Primary Multiple Myeloma Samples Research Articles

MYC Inhibition Potentiates CD8+ T Cells Against Multiple Myeloma and Overcomes Immunomodulatory Drug Resistance.

Immunomodulatory drugs (IMiDs), such as lenalidomide and pomalidomide, are a cornerstone of multiple myeloma (MM) therapies, yet the disease inevitably becomes refractory. IMiDs exert cytotoxicity by inducing cereblon-dependent proteasomal degradation of IKZF1 and IKZF3, resulting in downregulation of the oncogenic transcription factors IRF4 and MYC. To date, clinical IMiD resistance independent of cereblon or IKZF1/3 has not been well explored. Here, we investigated the roles of IRF4 and MYC in this context. Using bone marrow aspirates from patients with IMiD-naïve or refractory MM, we examined IKZF1/3 protein levels and IRF4/MYC gene expression following ex vivo pomalidomide treatment via flow cytometry and qPCR. We also assessed exvivo sensitivity to the MYC inhibitor MYCi975 using flow cytometry. We discovered that although pomalidomide frequently led to IKZF1/3 degradation in MM cells, it did not affect MYC gene expression in most IMiD-refractory samples. We subsequently demonstrated that MYCi975 exerted strong anti-MM effects in both IMiD-naïve and -refractory samples. Unexpectedly, we identified a cluster of differentiation 8+ (CD8+ T) cells from patients with MM as crucial effectors of MYCi975-induced cytotoxicity in primary MM samples, and we discovered that MYCi975 enhanced the cytotoxic functions of memory CD8+ T cells. We lastly observed synergy between MYCi975 and pomalidomide in IMiD-refractory samples, suggesting that restoring MYC downregulation can re-sensitize refractory MM to IMiDs. Our study supports the concept that MYC represents an Achilles' heel in MM across disease states and that MYCi975 may be a promising therapeutic for patients with MM, particularly in combination with IMiDs.

MUC20 regulated by extrachromosomal circular DNA attenuates proteasome inhibitor resistance of multiple myeloma by modulating cuproptosis

BackgroundProteasome inhibitors (PIs) are one of the most important classes of drugs for the treatment of multiple myeloma (MM). However, almost all patients with MM develop PI resistance, resulting in therapeutic failure. Therefore, the mechanisms underlying PI resistance in MM require further investigation.MethodsWe used several MM cell lines to establish PI-resistant MM cell lines. We performed RNA microarray and EccDNA-seq in MM cell lines and collected human primary MM samples to explore gene profiles. We evaluated the effect of MUC20 on cuproptosis of PI-resistant MM cells using Co-immunoprecipitation (Co-IP), Seahorse bioenergetic profiling and in vivo assay.ResultsThis study revealed that the downregulation of Mucin 20 (MUC20) could predict PI sensitivity and outcomes in MM patients. Besides, MUC20 attenuated PI resistance in MM cells by inducing cuproptosis via the inhibition of cyclin-dependent kinase inhibitor 2 A expression (CDKN2A), which was achieved by hindering MET proto-oncogene, receptor tyrosine kinase (MET) activation. Moreover, MUC20 suppressed MET activation by repressing insulin-like growth factor receptor-1 (IGF-1R) lactylation in PI-resistant MM cells. This study is the first to perform extrachromosomal circular DNA (eccDNA) sequencing for MM, and it revealed that eccDNA induced PI resistance by amplifying kinesin family member 3 C (KIF3C) to reduce MUC20 expression in MM.ConclusionOur findings indicated that MUC20 regulated by eccDNA alleviates PI resistance of MM by modulating cuproptosis, which would provide novel strategies for the treatment of PI-resistant MM.

Identification of potential therapeutic targets for multiple myeloma by gene expression analysis

New treatments are needed for multiple myeloma (MM), an almost always incurable cancer of plasma cells. Cancer therapies might have fewer side effects if they are aimed at specific protein targets expressed by cancer cells. We hypothesized that differences in gene expression between multiple myeloma cells and normal cells could be used to identify new targets for MM therapies. RNA sequencing (RNAseq) is a technique for quantifying gene expression in cells. We analyzed RNAseq data from the Cancer Cell Line Encyclopedia to identify genes with high expression in MM cell lines versus cell lines derived from cancers other than MM. Next, the 200 genes with the highest expression in MM cell lines versus other cell lines were evaluated for RNA expression in primary MM samples from patients. Of these genes, 72 were expressed in at least 23 of 25 primary samples from patients. We evaluated the 72 genes expressed at high levels in MM cell lines and expressed in primary MM cells for expression in normal human tissues. We evaluated gene expression in normal human tissues by using publicly available RNAseq data. Genes with high expression in normal tissues were eliminated as potential targets to decrease the chance of side effects caused by therapies targeting proteins expressed by normal tissues. We identified 19 genes with high expression in MM and low expression in normal tissues. The proteins encoded by these genes are promising therapeutic targets for MM and include Prepronociceptin (PNOC) and Interferon Regulatory Factor 4 (IRF4).

BCMAxCD3 Bispecific Antibody Elranatamab Is Effective in Patient Myeloma Relapsed after BCMA CAR-T

Introduction: In recent years, therapies targeting the plasma cell-specific protein BCMA have been widely adopted into the treatment of patients with relapsed refractory multiple myeloma (MM). Agents include chimeric antigen receptor t cell therapies (CAR-Ts) and bispecific T cell engaging antibodies (bsAbs). Targeting BCMA is effective in late-stage, heavily pretreated patients, and studies are examining if these therapies work even better earlier in disease progression. As the number of available BCMA agents has rapidly expanded, guidance is lacking on how to sequence these therapies to maximize patient benefits. Therefore, investigation is needed to determine if prior exposure to BCMA agents affects BCMA-targeted re-treatment. Here, we use Myeloma Drug Sensitivity Testing (My-DST, Walker et al, Blood Adv. 2020) to compare efficacy of the anti-BCMA bsAb elranatamab from diagnosis to post-CAR-T settings using primary MM samples that include the patients' own T cells. Methods: We obtained bone marrow and peripheral blood samples from patients after informed consent and Institutional Review Board approval. Mononuclear cells were incubated with or without elranatamab in triplicate for 48 hours. Flow cytometry measuring MM survival was analyzed with fluorescent antibodies against MM markers CD38, CD138, BCMA, CD46, CD56, CD45, CD19, and CD28, and T cell phenotype using antibodies against CD3, CD4, CD107a, CD107b, CD28, CD38, CD16, CD8, and CD56. To compare patient T cell effectiveness from different timepoints and versus healthy donors (HD), anti-human CD3 MicroBeads and MS columns were used to for T cell isolation. HD or MM patient-derived T cells were then added back to CD3- samples or the H929 MM cell lines and elranatamab My-DST was performed. Viability was normalized to untreated controls with matched source T cells. Results: Dose response titrations of elranatamab identified a mean EC50 of 52 pM and approximate EC90 of 1nM. The EC90 was used to screen for resistance in 16 primary MM samples, including 9 NDMM, 4 pre-BCMA CAR-T samples and 3 post-BCMA CAR-T (Fig 1A). MM viability of 80% or less was considered the sensitivity threshold, as cytotoxicity beyond this level achieved statistical significance across samples. Across all settings, 87.5% (14/16) met this sensitivity threshold to elranatamab. Notably, 3/3 samples taken from post-CAR-T patients were sensitive. MM cells from all samples tested expressed BCMA. However, 1/2 resistant samples (HTB-1802) had a low E:T ratio. We next examined the impact of T cell source on elranatamab-directed T cell killing of MM. We found that elranatamab treatment led to a greater reduction in primary MM viability when cultured with healthy donor-derived T cells than with MM patient-derived T cells (Fig 1B). Next, we isolated T cells before infusion with CAR-T and after relapse to CAR-T and cultured them with the MM cell line H929 with or without elranatamab. We found no difference in elranatamab-directed cytotoxicity between pre-CAR-T T cells and post-CAR-T T cells, implying T cell health was not adversely affected by CAR-T. Conclusion: These data demonstrate that My-DST can identify drug resistance to bispecific antibodies with ex vivo cultures that contain the patients' own T cells. The results suggest that most cases of bispecific antibody resistance are due to T cell health or number, rather than low BCMA expression. This supports BCMA-targeted re-treatment in patients with adequate T cell health. Future investigation will aim to further evaluate the critical elements of T cell health with a larger sample size. Ultimately, My-DST has potential as a precision medicine tool to guide bispecific antibody treatment and inform how best to sequence them with CAR-T.

