Mechanisms In Multiple Myeloma Research Articles

Abstract P33: Leveraging probabilistic causal disease models to understand molecular pathways and target resistance mechanisms in Multiple Myeloma with the CREBBP and EP300 bromodomain inhibitor, pocenbrodib

Abstract To explore and devise innovative approaches for the treatment of Multiple Myeloma (MM) – a disease often deemed incurable due to the development of resistance mechanisms against available therapies – we adopted an integrative data approach, constructing causal AI models of MM that integrate co-expression relationships, DNA variation, longitudinal clinical data, and expansive knowledge graphs. Diverging from traditional methods, our approach centers on constructing probabilistic causal models to elucidate the regulatory mechanisms of disease and capture existing known causal relationships published in the scientific literature or curated in pathway databases. We employ generative AI and other advanced machine learning approaches to stratify patients into mechanistically-informed subtypes that we can directly associate with clinical outcomes. These models, and the ability to map driver mechanisms to outcomes, serve as a foundation for analyzing our real-world data, a dataset made up of more than 200,000 cancer patients with multimodal molecular data generated on tumor tissues and matched to longitudinal clinical data. Finally, we can validate and refine therapeutically relevant hypotheses using a scalable high-content organoid and cell line screening platform to characterize drug and/or targeted genetic perturbation signatures.We analyzed our models in MM to reveal sub-populations of relapsed refractory patients that could be susceptible to specific pathway inhibition. This analysis revealed a focused causal subnetwork that predicts poor overall survival in standard MM treatment protocols. We demonstrate that not only does this subnetwork show promise in predicting response to a novel CREBBP and EP300 bromodomain inhibitor, pocenbrodib, but it sheds light on the molecular underpinnings of MM, beyond the currently known IRF4, MYC, and immune cell regulation mechanisms that the inhibition of CREBBP/EP300 is known to target. Our network models reveal that this novel subnetwork is intricately linked to a range of driver mechanisms that strongly predict disease progression and are associated with known mechanisms of resistance. Notably, the signature persists across various MM stages, from initial diagnosis to relapsed/refractory states, and intensifies with each additional line of therapy. Further, signature scores are higher in patients with known high-risk cytogenetic abnormalitiesOur findings suggest that stratifying patients based on this gene signature could be instrumental in identifying individuals at high risk of MM progression. Such stratification could transform the selection process for pocenbrodib treatment. By leveraging an integrated, AI-driven approach, we advance the understanding of MM and pave the way for more targeted and effective therapeutic strategies. Citation Format: Allison E Roder, Alice M Walsh, Taylor K Krebs, Veronica Calvo, Bonnie V Dougherty, Preston Smith, Jens Renstrup, Eric E Schadt. Leveraging probabilistic causal disease models to understand molecular pathways and target resistance mechanisms in Multiple Myeloma with the CREBBP and EP300 bromodomain inhibitor, pocenbrodib [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P33.

Bortezomib modulated the autophagy-lysosomal pathway in a TFEB-dependent manner in multiple myeloma

ObjectiveTo explore the involvement of TFEB-mediated autophagy-lysosomal mechanisms in multiple myeloma (MM) during bortezomib treatment. MethodsMM cells were exposed to bortezomib or subjected to TFEB knockdown. CCK assay was used to assess the cell proliferation. Western blotting and fluorescent staining were conducted to examine autophagy and lysosomes. The TFEB expression pattern was analyzed, and whole transcriptome sequencing was carried out. Additionally, TFEB target genes were predicted using the GTRD(http://gtrd.biouml.org/) website, and pathway analysis was performed. ResultsBortezomib demonstrated a dose-dependent and time dependent inhibition of cell proliferation. In MM cells treated with bortezomib, LC3B, Beclin-1, TFEB, and Lamp1 exhibited upregulation in a time- and concentration-dependent manner. LysoTracker dye labeling showed an increase in lysosomes in the bortezomib-treated group. Moreover, bortezomib elevated the expression of lysosome-associated factor Lamp1. Bortezomib promoted the nuclear translocation of TFEB, leading to decreased cytoplasmic TFEB and increased nuclear TFEB. TFEB gene silencing reversed bortezomib's inhibitory effect on MM cell lines, significantly reducing autophagosome expression and lysosome numbers. Furthermore, bioinformatic analysis identified the MAPK pathway as a potential downstream target of TFEB. ConclusionBortezomib effectively inhibits MM cell proliferation and induces autophagy, partly through TFEB-mediated mechanisms, with potential involvement of the MAPK pathway.

