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Journal of Pharmaceutical Sciences Oct 2023NDec is a novel combination of oral decitabine and tetrahydrouridine that is currently under clinical development for the treatment of sickle cell disease (SCD). Here,...
NDec is a novel combination of oral decitabine and tetrahydrouridine that is currently under clinical development for the treatment of sickle cell disease (SCD). Here, we investigate the potential for the tetrahydrouridine component of NDec to act as an inhibitor or substrate of key concentrative nucleoside transporters (CNT1-3) and equilibrative nucleoside transporters (ENT1-2). Nucleoside transporter inhibition and tetrahydrouridine accumulation assays were performed using Madin-Darby canine kidney strain II (MDCKII) cells overexpressing human CNT1, CNT2, CNT3, ENT1, and ENT2 transporters. Results showed that tetrahydrouridine did not influence CNT- or ENT-mediated uridine/adenosine accumulation in MDCKII cells at the concentrations tested (25 and 250 µM). Accumulation of tetrahydrouridine in MDCKII cells was initially shown to be mediated by CNT3 and ENT2. However, while time- and concentration-dependence experiments showed active accumulation of tetrahydrouridine in CNT3-expressing cells, allowing for estimation of K (3,140 µM) and V (1,600 pmol/mg protein/min), accumulation of tetrahydrouridine was not observed in ENT2-expressing cells. Potent CNT3 inhibitors are a class of drugs not generally prescribed to patients with SCD, except in certain specific circumstances. These data suggest that NDec can be administered safely with drugs that act as substrates and inhibitors of the nucleoside transporters included in this study.
Topics: Humans; Animals; Dogs; Nucleoside Transport Proteins; Nucleosides; Tetrahydrouridine; Equilibrative Nucleoside Transporter 1; Membrane Transport Proteins
PubMed: 37364771
DOI: 10.1016/j.xphs.2023.06.012 -
International Journal of Cancer Nov 2022Glial-lineage malignancies (gliomas) recurrently mutate and/or delete the master regulators of apoptosis p53 and/or p16/CDKN2A, undermining apoptosis-intending... (Review)
Review
Glial-lineage malignancies (gliomas) recurrently mutate and/or delete the master regulators of apoptosis p53 and/or p16/CDKN2A, undermining apoptosis-intending (cytotoxic) treatments. By contrast to disrupted p53/p16, glioma cells are live-wired with the master transcription factor circuits that specify and drive glial lineage fates: these transcription factors activate early-glial and replication programs as expected, but fail in their other usual function of forcing onward glial lineage-maturation-late-glial genes have constitutively "closed" chromatin requiring chromatin-remodeling for activation-glioma-genesis disrupts several epigenetic components needed to perform this work, and simultaneously amplifies repressing epigenetic machinery instead. Pharmacologic inhibition of repressing epigenetic enzymes thus allows activation of late-glial genes and terminates glioma self-replication (self-replication = replication without lineage-maturation), independent of p53/p16/apoptosis. Lineage-specifying master transcription factors therefore contrast with p53/p16 in being enriched in self-replicating glioma cells, reveal a cause-effect relationship between aberrant epigenetic repression of late-lineage programs and malignant self-replication, and point to specific epigenetic targets for noncytotoxic glioma-therapy.
Topics: Brain Neoplasms; Chromatin; Cyclin-Dependent Kinase Inhibitor p16; Glioma; Humans; Transcription Factors; Tumor Suppressor Protein p53
PubMed: 35603902
DOI: 10.1002/ijc.34131 -
Seminars in Hematology Apr 2018Fetal hemoglobin (HbF) inhibits the root cause of sickle pathophysiology, sickle hemoglobin polymerization. Individuals who naturally express high levels of HbF beyond... (Review)
Review
Fetal hemoglobin (HbF) inhibits the root cause of sickle pathophysiology, sickle hemoglobin polymerization. Individuals who naturally express high levels of HbF beyond infancy thus receive some protection from sickle complications. To mimic this natural genetic experiment using drugs, one guiding observation was that HbF is increased during recovery of bone marrow from extreme stress. This led to evaluation and approval of the cytotoxic (cell killing) drug hydroxyurea to treat sickle cell disease. Cytotoxic approaches are limited in potency and sustainability, however, since they require hematopoietic reserves sufficient to repeatedly mount recoveries from stress that destroys their counterparts, and such reserves are finite. HbF induction even by stress ultimately involves chromatin remodeling of the gene for HbF (HBG), therefore, a logical alternative approach is to directly inhibit epigenetic enzymes that repress HBG-implicated enzymes include DNA methyltransferase 1, histone deacetylases, lysine demethylase 1, protein arginine methyltransferase 5, euchromatic histone lysine methyltransferase 2 and chromodomain helicase DNA-binding protein 4. Clinical proof-of-principle that this alternative, noncytotoxic approach can generate substantial HbF and total hemoglobin increases has already been generated. Thus, with continued careful attention to fundamental biological and pharmacologic considerations (reviewed herein), there is potential that rational, molecular-targeted, safe and highly potent disease-modifying therapy can be realized for patients with sickle cell disease, with the accessibility and cost-effective properties needed for world-wide effect.
