Ripretinib and MEK Inhibitors Synergize to Induce Apoptosis In

Published OnlineFirst May 4, 2021; DOI: 10.1158/1535-7163.MCT-20-0824

MOLECULAR CANCER THERAPEUTICS | SMALL MOLECULE THERAPEUTICS

Ripretinib and MEK Inhibitors Synergize to Induce Apoptosis in Preclinical Models of GIST and Systemic Mastocytosis A C Anu Gupta1, Jarnail Singh1, Alfonso García-Valverde2, César Serrano2,3, Daniel L. Flynn1, and Bryan D. Smith1

ABSTRACT ◥ The majority of gastrointestinal stromal tumors (GIST) harbor inhibitors in cell lines and mouse models. Ripretinib potently constitutively activating mutations in KIT tyrosine kinase. Imatinib, inhibits a broad spectrum of primary and drug-resistant KIT/ sunitinib, and regorafenib are available as ﬁrst-, second-, and third- PDGFRA mutants and is approved by the FDA for the treatment line targeted therapies, respectively, for metastatic or unresectable of adult patients with advanced GIST who have received previous KIT-driven GIST. Treatment of patients with GIST with KIT kinase treatment with 3 or more kinase inhibitors, including imatinib. Here inhibitors generally leads to a partial response or stable disease but we show that ripretinib treatment in combination with MEK most patients eventually progress by developing secondary resis- inhibitors is effective at inducing and enhancing the apoptotic tance mutations in KIT. Tumor heterogeneity for secondary resis- response and preventing growth of resistant colonies in both tant KIT mutations within the same patient adds further complexity imatinib-sensitive and -resistant GIST cell lines, even after long- to GIST treatment. Several other mechanisms converge and reac- term removal of drugs. The effect was also observed in systemic tivate the MAPK pathway upon KIT/PDGFRA–targeted inhibition, mastocytosis (SM) cells, wherein the primary drug–resistant KIT generating treatment adaptation and impairing cytotoxicity. To D816V is the driver mutation. Our results show that the combi- address the multiple potential pathways of drug resistance in GIST, nation of KIT and MEK inhibition has the potential to induce the KIT/PDGFRA inhibitor ripretinib was combined with MEK cytocidal responses in GIST and SM cells.

Introduction imatinib withdrawal (9, 10). GIST most often becomes imatinib- resistant after acquiring secondary mutations in the KIT kinase Gastrointestinal stromal tumors (GIST) originate from the inter- domain that directly abrogate the binding of imatinib to KIT or that stitial cells of Cajal and are the most common malignant mesenchymal lead to conformational escape to type I conformations to which tumors of the gastrointestinal tract (1). GIST is driven by mutually imatinib does not bind (11). Many secondary mutations are found exclusive mutations in two receptor tyrosine kinases (KIT and in the ATP-binding pocket or the activation loop. Sunitinib and PDGFRA), which result in their constitutive activation. Eighty percent regorafenib, approved as second- and third-line therapy for GIST, to 85% of GIST have mutations in KIT and an additional 5%–10% have respectively, target only a subset of secondary resistant mutations in PDGFRA (2). Imatinib mesylate, a small-molecule mutations (12–15). Different resistant mutations can arise in meta- tyrosine kinase inhibitor (TKI) effectively inhibits KIT activity, and static tumor sites, and individual patients often acquire multiple is used as a ﬁrst line of therapy for treatment of metastatic GIST (3). secondary mutations in KIT during the course of therapy (16). First-line imatinib has proven to be effective in stabilizing disease Although the majority of resistance mutations occur within the KIT progression in most GIST patients, but only 2% of patients experience gene, recent genomic data has shown that mutations arise in other complete regression (4). While median progression-free survival genes as well, such as SDH, KRAS, BRAF, NF1, PI3KCA, and (mPFS) in the ﬁrst-line setting is 18.9 months, most patients with PTEN (17–19). Imatinib treatment can activate the MAPK pathway imatinib-treated GIST experience progressive disease and no longer through receptor tyrosine kinase switch (20) or reactivate MAPK respond to imatinib treatment (5–8). Imatinib treatment does not signaling through a positive feedback circuit wherein MAPK activation induce tumor eradication and long-term treatment with imatinib downstream of KIT stabilizes the transcription factor ETV1, leading to results in a cellular quiescent state and tumor cells start to grow upon a positive feedback loop upregulating KIT expression (21). Combined targeting of ETV1 stability by imatinib and the MEK inhibitor binimetinib resulted in increased growth suppression in imatinib- 1 2 Deciphera Pharmaceuticals, LLC, Waltham, Massachusetts. Sarcoma Trans- sensitive disease (21). Hence, novel therapeutic approaches are needed lational Research Laboratory, Vall d’Hebron Institute of Oncology (VHIO), Barcelona, Spain. 3Department of Medical Oncology, Vall d’Hebron University to treat patients with KIT mutational heterogeneity and KIT inde- Hospital, Barcelona, Spain. pendent pathway mutations that result in reactivation of the MAPK pathway. Given the convergence of these mechanisms feeding into the Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). RAS/MAPK pathway, a potential treatment strategy would be to combine a broad-spectrum KIT inhibitor with an inhibitor of the Corresponding Author: Bryan D. Smith, Deciphera Pharmaceuticals, LLC, 200 MAPK pathway. In addition, combination therapy provides the Smith Street, Waltham, MA 02451. Phone: 785-830-2113; Fax: 785-830-2150; E-mail: [email protected] potential to increase cytocidal activity compared with cytostatic responses to KIT monotherapy. Mol Cancer Ther 2021;XX:XX–XX Ripretinib was designed to target a broad spectrum of primary and doi: 10.1158/1535-7163.MCT-20-0824 secondary drug-resistant mutations in KIT and PDGFRA and is 2021 American Association for Cancer Research. currently in clinical trials for advanced GIST (22, 23). Ripretinib is

