Abstract 2981: Loss of the small GTPase RHOA enhances lymphomagenesis and impairs the activity of lenalidomide in mantle cell lymphoma

Abstract

Abstract The small GTPase Ras homolog family member A (RHOA) is one of the most extensively investigated members of the Rho GTPase family, that acts as a molecular switch controlling a wide variety of signal transduction pathways. Although RHOA has long been implicated in malignant transformation in solid tumors, recent evidences have demonstrated its tumor suppressor activity in different subgroups of B-cell non-Hodgkin lymphoma (B-NHL). Here, using a panel of 11 cell lines covering the most common and/or aggressive B-NHL subtypes, we observed that the lowest mRNA and protein levels of RHOA are found in mantle cell lymphoma (MCL), the most aggressive entity with a median overall survival of 5-7 years. Depletion of RHOA expression and activity was carried out in the two RHOA+ MCL cell lines, REC-1 and Z-138, by CRISPR/Cas9-mediated gene edition followed by RHOA pulldown activation assay. In RHOA knockout (KO) subclones, cell proliferation was increased by 40%, in association with a 10-fold increase in mitotic index and a 3-fold potentiation of cell migration, as assessed respectively by CellTitter-Glo assay, histone H3-pSer10 labeling and Transwell assay, thus suggesting a tumor suppressor role of RHOA in MCL malignancy. Comparative RNA-seq analysis of RHOAWT and RHOAKO MCL subclones then highlighted a crucial role of RHOA and its downstream signaling in the control of MCL cell proliferation, cell cycle and cell migration. Interestingly, the immunomodulatory drug lenalidomide has previously shown to regulate such processes and to be clinically active in patients with relapsed/refractory MCL. In a co-culture system associating RHOAWT MCL cells with peripheral blood-derived effector cells, we observed that lenalidomide treatment facilitated the formation of immunological synapse, as revealed by simultaneous immunodetection of effector T cells, target B cells and perforin, and by quantification of lactate dehydrogenase (LDH) release. Conversely, lenalidomide exhibited minimal effect in the RHOAKO MCL co-cultures, as shown by a 30-40% decrease in the formation of immune synapse and a 4-fold decrease in LDH secretion. Taken together, these data provide the first insight into the tumor suppressive activity of RHOA in MCL and into its requirement for the immune-mediated mechanism of action of lenalidomide in this disease. Ongoing in vivo validation studies will be presented. Citation Format: Juliana C. Santos, Marcelo L. Ribeiro, Gael Roue. Loss of the small GTPase RHOA enhances lymphomagenesis and impairs the activity of lenalidomide in mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2981.