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Synergy of WEE1 and Mtor Inhibition in Mutant KRAS-Driven

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Synergy of WEE1 and Mtor Inhibition in Mutant KRAS-Driven Published OnlineFirst August 18, 2017; DOI: 10.1158/1078-0432.CCR-17-1098 Cancer Therapy: Preclinical Clinical Cancer Research Synergy of WEE1 and mTOR Inhibition in Mutant KRAS-Driven Lung Cancers Josephine Hai1,2, Shengwu Liu1,2, Lauren Bufe1, Khanh Do1,2, Ting Chen1,3, Xiaoen Wang1, Christine Ng4, Shuai Li1,2, Ming-Sound Tsao4, Geoffrey I. Shapiro1,2, and Kwok-Kin Wong1,2,3 Abstract Purpose: KRAS-activating mutations are the most common Results: We demonstrate that combined inhibition of oncogenic driver in non–small cell lung cancer (NSCLC), but mTOR and WEE1 induced potent synergistic cytotoxic efforts to directly target mutant KRAS have proved a formidable effects selectively in KRAS-mutant NSCLC cell lines, delayed challenge. Therefore, multitargeted therapy may offer a plau- human tumor xenograft growth and caused tumor regres- sible strategy to effectively treat KRAS-driven NSCLCs. Here, we sion in a murine lung adenocarcinoma model. Mechanis- evaluate the efficacy and mechanistic rationale for combining tically, we show that inhibition of mTOR potentiates mTOR and WEE1 inhibition as a potential therapy for lung WEE1 inhibition by abrogating compensatory activation of cancers harboring KRAS mutations. DNA repair, exacerbating DNA damage in KRAS-mutant Experimental Design: We investigated the synergistic effect of NSCLC, and that this effect is due in part to reduction in combining mTOR and WEE1 inhibitors on cell viability, apo- cyclin D1. ptosis, and DNA damage repair response using a panel of Conclusions: These findings demonstrate that compro- human KRAS-mutantandwildtypeNSCLCcelllinesand mised DNA repair underlies the observed potent synergy of patient-derived xenograft cell lines. Murine autochthonous and WEE1 and mTOR inhibition and support clinical evaluation human transplant models were used to test the therapeutic of this dual therapy for patients with KRAS-mutant lung efficacy and pharmacodynamic effects of dual treatment. cancers. Clin Cancer Res; 23(22); 6993–7005. Ó2017 AACR. Introduction crosstalk of highly complex and redundant signaling cascades leading to bypass pathways and negative feedback loops (8). Lung cancer is the leading cause of cancer-related deaths world- Complicating this, we previously showed that KRAS-mutant lung wide and non–small cell lung cancer (NSCLC) accounts for more tumors bearing TP53 or LKB1 co-mutations differ in their than 80% of those cases (1, 2). Kirsten RAS viral oncogene response to docetaxel with or without selumetinib, suggesting homolog (KRAS) is mutated in 25% to 30% of lung adenocarci- that co-mutations may also impact treatment response (9, 10). nomas (3). Unlike NSCLC patients harboring EGFR-activating Given greater molecular diversity in KRAS-mutant tumors mutations or ALK fusions, for which targeted inhibitors have compared with other actionable oncogenic targets in lung ade- achieved objective responses in up to 80% of cases, direct molec- nocarcinoma, one approach to improving the clinical efficacy of ular inhibition of mutant RAS has proved difficult owing to its inhibitors is to identify drug combinations that either target structure as well as picomolar affinity to GDP/GTP (3–6). Patients multiple RAS-driven pathways or circumvent resistance. Previ- with tumors harboring KRAS-mutations are among the most ously, Weisberg and colleagues (11) used a chemical screen in a difficult to treat and individual inhibitors targeting mutant RAS mutant NRAS-transformed Ba/F3 cell line to show that an inhib- downstream signaling pathways such as PI3K/AKT/mTOR and itor of WEE1, AZD1775, synergized with the mTOR inhibitor RAF/MEK/ERK have yielded limited response rates of less than Torin2 in acute leukemia. mTOR is a critical downstream effector 20% in clinical trials (7). This suggests that RAS is associated with of RAS in lung cancer; however, clinical testing of mTOR inhibi- tors alone have demonstrated limited efficacy (12). Preclinical studies have shown that mTOR inhibitors suppress homologous 1Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Mas- recombination-mediated DNA repair (HDR) and synergize with sachusetts. 2Department of Medicine, Harvard Medical School, Boston, Massa- PARP inhibitors in BRCA1-proficient triple-negative breast can- chusetts. 3Perlmutter Cancer Center, New York University Langone Medical 4 cers (13). AZD2014 is a novel small-molecule ATP-competitive Center, New York, New York. Princess Margaret Cancer Centre/University dual inhibitor of both mTORC1 and mTORC2 kinase that is well Health Network, Toronto, Ontario, Canada. tolerated in preclinical studies and is currently in phase II clinical Note: Supplementary data for this article are available at Clinical Cancer trials (14, 15). Research Online (http://clincancerres.aacrjournals.org/). WEE1 is a protein kinase that negatively regulates the G2–M Corresponding Author: K.