Targeting the CDK4/6-Rb Pathway Enhances Response to PI3K
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Published OnlineFirst August 9, 2018; DOI: 10.1158/1078-0432.CCR-18-0717 Translational Cancer Mechanisms and Therapy Clinical Cancer Research Targeting the CDK4/6-Rb Pathway Enhances Response to PI3K Inhibition in PIK3CA-Mutant Lung Squamous Cell Carcinoma Ruoshi Shi1,2, Ming Li1, Vibha Raghavan1, Shirley Tam1, Michael Cabanero1, Nhu-An Pham1, Frances A. Shepherd1,3, Nadeem Moghal1,2, and Ming-Sound Tsao1,2,4 Abstract Purpose: Lung squamous cell carcinoma (LUSC) is a major of LUSC PDX models identified PI3K pathway alterations in subtype of non–small cell lung cancer characterized by mul- over 50% of the models. In vivo screening using PI3K tiple genetic alterations, particularly PI3K pathway alterations inhibitors in 12 of these models identified PIK3CA mutation which have been identified in over 50% of LUSC cases. Despite as a predictive biomarker of response (<20% tumor growth being an attractive target, single-agent PI3K inhibitors have compared with baseline/vehicle). Combined inhibition of demonstrated modest response in LUSC. Thus, novel combi- PI3K and CDK4/6 in models with PIK3CA mutation resulted nation therapies targeting LUSC are needed. in greater antitumor effects compared with either mono- Experimental Design: PI3K inhibitors alone and in com- therapy alone. In addition, the combination of the two bination with CDK4/6 inhibitors were evaluated in previously drugs achieved targeted inhibition of the PI3K and cell-cycle established LUSC patient-derived xenografts (PDX) using an pathways. in vivo screening method. Screening results were validated with Conclusions: PIK3CA mutations predict response to PI3K in vivo expansion to 5 to 8 mice per arm. Pharmacodynamics inhibitors in LUSC. Combined PI3K and CDK4/6 inhibition studies were performed to confirm targeted inhibition of enhances response to either single agents alone. Our findings compounds. provide a rationale for clinical testing of combined PI3K Results: Consistent with results from The Cancer Genome and CDK4/6 inhibitors in PIK3CA-mutant LUSC. Clin Cancer AtlasanalysisofLUSC,genomicprofiling of our large cohort Res; 24(23); 5990–6000. Ó2018 AACR. Introduction (2–4). However, to date, there has yet been very little successful clinical translation of these discoveries. Although preclinical work Lung squamous cell carcinoma (LUSC) is a common subtype of has been reported on these molecular targets (2), none of the non–small cell lung cancer (NSCLC) affecting over 300,000 inhibitors tested in early-phase clinical trials has demonstrated individuals worldwide each year (1). The current standard of care high response rates. The major shortcomings of these clinical trials for LUSC is surgical resection for early stages and cytotoxic could include drug toxicities and the lack of robust predictive chemotherapy and immunotherapy for inoperable disease. To biomarkers to select patients who may benefit from these date, the identification of treatment strategies for targetable dri- therapies. vers in LUSC has remained an elusive goal (2), with the possible One particular pathway whose alteration has been investigated exception of immunotherapy, although its long-term effects is the PI3K pathway. PI3K belongs to a family of lipid kinases remain to be seen. The Cancer Genome Atlas and other consortia involved in the regulation of cell proliferation, survival, and have molecularly characterized LUSC in large cohort studies, metabolism (5). This pathway has been explored in LUSC largely identifying numerous potentially targetable alterations including due to the high frequency of perturbations, with PIK3CA muta- FGFR1 amplification, PI3K pathway mutations/copy-number tions, amplifications, and PTEN loss being identified in 10% to changes, DDR2 mutation, and NRF2-KEAP1 pathway aberrations 15%, 50%, and 20% to 30% of LUSC cases (3). These PI3K pathway alterations putatively confer constitutive activation of 1University Health Network, Ontario Cancer Institute/Princess Margaret Cancer the pathway, resulting in oncogenic transformation in normal Centre, Toronto, Ontario, Canada. 2Department of Medical Biophysics, breast epithelial cell lines in soft-agar colony formation assays, University of Toronto, Toronto, Ontario, Canada. 