapitulate the genetic evolution of MM in the bone marrow and reveals biologically and therapeutically relevant data when detectable. DisclosuresNo relevant conflicts of interest to declare.

Molecular Characterisation of TP53 Aberrations in 1,777 Myeloma Trial Patients

Molecular Characterisation of TP53 Aberrations in 1,777 Myeloma Trial Patients

Identification of a Patients-Derived CD34+ Myeloma Cell Population with Tumor-Propagating Capacity and Resistance to Anticancer Drugs

Novel therapeutic agents have substantially improved the clinical outcomes of multiple myeloma (MM), although the majority of MM patients eventually relapse and ultimately die from therapy-refractory disease. Early studies documented the presence of clonogenic populations with MM stem-like properties in MM patients, which presumably arise from post-germinal center B (GCB) cells and are resistant to current therapies. However, both biologic properties and their ability of these cells have remained unclear. In this study, we attempted to isolate these cells from phenotypically defined (CD38+138+19-45-) MM cells (hereafter PhMMs) in bone marrow (BM) and characterized them.BM samples were obtained from 48 MM patients, including 26 newly diagnosed (NDMM) and 22 with relapsed or refractory disease (RRMM) according to the protocol approved by the Institutional Review Board of the Kindai University Faculty of Medicine. Since the side population (SP) phenotype has been considered to represent the stem cell population in various types of normal and tumor tissues, we first analyzed the expression of HSC/HPCs markers (e.g. CD34, 49f, 90, 133) in the PhMMs SP population. Among the HSC/HPCs markers, the expression levels of CD34 was significant higher in SP cells compared with those in the main population (MP) cells (mean±SD, MFI of SP vs MP in PhMMs: 925.0±84.5 vs 29.2±4.2, n=5, p<0.01). The median count of CD34+ cells in PhMMs was 0.63% (interquartile range, 0.20), and a large number of these CD34+ PhMMs displayed the same Ig light chain with the unseparated myeloma cell population. Furthermore, FISH analyses on these cell populations confirmed the presence of cytogenetic abnormalities, such as t(11;14) and del(17p) in almost examined cells, while these abnormalities were not observed in the CD34+38-45dim HSC/HPCs subsets of the same patient, suggesting that CD34+ PhMMs contain clonotypic cells with genetic abnormalities. Next, we investigated the % of CD34+ PhMMs at various disease status. Notably, the % of CD34+ PhMMs was significantly higher in RRMM than in NDMM samples. Also, paired analysis showed that the % of CD34+ fraction was significantly higher after therapy than before therapy in PhMMs, indicating that CD34+ PhMMs are more resistant to anticancer drugs than CD34- PhMMs.Next, we performed a colony forming cell assay by platting CD34+ and CD34- PhMMs isolated from 12 patients (2.5×103/dish), respectively. While CD34- PhMMs developed no colony, CD34+ PhMMs vigorously generated many large colonies in all cases, demonstrating that CD34+ PhMMs but not CD34- PhMMs have strong clonogenic and proliferative capacities. When CD34+ and CD34- PhMMs from 4 MM patients were injected intravenously into sublethally irradiated NOD/shi-scid, IL-2RγKO Jic (NOG) mice (6.0×103-1.0×104 cells/mice), CD34+ PhMMs from all 4 patients were fund to be engrafted in the BM by 24 weeks after transplant, while none of the mice transplanted with CD34- PhMMs showed no engraftment by the same time point. Importantly, all engrafted cells from CD34+ PhMMs had the same Ig light chain with patient myeloma cells. Interestingly, both CD34+ and CD34- PhMMs were identified in BM of mice receiving CD34+ PhMMs. These results indicate that myeloma-propagating activity in PhMMs can be distinguished based on the CD34 expression. To gain insights into the cell origin of CD34+ PhMMs, we quantify the expression levels of selected markers associated with the development of B cell (ex, GATA2, PU1, EBF1, OCT2, BCL6, PAX5, IRF4, BLIMP1, and XBP1) by qRT-PCR analysis using purified CD34+ and CD34- PhMM pairs from 6 patients. Gene expression profiling revealed that CD34- PhMMs had a plasma cell signature (down-regulation of Pax5, and up-regulation of IRF4, BLIMP1, and XBP1), whereas CD34+ PhMMs demonstrated a more immature pre-GCB signature(down-regulation of BCL6, IRF4, and XBP1), except for the aberrant suppression of PAX5 and the expression of GATA2 .In conclusion, this study revealed that phenotypically restricted cell population (PhMMs) contains a minor CD34+ myeloma cell fraction that originate from pre-GCB cells and possess myeloma-propagating activities, as well as resistance to anticancer drugs. Thus, the identification of these clonogenic MM propagating cells provides a basis for better understanding the pathogenesis of MM and for designing novel therapeutic strategies aimed to eradicate total MM cells. DisclosuresNo relevant conflicts of interest to declare.

