OPM2 Cells Research Articles

Abstract LB157: Discovery of HP537, a potent and selective p300/CBP inhibitor for the treatment of hematologic malignancies

Abstract E1A-binding protein P300 (p300) and its paralog CREB binding protein (CBP) are key acetyl transferases (HAT) and transcriptional co-factors. p300/CBP bromodomain (BRD) inhibition reduces acetylation of H3K27 at enhancers, leading to suppression of enhancer-driven gene expression, such as MYC and IRF4 that are critical for the initiation, progression, and chemoresistance of hematological malignancies. Thus, p300/CBP inhibition is a highly potential therapeutic strategy for the treatment of hematological malignancies, including multiple myeloma (MM), non-Hodgkin lymphoma (NHL) and leukemia. We have discovered HP537 that competitively inhibits an acetylated peptide substrate to bind BRD of p300/CBP with IC50 < 5 nM. HP537 does not inhibit the enzymatic activity of HAT domain of p300/CBP and HAT1, nor has binding affinity for other BRD containing proteins including BRD4 and BRD9. HP537 inhibits proliferation of a wide range of cell lines derived from MM, leukemia and lymphoma with IC50 values ranged from 0.01 μM to 0.4 μM. Protein expression levels of acetylated H3K27, c-Myc and IRF4 are significantly decreased by HP537 in MM, NHL and acute myelogenous leukemia (AML) cell lines in a dose- and time-dependent manner. HP537 robustly and dose-dependently inhibits tumor growth in OPM-2 (MM), MOLM-16 (AML), and KARPAS-422 (NHL) cell line derived tumor xenograft (CDX) mouse models. Down-regulation of c-Myc and IRF4 are also observed in the tumor samples. In conclusion, HP537 is a novel, potent and selective inhibitor of p300/CBP BRD. The pre-clinical data supports the clinical development of HP537 for the treatment of hematological malignancies. A FIH study of HP537 in cancer patients is planned to start in 2024. HP537 structure will not be disclosed. Citation Format: Jing Li, Zhilin Tu, Jian Peng, Lei Fan, Hua Yu, Fei Wang, Xinghai Li. Discovery of HP537, a potent and selective p300/CBP inhibitor for the treatment of hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB157.

Abstract 1318: Targeting multiple myeloma with BCMA-CAR NK cells expressing a GPRC5D-NKG2D bispecific antibody

Abstract Recently developed therapeutics for multiple myeloma (MM) include targeted therapy, immunomodulatory drugs, and immunotherapy, the latter of which consists of B-cell maturation antigen (BCMA)-CAR cells, bispecific antibodies (BsAbs) to engage T cells. However, challenges including relapse and toxicity still exist. The orphan G protein-coupled receptor, class C group 5 member D (GPRC5D), emerged as a novel target for MM therapy. Therefore, following our established system [Cancer Immunol Res PMID: 29769244; Leukemia, PMID:24067492], we generated a BsAb by fusing anti-GPCR5D single-chain variable fragment (scFv) and an anti-NKG2D scFv (GPRC5D-NKG2D BsAb) in a human IgG4 platform and expressed it from BCMA-CAR and soluble IL15 natural killer (NK) cells (s15.BsAb.BCMA-CAR NK) to address the current known limitations. Our data showed that s15.BsAb.BCMA-CAR NK cells secreted GPRC5D-NKG2D BsAb both in vitro and in vivo. To evaluate the function of GPRC5D- NKG2D BsAb expressed from NK cells in vivo, we treated MM tumor-bearing mice with the purified antibody. The data showed that GPRC5D-NKG2D BsAb demonstrated a significantly better survival effect than saline (P≤0.01). We tested s15.BsAb.BCMA-CAR NK cells for cytotoxicity against a homogenous tumor cell mode (BCMA + GPRC5D + ) and a heterogeneous MM tumor cell model, including 50% BCMA + GPRC5D + , 25% BCMA ─ GPRC5D + , and 25% BCMA + GPRC5D ─ cells. Real-time cell analysis (RTCA) data showed s15.BsAb.BCMA-CAR NK cells killed tumor cells in vitro better than both BCMA-CAR NK cells and empty vector-transduced NK cells in an antigen-dependent manner. To test the efficacy of s15.BsAb.BCMA-CAR NK cells in vivo, we established a heterogeneous MM tumor cell mouse model, by mixing BCMA + GPRC5D + OPM2 cells and BCMA + GPRC5D ─ OPM2 cells at the ratio of 2:1. MM mice were treated with “off-the-shelf” different construct NK cells. The results showed that s15.BsAb.BCMA-CAR NK cells significantly prolonged survival compared to other NK cells(s15.BsAb.BCMA-CAR vs. BCMA-CAR P = 0.0012). Our data demonstrated that this all-in-one, multifaceted modality provides 2 ready-to-use biologics simultaneously, CAR NK cells and BsAb, for MM treatment. Citation Format: Lei Tian, Victoria Chen, Tasha Barr, Zhiyao Li, Yuqing Chen, Jiaying Zhang, Michael A. Caligiuri, Jianhua Yu. Targeting multiple myeloma with BCMA-CAR NK cells expressing a GPRC5D-NKG2D bispecific antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1318.

Acute suppression of translation by hyperthermia enhances anti-myeloma activity of carfilzomib.

Hyperthermia is a unique treatment option for cancers. Multiple myeloma (MM) remains incurable and innovative therapeutic options are needed. We investigated the efficacy of hyperthermia and carfilzomib in combination against MM cells. Although MM cell lines exhibited different susceptibilities to pulsatile carfilzomib treatment, mild hyperthermia at 43℃ induced MM cell death in all cell lines in a time-dependent manner. Hyperthermia and carfilzomib cooperatively induced MM cell death even under suboptimal conditions. The pro-survival mediators PIM2 and NRF2 accumulated in MM cells due to inhibition of their proteasomal degradation by carfilzomib; however, hyperthermia acutely suppressed translation in parallel with phosphorylation of eIF2α to reduce these proteins in MM cells. Recovery of β5 subunit enzymatic activity from its immediate inhibition by carfilzomib was observed at 24h in carfilzomib-insusceptible KMS-11, OPM-2, and RPMI8226 cells, but not in carfilzomib-sensitive MM.1S cells. However, heat treatment suppressed the recovery of β5 subunit activity in these carfilzomib-insusceptible cells. Therefore, hyperthermia re-sensitized MM cells to carfilzomib. Our results support the treatment of MM with hyperthermia in combination with carfilzomib. Further research is warranted on hyperthermia for drug-resistant extramedullary plasmacytoma.

Bortezomib-encapsulated metal-phenolic nanoparticles for intracellular drug delivery.