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a “switch-control” kinase inhibitor designed to force even aggressively Western blot assays activated mutants into an inactive conformation. It inhibits KIT Cells were treated with drugs for 48 hours and cell lysates were activity of highly resistant KIT mutations including D816V (22). In prepared as described previously (22). Electrophoresis was carried out the randomized, double-blind, placebo-controlled INVICTUS phase in 10% polyacrylamide gels and transferred to nitrocellulose mem- III clinical study of ripretinib in patients with advanced GIST previ- branes. Bands were detected by incubating with Immobilon Forte ously treated with ≥3 prior TKIs, ripretinib demonstrated a significant Western HRP substrate (Millipore-MERCK KGaA) and captured by improvement in mPFS compared with placebo [6.3 vs. 1 months, chemiluminescence with Amersham Imager 600 (GE Healthcare Life respectively; HR, 0.15 (95% confidence interval (CI), 0.09–0.25); P < Science). Primary antibodies to phospho-KIT Y703 (3073; RRID: 0.0001] and had a favorable overall safety profile; the ORR was 9.4% vs. AB_1147635), Bim (2819; RRID:AB_10692515), phospho-Histone 0.0% (P ¼ 0.0504) for ripretinib and placebo, respectively H2A.X Ser139 (9718; RRID:AB_2118009), phospho-ERK Thr202/ (NCT0335373; ref. 23). In May 2020, the FDA approved ripretinib Tyr204 (9101; RRID:AB_331646), total ERK (9102; RRID: for the treatment of adult patients with advanced GIST who have AB_330744) were purchased from Cell Signaling Technology. Total received prior treatment with 3 or more kinase inhibitors, including KIT antibody (A4502; RRID:AB_2335702) was from DAKO and actin imatinib (24). (A4700; RRID:AB_476730) was from Sigma-Aldrich-MERCK Herein, we studied whether ripretinib, like other KIT-targeted (KGaA). therapies, has cytostatic activity and if it can be turned into cytotoxic response by combination therapy. We used ripretinib in combination Annexin V staining with three FDA approved MEK inhibitors (MEKi) in cellular assays of Cells were treated with various concentrations of drugs as shown in growth and apoptosis in both imatinib-sensitive and -resistant GIST figures for 24, 48, or 72 hours. A total of 1 106 cells were resuspended cell lines. GIST cell lines have been shown to be excellent models of in Hanks balanced salt solution (HBSS, Thermo Fisher Scientific, GIST tumor growth and response to drug treatment in vivo (14, 15, 25). 14025–092) and incubated with annexin V-FITC antibody (BD Bio- We also investigated the prevention of drug resistance in a cellular sciences, 550475; RRID:AB_2868885) for 15 minutes in the dark at model and in vivo efficacy of the ripretinib and MEKi combination. room temperature. Cells were incubated with propidium iodide at a Imatinib was used as a comparator in these studies. We also evaluated final concentration of 10 mg/mL (Sigma-Aldrich, P4170) for 5 minutes this combination in the systemic mastocytosis (SM) cell line HMC1.2. at room temperature and immediately analyzed by flow cytometry SM is driven by a KIT D816V mutation in 90% of patients and is with BD FACSCalibur from BD Biosciences. 10,000 cells were resistant to most KIT inhibitors in clinical practice (26, 27). The recorded and analyzed in all conditions. ripretinib/MEKi combination was also evaluated in HMC1.2 cells stably transfected with mutant NRASG12D, an acquired mutation Caspase assays found in advanced SM (28, 29). GIST cells at 10,000 cells/well were plated in complete media in 96-well white flat-bottom plates and drugs were added 24 hours later. Apoptosis was evaluated by Caspase Glo-3/7 assay system (Promega, Materials and Methods G8092) after 24 hours of treatment with inhibitors as per manu- Cell lines facturer’s instructions. A matrix of various concentrations of drugs GIST-T1 (RRID:CVCL_4976) and its derivative cell lines GIST-T1/ was used to measure caspase activity (10) and the BLISS synergy score 816 (with D816E mutation; RRID:CVCL_A9N0) and GIST-T1/670 was calculated with Combenefit software using 1/(fold caspase induc- (with T670I mutation; RRID:CVCL_A9M9) were obtained and cul- tion) with 100% being no induction and 0% being maximum induction tured as described previously (22). HMC1.2 (KIT-V560G/D816V) cell of caspase activity. line was obtained from Millipore-Sigma (SCC062; RRID: CVCL_H205). The Baf3-KIT-V560D cell line has been described Clonogenic assays previously (22). Cells were not further authenticated upon receipt. GIST-T1 and GIST-T1/D816E cells at 100 cells/well and GIST-T1/ Mycoplasma tests were performed using the MycoAlert Mycoplasma T670I cells at 500 cells/well were seeded in 6-well plates and treated Detection Kit (Lonza, LT07–318). Cells were expanded and frozen at with various concentrations of drugs alone or in combination for low passage number and were passaged <40 times before thawing a 2 weeks. The drugs were washed off with PBS twice and fresh complete fresh vial. Cells were grown in Iscove’s Modified Dulbecco’s Medium media without drug was added to the wells and incubated for 10 days to (IMDM; Life Technologies, 12240–046) with 10% FBS (Life Technol- check for any outgrowth of colonies. If there were no visible colonies, ogies, 16000–044) and penicillin–streptomycin-L-Glutamine (Life plates were incubated for additional 10 days with change of fresh Technologies, 10378–016). NRASG12D was cloned in PLVX-IRES media. (Clontech Laboratories, 632183) and virus was packaged in HEK293T cells with pVSVG and pCMVdR8.2 packaging plasmids. GIST-T1 and Soft-agar colony assays HMC1.2 cells were infected with lentivirus containing either empty Soft-agar colony formation assay was set up in 6-well plates with vector (EV) or NRASG12D and selected in media containing 1 mg/mL 2 mL base layer of 1% Difco noble agar (BD Biosciences, 214220) and puromycin (Sigma, P9620). 2 mL top layer of 0.36% agar with 10,000 cells/well in IMDM media. Agar was allowed to solidify for 2 hours at RT and 2 mL of IMDM Chemicals complete media with or without drug was added to each well. The Imatinib mesylate, ripretinib, and regorafenib (synthesized inter- media was carefully removed after 10 days of incubation. Wells were nally at Deciphera Pharmaceuticals according to published protocols), washed twice with PBS and fresh media without drug was added and sunitinib (LC Laboratories, S-8803), trametinib (LC Laboratories, incubated for 5 days. If no colonies were observed under the micro- T-8123), cobimetinib (Selleck Biochemicals, S8041), and binimetinib scope, cells were incubated for additional 8 days with fresh change of (Selleck Biochemicals, S7007) were prepared as 10 mmol/L stocks in media. Cells were stained with crystal violet (0.01% w/v in methanol) DMSO. and colonies were photographed and counted.

