Mechanisms of sensitivity and resistance to CDK4/CDK6 inhibitors in hormone receptor-positive breast cancer treatment.

Cell cycle dysregulation is a hallmark of cancer that promotes eccessive cell division. Cyclin-dependent kinase 4 (CDK4) and cyclin-dependent kinase 6 (CDK6) are key molecules in the G1-to-S phase cell cycle transition and are crucial for the onset, survival, and progression of breast cancer (BC). Small-molecule CDK4/CDK6 inhibitors (CDK4/6i) block phosphorylation of tumor suppressor Rb and thus restrain susceptible BC cells in G1 phase. Three CDK4/6i are approved for the first-line treatment of patients with advanced/metastatic hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) BC in combination with endocrine therapy (ET). Though this has improved the clinical outcomes for survival of BC patients, there is no established standard next-line treatment to tackle drug resistance. Recent studies suggest that CDK4/6i can modulate other distinct effects in both BC and breast stromal compartments, which may provide new insights into aspects of their clinical activity. This review describes the biochemistry of the CDK4/6-Rb-E2F pathway in HR+ BC, then discusses how CDK4/6i can trigger other effects in BC/breast stromal compartments, and finally outlines the mechanisms of CDK4/6i resistance that have emerged in recent preclinical studies and clinical cohorts, emphasizing the impact of these findings on novel therapeutic opportunities in BC.

Dun1, a Chk2-related kinase, is the central regulator of securin-separase dynamics during DNA damage signaling.

The DNA damage checkpoint halts cell cycle progression in G2 in response to genotoxic insults. Central to the execution of cell cycle arrest is the checkpoint-induced stabilization of securin-separase complex (yeast Pds1-Esp1). The checkpoint kinases Chk1 and Chk2 (yeast Chk1 and Rad53) are thought to critically contribute to the stability of securin-separase complex by phosphorylation of securin, rendering it resistant to proteolytic destruction by the anaphase promoting complex (APC). Dun1, a Rad53 paralog related to Chk2, is also essential for checkpoint-imposed arrest. Dun1 is required for the DNA damage-induced transcription of DNA repair genes; however, its role in the execution of cell cycle arrest remains unknown. Here, we show that Dun1's role in checkpoint arrest is independent of its involvement in the transcription of repair genes. Instead, Dun1 is necessary to prevent Pds1 destruction during DNA damage in that the Dun1-deficient cells degrade Pds1, escape G2 arrest and undergo mitosis despite the presence of checkpoint-active Chk1 and Rad53. Interestingly, proteolytic degradation of Pds1 in the absence of Dun1 is mediated not by APC but by the HECT domain-containing E3 ligase Rsp5. Our results suggest a regulatory scheme in which Dun1 prevents chromosome segregation during DNA damage by inhibiting Rsp5-mediated proteolytic degradation of securin Pds1.

Replication stress-induced endogenous DNA damage drives cellular senescence induced by a sub-lethal oxidative stress.

Although oxidative stress has been shown to induce senescence and replication stress independently, no study has implicated unresolved replication stress as the driver for cellular senescence in response to oxidative stress. Using cells exposed to increasing concentrations of hydrogen peroxide, we show that sub-lethal amount of exogenous hydrogen peroxide induces two waves of DNA damage. The first wave is rapid and transient while the second wave coincides with the cells transition from the S to the G2/M phases of cell cycle. Subsequently, cells enter growth arrest accompanied by the acquisition of senescence-associated characteristics. Furthermore, a p53-dependent decrease in Rad51, which is associated with the formation of DNA segments with chromatin alterations reinforcing senescence, and Lamin B1 that is involved in chromatin remodeling, is observed during the establishment of the senescent phenotype. On the other hand, increase in senescence associated-ß-Gal activity, a classical marker of senescence and HMGA2, a marker of the senescence-associated heterochromatin foci, is shown to be independent of p53. Together, our findings implicate replication stress-induced endogenous DNA damage as the driver for the establishment of cellular senescence upon sub-lethal oxidative stress, and implicate the role of p53 in some but not all hallmarks of the senescent phenotype.

