Targeting RAS Driven Human Cancer Cells with Antibodies to Upregulated and 1 Essential Cell-Surface Proteins

19 20 While there have been tremendous efforts to target oncogenic RAS signaling 21 from inside the cell, little effort has focused on the cell-surface. Here, we used 22 quantitative surface proteomics to reveal a signature of proteins that are 23 upregulated on cells transformed with KRASG12V, and driven by MAPK pathway 24 signaling. We next generated a toolkit of recombinant antibodies to seven of these 25 RAS induced proteins. We found that five of these proteins are broadly distributed 26 on cancer cell lines harboring RAS mutations. In parallel, a cell-surface CRISPRi 27 screen identified integrin and Wnt signaling proteins as critical to RAS transformed 28 cells. We show that antibodies targeting CDCP1, a protein common to our 29 proteomics and CRISPRi datasets, can be leveraged to deliver cytotoxic and 30 immunotherapeutic payloads to RAS transformed cancer cells and report for RAS 31 signaling status in vivo. Taken together, this work presents a technological platform 32 for attacking RAS from outside the cell. 33 34 Introduction: 35 36 RAS is a family of three ubiquitously expressed small GTPases found in all 37 animal cell types. RAS is localized to the intracellular leaflet of the cell membrane 38 where it serves as a major communication hub that relays extracellular growth 39 factor-dependent signaling to as many as a dozen different intracellular signaling 40 pathways, including the classically studied MAPK and PI3K pathways (Downward, 41 2003). Collectively, these pathways induce dramatic changes to cells including 42 transcriptional reprogramming, promotion of cell survival, suppression of 43 apoptosis, metabolic rewiring, promotion of proliferation, and increased cell 44 invasiveness (Pylayeva-Gupta et al., 2011). Many of these phenotypes are well45 known hallmarks of cancer survival (Hanahan and Weinberg, 2011). Thus, it is not 46 surprising that nearly one third of all human malignancies have been found to be 47 driven by mutational activation of one of the three RAS isoforms: KRAS, NRAS and 48 HRAS (Roberts and Der, 2007). Hence oncogenic RAS has been an important focus of 49 the cancer biology and drug discovery communities for several decades (Ledford, 5