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Mechanisms of sorafenib resistance in AML
RAS-GTPases.46,47 Consistent with our results, loss of LZTR1 function has recently been shown to cause resist- ance to tyrosine kinase inhibitors including several FLT3 inhibitors (tandutinib, quizartinib, and ponatinib) in AML cell lines.36 LZTR1 loss-of-function mutations have been observed in other cancers including glioblastoma multi- forme, adrenocortical cancer, and pancreatic cancer, and have also recently been reported in hepatocellular carcino- ma, a cancer for which sorafenib is a first-line therapy.48,49 The screen hits included components of the TSC and the GATOR complexes, which have not been previously iden- tified as modulators of resistance to FLT3 inhibitors. Our screen also identified two negative regulators of RAS: NF1 and RASA2. Loss-of-function mutations in NF1 have been associated with poor prognosis in AML patients,50 suggest-
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ing that patients with NF1 mutations would have poor sensitivity to FLT3 inhibitors.
TSC2 and TBC1D7 along with TSC1 form the TSC complex, which acts as a GTPase activating protein for RHEB, a small G-protein upstream of MTOR complex 1 (MTORC1).36,51-53 MTORC1 is activated by another small G-protein, RAG, which is negatively regulated by the GATOR1 complex, comprising NPRL2, NPRL3, and DEPDC5 proteins.37 Our screen identified TSC1 and com- ponents of the GATOR1 complex, DEPDC5 and NPRL2. Loss-of-function variants of TSC1 and TSC2 are found in ~16% of patients with hepatocarcinoma and are associat- ed with an aggressive form of this malignancy.54 Our data suggest that the roles of TSC1 and TSC2 are complex; we note that TSC1-, but not TSC2-deficient MOLM13 cells
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Figure 4. Evaluation of sensitivity to MTOR and MEK inhibitors in combination with FLT3 inhibitors. (A) Parental and FLT3- inhibitor-resistant MOLM13 cell lines described in Figure 3A were tested for sensitivity to sorafenib, trametinib and PP242 by 72 h drug sensitivity assays. Cell viability was measured in triplicate using the MTS assay with seven-point escalating drug concentrations. Drug combinations were tested at equimolar concentrations. (B) Immunoblot of parental MOLM13 cells treated with sorafenib, trametinib, PP4242 or combinations at 10 nM for 24 h performed with the indicated antibodies. Vinculin immunoreactivity served as a loading con- trol.
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