(I-356) Effects of Pancreas Cancer Cell K-Ras G12D Inhibition on Cancer-Associated Fibroblast Heterogeneity in Hypoxic Conditions 

Introduction: Pancreatic ductal adenocarcinoma (PDAC) accounts for 90% of all pancreatic cancer diagnoses and is a leading cause of cancer-related deaths worldwide [1]. Cancer-associated fibroblasts (CAFs) are a key component of the PDAC microenvironment and are heterogeneous with regards to their origin, spatial distribution, and functional phenotypes. Among the subset populations of CAFs, inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs) have been widely studied for their tumor-promoting or tumor-suppressing functions [2]. Several studies have established that hypoxia alters the crosstalk between cancer cells and CAFs to promote an inflammatory CAF phenotype [3-4]. Recently, Kemp et al. [5] demonstrated that MRTX1133, a novel small-molecule K-Ras G12D inhibitor, promotes the myCAF phenotype in an autochthonous mouse model of PDAC. While these results suggest a relationship between K-Ras inhibition and CAF heterogeneity, how K-Ras inhibition affects CAF heterogeneity under conditions of hypoxia, a feature of the PDAC microenvironment, is not understood. Our objective is to investigate the effect of K-Ras G12D inhibition on CAF heterogeneity in cocultures of CAFs and PDAC cells under hypoxic conditions. We hypothesize that K-Ras G12D inhibition will promote a more myCAF-like phenotype in CAFs by interrupting cancer cell signaling to CAFs, rather than through a direct effect on CAFs.

Materials and Methods: Immortalized CAFs derived from a human pancreas tumor and HPAF-II human pancreas cancer cells were cultured in RPMI supplemented with 10% FBS.  CAFs expressing red fluorescent protein (RFP) and HPAF-II cells were cultured separately (monoculture) or together (coculture) at a 1:1 ratio at 21 or 1% O₂ at 37˚C in 96-well plates. Monocultures and cocultures were treated with up to 40 nM MRTX1133 or DMSO (vehicle) for 72 hr. Cells were then fixed with 4% paraformaldehyde in PBS for 20 min and permeabilized with 0.25% Triton-X 100 in PBS for 5 min. Cells were incubated with primary antibodies in a humidified chamber overnight at 4˚C. Following five washes with 0.1% Tween 20 in PBS, cells were incubated for 1 hr at 37˚C in a humidified chamber with Alexa Fluor secondary antibodies and Hoechst nuclear stain. Cells were imaged with a 10× objective on a BioTek Cytation5 to quantify nuclei, phospho-ERK (pERK), RFP, and IL-6 expression. Image analysis was performed using CellProfiler. RFP was used to distinguish CAFs from HPAF-II cells.

Results and Discussion: MRTX1133 substantially reduced HPAF-II cell proliferation but had no effect on CAF proliferation (Fig 1A and 1B), as expected. In HPAF-II cells, MRTX1133 and trametinib (a MEK inhibitor) reduced pERK levels, validating the anticipated biochemical effects of MRTX1133. Finally, in hypoxic cocultures of CAFs and HPAF-II cells, MRTX1133 antagonized the expression of the iCAF marker IL6 in CAFs.

Conclusion: Our findings confirm the specificity and efficacy of MRTX1133 in K-Ras G12D-expressing PDAC cells and further suggest that K-RAS G12D inhibition interferes with the ability of PDAC cells to promote the iCAF phenotype under hypoxic conditions.

Acknowledgments: University of Virginia NSF REU program Award #1950374, NCI U01 CA243007 (MJL)

References: [1] Sarantis P, World J Gastrointest Oncol. 2020; 12(2): 173-181 [2] Zhang T, Cell Death Dis 2022; 13, 897 [3] Mello, A.M., Oncogenesis 2022; 11, 56 [4] Schworer S, Cancer Res 2023; 83(10): 15961610  

[5] Kemp S.B., Cancer Discov 2023; 13 (2): 298–311