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Combined LSD1 and ATRA in AML
Primary acute myeloid leukemia blast colony
Clonogenic progenitors from primary AML patients' samples were assessed in a semi-solid methylcellulose-based medium (ColonyGelTM, ReachBio) containing recombinant human (rh) SCF (50 ng/mL), rhIL-3 (10 ng/mL), rhGM-CSF (10 ng/mL), and rhEpo (3 U/mL). Each concentration of compound was tested with three technical replicates. Values were expressed as percent of vehicle and graphed as dose response curves (XLfit, IDBS). All known patients' sample characteristics are provided in Online Supplementary Table S1A.
RNA-sequencing
Methods for RNA-sequencing can be found in the Online Supplementary Appendix.
Results
Lysine specific demethylase 1 inhibition reduces growth and promotes differentiation of acute myeloid leukemia cells
To investigate the role of LSD1 activity on the growth of AML cells, AML cell lines were treated with LSD1 inhibitors. As shown previously, GSK2879552 and a struc- turally similar compound, GSK-LSD1, are both potent, selective inactivators of LSD1.11 GSK2879552 was ana- lyzed for its ability to inhibit tumor cell growth in a 10- day proliferation assay using CellTiter-Glo (Figure 1A). The concentration required to achieve half of the maximal observed effect (EC50) as well as the maximum percent
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Figure 2. Sensitivity of acute myeloid leukemia (AML) cells to GSK2879552 increases with addition of all-trans retinoic acid (ATRA). (A) Dose response curves of MOLM-13 (left) and OCI-AML3 (right) cells treated with a titration of GSK2879552 ± 1, 10, 100, or 1000 nM ATRA for four days. Values were graphed relative to the appropriate ATRA alone concentration for each curve. (B) Percent maximum inhibition (blue bars) ± Standard Error and IC50 values (red circles) ± Standard Error for MOLM-13 (left) and OCI-AML3 (right) cells from (A). *Significance (P<0.05) between dimethyl sulfoxide (DMSO) and ATRA treatments for maximum inhibition; †signif- icance (P<0.05) between dimethyl sulfoxide (DMSO) and ATRA treatments for IC50. (C) Synergy scores (± Standard Error) for seven AML cell lines treated with a titra- tion of GSK2879552 ± 1, 10, 100, or 1000 nM ATRA for four days. (D) Synergy scores (± Standard Error) for seven AML cell lines treated with a titration of GSK2879552 ± 1, 10, 100, or 1000 nM ATRA for six days.
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