INK4 Proteins Research Articles

Phylogenetic analysis of the MCL1 BH3 binding groove and rBH3 sequence motifs in the p53 and INK4 protein families.

B-cell lymphoma 2 (Bcl-2) proteins are central, conserved regulators of apoptosis. Bcl-2 family function is regulated by binding interactions between the Bcl-2 homology 3 (BH3) motif in pro-apoptotic family members and the BH3 binding groove found in both the pro-apoptotic effector and anti-apoptotic Bcl-2 family members. A novel motif, the reverse BH3 (rBH3), has been shown to interact with the anti-apoptotic Bcl-2 homolog MCL1 (Myeloid cell leukemia 1) and have been identified in the p53 homolog p73, and the CDK4/6 (cyclin dependent kinase 4/6) inhibitor p18INK4c, (p18, cyclin-dependent kinase 4 inhibitor c). To determine the conservation of rBH3 motif, we first assessed conservation of MCL1's BH3 binding groove, where the motif binds. We then constructed neighbor-joining phylogenetic trees of the INK4 and p53 protein families and analyzed sequence conservation using sequence logos of the rBH3 locus. This showed the rBH3 motif is conserved throughout jawed vertebrates p63 and p73 sequences and in chondrichthyans, amphibians, mammals, and some reptiles in p18. Finally, a potential rBH3 motif was identified in mammalian and osteichthyan p19INK4d (p19, cyclin dependent kinase 4 inhibitor d). These findings demonstrate that the interaction between MCL1 and other cellular proteins mediated by the rBH3 motif may be conserved throughout jawed vertebrates.

INK4 cyclin-dependent kinase inhibitors as potential prognostic biomarkers and therapeutic targets in hepatocellular carcinoma.

The INK4 family is an important family of cyclin-dependent kinase inhibitors (CDKIs) and consists of CDKN2A, CDKN2B, CDKN2, and CDKN2D. Abnormal expression of CDKN2A has been reported in hepatocellular carcinoma (HCC) and is associated with the prognosis of patients and infiltration of immune cells. However, there is a lack of systematic research on the roles of the other INK4 family members in the diagnosis, prognosis, and immune regulation of HCC. Using online public databases and clinical samples, we comprehensively analyzed the INK4 family in HCC. All four INK4 proteins were overexpressed in HCC and correlated with advanced cancer stage and poor prognosis. INK4 expression accurately distinguished tumor from normal tissue, particularly CDKN2A and CDKN2C. The INK4 family participated in cell-cycle regulation and the DNA damage repair pathway, which inhibited genotoxic-induced apoptosis in tumorigenesis. INK4 proteins were positively correlated with the infiltration of immune cells (B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells) and immune checkpoints (CTLA-4, PD1, and PD-L1). CDKN2D had the highest correlation (correlation coefficient >0.3) with all the above-mentioned infiltrating immune cells and immune checkpoints, indicating that it may be useful as an immunotherapy target. The INK4 family was valuable for diagnosis and predicting the prognosis of HCC and participated in the occurrence, progression, and immune regulation of HCC, demonstrating its potential as a diagnostic and prognostic biomarker and therapeutic target in HCC.

