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ONC201/TIC10 activates TRAIL signaling through ATF4 and the integrated stress response (ISR). ONC201 demonstrated tumor regressions and disease stability in patients with histone H3K27M-mutated midline-glioma. H3K27M-mutation prevents H3K27-methylation on the mutated allele. EZH2 inhibitors (EZH2i) reduce H3K27 methylation and have anti-tumor effects. We hypothesized ONC201 sensitivity and tumor apoptosis may increase by reducing H3K27-methylation with EZH2i or HDACi as mimics of H3K27M-mutation. EZH2i EPZ-6438 (tazemetostat) or PF-06821497 and HDACi vorinostat were combined with ONC201 to treat multiple cancer cell lines and cell viability and histone modifications were analyzed. We observed synergistic effects towards cell viability in multiple cancers by EPZ-6438 or PF-06821497 plus ONC201 or triple therapy with vorinostat, EPZ-6438, and ONC201. EPZ-6438 and vorinostat synergized with ONC201 to enhance apoptosis. Activation of the ISR and TRAIL-DR5 were observed in cells treated with ONC201 -/+ epigenetic modulators. Knockdown of ATF4 reduced DR5 induction and apoptosis following EZH2i and ONC201 treatment of U251 glioma cells. mRNA expression of dopamine-receptors did not correlate with ONC201 sensitivity in the tumor cell lines tested (N = 12), including changes after epigenetic drugs. Dopamine did not rescue apoptosis by ONC201 in different tumor cell lines (N = 10) including 2 GBM, 3 DIPG and did not prevent DR5 activation or apoptosis. DRD2 agonist sumanirole did not protect brain tumor cells (N = 6 including 4 DIPG cell lines) from ONC201 reduction in viability. Although synergy was observed with ONC201 and vorinostat, there was no significant increase in H3K27 acetylation in cell lines including DIPG as compared to vorinostat alone, and in some cases the acetylation was less than vorinostat alone at 72 H. H3K27 methylation reduction correlated with synergy from combinations of either EPZ-6438 or vorinostat with ONC201 or triple combination. Our findings provide a rationale for combination of ONC201 and epigenetic modulators including triple therapy for in vivo and clinical testing in treatment of human malignancies including brain tumors and DIPG.

ONC201 was originally discovered as TNF-Related Apoptosis Inducing Ligand (TRAIL)-inducing compound TIC10. ONC201 appears to act as a selective antagonist of the G protein coupled receptor (GPCR) dopamine receptor D2 (DRD2), and as an allosteric agonist of mitochondrial protease caseinolytic protease P (ClpP). Downstream of target engagement, ONC201 activates the ATF4/CHOP-mediated integrated stress response leading to TRAIL/Death Receptor 5 (DR5) activation, inhibits oxidative phosphorylation via c-myc, and inactivates Akt/ERK signaling in tumor cells. This typically results in DR5/TRAIL-mediated apoptosis of tumor cells; however, DR5/TRAIL-independent apoptosis, cell cycle arrest, or antiproliferative effects also occur. The effects of ONC201 extend beyond bulk tumor cells to include cancer stem cells, cancer associated fibroblasts and immune cells within the tumor microenvironment that can contribute to its efficacy. ONC201 is orally administered, crosses the intact blood brain barrier, and is under evaluation in clinical trials in patients with advanced solid tumors and hematological malignancies. ONC201 has single agent clinical activity in tumor types that are enriched for DRD2 and/or ClpP expression including specific subtypes of high-grade glioma, endometrial cancer, prostate cancer, mantle cell lymphoma, and adrenal tumors. Synergy with radiation, chemotherapy, targeted therapy and immune-checkpoint agents has been identified in preclinical models and is being evaluated in clinical trials. Structure-activity relationships based on the core pharmacophore of ONC201, termed the imipridone scaffold, revealed novel potent compounds that are being developed. Imipridones represent a novel approach to therapeutically target previously undruggable GPCRs, ClpP, and innate immune pathways in oncology.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease characterized by early metastasis, late detection, and poor prognosis. Progress towards effective therapy has been slow despite significant efforts. Novel treatment approaches are desperately needed and autophagy, an evolutionary conserved process through which proteins and organelles are recycled for use as alternative energy sources, may represent one such target. Although incompletely understood, there is growing evidence suggesting that autophagy may play a role in PDAC carcinogenesis, metastasis, and survival. Early clinical trials involving autophagy inhibiting agents, either alone or in combination with chemotherapy, have been disappointing. Recently, evidence has demonstrated synergy between the MAPK pathway and autophagy inhibitors in PDAC, suggesting a promising therapeutic intervention. In addition, novel agents, such as ONC212, have preclinical activity in pancreatic cancer, in part through autophagy inhibition. We discuss autophagy in PDAC tumorigenesis, metabolism, modulation of the immune response, and preclinical and clinical data with selected autophagy modulators as therapeutics.

