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Since their discovery as drivers of proliferation, cyclin-dependent kinases (CDKs) have been considered therapeutic targets. Small molecule inhibitors of CDK4/6 are used and tested in clinical trials to treat multiple cancer types. Despite their clinical importance, little is known about how CDK4/6 inhibitors affect the stability of CDK4/6 complexes, which bind cyclins and inhibitory proteins such as p21. We develop an assay to monitor CDK complex stability inside the nucleus. Unexpectedly, treatment with CDK4/6 inhibitors—palbociclib, ribociclib, or abemaciclib—immediately dissociates p21 selectively from CDK4 but not CDK6 complexes. This effect mediates indirect inhibition of CDK2 activity by p21 but not p27 redistribution. Our work shows that CDK4/6 inhibitors have two roles: non-catalytic inhibition of CDK2 via p21 displacement from CDK4 complexes, and catalytic inhibition of CDK4/6 independent of p21. By broadening the non-catalytic displacement to p27 and CDK6 containing complexes, next-generation CDK4/6 inhibitors may have improved efficacy and overcome resistance mechanisms. Clinical CDK4/6 inhibitors are used and tested to treat a variety of cancer types. Here, the authors identify that these drugs work in two ways, a known catalytic role to inhibit kinase activity and a newly discovered noncatalytic role to displace CDK inhibitor p21 from CDK4 but not CDK6 complexes.

To clarify the roles of vitamin D and calcium as potential chemopreventive agents against colorectal cancer in humans, and to develop \"treatable\", pre-neoplastic, phenotypic biomarkers of risk for colorectal neoplasms, we estimated the effects of supplemental vitamin D3 (1,000 IU/day [25 μg/day]) and calcium (1,200 mg/day), alone and in combination, on biomarkers of proliferation (mib-1), differentiation (p21), and apoptosis (bax [apoptosis-promoting] and bcl-2 [apoptosis-inhibiting]), in the normal-appearing rectal mucosa in a subsample of participants (n = 104) in a larger randomized, double-blind, placebo-controlled clinical trial among colorectal adenoma patients. The biomarkers were measured in rectal biopsies at baseline and after one year of follow up, using automated immunohistochemistry and quantitative image analysis. In the vitamin D plus calcium group relative to control, in the crypt differentiation zone (upper 40% of crypts), mib-1 expression decreased 24% (P = 0.28); p21 expression alone and relative to mib-1 expression increased 29% (P = 0.06) and 73% (P = 0.06), respectively; and bax expression relative to mib-1 expression increased 58% (P = 0.21). The estimated vitamin D alone treatment effects were similar but of lesser magnitudes, and those for calcium alone were mixed. All estimated treatment effects on bcl-2 expression were close to the null. These pilot study results support further investigation of whether 1) vitamin D and calcium promote colorectal epithelial cell differentiation, reduce proliferation, and promote apoptosis in the normal-appearing human colorectal mucosa, 2) vitamin D and calcium act as chemopreventive agents against colorectal neoplasms, and 3) mib-1, p21, and bax are potential \"treatable\", pre-neoplastic, biomarkers of risk for colorectal neoplasms.

The retinoblastoma protein RB and the transcription factor p53 are central tumor suppressors. They are often found inactivated in various tumor types. Both proteins play central roles in regulating the cell division cycle. RB forms complexes with the E2F family of transcription factors and downregulates numerous genes. Among the RB-E2F target genes, a large number code for key cell cycle regulators. Their transcriptional repression by the RB-E2F complex is released through phosphorylation of RB, leading to expression of the cell cycle regulators. The release from repression can be prevented by the cyclin-dependent kinase inhibitor p21/CDKN1A. The CDKN1A gene is transcriptionally activated by p53. Taken together, these elements constitute the p53-p21-RB signaling pathway. Following activation of p53, for example by viral infection or induction of DNA damage, p21 expression is upregulated. High levels of p21 then result in RB-E2F complex formation and downregulation of a large number of cell cycle genes. Thus, p53-dependent transcriptional repression is indirect. The reduced expression of the many regulators leads to cell cycle arrest. Examination of the p53-p21-RB targets and genes controlled by the related p53-p21-DREAM signaling pathway reveals that there is a large overlap of the two groups. Mechanistically this can be explained by replacing RB-E2F complexes with the DREAM transcriptional repressor complex at E2F sites in target promoters. In contrast to RB-E2F, DREAM can downregulate genes also through CHR transcription factor binding sites. This results in a distinct gene set controlled by p53-p21-DREAM signaling independent of RB-E2F. Furthermore, RB has non-canonical functions without binding to E2F and DNA. Such a role of RB supporting DREAM formation may be exerted by the RB-SKP2-p27-cyclin A/E-CDK2-p130-DREAM link. In the current synopsis, the mechanism of regulation by p53-p21-RB signaling is assessed and the overlap with p53-p21-DREAM signaling is examined.