P453: CDK6 DEGRADATION IS IMPEDED BY P16INK4A AND P18INK4C IN AML

Background: Acute myeloid leukemia (AML) is a highly aggressive disease that comprises a heterogeneous group of genetically distinct subtypes. AML is initiated by leukemic cells, which are able to self-renew and give rise to malignant myeloid blasts. A key regulator and prognostic biomarker for AML is the cell cycle regulator Cyclin-dependent kinase 6 (CDK6) which represents a novel therapeutic target for the treatment of certain subtypes of AML. CDK4/6 kinase inhibitors are extensively studied in several cancer types but the beneficial effects may be limited by primary and secondary resistance mechanisms. Pharmacological CDK6 degraders, which eliminate kinase -dependent and -independent effects, represent an alternative therapeutic option. The exact mechanism and efficacy of CDK6 degraders in AML subtypes remain unknown. The involvement of p16INK4A in Palbociclib resistance and the role of INK4 proteins in AML disease progression require a systematic investigation of INK4 proteins in the context of CDK6 degradation in distinct AML subtypes. Aims: We aim to elucidate CDK6 degrader efficacy in two CDK6-dependent AML subtypes harboring either MLL-AF9 or AML1-ETO. We hypothesize an involvement of INK4 proteins on CDK6 degrader efficacy. Understanding the exact mechanisms of the CDK6 degrader will help to determine AML subtypes that would benefit from CDK6-targeted therapy. Methods: We took advantage of a novel hematopoietic progenitor cell model (HPCLSKs) to study CDK6 degrader efficacy in a genetically defined setting of AML subtypes. We analyzed INK4 RNA expression of human AML patients and the HPCLSK cells using RNA-Seq and Microarray data sets. We analyzed cell proliferation and cell cycle changes using flow cytometry. CDK6 Co-immunoprecipitation followed by Mass Spectrometry was performed to screen for CDK6 interaction partners that are enriched upon CDK6 Degrader treatment. These findings were validated via immunoblotting. Human AML cell lines were used to further validate the results in a different in vitro system and to translate them to human settings. Results: We show that efficacy of the CDK6 specific protein degrader varies among AML subtypes and depends on low expression of the INK4 proteins p16INK4A and p18INK4C. INK4 protein levels are significantly elevated in MLL-AF9+ compared to AML1-ETO+ cells, contributing to the different CDK6 degradation efficacy. We demonstrate that CDK6 complexes containing p16INK4A or p18INK4C are protected from pharmacological degradation and that INK4 levels define the proliferative response to CDK6 degradation. These findings define INK4 proteins as predictive marker for CDK6 degradation – targeted therapies in AML. Summary/Conclusion: We here identify p16INK4A and p18INK4C as dominant CDK6 binding partners that counteract pharmacological protein degradation in AML. Our data highlight the need for novel CDK6 specific therapies targeting CDK6 sites not competing with INK4 or CIP/KIP binding to overcome therapeutic limitations.

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

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.

CDK6 Degradation Is Counteracted by p16INK4A and p18INK4C in AML.

Simple SummaryCDK4/6 kinase inhibitors show promising results in various subtypes of AML, which has been primarily assigned to the inhibition of CDK6. To bypass therapeutic resistances and tackle the kinase-dependent, as well as kinase-independent, functions of CDK6, new CDK6 degraders have been developed. Here, we present insights into the mechanistic requirements for the efficacy of a CDK6-specific degrader in AML. We show that the presence and levels of the INK4 proteins p16INK4A and p18INK4C determine the extent of CDK6 degradation. Our study reveals the importance of INK4 protein levels as predictive markers for CDK6-targeted therapy in AML.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.

INK4 Tumor Suppressor Proteins Mediate Resistance to CDK4/6 Kinase Inhibitors.

Cyclin-dependent kinases 4 and 6 (CDK4/6) represent a major therapeutic vulnerability for breast cancer. The kinases are clinically targeted via ATP competitive inhibitors (CDK4/6i); however, drug resistance commonly emerges over time. To understand CDK4/6i resistance, we surveyed over 1,300 breast cancers and identified several genetic alterations (e.g., FAT1, PTEN, or ARID1A loss) converging on upregulation of CDK6. Mechanistically, we demonstrate CDK6 causes resistance by inducing and binding CDK inhibitor INK4 proteins (e.g., p18INK4C). In vitro binding and kinase assays together with physical modeling reveal that the p18INK4C-cyclin D-CDK6 complex occludes CDK4/6i binding while only weakly suppressing ATP binding. Suppression of INK4 expression or its binding to CDK6 restores CDK4/6i sensitivity. To overcome this constraint, we developed bifunctional degraders conjugating palbociclib with E3 ligands. Two resulting lead compounds potently degraded CDK4/6, leading to substantial antitumor effects in vivo, demonstrating the promising therapeutic potential for retargeting CDK4/6 despite CDK4/6i resistance. SIGNIFICANCE: CDK4/6 kinase activation represents a common mechanism by which oncogenic signaling induces proliferation and is potentially targetable by ATP competitive inhibitors. We identify a CDK6-INK4 complex that is resilient to current-generation inhibitors and develop a new strategy for more effective inhibition of CDK4/6 kinases.This article is highlighted in the In This Issue feature, p. 275.