Cdk1 promotes kinetochore bi-orientation and regulates Cdc20 expression during recovery from spindle checkpoint arrest.

The spindle assembly checkpoint (SAC), an evolutionarily conserved surveillance pathway, prevents chromosome segregation in response to conditions that disrupt the kinetochore-microtubule attachment. Removal of the checkpoint-activating stimulus initiates recovery during which spindle integrity is restored, kinetochores become bi-oriented, and cells initiate anaphase. Whether recovery ensues passively after the removal of checkpoint stimulus, or requires mediation by specific effectors remains uncertain. Here, we report two unrecognized functions of yeast Cdk1 required for efficient recovery from SAC-induced arrest. We show that Cdk1 promotes kinetochore bi-orientation during recovery by restraining premature spindle elongation thereby extinguishing SAC signalling. Moreover, Cdk1 is essential for sustaining the expression of Cdc20, an activator of the anaphase promoting complex/cyclosome (APC/C) required for anaphase progression. We suggest a model in which Cdk1 activity promotes recovery from SAC-induced mitotic arrest by regulating bi-orientation and APC/C activity. Our findings provide fresh insights into the regulation of mitosis and have implications for the therapeutic efficacy of anti-mitotic drugs.

Transcription factor oscillations induce differential gene expressions.

Intracellular protein levels of diverse transcription factors (TFs) vary periodically with time. However, the effects of TF oscillations on gene expression, the primary role of TFs, are poorly understood. In this study, we determined these effects by comparing gene expression levels induced in the presence and in the absence of TF oscillations under same mean intracellular protein level of TF. For all the nonlinear TF transcription kinetics studied, an oscillatory TF is predicted to induce gene expression levels that are distinct from a nonoscillatory TF. The conditions dictating whether TF oscillations induce either higher or lower average gene expression levels were elucidated. Subsequently, the predicted effects from an oscillatory TF, which follows sigmoid transcription kinetics, were applied to demonstrate how oscillatory dynamics provide a mechanism for differential target gene transactivation. Generally, the mean TF concentration at which oscillations occur relative to the promoter binding affinity of a target gene determines whether the gene is up- or downregulated whereas the oscillation amplitude amplifies the magnitude of the differential regulation. Notably, the predicted trends of differential gene expressions induced by oscillatory NF-κB and glucocorticoid receptor match the reported experimental observations. Furthermore, the biological function of p53 oscillations is predicted to prime the cell for death upon DNA damage via differential upregulation of apoptotic genes. Lastly, given N target genes, an oscillatory TF can generate between (N-1) and (2N-1) distinct patterns of differential transactivation. This study provides insights into the mechanism for TF oscillations to induce differential gene expressions, and underscores the importance of TF oscillations in biological regulations.

DNA stretching in the nucleosome facilitates alkylation by an intercalating antitumour agent.

DNA stretching in the nucleosome core can cause dramatic structural distortions, which may influence compaction and factor recognition in chromatin. We find that the base pair unstacking arising from stretching-induced extreme minor groove kinking near the nucleosome centre creates a hot spot for intercalation and alkylation by a novel anticancer compound. This may have far reaching implications for how chromatin structure can influence binding of intercalator species and indicates potential for the development of site selective DNA-binding agents that target unique conformational features of the nucleosome.

DNA damage checkpoint execution and the rules of its disengagement.

Chromosomes are susceptible to damage during their duplication and segregation or when exposed to genotoxic stresses. Left uncorrected, these lesions can result in genomic instability, leading to cells' diminished fitness, unbridled proliferation or death. To prevent such fates, checkpoint controls transiently halt cell cycle progression to allow time for the implementation of corrective measures. Prominent among these is the DNA damage checkpoint which operates at G2/M transition to ensure that cells with damaged chromosomes do not enter the mitotic phase. The execution and maintenance of cell cycle arrest are essential aspects of G2/M checkpoint and have been studied in detail. Equally critical is cells' ability to switch-off the checkpoint controls after a successful completion of corrective actions and to recommence cell cycle progression. Interestingly, when corrective measures fail, cells can mount an unusual cellular response, termed adaptation, where they escape checkpoint arrest and resume cell cycle progression with damaged chromosomes at the cost of genome instability or even death. Here, we discuss the DNA damage checkpoint, the mitotic networks it inhibits to prevent segregation of damaged chromosomes and the strategies cells employ to quench the checkpoint controls to override the G2/M arrest.

