MutT Homolog 1 Inhibitors Research Articles

MTH1 inhibition synergizes with ROS-inducing agents to trigger cervical cancer cells undergoing parthanatos

Cervical cancer cells possess high levels of reactive oxygen species (ROS); thus, increasing oxidative stress above the toxicity threshold to induce cell death is a promising chemotherapeutic strategy. However, the underlying mechanisms of cell death are elusive, and efficacy and toxicity issues remain. Within DNA, 8-oxo-7,8-dihydroguanine (8-oxoG) is the most frequent base lesion repaired by 8-oxoguanine glycosylase 1 (OGG1)-initiated base excision repair. Cancer cells also express high levels of MutT homolog 1 (MTH1), which prevents DNA replication-induced incorporation of 8-oxoG into the genome by hydrolyzing 8-oxo-7,8-dihydro-2′-deoxyguanosine 5′-triphosphate (8-oxo-dGTP). Here, we revealed that ROS-inducing agents triggered cervical cancer to undergo parthanatos, which was mainly induced by massive DNA strand breaks resulting from overwhelming 8-oxoG excision by OGG1. Furthermore, the MTH1 inhibitor synergized with a relatively low dose of ROS-inducing agents by enhancing 8-oxoG loading in the DNA. In vivo, this drug combination suppressed the growth of tumor xenografts, and this inhibitory effect was significantly decreased in the absence of OGG1. Hence, the present study highlights the roles of base repair enzymes in cell death induction and suggests that the combination of lower doses of ROS-inducing agents with MTH1 inhibitors may be a more selective and safer strategy for cervical cancer chemotherapy.

Targeting MutT Homolog 1 (MTH1) for Breast Cancer Suppression by Using a Novel MTH1 Inhibitor MA-24 with Tumor-Selective Toxicity.

Breast cancer is a commonly diagnosed cancer worldwide. Human MutT homolog 1 (MTH1) is found to be elevated in breast tumors and cancer cells need MTH1 for survival. Pharmacological inhibition of MTH1 may be potentially beneficial in the treatment of breast cancer. MA-24 was screened by malachite green colorimetric assay for MTH1 inhibitors and the kinetic characteristics of MA-24 were assessed. The features of MA-24's binding with MTH1 were ascertained through molecular docking, and the cytotoxic activity of MA-24 was validated in vitro and in vivo. Target engagement assays, comet assay, and Western blot confirmed the intracellular target and mechanism of MA-24. MA-24 shows potent antitumor bioactivity both in vitro and in vivo. MA-24 competitively inhibited the MTH1 and further induced DNA strand breaks, leading to increased apoptosis of cancer cells depending on the upregulation of the cleaved-caspase 3-cleaved-PARP axis. In particular, MA-24 exhibited a powerful efficacy and safety in vivo (tumor growth inhibition rate: 61.8%). MA-24 possesses a broad spectrum of breast cancer cytotoxicity and offered valuable insights for overcoming the challenges of chemotherapy-related toxicity, which holds great potential for the further development MA-24 as an anti-cancer drug.

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.

Inhibitor development of MTH1 via high-throughput screening with fragment based library and MTH1 substrate binding cavity

MutT Homolog 1 (MTH1) has been proven to hydrolyze oxidized nucleotide triphosphates during DNA repair. It can prevent the incorporation of wrong nucleotides during DNA replication and mitigate cell apoptosis. In a cancer cell, abundant reactive oxygen species can lead to substantial DNA damage and DNA mutations by base-pairing mismatch. MTH1 could eliminate oxidized dNTP and prevent cancer cells from entering cell death. Therefore, inhibition of MTH1 activity is considered to be an anti-cancer therapeutic target. In this study, high-throughput screening techniques were combined with a fragment-based library containing 2,313 compounds, which were used to screen for lead compounds with MTH1 inhibitor activity. Four compounds with MTH1 inhibitor ability were selected, and compound MI0639 was found to have the highest effective inhibition. To discover the selectivity and specificity of this action, several derivatives based on the MTH1 and MI0639 complex structure were synthesized. We compared 14 complex structures of MTH1 and the various compounds in combination with enzymatic inhibition and thermodynamic analysis. Nanomolar-range IC50 inhibition abilities by enzyme kinetics and Kd values by thermodynamic analysis were obtained for two compounds, named MI1020 and MI1024. Based on structural information and compound optimization, we aim to provide a strategy for the development of MTH1 inhibitors with high selectivity and specificity.

