Role of Ras-Related C3 Botulinus Toxin Substrate 1 in p53-Related Proliferation and Drug Sensitivity in Multiple Myeloma

Introduction: TP53 is a tumor suppressor gene located on chromosome 17p 13.1 that plays a critical role in preventing and reducing the aggressiveness of many tumor types, including multiple myeloma (MM). 17p deletions and TP53 mutations are associated with poor clinical outcomes in patients with MM. Rho GTPase signaling is involved in cancer progression, dissemination, and chemoresistance. Among the three major Rho-GTPases (RhoA, Rac1, and Cdc42) we selected Rac1 because a previous study on lymphoma demonstrated an interaction between RaC1 and p53. In this study, we evaluated the role of Rac1 in MM, to inform novel therapy development.

Materials and Methods: Bone marrow plasma cells obtained with informed consent from 114 MM patients, 70 MGUS patients, and 15 controls, and purified via anti-CD138 antibody and magnetic beads were included in this study. The study was approved by Gunma University's IRB, and followed the Declaration of Helsinki guidelines. Three human myeloma cell lines (HMCLs), KMS11, KMS26, and MM.1S, whose respective TP53 statuses were deficient, mutated, and wild type, were used. KMS26 and KMS11 cells expressing DOX-inducible wild type (WT) p53 (KMS26/Tet-on p53 and KMS11/Tet-on p53, respectively) and p53 MM.1S knockdown by shp53 were used. RAC1 mRNA levels were assessed by RT-qPCR, with ACTB serving as an endogenous control, and MM.1S as a reference sample. Protein expression levels of p53, p21, Mdm2, and Rac1 were determined via western blotting. To quantify cell proliferation or induce cell death, EdU and Annexin V assays were performed. RNA-seq was performed using an Illumina Next Seq 500.

Results: KMS11/Tet-on p53 cell proliferation was not affected by p53 induction, while that of KMS26/Tet-on p53 cells was significantly reduced. Gene Ontology (GO) analysis using RNA-seq data demonstrated enhanced Rho-GTPase signaling in KMS11 compared to KMS26. RAC1 mRNA levels in purified BM plasma cells were significantly higher in patients with NDMM than in controls (p < 0.01). Rac1 inhibitor 1A-116 (50 μM) significantly reduced survival rates of both KMS11 and KMS26 cells at 72 h. This effect was more prominent in KMS11 cells than in KMS26 cells. 1A-116 also significantly reduced MM.1S cell survival at 72 h with p53 induction by MDM2 inhibitor Nutlin-3 (1 µM), or p53 knockdown by Shp53, although knockdown of WT p53 alone or Nutlin-3 alone did not affect survival. In KMS11/Tet-on p53, KMS26/Tet-on p53, and MM.1S cells, cotreatment with Nutlin-3 and 1A-116 did not increase p53, p21, or Mdm2 protein expression. In MM.1S cells with p53 knockdown, treatment with 1A-116 did not increase p53 or Mdm2 protein expression, but did increase that of p21. 1A-116 treatment significantly reduced EdU incorporation in all three HMCLs, indicating that Rac1 inhibition arrests the cell cycle, but the magnitude of this effect was attenuated in KMS11 cells. Apoptotic and dead cells, defined by annexin V-positivity and 7-AAD incorporation, were significantly increased after 1A-116 treatment in KMS11 and MM.1S cells. KMS11 and KMS26 cell survival at 72 h after treatment declined when CRBN modulators lenalidomide, pomalidomide, and iberdomide were combined with the Rac1 inhibitor 1A-116 (25µM) compared with CRBN modulator treatment alone. In contrast, Rac1 inhibitor showed no additive effect on cell survival after 24 h of bortezomib treatment in HMCLs. Median overall survival (OS) time of patients with high RAC1 mRNA expression (above median value) was significantly shortened (4.3 years vs not reached; p = 0.01), although progression free survival (PFS) was not significantly different (2.0 years vs 3.1 years; p = 0.21). In multivariate analysis, ASCT and RAC1 mRNA expression were independent prognostic factors for OS (hazard ratio [HR], 0.409; p = 0.04; RAC1 mRNA high: HR, 2.211; p = 0.02). Among patients who underwent ASCT, both OS and PFS were significantly lower in those with high RAC1 mRNA expression than in those with low RAC1 expression (OS: 5.1 years vs not reached, p = 0.02; PFS: 2.7 years vs not reached, p = 0.01).

Conclusions: Rac1 affects HMCL survival regardless of p53 status and Rac1 associates with CRBN modulator sensitivity. High RAC1 mRNA expression in intramedullary plasma cells of patients with NDMM is associated with worse prognosis. Our research provides new insights for development of novel therapies targeting the Rac1 pathway to improve MM patient prognosis, including patients with p53 dysfunction.

Ogawa:Chugai Pharmaceutical Co.: Honoraria. Handa:AbbVie: Research Funding; GSK: Research Funding; Pfizer: Research Funding; Takeda Pharmaceuticals: Honoraria; Janssen Pharmaceutical: Honoraria, Research Funding; Amgen: Research Funding; Sanofi: Research Funding; Bristol Myers Squibb: Honoraria; Ono: Honoraria.