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Diffuse gastric cancer (DGC) is a lethal malignancy lacking effective systemic therapy. Among the most provocative recent results in DGC has been that of highly recurrent missense mutations in the GTPase RHOA. The function of these mutations has remained unresolved. We demonstrate that RHOAY42C, the most common RHOA mutation in DGC, is a gain-of-function oncogenic mutant, and that expression of RHOAY42C with inactivation of the canonical tumor suppressor Cdh1 induces metastatic DGC in a mouse model. Biochemically, RHOAY42C exhibits impaired GTP hydrolysis and enhances interaction with its effector ROCK. RHOAY42C mutation and Cdh1 loss induce actin/cytoskeletal rearrangements and activity of focal adhesion kinase (FAK), which activates YAP–TAZ, PI3K–AKT, and β-catenin. RHOAY42C murine models were sensitive to FAK inhibition and to combined YAP and PI3K pathway blockade. These results, coupled with sensitivity to FAK inhibition in patient-derived DGC cell lines, nominate FAK as a novel target for these cancers.Significance:The functional significance of recurrent RHOA mutations in DGC has remained unresolved. Through biochemical studies and mouse modeling of the hotspot RHOAY42C mutation, we establish that these mutations are activating, detail their effects upon cell signaling, and define how RHOA-mediated FAK activation imparts sensitivity to pharmacologic FAK inhibitors.See related commentary by Benton and Chernoff, p. 182.This article is highlighted in the In This Issue feature, p. 161

Supplementary Methods

List of Models

Supplementary Video S2

Oncogenic KRAS drives cancer growth by activating diverse signaling networks, not all of which have been fully delineated. We set out to establish a system-wide profile of the KRAS-regulated kinase signaling network (kinome) in KRAS-mutant pancreatic ductal adenocarcinoma (PDAC). We knocked down KRAS expression in a panel of six cell lines, and then applied Multiplexed Inhibitor Bead/Mass Spectrometry (MIB/MS) chemical proteomics to monitor changes in kinase activity and/or expression. We hypothesized that depletion of KRAS would result in downregulation of kinases required for KRAS-mediated transforming activities, and in upregulation of other kinases that could potentially compensate for the deleterious consequences of the loss of KRAS. We identified 15 upregulated and 13 downregulated kinases in common across the panel. In agreement with our hypothesis, all 15 of the upregulated kinases have established roles as cancer drivers (e.g., SRC, TGFBR1, ILK), and pharmacologic inhibition of the upregulated kinase, DDR1, suppressed PDAC growth. Interestingly, 11 of the 13 downregulated kinases have established driver roles in cell cycle progression, particularly in mitosis (e.g., WEE1, Aurora A, PLK1). Consistent with a crucial role for the downregulated kinases in promoting KRAS-driven proliferation, we found that pharmacologic inhibition of WEE1 also suppressed PDAC growth. The unexpected paradoxical activation of ERK upon WEE1 inhibition led us to inhibit both WEE1 and ERK concurrently, which caused further potent growth suppression and enhanced apoptotic death than WEE1 inhibition alone. We conclude that system-wide delineation of the KRAS-regulated kinome can identify potential therapeutic targets for KRAS-mutant pancreatic cancer.

SUMMARY To identify therapeutic targets for KRAS mutant pancreatic cancer, we conduct a druggable genome small interfering RNA (siRNA) screen and determine that suppression of BCAR1 sensitizes pancreatic cancer cells to ERK inhibition. Integrative analysis of genome-scale CRISPR-Cas9 screens also identify BCAR1 as a top synthetic lethal interactor with mutant KRAS. BCAR1 encodes the SRC substrate p130Cas. We determine that SRC-inhibitor-mediated suppression of p130Cas phosphorylation impairs MYC transcription through a DOCK1-RAC1-β-catenin-dependent mechanism. Additionally, genetic suppression of TUBB3, encoding the βIII-tubulin subunit of microtubules, or pharmacological inhibition of microtubule function decreases levels of MYC protein in a calpain-dependent manner and potently sensitizes pancreatic cancer cells to ERK inhibition. Accordingly, the combination of a dual SRC/tubulin inhibitor with an ERK inhibitor cooperates to reduce MYC protein and synergistically suppress the growth of KRAS mutant pancreatic cancer. Thus, we demonstrate that mechanistically diverse combinations with ERK inhibition suppress MYC to impair pancreatic cancer proliferation., Graphical Abstract, In brief Using integrated analysis of genome-scale screens, Waters et al. identify BCAR1 and TUBB3, encoding p130Cas and βIII-tubulin, as two pancreatic cancer vulnerabilities that, when inhibited, sensitize KRAS mutant PDAC cells to ERK inhibition. Mechanistically, inhibition of p130Cas, βIII-tubulin, and ERK converges on and drives MYC loss to inhibit pancreatic cancer growth.

