Abstract LB217: ROBO1 promotes homing, dissemination, and survival of multiple myeloma within the bone marrow microenvironment
Authors: Bianchi Giada , Czarnecki Peter G. , Ho Matthew , Roccaro Aldo M. , Sacco Antonio , Kawano Yawara , Gulla Annamaria , Samur Anil Aktas , Cheng Tianzeng , Wen Kenneth , Tai Yu-Tzu , Moscvin Maria , Wu Xinchen , Camci-Unal Gulden , Da VIa Matteo Claudio , Bolli Niccolo , Carrasco Ruben D. , Ghobrial Irene M. , Anderson Kenneth C. 
Published: July 2021

Abstract
Introduction: The bone marrow (BM) niche promotes multiple myeloma (MM) growth, survival and drug resistance. Therapies targeting both cancer cells and the microenvironment are highly effective. We were interested in identifying novel signaling pathways supporting MM pathogenesis through MM-BM crosstalk. The transmembrane receptor Roundabout 1 (ROBO1) plays a role in growth and dissemination of solid tumors, however its function in MM is unknown. Material and Methods: We analyzed ROBO1 expression in cell lines and primary samples via western blot, immunohistochemistry (IHC) and gene expression profiling. We used short hairpin RNA and CRISPR-Cas9 for ROBO1 knock down (KD) and knock out (KO), respectively. For protein structure-function and rescue studies, we stably expressed full-length (FL) or mutant ROBO1 devoid of extracellular (Cyt) or intracellular domain (DeltaCyt), including patient-derived G674* truncation, with a C-terminus FLAG tag. We used a 3D hydrogel encapsulation system to study proliferation; FLAG immunoprecipitation (IP) followed by mass spectrometry to identify ROBO1 interacting partners; and immunofluorescence to detect ROBO1 localization. To study tumor growth in vivo, we performed PET-CT of mice inoculated subcutaneously or intramedullary with WT or ROBO1 KO MM cells and retrieved tumors for RNA sequencing. To study dissemination and homing, KO and FL addback MM cells were injected intravenously in SCID mice. Mice were monitored for development of tumors or hindlimb paralysis and femora/tumors harvested once mice reached endpoint. Results: ROBO1 is highly expressed in MM cell lines and primary cells but low/absent in normal plasma cells and other hematologic cancer cell lines. ROBO1 KD is specifically cytotoxic for MM cells and ROBO1 KO decreases proliferation, a phenotype fully rescued by FL ROBO1. Compared to WT, ROBO1 KO significantly decreases intramedullary (mean tumor volume (MTV): 1323 vs 457 mm3, p value= 0.02) and extramedullary (MTV: 2684 vs 823 mm3, p value= 0.001) tumor growth in vivo. We further discovered that ROBO1 KO decreases adhesion of MM to BM endothelial and BMSC, which is fully rescued by FL ROBO1. In a disseminated mouse model, ROBO1 KO cells generate bone plasmacytoma with reduced BM invasion, as compared to the extensive BM infiltration observed with ROBO1 FL cells. Consistently, in primary samples from patients, we detected ROBO1 expression only in 1 out of 10 solitary plasmacytoma (dim staining) as compared to 14 out of 14 MM bone marrow biopsy samples tested (11 strong, 3 dim, p value= 0.0001). Mechanistically, we show for the first time that ROBO1 C-terminus is cleaved in a ligand-independent fashion; translocates to the nucleus; and is necessary and sufficient to rescue ROBO1 KO proliferative defect. Viceversa, mutants lacking the cytoplasmic domain, including the G674* truncation, act dominantly negative. Interactomic and RNA sequencing studies point to a previously unknown function of ROBO1 in RNA processing, setting the bases for future studies. Conclusions: We show for the first time that ROBO1 is necessary for MM growth and homing to the BM. Cleaved ROBO1 cytosolic domain translocates to the nucleus and is necessary and sufficient to rescue ROBO1 KO proliferative defect, possibly by participating in RNA processing. These data suggest that ROBO1 C-terminus may be a novel molecular target in MM.
Citation Format: Giada Bianchi, Peter G. Czarnecki, Matthew Ho, Aldo M. Roccaro, Antonio Sacco, Yawara Kawano, Annamaria Gulla, Anil Aktas Samur, Tianzeng Cheng, Kenneth Wen, Yu-Tzu Tai, Maria Moscvin, Xinchen Wu, Gulden Camci-Unal, Matteo Claudio Da VIa, Niccolo Bolli, Ruben D. Carrasco, Irene M. Ghobrial, Kenneth C. Anderson. ROBO1 promotes homing, dissemination, and survival of multiple myeloma within the bone marrow microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB217.