Lung cancer is the leading cause of cancer-related deaths worldwide. In non-smokers, this disease is usually caused by a mutation in a protein found on the surface of a cell, called EGFR. In healthy lung cells, these proteins trigger a chain of chemical signals that tell the cells to multiply. However, faulty forms of EFGR make the cells grow uncontrollably, leading to the formation of tumors.

Current treatments use EGFR inhibitors that block the activity of these proteins. But cancer cells often become resistant to these treatments by activating other types of growth proteins. One way to overcome this resistance has been by targeting the signaling pathways within individual tumors. But since those pathways differ between tumors, it has been challenging to find a single therapy that can treat all drug-resistant cancer cells.

Now, Theard et al. assessed the therapeutic effects of blocking a specific protein inside lung cells, called SOS1, which is involved in growth signaling in all tumor cells. Six different types of human lung cancer cells were used, all of which had faulty forms of EGFR, with three of the cell types showing drug resistance to current therapies. The cancer cells were either exposed to EGFR inhibitors only or to a combination of EGFR and SOS1 inhibitors. The most effective treatment was found to be through combinational therapy, with enhanced killing of drug-resistant cells.

Theard et al. further assessed the effect of combinational therapy using cells kept in two different ways. Cancer cells were either grown in a two-dimensional format, with cells forming a single cell layer, or in a three-dimensional format, where cells were multi-layered and grew on top of each other as self-aggregating spheroids. Combinational therapy treatment was only successful when the cells where grown in a three-dimensional format.

These findings highlight that future drug development studies should give consideration to the way cells are grown, as it can impact the results. They also provide a steppingstone towards tackling drug resistance in lung cancers that arise from EGFR mutations.

Lung cancer is the leading cause of cancer-related death worldwide; adenocarcinomas are the most common subtype of lung cancer. Oncogenic driver mutations in the RTK/RAS pathway are found in over 75% of lung adenocarcinomas (Cancer Genome Atlas Research Network, 2014). Activating EGFR mutations occur in 10–30% of lung adenocarcinomas and are the major cause of lung cancer in never-smokers. In patients whose tumors harbor either an L858R mutation or an exon 19 deletion (85% of EGFR mutated tumors), first-generation EGFR-tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib enhance progression-free survival (Mok et al., 2009; Yang et al., 2017; Eberhard et al., 2005). However, resistance to first generation EGFR-TKIs invariably occurs. In most cases, acquired resistance to first generation EGFR-TKIs occurs via either a secondary EGFR ‘gatekeeper mutation’ (T790M, 50–60% of cases) that renders the receptor insensitive to first generation EGFR-TKIs or oncogenic shift to alternative RTKs (15–30%). To treat patients with T790M-mutated resistant tumors, the third generation EGFR-TKI osimertinib, which selectively targets activating EGFR mutant proteins including T790M but spares wild-type EGFR, was developed (Jänne et al., 2015; Cross et al., 2014). However, despite further enhancing survival of patients with EGFR-mutant tumors, resistance again emerges.

Unlike first-generation EGFR-TKIs, mechanisms driving osimertinib resistance are more variable, including both EGFR-dependent (10–30%) and EGFR-independent mechanisms (Mancini et al., 2018; Romaniello et al., 2018; La Monica et al., 2017; Eberlein et al., 2015). The most common EGFR-independent resistance mechanisms involve reactivation of the RTK/RAS/effector pathway (Eberlein et al., 2015), often via enhanced signaling through parallel RTKs (Mancini et al., 2018; Romaniello et al., 2018; La Monica et al., 2017; Shi et al., 2016; Park et al., 2016; Kim et al., 2019; Taniguchi et al., 2019; Jimbo et al., 2019; Namba et al., 2019). Here, combining osimertinib with individual RTK inhibitors can both inhibit the development of resistance through the inhibited RTK and kill cancer cells with resistance driven by the specific RTK being inhibited. However, simultaneous inhibition of multiple RTKs with osimertinib may be required to eliminate oncogenic shift to alternative RTKs (Romaniello et al., 2018). Downstream 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 (Tricker et al., 2015; Jacobsen et al., 2017; Ku et al., 2018; Ichihara et al., 2017). 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 (Nunes et al., 2019; Langhans, 2018). 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 (Hao et al., 2019; Kim et al., 2011; Riedl et al., 2017; Jones et al., 2019). These differences may be particularly relevant in EGFR-mutated NSCLC. EGFR-mutated cells show differential RTK expression and phosphorylation in 3D versus 2D conditions (Ekert et al., 2014). 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 (Jacobi et al., 2017). 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 (Vigil et al., 2010), 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 (Jeng et al., 2012; Sheffels et al., 2018; Sheffels et al., 2019), and a specific SOS1 inhibitor (BAY-293) has recently been identified (Hillig et al., 2019). Here, we investigate SOS1 and SOS2 as potential therapeutic targets in EGFR-mutated lung adenocarcinoma cells. Using two distinct measures of pharmacologic synergy, we demonstrate that SOS1 inhibition using BAY-293 synergizes with osimertinib only under 3D spheroid culture conditions, and in doing so add to the growing evidence that pharmacologic assessment of novel therapeutics designed to treat cancer must be performed under 3D culture conditions (Ekert et al., 2014; Sheffels et al., 2018; Nunes et al., 2019; Janes et al., 2018; Jacobi et al., 2017). By assessing the pharmacologic landscape of EGFR/RAS pathway inhibitors, we demonstrate that inhibition of proximal signaling is required to synergize with osimertinib, and that combined EGFR and SOS1 inhibition synergizes to inhibit RAS effector signaling in 3D culture. These findings have significant therapeutic implications for the development of combination therapies to treat EGFR-mutated lung adenocarcinoma.

