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Supplementary Fig S7 describes western blot and RTPCR of PARG in modified shPARG cells.

Patients with metastatic pancreatic ductal adenocarcinoma (PDAC) have an average survival of less than 1 year, underscoring the importance of evaluating novel targets with matched targeted agents. We recently identified that poly (ADP) ribose glycohydrolase (PARG) is a strong candidate target due to its dependence on the pro-oncogenic mRNA stability factor HuR (ELAVL1). Here, we evaluated PARG as a target in PDAC models using both genetic silencing of PARG and established small-molecule PARG inhibitors (PARGi), PDDX-01/04. Homologous repair–deficient cells compared with homologous repair–proficient cells were more sensitive to PARGi in vitro. In vivo, silencing of PARG significantly decreased tumor growth. PARGi synergized with DNA-damaging agents (i.e., oxaliplatin and 5-fluorouracil), but not with PARPi therapy. Mechanistically, combined PARGi and oxaliplatin treatment led to persistence of detrimental PARylation, increased expression of cleaved caspase-3, and increased γH2AX foci. In summary, these data validate PARG as a relevant target in PDAC and establish current therapies that synergize with PARGi.Significance:PARG is a potential target in pancreatic cancer as a single-agent anticancer therapy or in combination with current standard of care.

Supplementary Table 1 describes BRCA and KRAS status of PDX models and IC50 of PDDX-001 and olaparib in PDAC/PDX lines

DNA methylation, a key epigenetic driver of transcriptional silencing, is universally dysregulated in cancer. Reversal of DNA methylation by hypomethylating agents, such as the cytidine analogs decitabine or azacytidine, has demonstrated clinical benefit in hematologic malignancies. These nucleoside analogs are incorporated into replicating DNA where they inhibit DNA cytosine methyltransferases DNMT1, DNMT3A and DNMT3B through irreversible covalent interactions. These agents induce notable toxicity to normal blood cells thus limiting their clinical doses. Herein we report the discovery of GSK3685032, a potent first-in-class DNMT1-selective inhibitor that was shown via crystallographic studies to compete with the active-site loop of DNMT1 for penetration into hemi-methylated DNA between two CpG base pairs. GSK3685032 induces robust loss of DNA methylation, transcriptional activation and cancer cell growth inhibition in vitro. Due to improved in vivo tolerability compared with decitabine, GSK3685032 yields superior tumor regression and survival mouse models of acute myeloid leukemia.

Pulmonary hypertension (PH) is a disease of hyperplasia of pulmonary vascular cells. The pentose phosphate pathway (PPP)—a fundamental glucose metabolism pathway—is vital for cell growth. Because treatment of PH is inadequate, our goal was to determine whether inhibition of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the PPP, prevents maladaptive gene expression that promotes smooth muscle cell (SMC) growth, reduces pulmonary artery remodeling, and normalizes hemodynamics in experimental models of PH. PH was induced in mice by exposure to 10% oxygen (Hx) or weekly injection of vascular endothelial growth factor receptor blocker [Sugen5416 (SU); 20 mg kg−1] during exposure to hypoxia (Hx + SU). A novel G6PD inhibitor (N-[(3β,5α)-17-oxoandrostan-3-yl]sulfamide; 1.5 mg kg−1) was injected daily during exposure to Hx. We measured right ventricle (RV) pressure and left ventricle pressure-volume relationships and gene expression in lungs of normoxic, Hx, and Hx + SU and G6PD inhibitor–treated mice. RV systolic and end-diastolic pressures were higher in Hx and Hx + SU than normoxic control mice. Hx and Hx + SU decreased expression of epigenetic modifiers (writers and erasers), increased hypomethylation of the DNA, and induced aberrant gene expression in lungs. G6PD inhibition decreased maladaptive expression of genes and SMC growth, reduced pulmonary vascular remodeling, and decreased right ventricle pressures compared with untreated PH groups. Pharmacologic inhibition of G6PD activity, by normalizing activity of epigenetic modifiers and DNA methylation, efficaciously reduces RV pressure overload in Hx and Hx + SU mice and preclinical models of PH and appears to be a safe pharmacotherapeutic strategy. SIGNIFICANCE STATEMENT The results of this study demonstrated that inhibition of a metabolic enzyme efficaciously reduces pulmonary hypertension. For the first time, this study shows that a novel inhibitor of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme in the fundamental pentose phosphate pathway, modulates DNA methylation and alleviates pulmonary artery remodeling and dilates pulmonary artery to reduce pulmonary hypertension.

Pharmacological induction of fetal hemoglobin (HbF) expression is an effective therapeutic strategy for the management of beta-hemoglobinopathies such as sickle cell disease. DNA methyltransferase (DNMT) inhibitors 5-azacytidine (5-aza) and 5-aza-2′-deoxycytidine (decitabine) have been shown to induce fetal hemoglobin expression in both preclinical models and clinical studies, but are not currently approved for the management of hemoglobinopathies. We report here the discovery of a novel class of orally bioavailable DNMT1-selective inhibitors as exemplified by GSK3482364. This molecule potently inhibits the methyltransferase activity of DNMT1, but not DNMT family members DNMT3A or DNMT3B. In contrast with cytidine analog DNMT inhibitors, the DNMT1 inhibitory mechanism of GSK3482364 does not require DNA incorporation and is reversible. In cultured human erythroid progenitor cells (EPCs), GSK3482364 decreased overall DNA methylation resulting in de-repression of the gamma globin genes HBG1 and HBG2 and increased HbF expression. In a transgenic mouse model of sickle cell disease, orally administered GSK3482364 caused significant increases in both HbF levels and in the percentage HbF-expressing erythrocytes, with good overall tolerability. We conclude that in these preclinical models, selective, reversible inhibition of DNMT1 is sufficient for the induction of HbF, and is well-tolerated. We anticipate that GSK3482364 will be a useful tool molecule for the further study of selective DNMT1 inhibition both in vitro and in vivo.

[Image: see text] A combination of focused library and virtual screening, hit expansion, and rational design has resulted in the development of a series of inhibitors of RET(V804M) kinase, the anticipated drug-resistant mutant of RET kinase. These agents do not inhibit the wild type (wt) isoforms of RET or KDR and therefore offer a potential adjunct to RET inhibitors currently undergoing clinical evaluation.

We report the development of a large scale process for heat inactivation of clinical COVID-19 samples prior to laboratory processing for detection of SARS-CoV-2 by RT-qPCR. With more than 120 million confirmed cases, over 3.8 million deaths already recorded at the time of writing, COVID-19 continues to spread in many parts of the world. Consequently, mass testing for SARS-CoV-2 will remain at the forefront of the COVID-19 response and prevention for the near future. Due to biosafety considerations the standard testing process requires a significant amount of manual handling of patient samples within calibrated microbiological safety cabinets. This makes the process expensive, effects operator ergonomics and restricts testing to higher containment level laboratories. We have successfully modified the process by using industrial catering ovens for bulk heat inactivation of oropharyngeal/nasopharyngeal swab samples within their secondary containment packaging before processing in the lab to enable all subsequent activities to be performed in the open laboratory. As part of a validation process, we tested greater than 1200 clinical COVID-19 samples and showed less than 1 Cq loss in RT-qPCR test sensitivity. We also demonstrate the bulk heat inactivation protocol inactivates a murine surrogate of human SARS-CoV-2. Using bulk heat inactivation, the assay is no longer reliant on containment level 2 facilities and practices, which reduces cost, improves operator safety and ergonomics and makes the process scalable. In addition, heating as the sole method of virus inactivation is ideally suited to streamlined and more rapid workflows such as ‘direct to PCR’ assays that do not involve RNA extraction or chemical neutralisation methods.

