The impact of SBF2 on taxane-induced peripheral neuropathy.

Taxane-induced peripheral neuropathy (TIPN) is a devastating survivorship issue for many cancer patients. In addition to its impact on quality of life, this toxicity may lead to dose reductions or treatment discontinuation, adversely impacting survival outcomes and leading to health disparities in African Americans (AA). Our lab has previously identified deleterious mutations in SET-Binding Factor 2 (SBF2) that significantly associated with severe TIPN in AA patients. Here, we demonstrate the impact of SBF2 on taxane-induced neuronal damage using an ex vivo model of SBF2 knockdown of induced pluripotent stem cell-derived sensory neurons. Knockdown of SBF2 exacerbated paclitaxel changes to cell viability and neurite outgrowth while attenuating paclitaxel-induced sodium current inhibition. Our studies identified paclitaxel-induced expression changes specific to mature sensory neurons and revealed candidate genes involved in the exacerbation of paclitaxel-induced phenotypes accompanying SBF2 knockdown. Overall, these findings provide ex vivo support for the impact of SBF2 on the development of TIPN and shed light on the potential pathways involved.

Germline predictors for bevacizumab induced hypertensive crisis in ECOG-ACRIN 5103 and BEATRICE.

BACKGROUND: Bevacizumab is a beneficial therapy in several advanced cancer types. Predictive biomarkers to better understand which patients are destined to benefit or experience toxicity are needed. Associations between bevacizumab induced hypertension and survival have been reported but with conflicting conclusions. METHODS: We performed post-hoc analyses to evaluate the association in 3124 patients from two phase III adjuvant breast cancer trials, E5103 and BEATRICE. Differences in invasive disease-free survival (IDFS) and overall survival (OS) between patients with hypertension and those without were compared. Hypertension was defined as systolic blood pressure (SBP) ≥ 160 mmHg (n = 346) and SBP ≥ 180 mmHg (hypertensive crisis) (n = 69). Genomic analyses were performed to evaluate germline genetic predictors for the hypertensive crisis. RESULTS: Hypertensive crisis was significantly associated with superior IDFS (p = 0.015) and OS (p = 0.042), but only IDFS (p = 0.029; HR = 0.28) remained significant after correction for prognostic factors. SBP ≥ 160 mmHg was not associated with either IDFS or OS. A common single-nucleotide polymorphism, rs6486785, was significantly associated with hypertensive crisis (p = 8.4 × 10-9; OR = 5.2). CONCLUSION: Bevacizumab-induced hypertensive crisis is associated with superior outcomes and rs6486785 predicted an increased risk of this key toxicity.

Impact of African ancestry on the relationship between body mass index and survival in an early-stage breast cancer trial (ECOG-ACRIN E5103).

BACKGROUND: African ancestry (AA) and obesity are associated with worse survival in early-stage breast cancer. Obesity disproportionately affects women of AA; however, the intersection between ancestry and obesity on breast cancer outcomes remains unclear. METHODS: A total of 2854 patients in the adjuvant trial E5103 were analyzed. Genetic ancestry was determined using principal components from a genome-wide array. The impact of continuous or binary body mass index (BMI) on disease-free survival (DFS) and overall survival (OS) was evaluated by multivariable Cox proportional hazards models in AA patients and European ancestry (EA) patients. RESULTS: There were 2471 EA patients and 383 AA patients. Higher BMI was significantly associated with worse DFS and OS only in AA patients (DFS hazard ratio [HR], 1.25; 95% CI, 1.07-1.46; OS HR, 1.38; 95% CI, 1.10-1.73), not in EA patients (DFS HR, 0.97; 95% CI, 0.90-1.05; OS HR, 1.03; 95% CI, 0.93-1.14). Severe obesity (BMI ≥40) was significantly associated with worse survival in AA patients (DFS HR, 2.04; 95% CI, 1.21-3.43; OS HR, 2.21; 95% CI, 1.03-4.75) but had no impact on that of EA patients. In the estrogen receptor-positive (ER+) and triple-negative breast cancer subgroups, BMI was significantly associated with worse outcomes only in those AA patients with ER+ disease. Within the AA group, BMI remained associated with worse survival regardless of the AA proportion. CONCLUSIONS: Higher BMI was statistically significantly associated with worse breast cancer outcomes in AA but not EA patients. This association was most significant for severe obesity and those with ER+ disease. These observations help define optimal populations for weight change interventions designed to affect disparities and survival in early-stage breast cancer. LAY SUMMARY: African ancestry and obesity are both risk factors for worse survival after early-stage breast cancer. Women of African descent are also disproportionately affected by obesity; however, it is unclear what impact body weight has on racial disparities in breast cancer. Data from a large phase 3 clinical trial in high-risk, early-stage breast cancer were used to determine how body weight affects survival outcomes in European versus African Americans. Study results demonstrate that a higher body mass index is associated with increased risk of breast cancer recurrence and worse survival in women of African ancestry but not in women of European ancestry.

