The Effect of CDK6 Expression on DNA Methylation and DNMT3B Regulation.

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.

The interplay of CDK4 and CDK6 in melanoma.

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.

Cdk6: At the interface of Rb and p53.

In cancer the activity of cyclin-dependent kinase 4-(CDK4) and cyclin-dependent kinase 6 (CDK6)-cyclin complexes are frequently altered with enhanced CDK6 expression found in hematopoietic malignancies. Our latest findings show a so far unknown role of Cdk6 during oncogene-induced stress and transformation. Therein Cdk6 antagonizes p53 responses and subsequently shapes the critical decision between survival and apoptosis in pre-leukemic cells.

CDK6 Antagonizes p53-Induced Responses during Tumorigenesis.

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.

Palbociclib treatment of FLT3-ITD+ AML cells uncovers a kinase-dependent transcriptional regulation of FLT3 and PIM1 by CDK6.

Up to 30% of patients with acute myeloid leukemia have constitutively activating internal tandem duplications (ITDs) of the FLT3 receptor tyrosine kinase. Such mutations are associated with a poor prognosis and a high propensity to relapse after remission. FLT3 inhibitors are being developed as targeted therapy for FLT3-ITD(+) acute myeloid leukemia; however, their use is complicated by rapid development of resistance, which illustrates the need for additional therapeutic targets. We show that the US Food and Drug Administration-approved CDK4/6 kinase inhibitor palbociclib induces apoptosis of FLT3-ITD leukemic cells. The effect is specific for FLT3-mutant cells and is ascribed to the transcriptional activity of CDK6: CDK6 but not its functional homolog CDK4 is found at the promoters of the FLT3 and PIM1 genes, another important leukemogenic driver. There CDK6 regulates transcription in a kinase-dependent manner. Of potential clinical relevance, combined treatment with palbociclib and FLT3 inhibitors results in synergistic cytotoxicity. Simultaneously targeting two critical signaling nodes in leukemogenesis could represent a therapeutic breakthrough, leading to complete remission and overcoming resistance to FLT3 inhibitors.

Identification of RISC-associated adenoviral microRNAs, a subset of their direct targets, and global changes in the targetome upon lytic adenovirus 5 infection.

UNLABELLED: Adenoviruses encode a set of highly abundant microRNAs (mivaRNAs), which are generated by Dicer-mediated cleavage of the larger noncoding virus-associated RNAs (VA RNAs) I and II. We performed deep RNA sequencing to thoroughly investigate the relative abundance of individual single strands of mivaRNA isoforms in human A549 cells lytically infected with human adenovirus 5 (Ad5) at physiologically relevant multiplicities of infection (MOIs). In addition, we investigated their relative abundance in the endogenous RNA-induced silencing complexes (RISCs). The occupation of endogenous RISCs by mivaRNAs turned out to be pronounced but not as dominant as previously inferred from experiments with AGO2-overexpressing cells infected at high MOIs. In parallel, levels of RISC-incorporated mRNAs were investigated as well. Analysis of mRNAs enriched in RISCs in Ad5-infected cells revealed that only mRNAs with complementarity to the seed sequences of mivaRNAs derived from VA RNAI but not VA RNAII were overrepresented among them, indicating that only mivaRNAs derived from VA RNAI are likely to contribute substantially to the posttranscriptional downregulation of host gene expression. Furthermore, to generate a comprehensive picture of the entire transcriptome/targetome in lytically infected cells, we determined changes in cellular miRNA levels in both total RNA and RISC RNA as well, and bioinformatical analysis of mRNAs of total RNA/RISC fractions revealed a general, genome-wide trend toward detargeting of cellular mRNAs upon infection. Lastly, we identified the direct targets of both single strands of a VA RNAI-derived mivaRNA that constituted one of the two most abundant isoforms in RISCs of lytically infected A549 cells. IMPORTANCE: Viral and cellular miRNAs have been recognized as important players in virus-host interactions. This work provides the currently most comprehensive picture of the entire mRNA/miRNA transcriptome and of the complete RISC targetome during lytic adenovirus infection and thus represents the basis for a deeper understanding of the interplay between the virus and the cellular RNA interference machinery. Our data suggest that, at least in the model system that was employed, lytic infection by Ad5 is accompanied by a measurable global net detargeting effect on cellular mRNAs, and analysis of RISC-associated viral small RNAs revealed that the VA RNAs are the only source of virus-encoded miRNAs. Moreover, this work allows to assess the power of individual viral miRNAs to regulate cellular gene expression and provides a list of proven and putative direct targets of these miRNAs, which is of importance, given the fact that information about validated targets of adenovirus-encoded miRNAs is scarce.