Objective Short-term intensive insulin therapy (IIT) early in the course of type 2 diabetes acutely improves beta-cell function with long-lasting effects on glycemic control. However, conventional measures cannot determine which patients are better suited for IIT, and little is known about the molecular mechanisms determining response. Therefore, this study aimed to develop a model that could accurately predict the response to IIT and provide insight into molecular mechanisms driving such response in humans. Methods Twenty-four patients with early type 2 diabetes were assessed at baseline and four weeks after IIT, consisting of basal detemir and premeal insulin aspart. Twelve individuals had a beneficial beta-cell response to IIT (responders) and 12 did not (nonresponders). Beta-cell function was assessed by multiple methods, including Insulin Secretion-Sensitivity Index-2. MicroRNAs (miRNAs) were profiled in plasma samples before and after IIT. The response to IIT was modeled using a machine learning algorithm and potential miRNA-mediated regulatory mechanisms assessed by differential expression, correlation, and functional network analyses (FNA). Results Baseline levels of circulating miR-145-5p, miR-29c-3p, and HbA1c accurately (91.7%) predicted the response to IIT (OR = 121 [95% CI: 6.7, 2188.3]). Mechanistically, a previously described regulatory loop between miR-145-5p and miR-483-3p/5p, which controls TP53-mediated apoptosis, appears to also occur in our study population of humans with early type 2 diabetes. In addition, significant (fold change > 2, P 0.4, P, Graphical abstract Image 1, Highlights • Baseline levels of miR-145 and miR-29c predict short-term IIT response. • miR-138, -192, -195, -320b, and let-7a change in response to insulin therapy in patients with diabetes. • Changes in miRNAs correlate with changes in beta cell function and insulin sensitivity. • A network of “miRNA-overtargeted” genes regulates cell death and proliferation.

ONC201 is a promising anti-cancer agent that kills tumor cells by triggering an integrated stress response (ISR) dependent on ATF4. ONC201 demonstrated tumor regression and prolonged disease stability in patients with histone H3K27M-mutated midline glioma. The Enhancer of Zeste Homolog 2 (EZH2), a subunit of the polycomb repressive complex 2 (PRC2), is a histone methyltransferase that tri-methylates H3K27 (H3K27me3) and silences target genes. EZH2 inhibitors (EZH2i) reduce global H3K27 methylation. Based on the fact that the H3K27 mutation reduces H3K27 dimethylation (H3K27me2) and trimethylation (H3K27me3), we hypothesized that ONC201 sensitivity and tumor cell death may be enhanced by reducing H3K27 methylation with EZH2i as a mimic of H3K27M-mutation and by increasing H3K27 acetylation with histone deacetylase inhibitors (HDACi). We evaluated synergy of EZH2i EPZ-6438 or HDACi vorinostat with ONC201 against GBM cell lines, U251 and T98G-1 and DMG cell line, SF8638. Cell viability was determined with the Cell Titer Glo assay. Apoptosis was evaluated through immunoblotting of cleaved PARP and flow cytometry analysis of cell distribution. ISR activity was evaluated using immunoblotting of ATF4. Our result demonstrate that ONC201 synergistically reduced cell viability with vorinostat in U251, T98G-1 and SF8628 cell lines, induced apoptosis in combination with vorinostat in U251 and SF8628. ONC201 synergistically reduced cell viability and induced apoptosis with EPZ-6438 in U251. The immunoblotting detected no enhancement of ATF4 by addition of EPZ-6438 to ONC201. Immunoblotting analysis showed that EPZ-6438 reduced H3K27me3 in U251. Our results unravel potent synergy between ONC201 and EZH2i or HDACi in GBM and DMG cell lines, and provide further insights into the role of H3K27me3 in ONC201 drug sensitivity.

COVID-19 affects vulnerable populations including elderly individuals and patients with cancer. Natural Killer (NK) cells and innate-immune TRAIL suppress transformed and virally-infected cells. ACE2, and TMPRSS2 protease promote SARS-CoV-2 infectivity, while inflammatory cytokines IL-6, or G-CSF worsen COVID-19 severity. We show MEK inhibitors (MEKi) VS-6766, trametinib and selumetinib reduce ACE2 expression in human cells. Chloroquine or hydroxychloroquine increase cleaved active SP-domain of TMPRSS2, and this is potentiated by MEKi. In some human cells, remdesivir increases ACE2-promoter luciferase-reporter expression, ACE2 mRNA and protein, and ACE2 expression is attenuated by MEKi. We show elevated cytokines in COVID-19- (+) patient plasma (N=9) versus control (N=11). TMPRSS2, inflammatory cytokines G-CSF, M- CSF, IL-1a, IL-6 and MCP-1 are suppressed by MEKi alone or in combination with remdesivir. MEKi enhance NK cell (but not T-cell) killing of target-cells, without suppressing TRAIL-mediated cytotoxicity. We generated a pseudotyped SARS-CoV-2 virus with a lentiviral core but with the SARS-CoV-2 D614 or G614 SPIKE (S) protein on its envelope and used VSV-G lentivirus as a negative control. Our results show infection of human bronchial epithelial cells or lung cancer cells and that MEKi suppress infectivity of the SARS-CoV-2-S pseudovirus following infection. We show a drug class-effect with MEKi to promote immune responses involving NK cells, inhibit inflammatory cytokines and block host-factors for SARS-CoV-2 infection leading also to suppression of SARS-CoV-2-S pseudovirus infection of human cells in a model system. MEKi may attenuate coronavirus infection to allow immune responses and antiviral agents to control COVID-19 disease progression and severity.

