A Comprehensive Investigation of MTH1 as a Novel Therapeutic Target in RAS-Driven Cancers.

The nucleotide pool is significantly more susceptible to oxidative damage induced by reactive oxygen species (ROS) compared to genomic DNA, due to DNA being more protected by the double helix and nucleosome packing. The NUDIX pyrophosphatase, MutT Homolog 1 (MTH1), maintains nucleotide pool integrity by hydrolyzing oxidized purines thus preventing their genomic incorporation. If incorporated into DNA the accumulation of these oxidized purines can lead to DNA strand breaks, genomic instability, and ultimately cell death. Our prior work has shown that MTH1 is critical in facilitating multiple pro-tumorigenic phenotypes in oncogenic RAS-mutated cancer cells, with its depletion leading to decreased tumor formation in lung cancer xenograft models. As MTH1 loss is well tolerated in normal tissues, it was predicted to be an excellent anti-cancer therapeutic target with a wide therapeutic index; yet the first wave of MTH1 chemical inhibitors have yielded inconsistent results in their tumoricidal effects. Critical contributing factors to these discrepancies are 1) lack of an assay to measure endogenous cell/tissue-specific MTH1 8-oxo-dGTPase enzymatic activity, and 2) till-date unexplored biological redundancy in cellular 8-oxo-dGTPase activity that can compensate in the absence of functional MTH1. In order to address these critical gaps in the field, in collaboration with a synthetic chemistry group at Stanford, I utilized a novel ATP-releasing guanine-oxidized (ARGO) probe-based assay to measure endogenous 8-oxo-dGTPase activity in cancer cell lines and tissue specimens. I did an extensive comparative study on the effects of five independently developed small molecule MTH1 inhibitors on 8-oxo-dGTPase activity, oxidative DNA damage and cell viability in multiple cancer cell lines. My research helped establish that cancer cell lines and patient tissues possess variable levels of 8-oxo-dGTPase activity that cannot be decreased either by MTH1 shRNA-mediated depletion or the first-in-class MTH1 inhibitors, indicating previously unidentified redundancy in this function. My work additionally identified that the reported cytotoxicity from the first-in-class MTH1 inhibitors occurs through off-target mechanisms, implicating polypharmacology as their mechanism of action. Given this complication in MTH1 therapeutic targeting, in order to comprehensively investigate the biology of MTH1 in cancer initiation and progression, I generated the first mouse models of spontaneous KRAS-driven tumorigenesis in the background of MTH1 loss. I identified that the MTH1 null mouse also exhibits robust redundancy in the 8-oxodGTPase pathway. I next assessed how germline MTH1-knockout (KO) affected spontaneous in vivo oncogenic KRAS-driven pancreatic ductal adenocarcinoma and have found evidence that the functional 8-oxo-dGTPase activity redundancy existing in the MTH1-KO animals impacts the development of aggressive pancreatic cancer relative to the MTH1-wildtype animals. Our identification of MTH1-independent 8-oxo-dGTPase activity unveils previously undiscovered complexity in 8-oxo-dGTPase biology and its role in cancer. Identifying the source of this MTH-independent 8-oxo-dGTPase activity will be critical in understanding potential resistance mechanisms to the on-target effects of MTH1 inhibition. As the cytotoxic MTH1 inhibitors appear to exert their effects independent of MTH1 inhibition, through polypharmacology, our data imply that the utility of the on-target MTH1 inhibitors in the clinic may lie in combinatorial treatment regimens rather than as a monotherapy. Our findings have strong clinical implications, as the first-in-man clinical trial with Karonudib, an MTH1 inhibitor based on the same drug scaffold as the first-in-class inhibitors, is currently underway in patients with advanced solid tumors.</p