Topics: Antineoplastic Agents; Epigenomics; Fetal Hemoglobin; Humans; Hydroxyurea; Transcription Factors
PubMed: 29958562
DOI: 10.1053/j.seminhematol.2018.04.008 -
Organic & Biomolecular Chemistry Jul 2021The tolerance of cytidine deaminase (CDA) to expanded heterocycles is explored via three fluorescent cytidine analogues, where the pyrimidine core is fused to three...
The tolerance of cytidine deaminase (CDA) to expanded heterocycles is explored via three fluorescent cytidine analogues, where the pyrimidine core is fused to three distinct five-membered heterocycles at the 5/6 positions. The reaction between CDA and each analogue is followed by absorption and emission spectroscopy, revealing shorter reaction times for all analogues than the native substrate. Pseudo-first order and Michaelis-Menten kinetic analyses provide insight into the enzymatic deamination reactions and assist in drawing comparison to established structure activity relationships. Finally, inhibitor screening modalities are created for each analogue and validated with zebularine and tetrahydrouridine, two known CDA inhibitors.
Topics: Cytidine Deaminase
PubMed: 34019616
DOI: 10.1039/d1ob00705j -
Blood Mar 2021In this issue of , guided by clinical observations and needs, Gong et al have identified a germline missense mutation in DNA methyltransferase 1 (), a ubiquitously...
In this issue of , guided by clinical observations and needs, Gong et al have identified a germline missense mutation in DNA methyltransferase 1 (), a ubiquitously expressed key epigenetic regulator, as a cause of hereditary persistence of fetal hemoglobin (HPFH). HPFH protects against β-thalassemia and sickle cell disease (the β-hemoglobinopathies). Discussed here is how these findings by Gong et al continue the pioneering role of the β-hemoglobinopathies as a model of discovery for all biomedicine. Sickle cell disease, after all, is the “first molecular disease”: altered migration of sickle vs normal hemoglobin in gel electrophoresis demonstrated, for the first time, that the structure–chemical basis for disease is discoverable and knowable.
Topics: Hemoglobinopathies; Humans; beta-Globins
PubMed: 33764430
DOI: 10.1182/blood.2020009961 -
Journal For Immunotherapy of Cancer Nov 2022Novel therapies are urgently needed for ovarian cancer (OC), the fifth deadliest cancer in women. Preclinical work has shown that DNA methyltransferase inhibitors...
BACKGROUND
Novel therapies are urgently needed for ovarian cancer (OC), the fifth deadliest cancer in women. Preclinical work has shown that DNA methyltransferase inhibitors (DNMTis) can reverse the immunosuppressive tumor microenvironment in OC. Inhibiting DNA methyltransferases activate transcription of double-stranded (ds)RNA, including transposable elements. These dsRNAs activate sensors in the cytoplasm and trigger type I interferon (IFN) signaling, recruiting host immune cells to kill the tumor cells. Adenosine deaminase 1 (ADAR1) is induced by IFN signaling and edits mammalian dsRNA with an A-to-I nucleotide change, which is read as an A-to-G change in sequencing data. These edited dsRNAs cannot be sensed by dsRNA sensors, and thus ADAR1 inhibits the type I IFN response in a negative feedback loop. We hypothesized that decreasing ADAR1 editing would enhance the DNMTi-induced immune response.
