Small Molecule–Mediated Activation of RAS Elicits Biphasic Modulation of Phospho-ERK Levels That Are Regulated Through Negative Feedback on SOS1 Jennifer E

Published OnlineFirst February 13, 2018; DOI: 10.1158/1535-7163.MCT-17-0666

Cancer Biology and Translational Studies Molecular Cancer Therapeutics Small Molecule–Mediated Activation of RAS Elicits Biphasic Modulation of Phospho-ERK Levels that Are Regulated through Negative Feedback on SOS1 Jennifer E. Howes, Denis T. Akan, Michael C. Burns, Olivia W. Rossanese, Alex G. Waterson, and Stephen W. Fesik

Abstract

Oncogenic mutation of RAS results in aberrant cellular inhibit the association between SOS1 and GRB2 and disrupt signaling and is responsible for more than 30% of all human SOS1 membrane localization. Consistent with this, we show tumors. Therefore, pharmacologic modulation of RAS has that wild-type SOS1 and GRB2 dissociated in a time-depen- attracted great interest as a therapeutic strategy. Our laboratory dentfashioninresponsetocompound treatment, and con- has recently discovered small molecules that activate Son of versely, this interaction was enhanced with the expression of Sevenless (SOS)–catalyzed nucleotide exchange on RAS and an S1178A SOS1 mutant. Furthermore, in cells expressing inhibit downstream signaling. Here, we describe how phar- either S1178A SOS1 or a constitutively membrane-bound macologically targeting SOS1 induced biphasic modulation of CAAX box tagged SOS1 mutant, we observed elevated RAS- RAS-GTP and ERK phosphorylation levels, which we observed GTP levels over time in response to compound, as compared in a variety of cell lines expressing different RAS-mutant iso- with the biphasic changes in RAS-GTP exhibited in cells forms. We show that compound treatment caused an increase expressing wild-type SOS1. These results suggest that small in phosphorylation at ERK consensus motifs on SOS1 that was molecule targeting of SOS1 can elicit a biphasic modulation of not observed with the expression of a non-phosphorylatable RAS-GTP and phospho-ERK levels through negative feedback S1178A SOS1 mutant or after pretreatment with an ERK on SOS1 that regulates the interaction between SOS1 and inhibitor. Phosphorylation at S1178 on SOS1 is known to GRB2. Mol Cancer Ther; 17(5); 1051–60. 2018 AACR.

Introduction (GRB2), that recruit SOS1 to the membrane and facilitate its interaction with RAS (3, 4), stimulating downstream effector RAS proteins are small GTPases that serve as signaling trans- pathway signaling events. ducers by coupling extracellular receptor activation to intracellu- Mutationally activated RAS protein is implicated in the onco- lar effector pathway signaling. Canonically, RAS proteins cycle genic progression of three of the four most prevalent cancers in the from a GDP-bound "inactive" state to a GTP-bound "active" state. United States (colon, lung, and pancreas; ref. 5). Hyperactivation The transition between the inactive to the active GTP-bound state of RAS signaling confers capabilities, known as the Hallmarks of is catalytically regulated by guanine nucleotide exchange factors Cancer (6) that cause the cell to become malignant (7). Inacti- (GEF), such as Son of Sevenless 1 (SOS1; ref. 1). Membrane vation of oncogenic RAS leads to dramatic tumor regressions in localization of SOS1 enhances its local protein concentration and multiple mouse models, making RAS an important therapeutic enables the activation of RAS (2). Although RAS is tethered to the target (8–10). Early work in our laboratory led to the discovery of membrane through a C-terminal CAAX box motif (1), SOS1 is not small molecules that bind directly to KRAS and inhibit SOS- constitutively membrane bound and relies on posttranslational mediated nucleotide exchange (11). Analogously, a group from modifications and protein–protein interactions to facilitate mem- Genentech identified small molecules that bind directly to RAS brane recruitment (2). Classically, autophosphorylation of and also inhibit SOS-mediated nucleotide exchange (12). Using a ligand-activated growth factor receptors results in the binding of different strategy, the Shokat group discovered small molecules adaptor proteins, such as growth factor receptor–bound protein-2 that irreversibly bind to and inhibit the oncogenic G12C mutant form of KRAS. Efforts to indirectly target RAS include the use of farnesyl- and geranylgeranyl-transferase inhibitors to interfere Department of Biochemistry, Vanderbilt University, Nashville, Tennessee. with RAS membrane association (13), RAF, MEK, and ERK inhi- Note: Supplementary data for this article are available at Molecular Cancer bitors to block RAS-mediated downstream signaling events (14) Therapeutics Online (http://mct.aacrjournals.org/). and synthetic lethal strategies, such as the use of proteasome Corresponding Author: Stephen W. Fesik, Vanderbilt University, 2215 Garland inhibitors in the context of mutant RAS-driven tumors (15, 16). Avenue, 607 Light Hall, Nashville, TN 37232-0146. Phone: 615-322-6303; Fax: Our laboratory has recently discovered compounds that indi- 615-875-3236; E-mail: [email protected] rectly target RAS through the activation of SOS-mediated nucle- doi: 10.1158/1535-7163.MCT-17-0666 otide exchange (17). We identified aminopiperidine indole com- 2018 American Association for Cancer Research. pounds (henceforth referred to as RAS activator compounds) that