Abstract 4413: CDK6 overexpression promotes resistance to CDK4/6 inhibitors in breast cancer

Abstract CDK4/6 inhibitors (CDK4/6i) are highly active therapies in estrogen receptor-positive (ER+) breast cancer. Unfortunately, tumor relapse on these agents is frequently encountered in clinical practice with limited data on the biologic mechanisms that can mediate resistance. We utilized cell line models, patient derived xenografts and patient samples to interrogate mechanisms of resistance to CDK4/6 inhibitors. A survey of genomic alterations associated with limited benefit of CDK4/6iidentified several alterations involving the Hippo pathway which could potentially impact CDK6 kinase expression. Immunohistochemistry from these patients revealed increased YAP nuclear translocation and high levels of CDK6 protein specifically in the group of patients with short progression-free survival (PFS) on CDK4/6i. A PDX model of acquired resistance to CDK4/6i also showed that prolonged treatment with CDK4/6i led to a subset of tumors that developed resistance, all of which showed Hippo pathway suppression and CDK6 upregulation. Finally, cell lines models exposed to CDK4/6i showed multiple clones that featured upregulation of CDK6 through suppression of the Hippo pathway or amplification of the CDK6 gene. Identifying CDK6 overexpression as a recurrent feature of CDK4/6i resistant tumors, we next investigated the function(s) of CDK6 in our models. We performed knockdown of CDK6 by shRNA and found its overexpression to be necessary for drug resistance in these models while reinforced CDK6 could restore resistance or confer resistance when overexpressed in naïve cells. Given the close homology of CDK4 and CDK6, we asked whether any differences might exist between CDK4 and CDK6 complexes, performing immunoprecipitation and mass spectrometry (IP-MS) and found CDK6 to bind a distinct partition of proteins from CDK4 including INK4 proteins. In vitro kinase assays revealed that INK4 proteins impaired the ability of CDK4/6i to block CDK6 activity. INK4 proteins were found to cooperate with CDK6 in mediating drug resistance as their knockdown restored drug sensitivity to resistant cells, while INK4 overexpression promoted resistance in CDK6-high cells. Taken together, the data revealed that high levels of a CDK6-INK4 complex to be a recurrent mechanism of resistance to CDK4/6i and suggest novel CDK6 inhibitors as a strategy to overcome resistance. Citation Format: Qing Li, Pedram Razavi, Zhiqiang Li, Arnaud F. Da Cruz Paula, Edi Brogi, Maurizio Scaltriti, Jorge S. Reis-Filho, Sarat Chandarlapaty. CDK6 overexpression promotes resistance to CDK4/6 inhibitors in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4413.

P18 inhibits reprogramming through inactivation of Cdk4/6.

Pluripotent stem cells (PSCs), including embryonic and induced pluripotent stem cells (iPSCs), show atypical cell cycle regulation characterized by a high proliferation rate and a shorter G1 phase compared with somatic cells. The mechanisms by which somatic cells remodel their cell cycle to achieve the high proliferation rate of PSCs during reprogramming are unclear. Here we identify that the Ink4 protein p18, which is expressed at high levels in somatic cells but at low levels in PSCs, is a roadblock to successful reprogramming. Mild inhibition of p18 expression enhances reprogramming efficiency, while ectopic expression of p18 completely blocks reprogramming. Mechanistic studies show that expression of wild-type p18, but not a p18D68N mutant which cannot inhibit Cdk4/6, down-regulates expression of Cdk4/6 target genes involved in DNA synthesis (TK, TS, DHFR, PCNA) and cell cycle regulation (CDK1 and CCNA2) and thus inhibits reprogramming. These results indicate that p18 blocks reprogramming by targeting Cdk4/6-mediated cell cycle regulation. Taken together, our results define a novel pathway that inhibits somatic cell reprogramming, and provide a new target to enhance reprogramming efficiency.