Patients with metastatic pancreatic ductal adenocarcinoma (PDAC) have an average survival of less than 1 year, underscoring the importance of evaluating novel targets with matched targeted agents. We recently identified that poly (ADP) ribose glycohydrolase (PARG) is a strong candidate target due to its dependence on the pro-oncogenic mRNA stability factor HuR (ELAVL1). Here, we evaluated PARG as a target in PDAC models using both genetic silencing of PARG and established small-molecule PARG inhibitors (PARGi), PDDX-01/04. Homologous repair–deficient cells compared with homologous repair–proficient cells were more sensitive to PARGi in vitro. In vivo, silencing of PARG significantly decreased tumor growth. PARGi synergized with DNA-damaging agents (i.e., oxaliplatin and 5-fluorouracil), but not with PARPi therapy. Mechanistically, combined PARGi and oxaliplatin treatment led to persistence of detrimental PARylation, increased expression of cleaved caspase-3, and increased γH2AX foci. In summary, these data validate PARG as a relevant target in PDAC and establish current therapies that synergize with PARGi. Significance: PARG is a potential target in pancreatic cancer as a single-agent anticancer therapy or in combination with current standard of care.

On 11th March 2020, the UK government announced plans for the scaling of COVID-19 testing, and on 27th March 2020 it was announced that a new alliance of private sector and academic collaborative laboratories were being created to generate the testing capacity required. The Cambridge COVID-19 Testing Centre (CCTC) was established during April 2020 through collaboration between AstraZeneca, GlaxoSmithKline, and the University of Cambridge, with Charles River Laboratories joining the collaboration at the end of July 2020. The CCTC lab operation focussed on the optimised use of automation, introduction of novel technologies and process modelling to enable a testing capacity of 22,000 tests per day. Here we describe the optimisation of the laboratory process through the continued exploitation of internal performance metrics, while introducing new technologies including the Heat Inactivation of clinical samples upon receipt into the laboratory and a Direct to PCR protocol that removed the requirement for the RNA extraction step. We anticipate that these methods will have value in driving continued efficiency and effectiveness within all large scale viral diagnostic testing laboratories.

Background HPK1, a member of the MAP4K family of protein serine/threonine kinases, is involved in regulating signal transduction cascades in cells of hematopoietic origin. Recent data from HPK1 knockout animals and kinase-inactive knock-in animals underscores the role of HPK1 in negatively regulating immune cell activation. This negative-feedback role of HPK1 combined with its restricted expression in cells of hematopoietic origin, make it a compelling drug target for enhancing anti-tumor immunity. Methods A structure-based drug design approach was used to identify potent and selective inhibitors of HPK1. Biochemical assays, as well as primary human and mouse immune cell-based activation assays, were utilized for multiple iterations of structure-activity relationship (SAR) studies. In vivo efficacy, target engagement and pharmacodynamic data were generated using murine syngeneic tumor models. Results A highly potent, HPK1 inhibitor was identified, that showed high selectivity against T cell-specific kinases and kinases in the MAP4K family. In vitro, HPK1 small molecule inhibition resulted in enhanced IL-2 production in primary mouse and human T cells, enhanced IL-6 and IgG production in primary human B cells, and enhanced mouse dendritic cell activation and antigen presentation capacity. Furthermore, HPK1 inhibition alleviated the immuno-suppressive effects of PGE2 on naive human T cells and restored the proliferative capacity of exhausted human T cells. In vivo, HPK1 inhibitionHPK1 inhibition abrogated T cell receptor-stimulated phospho-SLP-76, enhanced cytokine production, and mediated robust tumor growth inhibition in a murine syngeneic tumor model. Conclusions Pharmacological blockade of HPK1 kinase activity represents a novel and potentially valuable immunomodulatory approach for anti-tumor immunity.

Metabolic reprogramming is considered important in the pathogenesis of the occlusive vasculopathy observed in pulmonary hypertension (PH). However, the mechanisms that link reprogrammed metabolism to aberrant expression of genes, which modulate functional phenotypes of cells in PH, remain enigmatic. Herein, we demonstrate that, in mice, hypoxia-induced PH was prevented by glucose-6-phosphate dehydrogenase deficiency (G6PDDef), and further show that established severe PH in Cyp2c44−/−mice was attenuated by knockdown with G6PD shRNA or by G6PD inhibition with an inhibitor (N-ethyl-N′-[(3β,5α)-17-oxoandrostan-3-yl]urea, NEOU). Mechanistically, G6PDDef, knockdown and inhibition in lungs: 1) reduced hypoxia-induced changes in cytoplasmic and mitochondrial metabolism, 2) increased expression of Tet methylcytosine dioxygenase 2 ( Tet2) gene, and 3) upregulated expression of the coding genes and long noncoding (lnc) RNA Pint, which inhibits cell growth, by hypomethylating the promoter flanking region downstream of the transcription start site. These results suggest functional TET2 is required for G6PD inhibition to increase gene expression and to reverse hypoxia-induced PH in mice. Furthermore, the inhibitor of G6PD activity (NEOU) decreased metabolic reprogramming, upregulated TET2 and lncPINT, and inhibited growth of control and diseased smooth muscle cells isolated from pulmonary arteries of normal individuals and idiopathic-PAH patients, respectively. Collectively, these findings demonstrate a previously unrecognized function for G6PD as a regulator of DNA methylation. These findings further suggest that G6PD acts as a link between reprogrammed metabolism and aberrant gene regulation and plays a crucial role in regulating the phenotype of cells implicated in the pathogenesis of PH, a debilitating disorder with a high mortality rate.

There is mounting evidence of androgen receptor signaling inducing genome instability and changing DNA repair capacity in prostate cancer cells. Expression of genes associated with base excision repair (BER) is increased with prostate cancer progression and correlates with poor prognosis. Poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) are key enzymes in BER that elongate and degrade PAR polymers on target proteins. While PARP inhibitors have been tested in clinical trials and are a promising therapy for prostate cancer patients with TMPRSS2-ERG fusions and mutations in DNA repair genes, PARG inhibitors have not been evaluated. We show that PARG is a direct androgen receptor (AR) target gene. AR is recruited to the PARG locus and induces PARG expression. Androgen ablation combined with PARG inhibition synergistically reduces BER capacity in independently derived LNCaP and LAPC4 prostate cancer cell lines. A combination of PARG inhibition with androgen ablation or with the DNA damaging drug, temozolomide, significantly reduces cellular proliferation and increases DNA damage. PARG inhibition alters AR transcriptional output without changing AR protein levels. Thus, AR and PARG are engaged in reciprocal regulation suggesting that the success of androgen ablation therapy can be enhanced by PARG inhibition in prostate cancer patients.