And-Gate CAR T-Cells to Improve Tumour Specificity and Targeting of Low-Expression Antigens in Multiple Myeloma

Whilst chimeric antigen receptor (CAR) T cells have shown remarkable success, further novel targets are required. However, our previous cell surface proteomics work in multiple myeloma (MM) showed that target choice is highly limited by an absence of truly tumour-specific antigens, and often very low on-tumour expression of target proteins in some primary samples. One approach to improve tumour specificity is to use combinatorial antigen recognition, or “AND-gates”, whereby a CAR T cell must recognise two different antigens for full activation and target cell elimination. It has also been reported that this approach might overcome low target expression. We therefore decided to explore the potential for AND-gate targeting in MM and identify a suitable target pairing. To determine a comprehensive list of viable AND-gate combinations for MM, we first integrated our MM proteomics data with a normal tissue proteomics dataset. From 777 extracellular proteins expressed in primary MM, we identified a total of 287,890 possible pairwise combinations. We next excluded any pairs with overlapping expression in vital, healthy tissues, reducing potential combinations to 664. Additional filtering steps to remove difficult-to-target proteins and combinations where either target was expressed on T cells - thus avoiding T cell fratricide - resulted in a final list of 92 AND-gate targets. From our prioritised candidates, we selected TNFRSF8 and TMPRSS11E as an exemplar pairing. By mass spectrometry, both TNFRSF8 and TMPRSS11E were expressed across all 8 profiled primary MM samples at comparable levels to existing CAR T cell targets, such as GPRC5D, and had no predicted overlapping expression in normal tissue. To test our AND-gate combination, we designed a ‘split-CAR’ system in which TNFRSF8 was targeted by a 1 st generation CAR with an intracellular signalling domain, but without a co-stimulatory receptor (CAR-only construct), and TMPRSS11E was targeted by a construct with a chimeric co-stimulatory receptor (CCR), but lacking an intracellular signalling domain (CCR-only construct). Constructs were initially validated in a Jurkat cell line model of T cell activation. Jurkats were transformed with CCR-only, CAR-only, or both constructs, as well as with full 2 nd generation CARs targeting TNFRSF8 or TMPRSS11E. These cells were co-cultured with the TNFRSF8+TMPRSS11E+ human MM cell line XG1 or the TNFRSF8+TMPRSS11E- erythroleukemic cell line K562. As anticipated, neither the CCR-only nor the CAR-only Jurkats were activated, with no production of IFNγ or IL2 ( Fig. 1A). Whilst Jurkats expressing the full 2 nd generation TMPRSS11E CAR were activated in co-culture with XG1, Jurkats expressing the TNFRSF8 2 nd generation CAR were not, despite expression of TNFRSF8 on XG1. Jurkats co-expressing both components of the split-CAR were markedly activated when co-cultured with TNFRSF8+TMPRSS11E+ XG1 cells (but not with TNFRSF8+TMPRSS11E- K562 cells), as demonstrated by a large increase in expression of CD69, IFNγ, and IL2 ( Fig. 1A). Thus, the split-CAR system showed a very high degree of specificity, requiring both antigens to be co-expressed on the target cell. Moreover, it was activated even when full 2 nd generation CARs against TNFRSF8 were not. Next, we sought to investigate the cytolytic activity of our split-CAR constructs in donor T cells. In a cytotoxicity assay, T cells expressing the CAR-only or CCR-only construct did not exhibit significant killing of XG1 cells ( Fig. 1B). In parallel with the Jurkat reporter assays, only 2 nd generation TMPRSS11E CAR T cells, and not 2 nd generation TNFRSF8 CAR T cells, were cytotoxic to XG1. Finally, donor T cells expressing both components of the split-CAR demonstrated potent killing of dual-positive XG1 cells ( Fig. 1B), but not single-positive K562 cells. We have thus developed an analytical pipeline for the discovery of AND-gate CAR T cell targets for MM, also applicable to other tumours. This approach led to the development of an extremely potent, but highly selective, split-CAR targeting TNFRSF8 and TMPRSS11E. Importantly, our split-CAR targeted very low levels of TNFRSF8 antigen that were insufficient to trigger activation of a 2 nd generation TNFRSF8 CAR-T. In summary, our work suggests that AND-gate targeting can increase not just specificity, but potency, even against very low expression tumour targets in MM. Link to preprint https://doi.org/10.1101/2023.04.04.535580

CD38 KO/CD38-CAR Human Primary Natural Killer Cells Enhance Cytotoxicity Against CD38-Expressing Primary Lymphoma, Leukemia, and Myeloma

Natural killer (NK) cells have strong cancer immunotherapeutic potential that can be further augmented by engineering with a chimeric antigen receptor (CAR). CD38, a transmembrane glycoprotein involved in signal transduction and cellular adhesion, is highly expressed on the surface of the hematologic malignancies multiple myeloma (MM), acute myeloid leukemia (AML), Burkitt Lymphoma (BL) and T-cell acute lymphoblastic leukemia (T-ALL). FDA approved CD38 monoclonal antibodies (mAb) daratumumab and isatuximab are used to treat MM in patients, and have been tested to treat other CD38 expressing malignancies. NK cells play an imperative role in the efficacy of these antibodies and have natural antitumor activity against these cancers. However, because NK cells also highly express CD38, anti-CD38 mAb can cause NK cell fratricide, limiting their efficacy. Here, we generated multiple CD38 scFv based on isatuximab sequences and generated fratricide-resistant primary CD38-CAR NK cells using CRISPR/Cas9 genome editing and AAV6 gene delivery. Using this approach, we inserted the CD38-CAR gene into the CD38 locus to efficiently generate simultaneous CD38 KO and CD38-CAR KI with a mean expression level of %60. The CAR-expressing NK cells could be expanded on mbIL21-expressing feeder cells to a clinically relevant number of cells with no decline in CAR-expression level during two weeks of expansion confirming the lack of CAR mediated fratricide. We evaluated transduction efficiency and expression of CD38-CAR, and compared the cytotoxic function of two constructs with CD8 transmembrane (TM)/stalk and 41BB signaling domains and reverse heavy and light chains against AML, MM, T-ALL and BL immortalized cell lines and primary patient samples. We enhanced CD38 expression in target cancer cells by pre-treatment with all-trans retinoic acid (ATRA), a metabolite of vitamin A that pushes cells to maturation, consequently upregulating CD38. The sequential treatment of target cells with 10nM ATRA for 48 hours enhanced the cytotoxic activity of the CD38 KO/CD38-CAR-NK cells against primary MM, BL and AML samples. This would be particularly important for patients with low expression of CD38 on their cancers. The enhanced anti-tumor activity of the CAR was also studied and observed by CyTOF. Additionally, to better understand the therapeutic potential of CD38-CAR NK cells compared to CD38-CAR T cells we generated CD38 KO/CD38-CAR T cells using the same engineering approach and comparing their cytotoxic function. We showed that both CD38-CAR-T and CD38-CAR-NK cells have comparable anti-tumor activity compare to their paired non-edited wild type cells. Finally, we investigated the need for a CD38 KO to develop a fratricide-resistant therapy by comparing the cytolytic and metabolic functions of CD38 +/AAVS1 KO and CD38 KO CD38-CAR NK cells. We conclusively report a CD38-CAR NK cell therapy with enhanced metabolism and cytotoxicity toward a variety of hematologic malignancies, which can be further augmented by combination treatment with ATRA to target CD38-low malignancies.

