TY  - THES
AU  - Bjelosevic, Stefan
Y2  - 2021/05/30
Y1  - 2021
UR  - http://hdl.handle.net/11343/274831
AB  - Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene are among the most frequently occurring somatic mutations in acute myeloid leukaemia (AML), a disease that presents with devastating prognosis. FLT3 is primarily expressed on hematopoietic progenitor cells, and during early haematopoiesis coordinates a ligand-dependent signalling cascade that regulates the proliferation and maturation of the progenitor pool. FLT3 internal tandem duplication (FLT3-ITD), the most common type of FLT3 mutation, promotes constitutive FLT3 kinase activity and hyperactivation of downstream signalling pathways including STAT5, PI3K/mTOR and MAPK. Though our understanding of the molecular pathways downstream of mutant FLT3 have greatly improved in the modern genomic sequencing era, the repertoire of molecular signalling events induced by mutant-FLT3 to drive leukaemogenesis has not been fully characterised.

In this thesis, a murine model of MLL-rearranged AML harbouring inducible FLT3-ITD expression was developed and used, along with human AML cell lines, to demonstrate that FLT3-ITD promotes serine uptake and serine biosynthesis via transcriptional regulation of neutral amino acid transporters (SLC1A4 and SLC1A5) and genes in the de novo serine biosynthesis pathway (PHGDH and PSAT1). Mechanistically, dysregulation of serine metabolism in FLT3-ITD-driven AML is dependent on the mTORC1-ATF4 axis, which drives RNA-Pol II occupancy at PHGDH, PSAT1, SLC1A4 and SLC1A5. Genetic or pharmacological inhibition of the de novo serine biosynthesis pathway, in vitro and in vivo, selectively inhibited the proliferation of FLT3-ITD-driven AML cells. Pharmacological inhibition of the de novo serine biosynthesis pathway using WQ-2101, an inhibitor of PHGDH, the first rate-limiting enzyme of the de novo serine biosynthesis pathway, sensitises FLT3-ITD-driven AML cells, including primary patient samples, to the standard of care chemotherapy agent cytarabine via exacerbation of DNA damage. 

Given that transcriptional activation of de novo serine biosynthesis and serine uptake was mediated by mTORC1 (a master regulator of biomass production and cellular metabolism), and that therapeutic responses in vitro and in vivo to mTORC1 inhibitors are poor, a small molecule compound screen utilising 181 epigenetic inhibitory compounds to determine novel synthetic lethal interactions between epigenetic regulators and mTORC1 inhibition was performed. This analysis revealed that inhibition of lysine-specific methyltransferase SETD8, the only known enzyme that catalyses H4K20 monomethylation (H4K20me1), synergised with mTORC1 inhibition. Transcriptional profiling suggested dual SETD8/mTORC1 inhibition preferentially suppressed mTORC1 target genes mediating amino acid biosynthesis and transamination (including de novo serine biosynthesis) to a greater extent than either single agent SETD8 or mTORC1 inhibition alone. Importantly, these observations were independent of global transcriptional repression induced by impaired cell viability or suppression of global transcription. Thus, this preliminary work suggests mTORC1 and/or its target genes and pathways may be dependent on SETD8 and/or H4K20me1 or, alternatively, mTORC1 functionally regulates SETD8.

Collectively, the results presented herein provide novel insights into FLT3-ITD-induced metabolic reprogramming events in AML and identify a targetable metabolic dependency in this poor prognosis subtype of disease. In addition, these results provide the preliminary basis of a SETD8/mTORC1 synthetic dependency that can be exploited in FLT3-ITD-driven AML.
KW  - Acute myeloid leukaemia
KW  - FLT3-ITD
KW  - Serine metabolism
KW  - Metabolic reprogramming
KW  - Purine metabolism
KW  - Epigenetics
KW  - SETD8
T1  - Investigating oncogenic FMS-like tyrosine kinase 3 (FLT3)-induced metabolic reprogramming in acute myeloid leukaemia
L1  - /bitstream/handle/11343/274831/136f5fca-222d-eb11-94d0-0050568d7800_Stefan_Bjelosevic_PhD_Thesis_REVISED.pdf?sequence=1&isAllowed=n
ER  -