A new autophagy-related nomogram and mechanism in multiple myeloma

A new autophagy-related nomogram and mechanism in multiple myeloma

OA-23 Resolving therapy resistance mechanisms in multiple myeloma by multi-omics subclone analysis

OA-23 Resolving therapy resistance mechanisms in multiple myeloma by multi-omics subclone analysis

Resolving therapy resistance mechanisms in multiple myeloma by multiomics subclone analysis

AbstractIntratumor heterogeneity as a clinical challenge becomes most evident after several treatment lines, when multidrug-resistant subclones accumulate. To address this challenge, the characterization of resistance mechanisms at the subclonal level is key to identify common vulnerabilities. In this study, we integrate whole-genome sequencing, single-cell (sc) transcriptomics (scRNA sequencing), and chromatin accessibility (scATAC sequencing) together with mitochondrial DNA mutations to define subclonal architecture and evolution for longitudinal samples from 15 patients with relapsed or refractory multiple myeloma. We assess transcriptomic and epigenomic changes to resolve the multifactorial nature of therapy resistance and relate it to the parallel occurrence of different mechanisms: (1) preexisting epigenetic profiles of subclones associated with survival advantages, (2) converging phenotypic adaptation of genetically distinct subclones, and (3) subclone-specific interactions of myeloma and bone marrow microenvironment cells. Our study showcases how an integrative multiomics analysis can be applied to track and characterize distinct multidrug-resistant subclones over time for the identification of molecular targets against them.

MiR-203a-5p Inhibits Multiple Myeloma Cell Proliferation and Cell Cycle Progression via Targeting JAG1

To investigate the biological function of miR-203a-5p and the underlying mechanism in multiple myeloma (MM). Three miRNA expression profiles (GSE16558, GSE24371 and GSE17498) were downloaded from the GEO database. The three miRNA expression profiles contained 131 MM samples and 17 normal plasmacyte samples. The robust rank aggregation (RRA) method was used to identify the differentially expressed miRNAs between MM and normal plasmacytes. In order to carry out cytological experiments, MM cell line with stable over-expression of miR-203a-5p was constructed with lentivirus. Expression levels of miR-203a-5p in MM cells were quantified by qRT-PCR. The effects of miR-203a-5p on MM cells were investigated using assays of cell viability and cell cycle. Cell proliferation was measured using the Cell Counting kit (CCK)8 assay. The percentage of cells in each cell cycle was measured with a FACSCalibur system. Xenograft tumor models were established to evaluate the role of miR-203a-5p in tumorigenesis in vivo . To elucidate the underlying molecular mechanisms of miR-203a-5p in mediating cell proliferation inhibition and cell cycle arrest in MM, we used TargetScan and miRanda to predict the candidate targets of miR-203a-5p. The potential target of miR-203a-5p in MM cells was explored using the luciferase reporter assay, qRT-PCR, and Western blot. An integrated analysis of three MM miRNA expression datasets showed that the levels of miR-203a-5p in MM were notably downregulated compared with those in normal plasmacytes. Accordingly, the relative expression levels of miR-203a-5p were decreased in MM cell lines. In addition, overexpression of miR-203a-5p inhibited the proliferation and cell cycle progression of RPMI8226 and U266 cells. In vivo experiments demonstrated that upregulation of miR-203a-5p expression could significantly inhibit the tumorigenesis of subcutaneous myeloma xenografts in nude mice. Mechanistic investigation led to the identification of Jagged 1 (JAG1) as a novel and direct downstream target of miR-203a-5p. Interestingly, the reintroduction of JAG1 abrogated miR-203a-5p-induced MM cell growth inhibition and cell cycle arrest. Our data demonstrate that miR-203a-5p inhibits cell proliferation and cell cycle progression in MM cells by targeting JAG1, supporting the utility of miR-203a-5p as a novel and potential therapeutic agent for miRNA-based MM therapy.