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Saturation mutagenesis activity in GIST-T1/D816E and GIST-T1/T670I cells, consistent with Ba/F3 cells stably transfected with V560D KIT kinase were treated its largely cytostatic activity in preclinical studies (22). The combina- with the mutagen ethylnitrosourea (ENU) for 18 hours (15). Cells were tion of ripretinib with trametinib exhibited cytocidal activity, showing washed several times, then 10,000 cells/well were plated in 96-well a dose-dependent increase (1.2–1.8-fold) in apoptosis in GIST-T1/ plates containing various concentrations of ripretinib or imatinib D816E and 2–4 fold increase in apoptosis in GIST-T1/T670I cells either alone or with 10 nmol/L of trametinib for 28 days. Wells (Fig. 1B and C). The combination of ripretinib and trametinib was showing cell growth were sequenced for KIT mutations as described synergistic in inducing apoptosis in both cell lines while combination previously (22). of imatinib and trametinib did not show synergy (Fig. 1D). Similarly, combinations of ripretinib with MEKi cobimetinib or binimetinib Mouse xenografts exhibited significant synergy in GIST-T1/D816E and GIST-T1/T670I Mouse studies were performed in compliance with all the laws, cells, whereas combinations with imatinib did not exhibit synergy regulations, and guidelines of the NIH and with the approval of the (GIST-T1), modest synergy (GIST-T1/T670I), or led to antagonism Animal Care and Use Committee of Molecular Imaging, Inc. or MI (GIST-T1/D816E; Supplementary Fig. S1). Bioresearch, AAALAC accredited facilities. Mice were housed in Apoptosis was also evaluated by Annexin V/PI staining in GIST-T1 Innovive disposable ventilated caging with corn cob bedding inside and GIST-T1/T670I cell lines. GIST-T1 showed an increase (18% ! Biobubble Clean Rooms, with 1–5 mice per cage. The light cycle was 40%) in apoptotic cells with imatinib or ripretinib alone after 72 hours 12-hour light/12-hour dark, with temperature at 70 2 F, and of treatment compared with DMSO control, and apoptosis increased humidity at 30%–70%. Mouse studies were done as described previ- to approximately 50% in combination with 25 nmol/L of trametinib ously (22). Briefly, tumors were staged for 5–10 days depending on with both drugs (Fig. 1E). tumor model. In the GIST T1 model, groups were treated from days Imatinib or ripretinib alone did not induce apoptosis in the ima- 10–27 as follows: Vehicle control diet (n ¼ 8); ripretinib formulated tinib-resistant GIST-T1/T670I cell line, and the imatinib/trametinib into the mouse diet to achieve approximately 100 mg/kg/day of combination failed to induce apoptosis as well, whereas the ripretinib/ ripretinib (n ¼ 10); or ripretinib formulated into the mouse diet to trametinib combination significantly increased apoptosis to 56% achieve approximately 25 mg/kg/day of ripretinib (n ¼ 10); trametinib compared with 18% in DMSO control (Fig. 1F). administered at 1 mg/kg PO twice a day (n ¼ 10) or combination MAPK activation is a major component of the KIT signaling groups (n ¼ 10 each). On day 27, all animals were placed on control pathway and is important for GIST cell survival. To corroborate the diet to monitor tumor regrowth. For the P815 allograft model, groups enhanced apoptotic effect of the combination of ripretinib and tra- were treated from days 5–28 as follows: Vehicle control diet (n ¼ 10); metinib, we evaluated the phosphorylation of ERK1/2 and levels of ripretinib formulated into the mouse diet to achieve approximately proapoptotic proteins BimEL and p-H2Ax. Phosphorylated H2Ax has 100 mg/kg/day of ripretinib (n ¼ 10); trametinib administered at been shown to be upregulated after imatinib-induced apoptosis in 0.5 mg/kg orally twice a day (5 days on/2 days off) (n ¼ 10); or GIST cells (30). Ripretinib treatment resulted in greater inhibition of ripretinib and trametinib in combination (n ¼ 10). For the HMC1.2 KIT and ERK1/2 phosphorylation (pERK) in both imatinib-resistant model, groups were treated from days 10–28 as follows: Vehicle control cell lines GIST-T1/D816E and GIST-T1/T670I compared with ima- diet (n ¼ 10); ripretinib formulated into the mouse diet to achieve tinib (Fig. 1G). Likewise, ripretinib as single agent induced higher approximately 100 mg/kg/day of ripretinib (n ¼ 10); trametinib apoptosis induction, as assessed by the expression of BimEL and administered at 0.3 mg/kg PO twice a day (n ¼ 10); or ripretinib and p-H2AX in imatinib-resistant cell lines. The addition of trametinib, trametinib in combination (n ¼ 10). in contrast to the imatinib and trametinib combination, increased levels of both BimEL and p-H2AX apoptosis markers in the imatinib- Results resistant cell lines. Combination of ripretinib and MEKi induces apoptosis in GIST Combination of ripretinib and MEKi inhibits colony outgrowth in cell lines GIST cell lines To investigate whether blockade of the MAPK and KIT signaling To further confirm that combination treatment is inducing cell pathways induces apoptosis, cells were treated with the FDA-approved death in GIST cell lines, we performed a long-term clonogenic MEKi trametinib, cobimetinib and binimetinib as single agents and in assay. Fig. 2A shows colony outgrowth for GIST-T1 with imatinib combination with ripretinib. Imatinib was used as a comparator. A treatment at 250 nmol/L and ripretinib at 100 nmol/L for 14 days, multi-dose combination matrix was used for each drug combination followed by drug removal for 10 days, with wells exhibiting no colony (as shown in Fig. 1; Supplementary S1) and caspase activity was outgrowth further monitored for an additional 10 days without drug. measured after 24 hours of treatment. Ripretinib or trametinib as a Ripretinib or imatinib each inhibited the number of outgrowth single agent induced a dose-dependent increase (up to 1.95-fold) colonies by approximately 80% of vehicle control and trametinib in caspase activity in GIST-T1 cells. Combination with trametinib alone also inhibited the colony number by 26%–48%. Combination led to further increase in caspase activity (>2-fold up to 2.65-fold, treatment with 100 nmol/L ripretinib and either 50 nmol/L or P < 0.001) with all concentrations of ripretinib tested (Fig. 1A). 100 nmol/L trametinib led to eradication of GIST-T1 with no visible Synergy scores were calculated for ripretinib (10–500 nmol/L) or outgrowth of colonies even after an extra 10 days of incubation (a total imatinib (1–500 nmol/L) with trametinib (0.1–250 nmol/L). Surface of 20 days after drug removal) in normal culture media, indicating that mapping of the combination dose response showed strong synergy in ripretinib and trametinib combination eradicated the GIST-T1 cells GIST-T1 cells with ripretinib and trametinib, whereas no significant within the limit of detection (Fig. 2B, arrows). In contrast, treatment synergy was observed with the imatinib and trametinib combination with 250 nmol/L of imatinib and either 50 nmol/L or 100 nmol/L (Fig. 1D). Similar trends were observed for comparison of ripretinib trametinib did not lead to complete eradication of colonies and versus imatinib in combination with cobimetinib or binimetinib approximately 15%–18% colonies outgrew after 10 days of revival (Supplementary Fig. S1). Ripretinib alone did not induce caspase compared with vehicle control (Fig. 2A and B).

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Figure 1. Ripretinib and trametinib combinations induce apoptosis in GIST cell lines. Heatmap of caspase activity in GIST-T1 (A), GIST-T1/D816E (B), GIST-T1/T670I (C) cell lines with ripretinib and trametinib combination after 24 hours of treatment. Values represent average of three replicates. P values for drug combination versus each drug alone were <0.001. BLISS scoring combination synergy graphs (D) of ripretinib (top) or imatinib (bottom) in combination with trametinib in GIST-T1, GIST-T1/D816E and GIST-T1/T670I cell lines. Apoptosis induction in GIST-T1 (E) and GIST-T1/T670I (F) cells as measured by AnnexinV/PI staining. Cells were treated with compounds at indicated concentrations for 24, 48, and 72 hours and stained with Annexin V and PI. G, Immunoblot of pKIT, pERK, BimEL, and pH2AX levels in imatinib-sensitive (GIST-T1) and imatinib-resistant (GIST-T1/D816E and GIST-T1/T670I) cells treated with imatinib (IM, 100 nmol/L) or ripretinib (Ripr, 100 nmol/L) in the absence or presence of trametinib (Tram, 10 nmol/L) for 48 hours. BL, baseline sample.

