RUNX1 aberrations in blast-phase CML induce the RBP SPATS2L which promotes growth, survival and stress granule assembly

The RUNX1 transcription factor is a critical regulator of hematopoiesis and frequently mutated in myeloid malignancies. In the myeloproliferative neoplasm, chronic myeloid leukemia (CML), secondary somatic RUNX1 mutations and RUNX1::MECOM/EVI1, are associated with tyrosine kinase inhibitor (TKI) resistance and progression to the blast-phase (BP-CML). Research has predominantly focussed on transcriptional dysregulation mediated by RUNX1 mutations in myeloid malignancies, whilst post-transcriptional dysregulation remains comparatively unexplored. To address this, we used orthogonal organic phase separation (OOPS), to characterise the RNA-binding proteome of RUNX1 deficient BP-CML cells. RUNX1 depleted BP-CML cells exhibited significant alterations to RBP abundance involved in stress response pathways and translation/ribosome-biogenesis (RiBi). Furthermore, RUNX1 depletion or expression of RUNX1::EVI1 in BP-CML cells induced expression and RNA binding activity of SPATS2L, a component of stress granules (SG); membraneless cytoplasmic condensates protecting mRNAs from degradation, promoting survival under stress. Whilst RUNX1 depletion increased SG-assembly, SPATS2L depletion reduced SG-assembly in BP-CML cells and inhibited the growth and survival of multiple BP-CML cell lines. The translation inhibitor homoharringtonine (HHT), used historically in TKI-resistant CML, ablated SG-assembly in BP-CML cells with RUNX1 depletion, and, primary BP-CML cells with LOF/hypomorphic RUNX1 mutations (characterised by defective DNA-binding/CBF{beta}-interaction) were preferentially sensitised to HHT. Finally, suppressing SPATS2L expression induced by RUNX1 depletion, increased the HHT-sensitivity of RUNX1 depleted BP-CML cells, suggesting SPATS2L contributes to therapeutic resistance in CML with RUNX1 mutations. This study suggests that SPATS2L and SG induction could be critical to RUNX1-mutant leukemias, and, provides preliminary evidence for a mutationally-targeted approach in CML with RUNX1 aberrations.