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bind to the RAS:SOS:RAS ternary complex in a pocket on the obtained from Cell Signaling Technology. Molecular weight CDC25 domain of SOS1, adjacent to the switch II region of RAS. A markers are indicated in kDa on each blot. group from AstraZeneca also identified fragments that bind at this site (18). Although compound-mediated activation of RAS to a Immunoprecipitation GTP-bound state seems like a counterintuitive strategy, we Cells were seeded and incubated for 24 hours to reach 70% observed that compounds that activated SOS1-mediated nucle- confluency. Subsequently, cells were transfected with an otide exchange on RAS actually caused a striking inhibition of HA-SOS1WT plasmid (a kind gift from Dr. Bar-Sagi, NYU School downstream signaling in the MAPK signaling pathway. Specifi- of Medicine, New York, Addgene #32920; ref. 21), or with a cally, we observed biphasic changes in ERK phosphorylation V5-SPRY2 plasmid (donor vector was a kind gift from Dominic characterized by an increase in phospho-ERK1/2T202/Y204 at lower Esposito, NIH National Cancer Institute, Frederick, Addgene compound concentrations and a decrease at higher concentra- #70614) using Lipofectamine 2000 (Life Technologies) and incu- tions (17). To follow up on this exciting discovery, we sought to bated overnight. Mock control cells were transfected with lipid understand the biological mechanism of RAS activator com- only. Cells were treated with compound or vehicle control alone pound action on intracellular ERK signaling. for the times and concentrations indicated. Cells were lysed on ice using lysis buffer [150 mmol/L NaCl, 10 mmol/L Tris-HCl pH 7.4, Materials and Methods 1% (v/v) Triton X-100, 10% (v/v) glycerol, 1 mmol/L EDTA, and Cell culture and compounds supplemented with protease and phosphatase inhibitors HeLa (ATCC CCL-2), HEK-293 (ATCC CRL-1573), NCI-H358 (Roche)]. Protein concentration of each sample was assessed fi (ATCC CRL-5807), NCI-H1792 (ATCC CRL-5895), HCT 116 using the Pierce BCA Protein Assay Kit (Thermo Fisher Scienti c #23225). Lysates of equal protein concentration were incubated (ATCC CCL-247), and NCI-H1299 (ATCC CRL-5803) cells were obtained from the ATCC and cultured in DMEM or RPMI sup- at 4 C for 1 hour with anti-HA magnetic beads for SOS1 immu- plemented with 10% (v/v) FBS where appropriate. Cell lines were noprecipitation (Cell Signaling Technology #11846), or anti-V5 authenticated by STR profiling using PowerPlex 16HS technology magnetic beads for SPRY2 immunoprecipitation (MBL #M167- 11). Samples were washed using PBS with 0.1% TWEEN 20 (v/v), and were tested negative for mycoplasma using the eMYCO Plus Kit PCR test in October 2017 (Genetica DNA Laboratories). eluted in SDS loading buffer, and incubated at 95 C for 10 After thawing from liquid nitrogen, cells were passaged at least minutes with 10 mmol/L DTT. For the SOS1-GRB2 coimmuno- twice before use in experiments and passaged for a maximum of precipitation, samples were eluted from the beads in SDS loading buffer and incubated at 50C for 10 minutes, prior to boiling the 25 times. Compound 1 was synthesized as described previously (17). eluent at 95 C with 10 mmol/L DTT. Input VC-treated samples HSP90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin were run on the immunoprecipitation Western blots to compare (17-AAG; ref. 19) and ERK1/2 inhibitor SCH772984 (20) were protein levels between the input and immunoprecipitation purchased from Selleckchem. EGF was purchased from R&D samples. Systems. Site-directed mutagenesis and cloning Active RAS-GTP pull-down and Western blotting The HA-SOS1S1178A construct was made using the QuikChange Cells were seeded to reach 70% confluency after 24-hour II Site Directed Mutagenesis Kit (Agilent #200523) by following incubation and subsequently treated as indicated. The vehicle the manufacturer's instructions. Mutations were introduced to the control (VC) for compound 1 was DMSO, and the vehicle control HA-SOS1WT plasmid. The primers used for mutagenesis were as for EGF was PBS. Lysates were resolved by SDS-PAGE and trans- follows: ferred onto Immobilon-FL PVDF membranes (Millipore). Mem- Forward: 50-ATTATGTCTAAGCACTTGGACGCCCCCCCAGC- branes were probed with primary antibodies as indicated, incu- 30; reverse: 50-GCTGGGGGGGCGTCCAAGTGCTTAGACATAAT- bated with labeled secondary antibody, and scanned using an 30. Primers were PAGE purified and synthesized by Eurofins Odyssey imager (LiCor). Multiple independent blots were run Genomics. using the same lysate from each experiment. Membranes were cut The HA-SOS1CAAX construct was made by cloning the hyper- to allow probing for multiple molecular weight proteins. Where variable region of KRAS 4B onto the C-terminus of the HA- appropriate, pull-down blots were probed with two different SOS1WT construct (protein sequence: KMSKDGKKKKKKSKTK- species of antibodies, using multiple colors to detect exactly the CVIM). The HA-SOS1WT plasmid was double digested using BsmI same molecular weight on the same membrane, for example, pSPP and SbfI (New England Biolabs). The CAAX box was ligated in the (rabbit ¼ green) and HA (mouse ¼ red). Levels of RAS-GTP were HA-SOS1WT plasmid using two preannealed oligomers. The oli- determined using an Active RAS Pull-Down and Detection Kit gomers used to insert KRAS 4B CAAX box onto HA-SOS1WT were according to the manufacturer's instructions (Thermo Fisher Sci- as follows: entific #16117). Primary antibodies used were anti-HA (BioLegend Forward 50-CATTCTTCCAAGATGAGCAAAGATGGTAAAAA- #901501); anti-SOS1 (Santa Cruz Biotechnology #SC-256); anti- GAAGAAAAAGAAGTCAAAGACAAAGTGTGTAATTATGTAACCT- SPRY4 (Abcam #ab103114); anti-V5 (Invitrogen #46-0705); and GCA-30, reverse: 50-GGTTACATAATTACACACTTTGTCTTTGAC- antibodies targeting pan-RAS (K-, N-, and HRAS isoforms; #3339), TTCTTTTTCTTCTTTTTACCATCTTTGCTCATCTTGGAAGAATGG- total ERK1/2 (#9102), phospho-ERK1/2T202/Y204 (#9106), total G-30. Oligomers were synthesized by Eurofins Genomics. AKT (#2920), phospho-AKTS473 (#4060), phospho-AKTT308 The V5-SPRY2 construct was made using Gateway Cloning (#4056) GRB2 (#3972), phospho-RSKS380 (#11989), phospho- (Invitrogen). The SPRY2 coding region from the donor vector RSKT359 (#8753), phospho-RSKT573 (#9346), total RSK (#9355), was recombined into the pcDNA-DEST40 vector for expression in DUSP4 (#5149), DUSP16 (#5523), SPRY2 (#14954), phospho- mammalian cells. All constructs were validated by sequencing SPP (#14390), phospho-Y (#8954), and GAPDH (#2118) were prior to use.

