D-type Cyclins Research Articles

Genomic Aberrations that Activate D-type Cyclins Are Associated with Enhanced Sensitivity to the CDK4 and CDK6 Inhibitor Abemaciclib

Most cancers preserve functional retinoblastoma (Rb) and may, therefore, respond to inhibition of D-cyclin-dependent Rb kinases, CDK4 and CDK6. To date, CDK4/6 inhibitors have shown promising clinical activity in breast cancer and lymphomas, but it is not clear which additional Rb-positive cancers might benefit from these agents. No systematic survey to compare relative sensitivities across tumor types and define molecular determinants of response has been described. We report a subset of cancers highly sensitive to CDK4/6 inhibition and characterized by various genomic aberrations known to elevate D-cyclin levels and describe a recurrent CCND1 3'UTR mutation associated with increased expression in endometrial cancer. The resultssuggest multiple additional classes of cancer that may benefit from CDK4/6-inhibiting drugs such as abemaciclib.

MOS1 functions closely with TCP transcription factors to modulate immunity and cell cycle in Arabidopsis.

Emerging evidence indicates a close connection between cell-cycle progression and the plant immune responses. In Arabidopsis, MODIFIER OF snc1-1 (MOS1) modulates a number of processes including endoreduplication and plant disease resistance, but the molecular mechanism underlying this modulation was not fully understood. Here, we provide biochemical and genetic evidence that TEOSINTE BRANCHED 1, CYCLOIDEA, PCF1 (TCP) transcription factors TCP15 and its homologues are mediators of MOS1 function in the immune response and are likely to be also involved in cell-cycle control. MOS1 and TCP proteins have a direct physical interaction. They both bind to the promoter of the immune receptor gene SUPRESSOR OF npr1-1, CONSTITUTIVE 1 (SNC1) and modulate its expression and consequently immune responses. MOS1 and TCP15 both affect the expression of cell-cycle genes D-type CYCLIN 3;1 (CYCD3;1), which may mediate the MOS1 function in cell-cycle modulation. In addition, CYCD3;1 overexpression upregulates immune responses, and SNC1 expression. This study investigated and revealed a role for MOS1 in transcriptional regulation through TCP15 and its homologues. This finding suggests the coordination of cell-cycle progression and plant immune responses at multiple levels.

Differential Regulation of G1 CDK Complexes by the Hsp90-Cdc37 Chaperone System

SummarySelective recruitment of protein kinases to the Hsp90 system is mediated by the adaptor co-chaperone Cdc37. We show that assembly of CDK4 and CDK6 into protein complexes is differentially regulated by the Cdc37-Hsp90 system. Like other Hsp90 kinase clients, binding of CDK4/6 to Cdc37 is blocked by ATP-competitive inhibitors. Cdc37-Hsp90 relinquishes CDK6 to D3- and virus-type cyclins and to INK family CDK inhibitors, whereas CDK4 is relinquished to INKs but less readily to cyclins. p21CIP1 and p27KIP1 CDK inhibitors are less potent than the INKs at displacing CDK4 and CDK6 from Cdc37. However, they cooperate with the D-type cyclins to generate CDK4/6-containing ternary complexes that are resistant to cyclin D displacement by Cdc37, suggesting a molecular mechanism to explain the assembly factor activity ascribed to CIP/KIP family members. Overall, our data reveal multiple mechanisms whereby the Hsp90 system may control formation of CDK4- and CDK6-cyclin complexes under different cellular conditions.

The WUSCHEL-related homeobox 5a (PtoWOX5a) is involved in adventitious root development in poplar.