Pulmonary hypertension (PH) is a multicellular and progressive disease with a high mortality rate. Among many cell types, hematopoietic stem cells (HSCs) are incriminated in the pathogenesis of PH. However, our understanding of the mechanisms that increase HSCs in blood and lungs of hypertensive animals or patients and the role played by HSCs in the pathogenesis of PH remains elusive. Studies suggest that glycolysis is critical for the survival and growth of HSCs. In various cell types from hypertensive lungs of animals and patients, glycolysis and the glucose-6-phosphate dehydrogenase (G6PD) activity are increased. Herein, we demonstrated in mice that chronic hypoxia increased HSCs (CD34+, CD117+, CD133+, CD34+/CD117+, and CD34+/CD133+) in bone marrow and blood and around hypertensive pulmonary arteries in a time-dependent manner. Intriguingly, we found fewer CD133+ cells in the bone marrow of C57BL/6 mice compared with Sv129J mice, and C57BL mice developed less severe chronic hypoxia-elicited PH and heart failure than Sv129J mice. Similarly, the numbers of CD34+ and CD117+ cells in blood of patients with pulmonary arterial hypertension (PAH) were higher (>3-fold) compared with healthy individuals. By allogeneic bone marrow transplantation, we found that GFP+ bone marrow cells infiltrated the lungs and accumulated around the pulmonary arteries in lungs of hypoxic mice, and these cells contributed to increased α-adrenergic receptor-mediated contraction of the pulmonary artery cultured in hypoxia. Inhibition of G6PD activity with (3β,5α)-3,21-dihydroxypregnan-20-one, a novel and potent G6PD inhibitor, decreased HSCs in bone marrow, blood, and lungs of hypoxic mice and reduced α-agonist-induced contraction of the pulmonary artery and established hypoxia-induced PH. We did not observe CD133+ cells around the pulmonary arteries in the lungs of chronically hypoxic G6PD-deficient mice. Furthermore, knockdown of G6PD and inhibition of G6PD activity: 1) downregulated canonical and noncanonical Wnt and Fzd receptors genes; 2) upregulated Bmpr1a; 3) decreased Cxcl12, and 4) reduced HSC (CD117+ and CD133+) numbers. In all, our findings demonstrate unexpected function for bone marrow-derived HSCs in augmenting α-adrenergic receptor-mediated contraction of pulmonary arteries and remodeling of pulmonary arteries that contribute to increase pulmonary vascular resistance in PAH patients and hypoxic mice and suggest that G6PD, by regulating expression of genes in the WNT and BMPR signaling, contributed to increase and release of HSCs from the bone marrow in response to hypoxic stimuli.

International audience; The synthesis of poly(ADP-ribose) (PAR) reconfigures the local chromatin environment and recruits DNA-repair complexes to damaged chromatin. PAR degradation by poly(ADP-ribose) glycohydrolase (PARG) is essential for progression and completion of DNA repair. Here, we show that inhibition of PARG disrupts homology-directed repair (HDR) mechanisms that underpin alternative lengthening of telomeres (ALT). Proteomic analyses uncover a new role for poly(ADP-ribosyl)ation (PARylation) in regulating the chromatin-assembly factor HIRA in ALT cancer cells. We show that HIRA is enriched at telomeres during the G2 phase and is required for histone H3.3 deposition and telomere DNA synthesis. Depletion of HIRA elicits systemic death of ALT cancer cells that is mitigated by re-expression of ATRX, a protein that is frequently inactivated in ALT tumors. We propose that PARylation enables HIRA to fulfill its essential role in the adaptive response to ATRX deficiency that pervades ALT cancers.

In order to exploit the complex tumour microenvironment, immunoreactivity in a multitude of cell-based assays is required to predict mode of action (MOA), pharmacodynamics, and efficacy downstream. We have developed an integrated approach starting with single cell and co-culture cell-based assays in 2D and 3D formats, followed by an in vivo set-up based on humanized mice to monitor an immunotherapeutic's interaction with specific cell types such as T cells including CAR-T cells, natural killer cells, macrophages, dendritic cells, neutrophils, and fibroblasts. Rituximab, a monoclonal antibody against CD20, is mainstay in the therapy for a broad variety of B-cell malignancies. Its MOA is not fully understood as direct signalling, complement dependent cellular cytotoxicity and antibody dependent cellular cytotoxicity (ADCC) all seem to have an impact. To elucidate the share and effect each of those mechanism has, we tested Rituximab in our immuno-oncology translational platform. Furthermore, our immuno-oncology platform was utilized to assess efficacy, bystander effects and off-target toxicity of a CD19 CAR-T cell therapy. In vitro assays for efficacy and bystander effect involved co-culture of the CAR-T cells with a mixture of non-target expressing cells and mid to high target antigen expressing cells, where T cell activation and cell death of both target cell types was quantified. Beyond efficacy the assay platforms were used to examine the influence of the therapy, antibodies or CAR-T cells, on normal human cells. These in vitro assays utilizing primary human cells from healthy tissue and/or differentiated iPCS-derived cells were used to assess cytotoxicity. The primary tissues were selected based on their potential safety risk by either expressing low levels of the target antigen or being major organs to de-risk for off-target effects. The results from our in vitro screen were validated in a xenograft in vivo system in the presence and absence of human immune cells. Four cell line derived xenografts were tested in vivo in immunocompromised mice subcutaneously and disseminated. The presence of human immune cells enhanced the antitumoral activity of Rituximab markedly, although the targeted effect of Rituximab induced already a significant prolongation in the overall survival (p< 0.005; Log-rank (Mantel-Cox) test).Over the last 30 years IO has progressed considerably with approvals for the use of various IO therapeutics including vaccines, cytokines, tumor-directed monoclonal antibodies, immune checkpoint inhibitors as well as Chimeric antigen receptor (CAR) and T cell receptor (TCR) engineered T cell therapies. Comprehensive characterization of novel IO compounds using a multitude of such in vitro and in vivo assays is essential to provide insights and understanding of the detailed mechanism of action and identify potential toxicity issues to ultimately improve translation to the clinic. Citation Format: Shilina Roman, Julia Schüler, Sanne Holt, Jeroen DeGroot, Omar Aziz, Ian Waddell. An integrated in vitro and in vivo approach providing insights in immune modulation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6705.

CAR-T-cell therapies represent a new area of synthetic biology success in cancer immunotherapy. However, the complexity of their adverse events related to their activity are still a major challenge. Using our in-house fully human computationally optimized phage display library, we established a novel cell-based ultra-high throughput panning and screening CAR-T platform. Our proprietary CAR-T vector is designed as a series of cassettes incorporating our SuperHuman phage library with an optimal backbone. This allows us to design CAR-T with desired properties, including: Range of Affinities, Humanness, Epitope Diversity, Thermostability, Immunogenicity, Tonic activation, Toxicity, and Functional Activity. Our library's encoding ensures a single CAR-T construct per one reporter cell. Moreover, to confirm CAR-T-specific-activation, we incorporated three different markers that will be upregulated simultaneously. We used CD19 and BCMA to validate our platform selecting target-specific functionally active scFv candidates against both targets, which are selective to activation conditions such as tonic signaling, and tissue specific silencing. Collectively we show that our platform can be readily used to characterize CAR-T in high-speed and - throughput fashion. Citation Format: Sawsan Youssef, Joyce Chou, Devin Pineda, Sarah Ives, Valerie Chiou, Jean-Philippe Buerckert, Raymond Newland, Shahrad Daraeikia, Sindy Liao, Chelsea Jones, Christina Pettus, Jessica Salas, Emelia Padilla, Christopher Smith, Giles Day, David Maurer, Ian Waddell, Jacob Glanville. Advanced engineering using CAR-T display libraries: Ultra-high throughput functional screen [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 523.

Graphical abstract, Fluorination of metabolic hotspots in a molecule is a common medicinal chemistry strategy to improve in vivo half-life and exposure and, generally, this strategy offers significant benefits. Here, we report the application of this strategy to a series of poly-ADP ribose glycohydrolase (PARG) inhibitors, resulting in unexpected in vivo toxicity which was attributed to this single-atom modification.

DNA damage repair enzymes are promising targets in the development of new therapeutic agents for a wide range of cancers and potentially other diseases. The enzyme poly(ADP-ribose) glycohydrolase (PARG) plays a pivotal role in the regulation of DNA repair mechanisms; however, the lack of potent drug-like inhibitors for use in cellular and in vivo models has limited the investigation of its potential as a novel therapeutic target. Using the crystal structure of human PARG in complex with the weakly active and cytotoxic anthraquinone 8a, novel quinazolinedione sulfonamides PARG inhibitors have been identified by means of structure-based virtual screening and library design. 1-Oxetan-3-ylmethyl derivatives 33d and 35d were selected for preliminary investigations in vivo. X-ray crystal structures help rationalize the observed structure–activity relationships of these novel inhibitors.

