Ribociclib

A375, SK-Mel5, MCF7 cells were purchased from ATCC. YUMM10.1 and YUMM1.7 cells were provided by Marcus Bosenberg (Yale University). Cells were cultured in DMEM/F12 medium supplemented with 10% FBS and 1% penicillin/streptomycin (all Gibco). HDM201, CGM097, ribociclib, and buparlisib were provided by Novartis. GDC-0994 and ipatasertib were purchased from Medkoo. Palbociclib and roscovitine were purchased from LC laboratories. Nutlin-3a was synthesized as described previously (70 (link)). Working concentrations of CDK4/6 inhibitors ribociclib and Palbociclib used for in vitro experiments were chosen based on previously reported human pharmacokinetics data and kinase-specificity assays (71 (link)-74 (link)). Antibodies used in this study are indicated in table S2.

This was a multicenter, phase II study in patients with foregut WDNETs (ClinicalTrials.gov study NCT03070301). The study was reviewed and approved by the Memorial Sloan Kettering Cancer Center and Dana-Farber Cancer Institute Institutional Review Boards and was conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice guidelines. Written informed consent was obtained from each patient after full explanation of the purpose and nature of all procedures used. Both ribociclib and everolimus were provided by Novartis.

All cell lines were cultured at 37°C in a humidified atmosphere containing 5% CO2. A673, SKNEP1, SKNMC, and the SKPNDW lines were grown in Dulbecco’s Modified Eagle’s Media (Life Technologies) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich). A673 cells were supplemented with 1 mmol/L of sodium pyruvate (Life Technologies). The CADOES1 and TC32 lines were cultured in RPMI1640 (Sigma-Aldrich) with 15% and 10% FBS, respectively. All cells were grown in the presence of 10 units/mL of penicillin, 10 μg/mL streptomycin and 30 μg/mL of L-Glutamine, PSQ (Thermo Fisher Scientific). Whole exome sequencing, RNA sequencing and STR genotyping of all Ewing sarcoma cell lines used in these studies and validated cell line identity.(20 (link)) Cell lines were regularly tested for Mycoplasma by PCR (Sigma Aldrich). All cell lines were passaged between 4–12 weeks between thawing and use in the described experiments. All cell lines were a kind gift from Todd Golub (Broad Institute, Cambridge, MA), Nathanael Gray (Dana-Farber Cancer Institute, Boston, MA). Ewing sarcoma cell lines were treated with the CDK4/6 inhibitor ribociclib and the IGF1R inhibitor AEW541 kindly provided by Novartis Oncology. Palbociclib (Cat. No S1116), linsitinib (Cat. No S1091), and GDC0941 (Cat. No S1065) were obtained from Selleck Chemicals. THZ1 was a kind gift from Nathanael Gray.