ABL1 kinase plays an important role in spontaneous and chemotherapy-induced genomic instability in multiple myeloma.

ABSTRACT: Genomic instability contributes to cancer progression and is at least partly due to dysregulated homologous recombination (HR). Here, we show that an elevated level of ABL1 kinase overactivates the HR pathway and causes genomic instability in multiple myeloma (MM) cells. Inhibiting ABL1 with either short hairpin RNA or a pharmacological inhibitor (nilotinib) inhibits HR activity, reduces genomic instability, and slows MM cell growth. Moreover, inhibiting ABL1 reduces the HR activity and genomic instability caused by melphalan, a chemotherapeutic agent used in MM treatment, and increases melphalan's efficacy and cytotoxicity in vivo in a subcutaneous tumor model. In these tumors, nilotinib inhibits endogenous as well as melphalan-induced HR activity. These data demonstrate that inhibiting ABL1 using the clinically approved drug nilotinib reduces MM cell growth, reduces genomic instability in live cell fraction, increases the cytotoxicity of melphalan (and similar chemotherapeutic agents), and can potentially prevent or delay progression in patients with MM.

Differentiation of BCMA-specific induced pluripotent stem cells into rejuvenated CD8αß+ T cells targeting multiple myeloma.

ABSTRACT: A major hurdle in adoptive T-cell therapy is cell exhaustion and failure to maintain antitumor responses. Here, we introduce an induced pluripotent stem cell (iPSC) strategy for reprogramming and revitalizing precursor exhausted B-cell maturation antigen (BCMA)-specific T cells to effectively target multiple myeloma (MM). Heteroclitic BCMA72-80 (YLMFLLRKI)-specific CD8+ memory cytotoxic T lymphocytes (CTL) were epigenetically reprogrammed to a pluripotent state, developed into hematopoietic progenitor cells (CD34+ CD43+/CD14- CD235a-), differentiated into the T-cell lineage and evaluated for their polyfunctional activities against MM. The final T-cell products demonstrated (1) mature CD8αß+ memory phenotype, (2) high expression of activation or costimulatory molecules (CD38, CD28, and 41BB), (3) no expression of immune checkpoint and senescence markers (CTLA4, PD1, LAG3, and TIM3; CD57), and (4) robust proliferation and polyfunctional immune responses to MM. The BCMA-specific iPSC-T cells possessed a single T-cell receptor clonotype with cognate BCMA peptide recognition and specificity for targeting MM. RNA sequencing analyses revealed distinct genome-wide shifts and a distinctive transcriptional profile in selected iPSC clones, which can develop CD8αß+ memory T cells. This includes a repertoire of gene regulators promoting T-cell lineage development, memory CTL activation, and immune response regulation (LCK, IL7R, 4-1BB, TRAIL, GZMB, FOXF1, and ITGA1). This study highlights the potential application of iPSC technology to an adaptive T-cell therapy protocol and identifies specific transcriptional patterns that could serve as a biomarker for selection of suitable iPSC clones for the successful development of antigen-specific CD8αß+ memory T cells to improve the outcome in patients with MM.

RAS/RAFlandscape in monoclonal plasma cell conditions.

Multiple Myeloma (MM) is characterized by a huge heterogeneity at the molecular level. The RAS/RAF pathway is the most frequently mutated, in about 50% of the patients. However, these mutations are frequently subclonal, suggesting a secondary event. Since these genes are part of our routine next-generation sequencing (NGS) panel, we analyzed >10,000 patients with different plasma cell disorders in order to describe the RAS/RAF landscape. In this large cohort of patients, almost 61% of the patients presented a RAS/RAF mutation at diagnosis or relapse, but much lower frequencies in pre-symptomatic cases. Of note, the mutations were different from that observed in solid tumors (higher proportions of Q61 mutations). In 29 patients with two different mutations, we were able to perform single cell sequencing, showing that in most cases, mutations occurred in different subclones, suggesting an ongoing mutational process. These findings suggest that RAS/RAF pathway is not an attractive target, both on therapeutic and residual disease assessment points of vue.

Loss of GABARAP mediates resistance to immunogenic chemotherapy in multiple myeloma.