Adventitious rooting is an essential step in vegetative propagation. Currently, the mechanism that regulates adventitious root (AR) development in woody plants is poorly understood. This work demonstrates that Populus tomentosa WUSCHEL-related homeobox 5a (PtoWOX5a) transcription factor is involved in AR development in poplar. PtoWOX5a was specifically expressed in the AR tip and lateral root tip during AR and lateral root regeneration from the stem segment. Phenotypic complementation experiments indicated that the PtoWOX5a can functionally complement AtWOX5 in quiescent center (QC) cells. Overexpression of PtoWOX5a introduces significant developmental phenotypes in roots and leaves, such as increased AR number, decreased AR length, swollen AR tip and lateral root tip, and decreased leaf number and area. The conserved mechanism of D-type cyclins (CYCD) repression mediated by WOX5 was confirmed in poplar. The co-expression network of PtWOX5a was constructed, which provided clues to reveal the molecular mechanism of PtoWOX5a in AR development in poplar. Taken together, our results suggest that the PtoWOX5a is involved in AR development though cooperating with a series of functional genes.

Neuromedin B and its receptor silencing suppresses osteoclast generation by modulating precursor proliferation via M-CSF/c-Fms/D-type cyclins

Neuromedin B (NMB), a mammalian bombesin-like peptide, regulates diverse physiological processes, such as energy metabolism, memory and fear behavior, and cellular growth, through its cognate receptor, NMBR. In this study, we report that NMB expression was upregulated during osteoclast development and that silencing NMB or NMBR attenuated osteoclast generation mediated by macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). We found that knockdown of NMB or NMBR using a small hairpin RNA suppressed M-CSF-induced proliferation of osteoclast precursor cells without altering osteoclast differentiation. Interestingly, NMB or NMBR knockdown reduced the expression of the M-CSF receptor, c-Fms, which is an important modulator of osteoclast development. Consequently, NMB or NMBR silencing inhibited M-CSF/c-Fms-mediated downstream signaling pathways like activation of ERK and Akt and induction of D-type cyclins, cyclin D1 and D2. Moreover, knockdown of NMB or NMBR accelerated apoptosis in osteoclast lineage cells by inducing caspase-3, caspase-9, and Bim expression. In summary, our study demonstrates that the NMB/NMBR axis plays a pivotal role in osteoclast generation by modulating the proliferation and survival of osteoclast lineage cells.

Multitype Network-Guided Target Controllability in Phenotypically Characterized Osteosarcoma: Role of Tumor Microenvironment.

This study highlights the relevance of network-guided controllability analysis as a precision oncology tool. Target controllability through networks is potentially relevant to cancer research for the identification of therapeutic targets. With reference to a recent study on multiple phenotypes from 22 osteosarcoma (OS) cell lines characterized both in vitro and in vivo, we found that a variety of critical proteins in OS regulation circuits were in part phenotype specific and in part shared. To generalize our inference approach and match cancer phenotypic heterogeneity, we employed multitype networks and identified targets in correspondence with protein sub-complexes. Therefore, we established the relevance for diagnostic and therapeutic purposes of inspecting interactive targets, namely those enriched by significant connectivity patterns in protein sub-complexes. Emerging targets appeared with reference to the OS microenvironment, and relatively to small leucine-rich proteoglycan members and D-type cyclins, among other collagen, laminin, and keratin proteins. These described were evidences shared across all phenotypes; instead, specific evidences were provided by critical proteins including IGFBP7 and PDGFRA in the invasive phenotype, and FGFR3 and THBS1 in the colony forming phenotype.

Cyclin D3 deficiency inhibits skin tumor development, but does not affect normal keratinocyte proliferation.