We profiled gene expression signatures to distinguish rheumatoid arthritis (RA) from non-inflammatory arthralgia (NIA), self-limiting arthritis (SLA), and undifferentiated arthritis (UA) as compared to healthy controls as novel potential biomarkers for therapeutic responsiveness. Global gene expression profiles of PBMCs from 43 drug-naïve patients presenting with joint symptoms were evaluated and differentially expressed genes identified by comparative analysis with 24 healthy volunteers. Patients were assessed at presentation with follow up at 6 and 12 months. Gene ontology and network pathway analysis were performed using DAVID Bioinformatics Resources v6.7. Gene expression profiles were also determined after disease-modifying anti-rheumatic drug (DMARD) treatment in the inflammatory arthritis groups (i.e. RA and UA) and confirmed by qRT-PCR. Receiver operating characteristic (ROC) curves analysis and Area Under the Curve (AUC) estimation were performed to assess the diagnostic value of candidate gene expression signatures. A type I interferon (IFN) gene signature distinguished DMARD-naïve patients who will subsequently develop persistent inflammatory arthritis (i.e. RA and UA) from those with NIA. In patients with RA, the IFN signature is characterised by up-regulation of SIGLEC1 (p = 0.00597) and MS4A4A (p = 0.00000904). We also identified, EPHB2 (p = 0.000542) and PDZK1IP1 (p = 0.0206) with RA-specific gene expression profiles and elevated expression of the ST6GALNAC1 (p = 0.0023) gene in UA. ROC and AUC risk score analysis suggested that MSA4A (AUC: 0.894, 0.644, 0.720), PDZK1IP1 (AUC: 0.785, 0.806, 0.977), and EPHB2 (AUC: 0.794, 0.723, 0.620) at 0, 6, and 12 months follow-up can accurately discriminate patients with RA from healthy controls and may have practical value for RA diagnosis. In patients with early inflammatory arthritis, ST6GALNAC1 is a potential biomarker for UA as compared with healthy controls whereas EPHB2, MS4A4A, and particularly PDZK1IP1 may discriminate RA patients. SIGLEC1 may also be a useful marker of disease activity in UA.

Roux-en-Y gastric bypass (RYGB) surgery improves insulin sensitivity (SI) and β-cell function in obese non-diabetic subjects. Exercise also improves SI and may be an effective adjunct therapy to RYGB surgery. However, the mechanisms by which exercise or weight loss improve peripheral SI after RYGB surgery are unclear. We hypothesized that microRNAs (miRNAs) mediate at least some of the regulatory processes driving such mechanisms. Consequently, this work aimed at profiling plasma miRNAs in participants of the Physical Activity Following Surgery Induced Weight Loss study (clinicaltrials.gov identifier: NCT00692367), to assess whether miRNA levels track with improvements in SI and cardiometabolic risk factors. Ninety-four miRNAs implicated in metabolism were profiled in plasma samples from 22 severely obese subjects who were recruited 1–3 months after RYGB surgery and followed for 6 months of RYGB surgery-induced weight loss, with (exercise program (EX), N=11) or without (CON, N=11) an exercise training intervention. The subjects were selected, considering a priori sample size calculations, among the participants in the parent study. Mixed-effect modeling for repeated measures and partial correlation analysis was implemented in the R environment for statistical analysis. Mirroring results in the parent trial, both groups experienced significant weight loss and improvements in cardiometabolic risk. In the CON group, weight loss significantly altered the pattern of circulating miR-7, miR-15a, miR-34a, miR-106a, miR-122 and miR-221. In the EX group, a distinct miRNA signature was altered: miR-15a, miR-34a, miR-122, miR-135b, miR-144, miR-149 and miR-206. Several miRNAs were significantly associated with improvements in acute insulin response, SI, and other cardiometabolic risk factors. These findings present novel insights into the RYGB surgery-induced molecular changes and the effects of mild exercise to facilitate and/or maintain the benefits of a ‘comprehensive’ weight-loss intervention with concomitant improvements in cardiometabolic functions. Notably, we show a predictive value for miR-7, miR-15a, miR-106b and miR-135b.

First-in-class small molecule ONC201 is an anti-cancer agent that activates the TRAIL pathway through ATF4 and the integrated stress response (ISR). ONC201 demonstrated durable tumor regressions and prolonged disease stabilization in some patients with histone H3K27M-mutated diffuse midline glioma (DMG). The H3K27M-mutation reduces H3K27 methylation. EZH2 inhibitors (EZH2i) reduce global H3K27 methylation. We hypothesized that ONC201 sensitivity and tumor cell death may be enhanced by reducing H3K27 methylation with EZH2i as a mimic of the H3K27M-mutation, particularly in tumor types beyond DMG. We treated cancer cells from different tissue origins including breast cancer, pancreatic cancer, colorectal cancer, glioblastoma, and DMG with single agent ONC201, EZH2 inhibitor EPZ-6438 or PF-06821497, or the combination of ONC201 plus EPZ-6438 or ONC201 plus PF-06821497. Cell viability was determined with the Cell Titer-Glo assay. Integrated stress response (ISR) activation widely observed in ONC201-treated tumor cells was evaluated using Western blot analysis of ATF4. We determined TRAIL pathway activity by analysis of DR5, and apoptosis by analysis of PARP cleavage. The effect of EPZ-6438 or PF-06821497on the target was validated by immunoblotting of H3K27me3. We knocked down ATF4 with siRNA to evaluate the role of ISR in cell death. Our results demonstrate that ONC201 synergistically reduced cell viability in combination with EZH2i, induced integrated stress response and apoptosis in combination with EZH2i. There was greater induction of ATF4 and DR5 in combinatorial therapy-treated tumor cells versus monotherapy. Knockdown of ATF4 reduced the apoptosis induced by combination therapy. EPZ-6438 or PF-06821497 decreased H3K27 tri-methylation. Our study results unravel potent synergy between ONC201 and EZH2i, highlight the role of ISR in the synergy, and provide further insights into the role of H3K27me3 in ONC201 drug sensitivity. Citation Format: Yiqun Zhang, Lanlan Zhou, Varun V. Prabhu, Joshua E. Allen, Attila A. Seyhan, Wafik S. El-Deiry. The integrated stress response (ISR) is involved in the synergistic combinatorial efficacy of ONC201 and epigenetic modulators in brain tumor cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1156.