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.

MutT Homolog1 has multifaceted role in glioma and is under the apparent orchestration by Hypoxia Inducible factor1 alpha

AimsThe study focused on the expression and role of a recent potential cancer therapeutic target protein, MutT Homolog1 (MTH1). MTH1 gets activated in an increased reactive oxygen species (ROS) environment and removes the oxidized nucleotides from the cell. The study aimed to check the role of MTH1 in DNA damage and apoptosis, migration and angiogenesis and also to examine its regulation in glioma. Main methodsThe experiments were carried out in human glioma tissue samples and brain tissues of epilepsy patients (non-tumor control). We used two human glioblastomas cell lines, U87MG and U251MG cells. In order to study the role of MTH1 in glioma and to analyze the relation of MTH1 with Hif1α, we have used MTH1 siRNA and Hif1α siRNA respectively. Key findingsWe found an increased expression of MTH1 in glioma tissues compared to the non-tumor brain tissues. Correlation analysis revealed that those samples showing reduced expression of MTH1 also had high levels of DNA damage and apoptotic markers, while diminished expression of angiogenesis regulators and levels of migration. MTH1 knockdown in vitro by siRNA in tumor cell lines corroborates the above observation. This justifies the emergence of MTH1 inhibitors as potential first-in-class drugs. Mechanistically, our observations suggest that Hif1α may modulate MTH1 expression. SignificanceWe found elevated MTH1 expression in glioma irrespective of their grades, while its inhibition affects multiple tumor progression pathways, and that targeting Hif1α could simulate the same.

Direct Measurement of the Intracellular Concentration of 8-Oxo-2'-Deoxyguanosine-5'-Triphosphate by LC-MS/MS

Exposure to reactive oxygen species can result in formation of oxidized nucleotides which increase the frequency of mutations, DNA damage, and cell death. MutT Homolog 1 (MTH1) is an enzyme that catalyzes removal of pyrophosphate from the highly mutagenic oxidized nucleoside triphosphate 8-oxo-2'-deoxyguanosine-5'-triphosphate (8-oxo-dGTP). Interest in MTH1 as a potential new target for cancer therapy surged due to reports of MTH1 inhibitors causing DNA damage and inducing cell death in cancer cells. However, questions have been raised about MTH1 target validation. One critical piece of information that is currently lacking is a quantitative understanding of the levels of 8-oxo-dGTP in cancer cells and the effect of MTH1 inhibition on them. In this study, we developed a sensitive and selective method to simultaneously measure the intracellular concentrations of 8-oxo guanosine nucleotides and their unmodified counterparts using liquid chromatography coupled to tandem mass spectrometry (LC-MS/ MS). An ion-pairing reversed phase liquid chromatography method using dimethylhexylamine was employed to quantify the highly polar analytes. The method was validated fit for purpose using a combination of authentic standards and cell samples. The intracellular oxo-nucleotide concentrations in human bone osteosarcoma cell line U2OS were determined using the method. We observed very low levels of 8-oxo nucleotides in untreated and hydrogen peroxide treated cells and MTH1 knockdown in these cells had minimal if any effect on 8-oxo nucleotide levels. This method will allow for a more comprehensive understanding of oxidized nucleotide detoxification pathways and MTH1 as a target for cancer therapy.

TH1579, MTH1 inhibitor, delays tumour growth and inhibits metastases development in osteosarcoma model.