Potential Therapeutics Targeting Upstream Regulators and Interactors of EHMT1/2.

Euchromatin histone lysine methyltransferases (EHMTs) are epigenetic regulators responsible for silencing gene transcription by catalyzing H3K9 dimethylation. Dysregulation of EHMT1/2 has been reported in multiple cancers and is associated with poor clinical outcomes. Although substantial insights have been gleaned into the downstream targets and pathways regulated by EHMT1/2, few studies have uncovered mechanisms responsible for their dysregulated expression. Moreover, EHMT1/2 interacting partners, which can influence their function and, therefore, the expression of target genes, have not been extensively explored. As none of the currently available EHMT inhibitors have made it past clinical trials, understanding upstream regulators and EHMT protein complexes may provide unique insights into novel therapeutic avenues in EHMT-overexpressing cancers. Here, we review our current understanding of the regulators and interacting partners of EHMTs. We also discuss available therapeutic drugs that target the upstream regulators and binding partners of EHMTs and could potentially modulate EHMT function in cancer progression.

Cryo-ET detects bundled triple helices but not ladders in meiotic budding yeast.

In meiosis, cells undergo two sequential rounds of cell division, termed meiosis I and meiosis II. Textbook models of the meiosis I substage called pachytene show that nuclei have conspicuous 100-nm-wide, ladder-like synaptonemal complexes and ordered chromatin loops. It remains unknown if these cells have any other large, meiosis-related intranuclear structures. Here we present cryo-ET analysis of frozen-hydrated budding yeast cells before, during, and after pachytene. We found no cryo-ET densities that resemble dense ladder-like structures or ordered chromatin loops. Instead, we found large numbers of 12-nm-wide triple-helices that pack into ordered bundles. These structures, herein called meiotic triple helices (MTHs), are present in meiotic cells, but not in interphase cells. MTHs are enriched in the nucleus but not enriched in the cytoplasm. Bundles of MTHs form at the same timeframe as synaptonemal complexes (SCs) in wild-type cells and in mutant cells that are unable to form SCs. These results suggest that in yeast, SCs coexist with previously unreported large, ordered assemblies.

Blau Syndrome Associated with Nucleotide-binding Oligomerization Domain Containing 2 Mutation in a Baby from Malaysia.

Blau syndrome (BS) is a very rare autosomal dominant juvenile inflammatory disorder caused by mutation in nucleotide-binding oligomerization domain containing 2 (NOD2). Usually, dermatitis is the first symptom that appears in the 1st year of life. About 220 BS cases with confirmed NOD2 mutation have been reported. However, the rarity and lack of awareness of the disease, especially in the regions where genetic tests are very limited, often result in late diagnosis and misdiagnosis. Here, we report a de novo BS case from Malaysia, which may be the first report from southeast Asia. PCR and DNA sequencing of peripheral blood mononuclear cells were performed to screen the entire coding region of NOD2 gene. A heterozygous c.1000C>T transition in exon 4, p. R334W, of the NOD2 gene was identified in the patient. This report further reaffirms the ubiquitousness of the disease and recurrency of p. R334W mutation.

Electron cryotomography analysis of Dam1C/DASH at the kinetochore-spindle interface in situ.

In dividing cells, depolymerizing spindle microtubules move chromosomes by pulling at their kinetochores. While kinetochore subcomplexes have been studied extensively in vitro, little is known about their in vivo structure and interactions with microtubules or their response to spindle damage. Here we combine electron cryotomography of serial cryosections with genetic and pharmacological perturbation to study the yeast chromosome segregation machinery in vivo. Each kinetochore microtubule has one (rarely, two) Dam1C/DASH outer kinetochore assemblies. Dam1C/DASH contacts the microtubule walls and does so with its flexible "bridges"; there are no contacts with the protofilaments' curved tips. In metaphase, ∼40% of the Dam1C/DASH assemblies are complete rings; the rest are partial rings. Ring completeness and binding position along the microtubule are sensitive to kinetochore attachment and tension, respectively. Our study and those of others support a model in which each kinetochore must undergo cycles of conformational change to couple microtubule depolymerization to chromosome movement.