Bevacizumab-induced hypertension and proteinuria: a genome-wide study of more than 1000 patients.

BACKGROUND: Hypertension and proteinuria are common bevacizumab-induced toxicities. No validated biomarkers are available for identifying patients at risk of these toxicities. METHODS: A genome-wide association study (GWAS) meta-analysis was performed in 1039 bevacizumab-treated patients of European ancestry in four clinical trials (CALGB 40502, 40503, 80303, 90401). Grade ≥2 hypertension and proteinuria were recorded (CTCAE v.3.0). Single-nucleotide polymorphism (SNP)-toxicity associations were determined using a cause-specific Cox model adjusting for age and sex. RESULTS: The most significant SNP associated with hypertension with concordant effect in three out of the four studies (p-value <0.05 for each study) was rs6770663 (A > G) in KCNAB1, with the G allele increasing the risk of hypertension (p-value = 4.16 × 10-6). The effect of the G allele was replicated in ECOG-ACRIN E5103 in 582 patients (p-value = 0.005). The meta-analysis of all five studies for rs6770663 led to p-value = 7.73 × 10-8, close to genome-wide significance. The most significant SNP associated with proteinuria was rs339947 (C > A, between DNAH5 and TRIO), with the A allele increasing the risk of proteinuria (p-value = 1.58 × 10-7). CONCLUSIONS: The results from the largest study of bevacizumab toxicity provide new markers of drug safety for further evaluations. SNP in KCNAB1 validated in an independent dataset provides evidence toward its clinical applicability to predict bevacizumab-induced hypertension. ClinicalTrials.gov Identifier: NCT00785291 (CALGB 40502); NCT00601900 (CALGB 40503); NCT00088894 (CALGB 80303) and NCT00110214 (CALGB 90401).

Using VBIM Technique to Discover ARMC4/ODAD2 as a Novel Negative Regulator of NF-κB and a New Tumor Suppressor in Colorectal Cancer.

Since nuclear factor (NF) κB plays pivotal roles in inflammation and cancer, understanding its regulation holds great promise for disease therapy. Using the powerful validation-based insertional mutagenesis (VBIM) technique established by us previously, we discovered armadillo repeat-containing protein 4 (ARMC4)/outer dynein arm docking complex subunit 2 (ODAD2), a rarely studied protein known to date, as a novel negative regulator of NF-κB in colorectal cancer (CRC). High expression of ARMC4 downregulated the expression of NF-κB-dependent genes, dramatically reduced NF-κB activity, cellular proliferation, anchorage-independent growth, and migratory ability in vitro, and significantly decreased xenograft tumor growth in vivo. Co-immunoprecipitation experiments demonstrated that ARMC4 forms a complex with NF-κB. Importantly, the lower ARMC4 expression in patient tumors than normal tissues indicates its potential tumor suppressor function in CRC. Collectively, we uncovered a completely new facet of ARMC4 function by identifying it as a novel NF-κB negative regulator, thus uncovering ARMC4 as a potential new therapeutic target in CRC.

Regulation of a PRMT5/NF-κB Axis by Phosphorylation of PRMT5 at Serine 15 in Colorectal Cancer.

