CIP/KIP and INK4 families of Cyclin-dependent kinase inhibitors (CKIs) are well-established cell cycle regulatory proteins whose canonical function is binding to Cyclin-CDK complexes and altering their function. Initial experiments showed that these proteins negatively regulate cell cycle progression and thus are tumor suppressors in the context of molecular oncology. However, expanded research into the functions of these proteins showed that most of them have non-canonical functions, both cell cycle-dependent and independent, and can even act as tumor enhancers depending on their posttranslational modifications, subcellular localization, and cell state context. This review aims to provide an overview of canonical as well as non-canonical functions of CIP/KIP and INK4 families of CKIs, discuss the potential avenues to promote their tumor suppressor functions instead of tumor enhancing ones, and how they could be utilized to design improved treatment regimens for cancer patients.

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.

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.

Cyclin-dependent kinase inhibitors (CDKIs) are important cell cycle regulators. The CDKI family is composed of the INK4 family and the CIP/KIP family. p19INK4d belongs to the INK4 gene family and is involved in a series of normal physiological activities and the pathogenesis of diseases. Many factors play regulatory roles in the p19INK4d gene expression at the transcriptional and posttranscriptional levels. p19INK4d not only regulates the cell cycle but also plays regulatory roles in apoptosis, DNA damage repair, cell differentiation of hematopoietic cells, and cellular senescence. In this review, the regulatory network of the p19INK4d gene expression and its biological functions are summarized, which provides a basis for further study of p19INK4d as a drug target for disease treatment.

Adult mammalian cardiomyocytes (CMs) exhibit limited proliferative capacity, as cell cycle activity leads to an increase in DNA content, but mitosis and cytokinesis are infrequent. This makes the heart highly inefficient in replacing with neoformed cardiomyocytes lost contractile cells as occurs in diseases such as myocardial infarction and dilated cardiomyopathy. Regenerative therapies based on the implant of stem cells of diverse origin do not warrant engraftment and electromechanical connection of the new cells with the resident ones, a fundamental condition to restore the physiology of the cardiac syncytium. Consequently, there is a growing interest in identifying factors playing relevant roles in the regulation of the CM cell cycle to be targeted in order to induce the resident cardiomyocytes to divide into daughter cells and thus achieve myocardial regeneration with preservation of physiologic syncytial performance. Despite the scientific progress achieved over the last decades, many questions remain unanswered, including how cardiomyocyte proliferation is regulated during heart development in gestation and neonatal life. This can reveal unknown cell cycle regulation mechanisms and molecules that may be manipulated to achieve cardiac self-regeneration. We hereby revise updated data on CM cell cycle regulation, participating molecules and pathways recently linked with the cell cycle, as well as experimental therapies involving them.

OBJECTIVE: Prostate cancer and benign prostate hyperplasia (BPH) are heterogeneous disorders with high prevalence among men. The antisense noncoding RNA in the INK4 locus codes for a long noncoding RNA whose participation in cancer has been elucidated.
METHODS: We analyzed rs1333045, rs4977574, rs1333048 and rs10757278 genotypes from this locus in 125 prostate cancer patients, 125 BPH patients as well as 220 normal age-matched subjects by means of tetra-primer amplification refractory mutation system PCR method.
RESULTS: The rs1333045 showed no significant difference in allele or genotype frequencies between three groups. However, the other three single nucleotide polymorphisms have been shown to be associated with BPH and prostate cancer risk.
CONCLUSIONS: Antisense noncoding RNA in the INK4 locus possibly participates in the pathogenesis of these disorders.

The naked mole rat (Heterocephalus glaber) is a long-lived and tumor-resistant rodent. Tumor resistance in the naked mole rat is mediated by the extracellular matrix component hyaluronan of very high molecular weight (HMW-HA). HMW-HA triggers hypersensitivity of naked mole rat cells to contact inhibition, which is associated with induction of the INK4 (inhibitors of cyclin dependent kinase 4) locus leading to cell-cycle arrest. The INK4a/b locus is among the most frequently mutated in human cancer. This locus encodes three distinct tumor suppressors: p15(INK4b), p16(INK4a), and ARF (alternate reading frame). Although p15(INK4b) has its own ORF, p16(INK4a) and ARF share common second and third exons with alternative reading frames. Here, we show that, in the naked mole rat, the INK4a/b locus encodes an additional product that consists of p15(INK4b) exon 1 joined to p16(INK4a) exons 2 and 3. We have named this isoform pALT(INK4a/b) (for alternative splicing). We show that pALT(INK4a/b) is present in both cultured cells and naked mole rat tissues but is absent in human and mouse cells. Additionally, we demonstrate that pALT(INK4a/b) expression is induced during early contact inhibition and upon a variety of stresses such as UV, gamma irradiation-induced senescence, loss of substrate attachment, and expression of oncogenes. When overexpressed in naked mole rat or human cells, pALT(INK4a/b) has stronger ability to induce cell-cycle arrest than either p15(INK4b) or p16(INK4a). We hypothesize that the presence of the fourth product, pALT(INK4a/b) of the INK4a/b locus in the naked mole rat, contributes to the increased resistance to tumorigenesis of this species.