CDK6: an attractive therapeutic target for T-ALL/LBL.

INTRODUCTION: Human T-cell acute lymphoblastic leukemia/T-cell lymphoblastic lymphoma (T-ALL/LBL) is a type of cancer that originates from the bone marrow and spreads quickly to other organs. Long-term survival rate with current available chemotherapy is less than 20%. Despite the potentially huge market, a truly effective and safe therapy for T-ALL/LBL is elusive. Thus, it is imperative to identify new therapeutic ways to target essential pathways in T-ALL that regulate the proliferation and survival of these cancer cells. AREAS COVERED: The role of the Cyclin-dependent kinase 6 (CDK6) pathway in human T-ALL is of significant interest with major clinical/translational relevance. This review covers the recent advances in elucidating the essential roles of CDK6 and its closely regulated networks in proliferation, survival, and metabolism of T-ALL cells, with new insight into its mechanisms of action which hopefully could trigger the identification of new therapeutic avenues. EXPERT OPINION: Animal models showed that inhibition of CDK6 and its related networks blocked initiation, growth, and survival of T-ALL in vivo. Numerous clinical trials of CDK4/6 inhibitors are ongoing in T-ALL. Specific CDK6 inhibitors alone or novel combination regimens may hopefully delay the progression, or even reverse the symptoms of T-ALL, leading to disease eradication and cure.

Kaempferol regulates the thermogenic function of adipocytes in high-fat-diet-induced obesity via the CDK6/RUNX1/UCP1 signaling pathway.

Activation of adipose tissue thermogenesis is a promising strategy in the treatment of obesity and obesity-related metabolic disorders. Kaempferol (KPF) is a predominant dietary flavonoid with multiple pharmacological properties, such as anti-inflammatory and antioxidant activities. In this study, we sought to characterize the role of KPF in adipocyte thermogenesis. We demonstrated that KPF-treated mice were protected from diet-induced obesity, glucose tolerance, and insulin resistance, accompanied by markedly increased energy expenditure, ex vivo oxygen consumption of white fat, and increased expression of proteins related to adaptive thermogenesis. KPF-promoted beige cell formation is a cell-autonomous effect, since the overexpression of cyclin-dependent kinase 6 (CDK6) in preadipocytes partially reversed browning phenotypes observed in KPF-treated cells. Overall, these data implicate that KPF is involved in promoting beige cell formation by suppressing CDK6 protein expression. This study provides evidence that KPF is a promising natural product for obesity treatment by boosting energy expenditure.

CDK6 increases glycolysis and suppresses autophagy by mTORC1-HK2 pathway activation in cervical cancer cells.

Cervical carcinoma is a leading malignant tumor among women worldwide, characterized by the dysregulation of cell cycle. Cyclin-dependent kinase 6 (CDK6) plays important roles in the cell cycle progression, cell differentiation, and tumorigenesis. However, the role of CDK6 in cervical cancer remains controversial. Here, we found that loss of CDK6 in cervical adenocarcinoma HeLa cell line inhibited cell proliferation but induced apoptosis as well as autophagy, accompanied by attenuated expression of mammalian target of rapamycin complex 1 (mTORC1) and hexokinase 2 (HK2), reduced glycolysis, and production of protein, nucleotide, and lipid. Similarly, we showed that CDK6 knockout inhibited the survival of CDK6-high CaSki but not CDK6-low SiHa cervical cancer cells by regulation of glycolysis and autophagy process. Collectively, our studies indicate that CDK6 is a critical regulator of human cervical cancer cells, especially with high CDK6 level, through its ability to regulate cellular apoptosis and metabolism. Thus, inhibition of CDK6 kinase activity could be a powerful therapeutic avenue used to treat cervical cancers.

Proliferation of hippocampal progenitors relies on p27-dependent regulation of Cdk6 kinase activity.

