The safety and efficacy outcomes of Minnelide given alone or in combination with paclitaxel in advanced gastric cancer: A phase I trial.

Minnelide is a water-soluble disodium salt variant of triptolide, an HSP70 inhibitor that can prevent tumor progression and induce apoptosis. Maximum tolerated dose (MTD), safety, and antitumor activity of Minnelide alone and its combination with paclitaxel were evaluated in this open-label, single-center, dose-escalation phase I study (NCT05566834) in patients who were previously treated for advanced gastric cancer (AGC). Minnelide was administered orally using a 3 + 3 dose-escalation design as monotherapy (Regimen A), and in combination with paclitaxel (Regimen B & C). Our results show that no patients experienced dose limiting toxicity (DLT) in the combination group (Regimen B& C) while 2 patients experienced DLT from the Regimen A group (n = 11) (Minnelide 1.5 mg). The MTD was Minnelide 1.25 mg once daily for 21days Q4 weeks as monotherapy. The most common Grade ≥3 AEs were neutropenia (19.4 %) and abdominal pain (11.1 %). In Regimen C, 71.5 % achieved either a partial response or a stable disease with the median PFS of 4.5 months, and the median OS of 10.7 months. The combination of Minnelide plus paclitaxel as salvage treatment in AGC patients showed meaningful clinical activity with a manageable safety profile. Based on these encouraging results, a phase II study is being initiated to test the effectiveness of the combination regimen in patients with advanced gastric cancer.

Role of epigenetics in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, associated with poor survival outcomes. Lack of early diagnosis, resistance to conventional therapeutic treatments (including immunotherapy) and recurrence are some of the major hurdles in PDAC and contribute to its poor survival rate. While the risk of genetic predisposition to cancers is widely acknowledged and understood, recent advances in whole-genome and next-generation sequencing techniques have led to the acknowledgment of the role played by epigenetics, especially in PDAC. Epigenetic changes are heritable genetic modifications that influence gene expression without altering the DNA sequence. Epigenetic mechanisms (e.g., DNA methylation, post-translational modification of histone complexes and ncRNA) that result in reversible changes in gene expression are increasingly understood to be responsible for tumor initiation, development and even escape from immune surveillance. Our review seeks to highlight the various components of the epigenetic machinery that are known to be implicated in PDAC initiation and development and the feasibility of targeting these components to identify novel pharmacological strategies that could potentially lead to breakthroughs in PDAC treatment.

Despite advances in detection and treatment, pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) remains one of the most aggressive malignancies known to mankind, with one of the lowest 5-year survival rates (11%). Due to the lack of distinctive symptoms and the tumors' aggressive ability to metastasize quickly, more than 50% of patients miss the opportunity to seek treatment at an early stage. While the role of genetics in cancer is well known, it is only recently that efforts have been made in identifying the role of heritable changes, regulated by epigenetic mechanisms, in the initiation and metastasis of cancer in individuals. Epigenetics refers to the phenomenon of alteration of gene expression through modifications of the chromatin landscape by DNA methylation, histone modifications and chromatin remodeling (due to ncRNA etc.) which is known to contribute to tumor heterogeneity. By identifying the epigenomic landscapes of patients' tumors, we can identify the components of the epigenetic machinery that influence PDAC initiation and development. These proteins and enzymes could be excellent targets for developing novel pharmacological strategies that could potentially lead to breakthroughs in PDAC treatment.

Selective CDK9 Inhibition by Natural Compound Toyocamycin in Cancer Cells.

Aberrant transcription in cancer cells involves the silencing of tumor suppressor genes (TSGs) and activation of oncogenes. Transcriptomic changes are associated with epigenomic alterations such as DNA-hypermethylation, histone deacetylation, and chromatin condensation in promoter regions of silenced TSGs. To discover novel drugs that trigger TSG reactivation in cancer cells, we used a GFP-reporter system whose expression is silenced by promoter DNA hypermethylation and histone deacetylation. After screening a natural product drug library, we identified that toyocamycin, an adenosine-analog, induces potent GFP reactivation and loss of clonogenicity in human colon cancer cells. Connectivity-mapping analysis revealed that toyocamycin produces a pharmacological signature mimicking cyclin-dependent kinase (CDK) inhibitors. RNA-sequencing revealed that the toyocamycin transcriptomic signature resembles that of a specific CDK9 inhibitor (HH1). Specific inhibition of RNA Pol II phosphorylation level and kinase assays confirmed that toyocamycin specifically inhibits CDK9 (IC50 = 79 nM) with a greater efficacy than other CDKs (IC50 values between 0.67 and 15 µM). Molecular docking showed that toyocamycin efficiently binds the CDK9 catalytic site in a conformation that differs from other CDKs, explained by the binding contribution of specific amino acids within the catalytic pocket and protein backbone. Altogether, we demonstrated that toyocamycin exhibits specific CDK9 inhibition in cancer cells, highlighting its potential for cancer chemotherapy.