LncRNA HCP5 acts as a miR-128-3p sponge to promote the progression of multiple myeloma through activating Wnt/β-catenin/cyclin D1 signaling via PLAGL2.

Although long non-coding RNA (lncRNA) HCP plays essential roles in human cancers, its function and mechanism in multiple myeloma (MM) have not crystallized. HCP5 level in MM was assessed through qRT-PCR. A series of functional investigations were conducted to evaluate the influences of HCP5 on proliferation and apoptosis. Bioinformatics analysis and RIP/RNA pull-down assays were carried out to determine the relationships among HCP5, miR-128-3p, and PLAGL2. Relative protein level was determined through Western blot. A xenograft tumor model was applied for validating the roles of HCP5/miR-128-3p/PLAGL2 axis in vivo. HCP5 was significantly increased in MM. HCP5 knockdown effectively thwarted the proliferative rate and cell cycle of MM cell lines and suppressed tumor growth. HCP5 regulated PLAGL2 expression by sponging miR-128-3p. PLAGL2 overexpression effectively rescued cells from influences by sh-HCP5 on cell proliferative and apoptotic rates. Additionally, HCP5 knockdown significantly inhibited Wnt/β-catenin/cyclin D1 signaling, and these effects were eliminated by PLAGL2 overexpression. Our study revealed that HCP5/miR-128-3p/PLAGL2 is closely correlated to MM development by modulating Wnt/β-catenin/cyclin D1 signaling. HCP5 promoted cell proliferation and tumor formation of MM cells by activating the Wnt/β-catenin/CCND1 signaling pathway by sponging miR-128-3p to increase PLAGL2 expression.

Prognostic model and immune-infiltrating cell landscape based on differentially expressed autophagy-related genes in TP53-mutated multiple myeloma

IntroductionAutophagy functions as a prosurvival mechanism in multiple myeloma (MM).The objective of this research was to establish an autophagy-related gene (ARG) signature for predicting the survival outcomes of MM patients with TP53 mutations.Material and methodsInformation about MM patients with TP53 mutations was downloaded from Gene Expression Omnibus (GEO) database. Cox proportional hazard regression was employed to determine the independent prognostic ARG and construct a risk signature. Time-dependent receiver-operating characteristic (t ROC) curve was used to explore the predictive accuracy of the prognostic model. A nomogram was constructed to give a more precise prediction of the probability of 5-year, 8-year and 10-year overall survival (OS). In addition, we utilized the CIBERSORT algorithm to explore the distribution difference of 22 immune-infiltrating cells.ResultsThree differentially expressed ARGs (CASP8, MAPK8, RB1CC1) were finally incorporated to construct the risk model. Area under the curve (AUC) of corresponding tROC curve for 5-year,8-year and 10-year OS were 0.735, 0.686 and 0.662, respectively. MM patients were categorized into high and low-risk group in accordance with the median threshold value (-1.724549). ARG-based risk score model was an independent prognostic element correlated with OS, giving an hazard ratio (HR) of 3.29 (95%CI 2.35-4.60, P<0.001). 13 immune infiltrating cells were found to have distribution differences between the two groups.ConclusionsWe established a three-ARGs risk signature which manifested an independent prognostic factor. The nomogram was testified to perform well in forecasting the long-term survival of TP53-mutated MM patients.

The paradoxical pharmacological mechanisms of lenalidomide and bortezomib in the treatment of multiple myeloma.

The combination of bortezomib (Velcade, PS-341) and lenalidomide (Revlimid) for the treatment of multiple myeloma was proved by USA Food and Drug Administration in 2006. Lenalidomide prevents the proliferation of multiple myeloma cells through binding to cereblon and promoting the ubiquitinational degradation of IKZF1 (Ikaros)/IKZF3 (Aiolos). However, the proteasome inhibitor bortezomib would inhibit the ubiquitinational degradation of IKZF1/IKZF3. How bortezomib could not block the antiproliferative effect of lenalidomide on multiple myeloma cells, which is the paradoxical pharmacological mechanisms in multiple myeloma. In this review, we summarized recent advances in molecular mechanisms underlying the combination of bortezomib and lenalidomide for the treatment multiple myeloma, discussed the paradoxical pharmacological mechanisms of lenalidomide and bortezomib in the treatment of multiple myeloma.

