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Direct CDKN2 Modulation of CDK4 Alters Target Engagement of CDK4 Inhibitor Drugs Jennifer L

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Direct CDKN2 Modulation of CDK4 Alters Target Engagement of CDK4 Inhibitor Drugs Jennifer L Published OnlineFirst March 5, 2019; DOI: 10.1158/1535-7163.MCT-18-0755 Cancer Biology and Translational Studies Molecular Cancer Therapeutics Direct CDKN2 Modulation of CDK4 Alters Target Engagement of CDK4 Inhibitor Drugs Jennifer L. Green1, Eric S. Okerberg1, Josilyn Sejd1, Marta Palafox2, Laia Monserrat2, Senait Alemayehu1, Jiangyue Wu1, Maria Sykes1, Arwin Aban1, Violeta Serra2, and Tyzoon Nomanbhoy1 Abstract The interaction of a drug with its target is critical to achieve bition is not possible by traditional kinase assays. Here, we drug efficacy. In cases where cellular environment influences describe a chemo-proteomics approach that demonstrates target engagement, differences between individuals and cell high CDK4 target engagement by palbociclib in cells without types present a challenge for a priori prediction of drug efficacy. functional CDKN2A and attenuated target engagement when As such, characterization of environments conducive to CDKN2A (or related CDKN2/INK4 family proteins) is abun- achieving the desired pharmacologic outcome is warranted. dant. Analysis of biological sensitivity in engineered isogenic We recently reported that the clinical CDK4/6 inhibitor pal- cells with low or absent CDKN2A and of a panel of previously bociclib displays cell type–specific target engagement: Palbo- characterized cell lines indicates that high levels of CDKN2A ciclib engaged CDK4 in cells biologically sensitive to the drug, predict insensitivity to palbociclib, whereas low levels do not but not in biologically insensitive cells. Here, we report a correlate with sensitivity. Therefore, high CDKN2A may pro- molecular explanation for this phenomenon. Palbociclib tar- vide a useful biomarker to exclude patients from CDK4/6 get engagement is determined by the interaction of CDK4 with inhibitor therapy. This work exemplifies modulation of kinase CDKN2A, a physiologically relevant protein inhibitor of target engagement by endogenous proteinaceous regulators CDK4. Because both the drug and CDKN2A prevent CDK4 and highlights the importance of cellular context in predicting kinase activity, discrimination between these modes of inhi- inhibitor efficacy. Introduction upon CDKN2A–CCND1–CDK4–RB signaling, and initial evalua- tions of individual pathway biomarkers (RB1 expression, CCND1 The eukaryotic cell cycle is tightly regulated to prevent excessive overexpression, and CDKN2A loss) have not yet further refined replication or propagation of cells with damaged DNA. Retino- predictions of patient response to CDK4/6 inhibitor therapies in blastoma protein (pRB, commonly referred to as RB), encoded by this group (2, 3, 5–7). Clinical utility for CDK4/6 inhibitors may the gene RB1, is a negative regulator that blocks the G –S phase þ 1 extend beyond ER advanced breast cancer and clinical trials are transition. Cyclin-dependent kinase 4 (CDK4) and the homolo- ongoing for a variety of other cancer types, including triple- gous CDK6, in complex with D-type cyclins (e.g., CCND1), À À À negative breast cancer (ER /PR /HER-2 ). In such cases, where overcome the RB cell-cycle checkpoint by phosphorylating and ER cannot be used to guide patient selection, additional biomar- inactivating RB. Cyclin-dependent kinase inhibitor 2A (CDKN2A, kers to prospectively identify CDK4/6 inhibitor responsive also known as p16 or INK4a) is another negative regulator of the patients are needed. cell cycle, which binds to and inhibits CDK4/6. CDKN2A, We recently demonstrated that palbociclib occupies the active CDKN2B (p15/INK4b), CDKN2C (p18/INK4c), and CDKN2D sites of native CDK4 and CDK6 in both cellular lysates and live (p19/INK4d) comprise the CDKN2/INK4 family and are struc- cells (target engagement) in a cell type–specific manner (8). turally and functionally similar, but display different expression Although cellular permeability was confirmed in all cells by patterns (1). Disruptions in the CDKN2A–CCND1–CDK4–RB inhibition of established off-targets, CDK4/6 target engagement axis are associated with diverse malignancies (2–4). was only observed in cell lines that are sensitive to growth- Three ATP-competitive CDK4/6 inhibitors (palbociclib, ribo- inhibition by palbociclib. Surprisingly, no CDK4 or CDK6 target ciclib, and abemaciclib) are approved by the FDA as therapies for engagement was observed in palbociclib-insensitive cell lines, advanced hormone receptor-positive, HER2-negative breast can- suggesting that additional mechanisms for CDK4/6 regulation cer either alone or in combination with hormone therapy. Estro- were likely in these cells. Here, we report that CDKN2/INK4 gen receptor (ER) provides a surrogate marker for dependence proteins specifically prevent both nucleotide probe binding to CDK4 and target engagement by small molecule inhibitors. This work provides an example of how regulatory proteins modulate 1ActivX Biosciences, La Jolla, California. 