ABSTRACT: Immunogenic cell death (ICD) is a form of cell death by which cancer treatments can induce a clinically relevant antitumor immune response in a broad range of cancers. In multiple myeloma (MM), the proteasome inhibitor bortezomib is an ICD inducer and creates durable therapeutic responses in patients. However, eventual relapse and resistance to bortezomib appear inevitable. Here, by integrating patient transcriptomic data with an analysis of calreticulin (CRT) protein interactors, we found that GABA type A receptor-associated protein (GABARAP) is a key player whose loss prevented tumor cell death from being perceived as immunogenic after bortezomib treatment. GABARAP is located on chromosome 17p, which is commonly deleted in patients with high risk MM. GABARAP deletion impaired the exposure of the eat-me signal CRT on the surface of dying MM cells in vitro and in vivo, thus reducing tumor cell phagocytosis by dendritic cells and the subsequent antitumor T-cell response. Low GABARAP was independently associated with shorter survival in patients with MM and reduced tumor immune infiltration. Mechanistically, we found that GABARAP deletion blocked ICD signaling by decreasing autophagy and altering Golgi apparatus morphology, with consequent defects in the downstream vesicular transport of CRT. Conversely, upregulating autophagy using rapamycin restored Golgi morphology, CRT exposure, and ICD signaling in GABARAPKO cells undergoing bortezomib treatment. Therefore, coupling an ICD inducer, such as bortezomib, with an autophagy inducer, such as rapamycin, may improve patient outcomes in MM, in which low GABARAP in the form of del(17p) is common and leads to worse outcomes.

Triplet Therapy, Transplantation, and Maintenance until Progression in Myeloma.

BACKGROUND: In patients with newly diagnosed multiple myeloma, the effect of adding autologous stem-cell transplantation (ASCT) to triplet therapy (lenalidomide, bortezomib, and dexamethasone [RVD]), followed by lenalidomide maintenance therapy until disease progression, is unknown. METHODS: In this phase 3 trial, adults (18 to 65 years of age) with symptomatic myeloma received one cycle of RVD. We randomly assigned these patients, in a 1:1 ratio, to receive two additional RVD cycles plus stem-cell mobilization, followed by either five additional RVD cycles (the RVD-alone group) or high-dose melphalan plus ASCT followed by two additional RVD cycles (the transplantation group). Both groups received lenalidomide until disease progression, unacceptable side effects, or both. The primary end point was progression-free survival. RESULTS: Among 357 patients in the RVD-alone group and 365 in the transplantation group, at a median follow-up of 76.0 months, 328 events of disease progression or death occurred; the risk was 53% higher in the RVD-alone group than in the transplantation group (hazard ratio, 1.53; 95% confidence interval [CI], 1.23 to 1.91; P<0.001); median progression-free survival was 46.2 months and 67.5 months. The percentage of patients with a partial response or better was 95.0% in the RVD-alone group and 97.5% in the transplantation group (P = 0.55); 42.0% and 46.8%, respectively, had a complete response or better (P = 0.99). Treatment-related adverse events of grade 3 or higher occurred in 78.2% and 94.2%, respectively; 5-year survival was 79.2% and 80.7% (hazard ratio for death, 1.10; 95% CI, 0.73 to 1.65). CONCLUSIONS: Among adults with multiple myeloma, RVD plus ASCT was associated with longer progression-free survival than RVD alone. No overall survival benefit was observed. (Funded by the National Heart, Lung, and Blood Institute and others; DETERMINATION ClinicalTrials.gov number, NCT01208662.).

Single-cell profiling in multiple myeloma: insights, problems, and promises.

In a short time, single-cell platforms have become the norm in many fields of research, including multiple myeloma (MM). In fact, the large amount of cellular heterogeneity in MM makes single-cell platforms particularly attractive because bulk assessments can miss valuable information about cellular subpopulations and cell-to-cell interactions. The decreasing cost and increasing accessibility of single-cell platform, combined with breakthroughs in obtaining multiomics data for the same cell and innovative computational programs for analyzing data, have allowed single-cell studies to make important insights into MM pathogenesis; yet, there is still much to be done. In this review, we will first focus on the types of single-cell profiling and the considerations for designing a single-cell profiling experiment. Then, we will discuss what have learned from single-cell profiling about myeloma clonal evolution, transcriptional reprogramming, and drug resistance, and about the MM microenvironment during precursor and advanced disease.