Bortezomib (BTZ) is an important boronate proteasome inhibitor that is widely used in cancer therapy. However, the clinical application of BTZ suffers from poor stability and serious adverse effects. Herein, we fabricated metal-polyphenol nanoparticles for the covalent encapsulation of BTZ. BTZ-encapsulated tannic acid (TA)-Fe3+ nanoparticles can be prepared by mixing BTZ, TA, and ferric chloride owing to the formation of metal-polyphenol coordination interaction and dynamic boronate ester bonds. The BTZ-encapsulated TA-Fe3+ nanoparticles (BTZ NPs) are stable in physiological environment (pH 7.4) with minimal drug leakage. However, BTZ NPs can be disassembled in an acidic environment. Therefore, BTZ can be rapidly released from BTZ NPs in an acidic environment (pH 5.0). More than 50% BTZ can be released from BTZ NPs after 8 h incubation at pH 5.0. BTZ NPs exhibited high cytotoxicity against human osteosarcoma Saos-2 cells and human multiple myeloma OPM-2 cells. The metal-polyphenol nanoparticles can be a promising nanoplatform for the delivery of BTZ with simultaneously enhanced therapeutic efficacy and reduced side effects.

A Functional Genomic Screen to Identify Novel Genes Involved in Multiple Myeloma Tumour Development

Multiple myeloma (MM) is an incurable malignancy characterised by uncontrolled proliferation of plasma cells (PCs) in the bone marrow (BM). MM is a genetically heterogeneous disease with each patient's PCs harbouring unique genetic mutations, however the development of MM tumours is not only dependent on the underlying genetics but also on the selective pressures applied by the BM microenvironment. Hence, we hypothesise that identifying the dependencies which promote MM cell outgrowth in the BM microenvironment will allow for the identification of new drug targets. This project aims to use an in vivo functional screen, combining CRISPR-Cas9 gene editing, using a single guide RNA (sgRNA) library targeting thousands of genes, with a murine model of MM, to identify novel genes involved in MM tumour development. The Bassik human apoptosis and cancer CRISPR knockout library (Addgene #101926) was used to transduce MM cells with 31,324 unique sgRNAs targeting 3,015 genes and 1,500 control regions. The human MM cell line OPM2 was transduced with the Cas9 transgene (FuCas9GFP), followed by the sgRNA expression vector (pMCB320) generating OPM2-Cas9-sgRNA cells. These cells were then injected (5x10 5 cells/mouse, n=8 mice + 1 tumour-naive control) into the tibiae of immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl/SzJ (NSG) mice. Four weeks post-injection, the primary tumour within the injected tibia was isolated and sgRNA frequencies were assessed via next generation sequencing and compared with that of the initial library and the injected cells using the MAGeCK algorithm. To identify genes that play roles in MM tumour formation in patients, we assessed the expression of our identified genes in CD138-selected BM PCs from newly diagnosed MM patients (n=155) when compared with normal PCs from healthy donors (n=5) in microarray dataset E-MTAB-363 (ArrayExpress). Furthermore, to identify potential pathway involvement of our identified genes we performed gene set enrichment analysis (GSEA) using the University of California San Diego and Broad Institute developed GSEA software, comparing to Hallmark Gene Sets. Comparison of the in vitro cultured cells with the initial library identified 34 genes with undetectable (p<0.05) sgRNAs post in vitro culture, suggesting these genes play key roles in the survival of OPM2 cells. This list contained a number of genes known to control MM cell survival, such as IRF4 and CCND2. Additionally, sgRNAs targeting a further 115 genes were significantly depleted (p<0.05) in vitro and were completely undetectable in vivo, suggesting that these are implicated in cell proliferation and tumour formation in vivo. This gene list contained some already known MM drivers such as MYC, KRAS and DIS3. Furthermore, we identified 28 genes where CRISPR knockout had no significant effect in vitro but were critical dependencies for tumour development, with sgRNAs being undetectable (p<0.05) in vivo. This list contained genes known to be important for MM and BM stromal cell interaction such as ALCAM and SPP1. Notably, 25% of the genes identified as critical dependencies for OPM2 cells in vitro and/or in vivo are significantly upregulated >2-fold (p<0.05; limma) in MM patient PCs when compared with normal PCs (E-MTAB-363), suggesting these may play a role in tumour development in patients. Gene set enrichment analysis revealed that the genes that were essential in vitro were predominantly involved in cell survival related pathways such as cell cycle regulation (G2M checkpoint) and DNA replication/repair; however, the uniquely in vivo genes are predominantly involved in cell metabolism pathways such as MTORC1 signalling, oxidative phosphorylation and glycolysis. Overall, this study has successfully used a functional genetic screen to identify novel gene dependencies required for in vitro MM cell growth and in vivo MM tumour development. A secondary genetic screen will be performed in vivo to validate and refine this list of candidate genes. These validated genes will be further investigated using patient derived data sets to determine if they represent common vulnerabilities in patients and in multiple MM cell lines to investigate their specific mechanistic and pathway involvement. Identification and understanding the driving dependencies which promote MM cell outgrowth in the BM microenvironment will allow for the identification and development of new drug targets to improve patient outcomes.

Inhibiting FOXM1 Sensitizes Myeloma Cells to BCL2 Inhibitor Venetoclax By Repressing MYC Pathway