Chromatin activation as a unifying principle underlying pathogenic mechanisms in multiple myeloma.

Multiple myeloma (MM) is a plasma cell neoplasm associated with a broad variety of genetic lesions. In spite of this genetic heterogeneity, MMs share a characteristic malignant phenotype whose underlying molecular basis remains poorly characterized. In the present study, we examined plasma cells from MM using a multi-epigenomics approach and demonstrated that, when compared to normal B cells, malignant plasma cells showed an extensive activation of regulatory elements, in part affecting coregulated adjacent genes. Among target genes up-regulated by this process, we found members of the NOTCH, NF-kB, MTOR signaling, and TP53 signaling pathways. Other activated genes included sets involved in osteoblast differentiation and response to oxidative stress, all of which have been shown to be associated with the MM phenotype and clinical behavior. We functionally characterized MM-specific active distant enhancers controlling the expression of thioredoxin (TXN), a major regulator of cellular redox status and, in addition, identified PRDM5 as a novel essential gene for MM. Collectively, our data indicate that aberrant chromatin activation is a unifying feature underlying the malignant plasma cell phenotype.

Differential RNA splicing as a potentially important driver mechanism in multiple myeloma.

Disruption of the normal splicing patterns of RNA is a major factor in the path o genesis of a number of diseases. Increasingl y research has shown the strong influence that splicing patterns can have on cancer progression. Multiple myeloma is a molecularly heterogeneous disease classified by the presence of key translocations, gene expression profiles and mutations but the splicing patterns in MM remains largely unexplored. We take a multifaceted approach to define the extent and impact of alternative splicing in MM. We looked at the spliceosome component, SF3B1, with hotspot mutations (K700E and K666T/Q) shown to result in an increase in alternative splicing in other cancers. We discovered a number of differentially spliced genes in comparison of the SF3B1 mutant and wild type samples that included, MZB1, DYNLL1, TMEM14C and splicing related genes DHX9, CLASRP, and SNRPE. We identified a broader role for abnormal splicing showing clear differences in the extent of novel splice variants in the different translocation groups. We show that a high number of novel splice loci is associated with adverse survival and an ultra-high risk group. The enumeration of patterns of alternative splicing has the potential to refine MM classification and to aid in the risk stratification of patients.

Knockdown of long non-coding RNA plasmacytoma variant translocation 1 inhibits cell proliferation while promotes cell apoptosis via regulating miR-486-mediated CDK4 and BCAS2 in multiple myeloma.

This study aimed to investigate the effect of long non-coding RNA-plasmacytoma variant translocation 1 (lnc-Pvt1) knockdown on regulating cell proliferation and apoptosis, and to explore its molecular mechanism in multiple myeloma (MM). Lnc-Pvt1 expression was detected in MM cell lines (NCI-H929, U-266, LP-1 and RPMI-8226 cell lines) and human normal plasma cells. In U-266 cells and LP-1 cells, control shRNA and lnc-Pvt1 shRNA plasmids were transferred. Rescue experiments were further performed by transfection of lnc-Pvt1 shRNA alone and lnc-Pvt1 shRNA and miR-486 shRNA plasmids. Cells proliferation, apoptosis, RNA expression, and protein expression were determined by cell counting kit-8, annexin V-FITC-propidium iodide, quantitative polymerase chain reaction, and Western blot assays, respectively. Lnc-Pvt1 expression was increased in MM cell lines (NCI-H929, U-266 and LP-1 cell lines) compared with human normal plasma cells. In U-266 cells, lnc-Pvt1 shRNA suppressed cell proliferation while enhanced cell apoptosis compared with control shRNA. Also, lnc-Pvt1 shRNA increased miR-486 expression compared with control shRNA. Further rescue experiment revealed that miR-486 shRNA did not change lnc-Pvt1 level, but increased CDK4 and BCAS2 expressions in lnc-Pvt1 knockdown-treated cells. In addition, miR-486 shRNA promoted cell proliferation while inhibited cell apoptosis in lnc-Pvt1 knockdown-treated cells. These results were further validated in LP-1 cells. Lnc-Pvt1 knockdown inhibits cell proliferation and induces cell apoptosis through potentially regulating miR-486-mediated CDK4 and BCAS2 in MM.