Rearrangement and amplification of the D-type cyclin genes have been reported in human cancer. Previous studies have demonstrated that Ras-mediated skin tumorigenesis depends on pathways that act through cyclin D1 and D2; however, the role of cyclin D3 remains unknown. The present study demonstrates that cyclin D3 ablation does not affect keratinocyte proliferation, but instead increases apoptosis levels in the bulge region of the hair follicle. Consequently, cyclin D3 ablation reduces skin papilloma development in a Ras-dependent carcinogenesis model. Previous results revealed that cyclin D3 preferentially binds to cyclin-dependent kinase 6 (CDK6) in mouse keratinocytes and transgenic expression of CDK6 (K5CDK6 mice) inhibits skin tumor development. Thus, we hypothesized that the inhibitory effect of CDK6 is dependent on cyclin D3 expression. To test this hypothesis, a mouse model that overexpresses CDK6 and does not express cyclin D3 (K5CDK6/cyclin D3-/− compound mouse) was developed. Biochemical analysis of the epidermis of K5CDK6/cyclin D3-/− mice revealed that cyclin D3 ablation resulted in increased expression of cyclin D1 protein, with a consequent elevation in the level of CDK6/cyclin D1 and CDK4/cyclin D1 complexes. These findings were concurrent with the increase skin papilloma malignant progression observed in K5CDK6/cyclin D3-/− mice. In summary the absence of cyclin D3 led to fewer number of papillomas in cyclin D3-ablated mice than in the wild-type owing to increased apoptosis, suggesting that alterations in cell survival are a crucial mechanism for crippling cellular defense against neoplasia. The results of the current study also suggest that although cyclin D3 expression does not alter the tumor suppressive role of CDK6 in skin carcinogenesis, the compensatory increase in cyclin D1 can shift the balance towards malignant progression.

Abstract 310: Enhanced stability of D-type cyclins correlates with glioblastoma resistance to ionizing radiation

Abstract Transition from G1 to S phase of the cell cycle is promoted by D-type cyclins (D1, D2 and D3), regulatory subunits critical for the activation of cyclin dependent kinases 4 and 6 (CDK4/6), which phosphorylate and inactivate the retinoblastoma protein (pRb) and activate the transcription factor E2F1 to promote the expression of genes essential for DNA synthesis. Deregulated expression of D-type cyclins is frequently observed in human malignancies and correlates with increased proliferation. Cyclin Ds overexpression is also associated with radioresistance, leading to radiotherapy failure and disease recurrence. Cyclin D1 undergoes ubiquitin-dependent degradation following the exposure of cancer cells to ionizing radiation (IR), and this has been shown to be critical for IR-induced G1 growth arrest and sensitivity of cancer cells to IR. While the role of cyclin D1 overexpression in drug resistance is already well documented, less is known about the role of cyclins D2 and D3 in radio-resistance in human cancer cells, particularly in brain neoplasms. In the present study, we investigated the effect of IR on different D-type cyclins (D1, D2, D3) in human glioblastoma cell lines as well as in tumor-derived mouse oligodendrocytes progenitor cells (OPCs). Here, we show that exposure of human astrocytes to increasing levels of IR led to a rapid proteasomal degradation of all three D-type cyclins. On the other hand, IR failed to induce significant cyclin Ds degradation in a number of human glioblastoma cells (U87, A172, U251, Snb19). Analysis of cell cycle profile of all glioblastoma cells tested and exposed to IR showed prominent cell cycle arrest in G1 starting at 24 hours following exposure. Furthermore, we show that the steady-state levels of D-type cyclin is higher in OPCs compared to mouse NIH3T3 cells and fail to undergo proteasomal degradation following IR exposure. Collectively, these results suggest that the degradation of D-type cyclins is not critical for IR-induced G1 cell cycle arrest in GMB and may underlie their resistance to IR. Understanding the role of cyclin Ds in promoting radioresistance may lead to a useful pharmacological strategy to enhance radiotherapy outcome and help implement a more effective treatment modality. Citation Format: Fadila Guessous, Mouadh Benamar, Hui Zhong, Tarek Abbas. Enhanced stability of D-type cyclins correlates with glioblastoma resistance to ionizing radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 310. doi:10.1158/1538-7445.AM2017-310

Abstract 2346: Preclinical selectivity profile of the CDK4/6 inhibitor ribociclib (LEE011) compared with that of palbociclib and abemaciclib

