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Venetoclax response and maturation stage in AML
this can be time consuming and enrichment might deplete cell populations such as monocytes that secrete cytokines important for blast cell survival and drug responses.11–13
To evaluate the ex vivo sensitivity of AML patient sam- ples at a cell population level, we applied a multiplexed, 96-well format flow cytometry (FC)-based drug sensitivity assay. We compared this approach with the CTG-based cell viability assay to study potential inconsistencies between these two methods. Furthermore, we aimed to identify drugs and drug combinations that could effective- ly target leukemic blasts in physiologically relevant con- centrations. In addition to standard of care drugs, cytara- bine and idarubicin, we selected five Food and Drug Administration approved targeted small molecule inhibitors that have shown AML-selective responses in our earlier studies:14,15 MEK inhibitor (trametinib), JAK1/2 inhibitor (ruxolitinib), mTORC1 inhibitor (everolimus), FLT3/broad range tyrosine kinase inhibitor (TKI, sunitinib) and Bcl-2 inhibitor (venetoclax). Most importantly, we demonstrate that targeted agents, particularly venetoclax, have different efficacies towards AML cells at distinct stages of myeloid differentiation.
Methods
Methods are described in more detail in the Online Supplementary Material and Methods.
Patient samples
BM samples from 34 AML patients and three healthy volunteers were obtained from the Helsinki University Hospital Comprehensive Cancer Center after informed consent (permit numbers 239/13/03/00/2010, 303/13/03/01/2011, Helsinki University Hospital Ethics Committee) and in compliance with the Declaration of Helsinki. The patient characteristics are presented in the Online Supplementary Table S1.
Preparation of drug plates
The compounds (Online Supplementary Table S2) were dispensed on 96-well V-bottom plates (Thermo Fisher Scientific, Carlsbad, CA) and 384-well plates (Corning, Corning, NY, USA) using an acoustic liquid handling device Echo 550 (Labcyte, Sunnyvale, CA). Drug plate layouts and concentrations are presented in Online Supplementary Figure S1. BM mononuclear cells (BM-MNC) were isolated using Ficoll-Paque Premium (GE Healthcare, Little Chalfont, Buckinhamshire, UK) density gradient centrifugation. Fresh or frozen BM-MNC were suspended in mononuclear cell
medium (MCM; PromoCell, Heidelberg, Germany) supplemented with 10 mg/mL gentamicin and 2.5 mg/mL amphotericin B and plated in parallel on pre-drugged 96-well plates (100,000 cells/well in 100 ml) for FC analysis and 384-well plates (10,000 cells/well in 25 ml) for CellTiter-Glo® (CTG)-based cell viability assay. The cells were incubated with the drugs for 72 hours at 37°C and 5% CO2.
FC-based readouts
Following the 72-hour incubation with the drugs, cells were stained with an antibody mix (CD33, CD45, CD14, CD38 and CD34) followed by apoptosis (Annexin-V) and dead (7-AAD) cell staining. A detailed description of the methods is presented in Online Supplementary Material and Methods and the gating strategy is illustrated in Figure 1B.
Cell viability analysis using CellTiter-Glo®
Parallel to FC analysis, cell viability was measured with CellTiter-Glo® (CTG; Promega, Madison, WI) in 384-well plates as described earlier.14 After the 72-hour incubation with the drugs, 25 mL CTG was added to each well. The luminescence signal was measured using a PHERAstar plate reader (BMG LABTECH, Ortenberg, Germany).
Calculation of the drug sensitivity (DSS) and drug combination scores
Ex vivo drug sensitivity of AML and healthy BM cells to the test- ed drugs was calculated using a DSS as previously described.16 Drug combination efficacies were calculated as the difference between observed and expected values. The expected value is computed using the Bliss independence model17 as reference, which assumes that two drugs exhibit their effect independently.18
Gene expression and pathway analysis
Publicly available microarray data from the Hemap data set19,20 (http://hemap.uta.fi/) and RNA-seq data (RSEM values) from the TCGA Research Network21 (http://cancergenome.nih.gov/) also included in the Hemap resource were used for gene expression and pathway analysis. Beat AML data22 was used to assess the cor- relation between venetoclax drug sensitivity and BCL2 family and monocytic/granulocytic differentiation marker gene expression. For the analysis of gene expression in healthy hematopoietic cell types Differentiation Map data was used.23 Detailed methods are described in the Online Supplementary Material and Methods.
Statistical analysis
Statistical analysis was conducted with Graph Prism version 7.0 (GraphPad Software, San Diego, CA). Differences between drug responses were analyzed by Mann-Whitney U test, and for multi-
Table 1. Median drug sensitivity score (DSS) and IC50 values of the seven tested drugs against different cell populations.
Blasts (n=33) Monocytes (n=18) Lymphocytes (n=31) Granulocytes (n=5) DSS IC50 (nM) DSS IC50 (nM) DSS IC50 (nM) DSS IC50 (nM)
Median (Range) Median (Range) Median (Range) Median (Range) Median (Range) Median (Range) Median (Range)Median (Range)
Venetoclax
Idarubicin Cytarabine Ruxolitinib Trametinib Sunitinib Everolimus
27.1 (0-43)
22.0 (0-40.0) 9.7 (0-36) 5.0 (0-32) 3.0 (0-27) 1.0 (0-17) 0.0 (0-19)
3.0 (1-1000)
28.7 (2-212) 894.2 (50-10000) 302.7 (50-3000) 18.9 (1-250) 321.1 (8-1000) 55.6 (1-100)
7.1 (0-29)
16.1 (6-34) 7.5 (0-23) 17.2 (0-37) 25.9 (0-42) 5.7 (3-22) 4.6 (0-28)
122.0 (1-1000)
78.7 (13-390) 1071 (20-10000) 93.3 (60-2896) 2.4 (1-250) 223.7 (71-423) 7.5 (3-28)
18.1 (9-30)
16.5 (9-28) 4.8 (0-10) 0 (0-8) 0 (0-1) 0 (0-4) 0 (0-10)
20.3 (2-84)
84.0 (18-227) 2550 (43-10000) 2511 (99-10000) > 250 (7-250) > 1000 (6-492) > 100 (2.5-100)
5.7 (0.3-9)
19.0 (12-24) 9.5 (4-19) 0 (0.0-7) 1.1 (0.0-7) 4.4 (1-11) 0 (0.0-3)
113 (11-220)
41.1 (26-154) 953 (305-1189) 2476 (246-3 000) 165 (15-250) 352 (92-434) > 100 (33-100)
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