Extensive preclinical validation of combined RMC-4550 and LY3214996 supports clinical investigation for KRAS mutant pancreatic cancer

Abstract

Over 90% of pancreatic cancers present mutations in KRAS, one of the most common oncogenic drivers overall. Currently, most KRAS mutant isoforms cannot be targeted directly. Moreover, targeting single RAS downstream effectors induces adaptive resistance mechanisms. We report here on the combined inhibition of SHP2, upstream of KRAS, using the allosteric inhibitor RMC-4550 and of ERK, downstream of KRAS, using LY3214996. This combination shows synergistic anti-cancer activity in vitro, superior disruption of the MAPK pathway, and increased apoptosis induction compared with single-agent treatments. In vivo, we demonstrate good tolerability and efficacy of the combination, with significant tumor regression in multiple pancreatic ductal adenocarcinoma (PDAC) mouse models. Finally, we show evidence that 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) can be used to assess early drug responses in animal models. Based on these results, we will investigate this drug combination in the SHP2 and ERK inhibition in pancreatic cancer (SHERPA; ClinicalTrials.gov: NCT04916236) clinical trial, enrolling patients with KRAS-mutant PDAC.

Keywords: ERK; Kras; MAPK; PDAC; SHP2; combination therapy; inhibitors; pancreatic cancer; preclinical models; targeted therapies.

Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.

Figures

Graphical abstract

Figure 1

Evaluation of the combined effects of RMC-4550 (SHP2i) and LY3214996 (ERKi) administration in murine and human KRAS-mutant pancreatic cancer cell lines (A) Western blot analysis with murine cancer cell line KCP_K2101 derived from KCP mouse model (KRASG12D) of spontaneous tumor formation and in human cancer cell lines: MiaPaCa-2 (KRASG12C) and Panc10.05 (KRASG12D). Cells were treated as depicted and collected for lysis at the indicated time points. Protein extracts were probed with specific antibodies against total RSK-1, phosphorylated RSK-1 (pRSK-1), and alpha-tubulin (as loading control). Numerical values indicate the pRSK-1/RSK-1 ratio quantified by densitometry. The blots are representative of at least three independent experiments. RSK-1, ribosomal S6 kinase 1. (B) Synergistic effects of SHP2i and ERKi administration were evaluated by colony-formation assay in the KRAS-mutant cell lines used in (A). SHP2i and ERKi were combined at the indicated concentrations. Representative crystal violet staining of cells is shown (top panel). Box matrices below the plate scans depict quantification of growth inhibition in relation to control wells (middle panel). Bottom panel: calculation of the combination index (CI) scores from the growth inhibition values (shown above) via CompuSyn software demonstrating strong synergism between SHP2i and ERKi across a wide range of combinatorial concentrations. CI <0.75 (shades of green) indicates synergism, CI = 0.75–1.25 (shades of blue) indicates additive effects, and CI >1.25 (shades of red) indicates antagonism. Experiments were repeated independently at least three times each, with similar results. (C) Apoptosis was analyzed in cell lines treated with either DMSO, SHP2i alone, ERKi alone, or a combination of SHP2i and ERKi at the indicated concentration in real time (top panel). GFP signal coupled to cleaved caspase 3 was quantified as readout. Bar plots for selected time points (48 h for KCP_K2101 and 76 h for MiaPaCa-2 and Panc10.05) show the fraction of GFP-positive cells (AU) (top panel) and the fold change GFP signal (bottom panel). AU, arbitrary units; GFP, green fluorescent protein. Experiments were repeated independently at least three times each. Results represent mean ± SD. ∗p 

Figure 2

MTD study design (A) Treatment schedule. Non-tumor-bearing wild-type and NOD scid gamma (NSG) mice were treated with the combination of RMC-4550 (SHP2i) and LY3214996 (ERKi) once per day via oral gavage for 14 consecutive days. (B) Graphical representation of dose combinations. SHP2i and ERKi were combined in different concentrations to make up 9 combined doses. (C) An illustration of the modified “3 + 3” study design. Each box represents a cohort comprising the indicated number of mice treated at a given dose level. DLT, dose-limiting toxicity; MTD, maximum tolerated dose. (D) Individual body weight-time profile of the treatment groups in male wild-type (WT) mice: d5 (n = 3), d8 (n = 3), and d9 (n = 6). (E) Individual body weight-time profile of the treatment groups in male NSG mice: d5 (n = 3), d8 (n = 3), and d9 (n = 3).