Abstract A hallmark of cancer is unchecked cell division. Retinoblastoma protein (Rb) is a human tumor suppressor that guards a cell’s entry into S phase by binding E2F transcription factors and keeping them inactive. Many growth-promoting stimuli increase expression of D-type cyclins, which bind to and activate cyclin-dependent kinases 4 and 6 (CDK4/6). The cyclin D–bound CDK4/6 holoenzymes phosphorylate Rb, resulting in release of E2F, which in turn activates genes required for S phase entry and DNA replication. Numerous oncogenic aberrations converge at the CDK4/6–Rb pathway, providing a strong rationale for developing CDK4/6 inhibitors as cancer therapeutics. Ribociclib (LEE011) is a selective CDK4/6 inhibitor that has received FDA breakthrough therapy and priority review designations for treatment of hormone receptor–positive breast cancer in combination with letrozole and is being tested in additional clinical trials. Here we describe the preclinical selectivity profile of ribociclib in biochemical and cellular assays. Ribociclib inhibits both CDK4–cyclin D1 and CDK6–cyclin D3 kinase activity with nanomolar IC50s in biochemical assays. To comprehensively address the selectivity of ribociclib in direct comparison with 2 other clinical CDK4/6 inhibitors, palbociclib and abemaciclib, we made use of the KINOMEscan assay consisting of >450 kinase active site–directed competition-binding assays. We adjusted the test concentrations in the kinase-selectivity panel per the binding constants for CDK4 and CDK6 to account for the higher potency of abemaciclib. Data showed that both ribociclib and palbociclib have high selectivity for CDK4 (CDK6 was not covered in the panel), with very few distinct additional binding events detected. In contrast, abemaciclib is a much more promiscuous kinase inhibitor. Next, we sought to determine the relative potencies of the 3 inhibitors against CDK4 vs CDK6 in cellular assays. When testing different routinely used readouts of cellular viability, we found that assays that measured metabolic activity (eg, CTG) tended to underestimate the effects of CDK4/6 inhibition; thus, assays that either directly or indirectly assessed cell number were used instead. We first identified cancer cell lines primarily dependent on either CDK4 or CDK6 as judged by combined RNA expression analysis and shRNA or CRISPR-based functional assays. When determining IC50s of the 3 CDK4/6 inhibitors in these cell lines, we found that ribociclib and abemaciclib demonstrated greater activity in CDK4-dependent cells vs CDK6-dependent cells, whereas palbociclib was similarly active in both cell types. The high degree of CDK4 selectivity of ribociclib suggests that off-target kinase inhibition is an unlikely complication in patients. Moreover, the apparent preference for CDK4 over CDK6 could be an advantage in certain cancer types that are primarily dependent on CDK4. Citation Format: Ralph Tiedt, Scott Delach, Steven Kovats, Thomas Horn, Michael Acker, Barbara Schacher Engstler, Giordano Caponigro, Fei Su. Preclinical selectivity profile of the CDK4/6 inhibitor ribociclib (LEE011) compared with that of palbociclib and abemaciclib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2346. doi:10.1158/1538-7445.AM2017-2346

The metabolic function of cyclin D3-CDK6 kinase in cancer cell survival.

D-type cyclins (D1, D2 and D3) and their associated cyclin-dependent kinases (CDK4 and CDK6) are components of the core cell cycle machinery that drives cell proliferation. Inhibitors of CDK4 and CDK6 are currently being tested in clinical trials for patients with several cancer types, with promising results. Here, using human cancer cells and patient-derived xenografts in mice, we show that the cyclin D3-CDK6 kinase phosphorylates and inhibits the catalytic activity of two key enzymes in the glycolytic pathway, 6-phosphofructokinase and pyruvate kinase M2. This re-directs the glycolytic intermediates into the pentose phosphate (PPP) and serine pathways. Inhibition of cyclin D3-CDK6 in tumour cells reduces flow through the PPP and serine pathways, thereby depleting the antioxidants NADPH and glutathione. This, in turn, increases the levels of reactive oxygen species and causes apoptosis of tumour cells. The pro-survival function of cyclin D-associated kinase operates in tumours expressing high levels of cyclin D3-CDK6 complexes. We propose that measuring the levels of cyclin D3-CDK6 in human cancers might help to identify tumour subsets that undergo cell death and tumour regression upon inhibition of CDK4 and CDK6. Cyclin D3-CDK6, through its ability to link cell cycle and cell metabolism, represents a particularly powerful oncoprotein that affects cancer cells at several levels, and this property can be exploited for anti-cancer therapy.