Discovery and Development of CD70 As a Cellular Therapy Target in High Risk Multiple Myeloma

Background: Even in the BCMA CAR-T era, multiple myeloma (MM) patients with high-risk genotypes have the poorest outcomes. We thus leveraged MMRF CoMMpass data to identify potential novel surface immunotherapeutic targets in high-risk MM. As described below, we focused on CD70, an immunotherapeutic target known to be upregulated on many hematologic and solid tumors but minimally expressed on normal tissue. While CD70 was previously explored in MM (McEarchern et al, Clin Cancer Res (2008)), it was not further investigated due to heterogeneous expression in newly diagnosed samples. Here we revisit this target to suggest cellular therapies against CD70 could be highly beneficial in high-risk MM patients, currently the greatest unmet need in the field. Methods: Primary patient samples were obtained under an IRB-approved protocol. CAR-T lentiviral transduction was performed on paired CD4/CD8 human T cells from at least 3 healthy donors. Effector function was assessed by cytotoxicity against MM cell lines and CAR-T profiling for memory and exhaustion markers. In vivo efficiency was assessed using bioluminescence. ATAC-seq peaks were called using Promo and HOMER (data from Jin et al. Blood (2018)). Machine learning was performed using XGBoost approach applied to CoMMpass RNA-seq data with 80% of samples for training and 20% as test set. Results: Analyzing CoMMpass mRNA data (release IA19) we first evaluated surface protein encoding-genes upregulated in tumors at relapse compared to diagnosis. Among >100 significantly upregulated genes, we focused on CD70 given known promise as an immunotherapy target. Further analysis across genomic subtypes revealed a specific, marked upregulation of CD70 in t(4;14) and gain 1q ( Fig. 1), as well as R-ISS 3 at diagnosis. Flow cytometry profiling of 8 cell line models, including t(4;14) models KHM11, KMS26, and LP1, revealed consistent CD70 protein expression with antigen density similar to BCMA. Profiling of relapsed primary MM samples confirmed that 19 of 28 samples had CD138+ plasma cells positive for CD70 expression. Of 5 patients in whom both BCMA and CD70 were measured, antigen densities were also similar. To target CD70, we then designed 10 unique anti-CD70 CAR-T constructs. 9 of these were scFv-based binders derived from therapeutic antibody fragments currently in preclinical or clinical development. By a structure-guided design based on the CD70:CD27 co-crystal structure (PDB: 7KX0), we also derived a unique truncated fragment of CD27 as an alternative CD70 binding element. All CD70 CARs were efficacious in in vitro cytotoxicity assays targeting MM cell lines AMO1, MM1.S, LP1, and RPMI8226. We then selected the top 3 performing scFv based-CARs as well as our natural ligand (CD27) CAR-T for in vivo studies. All 4 CD70 CAR-Ts prolonged survival of MM.1S-bearing mice compared to empty CAR ( n=5 mice/arm, p<0.005 by log-rank), similar to BCMA CAR-T controls. Remarkably, the CD27-based CAR showed superior expansion (50-85x, p< 0.0001 by ANOVA) and persistence compared to scFv-based ( Fig. 2). These results underscore that targeting CD70 using a natural ligand-based CAR-T represents a promising preclinical candidate for treatment of relapsed and high risk MM. We further investigated why CD70 may be upregulated in high risk MM. Analysis of ChIP-seq in ENCODE and primary MM tumor ATAC-seq was used to nominate a set of transcription factors that bind the CD70 locus. Extending from CoMMpass data, we built a predictive machine learning model for CD70 expression as a function of transcription factor gene expression, nominating key transcription factors TFAP2A, LEF1, MYB, and PAX5 as CD70 regulators; validation is now underway. Additionally, we showed that knockout of NSD2 in t(4;14) models KMS34 and KMS11 led to significantly decreased CD70 surface expression, implicating this central epigenetic driver in CD70 overexpression in this high-risk MM subtype. Finally, we also demonstrated that treatment with the hypomethylating agent azacytidine can drive increased CD70 expression, similar to that shown in AML models. Conclusion: Taken together, targeting CD70 appears to be a highly promising cellular therapeutic strategy for MM, with particularly high utility in high-risk patients after BCMA CAR-T relapse. We are continuing preclinical and future clinical development of this CAR-T strategy, using our natural ligand CD27 design with extended in vivo persistence.

Multiple Myeloma B Cells and Pre-Plasma Cells Are Important Reservoirs for Myeloma Relapse Following Plasma Cell-Directed Therapy and Prevent Cure with Standard Therapies

Multiple Myeloma (MM) is a B cell neoplasm characterized by the accumulation of mature plasma cells (PCs) within the bone marrow (BM). Despite treatment advances, MM remains incurable in the vast majority of patients. Disease relapse typically occurs regardless of treatment with proteasome inhibitors (such as bortezomib and carfilzomib), immunomodulatory drugs (such as lenalidomide and pomalidomide), anti-CD38 monoclonal antibodies (such as daratumumab and isatuximab) or myeloablative melphalan and autologous stem cell transplantation, and also occurs following treatment with immunotherapeutics such as bispecific antibodies and/or CAR-T cells (targeting BCMA, FcRH5 or GPRC5D). This failure to achieve reliable cure in MM, despite the routine attainment of deep treatment responses against PCs, indicates the existence of important intra-tumor heterogeneity and the presence of rare drug-resistant MM cells with full tumorigenic capacity. To identify intra-clonal MM cell subpopulations, we examined BM samples from MM patients using a combination of FACS, single cell resolved immunoflourescence-FISH (IF-FISH), whole exome sequencing (WES), custom-capture targeted deep sequencing (CC-Seq) and single cell RNA sequencing (scRNAseq). We sought to characterize the genomic landscape and to track the dynamic interconnectedness of these intra-clonal subpopulations in patients over time. Using sequential FACS-IF-FISH studies of MM BM samples (n=140) we identified MM clone cellular subpopulations within the BM of MM patients that recapitulate the normal maturation stages between non-malignant post germinal centre B cells and mature PCs. These subpopulations include rare MM cells that resemble CD20 +CD38 -CD138 -Bcma -Irf4 - Xbp1s - B cells, CD20 -CD38 -CD138 - Bcma -Irf4 -Xbp1s - pre-plasmablasts and CD38 +CD138 -Bcma -/lowIrf4 -/+Xbp1s + pre-plasma cells, as well as predominating tumor-bulk CD38 +CD138 +Irf4 +Bcma +Xbp1s + plasma cells. Importantly, MM progenitor cells were typically found to possess all of the chromosomal translocations and copy number variations (CNV) present in MM PCs including whole chromosome ploidy changes, translocations such as t(11;14), t(4;14) and t(14;16), and secondary aberrations such as gain(1q), del(1p) and del(17p). To examine if MM progenitor cells also possess the single nucleotide variations (SNV) present within MM PCs, and are thus fully malignant, and to track MM progenitor subpopulations and their inter-relatedness within patients over time, we performed WES (n=60) plus targeted deep CC-Seq (n=210) on purified cell subpopulations from 17 patients, including 5 patients whom we sampled serially over >3 years. A median of 5 cellular subpopulations were isolated from each BM sample, enriched by factors of up to 1000-fold or more, and each subpopulation was then sequenced to a depth of 4,000-20,000x. Significantly, SNVs that were present in tumor-bulk MM PCs were also regularly detected in MM progenitor cells, although the low frequency of the clonal progenitor cells often prevented capture of complete SNV profiles. At the same time, analyses of serial BM samples from repeat donor patients taken pre- and post-treatment demonstrated that relapsing MM PCs typically did not appear to derive linearly from pre-treatment PCs, as the relapsing PCs commonly lacked a number of SNVs that had been present in pre-treatment PCs. Importantly, we instead observed that relapsed MM plasma cells appeared to consistently derive from MM progenitor cell populations, frequently B cells, as emergent relapse-specific SNVs that were exclusively detected in relapsing post-treatment PCs (but in not pre-treatment PCs) could often also be detected within pre-treatment CD138- CD38- progenitor cells isolated from BM samples that had been collected up to 3 years prior to relapse. We conclude that MM B cells and pre-plasma cell progenitors possess all of the clonal genomic aberrations required for full malignant potential. Tracking of these cells in vivo in patients over time by their SNV profile implicates them as the cellular origin of PC relapse and recurrent MM disease. We propose that cure of MM requires eradication of MM progenitor cells alongside plasma cells. We are currently conducting scRNA-seq and CITE-seq studies of primary MM samples to further characterize MM progenitor cells for gene and protein expression in order to define their optimal therapeutic targets.