Background: Despite improved 5-year survival in multiple myeloma (MM), relapsed and/or refractory multiple myeloma (RRMM) remains a big challenge. Forkhead box transcription factor FOXM1 is a key regulator of metabolism and cell cycle progression in RRMM. FOXM1 is highly expressed in OPM2 and Delta47 cells compared to 9 other myeloma cell lines. Inhibiting FOXM1 function by deleting the FOXM1 gene or using the small-molecule FOXM1 inhibitor, NB73, suppresses OPM2 and Delta47 cells in vitro and in vivo. We hypothesized that FOXM1-targeted inhibition of RRMM may be deepened by combining NB73 with established or candidate myeloma drugs. Materials and methods: We used 3 tools to develop FOXM1-targeted combinatorial therapies by: (1) combining NB73 with 6 widely used myeloma drugs; (2) repurposing screens of cancer drugs in FOXM1-knockout (FOXM1 KO) vs FOXM1-proficient (FOXM1 WT) OPM2 cells; and (3) combining NB73 with compounds recommended by CMap analysisof FOXM1 KO vs FOXM1 WTcells. RNA seq analysis was used to elucidate pathways underlying drug synergy. Results: We adopted ZIP drug synergy scoring assay to examine the interactions between NB73 and the 6 myeloma drugs in MM cells. ZIP score takes the merits of both Loewe score that appraises drugs targeting the same pathway and Bliss score that appraises drugs targeting different pathways. Venetoclax, a BCL2 inhibitor treating MM with t(11;14) translocation, synergized with NB73 in killing OPM2 and Delta47 cells. In contrast, taking advantage of FOXM1 WT and FOXM1 KO MM cells, we conducted repurposing screens of FDA-approved anti-cancer drug library (NCI AOD X) in these cells. We identified Dasatinib and Panobinostat that killed FOXM1 KO OPM2 cells much more than FOXM1 WT cells. We also identified 3 drugs from the repurposing screens in FOXM1 WT and FOXM1 KO Delta47 cells. Thirdly, CMap, a large-scale compendium of functional perturbations in cultured human cells coupled to a gene expression read-out, implicated a synergy between NB73 and Thapsigargin that was verified by ZIP drug synergy scoring assay. Collectively, we have identified a total of 7 drugs (6 FDA-approved) synergizing with NB73 in killing OPM2 and/or Delta47 cells in which FOXM1 is highly expressed. Since Venetoclax is a current myeloma drug, we have focused on how inhibiting FOXM1 sensitizes MM cells to Venetoclax. Firstly, we showed the synergy between NB73 and Venetoclax in inducing apoptosis in OPM2 and Delta47 cells. We used the experimental condition in apoptosis assay to treat OPM2 cells for RNA sequencing study. Principal Component Analysis of RNA seq data indicated that the clusters of NB73 and the combo were well separated from each other and from the clusters of DMSO and Venetoclax. Gene Set Enrichment Analysis of these 4 groups presented many changes in tumor-associated pathways. Pathways regulated by a single drug ( e.g. MYC, p53, IFN-α) were further aggravated by the combo. Pathways ( e.g. Apoptosis) were not enriched by NB73 or Venetoclax, but enriched by the combo. Hyperactivation of MYC pathway is prevalent in MM. The combo significantly repressed MYC, PLK1, CCNA2 and CDC20 in MYC pathway through several mechanisms. For example, both NB73 and Venetoclax decreased MYC RNA, and the combo further lowered MYC, showing a “sum-up” effect. In contrast, the single drug lightly increased CDC20, but the combo significantly decreased it, presenting a “ de novo” effect. Since these 4 genes are important tumor promoters and inhibiting any one markedly represses tumor growth, we are addressing how the combo inhibits them. Ongoing experiments: We transplanted OPM2 cells into NSG mice and established the MM xenograft for in vivo test. NB73 is being given via sub-Q and Venetoclax is being given via oral gavage (n=5/group, 4 groups). The tumor burden will be monitored by live imaging every 2 weeks because OPM2 cells express Luciferase and the survival curve will be drawn. We are recruiting MM patients for bone marrow cells in order to develop a simplified protocol of culturing MM cells ex vivo that will be used to test new drug combinations targeting FOXM1 or other molecular vulnerabilities in MM. Conclusion: We identified FOXM1-targeted therapies consisting of novel FOXM1 inhibitor NB73 and FDA-approved drugs in vitro. We are addressing how the combo represses MYC pathway to sensitize MM cells to BCL2 inhibitor Venetoclax.

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.

A CD38/CD28xCD3 Trispecific T-Cell Engager (TCE) As a Potentially Active Agent in Multiple Myeloma Patients Relapsed and/or Refractory (RRMM) to Anti-CD38 Monoclonal Antibodies (mAbs)

Background: Despite great advances in the treatment of RRMM, including anti-CD38 mAbs, patients become resistant and therapeutic options are limited. Some factors involved in resistance to anti-CD38 mAbs have been described. On the one hand, their efficacy is partially dependent on baseline CD38 expression in MM plasma cells (MMPC), but the treatment induces a rapid and sustained CD38 downregulation. On the other hand, their tumor-killing activity relies on NK cells, but its frequency rapidly decreases following initiation of treatment, and persistent NK cells exhibit a dysfunctional phenotype. Given the reduction of CD38 levels in MMPC and the impaired NK-cell activity in resistance to anti-CD38 mAbs, we hypothesized that RRMM patients previously treated with these agents could benefit from T-cell-based immunotherapeutic strategies that depend less on CD38 antigen density and NK cell function. Aim: Evaluate a CD38/CD28xCD3 trispecific TCE (SAR442257) as a potential therapeutic agent in the RRMM setting. Results: CD38 expression in MMPC and NK-cell frequency in bone marrow were significantly reduced in 29 RRMM patients previously exposed to anti-CD38 mAbs vs 45 newly-diagnosed MM (NDMM) patients ( P <.001 and P =.013, respectively). Moreover, the percentage of CD57+ NK cells was significantly lower in RRMM than NDMM patients ( P =.006), suggesting that both CD38 downregulation and a contraction of cytotoxic NK cells may be associated with resistance to anti-CD38 mAbs. By contrast, T cells did not change significantly between stages, which ignited further interest in the preclinical efficacy of the CD38/CD28xCD3 TCE. First, we observed that, contrary to isatuximab and daratumumab, CD38/CD28xCD3 TCE did not reduce surface CD38 levels in the MOLP 8, RPMI 8226 and KMS 12 BM cell lines. Fluorescence microscopy imaging revealed that CD38 was present in cell membrane up to one week of treatment with CD38/CD28xCD3 TCE, while isatuximab induced an internalization of CD38 at 24h. Next, in a co-culture of the OPM-2 cell line with purified T or NK cells from healthy individuals (E:T ratio 2:1), we confirmed that the activity of CD38/CD28xCD3 TCE relied on T but not NK cells. Namely, no direct NK-cell stimulation was found whereas a significant T-cell activation was observed upon CD38/CD28xCD3 TCE treatment. Surprisingly, when OPM-2 were co-cultured with freshly-isolated peripheral blood mononuclear cells (PBMCs, E:T ratio 10:1), CD38/CD28xCD3 TCE induced activation of T- and NK-cells, suggesting a bystander NK-cell activation. This dual stimulation resulted in a significant reduction of OPM-2 cell viability at 48h. Interestingly, we found a comparable tumor-killing activity and T-cell activation in the presence of the CD38 null trispecific isotype (CD3xCD28 ΔCD38) compared with CD38/CD28xCD3 TCE, suggesting that the latter targets CD28 not only in T-cells, but also in OPM-2 cells, which express high CD28 basal levels. Altogether, these data demonstrate the potential dual CD38/CD28 targeting in RRMM patients previously exposed to anti-CD38 mAbs, particularly in those with double positive MMPC. Additionally, CD38 regulation was analyzed in the co-culture system (measured with a non-competing anti-CD38 antibody), and an unaltered and increased CD38 expression was observed in OPM-2 and T-cells, respectively, indicating again T-cell stimulation. We tested the ex vivo efficacy of CD38/CD28xCD3 TCE (48h, 700 pM) in primary bone marrow samples from eight RRMM patients, which were collected after a median of 10 months since the last anti-CD38 treatment (range, 0.5 - 37). T-cell activation and MMPC lysis were found in six and four samples, respectively, which was accompanied by a reduction in the frequency of CD4 T cells (from 100% to 76%; P =.014). A positive and borderline-significant correlation (r = 0.764, P =.057) was found between the response to CD38/CD28xCD3 TCE and the time since last anti-CD38 treatment, and no differences in CD38 expression were observed between CD38/CD28xCD3 TCE responders and non-responders. Conclusions: We observed a reduction in CD38 levels in MMPC and an impaired cytotoxic activity of NK cells in RRMM patients previously exposed to anti-CD38 mAbs. We propose a second targeting of CD38 using T-cell-based immunotherapeutic agents whose efficacy depends less on CD38 antigen density, especially after longer washout periods, as a potential therapeutic strategy in the RRMM setting.