The E3 ligase HUWE1 inhibition as a therapeutic strategy to target MYC in multiple myeloma

Proteasome inhibitors have provided a significant advance in the treatment of multiple myeloma (MM). Consequently, there is increasing interest in developing strategies to target E3 ligases, de-ubiquitinases, and/or ubiquitin receptors within the ubiquitin proteasome pathway, with an aim to achieve more specificity and reduced side-effects. Previous studies have shown a role for the E3 ligase HUWE1 in modulating c-MYC, an oncogene frequently dysregulated in MM. Here we investigated HUWE1 in MM. We identified elevated expression of HUWE1 in MM compared with normal cells. Small molecule-mediated inhibition of HUWE1 resulted in growth arrest of MM cell lines without significantly effecting the growth of normal bone marrow cells, suggesting a favorable therapeutic index. Studies using a HUWE1 knockdown model showed similar growth inhibition. HUWE1 expression positively correlated with MYC expression in MM bone marrow cells and correspondingly, genetic knockdown and biochemical inhibition of HUWE1 reduced MYC expression in MM cell lines. Proteomic identification of HUWE1 substrates revealed a strong association of HUWE1 with metabolic processes in MM cells. Intracellular glutamine levels are decreased in the absence of HUWE1 and may contribute to MYC degradation. Finally, HUWE1 depletion in combination with lenalidomide resulted in synergistic anti-MM activity in both in vitro and in vivo models. Taken together, our data demonstrate an important role of HUWE1 in MM cell growth and provides preclinical rationale for therapeutic strategies targeting HUWE1 in MM.

The Necessary for Long-Term Follow-up of Mutated Genes and Clonal Evolutions in Multiple Myeloma Combined with cfDNA Assay

