Molecular and Functional Identification of Tumour Suppressor Genes Involved in Mouse Skin Carcinogenesis

Mouse skin tumourigenesis occurs through a series of discrete steps associated with specific genetic alterations, from an initiated cell, to a benign papilloma, and subsequently to a malignant squamous carcinoma. A proportion of these malignant tumours undergo a dramatic change in cell phenotype, which is accompanied by substantial alterations in the expression of several markers of epithelial differentiation. These spindle cell carcinomas have been well characterised biologically, but the genetic events which are responsible for the transition to these invasive tumours are not well understood. The work presented in this thesis was designed to address the genetic basis of the spindle transition by cytogenetic, molecular genetic and functional approaches. Amplification of the mutant H-ra5 allele and / or loss of the normal W-ras allele are consistently found in spindle cell carcinomas, which also frequently show imbalances of chromosome 7. The mechanistic basis for these ras / chromosome 7 changes was investigated by Fluorescence in situ Hybridisation using chromosome paints. In the squamous carcinoma cell line, B9, increases in mutant W-ras were caused by whole chromosome duplication, while in the clonally-related spindle cell lines, A5 and D3, a further increase in expression was achieved by localised amplification of the mutant W-ras gene on double minute chromosomes. To establish the nature of the gene(s) lost at the squamous-spindle transition, somatic cell fusions were carried out between the clonally-related B9 and A5 cells. The hybrids were epithelial in morphology and expressed characteristic epithelial proteins, such as E- cadherin and the keratins. In addition, they were found to be suppressed in their ability to form tumours following injection into nude mice. These experiments demonstrate that spindle cells arise by a mechanism involving loss of a tumour suppressor gene. To identify putative tumour suppressor loci, we developed an approach using hybrid cells generated between a keratinocyte cell line, C5N, and the spindle carcinoma cell line, carB. Tumourigenicity was initially suppressed in these hybrids, but the tumours which did arise, after a long latency, were poorly differentiated squamous carcinomas or undifferentiated spindle carcinomas. Allelotype analysis of the tumours enabled us to identify regions on mouse chromosomes 4 and 7, which harbour putative tumour suppressor genes involved in mouse skin tumourigenesis. In order to determine the function and relevance of each of these loci in the acquisition of the spindle phenotype, the syntenic regions from the human genome were introduced by microcell-mediated monochromosome transfer into spindle carcinoma cell lines. This strategy, involving a single human chromosome on a mouse background, facilitates finer mapping of the loci in revertant clones. Using this approach, we have identified a locus on chromosome 15 corresponding to the locus identified on mouse chromosome 7, which may cause growth inhibition of A5 cells. Introduction of human chromosome 9 into A5 and carB cells caused a partial reversion to the squamous phenotype and tumour suppression. The loss of the pi6 tumour suppressor gene located on human chromosome 9p21, is know n to be associated with the loss of differentiation which occurs in the transition to the spindle phenotype. Therefore the effect of human chromosome 9 on spindle cells may be partly explained by the consequences of introducing pi6. However, several lines of evidence point to the existence of a second locus on human chromosome 9 which may be important in the conversion to spindle carcinomas. Future work will focus on the identification of this gene(s) and the role it plays in this last stage of mouse skin tumourigenesis

Fadraciclib (CYC065), a novel CDK inhibitor, targets key pro-survival and oncogenic pathways in cancer

Cyclin-dependent kinases (CDKs) contribute to the cancer hallmarks of uncontrolled proliferation and increased survival. As a result, over the last two decades substantial efforts have been directed towards identification and development of pharmaceutical CDK inhibitors. Insights into the biological consequences of CDK inhibition in specific tumor types have led to the successful development of CDK4/6 inhibitors as treatments for certain types of breast cancer. More recently, a new generation of pharmaceutical inhibitors of CDK enzymes that regulate the transcription of key oncogenic and pro-survival proteins, including CDK9, have entered clinical development. Here, we provide the first disclosure of the chemical structure of fadraciclib (CYC065), a CDK inhibitor and clinical candidate designed by further optimization from the aminopurine scaffold of seliciclib. We describe its synthesis and mechanistic characterization. Fadraciclib exhibits improved potency and selectivity for CDK2 and CDK9 compared to seliciclib, and also displays high selectivity across the kinome. We show that the mechanism of action of fadraciclib is consistent with potent inhibition of CDK9-mediated transcription, decreasing levels of RNA polymerase II C-terminal domain serine 2 phosphorylation, the pro-survival protein Myeloid Cell Leukemia 1 (MCL1) and MYC oncoprotein, and inducing rapid apoptosis in cancer cells. This cellular potency and mechanism of action translate to promising anti-cancer activity in human leukemia mouse xenograft models. Studies of leukemia cell line sensitivity identify mixed lineage leukemia (MLL) gene status and the level of B-cell lymphoma 2 (BCL2) family proteins as potential markers for selection of patients with greater sensitivity to fadraciclib. We show that the combination of fadraciclib with BCL2 inhibitors, including venetoclax, is synergistic in leukemic cell models, as predicted from simultaneous inhibition of MCL1 and BCL2 pro-survival pathways. Fadraciclib preclinical pharmacology data support its therapeutic potential in CDK9- or CDK2-dependent cancers and as a rational combination with BCL2 inhibitors in hematological malignancies. Fadraciclib is currently in Phase 1 clinical studies in patients with advanced solid tumors (NCT02552953) and also in combination with venetoclax in patients with relapsed or refractory chronic lymphocytic leukemia (CLL) (NCT03739554) and relapsed refractory acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) (NCT04017546)

