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A.K. Abdel-Aziz et al.
mTORC2 (evaluated by assessing AKT phosphorylation at S473) was not consistently modulated in response to LSD1i in resistant and sensitive AML cells (Figure 1D). Collectively, these findings suggest that distinct fine-tun- ing of mTORC1 activity correlates with the differential responsiveness of AML cells to DDP38003.
Mirroring the response to DDP38003, mTORC1 was induced in resistant AML cells and inhibited in sensitive AML cells following their treatment with MC2580 (anoth- er selective LSD1i previously referred to as Compound 14e27) (Online Supplementary Figure S1D-G). Finally, to con- firm that LSD1 was the key molecular target for the phe- notypic/molecular responses and exclude potential off-tar- get effects associated with pharmacological inhibition, two different LSD1-targeting small hairpin RNA (shRNA) were used. Consistently, while LSD1 knockdown sharply affected the proliferation of KASUMI-1 cells resulting in preferential counter-selection of one of the shRNA against wild-type cells, THP-1 and OCI-AML3 cells tolerated LSD1 knockdown (Figure 2A-C). shRNA against LSD1 efficiently reduced LSD1 mRNA and protein levels, and subsequently upregulated the transcription of a direct tar- get gene of LSD1 (CD11b) which was also modulated by DDP38003 (Figure 2D-L). Recapitulating the effect of pharmacological LSD1i, mTORC1 was inhibited in sensi- tive AML cells and induced in resistant AML cells follow- ing LSD1 knockdown (Figure 2J-L). Altogether, our data demonstrate that the sensitivity/resistance of AML cells to LSD1 inhibition is associated with distinctive modulation of mTORC1 activity.
Abrogating mTOR signaling counteracts the resistance of AML cells to LSD1 inhibition
Next, we explored the effect of inactivating mTOR on the response of AML cells resistant to LSD1 inhibition using different strategies. Inhibiting mTOR pharmacolog- ically using either rapamycin (allosteric mTOR inhibitor) or AZD8055 (ATP competitive mTOR kinase inhibitor) sensitized resistant THP-1 cells to pharmacological LSD1i or genetic knockdown of LSD1 (Figure 3A-H). Mimicking nutritional stress using 2-deoxyglucose (2DG, a non- metabolizable glucose analogue)28 also counteracted DDP38003-mediated mTOR activation and rendered THP-1 cells responsive to LSD1i (Online Supplementary Figure S2A-C).
A substantial proportion of initially responder cancer patients eventually relapses/progresses. Trying to simulate this scenario, parental KASUMI-1 cells (designated as KASUMI-1/P) were continuously exposed to increasing concentrations of DDP38003 for 12 months until they started to proliferate in the presence of DDP38003. Resistant descendent cells (named KASUMI-1/R) demon- strated a resistance index (RI) of 21 against DDP38003, and were also cross-resistant to MC2580 (RI of ~10) (Online Supplementary Figure S4A-C). As shown in the Online Supplementary Figure S4D, mTOR signaling was activated in KASUMI-1/R cells compared to their parental counter-part, and treatment with DDP38003, despite reducing the extent of mTOR activation, did not reach the low levels observed in parental cells. Besides boosting the responses of KASUMI-1/P cells to LSD1i, inhibiting mTOR dramatically reversed the acquired resistance of KASUMI-1/R cells to LSD1i via triggering apoptosis and this thereby indicates that compensatory mTOR activa- tion protects AML cells against LSD1i-induced apoptotic
cell death (Online Supplementary Figure S4E-G). Collectively, our data suggest that targeting mTOR coun- teracts both intrinsic and acquired resistance of AML cells to LSD1i in vitro.
IRS1 and ERK1/2 signaling are involved in mTOR regulation by LSD1
We attempted to gain insights into the mechanism(s) through which LSD1 regulates mTOR. AMP activated protein kinase (AMPK) is a key negative regulator of mTOR.29 Following LSD1i, AMPK activity was increased in both sensitive and resistant AML, as reflected by increased phosphorylation of AMPK and its downstream target, acetyl CoA carboxylase (ACC) (Online Supplementary Figure S5A-D). The levels of Raptor, a regu- lator and component of mTORC1, were not consistently modulated in response to LSD1 inhibition (Online Supplementary Figure S5E-G). Taken together, these results suggest that these mechanisms might not be critical for the observed modulatory effects on mTOR. We then monitored the activity of mTORC1 signaling in sensitive KASUMI-1 and resistant THP-1 cells at different time points following LSD1i. Even though six hours (h) of DDP38003 treatment were not enough to elicit any remarkable effects on the proliferation of sensitive KASU- MI-1 cells, mTORC1 was dramatically inactivated (Online Supplementary Figure S6A). Conversely, 24 h post-LSD1i in resistant THP-1 cells, mTORC1 was robustly triggered (Online Supplementary Figure S6B). Such modulatory effects were maintained throughout the subsequent time points of treatment. Accordingly, we decided to perform tran- scriptomic analysis at the earliest detected and last tested time points in which mTORC1 activity was modulated secondary to LSD1i (i.e. 6 and 72 h in KASUMI-1 cells and 24 and 72 h in THP-1 cells). Consistent with the results of cell viability assays (Figure 1A-B and Online Supplementary Figure S1A-B), ingenuity pathway analysis (IPA) showed a significant modulation of gene sets involved in “cellular growth and proliferation” in sensitive KASUMI-1 but not resistant THP-1 cells post-LSD1i treatment (Online Supplementary Figure S7A-B). Notably, IPA predicted extra- cellular-signal regulated kinases 1 and 2 (ERK1/2) to be activated in resistant but not in sensitive AML following LSD1i (Online Supplementary Table S1A-4). ERK1/2 is reported to be an upstream activator of mTOR.26 In paral- lel with mTOR modulation, DDP38003 inhibited ERK1/2 in sensitive AML cells (KASUMI-1) and activated ERK1/2 in resistant AML cells (THP-1 and NB4) cells (Online Supplementary Figure S6A-C). Inhibiting ERK1/2 using sev- eral unrelated selective MEK1/2 inhibitors as U0126, pimasertib and trametinib rendered resistant AML cells more vulnerable to LSD1 inhibition (Online Supplementary Figure S6D-H). These findings all suggest that ERK1/2 acts upstream of mTOR dysregulation by LSD1.
To further investigate how LSD1 regulates ERK1/2 and mTOR, we analyzed our RNA-Seq data which revealed that a subset of genes was differentially modulated in resistant versus sensitive AML following LSD1i (Figure 4A). Among those differentially expressed genes, insulin receptor substrate 1 (IRS1) was upregulated in resistant but not responsive AML cells after DDP38003 treatment (Figure 4A-B and Online Supplementary Table S5). IRS1 is an adaptor protein which regulates various pathways includ- ing ERK1/2 and mTOR.30 Confirming RNA sequencing (RNA-Seq) data, treatment with pharmacological LSD1i or
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