PS1567 UNCOUPLING INK PROTEINS FROM CDK4/CDK6 IN HSCS – UNCOUPLING PROLIFERATION FROM SENESCENCE

Abstract

Background:Cyclin‐dependent kinase (CDK) 4 and CDK6 are highly related cell‐cycle kinases that regulate progression from G1 to S‐phase. CDK4/6 are activated upon binding of D‐type cyclins and subsequently phosphorylate the retinoblastoma protein (Rb). CDK4/6 inhibitor proteins, the INK4 family (p16INK4a, p15INK4b, p18INK4c, and p19INK4d) as major player, adds another layer of complexity. Hyperactivity of CDK4 or CDK6 is frequently observed in hematopoietic malignancies due to loss of p16INK4a. Besides, CDK6 has also been identified as transcriptional regulator driving complex transcriptional programs. CDK4/6 inhibitors are approved in breast cancer treatment and are currently tested in hematopoietic malignancies.Aims:We aimed to unravel the consequences of the loss of these inhibitory INK‐dependent mechanisms for hematopoiesis.Methods:We used knock‐in mice expressing a CDK6 (CDK6R31C, Cdk6 R/R ) and/or a CDK4 (CDK4R24C, Cdk4 R/R ) mutant insensitive to INK4‐mediated inhibition and analyzed stem cell populations. Further we tested the self‐renewal capacity of mutant hematopoietic progenitors under conditions of hematopoietic stress.Results:Despite comparable bone marrow cellularity, we found a significant reduction in LSK cells and HSC populations in mice harboring Cdk4 R/R or Cdk6 R/R mutation. The combination of Cdk4 R/R /Cdk6 R/R in double‐knock‐in mice rescued these defects in all progenitor stages. Serially transplanted Cdk4 R/R /Cdk6 R/R double‐knock‐in HSCs display enhanced stem cell proliferation. They maintain the ability to give rise to both myeloid and lymphoid lineages. In response to hematopoietic stress (5‐FU, serial and competitive total bone marrow transplantation), CDK4R24C/CDK6R31C prevents stem cell exhaustion and show robust recovery in stem cell numbers. Low‐input RNAseq of long‐term stress‐triggered HSCs will unravel mechanisms uncoupling proliferation from senescence.Summary/Conclusion:HSCs with disrupted CDK4/6 – INK4 binding show enhanced proliferation, reduced exhaustion and improved transplant ability. Analysis of the underlying molecular alterations may open novel therapeutic options for CDK4/6 hyperactive hematopoietic malignancies.