2Vall dHebron Institute of Oncology the susceptibility of a protein kinase to small molecule inhibition. (VHIO), Barcelona, Spain. Corresponding Author: Tyzoon Nomanbhoy, ActivX Biosciences, 11025 N. Torrey Pines Road, Suite 120, La Jolla, CA 92037. Phone: 858-526-2516; Fax: 858- Materials and Methods 587-4878; E-mail: [email protected] Cell culture and proliferation assays doi: 10.1158/1535-7163.MCT-18-0755 All cells were cultured in media supplemented with 10% FBS Ó2019 American Association for Cancer Research. and 1% penicillin/streptomycin in a 37 C incubator with 5% www.aacrjournals.org 771 Downloaded from mct.aacrjournals.org on October 2, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst March 5, 2019; DOI: 10.1158/1535-7163.MCT-18-0755 Green et al. CO2. Basal media was EMEM (MCF7, SK-MEL-28), DMEM centration of 20 mmol/L and incubated for an additional 15 (MDA-MB-231, MDA-MB-468), or RPMI (DU-145, HCC- minutes. Samples were prepared for MS analysis as described 1806). The above cell lines were obtained directly from the ATCC previously (9, 10). Briefly, probe-labeled lysates were denatured and passaged less than 6 months. The HAP-1 WT and HAP-1 DKO and reduced (6 M urea, 10 mmol/L DTT, 65C, 15 minutes), cell lines were obtained from Horizon and cultured in Iscoves's alkylated (40 mmol/L iodoacetamide, 37C, 15 minutes), and gel Modified Dulbecco's medium (IMDM; Gibco) without phenol filtered (Bio-Rad Econo-Pac 10G) into 2 M urea, 5 mmol/L red supplemented with 10% charcoal stripped FBS (Gibco) and methionine, 1M Tris. The desalted protein mixture was digested 1% of penicillin/streptomycin. HAP-1 knockouts were engineered with trypsin (0.015 mg/mL) for 1 hour at 37C, and desthiobio- using the CRISPR-Cas9 system to introduce frameshift mutations tinylated peptides were captured using 12.5 mL high-capacity into the coding sequence of genes of interest. All HAP-1 knockout streptavidin resin (ThermoFisher Scientific ). Captured peptides cell lines were validated by PCR amplification and Sanger were then washed extensively, and probe-labeled peptides eluted Sequencing to confirm the mutation at the genomic level by from the streptavidin beads using two 35 mL washes of a 50% Horizon. CH3CN/water mixture containing 0.1% TFA at 20 to 25 C. HAP-1 proliferation assays were conducted in 96-well plates, with 1,500 cells seeded per well. Twenty-four hours after seeding, LC/MS-MS cells were exposed to vehicle or palbociclib at the indicated Samples were analyzed by LC/MS-MS as described previous- concentrations, followed by growth in media for three cycles. ly (10). Samples were analyzed on Thermo LTQ ion trap mass fi Then cells were xed with 4% glutaraldehyde in PBS, stained with spectrometers coupled with Agilent 1100 series micro-HPLC 0.1% of crystal violet in methanol, and solubilized with 10% of systems with autosamplers, essentially as described (11), using acetic acid in PBS. Absorbance of each well was measured at 560 a custom target list comprising 269 unique kinase peptides that nm. For MCF7 proliferation assays, transfected cells were seeded had been previously identified during proteome characterization in 96-well plates at 1,000 cells per well (the day after transfection) in data-dependent mode (10). and exposed to vehicle or palbociclib at the indicated concentra- tions. After growth in media for 5 days, the CellTiter-Glo assay was Data analysis performed (Promega G7570). For signal extraction and quantitation, typically up to four ions were selected based on their presence, intensity, and correlation to Transfections and siRNA the reference MS-MS spectrum. The resulting chromatographic Cells were transfected in 10 cm plates at 80% confluency with 9 peaks from each run were then integrated and the integrated peak mg DNA using the Fugene HD transfection reagent (Promega) areas were used to determine relative probe ratios between treated according to the manufacturer's instructions. All expression plas- samples and controls. The number of replicates and statistical tests mids were purchased from Origene: PS100010 (GFP), RC220937 used are indicated in the figure legends. (CDKN2A), RC204895 (CDKN2B), RC213083 (CDKN2C), RC214065 (CDKN2D), RC201817 (CDKN1A), RC01661 (CDKN1B), and RC209840 (CDKN1C). After brief expansion, transfected cells were divided into vessels for immunoblotting or Results KiNativ processing and harvested 3 days posttransfection. CDKN2 proteins reduce nucleotide probe binding of CDK4 CDKN2A siRNA (Dharmacon SMARTpool, 1029) or nontarget- Crystal structures of CDKN2A and CDKN2D proteins bound to ing control was delivered to MDA-MB-468 cells by nucleofection CDK6 showed that CDKN2 binding induces conformational (Lonza Nucleofector 4D, SE, DS-120). changes in the kinase that interfere with ATP binding (12, 13). These reports prompted our investigation into the influence of Immunoblotting CDKN2 on nucleotide acyl-phosphate probe labeling of CDK4 Cells were seeded into six-well plates at 400,000/well in com- and CDK6. We used desthiobiotin-linked nucleotide (ATP or plete media. The following day, cells were rinsed with PBS then ADP) probes to covalently modify active-site lysine residues in scraped directly in 1Â sample buffer with BME. Samples were protein kinases (9, 10); a method widely used to profile both sonicated and boiled immediately prior to SDS-PAGE.
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