The lack of effective RAS inhibition represents a major unmet medical need in the treatment of pancreatic ductal adenocarcinoma (PDAC). Here, we investigate the anticancer activity of RRSP-DTB, an engineered biologic that cleaves the Switch I of all RAS isoforms, in KRAS-mutant PDAC cell lines and patient-derived xenografts (PDX). We first demonstrate that RRSP-DTB effectively engages RAS and impacts downstream ERK signaling in multiple KRAS-mutant PDAC cell lines inhibiting cell proliferation at picomolar concentrations. We next tested RRSP-DTB in immunodeficient mice bearing KRAS-mutant PDAC PDXs. Treatment with RRSP-DTB led to ≥95% tumor regression after 29 days. Residual tumors exhibited disrupted tissue architecture, increased fibrosis and fewer proliferating cells compared with controls. Intratumoral levels of phospho-ERK were also significantly lower, indicating in vivo target engagement. Importantly, tumors that started to regrow without RRSP-DTB shrank when treatment resumed, demonstrating resistance to RRSP-DTB had not developed. Tracking persistence of the toxin activity following intraperitoneal injection showed that RRSP-DTB is active in sera from immunocompetent mice for at least 1 hour, but absent after 16 hours, justifying use of daily dosing. Overall, we report that RRSP-DTB strongly regresses hard-to-treat KRAS-mutant PDX models of pancreatic cancer, warranting further development of this pan-RAS biologic for the management of RAS-addicted tumors.

Ras-specific proteases to degrade RAS within cancer cells are under active development as an innovative strategy to treat tumorigenesis. The naturally occurring biological toxin effector called RAS/RAP1-specific endopeptidase (RRSP) is known to cleave all RAS within a cell, including HRAS, KRAS, NRAS and mutant KRAS G13D. Yet, our understanding of the mechanisms by which RRSP drives growth inhibition are unknown. Here, we demonstrate, using isogenic mouse fibroblasts expressing a single isoform of RAS or mutant KRAS, that RRSP equally inactivates all isoforms of RAS as well as the major oncogenic KRAS mutants. To investigate how RAS processing might lead to varying outcomes in cell fate within cancer cells, we tested RRSP against four colorectal cancer cell lines with a range of cell fates. While cell lines highly susceptible to RRSP (HCT116 and SW1463) undergo apoptosis, RRSP treatment of GP5d and SW620 cells induces G1 cell cycle arrest. In some cell lines, growth effects were dictated by rescued expression of the tumor suppressor protein p27 (Kip1). The ability of RRSP to irreversibly inhibit cancer cell growth highlights the antitumor potential of RRSP, and further warrants investigation as a potential anti-tumor therapeutic.

RAS is one of the most frequently mutated oncogenes in cancer with ~30% of all human tumors harboring a mutation in either HRAS, NRAS, or KRAS isoforms. Despite countless efforts for development of small molecule inhibitors for RAS, it remains an elusive target in the clinic. Here we demonstrated that the pan-RAS biological inhibitor RAS/RAP1-specific endopeptidase (RRSP) has proteolytic activity in ‘Ras-less’ mouse embryonic fibroblasts expressing human RAS isoforms (H/N/KRAS) or major oncogenic KRAS mutants (G12C, G12V, G12D, G13D, and Q61R). The cleavage of RAS inhibited phosphorylation of ERK and cell proliferation. To investigate how RAS processing affects colon cancer cells, we tested RRSP against KRAS-dependent (SW620 and GP5d) and KRAS-independent (HCT-116, SW1463, and HT-29) cell lines and found that RRSP inhibited growth. The cleavage of RAS was cytotoxic in some cell lines and induced either irreversible cell cycle arrest or uncharacterized growth inhibition in others. The G1 cell cycle arrest in some colon cancer cells was mediated through rescue of p27 (Kip1) protein expression resulting in reduced phosphorylation of retinoblastoma protein. Together, this work demonstrated that complete ablation of RAS in cells induces growth inhibition, but the mechanism of inhibition can vary in different tumor cell lines. This ability of RAS processing to halt cell proliferation by multiple strategies highlights RRSP both as a potential anti-tumor therapy and as a tool for studying RAS signaling across tumor types.

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Supplementary Figure from Proteolytic pan-RAS Cleavage Leads to Tumor Regression in Patient-derived Pancreatic Cancer Xenografts