SCNP assay
SCNP assays were performed as described previously [
8]. Aliquots of cryopreserved cells were thawed at 37°C, washed, resuspended in RPMI-1640 medium supplemented with 60% fetal bovine serum (FBS), and live mononuclear cells isolated via ficoll density gradient. After a second washing step with RPMI-1640 60% FBS, cells were washed in RPMI-1640 10% FBS, counted, filtered, re-suspended in RPMI-1640 10% FBS, then aliquoted (100,000 cells/condition for primary AML cells or 50,000 cells/condition for cell lines) and rested for 30 minutes at 37°C before addition of therapeutic agents (each tested at a clinically relevant dose ranging between Cmax and trough level as reported in pharmacokinetic studies [
9‐
11]). For all conditions, following incubation with drugs, cells were stained with amine aqua viability dye (Life Technologies, Carlsbad, CA) to distinguish non-viable cells, fixed with 1.6% paraformaldehyde for 10 minutes at 37°C, pelleted, permeabilized with 100% ice-cold methanol, and stored at -80°C. For antibody staining, cells were washed with FACS buffer (PBS, 0.5% BSA, 0.05% NaN
3), pelleted, and stained with unlabeled antibody cocktails followed by fluorochrome conjugated goat anti mouse or goat anti rabbit secondary antibodies (Life Technologies and Jackson Immunoresearch, West Grove, PA), then blocked with normal rabbit serum and normal mouse serum (Life Technologies) and stained with cockails of fluorochrome-conjugated antibodies. Cocktails included antibodies against cell surface markers for cell gating of AML cells [e.g. CD45, CD11b (Beckman Coulter, Brea, CA), CD34 and CD33 (BD Biosciences, San Jose, CA)] and up to 3 antibodies against intracellular signaling molecules (detailed below) for 6- 8-color flow cytometry assays.
Data was acquired on an LSR II flow cytometer using the FACS DIVA software (BD Biosciences). All flow cytometry data were analyzed with FlowJo (TreeStar Software, Ashland, OR) or WinList (Verity House Software, Topsham, ME). Daily QC of the LSRII cytometers was performed as previously described [
12]. Dead cells and debris were excluded by forward and side scatter properties combined with amine aqua viability dye exclusion. For AML samples, “all” non-apoptotic leukemic cells were identified based on expression of CD45 and side-scatter properties and lack of the apoptosis marker cleaved PARP (cPARP, BD Biosciences) as previously described [
8,
13], while CyclinA2 (Beckman Coulter) staining discriminated CyclinA2- and CyclinA2+ subsets. Similarly, normal lymphocytes within AML samples were identified by low side scatter and high CD45 expression as previously described [
8,
13]. For cell lines, forward scatter, side scatter, amine aqua, and cleaved PARP similarly identified live non-apoptotic (healthy) cells and CyclinA2 staining discriminated CyclinA2- and CyclinA2+ subsets. Specific drug treatments and readouts examined were as follows:
a) For experiments measuring multiple DDR readouts after etoposide treatment, cell lines (Cell line panel 1) or primary AML samples were treated with 30 μg/mL etoposide (Sigma, St. Louis, MO) for 2 h or 6 h and assayed for p-BRCA1 (S1423) (Novus, Littleton, CO), pDNA-PKcs (T2609) (Biolegend, San Diego, CA), p-53BP1 (S1778), p-ATM (S1981), p-p53 (S15), p-Chk2 (T68), and p-H2AX (S139) (Cell Signaling Technologies, Danvers, MA).
b) For experiments showing magnitude and reproducibility of multiple AZD2281+/- temozolomide-induced DDR readouts and the ability of these readouts to stratify for HRR status, BRCA1 +/+ , BRCA1 +/- , and BRCA2 -/- cell lines (Cell line panel 2) were treated with 6 μg/mL AZD2281 (Selleck, Houston, TX) +/- 2 μg/mL temozolomide (Sigma) for 48-72 h and assayed for p-H2AX (S139), p-RPA2/32 (T21) (Abcam, Cambridge, MA), p-DNA-PKcs (T2609), p21, p-ATM (S1981) and p-BRCA1 (S1423) in CyclinA2- and CylcinA2+ cells. In these experiments a 2 μg/mL dose of temozolomide was used due to excessive apoptosis observed with the combination of AZD2281 with higher doses of temozolomide (10 μg/mL) at later timepoints (data not shown).
c) For experiments measuring the cell cycle selectivity of individual genotoxic agents, cell lines in culture or AML samples pre-treated for 48 h with a panel of myeloid growth factors to induce proliferation in all AML subtypes [20 ng/mL of IL-3 (BD Biosciences), TPO, SCF (R&D Systems, Minneapolis, MN) and FLT3L (ebiosciences, San Diego, CA)] were challenged for 6 h with etoposide (30 μg/mL), PARP inhibitor AZD2281 (6 μg/mL), temozolomide (10 μg/mL), or the combination of AZD2281 + temozolomide (6 μg/mL + 10 μg/mL) and assessed for induced p-H2AX in distinct subsets of cells including CyclinA2+ cells, CyclinA2- cells, or “All” live, cPARP- cells regardless of CyclinA2 status.