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Figure 2. Combination of ripretinib with trametinib synergize to inhibit colony outgrowth of GIST cells. One-hundred cells for GIST-T1 and GIST-T1/D816E and 500 cells for GIST-T1/T670I were plated in 6-well plates and treated with compounds for 2 weeks. Drugs were washed off and cells were grown in complete media for an additional 10 days. If no colonies were visible, cells were revived for an extra 10 days. Colonies were stained with crystal violet and counted. A, Colony outgrowth in GIST-T1 cell line treated with ripretinib or imatinib in combination with trametinib. B, Average colony counts from three wells. Arrows indicate no visually determined colony outgrowth. C, Colony outgrowth in GIST-T1/D816E cell line treated with ripretinib or imatinib in combination with trametinib. D, Average colony counts from three wells. Arrows indicate no visually determined colony outgrowth. E, Colony outgrowth in GIST-T1/T670I cell line treated with ripretinib or imatinib in combination with trametinib. F, Average colony counts from three wells. Arrows indicate no visually determined colony outgrowth. G, Average colony counts for GIST-T1 cell line stably transfected with empty vector or NRASG12D. Colony outgrowth studies were done in presence of 100 nmol/L of imatinib or 50 nmol/L of ripretinib in combination with 50 or 100 nmol/L of trametinib. The values represent average from three wells. Arrows indicate no visually determined colony outgrowth.

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Both imatinib (250 nmol/L) and ripretinib (100 nmol/L) as single shown in Fig. 3C and D, single-agent treatment with ripretinib or agents demonstrated a lack of cytocidal activity in GIST T1/D816E trametinib resulted in colony outgrowth at the tested concentra- leading to approximately 61%–72% colony growth compared with tions after removal of drug. Ripretinib did not significantly inhibit vehicle control (Fig. 2C and D). A combination of ripretinib the colony outgrowth, but trametinib alone inhibited the growth of (100 nmol/L) and trametinib (100 nmol/L) completely inhibited the HMC1.2 by approximately 60% at 10 nmol/L and approximately outgrowth of colonies and only 2 colonies outgrew with extra revival 80% at 25 nmol/L. Combination of ripretinib (25 nmol/L) with of 10 days (a total of 20 days), whereas the imatinib/trametinib trametinib (25 nmol/L) resulted in a complete eradication of combination showed a modest decrease in outgrowth of colonies by colony outgrowth after 5 days of drug removal and approximately approximately 40%–60% (Fig. 2C and D). 15–20 colonies outgrew after extra 8 days of revival (a total of In GIST-T1/T670I, neither imatinib (250 nmol/L) nor ripretinib 13 days without drug). Combination of 50 nmol/L ripretinib with (100 nmol/L) as single agents led to a significant reduction in colony 10 nmol/L or 25 nmol/L of trametinib resulted in eradication of outgrowth after removal of drug. Treatment with 100 nmol/L ripre- outgrowth of viable HMC1.2 cells after 5 days of drug removal tinib in combination with 100 nmol/L trametinib eradicated colony (Fig. 3C and D, see arrows) and this inhibition was maintained outgrowth, with no detectable colonies even after 20 days of revival even after 8 additional days of drug removal (Fig. 3C). This result (Fig. 2E and F, arrow). Imatinib (250 nmol/L) in combination with demonstrated that the combination of ripretinib with trametinib trametinib (100 nmol/L) resulted in a significant reduction of colony synergizes to induce apoptosis in HMC1.2 cells, inhibiting colony outgrowth, but did not lead to complete inhibition and approximately outgrowth even after long-term removal of drug. 8%–10% colonies still outgrew after a total of 20 days of revival Similarly, ripretinib in combination with binimetinib also compared with vehicle control. Collectively, these results demonstrat- showed synergy in inducing caspase activity and the combination ed that the combination of ripretinib is more effective than a com- induced higher levels of caspase-3/7 activity compared with single- bination with imatinib in eradicating tumor cell outgrowth in all GIST agent treatments (Supplementary Fig. S3A and S3B). Although cell lines tested. binimetinib (500 nmol/L) as a single agent caused significant To mimic potential resistance due to MAPK reactivation, colony inhibition, ripretinib (25 nmol/L) when used with 250 nmol/L outgrowth experiments were also performed in GIST-T1 stably trans- and 500 nmol/L of binimetinib, led to greater inhibition in colony fected with NRASG12D. In these cells, combined treatment with formation assays (Supplementary Fig. S3C and S3D, see arrows). 50 nmol/L of ripretinib and 50 nmol/L or 100 nmol/L trametinib led The higher concentration of ripretinib (50 nmol/L) in combination to complete or near complete inhibition of colony outgrowth after with binimetinib (250 nmol/L or 500 nmol/L) also showed erad- 10 days after drug removal (Fig. 2G, right panel arrows). Although ication of viable cells, even after extended period of revival. Similar colonies formed were bigger in size in NRASG12D-transfected cells, no results were obtained with the ripretinib/cobimetinib combination significant difference in colony count was seen when compared with (Supplementary Fig. S3E–S3H). EV-transfected cells. Studies have shown that NRAS is frequently mutated in aggressive Other MEKi inhibitors, cobimetinib and binimetinib, were also SM and might precede KIT D816V in clonal development (29). We used in combination with imatinib and ripretinib. Ripretinib exhibited hypothesized that combination of ripretinib with trametinib might greater activity than imatinib in combination with cobimetinib and show better efficacy than either single agent in mast cells harboring binimetinib (Supplementary Fig. 2SA–2SL). Of the MEK inhibitors both KIT D816V and NRAS mutations. To test this hypothesis, tested, trametinib showed greater efficacy than binimetinib, followed NRASG12D was stably transfected into HMC1.2 cells and treated with by cobimetinib. various concentrations of drugs as shown in Fig. 4A. The combination of ripretinib with trametinib led to a synergistic increase in apoptosis in Combination of ripretinib and MEKi synergistically increases EV-transfected and in NRASG12D transfected HMC1.2 cells. Surpris- apoptosis in the HMC1.2 mast cell line ingly, apoptosis was higher in NRASG12D-transfected cells than Systemic mastocytosis is a rare neoplasm wherein KIT D816V is EV-transfected cells (Fig. 4A). found in 90% of patients, and associated with an aggressive clinical Ripretinib (25 nmol/L and 50 nmol/L) and trametinib (1, 10, or manifestation (14). The HMC1.2 cell line has a primary mutation in 25 nmol/L) combination also led to decreased colony outgrowth of exon 11 at V560G and a secondary mutation in exon 17 at D816V. To NRASG12D-transfected HMC1.2 cells, whereas the single agent treat- determine whether the ripretinib and trametinib combination induces ments resulted in colony outgrowth at all concentrations (Fig. 4B and apoptosis in these cells, cells were treated with each drug either alone C). Combination of ripretinib (50 nmol/L) with trametinib (1 nmol/L) or in combination at the concentrations shown in Fig. 3A. Ripretinib resulted in a significant reduction in colony outgrowth compared and trametinib as single agents induced 2– to 4-fold increases in with single-agent treatment of either drug. Combination of ripretinib caspase-3/7 activity at higher concentrations. An increase of caspase (50 nmol/L) with trametinib (10–25 nmol/L) dramatically led to activity (up to 12-fold) was seen with various ripretinib/trametinib eradication of outgrowth of viable HMC1.2- NRASG12D cells to the combinations in a dose-dependent manner (P < 0.001). A significant limit of detection as determined by visualization with 5X objective increase in apoptosis was seen even with 1 nmol/L trametinib in microscopy after 5 days of drug removal (Fig. 4B and C, arrows). combination with ripretinib, suggesting that much lower concentra- Further extension of colony outgrowth for 8 additional days (total of tions of each drug can be used in combination to induce apoptosis in 13 days after drug removal) still maintained the inhibitory effect. these cells (Fig. 3A). Ripretinib and trametinib exhibited synergy in Similar results were obtained in EV transfected cells except that this assay (Fig. 3B) combination of 50 nmol/L ripretinib and 10 nmol/L of trametinib showed approximately 20–25 colonies after extended period of revival. Combination of ripretinib and MEKi synergizes to eradicate NRASG12D-transfected cells were also more sensitive to colony growth colony outgrowth in HMC1.2 cells inhibition than EV-transfected cells. These results demonstrated that Because HMC1.2 cells grow in suspension, soft-agar colony both KIT-dependent and -independent mechanisms can be effectively formation assays were performed to confirm cytocidal activity. As inhibited with combination treatment of ripretinib and trametinib.

