Cereblon is the direct binding target of the immunomodulatory drugs (IMiDs) that are commonly used to treat multiple myeloma (MM), the second most frequent hematologic malignancy. Patients respond well to initial treatment with IMiDs, but virtually all patients develop drug resistance over time, and the underlying mechanisms are poorly understood. We identified an as yet undescribed DNA hypermethylation in an active intronic CRBN enhancer. Differential hypermethylation in this region was found to be increased in healthy plasma cells, but was more pronounced in IMiD-refractory MM. Methylation significantly correlated with decreased CRBN expression levels. DNA methyltransferase inhibitor (DNTMi) in vitro experiments induced CRBN enhancer demethylation, and sensitizing effects on lenalidomide treatment were observed in 2 MM cell lines. Thus, we provide first evidence that aberrant CRBN DNA methylation is a novel mechanism of IMiD resistance in MM and may predict IMiD response prior to treatment.

Purpose: Proteasome inhibitors (PI) are the backbone of various treatment regimens in multiple myeloma. We recently described the first in-patient point mutations affecting the 20S subunit PSMB5 underlying PI resistance. Notably, in vivo, the incidence of mutations in PSMB5 and other proteasome encoding genes is too low to explain the development of resistance in most of the affected patients. Thus, additional genetic and epigenetic alterations need to be explored. Experimental Design: We performed DNA methylation profiling by Deep Bisulfite Sequencing in PSMB5, PSMC2, PSMC5, PSMC6, PSMD1, and PSMD5, a subset of proteasome subunits that have hitherto been associated with PI resistance, recruited from our own previous research, the literature, or a meta-analysis on the frequency of somatic mutations. Methylation was followed up on gene expression level and by dual-luciferase reporter assay. The KMS11 cell line served as a model to functionally test the impact of demethylating agents. Results: We identified PSMD5 promoter hypermethylation and subsequent epigenetic gene silencing in 24% of PI refractory patients. Hypermethylation correlated with decreased expression and the regulatory impact of this region was functionally confirmed. In contrast, patients with newly diagnosed multiple myeloma, along with peripheral blood mononuclear cells and CD138+ plasma cells from healthy donors, generally show unmethylated profiles. Conclusions: Under the selective pressure of PI treatment, multiple myeloma cells acquire methylation of the PSMD5 promoter silencing the PSMD5 gene expression. PSMD5 acts as a key orchestrator of proteasome assembly and its downregulation was described to increase the cell's proteolytic capacity. PSMD5 hypermethylation, therefore, represents a novel mechanism of PI tolerance 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.

Super-resolution microscopy in combination with genetic labeling methods allows imaging of single proteins in cells. However, visualizing endogenous proteins on primary cells remains challenging due to the use of sterically demanding antibodies for labeling. Here, we demonstrate how immunolabeling conditions and antibody cross-linking influence the quantification and identification of membrane receptor stoichiometry on cells using single-molecule localization microscopy. We developed an optimized immunolabeling and analysis protocol and demonstrate the performance of the approach by resolving the molecular organization of endogenous CD45, CD69, and CD38 on Jurkat T cells. To demonstrate the usefulness of the method for immunotherapy applications, we investigated the interaction of primary multiple myeloma cells with the therapeutic monoclonal antibodies daratumumab and isatuximab and a polyclonal anti-CD38 antibody. Our approach might lay the foundation for improved personalized diagnostics and treatment with therapeutic antibodies.

Immunotherapies targeting surface antigens have transformed the treatment landscape of multiple myeloma (MM), with GPRC5D emerging as a promising therapeutic target. Monoallelic loss of GPRC5D is frequently observed in newly diagnosed MM patients, and the incidence of acquired GPRC5D alterations increases following exposure to GPRC5D-directed therapies. However, the functional consequences of both baseline monoallelic and therapy-induced biallelic GPRC5D alterations remain poorly understood. In this study, we modeled monoallelic versus biallelic loss of GPRC5D to investigate their impact on MM cell biology and responsiveness to GPRC5D-targeted immunotherapies. Our results demonstrate that monoallelic GPRC5D loss in OPM-2 cells reduces surface expression of the antigen and confers resistance to GPRC5D-directed therapies. Complete loss of GPRC5D alters the transcriptional state of MM cells and promotes reprogramming of the phosphoproteomic circuitry ultimately resulting in a pro-proliferative chemokine environment. As a result, GPRC5D deficiency increases the basal proliferation rate of MM cells thereby providing a competitive advantage which may further be amplified by selecting these aggressive phenotypes during ongoing treatment with anti-GPRC5D immunotherapies.

Proteasome inhibitors (PIs) are frontline therapies for multiple myeloma (MM). Although MM patients initially respond to PIs, resistance frequently emerges. While all PIs nominally target the same proteasomal catalytic subunit (PSMB5), the extent to which resistance mechanisms are the same or different among different PIs or between patients is poorly understood. To address this, we performed proteome and phosphoproteome profiling of 12 MM cell line models, comprising four parental lines (AMO-1, ARH77, L363, and RPMI8226) paired with lines that acquired resistance to bortezomib (BTZ) or carfilzomib (CFZ). Over 7,000 proteins and up to 10,000 phosphopeptides were identified per cell line, enabling a comprehensive comparative analysis of shared and cell line-specific resistance signatures at the protein level. Data analysis revealed surprisingly few changes in the phosphoproteome but substantial reprogramming of the proteome in most models. Beyond known adaptations such as the overexpression of the PI target PSMB5 and the drug efflux transporter ABCB1, we identified the oxidoreductases NQO1 and NQO2 as significantly upregulated proteins under chronic proteotoxic stress across several models. Pharmacological follow up in PI resistant AMO-1 cells showed that NQO2 inhibition by imatinib fully restored CFZ sensitivity, validating NQO2 as a contributor to resistance formation in this model system.