Bone marrow adipocytes and multiple myeloma

Multiple myeloma originating from clonal proliferation of plasma cells in the bone marrow is one of the most prevalent hematological malignancies worldwide. The pathogenetic mechanisms of multiple myeloma are far from being elucidated. Nevertheless, it is known that the adipocytes as the prevalent cellular component of bone marrow microenvironment contribute significantly to multiple myeloma growth and progression. The review discloses the recent data on the interactions between bone marrow adipocytes and myeloma cells, hematopoietic stemcells, hematopoietic progenitor cells, mesenchimal stem cells, osteoblasts, osteoclasts, endothelial cells, and cells of immune system. Also, the review places special emphasis on bone marrow adipocyte-produced adipokines, growth factors, cytokines, chemokines, and fatty acids providing the conditions for the preferential growth and migration of malignant plasma cells and contributing to hematopoiesis supression, bone tissue resorption, angiogenesis activation and immunosuppression.

Long noncoding RNA TUG1 promotes proliferation and inhibits apoptosis in multiple myeloma by inhibiting miR-29b-3p.

Background: Long non-coding RNA taurine up-regulated gene 1 (TUG1) was reportedly involved in initiation and development of several cancers. However, its function and molecular mechanisms in multiple myeloma (MM) are still unclear. The present study aimed to determine the expression status, biological function, and potential mechanisms of TUG1 in the progression of MM.Materials and methods: The expression levels of TUG1 were examined in MM samples and cell lines by real-time quantitative PCR. The effects of TUG1 on MM cells proliferation and apoptosis were assessed using Cell Counting Kit-8 assay and flow cytometry respectively. MiRNAs-targeted sites in TUG1 were screened by Starbase2.0 and were identified by RNA immunoprecipitation assay combined with luciferase reporter assay.Results: The expression levels of TUG1 were markedly increased in MM samples and cell lines. Knockdown of TUG1 significantly suppressed the proliferation, induced cell cycle arrest at G1/G0 phase, and promoted apoptosis of MM cells. In exploring the regulatory mechanism, miR-29b-3p was confirmed to be a direct target of TUG1, and repression of miR-29b-3p could partially rescue the effect TUG1 knockdown on MM cell proliferation, cycle, and apoptosis. In addition, TUG1 positively modulated histone deacetylases 4 (HDAC4, a target of miR-29b-3p) expression through sponging of miR-29b-3p in MM cells.Conclusion: These findings suggested that TUG1 exerted an oncogenic role in MM by acting as a competing endogenous RNA of miR-29b-3p, and implied the potential application of TUG1 in treatment for MM.

MicroRNA-26a inhibits multiple myeloma cell growth by suppressing cyclin-dependent kinase 6 expression.

MicroRNA-26a (miR-26a) has been reported to be involved in the tumorigenesis of several tumors, but its biological function and molecular mechanism in multiple myeloma (MM) are still unknown. In this study, we found that overexpression of miR-26a obviously inhibited MM cell growth, and delayed tumor growth in xenografts. Further studies showed that overexpression of miR-26a induced cell cycle arrest at G0/G1 phase in MM cells. MiR-26a mimic down-regulated the expression levels of CDK6 and E2F1, but up-regulated p53 and p21 expression. In contrast, overexpression of CDK6 decreased the effect of miR-26a mimic on MM cell survival. Moreover, miR-26a targeted CDK6 mRNA and thus suppressed CDK6 protein expression. Overexpression of miR-26a also enhanced the cytotoxic action of doxorubicin against MM. These results demonstrated that miR-26a was involved in the development of MM through regulating CDK6 signaling pathway, and indicated that miR-26a could be as a novel target for anti-tumor therapy in clinic as a single strategy or in combination with other anti-tumor drugs in MM.

