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Combined LSD1 and ATRA in AML
(Figure 1C). These data indicate that the growth inhibitory effects of LSD1 inhibitors are due to a slowing of cell divi- sion and not significant killing of tumor cells.
Lysine specific demethylase 1 is a known regulator of normal hematopoiesis whereby it maintains hematopoiet- ic stem and progenitor cell populations in a quiescent state through GFI1-mediated transcriptional repression of HoxA9 and Meis1.22 Additionally, several reports have shown that LSD1 inhibition promotes differentiation of leukemic cells.10 Knockdown of LSD1 leads to an increase in markers of differentiation, and this effect has been reca- pitulated using small molecule inhibitors of LSD1.1,12 To determine if growth inhibition achieved with GSK2879552 occurs in association with cellular differenti- ation, MOLM-13 cells were treated for one day with a titration of GSK2879552, and expression of myeloid dif- ferentiation marker genes, ITGAM (CD11b) and CD86, was measured by RT-PCR (Figure 1D). Higher expression of CD11b is indicative of differentiation from an imma- ture myelo/monoblast to a more mature myeloid cell, and CD86 is co-expressed with CD80 on mature macrophages and dendritic cells where it is poised to interact with CD4+ T cells.23,24 GSK2879552 increased expression of CD11b and CD86 genes with average EC50 values of 31±1 nM and 28±6 nM, respectively. To determine if these increases in gene expression translate to increased protein levels, cell surface expression of CD11b and CD86 were measured in THP-1 and MOLM-13 cells by flow cytometry (Figure 1E and Online Supplementary Figure S1D and E). The percent of cells positive for each surface marker was determined by gating against an isotype control. Cells treated with a titration of GSK2879552 for one day showed a dose- dependent increase in protein expression with average EC50 values of 23±4 nM and 44±4 nM for THP-1 and MOLM-13 cells, respectively.
To determine if the effect of GSK2879552 on differenti- ation markers occurs across cell lines that represent a range of AML subtypes, a panel of cell lines was evaluated by flow cytometry after treatment with either GSK2879552 or GSK-LSD1 (Figure 1F). Thirteen of 16 AML cell lines tested had at least a 10% increase in CD86 after three days of treatment, while 10 of 16 had at least a 10% increase in CD11b. Increases for both markers occurred in all five subtypes tested. Overall, the lowest increases in CD11b and CD86 occurred in the M6 sub- type-derived AML cell lines (Online Supplementary Figure S1F). This is not surprising given that M6 AMLs are ery- throleukemias, and differentiation of this subtype, similar to hematopoietic progenitor cells that undergo ery- thopoiesis to become mature red blood cells, may not involve increases in expression of CD11b or CD86.22 To better understand the effects on M6 subtype AML cells, another marker of hematopoietic progenitor cells, trans- ferrin receptor (CD71), was evaluated.25 CD71 decreases as progenitor cells mature and, indeed, five of six M6 sub- type AML cell lines showed at least a 10% decrease in expression of CD71 after three days of treatment with GSK2879552 (Online Supplementary Figure S1F).
One characteristic of active macrophages and neu- trophils is their ability to produce superoxide anion in response to stimulation;26,27 therefore, the ability of GSK2879552 to induce superoxide anion production was evaluated as a functional readout of differentiation. Four AML cell lines were treated with GSK2879552 for 4, 7, 11, and 14 days. Upon stimulation with PMA, all four cell
lines released higher levels of superoxide anion when pre- treated with LSD1 inhibitor (range: 3- to 249-fold over DMSO treated cells) (Figure 1G). SKM-1 cells were treated with a titration of GSK2879552 for seven days, and upon PMA stimulation, superoxide anion production was induced with an EC50 of 222±103 nM (Figure 1H).
To determine if the anti-proliferative effect of LSD1 inhibition in AML cell lines translates to primary AML, patient-derived samples that had not been subjected to long-term cell culture were evaluated using a blast colony formation assay. The number of blast colony forming units was enumerated after treatment with a titration of GSK2879552 in 14 AML patient samples. Twelve of the 14 samples were sensitive, defined as ≥30% inhibition, to GSK2879552 (Figure 1I). The majority of samples showed reduced colony growth by less than 50% while two sam- ples reached complete reduction in blast colonies at the highest dose tested. These data suggest that the anti- tumor activity of GSK2879552 is not limited to cell lines and extends to AML patient-derived cells.
The combination of LSD1 inhibitor with all-trans retinoic acid enhances anti-tumor activity
Given that the effects of LSD1 inhibition alone on AML cells are largely cytostatic and, therefore, may not be suf- ficient to combat the disease clinically, efforts were made to explore combinations that could improve outcome. Previous reports suggested the combination potential of LSD1 inhibition and ATRA in non-APL AML.19,12 ATRA is used clinically for the treatment of APL and works by degrading the PML-RARα fusion, resulting in differentia- tion of blast cells and elimination of disease.16 Schenk et al. have shown enhanced reduction of cell growth when tranylcypromine, a much less potent, non-selective inhibitor of LSD1, is combined with ATRA.19,20 Therefore, studies were undertaken to determine if a similar combi- nation effect could be observed with the selective LSD1 inhibitor, GSK2879552. Seven AML cell lines were treated with a titration of GSK2879552 + 0, 1, 10, 100, and 1000 nM ATRA and growth was monitored for up to six days. The maximum inhibition achieved with the combination increased as the ATRA concentration increased in five of seven cell lines with a corresponding decrease in the IC50 value in four of seven cell lines on day 4 (Figure 2A and B and Online Supplementary Figure S2A). An additional cell line showed a decrease in IC50 value without a concomi- tant increase in maximum inhibition. Together these data indicate that the combination of GSK2879552 and ATRA results in enhanced growth inhibition of AML cell lines. To determine whether the effect observed was synergistic, a Bliss Independence model was used (Online Supplementary Table S2A and B).21 A value ≥100 indicates synergy by calculating a single score that summarizes the Bliss Independence values obtained.9 Synergistic growth inhibition is evident by day 6 in all seven AML cell lines evaluated upon treatment with GSK2879552 combined with ATRA (Figure 2C-D).
Combination of GSK2879552 with all-trans retinoic acid leads to enrichment of differentiation associated gene signatures in acute myeloid leukemia cells
To elucidate the mechanism by which GSK2879552 and ATRA combine to effect tumor cell growth, an RNA sequencing study to examine differential gene expression was conducted in six AML cell lines treated with vehicle,
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