Activating EGFR mutations are found in 10–30% of lung adenocarcinomas and are the major cause of lung cancer in never smokers. The third-generation EGFR-TKI osimertinib enhances both progression-free (Soria et al., 2018) and overall survival (Ramalingam et al., 2020) compared to first generation EGFR-TKIs and is now considered first-line treatment in EGFR-mutated NSCLC. Osimertinib resistance often develops via activation of parallel RTK pathways (Mancini et al., 2018; Romaniello et al., 2018; La Monica et al., 2017), and broad inhibition RTK signaling may enhance osimertinib efficacy and delay therapeutic resistance. Here, we demonstrate that inhibition of the common RTK signaling intermediate SOS1 using BAY-293 showed marked synergy with osimertinib in 3D spheroid-cultured EGFR-mutated NSCLC cells. Our observations that (i) osimertinib–BAY-293 synergy was only observed in 3D spheroids but not in adherent (2D) cultures and (ii) synergy between RTK-signaling intermediates and osimertinib was not broadly applicable to EGFR downstream signaling components but was limited to proteins upstream of RAS reveal novel insights into pharmacologic studies assessing therapeutics designed to treat NSCLC.

While most studies designed to identify or test therapeutic targets to treat cancer are done in 2D adherent culture, a growing body of evidence suggests that pharmacologic assessment of novel therapeutics must be performed in 3D culture systems (Nunes et al., 2019). Here, there are many different 3D model systems available that vary in both ease-of-use and complexity of the system. The simplest systems employ non-scaffold-dependent monoculture of cancer cell lines where spheroids are either generated using hanging-drop methodology, magnetic levitation, or using ultra-low attachment plates. More complex systems include embedding spheroids in an extracellular matrix (Matrigel, collagen, gelatin, or a synthetic hydrogel) either as a cancer cell line monoculture or in combination with cancer-derived fibroblasts, or using specialized microfluidics or culturing cancer-derived organoids. These methods are have been thoroughly reviewed elsewhere (Langhans, 2018). In the current study, we use ultra-low attachment plates of monoculture NSCLC cell lines as these have the advantage of recapitulating in vivo findings while allowing for dose-response studies done at scale (Mittler et al., 2017).