3Department of Medical increased tumorigenic potential in mice, and predictive response Oncology and Hematology, University of Toronto, Toronto, Ontario, Canada. to PI3K inhibition in in vitro preclinical models (6–8). Inhibitors 4 Department of Laboratory Medicine and Pathobiology, University of Toronto, of PI3K such as BKM120 and BYL719 have demonstrated efficacy Toronto, Ontario, Canada. in PIK3CA-mutant pan-cancer cell lines, and these agents now are Note: Supplementary data for this article are available at Clinical Cancer being evaluated in clinical trials (9). Research Online (http://clincancerres.aacrjournals.org/). LUSC patients harboring PI3K-activated tumors have poorer Corresponding Author: Ming-Sound Tsao, University Health Network, Princess prognosis than patients without PI3K aberrations (10). Unfortu- Margaret Cancer Centre, 101 College Street 14-305, Toronto, Ontario M5G 1L7, nately, despite the potential driver role of PI3K alterations in Canada. Phone: 416-340-4737; Fax: 416-340-5517; E-mail: [email protected] LUSC, results from the BASALT-1 clinical trial (NCT01820325) of doi: 10.1158/1078-0432.CCR-18-0717 BKM120 in stage IV NSCLC patients were not as promising as Ó2018 American Association for Cancer Research. anticipated (11). Thus, reliable predictive biomarkers of response 5990 Clin Cancer Res; 24(23) December 1, 2018 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst August 9, 2018; DOI: 10.1158/1078-0432.CCR-18-0717 Dual Targeting of PI3K and CDK4/6 in PIK3CA-Mutant LUSC filtering implemented for samples without a matched normal Translational Relevance using dbSNP, ExAC, and ESP (17, 18). Annovar was used to There is currently a lack of targeted therapies that have annotate all the final calls (19). For CNV, DNA from each demonstrated robust efficacy in lung squamous cell carcinoma sample was hybridized to either the HumanOmni 2.5 (LUSC). In our study, we determined through an in vivo BeadChip or the HumanOmni 1-Quad BeadChip SNP arrays screen that PIK3CA-mutated LUSC may better respond to (Illumina). The LogR and B allele frequency values were PI3K inhibition. In addition, combined PI3K and CDK4/6 extracted at each probe locus for all the samples, which was inhibition enhanced response to PI3K monotherapy in then used by Allele-Specific Copy Number Analysis of Tumors PIK3CA-mutated tumors compared with PI3K wild-type (20) to identify copy-number gains and losses within each LUSC. Our data support further clinical evaluation of com- sample. GISTIC v2.0.23 (21) was used to determine regions bined PI3K and CDK4/6 inhibitors in PIK3CA-mutated LUSC. that are significantly amplified or deleted across the samples. For IHC, PTEN IHC tissue microarray (TMA) study was reported previously (22). In vivo and combination therapies are needed to improve LUSC response drug treatment studies in vivo   to PI3K inhibitors. In addition, a combination therapy strategy in The 1 1 1 drug screening method was adopted from a previous study (23). Drug treatment began when tumors reached LUSC could potentially mitigate toxicity resulting from single- 3 agent PI3K inhibitor by reducing drug doses. Therefore, preclin- 200 to 300 mm . Tumors for each model were implanted into ical testing of PI3K inhibitors and combination therapy using 2 NOD/SCID mice, one treated with vehicle and the other with clinically relevant LUSC models is needed. drug via daily oral gavage for 21 days. Tumors were harvested at fi fi In the present study, we used chemotherapy-na€ve LUSC the experimental endpoint, formalin- xed paraf n-embedded patient-derived xenografts (PDX) as preclinical models to screen and snap frozen for subsequent histologic analysis and protein for sensitivity to PI3K inhibitors and potential combination isolation. Waterfall plots of response were generated by calculat- targeted drugs. We determined that PIK3CA mutation is a pre- ing the slope of growth at 10-day intervals compared with base- fi dictor of PI3K inhibitor response (<20% tumor growth compared line. Drug response was determined by the Modi ed RECIST (23). with baseline/vehicle). In addition, combined inhibition of Drug response was validated with an expansion study of 6 to CDK4/6 with PI3K inhibitors in PDXs harboring PIK3CA muta- 8 mice per arm in which tumors were treated for 25 to 30 days. tion and p16 loss resulted in enhanced tumor shrinkage which BKM120 (25 and 50 mg/kg), BYL719 (25 and 50 mg/kg), was not observed with single-agent treatments. We provide a palbociclib (150 and 75 mg/kg), and abemaciclib (50 mg/kg) rationale for clinical testing of combined PI3K and CDK4/6 were purchased from University Health Network Shanghai, Inc. inhibition in LUSC. BKM120, BYL719, palbociclib, and abemaciclib were dissolved in PEG300/NMP, 0.5% carboxymethylcellulose, 0.05N sodium ¼ Materials and Methods lactate (pH 4), and 1% carboxyethylcellulose, respectively (Sigma). Patient samples and PDX establishment Establishment of PDXs from surgically resected primary LUSC FISH tumors from early-stage LUSC patients was approved by the The Vysis LSI CDKN2A SpectrumOrange/Vysis CEP 9 University Health Network Human Research Ethics and Animal SpectrumGreen probes were used for FISH analysis of CDKN2A Care Committee (REB #09-0510-T). All