Introduction)Somatic mutations in multiple myeloma (MM) are strongly related to the clinical outcome and clonal evolution over the clinical course, and are a major problem. From a clinical viewpoint, although numerous novel drugs have been utilized, achieving long-lasting and complete remission remains difficult. Recent studies have elucidated the mutated genes using next-generation sequencing, and have examined how clonal change can be acquired in myeloma. In this study, we traced the transition of the somatic mutations of bone marrow tumor cells in patients with MM over a long-term follow-up. Furthermore, we compared the somatic mutations found in serum cell-free DNA (cfDNA) and mutated genes obtained from bone marrow myeloma cells. Material and Methods)Patients diagnosed with multiple myeloma who provided written informed consent to participate in the study were enrolled. Patients were treated by immuno-chemotherapy with or without radiation between 2000 and 2017 at our institute. Bone marrow aspiration and biopsy were performed at the time of diagnosis and upon disease progression. Around the time of bone marrow aspiration, serum was obtained from a peripheral blood sample for cfDNA analysis. Myeloma cells were separated from bone marrow samples with MicroBeads of CD138 antibody and genomic DNA was extracted. The peripheral blood samples derived from myeloma patients. The cfDNA was extracted from the serum using a Maxwell RSC cfDNA Plasma kit. Using genomic DNA derived from cfDNA and bone marrow, multiplex polymerase chain reaction (PCR) was performed, and a sequence library was then constructed with an Ion Custom Amplicon panel. The panel for the sequence library was designed using an Ion AmpliSeq DesignerTM. 126 targeted genes were selected. The genomes were sequenced using the Ion ProtonTM System. This protocol was approved by the institutional review board and the Genomic Review Board of the Japanese Foundation for Cancer Research. Result)We followed 7 patients' long term-clinical course and the transition of mutations (8.5 year average). The expression of myeloma driver genes, such as RAS, BRAF, and MYC, were not critical. We did, however, detect a relationship between an increase in the dominant mutated gene, such as TP53, DIS3, FAM46C, KDM6B, and EGR1 and poor prognosis in patients with myeloma. Next, we calculated the cfDNA concentrations from 34 cases. The cfDNA concentrations were significantly higher than 10 control cases (average 62.0 ng/mL (0-200 ng/mL) and 8.18 ng/mL (4.3-14.1 ng/mL), P=0.0046). The 2.5 year-progression free survival (PFS) during the first treatment of MM were tend to be poorer in the group with cfDNA&gt;50 ng/mL (72.9%) than the group with cfDNA&lt;50 ng/mL(25.9%), however there are no statistical significance (P = 0.15).We caluculated concordance rate of derived mutations from bone marrow MM cells and cfDNA in 7 cases. The somatic mutations found in serum cell-free DNA (cfDNA) and bone marrow MM cells were determined the correlation coefficients. However, there are few difference expression pattern in each source. In cfDNA assay, CREEP, EGR1, HDAC4, HDAC6, and JMJD1C were highly expressed as 57.1% (4/7) - 85.7% (6/7), and these results were almost the same as those for bone marrow MM cells. On the other hand, KDM1A (85.7%), PI3KCD (71.4%), and KDM3B (57.1%) were highly detected in cfDNA, although those were not frequently expressed in bone marrow. Discussion)Our data demonstrate the importance of the long-term follow-up of somatic mutations during the clinical course of myeloma. Serum cfDNA is a useful alternative source for detecting somatic mutations in MM patients during long-term follow-up. Disclosures Mishima: Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy. Yokoyama:Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy. Nishimura:Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy; Celgene K.K.: Honoraria. Hatake:Celgene K.K.: Research Funding; Janssen Pharmaceutical K.K.: Research Funding; Takeda Pharmaceutical Co.,Ltd.: Honoraria. Terui:Bristol-Myers Squibb K.K.: Research Funding; Bristol-Myers Squibb, Celgene, Janssen, Takeda, MSD, Eisai, Ono, and Chugai-Roche Pharmaceuticals Co.,Ltd.: Honoraria.

Targeting the Plasma Cell Specific Purine Metabolic Enzyme, AMPD1, Induces Multiple Myeloma Cell Death Accompanying NAD Depletion