CD38 x ICAM-1 Bispecific Antibody Is a Novel Approach for Treating Multiple Myeloma and Lymphoma.

The cluster of differentiation 38 (CD38) is a well-validated target for treating multiple myeloma. Although anti-CD38 mAbs have demonstrated outstanding initial responses in patients with multiple myeloma, nearly all patients eventually develop resistance and relapse. In addition, currently approved CD38 targeting therapies have failed to show monotherapy efficacy in lymphomas, where CD38 expression is present but at lower levels. To effectively target CD38 on tumor cells, we generated an antibody-dependent cellular cytotoxicity (ADCC) enhanced bispecific CD38 x intercellular cell adhesion molecule 1 (ICAM-1) antibody, VP301. This bispecific antibody targets unique epitopes on CD38 and ICAM-1 on tumor cells with reduced red blood cell binding compared with the benchmark CD38 antibody daratumumab. VP301 demonstrated potent ADCC and antibody-dependent cellular phagocytosis activities on a selected set of myeloma and lymphoma cell lines even those with low CD38 expression. In an ex vivo drug sensitivity assay, we observed responses to VP301 in multiple myeloma primary samples from relapsed/refractory patients. Moreover, VP301 demonstrated potent tumor inhibition activities in in vivo myeloma and lymphoma models. Interestingly, combination of VP301 with the immunomodulatory drug, lenalidomide, led to synergistic antitumor growth activity in an in vivo efficacy study. In conclusion, the CD38 x ICAM-1 bispecific antibody VP301 demonstrated promising efficacy and specificity toward CD38+ and ICAM-1+ tumor cells and represents a novel approach for treating multiple myeloma and lymphoma.

Integrated epigenetic and transcriptional single-cell analysis of t(11;14) multiple myeloma and its BCL2 dependency

AbstractThe translocation t(11;14) occurs in 20% of patients with multiple myeloma (MM) and results in the upregulation of CCND1. Nearly two-thirds of t(11;14) MM cells are BCL2 primed and highly responsive to the oral BCL2 inhibitor venetoclax. Although it is evident that this unique sensitivity to venetoclax depends on the Bcl-2 homology domain 3– proapoptotic protein priming of BCL2, the biology underlying t(11;14) MM dependency on BCL2 is poorly defined. Importantly, the epigenetic regulation of t(11;14) transcriptomes and its impact on gene regulation and clinical response to venetoclax remain elusive. In this study, by integrating assay for transposase-accessible chromatin by sequencing (ATAC-seq) and RNA-seq at the single-cell level in primary MM samples, we have defined the epigenetic regulome and transcriptome associated with t(11;14) MM. A B-cell–like epigenetic signature was enriched in t(11;14) MM, confirming its phylogeny link to B-cell rather than plasma cell biology. Of note, a loss of a B-cell–like epigenetic signature with a gain of canonical plasma cell transcription factors was observed at the time of resistance to venetoclax. In addition, MCL1 and BCL2L1 copy number gains and structural rearrangements were linked to venetoclax resistance in patients with t(11;14) MM. To date, this is the first study in which both single-cell (sc) ATAC-seq and scRNA-seq analysis are integrated into primary MM cells to obtain a deeper resolution of the epigenetic regulome and transcriptome associated with t(11;14) MM biology and venetoclax resistance.

Abstract 207: Ex vivo modeling of multiple myeloma: A novel drug sensitivity screening platform

Abstract Multiple Myeloma (MM) is a heterogeneous malignancy characterized by abnormal clonal plasma cell infiltration in the bone marrow. It is the second most common hematologic malignancy, after non-Hodgkin’s Lymphoma. This deadly disease globally affects 1 to 5 people in every 100,000 people each year. After initial diagnosis, the 5-year survival rate is 44% because there is no curative therapy, and most patients will eventually experience relapse, and some become refractory. Large inter-patient and intra-patient genetic heterogeneity limit the identification of universal drivers of MM. However, several oncogenic dependencies such as primary events related to driver gene mutations and primary translocations can be targeted for better treatment strategies for R/R multiple myeloma. To establish a robust drug sensitivity screening platform, both MM cell lines and cryopreserved primary MM patient samples were used. MM cell lines screen with H929, MM1R and RPMI-8226 was performed to narrow down optimal concentrations of investigative test agents. During assay development, a panel of FDA approved agents for relapsed MM including panobinostat, bortezomib, melphalan, omacetaxine, and selinexor were tested. The cells were seeded at 50,000 cells per well in triplicates in a 96-well plate on day 0. The test agents were added in 9 concentrations with the top concentration being 25 uM with 2-fold dilutions. After 6- days of incubation, a luminescent cell viability assay was performed to calculate relative viability with drug treatment and IC50 was computed by fitting data using a standard four-parameter logistic model. For primary multiple myeloma samples, bone marrow aspirates were collected from Multiple Myeloma patients procured from different providers. BM mononuclear (BMMNC) cells were separated by Ficoll-Hypaque density sedimentation. Unfractionated BMMNC cells were used in these assays to preserve intra-tumor heterogeneity. Primary cells were seeded in triplicates per dose point on day 0 in Champions’ proprietary media. The test agents were also added on day 0 and were incubated for 6 days. On Day 6, the relative cell viability was measured based on Cell Titer Glo readout. The cryopreserved primary MM patient samples were viable in culture for the entire assay duration validating assay feasibility. A panel of FDA approved, standard of care drugs were tested in numerous clinically well-annotated and diverse MM patient models. Primary model selection was based on disease burden including plasma cell count from Core Biopsy and BM aspirates. Multiparametric flow cytometric immunophenotyping was also performed in these primary samples using monoclonal antibodies against CD56, CD19, CD117, CD27, CD138, and CD38. For the first time, we demonstrate the feasibility of a novel, ex-vivo drug sensitivity screening platform with cryopreserved, primary multiple myeloma cells from diverse, clinically well-annotated patient samples. Citation Format: Vaishnavi Sambandam, Sharvari Inamdar, Haoting Hsu, Brandon Walling, Paolo Schiviani, Abhay Andar, Marianna Zipeto, Michael Ritchie, Karin Abarca Heidemann, Maria Mancini. Ex vivo modeling of multiple myeloma: A novel drug sensitivity screening platform [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 207.