Oncogenic KRAS drives cancer growth by activating diverse signaling networks, not all of which have been fully delineated. We set out to establish a system-wide profile of the KRAS-regulated kinase signaling network (kinome) in KRAS-mutant pancreatic ductal adenocarcinoma (PDAC). We knocked down KRAS expression in a panel of six cell lines and then applied multiplexed inhibitor bead/MS to monitor changes in kinase activity and/or expression. We hypothesized that depletion of KRAS would result in downregulation of kinases required for KRAS-mediated transformation and in upregulation of other kinases that could potentially compensate for the deleterious consequences of the loss of KRAS. We identified 15 upregulated and 13 downregulated kinases in common across the panel of cell lines. In agreement with our hypothesis, all 15 of the upregulated kinases have established roles as cancer drivers (e.g., SRC, TGF-β1, ILK), and pharmacological inhibition of one of these upregulated kinases, DDR1, suppressed PDAC growth. Interestingly, 11 of the 13 downregulated kinases have established driver roles in cell cycle progression, particularly in mitosis (e.g., WEE1, Aurora A, PLK1). Consistent with a crucial role for the downregulated kinases in promoting KRAS-driven proliferation, we found that pharmacological inhibition of WEE1 also suppressed PDAC growth. The unexpected paradoxical activation of ERK upon WEE1 inhibition led us to inhibit both WEE1 and ERK concurrently, which caused further potent growth suppression and enhanced apoptotic death compared with WEE1 inhibition alone. We conclude that system-wide delineation of the KRAS-regulated kinome can identify potential therapeutic targets for KRAS-mutant pancreatic cancer.

To identify therapeutic targets for KRAS-mutant pancreatic cancer, we conducted a druggable genome siRNA screen and determined that suppression of BCAR1 sensitizes pancreatic cancer cells to ERK inhibition. Integrative analysis of genome-scale CRISPR-Cas9 screens also identified BCAR1 as a top synthetic lethal interactor with mutant KRAS. BCAR1 encodes the SRC substrate p130Cas. We determined that SRC inhibitor-mediated suppression of p130Cas phosphorylation impairs MYC transcription through a DOCK1-RAC1-beta-catenin dependent mechanism. Additionally, genetic suppression of TUBB3, encoding the betaIII-tubulin subunit of microtubules, or pharmacological inhibition of microtubule function, decreased levels of MYC protein and potently sensitized pancreatic cancer cells to ERK inhibition. Accordingly, the combination of a dual SRC/tubulin inhibitor with an ERK inhibitor cooperated to reduce MYC protein and to synergistically suppress the growth of KRAS-mutant pancreatic cancer. Thus, we demonstrate that mechanistically diverse combinations with ERK inhibition suppress MYC to impair pancreatic cancer proliferation.

Diffuse gastric cancer (DGC) is a lethal malignancy lacking effective systemic therapy. Among the most provocative recent results in DGC has been that of highly recurrent missense mutations in the GTPase RHOA. The function of these mutations has remained unresolved. We demonstrate that RHOAY42C, the most common RHOA mutation in DGC, is a gain-of-function oncogenic mutant, and that expression of RHOAY42C with inactivation of the canonical tumor suppressor Cdh1 induces metastatic DGC in a mouse model. Biochemically, RHOAY42C exhibits impaired GTP hydrolysis and enhances interaction with its effector ROCK. RHOAY42C mutation and Cdh1 loss induce actin/cytoskeletal rearrangements and activity of focal adhesion kinase (FAK), which activates YAP–TAZ, PI3K–AKT, and β-catenin. RHOAY42C murine models were sensitive to FAK inhibition and to combined YAP and PI3K pathway blockade. These results, coupled with sensitivity to FAK inhibition in patient-derived DGC cell lines, nominate FAK as a novel target for these cancers. Significance: The functional significance of recurrent RHOA mutations in DGC has remained unresolved. Through biochemical studies and mouse modeling of the hotspot RHOAY42C mutation, we establish that these mutations are activating, detail their effects upon cell signaling, and define how RHOA-mediated FAK activation imparts sensitivity to pharmacologic FAK inhibitors. See related commentary by Benton and Chernoff, p. 182. This article is highlighted in the In This Issue feature, p. 161