Neural stem cells give rise to granule dentate neurons throughout life in the hippocampus. Upon activation, these stem cells generate fast proliferating progenitors that complete several rounds of divisions before differentiating into neurons. Although the mechanisms regulating the activation of stem cells have been intensively studied, little attention has been given so far to the intrinsic machinery allowing the expansion of the progenitor pool. The cell cycle protein Cdk6 positively regulates the proliferation of hippocampal progenitors, but the mechanism involved remains elusive. Whereas Cdk6 functions primarily as a cell cycle kinase, it can also act as transcriptional regulator in cancer cells and hematopoietic stem cells. Using mouse genetics, we show here that the function of Cdk6 in hippocampal neurogenesis relies specifically on its kinase activity. The present study also reveals a specific regulatory mechanism for Cdk6 in hippocampal progenitors. In contrast to the classical model of the cell cycle, we observe that the Cip/Kip family member p27, rather than the Ink4 family, negatively regulates Cdk6 in the adult hippocampus. Altogether, our data uncover a unique, cell type-specific regulatory mechanism controlling the expansion of hippocampal progenitors, where Cdk6 kinase activity is modulated by p27.

CDK6 inhibits white to beige fat transition by suppressing RUNX1.

Whereas white adipose tissue depots contribute to the development of metabolic diseases, brown and beige adipose tissue has beneficial metabolic effects. Here we show that CDK6 regulates beige adipocyte formation. We demonstrate that mice lacking the CDK6 protein or its kinase domain (K43M) exhibit significant increases beige cell formation, enhanced energy expenditure, better glucose tolerance, and improved insulin sensitivity, and are more resistant to high-fat diet-induced obesity. Re-expression of CDK6 in Cdk6 -/- mature or precursor cells, or ablation of RUNX1 in K43M mature or precursor cells, reverses these phenotypes. Furthermore, RUNX1 positively regulates the expression of Ucp-1 and Pgc1α by binding to proximal promoter regions. Our findings indicate that CDK6 kinase activity negatively regulates the conversion of fat-storing cells into fat-burning cells by suppressing RUNX1, and suggest that CDK6 may be a therapeutic target for the treatment of obesity and related metabolic diseases.

Plexin-B2 Mediates Physiologic and Pathologic Functions of Angiogenin.

Angiogenin (ANG) is a secreted ribonuclease (RNase) with cell-type- and context-specific roles in growth, survival, and regeneration. Although these functions require receptor-mediated endocytosis and appropriate subcellular localization, the identity of the cell surface receptor remains undefined. Here, we show that plexin-B2 (PLXNB2) is the functional receptor for ANG in endothelial, cancer, neuronal, and normal hematopoietic and leukemic stem and progenitor cells. Mechanistically, PLXNB2 mediates intracellular RNA processing that contribute to cell growth, survival, and regenerative capabilities of ANG. Antibodies generated against the ANG-binding site on PLXNB2 restricts ANG activity in vitro and in vivo, resulting in inhibition of established xenograft tumors, ANG-induced neurogenesis and neuroprotection, levels of pro-self-renewal transcripts in hematopoietic and patient-derived leukemic stem and progenitor cells, and reduced progression of leukemia in vivo. PLXNB2 is therefore required for the physiological and pathological functions of ANG and has significant therapeutic potential in solid and hematopoietic cancers and neurodegenerative diseases.

Angiogenin Promotes Hematopoietic Regeneration by Dichotomously Regulating Quiescence of Stem and Progenitor Cells.

Regulation of stem and progenitor cell populations is critical in the development, maintenance, and regeneration of tissues. Here, we define a novel mechanism by which a niche-secreted RNase, angiogenin (ANG), distinctively alters the functional characteristics of primitive hematopoietic stem/progenitor cells (HSPCs) compared with lineage-committed myeloid-restricted progenitor (MyePro) cells. Specifically, ANG reduces the proliferative capacity of HSPC while simultaneously increasing proliferation of MyePro cells. Mechanistically, ANG induces cell-type-specific RNA-processing events: tRNA-derived stress-induced small RNA (tiRNA) generation in HSPCs and rRNA induction in MyePro cells, leading to respective reduction and increase in protein synthesis. Recombinant ANG protein improves survival of irradiated animals and enhances hematopoietic regeneration of mouse and human HSPCs in transplantation. Thus, ANG plays a non-cell-autonomous role in regulation of hematopoiesis by simultaneously preserving HSPC stemness and promoting MyePro proliferation. These cell-type-specific functions of ANG suggest considerable therapeutic potential.

Angiogenin mediates androgen-stimulated prostate cancer growth and enables castration resistance.