Molecular genetics of aggressive B-cell lymphoma.

Molecular genetics of aggressive B-cell lymphoma.

Assessment of endocrine molecular entities targeted by inflammation that are involved in the onset of autoimmune diabetes.

Abstract Type 1 diabetes (T1D) is an autoimmune disease caused by pancreatic beta cells demise due to the attack of self-lymphocyte repertoire. We aim to identify molecular entities targeted by the autoimmune assault to pancreatic beta cells that are causally related to T1D progression. The proinflammatory biological niche in which beta cells are immersed during the autoimmune insult promotes deep phenotypical changes crucial for the pathogenic process. These changes lead mainly to beta cell fitness impairment, cell cycle intervention and apoptosis triggering. By using the Microarray technology we identified a series of genes the expression of which is altered in the islet endocrine cells prior to diabetes onset in the NOD (Non Obese Diabetic) mice. One of those genes encodes for cyclin D3 that triggers cell cycle progression through G1 phase towards the S phase. Cyclin D3 can also bind certain transcription factors and activate inflammation process and development of the T cells (NFκB, GATA). The cyclin D3 promoter has binding sequences to NFκB a transcription factor linked to the action of T1D-related proinflammatory cytokines such as IL-1beta and TNFalpha. We found that cyclin D3 is the only D-type cyclin the expression of which is regulated by inflammation in NOD endocrine islet cells. Moreover we found that cyclin D3 protects beta cells against cytokine-induced apoptosis and is required for proper beta cell function. Moreoever, we observed that CDK11, another cell-cycle related gene that interacts with cyclin D3, is also regulated by inflammation in endocrine islet cells. We have assessed whether there is a causal relationship between the coordinated differential expression of both genes during the insulitic assault and diabetes onset

Identification and functional analysis of the ICK gene family in maize

Inhibitors of cyclin-dependent kinases (ICKs) are key regulators of cyclin-dependent kinase activities and cell division. Herein, we identified eight ICKs in maize, which we named Zeama;ICKs (ZmICKs). Primary sequencing and phylogenetic analyses were used to divide the ZmICK family into two classes: group B and group C. Subcellular localization analysis of ZmICK:enhanced green fluorescent protein (eGFP) fusion constructs in tobacco leaf cells indicated that ZmICKs are principally nuclear. Co-localization analysis of the ZmICKs and maize A-type cyclin-dependent kinase (ZmCDKA) was also performed using enhanced green fluorescent protein (eGFP) and red fluorescent protein (RFP) fusion constructs. The ZmICKs and ZmCDKA co-localized in the nucleus. Semi-quantitative RT-PCR analysis of the ZmICKs showed that they were expressed at different levels in all tissues examined and shared similar expression patterns with cell cycle-related genes. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that ZmICK1, ZmICK2, ZmICK3, and ZmICK4 interact with ZmCDKA1 and ZmCDKA3. Interestingly, ZmICK7 interacts with D-type cyclins. Transformed and expressed ZmCDKA1 and ZmICKs together in fission yeast revealed that ZmICK1, ZmICK3, and ZmICK4 can affect ZmCDKA1 function. Moreover, the C-group of ZmICKs could interact with ZmCDKA1 directly and affect ZmCDKA1 function, suggesting that C-group ZmICKs are important for cell division regulation.

G1 cyclins link proliferation, pluripotency and differentiation of embryonic stem cells.

Progression of mammalian cells through the G1 and S phases of the cell cycle is driven by the D-type and E-type cyclins. According to the current models, at least one of these cyclin families must be present to allow cell proliferation. Here, we show that several cell types can proliferate in the absence of all G1 cyclins. However, following ablation of G1 cyclins, embryonic stem (ES) cells attenuated their pluripotent characteristics, with the majority of cells acquiring the trophectodermal cell fate. We established that G1 cyclins, together with their associated cyclin-dependent kinases (CDKs), phosphorylate and stabilize the core pluripotency factors Nanog, Sox2 and Oct4. Treatment of murine ES cells, patient-derived glioblastoma tumour-initiating cells, or triple-negative breast cancer cells with a CDK inhibitor strongly decreased Sox2 and Oct4 levels. Our findings suggest that CDK inhibition might represent an attractive therapeutic strategy by targeting glioblastoma tumour-initiating cells, which depend on Sox2 to maintain their tumorigenic potential.

