Venetoclax resistance leads to broad resistance to standard-of-care anti-MM agents, but not to immunotherapies.

Venetoclax is the first example of personalized medicine for multiple myeloma (MM), with meaningful clinical activity as a monotherapy and in combination in myeloma patients harboring the t(11:14) translocation. However, despite the high response rates and prolonged PFS, a significant proportion of patients eventually relapse. Here, we aimed to study adaptive molecular responses after the acquisition of venetoclax resistance in sensitive t(11:14) MM cell models. We therefore generated single-cell venetoclax-resistant t(11:14) MM cell lines and investigated the mechanisms contributing to resistance as well as the cells' sensitivity to other treatments. Our data suggests that acquired resistance to venetoclax is characterized by reduced mitochondrial priming and changes in BCL-2 family proteins' expression in MM cells, conferring broad resistance to standard-of-care anti-myeloma drugs. However, our results show that the resistant cells are still sensitive to immunotherapeutic treatments, highlighting the need to consider appropriate sequencing of these treatments following venetoclax-based regimens.

Bone marrow stromal cells induce chromatin remodeling in multiple myeloma cells leading to transcriptional changes.

The natural history of multiple myeloma is characterized by its localization to the bone marrow and its interaction with bone marrow stromal cells. The bone marrow stromal cells provide growth and survival signals, thereby promoting the development of drug resistance. Here, we show that the interaction between bone marrow stromal cells and myeloma cells (using human cell lines) induces chromatin remodeling of cis-regulatory elements and is associated with changes in the expression of genes involved in the cell migration and cytokine signaling. The expression of genes involved in these stromal interactions are observed in extramedullary disease in patients with myeloma and provides the rationale for survival of myeloma cells outside of the bone marrow microenvironment. Expression of these stromal interaction genes is also observed in a subset of patients with newly diagnosed myeloma and are akin to the transcriptional program of extramedullary disease. The presence of such adverse stromal interactions in newly diagnosed myeloma is associated with accelerated disease dissemination, predicts the early development of therapeutic resistance, and is of independent prognostic significance. These stromal cell induced transcriptomic and epigenomic changes both predict long-term outcomes and identify therapeutic targets in the tumor microenvironment for the development of novel therapeutic approaches.

RAS/RAF landscape in monoclonal plasma cell conditions.

ABSTRACT: Multiple myeloma is characterized by a huge heterogeneity at the molecular level. The RAS/RAF pathway is the most frequently mutated, in ∼50% of the patients. However, these mutations are frequently subclonal, suggesting a secondary event. Because these genes are part of our routine next-generation sequencing panel, we analyzed >10 000 patients with different plasma cell disorders 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 occurred in presymptomatic cases. Of note, the mutations were different from that observed in solid tumors (higher proportions of Q61 mutations). In 29 patients with 2 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 the RAS/RAF pathway is not an attractive target, both on therapeutic and residual disease assessment points of view.

Mechanisms of resistance to bispecific T-cell engagers in multiple myeloma and their clinical implications.

ABSTRACT: Bispecific T-cell engagers (TCEs) are revolutionizing patient care in multiple myeloma (MM). These monoclonal antibodies, that redirect T cells against cancer cells, are now approved for the treatment of triple-class exposed relapsed/refractory MM (RRMM). They are currently tested in earlier lines of the disease, including in first line. Yet, primary resistance occurs in about one-third of patients with RRMM, and most responders eventually develop acquired resistance. Understanding the mechanisms of resistance to bispecific TCE is thus essential to improve immunotherapies in MM. Here, we review recent studies investigating the clinical and molecular determinants of resistance to bispecific TCE. Resistance can arise from tumor-intrinsic or tumor-extrinsic mechanisms. Tumor-intrinsic resistance involves various alterations leading to the loss of the target antigen, such as chromosome deletions, point mutations, or epigenetic silencing. Loss of major histocompatibility complex (MHC) class I, preventing MHC class I: T-cell receptor (TCR) costimulatory signaling, was also reported. Tumor-extrinsic resistance involves abundant exhausted T-cell clones and several factors generating an immunosuppressive microenvironment. Importantly, some resistance mechanisms impair response to 1 TCE while preserving the efficacy of others. We next discuss the clinical implications of these findings. Monitoring the status of target antigens in tumor cells and their immune environment will be key to select the most appropriate TCE for each patient and to design combination and sequencing strategies for immunotherapy in MM.

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.

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.

Immunomodulation of NK, NKT and B/T cell subtypes in relapsed/refractory multiple myeloma patients treated with pomalidomide along with velcade and dexamethasone and its association with improved progression-free survival.

Background: Multiple Myeloma (MM) patients exhibit dysregulated immune system, which is further weakened by chemotherapeutic agents. While cereblon-modulating agents, such as pomalidomide and lenalidomide, have been found to improve the immune profile, the efficacy of their impact in combination with other treatments is yet unknown. Methods: We conducted an immune-profiling of a longitudinal cohort of 366 peripheral blood samples from the CC4047-MM-007 (OPTIMISMM, NCT01734928) study. This study followed relapsed/refractory Multiple Myeloma (RRMM) patients who were treated with Velcade + dexamethasone (Vd), or Vd with pomalidomide (PVd). 366 blood samples from 186 patients were evaluated using multi-color flow cytometry at 3 timepoints: screening, day 8 of cycle 1, and cycle 3. Results: Among NK and NKT cell populations, adding pomalidomide showed no inhibition in the frequency of NK cells. When expression of double positivity for activation markers like, p46/NKG2D, on NK cells was higher than the median, PVd treated patients showed significantly better (p=0.05) progression-free survival (PFS) (additional 15 months) than patients with lower than the median expression of p46/NKG2D on NK cells. PVd treated patients who expressed CD158a/b below the median at cycle 1 demonstrated a significantly better PFS (more than 18months). Among B cell subtypes, PVd treatment significantly increased the abundance of B1b cells (p<0.05) and decreased Bregs (p<0.05) at day 8 of both cycle 1 and cycle 3 when compared to screening samples. Of all the B cell-markers evaluated among paired samples, a higher expression of MZB cells at day 8 of cycle 1 has resulted in enhanced PFS in PVd treated patients. Within T cells, pomalidomide treatment did not decrease the frequency of CD8 T cells when compared with screening samples. The higher the surface expression of OX-40 on CD8 T cells and the lower the expression of PD-1 and CD25 on CD4 T cells by PVd treatment resulted in improved PFS. Conclusion: The prognostic significance for the number of immune markers is only seen in the PVd arm and none of these immune markers exhibit prognostic values in the Vd arm. This study demonstrates the importance of the immunomodulatory effects and the therapeutic benefit of adding pomalidomide to Vd treatment.