Minnelide synergizes with conventional chemotherapy by targeting both cancer and associated stroma components in pancreatic cancer.

Addition of nab-paclitaxel to gemcitabine offers a survival benefit of only 6 weeks over gemcitabine alone at a cost of increased toxicity in PDAC. The goal of the present study is to evaluate the efficacy of Minnelide, a water-soluble prodrug of triptolide, in combination with the standard of care regimen for chemotherapy with the added advantage of reducing the doses of these drugs to minimize toxicity. Pancreatic cancer cell lines were implanted subcutaneously or orthotopically in athymic nude or C57BL/6J mice. Subsequently, animals were randomized and received saline or minnelide or full dose chemotherapy or low dose chemotherapy or minnelide in combination with low dose chemotherapy. Our results show that a combination of low doses of Minnelide with Gemcitabine + nab-paclitaxel significantly inhibited tumor progression and increased the survival of tumor-bearing mice in comparison with conventional chemotherapy alone. Moreover, combination therapy significantly reduced cancer-related morbidity by decreasing ascites and metastasis and effectively targeted both cancer and the associated stroma. In vitro studies with a combination of low doses of triptolide and paclitaxel significantly decreased the cell viability, increased apoptosis and led to significantly increased M-phase cell cycle arrest in various pancreatic cancer cell lines as compared to either drug alone. Our results show that Minnelide synergizes with conventional chemotherapy leading to a significant reduction in the doses of these toxic drugs, all the while achieving better efficacy in the treatment of PDAC. This combination effectively targeted both the cancer and the associated stromal components of pancreatic cancer.

Identification of Novel MeCP2 Cancer-Associated Target Genes and Post-Translational Modifications.

Abnormal regulation of DNA methylation and its readers has been associated with a wide range of cellular dysfunction. Disruption of the normal function of DNA methylation readers contributes to cancer progression, neurodevelopmental disorders, autoimmune disease and other pathologies. One reader of DNA methylation known to be especially important is MeCP2. It acts a bridge and connects DNA methylation with histone modifications and regulates many gene targets contributing to various diseases; however, much remains unknown about how it contributes to cancer malignancy. We and others previously described novel MeCP2 post-translational regulation. We set out to test the hypothesis that MeCP2 would regulate novel genes linked with tumorigenesis and that MeCP2 is subject to additional post-translational regulation not previously identified. Herein we report novel genes bound and regulated by MeCP2 through MeCP2 ChIP-seq and RNA-seq analyses in two breast cancer cell lines representing different breast cancer subtypes. Through genomics analyses, we localize MeCP2 to novel gene targets and further define the full range of gene targets within breast cancer cell lines. We also further examine the scope of clinical and pre-clinical lysine deacetylase inhibitors (KDACi) that regulate MeCP2 post-translationally. Through proteomics analyses, we identify many additional novel acetylation sites, nine of which are mutated in Rett Syndrome. Our study provides important new insight into downstream targets of MeCP2 and provide the first comprehensive map of novel sites of acetylation associated with both pre-clinical and FDA-approved KDACi used in the clinic. This report examines a critical reader of DNA methylation and has important implications for understanding MeCP2 regulation in cancer models and identifying novel molecular targets associated with epigenetic therapies.

Targeting CDK9 Reactivates Epigenetically Silenced Genes in Cancer.

Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.

Functional assessment of MeCP2 in Rett syndrome and cancers of breast, colon, and prostate.

Ever since the first report that mutations in methyl-CpG-binding protein 2 (MeCP2) causes Rett syndrome (RTT), a severe neurological disorder in females world-wide, there has been a keen interest to gain a comprehensive understanding of this protein. While the classical model associated with MeCP2 function suggests its role in gene suppression via recruitment of co-repressor complexes and histone deacetylases to methylated CpG-sites, recent discoveries have brought to light its role in transcription activation, modulation of RNA splicing, and chromatin compaction. Various post-translational modifications (PTMs) of MeCP2 further increase its functional versatility. Involvement of MeCP2 in pathologies other than RTT, such as tumorigenesis however, remains poorly explored and understood. This review provides a survey of the literature implicating MeCP2 in breast, colon and prostate cancer.

A novel MeCP2 acetylation site regulates interaction with ATRX and HDAC1.