The location of the t(4;14) translocation breakpoint within the NSD2 gene identifies a subset of patients with high-risk NDMM.

Although translocation events between chromosome 4 (NSD2 gene) and chromosome 14 (immunoglobulin heavy chain [IgH] locus) (t(4;14)) is considered high risk in newly diagnosed multiple myeloma (NDMM), only ∼30% to 40% of t(4;14) patients are clinically high risk. We generated and compared a large whole genome sequencing (WGS) and transcriptome (RNA sequencing) from 258 t(4;14) (n = 153 discovery, n = 105 replication) and 183 non-t(4;14) NDMM patients with associated clinical data. A landmark survival analysis indicated only ∼25% of t(4;14) patients had an overall survival (OS) <24 months, and a comparative analysis of the patient subgroups identified biomarkers associated with this poor outcome, including translocation breakpoints located in the NSD2 gene and expression of IgH-NSD2 fusion transcripts. Three breakpoints were identified and are designated as: "no-disruption" (upstream of NSD2), "early-disruption" (in the 5' UTR), and "late-disruption" (within the NSD2 gene). Our results show a significant difference in OS based on the location of DNA breakpoints (median OS 28.6 "late-disruption" vs 59.2 "early disruption" vs 75.1 months "no disruption"). These findings have been replicated in an independent replication dataset. Also, univariate and multivariate analysis suggest high-risk markers such as del17p, 1p independently contribute to poor outcome in t(4;14) MM patients.

Ubiquitin receptor PSMD4/Rpn10 is a novel therapeutic target in multiple myeloma.

PSMD4/Rpn10 is a subunit of the 19S proteasome unit that is involved with feeding target proteins into the catalytic machinery of the 26S proteasome. Because proteasome inhibition is a common therapeutic strategy in multiple myeloma (MM), we investigated Rpn10 and found that it is highly expressed in MM cells compared with normal plasma cells. Rpn10 levels inversely correlated with overall survival in patients with MM. Inducible knockout or knockdown of Rpn10 decreased MM cell viability both in vitro and in vivo by triggering the accumulation of polyubiquitinated proteins, cell cycle arrest, and apoptosis associated with the activation of caspases and unfolded protein response-related pathways. Proteomic analysis revealed that inhibiting Rpn10 increased autophagy, antigen presentation, and the activation of CD4+ T and natural killer cells. We developed an in vitro AlphaScreen binding assay for high-throughput screening and identified a novel Rpn10 inhibitor, SB699551 (SB). Treating MM cell lines, leukemic cell lines, and primary cells from patients with MM with SB decreased cell viability without affecting the viability of normal peripheral blood mononuclear cells. SB inhibited the proliferation of MM cells even in the presence of the tumor-promoting bone marrow milieu and overcame proteasome inhibitor (PI) resistance without blocking the 20S proteasome catalytic function or the 19S deubiquitinating activity. Rpn10 blockade by SB triggered MM cell death via similar pathways as the genetic strategy. In MM xenograft models, SB was well tolerated, inhibited tumor growth, and prolonged survival. Our data suggest that inhibiting Rpn10 will enhance cytotoxicity and overcome PI resistance in MM, providing the basis for further optimization studies of Rpn10 inhibitors for clinical application.

Whole-genome analysis identifies novel drivers and high-risk double-hit events in relapsed/refractory myeloma.

Large-scale analyses of genomic data from patients with newly diagnosed multiple myeloma (ndMM) have been undertaken, however, large-scale analysis of relapsed/refractory MM (rrMM) has not been performed. We hypothesize that somatic variants chronicle the therapeutic exposures and clonal structure of myeloma from ndMM to rrMM stages. We generated whole-genome sequencing (WGS) data from 418 tumors (386 patients) derived from 6 rrMM clinical trials and compared them with WGS from 198 unrelated patients with ndMM in a population-based case-control fashion. We identified significantly enriched events at the rrMM stage, including drivers (DUOX2, EZH2, TP53), biallelic inactivation (TP53), noncoding mutations in bona fide drivers (TP53BP1, BLM), copy number aberrations (CNAs; 1qGain, 17pLOH), and double-hit events (Amp1q-ISS3, 1qGain-17p loss-of-heterozygosity). Mutational signature analysis identified a subclonal defective mismatch repair signature enriched in rrMM and highly active in high mutation burden tumors, a likely feature of therapy-associated expanding subclones. Further analysis focused on the association of genomic aberrations enriched at different stages of resistance to immunomodulatory agent (IMiD)-based therapy. This analysis revealed that TP53, DUOX2, 1qGain, and 17p loss-of-heterozygosity increased in prevalence from ndMM to lenalidomide resistant (LENR) to pomalidomide resistant (POMR) stages, whereas enrichment of MAML3 along with immunoglobulin lambda (IGL) and MYC translocations distinguished POM from the LEN subgroup. Genomic drivers associated with rrMM are those that confer clonal selective advantage under therapeutic pressure. Their role in therapy evasion should be further evaluated in longitudinal patient samples, to confirm these associations with the evolution of clinical resistance and to identify molecular subsets of rrMM for the development of targeted therapies.

