Beta-Arrestin Isoforms Correlate with Clinical Outcomes in Patients with Multiple Myeloma and Alter PD-1/PD-L1 Expression in Murine T Cells

Intro:

Beta-arrestins are multifunctional adaptor proteins involved in the desensitization and trafficking of G-protein-coupled receptors (GPCRs). Two isoforms, β-arrestin1 (βarr1) and β-arrestin2 (βarr2), are ubiquitously expressed in human cells. Understanding their actions is critical to pharmacology and drug development broadly, as GPCRs are modulated/targeted by many common drugs. Prior studies have implicated β-arrestins in a range of critical downstream processes, including cell proliferation, survival and apoptosis, cell migration/chemotaxis, and modulation of the immune response. The role of β-arrestins in the pathogenesis of multiple myeloma (MM) has not been elucidated. This study investigated the expression of βarr1 and βarr2 in bone marrow samples from MM patients and their correlation with clinical outcomes. We also determined the effect of βarr2 in the regulation of the PD-1/PD-L1 checkpoints in an animal model of MM.

Methods:

The GSE13591 and APEX microarray datasets were analyzed for βarr1 and βarr2 expression. Archived bone marrow biopsy (BMBx) samples from patients (n= 45) with newly diagnosed MM were sectioned and stained with immunofluorescent-labeled βarr1 antibody (ab32099, 1:500) or βarr2 antibody (A2CT, 1:500) in addition to Hoechst (H3570, 1:1000). Immunofluoresence was measured using a Zeiss laser-scanning microscope and and β-arrestin expression was quantified using Imaris 8.1 software and normalized to isotype control and Hoechst expression. Patient characteristics, progression free survival (PFS) and overall survival (OS) were extracted from electronic medical records. To determine the effects of βarr2 in the regulation of PD-1/PD-L1 expression in murine cells, T cells were isolated from the spleen of βarr1 knockout (KO) mice, βarr2 KO mice or wild-type (WT) mice, cultured in vitro with CD3 and CD28 antibodies and PD-1 level was measured by flow cytometry. Additionally, splenic CD3+ T cells and myeloid derived suppressor cells (MDSCs) were harvested from WT mice, treated with βarr2 specific siRNAs, and PD-1 and PD-L1 expression was measured. S1PR1, a GPCR, was used as a positive control.

Results:

βarr1 expression was significantly lower in myeloma plasma cells compared to normal plasma cells (GSE13591, p=0.016), while βarr2 expression was significantly higher in MM patients resistant to bortezomib treatment compared to those who responded (APEX, p=0.0183). Consistent with these microarray results, higher βarr1 expression in BMBx samples was associated with improved PFS and OS: PFS was 36 months in patients with highest quartile (Q1) of βarr1 vs 14 months in patients with lowest quartile (Q4) of βarr1 (p=0.04); OS was 93 months in Q1 βarr1 vs 29.1 months in Q4 βarr1 (p=0.0448). In contrast, higher βarr2 expression in BM was associated with inferior PFS (22 months in high βarr2 vs 30.13 in low βarr2, p=0.0385) and OS (86.4 months in high βarr2 vs 107.3 in low βarr2, p=0.0301). Next, to assess differential βarr1 and βarr2 effects on the tumor microenvironment (TME), we assessed PD-1 expression in CD3 T cells from βarr1 KO mice, βarr2 KO mice, and WT mice after T cell activation. CD3 T cells isolated from βarr1 KO mice showed significantly increased PD-1 expression, while CD3 T cells isolated from βarr2 KO mice had reduced PD-1 expression. Furthermore, knockdown of βarr2 in CD3 T cells and MDSCs resulted in down-regulation of PD-1 expression in murine CD3 T cells and PD-L1 expression in murine MDSCs, respectively. These findings suggest distinct roles of these two distinct β-arrestin isoforms in MM pathogenesis, potentially mediated via modulation of immune checkpoints in the TME.

Conclusion:

This is the first study investigating the association of βarr1 and βarr2 expression with clinical outcomes in patients with MM and the role of β-arrestins in the regulation of PD-1/PD-L1, a novel finding given that PD-1 and PD-L1 are not GPCRs. Our study provides new evidence that β-arrestins may also regulate non-GPCRs and has important implications in our understanding of MM pathobiology and response to therapy, as well as immunotherapy and resistance to PD-1/PD-L1 blockage more broadly.

Rein:Abbvie: Consultancy; Sobi: Consultancy; DAVA Oncology: Other: Speaker, conference participant; Incyte: Consultancy, Other: Site Principal Investigator for clinical trial, Research Funding; Morphpsys: Consultancy; Novartis: Consultancy, Honoraria, Other: Site Principal Investigator for clinical trial, Research Funding; Sumitomo Dainippon Pharma Oncology: Consultancy, Other: Site Principal Investigator for clinical trial, Research Funding; Cogent Biosciences: Other: Site Principal Investigator for clinical trial, Research Funding; Protagonist: Other: Site Principal Investigator for clinical trial, Research Funding; Geron: Other: Site Principal Investigator for clinical trial, Research Funding; Blueprints Medicine: Other: Site Principal Investigator for clinical trial, Research Funding; Merck: Other: Site Principal Investigator for clinical trial, Research Funding; PharmaEssentia: Other: Site Principal Investigator for clinical trial, Research Funding; Karyopharm: Other: Site Principal Investigator for clinical trial, Research Funding; Telios Pharma: Research Funding; Silence Therapeutics: Research Funding.