The ATM Ser49Cys Variant Effects ATM Function as a Regulator of Oncogene-Induced Senescence.

An apical component of the cell cycle checkpoint and DNA damage repair response is the ataxia-telangiectasia mutated (ATM) Ser/Thr protein kinase. A variant of ATM, Ser49Cys (rs1800054; minor allele frequency = 0.011), has been associated with an elevated risk of melanoma development; however, the functional consequence of this variant is not defined. ATM-dependent signalling in response to DNA damage has been assessed in a panel of patient-derived lymphoblastoid lines and primary human melanocytic cell strains heterozygous for the ATM Ser49Cys variant allele. The ATM Ser49Cys allele appears functional for acute p53-dependent signalling in response to DNA damage. Expression of the variant allele did reduce the efficacy of oncogene expression in inducing senescence. These findings demonstrate that the ATM 146C>G Ser49Cys allele has little discernible effect on the acute response to DNA damage but has reduced function observed in the chronic response to oncogene over-expression. Analysis of melanoma, naevus and skin colour genomics and GWAS analyses have demonstrated no association of this variant with any of these outcomes. The modest loss of function detected suggest that the variant may act as a modifier of other variants of ATM/p53-dependent signalling.

Dysregulated G2 phase checkpoint recovery pathway reduces DNA repair efficiency and increases chromosomal instability in a wide range of tumours.

Defective DNA repair is being demonstrated to be a useful target in cancer treatment. Currently, defective repair is identified by specific gene mutations, however defective repair is a common feature of cancers without these mutations. DNA damage triggers cell cycle checkpoints that are responsible for co-ordinating cell cycle arrest and DNA repair. Defects in checkpoint signalling components such as ataxia telangiectasia mutated (ATM) occur in a low proportion of cancers and are responsible for reduced DNA repair and increased genomic instability. Here we have investigated the AURKA-PLK1 cell cycle checkpoint recovery pathway that is responsible for exit from the G2 phase cell cycle checkpoint arrest. We demonstrate that dysregulation of PP6 and AURKA maintained elevated PLK1 activation to promote premature exit from only ATM, and not ATR-dependent checkpoint arrest. Surprisingly, depletion of the B55α subunit of PP2A that negatively regulates PLK1 was capable of overcoming ATM and ATR checkpoint arrests. Dysregulation of the checkpoint recovery pathway reduced S/G2 phase DNA repair efficiency and increased genomic instability. We found a strong correlation between dysregulation of the PP6-AURKA-PLK1-B55α checkpoint recovery pathway with signatures of defective homologous recombination and increased chromosomal instability in several cancer types. This work has identified an unrealised source of G2 phase DNA repair defects and chromosomal instability that are likely to be sensitive to treatments targeting defective repair.

Mammary mechanobiology - investigating roles for mechanically activated ion channels in lactation and involution.

The ability of a mother to produce a nutritionally complete neonatal food source has provided a powerful evolutionary advantage to mammals. Milk production by mammary epithelial cells is adaptive, its release is exquisitely timed, and its own glandular stagnation with the permanent cessation of suckling triggers the cell death and tissue remodeling that enables female mammals to nurse successive progeny. Chemical and mechanical signals both play a role in this process. However, despite this duality of input, much remains unknown about the nature and function of mechanical forces in this organ. Here, we characterize the force landscape in the functionally mature gland and the capacity of luminal and basal cells to experience and exert force. We explore molecular instruments for force-sensing, in particular channel-mediated mechanotransduction, revealing increased expression of Piezo1 in mammary tissue in lactation and confirming functional expression in luminal cells. We also reveal, however, that lactation and involution proceed normally in mice with luminal-specific Piezo1 deletion. These findings support a multifaceted system of chemical and mechanical sensing in the mammary gland, and a protective redundancy that ensures continued lactational competence and offspring survival.

Multiscale imaging of basal cell dynamics in the functionally mature mammary gland.