Prolonged Maintenance of Ex Vivo Functional Vk*MYC Multiple Myeloma Cells Allows for in Vitro Manipulation and Subsequent In Vivo Analysis

Human multiple myeloma (MM) cell lines are easy to culture but only partially reflect the heterogeneity of primary MM samples as most cell lines are derived from primary human plasma-cell leukemia samples and have often lost their dependence on the marrow microenvironment. In contrast, maintaining primary human MM samples ex vivo and in vivo is very challenging. To bridge this gap, the immunocompetent Vk*MYC transgenic MM model was developed. It has high biological fidelity to the human disease characteristics and features easy in vivo engraftment and serial transplantation. However, Vk*MYC cells are considered notoriously difficult to grow and manipulate in vitro, limiting their usage for functional studies. Aim To overcome these limitations, we aimed to develop a robust culture system which promotes survival and function of primary Vk*MYC MM cells in culture as a research tool for in vitro manipulation and subsequent in vivo transplantation. Method Splenocytes from tVk*MYC (Vk12598 clone) engrafted mice were routinely maintained ex vivo and conditioned media collected for M-spike quantification. Analysis of cultures by flow cytometry allowed for Vk*MYC population quantification over time. Our novel plating method includes the following components to support Vk*MYC cell survival: 3D plating as hanging drops, low (6.5%) oxygen culture, high density cultures and a cytokine cocktail. Results The culture method described here routinely results in maintenance or slight enrichment of Vk*MYC (CD138+B220-) splenocyte fraction. Monoclonal immunoglobulin (M spike) accumulation in conditioned culture media was assessed after 7 days culture as a clinically relevant, functional output marker. Culture media displayed the same immunoglobulin isotype as parent Vk*MYC (Vk12598 clone). Figure 1 shows previously frozen donor Vk*MYC splenocytes (day 0) assessed by flow cytometry and either injected directly, or maintained 7 days ex vivo, prior to IV injection into naive C57BL/6 recipient mice. Culture-recipient mice developed Vk*MYC MM disease with the same serum M-spike isotype band, similar engraftment ratio, similar time to evidence of biochemical disease and similar overall survival time as mice transplanted with freshly defrosted Vk*MYC splenocytes. To exemplify in vitro manipulation, Vk*MYC splenocytes were cultured as described and treated with Cy3-labelled liposomal nanoparticles (LNP), a non-toxic way to deliver treatment loads e.g. siRNAs, then maintained in culture for up to 7 days after treatment. Flow analysis showed >95% of viable CD138+ cells were Cy3 positive as a marker of LNP uptake. In addition, Vk*MYC splenocytes were transduced with GFP-encoding lentivirus, then maintained in culture for up to 7 days. Flow analysis showed viable, CD138+B220- cells expressing GFP as a marker of lentiviral transduction, highlighting the potential of this culture method to allow manipulation of Vk*MYC cells in vitro. Discussion This innovative culture method allows for survival and enrichment, but not expansion of Vk*MYC splenocytes in culture. Our seven-day culture protocol allows in vitro manipulation with liposomal nanoparticles as well as viral vectors and subsequent in vivo investigations. Figure 1. Experimental setup. A) (Day 0) donor tVk*MYC spleen was defrosted and assessed for MM burden by flow (CD138+ B220-). Splenocytes were maintained for 7 days ex vivo in 3D hanging drop cultures. At day 7, conditioned media was assessed by modified SPEP method for evidence of biochemical disease, and cultured cells assessed by flow or injected into naive C57BL/6 recipient mice. B) After 4 weeks, evidence of biochemical disease (M-spike) was confirmed by SPEP and mouse spleen Vk*MYC burden assessed by flow cytometry. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting an MDM2/MYC Axis to Overcome Drug Resistance in Multiple Myeloma.

Simple SummaryWe show that high expression of MDM2 is associated with poor prognosis and enhanced drug resistance in human myeloma cell lines (HMCLs). Inhibition of MDM2 by RNAi or by the MDM2/XIAP dual inhibitor MX69 significantly increased the sensitivity of resistant HMCLs and primary MM samples to bortezomib and other anti-myeloma drugs, demonstrating that MDM2 can modulate drug response. We uncovered a novel oncogenic regulatory loop between MDM2 and c-Myc that mediated MM drug resistance. MDM2 inhibition resulted in a remarkable induction of apoptosis and suppression of relapsed MM cell growth. Mechanistically, MDM2 stabilized c-Myc mRNA to upregulate its expression, while c-Myc transcriptionally inducedMDM2. Targeting this regulatory loop with MX69 re-sensitized chemo-resistant MM cells to anti-myeloma agents, irrespective of TP53 tumor suppressor status, and prolonged survival in xenograft MM mouse model. These results establish a rationale for therapeutic targeting of the MDM2/c-Myc axis to improve the clinical outcome of patients with refractory/relapsed MM.Background: MDM2 is elevated in multiple myeloma (MM). Although traditionally, MDM2 negatively regulates p53, a growing body of research suggests that MDM2 plays several p53-independent roles in cancer pathogenesis as a regulator of oncogene mRNA stability and translation. Yet, the molecular mechanisms underlying MDM2 overexpression and its role in drug resistance in MM remain undefined. Methods: Both myeloma cell lines and primary MM samples were employed. Cell viability, cell cycle and apoptosis assays, siRNA transfection, quantitative real-time PCR, immunoblotting, co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), soft agar colony formation and migration assay, pulse-chase assay, UV cross-linking, gel-shift assay, RNA-protein binding assays, MEME-analysis for discovering c-Myc DNA binding motifs studies, reporter gene constructs procedure, gene transfection and reporter assay, MM xenograft mouse model studies, and statistical analysis were applied in this study. Results: We show that MDM2 is associated with poor prognosis. Importantly, its upregulation in primary MM samples and human myeloma cell lines (HMCLs) drives drug resistance. Inhibition of MDM2 by RNAi, or by the MDM2/XIAP dual inhibitor MX69, significantly enhanced the sensitivity of resistant HMCLs and primary MM samples to bortezomib and other anti-myeloma drugs, demonstrating that MDM2 can modulate drug response. MDM2 inhibition resulted in a remarkable suppression of relapsed MM cell growth, colony formation, migration and induction of apoptosis through p53-dependent and -independent pathways. Mechanistically, MDM2 was found to reciprocally regulate c-Myc in MM; MDM2 binds to AREs on c-Myc 3′UTR to increase c-Myc mRNA stability and translation, while MDM2 is a direct transcriptional target of c-Myc. MDM2 inhibition rendered c-Myc mRNA unstable, and reduced c-Myc protein expression in MM cells. Importantly, in vivo delivery of MX69 in combination with bortezomib led to significant regression of tumors and prolonged survival in an MM xenograft model. Conclusion: Our findings provide a rationale for the therapeutic targeting of MDM2/c-Myc axis to improve clinical outcome of patients with refractory/relapsed MM.