Bclxl Prevents Progressive Exhaustion in BCMA CAR T Cells with Maintenance of High TCF1 Expressing T Cells

Although BCMA targeted CAR T cell therapies are highly effective in the treatment of multiple myeloma (MM), patients with high disease burden have not fully benefited from this therapy with shorter disease remissions despite initial responses. It is postulated that high disease burden results in chronic antigenic stimulation of CAR T cells with the induction of exhaustion-associated gene signatures and CAR T cells contraction, limiting their in vivo potency. We have previously reported that BCL2L1 (gene encoding for BCLxL) armoring of BCMA CAR T cells enhanced CAR T cells functional fitness and improved proliferation and cytotoxicity after chronic antigenic stimulation in vitro, as well as superior tumor clearance in xenograft models of MM. In the current study we aimed to define the molecular mechanisms that mediates BCL2L1-armored CAR T cell and examine the effect of BCLxL variants on CAR T cells fitness. In in vivo studies, NSG mice were systemically injected with OPM2 cells (stably transduced with firefly luciferase) and led to develop large MM disease burden, treatment with BCLxL armored (BCMA_BCL2L1) vs unarmored CARs (BCMA_CAR) or control T cells completely eliminated the MM disease and significantly improved animal survival. In animals treated with armored CARs (BCMA_BCL2L1) no disease recurrence was noted 180 days post treatment (in contrast to unarmored CARs where disease recurrence was noted within 60 days). BCLxL also improved CARs persistence, with armored CAR T cells being detectable in the marrow of these mice 7 and 14 days post treatment. Of note, BCLxL armored CAR T cells did not demonstrate uncontrolled proliferation as they were no longer detectable on day 120 post-infusion. Furthermore, CITEseq profiling on harvested human T cells 7 days post CAR T cells infusion to diseased mice revealed a differential T cell repertoires and phenotypes between BCMA_CAR and BCMA_BCL2L1 CARs. While unarmored CARs were significantly enriched for exhausted T cells (CD8_Tex), BCMA_BCL2L1 CAR T were enriched with early activated non-exhausted CD8 T cells, precursor exhausted T cells (CD8_Tpex) and naïve CD4 and CD8 T cells. Importantly, single cell genes set enrichment analysis (ssGSEA) revealed a differential metabolic signature with higher oxidative phosphorylation in BCMA_BCL2L1 CAR T cells compared to unarmored CARs. These findings suggested that armoring CAR T cells with BCL2L1 maintain CAR T cells in an activated and precursor exhausted T cell states with improved mitochondrial and metabolic fitness, preventing terminal T cells exhaustion. To further evaluate the impact of BCLxL in suppressing BCMA CAR T cells' exhaustion, we engineered BCMA CAR T cells with armored expression of two BCLxL mutants (BCLxL Δ26-83 or BCLxL Δ46-83) harboring large or small deletions involving a BCLxL regulatory loop. These mutants were previously reported to enhance BCLxL function (Chang et al The EMBO Journal 1997,16:968-977). Under chronic antigenic stimulation (CAS) or hypoxic conditions, wild type or mutants BClxL overexpression in BCMA CAR T cells resulted in higher proliferation rates compared to unarmored CARs. Compared to wild type BClxL, BCLxL Δ26-83 and BCLxL Δ46-83 mutants demonstrated higher proliferation capacity post CARs or under hypoxic conditions. Intriguingly, majority of BCLxL-overexpressing BCMA CAR-T cells, particularly BCMA CAR-T BCLxL_Δ26-83 cells, maintained a central memory (T CM) phenotype following a long co-culture with MM tumor cells with lower levels of exhaustion markers. Importantly BCLxL-overexpressing BCMA CAR-T cells had substantially higher level of TCF1 with lower TOX/TOX2 expression compared to unarmored BCMA CAR T cells. TCF1 high population was also enriched in unstimulated BCLxL-overexpressing BCMA CAR T cells suggesting a positive regulatory impact of BCL2L1 on TCF1 expression, either through direct or indirect pathways. Expectedly, TCF1 high BCMA CAR-T cells overexpressing a BCLxL were mainly associated with the naive/T CM phenotype. In summary, we have identified a role for BCLxL and two mutant variants lacking a loop regulatory domain (BCLxL Δ26-83 or BCLxL Δ46-83), in maintenance of BCMA CAR T cells in central memory and precursor exhausted states with retained high TCF1 expression, even under hypoxic and chronic antigenic stimulation conditions. These studies further support the clinical development of a BCL2L1 armored CAR T cells for the treatment of MM.

