Targeting of REST with rationally-designed small molecule compounds exhibits synergetic therapeutic potential in human glioblastoma cells.

BACKGROUND: Glioblastoma (GBM) is an aggressive brain cancer associated with poor prognosis, intrinsic heterogeneity, plasticity, and therapy resistance. In some GBMs, cell proliferation is fueled by a transcriptional regulator, repressor element-1 silencing transcription factor (REST). RESULTS: Using CRISPR/Cas9, we identified GBM cell lines dependent on REST activity. We developed new small molecule inhibitory compounds targeting small C-terminal domain phosphatase 1 (SCP1) to reduce REST protein level and transcriptional activity in glioblastoma cells. Top leads of the series like GR-28 exhibit potent cytotoxicity, reduce REST protein level, and suppress its transcriptional activity. Upon the loss of REST protein, GBM cells can potentially compensate by rewiring fatty acid metabolism, enabling continued proliferation. Combining REST inhibition with the blockade of this compensatory adaptation using long-chain acyl-CoA synthetase inhibitor Triacsin C demonstrated substantial synergetic potential without inducing hepatotoxicity. CONCLUSIONS: Our results highlight the efficacy and selectivity of targeting REST alone or in combination as a therapeutic strategy to combat high-REST GBM.

Distinctive interactomes of RNA polymerase II phosphorylation during different stages of transcription.

During eukaryotic transcription, RNA polymerase II undergoes dynamic post-translational modifications on the C-terminal domain (CTD) of the largest subunit, generating an information-rich PTM landscape that transcriptional regulators bind. The phosphorylation of Ser5 and Ser2 of CTD heptad occurs spatiotemporally with the transcriptional stages, recruiting different transcriptional regulators to Pol II. To delineate the protein interactomes at different transcriptional stages, we reconstructed phosphorylation patterns of the CTD at Ser5 and Ser2 in vitro. Our results showed that distinct protein interactomes are recruited to RNA polymerase II at different stages of transcription by the phosphorylation of Ser2 and Ser5 of the CTD heptads. In particular, we characterized calcium homeostasis endoplasmic reticulum protein (CHERP) as a regulator bound by phospho-Ser2 heptad. Pol II association with CHERP recruits an accessory splicing complex whose loss results in broad changes in alternative splicing events. Our results shed light on the PTM-coded recruitment process that coordinates transcription.

Novel mitochondria-targeting compounds selectively kill human leukemia cells.

Acute myeloid leukemia (AML) is a heterogeneous group of aggressive hematological malignancies commonly associated with treatment resistance, high risk of relapse, and mitochondrial dysregulation. We identified six mitochondria-affecting compounds (PS compounds) that exhibit selective cytotoxicity against AML cells in vitro. Structure-activity relationship studies identified six analogs from two original scaffolds that had over an order of magnitude difference between LD50 in AML and healthy peripheral blood mononuclear cells. Mechanistically, all hit compounds reduced ATP and selectively impaired both basal and ATP-linked oxygen consumption in leukemic cells. Compounds derived from PS127 significantly upregulated production of reactive oxygen species (ROS) in AML cells and triggered ferroptotic, necroptotic, and/or apoptotic cell death in AML cell lines and refractory/relapsed AML primary samples. These compounds exhibited synergy with several anti-leukemia agents in AML, acute lymphoblastic leukemia (ALL), or chronic myelogenous leukemia (CML). Pilot in vivo efficacy studies indicate anti-leukemic efficacy in a MOLM14/GFP/LUC xenograft model, including extended survival in mice injected with leukemic cells pre-treated with PS127B or PS127E and in mice treated with PS127E at a dose of 5 mg/kg. These compounds are promising leads for development of future combinatorial therapeutic approaches for mitochondria-driven hematologic malignancies such as AML, ALL, and CML.

Mitochondrial metabolism as a target for acute myeloid leukemia treatment.

Acute myeloid leukemias (AML) are a group of aggressive hematologic malignancies resulting from acquired genetic mutations in hematopoietic stem cells that affect patients of all ages. Despite decades of research, standard chemotherapy still remains ineffective for some AML subtypes and is often inappropriate for older patients or those with comorbidities. Recently, a number of studies have identified unique mitochondrial alterations that lead to metabolic vulnerabilities in AML cells that may present viable treatment targets. These include mtDNA, dependency on oxidative phosphorylation, mitochondrial metabolism, and pro-survival signaling, as well as reactive oxygen species generation and mitochondrial dynamics. Moreover, some mitochondria-targeting chemotherapeutics and their combinations with other compounds have been FDA-approved for AML treatment. Here, we review recent studies that illuminate the effects of drugs and synergistic drug combinations that target diverse biomolecules and metabolic pathways related to mitochondria and their promise in experimental studies, clinical trials, and existing chemotherapeutic regimens.