Small-molecule inhibitors targeting INK4 protein p18(INK4C) enhance ex vivo expansion of haematopoietic stem cells.

Among cyclin-dependent kinase inhibitors that control the G1 phase in cell cycle, only p18 and p27 can negatively regulate haematopoietic stem cell (HSC) self-renewal. In this manuscript, we demonstrate that p18 protein is a more potent inhibitor of HSC self-renewal than p27 in mouse models and its deficiency promoted HSC expansion in long-term culture. Single-cell analysis indicated that deleting p18 gene favoured self-renewing division of HSC in vitro. Based on the structure of p18 protein and in-silico screening, we further identified novel smallmolecule inhibitors that can specifically block the activity of p18 protein. Our selected lead compounds were able to expand functional HSCs in a short-term culture. Thus, these putative small-molecule inhibitors for p18 protein are valuable for further dissecting the signalling pathways of stem cell self-renewal and may help develop more effective chemical agents for therapeutic expansion of HSC.

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

AbstractCdk4 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. Cdk6R/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 p16INK4a 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.

Regulation of T Cell Differentiation and Alloimmunity by the Cyclin-Dependent Kinase Inhibitor p18ink4c

Cellular proliferation in response to mitogenic stimuli is negatively regulated by the Cip/Kip and the Ink4 families of cyclin-dependent kinase (CDK) inhibitors. Several of these proteins are elevated in anergic T cells, suggesting a potential role in the induction or maintenance of tolerance. Our previous studies showed that p27kip1 is required for the induction of T cell anergy and transplantation tolerance by costimulatory blockade, but a role for Ink4 proteins in these processes has not been established. Here we show that CD4+ T cells from mice genetically deficient for p18ink4c divide more rapidly than wild-type cells in response to antigenic, costimulatory and growth factor signals. However, this gain of proliferative function was accompanied by a moderate increase in the rate of cell death, and was accompanied by an overall defect in the generation of alloreactive IFNγ-producing effector cells. Consistent with this, p18ink4c-deficient T cells were unable to induce graft-vs-host disease in vivo, and p18ink4c deficiency cooperated with costimulatory blockade to significantly increase the survival of fully mismatched allografts in a cardiac transplantation model. While both p18ink4c and p27kip1 act to restrict T cell proliferation, p18ink4c exerts an opposite effect from p27kip1 on alloimmunity and organ transplant rejection, most likely by sustaining T cell survival and the development of effector function. Our studies point to additional important links between the cell cycle machinery and the processes of T cell differentiation, survival and tolerance.

Expression of p15INK4b and p57KIP2 and Relationship with Clinicopathological Features and Prognosis in Patients with Vulvar Squamous Cell Carcinoma

BackgroundThe cyclin-dependent kinase inhibitors p15INK4b and p57KIP2 are important regulators of the cell cycle, and their abnormal expression has been detected in various tumors. However, little is known about the role of p15INK4b and p57KIP2 in the pathogenesis of vulvar carcinoma, and the prognostic impact is still unknown. In our current study, we examined the expression of p15INK4b and p57KIP2 in a large series of vulvar squamous cell carcinomas to elucidate the prognostic impact.MethodsExpression of p15INK4b and p57KIP2 were examined in 297 vulvar squamous cell carcinomas using immunohistochemistry. Both uni- and multivariate analysis of prognostic factors were performed, and correlations with clinicopathologic parameters were examined.ResultsCompared to the high levels of p15INK4b and p57KIP2 in normal vulvar squamous epithelium, low levels of p15INK4b and p57KIP2 were found in 82% and 44% of vulvar carcinomas, respectively. Low levels of p15INK4b and p57KIP2 correlated significantly with malignant features, including large tumor diameter (p = 0.03 and p = 0.001, respectively) and increased invasiveness (p = 0.003 and p = 0.04, respectively). Although p15INK4b and p57KIP2 levels could not be identified as prognostic markers, combined analysis of p14ARF/p15INK4b/p16INK4a showed that patients whose tumors expressed low levels of two or three of these INK4 proteins had a worse prognosis than those with only low levels of one or no protein (univariate analysis p = 0.02). The independent prognostic significance of these INK4 proteins was confirmed by multivariate analysis (p = 0.008).ConclusionsWe show for the first time that p15INK4b and p57KIP2 may be involved in the progression of vulvar carcinomas and the combined p14ARF/p15INK4b/p16INK4a status was a statistically independent prognostic factor.

