Pharmacological targeting of MTHFD2 suppresses acute myeloid leukemia by inducing thymidine depletion and replication stress.

The folate metabolism enzyme MTHFD2 (methylenetetrahydrofolate dehydrogenase/cyclohydrolase) is consistently overexpressed in cancer but its roles are not fully characterized, and current candidate inhibitors have limited potency for clinical development. In the present study, we demonstrate a role for MTHFD2 in DNA replication and genomic stability in cancer cells, and perform a drug screen to identify potent and selective nanomolar MTHFD2 inhibitors; protein cocrystal structures demonstrated binding to the active site of MTHFD2 and target engagement. MTHFD2 inhibitors reduced replication fork speed and induced replication stress followed by S-phase arrest and apoptosis of acute myeloid leukemia cells in vitro and in vivo, with a therapeutic window spanning four orders of magnitude compared with nontumorigenic cells. Mechanistically, MTHFD2 inhibitors prevented thymidine production leading to misincorporation of uracil into DNA and replication stress. Overall, these results demonstrate a functional link between MTHFD2-dependent cancer metabolism and replication stress that can be exploited therapeutically with this new class of inhibitors.

MTH1 Inhibitor TH1579 Induces Oxidative DNA Damage and Mitotic Arrest in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy, exhibiting high levels of reactive oxygen species (ROS). ROS levels have been suggested to drive leukemogenesis and is thus a potential novel target for treating AML. MTH1 prevents incorporation of oxidized nucleotides into the DNA to maintain genome integrity and is upregulated in many cancers. Here we demonstrate that hematologic cancers are highly sensitive to MTH1 inhibitor TH1579 (karonudib). A functional precision medicine ex vivo screen in primary AML bone marrow samples demonstrated a broad response profile of TH1579, independent of the genomic alteration of AML, resembling the response profile of the standard-of-care treatments cytarabine and doxorubicin. Furthermore, TH1579 killed primary human AML blast cells (CD45+) as well as chemotherapy resistance leukemic stem cells (CD45+Lin-CD34+CD38-), which are often responsible for AML progression. TH1579 killed AML cells by causing mitotic arrest, elevating intracellular ROS levels, and enhancing oxidative DNA damage. TH1579 showed a significant therapeutic window, was well tolerated in animals, and could be combined with standard-of-care treatments to further improve efficacy. TH1579 significantly improved survival in two different AML disease models in vivo. In conclusion, the preclinical data presented here support that TH1579 is a promising novel anticancer agent for AML, providing a rationale to investigate the clinical usefulness of TH1579 in AML in an ongoing clinical phase I trial. SIGNIFICANCE: The MTH1 inhibitor TH1579 is a potential novel AML treatment, targeting both blasts and the pivotal leukemic stem cells while sparing normal bone marrow cells.

Targeting OGG1 arrests cancer cell proliferation by inducing replication stress.

Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment.

Karonudib is a promising anticancer therapy in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common form of liver cancer and is generally caused by viral infections or consumption of mutagens, such as alcohol. While liver transplantation and hepatectomy is curative for some patients, many relapse into disease with few treatment options such as tyrosine kinase inhibitors, for example, sorafenib or lenvatinib. The need for novel systemic treatment approaches is urgent. METHODS: MTH1 expression profile was first analyzed in a HCC database and MTH1 mRNA/protein level was determined in resected HCC and paired paracancerous tissues with polymerase chain reaction (PCR) and immunohistochemistry. HCC cancer cell lines were exposed in vitro to MTH1 inhibitors or depleted of MTH1 by siRNA. 8-oxoG was measured by the modified comet assay. The effect of MTH1 inhibition on tumor growth was explored in HCC xenograft in vivo models. RESULTS: MTH1 protein level is elevated in HCC tissue compared with paracancerous liver tissue and indicates poor prognosis. The MTH1 inhibitor Karonudib (TH1579) and siRNA effectively introduce toxic oxidized nucleotides into DNA, 8-oxoG, and kill HCC cell lines in vitro. Furthermore, we demonstrate that HCC growth in a xenograft mouse model in vivo is efficiently suppressed by Karonudib. CONCLUSION: Altogether, these data suggest HCC relies on MTH1 for survival, which can be targeted and may open up a novel treatment option for HCC in the future.

Small-molecule inhibitor of OGG1 suppresses proinflammatory gene expression and inflammation.