The overexpression of PRMT5 is highly correlated to poor clinical outcomes for colorectal cancer (CRC) patients. Importantly, our previous work demonstrated that PRMT5 overexpression could substantially augment activation of the nuclear factor kappa B (NF-κB) via methylation of arginine 30 (R30) on its p65 subunit, while knockdown of PRMT5 showed the opposite effect. However, the precise mechanisms governing this PRMT5/NF-κB axis are still largely unknown. Here, we report a novel finding that PRMT5 is phosphorylated on serine 15 (S15) in response to interleukin-1ß (IL-1ß) stimulation. Interestingly, we identified for the first time that the oncogenic kinase, PKCι could catalyze this phosphorylation event. Overexpression of the serine-to-alanine mutant of PRMT5 (S15A), in either HEK293 cells or CRC cells HT29, DLD1, and HCT116 attenuated NF-κB transactivation compared to WT-PRMT5, confirming that S15 phosphorylation is critical for the activation of NF-κB by PRMT5. Furthermore, the S15A mutant when compared to WT-PRMT5, could downregulate a subset of IL-1ß-inducible NF-κB-target genes which correlated with attenuated promoter occupancy of p65 at its target genes. Additionally, the S15A mutant reduced IL-1ß-induced methyltransferase activity of PRMT5 and disrupted the interaction of PRMT5 with p65. Furthermore, our data indicate that blockade of PKCι-regulated PRMT5-mediated activation of NF-κB was likely through phosphorylation of PRMT5 at S15. Finally, inhibition of PKCι or overexpression of the S15A mutant attenuated the growth, migratory, and colony-forming abilities of CRC cells compared to the WT-PRMT5. Collectively, we have identified a novel PKCι/PRMT5/NF-κB signaling axis, suggesting that pharmacological disruption of this pivotal axis could serve as the basis for new anti-cancer therapeutics.

Serologic, but Not Genetic, Markers Are Associated With Impaired Anthropometrics at Diagnosis of Pediatric Crohn's Disease.

OBJECTIVES: Children with Crohn's disease may present with malnutrition and linear growth impairment, which can be secondary to insufficient caloric intake, chronic inflammation, malabsorption, and suppression of growth-promoting hormones. We evaluated clinical, serologic, and genetic data to determine risk factors for impaired anthropometrics in Crohn's disease at diagnosis. METHODS: Our study evaluated 772 children newly diagnosed with Crohn's disease, inflammatory phenotype, enrolled in the RISK Stratification Project to determine the factors associated with anthropometric impairment. Data were collected on demographics, growth parameters, disease location, serologic and immunologic markers, and disease severity. We performed a genome-wide association study of genetic polymorphisms associated with inflammatory bowel disease. Regression analysis determined associations between anthropometrics and clinical, serologic, and genetic variables. RESULTS: There were 59 (7%) children with height z score <-2, 126 (14%) with a weight z score <-2, and 156 (17%) with a body mass index z score <-2. Linear growth impairment was associated with hypoalbuminemia (P = 0.0052), elevated granulocyte-macrophage colony stimulating factor autoantibodies (P = 0.0110), and elevated CBir antibodies against flagellin (P = 0.0117). Poor weight gain was associated with female sex (P = 0.0401), hypoalbuminemia (P = 0.0162), and thrombocytosis (P = 0.0081). Malnutrition was associated with hypoalbuminemia (P = 0.0061) and thrombocytosis (P = 0.0011). Children with moderate or severe disease had lower weight (P = 0.02 and P = 1.16×10, respectively) and body mass index z scores (P = 2.7 × 10 and P = 1.01 × 10, respectively) than children with quiescent and mild disease. There was no association between age of diagnosis, Tanner stage, or disease location and having impaired anthropometrics. There was no genome-wide association between the genetic polymorphisms and the serologic variables and anthropometric measurements. CONCLUSIONS: This is the largest study evaluating growth in treatment-naïve children with Crohn's disease, inflammatory phenotype. It is the first study to use genome-wide sequencing to assess for genetic determinants of growth impairment. Granulocyte-macrophage colony stimulating factor autoantibodies and CBir antibodies are more likely to be elevated in children with growth impairment. Future investigations should evaluate the relationship between genetic polymorphisms, pathologic immune responses, and the biological pathways regulating growth.