High-dose melphalan treatment significantly increases mutational burden at relapse in multiple myeloma.

High-dose melphalan (HDM) improves progression-free survival in multiple myeloma (MM), yet melphalan is a DNA-damaging alkylating agent; therefore, we assessed its mutational effect on surviving myeloma cells by analyzing paired MM samples collected at diagnosis and relapse in the IFM 2009 study. We performed deep whole-genome sequencing on samples from 68 patients, 43 of whom were treated with RVD (lenalidomide, bortezomib, and dexamethasone) and 25 with RVD + HDM. Although the number of mutations was similar at diagnosis in both groups (7137 vs 7230; P = .67), the HDM group had significantly more mutations at relapse (9242 vs 13 383, P = .005). No change in the frequency of copy number alterations or structural variants was observed. The newly acquired mutations were typically associated with DNA damage and double-stranded breaks and were predominantly on the transcribed strand. A machine learning model, using this unique pattern, predicted patients who would receive HDM with high sensitivity, specificity, and positive prediction value. Clonal evolution analysis showed that all patients treated with HDM had clonal selection, whereas a static progression was observed with RVD. A significantly higher percentage of mutations were subclonal in the HDM cohort. Intriguingly, patients treated with HDM who achieved complete remission (CR) had significantly more mutations at relapse yet had similar survival rates as those treated with RVD who achieved CR. This similarity could have been due to HDM relapse samples having significantly more neoantigens. Overall, our study identifies increased genomic changes associated with HDM and provides rationale to further understand clonal complexity.

CDK7 controls E2F- and MYC-driven proliferative and metabolic vulnerabilities in multiple myeloma.

Therapeutic targeting of CDK7 has proven beneficial in preclinical studies, yet the off-target effects of currently available CDK7 inhibitors make it difficult to pinpoint the exact mechanisms behind MM cell death mediated by CDK7 inhibition. Here, we show that CDK7 expression positively correlates with E2F and MYC transcriptional programs in cells from patients with multiple myeloma (MM); its selective targeting counteracts E2F activity via perturbation of the cyclin-dependent kinases/Rb axis and impairs MYC-regulated metabolic gene signatures translating into defects in glycolysis and reduced levels of lactate production in MM cells. CDK7 inhibition using the covalent small-molecule inhibitor YKL-5-124 elicits a strong therapeutic response with minimal effects on normal cells, and causes in vivo tumor regression, increasing survival in several mouse models of MM including a genetically engineered mouse model of MYC-dependent MM. Through its role as a critical cofactor and regulator of MYC and E2F activity, CDK7 is therefore a master regulator of oncogenic cellular programs supporting MM growth and survival, and a valuable therapeutic target providing rationale for development of YKL-5-124 for clinical use.

A MIR17HG-derived long noncoding RNA provides an essential chromatin scaffold for protein interaction and myeloma growth.

Long noncoding RNAs (lncRNAs) can drive tumorigenesis and are susceptible to therapeutic intervention. Here, we used a large-scale CRISPR interference viability screen to interrogate cell-growth dependency to lncRNA genes in multiple myeloma (MM) and identified a prominent role for the miR-17-92 cluster host gene (MIR17HG). We show that an MIR17HG-derived lncRNA, named lnc-17-92, is the main mediator of cell-growth dependency acting in a microRNA- and DROSHA-independent manner. Lnc-17-92 provides a chromatin scaffold for the functional interaction between c-MYC and WDR82, thus promoting the expression of ACACA, which encodes the rate-limiting enzyme of de novo lipogenesis acetyl-coA carboxylase 1. Targeting MIR17HG pre-RNA with clinically applicable antisense molecules disrupts the transcriptional and functional activities of lnc-17-92, causing potent antitumor effects both in vitro and in vivo in 3 preclinical animal models, including a clinically relevant patient-derived xenograft NSG mouse model. This study establishes a novel oncogenic function of MIR17HG and provides potent inhibitors for translation to clinical trials.