BackgroundOsteosarcoma (OS) is the most common primary malignant bone tumour. Unfortunately, no new treatments are approved and over the last 30 years the survival rate remains only 30% at 5 years for poor responders justifying an urgent need of new therapies. The Mutt homolog 1 (MTH1) enzyme prevents incorporation of oxidized nucleotides into DNA and recently developed MTH1 inhibitors may offer therapeutic potential as MTH1 is overexpressed in various cancers.MethodsThe aim of this study was to evaluate the therapeutic benefits of targeting MTH1 with two chemical inhibitors, TH588 and TH1579 on human osteosarcoma cells. Preclinical efficacy of TH1579 was assessed in human osteosarcoma xenograft model on tumour growth and development of pulmonary metastases.FindingsMTH1 is overexpressed in OS patients and tumour cell lines, compared to mesenchymal stem cells. In vitro, chemical inhibition of MTH1 by TH588 and TH1579 decreases OS cells viability, impairs their cell cycle and increases apoptosis in OS cells. TH1579 was confirmed to bind MTH1 by CETSA in OS model. Moreover, 90 mg/kg of TH1579 reduces in vivo tumour growth by 80.5% compared to non-treated group at day 48. This result was associated with the increase in 8-oxo-dG integration into tumour cells DNA and the increase of apoptosis. Additionally, TH1579 also reduces the number of pulmonary metastases.InterpretationAll these results strongly provide a pre-clinical proof-of-principle that TH1579 could be a therapeutic option for patients with osteosarcoma.FundingThis study was supported by La Ligue Contre le Cancer, la SFCE and Enfants Cancers Santé.

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.

Vaccine & Immunotherapy Summit October 14-15, 2020 | Brussels, Belgium

Exposure to reactive oxygen species can result in formation of oxidized nucleotides which increase the frequency of mutations, DNA damage, and cell death. MutT Homolog 1 (MTH1) is an enzyme that catalyzes removal of pyrophosphate from the highly mutagenic oxidized nucleoside triphosphate 8-oxo-2'-deoxyguanosine-5'-triphosphate (8-oxo-dGTP). Interest in MTH1 as a potential new target for cancer therapy surged due to reports of MTH1 inhibitors causing DNA damage and inducing cell death in cancer cells. However, questions have been raised about MTH1 target validation. One critical piece of information that is currently lacking is a quantitative understanding of the levels of 8-oxo-dGTP in cancer cells and the effect of MTH1 inhibition on them. In this study, we developed a sensitive and selective method to simultaneously measure the intracellular concentrations of 8-oxo guanosine nucleotides and their unmodified counterparts using liquid chromatography coupled to tandem mass spectrometry (LC-MS/ MS). An ion-pairing reversed phase liquid chromatography method using dimethylhexylamine was employed to quantify the highly polar analytes. The method was validated fit for purpose using a combination of authentic standards and cell samples. The intracellular oxo-nucleotide concentrations in human bone osteosarcoma cell line U2OS were determined using the method. We observed very low levels of 8-oxo nucleotides in untreated and hydrogen peroxide treated cells and MTH1 knockdown in these cells had minimal if any effect on 8-oxo nucleotide levels. This method will allow for a more comprehensive understanding of oxidized nucleotide detoxification pathways and MTH1 as a target for cancer therapy.

Antitumor effects of MutT homolog 1 inhibitors in human bladder cancer cells.

As standard second-line regimen has not been established for patients who are refractory to or relapse with cisplatin-based chemotherapy, an effective class of novel chemotherapeutic agents is needed for cisplatin-resistant bladder cancer. Recent publications reported that MutT homolog 1 (MTH1) inhibitors suppress tumor growth and induce impressive therapeutic responses in a variety of human cancer cells. Few studies investigated the cytotoxic effects of MTH1 inhibitors in human bladder cancer. Accordingly, we investigated the antitumor effects and the possible molecular mechanisms of MTH1 inhibitors in cisplatin-sensitive (T24) and - resistant (T24R2) human bladder cancer cell lines. These results suggest that TH588 or TH287 may induce cancer cell suppression by off-target effects such as alterations in the expression of apoptosis- and cell cycle-related proteins rather than MTH1 inhibition in cisplatin-sensitive and - resistant bladder cancer cells.Abbreviations: MTH: MutT homolog; ROS: reactive oxygen species; CCK-8: cell counting kit-8; DCFH-DA: dichlorofluorescein diacetate; PARP: poly (ADP-ribose) polymerase.