The onset of inflammation is associated with reactive oxygen species and oxidative damage to macromolecules like 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA. Because 8-oxoguanine DNA glycosylase 1 (OGG1) binds 8-oxoG and because Ogg1-deficient mice are resistant to acute and systemic inflammation, we hypothesized that OGG1 inhibition may represent a strategy for the prevention and treatment of inflammation. We developed TH5487, a selective active-site inhibitor of OGG1, which hampers OGG1 binding to and repair of 8-oxoG and which is well tolerated by mice. TH5487 prevents tumor necrosis factor-α-induced OGG1-DNA interactions at guanine-rich promoters of proinflammatory genes. This, in turn, decreases DNA occupancy of nuclear factor κB and proinflammatory gene expression, resulting in decreased immune cell recruitment to mouse lungs. Thus, we present a proof of concept that targeting oxidative DNA repair can alleviate inflammatory conditions in vivo.

Development and validation of method for TH588 and TH287, potent MTH1 inhibitors and new anti-cancer agents, for pharmacokinetic studies in mice plasma.

MTH1 is a protein that is required for cancer cell survival and is overexpressed in cancer cells. TH588 and TH287 are two new compounds that inhibit the MTH1 protein. The inhibitors were tested in pharmacokinetic studies on mice. A bioanalytical method was developed and validated for determination in mice plasma. The method was based on protein precipitation followed by LC-MS/MS analysis. The separation was performed on an Ascentis Express RP-Amide C18 column. The mass spectrometer was operated in positive electrospray ionization mode and the analytes were determined with multiple reaction monitoring (MRM). Abundant monoisotopic fragments were used for quantification. Two additional fragments were used for conformational analysis. The recovery of the compounds in plasma varied between 61 and 91% and the matrix effects were low and ranged between -3% and +2%. The method showed to be selective, linear, accurate and precise, and applicable for preclinical pharmacokinetic studies of TH588 and TH287 in mouse plasma. Half-life (T1/2) was ≤3.5h and maximum concentration (Cmax) ranged between 0.82 and 338µM for the different administration routes and compounds.

MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool.

Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bind in the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.

Stereospecific targeting of MTH1 by (S)-crizotinib as an anticancer strategy.

Activated RAS GTPase signalling is a critical driver of oncogenic transformation and malignant disease. Cellular models of RAS-dependent cancers have been used to identify experimental small molecules, such as SCH51344, but their molecular mechanism of action remains generally unknown. Here, using a chemical proteomic approach, we identify the target of SCH51344 as the human mutT homologue MTH1 (also known as NUDT1), a nucleotide pool sanitizing enzyme. Loss-of-function of MTH1 impaired growth of KRAS tumour cells, whereas MTH1 overexpression mitigated sensitivity towards SCH51344. Searching for more drug-like inhibitors, we identified the kinase inhibitor crizotinib as a nanomolar suppressor of MTH1 activity. Surprisingly, the clinically used (R)-enantiomer of the drug was inactive, whereas the (S)-enantiomer selectively inhibited MTH1 catalytic activity. Enzymatic assays, chemical proteomic profiling, kinome-wide activity surveys and MTH1 co-crystal structures of both enantiomers provide a rationale for this remarkable stereospecificity. Disruption of nucleotide pool homeostasis via MTH1 inhibition by (S)-crizotinib induced an increase in DNA single-strand breaks, activated DNA repair in human colon carcinoma cells, and effectively suppressed tumour growth in animal models. Our results propose (S)-crizotinib as an attractive chemical entity for further pre-clinical evaluation, and small-molecule inhibitors of MTH1 in general as a promising novel class of anticancer agents.

SMG-1 suppresses CDK2 and tumor growth by regulating both the p53 and Cdc25A signaling pathways.

The DNA damage response is coordinated by phosphatidylinositol 3-kinase-related kinases, ATM, ATR, and DNA-PK. SMG-1 is the least studied stress-responsive member of this family. Here, we show that SMG-1 regulates the G 1/S checkpoint through both a p53-dependent, and a p53-independent pathway. We identify Cdc25A as a new SMG-1 substrate, and show that cells depleted of SMG-1 exhibit prolonged Cdc25A stability, failing to inactivate CDK2 in response to radiation. Given an increased tumor growth following depletion of SMG-1, our data demonstrate a novel role for SMG-1 in regulating Cdc25A and suppressing oncogenic CDK2 driven proliferation, confirming SMG-1 as a tumor suppressor.