Novel Serine 176 Phosphorylation of YBX1 Activates NF-κB in Colon Cancer.

Y box protein 1 (YBX1) is a well known oncoprotein that has tumor-promoting functions. YBX1 is widely considered to be an attractive therapeutic target in cancer. To develop novel therapeutics to target YBX1, it is of great importance to understand how YBX1 is finely regulated in cancer. Previously, we have shown that YBX1 could function as a tumor promoter through phosphorylation of its Ser-165 residue, leading to the activation of the NF-κB signaling pathway (1). In this study, using mass spectrometry analysis, we discovered a distinct phosphorylation site, Ser-176, on YBX1. Overexpression of the YBX1-S176A (serine-to-alanine) mutant in either HEK293 cells or colon cancer HT29 cells showed dramatically reduced NF-κB-activating ability compared with that of WT-YBX1, confirming that Ser-176 phosphorylation is critical for the activation of NF-κB by YBX1. Importantly, the mutant of Ser-176 and the previously reported Ser-165 sites regulate distinct groups of NF-κB target genes, suggesting the unique and irreplaceable function of each of these two phosphorylated serine residues. Our important findings could provide a novel cancer therapy strategy by blocking either Ser-176 or Ser-165 phosphorylation or both of YBX1 in colon cancer.

Meta-analysis of antiviral protection of white spot syndrome virus vaccine to the shrimp.

Currently, white spot syndrome virus (WSSV) is one of the most serious pathogens that impacts shrimp farming around the world. A WSSV vaccine provides a significant protective benefit to the host shrimp. Although various types of vaccines against WSSV have emerged, the immune effects among them were not compared, and it remains unclear which type of vaccine has the strongest protective effect. Meanwhile, due to the lack of effective routes of administration and immunization programs, WSSV vaccines have been greatly limited in the actual shrimp farming. To answer these questions, this study conducted a comprehensive meta-analysis over dozens of studies and compared all types WSSV vaccines, which include sub-unit protein vaccines, whole virus inactivated vaccines, DNA vaccines and RNA-based vaccines. The results showed that the RNA-based vaccine had the highest protection rate over the other three types of vaccines. Among the various sub-unit protein vaccines, VP26 vaccine had the best protective effects than other sub-unit protein vaccines. Moreover, this study demonstrated that vaccines expressed in eukaryotic hosts had higher protection rates than that of prokaryotic systems. Among the three immunization modes (oral administration, immersion and injection) used in monovalent protein vaccines, oral administration had the highest protection rate. In natural conditions, shrimp are mostly infected by the virus orally. These results provide a guide for exploration of a novel WSSV vaccine and help facilitate the application of WSSV vaccines in shrimp farming.

Discerning the clinical relevance of biomarkers in early stage breast cancer.

PURPOSE: Prior data suggest that breast cancer patients accept significant toxicity for small benefit. It is unclear whether personalized estimations of risk or benefit likelihood that could be provided by biomarkers alter treatment decisions in the curative setting. METHODS: A choice-based conjoint (CBC) survey was conducted in 417 HER2-negative breast cancer patients who received chemotherapy in the curative setting. The survey presented pairs of treatment choices derived from common taxane- and anthracycline-based regimens, varying in degree of benefit by risk of recurrence and in toxicity profile, including peripheral neuropathy (PN) and congestive heart failure (CHF). Hypothetical biomarkers shifting benefit and toxicity risk were modeled to determine whether this knowledge alters choice. Previously identified biomarkers were evaluated using this model. RESULTS: Based on CBC analysis, a non-anthracycline regimen was the most preferred. Patients with prior PN had a similar preference for a taxane regimen as those who were PN naïve, but more dramatically shifted preference away from taxanes when PN was described as severe/irreversible. When modeled after hypothetical biomarkers, as the likelihood of PN increased, the preference for taxane-containing regimens decreased; similarly, as the likelihood of CHF increased, the preference for anthracycline regimens decreased. When evaluating validated biomarkers for PN and CHF, this knowledge did alter regimen preference. CONCLUSIONS: Patients faced with multi-faceted decisions consider personal experience and perceived risk of recurrent disease. Biomarkers providing information on likelihood of toxicity risk do influence treatment choices, and patients may accept reduced benefit when faced with higher risk of toxicity in the curative setting.