The Epstein\u2013Barr virus nuclear antigen-1 upregulates the cellular antioxidant defense to enable B-cell growth transformation and immortalization

Epstein–Barr virus (EBV) immortalizes human B-lymphocytes and is implicated in the pathogenesis of lymphoid and epithelial cell malignancies. The EBV nuclear antigen (EBNA)-1 induces the accumulation of reactive oxygen species (ROS), which enables B-cell immortalization but causes oxidative DNA damage and triggers antiproliferative DNA damage responses. By comparing pairs of EBV-negative and -positive tumor cell lines we found that, while associated with the accumulation of oxidized nucleotides, EBV carriage promotes the concomitant activation of oxo-dNTP sanitization and purging pathways, including upregulation of the nucleoside triphosphatase mut-T homolog 1 (MTH1) and the DNA glycosylases 8-oxoguanine-glycosylase-1 (OGG1) and mut-Y homolog (MUTYH). Expression of EBNA1 was reversibly associated with transcriptional activation of this cellular response. DNA damage and apoptosis were preferentially induced in EBNA1-positive cell lines by treatment with MTH1 inhibitors, suggesting that virus carriage is linked to enhanced vulnerability to oxidative stress. MTH1, OGG1, and MUTYH were upregulated upon EBV infection in primary B-cells and treatment with MTH1 inhibitors prevented B-cell immortalization. These findings highlight an important role of the cellular antioxidant response in sustaining EBV infection, and suggests that targeting this cellular defense may offer a novel approach to antiviral therapy and could reduce the burden of EBV associated cancer.

Potent and specific MTH1 inhibitors targeting gastric cancer

Human mutT homolog 1(MTH1), the oxidized dNTP pool sanitizer enzyme, has been reported to be highly expressed in various malignant tumors. However, the oncogenic role of MTH1 in gastric cancer remains to be determined. In the current study, we found that MTH1 was overexpressed in human gastric cancer tissues and cells. Using an in vitro MTH1 inhibitor screening system, the compounds available in our laboratory were screened and the small molecules containing 5-cyano-6-phenylpyrimidine structure were firstly found to show potently and specifically inhibitory effect on MTH1, especially compound MI-743 with IC50 = 91.44 ± 1.45 nM. Both molecular docking and target engagement experiments proved that MI-743 can directly bind to MTH1. Moreover, MI-743 could not only inhibit cell proliferation in up to 16 cancer cell lines, especially gastric cancer cells HGC-27 and MGC-803, but also significantly induce MTH1-related 8-oxo-dG accumulation and DNA damage. Furthermore, the growth of xenograft tumours derived by injection of MGC-803 cells in nude mice was also significantly inhibited by MI-743 treatment. Importantly, MTH1 knockdown by siRNA in those two gastric cancer cells exhibited the similar findings. Our findings indicate that MTH1 is highly expressed in human gastric cancer tissues and cell lines. Small molecule MI-743 with 5-cyano-6-phenylpyrimidine structure may serve as a novel lead compound targeting the overexpressed MTH1 for gastric cancer treatment.

153 - Towards a better understanding of human MutT Homolog 1 (MTH1) as a therapeutic target in cancer

Oxidative stress as a consequence of oncogenic signaling-induced ROS is a targetable vulnerability in many cancers. Our previous work implicated the mammalian 8-oxodGTPase human MutT Homolog 1 (MTH1) as an oncogenic adaptation in RAS-driven lung cancers that facilitates tumorigenesis and inhibits oncogenic ROS-associated DNA breaks and tumor-suppressive responses. Although the first-in-class MTH1 small molecule inhibitors initially highlighted their therapeutic potential, more recent reports have questioned their efficacy against cancer cells. In this study, we have attempted to provide some clarity to the current controversy in the field. Here we utilize a newly-developed assay for accurately assessing MTH1 8-oxodGTPase activity in a panel of cancer cell lines as well as human tumor tissue to investigate the effects of first-in-class MTH1 inhibitors, TH588 and TH287, alongside IACS-4759, an independently developed second-generation MTH1 inhibitor that does not recapitulate the anti-tumor effects of the first-in-class inhibitors despite comparably inhibiting MTH1 8-oxodGTPase activity. Our findings show that while MTH1 expression is almost universally elevated in lung tumors relative to normal tissue, the association between MTH1-specific 8-oxodGTPase activity and MTH1 expression is not strong. Our results further indicate that efficacy of the first-in-class inhibitors correlate broadly with MTH1-specific but not total cellular 8-oxodGTPase activity in lung cancer lines, pointing to the existence of as-yet-unidentified redundant 8-oxodGTPases that function either alongside or in the absence of MTH1. Additionally when we compare the inhibitors against shRNA-mediated MTH1 depletion, our collective findings show the first-in-class inhibitors can recapitulate the effects of our previous shMTH1 studies but not IACS-4759, despite all three inhibiting MTH1-specific 8-oxodGTPase activity as effectively as the shRNA. An obvious implication of our findings is that the efficacy of the first-in-class inhibitors is due to some as-yet unidentified mechanism(s) of MTH1 action that appears to be independent from its 8-oxodGTPase activity. Thus a better understanding of noncanonical MTH1 function in cancer as well as of alternate 8-oxodGTPase mechanisms is needed for the development and use of more universally efficacious inhibitors.