Elevated APE1 Dysregulates Homologous Recombination and Cell Cycle Driving Genomic Evolution, Tumorigenesis, and Chemoresistance in Esophageal Adenocarcinoma.

BACKGROUND & AIMS: The purpose of this study was to identify drivers of genomic evolution in esophageal adenocarcinoma (EAC) and other solid tumors. METHODS: An integrated genomics strategy was used to identify deoxyribonucleases correlating with genomic instability (as assessed from total copy number events in each patient) in 6 cancers. Apurinic/apyrimidinic nuclease 1 (APE1), identified as the top gene in functional screens, was either suppressed in cancer cell lines or overexpressed in normal esophageal cells and the impact on genome stability and growth was monitored in vitro and in vivo. The impact on DNA and chromosomal instability was monitored using multiple approaches, including investigation of micronuclei, acquisition of single nucleotide polymorphisms, whole genome sequencing, and/or multicolor fluorescence in situ hybridization. RESULTS: Expression of 4 deoxyribonucleases correlated with genomic instability in 6 human cancers. Functional screens of these genes identified APE1 as the top candidate for further evaluation. APE1 suppression in EAC, breast, lung, and prostate cancer cell lines caused cell cycle arrest; impaired growth and increased cytotoxicity of cisplatin in all cell lines and types and in a mouse model of EAC; and inhibition of homologous recombination and spontaneous and chemotherapy-induced genomic instability. APE1 overexpression in normal cells caused a massive chromosomal instability, leading to their oncogenic transformation. Evaluation of these cells by means of whole genome sequencing demonstrated the acquisition of changes throughout the genome and identified homologous recombination as the top mutational process. CONCLUSIONS: Elevated APE1 dysregulates homologous recombination and cell cycle, contributing to genomic instability, tumorigenesis, and chemoresistance, and its inhibitors have the potential to target these processes in EAC and possibly other cancers.

IgM-MM is predominantly a pre-germinal center disorder and has a distinct genomic and transcriptomic signature from WM.

Immunoglobulin M (IgM) multiple myeloma (MM) is a rare disease subgroup. Its differentiation from other IgM-producing gammopathies such as Waldenström macroglobulinemia (WM) has not been well characterized but is essential for proper risk assessment and treatment. In this study, we investigated genomic and transcriptomic characteristics of IgM-MM samples using whole-genome and transcriptome sequencing to identify differentiating characteristics from non-IgM-MM and WM. Our results suggest that IgM-MM shares most of its defining structural variants and gene-expression profiling with MM, but has some key characteristics, including t(11;14) translocation, chromosome 6 and 13 deletion as well as distinct molecular and transcription-factor signatures. Furthermore, IgM-MM translocations were predominantly characterized by VHDHJH recombination-induced breakpoints, as opposed to the usual class-switching region breakpoints; coupled with its lack of class switching, these data favor a pre-germinal center origin. Finally, we found elevated expression of clinically relevant targets, including CD20 and Bruton tyrosine kinase, as well as high BCL2/BCL2L1 ratio in IgM-MM, providing potential for targeted therapeutics.

miR-15a/16-1 deletion in activated B cells promotes plasma cell and mature B-cell neoplasms.