AHSA1 is a promising therapeutic target for cellular proliferation and proteasome inhibitor resistance in multiple myeloma

BackgroundCurrently, multiple myeloma (MM) is still an incurable plasma cell malignancy in urgent need of novel therapeutic targets and drugs.MethodsBufalin was known as a highly toxic but effective anti-cancer compound. We used Bufalin as a probe to screen its potential targets by proteome microarray, in which AHSA1 was the unique target of Bufalin. The effects of AHSA1 on cellular proliferation and drug resistance were determined by MTT, western blot, flow cytometry, immunohistochemistry staining and xenograft model in vivo. The potential mechanisms of Bufalin and KU-177 in AHSA1/HSP90 were verified by co-immunoprecipitation, mass spectrometry, site mutation and microscale thermophoresis assay.ResultsAHSA1 expression was increased in MM samples compared to normal controls, which was significantly associated with MM relapse and poor outcomes. Furthermore, AHSA1 promoted MM cell proliferation and proteasome inhibitor (PI) resistance in vitro and in vivo. Mechanism exploration indicated that AHSA1 acted as a co-chaperone of HSP90A to activate CDK6 and PSMD2, which were key regulators of MM proliferation and PI resistance respectively. Additionally, we identified AHSA1-K137 as the specific binding site of Bufalin on AHSA1, mutation of which decreased the interaction of AHSA1 with HSP90A and suppressed the function of AHSA1 on mediating CDK6 and PSMD2. Intriguingly, we discovered KU-177, an AHSA1 selective inhibitor, and found KU-177 targeting the same site as Bufalin. Bufalin and KU-177 treatments hampered the proliferation of flow MRD-positive cells in both primary MM and recurrent MM patient samples. Moreover, KU-177 abrogated the cellular proliferation and PI resistance induced by elevated AHSA1, and decreased the expression of CDK6 and PSMD2.ConclusionsWe demonstrate that AHSA1 may serve as a promising therapeutic target for cellular proliferation and proteasome inhibitor resistance in multiple myeloma.

CD200 Expression on Multiple Myeloma Cells Induces Attenuation of T Cell-Mediated Cytotoxicity Via DOK2

IntroductionThe CD200/CD200 receptor (CD200R) axis is known to exert immunoregulatory effects in myeloid-derived cells and constitutes a putative immune checkpoint in hematological malignancies, in which CD200 expression is associated with poor prognosis. In multiple myeloma (MM), CD200 is expressed in the majority of patient-derived primary cells. However, its functional importance as well as the related downstream mechanism upon CD200 ligand binding to its CD200R on T cells are not well understood. In this study, we analyze the functional role of CD200 as a potential immune checkpoint in MM and decipher the mechanism of CD200-mediated immune escape.MethodsPrimary MM cells and MM cell lines were analyzed for CD200 surface expression by flow cytometry. To overexpress CD200 on non-expressing MM cell lines we used a Sleeping Beauty transposon vector system. CD200+/- MM cell lines L363, U266 and MM.1s were co-cultured with CD3/CD28-activated healthy donor T cells. T cell-mediated cytotoxicity in these co-cultures was assessed with flow cytometry and/or luciferase assay. Moreover, to analyze the effects of CD200R activation on downstream signaling pathways, activated T cells were treated with recombinant human CD200 (rhCD200) and/or anti-CD200 blocking antibody and subsequently, Western blotting was performed.ResultsOf n=120 primary MM samples (n=120) analyzed, CD200 protein expression could be detected in ca. 75 % of the cases. In contrast, all n=9 MM cell lines tested neither displayed surface nor cytoplasmic CD200 expression. Therefore, using a Sleeping Beauty transposon vector system we stably expressed CD200 on MM cell lines for further analyses. In the presence of CD200-expressing MM cells up to 50% decrease in CD3+ T cell-mediated cytotoxicity against MM cells was observed in flow cytometry and luciferase assay. Proliferation rates of MM cell lines remained unchanged regardless of the level of CD200 overexpression as determined by Alamar blue assays. In myeloid-derived cells, CD200R directly interacts with docking protein-2 (DOK2). In activated T cells, we observed DOK2 phosphorylation upon CD200 binding when treated with rhCD200 in a time- and concentration-dependent manner. Applying an anti-CD200 blocking antibody, this effect could be reversed, thus revealing a possible mechanism for the observed attenuation of T cell function.ConclusionOur study shows that anti-MM cytotoxicity from primary healthy donor CD3+ T cells is attenuated by CD200 expression on MM cells. We also demonstrate that this inhibitory mechanism in CD3+ T cells is mediated via DOK2, providing a potential target for immunotherapeutic approaches in MM. DisclosuresEinsele: Janssen, Celgene/BMS, Amgen, GSK, Sanofi: Consultancy, Honoraria, Research Funding.

Functional expression of aryl hydrocarbon receptor as a potential novel therapeutic target in human multiple myeloma

The etiology of multiple myeloma (MM) remains incompletely understood; however, epidemiologic studies have suggested a possible link between exposure to environmental aromatic hydrocarbons—which serve as exogenous ligands for the aryl hydrocarbon receptor (AHR), which has been implicated in cancer biology—and development of monoclonal gammopathy of undetermined significance (MGUS) and MM. Herein, we demonstrate the functional expression of AHR in MM cell lines and primary human MM samples. AHR is expressed in putative MM ‘stem cells’ and advanced clinical stages of MM, and functionally contributes to MM tumor cell phenotype and proliferation. Antagonism of AHR directly impairs MM cell viability and increases MM cell susceptibility to immune-mediated clearance. Furthermore, our findings indicate that AHR antagonism may represent an effective means to enhance the function of other drugs, such as anti-CD38 antibodies, in future clinical studies. Taken together, these data identify AHR as a novel target for MM therapy.

