Poster Session I: Acute myeloid leukemia - Biology & translational research
Acute myeloid leukemia (AML) is a heterogeneous disease characterized by various cytogenetic and molecular aberrations. About 2% of AML cases harbor a deletion of the long arm of chromosome 9 (del(9q)). A commonly deleted region (CDR) was identified and specified to a minimally deleted region (MDR), comprising seven annotated genes (GKAP1, KIF27, C9ORF64, HNRNPK, RMI1, SLC28A3, NTRK2) (Krönke, et al., Blood 2013, Naarmann-de Vries et al., Leuk Res 2019). However, the function of these genes and their impact on AML pathogenesis remain elusive. The HNRNPK-encoded DNA and RNA binding protein heterogeneous nuclear ribonucleoprotein K (hnRNP K) controls the expression of specific genes at the transcriptional and post-transcriptional level. It was shown that reduced hnRNP K expression contributes to leukemogenesis in mice (Gallardo et al., Cancer Cell 2015) and that the HNRNPK mRNA level is reduced in AML del(9q) (Naarmann-de Vries et al., Leuk Res 2019). On the other hand, overexpression of hnRNP K has been described in solid tumors (Barboro et al., Cancer Lett 2014). Therefore, hnRNP K can neither be classified as an oncogene nor a tumor suppressor, rather its functions seem to depend on its cellular context.
The aim of this study was the analysis of a potential function of hnRNP K in post-transcriptional regulation contributing to leukemogenesis. The functional characterization of hnRNP K will provide a basis for the development of new therapeutic strategies.
HnRNP K immunoprecipitation (IPP) from KG-1a cells followed by RNA-Seq was applied and identified hnRNP K target RNAs were validated by RT-qPCR. To functionally characterize the identified hnRNP K-RNA interactions, the expression of hnRNP K was reduced by RNA interference (RNAi). The effect on post-transcriptional regulation was analyzed by RT-qPCR, polysome fractionation and Western blot analysis.
RNA-Seq analysis of samples derived from the hnRNP K IPP revealed 1076 interacting RNAs. Panther Protein class annotation demonstrated an enrichment of mRNAs coding for nucleic acid binding proteins, especially transcription factors. Among this group, transcription factors implicated in hematopoiesis or AML development are enriched. This points to a role of hnRNP K in hematopoietic lineage commitment and suggests that disturbed hnRNP K function contributes to AML pathogenesis. We established an RNAi-based hnRNP K knock down strategy in KG-1a cells to characterize the function of the identified hnRNP K-RNA interactions. HnRNP K-depleted KG-1a cells display reduced C/EBPα expression and diminished SPI1 (PU.1) mRNA levels. C/EBPα and PU.1 are two important factors for myeloid differentiation (Koschmieder et al., Int J Hematol 2005). A possible function of hnRNP K on CEBPA and SPI1 mRNA translational regulation is currently examined. To study the hnRNP K-mediated post-transcriptional control in myelopoiesis, an induced differentiation approach will be employed.
Among the MDR genes, HNRNPK and the identified downstream targets are interesting candidates for the development of new therapeutic strategies. The identification of hnRNP K interacting RNAs and insights in their post-transcriptional regulation will improve the understanding of molecular mechanisms driving the pathogenesis of AML del(9q). The interaction of hnRNP K with CEBPA and SPI1 mRNAs suggests a potential role of hnRNP K as a modulator of myeloid lineage commitment. Complemented with differentiation studies, these results will provide a basis for new tailored therapies.