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RAS Activator Compound Regulates ERK via Feedback on SOS1

Results response, we observed a disconnect between RAS-GTP levels and Treatment with a RAS activator compound causes biphasic downstream signaling patterns in ERK phosphorylation, where an changes in RAS-GTP and phospho-ERK levels increase in RAS-GTP levels did not directly correspond to an To gain a mechanistic understanding of RAS activator–medi- increase in signaling downstream as anticipated (Fig. 1B). To ated signaling, we investigated how RAS activator compound 1 gain a better insight, we assessed the effects of 1 on RAS-GTP levels (compound 4 from ref. 17; Fig. 1A) modulated changes in RAS- and downstream ERK phosphorylation over a time course of 180 GTP levels and downstream phospho-ERK signaling events. minutes using an activating concentration of 1 (Fig. 1C). A rapid To determine that the effects on ERK phosphorylation elicited increase in RAS-GTP levels was observed after 5 minutes of by compound 1 treatment were not due to off-target kinase treatment with 1 that peaked after 10 to 20 minutes and remained inhibition, we tested compound 1 in a kinase activity panel and detectable until 90 minutes, then RAS-GTP levels were reduced did not identify any substantial inhibition against 342 protein back to baseline after 120 minutes of treatment (Fig. 1C). Sub- kinases (Supplementary Table S1). Next, we determined the sequent to heightened RAS-GTP levels, a peak in phospho-ERK effects of different concentrations of 1 on RAS-GTP and phos- protein levels occurred after 20 to 30 minutes of treatment with 1 pho-ERK1/2T202/Y204 levels in cancer cells. Consistent with our (Fig. 1C). Moreover, phospho-ERK levels decreased in conjunc- previous observations (17), RAS-GTP levels increased in response tion with RAS-GTP levels after 90 minutes of treatment with 1. to treatment with 50 to 100 mmol/L concentrations of 1 treatment Indicative of ERK activation (22), we also observed a similar (Fig. 1B). Furthermore, biphasic modulation of ERK phosphor- biphasic pattern in phosphorylation on RSK at S380 and T359 ylation was observed, characterized by an increase in phospho- (Fig. 1C). Phosphorylation at both of these sites on RSK was also ERK at lower concentrations and a decrease at higher concentra- induced by EGF stimulation. Phosphorylation on RSK at T573 tions of 1. Notably, when assessing the effects of 1 by dose was not induced by either 1 treatment or EGF stimulation

