The vital cell cycle machinery is tightly regulated and alterations of its central signaling hubs are a hallmark of cancer. The activity of CDK6 is controlled by interaction with several partners including cyclins and INK4 proteins, which have been shown to mainly bind to the amino-terminal lobe. We analyzed the impact of CDK6’s C-terminus on its functions in a leukemia model, revealing a central role in promoting proliferation. C-terminally truncated Cdk6 (Cdk6 ΔC) shows reduced nuclear translocation and therefore chromatin interaction and fails to enhance proliferation and disease progression. The combination of proteomic analysis and protein modeling highlights that Cdk6’s C-terminus is essential for protein flexibility and for its binding potential to cyclin D, p27Kip1 and INK4 proteins but not cyclin B. We demonstrate that the C-terminus is a unique and essential part of the CDK6 protein, regulating interaction partner binding and therefore CDK6’s functionality.

Metabolic reprogramming and cell cycle deregulation are hallmarks of cancer cells. The cell cycle kinase CDK6 has recently been implicated in a wide range of hematopoietic malignancies. We here investigate the role of CDK6 in the regulation of cellular metabolism in BCR::ABL1+ leukemic cells. Our study, using gene expression data and ChIP-Seq analysis, highlights the contribution of CDK6 kinase activity in the regulation of oxidative phosphorylation. Our findings imply a competition for promoter interaction of CDK6 with the master regulator of mitochondrial respiration, NRF-1. In line, cells lacking kinase active CDK6 display altered mitochondria morphology with a defective electron transport chain. The enhanced cytoplasm/mitochondria ATP ratio paralleled by high pyruvate and lactate levels indicate a metabolic switch to glycolysis. Accordingly, combinatorial treatment of leukemic cells including imatinib resistant cells with the CDK4/6 inhibitor palbociclib and the glycolysis inhibitor 2-deoxyglucose (2-DG) enhanced apoptosis, while blocking cell proliferation in leukemic cells. These data may open a new therapeutic avenue for hematologic malignancies with high CDK6 expression by exploiting metabolic vulnerabilities unmasked by blocking CDK6 kinase activity that might even be able to overcome imatinib resistance.

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. We here 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, proposing a concept where MAZ and 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.

Cyclin-dependent kinase 6 (CDK6) represents a novel therapeutic target for the treatment of certain subtypes of acute myeloid leukaemia (AML). CDK4/6 kinase inhibitors have been widely studied in many cancer types and their effects may be limited by primary and secondary resistance mechanisms. CDK4/6 degraders, which eliminate kinase-dependent and kinase-independent effects, have been suggested as an alternative therapeutic option. We show that the efficacy of the CDK6-specific protein degrader BSJ-03-123 varies among AML subtypes and depends on the low expression of the INK4 proteins p16INK4A and p18INK4C. INK4 protein levels are significantly elevated in KMT2A-MLLT3+ cells compared to RUNX1-RUNX1T1+ cells, contributing to the different CDK6 degradation efficacy. We demonstrate that CDK6 complexes containing p16INK4A or p18INK4C are protected from BSJ-mediated degradation and that INK4 levels define the proliferative response to CDK6 degradation. These findings define INK4 proteins as predictive markers for CDK6 degradation-targeted therapies in AML.

Studies of molecular mechanisms of hematopoiesis and leukemogenesis are hampered by the unavailability of progenitor cell lines that accurately mimic the situation in vivo. We now report a robust method to generate and maintain LSK (Lin-, Sca-1+, c-Kit+) cells, which closely resemble MPP1 cells. HPCLSKs reconstitute hematopoiesis in lethally irradiated recipient mice over >8 months. Upon transformation with different oncogenes including BCR/ABL, FLT3-ITD, or MLL-AF9, their leukemic counterparts maintain stem cell properties in vitro and recapitulate leukemia formation in vivo. The method to generate HPCLSKs can be applied to transgenic mice, and we illustrate it for CDK6-deficient animals. Upon BCR/ABLp210 transformation, HPCLSKsCdk6-/- induce disease with a significantly enhanced latency and reduced incidence, showing the importance of CDK6 in leukemia formation. Studies of the CDK6 transcriptome in murine HPCLSK and human BCR/ABL+ cells have verified that certain pathways depend on CDK6 and have uncovered a novel CDK6-dependent signature, suggesting a role for CDK6 in leukemic progenitor cell homing. Loss of CDK6 may thus lead to a defect in homing. The HPCLSK system represents a unique tool for combined in vitro and in vivo studies and enables the production of large quantities of genetically modifiable hematopoietic or leukemic stem/progenitor cells.

