Combination of farnesyl-transferase inhibition with KRAS G12D targeting breaks down therapeutic resistance in pancreatic cancer

Abstract

Pancreatic adenocarcinoma is one of the deadliest forms of cancer with no effective therapeutic options. A KRAS mutation can be found in up to 90% of all pancreatic tumors, making it a promising therapeutic target. The introduction of new KRAS inhibitors has been a milestone in the history of KRAS mutant tumors; however, therapeutic resistance limits their efficacy. Thus, new therapeutic options, including combination therapies, are urgently needed. Recently, we have shown that KRAS G12C inhibitors in combination with farnesyl-transferase inhibitors exert synergistic antitumor effects. Here, we provide evidence for the feasibility of this combinational approach to break down resistance in KRAS G12D mutant pancreatic cancer. Although we have shown that the 3D environment dramatically sensitizes cells to MRTX1133 treatment, the synergistic effect of this drug combination is present in both 2D and 3D in the PANC1 pancreatic adenocarcinoma model, which showed high resistance to MRTX1133 in 2D. The effects of the combination treatment show an association with the inhibition of farnesylated regulatory proteins, including HRAS and RHEB, along with the expression level of KRAS. Our study warrants further investigation for the potential applicability of KRAS G12D inhibitors in combination with farnesyl-transferase inhibitors for the treatment of KRAS mutant pancreatic adenocarcinoma.

Keywords: FTI; G12D mutant KRAS; KRAS inhibitor resistance; PDAC; combination therapy.

FIGURE 1

2D cell viability data of pancreatic adenocarcinoma cell lines. Cells were treated with various concentrations of MRTX1133 for 6 days. Control-normalized data show the resistant PANC1 cell line and the sensitive SW1990 and ASPC1 cells. Data were derived from three independent experiments and graphed using GraphPad Prism 5 (n = 3, average ± SEM). Asterisks mark significant differences (p < 0.05) measured by two-way ANOVA with Bonferroni post-test, comparing viability data of SW1990 and ASPC1 to PANC1.

FIGURE 2

Changes in cell signaling after MRTX1133 treatment. Cells were treated with 100 nM MRTX1133 for 48 h. RAS-related cell signaling was determined by Western blotting. (A) Representative bands of the proteins investigated. Activated, phosphorylated proteins are marked with the “p-” prefix. (B) Densitometric evaluation of the activation or expression of the represented proteins. Data were normalized to total protein level and expressed relative to control using GraphPad Prism 5 software (±SEM). Data are derived from three independent experiments. Asterisks mark significant differences (p < 0.05) as determined by an unpaired t-test.

FIGURE 3

Combination of farnesyl-transferase inhibition with KRAS G12D targeting in MRTX1133 resistant PANC1. 6-day combination therapy of tipifarnib and MRTX1133 was applied to 2D cultures of PANC1 cells. (A) Control-normalized viability values. Data are derived from three independent experiments and are expressed relative to control (n = 3, ±SEM) (B) Synergy map calculated from viability results using synergyfinder.org. In general, a synergy score greater than 10 is considered to have synergistic effects. Note that the most synergistic area is at low dose tipifarnib and high dose MRTX1133.

FIGURE 4

Combination of farnesyl-transferase inhibition with KRAS G12D targeting in PANC1 spheroids. A 6-day-long combination therapy of tipifarnib and MRTX1133 was applied to spheroid cultures of PANC1 cells. (A) Control-normalized viability values. Data are derived from three independent experiments and are expressed relative to control (n = 9, ±SEM) (B) Synergy map calculated from viability results using synergyfinder.org. Generally, a synergy score greater than 10 is considered to have synergistic effects. Note that the most synergistic area is at high dose tipifarnib and high dose MRTX1133.

FIGURE 5

Changes in cell signaling after MRTX1133 (M), tipifarnib (T), or combination treatment (M+T). PANC1 cells were treated with 100 nM MRTX1133 and 500 nM tipifarnib alone or in combination for 48 h. RAS-related cell signaling was determined by Western blotting. (A) Representative bands of the proteins investigated. Activated, phosphorylated proteins are marked with the “p-”prefix. (B) Densitometric evaluation of the activation of the represented proteins. Data were normalized to total protein and expressed relative to the control and graphed with GraphPad Prism 5 software (“C”, ±SEM). Data are derived from three independent experiments. Asterisks mark significant differences (p < 0.05) tested with one-way ANOVA followed by Bonferroni’s Multiple Comparison test comparing treatment groups to control samples.

FIGURE 6

Effects of MRTX1133 (M), tipifarnib (T), or combination(M+T) treatment on apoptosis, proliferation, and farnesylation. PANC1 cells were treated with 100 nM MRTX1133, 500 nM tipifarnib, or their combination for 48 h. RAS-related cell signaling was determined by Western blotting. (A) Representative bands of the proteins investigated. (B) Densitometric evaluation of the activation or expression of the represented proteins. Data were normalized to total protein and expressed relative to the control (C) and graphed with GraphPad Prism 5 software (n = 3, ±SEM). Data are derived from three independent experiments. Asterisks mark significant differences (p < 0.05) tested with one-way ANOVA followed by Bonferroni’s Multiple Comparison test comparing treatment groups to control samples.

FIGURE 7

Effects of MRTX1133 (M), tipifarnib (T), or combination (M+T) treatment on cell cycle distribution. Cells were treated with 100 nM MRTX1133, 500 nM tipifarnib, or their combination for 96 h (A) Changes in total cell number following treatment. (B) Cell cycle distribution of cells following treatment. MRTX1133 and combination therapy reduced the ratio of cells in the S-phase, although these changes are not statistically significant. (C) Morphology of cell nucleus following 96 h of treatment. Note the aberrant nuclear morphology after tipifarnib or combination treatment. Scale bar = 50 μm. Data shown in the graphs are derived from three independent experiments and are expressed as mean ± SEM. Asterisks mark significant differences (p < 0.05) tested by one-way ANOVA followed by Bonferroni’s Multiple Comparison test comparing treatment groups to control samples.