METHODS
Human OC cell lines were treated in vitro with DNMTi and then RNA-sequenced to measure RNA editing. Adar1 was stably knocked down in ID8 mouse OC cells. Control cells (shGFP) or shAdar1 cells were tested with mock or DNMTi treatment. Tumor-infiltrating immune cells were immunophenotyped using flow cytometry and cell culture supernatants were analyzed for secreted chemokines/cytokines. Mice were injected with syngeneic shAdar1 ID8 cells and treated with tetrahydrouridine/DNMTi while given anti-interferon alpha and beta receptor 1, anti-CD8, or anti-NK1.1 antibodies every 3 days.
RESULTS
We show that ADAR1 edits transposable elements in human OC cell lines after DNMTi treatment in vitro. Combining ADAR1 knockdown with DNMTi significantly increases pro-inflammatory cytokine/chemokine production and sensitivity to IFN-β compared with either perturbation alone. Furthermore, DNMTi treatment and Adar1 loss reduces tumor burden and prolongs survival in an immunocompetent mouse model of OC. Combining Adar1 loss and DNMTi elicited the most robust antitumor response and transformed the immune microenvironment with increased recruitment and activation of CD8+ T cells.
CONCLUSION
In summary, we showed that the survival benefit from DNMTi plus ADAR1 inhibition is dependent on type I IFN signaling. Thus, epigenetically inducing transposable element transcription combined with inhibition of RNA editing is a novel therapeutic strategy to reverse immune evasion in OC, a disease that does not respond to current immunotherapies.
Topics: Female; Humans; Animals; Mice; RNA Editing; Tumor Microenvironment; DNA Methylation; DNA Transposable Elements; RNA-Binding Proteins; RNA, Double-Stranded; Carcinoma, Ovarian Epithelial; Ovarian Neoplasms; Cytokines; Mammals
PubMed: 36343976
DOI: 10.1136/jitc-2022-004974 -
Cell Reports Aug 2023Small cell lung cancers (SCLCs) rapidly resist cytotoxic chemotherapy and immune checkpoint inhibitor (ICI) treatments. New, non-cross-resistant therapies are thus...
Small cell lung cancers (SCLCs) rapidly resist cytotoxic chemotherapy and immune checkpoint inhibitor (ICI) treatments. New, non-cross-resistant therapies are thus needed. SCLC cells are committed into neuroendocrine lineage then maturation arrested. Implicating DNA methyltransferase 1 (DNMT1) in the maturation arrests, we find (1) the repression mark methylated CpG, written by DNMT1, is retained at suppressed neuroendocrine-lineage genes, even as other repression marks are erased; (2) DNMT1 is recurrently amplified, whereas Ten-Eleven-Translocation 2 (TET2), which functionally opposes DNMT1, is deleted; (3) DNMT1 is recruited into neuroendocrine-lineage master transcription factor (ASCL1, NEUROD1) hubs in SCLC cells; and (4) DNMT1 knockdown activated ASCL1-target genes and released SCLC cell-cycling exits by terminal lineage maturation, which are cycling exits that do not require the p53/apoptosis pathway used by cytotoxic chemotherapy. Inhibiting DNMT1/corepressors with clinical compounds accordingly extended survival of mice with chemorefractory and ICI-refractory, p53-null, disseminated SCLC. Lineage commitment of SCLC cells can hence be leveraged into non-cytotoxic therapy able to treat chemo/ICI-refractory SCLC.
Topics: Animals; Mice; Tumor Suppressor Protein p53; Small Cell Lung Carcinoma; Cell Cycle; Cell Division; Lung Neoplasms
PubMed: 37597186
DOI: 10.1016/j.celrep.2023.113016 -
Clinical Epigenetics May 2023Targeting the epigenome of cancerous diseases represents an innovative approach, and the DNA methylation inhibitor decitabine is recommended for the treatment of...
BACKGROUND
Targeting the epigenome of cancerous diseases represents an innovative approach, and the DNA methylation inhibitor decitabine is recommended for the treatment of hematological malignancies. Although epigenetic alterations are also common to solid tumors, the therapeutic efficacy of decitabine in colorectal adenocarcinomas (COAD) is unfavorable. Current research focuses on an identification of combination therapies either with chemotherapeutics or checkpoint inhibitors in modulating the tumor microenvironment. Here we report a series of molecular investigations to evaluate potency of decitabine, the histone deacetylase inhibitor PBA and the cytidine deaminase (CDA) inhibitor tetrahydrouridine (THU) in patient derived functional and p53 null colon cancer cell lines (CCCL). We focused on the inhibition of cell proliferation, the recovery of tumor suppressors and programmed cell death, and established clinical relevance by evaluating drug responsive genes among 270 COAD patients. Furthermore, we evaluated treatment responses based on CpG island density.
