R-loops act as regulatory switches modulating transcription of COLD-responsive genes in rice.

COLD is a major naturally occurring stress that usually causes complex symptoms and severe yield loss in crops. R-loops function in various cellular processes, including development and stress responses, in plants. However, how R-loops function in COLD responses is largely unknown in COLD susceptible crops like rice (Oryza sativa L.). We conducted DRIP-Seq along with other omics data (RNA-Seq, DNase-Seq and ChIP-Seq) in rice with or without COLD treatment. COLD treatment caused R-loop reprogramming across the genome. COLD-biased R-loops had higher GC content and novel motifs for the binding of distinct transcription factors (TFs). Moreover, R-loops can directly/indirectly modulate the transcription of a subset of COLD-responsive genes, which can be mediated by R-loop overlapping TF-centered or cis-regulatory element-related regulatory networks and lncRNAs, accounting for c. 60% of COLD-induced expression of differential genes in rice, which is different from the findings in Arabidopsis. We validated two R-loop loci with contrasting (negative/positive) roles in the regulation of two individual COLD-responsive gene expression, as potential targets for enhanced COLD resistance. Our study provides detailed evidence showing functions of R-loop reprogramming during COLD responses and provides some potential R-loop loci for genetic and epigenetic manipulation toward breeding of rice varieties with enhanced COLD tolerance.

Interplay of DNA and RNA N 6 -methyladenine with R-loops in regulating gene transcription in Arabidopsis.

R-loops and covalent modifications of N 6 -methyladenine on DNA (D-6 mA) or RNA (R-m6A) have been documented to function in various cellular processes in eukaryotes. However, the relationships between R-loops and both covalent modifications are still elusive in plants. Here, we integrated existing ssDRIP-seq with D-6 mA and R-m6A data from Arabidopsis thaliana. We found that the presence of either of both modifications facilitates R-loop formation and transcription of overlapping genes. Interestingly, our study suggests that the presence of R-m6A is key to affect R-loop intensity and positively regulate gene transcription. Moreover, the presence of D-6 mA plays an additive role to facilitate the effect of R-m6A on R-loop intensity, however, D-6 mA may negatively regulate gene transcription when coexisted with R-m6A. Our analyses indicate that D-6 mA, R-m6A, or histone marks may act individually and cooperatively with R-loops in regulating gene transcription. Our study is the first to link R-loops with D-6 mA and R-m6A in plants, thereby providing new insights into interactions between R-loops with D-6 mA, R-m6A, and histone marks for regulating gene transcription. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01010-5.

Chemoselective Preparation of Clickable Aryl Sulfonyl Fluoride Monomers: A Toolbox of Highly Functionalized Intermediates for Chemical Biology Probe Synthesis.

Sulfonyl fluoride (SF)-based activity probes have become important tools in chemical biology. Herein, exploiting the relative chemical stability of SF to carry out a number of unprecedented SF-sparing functional group manipulations, we report the chemoselective synthesis of a toolbox of highly functionalized aryl SF monomers that we used to quickly prepare SF chemical biology probes. In addition to SF, the monomers bear an embedded click handle (a terminal alkyne that can perform copper(I)-mediated azide-alkyne cycloaddition). The monomers can be used either as fragments to prepare clickable SF analogues of drugs (biologically active compounds) bearing an aryl ring or, alternatively, attached to drugs as minimalist clickable aryl SF substituents.

Dose-dependent effects of small-molecule antagonists on the genomic landscape of androgen receptor binding.

