Umair Munawar

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.

DNA promoter methylation of a subset of proteasome subunit genes. Heatmap comparing methylation pattern of 35 MM patients and 30 PBMC samples. The methylation degree is given by a color code: red – methylated state, blue – unmethylated state. Each line represents an individual sample, each column a single CpG. The methylation degree is encoded by color with blue for an unmethylated and red for a methylated state. Artificially methylated controls were included. For PSMD5, hypermethylation was only present in MM cells, not in PBMCs. For PSMC2, hypermethylation occurred in both, MM cells and PBMCs, indicating a physiological state probably independent of disease development and progression. In the remaining proteasome subunit genes, no aberrant promoter methylation was detected.

Raw data measurements of the dual-luciferase reporter assay in the L363 cell line. All measurements were performed in duplicates on 20.000 cells.

Patient-derived mutations impair PI response to Carfilzomib and Ixazomib. Cytotoxicity assay and IC50 values of RPMI-8226 MM cell lines harboring two different stably expressed patient-derived mutations in proteasome subunits. The mutants were more resistant to the proteasome inhibitors Carfilzomib and Ixazomib compared to the controls (*) harboring lentivirally-encoded WT sequence of PSMC6/PSMD1. Shown are the means and standard deviations of three independent experiments (biological replicates) and three technical replicates in each experiment. The PSMC6 and PSMD1 expression levels were equivalent for the mutants and the controls.

Genetic variants in the Würzburg dataset.

Primers used for luciferase reporter assay.

Cell survival of DNMTi pretreated KMS11 cells under BTZ exposure. AlamarBlue assay after 72 hours of incubation with varying concentrations of BTZ (0.5 nM to 4 nM) on DNMTi pretreated cells (48 h 500 nM Aza, 72 h 100 nM Dec) and PI naïve KMS11. No resensitizing effect to PI treatment was observed.

Quality check of DBS assays with artificially methylated standard DNAs. DNA standards of 0%, 25%, 50%, 75% and 100% methylation were sequenced with the PSMB5, PSMC2, PSMC5, PSMC6, PSMD1 and PSMD5 DBS assays to confirm assay sensitivity. Results for all investigated DBS amplicons correlated well with the expected curves (P < 0.001) (A). The sequencing depths of the PSMD5 DBS assay was 10-3 (B).