Figure 1. Treatment with 1 causes modulation of RAS-GTP and phospho-ERK levels. A, Chemical structure of 1 (compound 4 from ref. 17). B, HeLa cells were treated with various concentrations of 1 for 30 minutes. C, HeLa cells were treated with 50 mmol/L 1 over a time course of up to 180 minutes. EGF (50 ng/mL for 5 minutes) was used as a positive control. D, HeLa cells were stimulated with EGF (50 ng/mL) over a time course of up to 60 minutes. Two biological repeats of each experiment were independently conducted.

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(Fig. 1C). Furthermore, as active ERK is known to induce the vated ERK inhibits the interaction between SOS1 and GRB2 (32) expression of DUSP and SPRY proteins that can negatively reg- and uncouples downstream activation of RAS (29). Due to the ulate RAS–MAPK signaling (23), we assessed the effects of com- phenotypic similarities between treatment with EGF and 1 (Fig. pound treatment on total protein levels of DUSP4, a nuclear 1C and D), we hypothesized that the same negative feedback phosphatase known to inactivate ERK1/2 in response to growth mechanism may be elicited in response to compound-mediated factor stimulation (24), DUSP16, a cytoplasmic phosphatase that induction of RAS-GTP and phospho-ERK levels. To test whether regulates JNK signaling and can sequester activated ERK from its treatment with 1 induced phosphorylation on SOS1 at ERK substrates (25), and SPRY2 (26) and SPRY4 (27), both antago- consensus motifs, we used an antibody to detect phosphorylated nists of receptor tyrosine kinase–mediated MAPK signaling. We serine residues at SPP motifs on immunoprecipitated SOS1 did not observe any increase in total protein expression of DUSP4, (Fig. 2A). We overexpressed an HA-tagged SOS1 construct and DUSP16, SPRY2, or SPRY4 in response to 1 treatment for up to treated cells with an activating concentration of 1 over a time 180 minutes in HeLa cells (Supplementary Fig. S1). course of up to 90 minutes. Subsequently, HA-SOS1 was isolated Importantly, we show that this biphasic modulation of both by immunoprecipitation and resolved by Western blot to assess RAS-GTP levels and ERK phosphorylation in response to 1 treat- phosphorylation of serine residues at any SPP motif on HA-SOS1 ment is also observed in cells that express mutant RAS isoforms (Fig. 2A). In response to treatment with 1, we observed an increase (Supplementary Fig. S2). We treated NCI-H358 (KRAS heterozy- over time in the levels of phospho-SPP in cells expressing HA- gous G12C), NCI-H1792 (KRAS heterozygous G12C), HCT 116 SOS1WT (Fig. 2A). Higher levels of phospho-SPP modifications (KRAS heterozygous G13D), and NCI-H1299 cells (NRAS het- were observed after 90 minutes of treatment with 1, concurrent with erozygous Q61K) with 1 over a time course of up to 180 minutes, heightened levels of phospho-ERK (Fig. 2A). Similarly, increases in and we observed various degrees of biphasic RAS-GTP and phos- SPP phosphorylation levels on HA-SOS1WT and downstream ERK pho ERK modulation. Interestingly, the degree of RAS-GTP fluc- phosphorylation levels were observed in response to EGF stimu- tuation in RAS-mutant cell lines was not as striking as in HeLa cells lation (Fig. 2B). A small shift in apparent molecular weight of SOS1 [KRAS homozygous wild-type (WT)]. However, EGF treatment was also detected after HA-SOS1WT–expressing cells were stimu- also did not markedly induce RAS-GTP levels in cells expressing lated with either compound 1 or EGF, suggestive of increased SOS1 mutant RAS isoforms. We hypothesized this is due to constitutive phosphorylation over time (Fig. 2A, B, and C). activation of RAS in RAS-mutant cell lines (28). Therefore, we Phosphorylation of SOS1 on S1178 (within an ERK consensus proceeded to explore the mechanism of compound action using motif) is known to disrupt the interaction between SOS1 and cell lines expressing WT KRAS. GRB2 and has been specifically implicated in the control of RAS The biphasic modulation of RAS-GTP and ERK phosphorylation activation in the context of growth factor stimulation (32, 33). that we observed in response to 1 treatment is similar to a pheno- Therefore, we tested whether S1178 on SOS1 was also phosphor- type reported with growth factor stimulation (29). Consistent with ylated in response to treatment with 1. Consistent with the the literature, after 1 minute of stimulation with EGF, we observed literature (32), after stimulation with EGF in cells overexpressing an increase in RAS-GTP, leading to heightened levels of phospho- a nonphosphorylatable mutant form of SOS1, HA-SOS1S1178A, ERK that returned to a level just above baseline after 10 to 15 SPP phosphorylation on SOS1 was not observed to the same minutes (Fig. 1D). Interestingly, an HSP90 chaperone inhibitor degree relative to HA-SOS1WT phospho-SPP levels (Fig. 2B). (17-AAG) can also induce similar fluctuations in phospho-ERK Although HA-SOS1S1178A cannot be phosphorylated at position signaling at high treatment concentrations through the induction of 1178, residual SPP phosphorylation on HA-SOS1S1178A was still a stress response in cells (30, 31). However, to our knowledge, the detected after EGF stimulation (Fig. 2B), likely because the C- effects of 17-AAG on RAS-GTP levels have not been assessed. terminus of SOS1 contains multiple other SPP motifs (32). Therefore, we treated HeLa cells with a high concentration of Interestingly, similarly to EGF stimulation, markedly less SPP 17-AAG over a time course of up to 180 minutes, to induce a stress phosphorylation was observed in response to 1 treatment in cells response (Supplementary Fig. S3). We show that there is a similarity expressing the HA-SOS1S1178A mutant, as compared to HA- between 1 treatment and 17-AAG treatment on phospho-ERK levels SOS1WT expressing cells. Under these circumstances, SOS1 phos- in HeLa cells. However, 17-AAG did not induce any changes in RAS- phorylation at SPP motifs did not increase over time, independent GTP levels, unlike that observed with 1 treatment. These data of phospho-ERK levels that behave in a similar manner to cells demonstrate that 1 treatment at higher concentrations was not expressing HA-SOS1WT (Fig. 2A). These data show that HA- simply causing biphasic phospho-ERK modulation due to a stress SOS1WT is phosphorylated in response to treatment with 1 and response because we also observed modulation of RAS-GTP; the led us to hypothesize that active phospho-ERK is the kinase anticipated phenotype of a SOS agonist. Furthermore, lower con- responsible for this phosphorylation event, as S1178 is at an ERK centrations of 1 treatment also caused changes in ERK phosphor- consensus SPP motif, and SPP SOS1 phosphorylation increases in ylation (Supplementary Fig. S4). parallel with elevated phospho-ERK levels (Fig. 2A). To test this, we pretreated cells expressing HA-SOS1WT with the ERK1/2 Treatment with 1 induces phosphorylation of SOS1 at S1178 inhibitor SCH772984 for 60 minutes and then assessed the levels within an ERK consensus motif of SPP phosphorylation on SOS1 in response to 1 treatment over a Due to the biphasic changes in RAS-GTP and phospho-ERK time course of up to 90 minutes (Fig. 2C). Following pretreatment levels observed in response to 1 treatment, we hypothesized that a with SCH772984 alone, we observed an anticipated decrease in negative feedback loop may be induced to dampen the effects of downstream phosphorylation of RSKT359 and, as previously increased signaling. In the context of growth factor signaling, reported, on ERK1/2 itself (20). Importantly, after combined ERK activated ERK phosphorylates the C-terminus of SOS1 on serine inhibitor pretreatment and treatment with 1, we observed less residues at the ERK consensus phosphorylation motif serine- phosphorylation of SPP motifs on SOS1, as compared with the proline-proline (SPP; ref. 32). Phosphorylation of SOS1 by acti- effects of 1 treatment alone. Similarly, pretreatment with an ERK

