Defining the roles of hnRNP family members in Acute Myeloid Leukaemia, with a focus on hnRNPA0 and hnRNPA3

Abstract

Acute myeloid leukaemia (AML) is a biologically heterogeneous malignancy characterised by genetic, epigenetic, transcriptional, and clinical diversity. Patients with AML have variable treatment responses and high relapse rates and therefore have a significant unmet clinical need regarding the availability of targeted therapies for all AML subtypes. While recurrent transcription factor alterations and oncogenic signalling pathways have been extensively studied, the contribution of post transcriptional gene regulation to AML pathobiology remains less well defined. RNA binding proteins, including members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, play central roles in coordinating mRNA stability, processing, and translation, enabling dynamic adaptation to cellular stress and differentiation cues. Here, analysis of nuclear proteomic data revealed a coordinated reduction in multiple heterogeneous nuclear ribonucleoproteins (hnRNP) in AML blasts, key regulators of mRNA processing, transport, and stability. Using publicly available mRNA data sets, this study examined the expression of hnRNPs family members during normal haematopoiesis and in AML patient blasts. hnRNPA0 and hnRNPA3 were prioritised for functional investigation. Functional studies using shRNA and CRISPR-Cas9 to reduce hnRNPA3 expression did not significantly impair the growth or survival of THP1 AML cells. In contrast, reduced hnRNPA0 expression in KG1a cells resulted in a marked reduction in cell growth and viability, as assessed by flow cytometry. This growth impairment was accompanied by a redistribution of the cell cycle, with an increased proportion of cells accumulating in the G₂/M phase. Further, an increase in apoptosis was observed using flow cytometry and Appotracker. To define the molecular programmes governed by hnRNPA0 to allow AML cells to survive RNA sequencing was performed in KG1a cells where hnRNPA0 was knocked down. Using a combination of gene ontology, KEGG and GSEA analysis, several pathways characterised by coordinated suppression of proliferative networks, including E2F regulated and G2/M checkpoint programmes were changed. In addition, an enrichment of stress responsive and apoptotic pathways were identified. Among the most prominently upregulated transcripts was the pro apoptotic effector BCL2L11 (BIM), suggesting hnRNPA0 knockdown may mediate AML cell survival by suppressing BIM expression. To support this data in an AML cell line, analysis of mRNA AML patient blasts demonstrated an inverse relationship between hnRNPA0 and BCL2L11 expression, supporting the relevance of hnRNPA0 dependent transcriptional programmes beyond in vitro models. In conclusion, this work positions hnRNPA0 as a post transcriptional regulator that sustains AML cell survival by maintaining proliferative and stress adaptive gene expression programmes. Rather than acting as a classical oncogenic driver, hnRNPA0 appears to modulate apoptosis.