CDK4/6 Inhibition in Breast Cancer: Mechanisms of Response and Treatment Failure.

To describe the role of D-type cyclins and CDKs 4 and 6 in breast cancer, and to discuss potential biomarkers for sensitivity or resistance to CDK4/6 inhibitors. A small number of preclinical and clinical studies have explored potential mechanisms of CDK4/6 inhibitor response and resistance in breast cancer. Putative markers of response include ER-positivity, luminal patterns of gene expression, high cyclin D1 levels, and low p16 levels. Possible resistance mechanisms include loss of Rb function, overexpression/amplification of cyclin E, and CDK6 amplification. Most these remain speculative and have not been validated in clinical specimens. If early successes with CDK4/6 inhibitors are to be capitalized upon, it is critical that our understanding of CDK4/6 biology in breast cancer extends beyond its current rudimentary state. Only then we will be able to develop rational therapeutic combinations that further enhance the efficacy of these agents.

Cyclin D mediates tolerance of genome-doubling in cancers with functional p53

BackgroundAneuploidy and chromosomal instability (CIN) are common features of human malignancy that fuel genetic heterogeneity. Although tolerance to tetraploidization, an intermediate state that further exacerbates CIN, is frequently mediated by TP53 dysfunction, we find that some genome-doubled tumours retain wild-type TP53. We sought to understand how tetraploid cells with a functional p53/p21-axis tolerate genome-doubling events.Methods We performed quantitative proteomics in a diploid/tetraploid pair within a system of multiple independently derived TP53 wild-type tetraploid clones arising spontaneously from a diploid progenitor. We characterized adapted and acute tetraploidization in a variety of flow cytometry and biochemical assays and tested our findings against human tumours through bioinformatics analysis of the TCGA dataset.ResultsCyclin D1 was found to be specifically overexpressed in early but not late passage tetraploid clones, and this overexpression was sufficient to promote tolerance to spontaneous and pharmacologically induced tetraploidy. We provide evidence that this role extends to D-type cyclins and their overexpression confers specific proliferative advantage to tetraploid cells. We demonstrate that tetraploid clones exhibit elevated levels of functional p53 and p21 but override the p53/p21 checkpoint by elevated expression of cyclin D1, via a stoichiometry-dependent and CDK activity-independent mechanism. Tetraploid cells do not exhibit increased sensitivity to abemaciclib, suggesting that cyclin D-overexpressing tumours might not be specifically amenable to treatment with CDK4/6 inhibitors.ConclusionsOur study suggests that D-type cyclin overexpression is an acute event, permissive for rapid adaptation to a genome-doubled state in TP53 wild-type tumours and that its overexpression is dispensable in later stages of tumour progression.

Opposing effects on the cell cycle of T lymphocytes by Fbxo7 via Cdk6 and p27

G1 phase cell cycle proteins, such as cyclin-dependent kinase 6 (Cdk6) and its activating partners, the D-type cyclins, are important regulators of T-cell development and function. An F-box protein, called F-box only protein 7 (Fbxo7), acts as a cell cycle regulator by enhancing cyclin D-Cdk6 complex formation and stabilising levels of p27, a cyclin-dependent kinase inhibitor. We generated a murine model of reduced Fbxo7 expression to test its physiological role in multiple tissues and found that these mice displayed a pronounced thymic hypoplasia. Further analysis revealed that Fbxo7 differentially affected proliferation and apoptosis of thymocytes at various stages of differentiation in the thymus and also mature T-cell function and proliferation in the periphery. Paradoxically, Fbxo7-deficient immature thymocytes failed to undergo expansion in the thymus due to a lack of Cdk6 activity, while mature T cells showed enhanced proliferative capacity upon T-cell receptor engagement due to reduced p27 levels. Our studies reveal differential cell cycle regulation by Fbxo7 at different stages in T-cell development.