The clock gene PER2 and sleep problems: association with alcohol consumption among Swedish adolescents.

BACKGROUND: Alcohol abuse is associated with sleep problems, which are often linked to circadian rhythm disturbances. Previous studies have separately examined the effects of mutations in the clock gene PER2 on alcohol consumption and sleep problems. Here we hypothesized that an allelic variation in the PER2 gene is associated with alcohol consumption in interaction with sleep problems among adolescents. METHODS: The Survey of Adolescent Life and Health in Västmanland 2006, a Swedish county, including 1254 students 17-18 years old, was used as a population-representative sample of adolescents. We investigated the PER2 Single Nucleotide polymorphism (SNP) 10870 (A/G) in the cohort together with an assessment of alcohol consumption according to the AUDIT-C questionnaire, and sleep problems using a survey consisting of 18 items. Furthermore, we carried out an exploratory analysis on the PER2 Single Nucleotide Polymorphism 10870 polymorphism in a group of severely alcoholic females. RESULTS: We found a significant association of the SNP 10870 in adolescent boys, where the genotype AA, in the presence of several and frequent sleep problems, was associated with increased alcohol consumption. Among adolescent girls, only sleep problems were related to alcohol consumption. A non-significant trend was observed among the severely alcoholic females, with the G allele being over-represented in the severely alcoholic females group in comparision to the control females. CONCLUSION: These results indicate that PER2 gene variation is associated with alcohol consumption in interaction with sleep problems among Swedish adolescent boys.

Two repeated maternal separation procedures differentially affect brain 5-hydroxytryptamine transporter and receptors in young and adult male and female rats.

Early environment is a known determinant for individual differences in vulnerability for adult psychopathology, e.g., ethanol addiction. One underlying mechanism could be dysfunction in serotonergic neurotransmission. This study focused on the methodological considerations regarding an animal model for studying effects of early environment, maternal separation (MS), using two different paradigms. Age- and sex-specific effects on brain stem 5-hydroxytryptamine (5-HT) transporter and receptors were examined. Male and female rat pups were assigned to either litter-wise MS for 15 or 360 min (MS15l or MS360l) or individual MS for 15 or 360 min (MS15i or MS360i) daily during postnatal days 1-21. Normal animal facility reared rats were used as controls. Analyses were performed in young and adult rats. As compared to the other males, MS15l males had lower 5-HT(1A) and 5-HT(2C) receptor mRNA expression at both ages, lower 5-HT(2A) receptor mRNA when young and lower 5-HTT mRNA expression when adult. In contrast, adult MS15l females had higher 5-HT(2C) receptor mRNA expression than other female rats. The strong impact of MS15l on 5-HT-related genes was either transient or persistent depending on sex and fewer effects on gene expression were observed in females than in males. This study shows the importance of tactile contact for the consequences of short but not prolonged MS, as evidenced by major differences between MS15l and MS15i. The results suggest that MS15i is less suitable than MS15l to simulate a protective environment in studies of, for instance, ethanol addiction processes.

Transcription factor AP2 beta involved in severe female alcoholism.

Susceptibility to alcoholism and antisocial behavior exhibits an evident link to monoaminergic neurotransmission. The serotonin system in particular, which is associated with regulation of mood and behavior, has an influence on personality characters that are firmly connected to risk of developing alcoholism and antisocial behavior, such as impulsiveness, and aggression. The transcription factor TFAP2b has repeatedly been shown to be involved in monoaminergic transmission, likely due to a regulatory effect on genes that are fundamental to this system, e.g. monoamine oxidase type A, and the serotonin transporter. Recent research has identified a functional polymorphism in the gene encoding TFAP2B that regulates its level of expression. In the present study we have compared a sample of female alcoholics (n=107), sentenced to institutional care for their severe addiction, contrasted against a control sample of adolescent females (n=875). The results showed that parental alcohol misuse was significantly more common among the alcoholic females, and also that parental alcohol misuse was associated with a reduction in age of alcohol debut. We also addressed the question of whether a functional TFAP2b polymorphism was associated with alcoholism. Results showed that the high-functioning allele was significantly more common among the female alcoholics, compared to the non-alcoholic controls. Furthermore, the results also indicated that psychosocial factors, in terms of parental alcohol misuse, depression or psychiatric disorder, had an influence on the association. It was observed that the genetic association was restricted to the subset of cases that had not experienced these negative psychosocial factors.