Figure 3

In vivo assessment of treatment response in a xenograft and in an orthotopic PDAC model (A) Schematic representation of the treatment schedule applied in a xenograft model of subcutaneously transplanted MiaPaCa-2 cell line and in a model of orthotopically transplanted KCPmut tumors. Cohort A: continuous treatment with RMC-4550 (SHP2i) alone daily (n = 15); cohort B: continuous treatment with LY3214996 (ERKi) alone daily (n = 15); and cohort C: continuous treatment with the combination of SHP2i and ERKi daily (n = 16). Control mice were continuously treated with vehicle (n = 15). (B) Evaluation of ERKi and SHP2i monotherapy treatments and combined administration of SHP2i and ERKi. For all the xenograft experiments, 5 × 106 MiaPaCa-2 cells were subcutaneously injected into the right flank of NSG mice. When tumors reached 200–250 mm3, mice were randomly assigned into cohorts and treated by oral gavage with inhibitors or vehicle according to treatment schedule for 21 days, after which tumors were resected. The y axis shows tumor volume change in percentage from baseline. Each bar represents the difference in pancreatic volume in an individual animal. According to the RECIST criteria, black indicates progressive disease, dark gray indicates stable disease, and light gray indicates partial response. Significance was determined by one-way ANOVA with Bonferroni’s multiple comparison test. (C and D) In vivo assessment of treatment response of orthotopically implanted tumors. ∼40 mm3 tumor pieces (KCPmut) were orthotopically implanted into the pancreata of 8-week-old male and female C57BL/6 mice. After 2 weeks, mice were either sacrificed as baseline (n = 12) or randomly assigned into cohorts and treated with inhibitors or vehicle according to the treatment schedule (A). (C) Tumor weight (mean ± SD) was determined after 14 days of therapy as indicated: baseline (n = 12), vehicle (n = 17), cohort A (n = 7), cohort B (n = 8), cohort C (n = 12). ∗∗∗∗p mut orthotopic mouse models. See also Figures S2 and S6.

Figure 4

In vivo assessment of optimal treatment regimen in a xenograft model (A) Schematic representation of the treatment schedule applied in MiaPaCa-2 xenograft model. Cohort A: continuous treatment with SHP2i alone daily; cohort B: continuous treatment with ERKi alone daily; cohort C: continuous treatment with the combination of SHP2i and ERKi daily; cohort D: intermittent treatment with the combination of SHP2i and ERKi 5 days on/2 days off; cohort E: semi-continuous treatment schedule with daily dosing of SHP2i and intermittent dosing with ERKi 5 days on/2 days off; cohort F: continuous treatment with SHP2i and on alternate days with ERKi; cohort G: intermittent dosing with SHP2i alone 5 days on/2 days off; cohort H: intermittent dosing with ERKi alone 5 days on/2 days off; and cohort I: treatment with ERKi alone on alternate days. Control mice were continuously treated with vehicle. For all the xenograft experiments, 5 × 106 cells were subcutaneously injected into the right flank of NSG mice. When tumors reached 200–250 mm3, mice were randomly assigned into cohorts and treated by oral gavage with inhibitors or vehicle according to treatment schedule. (B) Treatment response was assessed through tumor volume change using caliper measurements 3 times/week in MiaPaCa-2 (KRASG12C) xenograft model. Results represent mean ± SD. (C) Tumor volume change at time point day 21(n = 15 for vehicle cohort, n = 16 for all other cohorts). The y axis shows tumor volume change in percentage from baseline. Each bar represents the difference in tumor volume in an individual animal. According to the RECIST criteria, black indicates progressive disease, dark gray indicates stable disease, and light gray indicates partial response. Vehicle and cohort C data from Figure 3 B are reported again for comparison. Significance was determined by one-way ANOVA with Bonferroni’s multiple comparison test. See also Figure S2.