The failure of DNA ligases to complete their catalytic reactions generates cytotoxic adenylated DNA strand breaks. The APTX RNA‐DNA deadenylase protects genome integrity and corrects abortive DNA ligation arising during ribonucleotide excision repair and base excision DNA repair, and APTX human mutations cause the neurodegenerative disorder ataxia with oculomotor ataxia 1 (AOA1). How APTX senses cognate DNA nicks and is inactivated in AOA1 remains incompletely defined. Here, we report X‐ray structures of APTX engaging nicked RNA‐DNA substrates that provide direct evidence for a wedge‐pivot‐cut strategy for 5′‐AMP resolution shared with the alternate 5′‐AMP processing enzymes POLβ and FEN1. Our results uncover a DNA‐induced fit mechanism regulating APTX active site loop conformations and assembly of a catalytically competent active center. Further, based on comprehensive biochemical, X‐ray and solution NMR results, we define a complex hierarchy for the differential impacts of the AOA1 mutational spectrum on APTX structure and activity. Sixteen AOA1 variants impact APTX protein stability, one mutation directly alters deadenylation reaction chemistry, and a dominant AOA1 variant unexpectedly allosterically modulates APTX active site conformations.

Protein ADP-ribosylation is a conserved posttranslational modification that regulates many major cellular functions, such as DNA repair, transcription, translation, signal transduction, stress response, cell division, aging, and cell death. Protein ADP-ribosyl transferases catalyze the transfer of an ADP-ribose (ADPr) group from the β-nicotinamide adenine dinucleotide (β-NAD+) cofactor onto a specific target protein with the subsequent release of nicotinamide. ADP-ribosylation leads to changes in protein structure, function, stability, and localization, thus defining the appropriate cellular response. Signaling processes that are mediated by modifications need to be finely tuned and eventually silenced and one of the ways to achieve this is through the action of enzymes that remove (reverse) protein ADP-ribosylation in a timely fashion such as PARG, TARG1, MACROD1, and MACROD2. Here, we describe several basic methods used to study the enzymatic activity of de-ADP-ribosylating enzymes.

New targets for cancer treatment frequently emerge in literature, but the thorough target validation required to consider these targets for a drug discovery program is often lacking. In pharmacological or genetic perturbation studies using complex biological assays, undesired off-target effects cannot be easily distinguished from the intended mode of action at the desired target. This is especially evident in cancer drug development where it is important to discriminate on-target effects on cell viability from off-target effects resulting in non-specific loss of cellular fitness. Neglecting the possibility of being deceived by off-target effects can have tremendous scientific and financial impact on a drug discovery program. Ideally, confidence in a preclinical drug target and a modulating compound is boosted in an early stage by more extensive analysis and validation of the actual drug-target relationship. Rescue of a disease-relevant phenotype by genetic restoration of a target mutation is a gold standard approach in drug discovery by which target validation can be achieved. We aim to follow this approach targeting the BRAF V600E mutation in a number of well described melanoma lines as well as the MAP2K1 Q56P mutation in the non-small cell lung cancer cell line H1437. Target validation for both BRAF and MAP2K1 will be addressed by assessing viability, phenotypic changes and sensitivity to compound modulation upon CRISPR/Cas9 repair of the target mutation or by exogenous re-expression of the wild type variant. Compounds tested will be Vemurafenib for BRAF and Trametinib for MAP2K1. Further investigation into target validity will be done using a physiologically relevant 3D spheroid based co-culture system. Mimicking the tumor microenvironment increases the knowledge about “drug-ability” of a target and sustainability of the target modulation at an early time point in the development process. Such early in-depth validation of the relationship between a compound and the drug target is vital to mitigate the risk of failure at later steps of drug development. Citation Format: Laure Grandmoursel, Lieke Geerts, Geraldine Servant, Miranda van der Ham, Armin Maier, Jamil Aarbiou, Marijn Vlaming, Jeroen DeGroot, Julia Schuler, Ian Waddell, Anne-Marie Zuurmond. Validation of the interaction between a candidate compound and the intended drug target by a phenotypic rescue approach [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C021. doi:10.1158/1535-7163.TARG-19-C021

Recent Nobel awards in immune checkpoint inhibitor biology, phage display discovery and protein evolution highlight our current golden age of immune oncology research. While it took 15 years to bring anti-CTLA4 and anti-PD1 therapeutics from conception to global patient populations, new advances in synthetic and data driven immune engineering are enabling new therapeutics to be discovered and engineered in weeks rather than years. Here we review ten years of progress in computational optimization of antibody discovery libraries through the lens of high-throughput data collection technologies, with specific case studies of modern repertoire design principles applied to a panel of immune checkpoint targets. We emphasize the case study of PD1, where a computationally optimized library generated over 6500 unique anti-PD1 antibodies in two weeks, including picomolar binders, mouse/cyno/human triple cross-reactive epitopes, antagonists, agonists, and saturated epitope coverage of the target. Citation Format: Sarah Ives, David Maurer, Christina Pettus, Raymond Newland, Valerie Chiou, Shahrad Daraeikia, Chelsea Jones, Kieran Hervold, Giles Day, Chris Smith, Ian D. Waddell, Sawsan Youssef, Jacob Glanville. Bruteforcing immune oncology discovery with computational immuno-engineering [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1550.

Charles River (CR) has developed many disease-relevant cellular models to screen small molecules and genetic modifiers for target and phenotypic based drug discovery. Our expertise and portfolio in bespoke oncology model development (e.g. patient derived xenografts) covers a wide variety of target classes such as inhibitors of cell cycle, cellular motility and migration and transcription factor translocation. Complex cellular models derived from primary cells, patient derived embryonic stem cells and induced pluripotent stem cells have been adapted for phenotypic screening. Models have been further developed to produce physiologically relevant multicellular structure co-cultures. Combining complex biological systems with multiparametric high content analysis provides detailed single cell quantification of cellular and subcellular biology. Such validated High Content assays can be applied to both target and phenotypic based drug-discovery platforms to support preclinical drug discovery and improve translation of targets and compounds to the clinic. Citation Format: Servane Lachize, Rhea van de Bospoort, Marta da Silva da Silva, Niki van der Steentraten, Ian Gowers, Roger Clark, Jeroen DeGroot, Ian Waddell, Hajo Schiewe, Shushant Jain. High content analysis of 2D and 3D oncology models for target and phenotypic drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 986.

The recently discovered enzyme tyrosyl-DNA phosphodiesterase 2 (TDP2) has been implicated in the topoisomerase-mediated repair of DNA damage. In the clinical setting, it has been hypothesized that TDP2 may mediate drug resistance to topoisomerase II (topo II) inhibition by etoposide. Therefore, selective pharmacological inhibition of TDP2 is proposed as a novel approach to overcome intrinsic or acquired resistance to topo II-targeted drug therapy. Following a high-throughput screening (HTS) campaign, toxoflavins and deazaflavins were identified as the first reported sub-micromolar and selective inhibitors of this enzyme. Toxoflavin derivatives appeared to exhibit a clear structure-activity relationship (SAR) for TDP2 enzymatic inhibition. However, we observed a key redox liability of this series, and this, alongside early in vitro drug metabolism and pharmacokinetics (DMPK) issues, precluded further exploration. The deazaflavins were developed from a singleton HTS hit. This series showed distinct SAR and did not display redox activity; however low cell permeability proved to be a challenge.