PIKfyve Inhibitors for the Treatment of Multiple Myeloma

IntroductionUsing a high throughput screening approach employing ~80 FDA approved, late stage clinical trial, or drugs of interest to multiple myeloma (MM), we examined single agent drug sensitivity of 35 MM and lymphoma cell lines and in 65 primary MM samples. We identified APY-0201, a PIKfyve inhibitor (P5I), as lethal at nanomolar concentrations in 48% of human myeloma cell lines (HMCLs) and 28.6% of primary MM samples after 24 hour of drug exposure. In 20% of patient samples, in vitro sensitivity was less than 100nM EC50. PIKfyve is a lipid and protein kinase that controls pleiotropic cellular functions, notably regulation of endomembrane homeostasis; mechanisms of sensitivity and resistance were explored.MethodsApilimod, a key P5I initially developed as an interleukin (IL)-12 and IL-23 inhibitor, has been used as an immunomodulatory agent in the treatment of inflammatory diseases and has been recently suggested for the treatment of neoplastic diseases, including B-cell Non-Hodgkin's Lymphoma (NHL). YM201636 has also been described as a potent P5I. The cellular efficacies of APY-0201, apilimod, and YM201636 in HMCLs were assessed using the CellTiter Glo assay (Promega), in 20-point 2-fold dilutions of drug concentrations at 72 hours; and HMCL P5I sensitivity was correlated with RNA-SEQ data publically available at www.keatslab.org. Cell lysates were extracted for western blot expression analysis of transcription factor EB (TFEB), cathepsin D, caspase 3, poly (ADP-ribose) polymerase (PARP), at different drug exposure time points (3, 6, 12, 24, 48, and 72 hours). Ex vivo sensitivity to APY-0201, the most potent studied P5I, was measured in CD138+ primary cells of 56 primary MM patients at 24 hours. In vivo activity of apilimod at 100-150 mg/kg and APY-0201 at 30-45 mg/kg was evaluated over 2 weeks in the Vk*MYC transgenic mouse model of MM. Tumor burden was measured by weekly evaluation of M-spike serum levels.ResultsAPY-0201 was the most potent P5I, followed by YM201636 and apilimod. However, dose-dependent inhibition of cell viability was observed in all HMCLs. APY-0201 EC50 ranged from 2.8 to 266.7nM (mean 123.4nM), 63.8 to 8425.8nM (mean 1329nM) for YM201636, and 62.5 to 3561.2nM (mean 2026nM) for apilimod. APY-0201 maximum inhibition ranged from 68.2 to 100% (mean 87.2%), 33.2 to 100% (mean 64.6%) for YM201636, and 56.7 to 100% (mean 83.6%) for apilimod. A significant correlation between low RNA-SEQ expression of TFEB, which regulates lysosomal biogenesis and autophagy, and lower sensitivity for APY-0201 was found (Wilcoxon signed-rank test p-value = 0.017). No correlation with baseline OSTM1, CLCN7 or SNX10 levels was seen.APY-0201 dose dependent sensitivities were observed in 37.5% of ex vivo patient samples. Of the 21 patients demonstrating sensitivity, 2 were inhibited at an EC50<10nM (median 8.58nM), 10 samples presented EC50 between 10 and 100nM (median 64.67nM), and 9 samples presented EC50>100nM (median 1000.16nM). Thus, 12 (21.4%) primary MM patient samples exhibited lethality with less than 100nM concentration of APY-0201 after 24 hours. P5I activity profile was not obviously correlated with clinical findings, such as stage of disease or genetic subtype, in either cell lines or primary samples.As demonstrated previously in B-cell NHL, treatment of the most sensitive cell line, KMS26, with apilimod and APY-0201 was associated with activation of TFEB, found in its dephosphorylated state. An accumulation of the inactive precursor procathepsin D was also observed in KMS26 following treatment, demonstrating a decrease in maturation of lysosomal proteases. Furthermore, the cytotoxic activity of both compounds was shown by induction of apoptosis makers, i.e. caspase 3 and PARP. In ongoing studies, in vivo activity was evident in 2 of 6 Vk*MYC transgenic MM mice treated with a P5I; dose finding and longer treatment duration studies are underway.ConclusionsInhibition of PIKfyve surprisingly demonstrated promising anti-myeloma activity in vitro, ex vivo and in vivo, thus providing a rationale for further evaluation in the treatment of MM. As reported by others in NHL, a novel mechanism of action involving disruption of endolysosomal function is suggested in PIKfyve mediated cytotoxicity in MM.AcknowledgmentThis work was supported in part by the Multiple Myeloma Research Foundation. DisclosuresSharik:Phosplatin Therapeutics: Research Funding. Sepetov:Nanosyn Inc.: Employment. Romanov:Nanosyn Inc.: Employment. Bergsagel:Phosplatin Therapeutics: Research Funding. Chesi:Phosplatin Therapeutics: Research Funding. Stewart:Amgen: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Roche: Consultancy.

The Interaction of NEK2 with USP7 Causes Resistance to Proteasome Inhibitor in Multiple Myeloma

Background: Drug resistance is the major reason for treatment failure in multiple myeloma (MM). Our previous work has demonstrated that high expression of chromosomal instability (CIN) genes results in increased cell survival and drug resistance and is associated with poor outcome in MM and other cancers. NEK2 was the most significant CIN gene to induce drug resistance and poor outcome in MM. Though we have demonstrated that NEK2 induces drug resistance through activation of efflux drug pumps, the detail mechanisms remain unknown.Materials and Methods: Multiple MM cell lines, xenograft MM NOD-Rag1nullmice with both subcutaneous and by tail vein injection, and primary MM samples were included in this study to test NEK2 inhibitors and gene expression profiles (GEP). The tandem affinity purification - mass spectrometry (TAP-MS) and co-immunoprecipitation (Co-IP) were applied for the identification of NEK2 interacting partners. The dual-luciferase reporter assay and GEPs were performed to determine whether the interaction of NEK2 with USP7 activates the canonical NF-kB signaling pathway. Chromatin immunoprecipitation (ChIP)-qPCR and western blots were carried out to determine the direct target genes of the axis NEK2/USP7/NF-kB.Results: We demonstrate that NEK2 is stabilized by the de-ubiquitinating enzyme USP7, leading to accumulation of NEK2 within the cancer cells. Accumulation of NEK2 activates the canonical NF-kB through the AKT/IKK/IKB-a/p65 axis and, using this pathway as framework, we uncovered a novel bona fide NF-kB target, heparanase, a protein that is known to promote bone destruction in MM patients. A NF-kB or USP7 inhibitor could overcome NEK2-induced bortezomib resistance. The NEK2 inhibitor, INH1, showed great therapeutic effects on MM in vitro and in vivo . To corroborate our findings, we subcutaneously injected NEK2-OE ARP1 MM cells into NOD.Cg-Rag1 mice and targeted NEK2 with the small molecule drugs INH1 or P5091 (USP7 inhibitor) alone or in combination with bortezomib. As expected bortezomib had very little effect in reducing tumor burden since NEK2-OE cells are highly resistant to this drug. Inhibition of NEK2 with P5091 or INH1 alone had a strong anti-tumor effect and a synergistic effect was observed when combined with bortezomib.Conclusion: Our data present a novel mechanism for the interaction of NEK2 with USP7 driving drug resistance in MM. We conclude that (1) USP7 in MM cells leads to the de-ubiquitination and escape of proteasomal degradation of NEK2, leading to its accumulation in cancer cells. (2) Accumulated NEK2 binds and phosphorylates PP1a, suppressing its phosphatase activity thus promoting AKT overactivation. (3) AKT then phosphorylates the IKKa/b complex, triggering the canonical NF-kB cascade which phosphorylates and targets IKBa for proteasomal degradation, releasing the sequestered p65 to be phosphorylated at S536 and translocation to the nucleus where it drives bortezomib resistance by activating its target genes. Our data also show that INH1 and P5091 can deplete NEK2 protein in vitro and in vivo and suppress NEK2 downstream targets resulting in overcoming bortezomib resistance. Our findings should facilitate novel targeted approaches to the therapy and prevention of myeloma progression and relapse. DisclosuresNo relevant conflicts of interest to declare.

“Direct to Drug” Screening As a Route to Individualized Therapy in Multiple Myeloma

