Abstract 888: Exploring the signaling and activity interactome between oncogenic RAS and the nucleotide pool-detoxifying enzyme MTH1

Abstract MutT Homolog 1 (MTH1) is a NUDIX pyrophosphorylase that hydrolyzes oxidized purine nucleoside triphosphates in the nucleotide pool, thus preventing their incorporation into DNA. Our prior work has shown that MTH1 is critical for the maintenance of multiple pro-tumorigenic phenotypes in oncogenic RAS-driven cancer cells, with its depletion leading to decreased tumor formation in vivo. Our subsequent analyses of TCGA patient datasets showed elevated MTH1 expression to be significantly associated with poorer disease-free survival in RAS-mutated cancers, such as that of the lung and pancreas. We found that MTH1 mRNA levels were positively correlated with KRAS levels even in early-stage non-small cell lung cancer patient tissues, and that the introduction of oncogenic KRAS was sufficient to upregulate MTH1 mRNA and protein levels. The aim of this study is to identify RAS-effector signaling intermediates affecting MTH1 expression and activity. Chemical inhibitors of the MAPK/ERK, PI3K/AKT and NOX pathways, plus oncogenic RASV12-effector domain mutants (RASV12- S35/ E38/ G37/ C40), were used to identify key signaling molecular mediators of MTH1 expression in the distinct RAS isoforms (H- and K-RAS). The dependencies of the different KRASG12-mutant polymorphisms (KRASG12- C/ D/ V) on MTH1 expression and activity, as well as candidate transcription factors regulating MTH1 expression, were evaluated. Our work shows MTH1 at the nexus of crosstalk between different effector pathways activated downstream of RAS. Dissecting these signaling intermediates are important in identifying alternate pathways of MTH1 regulation, which may manifest as resistance mechanisms to standard-of-care cancer treatments. Our work will also help understand how to best leverage MTH1 as a therapeutic target in oncogenic RAS-driven cancers driven by the different isoforms, and their respective mutant polymorphisms. Citation Format: Govindi J. Samaranayake, Clara I. Troccoli, Christina Jayaraj, Brittany C. Durden, Nagaraj Nagathihalli, Nipun Merchant, Priyamvada Rai. Exploring the signaling and activity interactome between oncogenic RAS and the nucleotide pool-detoxifying enzyme MTH1 [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 888

Abstract 5473: Towards a better understanding of MTH1 as a therapeutic target in RAS-driven cancer

Abstract Oncogenic RAS signaling-generated reactive oxygen species (ROS) drive tumor progression by the hyperactivation of proliferative, anti-apoptotic, and metastatic pathways. Elevated ROS levels however can also cause oxidative DNA damage, leading to oncogene-induced senescence (OIS) and cell death. To avoid such tumor-suppressive outcomes, RAS-driven tumors often upregulate redox-protective proteins. The collective research from our group over the last few years has shown that oncogenic RAS elevates expression of the mammalian 8-oxo-dGTPase MutT Homolog 1 (MTH1), which in turn enables evasion of OIS, promotes transformation efficiency, and facilitates tumorigenicity. Our prior work has therefore demonstrated that MTH1 is beneficial to the spectrum of RAS-driven transformation, and that its shRNA-mediated targeting in oncogenic RAS-harboring lung cancer cells produces robust tumor-suppressive outcomes. However the first wave of chemical MTH1 inhibitors has led to controversial and conflicting results regarding MTH1 as a chemotherapeutic target. Here we evaluate the benefit of MTH1 inhibitors in wildtype. vs. oncogenic KRAS expressing cells, and directly assess the effects of oncogenic KRAS as well as three recently developed inhibitors on MTH1 8-oxo-dGTPase activity. Our results support that introduction of oncogenic KRAS elevates both MTH1 expression and activity, presumably through its elevation of ROS levels, and thus sensitizes cells to MTH1 inhibitors. The degree of this sensitization has a complex dependence on residual 8-oxodGTPase activity in the different cells following treatment with the MTH1 inhibitors, alternate antioxidant responses, and induction of different tumor suppressor pathways. Our data therefore highlight the importance of evaluating the molecular contexts and outcomes of MTH1 inhibition when determining its utility as a chemotherapeutic target. Citation Format: Govindi J. Samaranayake, Clara I. Troccoli, Mai Q. Huynh, Andrew Win, Debin Ji, Eric T. Kool, Priyamvada Rai. Towards a better understanding of MTH1 as a therapeutic target in RAS-driven cancer [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 5473. doi:10.1158/1538-7445.AM2017-5473</jats:p

Abstract 5479: Redox stress as a therapeutic Achilles heel in castration-resistant prostate cancer