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

Abstract 2321: Reactive oxygen species dictate the apoptotic response of melanoma cells to TH588

Abstract Cancer cells commonly contain elevated levels of reactive oxygen species (ROS) resulting from oncogenic stimulation. On one hand, ROS promote cancer cell survival, proliferation, and metastasis. On the other, high levels of ROS suppress tumour growth through inhibition of proliferation and induction of apoptosis and senescence via damage to DNA. Incorporation of oxidized dNTPs such as 8-oxo-deoxy-guanine (8-oxo-dGTP) and 2-OH-deoxy-adenosine (2-OH-dATP) into genomic DNA plays an important role in apoptosis induced by ROS. Human MutT homolog 1(MTH1) is an enzyme that sanitizes oxidized dNTP pools through converting 8-oxo-dGTP and 2-OH-dATP into monophosphates, thus preventing their incorporation into genomic DNA. Inhibition of MTH1 by small molecule inhibitors has been suggested to be a promising approach in cancer treatment. However, we have found that while silencing of MTH1 does not affect survival of melanoma cell, TH588, one of the first-in-class MTH1 inhibitors, kills melanoma cells through apoptosis independently of its inhibitory effect on MTH1. Induction of apoptosis by TH588 was not alleviated by MTH1 overexpression or introduction of the bacterial homologue of MTH1 that has 8-oxodGTPase activity but cannot be inhibited by TH588, indicating that MTH1 inhibition is not the cause of TH588-induced killing of melanoma cells. Although knockdown of MTH1 did not impinge on the viability of melanoma cells, it rendered melanoma cells sensitive to apoptosis induced by the oxidative stress inducer elesclomol. Of note, treatment with elesclomol also enhanced TH588-induced apoptosis, whereas a ROS scavenger or an antioxidant attenuated apoptosis triggered by TH588. Indeed, the sensitivity of melanoma cells to TH588 was correlated with endogenous levels of ROS. Collectively, these results suggest that: 1) TH588-induced apoptosis of melanoma cells is not associated with its inhibitory effect on MTH1; 2) TH588 remains a promising candidate for the treatment of melanoma; 3) MTH1 inhibition in combination with oxidative stress inducers may be a useful approach in melanoma treatment; and 4) the endogenous levels of ROS are a potential biomarker for prediction of the response of melanomas to TH588 and MTH1 inhibition in combination with oxidative stress inducers. Citation Format: jiayu wang, Jin Lei, Xu Guang yan, Simonne Sherwin, Margaret Farrelly, Yuan Yuan Zhang, Fen Liu, Chun Yan Wang, Su Tang Guo, Hamed Yari, Ting La, Jennifer McFarlane, Fu Xi Lei, Hessam Tabatabaee, Jie Zhong chen, Amanda Croft, chen chen Jiang, Xu Dong Zhang. Reactive oxygen species dictate the apoptotic response of melanoma cells to TH588 [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 2321. doi:10.1158/1538-7445.AM2017-2321

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.