MiR-125b Upregulates miR-34a and Sequentially Activates Stress Adaption and Cell Death Mechanisms in Multiple Myeloma

miR-125b, ubiquitously expressed and frequently dysregulated in several tumors, has gained special interest in the field of cancer research, displaying either oncogenic or oncosuppressor potential based on tumor type. We have previously demonstrated its tumor-suppressive role in multiple myeloma (MM), but the analysis of molecular mechanisms needs additional investigation. The purpose of this study was to explore the effects of miR-125b and its chemically modified analogs in modulating cell viability and cancer-associated molecular pathways, also focusing on the functional aspects of stress adaptation (autophagy and senescence), as well as programmed cell death (apoptosis). Based on the well-known low microRNA (miRNA) stability in therapeutic application, we designed chemically modified miR-125b mimics, laying the bases for their subsequent investigation in in vivo models. Our study clearly confirmed an oncosuppressive function depending on the repression of multiple targets, and it allowed the identification, for the first time, of miR-125b-dependent miR-34a stimulation as a possible consequence of the inhibitory role on the interleukin-6 receptor (IL-6R)/signal transducer and activator of transcription 3 (STAT3)/miR-34a feedback loop. Moreover, we identified a pattern of miR-125b-co-regulated miRNAs, shedding light on possible new players of anti-MM activity. Finally, functional studies also revealed a sequential activation of senescence, autophagy, and apoptosis, thus indicating, for the first two processes, an early cytoprotective and inhibitory role from apoptosis activation.

MicroRNA-338-3p Inhibits Proliferation and Promotes Apoptosis of Multiple Myeloma Cells Through Targeting Cyclin-Dependent Kinase 4.

MicroRNA-338-3p (miR-338-3p) has been reported to be a tumor suppressor in multiple cancer types. However, the biological role of miR-338-3p and its underlying mechanism in multiple myeloma (MM) remain unclear. In the present study, we investigated the biological role and potential of miR-338-3p in MM. We found that miR-338-3p was significantly decreased in newly diagnosed and relapsed MM tissues and cell lines. Overexpression of miR-338-3p in MM cells significantly inhibited proliferation and promoted apoptosis, caspase 3, and caspase 8 activity. Bioinformatics algorithm analysis predicted that cyclin-dependent kinase 4 (CDK4) was a direct target of miR-338-3p, and this was experimentally verified by a dual-luciferase reporter assay. Furthermore, overexpression of miR-338-3p inhibited CDK4 expression on mRNA and protein levels. Of note, the restoration of CDK4 expression markedly abolished the effect of miR-338-3p overexpression on cell proliferation, apoptosis, caspase 3, and caspase 8 activities in MM cells. Taken together, the present study is the first to demonstrate that miR-338-3p functions as a tumor suppressor in MM through inhibiting CDK4. This finding implies that miR-338-3p is a potential therapeutic target for the treatment of MM.