The mammary epithelium is indispensable for the continued survival of more than 5,000 mammalian species. For some, the volume of milk ejected in a single day exceeds their entire blood volume. Here, we unveil the spatiotemporal properties of physiological signals that orchestrate the ejection of milk from alveolar units and its passage along the mammary ductal network. Using quantitative, multidimensional imaging of mammary cell ensembles from GCaMP6 transgenic mice, we reveal how stimulus evoked Ca2+ oscillations couple to contractions in basal epithelial cells. Moreover, we show that Ca2+-dependent contractions generate the requisite force to physically deform the innermost layer of luminal cells, compelling them to discharge the fluid that they produced and housed. Through the collective action of thousands of these biological positive-displacement pumps, each linked to a contractile ductal network, milk begins its passage toward the dependent neonate, seconds after the command.

Class IIa Histone Deacetylases Drive Toll-like Receptor-Inducible Glycolysis and Macrophage Inflammatory Responses via Pyruvate Kinase M2.

Histone deacetylases (HDACs) drive innate immune cell-mediated inflammation. Here we identify class IIa HDACs as key molecular links between Toll-like receptor (TLR)-inducible aerobic glycolysis and macrophage inflammatory responses. A proteomic screen identified the glycolytic enzyme pyruvate kinase M isoform 2 (Pkm2) as a partner of proinflammatory Hdac7 in murine macrophages. Myeloid-specific Hdac7 overexpression in transgenic mice amplifies lipopolysaccharide (LPS)-inducible lactate and promotes a glycolysis-associated inflammatory signature. Conversely, pharmacological or genetic targeting of Hdac7 and other class IIa HDACs attenuates LPS-inducible glycolysis and accompanying inflammatory responses in macrophages. We show that an Hdac7-Pkm2 complex acts as an immunometabolism signaling hub, whereby Pkm2 deacetylation at lysine 433 licenses its proinflammatory functions. Disrupting this complex suppresses inflammatory responses in vitro and in vivo. Class IIa HDACs are thus pivotal intermediates connecting TLR-inducible glycolysis to inflammation via Pkm2.

Melanoma mutations modify melanocyte dynamics in co-culture with keratinocytes or fibroblasts.

Melanocytic cell interactions are integral to skin homeostasis, and affect the outcome of multiple diseases, including cutaneous pigmentation disorders and melanoma. By using automated-microscopy and machine-learning-assisted morphology analysis of primary human melanocytes in co-culture, we performed combinatorial interrogation of melanocyte genotypic variants and functional assessment of lentivirus-introduced mutations. Keratinocyte-induced melanocyte dendricity, an indicator of melanocyte differentiation, was reduced in the melanocortin 1 receptor (MC1R) R/R variant strain and by NRAS.Q61K and BRAF.V600E expression, while expression of CDK4.R24C and RAC1.P29S had no detectable effect. Time-lapse tracking of melanocytes in co-culture revealed dynamic interaction phenotypes and hyper-motile cell states that indicated that, in addition to the known role in activating mitogenic signalling, MEK-pathway-activating mutations may also allow melanocytes to escape keratinocyte control and increase their invasive potential. Expanding this combinatorial platform will identify other therapeutic target mutations and melanocyte genetic variants, as well as increase understanding of skin cell interactions.

Combined use of subclinical hydroxyurea and CHK1 inhibitor effectively controls melanoma and lung cancer progression, with reduced normal tissue toxicity compared to gemcitabine.

Drugs such as gemcitabine that increase replication stress are effective chemotherapeutics in a range of cancer settings. These drugs effectively block replication and promote DNA damage, triggering a cell cycle checkpoint response through the ATR-CHK1 pathway. Inhibiting this signalling pathway sensitises cells to killing by replication stress-inducing drugs. Here, we investigated the effect of low-level replication stress induced by low concentrations (> 0.2 mm) of the reversible ribonucleotide reductase inhibitor hydroxyurea (HU), which slows S-phase progression but has little effect on cell viability or proliferation. We demonstrate that HU effectively synergises with CHK1, but not ATR inhibition, in > 70% of melanoma and non-small-cell lung cancer cells assessed, resulting in apoptosis and complete loss of proliferative potential in vitro and in vivo. Normal fibroblasts and haemopoietic cells retain viability and proliferative potential following exposure to CHK1 inhibitor plus low doses of HU, but normal cells exposed to CHK1 inhibitor combined with submicromolar concentrations of gemcitabine exhibited complete loss of proliferative potential. The effects of gemcitabine on normal tissue correlate with irreversible ATR-CHK1 pathway activation, whereas low doses of HU reversibly activate CHK1 independently of ATR. The combined use of CHK1 inhibitor and subclinical HU also triggered an inflammatory response involving the recruitment of macrophages in vivo. These data indicate that combining CHK1 inhibitor with subclinical HU is superior to combination with gemcitabine, as it provides equal anticancer efficacy but with reduced normal tissue toxicity. These data suggest a significant proportion of melanoma and lung cancer patients could benefit from treatment with this drug combination.