Novel therapies are much needed in treating pediatric sarcomas, such as Ewing sarcoma. In the setting of metastatic Ewing sarcoma, survival expectancy ranges around 10%. A novel class of small molecules, imipridones, target G protein-coupled receptors and mitochondrial protease ClpP. Imipridones inactivate cell proliferation kinases Akt/ERK and induce cell death through the pro-apoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor DR5. In pre-clinical and clinical trials, imipridone ONC201 shows increased activity against tumors with mutations in histones, specifically histone H3 at the location of lysine 27 (H3K27). Changes in histone acetylation and the epigenetic effects of imipridones remain under investigation. Most commonly caused by a fusion of the EWS-FLI1 genes, Ewing sarcoma exhibits epigenetic modifications with the EWS-FLI1 protein acting as a transcription factor that causes covalent modifications of histone H3, including acetylation and activation of H3K27. Ewing sarcomas have also shown in vitro response to ERK pathway inhibition, leading to apoptosis and inhibition of metastasis formation. We hypothesized that Ewing sarcoma cell lines, with increased H3K27 acetylation due to EWS-FLI1 acting as an activating transcription factor, would respond to the combination of imipridones with histone deacetylase inhibitors. We performed combinatorial drug treatment on Ewing sarcoma cell lines SK-N-MC and RD-ES with 3 imipridones (ONC201, ONC206, and ONC212) and 3 HDAC inhibitors (vorinostat, entinostat, and panobinostat). Cell viability was measured after drug treatment in vitro. We investigated markers of cell death in established pediatric sarcoma cell lines via protein analysis and flow cytometry analysis. Using protein quantification studies and downstream target analysis on combination drug-treated cells, we investigated the mechanisms of how these two targeted therapies interact synergistically to cause cell death. We show that imipridones inactivate cellular proliferation through the Akt/ERK pathway and induce cell death through the TRAIL pathway, with ONC212 exhibiting the highest cell killing potency. Protein quantification by Western blotting revealed treatment with imipridone ONC201 affected the mitochondrial Clp protease complex by decreasing the chaperone subunit ClpX. Cell viability studies and analysis with Compusyn demonstrate potent synergistic effects causing tumor cell death when imipridones are combined with HDAC inhibitors. Our work presents a novel therapeutic combination for the treatment of Ewing sarcoma. Citation Format: Wen-i Chang, Lanlan Zhou, Attila Seyhan, Varun V. Prabhu, Wafik S. El-Deiry. Combinatorial therapy of imipridones and histone deacetylase inhibitors in Ewing sarcoma cell lines demonstrates synergistic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1060.

Patients diagnosed with pediatric sarcomas have poor outcomes with current standard therapy in the setting of relapse, progressive disease, or upfront metastasis. Imipridones, a class of novel small molecules, specifically ONC201, have shown pre-clinical and clinical efficacy in adult malignancies and in brain tumors with aberrant methylation of histone H3 on lysine 27 (associated with H3K27M mutation). ONC201 is an antagonist of dopamine receptor D2 (DRD2), which is a G protein-coupled receptor that is overexpressed in several malignancies. ONC201 inactivates cell proliferation kinases Akt/ERK and induces cell death through the pro-apoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor DR5. Prior in vitro studies of pediatric sarcomas have produced promising data with ERK pathway inhibition, leading to apoptosis and inhibition of metastasis formation. Some pediatric sarcomas, such as malignant peripheral nerve sheath tumors and fusion-positive sarcomas, have been noted to have aberrant activation or modifications of H3K27. H3K27 can be methylated or acetylated and both modifications are abrogated in H3K27M mutated alleles. Changes in histone acetylation and the epigenetic effects of imipridones remain under investigation. We hypothesized that sarcomas with aberrant H3K27 modifications may respond to the novel small molecule similarly to H3K27-mutated brain tumors, and that the combination of ONC201 with histone deacetylase inhibitors (HDACi) could lead to a synergistic effect on cell death mechanisms, potentially through effects of histone acetylation. We performed drug treatment with ONC201 on established pediatric sarcoma cell lines and applied combinatory drug treatment with HDACi, including vorinostat (SAHA). Cell viability was measured after drug treatment in vitro. We investigated markers of cell death in established pediatric sarcoma cell lines via protein analysis and flow cytometry analysis. Using protein quantification studies and downstream target analysis on combination drug-treated cells, we investigated the mechanisms of how these two targeted therapies interact synergistically to cause cell death. We show that ONC201 inactivates cellular proliferation in pediatric sarcomas through the Akt/ERK pathway and induces cell death through the TRAIL pathway. We demonstrated that treatment of cells with ONC201 modified the mitochondrial Clp protease complex, by activating the ClpP protease and by decreasing the chaperone subunit ClpX. Our studies demonstrate that ONC201 induces cell death in pediatric sarcomas, and that ONC201 has potent synergistic effects causing tumor cell death when combined with HDACi. Further studies are underway in elucidating the combinatory epigenetic effects of HDACi and ONC201 in H3K27-aberrant sarcomas, leading to synergistic cell death mechanisms. Our studies present a novel therapeutic combination for pediatric sarcomas that have relapsed or are refractory to standard chemotherapy. Citation Format: Wen-I Chang, Lanlan Zhou, Attila A. Seyhan, Yiqun Zhang, Wafik S. El-Deiry. Novel therapeutic targeting of epigenetic aberrations in pediatric sarcomas through combination of ONC201 and HDAC inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3902.