MTH1 as a Chemotherapeutic Target: The Elephant in the Room

Many tumors sustain elevated levels of reactive oxygen species (ROS), which drive oncogenic signaling. However, ROS can also trigger anti-tumor responses, such as cell death or senescence, through induction of oxidative stress and concomitant DNA damage. To circumvent the adverse consequences of elevated ROS levels, many tumors develop adaptive responses, such as enhanced redox-protective or oxidatively-generated damage repair pathways. Targeting these enhanced oxidative stress-protective mechanisms is likely to be both therapeutically effective and highly specific to cancer, as normal cells are less reliant on such mechanisms. In this review, we discuss one such stress-protective protein human MutT Homolog1 (MTH1), an enzyme that eliminates 8-oxo-7,8-dihydro-2’-deoxyguanosine triphosphate (8-oxodGTP) through its pyrophosphatase activity, and is found to be elevated in many cancers. Our studies, and subsequently those of others, identified MTH1 inhibition as an effective tumor-suppressive strategy. However, recent studies with the first wave of MTH1 inhibitors have produced conflicting results regarding their cytotoxicity in cancer cells and have led to questions regarding the validity of MTH1 as a chemotherapeutic target. To address the proverbial "elephant in the room" as to whether MTH1 is a bona fide chemotherapeutic target, we provide an overview of MTH1 function in the context of tumor biology, summarize the current literature on MTH1 inhibitors, and discuss the molecular contexts likely required for its efficacy as a therapeutic target

Abstract A38: MTH1 Inhibition as an effective tumor-suppressive strategy in RAS-driven cancer

Abstract Oncogenic RAS mutations confer multiple malignant traits to cancer cells, many of which are mediated by oncogene-induced reactive oxygen species (ROS). However, oncogenic ROS also create vulnerabilities in the cancer cells, sensitizing them to oxidative DNA damage and strand breaks which can trigger cellular senescence or cell death. Thus RAS-driven tumor cells require adaptive redox protective mechanisms to inhibit ROS-associated tumor suppression. We previously reported that MutT Homolog 1 (MTH1), the mammalian 8-oxodGTPase, comprises one such critical adaptation. We have shown MTH1 is important for facilitating the full gamut of RAS-driven malignancy by promoting evasion of the first barrier to transformation, viz. oncogene-induced senescence (OIS), enhancing RAS-mediated transformation and related pro-malignant traits, and maintaining proliferation and tumorigenicity in established RAS-driven tumor cells. Recently chemical inhibitors against MTH1 have been developed, although there is some controversy surrounding their efficacy and mode of action. Our work here discusses RAS regulation of MTH1 expression, and the molecular contexts under which MTH1 inhibition can synergize with oncogenic oxidative stress in RAS-driven tumor cells for optimal tumor suppressive response. Citation Format: Govindi Jayanika Samaranayake, Clara Issabella Troccoli, Tyler Andrew Cunningham, Priyamvada Rai. MTH1 Inhibition as an effective tumor-suppressive strategy in RAS-driven cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A38

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 2076: Low OGG1 protects against the DNA damage induced by MTH1 inhibition

