Functional Characterization of GPRC5D alteration and Its Impact on Talquetamab Resistance in Relapsed/ Refractory Multiple Myeloma

Abstract

Background T-cell engaging (TCE) antibodies show unprecedented efficacy in heavily pretreated penta-refractory multiple myeloma (MM). Responses however are often not durable as resistance mechanisms can arise despite the initial absence of resistance-conferring mutations. Biallelic loss of GPRC5D on chr.12p is a rare, yet well-established mechanism of acquired resistance to the first-in-class anti-GPRC5D directed TCE talquetamab (Lee et al., 2022). At the same time, monoallelic GPRC5D frameshift and missense mutations are much more common and occur in up to 15% of MM patients (Truger et al., 2021). In this study, we investigated the functional relevance of monoallelic GPRC5D alterations and their impact on talquetamab resistance. Methods GPRC5D alterations were modeled in vitro by using CRISPR-Cas9 editing in the OPM-2 MM cell line. The bioluminescence-based cytotoxic assay was performed using PBMCs as effector cells to measure the resistance to talquetamab. Quantitative PCR (qPCR), immunohistochemistry, bulk whole genome sequencing (WGS), and whole genome bisulfite sequencing (WGBS) were performed for molecular, genomic, and epigenetic characterization in a patient with acquired talquetamab resistance. Results Using CRISPR-Cas9, we generated cell line models with mono- vs. biallelic deletions in the GPRC5D gene. Clones with biallelic alterations (OPM-2 Del/Del) displayed a 96.4%, reduced expression of GPRC5D ( p < .0001), leading to in vitro resistance to talquetamab. GPRC5D expression levels in monoallelic models (OPM-2 Wt/Del) decreased by 57.4% ( p = .0004) compared to the wild type. Sensitivity to talquetamab in these clones was equally impaired, however, less pronounced than in the biallelic knock-outs. To assess the impact of monoallelic GPRC5D deletions in the clinic, we next investigated the case of a heavily pretreated 58-year-old patient with lambda light-chain MM treated at our institution with talquetamab in his 7th line of therapy. qPCR was used to measure GPRC5D expression in CD138+ MM cells from the bone marrow of this patient before and at the time of relapse to talquetamab after 15 months of treatment (best response: CR). At the time of relapse, we observed a 94.0% ( p < .0001) decrease in GPRC5D expression upon qPCR assessment as compared to the pre-treatment time point which was confirmed by immunohistochemistry. WGS revealed a novel monoallelic loss of chr.12p encompassing GPRC5D in talquetamab-resistant CD138+ MM cells. Since monoallelic alterations seem unlikely as the sole drivers of resistance, we hypothesized that epigenetic modifications may have contributed to talquetamab resistance in this patient. To this end, WGBS was performed and uncovered a significant shift towards hypermethylation of two regulatory regions of GPRC5D, most likely resulting in the silencing of the second GPRC5D allele. Time-course experiments are currently ongoing to explore the methylation landscape of our CRISPR-Cas9-generated clones with monoallelic GPRC5D deletions. Data for these validation experiments will be presented at the meeting. Conclusions Our study provides the first evidence of the functional impact of monoallelic GPRC5D alterations resulting in relative resistance to talquetamab in vitro. We further report on epigenetic regulation as a potential second-hit mechanism conferring clinical resistance to anti-GPRC5D-directed TCE therapy. This data suggests that patients with monoallelic GPRC5D deletions may be at particular risk for developing resistance to talquetamab.