Functional and Molecular Characterisation of Prospectively Isolated Subclones in Individual AML Patients Reveals Distinct Gene Regulatory Networks Linked to Targetable Metabolic Vulnerabilities

Clonal heterogeneity - even within individual patients - is a major problem in the treatment of acute myeloid leukaemia (AML). In about 80% of the patients a relapse occurs within 3 years after complete remission, and it is currently difficult to predict which subclone(s) escape therapy and re-initiate disease. Such subclones are genetically distinct: they carry similar founder mutations but secondary driver mutations are different, and therefore their biology and drug sensitivities most likely also differ. To improve our understanding of the biology of subpopulations and clonal evolution our lab developed a CombiFlow pipeline, which is a combinatorial flow cytometry-based approach based on differential plasma membrane protein expression profiles (de Boer et al., 2018; Houtsma et al., 2021; Houtsma et al., 2022). With this approach, genetically distinct subclones can be prospectively isolated as viable cell populations, which is obviously a prerequisite for in-depth functional studies. Here, we used this newly developed tool to characterise subclones in a large cohort of AML patients (n=12) at the functional and molecular level. This included genome, proteome, transcriptome, metabolome, epigenome and chromatin accessibility studies. In 5 patients harbouring FLT3 mutations, FLT3-ITD subclones could be distinguished from FLT3-wt subclones whereby initial founder mutations were similar. DNase-sequencing showed differences in chromatin accessibility between the FLT3-ITD and wild type clones with specific sites for transcription factors AP-1 and RUNX in the ITD population and specific sites for transcription factor GATA in the wt-clone. We constructed gene regulatory networks (GRNs, (Assi et al., 2019)) to link transcription factor occupancy to target gene expression determined by RNAseq and LC/MS-MS-based proteomics. These comprehensive genome-wide studies revealed striking differences in signaling network activities linked to gene expression control. Upregulated genes in the FLT3-ITD population were enriched in processes related to cell proliferation, L-GMP signatures, mitochondrial activity and fatty acid oxidation (FAO), whereas genes in the FLT3-WT population were more associated with histone modifications, stemness signatures and regulation of immune response. Functionally, we identified that FLT3-ITD clones were higher in OXPHOS-driven mitochondrial metabolism and had higher ROS levels. In some cases this was linked to enhanced mitochondrial electron transport chain (ETC) complex II activity as a consequence of upregulation of SULG1/2 and SDH, while in other cases this was rewired via enhanced FAO via the fatty acid transporter CD36. These insights into AML-specific mutation-driven signaling networks controlling metabolic programs allowed us to design focussed in vitro inhibitor screens to screen to identify subclone-specific targetable vulnerabilities. In conclusion, our studies indicate that genetically distinct subclones within individual AML patients differ at various levels, which should be taken into account to further improve treatment modalities.

Assi, S.A. et al. (2019). Subtype-specific regulatory network rewiring in acute myeloid leukemia. Nat Genet 51, 151-162.

de Boer, B. et al. (2018). Prospective Isolation and Characterization of Genetically and Functionally Distinct AML Subclones. Cancer Cell 34, 674-689 e678.

Houtsma, R. et al. (2021). CombiFlow: Flow cytometry-based identification and characterization of genetically and functionally distinct AML subclones. STAR Protoc 2, 100864.

Houtsma, R. et al. (2022). CombiFlow: Combinatorial AML-specific plasma membrane expression profiles allow longitudinal tracking of clones. Blood Adv 6, 2129-2143.

No relevant conflicts of interest to declare.