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Figure 3. Combination treatment induces apoptosis and inhibits colony outgrowth in HMC1.2 (V560G/D816V KIT) cells. A, Heatmap of caspase activity in HMC1.2 cells treated with ripretinib alone or in combination with trametinib at indicated concentrations for 24 hours. Values represent an average of three replicates each from two different experiments. B, BLISS synergy graph of ripretinib and trametinib combination at indicated concentrations. C, HMC1.2 cells were grown in soft agar for 10 days in the presence of ripretinib or trametinib alone and in combination. Drug treatments were washed off and cells were grown for 5 days in complete media. If no colonies were visible, cells were revived for extra 8 days. Colonies were stained and photographed. D, Average colony counts from three wells. Arrows show no colony outgrowth.

Similar results were observed with ripretinib in combination with In vivo antitumor studies demonstrate in vivo efficacy of cobimetinib (Supplementary Fig. S4A–S4C). ripretinib þ trametinib combination GIST-T1 tumors were staged for 10 days and then mice were fed on Ripretinib/trametinib combination prevents emergence of formulated diet containing ripretinib or control diet until day 27 and drug-resistant colonies then monitored for additional 40 days on control diet. In mice treated Ba/F3 cells stably transfected with KIT-V560D were treated with with single-agent trametinib (0.5 mg/kg orally, twice daily), no sig- the mutagen ethylnitrosourea (ENU) to induce random mutations. nificant difference in tumor growth was observed when compared with Ripretinib as a single agent showed outgrowth of a few colonies at control. Mice on single-agent ripretinib showed significant tumor 25 and 100 nmol/L Fig. 5A, left), although no secondary KIT muta- regression at both doses (Fig. 6A). At the high dose of ripretinib, 6 tions were observed (Fig. 5A, right), indicating that the outgrowth of of 10 mice had complete tumor regression, while the remaining 4 of 10 colonies with single-agent ripretinib treatment was due to another mice had partial tumor regression during the dosing period. At the low mechanism, likely involving signaling pathway-induced resistance. dose, 2 of 10 mice had complete tumor regression, and 8 of 10 had Ripretinib (25 nmol/L) in combination with trametinib (10 nmol/L) partial tumor regression. Combination treatment with the higher dose led to the outgrowth of only 2 colonies, whereas combination of of ripretinib and trametinib showed complete regression in 10 of 10 trametinib (10 nmol/L) with ripretinib (100 or 250 nmol/L) eliminated mice after 18 days of dosing; however, combination treatment at the outgrowth of any resistant colonies. In contrast, secondary resistant high dose resulted in significant body weight loss that was recovered mutations in KIT were identified in cells treated with imatinib alone at quickly after the end of treatment. Combination treatment at the lower all three concentrations tested (100, 250, and 500 nmol/L) as shown dose was well tolerated. Tumors exhibited slow regrowth after the end here and described previously (22). With imatinib/trametinib com- of the dosing period with ripretinib alone and slower regrowth with the bination, approximately 25–30 colonies outgrew at all concentrations combination of ripretinib and trametinib. Four mice had partial of imatinib tested, but no secondary resistance mutations in KIT were regression and one mouse showed complete regression after 40 days observed (Fig. 5B, right). of revival period in the higher dose combination group, versus two

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Figure 4. Combination treatment inhibits colony outgrowth in NRASG12D-transfected HMC1.2 cells. A, Caspase activity in EV (left) and NRASG12D (right) transfected cells in combination with ripretinib or trametinib alone and in combination at indicated concentrations. B, HMC1.2 empty vector (EV) control and NRASG12D-transfected cells were grown in soft agar for 10 days in the presence of ripretinib or trametinib and in combination. Drug treatments were washed off and cells were grown for 5 days in complete media. If no colonies were visible, cells were revived for extra 8 days. Colonies were stained and photographed. C, Average colony counts from three wells. Arrows show no colony growth.

partial regressions in the ripretinib high dose group and no partial tolerated though body weight loss was observed in the combination regressions in the trametinib group. Body weight changes for ripretinib group in both models (Fig. 6E and F). Body weight changes for the treated group were similar to vehicle. ripretinib single-agent–treated groups were similar to vehicle. In two imatinib-resistant KIT-mutant mastocytosis models, (P815 mouse D814Y KIT and HMC1.2 human V560D/D816V KIT), the ripretinib combination with trametinib also showed greater tumor growth Discussion inhibition than either single agent (Fig. 6C and D), with the combination The development of secondary resistance mutations in KIT has showing synergistic activity in the P815 allograft model. Treatments were been a major challenge for the treatment of patients with GIST. These

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Ripretinib and MEKi Synergize in Preclinical Models