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RAS Activator Compound Regulates ERK via Feedback on SOS1

Figure 2. SOS1 is phosphorylated at ERK consensus sites in response to stimulation with 1 and EGF. Levels of phospho-Ser Pro Pro peptide in HA-SOS1 immunoprecipitation (IP) eluent from HeLa cells overexpressing either an HA-tagged WT SOS1 construct (HA-SOS1WT) or an HA-tagged S1178A SOS1 construct (HA-SOS1S1178A). Cells were treated with 1 (A;50mmol/L) over a time course of up to 90 minutes, EGF (B; 50 ng/mL) over a time course of up to 20 minutes, and pretreatment with 1 mmol/L ERK inhibitor SCH772984 for 60 minutes followed by 1 treatment (50 mmol/L) over a time course of up to 90 minutes (C). Two biological repeats of each experiment were independently conducted. inhibitor caused less SPP phosphorylation on SOS1 in response to between SOS1 and GRB2 may be disrupted in response to EGF stimulation, as compared to the effects of EGF stimulation treatment with 1. To test this, we assessed the effects of treatment alone (Fig. 2C). These data show that ERK is the kinase responsible with 1 over time on the interaction between SOS1 and GRB2 using for phosphorylation of SPP motifs on SOS1 at S1178 in the coimmunoprecipitation. We used 293 cells due to their high level context of 1 treatment. of GRB2 expression. FBS stimulation was used as a negative control for the association between SOS1 and GRB2 (34), and Treatment with 1 causes dissociation between SOS1 and GRB2 consistent with the literature, FBS stimulation inhibited the that is mediated through SOS1 phosphorylation at S1178 interaction between HA-SOS1WT and GRB2 below the baseline After discovering that treatment with 1 caused phosphorylation levels of the vehicle control (Fig. 3A). The HA-SOS1WT–GRB2 on S1178 of SOS1 (Fig. 2A), we hypothesized that the interaction interaction decreased over time in response to treatment with 1.