Introduction. Novel agents have improved the prognosis of multiple myeloma (MM) patients. However, MM is still an incurable disease. In order to achieve a cure, it is necessary to develop novel therapeutic agents that are highly specific to MM cells and target different pathways from the present anti-MM agents used in the clinic. In the present study, we attempt to identify a specific molecule which is specifically expressed in plasma cells and MM cells, and to examine whether it can be a novel target for MM therapy. Materials and methods. Public available gene expression websites, GenomicScape and Genevestigator were utilized to study genes specifically expressed in human plasma cells and MM cells. To examine gene expression in MM cell lines, gene expression data set from the Cancer Cell Line Encyclopedia (CCLE) was analyzed. AMPD1 (AMP deaminase 1) gene expression in normal leukocytes and hematological malignancies were analyzed by RT-PCR. AMPD1 protein expression in MM cell lines and patient derived MM cells were examined by western blot and immunohistochemistry. Genes co-expressed with AMPD1 in MM cells were identified using public available gene expression datasets. Molecular pathway analysis was conducted using Molecular Signatures Database. Cell viability of MM cell lines, peripheral blood mononuclear cells (PBMCs) and patient derived bone marrow mononuclear cells treated by AMPD1 inhibitor (compound #3) (Admyre T et al. Chemistry &amp; Biology. 2014; 21: 1486-1496.) were analyzed by flow-cytometry after staining with 7AAD. Intracellular NAD and NADH concentrations in MM cell lines were analyzed using NAD / NADH assay kit. Detection of apoptosis in MM cell lines were examined by Annexin V and PI staining followed by flow cytometry analysis. Z-VAD-FMK (Caspase inhibitor) and Nec-1 (RIP1 kinase inhibitor) were used in combination with compound #3 to study the mechanism of AMPD1 inhibition induced MM cell death. Results. We identified several genes specifically expressed in human plasma cells and MM cells using public available gene expression websites. Among the identified genes, we focused on AMPD1, a purine metabolic enzyme that converts adenosine monophosphate (AMP) to inosine monophosphate (IMP), since this gene has not been previously studied in MM. We found that AMPD1 gene expression was limited to MM cell lines and patient derived MM cells through CCLE analysis and RT-PCR. AMPD1 protein expression was detected only in MM cell lines, bone marrow MM cells and extramedullary plasmacytomas. Genes associated with AMPD1 expression were related to hypoxic pathways. MM cell lines cultured under hypoxic condition had significantly higher AMPD1 expression compared to those cultured under normoxia, indicating that AMPD1 plays a significant role in MM cell surviving under hypoxic condition such as the bone marrow microenvironment. Compound #3 induced cell death in MM cell lines and patient derived MM cells, while toxicity against PBMCs and non-MM cells were minimal. Compound #3 was more effective against MM cell lines cultured under hypoxia compared to those under normoxia, reflecting the higher AMPD1 expression under hypoxia. Since, AMPD1 is associated with purine metabolism, we analyzed the intracellular concentration of NAD and NADH, which are major cellular metabolites, in MM cell lines post compound #3 treatment. Marked decrease of NAD concentration and NAD / NADH ratio was observed in compound #3 treated MM cell lines compared to control, demonstrating that AMPD1 inhibition depletes intracellular NAD leading to MM cell death. Compound #3 treated MM cell lines showed increase in Annexin V- PI+ and Annexin V+ PI+ fractions. Additionally, Z-VAD-FMK and Nec-1 treatment did not reverse cell death induced by compound #3, indicating a distinct mechanism of cytotoxicity by compound #3 from other anti-MM agents inducing apoptosis and necroptosis. Conclusions. This is the first report so far that AMPD1 is specifically expressed in MM cells. AMPD1 inhibitor showed specific cytotoxicity against MM cells, while toxicities against non-MM cells were minimal. Additionally, AMPD1 inhibitor was more effective under hypoxic condition, suggesting that AMPD1 inhibition works more efficiently in the bone marrow microenvironment. Considering the specificity against MM cells and its distinct mechanism of action from the present anti-MM agents, AMPD1 inhibition is a potent novel therapeutic strategy for MM. Disclosures Matsuoka: Bristol-Myers Squibb Corp.: Research Funding; Kyowa Kirin Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria.

Proteasome Inhibitors Impair the Innate Antiviral Immune Response and Potentiate Pelareorep-Based Viral Therapy in Multiple Myeloma