UNLABELLED: The androgen receptor (AR) is a critical effector of prostate cancer development and progression. Androgen-dependent prostate cancer is reliant on the function of AR for growth and progression. Most castration-resistant prostate cancer (CRPC) remains dependent on AR signaling for survival and growth. Ribosomal RNA (rRNA) is essential for both androgen-dependent and castration-resistant growth of prostate cancer cells. During androgen-dependent growth of prostate cells, androgen-AR signaling leads to the accumulation of rRNA. However, the mechanism by which AR regulates rRNA transcription is unknown. Here, investigation revealed that angiogenin (ANG), a member of the secreted ribonuclease superfamily, is upregulated in prostate cancer and mediates androgen-stimulated rRNA transcription in prostate cancer cells. Upon androgen stimulation, ANG undergoes nuclear translocation in androgen-dependent prostate cancer cells, where it binds to the rDNA promoter and stimulates rRNA transcription. ANG antagonists inhibit androgen-induced rRNA transcription and cell proliferation in androgen-dependent prostate cancer cells. Interestingly, ANG also mediates androgen-independent rRNA transcription through a mechanism that involves its constitutive nuclear translocation in androgen-insensitive prostate cancer cells, resulting in a constant rRNA overproduction and thereby stimulating cell proliferation. Critically, ANG overexpression in androgen-dependent prostate cancer cells enables castration-resistant growth of otherwise androgen-dependent cells. Thus, ANG-stimulated rRNA transcription is not only an essential component for androgen-dependent growth of prostate cancer but also contributes to the transition of prostate cancer from androgen-dependent to castration-resistant growth status. IMPLICATIONS: The ability of angiogenin to regulate rRNA transcription and prostate cancer growth makes it a viable target for therapy.

Loss of ARF sensitizes transgenic BRAFV600E mice to UV-induced melanoma via suppression of XPC.

Both genetic mutations and UV irradiation (UVR) can predispose individuals to melanoma. Although BRAF(V600E) is the most prevalent oncogene in melanoma, the BRAF(V600E) mutant is not sufficient to induce tumors in vivo. Mutation at the CDKN2A locus is another melanoma-predisposing event that can disrupt the function of both p16(INK4a) and ARF. Numerous studies have focused on the role of p16(INK4a) in melanoma, but the involvement of ARF, a well-known p53 activator, is still controversial. Using a transgenic BRAF(V600E) mouse model previously generated in our laboratory, we report that loss of ARF is able to enhance spontaneous melanoma formation and cause profound sensitivity to neonatal UVB exposure. Mechanistically, BRAF(V600E) and ARF deletion synergize to inhibit nucleotide excision repair by epigenetically repressing XPC and inhibiting the E2F4/DP1 complex. We suggest that the deletion of ARF promotes melanomagenesis not by abrogating p53 activation but by acting in concert with BRAF(V600E) to increase the load of DNA damage caused by UVR.

Ribonuclease 4 protects neuron degeneration by promoting angiogenesis, neurogenesis, and neuronal survival under stress.

Altered RNA processing is an underlying mechanism of amyotrophic lateral sclerosis (ALS). Missense mutations in a number of genes involved in RNA function and metabolisms are associated with ALS. Among these genes is angiogenin (ANG), the fifth member of the vertebrate-specific, secreted ribonuclease superfamily. ANG is an angiogenic ribonuclease, and both its angiogenic and ribonucleolytic activities are important for motor neuron health. Ribonuclease 4 (RNASE4), the fourth member of this superfamily, shares the same promoters with ANG and is co-expressed with ANG. However, the biological role of RNASE4 is unknown. To determine whether RNASE4 is involved in ALS pathogenesis, we sequenced the coding region of RNASE4 in ALS and control subjects and characterized the angiogenic, neurogenic, and neuroprotective activities of RNASE4 protein. We identified an allelic association of SNP rs3748338 with ALS and demonstrated that RNASE4 protein is able to induce angiogenesis in in vitro, ex vivo, and in vivo assays. RNASE4 also induces neural differentiation of P19 mouse embryonal carcinoma cells and mouse embryonic stem cells. Moreover, RNASE4 not only stimulates the formation of neurofilaments from mouse embryonic cortical neurons, but also protects hypothermia-induced degeneration. Importantly, systemic treatment with RNASE4 protein slowed weight loss and enhanced neuromuscular function of SOD1 (G93A) mice.

Cyclin D1 activity regulates autophagy and senescence in the mammary epithelium.