Objective. We explored the relationships among microRNAs (miRNAs) and SFRP4, as they relate to adipose tissue functions including lipolysis, glucose and glycerol turnover, and insulin sensitivity. Methods. Abdominal adipose tissue (AbdAT) levels of thirteen microRNAs (miRNAs), SFRP4, and VEGF in lean nondiabetic subjects (n=7), subjects with obesity (n=5), and subjects with obesity and type 2 diabetes (T2DM) (n=5) were measured by qPCR. Insulin sensitivity was measured by the euglycemic-hyperinsulinemic clamp. Osmium fixation and Coulter counting were used for adipocyte sizing. Data were analyzed using generalized linear models that adjusted for age, gender, and ethnicity. Results. AbdAT miR-24, miR-30d, and miR-146a were elevated in subjects with obesity (P<0.05) and T2DM (P<0.1) and positively correlated with measures of percent body fat by DXA (rmiR.24=0.894, rmiR.146a=0.883, P<0.05), and AbdAT SFRP4 (rmiR.30=0.93, rmiR.146a=0.88, P<0.05). These three miRNAs additionally correlated among themselves (rmiR.24~miR.146a=0.90, rmiR.30~miR.146a=0.85, P<0.01). Conclusions. This study suggests a novel association between the elevated levels of miRNAs miR-24, miR-30d, and miR-146a (apparently coregulated) and the level of SFRP4 transcript in AbdAT of subjects with obesity and T2DM. These molecules might be part of a regulatory loop involved in AbdAT remodeling/adiposity and systemic insulin resistance. This trial is registered with NCT00704197.

Vitamin D may play an important role in modifying the risk of type 2 diabetes. Supplementation with cholecalciferol has been shown to improve β cell function and to attenuate the rise in glycated hemoglobin in people at high risk of diabetes. We examined whether circulating microRNAs (miRNAs) reflect disease progression and/or respond to vitamin D supplementation. We measured plasma levels of select miRNAs implicated in diabetes in people with prediabetes treated either with placebo (n=21) or 2000 U of cholecalciferol daily (n=21) for 4 months in the Calcium and Vitamin D for Diabetes Mellitus trial and compared the baseline-adjusted changes after correcting for age, body mass index, race, time of study entry (season) and baseline disposition index. Circulating levels of miR-7 (sixfold reduction, P=.01), miR-152 (1.5-fold increase, P=.03), and miR-192 (1.7-fold reduction, P=.026) displayed significant treatment-by-time interactions between the placebo- and the vitamin-D-treated groups. Plasma levels of miR-7 were reduced in the vitamin D and increased in the placebo group. The change in miR-152 positively correlated with the change in levels of the circulating metabolite 25-hydroxyvitamin D (r=0.33, P=.046) and negatively correlated with the change in glycated hemoglobin (r=-0.37, P=.024). The change in miR-192 positively correlated with the change in fasting glucose (r=0.41, P

Objectives Rarefaction and inflammation of adipose tissue contributes to insulin resistance in obesity. It was hypothesized that angiostatic secreted frizzled-related protein 4 (SFRP4) causes adipose tissue rarefaction and leads to inflammation and ultimately insulin resistance in obese patients. Methods Abdominal subcutaneous adipose tissue (AbdAT), gluteal subcutaneous adipose tissue (GlutAT), and blood from 15 lean and obese subjects were collected. Circulating-SFRP4 was measured by ELISA. Body composition was measured by DEXA and insulin sensitivity by the euglycemic hyperinsulinemic clamp. Adipose tissue was analyzed using qRT-PCR for mRNA gene expression, Luminex system for tissue cytokine release, immunohistochemistry for labeling adipose capillaries, and osmium fixation and Coulter counting for adipocyte sizing. Results Circulating-SFRP4 was higher in obese vs. lean subjects (137.8 ± 33.6 ng ml−1 vs. 64.1 ± 23.8 ng ml−1, P