Topoisomerases regulate DNA topology by the transient cleavage and religation of DNA during transcription and replication. Topoisomerase II (Topo II) poisons such as etoposide can induce abortive DNA strand breaks in which Topo II remains covalently bound to a 5′ DNA strand terminus via a phosphotyrosyl linker. Tyrosyl DNA phosphodiesterase 2 (Tdp2) is a recently discovered human 5′-tyrosyl DNA phosphodiesterase that repairs this topoisomerase-mediated DNA damage, thereby playing a central role in maintaining normal DNA topology in cells. Cellular depletion of Tdp2 has been shown to result in increased susceptibility and sensitivity to Topo II-induced DNA double-strand breaks, thereby revealing Tdp2 as a potentially attractive anticancer target. No drug-like inhibitors of Tdp2 have been identified to date, and assays suitable for high-throughput screening (HTS) have not been widely reported. Here we have identified a new and effective chromogenic substrate for Tdp2 and developed a homogeneous and robust HTS assay. A second novel Tdp2 assay was also developed to cross-validate hit matter identified from an HTS. In addition, a new and specific Tdp2 antibody is described. Together, these new tools will aid in the identification of novel Tdp2 inhibitors and the investigation of the role of Tdp2 in cancer.

We present IncucyteDRC, an R package for the analysis of data from live cell imaging cell proliferation experiments carried out on the Essen Biosciences IncuCyte ZOOM instrument. The package provides a simple workflow for summarising data into a form that can be used to calculate dose response curves and EC50 values for small molecule inhibitors. Data from different cell lines, or cell lines grown under different conditions, can be normalised as to their doubling time. A simple graphical web interface, implemented using shiny, is provided for the benefit of non-R users. The software is potentially useful to any research group studying the impact of small molecule inhibitors on cell proliferation using the IncuCyte ZOOM.

Poly(ADP-ribose) (PAR) polymers are transient post-translational modifications, and their formation is catalyzed by poly(ADP-ribose) polymerase (PARP) enzymes. A number of PARP inhibitors are in advanced clinical development for BRCA-mutated breast cancer, and olaparib has recently been approved for BRCA-mutant ovarian cancer; however, there has already been evidence of developed resistance mechanisms. Poly(ADP-ribose) glycohydrolase (PARG) catalyzes the hydrolysis of the endo- and exo-glycosidic bonds within the PAR polymers. As an alternative strategy, PARG is a potentially attractive therapeutic target. There is only one PARG gene, compared with 17 known PARP family members, and therefore a PARG inhibitor may have wider application with fewer compensatory mechanisms. Prior to the initiation of this project, there were no known existing cell-permeable small molecule PARG inhibitors for use as tool compounds to assess these hypotheses and no suitable high-throughput screening (HTS)-compatible biochemical assays available to identify start points for a drug discovery project. The development of this newly described high-throughput homogeneous time-resolved fluorescence (HTRF) assay has allowed HTS to proceed and, from this, the identification and advancement of multiple validated series of tool compounds for PARG inhibition.

Novel derivatives of the steroid DHEA 1, a known uncompetitive inhibitor of G6PD, were designed, synthesized, and tested for their ability to inhibit this dehydrogenase enzyme. Several compounds with approximately 10-fold improved potency in an enzyme assay were identified, and this improved activity translated to efficacy in a cellular assay. The SAR for steroid inhibition of G6PD has been substantially developed; the 3β-alcohol can be replaced with 3β-H-bond donors such as sulfamide, sulfonamide, urea, and carbamate. Improved potency was achieved by replacing the androstane nucleus with a pregnane nucleus, provided a ketone at C-20 is present. For pregnan-20-ones incorporation of a 21-hydroxyl group is often beneficial. The novel compounds generally have good physicochemical properties and satisfactory in vitro DMPK parameters. These derivatives may be useful for examining the role of G6PD inhibition in cells and will assist the future design of more potent steroid inhibitors with potential therapeutic utility.

Poly(ADP-ribose) glycohydrolase (PARG) hydrolyzes poly(ADP-ribose) (PAR) chains that are polymerized by PARP enzymes, completing the PAR cycle. Small molecule inhibitors of PARG result in a dose dependent increase in cellular PAR after DNA damage. Here we demonstrate that depletion of XRCC1, a scaffolding protein with an essential role in base-excision repair (BER), sensitizes cancer cells to PARG inhibition. XRCC1 deficient cells exhibit increased nuclear PAR foci in response to PARG inhibition even in the absence of DNA damaging agents. Inhibition of PARP1 with RNAi or small molecule inhibitors rescues cell growth inhibition and reduces the amount of cellular PAR accumulation in PARG inhibitor treated cells. This indicates that the cellular growth inhibition is dependent upon cellular PAR levels, demonstrating selectivity of the small molecule inhibitors for PARG. We hypothesized that inhibition of PAR hydrolysis could result in depletion of cellular NAD as this could prevent recycling of PAR to NAD. Consistent with this hypothesis, PARG inhibition enhanced NAD depletion after treatment of cells with the DNA damaging agent methyl methanesulfonate (MMS). Live cell imaging of XRCC1 depleted cells treated with a PARG inhibitor revealed that cells have large membrane protrusions, similar to the morphology of cells that have been treated with a NAMPT inhibitor, which results in depletion of cellular NAD. Furthermore, addition of the NAD precursor, nicotinamide mononucleotide (NMN) rescued proliferation of PARG inhibited cells. Taken together, these data support a hypothesis in which PARG inhibitors are cytotoxic to sensitive cancer cells via depletion of NAD, ultimately starving the cell of ATP. Thus, PARG inhibition is a novel strategy for exploiting synthetic lethality in cancer cells. The defects that sensitize cancer cells to PARG inhibition are distinct from those that sensitize to PARP inhibitors, namely defects in homology directed repair. Approximately 15% of breast cancer samples exhibit low or no XRCC1 by IHC. A subset (approximately 35%) of the XRCC1 low patient samples also have defects in BRCA1, suggesting that the majority of XRCC1 low tumors may not be responsive to PARP inhibitors. Small molecule PARG inhibitors are currently being evaluated for efficacy in XRCC1 low xenograft models. Citation Format: Leenus Martin, Tzuling Cheng, Dominic I. James, Habiba Begum, Kate M. Smith, Allan Jordan, Ian Waddell, Kedar Vaidya, Marcus Fischer, Bing Yao, Jason Drummond, Leah Cleary, Ruben Martinez, James Sutton, Nandini Ravindran, James Joseph, Eleni Venetsanakos, Michael Dillon, Jeffrey H. Hager, Lisa D. Belmont. PARG inhibitors exhibit synthetic lethality with XRCC1 deficiency and a cellular mechanism of action that is distinct from PARP inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1943.

