Atypical Kinase RIOK2 Is a Master Regulator of Hematopoietic Cell Fate
Authors: Ghosh Shrestha , Raundhal Mahesh , Myers Samuel A. , Carr Steven A. , Chen Xi , Petsko Gregory A. , Glimcher Laurie H. 
Published: November 2021

Abstract
Here we report the discovery of a new master regulator of cell fate during hematopoietic differentiation, one whose function has major implications for the treatment of blood disorders such as anemia. Anemia is a major comorbidity in aging, chronic diseases such as renal failure and inflammation, bone marrow failure disorders and in hematologic neoplasms such as myelodysplastic syndromes (MDS), affecting roughly one third of the world population. Anemia is also often diagnosed in patients treated with chemotherapy or other cytotoxic agents. The comorbidities of peripheral blood cytopenias especially in elderly patients with MDS often outweigh the treatment benefits from allogeneic stem cell transplants leaving only a handful of FDA-approved drugs/therapies for treatment of such disorders. There is thus a dire need to revisit the origins of hematopoietic differentiation defects underlying these hematologic disorders to identify additional targets for novel therapies in treating anemia.
We present evidence establishing that right open reading frame kinase 2 (RIOK2), an understudied atypical kinase associated with pre-40S ribosome biogenesis (Ferreira-Cerca et al., Nat. Str. Biol. 2012), is also a master transcriptional regulator of hematopoietic lineage commitment that simultaneously drives erythroid differentiation and represses myeloid and megakaryocytic lineages. We show that ablation of RIOK2 expression leads to hematopoietic differentiation defects in primary human hematopoietic stem and progenitor cells, the cells of origin for hematologic neoplasms.
We identity RIOK2 as an integral player in governing major blood cell differentiation processes: erythropoiesis, megakaryopoiesis and myelopoiesis. Analyses in primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) revealed that CRISPR/Cas9-mediated depletion of RIOK2 led to impaired erythropoiesis and a concomitant elevation in megakaryopoiesis and myelopoiesis. A more comprehensive analysis revealed that RIOK2 regulates the transcriptomic profiles of several key transcription factors that determine hematopoietic cell fate, including GATA1, GATA2, SPI1, RUNX3 and KLF1. Most importantly, we also observed a significant correlation between mRNA levels of RIOK2 and GATA1, GATA2, RUNX3 and KLF1 in MDS patient-derived bone marrow cells.
We also demonstrate that loss of RIOK2 causes massive alterations in chromatin accessibility, both globally and specifically at the promoters of its putative target genes. This places RIOK2 at the apex of a transcriptional regulatory network controlling hematopoietic differentiation.
We identify a previously unappreciated DNA-binding winged helix-turn-helix (wHTH) domain in RIOK2 conferring the protein with the properties and activities of a transcription factor. Transcriptomic profiling, structural modeling, chromatin immunoprecipitation-sequencing and a range of domain-deleted mutants reveal that RIOK2 functions as a bona-fide master transcription factor in hematopoiesis. We also identify two transactivation domains within the wHTH motif of RIOK2 that play integral roles in associating with the core transcriptional complex at promoter regions of genes. To the best of our knowledge, we present the first evidence of a protein that not only controls 40S ribosome biogenesis governing translation but also functions in the nucleus as a master transcription factor by regulating the expression of key transcription factors that determine hematopoietic cell fate.
Our discovery of a novel master transcriptional regulator governing a multitude of hematopoietic lineages significantly advances our current understanding of the transcriptomic landscape underlying hematopoietic differentiation. We hope that our findings may lead to new approaches to target these newly identified regulatory networks in hematopoiesis that may be relevant not just for malignancies, but for other hematologic disorders as well, such as the anemia of aging, chronic and inflammatory diseases and aplastic anemias. We are hopeful that this study will also lay a foundation to discovering how proteins, like RIOK2, may integrate transcriptional processes with translational outcomes to drive cellular functions.

Disclosures
Raundhal: Jnana Therapeutics: Current Employment. Petsko: Amicus Therapeutics, MeiraGTx, Annovis Bio, Retromer Therapeutics, and Proclara Bioscience: Membership on an entity's Board of Directors or advisory committees; Denali Therapeutics, MeiraGTx, Annovis Bio, Retromer Therapeutics and Proclara Biosciences: Current equity holder in publicly-traded company. Glimcher: Kaleido Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Other: Former Director; Repare Therapeutics: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Abpro Therapeutics: Membership on an entity's Board of Directors or advisory committees.