ONC201, the founding member of the imipridone class of small molecules, has broad spectrum anti-tumor activity and achieved tumor regressions and prolonged disease stability in clinical trials, especially in patients with histone H3 K27M mutated glioma. Levels of H3K27 dimethylation (H3K27me2) and trimethylation (H3K27me3) are reduced globally in H3 K27M patient samples. EZH2 is the catalytic component of the Polycomb Repressor Complex 2 (PRC2), a conserved multi-subunit that represses gene transcription by methylating lysine 27 on Histone H3 (H3K27). EZH2 is frequently overexpressed or mutated (activating) in numerous human malignancies and EZH2 inhibitors represent a promising therapeutic strategy. We hypothesized that cancer cells could be sensitized to ONC201 by EZH2 inhibition through suppression of H3K27 methylation that may mimic tumors such as DIPG with H3K27M mutations which are known to respond to ONC201 in the clinic and are defective in H3K27 methylation. We treated cancer cells from different tissue origins including breast cancer, pancreatic cancer, colorectal cancer, GBM, and DIPG with single agent ONC201, EZH2 inhibitor EPZ-6438 or the combination of ONC201 plus EPZ-6438. Cell viability was determined with the Cell Titer-Glo assay. Integrated stress response (ISR) activity widely observed in ONC201-treated tumor cells was evaluated using Western blot analysis of ATF4 and apoptosis by analysis of PARP cleavage. Our results demonstrate that ONC201 synergistically reduced cell viability, induced integrated stress response and apoptosis in combination with EZH2 inhibitor EPZ-6438. There was greater induction of ATF4 in combination therapy-treated tumor cells versus monotherapy. Intriguingly, U-251 GBM cells that were resistant to ONC201 under hypoxia could be sensitized to ONC201 by EZH2 inhibitor EPZ-6438. Our findings provide a preclinical rationale for developing ONC201 and EZH2 inhibitors as a novel synergistic combinatorial regimen with the potential for clinical translation in the treatment of several human malignancies including GBM, pancreatic cancer, colorectal cancer, and breast cancer. Citation Format: Yiqun Zhang, Lanlan Zhou, Shengliang Zhang, Robyn Borsuk, Howard P. Safran, Attila A. Seyhan, Wafik S. El-Deiry. Preclinical synergistic anti-tumor effect against multiple cancer types by EZH2 inhibitor EPZ-6438 plus imipridone ONC201 to mimic H3K27M mutation observed in DIPG tumors that respond to ONC201 in the clinic [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-C10. doi:10.1158/1535-7163.TARG-19-LB-C10

Today obesity and type 2 diabetes (T2D) have both reached epidemic proportions. However, our current understanding of the primary mechanisms leading to these diseases is still limited due to the complex multifactorial nature of the underlying phenomena. We hypothesize that the levels of specific cytokines and miRNAs vary across the diabetes spectrum and unique signatures associated with them may serve as early biomarkers of the disease and provide insights into respective pathogenetic mechanisms. In this study, we measured the circulating levels of cytokines and microRNAs (miRNAs) in lean and obese humans with prediabetes (n = 21), T2D (n = 17), and healthy controls (n = 20) (ORIGINS trial, NCT02226640). Data were analyzed by fitting linear models adjusted for confounding variables (BMI, age, and gender in the diabetes context and age, gender, and diabetes status in the obesity context) and implementing nonparametric randomization-based tests for statistical inference. Group differences and correlations (r > 0.3) between variables with P < 0.05 were considered significant. False discovery rates (FDR) correcting for multiple testing were calculated using the Benjamini–Hochberg correction. We found a number of circulating cytokines and miRNAs deregulated in subjects with obesity, prediabetes, and T2D. Specifically, cytokines IL-6, IL-8, IL-10, IL-12, and SFRP4, as well as miRNAs miR-21, miR-24.1, miR-27a, miR-28-3p, miR-29b, miR-30d, miR-34a, miR-93, miR-126, miR-146a, miR-148, miR-150, miR-155, and miR-223, significantly changed across the diabetes spectrum, and were associated with measures of pancreatic islet β cell function and glycemic control, among others. Notably, SFRP4 was the only studied cytokine that was significantly associated with obesity, prediabetes, and T2D, which underscores the important role of this molecule during disease development and progression. Our data suggest that changes in circulating miRNAs and cytokines may have clinical utility as biomarkers of prediabetes.

The clinical presentation of diabetes sometimes overlaps, contributing to ambiguity in the diagnosis. Thus, circulating pancreatic islet-enriched microRNAs (miRNAs) might be useful biomarkers of β-cell injury/dysfunction that would allow more accurate subtyping of diabetes. We measured plasma levels of selected miRNAs in subjects with prediabetes (n = 12), type 2 diabetes (T2D, n = 31), latent autoimmune diabetes of adults (LADA, n = 6) and type 1 diabetes (T1D, n = 16) and compared them to levels in healthy control subjects (n = 27). The study was conducted at the Translational Research Institute for Metabolism and Diabetes (TRI-MD), Florida Hospital. MiRNAs including miR-375 (linked to β-cell injury), miR-21 (associated with islet inflammation), miR-24.1, miR-30d, miR-34a, miR-126, miR-146, and miR-148a were significantly elevated in subjects with various forms of diabetes compared to healthy controls. Levels of several miRNAs were significantly correlated with glucose responses during oral glucose tolerance testing, HbA[subscript 1c], β-cell function, and insulin resistance in healthy controls, prediabetes, and T2D. These data suggest that miRNAs linked to β-cell injury and islet inflammation might be useful biomarkers to distinguish between subtypes of diabetes. This information could be used to predict progression of the disease, guide selection of optimal therapy and monitor responses to interventions, thus improving outcomes in patients with diabetes., Translational Research Institute for Metabolism and Diabetes (TRI)

