Targeting the DNA damage response protein ATR kinase in pancreatic cancer

Therapeutic targeting of the DNA damage response (DDR), coupled with the implementation of personalised treatment strategies, has the potential to improve the poor survival outcomes of pancreatic ductal adenocarcinoma (PDAC). Many common genetic alterations in PDAC augment replication-associated DNA damage, or replication stress (RS), which increases reliance on the RS-response factor, Ataxia Telangiectasia and Rad3-related (ATR) kinase. Furthermore, many chemotherapies used in the treatment of PDAC increase RS, leading to ATR activation which limits the efficacy of these therapies. Therefore, there is good rationale for targeting ATR as a strategy for treatment of patients with PDAC. In this thesis, I report on investigations into the therapeutic potential of combining the ATR inhibitor, AZD6738, with DNA-damaging drugs and DDR-targeted agents in preclinical models of PDAC. My primary focus was on maximising the efficacy of combined ATR inhibition and gemcitabine (ATRi/gem). I hypothesised that ATRi/gem would most likely benefit specific sub-groups of patients with pre-existing aberrations in DDR pathways (i.e. a precision medicine approach to patient selection). Accordingly, I investigated the potential for deficiency in the double-strand-break master-regulator, Ataxia Telangiectasia Mutated (ATM), to sensitise PDAC models to ATRi/gem. I found that complete loss of ATM function - through kinase inhibition or through CRISPR knockout, but not ATM depletion by RNA interference - sensitised to ATRi and ATRi/gem in PDAC models. Using flow cytometry and quantitative image-based cytometry, I gained insight into the mechanisms by which AZD6738 and gemcitabine synergise, finding evidence for replication catastrophe that was significantly augmented in ATM-deficient cells. In vivo, I demonstrated that low-dose gemcitabine can be used to sensitise to AZD6738, much more effectively in an ATM-deficient setting. My results suggest that ATM-deficiency augments the replication catastrophe-mediated cell death induced by ATRi/gem, thus ATM-loss could predict response to this combination. The preclinical assessments of AZD6738 and gemcitabine that formed part of this project have led to the launch of a phase-I clinical trial (ATRiUM; NCT03669601). My data indicate that ATM status should be carefully assessed in tumours from patients with PDAC, since the distinction between ATM-low and ATM-null could be crucial in maximising the success of trials using ATM expression as a predictive biomarker. In this dissertation, I also present my investigations into the combination of AZD6738 with the poly(ADP-ribose) polymerase (PARP) inhibitor, olaparib. While AZD6738 and olaparib synergistically inhibited the growth of PDAC cells proficient in homologous recombination (HR) in vitro, I identified no anti-tumour effect in HR-proficient in vivo models. This could indicate that a DDR deficiency would be necessary for this combination to be most effective in PDAC, a hypothesis that will be tested in the clinic in the Precision-Panc trial, PRIMUS-004.CRUK, Pancreatic Cancer U

William H. Calvin, How Brains Think: Evolving Intelligence, Then and Now

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43244/1/11023_2004_Article_184637.pd

Performatives and dream skepticism

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43378/1/11098_2004_Article_BF00375718.pd

Dreams, skepticism, and scientific research

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47971/1/11406_2006_Article_BF02379248.pd

Wittgenstein on sensation and ‘seeing-as’

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43832/1/11229_2004_Article_BF00485563.pd

Reconciling Bayesian and Perimeter Regularization for Binary Inversion

A central theme in classical algorithms for the reconstruction of discontinuous functions from observational data is perimeter regularization via the use of total variation. On the other hand, sparse or noisy data often demand a probabilistic approach to the reconstruction of images, to enable uncertainty quantification; the Bayesian approach to inversion, which itself introduces a form of regularization, is a natural framework in which to carry this out. In this paper the link between Bayesian inversion methods and perimeter regularization is explored. In this paper two links are studied: (i) the maximum a posteriori objective function of a suitably chosen Bayesian phase-field approach is shown to be closely related to a least squares plus perimeter regularization objective; (ii) sample paths of a suitably chosen Bayesian level set formulation are shown to possess a finite perimeter and to have the ability to learn about the true perimeter

A Simple and Robust Single-Step Method for CAR-Vδ1 γδT Cell Expansion and Transduction for Cancer Immunotherapy

The γδT cell subset of peripheral lymphocytes exhibits potent cancer antigen recognition independent of classical peptide MHC complexes, making it an attractive candidate for allogeneic cancer adoptive immunotherapy. The Vδ1-T cell receptor (TCR)-expressing subset of peripheral γδT cells has remained enigmatic compared to its more prevalent Vγ9Vδ2-TCR and αβ-TCR-expressing counterparts. It took until 2021 before a first patient was dosed with an allogeneic adoptive Vδ1 cell product despite pre-clinical promise for oncology indications stretching back to the 1980s. A contributing factor to the paucity of clinical progress with Vδ1 cells is the lack of robust, consistent and GMP-compatible expansion protocols. Herein we describe a reproducible one-step, clinically translatable protocol for Vδ1-γδT cell expansion from peripheral blood mononuclear cells (PBMCs), that is further compatible with high-efficiency gene engineering for immunotherapy purposes. Briefly, αβTCR- and CD56-depleted PBMC stimulation with known-in-the-art T cell stimulators, anti-CD3 mAb (clone: OKT-3) and IL-15, leads to robust Vδ1 cell expansion of high purity and innate-like anti-tumor efficacy. These Vδ1 cells can be virally transduced to express chimeric antigen receptors (CARs) using standard techniques, and the CAR-Vδ1 exhibit antigen-specific persistence, cytotoxicity and produce IFN-γ. Practicable, GMP-compatible engineered Vδ1 cell expansion methods will be crucial to the wide-spread clinical testing of these cells for oncology indications

Hereditary predisposition to ovarian cancer, looking beyond BRCA1/BRCA2

AbstractObjectiveGenetic predisposition to ovarian cancer is well documented. With the advent of next generation sequencing, hereditary panel testing provides an efficient method for evaluating multiple genes simultaneously. Therefore, we sought to investigate the contribution of 19 genes identified in the literature as increasing the risk of hereditary breast and ovarian cancer (HBOC) in a BRCA1 and BRCA2 negative population of patients with a personal history of breast and/or ovarian cancer by means of a hereditary cancer panel.MethodsSubjects were referred for multi-gene panel testing between February 2012 and March 2014. Clinical data was ascertained from requisition forms. The incidence of pathogenic mutations (including likely pathogenic), and variant of unknown significance were then calculated for each gene and/or patient cohort.ResultsIn this cohort of 911 subjects, panel testing identified 67 mutations. With 7.4% of subjects harboring a mutation on this multi-gene panel, the diagnostic yield was increased, compared to testing for BRCA1 and BRCA2 mutations alone. In the ovarian cancer probands, the most frequently mutated genes were BRIP1 (n=8; 1.72%) and MSH6 (n=6; 1.29%). In the breast cancer probands, mutations were most commonly observed in CHEK2 (n=9; 2.54%), ATM (n=3; 0.85%), and TP53 (n=3; 0.85%).ConclusionsAlthough further studies are needed to clarify the exact management of patients with a mutation in each gene, this study highlights information that can be captured with panel testing and provides support for incorporation of panel testing into clinical practice