Metastatic pancreatic ductal adenocarcinoma (PDA) has an average survival of less than one year. There is a pressing need to identify patient subgroups for treatment with novel targeted agents and the necessity to combat increasing incidence of therapeutic resistance. In a previous study, we identified that poly(ADP) ribose glycohydrolase (PARG) is a critical player in mediating resistance to PARP inhibitor (PARPi); therefore targeting PARG is a strategy to enhance PARPi therapy in PDA and can be optimized to benefit patients with or without homologous repair (HR) deficiencies. We developed and characterized multiple PARG inhibition models in both DNA- repair proficient (MIA PaCa-2) and deficient (Hs766t) PDA lines; doxycycline-inducible shPARG knockdown, CRISPR- mediated PARG knockout and small molecule inhibition via a series of potent first-in-class, cell-active PARG inhibitors (PARGi). Our data show that PARG inhibition is synthetic lethal with DNA damage repair deficiency in PDA cells. This was further validated in isogenic colorectal cell lines with varying DNA repair functionality: DLD1 lines with BRCA2 (+/+, +/-, -/-) and RKO lines with FANCC (+/+, +/-, -/-). We have also shown that PARG inhibition enhances PARPi sensitivity through increased accumulation of DNA damage, apoptosis and persistence of detrimental PARylation. Moreover, PARP1 was trapped on the chromatin in response to both PARPi treatment as well as DNA damaging agents such as oxaliplatin. Complementary xenograft experiments were performed wherein MIA.shPARG cells were injected in nude female athymic mice. At an average tumor volume of 50mm3, respective groups were fed DOX- chow to induce PARG knockdown and treated with olaparib intraperitoneally at 100mg/kg five times a week. PARG inhibition by doxycycline induction significantly decreased tumor volumes (50% decrease, p-value 0.0165), which was further enhanced with olaparib treatment (70% decrease, p- value 0.0004), when compared with control arms. Similar results were obtained when DOX-fed mice with MIA.shPARG cells were treated with olaparib at 50mg/kg. Furthermore, in an attempt to mimic and break long-term in vivo PARPi resistance, doxycycline-mediated PARG inhibition was induced in the olaparib treatment arm on day 56 (with established tumors, and exposed to olaparib for 3weeks i.e. 15 injections). This resulted in a significant decrease in tumor volume when compared to control untreated arm (46% decrease, p-value 0.0025) and the olaparib only treatment arm (25% decrease, p-value 0.0124). We are currently validating these results in a DDR-deficient HST.shPARG cell line, as well as with CRISPR knockouts of PARG. Together these studies validate PARG as a therapeutically relevant and “druggable” target in both HR-proficient and deficient PDA cells, and lays the groundwork to optimize PARPi-based as well as other DNA targeted therapies in the treatment of PDA. Citation Format: Saswati N. Chand, AnnJosette Ramirez, Aditi Jain, Avinoam Nevler, Cinthya Yabar-Lowder, Joseph A. Cozzitorto, Dominic I. James, Allan Jordan, Kate M. Smith, Ian Waddell, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. Targeting PARG in pancreatic cancer: Implications for synthetic lethal therapeutic strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1973.

DNA Methyltransferases (or DNMTs) play an important role in genomic integrity. The DNMT family of enzymes catalyse the transfer of methyl groups to specific CpG structures on DNA, with DNMT1 acting as a maintenance methyltransferase and DNMT3a and 3b acting as de novo methyltransferases. Upon cell division, epigenetic profiles within DNA strands are copied, with DNMT1 ensuring that methyl marks are transferred correctly to the newly synthesised DNA strand, thereby conferring heritability. Aberrant DNA hypermethylation within DNA promoter regions can lead to gene silencing, which is a hallmark of human cancer. Treatment with hypomethylating agents such as azacytidine and decitabine reverse methyl marks and show clinical benefit for the treatment of Acute Myeloid Leukemia (AML) and Chronic Myelomonocytic Leukemia (CMML) and are also the standard of care for Myelodysplastic Syndrome (MDS). However, these agents also show dose limiting toxicity due to their irreversible and non-selective mechanism of action and generally poor pharmacokinetic properties. Over the past decades both the Pharma and Biotech industries have deployed significant resource in a bid to identify and develop potent, selective DNMT1 inhibitors. These attempts have delivered little, if any, success until now. Collaboration between the CRUK Manchester Institute and GSK has resulted in the successful development of a screening cascade to identify compounds that selectively inhibit DNMT1. A collection of over 1 million compounds was screened at GSK using a radioactive scintillation proximity assay (SPA). Preliminary hits were identified and triaged through our screening cascade to determine genuine hits from false positive results. Firstly, ‘hits' from the SPA screen were tested in rapid-fire mass spectrometry and also in fluorescence intensity coupled ‘break-light' assays. Though these orthogonal screens apparently confirmed activity, IC50 screening of many of these compounds revealed high Hill slopes and high maximal inhibition profiles which are indicative of non-specific inhibition mechanisms. Detailed mechanism of inhibition studies alongside differential scanning fluorimetry was employed to rule out those compounds that were binding non-specifically to the oligonucleotide substrate. One series of compounds displayed IC50 profiles consistent with specific inhibition of DNMT1, with a 1:1 stoichiometry and no evidence for non-specific binding to the oligonucleotide substrate. Selectivity screening against DNMT3a and 3b also showed that within the DNMT family this series of compounds was selective for DNMT1 only. All compounds that displayed reasonable biochemical potency, (sub 1µΜ), were tested further in cellular assays to confirm target engagement. Through our extensive range of assays and thorough profiling of hit compounds, we were able to quickly de-validate the significant number of false positive hits that have plagued previous drug discovery screens in this area and identify a series of genuine, selective, small molecule inhibitors of DNMT1. Citation Format: Alexandra Stowell, Graeme Thomson, Mark Cockerill, Charlotte Burt, Emma Fairweather, Ian Waddell, Ali Raoof, Allan Jordan, Donald 0gilvie, Melissa Pappalardi, Juan Luengo, Mehul Patel, Ryan Kruger, Chris Carpenter. Development of a screening cascade to identify selective small molecule inhibitors of DNMT1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-068.

Charles River Laboratories (CRL) has established a powerful translational immuno-oncology platform with the capability of progressing biologics or small-molecule modulators of immune response from in vitro to in vivo assays using human and mouse variants of current checkpoint inhibitors and small molecules. Utilizing a blood donor panel, our in vitro platform includes primary human immune cell assays that profile T-cell activation, T cell mediated-cancer cell kill, expansion of T-cell populations, mixed lymphocyte reactions (MLR), T-cell invasion, and antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The platform is currently being expanded to determine the effect of activated immune cell populations on tumor cell spheroid cultures. It has been validated with standard-of-care chemotherapeutics, including anti-CTLA4, anti-PD1 and a selection of small-molecule inhibitors of targets known to modulate immune responses including IDO inhibitors. Ex vivo analysis of activated mouse splenocyte response to checkpoint inhibitors, measured as cytokine release, and modulation of immune cell populations, as determined by flow cytometry, supports the translation of important compounds from the bench to preclinical models. Syngeneic mouse tumor models have frequently been used to profile immune responses in tumors; CRL has optimized and profiled existing checkpoint inhibitors to support immuno-oncology drug discovery using mouse and rat antibody variants of anti-CTLA4 and anti-PD1. To confirm the translational development of our platform in human tissue, CRL has developed and optimized humanized mouse models using subcutaneous implanted patient-derived xenografts (PDX) with human engraftment via CD34+ haematopoeitic stem cells in NOG mice, which were treated with anti-CTLA4 and anti-PD1. Infiltration of human immune cells and PDL-1 expression was detected by flow cytometry (FC) and immunohistochemistry (IHC) in hematopoietic organs and tumor tissue, supporting the initial in vitro response in primary immune cells. Here we present a screening platform, validated with standard-of-care chemotherapeutics, which will support translation of compounds from in vitro primary immune cell assays to modulation of mouse immune cell population in spleen and tumors, resulting in efficacy and tumor immune cell activation in humanized mouse models. Citation Format: Martin O'Rourke, Shilina Roman, Gary Salmon, Ian Waddell, Julia Schueler, Edgar Wood. A translational platform using primary human immune cells in vitro, syngeneic and humanized models in vivo to support and advance immuno-oncology drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1730.

