Combination of 5-FU plus KRAS G12D inhibitor MRTX1133 against human colorectal and pancreatic cancer cells and the affects on inhibition of pERK and immune-stimulatory cytokine patterns in in KRAS G12D and KRAS G12V tumor cells.
Authors: Tajiknia Vida , El-Deiry Wafik S. , Schwermann Maximilian , Huntington Kelsey , Zhou Lanlan , Srinivasan Praveen 
Published: June 2023

e16301

Background: Colorectal cancer (CRC) & pancreatic cancer are among deadliest cancers with great disease burden. The most commonly mutated oncogenic alteration in human cancers is KRAS, which is prevalent in pancreatic, colorectal malignancies. KRAS is considered an especially difficult, if not "undruggable" target. The KRAS G12D is present in more than 40% of PDAC & 34.2% of CRC cases. KRAS mutation subtypes are associated with different downstream pathways and varied clinical outcomes in cancer. MRTX1133 has been identified as a noncovalent, potent, and selective inhibitor of KRASG12D which has been shown to suppress KRASG12D signaling in cells and in vivo. We hypothesized that combination of MRTX1133 with 5-FU will be more effective at slowing or stopping tumor growth, and prolonging overall survival time for patients while allowing lower dose 5-FU with less side effects. Methods: We investigated synergistic effects on pERK inhibition and changes in immune tumor killing with 5-FU plus KRAS G12D inhibitor MRTX1133 in pancreatic and colon cancer cell lines. Human CRC (N=6) and pancreatic (N=4) cancer cell lines with different KRAS mutations were used in experiments evaluating drug effects on pERK at 6 and 24 hours. We evaluated changes at 48 hours in cytokine profiles in treated cells using a custom panel of 62 cytokines, chemokines, and growth factors associated with tumor growth, immune stimulation or immune suppression. Results: Colorectal and pancreatic cancer cell lines had IC50 sensitivities ranging from 7 to 12 microM 5-FU, and >100 nM to >5 microM for MRTX1133 (G12D N=4: LS513, 120 nM; HPAF-II, 1.8 microM; SNUC2B, 5.7 microM; PANC-1, 2.8 microM). For non-G12D, the range of IC50 for MRTX1133 was in the range of 2 to 9 microM for CRC lines with G12V, G13D, or wild- type KRAS (N=7). Synergies between 5-FU and MRTX1133 were observed regardless of presence of KRAS G12D mutation with combination indices of <0.5 indicating strong synergy. pERK was suppressed by MRTX1133 treatment of tumor cells regardless of KRAS G12D mutation. IL8/CXCL8, MICA, Angiopoietin 2, VEGF and TNF-alpha were reduced while IL-18/IL-1F4 increased. Conclusions: Our studies reveal strong synergy between MRTX1133 & 5-FU in human pancreatic & colon cancer models at much lower than IC50 dosage which is important for avoiding side effects. This is the first description that effect of KRAS G12D inhibitor MRTX1133 is active on KRAS G12V. The surprising synergy in KRAS G12V samples with combination therapy and the important synergistic change in cytokine patterns suggests potential strong immune stimulatory anti-cancer effects of MRTX1133 & 5FU against mCRC and pancreatic cancer cells regardless of KRAS G12D mutation which should be considered when including patients with respective mutations in clinical trials.