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K.N. Smitheman et al.
1000 nM ATRA for up to six days and caspase 3/7 induc- tion was measured. Similar to the method used in analysis of the cell surface marker data (see above), on the day of peak caspase induction an additivity threshold was deter- mined for each combination by adding the fold caspase induction values obtained with each individual treatment condition. By comparing the observed fold induction achieved to the additive threshold, synergistic caspase activation is revealed with the combination at ATRA con- centrations as low as 100 nM in MOLM-13 and OCI- AML3 cells and as low as 10 nM ATRA in SIG-M5, MV-4- 11, and HL-60 cells (Figure 5C and Online Supplementary Figure S5B). Additionally, annexin V staining was per- formed on MOLM-13 cells treated for three days with GSK-LSD1±1000 nM ATRA. Annexin V positive cells increased with the combination to a greater extent than each compound alone, providing supporting evidence for the cell death caused by the combination (Online Supplementary Figure S5C). Importantly, the cells that are positive for Annexin V are also positive for CD11b, demonstrating that the cells that differentiate also undergo apoptosis (Online Supplementary Figure S5D).
Using an approach similar to that utilized in the cell sur- face marker assessment, the difference between the addi- tive threshold of caspase activation and the observed value was determined for each cell line and a heatmap was generated (Figure 5B). The combination of GSK2879552 plus ATRA induced caspase 3/7 in five out of seven AML cell lines tested. Importantly, the caspase induction in the combination reached levels greater than 10-fold above the additive threshold in all five of the pos- itive cell lines at 100 nM ATRA, a concentration that cor- responds to clinically achieved exposures.31,32
Acute myeloid leukemia blast colony forming ability is impaired in patient-derived samples treated with
a combination of GSK2879552 plus all-trans retinoic acid
Translating a combination effect beyond cell lines is important in understanding the potential for success in clinical trials. To this end, we assessed GSK2879552 com- bined with 100 nM ATRA in blast cells derived from BM of nine different AML patients (Online Supplementary Supplementary Table S1A). Dose response curves for GSK2879552, ATRA, and the combination of GSK2879552 plus 100 nM ATRA are shown for two patients (Figure 6A and D). For patient AML0007, the maximum inhibition of blast colony formation achieved with GSK2879552 was 39% while 100 nM ATRA had no effect on colony count. The combination of GSK2879552 and ATRA reached almost complete inhibition, a >50% decrease in AML blast colony forming ability (Figure 6B). Similarly, the IC50 improved >2-fold from 105 nM with GSK2879552 to 43 nM with the combination (Figure 6C). A second patient-derived sample, 4031113SH, had a mod- est improvement in maximum inhibition (54% inhibition with GSK2879552, 4% inhibition with ATRA, 69% inhi- bition with the combination); however, the IC50 decreased >10 fold from 150 nM with GSK2879552 to 11 nM with the combination (Figure 6E and F). Overall, a combination effect was observed in seven out of nine patient samples analyzed in at least one of the two parameters measured (Online Supplementary Figure S6A and B). An increase in maximum inhibition of at least 15% with the combina- tion was achieved in five out of eight samples and of at
least 30% in three out of eight samples. One sample achieved 100% inhibition with GSK2879552 alone so an improvement in inhibition was not possible with the combination in this sample. A decrease in IC50 of at least 2-fold with the combination was achieved in six out of nine samples and of at least 5-fold in two out of nine sam- ples. Together, these data suggest that the combination effects are not limited to cell lines and that a lower dose of LSD1 inhibitor than is maximally efficacious as single agent may be sufficient to elicit the combination response.
Discussion
Acute myeloid leukemia is a deadly cancer character- ized by accumulation of immature myeloid cells in the BM that undergo replication in an uncontrolled manner. Treatments such as chemotherapy focus on de-bulking the tumor by killing fast-growing cells. Unfortunately, it has been demonstrated that even after treatment some refrac- tory cells remain that can resume growth resulting in dis- ease relapse.33 A recent study has shown that inhibition of LSD1 can affect the leukemia-initiating population sug- gesting a potential clinical opportunity for LSD1 inhibitors in combination with de-bulking agents, even if these are not obviously beneficial as monotherapy.13
Another treatment option that has been more than 90% effective in APL involves the use of ATRA. ATRA has been shown to promote differentiation of myeloid cells and, therefore, the cells no longer possess replication poten- tial.16 Applying this methodology more broadly to AML has thus far been unsuccessful because ATRA is unable to elicit a differentiation phenotype in non-APL AML.18 Interestingly, a new differentiation therapy, Enasidenib, was recently approved in the USA for the treatment of IDH2 mutant AML, demonstrating the potential of differ- entiation therapies in the non-APL AML setting.34
The results presented here indicate that features consis- tent with differentiation in AML cells can be achieved by inhibition of LSD1. Changes in cell surface expression of myeloid maturation markers is evident in all AML sub- types tested. LSD1 inhibition also increases superoxide anion production, indicative of a more granulocytic phe- notype, in AML cells. These results combined with the decrease in blast cells of primary AML patient samples treated with GSK2879552 suggest the therapeutic oppor- tunity for LSD1 inhibition in treating non-APL AML. The addition of ATRA synergistically enhanced the differenti- ation effect achieved with GSK2879552 through increased expression of myeloid cell surface marker CD11b. This is achieved at lower GSK2879552 concentrations than required when AML cells are treated without ATRA thus potentially widening the therapeutic index of LSD1 inhi- bition. Importantly this effect is observed across AML subtypes and is not exclusive to APL. Gene expression analysis revealed enrichment of the hematopoietic and CAM pathways further indicating that a primary mecha- nism in association with this combination involves differ- entiation. Pushing AML cells to differentiate with a com- bination of GSK2879552 and ATRA may offer profound implications for the success of AML treatment options for patients beyond APL or those with IDH2 mutations.
While enhancing differentiation provides an attractive rationale for pursuing the combination of LSD1 inhibitors with ATRA in the clinic, the observation that the combi-
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haematologica | 2019; 104(6)