A novel oral inhibitor for one-carbon metabolism and checkpoint kinase 1 inhibitor as a rational combination treatment for breast cancer.

Patients with triple-negative breast cancer have a poor prognosis as only a few efficient targeted therapies are available. Cancer cells are characterized by their unregulated proliferation and require large amounts of nucleotides to replicate their DNA. One-carbon metabolism contributes to purine and pyrimidine nucleotide synthesis by supplying one carbon atom. Although mitochondrial one-carbon metabolism has recently been focused on as an important target for cancer treatment, few specific inhibitors have been reported. In this study, we aimed to examine the effects of DS18561882 (DS18), a novel, orally active, specific inhibitor of methylenetetrahydrofolate dehydrogenase (MTHFD2), a mitochondrial enzyme involved in one-carbon metabolism. Treatment with DS18 led to a marked reduction in cancer-cell proliferation; however, it did not induce cell death. Combinatorial treatment with DS18 and inhibitors of checkpoint kinase 1 (Chk1), an activator of the S phase checkpoint pathway, efficiently induced apoptotic cell death in breast cancer cells and suppressed tumorigenesis in a triple-negative breast cancer patient-derived xenograft model. Mechanistically, MTHFD2 inhibition led to cell cycle arrest and slowed nucleotide synthesis. This finding suggests that DNA replication stress occurs due to nucleotide shortage and that the S-phase checkpoint pathway is activated, leading to cell-cycle arrest. Combinatorial treatment with both inhibitors released cell-cycle arrest, but induced accumulation of DNA double-strand breaks, leading to apoptotic cell death. Collectively, a combination of MTHFD2 and Chk1 inhibitors would be a rational treatment option for patients with triple-negative breast cancer.

BRCA2 associates with MCM10 to suppress PRIMPOL-mediated repriming and single-stranded gap formation after DNA damage.

The BRCA2 tumor suppressor protects genome integrity by promoting homologous recombination-based repair of DNA breaks, stability of stalled DNA replication forks and DNA damage-induced cell cycle checkpoints. BRCA2 deficient cells display the radio-resistant DNA synthesis (RDS) phenotype, however the mechanism has remained elusive. Here we show that cells without BRCA2 are unable to sufficiently restrain DNA replication fork progression after DNA damage, and the underrestrained fork progression is due primarily to Primase-Polymerase (PRIMPOL)-mediated repriming of DNA synthesis downstream of lesions, leaving behind single-stranded DNA gaps. Moreover, we find that BRCA2 associates with the essential DNA replication factor MCM10 and this association suppresses PRIMPOL-mediated repriming and ssDNA gap formation, while having no impact on the stability of stalled replication forks. Our findings establish an important function for BRCA2, provide insights into replication fork control during the DNA damage response, and may have implications in tumor suppression and therapy response.

USP11 controls R-loops by regulating senataxin proteostasis.

R-loops are by-products of transcription that must be tightly regulated to maintain genomic stability and gene expression. Here, we describe a mechanism for the regulation of the R-loop-specific helicase, senataxin (SETX), and identify the ubiquitin specific peptidase 11 (USP11) as an R-loop regulator. USP11 de-ubiquitinates SETX and its depletion increases SETX K48-ubiquitination and protein turnover. Loss of USP11 decreases SETX steady-state levels and reduces R-loop dissolution. Ageing of USP11 knockout cells restores SETX levels via compensatory transcriptional downregulation of the E3 ubiquitin ligase, KEAP1. Loss of USP11 reduces SETX enrichment at KEAP1 promoter, leading to R-loop accumulation, enrichment of the endonuclease XPF and formation of double-strand breaks. Overexpression of KEAP1 increases SETX K48-ubiquitination, promotes its degradation and R-loop accumulation. These data define a ubiquitination-dependent mechanism for SETX regulation, which is controlled by the opposing activities of USP11 and KEAP1 with broad applications for cancer and neurological disease.

Xanthine Derivatives Reveal an Allosteric Binding Site in Methylenetetrahydrofolate Dehydrogenase 2 (MTHFD2).

Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) plays an important role in one-carbon metabolism. The MTHFD2 gene is upregulated in various cancers but very low or undetectable in normal proliferating cells, and therefore a potential target for cancer treatment. In this study, we present the structure of MTHFD2 in complex with xanthine derivative 15, which allosterically binds to MTHFD2 and coexists with the substrate analogue. A kinetic study demonstrated the uncompetitive inhibition of MTHFD2 by 15. Allosteric inhibitors often provide good selectivity and, indeed, xanthine derivatives are highly selective for MTHFD2. Moreover, several conformational changes were observed upon the binding of 15, which impeded the binding of the cofactor and phosphate to MTHFD2. To the best of our knowledge, this is the first study to identify allosteric inhibitors targeting the MTHFD family and our results would provide insights on the inhibition mechanism of MTHFD proteins and the development of novel inhibitors.

Catalytically inactive, purified RNase H1: A specific and sensitive probe for RNA-DNA hybrid imaging.

R-loops are three-stranded nucleic acid structures with both physiological and pathological roles in cells. R-loop imaging generally relies on detection of the RNA-DNA hybrid component of these structures using the S9.6 antibody. We show that the use of this antibody for imaging can be problematic because it readily binds to double-stranded RNA (dsRNA) in vitro and in vivo, giving rise to nonspecific signal. In contrast, purified, catalytically inactive human RNase H1 tagged with GFP (GFP-dRNH1) is a more specific reagent for imaging RNA-DNA hybrids. GFP-dRNH1 binds strongly to RNA-DNA hybrids but not to dsRNA oligonucleotides in fixed human cells and is not susceptible to binding endogenous RNA. Furthermore, we demonstrate that purified GFP-dRNH1 can be applied to fixed cells to detect hybrids after their induction, thereby bypassing the need for cell line engineering. GFP-dRNH1 therefore promises to be a versatile tool for imaging and quantifying RNA-DNA hybrids under a wide range of conditions.

The folate cycle enzyme MTHFD2 induces cancer immune evasion through PD-L1 up-regulation.

Metabolic enzymes and metabolites display non-metabolic functions in immune cell signalling that modulate immune attack ability. However, whether and how a tumour's metabolic remodelling contributes to its immune resistance remain to be clarified. Here we perform a functional screen of metabolic genes that rescue tumour cells from effector T cell cytotoxicity, and identify the embryo- and tumour-specific folate cycle enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2). Mechanistically, MTHFD2 promotes basal and IFN-γ-stimulated PD-L1 expression, which is necessary for tumourigenesis in vivo. Moreover, IFN-γ stimulates MTHFD2 through the AKT-mTORC1 pathway. Meanwhile, MTHFD2 drives the folate cycle to sustain sufficient uridine-related metabolites including UDP-GlcNAc, which promotes the global O-GlcNAcylation of proteins including cMYC, resulting in increased cMYC stability and PD-L1 transcription. Consistently, the O-GlcNAcylation level positively correlates with MTHFD2 and PD-L1 in pancreatic cancer patients. These findings uncover a non-metabolic role for MTHFD2 in cell signalling and cancer biology.

Identification of theranostic factors for patients developing metastasis after surgery for early-stage lung adenocarcinoma.

Rationale: Lung adenocarcinoma (LUAD) is an aggressive disease with high propensity of metastasis. Among patients with early-stage disease, more than 30% of them may relapse or develop metastasis. There is an unmet medical need to stratify patients with early-stage LUAD according to their risk of relapse/metastasis to guide preventive or therapeutic approaches. In this study, we identified 4 genes that can serve both therapeutic and diagnostic (theranostic) purposes. Methods: Three independent datasets (GEO, TCGA, and KMPlotter) were used to evaluate gene expression profile of patients with LUAD by unbiased screening approach. Upon significant genes uncovered, functional enrichment analysis was carried out. The predictive power of their expression on patient prognosis were evaluated. Once confirmed their theranostic roles by integrated bioinformatics, we further conducted in vitro and in vivo validation. Results: We found that four genes (ADAM9, MTHFD2, RRM2, and SLC2A1) were associated with poor patient outcomes with an increased hazard ratio in LUAD. Knockdown of them, both separately and simultaneously, suppressed lung cancer cell proliferation and migration ability in vitro and prolonged survival time in metastatic tumor mouse models. Moreover, these four biomarkers were found to be overexpressed in tumor tissues from LUAD patients, and the total immunohistochemical staining scores correlated with poor prognosis. Conclusions: These results suggest that these four identified genes could be theranostic biomarkers for stratifying high-risk patients who develop relapse/metastasis in early-stage LUAD. Developing therapeutic approaches for the four biomarkers may benefit early-stage LUAD patients after surgery.