Figure 5. Combination of ripretinib and trametinib prevents emergence of drug resistance mediated by KIT independent mechanisms. Ba/F3 cells stably expressing V560D KIT were treated with ENU and grown in the presence of ripretinib trametinib (A) or imatinib trametinib (B) for 28 days. Wells exhibiting outgrowth with no identified secondary KIT mutations (“V560D native”) are shown in the left panels. Wells exhibiting outgrowth with identified secondary KIT mutations are shown in the right panels. Arrows indicate no outgrowth. mutations often arise in exons 13, 14, 17, and 18 after treatment with receptor tyrosine kinases such as FGFR1 or FGFR2, AXL and c-MET imatinib, sunitinib, and/or regorafenib. A major focus of research in can also induce adaptation to imatinib treatment (37, 38). These the GIST field has been to develop novel and more potent inhibitors studies demonstrate that both KIT-dependent and -independent that can target most, if not all, of the possible combination of primary pathways are active in some cases of GIST and these patients may and secondary mutations. Other mechanisms of resistance have also not respond to KIT inhibition alone. Recent studies have shown that been reported, such as cells entering quiescent state indefinitely by transcription factor ETV1 activates a positive feedback mechanism for undergoing autophagy (10) or existence of KITlow stem/progenitor KIT transcription and its levels are maintained by MAPK signaling tumor cells that fail to respond to KIT inhibitors (31). With the advent pathway and PDGFRA signaling in KIT-mutant GIST (21, 39, 40). of next-generation sequencing of GIST tumors in the clinic and in Because most of the KIT-independent pathways appear to act via clinical trials, a new landscape for GIST has emerged. It has become MAPK signaling, identifying new strategies that target the full range of evident that in some cases, GISTs can also become dependent on other KIT/PDGFRA mutations and MAPK signaling pathway are of para- signaling pathways besides KIT/PDGFRA (18, 32). Activating muta- mount importance. tions have been found in PI3K/AKT/mTOR and RAS/RAF/MAPK In this study, ripretinib or imatinib was used in combination with signaling pathways in KIT mutated or in wild-type GIST and in FDA-approved MEK inhibitors in imatinib-sensitive and -resistant patients on later lines of therapy (17–19, 32–35). Mutations in BRAF, GIST cell lines. The data showed that combining imatinib with MEKi KRAS,orPIK3CA, which encode downstream effectors of KIT/ has a distinct advantage over treatments with imatinib alone in killing PDGFRA, are associated with poor prognosis and patients progress the cancer cells. This is in accordance with recent reports where on imatinib (34–36). Cell lines with KRAS or BRAF mutations imatinib or pexidartinib (a multikinase inhibitor including KIT) used concomitant with KIT/PDGFRA mutations failed to respond to ima- in combination with binimetinib has shown better efficacy in advanced tinib (35). Activation of the ERK signaling pathway through alternate GIST tumors (21, 41). Ripretinib showed superior efficacy to imatinib

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Figure 6. Ripretinib and trametinib combination treatment in KIT mutant models in mice. A, Mice bearing GIST T1 tumors were treated as indicated. Dosing started on day 10 and ended on day 27. Tumor growth was monitored for additional 40 days. B, Body weight change in mice on study. Data are represented as mean SEM. C, Mice bearing P815 tumors were treated as indicated. Dosing started on day 5 and ended on day 28. D, Mice bearing HMC1.2 tumors were treated as indicated. Dosing started on day 10 and ended on day 28. E, Body weight change in mice on P815 study. Data are represented as mean SEM. F, Body weight change in mice on HMC1.2 study. Data are represented as mean SEM.

in combination with MEKi in the imatinib-sensitive and particularly, or mutations in other genes that would lead to resistance would be in the imatinib-resistant cell lines tested. Ripretinib combined with generated. However, resistant clones were isolated following imatinib MEKi completely abrogated the residual MAPK signaling that was not treatment, suggesting that ripretinib/MEKi combination treatment inhibited by KIT inhibitors alone and exhibited strong synergy in warrants further study in clinic as a method for slowing disease inducing apoptosis. The ripretinib-MEKi combination was also better progression due to drug resistance in patients. Combination of ima- than imatinib-MEKi combination at inhibiting colony outgrowth even tinib/trametinib resulted in outgrowth of several resistant colonies, after long-term removal of drug. Ripretinib/MEKi combinations were possibly due of activation of other pathways (42) or overexpression of also tested in GIST-T1 stably transfected with NRASG12D to mimic the certain transcription or translation factors. On the basis of these data, mutations found in some patients. Colony outgrowth assay showed imatinib could be a weaker inhibitor of KIT compared with ripretinib, NRASG12D-transfected cells were efﬁciently inhibited by combination and therefore might allow some colonies to go into quiescent or treatment. cytostatic phase, leading to later stage activation of different pathways. The combination of ripretinib with trametinib completely pre- Another disease which is dependent on KIT is SM, where 90% of the vented outgrowth of resistant colonies in saturation mutagenesis indolent and aggressive SM patients carry the KIT D816V muta- studies, although there are limitations to the experiment. Ethylnitro- tion (27). These patients are at a greater risk for disease progres- surea treatment leads to predominantly A to G transitions, and based sion (29). So far, the only FDA approved D816V KIT inhibitor for SM on the number of cells mutagenized, not all secondary KIT mutations is midostaurin (43). Advanced SM patients also can have NRAS

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mutations (29, 44), which may indicate the need to target both driver nation therapy, if successful in clinical trials, could be used as a first- mutations. We demonstrate here that ripretinib and MEKi combina- or second-line of therapy for patients with GIST. tion synergistically induced apoptosis in HMC1.2 cells with complete inhibition of colony outgrowth observed at much lower concentrations Authors’ Disclosures of each drug in long term assays. The combination was more potent in A. Gupta reports a patent for US 16/943,821 pending and a patent for G12D HMC1.2 NRAS cells at lower concentrations, demonstrating that US 16/943,871 pending. C. Serrano reports grants from Deciphera Pharmaceu- combination warrants further study in the clinic as an option for ticals, Pfizer, and Bayer; personal fees from Blueprint Medicines, Immunicum AB, advanced SM patients. and personal fees from Deciphera Pharmaceuticals during the conduct of the fi Because MEKi have shown some toxicity in the clinic (45, 46), lower study; non- nancial support from Pharmamar, Novartis, Lilly, Bayer, and non- financial support from Pfizer outside the submitted work. D.L. Flynn reports doses needed for combination treatment might be better tolerated. The other from Deciphera Pharmaceuticals outside the submitted work; in addition, high-dose combination of ripretinib with trametinib led to body D.L. Flynn has a patent for US 8,461,179 issued; and Deciphera Pharmaceuticals weight loss in mouse studies, but treatment cessation led to recovery has a relationship with the following GIST support groups: (i) The Life Raft in body weights within 2 days in the GIST T1 model. Alternatively, Group; (ii) Gist Support International. B.D. Smith reports other from Deciphera binimetinib could be a better combination partner with ripretinib Pharmaceuticals outside the submitted work; in addition, B.D. Smith has a patent because of its higher clearance compared to trametinib, and no toxicity for US8461179 issued, a patent for US8188113 issued, a patent for US: 16/943821 pending, and a patent for US: 16/943871 pending. No disclosures were reported has been reported in GIST T1 xenograft studies when used in by the other authors. combination with imatinib (41). In summary, our data suggest that further clinical study of Authors’ Contributions combination therapy is warranted for advanced GIST and SM A. Gupta: Conceptualization, data curation, investigation, methodology, writing– patients. Ripretinib potently inhibits a broad spectrum of primary original draft, writing–review and editing. J. Singh: Data curation, investigation, and drug-resistant KIT/PDGFRAmutantsandisapprovedbythe methodology, writing–review and editing. A. García-Valverde: Data curation, inves- FDA for the treatment of advanced GIST who have received tigation, methodology. C. Serrano: Conceptualization, data curation, investigation, – previous treatment with 3 or more kinase inhibitors, including methodology, writing review and editing. D.L. Flynn: Conceptualization, methodology, writing–original draft, writing–review and editing. B.D. Smith: Conceptual- imatinib. Studies from several clinical trials have shown that some ization, data curation, investigation, methodology, writing–original draft, writing– KIT patients with exon 11 mutations do not respond to imatinib review and editing. treatment, and analysis of KRAS and BRAF mutations together with KIT mutations has been suggested for profiling patients (35). In that Acknowledgments respect, dual inhibition of KIT and the MAPK pathway may be The authors thank B. Rubin for GIST T1/T670I, GIST T1/D816E, and GIST T1 highly beneficial for both imatinib-na€ve and heavily treated KIT-mutant cells. patients. This strategy has the potential to overcome the MAPK pathway reactivation compensatory resistance mechanisms in this The costs of publication of this article were defrayed in part by the payment of page advertisement continuously evolving disease. One major advantage of using charges. This article must therefore be hereby marked in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ripretinib as a combination partner with MEKi over imatinib is that ripretinib treatment did not result in the generation of sec- Received September 22, 2020; revised February 10, 2021; accepted April 19, 2021; ondary KIT resistance mutations in preclinical studies. A combi- published first May 4, 2021.