Molecular Resistance Mechanisms in Multiple Myeloma

Mechanisms of drug resistance in Multiple Myeloma (MM) are poorly understood. Mutations and/or changes in the protein expression of the CRBN pathway and proteasome subunits have been identified to induce resistance to IMiDs and PIs. However, only few patients are affected by these alterations. To determine the specific genomic fingerprint of MM relapse we selected 57 MM patients from the CoMMpass trial (version IA11) that have genomic data of paired samples available (diagnosis/relapse). 35 of them have also sequential FISH-seq data. We focused on acquired mutations in first relapse and filtered all mutations and genetic alterations already present at diagnosis. Doing so, we found 1.274 mutations, representing an average of 23 new mutations/patient (range; 2-76). Of interest, 66% of the acquired mutations were present in a sub-clonal level (Variant read frequency (VRF) < 25%).Most common mutations include known hotspots of the RAS pathway (NRAS 12%, KRAS 7% and BRAF 4%). Notably, all 7 NRAS mutations in relapse were located at Q61K, suggesting a functional role of disease progression for this specific and known hotspot location. In total 5 of 35 cases (14%) with FISH-seq data developed a 17p13 deletion in relapse. Of these, three patients acquired a bi-allelic alteration in addition to a preexisting TP53 mutation and one developed a biallelic inactivation of TP53 (VRF = 100%), through parallel acquisition of del17p and TP53 mutation. Gain of 1q21 was observed in relapse in 5 of 35 (14%) cases, and one 1q gain was lost from diagnosis to relapse. Two cases (4%) presented mutations in IMiD treatment related genes, with two mutations in the CRBN pathway. One harbored a missense mutation in the Lenalidomide (LEN) degron sequence of IKZF3 (G159A) (VRF = 36%), known to be essential for the IMiD action in vitro, 45 months after continuous exposition to LEN . The other case presented two subclonal frameshift mutations in CUL4B (VRF = 5% and 32%), detected after more than three years of LEN containing therapy. We functionally validated in vitro LEN resistance through CRISPR/Cas9 knockout of CUL4B, suggesting a resistance inducing effect of the acquired CUL4B mutations. Six cases (11%) harbored acquired mutations in proteasome subunit genes (PSMC2, PSMC6, PSMD8, PSME4, PSMB9 (two mutations)), all of them had undergone prior proteasome inhibitor (PI) containing therapy. We validated earlier the 19S protein subunits PSMC6 and PSMC2 (KO and/or point mutations) as inducers of PI resistance in vitro, thus we hypothesize contribution to resistance induction / disease progression through these 19s mutations.Remarkably ubiquitin (E3, E2 and SUBs) and histone related genes (histones and histone methylases and deacetylases) were found mutated in 51% and 19% of the relapsed patients. Genes for drug transporters (ATP-binding cassette (ABC) and Solute Carrier (SLC) transporters) were hit in 32% of cases and genes for mucins (previously related with genotoxic agents and immunotherapy resistance) in 19%. Notably, RRBP1 presented 10 mutations in 6 patients (11%) with the mutations clustering within 30 amino-acids (aa) of exons 9 and 10 and 3 hotspots (2 patients each) in aa Q426P, K430R and Q436P. RRBP1 is involved in the binding of the ribosome to the endoplasmic reticulum (ER) and is related with the unfolded protein response and ER stress via GRP78. All the patients with RRBP1 mutations were pretreated with PI inhibitors and exhibited worst survival outcome affecting PFS (Pval<0.001) and OS (Pval=0.0016) in this limited dataset. The mutations were detected on average 433 days (range: 258-568) after diagnosis. Five of the 6 patients died on average 180 days after RRBP1 mutation detection (range: 18-446) further suggesting high risk features of such acquired mutations.In summary, we observe clonal selection of known high-risk related alterations like TP53 mutations, 17p deletions or 1q13 in early relapse data of the CoMMpass trial. Furthermore we identify RRBP1 mutations as a new acquired high-risk biomarker of MM. Alterations are specifically related to subclonal selection by therapy, thus we suggest that the definition of high-risk disease in MM needs to be revisited and should also include clonal selection processes under anti-tumor therapy. DisclosuresNo relevant conflicts of interest to declare.

IL-10 inducible CD8+ regulatory T-cells are enriched in patients with multiple myeloma and impact the generation of antigen-specific T-cells.

Tumor-mediated immunosuppression via regulatory T-cells is a key player among the various immune-escape mechanisms in multiple myeloma. We analyzed the generation, distribution, function and immunophenotype of CD8+CD28- regulatory T-cells in patients with multiple myeloma. Functionality of CD8+CD28- T-cells was assessed by immunological assays using ex vivo generated antigen-specific T-cells from patients with plasma cell dyscrasias and healthy donors. Detailed analysis of distribution, immunophenotype and cytotoxic potential of CD8+CD28- T-cells was performed by flow cytometry and ELISA. We found that the amount of CD8+CD28- T-cells was directly correlated with the suppression of antigen-specific T-cell responses in patients with plasma cell dyscrasia. Analyzing the CD8+CD28- T-cells in detail, increased numbers of these cells were observed in the bone marrow (i.e., tumor microenvironment) of patients with plasma cell dyscrasia. Furthermore, we identified the expression of lymphocyte function-associated antigen 1 (LFA-1) as a marker of immunosuppression and defined the CD8+CD28-CD57+LFA-1high population as the relevant immunosuppressive compartment. These regulatory T-cells act as immunosuppressors via soluble factors and incubation with IL-10 augmented their immunosuppressive capacity. The immunosuppressive regulatory network of IL-10 and the CD8+CD28-CD57+LFA-1high regulatory T-cells show unique characteristics and contribute to the tumor immune escape mechanism in patients with multiple myeloma.

Drug targets and resistance mechanisms in multiple myeloma

Drug targets and resistance mechanisms in multiple myeloma