Overexpression of cyclin D1 is believed to endow mammary epithelial cells (MEC) with a proliferative advantage by virtue of its contribution to pRB inactivation. Accordingly, abrogation of the kinase-dependent function of cyclin D1 is sufficient to render mice resistant to breast cancer initiated by ErbB2. Here, we report that mouse cyclin D1(KE/KE) MECs (deficient in cyclin D1 activity) upregulate an autophagy-like process but fail to implement ErbB2-induced senescence in vivo. In addition, immortalized cyclin D1(KE/KE) MECs retain high rates of autophagy and reduced ErbB2-mediated transformation in vitro. However, highlighting its dual role during tumorigenesis, downregulation of autophagy led to an increase in senescence in cyclin D1(KE/KE) MECs. Autophagy upregulation was also confirmed in human mammary epithelial cells (HMEC) subjected to genetic and pharmacologic inhibition of cyclin D1 activity and, similar to our murine system, simultaneous inhibition of Cdk4/6 and autophagy in HMECs enhanced the senescence response. Collectively, our findings suggest a previously unrecognized function of cyclin D1 in suppressing autophagy in the mammary epithelium.

CDK6 kinase activity is required for thymocyte development.

Cyclin-dependent kinase-6 (CDK6) is required for early thymocyte development and tumorigenesis. To mechanistically dissect the role of CDK6 in thymocyte development, we generated and analyzed mutant knock-in mice and found that mice expressing a kinase-dead Cdk6 allele (Cdk6(K43M)) had a pronounced reduction in thymocytes and hematopoietic stem cells and progenitor cells (Lin⁻Sca-1⁺c-Kit⁺ [LSK]). In contrast, mice expressing the INK4-insensitive, hyperactive Cdk6(R31C) allele displayed excess proliferation in LSK and thymocytes. However, this is countered at least in part by increased apoptosis, which may limit progenitor and thymocyte expansion in the absence of other genetic events. Our mechanistic studies demonstrate that CDK6 kinase activity contributes to Notch signaling because inactive CDK6 kinase disrupts Notch-dependent survival, proliferation, and differentiation of LSK, with concomitant alteration of Notch target gene expression, such as massive up-regulation of CD25. Further, knockout of CD25 in Cdk6(K43M) mice rescued most defects observed in young mice. These results illustrate an important role for CDK6 kinase activity in thymocyte development that operates partially through modulating Notch target gene expression. This role of CDK6 as a downstream mediator of Notch identifies CDK6 kinase activity as a potential therapeutic target in human lymphoid malignancies.

Cyclin D1 kinase activity is required for the self-renewal of mammary stem and progenitor cells that are targets of MMTV-ErbB2 tumorigenesis.

Transplantation studies have demonstrated the existence of mammary progenitor cells with the ability to self-renew and regenerate a functional mammary gland. Although these progenitors are the likely targets for oncogenic transformation, correlating progenitor populations with certain oncogenic stimuli has been difficult. Cyclin D1 is required for lobuloalveolar development during pregnancy and lactation as well as MMTV-ErbB2- but not MMTV-Wnt1-mediated tumorigenesis. Using a kinase-deficient cyclin D1 mouse, we identified two functional mammary progenitor cell populations, one of which is the target of MMTV-ErbB2. Moreover, cyclin D1 activity is required for the self-renewal and differentiation of mammary progenitors because its abrogation leads to a failure to maintain the mammary epithelial regenerative potential and also results in defects in luminal lineage differentiation.

Neamine inhibits prostate cancer growth by suppressing angiogenin-mediated rRNA transcription.

PURPOSE: Angiogenin (ANG) undergoes nuclear translocation and stimulates rRNA transcription in both prostate cancer cells and endothelial cells. The purpose of this study is to assess the antitumor activity of neamine, a nontoxic degradation product of neomycin that blocks nuclear translocation of ANG. EXPERIMENTAL DESIGN: The anti-prostate cancer activity of neamine was first evaluated in a xenograft animal model. It was then examined in the murine prostate-restricted AKT transgenic mice that develop prostate intraepithelial neoplasia (PIN) owing to AKT transgene overexpression. RESULTS: Neamine inhibits xenograft growth of PC-3 human prostate cancer cells in athymic mice. It blocks nuclear translocation of ANG and inhibits rRNA transcription, cell proliferation, and angiogenesis. Neamine also prevents AKT-induced PIN formation as well as reverses fully developed PIN in murine prostate-restricted AKT mice, accompanied by a decrease in rRNA synthesis, cell proliferation, and angiogenesis and an increase in prostate epithelial cell apoptosis. CONCLUSION: We confirmed that ANG is a molecular target for cancer drug development and that blocking nuclear translocation of ANG is an effective means to inhibit its activity. Our results also suggested that neamine is a lead compound for further preclinical evaluation.