INTRODUCTION: Pelareorep is the infusible form of human reovirus (RV). Our single-agent phase 1 RV trial in relapsed multiple myeloma (MM) showed that pelareorep treatment selectively infected MM cells, as viral RNA was found in myeloma cells and not the bone marrow (BM) stroma. However, we did not observe apoptosis. Our ongoing phase 1 trial, which combines the proteasome inhibitor carfilzomib with RV, has demonstrated RV infection, apoptosis, and clinical responses. We investigated the molecular mechanisms behind the role of a PI (carfilzomib) in this setting. RESULTS: In all MM cell lines we tested (n=4), independently from their sensitivity to RV infection (Stiff et al., Mol Cancer Ther, 2016), viral replication and apoptosis was impaired when MM cells were directly exposed to PIs (carfilzomib and bortezomib). When this experiment was repeated in the setting of the bone marrow cellular fraction or peripheral blood mononuclear cells (PBMCs), it had the opposite effect, as the addition of PI to RV increased RV replication and apoptosis in MM cells. When we washed PBMCs after overnight exposure to RV+PI or to either single agent, then added MM cells, we observed higher infection and apoptosis in cancer cells co-cultured with RV+PI compared to levels from PBMCs treated with each of the single agents, suggesting that PIs increase the ability of PBMCs to serve as a reservoir for infectious reovirus. Monocytes (CD14+) can engage in phagocytosis of reovirus (Berkeley et al., Cancer Immunol Res, 2018), and accordingly we found that RV genome and capsid protein production were detected in CD14+ cells, but not in CD14-depleted PBMCs, and increased upon PI treatment compared to that in RV-treated CD14+ cells. Given that the NF-κB complex is a key proinflammatory signaling pathway associated with the early innate-antiviral immune response, and because PIs can block the degradation of the NF-κB inhibitor IκBα upon phosphorylation, we investigated the effect of the specific IκBα inhibitor Bay-11 in RV viral replication. Our data show that either PI or Bay-11 can inhibit RV-induced IκBα phosphorylation and its subsequent degradation upon RV infection in CD14+ cells, an effect associated with higher capsid formation in RV-treated CD14+ cells in combination with PI or Bay-11, compared to levels from RV alone. Cytokine profiling in PBMCs and CD14+ cells treated with RV in combination with either PI or Bay-11 showed a significant decrease in IFN-α and IFN-β (IFNs) levels and a concomitant increase in RV replication, in contrast to levels from RV alone (p&lt;0.001). We then decided to investigate whether CD14 depletion could affect RV delivery to the cancer cells invivo. Upon intra-femoral injection of 5TGM1 MM cells into syngeneic C57BL/KaLwRij mice, the mice in which the monocytes were depleted by clodronate-liposome treatment before intravenous RV injection showed lower capsid protein formation in the BM MM cells compared to that in mice where the monocytic population was intact. Because monocytes respond to infection by dividing into macrophages to eliminate pathogens, we wanted to investigate whether PI could impair this effect. Intriguingly, although higher levels of viral genome were detected in PI+RV-treated CD14+ cells compared to RV-treated cells (p&lt;0.01), PI abolished ex-vivo RV-induced monocyte differentiation (n=3, p&lt;0.001), without affecting the ability of RV-infected CD14+ cells to induce higher MM cell killing as compared to that from the CD14+ fraction treated with either single agent. Immune killing assays showed that RV-infected MM cells are more susceptible than non-infected cells to T cell effectors (p&lt;0.001), a mechanism that is further potentiated by the addition of PI. In fact, in contrast to the PI-induced blocking of monocyte expansion and activation in response to the virus (n=3, p&lt;0.001), our data suggest that the PI potentiate the expansion of CD8+ T cells, both ex vivo and in two RV+PI treated patients we analyzed so far. CONCLUSIONS: Here we report for the first time that PIs enhance pelareorep entry, infection, and killing of myeloma cells through its effect on the CD14+ fraction. Reovirus infection and replication within CD14+ cells are augmented by PI-induced NF-κB inhibition of the early innate pro-inflammatory immune response. We also report for the first time that carfilzomib induces direct T cell activation and potentiates T cell killing activity against RV-infected MM cells. Disclosures Krishnan: Takeda: Research Funding; Celgene, Z Predicta: Other: Stock Ownership; Amgen, Takeda: Speakers Bureau; Sutro BioPharma, zPredicta: Consultancy; Celgene, Janssen, Sanofi, BMS: Consultancy. Sborov:Celgene: Honoraria; Janssen: Consultancy. Hofmeister:Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Imbrium: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees.