References 1. Ducimetiere F, Lurkin A, Ranchere-Vince D, Decouvelaere AV, Peoc’hM, for research and treatment of cancer, italian sarcoma group, and australasian Istier L, et al. Incidence of sarcoma histotypes and molecular subtypes in a gastrointestinal trials group intergroup phase III randomized trial on imatinib at prospective epidemiological study with central pathology review and molec- two dose levels. J Clin Oncol 2017;35:1713–20. ular testing. PLoS One 2011;6:e20294. 8. Agaram NP, Besmer P, Wong GC, Guo T, Socci ND, Maki RG, et al. Pathologic 2. Heinrich MC, Corless CL, Demetri GD, Blanke CD, von Mehren M, Joensuu H, and molecular heterogeneity in imatinib-stable or imatinib-responsive gastro- et al. Kinase mutations and imatinib response in patients with metastatic intestinal stromal tumors. Clin Cancer Res 2007;13:170–81. gastrointestinal stromal tumor. J Clin Oncol 2003;21:4342–9. 9. Boichuk S, Parry JA, Makielski KR, Litovchick L, Baron JL, Zewe JP, et al. The 3. Rubin BP, Heinrich MC, Corless CL. Gastrointestinal stromal tumour. Lancet DREAM complex mediates GIST cell quiescence and is a novel therapeutic target 2007;369:1731–41. to enhance imatinib-induced apoptosis. Cancer Res 2013;73:5120–9. 4. Blanke CD, Demetri GD, von Mehren M, Heinrich MC, Eisenberg B, Fletcher 10. Gupta A, Roy S, Lazar AJ, Wang WL, McAuliffe JC, Reynoso D, et al. Autophagy JA, et al. Long-term results from a randomized phase II trial of standard- inhibition and antimalarials promote cell death in gastrointestinal stromal tumor versus higher-dose imatinib mesylate for patients with unresectable or (GIST). Proc Natl Acad Sci U S A 2010;107:14333–8. metastatic gastrointestinal stromal tumors expressing KIT. J Clin Oncol 11. Gajiwala KS, Wu JC, Christensen J, Deshmukh GD, Diehl W, DiNitto JP, et al. 2008;26:620–5. KIT kinase mutants show unique mechanisms of drug resistance to imatinib and 5. Bachet JB, Hostein I, Cesne AL, Brahimi S, Beauchet A, Tabone-Eglinger S, et al. sunitinib in gastrointestinal stromal tumor patients. Proc Natl Acad Sci U S A Prognosis and predictive value of KIT exon 11 deletion in GISTs. Br J Cancer 2009;106:1542–7. 2009;101:7–11. 12. Demetri GD, Reichardt P, Kang YK, Blay JY, Rutkowski P, Gelderblom H, et al. 6. Blay JY, Cesne AL, Ray-Coquard I, Bui B, Duffaud F, Delbaldo C, et al. Efﬁcacy and safety of regorafenib for advanced gastrointestinal stromal tumours Prospective multicentric randomized phase III study of imatinib in patients after failure of imatinib and sunitinib (GRID): an international, multicentre, with advanced gastrointestinal stromal tumors comparing interruption versus randomised, placebo-controlled, phase 3 trial. Lancet 2013;381:295–302. continuation of treatment beyond 1 year: the french sarcoma group. J Clin Oncol 13. Heinrich MC, Corless CL, Blanke CD, Demetri GD, Joensuu H, Roberts PJ, et al. 2007;25:1107–13. Molecular correlates of imatinib resistance in gastrointestinal stromal tumors. 7. Casali PG, Zalcberg J, Cesne AL, Reichardt P, Blay JY, Lindner LH, et al. Ten- J Clin Oncol 2006;24:4764–74. Year progression-free and overall survival in patients with unresectable or 14. Serrano C, Marino-Enriquez A, Tao DL, Ketzer J, Eilers G, Zhu M, et al. metastatic GI stromal tumors: long-term analysis of the european organisation Complementary activity of tyrosine kinase inhibitors against secondary kit

AACRJournals.org Mol Cancer Ther; 2021 OF11

Gupta et al.