Efficacy of Human iPSC-Derived CAR-NK Cells Targeting Multiple Myeloma Cells

NK cells armed with chimeric antigen receptors (CAR) enable NK cells specifically to target cancer cells by recognizing tumor associated antigens. Previous studies have proven that human iPSC-derived CAR-NK cells have enhanced anti-tumor activity. Targeted B-cell maturation antigen (BCMA) therapy for relapsed refractory MM (rrMM) has been widely used with antibody-drug conjugate (ADC), bispecific antibodies, and CART cells, which has produced a rapid response, but relapse is common. Recent work has revealed GPRC5D as an alternative target in MM for immunotherapy and patients who have previously received anti-BCMA therapy have responded to GPRC5D CART cells. To assess target GPRC5D therapy with CAR-NK cells, which may in future serve as allogeneic immunotherapy, we transduced anti-GPRC5D CAR into an iPSC line derived from a healthy donor using a piggybac transposon system. More than 97% of CAR inserted-iPSCs (CAR-iPSCs) expressed anti-GPRC5D scFV with a similar expression level identified in CAR-iPSC-derived-NK cells (CAR-iNK, 92.7%). The anti-GPRC5D CAR-iNK demonstrated high purity (>99.9% for CD45 + and CD56 +) and expressed a high level of CD16 (63.6%), the NK cell activating receptor NKG2D (96.3%) and NKp30 (98.7%), and the co-stimulatory receptors CD244 (99.6%) and CD226 (97.8%). Nonetheless expression of TCR⍺β and TCRƳ- was relatively low (<2%). Cytotoxicity assay revealed that anti-GPRC5D CAR-iNK had similar cytotoxicity against K562 cells (No GPRC5D antigen) to cord blood-derived NK cells (CB-NK), wide type (WT) iNK and anti-BCMA CAR-iNK. Cytotoxicity was dose-dependent indicating that these NK cells had similar innate anti-cancer cytotoxicity. When incubated at a ratio of 4:1 with NCI-H929 cells (MM) for 4 hours, which express a high level of both BCMA and GPRC5D, anti-GPRC5D CAR-iNK demonstrated an overwhelming advantage (approximately 90% killing rate) over the CB-NK (no killing) and WT iNK (around 10% killing rate). Furthermore, the anti-GPRC5D CAR-iNK also showed superior cytotoxicity to anti-BCMA CAR-iNK (Figure 1), indicating that GPRC5D may be a more efficient target for CAR-NK-based immunotherapy in MM. We further confirmed that anti-GPRC5D CAR-NK have acceptable cytotoxicity to NCI-H929 and OPM-2 cells, even when transported over a long distance (more than 2000 km) and following cryopreservation. The lower expression by OPM-2 cells of BCMA and GPRC5D compared with NCI-H929 indicates that both fresh and cryopreserved anti-GPRC5D CAR-iNK can specifically target MM with a higher and lower expression of GPRC5D. In addition, cryopreserved anti-GPRC5D CAR-iNK cells maintain comparable antigen-specific cytotoxicity in vitro, and demonstrate the ability to reduce tumor burden in an antigen-specific manner in an OPM-2 xenograft model. Our results demonstrate that genome-engineered human iPSCs can provide an unlimited source for manufacturing scalable, off-the-shelf, and cost-effective CAR-NK cells, and GPRC5D is a potent immunotherapeutic target in multiple myeloma.

Targeting Distinct Epitopes of FCRL5 Using CAR-T Generated with Sequences Derived from Newly Identified FCRL5 Humanized Antibodies Resulted in Potent Anti-Myeloma Activity in Vitro and In Vivo

Despite improvement in treatment options for multiple myeloma (MM) patients, including targeted immunotherapies, MM remains incurable. Given the potential for a single CAR-T treatment to generate long-term remission and the optimal expression profile of FCLR5 (expressed on most MM with limited expression on normal cells), we generated new FCLR5 antibodies and obtained single chain variable fragment (scFv) sequences to develop FCRL5 CAR-Ts. The extracellular domain of FCRL5 has 9 Ig domain subunits (D1-9). We immunized ATX mice (Alloy Therapeutics), genetically engineered to produce humanized antibodies, with human membrane proximal D9 of FCRL5 since cell surface membrane proximal epitopes enhance membrane synapse interactions. Engaging D1-8 could also be effective for targeting FCRL5 by CAR-T, so we immunized mice with full-length FCRL5 and screened for clones binding to D1-8. Because we found FCRL5 expression on primary MM cells was universal but modest and variable, we prioritized screening the 40 antibodies we identified (23 bound to D9 and 17 bound to D1-8) for binding to MOLP-2 cells, known to endogenously express low levels of FCRL5 using flow cytometry. The thirteen best binders (highest mean fluorescence intensity; MFI) were next tested for binding to the MM.1S cell line modified to overexpress FCRL5. All 13 antibodies bound with high efficiency. We generated CAR-T (4-1BB CD3z) for the top seven clones (four D9; three D1-8 binders) and assessed cytolytic activity on MOLP-2 target cells modified to express luciferase (MOLP2-CG) and OPM2 cells modified to express luciferase and high levels of full length human FCRL5 (OPM2-CG-FCRL5). Effector (E) CAR-T cells were incubated with target (T) cells at a range of E:T ratios for 48 hours and bioluminescence (BLI) was used to determine cytolytic activity. Most clones showed high killing activity. We tested clones 977 (D1-8), 942, 947 and 980 (D9 binders) for in vivo studies due to highest performance in vitro. NSG mice were engrafted with 1x10 6 OPM2-CG-FCRL5 cells i.v. into tail veins and treated 14 days later i.v. with 2x10 6 FCRL5 CAR-T or BCMA CAR-T, the current lead CAR-T target in MM as an independent positive control. We found significant extension of survival compared to controls (Kaplan Meier, 942: P<.001, 947 P<.003, 977 P<.0002, 980 P<.008 BCMA P<.001) and reduced tumor burden (longitudinal BLI) in all CAR-T treated mice compared to untreated and mice treated with non-transduced T cell controls in this aggressive tumor model ( Figure 1). Co-targeting BCMA and FCRL5 may prevent antigen escape since it targets cells expressing either or both FCRL5 and BCMA. We generated a bi-targeted CAR targeting both BCMA and FCRL5 (clone D1-8 977). To confirm each scFv was active, we ran in vitro killing assays using BCMA knockout (KO)-OPM2-CG FCRL5 (BCMA-FCRL5+), OPM2-CG (BCMA+ FCLR5-) targets and found killing of each cell type by our bi-targeted CAR-T, confirming both scFvs were active. BCMA-FCRL5 CAR-T killed OPM2-CG FCRL5 and MOLP-2-CG cells (both BCMA+FCRL5+) but not BCMA KO OPM2 (BCMA-FCLR5-) cells. The best performing BCMA CAR-T in the clinic targets two BCMA epitopes- our scFv's targeting different domains of FCLR5 opens the opportunity for bi-targeting the FCLR5 protein itself. We generated an FCRL5-FCRL5 bi-targeted CAR-T using clone 977 (D1-8) and 980 (D9) and demonstrate activity of this CAR-T on OPM2-CG-FCRL5 cells; ongoing studies will determine if each scFv is active and whether in vivo activity is superior to single epitope targeting in vivo. Finally, blocking studies of the D9 antibodies showed potential for multiple epitopes within that domain which may facilitate co-targeting D9. We tested four FCRL5 CAR-T (4-1BB CD3z) predicted to target distinct D9 epitopes and observed high cytolytic activity in killing assays using OPM2-CG-FCRL5 target cells. We tested each in vivo by injecting NSG mice in with OPM2-CG and 18 days later treated with 2x10 6 FCRL5 CAR-T. As above, we observed significant extension of survival and reduced tumor burden compared to controls. Future studies will assess feasibility of D9 co-targeting. Together, our data demonstrate efficacy of FCRL5 CAR-T using novel sequences targeting FCLR5 both within and outside of D9 and support development of an FCRL5 CAR-T clinical candidate for use in relapsed/refractory MM patients.

Inhibition of USP10 induces myeloma cell apoptosis by promoting cyclin D3 degradation.

