Page 71 - Haematologica - Vol. 105 n. 6 - June 2020

Innate drug responses in hematologic cell populations
CD3+CD8+), NK-T cells (NK-T, CD3+CD56+), and mono- cytes (CD14+) using three healthy blood samples to gener- ate a global view of response profiles. Unsupervised hier- archical clustering of DSS of the screened samples segre- gated in three major clusters based on cellular lineages (Figure 2A). Monocytes formed a single cluster and dis- played selective sensitivity to MEK/ERK inhibitors and the kinase inhibitor dasatinib (Figure 2A). The MEK inhibitor
trametinib was similarly active in BM derived CD14+ cells from healthy and AML samples (Online Supplementary Figure S4A). However, reduced efficacy of dasatinib in BM monocytes was noted compared to those derived from blood (Online Supplementary Figure S4B). B and NK cells showed similar drug response profiles with higher sensi- tivity to the glucocorticoid dexamethasone, BCL2 inhibitor venetoclax and pan-kinase inhibitor midostaurin
Figure 1. Overview of the study. Schematic diagram summarizing the study design, datasets and analytical framework of the study. Bone marrow (BM) and peripheral blood (PB) samples from both healthy individuals and cancer patients were subjected to drug sensitivity assessment. Single cell drug sensitivity assay using the iQue® Screener PLUS flow cytometer was performed in 96- and 384-well plates to monitor drug effects on ten and six hematopoietic cell subtypes, respectively. Immunophenotypic details and cellular proportions of the analyzed cell types are provid- ed in Online Supplementary Figure S1A-D, Figure S2 and Online Supplementary Table S3, respectively. 71 drugs in 384-well plates and six drugs in 96-well plates were tested. Proteomic analysis was performed on three cell subsets (monocytes, T and B cells) from two healthy individuals and four myeloma patients. Basal phosphorylation of nine signaling proteins involved in MAPK, JAK- STAT, PI3K-AKT-mTOR and NF-κB signaling was monitored in 14 samples. Healthy BM samples from four healthy individuals were subjected to CD34, CD3, CD14, CD19 and CD138 cell enrichment and tested against 71 small mole- cules with cell viability as the end point readout using the CellTiter- Glo® assay. A comparison of ex vivo drug response in healthy and corresponding malignant cell types was performed for six drugs in 281 primary patient samples representing different hematolog- ic malignancies. Samples included both published and unpublished datasets from chronic myeloid leukemia (CML, n=13),11,12, chronic myelomonocytic leukemia (CMML, n=11),12 myelodysplastic syn- dromes (MDS, n=4), acute myeloid leukemia (AML, n=145),9,12 B-cell acute lymphoblastic leukemia (B- ALL, n=14),13 chronic lymphocytic leukemia (CLL, n=4),12 T-cell pro- lymphocytic leukemia (T-PLL, n=40),14 multiple myeloma (MM, n=50),15 and other hematologic malignancies (n=6). PLL: prolym- phocytic leukemia.
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