Roundabout 1 (ROBO1)/SLIT2 Is a Novel Signaling Pathway in Multiple Myeloma Promoting Survival and Bone Marrow Niche Interaction
Authors: Bianchi Giada , Czarnecki Peter G. , Roccaro Aldo M. , Sacco Antonio , Kawano Yawara , Samur Mehmet K. , Gulla Annamaria , Tai Yu-Tzu , Ghobrial Irene M. , Carrasco Ruben D. , Anderson Kenneth C. 
Published: October 2019

Abstract
Introduction
Molecular mechanisms of multiple myeloma (MM) pathogenesis are unknown, but a role for bone marrow (BM) niche in supporting MM is well established. The transmembrane receptor Roundabout 1 (ROBO1) has been historically annotated as a tumor suppressor gene in solid cancer, and more recently myelodysplastic syndrome. High ROBO1 expression was identified as a poor prognostic factor in newly diagnosed MM; however, its function in MM is unknown.
Material and Methods
We analyzed ROBO1 and SLIT2 expression in human MM cell lines (HMCL) and a panel of human cancer cell lines via western blot (WB). ROBO1/SLIT2 expression in primary BM samples was assessed via immunohistochemistry and/or WB. We used short hairpin RNA (shRNA) for stable ROBO1 knock down (KD). We designed and cloned single guide RNA (sgRNA) into pSpCas9(BB)-2A-GFP vector and developed CRISPR-Cas9-mediated ROBO1 knock out (KO) 293T and OPM2 clones. WT and KO ROBO1 OPM2 cells were used for standard viability, proliferation, and adhesion assays. Full length (FL) and mutant ROBO1 isoforms devoid of extracellular or intracellular domain, with a C-terminus triple FLAG tag were designed and cloned into pCDH lentiviral vectors. WT and ROBO1 KO OPM2 were injected in the BM cavity of femoral bones harvested from donor SCID mice, and implanted subcutaneously into recipient SCID mice to study ROBO1 KO in vivo.
Results
HMCL, primary MM and BM stromal cells (MM-BMSC) from patients, but not healthy donor BM-resident plasma cells and BMSC, express ROBO1 and SLIT2. Across a panel of myeloid and lymphoid cancer cell lines, high ROBO1 expression was detected only in SU-DHL-4 and BCWM1 cell lines. In newly diagnosed MM patients enlisted in the IMF170 database, high ROBO1 portended worse overall survival compared to low ROBO1 expression (P=0.018). Sh-RNA mediated ROBO1 KD was profoundly and specifically cytotoxic to HMCL, with no cytotoxicity observed in high-ROBO1 expressing SH-SY5Y and HeLa cancer cell lines. Consistent with on target effect, ROBO1 shRNA was not cytotoxic for non-ROBO1 expressing cancer cell lines. To study the functional consequences of ROBO1 loss in MM, we performed CRISPR-Cas9-mediated ROBO1 KO in HMCL. We were unable to establish biallelic ROBO1 KO H929 or U266 clones, suggesting that biallelic loss may be lethal in these HMCL, but created two clones of ROBO1 KO OPM2 cells. In vitro, ROBO1 KO and WT OPM2 cells have comparable viability and proliferation rate, even in the presence of MM-BMSC. However, ROBO1 KO OPM2 cells have profound defects in adhesion to MM-BMSC and BM endothelial cell lines (BMEC). Lentiviral transduction of OPM2 ROBO1 KO cells with FL-ROBO1 addback lentiviral construct completely rescues the BMEC adhesion defect, confirming that loss of ROBO1 is responsible for the defective adhesive phenotype. Interestingly, treatment with heparin abolishes the rescue effect of FL-ROBO1, implying that surface heparan sulfate proteoglycans (HSPG) are necessary for ROBO1-mediated MM-BMEC adhesion. Consistent with a role for ROBO1 in MM-BM niche interaction, ROBO1 KO OPM2 cells show higher failure to engraft in the bone implants compared to ROBO1 WT OPM2 (bone implants without MM engraftment: 5/15 versus 1/15, respectively, P=0.067). PET-CT scan of study mice shows ROBO1 KO tumors to be significantly smaller compared to ROBO1 WT (mean volume 1.2 cm3 vs. 2.9 cm3, P=0.003), suggesting a profound in vivo proliferation/survival defect of ROBO1 KO OPM2.
Summary and Conclusions
Our data demonstrate that ROBO1 and SLIT2 are highly expressed in HMCL and in the BM of MM patients, but not healthy controls. ROBO1 expression correlates with worse OS in newly diagnosed MM patients; and ROBO1 KD is selectively cytotoxic for HMCL, but not high-ROBO1 expressing SH-SY5Y and HeLa cells. CRISPR-Cas9-mediated ROBO1 KO significantly impairs OPM2 cell adhesion to BMEC and MM-BMSC, which is completely reversed by adding back FL-ROBO1. In vivo, ROBO1 KO OPM2 cells show impaired engraftment to bone implants and significantly decreased growth rate compared to ROBO1 WT OPM2.
Altogether, our data demonstrate that ROBO1 is a novel pro-survival protein in MM, mediating MM-BM niche interaction and suggest the therapeutic potential of targeting ROBO1/SLIT2 pathway in MM. Proteomic and RNA-sequencing studies on the downstream signaling effectors of ROBO1/SLIT2 are currently ongoing to elucidate the molecular mechanisms of pathway activation in MM.

Disclosures
Roccaro: Takeda Pharmaceutical Company Limited: Honoraria. Ghobrial:Takeda: Honoraria; Novartis: Honoraria; Celgene: Honoraria, Research Funding; Noxxon: Honoraria; Amgen: Honoraria; BMS: Honoraria, Research Funding. Anderson:Oncoprep: Equity Ownership; Acetylon: Equity Ownership; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.