The cell cycle regulator cyclin D3 (CCND3) is highly expressed in multiple myeloma (MM) and it promotes MM cell proliferation. After a certain phase of cell cycle, CCND3 is rapidly degraded, which is essential for the strict control of MM cell cycle progress and proliferation. In the present study, we investigated the molecular mechanisms regulating CCND3 degradation in MM cells. By utilizing affinity purification-coupled tandem mass spectrometry, we identified the deubiquitinase USP10 interacting with CCND3 in human MM OPM2 and KMS11 cell lines. Furthermore, USP10 specifically prevented CCND3 from K48-linked polyubiquitination and proteasomal degradation, therefore enhancing its activity. We demonstrated that the N-terminal domain (aa.1-205) of USP10 was dispensable for binding to and deubiquitinating CCND3. Although Thr283 was important for CCND3 activity, it was dispensable for CCND3 ubiquitination and stability modulated by USP10. By stabilizing CCND3, USP10 activated the CCND3/CDK4/6 signaling pathway, phosphorylated Rb, and upregulated CDK4, CDK6 and E2F-1 in OPM2 and KMS11 cells. Consistent with these findings, inhibition of USP10 by Spautin-1 resulted in accumulation of CCND3 with K48-linked polyubiquitination and degradation that synergized withPalbociclib, a CDK4/6 inhibitor, to induce MM cell apoptosis. In nude mice bearing myeloma xenografts with OPM2 and KMS11 cells, combined administration of Spautin-l and Palbociclib almost suppressed tumor growth within 30 days. This study thusidentifies USP10 as the first deubiquitinase of CCND3and also finds that targeting the USP10/CCND3/CDK4/6 axis may be a novel modality for the treatment of myeloma.

Abstract 2505: PYCR1 inhibition in multiple myeloma-associated stroma limits tumor growth

Abstract Introduction Multiple myeloma (MM) is a hematological cancer, characterized by the accumulation of monoclonal plasma cells in the bone marrow. It remains an incurable cancer due to drug resistance, wherein the bone marrow microenvironment plays a crucial role. We have previously shown that glutamine-to-proline conversion is upregulated in MM cells upon hypoxic culture. Moreover, inhibition of proline production by blocking its converting enzyme PYCR1 successfully reduced proliferation and viability in vitro. Importantly, PYCR1 inhibition combined with standard-of-care agent bortezomib decreased tumor load in vivo. As PYCR1 is also highly expressed in stromal cells and proline an important component of extracellular matrix proteins, we investigated whether PYCR1 targeting in stromal cells affects MM viability and its structural microenvironment. Material and methods PYCR1 expression was investigated by the use of microarray data from the Heidelberg/Montpellier cohort. For in vitro experiments, the human MM cell line OPM-2 and human stromal cell line HS-5 were used. CD138+ and CD138- fractions from primary patient samples were separated by MACS. To obtain primary stromal cells, the CD138- fraction was plated out in fresh medium. After 48h, the medium was refreshed and all adherent cells were further cultured and used as primary stromal cells. PYCR1 expression in HS-5 cells was reduced through siRNA. Hypoxic culture (<1% O2) was established through chambers. RNA expression was measured by RT-qPCR, while protein expression was measured by western blot. Viability was assessed by CellTiterGlo assay. Results Gene expression analysis shows high RNA expression of PYCR1 in healthy bone marrow plasma cells (BMPCs), plasma cells from monoclonal gammopathy of undetermined significance (MGUS) and MM patients, but also in bone marrow stromal cells (BMSCs). PYCR1 expression was low or absent in other cell types, including T cells, osteoclasts, monocytic and granulocytic cells. On protein level, we also confirmed PYCR1 expression in MM cells (CD138+), CD138- fraction (including stromal cells) and cultured primary stromal cells. Moreover, PYCR1 expression increased upon hypoxic culture in primary stromal cells and stromal cell line HS-5. PYCR1 knockdown in stromal cell line HS-5 did not affect viability of the stromal cells, but its conditioned medium did reduce its protective effects when OPM-2 MM cells were treated with standard-of-care agent bortezomib. Further investigation revealed that knockdown of PYCR1 in HS-5 reduces RNA expression of structural proteins col1a1, col1a2, col3a1, ctgf and acta2 in HS-5. Conclusion PYCR1 is highly expressed in myeloma cells and stromal cells. PYCR1 inhibition in stromal cells reduces its proliferative effect on myeloma cells when combined with bortezomib. Preliminary data indicates a link between PYCR expression in stromal cells and rearrangement of extracellular matrix proteins. Citation Format: Inge Oudaert, Catharina Muylaert, Hatice Satilmis, Sylvia Faict, Kim De Veirman, Elke De Bruyne, Karin Vanderkerken, Eline Menu. PYCR1 inhibition in multiple myeloma-associated stroma limits tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2505.

BRD9 Is Essential for Ribosome Biogenesis and the Survival of Multiple Myeloma Cells