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Figure 3. The GRB2-SOS1 association is reduced over time after treatment with 1. Two hundred and ninety-three cells were transiently transfected with HA-SOS1WT (A), HA-SOS1S1178A (B), HA-SOS1CAAX (C), and treated with 25 mmol/L 1 over 90 minutes. D, 293 cells were transiently transfected with HA-SOS1WT and pretreated with 1 mmol/L ERK inhibitor SCH772984 or a vehicle control for 1 hour where indicated, followed by treatment with 25 mmol/L 1 for either 60 or 90 minutes. Where indicated, cells were treated with FBS (20% v/v) for 15 minutes. Two biological repeats of each experiment were independently conducted.

In parallel, ERK phosphorylation gradually increased over time Next, we used a SOS1 S1178A nonphosphorylatable mutant (Fig. 3A). This is consistent with the hypothesis that treatment (32) to prevent inhibition of the SOS1–GRB2 association through with 1 increases phospho-ERK levels that result in the negative ERK-mediated phosphorylation on SOS1. After overexpression of regulation of SOS1 activity through phosphorylation at S1178. HA-SOS1S1178A, some enhancement of the baseline interaction

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RAS Activator Compound Regulates ERK via Feedback on SOS1

between SOS1 and GRB2 was observed, compared with cells SOS1, and this is the primary mechanism modulating cellular overexpressing HA-SOS1WT (Fig. 3B). This is consistent with the responses to compound. literature showing that phosphorylation at S1178 of SOS1 negatively regulates the association between SOS1 and GRB2 Changes in RAS-GTP levels in response to treatment with 1 are (32). After overexpression of HA-SOS1S1178A, we hypothesized mediated through negative feedback on SOS1 at S1178 that there would be no disruption of the SOS1–GRB2 interac- SOS1 is localized to the membrane through interactions with tion in response to 1 treatment. After a 60- to 90-minute GRB2 (4). As RAS is constitutively membrane bound, membrane treatment with 1, we did not see marked inhibition of the localization of SOS1 facilitates activation of RAS to its GTP-bound HA-SOS1S1178A–GRB2 association below the vehicle control state (2, 4, 33). Because overexpression of HA-SOS1S1178A reduced baseline, as was observed in HA-SOS1WT cells (Fig. 3A and B). compound-mediated dissociation of SOS1 and GRB2 (Fig. 3B), Similarly, the GRB2–SOS1 association was maintained at we anticipated that in cells expressing HA-SOS1S1178A or consti- baseline after stimulation with FBS in cells overexpressing tutively membrane bound HA-SOS1CAAX, the biphasic changes in HA-SOS1S1178A (Fig.3B).Furthermore,weusedaSOS1CAAX RAS-GTP levels in response to 1 over time would be diminished, box construct (2) to constitutively localize SOS1 to the cell as compared with the response of cells expressing HA-SOS1WT.To membrane. The baseline SOS1–GRB2 association was lower in test this, we overexpressed HA-SOS1WT, HA-SOS1S1178A, and HA- cells expressing HA-SOS1CAAX as compared with cells expres- SOS1CAAX in HeLa cells and assessed RAS-GTP levels in response sing HA-SOS1WT, and this may have been due to the position of to an activating concentration of 1, in a time-dependent fashion. the CAAX tag on SOS1 interfering with C-terminal GRB2 After HA-SOS1WT overexpression, we observed a similar biphasic binding domain of SOS1 (Fig. 3C). Similarly to cells expressing pattern of RAS-GTP stimulation and phospho-ERK activation as HA-SOS1S1178A, we did not observe marked disruption of in HeLa cells without SOS1 overexpression (Figs. 4A and 1C). the GRB2-HA-SOS1CAAX association after stimulation with However, after overexpression of either HA-SOS1S1178A or HA- FBS or 1 treatment (Fig. 3C). Overall, these data show that 1 SOS1CAAX, RAS-GTP levels remained elevated at later time points, disrupts the GRB2–SOS1 interaction, and this may be due to independent of phospho-ERK levels (Fig. 4B and C). Consistent phosphorylation of S1178 on SOS1 by active ERK. with the literature (35), these data suggest that membrane local- As there was still a partial disruption of the HA-SOS1S1178A– ization of SOS1 either through CAAX box expression or consti- GRB2associationinresponseto1 treatment (Fig. 3B), we tutive GRB2 association is the key to maintaining RAS-GTP levels, hypothesized that active ERK-mediated