Abstract
hnRNP K (heterogeneous ribonucleoprotein K) is an RNA-binding protein that binds to conserved poly-C rich tracks in RNA and influences a diverse set of molecular pathways involved in tumorigenesis. Our previous studies identified hnRNP K overexpression in patients with diffuse large B-cell lymphoma (46/75, 61%) and acute myeloid leukemia (45/160, 28%). This overexpression correlates with dismal clinical outcomes and a lack of therapeutic responses to standard treatment. To explore hnRNP K's in vivo functions, we generated Hnrnpk-transgenic mouse models. These mice develop lymphoma phenotypes through activation of the c-Myc pathway. In pre-clinical settings, bromodomain inhibitors disrupted hnRNP K-mediated c-Myc activation, demonstrating that hnRNP K overexpression mediated-pathways are amenable to therapeutic intervention.
To further our studies, we used IP-mass spectrometry, RNA-sequencing, RNA immunoprecipitation, reverse phase protein analyses, and polysome profiling to identify novel pathways associated with changes in hnRNP K expression. Here, we observed that alterations in hnRNP K expression result in an impairment of ribosomal biogenesis and activation of pathways directly responsible for global translation. Using both knockdown and overexpression systems, we observed a direct correlation between hnRNP K expression and expression of S6, S6K, phosphorylated S6, eIF and mTOR pathways and uncovered defects in rRNA splicing. Collectively, these data indicate that impairment of cap-dependent loading and alterations in ribogenesis may be a driving force in the clinical manifestations of hnRNP K-driven malignancies. Furthermore, these results suggest that translational-inhibitors may be useful in exploiting hnRNP K-dependent vulnerabilities. To examine this aspect, we are currently using FDA-approved translation inhibitors and disruptors of ribogenesis (e.g. homoharringtonineand mTOR-inhibitors) and KTP- compounds, respectively.
While these indirect targeting strategies are interesting, our results indicate that hnRNP K also regulates cellular programs outside of translation. Thus, potential therapies that effectively target hnRNP K overexpression will require direct inhibition of its RNA binding functions. To this end, we used several screening assays including fluorescence anisotropy (FA), surface plasmon resonance, SYPRO-orange thermal shift assays, and cell proliferation assays to screen 80,000 small molecule compounds which led to the identification of 9 candidates that disrupt hnRNP K-mRNA interactions and cause cell death in an hnRNP K-dependent manner. Further, cellular thermal shift assays revealed these lead compounds engage hnRNP K within cells and most critically, result in reduced expression of hnRNP K targets in vivo. These candidate compounds as well as potentially more potent structural analogs are currently being evaluated.
Collectively, our results demonstrate that the oncogenic functions of hnRNP K are amenable to both indirect therapeutic intervention using FDA-approved agents as well as direct inhibition through newly identified small molecule compounds, signifying that there may be a roadmap to effective therapies for hnRNP K-dependent malignancies.
Disclosures
No relevant conflicts of interest to declare.
Syncrip/hnRNPQ is required for activity-induced Msp300/Nesprin-1 expression and new synapse formation