Loss of the tumor suppressor gene von Hippel-Lindau (VHL) plays a key role in the oncogenesis of clear cell renal cell carcinoma (CCRCC). The loss leads to stabilization of the HIF transcription complex, which induces angiogenic and mitogenic pathways essential for tumor formation. Nonetheless, additional oncogenic events have been postulated to be required for the formation of CCRCC tumors. Here, we show that the Notch signaling cascade is constitutively active in human CCRCC cell lines independently of the VHL/HIF pathway. Blocking Notch signaling resulted in attenuation of proliferation and restrained anchorage-independent growth of CCRCC cell lines. Using siRNA targeting the different Notch receptors established that the growth-promoting effects of the Notch signaling pathway were attributable to Notch-1 and that Notch-1 knockdown was accompanied by elevated levels of the negative cell-cycle regulators p21 Cip1 and/or p27 Kip1. Treatment of nude mice with an inhibitor of Notch signaling potently inhibited growth of xenotransplanted CCRCC cells. Moreover, Notch-1 and the Notch ligand Jagged-1 were expressed at significantly higher levels in CCRCC tumors than in normal human renal tissue, and the growth of primary CCRCC cells was attenuated upon inhibition of Notch signaling. These findings indicate that the Notch cascade may represent a novel and therapeutically accessible pathway in CCRCC.

Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) combined with endocrine therapy are the standard first-line treatment for hormone receptor-positive, HER2−negative (HR+/HER2−) metastatic breast cancer, but resistance inevitably develops. In triple-negative breast cancer (TNBC), the efficacy of CDK4/6i remains uncertain. Our study shows that the selective CDK2 inhibitor BLU-222, while effective alone, enhances synergistic activity when combined with CDK4/6i in resistant HR+/HER2− and TNBC models, leading to increased apoptosis and cell cycle arrest. In vivo, combining BLU-222 with palbociclib or ribociclib produced significant antitumor activity across eight resistant models, driving durable tumor regression and prolonged survival. Mechanistically, BLU-222, alone or with palbociclib, upregulated p21 and p27 expression, enhanced p21 binding to CDK2 as well as p21 and p27 binding to CDK4. CRISPR knockout of p21 or p27 in palbociclib-resistant cells eliminated this synergy. Further, RNA sequencing revealed that combination treatment upregulated senescence and interferon pathways, providing mechanistic insight into the observed therapeutic synergy. While CDK4/6 inhibitors (CDK4/6i) are often initially successful in many breast cancer subtypes, often resistance develops and other subtypes like triple-negative breast cancer (TNBC) fail to respond. Here, the authors demonstrate that the CDK2 inhibitor BLU-222, alone or with CDK4/6i, restores cell-cycle control via p21/p27 induction overcoming resistance in preclinical models of breast cancer, including TNBC.

