Sensitivity of Oncogenic KRAS-Expressing Cells to CDK9 Inhibition

Abstract

Oncogenic forms of KRAS proteins are known to be drivers of pancreatic, colorectal, and lung cancers. The goal of this study is to identify chemical leads that inhibit oncogenic KRAS signaling. We first developed an isogenic panel of mouse embryonic fibroblast (MEF) cell lines that carry wild-type RAS, oncogenic KRAS, and oncogenic BRAF. We validated these cell lines by screening against a tool compound library of 1402 annotated inhibitors in an adenosine triphosphate (ATP)-based cell viability assay. Subsequently, this MEF panel was used to conduct a high-throughput phenotypic screen in a cell viability assay with a proprietary compound library. All 126 compounds that exhibited a selective activity against mutant KRAS were selected and prioritized based on their activities in secondary assays. Finally, five chemical clusters were chosen. They had specific activity against SW620 and LS513 over Colo320 colorectal cancer cell lines. In addition, they had no effects on BRAF^V600E, MEK1, extracellular signal-regulated kinase 2 (ERK2), phosphoinositide 3-kinase alpha (PI3Kα), AKT1, or mammalian target of rapamycin (mTOR) as tested in in vitro enzymatic activity assays. Biophysical assays demonstrated that these compounds did not bind directly to KRAS. We further identified the mechanism of action and showed that three of them have CDK9 inhibitory activity. In conclusion, we have developed and validated an isogenic MEF panel that was used successfully to identify RAS oncogenic or wild-type allele-specific vulnerabilities. Furthermore, we identified sensitivity of oncogenic KRAS-expressing cells to CDK9 inhibitors, which warrants future studies of treating KRAS-driven cancers with CDK9 inhibitors.

Keywords: CDK9; KRAS; isogenic cell panel; phenotypic high-throughput screen; synthetic lethality.

Figure 1.

Validation of the isogenic mouse embryonic fibroblast (MEF) panel with a tool compound screen: (A) Sensitivities of 1402 inhibitors were compared between the KRAS-4B-expressing MEF and the HRAS-expressing MEF. (B) Sensitivities of the receptor tyrosine kinase (RTK) inhibitors within the tool compound library were compared among the isogenic MEF panel cell lines. (C) Farnesyl-transferase inhibitors (Lonafarnib, LB42708, and Tipifarnib) dose–response curves for the cell proliferation assay were constructed for the isogenic MEF panel cell lines. (D) AZD217 and (E) vemurafenib and dabrafenib dose–response curves for the cell proliferation assay were constructed for the isogenic MEF panel cell lines.

Figure 2.

Phase 1: High-throughput screening of the proprietary compound library with the isogenic mouse embryonic fibroblast (MEF) panel. The isogenic MEF panel was used to conduct high-throughput screening with Sanofi’s compound library (923,000 compounds in the primary screen and 8376 compounds in the backscreen). In Phase 1, 696 active compounds were identified. 19 clusters and 8 singletons were discarded based on the biological profile and drug likeness. 126 compounds (28 clusters, 10 singletons, and 1 natural product) were ultimately selected for secondary assays for Phase 2.

Figure 3.

Phase 2: Characterization of the specific actives and mechanism-of-action studies. Specific actives (126 compounds) were further tested in a series of cell-based, biochemical, and biophysical assays to identify potential mechanisms of action. Cell viability assays using KRAS-dependent and -independent human colorectal cancer cell lines were performed. Cell-based assays [pERK, pAKT, and pEGFR homogeneous time-resolved fluorescence (HTRF)] as well as biochemical assays for BRAF^V600E, MEK1, ERK2, phosphoinositide 3-kinase alpha (PI3Kα), AKT1, and mammalian target of rapamycin (mTOR) activities were performed to assess RAS-related pathway activities. Biophysical assays were also performed to assess direct compound KRAS^WT and KRAS^G12D binding.

Figure 4.

Dose–response curves of selected compounds in the cell viability assay: Mouse embryonic fibroblasts (MEFs) (72 h treatments) and colorectal cancer cell lines (96 h treatments) were treated with the indicated compounds and examined with the CellTiter-Glo assay. Data were normalized to DMSO-treated cells. Each experiment was performed at least twice, and the error bars represent the standard deviations.

Figure 5.

Three candidate chemical clusters demonstrated CDK9 inhibitory activity: KRAS^G12D-expressing mouse embryonic fibroblasts (MEFs) were treated with the indicated compounds for 6 h. Multiple doses (0.1 μM, 1 μM, and 10 μM) and multiple compounds from the same series were tested. (A) The CDK9 activity was assessed by measuring the phosphorylation level of Ser2-RNA polymerase II with Western blots. SNS032 is a CDK9 inhibitor and is used as the positive control. (B) The ratios of phospo-Ser2 to total RNA polymerase II were plotted.