S104: RBPS DYSREGULATION CAUSE HYPER-NUCLEOLI AND RIBOSOME GAIN-OF-FUNCTION DRIVING BONE MARROW FAILURE

Abstract

Background: Nucleoli and ribosome cross-talk regulates cell translation capacity. Its dysregulation and impairment drive ribosome and nucleolus stress (NS), related to the biological mechanism of cancer. Ribosomopathies are a group of diseases characterized by ribosome defects leading to complex syndromes that include bone marrow failure. hnRNP K is an RNA binding protein (RBP) in charge of processing nascent RNAs (nucleoli) into mature mRNAs (ribosome). Our research found a novel ribosome gain-of-function ribosomopathy phenotype by hyper-nucleoli generation due RBP hnRNP K dysregulation. Aims: We aim to elucidate how hnRNP K dysregulation impact on haematopoietic stem cell biology. Methods:Hnrnpk overexpression was established in MEFs using CRISPR/SAM (Konermann S. et al, Nature). Global protein synthesis was tested using a Click-iT OPP and proteasome function was evaluated by Proteasome 20S activity NS hallmarks were analyzed by confocal microscopy evaluating Ncl, NS sensor marker. To evoke NS in our cells, we used Actinomycin D insult. Cell cycle FACS analysis (DAPI) and senescence assays (β-galactosidase staining) were performed. Molecular mechanism underlying was elucidated by qRT-PCR and WB (p21, p16, c-Myc, and mTor). To study the impact of hnRNP K overexpression in vivo, we developed an inducible tamoxifen mouse model activated 30-60 days after birth (HnrnpkTg-Ubc-creERT2). Survival was evaluated by Kaplan-Meier, and phenotype described by symptoms, signs, CBC and bone marrow IHC panel (CD34, Gr1, B220, MPO). Results:Hnrnpk overexpressing cells led to an increment in protein and gene expression of Ncl, mTor and c-Myc (Figure 1A-B). Moreover, we found an increase in global protein synthesis (Figure 2C-D). Nevertheless, the elevation of hnRNP K inversely correlated with the proteasome function, which dropped significantly (Figure 1E). NS induction promoted higher hnRNP K expression. Additionally, hnRNP K overexpressing cells showed NS hallmarks associated with an increase of the number of nucleoli, and total area of the nucleoli and nucleus (Figure 1F-G). Cell cycle analysis confirmed an increment of arrested G2/M phase cells (Figure 1H-I), linked to an increment in p21 and p16 levels all leading towards a senescent cell phenotype (Figure 1J-L). HnrnpkTg-Ubc-creERT2 mice had widespread Hnrnpk overexpression (Figure 1M) and a reduction in lifespan (Figure 1N), mainly due to dysplastic and bone marrow failure phenotype, with dramatic reduction of CD34 and b-cells, leukopenia, anaemia and thrombocytopenia. (Figure 1O-Q). Image:Summary/Conclusion: The overexpression of hnRNP K drives to an increase in nucleoli activity, leading to ribosome biogenesis and higher global translation by the regulation of molecules involved in both systems: Ncl, c-Myc or mTor. Our work found that hnRNP K overexpression in vivo drives a bone marrow failure phenotype, promoting the exhaustion of haematopoietic stem cells by ribosome dysregulation that triggered cell senescence. Of note, this is the first time reported that a nucleoli/ribosome-gain-of-function induce bone marrow failure ribosomopathies phenotype. This work was financially supported by CRIS contra el Cancer Association (NGO) AES ISCIII (PI18/00295), ISCIII Miguel Servet (CP19/00140), Cancer Research UK [C355/A26819], FC AECC and AIRC under the Accelerator Award Program and National Cancer Institutes of Health Award (R01CA207204, SMP) Leukemia and Lymphoma Society (6577-19, SMP).