Dominant negative genetic disorders, in which a mutant allele of a gene causes disease in the presence of a second, normal copy, have been challenging since there is no cure and treatments are only to alleviate the symptoms. Current therapies involving pharmacological and biological drugs are not suitable to target mutant genes selectively due to structural indifference of the normal variant of their targets from the disease-causing mutant ones. In instances when the target contains single nucleotide polymorphism (SNP), whether it is an enzyme or structural or receptor protein are not ideal for treatment using conventional drugs due to their lack of selectivity. Therefore, there is a need to develop new approaches to accelerate targeting these previously inaccessible targets by classical therapeutics. Although there is a cooling trend by the pharmaceutical industry for the potential of RNA interference (RNAi), RNAi and other RNA targeting drugs (antisense, ribozyme, etc.) still hold their promise as the only drugs that provide an opportunity to target genes with SNP mutations found in dominant negative disorders, genes specific to pathogenic tumor cells, and genes that are critical for mediating the pathology of various other diseases. Because of its exquisite specificity and potency, RNAi has attracted a considerable interest as a new class of therapeutic for genetic diseases including amyotrophic lateral sclerosis, Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), spinocerebellar ataxia, dominant muscular dystrophies, and cancer. In this review, progress and challenges in developing RNAi therapeutics for genetic diseases will be discussed.

The development of RNA interference (RNAi)-mediated gene inhibition has changed the direction and speed of drug target discovery and validation. RNAi technology has already influenced strategies for the pharmacological treatment of many diseases including cancer, viral diseases, bacterial pathogens, inflammation, diseases of central nervous systems (CNS), and others. This technology provides a better understanding of the mechanisms which underlie disease pathogenesis and lead to the identification of novel factors that alter the disease phenotype. With the introduction of RNAi libraries in various formats, systematic loss-of-function screens can now be conducted to interrogate the biological functions of specific genes and pathways or an entire genome in various disease areas. The identification of novel mediators of cellular response to disease pathogenesis or treatment approaches may lead to the identification of novel drug targets, development of combinatorial treatment approaches, or pharmacodynamic and patient selection biomarkers and expand our understanding of disease pathogenesis. Here, we review the use of RNAi in various screening formats, and examine the types of targets pursued for oncology and other disease indications.

Biomarkers provide information on early detection of diseases, in determining individuals at risk of developing complications or subtyping individuals for disease phenotypes. In addition, biomarkers may lead to better treatment strategies, personalized therapy, and improved outcome. A major gap in the field of biomarker development is that we have not identified appropriate (minimally invasive, life-style independent and informative) biomarkers for the underlying disease process(es) that can be measured in readily accessible samples (e.g. serum, plasma, blood, urine). miRNAs function as regulators in wide ranging cellular and physiological functions and also participate in many physiopathological processes and thus have been linked to many diseases including diabetes, metabolic and cardiovascular diseases, cancer, neurodegenerative diseases, and autoimmunity. Many miRNAs have been shown to have predictive value as potential biomarkers in a variety of diseases including diabetes, which are detectable in some instances many years before the manifestation of disease. Although some technical challenges still remain, due to their availability in the circulation, relative stability, and ease of detection; miRNAs have emerged as a promising new class of biomarkers to provide information on early detection of disease, monitoring disease progression, in determining individual’s risk of developing complications or subtyping individuals for disease phenotypes, and to monitor response to therapeutic interventions. As a final note, most of the miRNAs reported in the literature have not yet been validated in sufficiently powered and longitudinal studies for specificity for that particular disease.

Methods most commonly used for producing small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) are chemical synthesis and intracellular expression from engineered vectors. For shRNAs, chemical synthesis is very costly and construction of vectors is laborious. Synthesis by phage RNA polymerases from their natural promoters results in a 5 -terminal triphosphate that can trigger an interferon (IFN) response. Moreover, due to the requirement of phage promoters for 5 - GPuPuPu sequences for transcription initiation, shRNA transcripts may have extra 5 -nucleotides that can constrain the sequences that can be targeted. Also, the 3 ends may have an additional n + 1 nucleotide not encoded by the template. Here we present a novel approach for synthesizing functional shRNAs via rolling circle transcription (RCT) of small (approximately 70 nt) single-stranded DNA circles using T7 RNA polymerase, which avoids these issues. Due to internal pairing, these circles are dumbbell-shaped. RCT produces large transcripts (>10 kb in length) consisting of multimers (>150 copies) of shRNAs in the absence of promoter, terminator, or primer sequences. Dumbbells targeting red fluorescent protein (DsRed), human tumor necrosis factor-alpha (TNF-alpha) and hepatitis C virus (HCV) internal ribosome entry site (IRES) were prepared and transcribed. The resulting long transcripts are substrates for Dicer. When introduced into 293FT and Huh7 cells, the multimeric transcripts inhibited their target genes at levels similar to an equivalent mass of monomeric shRNAs, indicating that they can enter the RNAi pathway. Thus, rolling circle transcription of small DNA dumbbells provides a new source of biologically active interfering RNA.

The ability of small hairpin RNAs (shRNAs) to inhibit hepatitis C virus internal ribosome entry site (HCV IRES)-dependent gene expression was investigated in cultured cells and a mouse model. The results indicate that shRNAs, delivered as naked RNA or expressed from vectors, may be effective agents for the control of HCV and related viruses.