Expression and function of MutT homolog 1 in distinct subtypes of breast cancer

Human MutT homolog 1 (MTH1) detoxifies the oxidized DNA precursor 8-oxo-2′-deoxyguanosine-5′-triphosphate and serves a tumor suppressive role in distinct types of cancer. In the present study, the expression of MTH1 was examined in various subtypes of breast cancer, and the effect of its suppression on breast cancer growth was characterized in vitro and in vivo. MTH1 mRNA and protein levels were assessed using the reverse transcription-quantitative polymerase chain reaction and immunohistochemistry. The effect of MTH1 expression on the proliferation of breast cancer cells was investigated in vitro using Cell Counting Kit-8 and colony formation assays, and in vivo using breast cancer cell line xenografts in mice. The toxicity of the MTH1 inhibitor TH588 was investigated in nude mice. A marked increase in MTH1 protein and mRNA levels was demonstrated in breast cancer tissues compared with the non-cancerous control. However, no apparent differences in MTH1 expression were observed between distinct molecular subtypes of breast cancer. MTH1 overexpression was demonstrated to be independent of patient age, tumor size and lymph node metastasis. Inhibition of MTH1 decreased cancer cell viability and the clonogenic potential of cancer cells in a dose-dependent manner. These results were confirmed by decreased in vivo proliferation of MCF7, MDA-MB-231 and MDA-MB-453 cancer cell lines, representing distinct subtypes of breast cancer. Although inhibition of MTH1 activity decreased xenograft growth in mice, no major adverse effects of TH588 were detected on the basis of blood biochemistry, and liver and kidney function. The results of the present study suggested that MTH1 is overexpressed in the majority of breast cancers, independent of the molecular identity and clinicopathological features of the tumor, including patient age, tumor size and lymph node metastasis. Inhibition of MTH1 activity suppressed the growth of three subtypes of breast cancer, including luminal, basal-like and human epidermal growth factor receptor 2-positive, in vitro and in vivo. Treatment with the MTH1 inhibitor appears to be safe; however, further studies are required prior to the clinical use of MTH1 inhibitors.

Combined multi-pharmacophore, molecular docking and molecular dynamic study for discovery of promising MTH1 inhibitors

MutT homolog 1 (MTH1), a nudix phosphohydrolase enzyme participates in the process of repairing of DNA damage by hydrolyzing oxidized deoxy-ribonucleoside triphosphate in cancer cells, is regarded as a potential target for anticancer therapy. In order to seek for promising inhibitor of MTH1, structured-based pharmacophore and 3D-QSAR pharmacophore hypotheses combine with the ADMET analysis and Lipinski's rule of five were used for screening the public molecules libraries (Asinex, Ibscreen and Natural). Then molecular docking studies were performed on screened hits via various docking programs (Glide SP, GOLD and Glide XP), five molecules with three scaffolds were picked out as potential inhibitors against MTH1. Eventually, 20 ns molecular dynamics simulation was implemented on the potential inhibitors. The RMSD (Root Mean Square Deviation) values were used to illustrate bind stability between potential molecules and MTH1. Therefore, the five hits may be considered as promising MTH1 inhibitors by all above studies.

Synergistic therapy of chemotherapeutic drugs and MTH1 inhibitors using a pH-sensitive polymeric delivery system for oral squamous cell carcinoma.

MutT homolog 1 (MTH1) is an essential sanitizer of the free nucleotide pool that prevents lethal DNA damage in cancer cells, which has been validated as an anticancer target in recent years. Small molecule TH287 potently and selectively inhibits the MTH1 protein in cells. Here, we developed an effective chemotherapeutic system for oral squamous cell carcinoma (OSCC) based on polymeric nanoparticles that achieve co-delivery of anticancer drug sodium arsenite (NaAsO2) and MTH1 inhibitor TH287. Cationic hyperbranched poly(amine-ester) (HPAE), an amphiphilic and pH-sensitive polymer with a highly branched structure, self-assembled into nanoparticles in aqueous solution. Both NaAsO2 and TH287 could be loaded into HPAE nanoparticles with the help of electrostatic attraction and hydrophobic interaction. The release of NaAsO2 and TH287 from HPAE(NaAsO2 + TH287) nanoparticles was pH-dependent. In vitro evaluation demonstrated that the HPAE(NaAsO2 + TH287) nanoparticles rapidly entered cancer cells and released NaAsO2 and TH287 in response to acidic intracellular environments. In comparison with NaAsO2, TH287, HPAE(NaAsO2) nanoparticles, HPAE(TH287) nanoparticles, and the physical mixture of HPAE(NaAsO2) nanoparticles and TH287, the HPAE(NaAsO2 + TH287) nanoparticles exhibited more effective inhibition of tumor cell proliferation, illustrating the synergistic effect of NaAsO2 and TH287. The experimental results show that TH287 is likely to inhibit MTH1 in tumor cells, rendering them more sensitive to NaAsO2.