Comprehensive comparison of molecular portraits between cell lines and tumors in breast cancer.

BACKGROUND: Proper cell models for breast cancer primary tumors have long been the focal point in the cancer's research. The genomic comparison between cell lines and tumors can investigate the similarity and dissimilarity and help to select right cell model to mimic tumor tissues to properly evaluate the drug reaction in vitro. In this paper, a comprehensive comparison in copy number variation (CNV), mutation, mRNA expression and protein expression between 68 breast cancer cell lines and 1375 primary breast tumors is conducted and presented. RESULTS: Using whole genome expression arrays, strong correlations were observed between cells and tumors. PAM50 gene expression differentiated them into four major breast cancer subtypes: Luminal A and B, HER2amp, and Basal-like in both cells and tumors partially. Genomic CNVs patterns were observed between tumors and cells across chromosomes in general. High C > T and C > G trans-version rates were observed in both cells and tumors, while the cells had slightly higher somatic mutation rates than tumors. Clustering analysis on protein expression data can reasonably recover the breast cancer subtypes in cell lines and tumors. Although the drug-targeted proteins ER/PR and interesting mTOR/GSK3/TS2/PDK1/ER_P118 cluster had shown the consistent patterns between cells and tumor, low protein-based correlations were observed between cells and tumors. The expression consistency of mRNA verse protein between cell line and tumors reaches 0.7076. These important drug targets in breast cancer, ESR1, PGR, HER2, EGFR and AR have a high similarity in mRNA and protein variation in both tumors and cell lines. GATA3 and RP56KB1 are two promising drug targets for breast cancer. A total score developed from the four correlations among four molecular profiles suggests that cell lines, BT483, T47D and MDAMB453 have the highest similarity with tumors. CONCLUSIONS: The integrated data from across these multiple platforms demonstrates the existence of the similarity and dissimilarity of molecular features between breast cancer tumors and cell lines. The cell lines only mirror some but not all of the molecular properties of primary tumors. The study results add more evidence in selecting cell line models for breast cancer research.

Fatty acid desaturase 1 gene polymorphisms control human hepatic lipid composition.

UNLABELLED: Fatty acid desaturase (FADS) genes and their variants have been associated with multiple metabolic phenotypes, including liver enzymes and hepatic fat accumulation, but the detailed mechanism remains unclear. We aimed to delineate the role of FADSs in modulating lipid composition in human liver. We performed a targeted lipidomic analysis of a variety of phospholipids, sphingolipids, and ceramides among 154 human liver tissue samples. The associations between previously genome-wide association studies (GWASs)-identified six FADS single-nucleotide polymorphisms (SNPs), and these lipid levels as well as total hepatic fat content (HFC) were tested. The potential function of these SNPs in regulating transcription of three FADS genes (FADS1, FADS2, and FADS3) in the locus was also investigated. We found that though these SNPs were in high linkage disequilibrium (r(2) > 0.8), the rare alleles of these SNPs were consistently and significantly associated with the accumulation of multiple long-chain fatty acids (LCFAs), with C47H85O13P (C36:4), a phosphatidylinositol (PI), and C43H80O8PN (C38:3), a phosphatidylethanolamine (PE), reached the Bonferroni corrected significance (P < 3 × 10(-4)). Meanwhile, these SNPs were significantly associated with increased ratios between the more saturated and relatively less saturated forms of LCFAs, especially between PEs, PIs, and phosphatidylcholines (PCs; P ≤ 3.5 × 10(-6)). These alleles were also associated with increased total HFC (P < 0.05). Further analyses revealed that these alleles were associated with decreased hepatic expression of FADS1 (P = 0.0018 for rs174556), but not FADS2 or FADS3 (P > 0.05). CONCLUSION: Our findings revealed critical insight into the mechanism underlying FADS1 and its polymorphisms in modulating hepatic lipid deposition by altering gene transcription and controlling lipid composition in human livers.