Folate-mediated one-carbon metabolism: a targeting strategy in cancer therapy.

Folate-mediated one-carbon metabolism (FOCM) supports vital events for the growth and survival of proliferating cells. Nucleotide synthesis and DNA methylation are the biochemical bases of cancers that are highly dependent on FOCM. Recent studies revealed that FOCM is connected with redox homeostasis and epigenetics in cancer. Furthermore, folate-metabolizing enzymes, such as serine hydroxymethyltransferase 2 (SHMT2) and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), are associated with the development of cancers, including breast cancer, highlighting their potential application in tumor-targeted therapy. Therefore, targeting metabolizing enzymes, especially SHMT2 and MTHFD2, provides a novel strategy for cancer treatment. In this review, we outline current understanding of the functions of SHMT2 and MTHFD2, discussing their expression, potential functions, and regulatory mechanism in cancers. Furthermore, we discuss examples of inhibitors of SHMT2 and MTHFD2.

Therapeutic Targeting of Mitochondrial One-Carbon Metabolism in Cancer.

One-carbon (1C) metabolism encompasses folate-mediated 1C transfer reactions and related processes, including nucleotide and amino acid biosynthesis, antioxidant regeneration, and epigenetic regulation. 1C pathways are compartmentalized in the cytosol, mitochondria, and nucleus. 1C metabolism in the cytosol has been an important therapeutic target for cancer since the inception of modern chemotherapy, and "antifolates" targeting cytosolic 1C pathways continue to be a mainstay of the chemotherapy armamentarium for cancer. Recent insights into the complexities of 1C metabolism in cancer cells, including the critical role of the mitochondrial 1C pathway as a source of 1C units, glycine, reducing equivalents, and ATP, have spurred the discovery of novel compounds that target these reactions, with particular focus on 5,10-methylene tetrahydrofolate dehydrogenase 2 and serine hydroxymethyltransferase 2. In this review, we discuss key aspects of 1C metabolism, with emphasis on the importance of mitochondrial 1C metabolism to metabolic homeostasis, its relationship with the oncogenic phenotype, and its therapeutic potential for cancer.

HMCES Maintains Replication Fork Progression and Prevents Double-Strand Breaks in Response to APOBEC Deamination and Abasic Site Formation.

5-Hydroxymethylcytosine (5hmC) binding, ES-cell-specific (HMCES) crosslinks to apurinic or apyrimidinic (AP, abasic) sites in single-strand DNA (ssDNA). To determine whether HMCES responds to the ssDNA abasic site in cells, we exploited the activity of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3A (APOBEC3A). APOBEC3A preferentially deaminates cytosines to uracils in ssDNA, which are then converted to abasic sites by uracil DNA glycosylase. We find that HMCES-deficient cells are hypersensitive to nuclear APOBEC3A localization. HMCES relocalizes to chromatin in response to nuclear APOBEC3A and protects abasic sites from processing into double-strand breaks (DSBs). Abasic sites induced by APOBEC3A slow both leading and lagging strand synthesis, and HMCES prevents further slowing of the replication fork by translesion synthesis (TLS) polymerases zeta (Polζ) and kappa (Polκ). Thus, our study provides direct evidence that HMCES responds to ssDNA abasic sites in cells to prevent DNA cleavage and balance the engagement of TLS polymerases.

Discovery of a Potent, Selective, and Orally Available MTHFD2 Inhibitor (DS18561882) with in Vivo Antitumor Activity.