Background Heterogeneity of clinical manifestations and underlying biology has confounded treatment development and optimal care of systemic lupus erythematosus (SLE) patients. About half of adult SLE patients tend to have stable, high expression levels of genes in the interferon (IFN) signaling pathways. We and others have previously reported differences in expression levels between IFN groups of genes in pathways other than IFN signaling pathways1,2,3,4,5. Objectives To determine how gene expression changes with disease flare in SLE patients with high versus low expression of IFN pathway genes. Methods The BOLD study enrolls patients with active disease who are withdrawn from potentially confounding background immunosuppressive therapy and given brief intramuscular steroids (depomedrol) to induce improvement (Improving Visit), and followed until flare (Flare Visit). An interim analysis was conducted by TaqMan® RT-PCR on 23 patients who completed the study. Expression levels of 272 genes (selected based on reported associations with lupus and/or inflammation) were compared between Improving Visit and Flare Visit. Results Striking expression differences between Improving Visit and Flare Visit were observed for some genes, predominantly associated with the IFN High Group. Expression of most (76%) genes tested did not change with flare (p>0.1). IFN groups did not differ in demographic features. There was a trend towards higher disease activity (CLASI score, p=0.06, BILAG scores p=0.1) at baseline in the IFN high group. View this table: Table 1 Conclusions This interim report suggests biomarkers in several pathways might be tested as sensitive indicators to guide patient selection and dosing in the development of treatments which affect the TLR/IFN pathway, and to optimize the eventual use of these agents in clinic. Given the heterogeneity of patients with SLE it could be that some of these downstream indicators could help differentiate between patients who are or are not intrinsically good candidates for targeting of IFN signaling pathways. 1. O'Toole et al., ACR 2011, Abstract 1407. 2. Baechler et al., PNAS 2003. 3. Bennett et al., J. Exp. Med 2003. 4. Kirou et al., Arthritis and Rheumatism 2005. 5. Petri et al, Lupus 2009. Disclosure of Interest A. Seyhan Employee of: Pfizer, M. O'Toole Employee of: Pfizer, Y. Zhang Employee of: Pfizer, F. Immermann Employee of: Pfizer, A. Hill Employee of: Pfizer, P. Reddy Employee of: Pfizer, J. Masferrer Employee of: Pfizer, T. Zhou Employee of: Pfizer, W. Mounts Employee of: Pfizer, M. Whitley Employee of: Pfizer, T. Walker Employee of: Pfizer, S. Kamp: None Declared, J. James: None Declared, S. Sridharan Employee of: Pfizer, J. Merrill: None Declared, M. Honczarenko Employee of: Pfizer

Neratinib (HKI-272) is a small molecule tyrosine kinase inhibitor of the ErbB receptor family currently in Phase III clinical trials. Despite its efficacy, the mechanism of potential cellular resistance to neratinib and genes involved with it remains unknown. We have used a pool-based lentiviral genome-wide functional RNAi screen combined with a lethal dose of neratinib to discover chemoresistant interactions with neratinib. Our screen has identified a collection of genes whose inhibition by RNAi led to neratinib resistance including genes involved in oncogenesis (e.g. RAB33A, RAB6A and BCL2L14), transcription factors (e.g. FOXP4, TFEC, ZNF), cellular ion transport (e.g. CLIC3, TRAPPC2P1, P2RX2), protein ubiquitination (e.g. UBL5), cell cycle (e.g. CCNF), and genes known to interact with breast cancer-associated genes (e.g. CCNF, FOXP4, TFEC, several ZNF factors, GNA13, IGFBP1, PMEPA1, SOX5, RAB33A, RAB6A, FXR1, DDO, TFEC, OLFM2). The identification of novel mediators of cellular resistance to neratinib could lead to the identification of new or neoadjuvant drug targets. Their use as patient or treatment selection biomarkers could make the application of anti-ErbB therapeutics more clinically effective.

ErbB2 is frequently activated in tumors, and influences a wide array of cellular functions, including proliferation, apoptosis, cell motility and adhesion. HKI-272 (neratinib) is a small molecule pan-kinase inhibitor of the ErbB family of receptor tyrosine kinases, and shows strong antiproliferative activity in ErbB2-overexpressing breast cancer cells. We undertook a genome-wide pooled lentiviral RNAi screen to identify synthetic lethal or enhancer (synthetic modulator screen) genes that interact with neratinib in a human breast cancer cell line (SKBR-3). These genes upon knockdown would modulate cell viability in the presence of subeffective concentrations of neratinib. We discovered a diverse set of genes whose depletion selectively impaired or enhanced the viability of SKBR-3 cells in the presence of neratinib. We observed diverse pathways including EGFR, hypoxia, cAMP, and protein ubiquitination that, when co-treated with RNAi and neratinib, resulted in arrest of cell proliferation. Examining the changes of these genes and their protein products also led to a rationale for clinically relevant drug combination treatments. Treatment of cells with either paclitaxel or cytarabine in combination with neratinib resulted in a strong antiproliferative effect. The identification of novel mediators of cellular response to neratinib and the development of potential drug combination treatments have expanded our understanding of neratinib's mode-of-action for the development of more effective therapeutic regimens. Notably, our findings support a paclitaxel and neratinib phase III clinical trial in breast cancer patients.