Cyclin-Dependent Kinase 4/6 Inhibitor Abemaciclib Exerts Dose-Dependent Cytostatic and Cytocidal Effects on Multiple Myeloma Cells Via Autophagy

Background:Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of abnormal plasma cells in the bone marrow. D-type cyclins (CCNDs), an important family of cell cycle regulators, are thought to be implicated in multiple myeloma (MM) development because CCNDs are commonly expressed in myeloma cells. CCND is known to positively regulate the cell cycle from G1 to S-phase initiation by binding to cyclin-dependent kinase (CDK) 4/6, resulting in potentiation of myeloma cell growth. These findings suggest a possible role for CDK4/6-targeting therapy in MM, yet the details remain incompletely understood. In this regard, we investigated the biological activity of abemaciclib, a potent, highly selective CDK4/6 inhibitor, in myeloma cell lines, to elucidate the mechanisms underlying the involvement of the CCND-CDK4/6 complex in cell cycle regulation and survival.Methods:The effects of abemaciclib on myeloma cells were investigated using three myeloma cell lines, KMS12-PE (CCND1-positive and CCND2-negative), RPMI8226 (CCND1-negative and CCND2-positive), and IM-9 (both CCND1- and CCND2-positive). Cell growth was assessed by trypan blue exclusion assay. Cell cycle analysis was performed using propidium iodide (PI) and apoptosis was measured using annexin V/PI staining via flow cytometry. Cell cycle regulated proteins, including p21 and p27, and phosphorylated proteins, including STAT1, STAT3, ERK, JNK, p38, and AKT, were evaluated using a phospho-flow method. Autophagy was assessed using CYTO-ID via flow cytometry. PARP cleavage was investigated via western blotting. Clarithromycin, an antibiotic agent belonging to the macrolide class, was used as an autophagy inhibitor.Results:Abemaciclib inhibited myeloma cell growth in a dose-dependent manner in all the cell lines evaluated, with significant differences seen at a concentration of 320 nM. Annexin V/PI staining revealed that 1 μM abemaciclib showed little or no effect on apoptosis, but 3.2 μM abemaciclib induced apparent myeloma cell apoptosis, with an increase in both the early and late apoptotic fractions. Therefore, 1 and 3.2 μM of abemaciclib were used in subsequent experiments for the assessment of cell growth and apoptosis, respectively. Cell cycle analyses revealed that 1 μM abemaciclib increased the fraction of cells in G0/G1 phase and decreased the fraction in S-G2/M phase. Furthermore, this effect was associated with the upregulation of p21 and p27 in the evaluated myeloma cells. PARP cleavage was observed in KMS12-PE cells treated with 3.2 μM abemaciclib, but not 1 μM, suggesting a close connection between the degree of PARP cleavage and apoptosis in myeloma cells. Importantly, abemaciclib induced autophagy in a dose-dependent manner. However, no apparent inhibitory effect on the autophagy-related phosphorylated proteins STAT1 (Y701), STAT3 (Y705), ERK (T202/Y204), JNK (T183/Y185), p38 (T180/Y182), or AKT (Y315) was observed in myeloma cells treated with 3.2 μM abemaciclib. To investigate the role of abemaciclib-induced autophagy on myeloma cell apoptosis, we further assessed the apoptotic effect of 3.2 μM abemaciclib or 50 μg/mL clarithromycin, alone or in combination. Clarithromycin did not induce apoptosis of myeloma cells. Importantly, clarithromycin treatment in combination with abemaciclib attenuated the apoptotic effect of abemaciclib.Discussion & Conclusions: Although the underlying mechanisms conferring the level of CCND expression are known to differ greatly (e.g., CCND translocation, hyperdiploidy, or activation of upstream pathways of CCND transcription), the results of the current study indicate that the CCND-CDK4/6 complex is closely involved in myeloma cell growth and survival regardless of the CCND family member present. In addition, we demonstrate that abemaciclib exerts multiple effects, such as myeloma cell apoptosis, via the PARP pathway or autophagy, as well as cell cycle regulation. Because abemaciclib in combination with clarithromycin inhibits myeloma cell apoptosis, the autophagy induced by abemaciclib is considered to have a critical role in the induction of apoptosis, so-called "autophagic cell death." These results provide novel insights into a possible therapeutic approach using abemaciclib to target CDK4/6 in patients with MM, and offer new possibilities for combination therapy with CDK4/6 inhibitors and autophagy regulators. DisclosuresIriyama:Novartis: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau. Hatta:Novartis Pharma: Honoraria.

