Distinct Roles for mTOR- and CDK1-Dependent Translation in FLT3-Mutated AML Cell Survival

Although FLT3 inhibitors (FLT3i) improve median survival of patients with Acute Myeloid Leukemia (AML), they will eventually acquire resistance and relapse. Clinical trials have revealed that AML blasts in the bone marrow (BM) were not effectively eliminated compared to AML blasts in peripheral blood, suggesting a protective role of the BM microenvironment that counteracts FLT3i therapy.

Our group discovered that BM stromal cells protect FLT3-mutated AML cells from the killing effect FLT3i through upregulation of Ataxia Telangiectasia Mutated (ATM), which promotes Mammalian Target of Rapamycin Complex 1 (mTORC1) activity (Park et al., 2022). Mechanistically, FLT3i inhibits mTOR and mTOR-dependent translation leading to cell death. However, in the presence of BM stromal factors, survival is restored despite FLT3i treatment by upregulating ATM/mTOR-dependent translation of essential oxidative phosphorylation (OXPHOS) genes. Moreover, we demonstrated that the combination of FLT3i and mTORC1 inhibition (mTORC1i) synergistically kills human FLT3-mutated AML cells in vitro and substantially reduces tumor burden and prevents relapse in mouse models transplanted with FLT3-mutated primary human AML.

We observed that inhibition of mTOR alone has a minor effect on cell viability and a modest reduction in translation as measured by O-Propargyl Puromycin (OPP)-translation assays. Hence, to determine how these cells maintain translation and survival despite mTOR inhibition, we sought to identify the key factors mediating mTOR-independent translation. We tested roles for multiple kinases including CDK1, GSK3β, and p38, which have each been previously shown to phosphorylate 4E-BP1, in mTOR-independent translation in FLT3-mutated AML cells. We performed OPP assays to assess the impact of inhibitors against these kinases on translation activity in FLT3-mutated AML cell lines in the presence and absence of BM stromal factors. This approach revealed CDK1 as a critical regulator of mTOR-independent translation, suggesting that AML cells can maintain survival through this mTOR-independent pathway.

Through immunoblotting, we demonstrate that a critical role for CDK1 in 4E-BP1 phosphorylation and protein translation in that combined inhibition of mTORC1 and CDK1 in FLT3-mutated AML cells leads to a near complete block in these activities even in the presence of BM stromal factors. Because of its canonical role in the cell cycle, we performed cell cycle assays in parallel with the OPP assays to assess the relevance of cell cycle state in CDK1-dependent translation. The results show that concentrations of CDK1 inhibitor that only modestly impact the cell cycle can still markedly inhibit translation.

These studies define a new role of CDK1 as a critical regulator of mTOR-independent translation and thus survival of FLT3-mutated AMLs. Further investigation of CDK1-dependent translation will leverage transcriptome/ translatome analysis to identify which mRNAs are being specifically translated in mTOR-dependent versus CDK1-dependent manner to sustain FLT3-mutated AML survival against FLT3i-based therapies. Ongoing studies are also dissecting the mechanism whereby ATM promotes mTOR expression and activity.

In all, understanding how translation and thus AML cell survival is maintained independently of mTOR is critical for evaluating efficacy of therapies targeting FLT3 and mTOR kinases, upfront AML resistance to these therapies, and the evolution of resistance.

No relevant conflicts of interest to declare.