Comprehensive characterisation of CDK4/6 inhibitor trilaciclib in acute myeloid Leukaemia using proteomics approaches

Cyclin-dependent kinases 4 and 6 (CDK4/6) are important mediators of cell cycle progression and are essential for the growth of many cancer types. Inhibition of these kinases can elicit varied phenotypic responses that are contingent on cell type, determining clinical utility. Acute myeloid leukaemia (AML) with FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive type of haematological malignancy that exhibits hypersensitivity to CDK4/6 inhibition pre-clinically, but the biological mechanism underlying this effect remains largely unexplored. Here, we aim to delineate the mode of action of trilaciclib, a CDK4/6 inhibitor, for repositioning in the treatment of AML through comprehensive proteomic analyses.

MOLM-13 cells (FLT3-ITD+) were treated with 1 µM trilaciclib or vehicle control for 24 hours, with sample collection at 0hr, 1hr, 6hr and 24 hr time points to investigate (i) initial responses by thermal proteome profiling, (ii) cellular reprogramming by phosphoproteomics, and (iii) expression proteomics reflecting the induced phenotype after 24 hours.

At 1hr, thermal proteome profiling was conducted to identify proteins exhibiting altered stability due to ligand-binding, post-translational modifications, and protein-protein interactions within live cells. Cells were aliquot, heated at 8 temperatures between 40-68 °C and then lysed by freeze-thawing. The soluble protein fraction was quantified, and equal amounts digested using 96-well S-Trap™ plates. Peptides (500ng) underwent single-shot analysis by 40SPD diaPASEF using Evosep One LC coupled to a TimsToF-HT mass spectrometer, identifying more than 6000 proteins. Thermal shifts in CDK4 and CDK6 from ligand binding were not detectable, but AP2 associated kinase 1 (AAK1) established a clear significant thermal shift in all four replicates.

To complement this, cell pellets were collected at 1hr, 6 hr and 24 hr and lysed with 500µL 5% SDS, 50mM TEAB. Protein (3 mg) was digested using S-Trap™ Midi-columns and 1 µg injected for whole-cell proteomic analysis. Phosphopeptides were enriched from the remaining digest using in-house TiO2-based microcolumns, then labelled using TMTpro 16plex (Fisher Scientific, UK), pooled, separated into 12 fractions by basic-pH reversed-phase offline-LC, and analysed with an Orbitrap Fusion Lumos Tribrid mass spectrometer. Raw DIA data was analysed using DIA-NN version 1.8 in library-free mode, while TMT-DDA MS files were analysed with MaxQuant. Trilaciclib treatment resulted in the downregulation of many proteins associated with S-Phase transition and DNA synthesis reflecting inhibited CDK4/6 activity and consequential cell cycle arrest in G1 phase. We highlight signalling pathways implicated in cellular response to CDK4/6 inhibition in leukaemia with constitutive FLT3 activity.

This study provides a detailed understanding of trilaciclib's effects in AML, elucidating its mode of action through advanced proteomic analyses.