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A. Damnernsawad et al.
showed greater sensitivity to trametinib relative to the parental cells (Figure 3C; Online Supplementary Figure S4A). Combinations of each FLT3 inhibitor with trame- tinib revealed enhanced efficacy in both resistant and parental AML cells (Figure 4A; Online Supplementary Figure S4B). Moreover, combinations of sorafenib and trametinib showed high synergy scores at several con- centrations as analyzed by R_SynergyFinder45 (average synergy [zero interaction potency] score for parental cells = 3.449 vs. 8.015 in sorafenib-resistant MOLM13 cells; synergy scores above 1 are significant). Similar synergy in sensitivity was obtained with trametinib in combina- tion with either crenolanib or quizartinib (Online Supplementary Figure S4D). MTOR inhibitors PP242 and PI-103 did not exhibit substantial cell killing as single agents in any of the FLT3-inhibitor-resistant cell lines (Figure 3C, bottom panel; Online Supplementary Figure S4A) and a combination of MTOR inhibitor and a FLT3 inhibitor resulted in a marginal decrease in cell viability (Figure 4A bottom panels; Online Supplementary Figure S4B, C). In contrast, MTOR inhibitors appeared to resen- sitize TSC1- and NPRL2-deficient cells to sorafenib (Online Supplementary Figure S5). The effect of PI-103 was more pronounced than that of PP242 which may reflect its dual targeting of MTOR and PI3K.
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Discussion
Sorafenib, as well as other FLT3 inhibitors, in combina- tion with standard-of-care chemotherapy prolongs the survival of AML patients with or without FLT3 mutations, although relapse caused by drug resistance remains a clin- ical challenge. To elucidate mechanisms of resistance to sorafenib, we subjected MOLM13 AML cells to genome- wide CRISPR screening to identify genes whose loss-of- function contributes to reduced drug sensitivity. The top screen hits indicated that resistance to FLT3 inhibitors in AML can occur via aberrant activation of the AKT/PI3K/MTOR and RAS/MAPK signaling pathways. Our results are consistent with findings from previous studies on resistance to FLT3 inhibitors that revealed aber- rant ERK and RAS signaling18,23 and extend these with the identification of a broad spectrum of genes regulating RAS/MAPK and, additionally, MTOR signaling pathways as modulators of resistance (Figure 4).
Analysis of the screen using the MAGeCK pipeline in combination with a tiering system developed previously32 identified LZTR1, TSC1/2, NPRL2, NF1, and TBC1D7 as significant hits. The identification of LZTR1 is not unex- pected as LZTR1 loss confers MAPK activation by dysreg- ulating RAS signaling;35,36 it also facilitates degradation of
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Figure 3. Acute myeloid leukemia cell lines made resistant to FLT3 inhibitors demonstrate activation of MAPK and/or MTOR pathways. (A) MOLM13 AML cell lines were made resistant to type I and type II FLT3 inhibitors by continuous exposure to crenolanib, quizartinib or sorafenib. Drug sensitivity assays were performed for 72 h on parental and FLT3-inhibitor-resistant MOLM13 cells. Cell viability was measured in triplicate using the MTS assay with seven-point escalating drug concen- trations. (B) Immunoblot of whole cell lysates from parental MOLM13 and MOLM13 cells resistant to crenolanib and sorafenib treated with dimethlysulfoxide or FLT3 inhibitors at 20 nM concentration for 4 h performed with indicated antibodies. (C) Dose response curves of trametinib and PP242 on parental and FLT3-inhibitor- resistant MOLM13 cells.
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haematologica | 2022; 107(1)