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Combined LSD1 and ATRA in AML
The union of the time points across all four cell lines shows that 593 genes are altered in all four cell lines in response to the combination, defining an ATRA + LSD1i combination signature (Figure 3C, bottom). The log2 trans- formed gene expression of the intersect genes for each time point (Figure 3C, top) is depicted in a heatmap that shows the row-mean centered data clustered hierarchical- ly (Figure 3D). A subset of genes was validated by qRT- PCR (Online Supplementary Figure S3C). Upon further examination of the combination treatment genes found in both time points (Figure 3C, 593 genes indicated in red) using gene set enrichment analyses, strong enrichment in KEGG pathways include hematopoietic cell lineage and cell adhesion molecule (CAMs) pathways (Figure 3E). This finding was validated using an additional methodology (Online Supplementary Figure S3D) as well as in a subset of cell lines assessed individually (Online Supplementary Figure S3E). CAMs are involved in immune cell function and, therefore, their expression is consistent with a more differ- entiated state. Together with the enriched expression of genes in the hematopoietic cell lineage pathway, enrich- ment in CAMs through the combined treatment of GSK2879552 and ATRA suggests enhanced differentiation of AML cells.
Gene set enrichment analyses revealed additional path- ways in the combination that are associated with immune cell function. The mTOR, focal adhesion, and lysosome pathways are involved in autophagy, an immune mecha- nism for clearing intra-cellular proteins and organelles usu- ally in response to cell stress (Figure 3E).28 Additionally, the systemic lupus erythematosus (SLE) pathway is highly enriched in the combination treatment. Given that SLE is an autoimmune disease, this finding is consistent with an immune-related functional effect.29 In conjunction with enrichment of hematopoietic cell lineage and CAMs sig- natures, perturbation of these additional pathways may provide further evidence of a molecular shift towards a gain of immune cell function.
The addition of all-trans retinoic acid enhances GSK2879552 induced differentiation effects on acute myeloid leukemia cells
Given that the GSK2879552 and ATRA combination affects hematopoiesis and cell adhesion molecule path- ways, studies to confirm these results by measuring differ- entiation in AML cell lines were performed. Myeloid dif- ferentiation cell surface marker expression was measured by flow cytometry on 4 AML cell lines treated for two days with GSK2879552. Representative histograms of CD11b expression on MOLM-13 and OCI-AML3 cells treated with DMSO, 1000 nM GSK2879552, 100 nM ATRA, or the combination of GSK2879552 and ATRA are shown (Figure 4A). In both cell lines, an increase in CD11b expression is evident with the combination. Cell lines were treated with a titration of GSK2879552 plus varying concentrations of ATRA. Percent of cells positive for CD11b were gated relative to isotype control and the val- ues were plotted as dose response curves (Figure 4C). The increase in CD11b was dose responsive both with GSK2879552 and as increasing concentrations of ATRA were combined with GSK2879552 (Online Supplementary Table S3A). To determine whether the impact on CD11b expression with the combination is synergistic, an additiv- ity threshold was calculated for each combination by adding together the increases obtained with each single
agent and was compared to the observed values achieved with the combination (Figure 4D and Online Supplementary Figure S4A). MOLM-13 and SIG-M5 cells treated with as little as 1 nM ATRA combined with GSK2879552 showed synergistic increases in CD11b. Synergistic effects were seen in OCI-AML3 cells with as little as 10 nM ATRA. THP-1 cells had a very robust response to GSK2879552 alone, reaching near 100% positive with as little as 12 nM GSK2879552, precluding assessment of the combination effect (Online Supplementary Figure S4A).
The difference between the additive threshold and the actual percent of CD11b expression achieved was deter- mined for each cell line (Figure 4B). Synergistic increases in CD11b expression were observed in three of four cell lines treated with the combination of ATRA and GSK2879552. Because GSK2879552 alone increased CD11b expression on nearly the entire population of THP-1 cells, median flu- orescence intensity (MFI) values were determined to quantify shifts in CD11b expression (Online Supplementary Figure S4B and C). Fold changes in CD11b MFI were deter- mined relative to DMSO-treated control and additivity threshold values were calculated by adding together the fold increases obtained with each single agent. Actual increases in CD11b MFI were synergistic with as little as 1 nM ATRA combined with GSK2879552.
A hallmark of functional differentiation involves the appearance of morphological changes.30 To investigate the ability of the combination to elicit an altered cell appear- ance, MOLM-13 cells were treated for three days with DMSO, GSK-LSD1, ATRA, or the combination of GSK- LSD1 plus ATRA. Morphology was assessed by May Grunwald/Giemsa stain (Online Supplementary Figure S4D). The combination treatment resulted in the presence of lobular nuclei and granulocytic cytoplasm; both are characteristics of differentiated myeloid cells. These changes were largely absent from the cells treated with a single agent. These observations suggest that morpholog- ical differentiation represents a functional consequence of the gene expression and surface marker changes in AML cells caused by treatment with LSD1 inhibitor plus ATRA.
The combination of GSK2879552 and all-trans retinoic acid promotes caspase-mediated cell death
In addition to inhibiting the growth of cells (Figure 2), a decrease in cell number relative to the number of cells at the start of the assay was observed in the combination (Figure 5A). By day 3, in MOLM-13 cells, the combination treatment produced a negative growth death index (GDI) value that continued to decrease with additional duration of treatment. Neither ATRA nor GSK2879552 alone achieved negative GDI values. By day 5, in OCI-AML3 cells, GSK2879552 plus 1000 nM ATRA achieved a GDI value of -50% compared to 3% with 1000 nM ATRA alone. A decrease in GDI value with the combination was observed in four of seven cell lines tested (Figure 5A and Online Supplementary Figure S5). These results indicate that, in addition to the improved inhibition of cell growth, AML cell death can be achieved with the combination of GSK2879552 and ATRA, and this cell death occurs at a time point after the observed increases in CD11b expres- sion.
To confirm that the combination kills AML cells, and better understand the mechanism associated with this cytotoxic response, AML cells were treated with a dose titration of GSK2879552 plus and minus 1, 10, 100, and
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