In NSCLC, multiple studies have now revealed the importance of 3D culture systems in order to recapitulate in vivo findings. EGFR-mutated cells show differential RTK expression and phosphorylation in 3D versus 2D conditions (Ekert et al., 2014) and respond more robustly to EGFR-TKIs in 3D cultures compared to 2D settings (Figure 1 and Jacobi et al., 2017); KRAS-mutated cell lines deemed ‘KRAS-independent’ in 2D culture (Balbin et al., 2013; Singh et al., 2009; Singh et al., 2012; Scholl et al., 2009; Lamba et al., 2014) still require KRAS for anchorage-independent growth (Fujita-Sato et al., 2015; Rotem et al., 2015; Zhang et al., 2006; McCormick, 2015), and some KRAS^G12C-mutated NSCLC cell lines respond to KRAS(G12C) inhibitors in 3D culture and in vivo but not in 2D adherent culture (Janes et al., 2018). The relevance of 3D culture systems extends to the identification of novel therapeutic targets and therapeutic combinations. We recently showed that SOS2 is specifically required for PI3K-dependent protection from anoikis in KRAS-mutated NSCLC cells (Sheffels et al., 2019) and SOS2 deletion synergizes with MEK inhibition to kill KRAS mutated cells only under 3D culture conditions (Sheffels et al., 2018). Here, we show marked synergy between vertical inhibition of EGFR and SOS1 in EGFR mutated cancer cells, but only under 3D culture conditions (Figure 2). CRISPR screens performed in spheroid cultures of KRAS- and EGFR-mutated NSCLC cell lines more accurately reproduce in vivo findings and identify drivers of oncogenic growth compared to screens performed in 2D cultures (Han et al., 2020). Intriguingly, in this study SOS1 was essential for 3D spheroid survival but not 2D spheroid growth of both EGFR- and KRAS-mutated cells, and a recently accepted publication assessing a novel SOS1 inhibitor showed that it was more effective in 3D compared to 2D culture (Hofmann et al., 2020). These data are in complete agreement with our data from Figure 1 showing the requirement for SOS1 in 3D transformation but not 2D proliferation, and support our conclusion that SOS1 is an important therapeutic target in EGFR-mutated NSCLC. We hypothesize the requirement for SOS1 (and SOS2) to promote oncogenic growth in 3D versus proliferation in 2D culture are due to the requirement for PI3K signaling to promote cell survival in 3D but not 2D. Downstream of EGFR activation, the threshold for Raf/MEK/ERK versus PI3K/AKT pathway activation are drastically different, so small amounts of EFGR signaling (in the presence of either SOS1 or SOS2) promote Raf/MEK/ERK signaling, whereas high levels of EGFR signaling are required to activate the PI3K/AKT pathway (Fortian and Sorkin, 2014). While this hypothesis remains to be tested, we speculate that depending on the specific oncogenic contexts, either SOS1 or SOS2 inhibition will be sufficient to modulate RTK signaling and change the threshold for PI3K signaling, thereby affecting oncogenic growth. These data suggest that future studies assessing novel therapeutics to treat lung adenocarcinomas must be performed in a 3D setting, and that SOS1 and SOS2 might be ubiquitous therapeutic targets in RTK-driven tumors.

Osimertinib resistant can occur via oncogenic shift to alternative RTKs including c-MET (Shi et al., 2016), HER2 and/or HER3 (Mancini et al., 2018; Romaniello et al., 2018; La Monica et al., 2017), IGF1R (Park et al., 2016), and AXL (Kim et al., 2019; Taniguchi et al., 2019; Jimbo et al., 2019; Namba et al., 2019). The variety of RTK bypass pathways that can lead to osimertinib resistance suggests that broad inhibition of RTK signaling may be a more effective therapeutic strategy than any individual RTK inhibitor to limit osimertinib resistance, whereas once resistance via oncogenic shift to an alternative RTK occurs then inhibition of the upregulated RTK would have therapeutic benefit. Toward this end, Phase I and II clinical trials are currently examining whether combining osimertinib with inhibitors of AXL (DS-1205c, NCT03255083) or c-MET (teponitib, NCT03940703; savolitinib, NCT03778229) are effective in patients who have progressed on osimertinib treatment.

Combining osimertinib with a MEK inhibitor can enhance osimertinib efficacy (Eberlein et al., 2015; Tricker et al., 2015; Ichihara et al., 2017; Shi et al., 2017; Della Corte et al., 2018) and Phase II clinical trials are currently underway to assess combining osimertinib with the MEK inhibitor selumetinib in EGFR-mutated NSCLC (NCT03392246), although resistance to combined osimertinib and MEK inhibition still occurs (Tricker et al., 2015). In a recent study designed to understand resistance to combined osimertinib and MEK inhibition, Kurppa et al., 2020 show that combining osimertinib with the MEK inhibitor trametinib results in EGFR-mutated cells entering a senescent state that is dependent on the activation of the Hippo pathway effector YAP and its transcription-factor-binding partner TEAD (Kurppa et al., 2020). Inhibition of YAP/TEAD signaling overcame this senescence and enhanced killing of EGFR-mutated cells (Kurppa et al., 2020). EGFR-signaling drives YAP nuclear translocation and transcriptional regulation through PI3K-PDK1 signaling (Fan et al., 2013; Xia et al., 2018; Tumaneng et al., 2012). This suggest that therapeutic combinations able to synergistically inhibit both Raf/MEK/ERK and PI3K/AKT effector signaling should overcome YAP-dependent senescence and treat EGFR-mutated NSCLC.

Here, we show that osimertinib does not broadly synergize with inhibitors of downstream EGFR/RAS/RAS effector signaling. Instead, we found that synergy was limited to combinations of osimertinib with inhibitors of proximal EGFR signaling intermediates SOS1 and SHP2 (Figure 7). Further, SOS1 inhibition significantly enhanced osimertinib-dependent inhibition of both Raf/MEK/ERK and PI3K/AKT signaling (Figure 6), whereas inhibition of individual downstream Raf/MEK/ERK or PI3K/AKT effector pathways did not synergize with osimertinib (Figure 7) to inhibit 3D spheroid growth. We hypothesize that these two findings are inexorably linked, so that any potential therapeutic must synergize with osimertinib to inhibit all downstream RAS effector signaling to show drug-drug synergy in 3D culture. In support of this idea, previous studies showed inhibition of SRC family kinases (SFK) potentiated osimertinib to a much greater extent than either MEK or PI3K inhibition (Ichihara et al., 2017), and that SFK inhibition synergized with osimertinib to inhibit both Raf/MEK/ERK and PI3K/AKT signaling (Ichihara et al., 2017; Watanabe et al., 2017).