Abstract Androgen deprivation therapy (ADT) initially suppresses prostate cancer (PC) progression. However, castration resistant PC (CRPC) cells inevitably emerge, resulting in incurable disease. We recently demonstrated that AD produces a form of cellular senescence leading to outgrowth of CRPC subpopulations from the AD-sensitive parental cells. Gene expression profiling studies comparing the parental LNCaP line to the senescence-resistant, early CRPC variant, LNCaP SB5, revealed an enrichment of thiol-based redox-protective proteins in the latter under AD conditions. This finding suggests that redox stress due to hyperactivated mitogenic/survival signaling or metabolic stresses may be an important and understudied Achilles heel in the progression to CRPC. Here we present the effects of inhibiting thioredoxin-1 (TRX1), a redox-protective protein that was identified as being elevated in our early CRPC LNCaP SB5 model. Suppression of TRX1 expression via shRNA led to profound growth suppression of CRPC cells, relative to their AD-sensitive counterparts, and significantly reduced CRPC xenograft tumor growth. Furthermore, under AD, TRX1 suppression promoted p53-induced cell death, which was accompanied by increased reactive oxygen species (ROS). These in vitro and in vivo results were recapitulated using a Phase II clinical trial-tested chemical TRX1 inhibitor. Thus our results point to TRX1 as a critical requirement for CRPC progression and provide a rationale for using TRX1 inhibitors in conjunction with ADT to limit CRPC. Citation Format: Clara I. Troccoli, Govindi J. Samaranayake, Mai Q. Huynh, Karen Kage, Deukwoo Kwon, Yuguang Ban, Xi S. Chen, Enrique R. Zarco, Merce Jorda, Kerry L. Burnstein, Priyamvada Rai. Redox stress as a therapeutic Achilles heel in castration-resistant prostate cancer [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 5479. doi:10.1158/1538-7445.AM2017-5479</jats:p

The Existence of MTH1-independent 8-oxodGTPase Activity in Cancer Cells as a Compensatory Mechanism against On-target Effects of MTH1 Inhibitors

Investigations into the human 8-oxodGTPase, MutT Homolog 1 (MTH1), have risen sharply since the first-in-class MTH1 inhibitors were reported to be highly tumoricidal. However, MTH1 as a cancer therapeutic target is currently controversial because subsequently developed inhibitors did not exhibit similar cytotoxic effects. Here, we provide the first direct evidence for MTH1-independent 8-oxodGTPase function in human cancer cells and human tumors, using a novel ATP-releasing guanine-oxidized (ARGO) chemical probe. Our studies show that this functionally redundant 8-oxodGTPase activity is not decreased by five different published MTH1-targeting small molecules or by MTH1 depletion. Significantly, while only the two first-in-class inhibitors, TH588 and TH287, reduced cancer cell viability, all five inhibitors evaluated in our studies decreased 8-oxodGTPase activity to a similar extent. Thus, the reported efficacy of the first-in-class MTH1 inhibitors does not arise from their inhibition of MTH1-specific 8-oxodGTPase activity. Comparison of DNA strand breaks, genomic 8-oxoguanine incorporation, or alterations in cellular oxidative state by TH287 versus the noncytotoxic inhibitor, IACS-4759, contradict that the cytotoxicity of the former results solely from increased levels of oxidatively damaged genomic DNA. Thus, our findings indicate that mechanisms unrelated to oxidative stress or DNA damage likely underlie the reported efficacy of the first-in-class inhibitors. Our study suggests that MTH1 functional redundancy, existing to different extents in all cancer lines and human tumors evaluated in our study, is a thus far undefined factor which is likely to be critical in understanding the importance of MTH1 and its clinical targeting in cancer

Increased MTH1-specific 8-oxodGTPase activity is a hallmark of cancer in colon, lung and pancreatic tissue

