Cyclophilin cis-trans prolyl isomerases, DNA damage and synthetic lethality.
Cyclosporine A (CsA) is a cyclic undecapeptide routinely clinically used as a prophylaxis against Graft-versus-Host disease, an important contributor to bone marrow and solid organ transplantation morbidity. The biological target of CsA are members of the Cyclophilin family of cis-trans prolyl isomerases. We found that CsA can induce DNA double strand breaks (DSBs) in human cells (O’Driscoll et al, Bone Marrow Transplantation 2008). CsA has an interesting documented history against a range of cancers. Non-immunosuppressive CsA analogues have been generated and are being developed as HIV and Hepatitis antivirals. We are currently investigating how Cyclophilin A loss and inhibition causes DNA breakage, complex DNA rearrangements and specific cell cycle abnormalities. We have generated a comprehenise Cyclophilin A interactome which has provided a clear routemap to direct these studies. Excitingly, we have also found that Cyclophilin A loss/inhibition is synthetically lethal in certain cancers with specific actionable genetic vulnerabilities.
Understanding the interplay between and dysfunctions of the RTK-PI3K-mTOR network and the DNA damage response.
Components of the mTOR pathway, specifically PKB/AKT are often over-expressed/hyperactive in malignancy and represent important drug targets for cancer treatment. In fact, mis-regulated mTOR function underlies several cancer predisposition syndromes including Cowden syndrome (PTEN mutations), Tuberous sclerosis (TSC1/2 mutations) and Peutz-Jeghers syndrome (LKB1 mutations). We have been involved in studies identifying novel congenital defects in components of the mTOR signal transduction network in close collaboration with colleagues in the US and Canada. We recently functionally characterised cell lines from patients with complex developmental conditions ('Megalencephaly-capillary malformation' and 'Megalencephaly-polymicrogyria-polydactyly-hydrocephalus' syndromes), with novel defects in AKT3, PIK3R2 and PIK3CA, showing that hyperactive signal transduction through the mTOR network underlay these disorders (Alcantara D et al Brain 2017, Riviere et al Nat Genet 2012).
Our continuing work in this area has also highlighted the complex clinical presentation of people with constitutional defects in this pathway such as PIK3CA (Di Donanto N et al Hum Mut 2015). Our collaborative contributions to elucidating the basis and natural history of these types of mosaic overgrowth and cancer predisposition conditions has lead to the identification of gain of function mutations in FGFR1 as the underlying cause of Encephalocraniocutaneous Lipomatosis (Bennett J et al Am J Hum Genet 2016). One potentially important implication of this could be the application of more targeted therapeutic strategies towards a particularly aggressive brain tumour (pilocytic astrocytoma) frequently found to occur in children with this condition.
We have also been involved in functional and genetic characterisation of SHORT syndrome. In this disorder we found hypo-activation of the mTOR network due to reduced function mutations in the gene encoding the regulatory subunit of PI3K (Dyment DA et al Am J Hum Genet 2013). We have identified PRKCE (PKCε) as an additional SHORT syndrome gene, in this instance through impairment of mTORC2-dependent AKT activation (Alcantara D et al Hum Mol Genet 2017) Furthermore, we played an active role in the molecular and genetic delineation of 'Microcephaly Capillary Malformation syndrome', a devastating developmental condition associated with progressive neurodegeneration and cutaneous vascular abnormalities (McDonell L et al Nat Genet 2013).
There are many examples of different variants within the same gene presenting as distinct clinical entities. We have contributed functional analysis in describing novel loss-of-function PDGFRB mutations (encoding PDGF-receptor β isoform) in Primary Familial Brain Calcification, a rare microvascular calcifying disorder involving motor, cognitive and neuropsychiatric symptoms (Ramos EM et al Eur J Hum Genet 2018). We have also shown that novel gain-of-function PDGFRB mutations in Fusifom Cerebral Aneurysms, a rare type of difficult to treat intracranial aneurysm (Karasozen Y et al Am J Hum Genet 2019). The latter instance offers the potential for therpeutic intervention through the repurposing of RTK inhibitors used to treat cancer.
Genomic Disorders.