ECOG-ACRIN EAZ171: Prospective Validation Trial of Germline Predictors of Taxane-Induced Peripheral Neuropathy in Black Women With Early-Stage Breast Cancer.

PURPOSE: Black women experience higher rates of taxane-induced peripheral neuropathy (TIPN) compared with White women when receiving adjuvant once weekly paclitaxel for early-stage breast cancer, leading to more dose reductions and higher recurrence rates. EAZ171 aimed to prospectively validate germline predictors of TIPN and compare rates of TIPN and dose reductions in Black women receiving (neo)adjuvant once weekly paclitaxel and once every 3 weeks docetaxel for early-stage breast cancer. METHODS: Women with early-stage breast cancer who self-identified as Black and had intended to receive (neo)adjuvant once weekly paclitaxel or once every 3 weeks docetaxel were eligible, with planned accrual to 120 patients in each arm. Genotyping was performed to determine germline neuropathy risk. Grade 2-4 TIPN by Common Terminology Criteria for Adverse Events (CTCAE) v5.0 was compared between high- versus low-risk genotypes and between once weekly paclitaxel versus once every 3 weeks docetaxel within 1 year. Patient-rated TIPN and patient-reported outcomes were compared using patient-reported outcome (PRO)-CTCAE and Functional Assessment of Cancer Therapy/Gynecologic Oncology Group-Neurotoxicity. RESULTS: Two hundred and forty of 249 enrolled patients had genotype data, and 91 of 117 (77.8%) receiving once weekly paclitaxel and 87 of 118 (73.7%) receiving once every 3 weeks docetaxel were classified as high-risk. Physician-reported grade 2-4 TIPN was not significantly different in high- versus low-risk genotype groups with once weekly paclitaxel (47% v 35%; P = .27) or with once every 3 weeks docetaxel (28% v 19%; P = .47). Grade 2-4 TIPN was significantly higher in the once weekly paclitaxel versus once every 3 weeks docetaxel arm by both physician-rated CTCAE (45% v 29%; P = .02) and PRO-CTCAE (40% v 24%; P = .03). Patients receiving once weekly paclitaxel required more dose reductions because of TIPN (28% v 9%; P < .001) or any cause (39% v 25%; P = .02). CONCLUSION: Germline variation did not predict risk of TIPN in Black women receiving (neo)adjuvant once weekly paclitaxel or once every 3 weeks docetaxel. Once weekly paclitaxel was associated with significantly more grade 2-4 TIPN and required more dose reductions than once every 3 weeks docetaxel.

Simultaneous inferences based on empirical Bayes methods and false discovery rates ineQTL data analysis.

BACKGROUND: Genome-wide association studies (GWAS) have identified hundreds of genetic variants associated with complex human diseases, clinical conditions and traits. Genetic mapping of expression quantitative trait loci (eQTLs) is providing us with novel functional effects of thousands of single nucleotide polymorphisms (SNPs). In a classical quantitative trail loci (QTL) mapping problem multiple tests are done to assess whether one trait is associated with a number of loci. In contrast to QTL studies, thousands of traits are measured alongwith thousands of gene expressions in an eQTL study. For such a study, a huge number of tests have to be performed (~10(6)). This extreme multiplicity gives rise to many computational and statistical problems. In this paper we have tried to address these issues using two closely related inferential approaches: an empirical Bayes method that bears the Bayesian flavor without having much a priori knowledge and the frequentist method of false discovery rates. A three-component t-mixture model has been used for the parametric empirical Bayes (PEB) method. Inferences have been obtained using Expectation/Conditional Maximization Either (ECME) algorithm. A simulation study has also been performed and has been compared with a nonparametric empirical Bayes (NPEB) alternative. RESULTS: The results show that PEB has an edge over NPEB. The proposed methodology has been applied to human liver cohort (LHC) data. Our method enables to discover more significant SNPs with FDR<10% compared to the previous study done by Yang et al. (Genome Research, 2010). CONCLUSIONS: In contrast to previously available methods based on p-values, the empirical Bayes method uses local false discovery rate (lfdr) as the threshold. This method controls false positive rate.

