P1412: UNCOUPLING INK4 PROTEINS FROM CDK4/CDK6 IN HEMATOPOIETIC STEM CELLS

Abstract

Background: Cell-cycle progression is governed by regulatory proteins including cyclin-dependent kinases (CDKs), cyclins and CDK inhibitors. The INK4 family comprises p16INK4a, p15INK4b, p18INK4c and p19INK4d which are CDK4/6 specific inhibitors. Different tumor types display deletion, mutation, or promoter hypermethylation of the INK4 proteins resulting in enhanced CDK4/6 activity. The INK4a-ARF-INK4b locus encodes for p16INK4a and p15INK4b and for the tumor suppressor protein p14ARF (p19ARF in the mouse) and is one of the most frequently mutated or epigenetically silenced site in human malignancies. CDK4R24C/CDK6R31C double knock-in mice, where R24C and R31C mutations make CDK4/6 fully insensitive to INK4 mediated inhibition, show a shortened survival caused by the onset of a variety of tumors including hematopoietic malignancies. This shows the vital importance of a tight controlled inhibition of CDK4/6 activity in hematopoiesis. Aims: We here explore the consequences of INK4 inhibitory loss on CDK4/6 on the hematopoietic stem cell (HSC) compartment using CDK4R24C/CDK6R31C mice. We propose that INK factors regulate important functions in HSCs by direct binding to CDK4/6. This knowledge is required to understand how to best employ CDK inhibitors in therapeutic settings. Methods: We analyzed the composition of the hematopoietic compartment of CDK4R24C/CDK6R31C double knock-in mice under homeostatic conditions. Long-term self-renewal capacity of CDK4R24C/CDK6R31C bone marrow (BM) cells was assessed by serial bone marrow transplantation (BMT). RNAseq was used to investigate the molecular mechanisms underlying the improved self-renewal of CDK4R24C/CDK6R31C HSCs. Using the stem/progenitor cell line HPCLSK enabled us to perform ChIP qPCR to understand the involvement of CDK4R24C/CDK6R31C for Id1 transcriptional regulation. Results: Upon inhibition of CDK4/6 binding to INK4 proteins, HSC numbers in the BM of CDK4R24C/CDK6R31C mice were reduced. Despite the cell number reduction, CDK4R24C/CDK6R31C HSCs displayed an enhanced self-renewal potential during serial BMT. Transcriptome analysis pointed to the involvement of Id1 in enhanced self-renewal. We indeed found Id1 down-regulated in CDK4R24C/CDK6R31C HSCs at the 3rd round of BMT. CDK6 ChIP experiments confirmed that INK4 binding alters binding of CDK6 to the Id1 promoter, which is significantly reduced in CDK6R31C cells. Summary/Conclusion: Despite reduced qHSCs/MPP1 content under homeostatic conditions, CDK4R24C/CDK6R31C HSCs possess an enhanced self-renewal potential elucidated in serial BMT. We propose that a reduced Id1 expression in qHSCs/MPP1 CDK4R24C/CDK6R31C cells explains this phenotype and underlines improved repopulation ability of CDK4R24C/CDK6R31C BM cells. Our data indicate that INK4 binding to CDK6 dictates its function in regulating transcription.