Methyl-CpG-binding protein-2 (MeCP2) regulates gene expression by recruiting SWI/SNF DNA helicase/ATPase (ATRX) and Histone Deacetylase-1 (HDAC1) to methylated gene regions and modulates heterochromatin association by interacting with Heterochromatin protein-1. As MeCP2 contributes to tumor suppressor gene silencing and its mutation causes Rett Syndrome, we investigated how novel post-translational-modification contributes to its function. Herein we report that upon pharmacological inhibition of SIRT1 in RKO colon and MCF-7 breast cancer cells, endogenous MeCP2 is acetylated at sites critical for binding to DNA and transcriptional regulators. We created an acetylation mimetic mutation in MeCP2 and found it to possess decreased binding to ATRX and HDAC1. Conditions inducing MeCP2 acetylation do not alter its promoter occupancy at a subset of target genes analyzed, but do cause decreased binding to ATRX and HDAC1. We also report here that a specific inhibitor of SIRT1, IV, can be used to selectively decrease H3K27me3 repressive marks on a subset of repressed target gene promoters analyzed. Lastly, we show that RKO cells over-expressing MeCP2 mutant show reduced proliferation compared to those over-expressing MeCP2-wildtype. Our study demonstrates the importance of acetylated lysine residues and suggests their key role in regulating MeCP2 function and its ability to bind transcriptional regulators.

Contrast-enhanced [18F] fluorodeoxyglucose-positron emission tomography-computed tomography as an initial imaging modality in patients presenting with metastatic malignancy of undefined primary origin.

BACKGROUND: To evaluate the advantages of contrast enhanced F-18-fluorodeoxyglucose (FDG) positron emission tomography-computed tomography (PET-contrast enhanced CT [CECT]) when used as an initial imaging modality in patients presenting with metastatic malignancy of undefined primary origin (MUO). MATERIALS AND METHODS: A total of 243 patients with fine needle aspiration cytology/biopsy proven MUO were included in this prospective study. Patients who were thoroughly evaluated for primary or primary tumor was detected by any other investigation were excluded from the analysis. Totally, 163 patients with pathological diagnosis of malignancy but no apparent sites of the primary tumor were finally selected for analysis. The site of probable primary malignancy suggested by PET-CECT was confirmed by biopsy/follow-up. RESULTS: PET-CECT suggested probable site of primary in 128/163 (78.52%) patients. In 30/35 remaining patients, primary tumor was not detected even after extensive work-up. In 5 patients, where PET-CECT was negative, primary was found on further extensive investigations or follow-up. The sensitivity, specificity, positive predictive value and negative predictive value of the study were 95.76%, 66.67%, 88.28% and 85.71% respectively. CONCLUSIONS: F-18 FDG PET-CECT aptly serves the purpose of initial imaging modality owing to high sensitivity, negative and positive predictive value. PET-CECT not only surveys the whole body for the primary malignancy but also stages the disease accurately. Use of contrast improves the diagnostic utility of modality as well as help in staging of the primary tumor. Although benefits of using PET-CECT as initial diagnostic modality are obvious from this study, there is a need for a larger study comparing conventional methods for diagnosing primary in patients with MUO versus PET-CECT.

Frizzled 7 expression is positively regulated by SIRT1 and ß-catenin in breast cancer cells.

The Wnt signaling pathway is often chronically activated in diverse human tumors, and the Frizzled (FZD) family of receptors for Wnt ligands, are central to propagating oncogenic signals in a ß-catenin-dependent and independent manner. SIRT1 is a class III histone deacetylase (HDAC) that deacetylates histone and non-histone proteins to regulate gene transcription and protein function. We previously demonstrated that SIRT1 loss of function led to a significant decrease in the levels of Dishevelled (Dvl) proteins. To further explore this connection between the sirtuins and components of the Wnt pathway, we analyzed sirtuin-mediated regulation of FZD proteins. Here we explore the contribution of sirtuin deacetylases in promoting constitutive Wnt pathway activation in breast cancer cells. We demonstrate that the use of small molecule inhibitors of SIRT1 and SIRT2, and siRNA specific to SIRT1, all reduce the levels of FZD7 mRNA. We further demonstrate that pharmacologic inhibition of SIRT1/2 causes a marked reduction in FZD7 protein levels. Additionally, we show that ß-catenin and c-Jun occupy the 7 kb region upstream of the transcription start site of the FZD7 gene, and SIRT1 inhibition leads to a reduction in the occupancy of both ß-catenin and c-Jun at points along this region. This work uncovers a new mechanism for the regulation of FZD7 and provides a critical new link between the sirtuins and FZD7, one of the earliest nodal points from which oncogenic Wnt signaling emanates. This study shows that inhibition of specific sirtuins may provide a unique strategy for inhibiting the constitutively active Wnt pathway at the level of the receptor.