Stabilization of the MYC oncoprotein by KRAS signaling critically promotes the growth of pancreatic ductal adenocarcinoma (PDAC). Thus, understanding how MYC protein stability is regulated may lead to effective therapies. Here, we used a previously developed, flow cytometry-based assay that screened a library of >800 protein kinase inhibitors and identified compounds that promoted either the stability or degradation of MYC in a KRAS-mutant PDAC cell line. We validated compounds that stabilized or destabilized MYC and then focused on one compound, UNC10112785, that induced the substantial loss of MYC protein in both two-dimensional (2D) and 3D cell cultures. We determined that this compound is a potent CDK9 inhibitor with a previously uncharacterized scaffold, caused MYC loss through both transcriptional and posttranslational mechanisms, and suppresses PDAC anchorage-dependent and anchorage-independent growth. We discovered that CDK9 enhanced MYC protein stability through a previously unknown, KRAS-independent mechanism involving direct phosphorylation of MYC at Ser62 Our study thus not only identifies a potential therapeutic target for patients with KRAS-mutant PDAC but also presents the application of a screening strategy that can be more broadly adapted to identify regulators of protein stability.

Diffuse gastric cancer (DGC) is characterized by frequent missense mutations in the small GTPase RHOA, but the effects of this mutation on enzyme activity and signaling have been widely debated. In this work, the authors show that the most common RHOA mutation in DGC, encoding RHOA(Y42C), represents a gain-of-function, that a mouse model incorporating this mutation in association with loss of the E-cadherin gene CDH1, recapitulates many aspects of DGC, and that rationally designed therapeutics based on our understanding of RHOA signaling are promising agents for treating DGC.

Missense mutations of the RAS homologous RHOA gene have recently been identified in ~25% of diffuse gastric cancer (DGC). Unexpectedly, in contrast to the gain-of-function hotspot mutations found in RAS in cancer, DGC-associated RHOA mutations (e.g., R5W, Y42C) are localized at different hotspots that instead suggest loss-of-function alterations. First, we utilized bacterially expressed recombinant proteins and determined the biochemical consequences of these mutations on RHOA function. We found that RHOA WT and Y42C, but not R5W nucleotide exchange, was catalyzed by the ECT2 RhoGEF. Similarly, WT and Y42C, but not R5W GTP hydrolysis activity, was stimulated by the p190RhoGAP. Surprisingly, Y42C and to a lesser degree R5W exhibited impaired intrinsic GTP hydrolysis activity. Interestingly, Y42C showed both loss- and gain-of-function interaction with effectors. Second, we ectopically expressed WT and mutant RHOA proteins in NIH/3T3 fibroblast, and surprisingly, found that Y42C and R5W, similar to the lab-generated constitutively GTP-bound Q63L mutant (analogous to RAS Q61L), stimulated actin stress fiber formation and focal adhesion assembly. Finally, when ectopically expressed in beta-catenin-deficient (another mutation found in gastric cancer) mouse gastric organoids, Y42C disrupted the 3D architecture and induced a DGC-like phenotype in vitro and in vivo. In summary, our studies suggest mutation-specific biochemical defects that act as gain-of-function oncogenic RHOA mutations. Citation Format: Antje Schaefer, Richard G. Hodge, Devon R. Blake, Haisheng Zhang, Adam J. Bass, Channing J. Der. The gastric cancer-associated mutations R5W and Y42C in the RAS homologous RHOA protein cause distinct biochemical alterations, exhibit gain-of-function signaling and oncogenic activities [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A29.

G-protein β subunits perform essential neuronal functions as part of G-protein βγ and Gβ5-regulators of G-protein signaling (RGS) complexes. Both Gβγ and Gβ5-RGS are obligate dimers that are thought to require the assistance of the cytosolic chaperonin CCT and a cochaperone, phosducin-like protein 1 (PhLP1) for dimer formation. To test this hypothesisin vivo, we deleted thePhlp1gene in mouse (Mus musculus) retinal rod photoreceptor cells and measured the effects on G-protein biogenesis and visual signal transduction. In the PhLP1-depleted rods, Gβγ dimer formation was decreased 50-fold, resulting in a >10-fold decrease in light sensitivity. Moreover, a 20-fold reduction in Gβ5and RGS9–1 expression was also observed, causing a 15-fold delay in the shutoff of light responses. These findings conclusively demonstratein vivothat PhLP1 is required for the folding and assembly of both Gβγ and Gβ5-RGS9.

Haptotaxis is the process by which cells respond to gradients of substrate-bound cues, such as extracellular matrix proteins (ECM); however, the cellular mechanism of this response remains poorly understood and has mainly been studied by comparing cell behavior on uniform ECMs with different concentrations of components. To study haptotaxis in response to gradients, we utilized microfluidic chambers to generate gradients of the ECM protein fibronectin, and imaged the cell migration response. Lamellipodia are fan-shaped protrusions that are common in migrating cells. Here, we define a new function for lamellipodia and the cellular mechanism required for haptotaxis – differential actin and lamellipodial protrusion dynamics lead to biased cell migration. Modest differences in lamellipodial dynamics occurring over time periods of seconds to minutes are summed over hours to produce differential whole cell movement towards higher concentrations of fibronectin. We identify a specific subset of lamellipodia regulators as being crucial for haptotaxis. Numerous studies have linked components of this pathway to cancer metastasis and, consistent with this, we find that expression of the oncogenic Rac1 P29S mutation abrogates haptotaxis. Finally, we show that haptotaxis also operates through this pathway in 3D environments.