Data sharing reveals complexity in the westward spread of domestic animals across Neolithic Turkey

This study presents the results of a major data integration project bringing together primary archaeozoological data for over 200,000 faunal specimens excavated from seventeen sites in Turkey spanning the Epipaleolithic through Chalcolithic periods, c. 18,000-4,000 cal BC, in order to document the initial westward spread of domestic livestock across Neolithic central and western Turkey. From these shared datasets we demonstrate that the westward expansion of Neolithic subsistence technologies combined multiple routes and pulses but did not involve a set 'package' comprising all four livestock species including sheep, goat, cattle and pig. Instead, Neolithic animal economies in the study regions are shown to be more diverse than deduced previously using quantitatively more limited datasets. Moreover, during the transition to agro-pastoral economies interactions between domestic stock and local wild fauna continued. Through publication of datasets with Open Context (opencontext.org), this project emphasizes the benefits of data sharing and web-based dissemination of large primary data sets for exploring major questions in archaeology (Alternative Language Abstract S1)

Further evidence that the tyrosine phosphorylation of glycogen synthase kinase-3 (GSK3) in mammalian cells is an autophosphorylation event.

Phosphorylation of the endogenous GSK3alpha (glycogen synthase kinase-3alpha) at Tyr279 and GSK3beta at Tyr216 was suppressed in HEK-293 or SH-SY5Y cells by incubation with pharmacological inhibitors of GSK3, but not by an Src-family inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4- d ]pyrimidine (PP2), or a general protein tyrosine kinase inhibitor (genistein). GSK3beta transfected into HEK-293 cells or Escherichia coli became phosphorylated at Tyr216, but catalytically inactive mutants did not. GSK3beta expressed in insect Sf 21 cells or E. coli was extensively phosphorylated at Tyr216, but the few molecules lacking phosphate at this position could autophosphorylate at Tyr216 in vitro after incubation with MgATP. The rate of autophosphorylation was unaffected by dilution and was suppressed by the GSK3 inhibitor kenpaullone. Wild-type GSK3beta was unable to catalyse the tyrosine phosphorylation of catalytically inactive GSK3beta lacking phosphate at Tyr216. Our results indicate that the tyrosine phosphorylation of GSK3 is an intramolecular autophosphorylation event in the cells that we have studied and that this modification enhances the stability of the enzyme

Phosphorylation of the regulatory subunit of smooth muscle protein phosphatase 1M at Thr850 induces its dissociation from myosin

AbstractRho kinase is known to control smooth muscle contractility by phosphorylating the 110 kDa myosin-targetting subunit (MYPT1) of the myosin-associated form of protein phosphatase 1 (PP1M). Phosphorylation of MYPT1 at Thr695 has previously been reported to inhibit the catalytic activity of PP1. Here, we show that the phosphorylation of Thr850 by Rho kinase dissociates PP1M from myosin, providing a second mechanism by which myosin phosphatase activity is inhibited

Interrogation of novel CDK2/9 inhibitor fadraciclib (CYC065) as a potential therapeutic approach for AML

Abstract Over the last 50 years, there has been a steady improvement in the treatment outcome of acute myeloid leukemia (AML). However, median survival in the elderly is still poor due to intolerance to intensive chemotherapy and higher numbers of patients with adverse cytogenetics. Fadraciclib (CYC065), a novel cyclin-dependent kinase (CDK) 2/9 inhibitor, has preclinical efficacy in AML. In AML cell lines, myeloid cell leukemia 1 (MCL-1) was downregulated following treatment with fadraciclib, resulting in a rapid induction of apoptosis. In addition, RNA polymerase II (RNAPII)-driven transcription was suppressed, rendering a global gene suppression. Rapid induction of apoptosis was observed in primary AML cells after treatment with fadraciclib for 6–8 h. Twenty-four hours continuous treatment further increased efficacy of fadraciclib. Although preliminary results showed that AML cell lines harboring KMT2A rearrangement (KMT2A-r) are more sensitive to fadraciclib, we found that the drug can induce apoptosis and decrease MCL-1 expression in primary AML cells, regardless of KMT2A status. Importantly, the diversity of genetic mutations observed in primary AML patient samples was associated with variable response to fadraciclib, confirming the need for patient stratification to enable a more effective and personalized treatment approach. Synergistic activity was demonstrated when fadraciclib was combined with the BCL-2 inhibitor venetoclax, or the conventional chemotherapy agents, cytarabine, or azacitidine, with the combination of fadraciclib and azacitidine having the most favorable therapeutic window. In summary, these results highlight the potential of fadraciclib as a novel therapeutic approach for AML