Mechanism of action of the third generation benzopyrans and evaluation of their broad anti-cancer activity in vitro and in vivo.

Successive rounds of chemical modification in three generations of benzopyran molecules have shown to select for different mechanisms of actions and progressive increases in anti-cancer activity. In this study, we investigated the mechanism of action of the third-generation benzopyran compounds, TRX-E-002-1 and TRX-E-009-1. High-content screening of a panel of 240 cancer cell lines treated with TRX-E-009-1 demonstrated it has broad anti-cancer potential. Within this screen, melanoma cell lines showed a range of sensitivities and subsequently a second independent panel of 21 melanoma 3D spheroid lines were assessed for their responses to both TRX-E-002-1 and TRX-E-009-1 compounds. Time-lapse microscopy illustrated both of these compounds caused mitotic delays in treated cells, resulting in either mitotic slippage or apoptosis. This finding along with immunostaining, in vitro polymerization assays, and animal experiments in both athymic and immunocompetent mice, demonstrates that these third-generation benzopyran compounds are potent tubulin polymerization inhibitors in vitro and in vivo, and this is the molecular basis of their anti-cancer activity in melanoma. These findings indicate these BP compounds may offer a novel anti-microtubule strategy for cancer intervention and provides the basis for further investigation into biomarkers of clinical sensitivity.

Endogenous Replication Stress Marks Melanomas Sensitive to CHEK1 Inhibitors In Vivo.

Purpose: Checkpoint kinase 1 inhibitors (CHEK1i) have single-agent activity in vitro and in vivo Here, we have investigated the molecular basis of this activity.Experimental Design: We have assessed a panel of melanoma cell lines for their sensitivity to the CHEK1i GNE-323 and GDC-0575 in vitro and in vivo The effects of these compounds on responses to DNA replication stress were analyzed in the hypersensitive cell lines.Results: A subset of melanoma cell lines is hypersensitive to CHEK1i-induced cell death in vitro, and the drug effectively inhibits tumor growth in vivo In the hypersensitive cell lines, GNE-323 triggers cell death without cells entering mitosis. CHEK1i treatment triggers strong RPA2 hyperphosphorylation and increased DNA damage in only hypersensitive cells. The increased replication stress was associated with a defective S-phase cell-cycle checkpoint. The number and intensity of pRPA2 Ser4/8 foci in untreated tumors appeared to be a marker of elevated replication stress correlated with sensitivity to CHEK1i.Conclusions: CHEK1i have single-agent activity in a subset of melanomas with elevated endogenous replication stress. CHEK1i treatment strongly increased this replication stress and DNA damage, and this correlated with increased cell death. The level of endogenous replication is marked by the pRPA2Ser4/8 foci in the untreated tumors, and may be a useful marker of replication stress in vivoClin Cancer Res; 24(12); 2901-12. ©2018 AACR.

Genome-Wide Overexpression Screen Identifies Genes Able to Bypass p16-Mediated Senescence in Melanoma.

Malignant melanomas often arise from nevi, which result from initial oncogene-induced hyperproliferation of melanocytes that are maintained in a CDKN2A/p16-mediated senescent state. Thus, genes that can bypass this senescence barrier are likely to contribute to melanoma development. We have performed a gain-of-function screen of 17,030 lentivirally expressed human open reading frames (ORFs) in a melanoma cell line containing an inducible p16 construct to identify such genes. Genes known to bypass p16-induced senescence arrest, including the human papilloma virus 18 E7 gene ( HPV18E7), and genes such as the p16-binding CDK6 with expected functions, as well as panel of novel genes, were identified, including high-mobility group box (HMGB) proteins. A number of these were further validated in two other models of p16-induced senescence. Tissue immunohistochemistry demonstrated higher levels of CDK6 in primary melanomas compared with normal skin and nevi. Reduction of CDK6 levels drove melanoma cells expressing functional p16 into senescence, demonstrating its contribution to bypass senescence.