Although more than 2,000 multiple myeloma (MM) genomes are now available, the expected era of genomic-driven precision medicine has not emerged since actionable mutations are uncommon, mostly subclonal, and, for now, lack matched therapeutics. Advances in high throughput screening and the rapid expansion of FDA approved targeted oncology drugs now raise the prospect of “direct to drug” screening as an alternate route to genomic-driven individualized therapy and as a source for chemogenomic interrogation of drug sensitivity and resistance.Using an in house bioassay of FDA approved or late stage clinical trial drugs, epigenetic and kinase inhibitors screened in MM and NHL cell lines and by collating clinical experience, we established a panel of ˜80 MM relevant FDA approved and late stage clinical trial drugs for our standardized screening platform. This custom drug panel was specifically designed for screening live CD138+ selected tumor cells from MM patients with immediately actionable results.Feasibility studies were conducted in human MM cell lines over 72h using a real time 384 well format, non-lytic cellular viability assay (Real Time Glo, Promega). These studies revealed an optimal cellular concentration per well, and that drug efficacy can be detected as early as 24 hours for most compounds, with the notable exception of immunomodulatory drugs (IMiDs). Further optimization using a lytic cellular viability assay (Cell Titer Glo, Promega) confirmed that a 24h drug exposure time point could be fixed for clinical relevance (72h for IMiDs). We then derived consensus conditions to screen the MM drug panel in live CD138+ sorted tumor cells collected from MM patients. Baseline clinical and cytogenetic characteristics are recorded, and a library of baseline DNA and RNA is assembled for further genomic interrogation.The MM ˜80 drug panel was first screened in 25 human MM and 10 lymphoma cell lines of known genetic subtype and drug dose dependency measured in duplicate for 7 drug doses covering a broad concentration range. All drugs exhibiting dose-dependent reduction of cellular viability were reported as hits in the screen and the data collated into a drug sensitivity map. At 24h, 10 drugs exhibited broad cytotoxicity across all cell lines, increasing to 22 drugs at 72h. These included all proteasome inhibitors, the CDK5 inhibitor dinaciclib, the CRM disruptor selinexor and the HDAC inhibitors panobinostat and romidepsin. Conversely, 16 drugs were completely inactive at 24h. As expected, IMiDs and other activities emerged at 72h and the number of inactives was reduced to 7 drugs, including erlotinib, vismodegib, enasidenib, nindetanib, plerixafor, tofacitinib, and the negative control of unmetabolized cyclophosphamide. Other drug classes, exemplified by venetoclax, presented differential activity profiles that correlated with genomic features, and novel target classes for MM were suggested (e.g. PIKfyve).To date, 65 primary patient samples have been screened, of which 23 were newly diagnosed and 42 received previous treatment (median of prior therapies = 5). Eleven drugs were active in less than 5% of primary patient samples, including: lenvatinib, tofacitinib, paclitaxel, pemetrexed, vismodegib, decitabine, and plerixafor. In contrast, 8 drugs were active in more than 70% of patients samples at nanomolar concentrations, including dinaciclib, bortezomib, carfilzomib, ixazomib, panobinostat, selinexor, venetoclax, romidepsin, and belinostat. The most potent drugs, with the majority of patient samples presenting EC50s lower than 10nM, were dinaciclib, carfilzomib, panobinostat, and romidepsin.Using bromodomain inhibitors (BETi) as an example, in 42 primary MM samples, 17 (40%) were responsive to all three BETis and 32 had dose-dependent response to at least 1 of the BETis. Of these 32 drug sensitive patients, 18 were genetically hyperdiploid and 5 had a FISH defined disruption at the Myc locus (71%), suggesting the expected correlation of Myc expression with responsiveness. Similar positive correlation was seen with venetoclax and presence of a t(11;14).As numbers of tested patients increase, a powerful dataset emerges of ex vivo drug sensitivities linked with clinical outcomes and genomic biomarkers, which can be harnessed for clinical trial enrichment, MOA discovery, and potentially patient care, enlightened by clinical and genomic annotation. DisclosuresMikhael:Abbvie: Research Funding; sanofi-aventis: Research Funding; Celgene Corp: Research Funding. Reeder:Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Research Funding; Millennium: Research Funding; Affimed: Research Funding. Bergsagel:Phosplatin Therapeutics: Research Funding. Fonseca:Celgene Corporation: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy; AMGEN: Consultancy; Bayer: Consultancy; Takeda: Consultancy; Jansen: Consultancy; Mayo Clinic & Dr Fonseca: Patents & Royalties: Prognostication of myeloma via FISH, ~$2000/year; Pharmacyclics: Consultancy; Sanofi: Consultancy; Merck: Consultancy; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees. Sepetov:Nanosyn Inc.: Employment. Romanov:Nanosyn Inc.: Employment. Stewart:Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Roche: Consultancy.

Charting Venetoclax Sensitivity and Synergism Ex Vivo in Multiple Myeloma

We have implemented a drug screening platform for evaluation of multiple myeloma (MM) relevant therapeutics alone or in combination, providing immediate actionable results. We assembled 80 assay-ready, MM relevant FDA approved drugs and clinical trial therapeutics. This panel was evaluated in 25 MM and 10 Non-Hodgkin lymphoma cell lines, and in 65 primary MM samples using a viability endpoint. The panel includes venetoclax, a selective BCL-2 inhibitor that is currently in clinical development for MM as a single agent or in combination with bortezomib and dexamethasone. Single agent clinical trial results have shown that venetoclax is active in 21% of all relapsed refractory MM patients, 40% of patients with a t(11;14) and only 9% of patients lacking this translocation. It is unclear why not all patients with this translocation respond and who the 9% of non-translocated responders are. We were interested to know if our assay exhibited the same targeted pattern of activity and in assembling a genomic database to interrogate these questions.The cellular efficacies of the MM drug panel, including venetoclax, were measured using the CellTiter Glo assay (Promega), in 25 MM cell lines using 7 drug doses covering a broad concentration range at both 24 and 72 hour time points. Concurrent with clinical observations, venetoclax presents a differential activity profile (Table 1). At 72h drug incubation, 75% of t(11;14) MM cell lines responded with an EC50<100nM but only 3 of 21 (14%) of translocation negative cell lines responded (overall response rate of 24%). Ex vivo sensitivity to venetoclax was also measured following 24h drug exposure in CD138+ selected tumor cells from 56 MM patients, 15 of which have a t(11;14). A majority of patient samples (85%) exhibit a dose response curve to venetoclax, but a broad EC50 range is observed (0.59 to 4900 nM). Twenty-seven patient samples (48% of all those tested) were inhibited by venetoclax at <100 nM concentrations, 10 of the 15 patients with t(11;14) responded at <100 nanomolar EC50, accounting for 67% of the samples with that translocation. Two patients with a t(6;14) also responded. For patients who lack either cyclin D1/D3 translocations (n=39), 15 responded with efficacies <100nM, or 38%. These trends are confirmed by median efficacies, with a median EC50 for t(11;14) samples of 29nM, much lower than the 118nM median observed for samples without this translocation. Nevertheless 17 of 41 patients lacking the t(11;14) are sensitive below 100nM EC50, a group which clearly deserves a deeper genomic analysis and clinical correlation.Using venetoclax as a base drug, we then studied all possible combinations with the 79 other agents of the MM drug panel in sensitive and resistant HMCLs. The cytotoxic response of the base and combined drugs was measured in 5-point 2-fold drug dilutions centered on their respective EC50s. Primary combination screen results were scored using consensus performance metrics from Chou-Talalay, Highest Single Agent (HSA), and Bliss independence models. Combinations predicted synergistic by at least 2 methods were confirmed in a matrix format. The screen combining venetoclax and the drug panel was designed to identify synergistic combinations that enhance venetoclax response in groups that lack t(11;14) and have either intermediate sensitivity (OPM2) or are resistant (AMO1). In OPM2, 25 drugs were found to synergize with venetoclax with a combination index CI < 0.2 (strong synergism), vs 1 non overlapping drug in the resistant HMCL, AMO1. The confirmation of these findings in a matrix format and in sensitive cell lines will be reported.In summary, our data corroborates the clinical observation of a strong pattern for venetoclax response and t(11;14) through drug screening in primary patient materials ex vivo with a 24 hour read out. Using this strategy we will begin to add drug combinations of high promise to our drug screening platform to develop the map of drug synergism in multiple myeloma.Acknowledgments: We thank the Multiple Myeloma Research Foundation for funding this project. [Display omitted] DisclosuresStewart:Bristol-Myers Squibb: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Roche: Consultancy.