Genetic Regulation of Human isomiR Biogenesis.

MicroRNAs play a critical role in regulating gene expression post-transcriptionally. Variations in mature microRNA sequences, known as isomiRs, arise from imprecise cleavage and nucleotide substitution or addition. These isomiRs can target different mRNAs or compete with their canonical counterparts, thereby expanding the scope of miRNA post-transcriptional regulation. Our study investigated the relationship between cis-acting single-nucleotide polymorphisms (SNPs) in precursor miRNA regions and isomiR composition, represented by the ratio of a specific 5'-isomiR subtype to all isomiRs identified for a particular mature miRNA. Significant associations between 95 SNP-isomiR pairs were identified. Of note, rs6505162 was significantly associated with both the 5'-extension of hsa-miR-423-3p and the 5'-trimming of hsa-miR-423-5p. Comparison of breast cancer and normal samples revealed that the expression of both isomiRs was significantly higher in tumors than in normal tissues. This study sheds light on the genetic regulation of isomiR maturation and advances our understanding of post-transcriptional regulation by microRNAs.

Critical Role of Novel O-GlcNAcylation of S550 and S551 on the p65 Subunit of NF-κB in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, with a mere 5-year survival of ~10%. This highlights the urgent need for innovative treatment options for PDAC patients. The nuclear factor κB (NF-κB) is a crucial transcription factor that is constitutively activated in PDAC. It mediates the transcription of oncogenic and inflammatory genes that facilitate multiple PDAC phenotypes. Thus, a better understanding of the mechanistic underpinnings of NF-κB activation holds great promise for PDAC diagnosis and effective therapeutics. Here, we report a novel finding that the p65 subunit of NF-κB is O-GlcNAcylated at serine 550 and 551 upon NF-κB activation. Importantly, the overexpression of either serine-to-alanine (S-A) single mutant (S550A or S551A) or double mutant (S550A/S551A) of p65 in PDAC cells impaired NF-κB nuclear translocation, p65 phosphorylation, and transcriptional activity, independent of IκBα degradation. Moreover, the p65 mutants downregulate a category of NF-κB-target genes, which play a role in perpetuating major cancer hallmarks. We further show that overexpression of the p65 mutants inhibited cellular proliferation, migration, and anchorage-independent growth of PDAC cells compared to WT-p65. Collectively, we discovered novel serine sites of p65 O-GlcNAcylation that drive NF-κB activation and PDAC phenotypes, thus opening new avenues by inhibiting the NF-κB O-GlcNAcylation enzyme, O-GlcNAc transferase (OGT), for PDAC treatment in the future.

Cytochrome P450 Oxidoreductase (POR) Associated with Severe Paclitaxel-Induced Peripheral Neuropathy in Patients of European Ancestry from ECOG-ACRIN E5103.

PURPOSE: Paclitaxel is a widely used anticancer therapeutic. Peripheral neuropathy is the dose-limiting toxicity and negatively impacts quality of life. Rare germline gene markers were evaluated for predicting severe taxane-induced peripheral neuropathy (TIPN) in the patients of European ancestry. In addition, the impact of Cytochrome P450 (CYP) 2C8, CYP3A4, and CYP3A5 metabolizer status on likelihood of severe TIPN was also assessed. EXPERIMENTAL DESIGN: Whole-exome sequencing analyses were performed in 340 patients of European ancestry who received a standard dose and schedule of paclitaxel in the adjuvant, randomized phase III breast cancer trial, E5103. Patients who experienced grade 3-4 (n = 168) TIPN were compared to controls (n = 172) who did not experience TIPN. For the analyses, rare variants with a minor allele frequency ≤ 3% and predicted to be deleterious by protein prediction programs were retained. A gene-based, case-control analysis using SKAT was performed to identify genes that harbored an imbalance of deleterious variants associated with increased risk of severe TIPN. CYP star alleles for CYP2C8, CYP3A4, and CYP3A5 were called. An additive logistic regression model was performed to test the association of CYP2C8, CYP3A4, and CYP3A5 metabolizer status with severe TIPN. RESULTS: Cytochrome P450 oxidoreductase (POR) was significantly associated with severe TIPN (P value = 1.8 ×10-6). Six variants were predicted to be deleterious in POR. There were no associations between CYP2C8, CYP3A4, or CYP3A5 metabolizer status with severe TIPN. CONCLUSIONS: Rare variants in POR predict an increased risk of severe TIPN in patients of European ancestry who receive paclitaxel.

