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A. Damnernsawad et al.
showed increased phospho-ERK activity. This observation may not be surprising given the structural difference between the two proteins; TSC1 lacks the kinase domain that is present in TSC2.36,51 It is possible that TSC1 exe- cutes functions outside of its interactions with TSC2 to regulate MAPK signaling. Consistent with results from the CRISPR screen, samples from AML patients harboring FLT3-ITD mutations with reduced RNA expression levels of LZTR1, NF1, and TSC2 exhibited less sensitivity to sorafenib.
Upregulation of phospho-MTORC1 (Ser2481) is observed in two FLT3-inhibitor-resistant cell lines, made by gradual exposure to higher concentrations of FLT3 inhibitors, supporting a role for MTORC1 in resistance to FLT3 inhibitors. Increased phospho-ERK levels in FLT3- resistant cells confirmed the importance of the MAPK pathway in FLT3 inhibitor resistance. Concordantly, the resistant cells demonstrated enhanced sensitivity to MEK inhibitors. We also showed that the connection between MAPK and MTOR pathways influenced FLT3 inhibitor resistance. We found enhanced levels of phospho-TSC2 at S664 and Y1571 in the resistant lines which are regulat- ed by phosphorylated ERK and AKT, respectively. These phosphorylation events inhibit the formation of the TSC complex, mimicking TSC1 and TCS2 loss-of-function hits as revealed by the CRISPR resistance screen. The combi- nation of FLT3 plus MEK inhibitors has synergistic effec- tiveness in both sensitive and resistant cells, a finding that is consistent with data from a recent study that demon- strated synergy for the combination of crenolanib with trametinib in Ba/F3 cells harboring PTPN11 A72D, FLT3 D835Y or double mutations of both.22 Similarly, the com- bination of sorafenib or pazopanib with trametinib showed strong synergy in MOLM13 cells.25 Moreover, the combination of sorafenib with the MEK inhibitor, PD0325901, showed synergy in MOLM14 and MV4;11 AML cell lines,23,55 and the combination of gilteritinib and trametinib had enhanced efficacy in MOLM14 cells with NRAS G12C and NRAS Q61K.18 Enhanced efficacy was observed in AML patients’ samples assayed ex vivo for sensitivity to a combination of an FLT3 inhibitor (quizar- tinib) and trametinib (Beat AML data, Online Supplementary Figure S4E). Evaluation of signaling in FLT3i-resistant cells and top hits from the CRISPR knock- out screen underscore activation of the MTOR pathway
in sorafenib resistance. Assessment of sensitivity to an MTOR inhibitor in combination with an FLT3 inhibitor showed a profound effect only in TSC1- and NPRL2-defi- cient cells. This result may indicate reliance on multiple pathways in FLT3-inhibitor-resistant cells.
Increased activity of JAK/STAT5 has also been implicat- ed in resistance to FLT3 inhibitors. Granulocyte- macrophage colony-stimulating factor and interleukin-3 mediate FLT3 resistance in AML cells via JAK/STAT5 and PIM/cytokine-activated JAK/STAT5 signaling.26 Although, we did not evaluate JAK/STAT5 pathways in this study, our CRISPR screen did identify several negative regula- tors of JAK/STAT5 pathways, including PTPN1, SUMO3, and PTPN6. Additionally, our screen identified several tier 1 hits involved in RNA metabolism and splicing, including DHX15, EBNA1BP2, LSMR, PUS7 and RPSA. DHX15, for example, encodes an RNA helicase that is commonly mutated in AML patients with RUNX1- RUNX1T1 fusions.56 It will be important to pursue these findings in subsequent studies given the roles of RNA metabolism in cancer pathogenesis.57-59
Disclosures
JWT has received research support from Agios, Aptose, Array, AstraZeneca, Constellation, Genentech, Gilead, Incyte, Janssen, Petra, Seattle Genetics, Syros, and Takeda. The other authors have no conflicts of interest to disclose.
Contributions
AD and TN performed experimentation, DB and CE per- formed data analyses, AD, SK, TN, and JT, wrote the manu- script, JT, SM, and TN supervised the study.
Acknowledgments
We thank the OHSU Massively Parallel Sequencing Shared Resource and Flow Cytometry Core for technical support, and Sunil K. Joshi for reviewing the manuscript.
Funding
This study was supported by grants from the National Cancer Institute (1U01CA217862, 1U54CA224019, 3P30CA069533- 18S5). JWT received grants from the V Foundation for Cancer Research, the Gabrielle’s Angel Foundation for Cancer Research, and the National Cancer Institute (1R01CA183947). TN is sup- ported by grant R50 CA251708-01.
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