Abstract Increased metabolic activity of cancer cells due to uncontrolled proliferation, often leads to the accumulation of reactive oxygen species (ROS). High ROS can produce tumor-suppressive oxidative DNA damage either directly in the genome or through oxidation of deoxynucleotides that can then be incorporated into DNA. The human 8-oxoguanine glycosylase 1 (OGG1), a base excision repair (BER) enzyme, protects cells from the former by excising genomic 8-oxodG; MutT Homolog 1 (MTH1), the mammalian 8-oxodGTPase, protects cells from the latter by degrading 8-oxo-dGTP. There has been much interest in increasing oxidative DNA damage in tumors as a therapeutic option. Accordingly, inhibitors are being developed that can target both MTH1 and 8-oxodG glycosylase 1 (OGG1) with the speculation that this co-targeting will significantly increase tumor suppression efficiency. Our lab has shown that MTH1 inhibition leads to DNA strand breaks and tumor-suppressive effects in cancers characterized by high MTH1enzymatic activity. Here we assessed whether co-inhibition of OGG1 would provide benefits in tumor-suppressive effects over MTH1 inhibition alone. We first compared OGG1 and MTH1 expression in paired normal versus tumor tissue from lung adenocarcinoma patients and found that, unlike MTH1, OGG1 tends to remain within normal range in cancerous tissue. Similarly, TCGA lung adenocarcinoma patients show minimal OGG1 elevations and a positive correlation between OGG1 and MTH1 mRNA levels even when both are low. This data suggests that tumors with low OGG1 and MTH1 are robustly represented in the tumor continuum. We then co-depleted OGG1/MTH1 in the A549 lung cancer cell line and found this co-depletion led to lower DNA strand breaks and decreased cell senescence over those caused by MTH1 depletion alone. The shMTH1-transduced cells were less sensitive to SU0268, a potent OGG1 inhibitor compared to shGFP-transduced counterparts. We also treated A549 cells with SU0383, a dual OGG1/MTH1 inhibitor. The dual-inhibitor did not consistently increase cytotoxicity either with duration or dose of treatment beyond single inhibitors or combined single MTH1 and OGG1 inhibitors. In conclusion, our findings suggest that the co-inhibition of OGG1 with MTH1 does not produce enhanced therapeutic benefit in tumor cells, and could instead diminish the tumor-inhibitory effects of MTH1 inhibition that arise from induction of DNA strand breaks and p53-induced senescence. Citation Format: Laura Misiara Lincheta, Ling Zhang, Govindi Samaranayake, Nisha Sharma, Dao Nguyen, Yu-Ki Tahara, Eric Kool, Priyamvada Rai. Low OGG1 protects against the DNA damage induced by MTH1 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2076

OGG1 co-inhibition antagonizes the tumor-inhibitory effects of targeting MTH1

Cancer cells develop protective adaptations against oxidative DNA damage, providing a strong rationale for targeting DNA repair proteins. There has been a high degree of recent interest in inhibiting the mammalian Nudix pyrophosphatase MutT Homolog 1 (MTH1). MTH1 degrades 8-oxo-dGTP, thus limiting its incorporation into genomic DNA. MTH1 inhibition has variously been shown to induce genomic 8-oxo-dG elevation, genotoxic strand breaks in p53-functional cells, and tumor-inhibitory outcomes. Genomically incorporated 8-oxo-dG is excised by the base excision repair enzyme, 8-oxo-dG glycosylase 1 (OGG1). Thus, OGG1 inhibitors have been developed with the idea that their combination with MTH1 inhibitors will have anti-tumor effects by increasing genomic oxidative DNA damage. However, contradictory to this idea, we found that human lung adenocarcinoma with low OGG1 and MTH1 were robustly represented in patient datasets. Furthermore, OGG1 co-depletion mitigated the extent of DNA strand breaks and cellular senescence in MTH1-depleted p53-wildtype lung adenocarcinoma cells. Similarly, shMTH1-transduced cells were less sensitive to the OGG1 inhibitor, SU0268, than shGFP-transduced counterparts. Although the dual OGG1/MTH1 inhibitor, SU0383, induced greater cytotoxicity than equivalent combined or single doses of its parent scaffold MTH1 and OGG1 inhibitors, IACS-4759 and SU0268, this effect was only observed at the highest concentration assessed. Collectively, using both genetic depletion as well as small molecule inhibitors, our findings suggest that OGG1/MTH1 co-inhibition is unlikely to yield significant tumor-suppressive benefit. Instead such co-inhibition may exert tumor-protective effects by preventing base excision repair-induced DNA nicks and p53 induction, thus potentially conferring a survival advantage to the treated tumors. [Display omitted] •Low MTH1/low OGG1 tumors are robustly represented in patient lung adenocarcinoma datasets but low MTH1/high OGG1 are not.•Co-depletion of OGG1 in lung adenocarcinoma cells mitigates shMTH1-induced DNA strand breaks and p53-induced senescence.•p53-null tumor cells have lower OGG1 vs. wt p53 counterparts and are more resistant to MTH1 loss-induced anti-tumor effects.•Pharmacologic co-inhibition of OGG1 and MTH1 does not enhance cytotoxicity over the respective single inhibitors