We have characterised the functional significance of copy number variation (CNV; deletion and/or duplication) of genes that are known to function in various aspects of cell cycle checkpoint control, DNA repair and DNA damage signalling, using material derived from individuals with diverse human Genomic Disorders (see Colnaghi et al Sem Cell & Dev Biol 2011 for an overview). To date, in association with colleagues in the US, Canada and Europe we have characterised novel and specific DNA damage response defects in cells from well-known Genomic Disorders including Miller-Dieker Lissencephaly and Williams-Beuren syndromes (O’Driscoll et al Am J Hum Genet ), 1q21.1 CNV syndrome (Harvard C et al Orphanet J Rare Diseases 2011), DiGeorge/Veleocardiofacial syndrome and rec(3) syndrome (Colnaghi R et al Sem Cell & Dev Biol 2011), as well as other well-known syndromes such as 17p13.3 duplication syndrome (Outwin E et al PLoS Genetics 2011), 2p15p16.1 microdeletion syndrome (Bagheri H et al J Clin Invest Insight 2016) and Wolf-Hirschhorn syndrome (Hart L et al Dis Models & Mech 2014 and Kerzendorfer et al Hum Mol Genet 2012). These findings offer new insight into the distinct pathomechanisms underlying the clinical presentation and progression of these conditions.
Defective ubiquitin pathway function neuropathologies and syndromal intellectual disability.
In this diverse group of disorders, we are interested in understanding how primary genetic defects in ubiquitin pathway components can impact on various aspects of the DNA damage response and how these combine to present clinically. For example, we have found that cells derived from patients with mutations in the E3-ubiquitinin ligase Cullin 4B are highly sensitive the topoisomerase I (Topo I) poison camptothecin (CPT) and that this sensitivity is likely a result of impaired CPT-mediated Topo I degradation suggesting Topo I to be a CUL4B-dependent substrate (Kerzendorfer et al Hum Mol Genet 2010). We are currently working on understanding other impacts of defective CUL4B function, on how these may contribute to the human disorder or could be exploited to sensitise cells to certain DNA damaging agents (Kerzendorfer et al Mech Ageing & Dev 2011). We are also working on understanding the pathophysiological impact of clinically relevant deficits of other syndromal mental retardation genes that encode ubiquitin pathway components with specific relevance to the DDR and genome stability networks. Our work on Microcephaly Capillary Malformation Syndrome (MIC-CAP) characterised the functional consequences of pathogenic defects in STAMBP/AMSH, which encodes a deubiquitinating isopeptidase (McDonell L et al Nat Genet 2013).
‘Novel’ genome stability disorders.
Many human DNA damage response/repair defective disorders exhibit the specific clinical combination of microcephaly and pre- and post-natal growth retardation. Provocatively, the medical literature abounds with clinical descriptions of genetically uncharacterised syndromes exhibiting similar features, many of which also appear to be cancer prone. Whether any of these disorders are caused by a defective response to DNA damage is unknown. Recently, we were involved in a study identify a pathogenic defect in ATR in an autosomal dominant oropharyngeal cancer syndrome family (Tanaka et al Am J Hum Genet 2012). This unexpected clinical presentation of an ATR-pathway defect illustrates how much we have yet to learn concerning the intricate biological connections between the DDR, genomic instability and normal human development.
Another aspect of our work concerns the identification and characterisation of novel DNA damage response disorders. This evolving aspect of our research has been greatly facilitated by the recent technological revolution in genomic array and next generation sequencing technologies, which can generate target candidate lists from which we can prioritise characterisation studies (e.g. see Raffan et al, Frontiers in Genomic Endocrinology 2011). Together with our collaborators from the UK, mainland Europe, North America and Canada, we are currently working on several novel congenital defects associated with fascinating human disorders concerning various aspects of the DDR, DNA replication and cell cycle control. For example, together with colleagues in the UK, we described and characterised novel defects in the SMC5-6-associated SUMO Ligase, NSE2 (mms21/NSMCE2), in individuals exhibiting primordial dwarfism and extreme insulin resistance (Payne, Colnaghi, Rocha & Seth et al, J Clin Invest 2014). This further highlights the functional cross-talk between genome stability pathways and those that control metabolic homeostasis. We have shown that the ciliopathy Oral-Facial-Digital syndrome Type I also exhibits compromised homologous recombination repair, further highlighting the cross-talk between distinct cellular networks (Abramowicz I et al Hum Mol Genet 2017).