This study lays the groundwork to target poly-ADP ribose glycohydrolase (PARG), a DNA repair enzyme that counteracts PARP-1 activity and is upregulated in response to PARP inhibitor (PARPi) treatment. The goal of this study is to optimize PARPi-based therapies for all pancreatic cancer patients, irrespective of their DNA repair status (i.e., homologous repair (HR) deficiency). Metastatic pancreatic ductal adenocarcinoma (PDA) is a lethal disease with an average survival of less than one year, which highlights the need for novel treatment regimens. Though PARPi therapy offers a personalized strategy in a subset of tumors with HR defects, initially responsive patients typically become resistant to therapy. Therefore, identifying and combating PARPi resistance mechanisms is a rising concern. We have previously shown that PARG is a critical player in mediating resistance; therefore we evaluated PARG as a candidate target in PDA. Importantly, we demonstrated that the pro-oncogenic protein, HuR (ELAVL1), upregulates PARG specifically in tumor microenvironment niches (e.g., drug exposure), thus providing a unique, cancer-specific therapeutic window for targeting PARG. We evaluated a series of potent first-in-class, cell-active small molecule PARG inhibitors; PDDX-01, PDDX02 and PDDX-04 in pre-clinical models of PDA. These PARGi effectively inhibited PARG activity, caused persistence of PAR polymers, increased accumulation of γH2AX foci and inhibited short and long-term growth of PDA cell lines. Similar to PARPi agents, PARG inhibition was also shown to be synthetic lethal; DNA damage repair (DDR)- deficient cell lines were more sensitive than the DDR- proficient PDA cell lines. This was further validated in isogenic colorectal cell lines with varying DNA repair functionality; DLD1 lines with BRCA2 (+/+, +/-, -/-) and RKO lines with FANCC (+/+, +/-, -/-) modulation. Using chromatin tethering assays, these PARGis effectively trapped PARP1 on DNA, alone as well as combination with the PARPi olaparib, and a DNA damaging agent oxaliplatin. We also developed and characterized a PARG inhibition model in vivo in a DNA- repair proficient PDA line, MIA PaCa2. The doxycycline-inducible shPARG knockdown was validated for PARG inhibition, PAR accumulation and increased PARPi sensitivity in vitro. Complementing xenograft experiments were performed wherein 3 million cells were injected per flank in nude female athymic mice. At an average tumor volume of 50mm3, respective groups were fed DOX- chow to induce PARG knockdown and treated with olaparib intraperitoneally at 50mg/kg five times a week. PARG inhibition by doxycycline induction significantly decreased tumor volumes (50% decrease, p-value 0.0165), which was further enhanced with olaparib treatment (70% decrease, p- value 0.0004), when compared with control arms. Taken together, these studies help define and break a potent PARPi resistance mechanism and also validate pursuing PARG as a valuable “druggable” target in both HR-proficient and deficient PDA cells. Citation Format: Saswati N. Chand, AnnJosette Ramirez, Aditi Jain, Avinoam Nevlar, Cinthya Yabar-Lowder, Joseph A. Cozzitorto, Dominic I. James, Ian D. Waddell, Charles J. Yeo, Michael J. Pishvaian, Jordan M. Winter, Jonathan R. Brody. Targeting poly-ADP ribose glycohydrolase: pursuing a synthetic lethal therapeutic strategy to treat all pancreatic cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A13.

Background: Constitutive activation of RET kinase activity following mutation or rearrangement can lead to the development of cancers such as medullary thyroid carcinoma and lung adenocarcinoma. The currently approved therapeutics for these diseases are significantly compromised due to dose-limiting toxicities associated with off-target activity vs KDR (VEGFR2) and lack of potency vs anticipated secondary resistance (e.g., gatekeeper) mutations. Consequently there is considerable interest in the development of highly selective inhibitors targeting diverse RET alterations including the putative resistance mutation, V804M. Methods: We have established a robust screening cascade complemented by structure-enabled drug design and effective medicinal chemistry. Biochemical activity vs RET, KDR, and RETV804M protein was assessed using a HTRF assay. Cellular activity was quantified in BaF3 cells dependent on activity of RET, KDR, or RETV804M for proliferation. Tumor growth inhibition and supporting PK/PD studies were carried out in a number of disease-relevant models including a KIF5B-RET lung cancer patient-derived xenograft (PDX) model, a medullary thyroid carcinoma (MZ-CRC-1) xenograft model, and a lung cancer control (Calu-6) xenograft model. Results: Using this optimized, robust platform, we have identified a number of selective compounds offering a range of interesting biochemical and cellular profiles, targeting either, or both, RET and the gatekeeper mutant, RETV804M. We believe certain examples of these compounds offer the first cell-active RETV804M-selective derivatives. More importantly perhaps, we have also delivered a highly selective preclinical candidate compound demonstrating potency vs both RET fusion and RETV804M. This compound is well tolerated in vivo after oral dosing at up to 80mg/kg bid and, in a KIF5B-RET lung cancer PDX model, demonstrates efficacy at much lower doses: 50% tumor regression at 20mg/kg bid and 92% tumor growth inhibition at 10mg/kg bid. Importantly, this agent shows no efficacy in the (non-RET driven) Calu-6 xenograft model, demonstrating selective inhibition of the RET kinase domain. Conclusions: We believe that the identification of well-tolerated, selective RET inhibitors with potent activity against diverse RET alterations (including the anticipated resistance mutation, V804M) offers a clear therapeutic advantage over the present clinically approved compounds. Our most advanced compound fulfills all of these challenging criteria and has now entered preclinical development. Citation Format: Mandy Watson, Rebecca Newton, Ben Acton, Helen Small, Habiba Begum, Samantha Hitchin, Paul Kelly, Donald Ogilvie, Ian Waddell, Allan Jordan. Delivery of a potent, selective, and efficacious RET inhibitor for the treatment of RET-driven lung adenocarcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A123.

The identification of novel oncology targets for small molecule drug discovery is becoming increasingly challenging despite 10-15% of the human genome estimated to be druggable. The literature is an important source of novel targets; however, several recent reports by pharma and academia have indicated that approximately 11-55% of published studies are irreproducible. Moreover, oncology drug attrition rates are extremely poor, with 66% of candidates in Phase III clinical trials not achieving approval. This is reflected by increasing failure rates of drug development projects in Phase II, from 72% in 2006-2007 to 82% in 2008-2009. These failures have been attributed to lack of efficacy, thus highlighting the critical requirement for new novel drug targets that demonstrate clear promise of clinical efficacy through predictive in vitro and in vivo models. Furthermore, it is important to prioritize and pursue targets that are most likely to bind a small molecule inhibitor with high infinity. We sought to utilize the wealth of unvalidated genomic data now available in the public domain by developing a bioinformatic pipeline founded on the collateral vulnerability hypothesis, which is based on the concept of synthetic lethality and exploits the co-occurrence of deletions of genes flanking tumor suppressors that are lost during the course of tumor evolution. We can then target the paralogs of these genes if together they belong to gene families with essential predicted function, thereby exploiting a vulnerability of the cancer cells with these losses. Here, we describe our industry-standard approaches to the target validation of the output generated from lung adenocarcinoma data, and present the validation and de-validation of several novel targets predicted by the collateral vulnerability pipeline, including SMARCA2, KMT2C, and SETD1B. Citation Format: H. Nikki March, Phil Chapman, Elizabeth Blaikley, Catherine A. Eberlein, Mark Cockerill, Samantha Hitchin, Ian D. Waddell, Donald Ogilvie. Discovery, validation and targeting of novel synthetic lethal interactions in academic drug discovery [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B05.