Contrasting Behavior of the p18INK4c and p16INK4a Tumor Suppressors in Both Replicative and Oncogene-Induced Senescence

The cyclin-dependent kinase (CDK) inhibitors, p18(INK4c) and p16(INK4a), both have the credentials of tumor suppressors in human cancers and mouse models. For p16(INK4a), the underlying rationale is its role in senescence, but the selective force for inactivation of p18(INK4c) in incipient cancer cells is less clear. Here, we show that in human fibroblasts undergoing replicative or oncogene-induced senescence, there is a marked decline in the levels of p18(INK4c) protein and RNA, which mirrors the accumulation of p16(INK4a). Downregulation of INK4c is not dependent on p16(INK4a), and RAS can promote the loss of INK4c without cell-cycle arrest. Downregulation of p18(INK4c) correlates with reduced expression of menin and E2F1 but is unaffected by acute cell-cycle arrest or inactivation of the retinoblastoma protein (pRb). Collectively, our data question the idea that p18(INK4c) acts as a backup for loss of p16(INK4a) and suggest that the apparent activation of p18(INK4c) in some settings represents delayed senescence rather than increased expression. We propose that the contrasting behavior of the two very similar INK4 proteins could reflect their respective roles in senescence versus differentiation.

P27Kip1 is required to maintain proliferative quiescence in the adult cochlea and pituitary

Cell cycle inhibitors, such as the cyclin-dependent kinase (Cdk) inhibitor proteins and retinoblastoma (Rb) family members, control exit from the cell cycle during the development of a variety of terminally differentiated tissues. It is unclear whether sustained expression of these proteins is required to prevent cell cycle re-entry in quiescent and terminally differentiated cells. The organ of Corti (cochlear sensory epithelium) and pars intermedia (intermediate lobe of the pituitary) are two tissues that share the characteristic of ongoing cell division in mice lacking either the p27Kip1 Cdk inhibitor, Ink4 proteins, or Rb. Here, we use tamoxifen-inducible mouse models to delete p27Kip1 in postnatal animals and show this is sufficient to induce proliferation in both the organ of Corti and pars intermedia. Thus, these tissues remain sensitive to the presence of p27Kip1 even after their developmental exit from the cell cycle. The neonatal cochlea displayed heightened sensitivity to changes in p27Kip1 expression, with a proliferative response higher than that of constitutive null mice. In adults, the proliferative response was reduced but was accompanied by increased cell survival. In contrast, re-establishment of normal p27Kip1 expression in animals with established pituitary tumors, in an inducible “knock-on” model, led to cessation of pituitary tumor growth, indicating the cells had maintained their susceptibility to p27-mediated growth suppression. Although restoration of p27Kip1 did not induce apoptosis, it did lead to resolution of pathological features and normalization of gene expression. Our data underscore the importance of p27Kip1 expression in the maintenance of cellular quiescence and terminal differentiation.

