Targeting KRAS-mutant pancreatic cancer through simultaneous inhibition of KRAS, MEK, and JAK2

Abstract

The Kirsten rat sarcoma (KRAS) oncogene was considered "undruggable" until the development of sotorasib, a KRAS^G12C selective inhibitor that shows favorable effects against lung cancers. MRTX1133, a novel KRAS^G12D inhibitor, has shown promising results in basic research, although its effects against pancreatic cancer are limited when used alone. Therefore, there is an urgent need to identify effective drugs that can be used in combination with KRAS inhibitors. In this study, we found that administration of the KRAS inhibitors sotorasib or MRTX1133 upregulated STAT3 phosphorylation and reactivated ERK through a feedback reaction. The addition of the MEK inhibitor trametinib and the JAK2 inhibitor fedratinib successfully reversed this effect and resulted in significant growth inhibition in vitro and in vivo. Analyses of sotorasib- and MRTX1133-resistant cells showed that trametinib plus fedratinib reversed the resistance to sotorasib or MRTX1133. These findings suggest that the JAK2-mediated pathway and reactivation of the MAPK pathway may play key roles in resistance to KRAS inhibitors in pancreatic cancers. Accordingly, simultaneous inhibition of KRAS, MEK, and JAK2 could be an innovative therapeutic strategy against KRAS-mutant pancreatic cancer.

Keywords: KRAS; MRTX1133; fedratinib; pancreatic cancer; sotorasib; trametinib.

Fig. 1

Feedback activation of ERK and STAT3 occurs following treatment with sotorasib in KRAS ^G12C pancreatic ductal adenocarcinoma (PDAC) cell lines. (A) KRAS ^G12C PDAC cell line MIA PaCa‐2 was treated with 5 nm sotorasib for 0, 3, 24, and 48 h. The expression of proteins involved in the RAS/MAPK, JAK2/STAT3, and PI3K/AKT pathways was evaluated using western blotting analysis. A representative image is shown (n = 4). (B) Dose–response curve of sotorasib, trametinib, fedratinib, and their combinations established using Cell Counting Kit‐8 (CCK‐8). S: sotorasib; T: trametinib; F: fedratinib (n = 8). (C, D) MIA PaCa‐2 was treated with sotorasib (5 nm), trametinib (1 nm), and/or fedratinib (250 nm). (C) Proliferation was assessed using CCK‐8 (n = 8). Statistical significance was evaluated using a one‐way ANOVA followed by Tukey's test. **P < 0.01, *P < 0.05. Error bars indicate standard deviations. (D) Cell lysates for western blotting analysis were collected 48 h after treatment. A representative image is shown (n = 3).

Fig. 2

Combination of MRTX1133, an MEK inhibitor, and a JAK2 inhibitor is effective in KRAS ^G12D mutant PDAC cell lines. (A) The KRAS ^G12D cell lines SUIT‐2, KP‐4, PANC‐1, and PK‐59 were treated with 5 nm MRTX1133 for 0, 3, 24, and 48 h. The expression of proteins involved in the RAS/MAPK, JAK2/STAT3, and PI3K/AKT pathways was evaluated using western blotting analysis. Representative image is shown (n = 3). (B) Dose–response curve of MRTX1133, trametinib, fedratinib, and their combinations established using Cell Counting Kit‐8 (CCK‐8). M: MRTX1133; T: trametinib; F: fedratinib. (C, D) SUIT‐2 was treated with MRTX1133 (5 nm), trametinib (1 nm), and/or fedratinib (500 nm) (n = 11). KP‐4 was treated with MRTX1133 (4 nm), trametinib (4 nm), and/or fedratinib (2 μm) (n = 5). (C) Proliferation of SUIT‐2 (n = 11) and KP‐4 (n = 5) was assessed using CCK‐8. Statistical significance was evaluated using a one‐way ANOVA followed by Tukey's test. **P < 0.01, *P < 0.05. Error bars indicate standard deviations. (D) Cell lysates were collected 48 h after treatment for western blotting analysis. A representative image is shown (n = 3).

Fig. 3

Phosphorylation of ERK and AKT is upregulated in sotorasib‐ and MRTX1133‐resistant cells. (A) Dose–response curve of sotorasib‐ and MRTX1133‐resistant cells and their parental cells established using Cell Counting Kit‐8 (CCK‐8) (n = 3). Error bars indicate standard deviations. (B, C) Whole cell lysates were collected for western blotting analysis of the expression of proteins involved in the RAS/MAPK, JAK2/STAT3, and PI3K/AKT pathways. Cells were incubated in sotorasib‐ or MRTX1133‐free media for seven days before the collection of cell lysates. Relative intensity of phosphorylated proteins to that of total proteins of MIA PaCa‐2 (n = 5), SUIT‐2 (n = 6), and KP‐4 (n = 4) were analyzed. Statistical significance was evaluated using the Student's T‐test. **P < 0.01, *P < 0.05. Error bars indicate standard deviations. (D) Dose–response curve of the combination of trametinib and fedratinib in sotorasib‐ or MRTX1133‐resistant cells and their parental cells established using CCK‐8 (n = 4). Error bars indicate standard deviations. (E) Proliferation was assessed using CCK‐8 (n = 6). Statistical significance was evaluated using the Student's T‐test. **P < 0.01. Error bars indicate standard deviations. (F) Western blotting analysis of cell lysates collected 48 h after drug administration. A representative image is shown (n = 3).

Fig. 4

The three‐drug combination has a significantly high tumor inhibition effect in vivo. (A) MRTX1133 (0.5 mg·kg⁻¹), trametinib (0.1 mg·kg⁻¹), and/or fedratinib (25 mg·kg⁻¹) were orally administered to BALB/cAJclFoxn1 ^nu mice daily (n = 6). Tumor width (W), length (L), and height (H) were measured twice a week. The formula W × L × H × π/6 was used to estimate tumor volume. Relative volume compared with that on day 1 is shown. Error bars indicate standard deviations. Mice were euthanized on day 22. A resected tumor is shown. M: MRTX1133; F: fedratinib; T: trametinib. (B) Relative tumor volume at day 22. Statistical evaluation was performed using one‐way ANOVA followed by Tukey's test. **P < 0.01, *P < 0.05. Error bars indicate standard deviations. (C) The resected tumor was stained for phosphorylated ERK, phosphorylated STAT3, Ki‐67, and TUNEL; a representative image is shown (n = 3). M: MRTX1133; F: fedratinib; T: trametinib.

Fig. 5

The three‐drug combination also has a strong tumor inhibition effect in MRTX1133‐resistant SUIT‐2 cells in vivo. (A) MRTX1133 (2.0 mg·kg⁻¹) and/or trametinib (0.1 mg·kg⁻¹) plus fedratinib (25 mg·kg⁻¹) was orally administered to BALB/cAJclFoxn1 ^nu mice daily (n = 4). Tumor width (W), length (L), and height (H) were measured twice a week. The formula W × L × H × π/6 was used to estimate tumor volume. Relative volume compared with that on day 1 is shown. Error bars indicate standard deviations. Mice were euthanized on day 22. A resected tumor is shown. M: MRTX1133; F: fedratinib; T: trametinib. (B) Relative tumor volume at day 22. Statistical significance was evaluated using the Student's T‐test. **P < 0.01. Error bars indicate standard deviations.