phosphorylation and based on these results, we suggest that phosphorylation of on SOS1 may not be the sole mechanism mediating the SOS1 at S1178 negatively regulates SOS1-mediated RAS activa- SOS1–GRB2associationinthepresenceof1.Totestthis,we tion, in the context of treatment with 1. pretreated cells expressing HA-SOS1WT with ERK1/2 inhibitor We propose a negative feedback mechanism through which SCH772984 for 60 minutes and then assessed the HA-SOS1WT– treatment with 1 leads to changes in RAS-GTP and phospho-ERK GRB2 association in the presence of 1 or FBS. Importantly, signaling over time (Fig. 5A and B). The responses to compound ERK inhibitor pretreatment prevented compound 1–induced are split into two main phases, the early and late responses. dissociation between SOS1 and GRB2 that we observed with 1 Initially, 1 binds to SOS1 and stimulates the exchange of GDP treatment or FBS treatment alone (Fig. 3D). However, there was for GTP on RAS, characterized by a rapid induction in RAS-GTP still partial disruption of the HA-SOS1WT–GRB2 association levels. Increased levels of RAS-GTP result in enhanced down- in the presence of the ERK inhibitor, indicating that other stream signaling, as evidenced by an increase in phospho-ERK negative feedback mechanisms may regulate this association. levels. Subsequently, a negative feedback loop causes SOS1 to be One possibility is that SPRY2 is phosphorylated at Y55 in phosphorylated at the C-terminus by the active form of ERK. response to ERK pathway activation (26). Phospho-SPRY2Y55 Phosphorylation of SOS1 at S1178 disrupts the interaction is known to negatively regulate RAS-GTP levels through inter- between GRB2 and SOS1, hence delocalizing SOS1 from the action with GRB2, resulting in sequestration of GRB2 and vicinity of RAS. This results in a gradual decrease over time in SOS1 from the membrane (26). Therefore, we assessed phos- RAS-GTP and consequently in phospho-ERK levels at later stages pho-SPRY2 levels in response to 1 treatment over a time course of response to compound. of up to 90 minutes. To achieve this, we overexpressed V5-SPRY2 alone (Supplementary Fig. S5) or in combination S1178A Discussion with HA-SOS1 (Supplementary Fig. S6) in 293 cells, immunoprecipitatedSPRY2usingtheV5tagandassessed Our laboratory has discovered compounds that bind to SOS1 tyrosine phosphorylation on SPRY2. We did not observe tyro- and activate RAS, resulting in biphasic signaling in the MAPK sine phosphorylation of SPRY2 in 293 cells, as has been pathway (17). The experiments outlined here help deﬁne the previously reported in HeLa cells (26). These data suggest that biological mechanism of action of a RAS activator compound. SPRY2 phosphorylation on tyrosine residues does not mediate Overall, our data show that a decrease in ERK phosphorylation is cellular response to 1 treatment in 293 cells. achieved via induction of a negative feedback loop to override We also assessed the effects of compound treatment on compound-mediated stimulation of RAS-GTP. Furthermore, DUSP16 and DUSP4 protein levels and saw small increases membrane localization of SOS1 may be important in the context in the levels of both proteins at later time points of 1 treatment of 1 treatment; substantiating the mechanistic hypothesis that in 293 cells, as compared with the vehicle control (Fig. 3D). compound-mediated phosphorylation of SOS1 by ERK can neg- Overall, although there may be multiple cell line–speciﬁc atively regulate its localization in proximity to RAS through mechanisms modulating molecular responses to 1 treatment, dissociation with GRB2. our data suggest that treatment with 1 causes dissociation of We propose that modulation of the association between SOS1 SOS1 and GRB2 through ERK-mediated phosphorylation of and GRB2 is the main mechanism mediating cell response to

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Figure 4. RAS-GTP levels are maintained above baseline in response to treatment with 1 in cells overexpressing S1178A and CAAX SOS1 mutants. HeLa cells were transiently transfected with HA-SOS1WT (A), HA-SOS1S1178A (B), HA-SOS1CAAX (C), and treated with 1 (50 mmol/L) over a time course of up to 150 minutes. EGF (50 ng/mL for 5 minutes) was used as a positive control. Two biological repeats of each experiment have been independently conducted.