We report the discovery of a potent and isozyme-selective MTHFD2 inhibitor, DS18561882 (2). Through investigation of the substituents on our tricyclic coumarin scaffold (1,2,3,4-tetrahydrochromeno[3,4-c]pyridin-5-one), MTHFD2 inhibitory activity was shown to be elevated by incorporating an amine moiety at the 8-position and a methyl group at the 7-position of the initial lead 1. X-ray structure analysis revealed that a key interaction for enhanced potency was salt bridge formation between the amine moiety and the diphosphate linker of an NAD+ cofactor. Furthermore, ortho-substituted sulfonamide in place of benzoic acid of 1 significantly improved cell permeability and cell-based growth inhibition against a human breast cancer cell line. The thus-optimized DS18561882 showed the strongest cell-based activity (GI50 = 140 nM) in the class, a good oral pharmacokinetic profile, and thereby tumor growth inhibition in a mouse xenograft model upon oral administration.

Modulation of Redox Homeostasis by Inhibition of MTHFD2 in Colorectal Cancer: Mechanisms and Therapeutic Implications.

BACKGROUND: Overcoming oxidative stress is a critical step for tumor progression; however, the underlying mechanisms in colorectal cancer (CRC) remain unclear. METHODS: We investigated nicotinamide adenine dinucleotide (phosphate) (NAD(P))-dependent enzyme methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) expression, clinical relevance, redox modification, and molecular mechanisms using the CRC cells and tissues (n = 462 paired samples). The antitumor effects of MTHFD2 inhibitor LY345899 on CRC tumorigenesis and metastasis were evaluated in vitro and in vivo. Data analysis used Kaplan-Meier, Pearson's correlation, and Student t test where appropriate. All statistical tests were two-sided. RESULTS: Here, we report that the patients with high expression of MTHFD2 have a shorter overall survival (HR = 1.62, 95% CI = 1.12 to 2.36, P = .01) and disease-free survival (HR = 1.55, 95% CI = 1.07 to 2.27, P = .02) than patients with low MTHFD2 expression. Suppression of MTHFD2 disturbs NADPH and redox homeostasis and accelerates cell death under oxidative stress, such as hypoxia or anchorage independence (P ≤ .01 for all). Also, genetic or pharmacological inhibition of MTHFD2 suppresses CRC cell growth and lung and peritoneal metastasis in cell-based xenografts (n = 5-8 mice per group). Importantly, LY345899 treatment statistically significantly suppresses tumor growth and decreases the tumor weight in CRC patient-derived xenograft models (n = 10 mice per group, mean [SD] tumor weight of the vehicle-treated group was 1.83 [0.19] mg vs 0.74 [0.30] mg for the LY345899-treated group, P < .001). CONCLUSIONS: Our study presents evidence that MTHFD2 confers redox homeostasis and promotes CRC cell growth and metastasis. The folate analog LY345899 as MTHFD2 inhibitor displays therapeutic activity against CRC and warrants further clinical investigation for CRC treatment.

MTHFD2 Overexpression Predicts Poor Prognosis in Renal Cell Carcinoma and is Associated with Cell Proliferation and Vimentin-Modulated Migration and Invasion.

BACKGROUND/AIMS: To investigate the role of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) in the clinical prognosis and cell biology of renal cell carcinoma (RCC). METHODS: A total of 137 RCC tissues were evaluated by immunohistochemistry. The relationship between MTHFD2 overexpression and clinical parameters and vimentin expression was assessed. Kaplan-Meier curves and the log-rank test were applied for survival analysis according to MTHFD2 and vimentin expression in RCC tissues. The expression of MTHFD2 mRNA and protein was examined by quantitative reverse transcription PCR and western blotting, respectively. To determine further the biological activity of MTHFD2 in RCC, 786-O cells were transfected with short hairpin RNA specifically targeting MTHFD2 (shMTHFD2) with or without tumor necrosis factor (TNF)-α stimulation. Cell proliferation, cell migration and invasion and drug sensitivity were subsequently assessed using Cell Counting Kit-8, wound healing, and Transwell assays. RESULTS: Immunohistochemical analysis demonstrated that both MTHFD2 and vimentin overexpression was positively associated with clinical staging, pathological grade, and poor overall survival (all P < 0.05). MTHFD2 expression was closely correlated with vimentin overexpression in RCC (r = 0.402, P < 0.001). After knocking down MTHFD2 expression in 786-O cells, decreased cell proliferation, migration, and invasion were observed and accompanied by the reduced expression of vimentin. The effects of MTHFD2 down-regulation could be partially restrained by TNF-α treatment. Vimentin expression and cell migration and invasion, but not cell proliferation, were reversed by TNF-α stimulation. Furthermore, treatment of 786-O cells with shMTHFD2 increased their sensitivity to chemotherapy drugs. CONCLUSION: The current results demonstrated that MTHFD2 was overexpressed in RCC and associated with poor clinical characteristics, vimentin expression, and cellular features connected to malignant disease, thus, implicating MTHFD2 as a potential target for RCC therapy.