The induction of myeloma cell death and DNA damage by tetrac, a thyroid hormone derivative

Multiple myeloma (MM) is a plasma cell malignancy in which involvement of the thyroid hormone-integrin αvβ3 pathway was shown, and pharmacologic inhibition of this pathway is a rational approach to disease management. A thyroid hormone derivative, tetraiodothyroacetic acid (tetrac), which inhibits l-thyroxine (T4) and 3,5,3'-triiodo-l-thyronine (T3) binding to αvβ3 integrin, was studied in five MM cell lines and primary bone marrow (BM) MM cells. Tetrac inhibited MM cell proliferation (absolute cell number/viability) and induced caspase-dependent apoptosis (annexin-V/PI and cell cycle). Activation of caspase-9 and caspase-3 was further demonstrated. Moreover, DNA damage markers, ataxia-telangiectasia-mutated (ATM) kinase, poly ADP-ribose polymerase (PARP-1) and histone γH2AX were induced by tetrac. The various tetrac-initiated effects were attenuated by Arg-Gly-Asp (RGD) peptide, suggesting integrin involvement. Primary BM mononuclear cells were harvested from MM patients (n = 39) at various disease stages. Tetrac-induced apoptosis (12/17 samples) and sensitized the cytotoxic action of bortezomib (6/9 samples). Lastly, expression of plasma membrane integrin αvβ3 was shown not only in the malignant plasma clone, but also in other cell populations within the BM samples (n = 25). Tetrac is anti-proliferative and pro-apoptotic in MM and cells may offer a therapeutic approach for this disease.

Anterior mediastinum invasion by multiple myeloma: A case report.

Multiple myeloma (MM) is a clonal proliferation of malignant plasma cells in the bone marrow (BM) that secretes monoclonal paraproteins in the blood serum and urine. Bone marrow MM cells can invade and damage the functions of other tissues and organs, such as the lungs, spleen, liver, pancreas, kidneys and lymph nodes. However, the invasion of MM cells primarily located in the BM to the anterior mediastinum at the site of the thymus is an extremely rare event. The current study reports the case of a 53-year-old female who presented with MM with involvement of the anterior mediastinum. The diagnosis was based on clinical imaging analyses and the results from BM and laboratory examinations, local biopsy pathology and immunohistochemistry. The patient was administered two courses of chemotherapy (epirubicin, dexamethasone and thalidomide). As a result, the tumor reduced in size, but the laboratory examination indicated no significant change. Next, the patient was switched to one course of PAD chemotherapy (bortezomib, epirubicin and dexamethasone). The original tumor was significantly reduced in size following this chemotherapy, and all the indicators improved. The present study suggests that invasion of the thymus by MM may lead to immune disturbance arising from the abnormal thymus gland. In the clinic, extramedullary plasmacytoma in the thymus should be carefully distinguished from thymoma.

An APRIL Based Chimeric Antigen Receptor to Simultaneously Target BCMA and TACI in Multiple Myeloma (MM) Has Potent Activity in Vitro and in Vivo