Chromosome 13q deletion [del(13q)], harboring the miR-15a/16-1 cluster, is one of the most common genetic alterations in mature B-cell malignancies, which originate from germinal center (GC) and post-GC B cells. Moreover, miR-15a/16 expression is frequently reduced in lymphoma and multiple myeloma (MM) cells without del(13q), suggesting important tumor-suppressor activity. However, the role of miR-15a/16-1 in B-cell activation and initiation of mature B-cell neoplasms remains to be determined. We show that conditional deletion of the miR-15a/16-1 cluster in murine GC B cells induces moderate but widespread molecular and functional changes including an increased number of GC B cells, percentage of dark zone B cells, and maturation into plasma cells. With time, this leads to development of mature B-cell neoplasms resembling human extramedullary plasmacytoma (EP) as well as follicular and diffuse large B-cell lymphomas. The indolent nature and lack of bone marrow involvement of EP in our murine model resembles human primary EP rather than MM that has progressed to extramedullary disease. We corroborate human primary EP having low levels of miR-15a/16 expression, with del(13q) being the most common genetic loss. Additionally, we show that, although the mutational profile of human EP is similar to MM, there are some exceptions such as the low frequency of hyperdiploidy in EP, which could account for different disease presentation. Taken together, our studies highlight the significant role of the miR-15a/16-1 cluster in the regulation of the GC reaction and its fundamental context-dependent tumor-suppression function in plasma cell and B-cell malignancies.

The JAK-STAT pathway regulates CD38 on myeloma cells in the bone marrow microenvironment: therapeutic implications.

Anti-CD38 monoclonal antibody (MoAb) treatments including daratumumab (DARA) are effective therapies for both newly diagnosed and relapsed multiple myeloma (MM). In this study, we examined the soluble factors that modulate CD38 expression and are associated with sensitivity to DARA-mediated antibody-dependent cellular cytotoxicity (ADCC) in the bone marrow (BM) microenvironment. Importantly, primary BM stromal cell (BMSC) culture supernatant (BMSC-sup) and interleukin-6 (IL-6) downregulated CD38 expression and reduced DARA-mediated ADCC. Both cytokine profiling of the BMSC-sup and genome-scale clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) knockout screening in MM cell lines identified and validated the JAK-STAT3 signaling pathway mediating CD38 downregulation, whereas the JAK-STAT1 pathway mediated CD38 upregulation. STAT3 knockdown abrogated BMSC-sup- and IL-6-induced CD38 downregulation on MM cell lines. We also confirmed that STAT3 and CD38 is negatively correlated in primary MM cells. To assess potential clinical relevance, pharmacological inhibition of the JAK-STAT pathway on BMSC-sup-induced CD38 downregulation was further examined. JAK inhibitor ruxolitinib inhibited STAT3 phosphorylation in MM cell lines, upregulated CD38 expression in MM cell lines and primary patient MM cells, and augmented DARA-mediated ADCC against MM cell lines. Taken together, our results suggest that CD38 expression on MM cells in the BM microenvironment is regulated by both STAT1 (positively) and STAT3 (negatively), and that inhibition of the JAK-STAT3 pathway represents a novel therapeutic option to enhance CD38 expression and anti-CD38 MoAb-mediated MM cytotoxicity.

Clinical features associated with COVID-19 outcome in multiple myeloma: first results from the International Myeloma Society data set.

The primary cause of morbidity and mortality in patients with multiple myeloma (MM) is an infection. Therefore, there is great concern about susceptibility to the outcome of COVID-19-infected patients with MM. This retrospective study describes the baseline characteristics and outcome data of COVID-19 infection in 650 patients with plasma cell disorders, collected by the International Myeloma Society to understand the initial challenges faced by myeloma patients during the COVID-19 pandemic. Analyses were performed for hospitalized MM patients. Among hospitalized patients, the median age was 69 years, and nearly all patients (96%) had MM. Approximately 36% were recently diagnosed (2019-2020), and 54% of patients were receiving first-line therapy. Thirty-three percent of patients have died, with significant geographic variability, ranging from 27% to 57% of hospitalized patients. Univariate analysis identified age, International Staging System stage 3 (ISS3), high-risk disease, renal disease, suboptimal myeloma control (active or progressive disease), and 1 or more comorbidities as risk factors for higher rates of death. Neither history of transplant, including within a year of COVID-19 diagnosis, nor other anti-MM treatments were associated with outcomes. Multivariate analysis found that only age, high-risk MM, renal disease, and suboptimal MM control remained independent predictors of adverse outcome with COVID-19 infection. The management of MM in the era of COVID-19 requires careful consideration of patient- and disease-related factors to decrease the risk of acquiring COVID-19 infection, while not compromising disease control through appropriate MM treatment. This study provides initial data to develop recommendations for the management of MM patients with COVID-19 infection.

YWHAE/14-3-3ε expression impacts the protein load, contributing to proteasome inhibitor sensitivity in multiple myeloma.