We have identified and functionally characterised hypomorphic pathogenic mutations in POLA1 as underlying an X-linked (male only) syndromal Intellectual Disability with associated severe growth restriction, microcephaly and hypogonadism: Van Esch-O'Driscoll Syndrome (OMIM: 301030) (Van Esch H et al Am J Hum Genet 2019). POLA1 encodes the catalytic subunit of DNA Polymerase α-Primase.
Understanding the molecular basis of human Microcephalic Primordial Dwarfism.
This family of conditions is characterised by profound short stature associated with a severely reduced head circumference denoting an extreme impairment of normal brain development (microcephaly: see Alcantara D & O'Driscoll M Am J Med Genet Part C 2014). Very often, the skeletal system is also affected in these disorders. The archetypal example of such conditions is Seckel syndrome, first described as a distinct clinical disorder in 1960 by Dr. Helmut Seckel in a monograph entitled “Bird-Headed Dwarfs. Studies in developmental anthropology including human proportions”. This condition is characterised by a dramatic proportionate dwarfism evident even in utero, a characteristic facial appearance (small chin, protruding nose, receding forehead), severe microcephaly (reduced head circumference) and isolated skeletal abnormalities. We characterised the first identified genetic defect in this disorder as a hypomorphic mutation in gene encoding ATR (ataxia telangiectasia and Rad3-related), a protein kinase that plays a central role in the DNA damage response (O’Driscoll et al Nat Genet 2003). A ‘humanised’ mouse model of this defect was created by Murga and colleagues (Murga et al, Nat Genet 2009) underscoring the importance of this DNA damage response pathway for normal development and maintenance of tissue homeostasis (O’Driscoll M, Nat Genet 2009, O’Driscoll M, DNA Repair 2009). Recently, we have been involved in a study describing novel ATR mutations in additional cases of Seckel syndrome as well as a novel defect in ATRIP, the ATR interacting protein (Ogi et al PLoS Genet 2012). This work helps consolidate the clinical presentation of impaired ATR-ATRIP function in humans.
We have also been involved in other studies that have identified novel defects in related conditions including Pericentrin (PCNT) and Origin Recognition Complex 1 (ORC1) in Microcephalic Primordial Dwarfism Type II and Meier-Gorlin syndrome respectively (Griffith et al Nat Genet 2008 and Bicknell et al Nat Genet 2011). These studies highlight the previously hitherto under-appreciated significance of defects in centrosome function and DNA replication licensing to normal human development. Interestingly, we have also identified specific ATR-pathway defects in cells from these conditions and are currently pursuing the characterisation of novel defects in these genetically heterogeneous disorders. Our work on human ORC-defects has also uncovered a pathomechanistic link between these DNA replication controlling factors and cilia formation and function (Stiff T et al PLoS Genet 2013). We have also been involved in the description of a novel kinetochore-based genetic defect (CENPE) causative of Microcephalic Primordial Dwarfism (Mirzaa GM et al Hum Genet 2014). These findings further expand the growing landscape of complex cellular pathomechanisms underlying these conditions.
Several isolated skeletal abnormalities are associated, to varying degrees, with Microcephalic Primordial Dwarfism syndromes. Specific examples include, fifth finger clinodactyly, thoracic kyphosis, joint dislocation, ivory epiphysis, delayed ossification, absent patella and dental malocclusion. The molecular aetiology of these clinical features is currently unclear. Using model cell-based systems for osteogenesis and chrondrogenesis we have been investigating how, at the molecular level, defects in genes associated with these conditions can impact upon these processes. Specifically with regard to chondrogenesis and chondroinduction, we have developed a differentiation based system that employs patient-derived material, thereby carrying the clinically relevant pathogenic defects (Stiff T et al PLoS Genet 2013). Interestingly, we have found that ATR dysfunction is not entirely restricted to genome instability and we have been involved in work identifying novel impacts of impaired ATR upon cilia signalling in G0 cells (Stiff T & Casar T el al Hum Mol Genet 2016). These potentially non-canonical roles of ATR expand upon our knowledge of what has hitherto been assumed to be exclusively a DNA damage response and cell cycle checkpoint factor.