KRAS and HRAS mutations confer resistance to MET targeting in preclinical models of MET-expressing tumor cells

Abstract

The MET receptor tyrosine kinase is often deregulated in human cancers and several MET inhibitors are evaluated in clinical trials. Similarly to EGFR, MET signals through the RAS-RAF-ERK/MAPK pathway which plays key roles in cell proliferation and survival. Mutations of genes encoding for RAS proteins, particularly in KRAS, are commonly found in various tumors and are associated with constitutive activation of the MAPK pathway. It was shown for EGFR, that KRAS mutations render upstream EGFR inhibition ineffective in EGFR-positive colorectal cancers. Currently, there are no clinical studies evaluating MET inhibition impairment due to RAS mutations. To test the impact of RAS mutations on MET targeting, we generated tumor cells responsive to the MET inhibitor EMD1214063 that express KRAS G12V, G12D, G13D and HRAS G12V variants. We demonstrate that these MAPK-activating RAS mutations differentially interfere with MET-mediated biological effects of MET inhibition. We report increased residual ERK1/2 phosphorylation indicating that the downstream pathway remains active in presence of MET inhibition. Consequently, RAS variants counteracted MET inhibition-induced morphological changes as well as anti-proliferative and anchorage-independent growth effects. The effect of RAS mutants was reversed when MET inhibition was combined with MEK inhibitors AZD6244 and UO126. In an in vivo mouse xenograft model, MET-driven tumors harboring mutated RAS displayed resistance to MET inhibition. Taken together, our results demonstrate for the first time in details the role of KRAS and HRAS mutations in resistance to MET inhibition and suggest targeting both MET and MEK as an effective strategy when both oncogenic drivers are expressed.

Keywords: MET; Mutations; RAS; Resistance; Small molecule inhibitors.

Figure 1

A. KRAS and HRAS mutant variants levels following stable transfection of NIH3T3 MET M1268T cells (left) and H1993 cells (right). Clones were chosen following puromycin selection. Parental cells are shown for comparison. A RAS binding domain (RBD) pull‐down was used to determine active RAS levels. RAS protein was detected using a non‐isoform specific p21/RAS (pan‐RAS) antibody. β Actin was used as a loading control. B. MET and ERK1/2 phosphorylation levels in NIH3T3 MET M1268T cells (left) and H1993 cells (right) stably transfected with mutated HRAS or KRAS proteins. The cells were treated with either vehicle or 50 nM EMD1214063 for 16 h. β Actin was used as a loading control.

Figure 2

A. XTT cell proliferation assays for parental NIH3T3 MET M1268T as well as cells stably expressing KRAS or HRAS mutants. Cells were treated with vehicle (untreated) or exposed to the MET inhibitor EMD1214063 (25 nM or 50 nM; E1 and E2, respectively), the MEK1/2 inhibitor AZD6244 (0.5 μM or 1 μM; A1 and A2, respectively), or to a combination of both inhibitors (E1/A1, E1/A2, E2/A1, E2/A2) for 72 h. B. XTT proliferation assay comparing the impact of EMD1214063 and the EMD1214063/AZD6244 combination on H1993 and H1993 KRAS G13D cells. Concentrations and protocols were the same as in A. C. Proliferation of parental NIH3T3 MET M1268T and NIH3T3 MET M1268T cells stably expressing KRAS G12D or HRAS G12V variants. Cells were treated with vehicle (untreated) or were exposed to 50 nM MET inhibitor EMD1214063, the MEK1/2 inhibitor UO126 (20 μM or 40 μM; U1 and U2, respectively), or to the combination of both for 72 h. The data represent mean ± standard deviation of three experiments. Statistical significance of the group means was measured by a one‐way ANOVA followed by Bonferroni's Multiple Comparison Test post‐hoc analysis. Overall P‐value is shown above the graph. The star (*) indicates statistical significance compared with untreated samples. The number of * indicates P‐values as described in the Materials and methods.

Figure 3

Impact of MET (EMD1214063) and MEK1/2 (AZD6244) inhibitors and their combination on morphology of parental and RAS‐mutated NIH3T3 MET M1268T cells. T0 – time 0 h (the time point immediately prior to the start of treatment), T24 – time 24 h (24 h following the start of the treatment). Representative pictures are shown; on the right side, magnified images of red squares are provided.

Figure 4

Impact of MET (EMD1214063) and MEK1/2 (AZD6244) inhibitors and their combination on MET‐driven cellular motility of parental and RAS‐mutated NIH3T3 MET M1268T cells using a wound healing assay. T0 – time 0 h (the time point immediately prior to the start of treatment), T24 – time 24 h (24 h following the start of the treatment). Representative pictures (top) and quantification of invaded area of 5 experiments (bottom) are shown.

Figure 5

Soft agarose colony‐formation capacity of parental and RAS‐mutated NIH3T3 MET M1268T and H1993 cell lines upon MET (EMD1214063) and/or MEK1/2 (AZD6244) inhibition. A. Representative pictures of colonies formed by parental and HRAS G12V NIH3T3 MET M1268T control cells (top) and cells treated with 50 nM MET inhibitor EMD1214063 (bottom). B. Impact of MET and MEK1/2 inhibitors on relative colony count in parental and RAS‐mutated H1993 cells. C. Impact of MET and MEK1/2 inhibitors on relative colony count in parental and RAS‐mutated NIH3T3 MET M1268T cells. The data represent mean ± standard deviation of three experiments. Statistical significance of the group means was measured by one‐way ANOVA followed by Bonferroni's Multiple Comparison Test post‐hoc analysis. Overall P‐value is shown above the graph. Star (*) indicates statistical significance compared with untreated. The number of stars (*) indicates P‐values as described in the Materials and Methods.

Figure 6

Impact of EMD1214063 on tumor growth in parental versus KRAS G13D NIH3T3 MET M1268T cells in an in vivo mouse model. A. Growth of parental and RAS‐mutated NIH3T3 MET M1268T‐derived tumors monitored daily by caliper measurement. EMD1214063 treatment was initiated on day 10 and continued daily. The data represent mean tumor volume ± SEM with 6 animals per group. B. Relative tumor sizes at day 15 (5 days after MET inhibitor treatment initiation). Comparison with vehicle‐treated controls was done using Student's t‐test.