CDK6 as a key regulator of hematopoietic and leukemic stem cell activation.

The cyclin-dependent kinase 6 (CDK6) and CDK4 have redundant functions in regulating cell-cycle progression. We describe a novel role for CDK6 in hematopoietic and leukemic stem cells (hematopoietic stem cells [HSCs] and leukemic stem cells [LSCs]) that exceeds its function as a cell-cycle regulator. Although hematopoiesis appears normal under steady-state conditions, Cdk6(-/-) HSCs do not efficiently repopulate upon competitive transplantation, and Cdk6-deficient mice are significantly more susceptible to 5-fluorouracil treatment. We find that activation of HSCs requires CDK6, which interferes with the transcription of key regulators, including Egr1. Transcriptional profiling of HSCs is consistent with the central role of Egr1. The impaired repopulation capacity extends to BCR-ABL(p210+) LSCs. Transplantation with BCR-ABL(p210+)-infected bone marrow from Cdk6(-/-) mice fails to induce disease, although recipient mice do harbor LSCs. Egr1 knock-down in Cdk6(-/-) BCR-ABL(p210+) LSKs significantly enhances the potential to form colonies, underlining the importance of the CDK6-Egr1 axis. Our findings define CDK6 as an important regulator of stem cell activation and an essential component of a transcriptional complex that suppresses Egr1 in HSCs and LSCs.

Cdk4 and Cdk6 cooperate in counteracting the INK4 family of inhibitors during murine leukemogenesis.

Cdk4 and Cdk6 are related protein kinases that bind d-type cyclins and regulate cell-cycle progression. Cdk4/6 inhibitors are currently being used in advanced clinical trials and show great promise against many types of tumors. Cdk4 and Cdk6 are inhibited by INK4 proteins, which exert tumor-suppressing functions. To test the significance of this inhibitory mechanism, we generated knock-in mice that express a Cdk6 mutant (Cdk6 R31C) insensitive to INK4-mediated inhibition. Cdk6(R/R) mice display altered development of the hematopoietic system without enhanced tumor susceptibility, either in the presence or absence of p53. Unexpectedly, Cdk6 R31C impairs the potential of hematopoietic progenitors to repopulate upon adoptive transfer or after 5-fluorouracil-induced damage. The defects are overcome by eliminating sensitivity of cells to INK4 inhibitors by introducing the INK4-insensitive Cdk4 R24C allele, and INK4-resistant mice are more susceptible to hematopoietic and endocrine tumors. In BCR-ABL-transformed hematopoietic cells, Cdk6 R31C causes increased binding of p16(INK4a) to wild-type Cdk4, whereas cells harboring Cdk4 R24C and Cdk6 R31C are fully insensitive to INK4 inhibitors, resulting in accelerated disease onset. Our observations reveal that Cdk4 and Cdk6 cooperate in hematopoietic tumor development and suggest a role for Cdk6 in sequestering INK4 proteins away from Cdk4.

Monitoring cellular stress responses to nanoparticles using a lab-on-a-chip.

As nanotechnology moves towards widespread commercialization, new technologies are needed to adequately address the potential health impact of nanoparticles (NPs). Assessing the safety of over 30,000 NPs through animal testing would not only be expensive, but it would also raise a number of ethical considerations. Furthermore, existing in vitro cell-based assays are not sufficient in scope to adequately address the complexity of cell-nanoparticle interactions including NP translocation, accumulation and co-transport of e.g. allergens. In particular, classical optical/fluorescent endpoint detection methods are known to provide irreproducible, inaccurate and unreliable results since these labels can directly react with the highly catalytic surfaces of NP. To bridge this technological gap we have developed a lab-on-a-chip capable of continuously and non-invasively monitoring the collagen production of primary human fibroblast cells (NHDF) using contactless dielectric microsensors. Human dermal fibroblast cells are responsible for the maintenance of soft tissue integrity, are found throughout the human body and their primary function is collagen expression. We show that cellular collagen production can be readily detected and used to assess cellular stress responses to a variety of external stimuli, including exposure to nanoparticles. Results of the study showed a 20% and 95% reduction of collagen production following 4 hour exposure to 10 µg mL(-1) gold and silver nanoparticles (dia.10 nm), respectively. Furthermore a prolonged perfusion of sub-toxic concentrations (0.1 µg mL(-1)) of silver NP reduced NHDF collagen production by 40% after 10 h indicating increased NP take up and accumulation. We demonstrate that the application of microfluidics for the tailored administration of different NP treatments constitutes a powerful new tool to study cell-nanoparticle interactions and nanoparticle accumulation effects in small cell populations.