-K. Wong, NYU Langone Medical Center, 550 1st checkpoint by inhibiting cyclin-dependent kinase (CDK) 1 via Avenue, Smilow 1011, New York, NY 10016. Phone: 212-263-9203; Fax: 617-632- tyrosine 15 phosphorylation (16). AZD1775 is a first-in-class, 7839; E-mail: [email protected] pyrazolo-pyrimidine derivative and potent small-molecule doi: 10.1158/1078-0432.CCR-17-1098 WEE1 kinase inhibitor (17). CDK1 is also necessary for Ó2017 American Association for Cancer Research. BRCA1-mediated S phase checkpoint activation and HDR (18). www.aacrjournals.org 6993 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst August 18, 2017; DOI: 10.1158/1078-0432.CCR-17-1098 Hai et al. RNAi Consortium (TRC; Broad Institute, Cambridge, MA; ref. 22). Translational Relevance Stable cell lines were isolated following viral transduction and Considering the absence of targeted therapies for KRAS- selection with puromycin antibiotics (1–2 mg/mL). driven cancers, the identification of combinatorial therapies remains an area of great clinical need. Here, we provide Human xenograft models mechanistic rationale for combining mTOR and WEE1 inhi- Nude mice were obtained from Charles River Laboratories KRAS bition to selectively target -mutant NSCLCs. We dem- (Wilmington, MA). All manipulations were performed under fi onstrate for the rst time that inhibition of mTOR potentiates sterile conditions in a laminar flow hood, in accordance with WEE1 inhibition by mitigating compensatory activation of procedures approved by the DFCI Animal Care and Use Com- DNA repair by homologous recombination, resulting in cyto- mittee. A427 (5 Â 106) and H1355 (5 Â 106) cells were injected toxic synergism. Thus, dual inhibition of mTOR and WEE1 in subcutaneously in the flanks of 6-week-old nude mice (n ¼ 8–10/ KRAS -driven NSCLCs presents a promising approach when group). For treatment, mice were randomized into groups with targeted therapies remain elusive in this patient population. similar mean tumor volumes of 100 to 150 mm3. Treatment began at Day 11 after implantation for A427 lines and Day 32 for H1355 lines. AZD2014 and AZD1775 was dissolved in 0.5% Although previous studies have shown that WEE1 inhibitors such hydroxypropyl methylcellulose (HPMC) and administered by as AZD1775 augment the effects of chemotherapy by driving oral gavage once a day at 15 mg/kg (AZD2014) and 40 mg/kg transformed cells to mitotic catastrophe, it is unclear how mTOR (AZD1775). inhibition potentiates the effects of AZD1775 (19). Mice were examined every 2 to 3 days, and tumor length and Here, we investigated the mechanism underlying KRAS-mutant width were measured using calipers. Tumor volume was calcu- Â 2 cell sensitivity to dual WEE1 and mTOR inhibition. We show that lated using the following formula: (length width )p/6. At fi WEE1 inhibition in KRAS-mutant cells induces DNA damage sacri ce, portions of tumors were snap-frozen and stored in liquid fi accumulation, which is further compounded by simultaneous nitrogen or were xed in 10% buffered formalin for routine compromise of DNA repair, mediated by mTOR inhibition. histopathologic processing. Importantly, oral administration of mTOR and WEE1 inhibitors caused significant reduction in human xenografted tumor growth Inducible mutant KRAS-mutant lung cancer as well as tumor regression in autochthonous lung adenocar- Mouse strains harboring a conditional activating mutation cinoma murine models. Our results support further clinical (G12D) at the endogenous KRAS locus were induced intrana- investigation of combined WEE1/mTOR inhibition as a poten- sally with 5 Â 107 p.f.u. adeno-Cre recombinase (University of tial KRAS-driven NSCLC therapy. Iowa adenoviral core). All experimental mice were maintained on a mixed genetic background (C57BL/6, BALB/c, and S129). Materials and Methods Upon detection of tumor burden at approximately 14 to -Cre Cell culture, CRISPR vectors, and stable isogenic cell line 16 weeks after adeno recombinase induction by analyzing generation MRI scans using 3D Slicer software, animals were randomly Human NSCLC cell lines (A427, NCI-H23, NCI-H1355, assigned to treatment groups (23). AZD2014 and AZD1775 NCI-H441, NCI-H2009, NCI-H358, Calu1, NCI-H460, HCC827, was dissolved in 0.5% HPMC and administered by oral gavage HCC4006, NCI-H1975, NCI-H3255, and NCI-H1650) were once a day at 10 mg/kg (AZD2014) and 20 mg/kg (AZD1775). obtained from the ATCC and cultured in RPMI-1650 media supplemented with 10% FBS (Thermo Fisher) and antibiotics. Western blot analysis, antibodies and immunohistochemistry Patient-derived xenograft
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