Genome-wide gain-of-function screen for genes that induce epithelial-to-mesenchymal transition in breast cancer.

Epithelial to mesenchymal transition (EMT) is a developmental program that has been implicated in progression, metastasis and therapeutic resistance of some carcinomas. To identify genes whose overexpression drives EMT, we screened a lentiviral expression library of 17000 human open reading frames (ORFs) using high-content imaging to quantitate cytoplasmic vimentin. Hits capable of increasing vimentin in the mammary carcinoma-derived cell line MDA-MB-468 were confirmed in the non-tumorigenic breast-epithelial cell line MCF10A. When overexpressed in this model, they increased the rate of cell invasion through Matrigel™, induced mesenchymal marker expression and reduced expression of the epithelial marker E-cadherin. In gene-expression datasets derived from breast cancer patients, the expression of several novel genes correlated with expression of known EMT marker genes, indicating their in vivo relevance. As EMT-associated properties are thought to contribute in several ways to cancer progression, genes identified in this study may represent novel targets for anti-cancer therapy.

Self-Renewal and High Proliferative Colony Forming Capacity of Late-Outgrowth Endothelial Progenitors Is Regulated by Cyclin-Dependent Kinase Inhibitors Driven by Notch Signaling.

Since the discovery of endothelial colony forming cells (ECFC), there has been significant interest in their therapeutic potential to treat vascular injuries. ECFC cultures display significant heterogeneity and a hierarchy among cells able to give rise to high proliferative versus low proliferative colonies. Here we aimed to define molecularly this in vitro hierarchy. Based on flow cytometry, CD34 expression levels distinguished two populations. Only CD34 + ECFC had the capacity to reproduce high proliferative potential (HPP) colonies on replating, whereas CD34- ECFCs formed only small clusters. CD34 + ECFCs were the only ones to self-renew in stringent single-cell cultures and gave rise to both CD34 + and CD34- cells. Upon replating, CD34 + ECFCs were always found at the centre of HPP colonies and were more likely in G0/1 phase of cell cycling. Functionally, CD34 + ECFC were superior at restoring perfusion and better engrafted when injected into ischemic hind limbs. Transcriptomic analysis identified cyclin-dependent kinase (CDK) cell cycle inhibiting genes (p16, p21, and p57), the Notch signaling pathway (dll1, dll4, hes1, and hey1), and the endothelial cytokine il33 as highly expressed in CD34 + ECFC. Blocking the Notch pathway using a γ-secretase inhibitor (DAPT) led to reduced expression of cell cycle inhibitors, increased cell proliferation followed by a loss of self-renewal, and HPP colony formation capacity reflecting progenitor exhaustion. Similarly shRNA knockdown of p57 strongly affected self-renewal of ECFC colonies. ECFC hierarchy is defined by Notch signalling driving cell cycle regulators, progenitor quiescence and self-renewal potential.

Aurora A Is Critical for Survival in HPV-Transformed Cervical Cancer.

Human papillomavirus (HPV) is the causative agent in cervical cancer. HPV oncogenes are major drivers of the transformed phenotype, and the cancers remain addicted to these oncogenes. A screen of the human kinome has identified inhibition of Aurora kinase A (AURKA) as being synthetically lethal on the background of HPV E7 expression. The investigational AURKA inhibitor MLN8237/Alisertib selectively promoted apoptosis in the HPV cancers. The apoptosis was driven by an extended mitotic delay in the Alisertib-treated HPV E7-expressing cells. This had the effect of reducing Mcl-1 levels, which is destabilized in mitosis, and increasing BIM levels, normally destabilized by Aurora A in mitosis. Overexpression of Mcl-1 reduced sensitivity to the drug. The level of HPV E7 expression influenced the extent of Alisertib-induced mitotic delay and Mcl-1 reduction. Xenograft experiments with three cervical cancer cell lines showed Alisertib inhibited growth of HPV and non-HPV xenografts during treatment. Growth of non-HPV tumors was delayed, but in two separate HPV cancer cell lines, regression with no resumption of growth was detected, even at 50 days after treatment. A transgenic model of premalignant disease driven solely by HPV E7 also demonstrated sensitivity to drug treatment. Here, we show for the first time that targeting of the Aurora A kinase in mice using drugs such as Alisertib results in a curative sterilizing therapy that may be useful in treating HPV-driven cancers.