CDK6 is frequently overexpressed in various cancer types and functions as a positive regulator of the cell cycle and as a coregulator of gene transcription. We provide evidence that CDK6 is involved in the process of DNA methylation, at least in ALL. We observe a positive correlation of CDK6 and DNMT expression in a large number of ALL samples. ChIP-seq analysis reveals CDK6 binding to genomic regions associated with DNA methyltransferases (DNMTs). ATAC-seq shows a strong reduction in chromatin accessibility for DNMT3B in CDK6-deficient BCR-ABL + Cdk6-/- cells, accompanied by lower levels of DNMT3B mRNA and less chromatin-bound DNMT3B, as shown by RNA-seq and chromatome analysis. Motif analysis suggests that ETS family members interact with CDK6 to regulate DNMT3B. Reduced representation bisulfite sequencing analysis uncovers reversible and cell line-specific changes in DNA methylation patterns upon CDK6 loss. The results reveal a function of CDK6 as a regulator of DNA methylation in transformed cells.

Studies of molecular mechanisms of hematopoiesis and leukemogenesis are hampered by the unavailability of progenitor cell lines that accurately mimic the situation in vivo . We now report a robust method to generate and maintain LSK (lin - , Sca-1 + , c-Kit + ) cells which closely resemble MPP1 cells. HPC LSK reconstitute hematopoiesis in lethally irradiated recipient mice over more than eight months. Upon transformation with different oncogenes including BCR/ABL, FLT3-ITD or MLL-AF9 their leukemic counterparts maintain stem cell properties in vitro and recapitulate leukemia formation in vivo . The method to generate HPC LSK can be applied to transgenic mice and we illustrate it for CDK6-deficient animals. Upon BCR/ABL p210 transformation, Cdk6 -/- HPC LSKs induce disease with a significantly enhanced latency and reduced incidence, showing the importance of CDK6 in leukemia formation. Studies of the CDK6 transcriptome in murine HPC LSK and human BCR/ABL + cells have verified that certain pathways depend on CDK6 and have uncovered a novel CDK6-dependent signature, suggesting a role for CDK6 in leukemic progenitor cell homing. Loss of CDK6 may thus lead to a defect in homing. The HPC LSK system represents a unique tool for combined in vitro and in vivo studies and enables the production of large quantities of genetically modifiable hematopoietic or leukemic stem/progenitor cells. Key points We describe the generation of murine cell lines (HPC LSK ) which reliably mimic hematopoietic/leukemic progenitor cells. Cdk6 -/- BCR/ABL p210 HPC LSKs uncover a novel role for CDK6 in homing.

The cyclin-dependent kinases CDK4 and CDK6 promote progression through the cell cycle, where their functions are considered to be redundant. Recent studies have identified an additional role for CDK6 in the transcriptional regulation of cancer-relevant genes such as VEGF-A and EGR1 in hematopoietic malignancies. We show that the CDK4/6 inhibitor PD0332991 causes a significant decrease in tumor growth in a xenotransplantation mouse model of human melanoma. shRNA knockdown of either CDK4 or CDK6 significantly reduces cell proliferation and impedes their migratory capacity in vitro, which translates into a strong inhibition of tumor growth in xenotransplantation experiments. CDK4/6 inhibition results not only in the pronounced reduction of cell proliferation but also in an impaired tumor angiogenesis. CDK6 knockdown in melanoma cell lines impairs VEGF-A expression and reduces the potential stimulation of endothelial cell growth. The knockdown of CDK4 ends in similar results. The effect is caused by changes of CDK6 localization, less CDK6 is detected on the VEGF-A promoter. Bioinformatic analysis of human melanoma patient data verifies the key role of CDK6 in tumor angiogenesis in melanoma. The results highlight the importance of the delicate balance between CDK4 and CDK6 in regulating the cell cycle and transcription.

Over 80% of patients with myeloproliferative neoplasms (MPNs) harbor the acquired somatic JAK2 V617F mutation. JAK inhibition is not curative and fails to induce a persistent response in most patients, illustrating the need for the development of novel therapeutic approaches. We describe a critical role for CDK6 in MPN evolution. The absence of Cdk6 ameliorates clinical symptoms and prolongs survival. The CDK6 protein interferes with 3 hallmarks of disease: besides regulating malignant stem cell quiescence, it promotes nuclear factor κB (NF-κB) signaling and contributes to cytokine production while inhibiting apoptosis. The effects are not mirrored by palbociclib, showing that the functions of CDK6 in MPN pathogenesis are largely kinase independent. Our findings thus provide a rationale for targeting CDK6 in MPN.