Immune based strategies have shown early promise in multiple myeloma (MM), and are likely to prove a valuable addition to the therapeutic platforms for these patients. B cell maturation antigen (BCMA) is a promising therapeutic target with selective expression on plasma cells, including MM cells. In the first report of BCMA targeting therapies, an anti-BCMA scFv-based chimeric antigen receptor (CAR) had clinical activity, albeit with short lived responses (Ali et al, 2016). Observed challenges included low expression level of BCMA, shedding of BCMA and potential loss of expression.In addition to BCMA, MM cells also express transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) which functions in plasma cell survival and differentiation. TACI is selectively expressed within lymphoid tissue and at reduced levels compared to BCMA, as measured by RT-PCR on normal tissues. TACI and BCMA share a common ligand called a proliferating ligand (APRIL), a compact self-protein forming a trimer and binding with nanomolar affinity to TACI and BCMA. The natural trimerization of APRIL would serve to enhance cell binding, especially important in the case of low levels of target antigen. Concurrent targeting of two tumour antigens, by increasing antigen density, would improve efficacy as well as reducing the risk of antigen negative disease escape.We quantified APRIL targets on primary MM cells by flow cytometry and observed BCMA expression in all of 41 patients tested (median 1051, range 105-8323 antibodies bound per cell, ABC) with most patients (78%) also expressing TACI (333, 0-21301 ABC). We designed an APRIL based 3rd generation chimeric antigen receptor (APRIL-CAR) comprising an engineered APRIL protein as antigen-binding domain. Peripheral blood mononuclear cells (PBMCs) were activated, transduced with APRIL-CAR and CD56-depleted before testing against cell lines expressing a wide range of BCMA and TACI (1005-1.5x10^5 and 2200-1.6x10^5 ABC respectively). We confirmed the cytolytic capability of APRIL-CAR against engineered targets, even at the low antigen levels observed in primary cells. Using standard chromium release, there was significant cytolysis of the lowest antigen expressers, with 40.9±3.8% (mean±SEM) and 42.6±5.7% cell death in SUPT1BCMAlo and SUPT1TACIlo respectively cf 15.0±5.8% for control SUPT1NT targets, at an E:T ratio of 4:1 (n=4, p<0.01 and p<0.05). When we lowered the E:T ratio, APRIL-CAR continued to show potent activity, with cytolysis of SUPT1BCMAlo/ SUPT1TACIlo targets at 1:10 (71.7±3.8% and 70.4±6.3% cell death respectively cf 21.6±5.8% for SUPT1NT, n=4, p<0.001 for both) and human myeloma cell lines at 1:32 (MM.1s and U266, 56.2±3.9% and 25.1±3.9% target cytolysis by APRIL-CAR and 4.7±11.6% and 6.6±3.9% by PBMCs NT respectively, n=5, p<0.01 for both).We confirmed cytolytic activity of APRIL-CAR against primary MM cells. Allogeneic PBMCs transduced with APRIL-CAR and CD56-depleted were co-cultured 1:1 with CD138+ bone marrow MM cells from 5 patients. Despite variable BCMA and TACI expression between patient samples (BCMA 1224-7728 and TACI 563-1213 ABC), tumour cytolysis was evident in all cases (APRIL-CAR 72.9±12.2% specific cell death cf media control 2.8±15.3%; p<0.05).Finally we tested the in vivo activity of APRIL-CAR in an intramedullary model established with tail vein injection of MM.1s.Fluc cells into NOD scid gamma (NSG) mice. Animals were analysed by bioluminescent imaging and FACS analysis of bone marrow for tumour cells. Complete clearance of established intramedullary disease was seen 5 days after infusion of 5x10^6 APRIL-CAR positive PBMCs in 12/12 animals. Elimination of tumour cells was confirmed in the bone marrow (p<0.01) with concurrent detection of APRIL-CAR T cells by FACS analysis.In summary we report for the first time a 3rd generation CAR utilising a natural ligand to simultaneously target two antigens found on MM cells. Our APRIL-based CAR demonstrated potent activity at the low levels of BCMA and TACI found in primary MM cells, at low (physiological) E:T ratios, and in an intramedullary MM model. This APRIL based CAR holds particular promise and deserves testing in clinical studies. DisclosuresLee:Bloodwise: Research Funding; Autolus Ltd: Patents & Royalties: APRIL based chimeric antigen receptor. Draper:Autolus Ltd: Equity Ownership, Patents & Royalties: APRIL based CAR. Chaplin:Autolus Ltd: Equity Ownership. Philip:Autolus: Equity Ownership. Thomas:Autolus Ltd: Employment. Kokalaki:Autolus Ltd: Employment. Francis:Autolus Ltd: Employment. Yong:Autolus Ltd: Equity Ownership, Patents & Royalties: APRIL based chimeric antigen receptor; Janssen: Research Funding. Pule:Autolus Ltd: Employment, Equity Ownership, Research Funding; UCL Business: Patents & Royalties; Amgen: Honoraria; Roche: Honoraria.