The transcription factor TFAP2B is associated with insulin resistance and adiposity in healthy adolescents.

Insulin resistance and central adiposity are strong risk indicators for type 2 diabetes and coronary heart disease. An important role for adipose tissue in the etiology and progression of these conditions has recently become more evident. A transcription factor, TFAP2B, has been shown to participate in the regulation of adipocyte metabolism, by facilitating glucose uptake and lipid accumulation, while simultaneously reducing insulin sensitivity, and recently a direct function for TFAP2B as an inhibitor of adiponectin expression was observed. In this study, we have investigated how insulin resistance, plasma adiponectin, and central adiposity, in a normal population of adolescents, are affected by genetic variability in TFAP2B. Our results show that both insulin sensitivity, as measured from levels of fasting glucose and insulin, and central adiposity, estimated by subscapular skinfold thickness, were significantly associated to genetic variability in TFAP2B. This association was restricted to males only, where carriers of the 4-repeat allele of intron 2 had higher insulin sensitivity and lower subscapular skinfold thickness. Levels of adiponectin did not show any association to the TFAP2B polymorphism, but was negatively correlated to central adiposity in females. These results suggest that reduction of TFAP2B expression could have a protective effect against future risk of complications associated with decreased insulin sensitivity and central adiposity, such as type 2 diabetes and coronary heart disease.

Serotonin transporter (5-HTTLPR) and monoamine oxidase (MAOA) promoter polymorphisms in women with severe alcoholism.

The serotonin system is known to play a pivotal role for mood, behaviour and psychic illness as e.g. alcoholism. Alcoholism in both males and females has been associated with polymorphisms in genes encoding for proteins of importance for central serotonergic function. Genotyping of two functional polymorphisms in the promoter region of the serotonin transporter and monoamine oxidase-A, respectively, (5-HTT-LPR and MAOA-VNTR), was performed in a group of women with severe alcohol addiction. A large sample of adolescent females from a normal population was used as controls. A significantly higher frequency of the LL 5-HTT genotype (high activity) was found in female addicts without a known co-morbid psychiatric disorder than in the controls. Genotype of the MAOA-VNTR polymorphism did not differ significantly between addicts and controls. However, within the group of alcoholics, when the patients with known co-morbid psychiatric disorders were excluded, aggressive anti-social behaviour was significantly linked to the presence of the high activity MAOA allele. The pattern of associations between genotypes of 5-HTT-LPR and MAOA-VNTR in women with severe alcoholism differs from most corresponding studies on males.

A novel method for quantification of the folding of elastic laminae in elastic arteries.

A transgenic mouse overexpressing the human form of semicarbazide-sensitive amine oxidase (SSAO) is known to have an abnormal structure of the elastic laminae and the elastic fibres in the aorta. Compared to the non-transgenic littermates, the elastic laminae are less folded. In order to quantify the undulation of this structure, an image analysis program that identified the elastic laminae was developed. The program measures the area fraction in different sectors from a plane parallel to the aorta wall. Images were taken from unstained aorta specimens where the elastic laminae were visualised with phase contrast microscopy. A contextual operation of the images produced a local orientation estimation for every linear structure. The image was then thresholded in eight sectors from 0 degrees to 180 degrees , with different orientation angles. The results show that the area fraction of the elastic laminae was significantly lower for the transgenic mouse in all sectors measured except for two. At 0-25 degrees , no difference was seen. In the sector at 160-180 degrees , parallel to the aorta wall, the area fraction of elastic laminae was instead significantly higher in the transgenic mouse. A novel method is presented, developed for detection and quantification of pathological changes in the elastic laminae in the aorta wall. The method gave reliable results and is considered to be a useful tool for morphometric studies of aorta with this kind of altered morphology concerning the elastic laminae. When compared with tangent count, the control group had a significantly larger mean curvature.

Macrovascular changes in mice overexpressing human semicarbazide-sensitive amine oxidase in smooth muscle cells.

