Mayer, Isabella M. (University of Veterinary Medicine Vienna) Kollmann, Karoline (University of Veterinary Medicine Vienna) Shaw, Lisa E. (Medical University of Vienna) Gebrail, Lea (University of Veterinary Medicine Vienna) Kunowska, Natalia (University of Graz) Zojer, Markus (University of Veterinary Medicine Vienna) Scheiblecker, Lisa (University of Veterinary Medicine Vienna) Schirripa, Alessia (University of Veterinary Medicine Vienna) Doma, Eszter (University of Veterinary Medicine Vienna) Klampfl, Thorsten (University of Veterinary Medicine Vienna) Prchal-Murphy, Michaela (University of Veterinary Medicine Vienna) Kollmann, Sebastian (University of Veterinary Medicine Vienna) Sexl, Veronika (University of Veterinary Medicine Vienna / Universität Innsbruck) Malumbres, Marcos (Vall d'Hebron Institute of Oncology / Spanish National Cancer Research Centre / Institució Catalana de Recerca i Estudis Avançats) Farlik, Matthias (Medical University of Vienna) Zebedin-Brandl, Eva (Medical University of Vienna) Heller, Gerwin (Medical University of Vienna) Grausenburger, Reinhard (University of Veterinary Medicine Vienna) Farr, Alex (Medical University of Vienna) Mann, Ulrike (Medical University of Vienna) Hematopoietic stem cells (HSCs) are characterized by the ability to self-renew and to replenish the hematopoietic system. The cell-cycle kinase cyclin-dependent kinase 6 (CDK6) regulates transcription, whereby it has both kinase-dependent and kinase-independent functions. Herein, we describe the complex role of CDK6, balancing quiescence, proliferation, self-renewal, and differentiation in activated HSCs. Mouse HSCs expressing kinase-inactivated CDK6 show enhanced long-term repopulation and homing, whereas HSCs lacking CDK6 have impaired functionality. The transcriptomes of basal and serially transplanted HSCs expressing kinase-inactivated CDK6 exhibit an expression pattern dominated by HSC quiescence and self-renewal, supporting a concept, in which myc-associated zinc finger protein (MAZ) and nuclear transcription factor Y subunit alpha (NFY-A) are critical CDK6 interactors. Pharmacologic kinase inhibition with a clinically used CDK4/6 inhibitor in murine and human HSCs validated our findings and resulted in increased repopulation capability and enhanced stemness. Our findings highlight a kinase-independent role of CDK6 in long-term HSC functionality. CDK6 kinase inhibition represents a possible strategy to improve HSC fitness. Kinase-inactivated CDK6 preserves the long-term functionality of adult hematopoietic stem cells application/pdf 2024 Blood 144(2), 156-170 (2024) Cyclin-Dependent Kinase 6 Metabolism Genetics; Animals; Hematopoietic Stem Cells Metabolism Cytology; Mice; Humans; Adult Stem Cells Metabolism Cytology; Cell Proliferation; Cell Differentiation; Mice, Inbred C57BL; Hematopoietic Stem Cell Transplantation; Cell Self Renewal Drug effects CC BY-NC-ND 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ article eng doi:10.1182/blood.2023021985 https://phaidra.vetmeduni.ac.at/o:3470 American Society of Hematology