While significant progress has been made in the treatment of acute myeloid leukemia (AML), not all patients can be cured. Mutated in about 1/3 of de novo AML, the FLT3 receptor tyrosine kinase is an attractive target for drug development, activating mutations of the FLT3 map to the juxtamembrane domain (internal tandem duplications, ITD) or the tyrosine kinase domain (TKD), most frequently at codon D835. While small molecule tyrosine kinase inhibitors (TKI) effectively target ITD mutant forms, those on the TKD are not responsive. Moreover, FLT3 inhibition fails to induce a persistent response in patients due to mutational resistance. More potent compounds with broader inhibitory effects on multiple FLT3 mutations are highly desirable. We describe a critical role of CDK6 in the survival of FLT3+ AML cells as palbociclib induced apoptosis not only in FLT3–ITD+ cells but also in FLT3–D835Y+ cells. Antineoplastic effects were also seen in primary patient-derived cells and in a xenograft model, where therapy effectively suppressed tumor formation in vivo at clinically relevant concentrations. In cells with FLT3–ITD or -TKD mutations, the CDK6 protein not only affects cell cycle progression but also transcriptionally regulates oncogenic kinases mediating intrinsic drug resistance, including AURORA and AKT—a feature not shared by its homolog CDK4. While AKT and AURORA kinase inhibitors have significant therapeutic potential in AML, single agent activity has not been proven overly effective. We describe synergistic combination effects when applying these drugs together with palbociclib which could be readily translated to patients with AML bearing FLT3–ITD or –TKD mutations. Targeting synergistically acting vulnerabilities, with CDK6 being the common denominator, may represent a promising strategy to improve AML patient responses and to reduce the incidence of selection of resistance-inducing mutations.

Tumor formation is a multistep process during which cells acquire genetic and epigenetic changes until they reach a fully transformed state. We show that CDK6 contributes to tumor formation by regulating transcriptional responses in a stage-specific manner. In early stages, the CDK6 kinase induces a complex transcriptional program to block p53 in hematopoietic cells. Cells lacking CDK6 kinase function are required to mutate TP53 (encoding p53) to achieve a fully transformed immortalized state. CDK6 binds to the promoters of genes including the p53 antagonists Prmt5, Ppm1d, and Mdm4 The findings are relevant to human patients: Tumors with low levels of CDK6 have mutations in TP53 significantly more often than expected.Significance: CDK6 acts at the interface of p53 and RB by driving cell-cycle progression and antagonizing stress responses. While sensitizing cells to p53-induced cell death, specific inhibition of CDK6 kinase activity may provoke the outgrowth of p53-mutant clones from premalignant cells. Cancer Discov; 8(7); 884-97. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 781.

Das hepatozellulare Leberkarzinom (HCC) ist eine der funf verbreitesten und eine der todlichsten Krebsarten weltweit. Die haufigste Ursache fur die Entstehung von HCC ist eine chronische Infektion mit dem Hepatitis B Virus (HBV). Andere, weniger verbreitete Risikofaktoren sind die Infektion mit dem Hepatis C Virus (HBC), Vergiftung durch Nahrungsverunreinigung (z.B. Aflatoxin), Alkoholismus, Fettsucht oder Metastasierung aus anderen Organen. Diese Risikofaktoren konnen anhaltend die Leber schadigen und so eine Umgebung schaffen, die Fibrose, Zirrhose und letztlich die Entstehung von HCC fordert. Wir etablierten ein Mausmodell fur HCC, bei dem wir immortalisierte p19ARF defiziente, Ha-Ras transformierte Hepatozyten verwendeten, die bei TGF-β Zugabe einen Ubergang vom epithelformigem zum mesenchymalen Zustand (EMT) durchmachen. Diese Zellen erfahren einen morphologischen Wandel, bei welchem sie sich von einem polarisiertem, epitheloiden Phanotyp zu einem fibroblastoiden Phanotyp transformieren. Dieser fibroblastoide Phanotyp besitzt eine veranderte Interaktion mit der extrazellularen Matrix (ECM), welche die Motilitat und die Invasivitat fordert. Eine Microarrayanalyse der Genexpression wahrend der EMT ergab, dass PDGF Ligand und Rezeptor hochreguliert sind, was auf eine mogliche Beteiligung an der Transformation schliessen lasst. Wir konstruierten einen dominant-negativen PDGF-α Rezeptor (dnPDGFR-α), um die Auswirkungen von gesteigerter PDGF Signaltransduktion zu untersuchen. Dadurch stellten wir eine Beteiligung von PDGF bei der Motilitat fest. Weiters konnten wir durch den Rezeptortyrosinkinaseinhibitor STI571 den PDGF Signaltransduktionsweg blockieren. Die Entdeckung von micro RNAs (miRs) als regulatives Netzwerk, und die Erkenntnis, dass PDGF ein miR-140 bindendes Motif im 3' UTR besitzt, sind ein aussichtsreicher Ansatz, um diesen Regulationsmechanismus zu verstehen und in bestehende Krebsmodelle, als auch in neue Strategien fur die Krebstherapie zu integrieren.