Inhibition of PRMT5 by market drugs as a novel cancer therapeutic avenue.

Market drugs, such as Food and Drug Administration (FDA) or European Medicines Agency (EMA)-approved drugs for specific indications provide opportunities for repurposing for newer therapeutics. This potentially saves resources invested in clinical trials that verify drug safety and tolerance in humans prior to alternative indication approval. Protein arginine methyltransferase 5 (PRMT5) overexpression has been linked to promoting the tumor phenotype in several cancers, including pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), and breast cancer (BC), making PRMT5 an important target for cancer therapy. Previously, we showed that PRMT5-mediated methylation of the nuclear factor (NF)-κB, partially contributes to its constitutive activation observed in cancers. In this study, we utilized an AlphaLISA-based high-throughput screening method adapted in our lab, and identified one FDA-approved drug, Candesartan cilexetil (Can, used in hypertension treatment) and one EMA-approved drug, Cloperastine hydrochloride (Clo, used in cough treatment) that had significant PRMT5-inhibitory activity, and their anti-tumor properties were validated using cancer phenotypic assays in vitro. Furthermore, PRMT5 selective inhibition of methyltransferase activity was confirmed by reduction of both NF-κB methylation and its subsequent activation upon drug treatment. Using in silico prediction, we identified critical residues on PRMT5 targeted by these drugs that may interfere with its enzymatic activity. Finally, Clo and Can treatment have exhibited marked reduction in tumor growth in vivo. Overall, we provide basis for pursuing repurposing Clo and Can as anti-PRMT5 cancer therapies. Our study offers potential safe and fast repurposing of previously unknown PRMT5 inhibitors into clinical practice.

Osteocyte Egln1/Phd2 links oxygen sensing and biomineralization via FGF23.

Osteocytes act within a hypoxic environment to control key steps in bone formation. FGF23, a critical phosphate-regulating hormone, is stimulated by low oxygen/iron in acute and chronic diseases, however the molecular mechanisms directing this process remain unclear. Our goal was to identify the osteocyte factors responsible for FGF23 production driven by changes in oxygen/iron utilization. Hypoxia-inducible factor-prolyl hydroxylase inhibitors (HIF-PHI) which stabilize HIF transcription factors, increased Fgf23 in normal mice, as well as in osteocyte-like cells; in mice with conditional osteocyte Fgf23 deletion, circulating iFGF23 was suppressed. An inducible MSC cell line ('MPC2') underwent FG-4592 treatment and ATACseq/RNAseq, and demonstrated that differentiated osteocytes significantly increased HIF genomic accessibility versus progenitor cells. Integrative genomics also revealed increased prolyl hydroxylase Egln1 (Phd2) chromatin accessibility and expression, which was positively associated with osteocyte differentiation. In mice with chronic kidney disease (CKD), Phd1-3 enzymes were suppressed, consistent with FGF23 upregulation in this model. Conditional loss of Phd2 from osteocytes in vivo resulted in upregulated Fgf23, in line with our findings that the MPC2 cell line lacking Phd2 (CRISPR Phd2-KO cells) constitutively activated Fgf23 that was abolished by HIF1α blockade. In vitro, Phd2-KO cells lost iron-mediated suppression of Fgf23 and this activity was not compensated for by Phd1 or -3. In sum, osteocytes become adapted to oxygen/iron sensing during differentiation and are directly sensitive to bioavailable iron. Further, Phd2 is a critical mediator of osteocyte FGF23 production, thus our collective studies may provide new therapeutic targets for skeletal diseases involving disturbed oxygen/iron sensing.