Targeting post-transcriptional dysregulation in RUNX1 mutated myeloid malignancies

Abstract

The RUNX1 transcription factor is essential for regulating myelopoiesis. RUNX1 mutations and RUNX1 fusion genes, are frequently identified across different myeloid malignancies, including the aggressive haematological neoplasm, acute myeloid leukaemia (AML). However, due to their heterogeneity, targeting RUNX1 mutations has proved challenging so far. Furthermore, the diverse mechanisms resulting from RUNX1 mutations that drive leukaemogenesis in different contexts, requires a greater functional understanding. Much research has previously focussed on the role of RUNX1 as a transcriptional-regulator, although RUNX1 also affects post transcriptional processes, including splicing and translation. More recently, it has been suggested RUNX1 can also directly regulate RNA. However, its role as a post-transcriptional regulator in myeloid malignancies remains less well characterised. To address this, we first aimed to establish how RUNX1 depletion modified the RNA-bound proteome (RBP). Using the model of myeloid blast-phase chronic myeloid leukaemia (CML), a disease enriched for somatic RUNX1 mutations with a similar presentation to AML, we used orthogonal organic phase separation (OOPS) coupled to TMT-labelled mass spectrometry (MS), to quantitatively characterise the ‘RNA-binding protein interactome’ in RUNX1 depleted KU812 and K562 cells. We found RUNX1 depletion induced stress response pathways and led to translational dysregulation/dysregulated ribosome biogenesis (RiBi); processes enriched for differentially regulated RBPs. Subsequently, we found BP-CML cells with RUNX1 depletion and RUNX1 mutations, which led to defective DNA binding/CBFb interaction, were sensitised to the translation-inhibitor homoharringtonine (HHT), which has a history of clinical use in CML. Further interrogation of our OOPS revealed the RBP, SPATS2L, was induced with RUNX1 depletion or expression of the RUNX1::EVI1 oncoprotein in BP-CML cells. SPATS2L depletion inhibited the growth and survival of BP-CML cells, and inhibited stress granule formation; critical cellular components which promote survival. BP-CML cells with RUNX1 depletion, had increased stress granules which were almost eradicated by HHT. The combined depletion of SPATS2Lin K562 cells with RUNX1 depletion, further sensitised these cells to HHT, indicating that SPATS2L may contribute to therapeutic resistance in CML with RUNX1 aberrations. Finally, we explored how different RUNX1 frameshift mutant-proteins led to post-transcriptional dysregulation in AML cells. Importantly, we found that frameshift mutant-proteins in the last-exon of RUNX1 create stable proteins, whereas RUNX1mutant-proteins occurring upstream of exons 7-8 are less stable, leading to induction of the integrated stress response and post-transcriptional dysregulation. In summary, this research provides evidence that the role of RUNX1 extends beyond transcriptional regulation, and, RUNX1 mutations in myeloid malignancies could be targeted through post-transcriptional dysregulation.

Date of Award	Mar 2026
Original language	English
Awarding Institution	University of Brighton
Supervisor	Tim Chevassut (Supervisor), Dr Rhys Morgan (Supervisor) & John Jones (Supervisor)