Our recent ERK inhibitor analyses in pancreatic ductal adenocarcinoma (PDAC) demonstrated that MYC protein loss is a marker of inhibitor sensitivity, and indicated that resistant cell lines possess KRASdependent, ERK-independent mechanisms maintaining MYC protein levels. In seeking to identify these mechanisms, we first determined the signaling networks by which mutant KRAS regulates MYC. Acute KRAS suppression caused rapid proteasome-dependent loss of MYC protein, through both ERK1/2-dependent and -independent mechanisms. Surprisingly, this MYC degradation was independent of both PI3K-AKTGSK3β signaling and the E3 ligase FBWX7. We then established and applied a high-throughput screen for MYC protein degradation, and performed a kinome-wide proteomics screen. We identified an ERK1/2- inhibition-induced feed-forward mechanism dependent on EGFR and SRC, leading to ERK5 activation and phosphorylation of MYC at S62, preventing MYC degradation. We further determined that concurrent inhibition of ERK1/2 and ERK5 disrupted this feed-forward compensatory mechanism, synergistically causing loss of MYC and suppressing PDAC growth.

G protein β subunits (Gβ) play essential roles in phototransduction as part of G protein βγ (Gβγ) and regulator of G protein signaling 9 (RGS9)-Gβ5 heterodimers. Both are obligate dimers that rely on the cytosolic chaperone CCT and its co-chaperone PhLP1 to form complexes from their nascent polypeptides. The importance of PhLP1 in the assembly process was recently demonstrated in vivo in a retinal rod-specific deletion of the Phlp1 gene. To test whether this is a general mechanism that also applies to other cell types, we disrupted the Phlp1 gene specifically in mouse cones and measured the effects on G protein expression and cone visual signal transduction. In PhLP1-deficient cones, expression of cone transducin (Gt2) and RGS9-Gβ5 subunits was dramatically reduced, resulting in a 27-fold decrease in sensitivity and a 38-fold delay in cone photoresponse recovery. These results demonstrate the essential role of PhLP1 in cone G protein complex formation. Our findings reveal a common mechanism of Gβγ and RGS9-Gβ5 assembly in rods and cones, highlighting the importance of PhLP1 and CCT-mediated Gβ complex formation in G protein signaling.

With the nearly 100% mutation frequency of KRAS in pancreatic ductal adenocarcinoma (PDAC), the development of therapeutic strategies to target KRAS is a high priority for the pancreatic cancer field. In the current study, we aimed to identify signaling changes caused by the acute suppression of mutant KRAS in PDAC cell lines. Strikingly, acute suppression of mutant KRAS in PDAC cell lines caused potent and rapid proteasome-dependent degradation of MYC protein. Ablation of MYC also suppressed PDAC growth both in vitro and in vivo, indicating a critical driver role for MYC in KRAS-dependent PDAC maintenance. A mechanism by which RAS effector signaling regulates MYC protein stability has been described, however we determined that this mechanism cannot fully account for how endogenous mutant KRAS stabilizes MYC protein in PDAC cells. We verified a role for the Raf-MEK-ERK but not the PI3K-AKT-GSK3β effector signaling pathway. Unexpectedly, we also excluded a role for MYC protein phosphorylation at MYC residue T58, and determined that ubiquitin ligases other than FBW7 are involved. These findings prompted us to search for additional KRAS-dependent protein kinases that facilitate MYC protein stability. We applied multiple screening strategies. First, we developed a novel fluorescence-based system to monitor real-time MYC protein degradation in PDAC cells and we adapted this system for use in a high-throughput flow-cytometry based assay. Second, we applied a mass spectrometry-based approach to profile the human kinome for KRAS-dependent changes in protein kinases. Finally, we applied gain-of-function (Cancer Toolkit) and loss-of-function (CRISPR/Cas9) genetic screens to identify signaling regulators of MYC protein stability. The results of these screens will be presented. Citation Format: Angelina V. Vaseva, Devon R. Blake, Salma H. Azam, Karim T. Gilbert, Chad V. Pecot, Kenneth H. Pearce, Laura E. Herring, Lee M. Graves, Peter J. Houghton, Channing J. Der. Regulation of MYC protein stability by mutant KRAS in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4458. doi:10.1158/1538-7445.AM2017-4458