[Display omitted] •ARGO assay uses a chimeric substrate for luminescent measurement of functional MTH1 activity.•MTH1-specific 8-oxodGTPase activity is higher in CRC, NSCLC and PDAC compared to normal tissue.•In CRC, MTH1 activity measured by ARGO assay correlates to MTH1 protein level but not mRNA expression. Cellular homeostasis is dependent on a balance between DNA damage and DNA repair mechanisms. Cells are constantly assaulted by both exogenous and endogenous stimuli leading to high levels of reactive oxygen species (ROS) that cause oxidation of the nucleotide dGTP to 8-oxodGTP. If this base is incorporated into DNA and goes unrepaired, it can result in G > T transversions, leading to genomic DNA damage. MutT Homolog 1 (MTH1) is a nucleoside diphosphate X (Nudix) pyrophosphatase that can remove 8-oxodGTP from the nucleotide pool before it is incorporated into DNA by hydrolyzing it into 8-oxodGMP. MTH1 expression has been shown to be elevated in many cancer cells and is thought to be a survival mechanism by which a cancer cell can stave off the effects of high ROS that can result in cell senescence or death. It has recently become a target of interest in cancer because it is thought that inhibiting MTH1 can increase genotoxic damage and cytotoxicity. Determining the role of MTH1 in normal and cancer cells is confounded by an inability to reliably and directly measure its native enzymatic activity. We have used the chimeric ATP-releasing guanine-oxidized (ARGO) probe that combines 8-oxodGTP and ATP to measure MTH1 enzymatic activity in colorectal cancer (CRC), non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) along with patient-matched normal tissue. MTH1 8-oxodGTPase activity is significantly increased in tumors across all three tissue types, indicating that MTH1 is a marker of cancer. MTH1 activity measured by ARGO assay was compared to mRNA and protein expression measured by RT-qPCR and Western blot in the CRC tissue pairs, revealing a positive correlation between ARGO assay and Western blot, but little correlation with RT-qPCR in these samples. The adoption of the ARGO assay will help in establishing the level of MTH1 activity in model systems and in assessing the effects of MTH1 modulation in the treatment of cancer

The soluble guanylyl cyclase pathway is inhibited to evade androgen deprivation-induced senescence and enable progression to castration resistance

Castration-resistant prostate cancer (CRPC) is fatal and therapeutically under-served. We describe a novel CRPC-restraining role for the vasodilatory soluble guanylyl cyclase (sGC) pathway. We discovered that sGC subunits are dysregulated during CRPC progression and its catalytic product, cyclic GMP (cGMP), is lowered in CRPC patients. Abrogating sGC heterodimer formation in castration-sensitive prostate cancer (CSPC) cells inhibited androgen deprivation (AD)-induced senescence, and promoted castration-resistant tumor growth. We found sGC is oxidatively inactivated in CRPC. Paradoxically, AD restored sGC activity in CRPC cells through redox-protective responses evoked to protect against AD-induced oxidative stress. sGC stimulation via its FDA-approved agonist, riociguat, inhibited castration-resistant growth, and the anti-tumor response correlated with elevated cGMP, indicating on-target sGC activity. Consistent with known sGC function, riociguat improved tumor oxygenation, decreasing the PC stem cell marker, CD44, and enhancing radiation-induced tumor suppression. Our studies thus provide the first evidence for therapeutically targeting sGC via riociguat to treat CRPC