Differential post-transcriptional regulation of two Ink4 proteins, p18Ink4c and p19Ink4d

Cyclin(-D-)-dependent kinase (Cdk) inhibitors of the Ink4 family specifically bind to Cdk4 and Cdk6, but not to other Cdks. Ink4c and Ink4d mRNAs are maximally and periodically expressed during the G2/M phase of the cell division cycle, but the abundance of their encoded proteins is regulated through distinct mechanisms. Both proteins undergo polyubiquitination, but the half life of p18Ink4c (~10 hours) is much longer than that of p19Ink4d (~2.5 hours). Lysines 46 and 112 are preferred sites of ubiquitin conjugation in p18Ink4c, although substitution of these and other lysine residues with arginine, particularly in combination, triggers protein misfolding and accelerates p18Ink4c degradation. When tethered to either catalytically active or inactive Cdk4 or Cdk6, polyubiquitination of p18Ink4c is inhibited, and the protein is further stabilized. Conversely, in competing with p18Ink4c for binding to Cdks, cyclin D1 accelerates p18Ink4c turnover. In direct contrast, polyubiquitination of p19Ink4d is induced by its association with Cdks, whereas cyclin D1 overexpression retards p19Ink4d degradation. Although it has been generally assumed that p18Ink4c and p19Ink4d are biochemically similar Cdk inhibitors, the major differences in their stability and turnover are likely key to understanding their distinct biological functions.

Genetic cooperation between p21Cip1 and INK4 inhibitors in cellular senescence and tumor suppression

Cell-cycle inhibitors of the Cip/Kip and INK4 families are involved in cellular senescence and tumor suppression. Some of these proteins, p21(Cip1), p16(INK4a) and p15(INK4b), are coexpressed in response to antiproliferative signals such as cellular senescence resulting in cell-cycle arrest. To understand the roles of these inhibitors and their synergistic effect, we have characterized the growth properties and senescent behavior of primary cells deficient in p21(Cip1) and expressing an endogenous Cdk4(R24C) (cyclin-dependent kinase) mutant (Cdk4(R24C) knock-in cells) insensitive to INK4 proteins. Inactivation of both p21(Cip1) and INK4 pathways strongly cooperate in suppressing cellular senescence in vitro. These double mutant cells behavior as immortal cultures and display high sensitivity to cellular transformation by oncogenes. Moreover, mice double mutant in the INK4 and p21(Cip1) pathways (Cdk4(R24C); p21(Cip1)-null mice) display an increased incidence of specific sarcomas, suggesting a significant cooperation between these two families of cell-cycle inhibitors in senescence responses and tumor suppression in vivo.

CDK4 and CDK6 Delay Senescence by Kinase-Dependent and p16INK4a-Independent Mechanisms

Replicative senescence of human diploid fibroblasts (HDFs) is largely implemented by the cyclin-dependent kinase (CDK) inhibitors p16(INK4a) and p21(CIP1). Their accumulation results in a loss of CDK2 activity, and cells arrest with the retinoblastoma protein (pRb) in its hypophosphorylated state. It has become standard practice to bypass the effects of p16(INK4a) by overexpressing CDK4 or a variant form that is unable to bind to INK4 proteins. Although CDK4 and CDK6 and their INK4-insensitive variants can extend the life span of HDFs, they also cause a substantial increase in the levels of endogenous p16(INK4a). Here we show that CDK4 and CDK6 can extend the life span of HDFs that have inactivating mutations in both alleles of INK4a or in which INK4a levels are repressed, indicating that overexpression of CDK4/6 is not equivalent to ablation of p16(INK4a). However, catalytically inactive versions of these kinases are unable to extend the replicative life span, suggesting that the impact of ectopic CDK4/6 depends on their ability to phosphorylate as yet unidentified substrates rather than to sequester CDK inhibitors. Since p16(INK4a) deficiency, CDK4 expression, and p53 or p21(CIP1) ablation have additive effects on replicative life span, our results underscore the idea that senescence is an integrated response to diverse signals.