BRD9 is a component of the noncanonical SWI/SNF (ncSWI/SNF) complex, which is a chromatin-remodeling complex that modulates genomic architecture and DNA accessibility. Since BRD9 has been shown to play a critical tumor-promoting role in various cancers, we investigated its biologic impact in multiple myeloma (MM) cells. We found that BRD9 was highly expressed in patient MM cells compared to normal plasma cells (GSE39754, P = 0.045). Moreover, higher BRD9 expression was associated with poor overall survival (GSE39754, P = 0.049) in MM patients. We next confirmed that genetic (shRNA) and pharmacological (dBRD9-A) depletion of BRD9 significantly reduced growth in a panel of MM cell lines and patient MM cells. Furthermore, dBRD9-A induced G1 cell-cycle arrest and apoptosis in OPM2 and H929 cells, even in the presence of anti-apoptotic cytokines (IL-6, IGF-1), or bone marrow stromal cells. In a murine xenograft model, dBRD9-A treatment significantly inhibited OPM2 tumor growth compared with vehicle control (P = 0.001 and P < 0.001, respectively) without significant body weight loss. Kaplan-Meier curves and log-rank analysis showed a significantly prolonged overall survival in the dBRD9-A treatment cohort compared with the vehicle control cohort (P < 0.001). Having nominated BRD9 as a potential therapeutic target, we next investigated the cellular mechanisms impacted by BRD9 depletion by RNA-seq analyses. After BRD9 knockdown (shRNA) or dBRD9-Aapplication, a total of 162 and 197genes were commonly up- or downregulated, respectively, compared with DMSO-treated control cells (Padj < 0.05). Importantly, we identified that ribosome biogenesis and rRNA processing were the most significantly downregulated pathways. Notably, target genes of MYC, the master regulator of ribosome biogenesis, were also markedly downregulated. Furthermore, we observed that the expression of representative proteins (i.e., MYC, RRS1, and BOP1) was also consistently decreased by dBRD9-A in vitro and in vivo. Indeed, we observed a significant decrease in total pre-rRNA levels and nucleolar number by dBRD9-A treatment in OPM2 and H929 cells, indicating ribosome biogenesis was positively regulated, at least in part, by BRD9. OPM2 cells treated with dBRD9-A had fewer ribosomal subunits and fewer actively translating polysomes compared with DMSO-treated controls, as determined by polysome profiling of cytosolic extracts. Furthermore, AgNOR staining of tumors harvested from our murine xenograft model demonstrated that dBRD9-A treatment reduced the number of nucleolar organizer regions in vivo. To understand how BRD9 regulates the expression of ribosome biogenesis genes, we next performed ChIP-seq. We found that many of the genes downregulated by dBRD9-A had BRD9 occupancy at their promoters compared to other intact genes. Notably, genes with BRD9 signals in their promoter regions were enriched for ribosome biogenesis and rRNA processing and were downregulated by dBRD9-A treatment. Although GLTSCR1, another ncBAF-specific component, was still able to form a complex even after BRD9 depletion, ChIP-seq revealed a significant loss of GLTSCR1 binding throughout the genome after treatment with dBRD9-A. Thus, we hypothesized that BRD9 is required for the localization of the ncSWI/SNF complex to target gene regions. Indeed, BRD9 co-immunoprecipitated with the positive transcription elongation factors, RNAPII, BRD4, and MYC in MM cells. Interestingly, the interaction between BRD9 and BRD4 was enhanced by an HDAC inhibitor, while BRD9 depletion attenuated the interaction between GLSTCR1 and BRD4, suggesting that BRD9 interacts with the acetylated form of BRD4, stabilizing its binding to the ncBAF complex and thereby supporting the initiation of transcription of target genes. Indeed, ChIP-seq analysis demonstrated that BRD9-bound sites preferentially co-localized with ChIP peaks of BRD4, MYC, and RNAPII to the promoter regions of all ribosome biosynthesis genes. In summary, our data identify a novel function for BRD9 in promoting ribosome biogenesis in MM. BRD9 depletion impairs ncBAF localization to its genomic targets, thereby disrupting ribosome biogenesis machinery, resulting in significant cell growth inhibition. Our study therefore provides the rationale for further evaluation of BRD9 degraders as a novel treatment strategy in MM.

Potent Pre-Clinical and Early Phase Clinical Activity of EP300/CBP Bromodomain Inhibitor CCS1477 in Multiple Myeloma

CCS1477 (also known as inobrodib) is a potent and selective inhibitor of the bromodomains of EP300 and CBP, two homologous histone acetyltransferases with critical roles in cellular growth and differentiation. In preclinical analyses we find that CCS1477 induces potent growth inhibition though inducing cell cycle arrest at low nanomolar concentrations (GI50<100nM) across a panel of 12 human myeloma cell lines, including lenalidomide refractory lines. In vivo, in an OPM-2 human myeloma cell line NOD-SCID xenograft model, there was dose dependent inhibition of tumour growth, with 20mg/kg daily eliciting tumour regression and sustained inhibition of tumour growth for two weeks beyond the end of treatment. RNA sequencing of tumours recovered from mice after five days of CCS1477 treatment revealed substantial down regulation of transcription factor genes including MYB, MYC, IRF4, E2F1 and FOXM1; and GSEA showed significant enrichment among down regulated genes of gene sets associated with both MYC and E2F targets, consistent with drug-induced proliferative arrest. In the OPM-2 model, ChIP sequencing showed that EP300 binding peaks were predominantly distributed over transcription factor binding motifs for IRF, Runt, bHLH, bZIP and MYB/SANT factor classes. Within six hours of 100nM CCS1477 treatment there was a localized but significant reduction of EP300 recruitment to intronic FGFR3 sites: FGFR3 is placed under the control of the active IGH promoter in the intergenic t(4;14) in OPM-2 cells. By 48 hours, in keeping with CCS1477-induced inactivation of IRF4 (a critical regulator of plasma cell differentiation required for myeloma cell growth) there was extensive genome-wide redistribution of EP300 binding away from sites with IRF4 motifs, while sites with other motifs remained occupied. In our ongoing phase I/IIa clinical trial investigating the safety and efficacy of CCS1477 in advanced haematological malignancies (NCT04068597), we have treated four heavily pre-treated relapsed/refractory myeloma patients at the recommended phase 2 dose (35mg PO twice daily, four days a week). Of these, two exhibited objective responses with substantial reductions in serum or urinary M-protein, with one patient achieving a PR and the other exhibiting a complete remission after four months. Both patients remain on treatment as of the submission of this abstract. Treatment was well tolerated, with moderate thrombocytopaenia being an anticipated on-target side effect. Finally, we evaluated the activity of CCS1477 in an OPM-2 xenograft model in combination with existing standard of care agents in myeloma and observed additive or synergistic growth inhibitory activity with each of bortezomib, lenalidomide and vorinostat in combination with CCS1477. These encouraging data suggest strong promise for the clinical utility of CCS1477 in the treatment of multiple myeloma as monotherapy and/or in combination with standard of care agents.

The efficacy of combination treatment with elotuzumab and lenalidomide is dependent on crosstalk between natural killer cells, monocytes and myeloma cells.

Patients with refractory relapsed multiple myeloma respond to combination treatment with elotuzumab and lenalidomide. The mechanisms underlying this observation are not fully understood. Furthermore, biomarkers predictive of response have not been identified to date. To address these issues, we used a humanized myeloma mouse model and adoptive transfer of human natural killer (NK) cells to show that elotuzumab and lenalidomide treatment controlled myeloma growth, and this was mediated through CD16 on NK cells. In co-culture studies, we showed that peripheral blood mononuclear cells from a subset of patients with refractory relapsed multiple myeloma were effective killers of OPM2 myeloma cells when treated with elotuzumab and lenalidomide, and this was associated with significantly increased expression of CD54 on OPM2 cells. Furthermore, elotuzumab- and lenalidomide-induced OPM2 cell killing and increased OPM2 CD54 expression were dependent on both monocytes and NK cells, and these effects were not mediated by soluble factors alone. At the transcript level, elotuzumab and lenalidomide treatment significantly increased OPM2 myeloma cell expression of genes for trafficking and adhesion molecules, NK cell activation ligands and antigen presentation molecules. In conclusion, our findings suggest that multiple myeloma patients require elotuzumab- and lenalidomide-mediated upregulation of CD54 on autologous myeloma cells, in combination with NK cells and monocytes to mediate an effective anti-tumor response. Furthermore, our data suggest that increased myeloma cell CD54 expression levels could be a powerful predictive biomarker for response to elotuzumab and lenalidomide treatment.