Cellular senescence has been recently shown to have an important role in opposing tumour initiation and promotion. Senescence induced by oncogenes or by loss of tumour suppressor genes is thought to critically depend on induction of the p19 Arf –p53 pathway. The Skp2 E3-ubiquitin ligase can act as a proto-oncogene and its aberrant overexpression is frequently observed in human cancers. Here we show that although Skp2 inactivation on its own does not induce cellular senescence, aberrant proto-oncogenic signals as well as inactivation of tumour suppressor genes do trigger a potent, tumour-suppressive senescence response in mice and cells devoid of Skp2 . Notably, Skp2 inactivation and oncogenic-stress-driven senescence neither elicit activation of the p19 Arf –p53 pathway nor DNA damage, but instead depend on Atf4, p27 and p21. We further demonstrate that genetic Skp2 inactivation evokes cellular senescence even in oncogenic conditions in which the p19 Arf –p53 response is impaired, whereas a Skp2–SCF complex inhibitor can trigger cellular senescence in p53/Pten-deficient cells and tumour regression in preclinical studies. Our findings therefore provide proof-of-principle evidence that pharmacological inhibition of Skp2 may represent a general approach for cancer prevention and therapy. Senescence kills tumours Recent studies suggest that cellular senescence — an irreversible form of cell-cycle arrest — can halt tumour growth in vitro . Hui-Kuan Lin et al . now identify a previously unknown pathway that drives senescence without the involvement of most of the known mediators of senescence. Instead, it signals via the transcription factor Atf6, and the cyclin-dependent kinase inhibitors p27 and p21. The pathway is uncovered by inactivation of the proto-oncogene Skp2 , but only in the context of oncogenic signalling. Targeting the Skp2 complex pharmacologically restricts tumorigenesis by inducing cellular senescence, suggesting that such drugs may be effective in cancer prevention and therapy. Cellular senescence — an irreversible cell-cycle arrest — has been implicated in suppressing tumour formation or growth. A new cellular signalling pathway that drives senescence has now been identified. This pathway does not involve most known mediators of senescence, and instead signals via the proteins Atf4, p27 and p21. Inactivating the proto-oncogene Skp2 in the context of oncogenic signalling can induce senescence through this new pathway, indicating that drugs that target Skp2 might be useful in cancer treatment.

Efficient entry into S phase of the cell cycle is necessary for embryonic development and tissue homoeostasis. However, unscheduled S phase entry triggers DNA damage and promotes oncogenesis, underlining the requirement for strict control. Here, we identify the NUCKS1-SKP2-p21/p27 axis as a checkpoint pathway for the G1/S transition. In response to mitogenic stimulation, NUCKS1, a transcription factor, is recruited to chromatin to activate expression of SKP2 , the F-box component of the SCF SKP2 ubiquitin ligase, leading to degradation of p21 and p27 and promoting progression into S phase. In contrast, DNA damage induces p53-dependent transcriptional repression of NUCKS1 , leading to SKP2 downregulation, p21/p27 upregulation, and cell cycle arrest. We propose that the NUCKS1-SKP2-p21/p27 axis integrates mitogenic and DNA damage signalling to control S phase entry. The Cancer Genome Atlas (TCGA) data reveal that this mechanism is hijacked in many cancers, potentially allowing cancer cells to sustain uncontrolled proliferation. Entry into S phase of the cell cycle is regulated positively by mitogens and negatively by DNA damage; however, how balance of these signals is achieved is not well known. Here the authors show that the NUCKS1-SKP2- p21/p27 axis integrates this information, where the NUCKS1 transcription factor affects levels of p21/p27 to readout the mitogen:DNA damage balance and regulate S phase entry decision.

Senescent cells accumulate with age in vertebrates and promote aging largely through their senescence‐associated secretory phenotype (SASP). Many types of stress induce senescence, including genotoxic stress. ERCC1‐XPF is a DNA repair endonuclease required for multiple DNA repair mechanisms that protect the nuclear genome. Humans or mice with reduced expression of this enzyme age rapidly due to increased levels of spontaneous, genotoxic stress. Here, we asked whether this corresponds to an increased level of senescent cells. p16Ink4a and p21Cip1 mRNA were increased ~15‐fold in peripheral lymphocytes from 4‐ to 5‐month‐old Ercc1−/∆ and 2.5‐year‐old wild‐type (WT) mice, suggesting that these animals exhibit a similar biological age. p16Ink4a and p21Cip1 mRNA were elevated in 10 of 13 tissues analyzed from 4‐ to 5‐month‐old Ercc1−/∆ mice, indicating where endogenous DNA damage drives senescence in vivo. Aged WT mice had similar increases of p16Ink4a and p21Cip1 mRNA in the same 10 tissues as the mutant mice. Senescence‐associated β–galactosidase activity and p21Cip1 protein also were increased in tissues of the progeroid and aged mice, while Lamin B1 mRNA and protein levels were diminished. In Ercc1−/Δ mice with a p16Ink4a luciferase reporter, bioluminescence rose steadily with age, particularly in lung, thymus, and pancreas. These data illustrate where senescence occurs with natural and accelerated aging in mice and the relative extent of senescence among tissues. Interestingly, senescence was greater in male mice until the end of life. The similarities between Ercc1−/∆ and aged WT mice support the conclusion that the DNA repair‐deficient mice accurately model the age‐related accumulation of senescent cells, albeit six‐times faster. Senescent cells contribute to aging and its associated morbidities. Senescent cells accumulate in vertebrates with aging. Here, we survey where (in what tissues) senescence occurs with aging in mice, by measuring p16Ink4a and p21Cip1 mRNA. A similar survey in Ercc1−/Δ mice illustrates where (in what tissues) senescence occurs in vivo as a consequence of endogenous DNA damage.