Expression of p16Ink4a Compensates for p18Ink4c Loss in Cyclin-Dependent Kinase 4/6–Dependent Tumors and Tissues

Cell cycle progression from G(1) to S phase depends on phosphorylation of pRb by complexes containing a cyclin (D type or E type) and cyclin-dependent kinase (e.g., cdk2, cdk4, or cdk6). Ink4 proteins function to oppose the action of cdk4/6-cyclin D complexes by inhibiting cdk4/6. We employed genetic and pharmacologic approaches to study the interplay among Ink4 proteins and cdk4/6 activity in vivo. Mouse embryo fibroblasts (MEF) lacking p16(Ink4a) and p18(Ink4c) showed similar growth kinetics as wild-type MEFs despite increased cdk4 activity. In vivo, germline deficiency of p16(Ink4a) and p18(Ink4c) resulted in increased proliferation in the intermediate pituitary and pancreatic islets of adult mice, and survival of p16(Ink4a-/-);p18(Ink4c-/-) mice was significantly reduced due to aggressive pituitary tumors. Compensation among the Ink4 proteins was observed both in vivo in p18(Ink4c-/-) mice and in MEFs from p16(Ink4a-/-), p18(Ink4c-/-), or p16(Ink4a-/-);p18(Ink4c-/-) mice. Treatment with PD 0332991, a specific cdk4/6 kinase inhibitor, abrogated proliferation in those compartments where Ink4 deficiency was associated with enhanced proliferation (i.e., islets, pituitary, and B lymphocytes) but had no effect on proliferation in other tissues such as the small bowel. These data suggest that p16(Ink4a) and p18(Ink4c) coordinately regulate the in vivo catalytic activity of cdk4/6 in specific compartments of adult mice.

Inhibition of the Extracellular Signal-regulated Kinase/Mitogen-activated Protein Kinase Pathway Decreases DNA Methylation in Colon Cancer Cells

The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) pathway is a critical intermediary for cell proliferation, differentiation, and survival. In the human colon cancer cell line SW1116, treatment with the DNA methyltransferase 1 (DNMT1) inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) or the ERK-MAPK inhibitors PD98059 or rottlerin, or transient transfection with the MAP/ERK kinase (MEK)1/2 small interfering RNA down-regulates DNMT1 and proliferating cell nuclear antigen levels. In this report, we found that drug treatment or small interfering RNA transfection of SW1116 cells induced promoter demethylation of the p16(INK4A) and p21(WAF1) genes, which up-regulated their mRNA and protein expression levels. Flow cytometry revealed that rottlerin treatment induced cell cycle arrest at phase G(1) (p < 0.05). Thus, the ERK-MAPK inhibitor treatment or siRNA-mediated knockdown of ERK-MAPK decreases DNA methylation via down-regulating DNMT1 expression and other unknown mediator(s) in SW1116 colon cancer cells.

INK4 proteins, a family of mammalian CDK inhibitors with novel biological functions

The cyclin D-Cdk4-6/INK4/Rb/E2F pathway plays a key role in controlling cell growth by integrating multiple mitogenic and antimitogenic stimuli. The members of INK4 family, comprising p16(INK4a), p15(INK4b), p18(INK4c), and p19(INK4d), block the progression of the cell cycle by binding to either Cdk4 or Cdk6 and inhibiting the action of cyclin D. These INK4 proteins share a similar structure dominated by several ankyrin repeats. Although they appear to be structurally redundant and equally potent as inhibitors, the INK4 family members are differentially expressed during mouse development. The striking diversity in the pattern of expression of INK4 genes suggested that this family of cell cycle inhibitors might have cell lineage-specific or tissue-specific functions. The INK4 proteins are commonly lost or inactivated by mutations in diverse types of cancer, and they represent established or candidate tumor suppressors. Apart from their capacity to arrest cells in the G1-phase of the cell cycle they have been shown to participate in an increasing number of cellular processes. Given their emerging roles in fundamental physiological as well as pathological processes, it is interesting to explore the diverse roles for the individual INK4 family members in different functions other than cell cycle regulation. Extensive studies, over the past few years, uncover the involvement of INK4 proteins in senescence, apoptosis, DNA repair, and multistep oncogenesis. We will focus the discussion here on these unexpected issues.