Aberrant Activation of the PI3K/mTOR Pathway Promotes Resistance to Sorafenib in AML
Authors: Lindblad Oscar , Cordero Eugenio , Puissant Alexandre , Macaulay Lucy , Kabir Nuzhat N. , Sun Jianmin , Haraldsson Karin , Borg Åke , Levander Fredrik , Stegmaier Kimberly , Pietras Kristian , Ronnstrand Lars , Kazi Julhash U. 
Published: October 2019

Abstract
Therapy directed against oncogenic FLT3 has been shown to induce response in patients with AML, but these responses are almost always transient. To address the mechanism of FLT3 inhibitor resistance, we generated two resistant MV4-11 and MOLM-13 cell lines by sustained treatment with the FLT3 inhibitor sorafenib. MV4-11 cells express only FLT3-ITD, while MOLM-13 cells express wild-type FLT3 and FLT3-ITD. Both cell lines are dependent on FLT3 activation as sorafenib, PKC-412, and AC220, but not imatinib, dasatinib, nilotinib or bosutinib, inhibit cell survival in both cell lines. After treatment with sorafenib for 90 days, we observed that both cell lines displayed resistance to sorafenib as well as to AC220 suggesting that sustained treatment with an FLT3 inhibitor results in acquired resistance to multiple FLT3 inhibitors.
To test whether sorafenib was still effective in FLT3 inhibition, we treated sensitive and resistant cells with sorafenib or DMSO and then stimulated with FLT3 ligand (FL). While DMSO-treated resistant cells responded to FL as expected, sorafenib-treated cells displayed poor FLT3 activation, suggesting that sorafenib was still capable of inhibiting FLT3 activation in the resistant cells. Surprisingly we observed that the resistant cells treated with DMSO had a much more robust response to ligand in terms of FLT3 activation. Similar results were observed with AKT and ERK activation, as sorafenib-treated cells poorly responded to FL-stimulation. Furthermore, resistant cells treated with sorafenib could still form colonies similar to DMSO-treated cells indicating that these cells were no longer dependent on FLT3 activation, although sorafenib could partially block FLT3 activation.
To determine whether secondary mutations occurred in FLT3, we sequenced the whole coding region using Sanger sequencing. Except for a mutation in the extracellular domain in all four cell lines no mutations were detected in the inhibitor binding site. Similar to Sanger sequencing, mass spectroscopic analysis of affinity-enriched FLT3 indicated no differences in the intracellular part of FLT3 between sensitive and resistant cells. Because we observed an unexpected activation of FLT3 in resistant cells stimulated with FL, we hypothesized that certain FLT3 residues remain hyper-tyrosine phosphorylated. To test that we used phospho-specific antibodies against known FLT3 tyrosine phosphorylation sites. Although we observed an increase in total FLT3 tyrosine phosphorylation, we were unable to identify a single site that was selectively hyper-phosphorylated. Instead, all sites remained slightly more phosphorylated compared to control cells.
Gene expression analysis of mRNA from sensitive and resistant cell lines using ANOVA and significance analysis of microarrays (SAM) suggested an enrichment of the PI3K/mTOR pathway in the resistant phenotype. In addition to pathway enrichment, using a phospho-protein antibody array, we found that phosphorylation of the mTOR substrates S6K and AKT were selectively increased in resistant cells. We also observed an increase in STAT3 phosphorylation. Elevated STAT3 phosphorylation was probably due to the previously described upregulation of JAK3 expression in sorafenib-resistant AML. We also observed enrichment of an mTOR signature in AML blasts from eight patients with sorafenib-resistant AML (GSE35907) and in AML blasts from patients expressing FLT3-ITD compared to those lacking FLT3-ITD (525 samples, GSE14468).
Furthermore, a selective PI3K/mTOR inhibitor, gedatolisib, efficiently blocked proliferation, colony and tumor formation, and induced apoptosis in the resistant cell lines. Treatment of cells with a higher concentration of gedatolisib did not alter the phosphorylation of other signaling proteins except for AKT and S6K which was detected by a phospho-specific antibody array. The array data was further verified with western blotting using phospho-specific antibodies against AKT, ERK1/2, p38 and S6K. These results suggest that gedatolisib efficiently blocks the downstream effectors PI3K/mTOR without affecting other signaling pathways.
Taken together, our data suggest that aberrant activation of the PI3K/mTOR pathway in FLT3-ITD dependent AML results in resistance to drugs targeting FLT3 and treatment with selective PI3K/mTOR inhibitors are likely to be effective in overcoming acquired resistance in AML patients.

Disclosures
Stegmaier: Novartis Pharmaceuticals: Consultancy.