Inhibition of the Protein Arginine Methyltransferase PRMT5 in High-Risk Multiple Myeloma as a Novel Treatment Approach.

Multiple myeloma (MM) is an incurable clonal plasma cell malignancy. Subsets of patients have high-risk features linked with dismal outcome. Therefore, the need for effective therapeutic options remains high. Here, we used bio-informatic tools to identify novel targets involved in DNA repair and epigenetics and which are associated with high-risk myeloma. The prognostic significance of the target genes was analyzed using publicly available gene expression data of MM patients (TT2/3 and HM cohorts). Hence, protein arginine methyltransferase 5 (PRMT5) was identified as a promising target. Druggability was assessed in OPM2, JJN3, AMO1 and XG7 human myeloma cell lines using the PRMT5-inhibitor EPZ015938. EPZ015938 strongly reduced the total symmetric-dimethyl arginine levels in all cell lines and lead to decreased cellular growth, supported by cell line dependent changes in cell cycle distribution. At later time points, apoptosis occurred, as evidenced by increased AnnexinV-positivity and cleavage of PARP and caspases. Transcriptome analysis revealed a role for PRMT5 in regulating alternative splicing, nonsense-mediated decay, DNA repair and PI3K/mTOR-signaling, irrespective of the cell line type. PRMT5 inhibition reduced the expression of upstream DNA repair kinases ATM and ATR, which may in part explain our observation that EPZ015938 and the DNA-alkylating agent, melphalan, have combinatory effects. Of interest, using a low-dose of mTOR-inhibitor, we observed that cell viability was partially rescued from the effects of EPZ015938, indicating a role for mTOR-related pathways in the anti-myeloma activity of EPZ015938. Moreover, PRMT5 was shown to be involved in splicing regulation of MMSET and SLAMF7, known genes of importance in MM disease. As such, we broaden the understanding of the exact role of PRMT5 in MM disease and further underline its use as a possible therapeutic target.

Discovery of novel benzimidazole derivatives as potent p300 bromodomain inhibitors with anti-proliferative activity in multiple cancer cells

Adenovirus E1A-associated 300-kD protein (p300) bromodomain, which regulates gene expression by recognizing acetylated lysine (KAc) of histone, is a promising target for the treatment of cancer. Herein, a series of potent p300 bromodomain inhibitors with novel CBP30-based scaffolds was discovered through bioisosterism and conformational restriction strategies. The most promising compound 1u showed more potent inhibitory activity (IC50 = 49 nM) against p300 bromodomain and anti-proliferative activity in various cancer cell lines compared to CBP30. Moreover, 1u suppressed the expression of c-Myc and induced G1/G0 phase arrest and apoptosis in OPM-2 cells more potently than CBP30. This study provides new lead compounds for further research on the biological functions of p300.

Mesenchymal Stem Cells Inhibit the Effects of Dexamethasone in Multiple Myeloma Cells.

Mesenchymal stem cells (MSCs) participate in the occurrence and development of multiple myeloma. This study is aimed at exploring whether the presence of MSCs affects dexamethasone's antitumor effects against multiple myeloma. Multiple myeloma cells (OPM-2 and RPMI8226 cells) were cocultured with MSCs with or without dexamethasone. Cell viability was determined by using cell number count, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and colony formation assay, respectively. Cell cycle distribution and cell apoptosis were evaluated by using flow cytometry. The mRNA and protein expressions of target genes were checked by using qRT-PCR and western blotting, respectively. It was found that cell viability of multiple myeloma cells increased in the presence of MSCs. Besides, the presence of MSCs suppressed cell apoptosis induced by dexamethasone via the regulation of BCL-2 (B cell lymphoma 2). The presence of MSCs also affected the effects of dexamethasone on cell cycle distribution. Similarly, LINC00461 overexpression suppressed the inhibition of cell proliferation, suppressed the induction of cell apoptosis, and affected the effects on cell cycle distribution induced by dexamethasone insult. However, LINC00461 knockdown enhanced the inhibitory effects on cell proliferation and the induction of cell apoptosis induced by dexamethasone. In summary, MSCs inhibited the effects of dexamethasone on multiple myeloma and its regulatory effects were associated with LINC00461.

Pyrroline-5-Carboxylate Reductase 1: a novel target for sensitizing multiple myeloma cells to bortezomib by inhibition of PRAS40-mediated protein synthesis

BackgroundMultiple myeloma (MM) remains an incurable cancer despite advances in therapy. Therefore, the search for new targets is still essential to uncover potential treatment strategies. Metabolic changes, induced by the hypoxic bone marrow, contribute to both MM cell survival and drug resistance. Pyrroline-5-carboxylate reductase 1 and 2 (PYCR1 and PYCR2) are two mitochondrial enzymes that facilitate the last step in the glutamine-to-proline conversion. Overexpression of PYCR1 is involved in progression of several cancers, however, its’ role in hematological cancers is unknown. In this study, we investigated whether PYCR affects MM viability, proliferation and response to bortezomib.MethodsCorrelation of PYCR1/2 with overall survival was investigated in the MMRF CoMMpass trial (653 patients). OPM-2 and RPMI-8226 MM cell lines were used to perform in vitro experiments. RPMI-8226 cells were supplemented with 13C-glutamine for 48 h in both normoxia and hypoxia (< 1% O2, by chamber) to perform a tracer study. PYCR1 was inhibited by siRNA or the small molecule inhibitor pargyline. Apoptosis was measured using Annexin V and 7-AAD staining, viability by CellTiterGlo assay and proliferation by BrdU incorporation. Differential protein expression was evaluated using Western Blot. The SUnSET method was used to measure protein synthesis. All in vitro experiments were performed in hypoxic conditions.ResultsWe found that PYCR1 and PYCR2 mRNA expression correlated with an inferior overall survival. MM cells from relapsed/refractory patients express significantly higher levels of PYCR1 mRNA. In line with the strong expression of PYCR1, we performed a tracer study in RPMI-8226 cells, which revealed an increased conversion of 13C-glutamine to proline in hypoxia. PYCR1 inhibition reduced MM viability and proliferation and increased apoptosis. Mechanistically, we found that PYCR1 silencing reduced protein levels of p-PRAS40, p-mTOR, p-p70, p-S6, p-4EBP1 and p-eIF4E levels, suggesting a decrease in protein synthesis, which we also confirmed in vitro. Pargyline and siPYCR1 increased bortezomib-mediated apoptosis. Finally, combination therapy of pargyline with bortezomib reduced viability in CD138+ MM cells and reduced tumor burden in the murine 5TGM1 model compared to single agents.Conclusions This study identifies PYCR1 as a novel target in bortezomib-based combination therapies for MM.