Biocatalysis with the milk protein ß-lactoglobulin: promoting retroaldol cleavage of α,ß-unsaturated aldehydes.

Enzymes with a hydrophobic binding site and an active site lysine have been suggested to be promiscuous in their catalytic activity. ß-Lactoglobulin (BLG), the principle whey protein found in milk, possesses a central calyx that binds non-polar molecules. Here, we report that BLG can catalyze the retro-aldol cleavage of α,ß-unsaturated aldehydes making it a naturally occurring protein capable of catalyzing retro-aldol reactions on hydrophobic substrates. Retroaldolase activity was seen to be most effective on substrates with phenyl or naphthyl side-chains. Use of a brominated substrate analogue inhibitor increases the product yield by a factor of three. BLG's catalytic activity and its ready availability make it a prime candidate for the development of commercial biocatalysts.

PolDIP2 interacts with human PrimPol and enhances its DNA polymerase activities.

Translesion synthesis (TLS) employs specialized DNA polymerases to bypass replication fork stalling lesions. PrimPol was recently identified as a TLS primase and polymerase involved in DNA damage tolerance. Here, we identify a novel PrimPol binding partner, PolDIP2, and describe how it regulates PrimPol's enzymatic activities. PolDIP2 stimulates the polymerase activity of PrimPol, enhancing both its capacity to bind DNA and the processivity of the catalytic domain. In addition, PolDIP2 stimulates both the efficiency and error-free bypass of 8-oxo-7,8-dihydrodeoxyguanosine (8-oxoG) lesions by PrimPol. We show that PolDIP2 binds to PrimPol's catalytic domain and identify potential binding sites. Finally, we demonstrate that depletion of PolDIP2 in human cells causes a decrease in replication fork rates, similar to that observed in PrimPol(-/-)cells. However, depletion of PolDIP2 in PrimPol(-/-)cells does not produce a further decrease in replication fork rates. Together, these findings establish that PolDIP2 can regulate the TLS polymerase and primer extension activities of PrimPol, further enhancing our understanding of the roles of PolDIP2 and PrimPol in eukaryotic DNA damage tolerance.

Synergistic control of sex hormones by 17ß-HSD type 7: a novel target for estrogen-dependent breast cancer.

17ß-hydroxysteroid dehydrogenase (17ß-HSD) type 1 is known as a critical target to block the final step of estrogen production in estrogen-dependent breast cancer. Recent confirmation of the role of dyhydroxytestosterone (DHT) in counteracting estrogen-induced cell growth prompted us to study the reductive 17ß-HSD type 7 (17ß-HSD7), which activates estrone while markedly inactivating DHT. The role of DHT in breast cancer cell proliferation is demonstrated by its independent suppression of cell growth in the presence of a physiological concentration of estradiol (E2). Moreover, an integral analysis of a large number of clinical samples in Oncomine datasets demonstrated the overexpression of 17ß-HSD7 in breast carcinoma. Inhibition of 17ß-HSD7 in breast cancer cells resulted in a lower level of E2 and a higher level of DHT, successively induced regulation of cyclinD1, p21, Bcl-2, and Bik, consequently arrested cell cycle in the G(0)/G(1) phase, and triggered apoptosis and auto-downregulation feedback of the enzyme. Such inhibition led to significant shrinkage of xenograft tumors with decreased cancer cell density and reduced 17ß-HSD7 expression. Decreased plasma E2 and elevated plasma DHT levels were also found. Thus, the dual functional 17ß-HSD7 is proposed as a novel target for estrogen-dependent breast cancer by regulating the balance of E2 and DHT. This demonstrates a conceptual advance on the general belief that the major role of this enzyme is in cholesterol metabolism.