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A.K. Abdel-Aziz et al.
(control [scrambled] or LSD1 [shLSD1 #1 and shLSD1 #2]) using real-time quantitative PCR (RT-qPCR). Data were statistically analyzed using one way ANOVA fol- lowed by Bonferrroni post hoc test.*: P<0.05 compared to control (scrambled). (H-K) Western blot analysis of lysates obtained from THP-1 (H), OCI-AML3 (I), NB4 (J) and SKNO-1 (K) cells treated with either vehicle or DDP38003 (0.5 mM). Vinculin served as a loading control. (L) Schematic outline of the chromatin immunoprecip- itation qPCR (ChIP-qPCR) primers designed to analyze the enrichment of LSD1 or histone marks on IRS1 promoter. (M) LSD1 ChIP-qPCR analyses were performed in THP-1 cells using antibody against LSD1 or IgG as a control. Enrichment values at the indicated sites (A–D) were normalized to input DNA. Values are means ± standard deviation (SD). *: P<0.05. (N-P) ChIP-qPCR analyses to assess H3K4me3 (N), H3K9Ac (O) and H3K27Ac (P) histone marks were performed in THP-1 cells 72 h following their treatment with either vehicle or DDP38003 (0.5 mM). Enrichment values at the indicated sites (A–D) were normalized to input DNA. Values are means ± standard deviation (SD). *: P<0.05.
ERK1/2 and mTOR, we investigated the effect of NT157, a selective IRS1/2 inhibitor.31 Co-treatment with NT157 reversed LSD1i-induced ERK/mTOR stimulation and sen- sitized tolerant AML cells to LSD1i, suggesting that IRS1 acts upstream of ERK/mTOR (Figure 5A-C).
All trans-retinoic acid (ATRA) has been reported to downregulate IRS1.32,33 Indeed, ATRA repressed IRS1 tran- scription and cooperated with LSD1i as previously report- ed (Online Supplementary Figure S9A-E and Figure 5D-F).4 Consistently, ATRA counteracted LSD1i-mediated induc- tion of IRS1/ERK/mTOR in resistant AML cells and led to reduced H3K4me2 and, to a greater extent, H3K27Ac accumulation on IRS1 promoter (Figure 5F and Online Supplementary Figure S9F-I). Collectively, our data indicate that differential dysregulation of IRS1/ERK signaling might contribute at least partly to the modulation of mTOR following LSD1i.
Targeting mTOR sensitizes resistant primary human AML blasts to LSD1 inhibition in vitro and in vivo
We then checked the effect of LSD1i on primary human cells. mTOR signaling was not modulated in primary human hematopoietic CD34+ progenitor cells which toler- ated DDP38003 (Online Supplementary Figure S10A-C). In contrast, mTOR was inhibited by LSD1i treatment in transduced human MLL-AF9 expressing CD34+ cells where LSD1i adversely affected their proliferation and clonogenicity while promoting myeloid differentiation (Online Supplementary Figure S11A-G).
Eventually, we explored the therapeutic value of co- inhibiting mTOR in primary patient derived AML blasts resistant to LSD1i (referred to as AML-IEO20, expressing the oncofusion protein MLL-AF9). Confirming the results seen in resistant AML cell lines, DDP38003 induced mTORC1 in resistant AML-IEO20 cells (Figure 6A-C). Inhibiting mTOR signaling sensitized AML-IEO20 cells to LSD1i (Figure 6D-F). This was associated with increased G0/G1 arrest and apoptotic cell death (Online Supplementary Figure S12A-B).
To validate our results in vivo, AML-IEO20 cells were transplanted into NSG mice. As shown in Figure 7A, one week post-transplantation, mice were randomly assigned into four cohorts and treated with: vehicle, DDP38003, rapamycin or their combination. After two weeks of treat- ment, DDP38003 as a monotherapy failed to lessen the percent of circulating human AML cells, while rapamycin caused a significant decrement (Figure 7C). DDP38003/rapamycin combinatorial regimen significant- ly reduced the percent of hCD45+ leukemic cells in the peripheral blood as compared to vehicle and DDP38003- treated groups (Figure 7C). Even though DDP38003/rapamycin co-treatment further lessened the percent of human AML cells by almost 60% compared to rapamycin alone, such a decrement was not statistically significant. At this stage of treatment, two mice from each
cohort were sacrificed, and spleen and bone marrow tis- sues were harvested. Rapamycin, but not DDP38003, reduced spleen and bone marrow infiltration by leukemic cells was comparable to the vehicle-treated group (Figure 7D-F). Notably, DDP38003/rapamycin combination elicit- ed an even stronger reduction of leukemic infiltration (Figure 7E-F). May Grunwald/Giemsa-stained cytospin preparations of blood smear, spleen and bone marrow, histopathological and immunohistochemical examina- tions further confirmed the superior antileukemic the activity of the LSD1i/mTORi combinatorial regimen (Online Supplementary Figure S12C-F and Table 1). After three weeks of treatment, circulating hCD45+ leukemic cells were present in the cohorts treated with vehicle, DDP38003 and rapamycin as monotherapies while they remained dramatically decreased by the combination treatment (Figure 7G). Indeed, the combination treatment significantly prolonged the survival of PDX mice as com- pared to vehicle (P=0.001), DDP38003 (P=0.0004) and rapamycin (P=0.0024)-treated groups (Figure 7J). Altogether, our results provide the first proof of principle demonstrating preclinical evidence for a therapeutic strat- egy to restore the efficacy of LSD1i in irresponsive AML patients based on co-inhibiting LSD1/mTOR.
Discussion
AML cells have been reported to elicit heterogeneous responses to LSD1i.12,17 Here, we explored the mechanisms of sensitivity and resistance of AML cells to LSD1 inhibi- tion. Initially, we ruled out the possibility that differential basal LSD1 levels might account for discrepant vulnerabil- ity of AML cells to LSD1i, consistent with what was described with T-cell lymphoblastic leukemias.34 Moreover, global transcriptomic changes in the target genes of LSD1 (as CD11b) did not correlate with the dis- crepant responses of AML cells to LSD1i. Intriguingly, we found that distinctive modulation of mTORC1 activity acts as a key mediator of the susceptibility of AML cells to LSD1i therapy (Figure 7K). We and others have previously demonstrated that mTORC1 contributes to the resistance of diverse types of tumours to targeted anticancer thera- pies, such as histone deacetylase (HDAC) and tyrosine kinase inhibitors.23,35,36 Likewise, mTORC1 signaling was robustly triggered in AML cells that tolerated LSD1i. In contrast, mTORC1 was inhibited in LSD1i-sensitive AML, as recently described using S2101, another LSD1i, in responsive ovarian carcinoma cells.37 Inhibiting mTOR via direct pharmacological inhibition, or mimicking energetic stress using the non-metabolizable glucose analogue, 2- deoxyglucose, reversed LSD1i-induced mTOR activation and counteracted the resistance of AML cells to LSD1i. Intriguingly, our findings with the glycolytic inhibitor, 2- deoxyglucose, could also be explained by Poulain et al.,
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haematologica | 2020; 105(8)