Cellular senescence, which is a major cause of tissue dysfunction with aging and multiple other conditions, is known to be triggered by p16Ink4a or p21Cip1, but the relative contributions of each pathway toward inducing senescence are unclear. Here, we directly addressed this issue by first developing and validating a p21‐ATTAC mouse with the p21Cip1 promoter driving a “suicide” transgene encoding an inducible caspase‐8 which, upon induction, selectively kills p21Cip1‐expressing senescent cells. Next, we used the p21‐ATTAC mouse and the established p16‐INK‐ATTAC mouse to directly compare the contributions of p21Cip1 versus p16Ink4a in driving cellular senescence in a condition where a tissue phenotype (bone loss and increased marrow adiposity) is clearly driven by cellular senescence—specifically, radiation‐induced osteoporosis. Using RNA in situ hybridization, we confirmed the reduction in radiation‐induced p21Cip1‐ or p16Ink4a‐driven transcripts following senescent cell clearance in both models. However, only clearance of p21Cip1+, but not p16Ink4a+, senescent cells prevented both radiation‐induced osteoporosis and increased marrow adiposity. Reduction in senescent cells with dysfunctional telomeres following clearance of p21Cip1+, but not p16Ink4a+, senescent cells also reduced several of the radiation‐induced pro‐inflammatory senescence‐associated secretory phenotype factors. Thus, by directly comparing senescent cell clearance using two parallel genetic models, we demonstrate that radiation‐induced osteoporosis is driven predominantly by p21Cip1‐ rather than p16Ink4a‐mediated cellular senescence. Further, this approach can be used to dissect the contributions of these pathways in other senescence‐associated conditions, including aging across tissues. We generated a new mouse model (p21‐ATTAC) for clearance of senescent cells expressing p21Cip1. Clearance of p21Cip1‐expressing senescent cells, but not of p16Ink4a‐expressing cells, prevented bone loss following focal radiation. The genetic approach described here can be used to dissect the contributions of p21Cip‐ versus p16Ink4a‐driven cellular senescence in other senescence‐associated conditions, including aging across tissues.

Therapeutic application of natalizumab, an anti-CD49d Ab, in patients with multiple sclerosis (MS) has been associated with increased levels of circulating CD34+ progenitors. We analyzed the frequency, phenotype and functional activity of CD34+ HSC in blood and BM of patients with MS who were treated with natalizumab. Compared with healthy controls and untreated MS patients, natalizumab treatment increased CD34+ cells in the peripheral blood 7-fold and in BM 10-fold. CD34+ cells derived from blood and marrow of natalizumab-treated patients expressed less of the stem cell marker CD133, were enriched for erythroid progenitors (CFU-E) and expressed lower levels of adhesion molecules than G-CSF-mobilized CD34+ cells. The level of surface CXCR-4 expression on CD34+ cells from patients treated with natalizumab was higher compared with that of CD34+ cells mobilized by G-CSF (median 43.9 vs 15.1%). This was associated with a more than doubled migration capacity toward a chemokine stimulus. Furthermore, CD34+ cells mobilized by natalizumab contained more mRNA for p21 and less for matrix metallopeptidase 9 compared with G-CSF-mobilized hematopoietic stem cell (HSC). Our data indicate that G-CSF and CD49d blockade mobilize different HSC subsets and suggest that both strategies may be differentially applied in specific cell therapy approaches.