mutations in imatinib-resistant gastrointestinal stromal tumours. Br J Cancer 31. Bardsley MR, Horvath VJ, Asuzu DT, Lorincz A, Redelman D, Hayashi Y, et al. 2019;120:612–20. Kitlow stem cells cause resistance to kit/platelet-derived growth factor alpha 15. Heinrich MC, Maki RG, Corless CL, Antonescu CR, Harlow A, Griffith D, et al. inhibitors in murine gastrointestinal stromal tumors. Gastroenterology 2010; Primary and secondary kinase genotypes correlate with the biological and 139:942–52. clinical activity of sunitinib in imatinib-resistant gastrointestinal stromal tumor. 32. Mavroeidis L, Metaxa-Mariatou V, Papoudou-Bai A, Lampraki AM, J Clin Oncol 2008;26:5352–9. KostadimaL,TsinokouI,etal.Comprehensive molecular screening by 16. Liegl B, Kepten I, Le C, Zhu M, Demetri GD, Heinrich MC, et al. Heterogeneity of next generation sequencing reveals a distinctive mutational profile of kinase inhibitor resistance mechanisms in GIST. J Pathol 2008;216:64–74. KIT/PDGFRA genes and novel genomic alterations: results from a 20-year 17. Li B, Garcia CS, Marino-Enriquez A, Grunewald S, Wang Y, Bahri N, et al. cohort of patients with GIST from north-western greece. ESMO Open 2018; Conjoined hyperactivation of the RAS and PI3K pathways in advanced GIST. 3:e000335. American Society of Clinical Oncology; 2016. 33. Hechtman JF, Zehir A, Mitchell T, Borsu L, Singer S, Tap W, et al. Novel 18. Muhlenberg T, Ketzer J, Heinrich MC, Grunewald S, Marino-Enriquez A, oncogene and tumor suppressor mutations in KIT and PDGFRA wild type Trautmann M, et al. KIT-Dependent and KIT-Independent genomic hetero- gastrointestinal stromal tumors revealed by next generation sequencing. geneity of resistance in gastrointestinal stromal tumors - TORC1/2 inhibition as Genes Chromosomes Cancer 2015;54:177–84. salvage strategy. Mol Cancer Ther 2019;18:1985–96. 34. Agaram NP, Wong GC, Guo T, Maki RG, Singer S, Dematteo RP, et al. Novel 19. Serrano C, Wang Y, Marino-Enriquez A, Lee JC, Ravegnini G, Morgan JA, et al. V600E BRAF mutations in imatinib-naive and imatinib-resistant gastrointes- KRAS and KIT gatekeeper mutations confer polyclonal primary imatinib tinal stromal tumors. Genes Chromosomes Cancer 2008;47:853–9. resistance in GI stromal tumors: relevance of concomitant phosphatidylinositol 35. Miranda C, Nucifora M, Molinari F, Conca E, Anania MC, Bordoni A, et al. 3-kinase/AKT dysregulation. J Clin Oncol 2015;33:e93–6. KRAS and BRAF mutations predict primary resistance to imatinib in gastro- 20. Boichuk S, Galembikova A, Dunaev P, Valeeva E, Shagimardanova E, Gusev O, intestinal stromal tumors. Clin Cancer Res 2012;18:1769–76. et al. A novel receptor tyrosine kinase switch promotes gastrointestinal stromal 36. Gao J, Li J, Li Y, Li Z, Gong J, Wu J, et al. Intratumoral KIT mutational tumor drug resistance. Molecules 2017;22:2152. heterogeneity and recurrent KIT/PDGFRA mutations in KIT/PDGFRA wild- 21. Ran L, Sirota I, Cao Z, Murphy D, Chen Y, Shukla S, et al. Combined inhibition of type gastrointestinal stromal tumors. Oncotarget 2016;7:30241–9. MAP kinase and KIT signaling synergistically destabilizes ETV1 and suppresses 37. Javidi-Sharifi N, Traer E, Martinez J, Gupta A, Taguchi T, Dunlap J, et al. GIST tumor growth. Cancer Discov 2015;5:304–15. Crosstalk between KIT and FGFR3 promotes gastrointestinal stromal tumor cell 22. Smith BD, Kaufman MD, Lu WP, Gupta A, Leary CB, Wise SC, et al. Ripretinib growth and drug resistance. Cancer Res 2015;75:880–91. (DCC-2618) is a switch control kinase inhibitor of a broad spectrum of 38. Boichuk S, Galembikova A, Dunaev P, Micheeva E, Valeeva E, Novikova M, et al. oncogenic and drug-resistant KIT and PDGFRA variants. Cancer Cell 2019; Targeting of FGF-signaling re-sensitizes gastrointestinal stromal tumors (GIST) 35:738–51. to imatinib in vitro and in vivo. Molecules 2018;23:2643. 23. Blay JY, Serrano C, Heinrich MC, Zalcberg J, Bauer S, Gelderblom H, et al. 39. Hayashi Y, Bardsley MR, Toyomasu Y, Milosavljevic S, Gajdos GB, Choi KM, Ripretinib in patients with advanced gastrointestinal stromal tumours et al. Platelet-derived growth factor receptor-alpha regulates proliferation of (INVICTUS): a double-blind, randomised, placebo-controlled, phase 3 trial. gastrointestinal stromal tumor cells with mutations in KIT by stabilizing ETV1. Lancet Oncol 2020;21:923–34. Gastroenterology 2015;149:420–32. 24. Dhillon S. Ripretinib: First Approval. Drugs 2020;80:1133–8. 40. Duensing A. Targeting ETV1 in gastrointestinal stromal tumors: tripping the 25. Heinrich MC, Owzar K, Corless CL, Hollis D, Borden EC, Fletcher CD, et al. circuit breaker in GIST? Cancer Discov 2015;5:231–3. Correlation of kinase genotype and clinical outcome in the north american 41. Rosenbaum E, Kelly C, D’Angelo SP, Dickson MA, Gounder M, Keohan ML, intergroup phase III trial of imatinib mesylate for treatment of advanced et al. A phase I study of binimetinib (MEK162) combined with pexidartinib gastrointestinal stromal tumor: CALGB 150105 study by Cancer and Leukemia (PLX3397) in patients with advanced gastrointestinal stromal tumor. Oncologist Group B and Southwest Oncology Group. J Clin Oncol 2008;26:5360–7. 2019;24:1309–e983. 26. Garcia-Montero AC, Jara-Acevedo M, Teodosio C, Sanchez ML, Nunez R, 42. Pierotti MA, Tamborini E, Negri T, Pricl S, Pilotti S. Targeted therapy in Prados A, et al. KIT mutation in mast cells and other bone marrow hemato- GIST: in silico modeling for prediction of resistance. Nat Rev Clin Oncol poietic cell lineages in systemic mast cell disorders: a prospective study of the 2011;8:161–70. spanish network on mastocytosis (REMA) in a series of 113 patients. Blood 2006; 43. Jawhar M, Schwaab J, Naumann N, Horny HP, Sotlar K, Haferlach T, 108:2366–72. et al. Response and progression on midostaurin in advanced systemic 27. Furitsu T, Tsujimura T, Tono T, Ikeda H, Kitayama H, Koshimizu U, et al. mastocytosis: KIT D816V and other molecular markers. Blood 2017;130: Identification of mutations in the coding sequence of the proto-oncogene c-kit 137–45. in a human mast cell leukemia cell line causing ligand-independent activation 44. Zorzan E, Hanssens K, Giantin M, Dacasto M, Dubreuil P. Mutational hotspot of of c-kit product. J Clin Invest 1993;92:1736–44. TET2, IDH1, IDH2, SRSF2, SF3B1, KRAS, and NRAS from human systemic 28. Pardanani A. Systemic mastocytosis in adults: 2019 update on diagnosis, risk mastocytosis are not conserved in canine mast cell tumors. PLoS One 2015;10: stratification and management. Am J Hematol 2019;94:363–77. e0142450. 29. Wilson TM, Maric I, Simakova O, Bai Y, Chan EC, Olivares N, et al. Clonal 45. Welsh SJ, Corrie PG. Management of BRAF and MEK inhibitor toxicities in analysis of NRAS activating mutations in KIT-D816V systemic mastocytosis. patients with metastatic melanoma. Ther Adv Med Oncol 2015;7:122–36. Haematologica 2011;96:459–63. 46. Infante JR, Fecher LA, Falchook GS, Nallapareddy S, Gordon MS, Becerra C, 30. Liu Y, Tseng M, Perdreau SA, Rossi F, Antonescu C, Besmer P, et al. Histone et al. Safety, pharmacokinetic, pharmacodynamic, and efficacy data for the H2AX is a mediator of gastrointestinal stromal tumor cell apoptosis following oral MEK inhibitor trametinib: a phase 1 dose-escalation trial. Lancet Oncol treatment with imatinib mesylate. Cancer Res 2007;67:2685–92. 2012;13:773–81.

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Ripretinib and MEK Inhibitors Synergize to Induce Apoptosis in Preclinical Models of GIST and Systemic Mastocytosis

Anu Gupta, Jarnail Singh, Alfonso García-Valverde, et al.

Mol Cancer Ther Published OnlineFirst May 4, 2021.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-20-0824

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