Figure 5

In vivo assessment of optimal treatment regimen in an endogenous murine PDAC model (A) Schematic representation of the treatment schedule applied in the endogenous (KPC) murine model of spontaneous tumor formation as well as the magnetic resonance imaging (MRI) time points applied. Cohort C: continuous treatment with the combination of SHP2i and ERKi daily; cohort D: intermittent treatment with the combination of SHP2i and ERKi 5 days on/2 days off; and cohort E: semi-continuous treatment schedule with daily dosing of SHP2i and intermittent dosing with ERKi 5 days on/2 days off. Control mice were treated with vehicle for 14 consecutive days. All treated mice were sacrificed on day 15, and tumors were resected for histological analysis. (B) Representative MRI scan slices depicting PDAC tumor sections of KCP mice treated with vehicle (n = 5), cohort C (n = 7), cohort D (n = 7), or cohort E (n = 6) at the indicated time points (days) following the start of therapy (pre), with similar results among the groups. Volumetric measurements indicate a decrease in pancreatic volume in mice treated with the combination of SHP2i and ERKi for 2 weeks compared with vehicle-treated mice. The y axis shows pancreatic volume change in percentage quantified by measurements of MRI scans. Each bar represents the difference in pancreatic volume in an individual animal from days 0 to 15. According to the RECIST criteria, black indicates progressive disease, dark gray indicates stable disease, and light gray indicates partial response. Significance was determined by one-way ANOVA with Bonferroni’s multiple comparison test. Volume-tracking curves for individual mice over the whole course of therapy are available in Figures S3B–S3E. (C) Macroscopic images of pancreas and spleen (top row). Representative H&E-stained sections of pancreata from mice, treated as indicated. Scale bars represent 1,000 (middle) and 200 μm (bottom). Mice numbers are indicated below. (D) Relative pancreatic weight was significantly lower in all groups treated with the combination of SHP2i and ERKi: cohort C (n = 7), cohort D (n = 6), and cohort E (n = 7) compared with vehicle-treated control mice (n = 9). Results represent mean ± SD. ∗p

Figure 6

Evaluation of treatment response by the combined administration of RMC-4550 (SHP2i) and LY3214996 (ERKi) in patient-derived xenograft (PDX) models (A) Treatment schedule. Mice were treated with the combination of RMC-4550 (SHP2i) and LY3214996 (ERKi) once per day via oral gavage for 14 consecutive days (cohort C) or 5 days on/2 days off (cohort D) or SHP2i continuously and ERKi5 days on/2 days off (cohort E). For PDX354 model, tumor pieces of 50 mm3 were subcutaneously implanted into both flanks of NSG mice (n = 7 mice per cohort). When tumors reached 200–250 mm3 (approximately 6–8 weeks after subcutaneous transplantation), mice were randomly assigned into cohorts and treated by oral gavage with inhibitors or vehicle according to treatment schedule for the indicated time, after which tumors were resected. (B and C) Treatment response was assessed through tumor volume changes using daily caliper measurements (B) and tumor weight at endpoint (C) in PDX354 model. Results represent mean ± SD. ∗∗∗∗p

Figure 7

Early non-invasive assessment of the treatment response in a subcutaneous tumor mouse model (A) Schematic representation of the treatment schedule applied in the subcutaneous tumor mouse model as well as the [18F]-FDG-PET imaging time points applied. 2.5 × 106–3 × 106 cells were injected subcutaneously into the left and right flank of 10- to 15-week-old non-tumor-bearing littermates. Two to three weeks after subcutaneous injection, mice were randomly assigned into cohorts and treated by oral gavage with inhibitors or vehicle for 7 consecutive days. [18F]-FDG-PET scans were obtained at baseline before commencement of therapy (day 0) and at days 3 and 7 during treatment. (B) Representative [18F]-FDG-PET images of tumor-bearing mice treated with vehicle or undergoing treatment with the combination of RMC-4550 (SHP2i) and LY3214996 (ERKi) once per day for 7 consecutive days (cohort C). Subcutaneous tumor areas are shown in dashed circles, and SUV of FDG uptake is indicated by color. White color indicates highest uptake, red color high uptake, yellow and green intermediate, and blue low uptake. [18F]-FDG-PET, 18-fluordesoxyglucose positron emission tomography; PET, positron emission tomography; SUV, standardized uptake value; FDG, fluorodeoxyglucose. (C) Upper panel: relative total lesion glycolysis (TLG) on days 0, 3, and 7 in vehicle versus cohort C (n = 8–9 animals/group). Lower panel: relative tumor volume on days 0, 3, and 7 in vehicle versus cohort C. Results represent mean ± SD. ∗∗∗p