A requirement for cyclin-dependent kinase 6 in thymocyte development and tumorigenesis.

Cyclin-dependent kinase 6 (CDK6) promotes cell cycle progression and is overexpressed in human lymphoid malignancies. To determine the role of CDK6 in development and tumorigenesis, we generated and analyzed knockout mice. Cdk6-deficient mice show pronounced thymic atrophy due to reduced proliferative fractions and concomitant transitional blocks in the double-negative stages. Using the OP9-DL1 system to deliver temporally controlled Notch receptor-dependent signaling, we show that CDK6 is required for Notch-dependent survival, proliferation, and differentiation. Furthermore, CDK6-deficient mice were resistant to lymphomagenesis induced by active Akt, a downstream target of Notch signaling. These results show a critical requirement for CDK6 in Notch/Akt-dependent T-cell development and tumorigenesis and strongly support CDK6 as a specific therapeutic target in human lymphoid malignancies.

Epithelial-mesenchymal transition induced by growth suppressor p12CDK2-AP1 promotes tumor cell local invasion but suppresses distant colony growth.

Epithelial-mesenchymal transition (EMT) has been considered essential for metastasis, a multistep process including local invasion, intravasation, extravasation, and proliferation at distant sites. However, controversy remains as to whether EMT truly happens and how important it is to metastasis. We studied the involvement of EMT in individual steps of metastasis and found that p12(CDK2-AP1), a down-stream effector of transforming growth factor beta, induced EMT of hamster cheek pouch carcinoma-1 cells by promoting the expression of Twist2. EMT cells have an increased invasive but decreased metastatic phenotype. When s.c. inoculated, both EMT and non-EMT cells established primary tumors, but only EMT cells invaded into the adjacent tissues and blood vessels; however, neither cells formed lung metastases. When i.v. inoculated, only non-EMT cells established lung metastases. Moreover, s.c. inoculation of a mixture of the two cell types resulted in intravasation of both cell types and formation of lung metastasis from non-EMT cells. Our results allowed us to propose a novel model for the role of EMT in cancer metastasis. We showed that EMT and non-EMT cells cooperate to complete the spontaneous metastasis process. We thus hypothesize that EMT cells are responsible for degrading the surrounding matrix to lead the way of invasion and intravasation. Non-EMT cells then enter the blood stream and reestablish colonies in the secondary sites.

Role of p12(CDK2-AP1) in transforming growth factor-beta1-mediated growth suppression.

p12(CDK2-AP1) (p12) is a growth suppressor isolated from normal keratinocytes. Ectopic expression of p12 in squamous carcinoma cells reversed the malignant phenotype of these cells, in part due an ability of p12 to bind to both DNA polymerase alpha/primase and to cyclin-dependent kinase 2 (CDK2), thereby inhibiting their activities. We report in this article that in normal epithelial cells, transforming growth factor beta1 (TGF-beta1) induces p12 expression transcriptionally, which, in turn, mediates the growth inhibitory activity of TGF-beta1. We created inducible p12 antisense HaCaT cell lines [ip12 (-) HaCaT] and showed that selective reduction of cellular p12 resulted in an increase in: (a) CDK2-associated kinase activity; (b) protein retinoblastoma (pRB) phosphorylation; and (c) [(3)H]thymidine incorporation, and partially reversed TGF-beta1-mediated inhibition of CDK2 kinase activity, pRB phosphorylation, and cell proliferation. Furthermore, we generated p12-deficient mouse oral keratinocytes (MOK(p12-/-)) and compared their growth characteristics and response to TGF-beta1 with that of wild-type mouse oral keratinocytes (MOK(WT)). Under normal culture conditions, the number of MOK(p12-/-) in S phase is 2-fold greater than that of MOK(WT). Concomitantly, fewer cells are in G(2) phase in MOK(p12-/-) than that in MOK(WT). Moreover, response to TGF-beta1-mediated growth suppression is compromised in MOK(p12-/-) cells. Mechanistic studies showed that MOK(p12-/-) have increased CDK2 activity and reduced sensitivity to inhibition by TGF-beta1. Collectively our data suggest that p12 plays a role in TGF-beta1-mediated growth suppression by modulating CDK2 activities and pRB phosphorylation.