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.

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.

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.

BRD9 Degradation Disrupts Ribosome Biogenesis in Multiple Myeloma.

PURPOSE: BRD9 is a defining component of the noncanonical SWI/SNF complex, which regulates gene expression by controlling chromatin dynamics. Although recent studies have found an oncogenic role for BRD9 in multiple cancer types including multiple myeloma, its clinical significance and oncogenic mechanism have not yet been elucidated. Here, we sought to identify the clinical and biological impact of BRD9 in multiple myeloma, which may contribute to the development of novel therapeutic strategies. EXPERIMENTAL DESIGN: We performed integrated analyses of BRD9 in vitro and in vivo using multiple myeloma cell lines and primary multiple myeloma cells in established preclinical models, which identified the molecular functions of BRD9 contributing to multiple myeloma cell survival. RESULTS: We found that high BRD9 expression was a poor prognostic factor in multiple myeloma. Depleting BRD9 by genetic (shRNA) and pharmacologic (dBRD9-A; proteolysis-targeting chimera; BRD9 degrader) approaches downregulated ribosome biogenesis genes, decreased the expression of the master regulator MYC, and disrupted the protein-synthesis maintenance machinery, thereby inhibiting multiple myeloma cell growth in vitro and in vivo in preclinical models. Importantly, we identified that the expression of ribosome biogenesis genes was associated with the disease progression and prognosis of patients with multiple myeloma. Our results suggest that BRD9 promotes gene expression by predominantly occupying the promoter regions of ribosome biogenesis genes and cooperating with BRD4 to enhance the transcriptional function of MYC. CONCLUSIONS: Our study identifies and validates BRD9 as a novel therapeutic target in preclinical models of multiple myeloma, which provides the framework for the clinical evaluation of BRD9 degraders to improve patient outcome.

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.

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.

Prolyl-tRNA synthetase as a novel therapeutic target in multiple myeloma.

Multiple myeloma (MM) is a plasma cell malignancy characterised by aberrant production of immunoglobulins requiring survival mechanisms to adapt to proteotoxic stress. We here show that glutamyl-prolyl-tRNA synthetase (GluProRS) inhibition constitutes a novel therapeutic target. Genomic data suggest that GluProRS promotes disease progression and is associated with poor prognosis, while downregulation in MM cells triggers apoptosis. We developed NCP26, a novel ATP-competitive ProRS inhibitor that demonstrates significant anti-tumour activity in multiple in vitro and in vivo systems and overcomes metabolic adaptation observed with other inhibitor chemotypes. We demonstrate a complex phenotypic response involving protein quality control mechanisms that centers around the ribosome as an integrating hub. Using systems approaches, we identified multiple downregulated proline-rich motif-containing proteins as downstream effectors. These include CD138, transcription factors such as MYC, and transcription factor 3 (TCF3), which we establish as a novel determinant in MM pathobiology through functional and genomic validation. Our preclinical data therefore provide evidence that blockade of prolyl-aminoacylation evokes a complex pro-apoptotic response beyond the canonical integrated stress response and establish a framework for its evaluation in a clinical setting.

Genetic Alterations in Members of the Proteasome 26S Subunit, AAA-ATPase (PSMC) Gene Family in the Light of Proteasome Inhibitor Resistance in Multiple Myeloma.

For the treatment of Multiple Myeloma, proteasome inhibitors are highly efficient and widely used, but resistance is a major obstacle to successful therapy. Several underlying mechanisms have been proposed but were only reported for a minority of resistant patients. The proteasome is a large and complex machinery. Here, we focus on the AAA ATPases of the 19S proteasome regulator (PSMC1-6) and their implication in PI resistance. As an example of cancer evolution and the acquisition of resistance, we conducted an in-depth analysis of an index patient by applying FISH, WES, and immunoglobulin-rearrangement sequencing in serial samples, starting from MGUS to newly diagnosed Multiple Myeloma to a PI-resistant relapse. The WES analysis uncovered an acquired PSMC2 Y429S mutation at the relapse after intensive bortezomib-containing therapy, which was functionally confirmed to mediate PI resistance. A meta-analysis comprising 1499 newly diagnosed and 447 progressed patients revealed a total of 36 SNVs over all six PSMC genes that were structurally accumulated in regulatory sites for activity such as the ADP/ATP binding pocket. Other alterations impact the interaction between different PSMC subunits or the intrinsic conformation of an individual subunit, consequently affecting the folding and function of the complex. Interestingly, several mutations were clustered in the central channel of the ATPase ring, where the unfolded substrates enter the 20S core. Our results indicate that PSMC SNVs play a role in PI resistance in MM.