Distinct mTOR- and CDK1-regulated translation circuits sustain FLT3-mutated AML survival under therapy Free

Background

Previously, we demonstrated that bone marrow stromal factors protect FLT3-mutant AML cells from FLT3 inhibitor (FLT3i)-induced death by restoring mTORC1-dependent translation of essential oxidative phosphorylation genes (Park et al., 2022). While FLT3 plus mTORC1 inhibition synergistically eliminates AML cells and prevents relapse in vivo, mTORC1 inhibition alone has modest effects on translation (~50%) and viability (<10%). This suggests that there are compensatory mTOR-independent mechanisms of translation. Our initial findings identified CDK1 as a key mediator of mTOR-independent translation. Importantly, CDK1 inhibition suppressed protein synthesis at drug concentrations that had minimal effects on the cell cycle, revealing a noncanonical role in translation. This study aims to define how AML cells maintain translation despite mTORC1 inhibition and whether this contributes to resistance against FLT3- and mTORC1-targeted therapies.

Methods

Pulse SILAC was performed on MOLM14 cells (FLT3-ITD AML cell line) to track nascent protein synthesis over a 5-hour window starting 4 hours after treatment, using Everolimus to inhibit mTORC1 and RO-3306 to inhibit CDK1. Pathway enrichment analyses were conducted using MetaboAnalyst and ENRICHR (Reactome, KEGG, WikiPathways). Global translation was quantified using O-propargyl-puromycin (OPP) incorporation, and cell viability/cycle status assessed via propidium iodide (PI) flow cytometry.

Results

To quantify nascent protein synthesis, or lack thereof, under CDK1 or mTORC1 inhibition, we performed pulse SILAC, a mass spectrometry-based method tracking incorporation of heavy-labeled amino acids into newly synthesized proteins, over a 5-hour window in MOLM14 cells, using RO-3306 and Everolimus to inhibit CDK1 and mTORC1, respectively. Prior OPP-incorporation assays showed that CDK1 inhibition reduces translation by ~40% within 3–6 hours, independent of cell cycle arrest, while by 9 hours, combined CDK1/mTORC1 inhibition nearly abolished translation, with modest effects on the cell cycle. We performed principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) from SILAC results, which revealed clear separation among vehicle, CDK1i, and mTORC1i treatment groups based on global profiles of nascent proteins. These distinct clustering patterns indicate significant shifts in protein expression induced by each treatment condition.

Pathway enrichment analyses of differences in nascent proteins revealed that CDK1 inhibition specifically suppressed pathways related to RNA splicing, nuclear export, and transcript maturation, implicating CDK1 as a key regulator of RNA processing and transcript stability. Interestingly, CDK1 inhibition also triggered compensatory upregulation of mTORC1 pathway proteins, suggesting feedback adaptation. In contrast, mTORC1 inhibition specifically promoted DNA repair pathways such as mismatch repair and homologous recombination, while repressing the protein expression of glycolysis, pyruvate metabolism, and apoptotic signaling components. These results suggest a coordinated shift toward a survival state under mTOR suppression.

Analysis of alterations in common for CDK1- and mTORC1- inhibited treatment groups revealed downregulation of RNA processing, splicing, and protein turnover, demonstrating non-redundant contributions to these key pathways. Meanwhile, analysis of overlapping but oppositely regulated proteins showed that CDK1 and mTORC1 also exert distinct, and at times antagonistic, control over stress response pathways such as DNA repair, mitochondrial biogenesis, and translation. Thus, their balance may shape how AML cells adapt under therapeutic pressure.

Conclusion

Our integrated proteomic and pathway analyses reveal that CDK1 and mTORC1 regulate distinct yet intersecting stress adaptation programs in FLT3-ITD AML. Importantly, our analyses reveal novel, noncanonical roles for CDK1 beyond its established function in cell cycle regulation in maintaining the protein expression of mRNA splicing, transcript maturation, and nuclear export components, highlighting a critical role beyond cell cycle control. These findings expose a critical vulnerability in the translational dependence of FLT3-ITD AML cells, highlighting the need to understand how translation is sustained independently of mTOR to anticipate resistance and guide future FLT3i and mTORC1i combination therapies aimed at preventing relapse.