Lung adenocarcinoma promotion by air pollutants

This research was conducted using the UK Biobank Resource under application number 82693. This work was supported by the Mark Foundation ASPIRE I Award (grant 21-029-ASP), the Lung Cancer Research Foundation Grant on Disparities in Lung Cancer, Advanced Grant (PROTEUS, grant agreement no. 835297), CRUK EDD (EDDPMA-Nov21\100034) and a Rosetrees Out-of-round Award (OoR2020\100009). W.H. is funded by an ERC Advanced Grant (PROTEUS, grant agreement no. 835297), CRUK EDD (EDDPMA-Nov21\100034), The Mark Foundation (grant 21-029-ASP) and has been supported by Rosetrees. E.L.L. receives funding from the NovoNordisk Foundation (ID 16584), The Mark Foundation (grant 21-029-ASP) and has been supported by Rosetrees. C.E.W. is supported by a RESPIRE4 fellowship from the European Respiratory Society and Marie-Sklodowska-Curie Actions. C.L. is supported by the Agency for Science, Technology & Research, Singapore and the Cancer Research UK City of London Centre and the City of London Centre Clinical Academic Training Programme. M.A. is supported by the City of London Centre Clinical Academic Training Programme (Year 3, SEBSTF-2021\100007). K.C. is supported by the Research Unit of Intelligence Diagnosis and Treatment in Early Non-small Cell Lung Cancer, the Chinese Academy of Medical Sciences (2021RU002), the National Natural Science Foundation of China (no. 82072566) and Peking University People’s Hospital Research and Development Funds (RS2019-01). T.K. receives grant support from JSPS Overseas Research Fellowships Program (202060447). S.-H.L. is supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (no. 2020R1A2C3006535), the National Cancer Center Grant (NCC1911269-3) and a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number HR20C0025). L.H.S. receives grant support from the Berta Kamprad Foundation, the Swedish Cancer Society and the Swedish Research Council. R.M. and S.L. acknowledge funding from the Terry Fox Research Institute. N.M. is a Sir Henry Dale Fellow, jointly funded by the Wellcome Trust and the Royal Society (grant number 211179/Z/18/Z) and receives funding from Cancer Research UK, the Rosetrees and the NIHR BRC at University College London Hospitals and the CRUK University College London Experimental Cancer Medicine Centre. J. DeGregori, M.G., Y.E.M., D.T.M. and R.L.K. receive funding from the American Association for Cancer Research/Johnson&Johnson (18-90-52-DEGR), and J. DeGregori is supported by the Courtenay C. and Lucy Patten Davis Endowed Chair in Lung Cancer Research and a Merit Award from the Veteran’s Administration (1 I01 BX004495). M.G., Y.E.M., D.T.M. and R.L.K. were supported by the National Cancer Institute (NCI) RO1 CA219893. E.J.E.J. was supported by a NCI Ruth L. Kirschstein National Research Service Award T32-CA190216 and the Blumenthal Fellowship from the Linda Crnic Institute for Down Syndrome. C.I.T. acknowledges funding from UC Anschutz (LHNC T32CA174648). The work at the University of Colorado was also supported by NCI Cancer Center Support Grant P30CA046934. K. Litchfield is funded by the UK Medical Research Council (MR/P014712/1 and MR/V033077/1), the Rosetrees Trust and the Cotswold Trust (A2437) and Cancer Research UK (C69256/A30194). M.J.-H. is a CRUK Career Establishment Awardee has received funding from Cancer Research UK, IASLC International Lung Cancer Foundation, the National Institute for Health Research, the Rosetrees Trust, UKI NETs and the NIHR University College London Hospitals Biomedical Research Centre. C.S. is a Royal Society Napier Research Professor (RSRP\R\210001). His work is supported by the Francis Crick Institute that receives its core funding from Cancer Research UK (CC2041), the UK Medical Research Council (CC2041), and the Wellcome Trust (CC2041). For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. C.S. is funded by Cancer Research UK (TRACERx (C11496/A17786), PEACE (C416/A21999) and CRUK Cancer Immunotherapy Catalyst Network); Cancer Research UK Lung Cancer Centre of Excellence (C11496/A30025); the Rosetrees Trust, Butterfield and Stoneygate Trusts; NovoNordisk Foundation (ID16584); Royal Society Professorship Enhancement Award (RP/EA/180007); National Institute for Health Research (NIHR) University College London Hospitals Biomedical Research Centre; the Cancer Research UK-University College London Centre; Experimental Cancer Medicine Centre; the Breast Cancer Research Foundation (US) (BCRF-22-157); Cancer Research UK Early Detection an Diagnosis Primer Award (grant EDDPMA-Nov21/100034); and The Mark Foundation for Cancer Research Aspire Award (grant 21-029-ASP). This work was supported by a Stand Up To Cancer‐LUNGevity-American Lung Association Lung Cancer Interception Dream Team Translational Research Grant (grant number: SU2C-AACR-DT23-17 to S.M. Dubinett and A.E. Spira). Stand Up To Cancer is a division of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, the Scientific Partner of SU2C. C.S. is in receipt of an ERC Advanced Grant (PROTEUS) from the European Research Council under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 835297). We acknowledge the PEACE Consortium (PEACE Consortium members are named below) for their expertise and support in putting together the healthy tissue sample cohorts. We thank the clinical and administrative team of the PEACE study for their assistance in data curation (S. Shepherd, Z. Tippu, B. Shum, C. Lewis, M. O’Flaherty, A. Lucanas, E. Carlyle, L. Holt, F. Williams); nursing and biospecimen coordinators for their assistance in sample curation (K. Edmonds, L. Grostate, K. Lingard, D. Kelly, J. Korteweg, L. Terry, J. Biano, A. Murra, K. Kelly, K. Peat, N. Hunter); A. H. -K. Cheung for assistance in pathology review; J. Asklin and C. Forsberg for logistical and technical assistance; staff at the Chang Gung Memorial Hospital for providing Chang Gung Research Database (CGRD) data; staff who provided support at the Flow Cytometry Unit, the Experimental Histopathology Unit, the Advanced Light Microscopy Facility, the Advanced Sequencing Facility and the Biological Resources Unit, especially N. Chisholm and Jay O’Brien, at the Francis Crick Institute; A. Yuen, A. Azhar, K. Lau, C. Schwartz, A. Lee and C. Rider for their logistical support for the human exposure study; and staff at the Centre d’expertise et de services Génome Québec for their sequencing services and support. Data for this study are based on patient-level information collected by the NHS, as part of the care and support of cancer patients. The data are collated, maintained and quality assured by the National Cancer Registration and Analysis Service, which is part of NHS England (NHSE). We extend our thanks to the skilled Cancer Registration Officers (CROs) within the National Disease Registration Service, who abstracted and registered the English tumour and molecular testing data.Peer reviewedPostprin