Activating gene fusions in the RET receptor tyrosine kinase have been found to drive 1-2% of lung adenocarcinomas and therefore offer an attractive target for targeted therapy. Whilst non-selective tyrosine kinase inhibitors with RET activity are efficacious in this setting, their use is generally limited by dose limiting toxicity associated with their more potent activity versus other targets, specifically KDR (VEGFR2) in the case of cabozantinib and vandetanib. Given this limitation, there is considerable interest in developing more selective inhibitors of RET kinase. Tyrosine kinase inhibitors are prone to early clinical failure due to mutations in the kinase ATPase binding domain, which render the kinase catalytically active but no longer sensitive to drug treatment. Such mutations often occur in the so-called “gatekeeper” region and in this specific case, resistance is predicted to arise from a Val-Met or Val-Leu mutation at residue 804. Through a combination of computational methods, structural biology and drug design, we have identified and further optimized a series of inhibitors of the V804M mutant RET kinase which show sub-micromolar cellular activity in cells driven by V804M RET. Moreover, these agents show excellent selectivity against the wtRET kinase and KDR. As such, these agents may offer valuable start-points for second-generation RET inhibitors for use in patents who relapse after treatment with first generation selective RET inhibitors. Citation Format: Allan M. Jordan, Rebecca Newton, Bohdan Waszkowycz, Richard Bayliss, Habiba Begum, Daniel Burschowsky, Aude Echalier, Samantha Hitchin, Colin Hutton, Shaun Johns, Stuart Jones, Li-Ying Lin, Mark Richards, Chitra Seewooruthun, Alex Stowell, Ian Waddell, Mandy Watson, Donald Ogilvie. Delivering selective and cell-active inhibitors of V804M mutant RET kinase through structure-guided drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3236. doi:10.1158/1538-7445.AM2017-3236

Background: The aim of this CRUK-MI Drug Discovery project is to deliver a RET-selective inhibitor for the treatment of cancers with RET activating mutations, which include 1-2% of lung adenocarcinomas and medullary thyroid cancers (MTC). Recent data supports the hypothesis that the efficacy of vandetanib and cabozantinib, clinically approved multi-kinase inhibitors, is limited by toxicities associated with potent activity against KDR. Therefore, a RET-selective inhibitor would represent a best-in-class agent for the treatment of these cancers. Methods: We have established a robust screening cascade to develop a potent, selective RET inhibitor and developed several in vivo models to evaluate compound PKPD and antitumor efficacy. Tumor growth inhibition and PKPD studies were carried out in BaF3 mouse allograft models overexpressing KIF5B-RET or RETV804M and other disease relevant models, including an MTC xenograft (MZ-CRC-1), a KIF5B-RET lung cancer patient derived xenograft (PDX) model (CTG-0838, Champions Oncology) and a lung cancer control xenograft (Calu-6). Results: Two orally bioavailable compounds displaying nanomolar RET potency and >10 fold selectivity over KDR in cellular assays were selected from the lead series and further evaluated in our in vivo PD and efficacy models. Both compounds demonstrated efficacy in the BaF3 KIF5B-RET driven model (71% and 103% tumor growth inhibition (TGI), respectively), accompanied by reduced levels of pRET in the tumor tissue. Following further lead optimisation; a compound displaying an improved DMPK profile and additional nanomolar potency versus the gatekeeper mutation (RETV804M) was identified and accelerated through our DMPK/in vivo cascade. We consider this additional activity versus RETV804M beneficial since mutations at the gatekeeper residue in other tyrosine kinases (e.g. EGFR) have been shown to mediate acquired drug resistance in the clinic. This compound demonstrated significant TGI of 58% and 82% respectively in the BaF3 KIF5B-RET and BaF3 RETV804M allograft models. Moreover, tumor growth in the lung cancer PDX model was strongly inhibited (95% TGI) and tumor regression induced in the MTC xenograft model (109% TGI). As expected, this potent and selective RET inhibitor was not active in the Calu-6 model, which is sensitive to KDR inhibition, whereas vandetanib, a potent KDR inhibitor, significantly inhibited tumor growth (84% TGI). Additional in vitro and in vivo DMPK analyses further support the nomination of this compound as a preclinical candidate. Conclusions: The identification of selective RET inhibitors with significant in vivo activity and minimal toxicity may overcome the limitations of the currently available clinical compounds. We have made considerable progress towards this goal and show here the compelling data supporting our nomination of a preclinical development compound. Citation Format: Mandy Watson, Helen Small, Ben Acton, Habiba Begum, Samantha Hitchin, Allan Jordan, Paul Kelly, Rebecca Newton, Ian Waddell, Gina Paris, Donald Ogilvie. A potent and selective RET inhibitor with efficacy in RET-driven mouse models of medullary thyroid carcinoma and lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2092. doi:10.1158/1538-7445.AM2017-2092

The contraction in research within pharma has seen a renaissance in drug discovery within the academic setting. Often, groups grow organically from academic research laboratories, exploiting a particular area of novel biology or new technology. However, increasingly, new groups driven by industrial staff are emerging with demonstrable expertise in the delivery of medicines. As part of a strategic review by Cancer Research UK (CR-UK), the drug discovery team at the Manchester Institute was established to translate novel research from the Manchester cancer research community into drug discovery programmes. From a standing start, we have taken innovative approaches to solve key issues faced by similar groups, such as hit finding and target identification. Herein, we share our lessons learnt and successful strategies.

Skeletal muscle atrophy and weakness are thought to be stimulated by tumor necrosis factor alpha (TNF-alpha) in a variety of chronic diseases. However, little is known about the direct effects of TNF-alpha on differentiated skeletal muscle cells or the signaling mechanisms involved. We have tested the effects of TNF-alpha on the mouse-derived C2C12 muscle cell line and on primary cultures from rat skeletal muscle. TNF-alpha treatment of differentiated myotubes stimulated time- and concentration-dependent reductions in total protein content and loss of adult myosin heavy chain (MHCf) content; these changes were evident at low TNF-alpha concentrations (1-3 ng/ml) that did not alter muscle DNA content and were not associated with a decrease in MHCf synthesis. TNF-alpha activated binding of nuclear factor kappaB (NF-kappaB) to its targeted DNA sequence and stimulated degradation of I-kappaBalpha, an NF-kappaB inhibitory protein. TNF-alpha stimulated total ubiquitin conjugation whereas a 26S proteasome inhibitor (MG132 10-40 microM) blocked TNF-alpha activation of NF-kappaB. Catalase 1 kU/ml inhibited NF-kappaB activation by TNF-alpha; exogenous hydrogen peroxide 200 microM activated NF-kappaB and stimulated I-kappaBalpha degradation. These data demonstrate that TNF-alpha directly induces skeletal muscle protein loss, that NF-kappaB is rapidly activated by TNF-alpha in differentiated skeletal muscle cells, and that TNF-alpha/NF-kappaB signaling in skeletal muscle is regulated by endogenous reactive oxygen species.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the hydrolysis of phosphodiester bonds between the DNA 3′-phosphate and tyrosine residues and plays a major role in the repair of stalled topoisomerase I-DNA covalent complexes. Given this role, Tdp1 is of interest as a potential target for anticancer therapy. Inhibiting Tdp1 in combination with clinically used Top1 inhibitors may potentiate the effects of the latter and help to overcome some of the chemoresistance issues currently observed. In addition, Tdp1 can function during DNA repair to remove a variety of other 3′ adducts from DNA such as phosphoglycolates and abasic or apurinic/apyrimidinic (AP) sites. Here we describe a new mix-and-read homogeneous fluorogenic assay for the measurement of the AP-site cleavage activity of Tdp1 that is compatible with high-throughput screening. The application of such an assay will open up further avenues for the discovery of novel Tdp1 inhibitors.