Resistance to anti-microtubule drug-induced cell death is determined by regulation of BimEL expression.

Anti-microtubule agents are frequently used as anticancer therapeutics. Cell death induced by these agents is considered to be due to sustained mitotic arrest caused by the activation of spindle assembly checkpoint (SAC). However, some cell types are resistant to mitotic cell death. Cells' ability to escape mitotic arrest (mitotic slippage) is thought to be a major mechanism contributing to this resistance. Here, we show that resistance to cell death induced by anti-mitotic agents is not linked to cells' capacity to undergo mitotic slippage as generally believed but is dependent on the state of BimEL regulation during mitosis. While transcriptional repression of BimEL in the mitotic death-resistant cells involves polycomb repressive complex 2 (PRC2)-mediated histone trimethylation, the BimEL protein is destabilized by cullin 1/4A-ßTrCP-dependent degradation involving activation of cullin 1/4A by neddylation. These results imply that pharmacological augmentation of BimEL activity in anti-microtubule drug-resistant tumors may have important therapeutic implications.

Solubilization and preformulation of poorly water soluble and hydrolysis susceptible N-epoxymethyl-1,8-naphthalimide (ENA) compound.

N-Epoxymethyl-1,8-naphthalimide (ENA) is a novel antiproliferative drug candidate with potent anticancer and antifungal activity. It has an aqueous solubility of 0.0116mg/mL and also exhibits hydrolytic instability with a first-order hydrolysis rate of 0.051 h(-1). The present preformulation study aimed to characterize the physicochemical properties of ENA and develop an early injectable solution formulation for preclinical studies. To minimize hydrolysis, ENA is proposed to be formulated as either lyophilized powders or nonaqueous solutions followed by solubilization/reconstitution prior to administration. ENA solubilization was investigated in both aqueous media (by cosolvency, micellization and complexation) and nonaqueous solutions (mixture of Cremophor EL and ethanol). It is found that none of the solubilization techniques in aqueous media could increase ENA solubility to a desired level of several hundreds microg/mL at pharmaceutically acceptable excipient concentrations (< or =10%). In contrast, a combination of 70% Cremophor EL and 30% ethanol (v/v) proved effective in solubilizing ENA at 4 mg/mL, which exhibited good physical and chemical stability on storage at both 4 degrees C and room temperature over 4 months. No precipitation was observed upon 5-20 times dilution by the saline; in addition, less than 5% of ENA was hydrolyzed in 4h for the saline-diluted aqueous solutions. This nonaqueous ENA formulation is thus proposed for further preclinical studies, which can be reconstituted, prior to administration, by the 5-20 times infusion fluids (saline, 5% dextrose, etc.) to the desired drug dosing concentration at the acceptable excipient level. The approach used in this work could serve as a useful reference in formulating nonpolar drugs with hydrolytic instability.

Mapping Mitotic Death: Functional Integration of Mitochondria, Spindle Assembly Checkpoint and Apoptosis.

Targeting the mitotic pathways of rapidly proliferating tumor cells has been an effective strategy in traditional cancer therapy. Chemotherapeutics such as taxanes and vinca alkaloids, which disrupt microtubule function, have enjoyed clinical success; however, the accompanying side effects, toxicity and multi drug resistance remain as serious concerns. The emerging classes of inhibitors targeting mitotic kinases and proteasome face their own set of challenges. It is hoped that elucidation of the regulatory interface between mitotic checkpoints, mitochondria and mitotic death will aid the development of more efficacious anti-mitotic agents and improved treatment protocols. The links between the spindle assembly checkpoint (SAC) and mitochondrial dynamics that control the progression of anti-mitotic agent-induced apoptosis have been under investigation for several years and the functional integration of these various signaling networks is now beginning to emerge. In this review, we highlight current research on the regulation of SAC, the death pathway and mitochondria with particular focus on their regulatory interconnections.

Conformational landscape of the epidermal growth factor receptor kinase reveals a mutant specific allosteric pocket.

