Drug treatment of 3D cancer spheroids more accurately reflects in vivo therapeutic responses compared to adherent culture studies. In EGFR-mutated lung adenocarcinoma, EGFR-TKIs show enhanced efficacy in spheroid cultures. Simultaneous inhibition of multiple parallel RTKs further enhances EGFR-TKI effectiveness. We show that the common RTK signaling intermediate SOS1 was required for 3D spheroid growth of EGFR-mutated NSCLC cells. Using two distinct measures of pharmacologic synergy, we demonstrated that SOS1 inhibition strongly synergized with EGFR-TKI treatment only in 3D spheroid cultures. Combined EGFR- and SOS1-inhibition markedly inhibited Raf/MEK/ERK and PI3K/AKT signaling. Finally, broad assessment of the pharmacologic landscape of drug-drug interactions downstream of mutated EGFR revealed synergy when combining an EGFR-TKI with inhibitors of proximal signaling intermediates SOS1 and SHP2, but not inhibitors of downstream RAS effector pathways. These data indicate that vertical inhibition of proximal EGFR signaling should be pursued as a potential therapy to treat EGFR-mutated tumors.

The RAS family of genes (HRAS, NRAS, and KRAS) is mutated in around 30% of human tumours. Wild-type RAS isoforms play an important role in mutant RAS-driven oncogenesis, indicating that RasGEFs may play a significant role in mutant RAS-driven transformation. We recently reported a hierarchical requirement for SOS2 in mutant RAS-driven transformation in mouse embryonic fibroblasts, with KRAS>NRAS>HRAS (Sheffels et al., 2018). However, whether SOS2 deletion differentially affects mutant RAS isoform-dependent transformation in human tumour cell lines has not been tested. After validating sgRNAs that efficiently deleted HRAS and NRAS, we showed that the differential requirement for SOS2 to support anchorage-independent (3D) growth, which we previously demonstrated in MEFs, held true in cancer cells. KRAS-mutant cells showed a high dependence on SOS2 for 3D growth, as previously shown, whereas HRAS-mutant cells did not require SOS2 for 3D growth. This differential requirement was not due to differences in RTKstimulated WT RAS activation, as SOS2 deletion reduced RTK-stimulated WT RAS/PI3K/AKT signalling in both HRAS and KRAS mutated cell lines. Instead, this differential requirement of SOS2 to promote transformation was due to the differential sensitivity of RAS-mutated cancer cells to reductions in WT RAS/PI3K/AKT signalling. KRAS mutated cancer cells required SOS2/PI3K signaling to protect them from anoikis, whereas survival of both HRAS and NRAS mutated cancer cells was not altered by SOS2 deletion. Finally, we present an integrated working model of SOS signaling in the context of mutant KRAS based on our findings and those of others.

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

Drug treatment of 3D cancer spheroids more accurately reflects in vivo therapeutic responses compared to adherent culture studies. In EGFR-mutated lung adenocarcinoma, EGFR-TKIs show enhanced efficacy in spheroid cultures. Simultaneous inhibition of multiple parallel RTKs further enhances EGFR-TKI effectiveness. We show that the common RTK signaling intermediate SOS1 was required for 3D spheroid growth of EGFR-mutated NSCLC cells. Using two distinct measures of pharmacologic synergy, we demonstrated that SOS1 inhibition strongly synergized with EGFR-TKI treatment only in 3D spheroid cultures. Combined EGFR-and SOS1-inhibition markedly inhibited Raf/MEK/ERK and PI3K/AKT signaling. Finally, broad assessment of the pharmacologic landscape of drug-drug interactions downstream of mutated EGFR revealed synergy when combining an EGFR-TKI with inhibitors of proximal signaling intermediates SOS1 and SHP2, but not inhibitors of downstream RAS effector pathways. These data indicate that vertical inhibition of proximal EGFR signaling should be pursued as a potential therapy to treat EGFR-mutated tumors.of RAS, co-targeting intermediates of the RAF/MEK/ERK and PI3K/AKT pathways enhances of osimertinib effectiveness, however, signaling through the uninhibited effector pathway may drive resistance (17)(18)(19)(20). Thus, it may be important for therapeutic combinations including osimertinib to stifle all downstream RTK/RAS signaling to be effective.Recent studies suggest that pharmacologic assessments of targeted therapeutics should be performed under 3D culture conditions rather than in 2D adherent cultures (21,22). 3D spheroids show altered growth characteristics, changes in cell surface proteins, altered metabolism, changes in activation of signaling pathways or altered responses to targeted pathway inhibitors, and are more resistant to drug-induced apoptosis compared to 2D adherent cultures signaling (23-26). These differences may be particularly relevant in EGFR-mutated NSCLC. EGFR-mutated cells show differential RTK expression and phosphorylation in 3D versus 2Dconditions (27). Further, EGFR-mutated cells respond more robustly to first-generation EGFR-TKIs in 3D cultures, and these responses more closely resemble responses seen in vivo (28).These data highlight the need for pharmacologic assessment of therapeutics designed to treat EGFR-mutated NSCLC under 3D culture conditions. The ubiquitously expressed RasGEFs (guanine nucleotide exchange factors) SOS1 and SOS2 (son of sevenless 1 and 2) are common signaling intermediates of RTK-mediated RAS activation. Although not initially considered as drug targets because of the low oncogenic potential of SOS (29), there has been renewed interest in SOS proteins as therapeutic targets for cancer treatment. We and others have shown that SOS1 and SOS2 may be important therapeutic targets in KRAS-mutated cancer cells (30-32), and a specific SOS1 inhibitor (BAY-293) has recently been identified (33). Here, we investigate SOS1 and SOS2 as potential therapeutic targets in EGFR-mutated l...

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