Intrinsic and acquired resistance limit the window of effectiveness for oncogene-targeted cancer therapies. Preclinical studies that identify synergistic combinations enhance therapeutic efficacy to target intrinsic resistance, however, methods to study acquired resistance in cell culture are lacking. Here, we describe a novel in situ resistance assay (ISRA), performed in a 96-well culture format, that models acquired resistance to RTK/RAS pathway targeted therapies. Using osimertinib resistance in EGFR-mutated lung adenocarcinoma (LUAD) as a model system, we show acquired resistance can be reliably modeled across cell lines using objectively defined osimertinib doses. Similar to patient populations, isolated osimertinib-resistant populations showed resistance via enhanced activation of multiple parallel RTKs so that individual RTK inhibitors did not re-sensitize cells to osimertinib. In contrast, inhibition of proximal RTK signaling using the SHP2 inhibitor RMC-4550 both re-sensitized resistant populations to osimertinib and prevented the development of osimertinib resistance as a primary therapy. Similar, objectively defined drug doses were used to model resistance to additional RTK/RAS pathway targeted therapies including the KRASG12C inhibitors adagrasib and sotorasib, the MEK inhibitor trametinib, and the farnesyl transferase inhibitor tipifarnib. These studies highlight the tractability of in situ resistance assays to model acquired resistance to targeted therapies and provide a framework for assessing the extent to which synergistic drug combinations can target acquired drug resistance.
KRAS is the most commonly mutated oncogene; targeted therapies have been developed against mediators of key downstream signaling pathways, predominantly components of the RAF/MEK/ERK kinase cascade. Unfortunately, single-agent efficacy of these agents is limited both by intrinsic and acquired resistance. Survival of drug-tolerant persister cells within the heterogeneous tumor population and/or acquired mutations that reactivate RTK/RAS signaling can lead to outgrowth of tumor initiating cells (TICs) and drive therapeutic resistance. Here, we show that targeting the key RTK/RAS pathway signaling intermediates SOS1 or KSR1 both enhances the efficacy of and prevents resistance to the MEK inhibitor trametinib in KRAS-mutated lung and colorectal adenocarcinoma cell lines depending on the specific mutational landscape. The SOS1 inhibitor BI-3406 enhanced the efficacy of trametinib and prevented trametinib resistance by targeting TICs, but only in KRASG12- or KRASG13-mutated LUAD and COAD cell lines that lacked PIK3CA co-mutations. Cell lines with KRASQ61 and/or PIK3CA mutations were insensitive to combination therapy with trametinib and BI-3406. In contrast, deletion of the RAF/MEK/ERK scaffold protein KSR1 prevented treatment-induced TIC upregulation and restored trametinib sensitivity across KRAS mutant cell lines in both PIK3CA-mutated and PIK3CA wildtype cancers. Our findings demonstrate that vertical targeting of RTK/RAS signaling is an effective strategy to target KRAS-mutated cancers, but the specific combination is dependent both on the specific KRAS mutant and underlying co-mutations. Thus, selection of optimal therapeutic combinations in KRAS-mutated cancers will require a detailed understanding of functional dependencies imposed by allele-specific KRAS mutations.
Son of Sevenless 1 and 2 (SOS1 and SOS2) are RAS guanine nucleotide exchange factors (RasGEFs) that mediate physiologic and pathologic RTK-dependent RAS activation. Here we show that SOS2 modulates the threshold of epidermal growth factor receptor (EGFR) signaling to regulate the efficacy of and resistance to the EGFR-TKI osimertinib in lung adenocarcinoma (LUAD). SOS2 deletion sensitized EGFR-mutated cells to perturbations in EGFR signaling caused by reduced serum and/or osimertinib treatment to inhibit PI3K/AKT pathway activation, oncogenic transformation, and survival. Bypass RTK reactivation of PI3K/AKT signaling represents a common resistance mechanism to EGFR-TKIs; SOS2 KO reduced PI3K/AKT reactivation to limit osimertinib resistance. In a forced HGF/MET-driven bypass model, SOS2 KO inhibited HGF-stimulated PI3K signaling to block HGF-driven osimertinib resistance. Using a long term in situ resistance model, a majority of osimertinib resistant cultures exhibited a hybrid epithelial/mesenchymal phenotype associated with reactivated RTK/AKT signaling. In contrast, RTK/AKT-dependent osimertinib resistance was markedly reduced by SOS2 deletion; the few SOS2 KO cultures that became osimertinib resistant primarily underwent non-RTK dependent EMT. Since bypass RTK reactivation and/or tertiary EGFR mutations represent the majority of osimertinib-resistant cancers, these data suggest that targeting
Lung cancer is the leading cause of cancer-related death worldwide; adenocarcinoma is the most common subtype of lung cancer. Oncogenic driver mutations in the RTK/RAS pathway occur in 75-90% of LUAD. Oncogene-targeted therapies substantially improve outcomes in LUAD; however, recalcitrant tumors invariably emerge necessitating novel therapeutic approaches that either delay therapeutic resistance or treat resistant cancers to enhance patient outcomes. Resistance to oncogene-targeted therapies in LUAD is commonly due to RTK pathway reactivation. Resistance to osimertinib in EGFR-mutated tumors, KRASG12C inhibitors (sotorasib, adagrasib) in KRASG12C-mutated tumors, or MEK inhibitors (trametinib) in KRAS (non-G12C)-mutated tumors often develops via activation of multiple RTKs. The multiplicity of RTKs that can be activated to drive resistance suggests that individual RTK inhibitors will be ineffective alone, while broad inhibition of RTK signaling can enhance the efficacy of and delay resistance to targeted therapies in LUAD. Tumor initiating cells (TICs) are a functional subset of LUAD cells that exhibit self-renewal and are recalcitrant to oncogene-targeted therapies; it has been hypothesized that therapy-resistant TICs are the sanctuary population within the bulk tumor responsible for therapeutic resistance. Targeted ablation of TICs has the potential to enhance the efficacy of and delay resistance to targeted therapies in LUAD. We found that proximal RTK inhibition (SOS1-I and/or SOS2 KO) enhanced the killing effect of oncogene-targeted therapies under 3D culture conditions. SOS1 inhibition and/or SOS2 KO further delayed resistance to oncogenic targeted therapies in both EGFR- and KRASG12C-mutated cell lines. Since TICs are proposed as driving therapeutic resistance, these data suggested that SOS1/2 may regulate TIC survival. TICs can be functionally defined by their ability to proliferate in 3D culture from a single cell. Using this functional definition, we found that 72-h treatment with the G12C-Is adagrasib or sotorasib enriched for functional TICs 2-3 fold in multiple KRASG12C-mutated cell lines, suggesting that TICs could act as a sanctuary population of G12C-I resistant cells. We further isolated cells based on ALDH activity and found isolated ALDHhigh TICs are markedly recalcitrant to short-term G12C-I treatment. In both cases, we found that SOS1-I and/or SOS2 KO inhibited the recalcitrance of TICs to G12C inhibitors to synergistically inhibit TIC survival. These data suggest that SOS1 inhibition and/or SOS2 KO impedes the development of resistance to oncogenic targeted therapy by targeting TIC outgrowth. Citation Format: Brianna Daley, Rob Kortum. Proximal RTK signaling regulates tumor initiating cell survival and therapeutic responsiveness in EGFR- and KRAS-mutated lung adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B007.
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