Activating mutations within the epidermal growth factor receptor (EGFR) kinase domain give rise to several cancers including Non-Small Cell Lung Cancer (NSCLC). Small molecule inhibitors targeted at these mutants have proven to be clinically successful drugs. These molecules are ATP competitive and rapidly result in the emergence of resistance. Recently Jia et al. [Nature, 2016, 534, 129-132] reported a small molecule inhibitor (called EAI045) that binds at an allosteric pocket, does not compete with ATP and displays high potency and selectivity towards certain activating mutants (L858R, T790M, L858R/T790M) of EGFR, with IC50 values ranging from 3 nM to 49 nM. We present here a study combining extensive molecular dynamics simulations with binding assays to provide a structural basis underlying the mechanism of binding of this molecule. It appears that in mutants, conformational destabilization of the short helix (that carries Leu858 in the wildtype), is key to the exposure of the allosteric pocket which otherwise is occluded by a set of sidechains including L858. We extend this hypothesis to show that a similar mechanism would enable the molecule to inhibit EGFRL861Q which is another oncogenic mutant and validate this with binding experiments. The screening of the human structural kinome revealed at least 12 other oncogenic kinases which carry at least one activating mutant in this disorder-prone region and hence would be amenable to allosteric inhibition by molecules such as EAI045. Our study characterizes a druggable allosteric pocket which appears to be specific to certain oncogenic mutants of the EGFR and holds therapeutic potential.

Inactivation of mitotic kinase triggers translocation of MEN components to mother-daughter neck in yeast.

Chromosome segregation, mitotic exit, and cytokinesis are executed in this order during mitosis. Although a scheme coordinating sister chromatid separation and initiation of mitotic exit has been proposed, the mechanism that temporally links the onset of cytokinesis to mitotic exit is not known. Exit from mitosis is regulated by the mitotic exit network (MEN), which includes a GTPase (Tem1) and various kinases (Cdc15, Cdc5, Dbf2, and Dbf20). Here, we show that Dbf2 and Dbf20 functions are necessary for the execution of cytokinesis. Relocalization of these proteins from spindle pole bodies to mother daughter neck seems to be necessary for this role because cdc15-2 mutant cells, though capable of exiting mitosis at semipermissive temperature, are unable to localize Dbf2 (and Dbf20) to the "neck" and fail to undergo cytokinesis. These cells can assemble and constrict the actomyosin ring normally but are incapable of forming a septum, suggesting that MEN components are critical for the initiation of septum formation. Interestingly, the spindle pole body to neck translocation of Dbf2 and Dbf20 is triggered by the inactivation of mitotic kinase. The requirement of kinase inactivation for translocation of MEN components to the division site thus provides a mechanism that renders mitotic exit a prerequisite for cytokinesis.

Identification of novel homozygous SLURP1 mutation in a Javanese family with Mal de Meleda.

BACKGROUND: Mal de Meleda (OMIM# 248300; keratosis palmoplantaris transgrediens) is an autosomal recessive form of palmoplantar keratoderma, clinically characterized by sharp demarcated erythema and hyperkeratosis of the palms and soles that progress with age and extend to the dorsal aspects of the hands and feet. The mal de Meleda is caused by mutations in the SLURP1 gene that encodes secreted lymphocyte antigen 6/urokinase-type plasminogen receptor-related protein 1 (SLURP1). To date no reported cases from Indonesia. The aims of the study were to describe the typical features of mal de Meleda cases in a Javanese family in Indonesia and identify the mutation in the ARS B gene which encodes SLURP1. PATIENTS AND METHODS: In this study, three Javanese patients, siblings from nonconsanguineous nonaffected parents, presented with classical symptoms of mal de Meleda. Genetic analysis screening SLURP1 gene was conducted for the specimens from the patients and other family members. RESULTS: A novel homozygous three-nucleotide deletion in exon 3, i.e. c.271-273TCTdel, was identified in the patients. Subcloning and sequencing revealed both parents (I.2 and I.3) and one of the father's siblings (I.1) carry heterozygous c.271-273TCTdel, while the other father's sibling (I.2), the mother's sister (I.4), and a healthy control matched the ethnicity of the family, showing normal sequence of the entire SLURP1. CONCLUSION: This is the first mal de Meleda case of Javanese ethnicity to be documented, and the unique mutation has not previously been reported. The finding supports the notion that despite the rarity, SLURP1 mutation causing mal de Meleda is ubiquitous.