An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity.

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes more complex, the goal of targeting mitochondria using various compounds that inflict mitochondrial damage (so-called mitocans) is becoming quite popular. Unfortunately, many existing cytotoxicity assays, such as those based on tetrazolium salts or resazurin require functional mitochondrial enzymes for their performance. The damage inflicted by compounds that target mitochondria often compromises the accuracy of these assays. Here, we describe a modified protocol based on differential staining with two fluorescent dyes, one of which is cell-permeant (Hoechst 33342) and the other of which is not (propidium iodide). The difference in staining allows living and dead cells to be discriminated. The assay is amenable to automated microscopy and image analysis, which increases throughput and reduces bias. This also allows the assay to be used in high-throughput fashion using 96-well plates, making it a viable option for drug discovery efforts, particularly when the drugs in question have some level of mitotoxicity. Importantly, results obtained by Hoechst/PI staining assay show increased consistency, both with trypan blue exclusion results and between biological replicates when the assay is compared to other methods.

Utilizing Synergistic Potential of Mitochondria-Targeting Drugs for Leukemia Therapy.

Acute myeloid leukemia (AML) is an aggressive group of cancers with high mortality rates and significant relapse risks. Current treatments are insufficient, and new therapies are needed. Recent discoveries suggest that AML may be particularly sensitive to chemotherapeutics that target mitochondria. To further investigate this sensitivity, six compounds that target mitochondria [IACS-010759, rotenone, cytarabine, etoposide, ABT-199 (venetoclax), and carbonyl cyanide m-chlorophenylhydrazone] were each paired with six compounds with other activities, including tyrosine kinase inhibitors (midostaurin and dasatinib), glycolytic inhibitors (2-deoxy-D-glucose, 3-bromopyruvate, and lonidamine), and the microtubule destabilizer vinorelbine. The 36 resulting drug combinations were tested for synergistic cytotoxicity against MOLM-13 and OCI-AML2 AML cell lines. Four combinations (IACS-010759 with vinorelbine, rotenone with 2-deoxy-D-glucose, carbonyl cyanide m-chlorophenylhydrazone with dasatinib, and venetoclax with lonidamine) showed synergistic cytotoxicity in both AML cell lines and were selective for tumor cells, as survival of healthy PBMCs was dramatically higher. Among these drug pairs, IACS-010759/vinorelbine decreased ATP level and impaired mitochondrial respiration and coupling efficiency most profoundly. Some of these four treatments were also effective in K-562, KU812 (chronic myelogenous leukemia) and CCRF-CEM, MOLT-4 (acute lymphoblastic leukemia) cells, suggesting that these treatments may have value in treating other forms of leukemia. Finally, two of the four combinations retained high synergy and strong selectivity in primary AML cells from patient samples, supporting the potential of these treatments for patients.

A mechanism for increased sensitivity of acute myeloid leukemia to mitotoxic drugs.

Mitochondria play a central and multifunctional role in the progression of tumorigenesis. Although many recent studies have demonstrated correlations between mitochondrial function and genetic makeup or originating tissue, it remains unclear why some cancers are more susceptible to mitocans (anticancer drugs that target mitochondrial function to mediate part or all of their effect). Moreover, fundamental questions of efficacy and mechanism of action in various tumor types stubbornly remain. Here we demonstrate that cancer type is a significant predictor of tumor response to mitocan treatment, and that acute myeloid leukemias (AML) show an increased sensitivity to these drugs. We determined that AML cells display particular defects in mitochondrial metabolism that underlie their sensitivity to mitocan treatment. Furthermore, we demonstrated that combinatorial treatment with a mitocan (CCCP) and a glycolytic inhibitor (2-deoxyglucose) has substantial synergy in AML cells, including primary cells from patients with AML. Our results show that mitocans, either alone or in combination with a glycolytic inhibitor, display anti-leukemia effects in doses much lower than needed to induce toxicity against normal blood cells, indicating that mitochondria may be an effective and selective therapeutic target.

A Proteomics Analysis Reveals 9 Up-Regulated Proteins Associated with Altered Cell Signaling in Colon Cancer Patients.

