Azd1775

AZD1775 was provided by AstraZeneca (Wilmington, DE). The chemical structure of AZD1775 has previously been described (14 (link)). Cytarabine and vincristine were purchased from Sigma-Aldrich (St. Louis, MO) and diluted in water. Vorinostat, panobinostat, and CPI-455 were purchased from Selleckchem (Houston, TX). TP-0906 was purchased from MedChemExpress (Monmouth Junction, NJ). JQ1 was a kind gift from the laboratory of Dr. Jay Bradner. Antibodies specific to actin, c-MYC, pCDK1 Tyr15, CDK1, pCHK1 Ser345, PARP, γH2AX, histone 3 were purchased from Cell Signaling Technology (Danvers, MA). The antibody against KDM5A was purchased from Bethyl Laboratories (Montgomery, TX), and the antibody against trimethylated histone 3 lysine 4 was purchased from Active Motif (Carlsbad, CA). Primers to detect levels of AXL expression relative to GAPDH were 5′- CAGCGCAGCCTGCATGT-3′ (Forward) and 5′- TTGGCGTTATGGGCTTCG-3′ (Reverse).

PDAC cell lines BxPc-3, MiaPaca-2, and Panc1 were purchased from ATCC and routinely screened for mycoplasma. The PDAC cell line L3.3 was a generous gift from John’s Hopkins University. All cells were cultured in DMEM (Corning) supplemented with 10% FBS (Atlas Biologicals), 1% penicillin-streptomycin, and 1% MEM nonessential amino acids (Corning). All PDAC cells tested were p53 mutants. Cells were maintained at 37˚C in an atmosphere containing 5% CO₂. AZD1775 was provided by AstraZeneca or purchased from MolPool (Hong Kong) depending on availability during the study. Irinotecan was purchased from the University of Colorado Hospital Pharmacy. The active metabolite of Irinotecan, SN38, was purchased from Sigma for in vitro analyses. Capecitabine and navitoclax were purchased from Active Biochem. The active metabolite of Capecitabine, 5-fluoruracil (5-FU), was purchased from the University of Colorado Hospital Pharmacy for in vitro analyses.

Cell line origins and their cell growth media can be found in Supplementary Methods Table S1. mESC, FaDu and A549 ATM-knockout cell lines were generated as described before (37 (link),38 (link)). mESC Cdc25a KO clones #4/5 were kindly provided by Oscar Fernández-Capetillo (24 (link)) and U2-OS T-Rex GFP-RNase H1(D210N) (RNH1(D210N)-GFP) or GFP-RNase H1 (RNH1-GFP) cells by Pavel Janscak (39 (link)). mESCs were cultured on 0.1% gelatin-coated tissue culture flasks, and Cas9-expressing cell lines were maintained in blasticidin (10 μg/ml mESC and U2-OS, 5 μg/ml HAP-1). AZD6738 and AZD1775 were made by AstraZeneca. DRB (5,6-dichloro-1-beta-ribo-fuanosyl benzimidazole), actinomycin D, aphidicolin, hydroxyurea, carboplatin and etoposide were obtained from Sigma-Aldrich. XL413 (S7547), VE-822 (S27102), LY2603618 (S2626) and LY2606368 (S7178) were obtained from SelleckChem. CX-5461 (509265) was obtained from Merck Millipore, and BRD-6989 (6438) from Tocris. Final assay DMSO concentrations were normalised to 1:1000 as required. Recombinant human IFNγ was obtained from Peprotech (300–02) and used at a final concentration of 2.5 ng/ml.

The data used to illustrate and implement these approaches examined the effect of the combination of AZD1775 provided by AstraZeneca or purchased from MolPool (Hong Kong) with Navitoclax purchased from Active Biochem (“drug AB”) versus a placebo treatment (“vehicle”). A subset of data from a past experiment on 1 PDX line for triple‐negative breast cancer (TNBC012) is used to illustrate the different methods where 2 tumors were planted into the hind flanks of each mouse as described previously.², ¹⁵, ¹⁶ These tumors were repeatedly measured at unequally spaced days. Tumors were excluded in the real‐world data analysis if the initial starting volume was less than 70 mm³ or greater than 500 mm³. Given the small sample size within each treatment group, a simplifying assumption that all tumors were independent of each other was made for the real‐world data.
The study was carried out in accordance with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals, and in a facility accredited by the American Association for Accreditation of Laboratory Animal Care. Approval from University of Colorado Animal Care and Use Committee was obtained before the initiation of experiments. All mice were female athymic nude mice.

AZD1775, a WEE1 inhibitor, and olaparib, a PARP inhibitor, and AZD6783 a ATR inhibitor were provided by AstraZeneca (Macclesfield, Cheshire, UK) and dissolved in dimethyl sulfoxide (DMSO) at 10 mmol/L to produce stock solutions.