An overview on the role of dietary phenolics for the treatment of cancers.

Plant derived phenolic compounds have been shown to inhibit the initiation and progression of cancers by modulating genes regulating key processes such as: (a) oncogenic transformation of normal cells; (b) growth and development of tumors; and (c) angiogenesis and metastasis. Recent studies focusing on identifying the molecular basis of plant phenolics-induced cancer cell death have demonstrated down-regulation of: (a) oncogenic survival kinases such as PI3K and Akt; (b) cell proliferation regulators that include Erk1/2, D-type Cyclins, and Cyclin Dependent Kinases (CDKs); (c) transcription factors such as NF-kβ, NRF2 and STATs; (d) histone deacetylases HDAC1 and HDAC2; and (e) angiogenic factors VEGF, FGFR1 and MIC-1. Furthermore, while inhibiting oncogenic proteins, the phenolic compounds elevate the expression of tumor suppressor proteins p53, PTEN, p21, and p27. In addition, plant phenolic compounds and the herbal extracts rich in phenolic compounds modulate the levels of reactive oxygen species (ROS) in cells thereby regulate cell proliferation, survival and apoptosis. Furthermore, recent studies have demonstrated that phenolic compounds undergo transformation in gut microbiota thereby acquire additional properties that promote their biological activities. In vitro observations, preclinical and epidemiological studies have shown the involvement of plant phenolic acids in retarding the cancer growth. However, to date, there is no clinical trial as such testing the role of plant phenolic compounds for inhibiting tumor growth in humans. More over, several variations in response to phenolic acid rich diets-mediated treatment among individuals have also been reported, raising concerns about whether phenolic acids could be used for treating cancers. Therefore, we have made an attempt to (a) address the key structural features of phenolic acids required for exhibiting potent anti-cancer activity; (b) review the reported findings about the mechanisms of action of phenolic compounds and their transformation by gut microbiota; and (c) update the toxicological aspects and anti-tumor properties of phenolic compounds and extracts containing phenolic compounds in animals.

Arabidopsis seed germination speed is controlled by SNL histone deacetylase-binding factor-mediated regulation of AUX1

Histone acetylation is known to affect the speed of seed germination, but the molecular regulatory basis of this remains ambiguous. Here we report that loss of function of two histone deacetylase-binding factors, SWI-INDEPENDENT3 (SIN3)-LIKE1 (SNL1) and SNL2, results in accelerated radicle protrusion and growth during seed germination. AUXIN RESISTANT 1 (AUX1) is identified as a key factor in this process, enhancing germination speed downstream of SNL1 and SNL2. AUX1 expression and histone H3 acetylation at lysines 9 and 18 is regulated by SNL1 and SNL2. The D-type cyclins encoding genes CYCD1;1 and CYCD4;1 display increased expression in AUX1 over-expression lines and the snl1snl2 double mutant. Accordingly, knockout of CYCD4;1 reduces seed germination speed of AUX1 over-expression lines and snl1snl2 suggesting the importance of cell cycling for radicle protrusion during seed germination. Together, our work identifies AUX1 as a link between histone acetylation mediated by SNL1 and SNL2, and radicle growth promoted by CYCD1;1 and CYCD4;1 during seed germination.