High protein load is a feature of multiple myeloma (MM), making the disease exquisitely sensitive to proteasome inhibitor (PIs). Despite the success of PIs in improving patient outcome, the majority of patients develop resistance leading to progressive disease; thus, the need to investigate the mechanisms driving the drug sensitivity vs resistance. With the well-recognized chaperone function of 14-3-3 proteins, we evaluated their role in affecting proteasome activity and sensitivity to PIs by correlating expression of individual 14-3-3 gene and their sensitivity to PIs (bortezomib and carfilzomib) across a large panel of MM cell lines. We observed a significant positive correlation between 14-3-3ε expression and PI response in addition to a role for 14-3-3ε in promoting translation initiation and protein synthesis in MM cells through binding and inhibition of the TSC1/TSC2 complex, as well as directly interacting with and promoting phosphorylation of mTORC1. 14-3-3ε depletion caused up to a 50% reduction in protein synthesis, including a decrease in the intracellular abundance and secretion of the light chains in MM cells, whereas 14-3-3ε overexpression or addback in knockout cells resulted in a marked upregulation of protein synthesis and protein load. Importantly, the correlation among 14-3-3ε expression, PI sensitivity, and protein load was observed in primary MM cells from 2 independent data sets, and its lower expression was associated with poor outcome in patients with MM receiving a bortezomib-based therapy. Altogether, these observations suggest that 14-3-3ε is a predictor of clinical outcome and may serve as a potential target to modulate PI sensitivity in MM.

Comprehensive genomic analysis of refractory multiple myeloma reveals a complex mutational landscape associated with drug resistance and novel therapeutic vulnerabilities.

The outcomes of patients with multiple myeloma (MM) refractory to immunomodulatory agents (IMiDs) and proteasome inhibitors (PIs) remain poor. In this study, we performed whole genome and transcriptome sequencing of 39 heavily pretreated relapsed/refractory MM (RRMM) patients to identify mechanisms of resistance and potential therapeutic targets. We observed a high mutational load and indications of increased genomic instability. Recurrently mutated genes in RRMM, which had not been previously reported or only observed at a lower frequency in newly diagnosed MM, included NRAS, BRAF, TP53, SLC4A7, MLLT4, EWSR1, HCFC2, and COPS3. We found multiple genomic regions with bi-allelic events affecting tumor suppressor genes and demonstrated a significant adverse impact of bi-allelic TP53 alterations on survival. With regard to potentially resistance conferring mutations, recurrently mutated gene networks included genes with relevance for PI and IMiD activity; the latter particularly affecting members of the Cereblon and the COP9 signalosome complex. We observed a major impact of signatures associated with exposure to melphalan or impaired DNA double-strand break homologous recombination repair in RRMM. The latter coincided with mutations in genes associated with PARP inhibitor sensitivity in 49% of RRMM patients; a finding with potential therapeutic implications. In conclusion, this comprehensive genomic characterization revealed a complex mutational and structural landscape in RRMM and highlights potential implications for therapeutic strategies.

Apoptosis reprogramming triggered by splicing inhibitors sensitizes multiple myeloma cells to Venetoclax treatment.

Identification of novel vulnerabilities in the context of therapeutic resistance is emerging as a key challenge for cancer treatment. Recent studies have detected pervasive aberrant splicing in cancer cells, supporting its targeting for novel therapeutic strategies. Here, we evaluated the expression of several spliceosome machinery components in multiple myeloma (MM) cells and the impact of splicing modulation on tumor cell growth and viability. A comprehensive gene expression analysis confirmed the reported deregulation of spliceosome machinery components in MM cells, compared to normal plasma cells from healthy donors, with its pharmacological and genetic modulation resulting in impaired growth and survival of MM cell lines and patient-derived malignant plasma cells. Consistent with this, transcriptomic analysis revealed deregulation of BCL2 family members, including decrease of anti-apoptotic long form of myeloid cell leukemia-1 (MCL1) expression, as crucial for "priming" MM cells for Venetoclax activity in vitro and in vivo, irrespective of t(11;14) status. Overall, our data provide a rationale for supporting the clinical use of splicing modulators as a strategy to reprogram apoptotic dependencies and make all MM patients more vulnerable to BCL2 inhibitors.