RESULTS
Decitabine caused marked repression of the DNMT1 protein. Conversely, PBA treatment of CCCL recovered acetylation of histone 3 lysine residues, and this enabled an open chromatin state. Unlike single decitabine treatment, the combined decitabine/PBA treatment caused > 95% inhibition of cell proliferation, prevented cell cycle progression especially in the S and G2-phase and induced programmed cell death. Decitabine and PBA differed in their ability to facilitate re-expression of genes localized on different chromosomes, and the combined decitabine/PBA treatment was most effective in the re-expression of 40 tumor suppressors and 13 genes typically silenced in cancer-associated genomic regions of COAD patients. Furthermore, this treatment repressed expression of 11 survival (anti-apoptotic) genes and augmented expression of X-chromosome inactivated genes, especially the lncRNA Xist to facilitate p53-mediated apoptosis. Pharmacological inhibition of CDA by THU or its gene knockdown prevented decitabine inactivation. Strikingly, PBA treatment recovered the expression of the decitabine drug-uptake transporter SLC15A1, thus enabling high tumor drug-loads. Finally, for 26 drug responsive genes we demonstrated improved survival in COAD patients.
CONCLUSION
The combined decitabine/PBA/THU drug treatment improved drug potency considerably, and given their existing regulatory approval, our findings merit prospective clinical trials for the triple combination in COAD patients.
Topics: Humans; Decitabine; Azacitidine; Histone Deacetylases; Cytidine Deaminase; Tumor Suppressor Protein p53; Prospective Studies; DNA Methylation; Histone Deacetylase Inhibitors; Tetrahydrouridine; Epigenesis, Genetic; Adenocarcinoma; Colorectal Neoplasms; Cell Line, Tumor; Tumor Microenvironment
PubMed: 37208732
DOI: 10.1186/s13148-023-01500-1 -
Journal of Neuro-oncology Jan 2016Chemotherapies active in preclinical studies frequently fail in the clinic due to lack of efficacy, which limits progress for rare cancers since only small numbers of...
Chemotherapies active in preclinical studies frequently fail in the clinic due to lack of efficacy, which limits progress for rare cancers since only small numbers of patients are available for clinical trials. Thus, a preclinical drug development pipeline was developed to prioritize potentially active regimens for pediatric brain tumors spanning from in vitro drug screening, through intracranial and intra-tumoral pharmacokinetics to in vivo efficacy studies. Here, as an example of the pipeline, data are presented for the combination of 5-fluoro-2'-deoxycytidine and tetrahydrouridine in three pediatric brain tumor models. The in vitro activity of nine novel therapies was tested against tumor spheres derived from faithful mouse models of Group 3 medulloblastoma, ependymoma, and choroid plexus carcinoma. Agents with the greatest in vitro potency were then subjected to a comprehensive series of in vivo pharmacokinetic (PK) and pharmacodynamic (PD) studies culminating in preclinical efficacy trials in mice harboring brain tumors. The nucleoside analog 5-fluoro-2'-deoxycytidine (FdCyd) markedly reduced the proliferation in vitro of all three brain tumor cell types at nanomolar concentrations. Detailed intracranial PK studies confirmed that systemically administered FdCyd exceeded concentrations in brain tumors necessary to inhibit tumor cell proliferation, but no tumor displayed a significant in vivo therapeutic response. Despite promising in vitro activity and in vivo PK properties, FdCyd is unlikely to be an effective treatment of pediatric brain tumors, and therefore was deprioritized for the clinic. Our comprehensive and integrated preclinical drug development pipeline should reduce the attrition of drugs in clinical trials.
Topics: Animals; Antineoplastic Agents; Apoptosis; Brain; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Deoxycytidine; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Epigenesis, Genetic; Mice; Mice, Nude; Tetrahydrouridine
PubMed: 26518542
DOI: 10.1007/s11060-015-1965-0