Effect of ROCK2 signaling on resistance to KRAS inhibitors in pancreatic cancer.

756 Background: Inhibitors targeting mutant and wildtype forms of KRAS, which is a key driver of pancreatic oncogenesis in humans, have entered the clinical arena. However, cancer cells can activate bypass pathways and develop resistance to KRAS inhibitors. Thus, there is a critical need to identify resistance mechanisms and combination regimens to enhance the efficacy of KRAS inhibitors in PDAC tumors. Methods: Pancreatic cells resistant to KRAS G12D inhibitor MRTX1133 or the pan-RAS inhibitor RMC-6236 were generated by exposing the cells to increasing concentrations of the drug. Parental or MRTX1133-resistant cells were implanted subcutaneously into the flanks of immunocompetent mice and treated daily with vehicle control or MRTX1133. Tumors were collected and analyzed by immunohistochemistry (IHC) for CD8+ T-cell and F4/80+ macrophages and by flow cytometry for macrophage polarization. To determine whether KRAS-resistant cells induce macrophage polarization, bone-marrow derived macrophages were co-cultured in collagen matrices with KRAS-resistant cells. Rho-associated kinase (ROCK) activity was evaluated by western blot and by immunofluorescence for p-MLC. Mice with implanted MRTX1133-inhibitor-resistant tumors were treated with MRTX1133 in combination with the FDA-approved ROCK2 inhibitor Belumosudil and tumor volume was measured over time until study-end point. Results: Syngeneic tumors established from MRTX1133-resistant and RMC-6236-resistant PDAC cells resemble mutant KRAS inhibitor-resistant human tumor immune profile. There is a scarcity of CD8+ T cells and polarization of macrophage to the M2 phenotype. Notably, the MRTX1133-resistant and the RMC-6236-resistant PDAC cells exhibit increased Rho-associated protein kinase 2 (ROCK2) signaling, and blocking ROCK2 activity prevents macrophage polarization towards the M2 phenotype in vitro and in vivo . Significantly, Belumosudil enhanced the anti-tumor efficacy of MRTX1133 in the MRTX1133-resistant tumors in mice. Conclusions: Our findings suggest that increased ROCK2 signaling mediates resistance to KRAS inhibitors in pancreatic cancer and targeting ROCK2 represents a promising approach to overcome resistance to KRAS inhibitors.