Colorectal cancer is the second most common cancer in women and third most common cancer in men. Cell signaling alterations in colon cancer, especially in aggressive metastatic tumors, require further investigations. The present study aims to compare the expression pattern of proteins associated with cell signaling in paired tumor and non-tumor samples of patients with colon cancer, as well as to define the cluster of proteins to differentiate patients with non-metastatic (Dukes' grade B) and metastatic (Dukes' grade C&D) colon cancer. Frozen tumor and non-tumor samples were collected after tumor resection from 19 patients with colon cancer. The Panorama™ Antibody Microarray-Cell Signaling kits were used for the analyses. The expression ratios of paired tumor/non-tumor samples were calculated for the each protein. We employed R packages 'samr', 'gplots', 'supclust' (pelora, wilma algorithms), 'glmnet' for the differential expression analysis, supervised clustering and penalized logistic regression. Significance analysis of microarrays revealed 9 significantly up-regulated proteins, including protein kinase C gamma, c-Myc, MDM2, pan cytokeratin, and 1 significantly down-regulated protein (GAP1) in tumoral mucosa. Pan-cytokeratin and APP were up-regulated in tumor versus non-tumor tissue, and were selected in the predictive cluster to discriminate colon cancer type. Higher levels of S-100b and phospho-Tau-pSer199/202 were confirmed as the predictors of non-metastatic colon cancer by all employed regression/clustering methods. Deregulated proteins in colon cancer are involved in oncogenic signal transduction, cell cycle control, and regulation of cytoskeleton/transport. Further studies are needed to validate potential protein markers of colon cancer development and metastatic progression.

Circulating levels of proinflammatory mediators as potential biomarkers of post-traumatic knee osteoarthritis development.

BACKGROUND: The identification of biomarkers of post-traumatic osteoarthritis (PTOA) progression is of clinical importance. The aims of this study were: (1) to assess the abilities of various soluble proinflammatory mediators in plasma to distinguish patients with knee PTOA from controls; (2) to determine the correlations between the mediators in plasma and those mediators in synovial fluid (SF); and (3) to explore the associations of the mediators with radiographic PTOA severity. MATERIALS AND METHODS: The concentrations of IL-1ß, IL-6, IL-18, TNFα, and leptin were measured using ELISA. Nitric oxide was determined as nitrite/nitrate (NO x ) using the Griess reaction. RESULTS: We included 171 subjects (134 PTOA patients and 37 controls) and excluded patients with rheumatoid arthritis or gout. The ROC curve of plasma NO x had the highest AUC, a specificity of 100%, and a sensitivity of 84.4%. The combination of IL-6 and leptin proved to be the most discriminatory, with an AUC value of 0.933, a specificity of 96.7%, and a sensitivity of 85.7%. The levels of NO x , IL-6, IL-18, and leptin in plasma were significantly correlated with their levels in SF. Leptin levels in both plasma (p = 0.036) and SF (p = 0.041) and the synovial IL-18 level (p = 0.045) were correlated with the Kellgren-Lawrence (KL) grade. Early-stage PTOA (KL 1-2) was associated with a high concentration of IL-1ß in plasma before and after (OR 6.235, 95% CI 1.362 to 28.552, p = 0.018) adjusting for age, gender, and BMI. CONCLUSIONS: Circulating NO x level and a combination of IL-6 and leptin permitted the strongest discrimination of patients with PTOA from controls. PTOA severity was correlated with leptin levels in plasma and SF and with the synovial IL-18 level. Early PTOA was associated with the circulating level of IL-1ß. LEVEL OF EVIDENCE: III (case-control study).

EGFR mutations and tumor metastases in patients with nonsmall cell lung cancer in the South of Russia.

PURPOSE: To assess the frequencies of somatic EGFR mutations in the tumor tissues of patients with non-small cell lung cancer (NSCLC) residing in the South of Russia (SR), and to define the relationship between genetic subtypes of NSCLC and the emergence of different types of metastases. METHODS: DNA was extracted from formalin-fixed parrafin embedded (FFPE) samples of 721 patients. A total of 29 somatic EGFR mutations were detected using commercial Therascreen EGFR RGQ PCR Kit. RESULTS: EGFR mutations were significantly more frequent in females and non-smokers even when considering the combination of both factors. The frequency of activating EGFR mutations across three age groups (<51, 51-61, >61 years) of women with NSCLC was significantly different (x2=10.94, p=0.004) and became higher with increasing age. Both activating and resistance mutations of EGFR were not associated with the frequency of regional or distant metastases. The frequencies of both regional and distant metastases were associated with higher disease stage (odds ratio/OR)=16.71; 95% confidence interval (CI): 9.5-29.38; p<0.0001, and OR=2.94; 95% CI: 2.22-3.88; p<0.0001, respectively) and adenocarcinona histology (OR=6.52; 95% CI: 2.03-20.92; p=0.002, and OR=1.99; 95% CI: 0.91-4.34; p=0.083, respectively) even when adjusted for age, gender, and smoking status. The risk for regional metastases development was associated with poor tumor differentiation (OR=2.91; 95% CI: 1.21-7.02; p=0.017). CONCLUSION: EGFR mutations were not associated with the frequency of regional or distant metastases in SR patients with NSCLC.