P325: A KINASE-DEAD VARIANT OF CDK6 IS ASSOCIATED WITH REDUCED TUMORIGENIC POTENTIAL AND A DISTINCT TRANSCRIPTIONAL REWIRING IN AN ALL MODEL

Background: The cell cycle kinase Cdk6 is a major regulator of cell cycle progression from G1 to S phase. Cdk4/6 kinase inhibitors are approved for the treatment of breast cancer and show promising results in pre-clinical studies of other cancers, including hematological malignancies. Cdk6 was also shown to exhibit kinase-independent functions including transcriptional regulation of gene expression. Aims: A systematic understanding of the kinase independent functions of Cdk6 should allow for a better comprehension of the mechanism of action of Cdk4/6 inhibitors. Methods: To model kinase-independent functions of Cdk6 in Acute Lymphoblastic Leukemia (ALL), mouse pre/pro B-cell lines were generated. Whole bone marrow from mice carrying a kinase-dead mutant of Cdk6 (Cdk6-K43M), Cdk6 knock-out (Cdk6-KO) or wildtype Cdk6 (Cdk6-WT) were retrovirally transduced with the BCR-ABL1p185 fusion gene. The resulting cell lines were analyzed in-vivo in transplant settings as well as on the molecular level where RNA-seq and ChIP-seq were performed. Results: Tail vain injection of Cdk6-K43M, Cdk6-KO and Cdk6-WT pre/pro B-cell lines into NSG mice led to the development of lymphoid malignancies albeit with different latencies depending on genotype. Mice injected with Cdk6-KO lines showed a significantly longer survival than mice injected with Cdk6-WT lines. The injection of Cdk6-K43M cells led to an intermediate phenotype with significantly shorter survival than Cdk6-KO, but significantly longer survival than Cdk6-WT. Subcutaneous injection of the cell lines also allowed for the formation of tumors, where tumor weight was largest in mice injected with Cdk6-WT cells, intermediate in the case of Cdk6-K43M cells and lowest in the case of Cdk6-KO cells. Therefore, while kinase-dead Cdk6 had reduced tumorigenic potential compared to Cdk6-WT, loss of kinase function alone exhibited stronger tumorigenic potential than the complete Cdk6 KO. Differential gene expression analysis between cell lines of the three genotypes revealed common alterations associated with Cdk6-K43M and Cdk6-KO compared to Cdk6-WT. Some of these changes could directly be attributed to the loss of kinase function, including for example the downregulation of E2F target genes. Additionally, changes specific for either Cdk6-K43M or Cdk6-KO were observed. As Cdk6-K43M retains kinase-independent functionality, which includes transcriptional regulation, we investigated the DNA binding profile of transcriptional complexes containing Cdk6-K43M. ChIP-seq analysis revealed that such complexes bind the DNA at largely similar sites as complexes containing Cdk6-WT. The majority of genes differentially expressed between Cdk6-WT and Cdk6-K43M were associated with transcriptional complexes harboring Cdk6. Summary/Conclusion: Pre/pro B-cell lines carrying kinase-dead Cdk6 (Cdk6-K43M) showed intermediate tumorigenic potential between lines with Cdk6-WT and Cdk6-KO. While Cdk6-K43M is associated with the DNA at similar sites as Cdk6-WT, there are distinct transcriptional changes associated with this mutation. The further elucidation of these alterations is expected to provide a better understanding of both, kinase independent functions of Cdk6 as well as the effects of its pharmacological inhibition.