Anti cancer compound library

3 × 10³ MEFs were plated in 96-well plates a day before treatment with chemicals. The cells were treated with dimethyl sulfoxide (DMSO; Biosesang, Korea) or Selleck Anti-Cancer Compound Library for 24 h. Cells treated with 1 µM Tg for 30 min served as positive control. The medium was replaced with that containing 12 μg/mL puromycin, and the cells were incubated for 10 min. The cells were then fixed with 4% paraformaldehyde for 15 min. After washing twice with phosphate-buffered saline (PBS), the cells were permeabilized in 0.3% Triton X-100 in 1% bovine serum albumin (BSA)/PBS for 45 min and treated with the primary anti-puromycin antibody (1:5000) at 4 °C overnight. After washing with PBS, the cells were incubated with Alexa Fluor^® 488 AffiniPure Goat Anti-Mouse IgG (1:400; Alexa Fluor 488-conjugated anti-mouse antibody) for 90 min. Cells were washed with PBS and incubated with Hoechst 33342-containing PBS (1:2500; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The fluorescence intensity of Alexa 488 was detected using a fluorescence confocal microscope (Operetta High-content analysis system; PerkinElmer, Waltham, MA, USA). The cell number and cytoplasmic intensity of Alexa Fluor 488 in the acquired images were determined using Harmony software.

Cells derived from 18 of the patients included in our study were screened for sensitivity to 349 drugs (anti-cancer compound library #L3000 from Selleck Chemicals) using DSRT^{16 (link)}.
The 349 compounds from the anti-cancer Selleck Chemical library were pre-aliquoted at five concentrations, increasing in 10-fold steps from 1 nM to 10 μM, in 384-well plates using an Echo Liquid Handler. Each plate also contained eight positives (benzethonium chloride, BzCl) and eight negative (DMSO only) controls. Fresh diagnostic (pre-treatment) BM or PB was lymphoprepped (15 BM and 8 PB), and cells were seeded directly into the plates containing the drugs at a concentration of 10.000 cells/well and incubated for 72 h in mononuclear cell media (MCM) media (promo cell C-28030) supplemented with 1% Penicillin + Streptomycin (PS) (Gibco,15140-122). Finally, cell viability was assessed by CellTiter-Glo Luminescent viability assay. All drugs were also tested against samples from five healthy donors (four BM and one PB).
All patient samples were normalized to the median of the positive (BzCl) and negative (DMSO) controls per plate and presented as selective Drug Sensitivity Score (sDSS), calculated using the online tool Breeze (https://breeze.fimm.fi/28489_mc43oty0nzywmcaxnje3nzgwnzaw/index.php#).

Specific antibodies against KLF4 (D1F2, 1:1,000), PARP (46D11, 1:1,000), Cleaved PARP (Asp214) (D64E10, 1:1,000), FLAG (D6W5B, 1:1,000), Phospho‐Histone H2A.X (Ser139, 1:1,000), 53BP1 (P550, 1:1,000), and BRCA1 (A8X9F, 1:1,000) were purchased from cell signaling (Beverly, MA). KLF4 (H‐180, 1:1,000), KLF4 (F‐8, 1:1,000), PARP1 (H‐300, 1:1,000), HA (F‐7, 1:1,000), and Myc tag (9E10, 1:1,000) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against β‐actin (AC‐15, 1:5,000) and FLAG (M2, 1:2,000) were from Sigma‐Aldrich. Antibodies against PAR were purchased from Trevigen (4336‐BPC‐100, 1:1,000). The anti‐FLAG M2 affinity gel was from Sigma‐Aldrich. The PARP1 inhibitor niraparib, olaparib, and rucaparib were purchased from Selleckchem (Houston, TA). Puromycin and blasticidin were from Invitrogen. Cycloheximide was from Sigma. The anti‐cancer compound library was purchased from Selleckchem. The compounds ABT‐263 and dasatinib used in animal model were purchased from Selleckchem.