There remain several open questions regarding SOS1 inhibition as a therapeutic strategy to limit osimertinib resistance. First, does SOS1 inhibition enhance osimertinib efficacy in vivo using xenograft studies? While BAY-293 shows tremendous specificity toward SOS1 (Figure 1—figure supplement 1 and 2, and Hillig et al., 2019) and is a very useful tool compound for in vitro studies, it has limited bioavailability making it unsuitable for in vivo use. Thus, new SOS1 inhibitors that can be used in vivo are needed to move SOS1 forward as a therapeutic target. Intriguingly, while this paper was under review Boehringer Ingelheim reported two orally available SOS1 inhibitors suitable for in vivo studies (Hofmann et al., 2020). They found that SOS1 inhibition could overcome MEK inhibitor resistance in KRAS-mutated cell lines and that the combination of SOS1 and MEK inhibition showed marked show efficacy in KRAS-mutated cell lines and xenograft models. They are now moving one of these compounds into Phase I safety trials for KRAS mutated solid tumors (BI-1701963, NCT04111458). It will be exciting to assess whether these new SOS1 inhibitors work in combination with osimertinib to limit the growth EGFR-mutated tumors. Further, these studies will be necessary to translate SOS1-targeted therapies for use in EGFR-mutated lung adenocarcinoma. Second, does SOS1 inhibition actually limit the development of osimertinib resistance? While outside the scope of the current paper, it will be intriguing to use in vitro models of EGFR-TKI resistance (Tricker et al., 2015) to assess whether SOS1 inhibition can block the development of osimertinib resistance. Third, while we have focused on the RAF/MEK/ERK and PI3K/AKT effector pathways as the major contributors to mutant EGFR-driven NSCLC, there are many different effector pathways downstream of RAS that may be SOS1-dependent and contribute to the oncogenic phenotype. Here, and unbiased approach at understanding the individual and combined effects of osimertinib and SOS1 inhibition on RAS activation (to validate relatively new SOS1 inhibitors) and RAS effector signaling would provide valuable insight into how these therapies alter EGFR-driven signaling in NSCLC.

Overall, our data suggest that inhibitors of proximal signaling may be the most efficacious therapeutics to combine with osimertinib to treat EGFR-mutated tumors. Toward this end, Phase I trials are currently underway assessing the combination of osimertinib and the SRC inhibitor dasatinib (NCT02954523) in EGFR-mutated NSCLC, and recently developed SOS1 (BI-1701963, NCT04111458) and SHP2 (JAB-3068, NCT03565003; RMC-4630, NCT03634982) inhibitors have entered Phase I safety trials. Our study provides a framework for the systematic, preclinical assessment of therapeutic combinations designed to treat EGFR-mutated cancer cells. We show both how to use basic pharmacologic principles to assess drug-drug synergy and that these combinations must be assessed under 3D culture conditions. Using this framework, we show that the combination of osimertinib and the SOS1 inhibitor BAY-293 shows marked efficacy in 3D spheroid culture and should be pursued as a therapeutic option to treat EGFR-mutated lung adenocarcinoma.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1, 2, 3, 4, 5, 6, 7, and Figure 1—figure supplement 2 and Figure 3—figure supplement 1.

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Author details

1. Patricia L Theard

   Department of Pharmacology and Molecular Therapeutics, Uniformed Services University of the Health Sciences, Bethesda, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing

   Competing interests

   No competing interests declared

2. Erin Sheffels

   Department of Pharmacology and Molecular Therapeutics, Uniformed Services University of the Health Sciences, Bethesda, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

3. Nancy E Sealover

   Department of Pharmacology and Molecular Therapeutics, Uniformed Services University of the Health Sciences, Bethesda, United States

   Contribution

   Conceptualization, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

4. Amanda J Linke

   Department of Pharmacology and Molecular Therapeutics, Uniformed Services University of the Health Sciences, Bethesda, United States

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

5. David J Pratico

   Department of Pharmacology and Molecular Therapeutics, Uniformed Services University of the Health Sciences, Bethesda, United States

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

6. Robert L Kortum

   Department of Pharmacology and Molecular Therapeutics, Uniformed Services University of the Health Sciences, Bethesda, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   robert.kortum@usuhs.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1634-4882

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