BACKGROUND: The catalytic activity of semicarbazide-sensitive amine oxidase (SSAO) is increased in diabetes, as well as in other disorders of cardiovascular origin. Our hypothesis is that SSAO is involved in the synthesis or maturation of elastin in vascular tissue. An increased SSAO activity can thereby be involved in the development of vascular damage. METHODS: Elastin quantification was performed in aorta of transgenic mice overexpressing the human form of SSAO, using electron microscopy. Furthermore, lung capacity was measured using a spirometry-mimicking method, developed for mice. The effect of vasoactive substances was estimated by measuring mean arterial pressure and pulse pressure under anesthesia. RESULTS: No differences in elastin quantity or lung capacity could be observed between transgenic or nontransgenic littermates. Pulse pressure was higher in transgenic mice, and electron microscopy of aorta showed elastin fibers parallel with the aorta wall (ie, straight fibers instead of folded compared with control mice). No difference in the response to adrenaline or sodium chloride was observed between the transgenic and control mice. The control mice had a clear decrease in blood pressure (BP) with a longer duration as a response to injection of a nitric oxide (NO) donor, sodium nitroprusside, compared with transgenic mice where only a minor response was observed. The SSAO activity in serum of control mice was elevated in response to injection of the NO donor, but not in response to a ganglion blocker. CONCLUSIONS: An elevated pulse pressure, together with an abnormal elastin structure in the aorta, suggests a rigidity of large arteries as a result of an elevated SSAO activity as well as a physiologic role for SSAO in elastin maturation.

Overexpression of semicarbazide-sensitive amine oxidase in smooth muscle cells leads to an abnormal structure of the aortic elastic laminas.

Elevated semicarbazide-sensitive amine oxidase (SSAO) activity has been observed in several human conditions, eg, diabetes, and it has been speculated that SSAO contributes to the development of vasculopathies associated with this disease. To investigate in vivo consequences of elevated expression of SSAO in vascular tissues, we have developed a transgenic model for overexpression of human SSAO in mice. A smooth muscle-specific promoter, smooth muscle alpha-actin promoter 8 (SMP8) was used. Transgenic expression of human SSAO in tissues with a high content of smooth muscle cells was confirmed by Northern blot analysis. Enzymatic analysis of homogenates from transgenic tissues showed elevated levels of SSAO activity compared to non-transgenic littermates. Furthermore, when plasma SSAO activity was analyzed, much higher activity was detected compared to plasma from control mice, indicating that plasma SSAO may originate from smooth muscle cells. Histopathological evaluation of aorta and renal artery from transgenic mice revealed an abnormal structure of the elastin tissue. Instead of the regularly folded elastic laminae normally found in tunica media of sacrificed mice, the elastic laminae were straight and unfolded with irregularly arranged elastic fibers, forming tangled webs, between the intercalating elastic laminae. These alterations of the elastin structures suggest that overexpression of SSAO has led to a reduced elasticity of the arteries. Moreover, the mean femoral arterial pressure of the SMP8 SSAO transgenic mice was significantly lower in comparison to non-transgenic littermates. This suggests that the transgenic mice have a defect in their ability to regulate blood pressure.

Semicarbazide-sensitive amine oxidase (SSAO) gene expression in alloxan-induced diabetes in mice.

BACKGROUND: Plasma activity of semicarbazide-sensitive amine oxidase (SSAO) has been reported to be significantly higher in diabetic patients compared to healthy controls. Due to the production of highly angiotoxic substances in SSAO-catalyzed reactions, it has been speculated that this could be a cause for the vascular complications frequently associated with diabetes. Little is known about how the enzyme activity is regulated, and why it is high in these patients. In the present study, we assessed the possibility of transcriptional regulation by analyzing SSAO activity and SSAO-mRNA levels in mice with alloxan-induced diabetes. MATERIALS AND METHODS: Diabetes was induced in NMRI mice by a single intravenous injection of alloxan. The enzyme activity was analyzed by a radiometric assay using (14) C-benzylamine as a substrate, and the mRNA-levels were analyzed by real-time PCR. RESULTS: We found that the enzyme activity was increased in lung and adipose tissue 1 day after induction, as the glucose levels start to rise. Seven days after the injection of alloxan, the activity in serum was increased, and this activity was positively correlated with blood glucose levels in the alloxan-treated animals. Although the enzyme activity was increased in adipose tissue as a result of the treatment, SSAO-mRNA levels in this tissue were decreased, possibly suggesting a negative feedback on the gene expression. CONCLUSIONS: The main conclusion from this study is that the increased enzyme activity observed in diabetes is not a result of increased SSAO gene transcription. We speculate that the enzyme activity is controlled by posttranslational modifications of the protein, and that the catalytic activity controls the gene expression.