Palbociclib

RPE1 cells were grown in Dulbecco’s Modified Eagle’s Medium F-12 (DMEM/F-12) (Nacalai Tesque, 11581-15) with 10% FBS (NICHIREI, 175012) and 1% penicillin/streptomycin (Nacalai Tesque, 09367-34). BJ-5ta cells were grown in a 4:1 mixture of DMEM (Nacalai Tesque, 08459-64) and Medium 199 (ThermoFisher, 11150059) with 10% FBS and 1% penicillin/streptomycin. All cell lines were cultured at 37 °C in a humidified 5% CO₂ incubator. For cell-cycle synchronization with palbociclib, RPE1 cells were treated with 300 nM palbociclib (Selleck, S1116) for 24 h and released by replacing with fresh media. palbociclib stock solution (100 µM) was prepared in DMSO and added 3 µL to 1 mL of fresh media. For the starvation and aphidicolin synchronization, cells were first serum starved with medium containing 0.1% FBS for 24 h and subsequently released by replacing with medium containing 20% FBS. After 4 h of the release, cells were treated with 1 µM aphidicolin (Sigma-Aldrich, A0781) for 18 h and released by replacing with fresh media. aphidicolin stock solution (1 mg/mL) was prepared in DMSO and added 0.34 µL to 1 mL of fresh media.

K562 cells were treated with 5 μM palbociclib (PD-0332991; Selleck, S1116) for 36 h before SpCas9 transfection and were further cultured in 5 μM palbociclib before cell harvest. For cell cycle analysis, cells were labeled with 50 μM BrdU for 60 min and fixed by paraformaldehyde (PFA) for 60 min at 4 °C followed by anti-BrdU (100×, BD Biosciences, 556028) incubation for 40 min. Next, cells were stained with 7-AAD (250×, BD Pharmingen, 559925) for 20 min and analyzed by FACS.

Melanoma cell lines YUSEEP, YUHIMO, YUWERA, and YUCRATE were obtained from Ruth Halaban (Yale University) in 2020. Melanoma cell lines WM4235, WM4324, WM9, 1205Lu, WM989, WM983B, WM4380 and WM4258, as well as the PDX model WM4223 were obtained from Meenhard Herlyn (Wistar Institute) in 2020. All patient samples were collected under institutional review board (IRB) approval [34 (link)]. Cell lines were tested for Mycoplasma biannually and authenticated using short-tandem repeat fingerprinting. All cell lines are cultured in RPMI-1640 (Corning,10-040-CM) supplemented with 5% fetal bovine serum (FBS; Cytiva, SH30109.03) in the presence of 5% CO₂ at 37 °C. Commercially purchased compounds include palbociclib (SelleckChem, S1116), ribociclib (SelleckChem, S7440), abemaciclib (Apex Biotechnology, A1794), trametinib (SelleckChem, S2673), AZD6244 (SelleckChem, S1008) and VX-11e (SelleckChem, S7709).

The aim of this study was to test an evolution-based strategy to treat ER+ endocrine-resistant breast cancer. We first evolved the estrogen-positive (ER+) breast cancer cell line MCF7 to be resistant to fulvestrant (a selective estrogen receptor modulator (SERM), Selleck Chemicals LLC, Houston, TX, USA) and palbociclib (a CDK4/6 inhibitor, Selleck Chemicals LLC, Houston, TX, USA), both of which are commonly used clinically for ER+ breast cancer. We then tested a variety of adaptive therapy protocols and compared them to constant MTD therapy as well as vehicle control (no therapy), measuring time until death as the primary outcome. In all cohorts, the mice were randomly assigned to the treatment and control groups presented in Table 1. The primary endpoint for the mouse experiments was time to death (see below). We monitored tumor burden by the Xenogen IVIS Spectrum (PerkinElmer, Waltham, MA, USA) in vivo imaging system.

Confocal images were acquired in a TCS-SP5 AOBS laser scanning confocal microscope (Leica Microsystems). For the detection of palbociclib fluorescence in live cells we used a 405 nm excitation laser and collected the emitted light between 500 and 550 nm. For the detection of ribociclib fluorescence in live cells we used a 405 nm excitation laser and collected the emitted light between 450 and 500 nm. Acridine orange and lysotracker red (both from Life Technologies) were added at 1 μg/ml to the culture media 30 min before image acquisition. To acquire the lysotracker signal live cells were excited with a 561 nm laser and the fluorescence was detected in the 580–650 nm range. Acridine Orange signal was acquired by excitation of live cells with an Argon laser (488 nm) and the emitted signal was collected in two separate fluorescence emission ranges: from 500 to 550 and from 660 to 750 nm. To analyze the effect of lysosomotropic compounds on the pH of acidic vesicles, 4 μM palbociclib (Sellekchem), 50 μM chloroquine (Sigma) or 40 nM bafilomycin (Calbiochem) were added to the cells 1 h before acridine orange addition. The fluorescence signal of palbociclib and lysotracker red in individual cells was quantified using a Defineas software. At least 100 cells of each conditions were quantified.

The breast cancer cell lines MCF‐7, MDA‐MB‐231, MDA‐MB‐468 and HCC1954 were maintained in RPMI‐1640 plus GlutaMAX‐1 (Gibco) supplemented with 10% fetal calf serum (FCS) and 10 µg/ml insulin. HEK293T cells were maintained in DMEM (Gibco) plus 10% FCS. JQ1, I‐BET762 and I‐BET151 were purchased from Cayman Chemical (Ann Arbor, MI, USA). Trametinib, olaparib, palbociclib, alisertib, dasatinib, 17‐AAG, lapatinib, trastuzumab, cycloheximide (CHX) and Mcl‐1 inhibitors (S63845 and TW‐37) were commercially sourced from Selleckchem (Houston, TX, USA).