Induced-Decay of Glycine Decarboxylase Transcripts as an Anticancer Therapeutic Strategy for Non-Small-Cell Lung Carcinoma.

Self-renewing tumor-initiating cells (TICs) are thought to be responsible for tumor recurrence and chemo-resistance. Glycine decarboxylase, encoded by the GLDC gene, is reported to be overexpressed in TIC-enriched primary non-small-cell lung carcinoma (NSCLC). GLDC is a component of the mitochondrial glycine cleavage system, and its high expression is required for growth and tumorigenic capacity. Currently, there are no therapeutic agents against GLDC. As a therapeutic strategy, we have designed and tested splicing-modulating steric hindrance antisense oligonucleotides (shAONs) that efficiently induce exon skipping (half maximal inhibitory concentration [IC50] at 3.5-7 nM), disrupt the open reading frame (ORF) of GLDC transcript (predisposing it for nonsense-mediated decay), halt cell proliferation, and prevent colony formation in both A549 cells and TIC-enriched NSCLC tumor sphere cells (TS32). One candidate shAON causes 60% inhibition of tumor growth in mice transplanted with TS32. Thus, our shAONs candidates can effectively inhibit the expression of NSCLC-associated metabolic enzyme GLDC and may have promising therapeutic implications.

An improved pre-clinical patient-derived liquid xenograft mouse model for acute myeloid leukemia.

BACKGROUND: Xenotransplantation of patient-derived AML (acute myeloid leukemia) cells in NOD-scid Il2rγ null (NSG) mice is the method of choice for evaluating this human hematologic malignancy. However, existing models constructed using intravenous injection in adult or newborn NSG mice have inferior engraftment efficiency, poor peripheral blood engraftment, or are difficult to construct. METHODS: Here, we describe an improved AML xenograft model where primary human AML cells were injected into NSG newborn pups intrahepatically. RESULTS: Introduction of primary cells from AML patients resulted in high levels of engraftment in peripheral blood, spleen, and bone marrow (BM) of recipient mice. The phenotype of engrafted AML cells remained unaltered during serial transplantation. The mice developed features that are consistent with human AML including spleen enlargement and infiltration of AML cells into multiple organs. Importantly, we demonstrated that although leukemic stem cell activity is enriched and mediated by CD34+CD117+ subpopulation, CD34+CD117- subpopulation can acquire CD34+CD117+ phenotype through de-differentiation. Lastly, we evaluated the therapeutic potential of Sorafenib and Regorafenib in this AML model and found that periphery and spleen AML cells are sensitive to these treatments, whereas BM provides a protective environment to AML. CONCLUSIONS: Collectively, our improved model is robust, easy-to-construct, and reliable for pre-clinical AML studies.

Essential tension and constructive destruction: the spindle checkpoint and its regulatory links with mitotic exit.

Replicated genetic material must be partitioned equally between daughter cells during cell division. The precision with which this is accomplished depends critically on the proper functioning of the mitotic spindle. The assembly, orientation and attachment of the spindle to the kinetochores are therefore constantly monitored by a surveillance mechanism termed the SCP (spindle checkpoint). In the event of malfunction, the SCP not only prevents chromosome segregation, but also inhibits subsequent mitotic events, such as cyclin destruction (mitotic exit) and cytokinesis. This concerted action helps to maintain temporal co-ordination among mitotic events. It appears that the SCP is primarily activated by either a lack of occupancy or the absence of tension at kinetochores. Once triggered, the inhibitory circuit bifurcates, where one branch restrains the sister chromatid separation by inhibiting the E3 ligase APC(Cdc20) (anaphase-promoting complex activated by Cdc20) and the other impinges on the MEN (mitotic exit network). A large body of investigations has now led to the identification of the control elements, their targets and the functional coupling among them. Here we review the emerging regulatory network and discuss the remaining gaps in our understanding of this effective mechanochemical control system.