GPRC5D Status Impacts CD38 Expression in Multiple Myeloma
Authors: Munawar Umair , Weingart Julia , Eiring Patrick , Nerreter Silvia , Vogt Cornelia , Kurian Shilpa , Han Seungbin , Steinhardt Max J , Zhou Xiang , Sauer Markus , Einsele Hermann , Rasche Leo , Waldschmidt Johannes , Kortüm K. Martin 
Published: December 2024

Background:
Treatment paradigm in Multiple Myeloma (MM) has shifted towards immunotherapies such as monoclonal antibodies,T cell engagers (TCE) and CART cells targeting surface antigens. While G protein-coupled receptor class C group 5 member D (GPRC5D) has emerged as a promising antigen for TCE, newly acquired GPRC5D alterations have been reported to convey resistance to the anti-GPRC5D-CD3 TCE talquetamab. Since surface epitome expression in MM cells is highly impacted by secondary genetic events, we investigated the role of GPRC5D alterations on the surface expression of common immunotherapy targets (BCMA, CD38, CD138) in MM.
Methods:
CRISPR-Cas9 technology was utilized to create GPRC5D knock out (KO) cell models using the MM cell line OPM-2. Direct stochastic optical reconstruction microscopy (dSTORM) was used for high-resolution receptor quantification and distribution estimation. Gene expression profiling at transcriptomic and protein level was performed utilizing Real time PCR (RT-PCR), RNA sequencing and Western blotting. MM cells were engineered with Sleeping Beauty transposons system for stable expression of luciferase to perform cytotoxicity, functional and clonal competition assays using immunotherapeutic agents in presence of effector T cells from healthy donors.
Results:
Digital droplet PCR (ddPCR) and Sanger sequencing were used to verify the generation of GPRC5D mono- and bi-allelic KO cells. Ultra-high resolution surfaceome screening via dSTORM confirmed a significant reduction of surface GPRC5D expression in KO cell models (0.032±0.007 clusters/µm2 vs 0.323±0.102 clusters/µm2 in WT cells, p=0.002). Surprisingly, GPRC5D altered cells also showed a significant reduction in CD38 surface expression: GPRC5DWt/Del cells had 5.745±0.432 CD38 clusters/µm2 compared to WT parental cells with 9.594±0.717 clusters/µm2, (p=0.04). CD38 cluster density was further reduced in GPRC5DDel/Del clones (2.226±0.203 clusters/µm2, p&lt;0,0001). Of note, no changes in other immunotherapeutic surface antigens (BCMA, SLAMF7, CD138) were observed, suggesting an exclusive impact of GPRC5D alterations on CD38 expression. Western blotting confirmed a reduction in CD38 expression at protein level. No changes at RNA levels were observed via bulk RNA sequencing and RT-PCR implying a post-transcriptional link between GPRC5D and CD38. To understand the functional impact of CD38 reduction in GPRC5D models, cells were treated with the CD38 targeting immunotherapeutic agents daratumumab (Dara) and isatuximab (Isa). GPRC5DWt/Del cells with reduced CD38 expression depicted a significant resistance towards CD38 targeting antibodies (8.01% specific lysis compared to 31.50% in WT cells, p &lt;0.0001). This resistance was further enhanced in GPRC5DDel/Del clones (6.387 % specific lysis, p &lt;0.0001), whereas no differences in sensitivity towards genotoxic drugs, proteasome inhibitors (PIs) and immunomodulatory drugs (IMiDs) were seen. Exogenous overexpression of GPRC5D in WT and KO cells did not impact CD38 at transcript level but an increase in CD38 protein level was observed via Western blotting. Patients with GPRC5D alterations are being screened for daratumumab sensitivity, notably one patient from our institution with biallelic GPRC5D loss did not respond to subsequent Dara-PACE therapy.
Summary:
This study suggests that GPRC5D alterations are associated with downregulation of CD38 at post transcriptomic level. Co-localization and clonal competition experiments are ongoing along with clinical analysis of anti-GPRC5D treated patients.