compound; however, our data also suggest that additional feed- SOS1–GRB2 association is important for mediating response to 1 back mechanisms may contribute to the biphasic signaling phe- in cells, and consistent with our ﬁndings, targeted inhibition of notype that we observe. There are multiple alternative molecular the SOS1–GRB2 interaction using a reactive peptide also causes mechanisms that regulate SOS1 membrane localization, such as inhibition of phospho-ERK levels (37). PIP2 binding via the pleckstrin homology domain of SOS1, and Here, we have shown that treatment with compound 1 can lead allosteric binding of RAS to the CDC25 domain of SOS1 (2, 36). to activation of RAS–GTP and of downstream ERK signaling. We show that total DUSP4 and DUSP16 protein levels were Although seemingly counterintuitive, we speculate that rapid increased in response to compound treatment in 293 cells, but hyperactivation of an oncogenic pathway may be a valid thera- not in HeLa cells, and so even though we observe similar biphasic peutic strategy. In support of this, early experiments using trans- signaling phenotypes in response to RAS activator compound forming retroviruses showed that overexpression of oncogenes treatment across different cell lines, multiple feedback mechan- can result in growth arrest and cell death, for example, activation isms may occur to achieve the same overall phenotype, and of RAS in rat embryonic ﬁbroblasts leads to growth arrest (38), these could include changes in DUSP and SPRY total- and phos- and overexpression of active RAS in glioblastoma cells results in pho-protein levels. Nonetheless, our experiments show that the autophagic cell death (39). Furthermore, a publication from the

1058 Mol Cancer Ther; 17(5) May 2018 Molecular Cancer Therapeutics

RAS Activator Compound Regulates ERK via Feedback on SOS1

Figure 5. Negative feedback model for modulation of RAS-GTP and phospho-ERK signaling by RAS activator compound 1. A, Early signaling responses to compound (up to 30 minutes posttreatment). Activator compound 1 (hexagon) binds to SOS1, stimulates exchange of RAS-GDP to RAS-GTP, and subsequently activates downstream signaling. Activated ERK phosphorylates SOS1 and can also increase DUSP protein expression levels in a cell line–speciﬁc manner. B, Late signaling responses to activator compounds (past 60 minutes treatment). Phosphorylation of SOS1 disrupts the interaction between GRB2, hence preventing association with RAS. RAS-GTP levels and downstream signaling are inhibited. DUSPs can also inhibit ERK activation.

Varmus laboratory showed that mutual exclusivity of oncogenic Analysis and interpretation of data (e.g., statistical analysis, biostatistics, KRAS and EGFR mutations is likely due to synthetic lethality computational analysis): J.E. Howes, D.T. Akan, M.C. Burns and not functional redundancy (40). In cells coexpressing Writing, review, and/or revision of the manuscript: J.E. Howes, D.T. Akan, M.C. Burns, O.W. Rossanese, A.G. Waterson, S.W. Fesik both EGFR and RAS mutations, concurrent increases in both Study supervision: A.G. Waterson, S.W. Fesik phospho-ERK levels and PARP cleavage were observed. On the basis of these results, it was suggested that signaling agonists could Acknowledgments be utilized therapeutically to provoke synthetic lethal events in the This work was supported by a Research Investigator Award from the Lust- presence of another mutant oncogene (40). Thus, it may be garten Foundation awarded to S.W. Fesik in 2015. possible that a RAS activator compound could provide a useful We thank our colleagues in the Fesik group (Vanderbilt University) for approach to therapeutically impair oncogenic signaling. helpful discussions, and we acknowledge P. Patel, J. Abbott, and A. Little (Vanderbilt University, Fesik Lab) for synthesizing RAS activator compound 1 and T. Sobolik (Vanderbilt University) for help with DNA preparation. We Disclosure of Potential Conﬂicts of Interest acknowledge B. Papke and C. Der (University of North Carolina, Chapel Hill) RAS activator compounds have been licensed to Boehringer Ingelheim. for discussions and experimental ideas.

Authors' Contributions The costs of publication of this article were defrayed in part by the payment of advertisement Conception and design: J.E. Howes, D.T. Akan, M.C. Burns, O.W. Rossanese, page charges. This article must therefore be hereby marked in A.G. Waterson, S.W. Fesik accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Development of methodology: J.E. Howes, D.T. Akan, M.C. Burns Acquisition of data (provided animals, acquired and managed patients, Received July 12, 2017; revised December 11, 2017; accepted January 11, provided facilities, etc.): J.E. Howes, D.T. Akan, M.C. Burns 2018; published ﬁrst February 13, 2018.

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1060 Mol Cancer Ther; 17(5) May 2018 Molecular Cancer Therapeutics

Small Molecule−Mediated Activation of RAS Elicits Biphasic Modulation of Phospho-ERK Levels that Are Regulated through Negative Feedback on SOS1

Jennifer E. Howes, Denis T. Akan, Michael C. Burns, et al.

Mol Cancer Ther 2018;17:1051-1060. Published OnlineFirst February 13, 2018.

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