Comprehensive Genetic Interrogation of Circulating Multiple Myeloma Cells at Single Cell Resolution

Continuous genomic evolution has been a major limitation to curative treatment of multiple myeloma (MM). Frequent monitoring of the genetic heterogeneity in MM from blood, rather than serial bone marrow (BM) biopsies, would therefore be desirable. We hypothesized that genomic characterization of circulating MM cells (CMMCs) recapitulates the genetics of MM in BM biopsies, enables MM classification, and is feasible in the majority of MM patients with active disease.Methods: To test these hypotheses, we developed a method to enrich, purify and isolate single CMMCs with a sensitivity of at least 1:10(5). We then performed DNA- and RNA-sequencing of single CMMCs and compared them to single BM-derived MM cells. We determined CMMC numbers in 24 randomly selected MM patient samples and compared them to numbers of circulating MM cells obtained by flow cytometry. We performed single-cell whole genome amplification of single cells from 10 MM patients, and targeted sequencing of the 35 most recurrently mutated loci in MM. A total of 568 single primary cells representing CMMCs, BM MM cells, CD19+ B lymphocytes, CD45+CD138- WBC from these patients were subjected to DNA-sequencing. By processing 80 single cells from four MM cell lines with known mutations we determined the mean sensitivity of mutation detection in single cells to be 93 ± 9%. In addition to DNA-sequencing we also isolated 57 single MM cells from the BM and peripheral blood of two MM patients and performed whole transcriptome single cell RNA-sequencing.Results: In 24 randomly selected MM patient samples we detected >12 CMMCs per 1ml of blood in all 24 patients. In comparison, by flow cytometry, we detected ≥10 CMMCs per 10(5) white blood cells in 10/24 cases (42%), ≥1 CMMC but ≤ 10 CMMCs in 13/24 cases (54%), and < 1 CTCs in 1/24 patients (4%). Mutational analysis of 35 recurrently mutated loci in 335 high quality single MM cells from the blood and BM of 10 patients, including one MGUS patient, revealed the presence of a total of 12 mutations (in KRAS, NRAS, BRAF, IRF4 and TP53). All targeted mutations that were detected by clinical-grade genotyping of bulk BM were also detected in single cell analysis of CMMCs. While in most patients, the fraction of mutated single cells was similar between blood and BM, in three patients, the proportion of MM cells harboring TP53 R273C, BRAF G469A and NRAS G13D mutations was significantly higher in the blood than in the BM, suggesting a different clonal composition. We developed an analytical model to predict whether a genetic locus underwent loss of heterozygosity, using the distribution of known allelic fractions of previously described mutations in MM cell lines as a benchmark. In two patients who simultaneously harbored two mutations, we predicted a BRAF G469E and a KRAS G12C mutation to be heterozygous, whereas the loci harboring a TP53 R273C and a TP53 R280T mutation were predicted to be associated with LOH with high statistical confidence. Whole transcriptome single cell RNA-sequencing of 57 MM cells from the BM and peripheral blood of two patients showed >3,700 transcripts per cell. Single-cell RNA-sequencing allowed for a clear distinction between normal plasma cells and MM cells, either based on analysis of CD45, CD27, and CD56 alone, or by unsupervised hierarchical clustering of detected transcripts in single cells. In addition, single cell CMMC expression analysis could be used to infer the existence of key MM chromosomal translocations. For example, CCND1 and CCND3 were highly upregulated in single MM cells from the blood and BM of two patients, whose MM was found by FISH analysis to harbor a t(11;14) and a t(6;14) translocation, respectively.Conclusion: We demonstrate that extensive genomic characterization of MM is feasible from very small numbers of CMMCs with single cell resolution. Interrogation of single CMMCs faithfully reproduces the pattern of somatic mutations present in MM in the BM, identifies actionable oncogenes, and reveals if somatic mutated loci underwent loss of heterozygosity. Single CMMCs also reveal mutations that are not detectable in the BM either by single cell sequencing or clinical grade bulk sequencing. Single cell RNA-sequencing of CMMCs provides robust transcriptomic profiling, allowing for class-differentiation and inference of translocations in MM patients. DisclosuresRaje:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Eli Lilly: Research Funding.