BACKGROUND: The androgen receptor plays a critical role throughout the progression of prostate cancer and is an important drug target for this disease. While chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-Seq) is becoming an essential tool for studying transcription and chromatin modification factors, it has rarely been employed in the context of drug discovery. RESULTS: Here we report changes in the genome-wide AR binding landscape due to dose-dependent inhibition by drug-like small molecules using ChIP-Seq. Integration of sequence analysis, transcriptome profiling, cell viability assays and xenograft tumor growth inhibition studies enabled us to establish a direct cistrome-activity relationship for two novel potent AR antagonists. By selectively occupying the strongest binding sites, AR signaling remains active even when androgen levels are low, as is characteristic of first-line androgen ablation therapy. Coupled cistrome and transcriptome profiling upon small molecule antagonism led to the identification of a core set of AR direct effector genes that are most likely to mediate the activities of targeted agents: unbiased pathway mapping revealed that AR is a key modulator of steroid metabolism by forming a tightly controlled feedback loop with other nuclear receptor family members and this oncogenic effect can be relieved by antagonist treatment. Furthermore, we found that AR also has an extensive role in negative gene regulation, with estrogen (related) receptor likely mediating its function as a transcriptional repressor. CONCLUSIONS: Our study provides a global and dynamic view of AR's regulatory program upon antagonism, which may serve as a molecular basis for deciphering and developing AR therapeutics.

SAM-Competitive PRMT5 Inhibitor PF-06939999 Demonstrates Antitumor Activity in Splicing Dysregulated NSCLC with Decreased Liability of Drug Resistance.

Protein arginine methyltransferase 5 (PRMT5) overexpression in hematologic and solid tumors methylates arginine residues on cellular proteins involved in important cancer functions including cell-cycle regulation, mRNA splicing, cell differentiation, cell signaling, and apoptosis. PRMT5 methyltransferase function has been linked with high rates of tumor cell proliferation and decreased overall survival, and PRMT5 inhibitors are currently being explored as an approach for targeting cancer-specific dependencies due to PRMT5 catalytic function. Here, we describe the discovery of potent and selective S-adenosylmethionine (SAM) competitive PRMT5 inhibitors, with in vitro and in vivo characterization of clinical candidate PF-06939999. Acquired resistance mechanisms were explored through the development of drug resistant cell lines. Our data highlight compound-specific resistance mutations in the PRMT5 enzyme that demonstrate structural constraints in the cofactor binding site that prevent emergence of complete resistance to SAM site inhibitors. PRMT5 inhibition by PF-06939999 treatment reduced proliferation of non-small cell lung cancer (NSCLC) cells, with dose-dependent decreases in symmetric dimethyl arginine (SDMA) levels and changes in alternative splicing of numerous pre-mRNAs. Drug sensitivity to PF-06939999 in NSCLC cells associates with cancer pathways including MYC, cell cycle and spliceosome, and with mutations in splicing factors such as RBM10. Translation of efficacy in mouse tumor xenograft models with splicing mutations provides rationale for therapeutic use of PF-06939999 in the treatment of splicing dysregulated NSCLC.

Identification of optimal dosing schedules of dacomitinib and osimertinib for a phase I/II trial in advanced EGFR-mutant non-small cell lung cancer.

Despite the clinical success of the third-generation EGFR inhibitor osimertinib as a first-line treatment of EGFR-mutant non-small cell lung cancer (NSCLC), resistance arises due to the acquisition of EGFR second-site mutations and other mechanisms, which necessitates alternative therapies. Dacomitinib, a pan-HER inhibitor, is approved for first-line treatment and results in different acquired EGFR mutations than osimertinib that mediate on-target resistance. A combination of osimertinib and dacomitinib could therefore induce more durable responses by preventing the emergence of resistance. Here we present an integrated computational modeling and experimental approach to identify an optimal dosing schedule for osimertinib and dacomitinib combination therapy. We developed a predictive model that encompasses tumor heterogeneity and inter-subject pharmacokinetic variability to predict tumor evolution under different dosing schedules, parameterized using in vitro dose-response data. This model was validated using cell line data and used to identify an optimal combination dosing schedule. Our schedule was subsequently confirmed tolerable in an ongoing dose-escalation phase I clinical trial (NCT03810807), with some dose modifications, demonstrating that our rational modeling approach can be used to identify appropriate dosing for combination therapy in the clinical setting.

Discovery of N-((3R,4R)-4-Fluoro-1-(6-((3-methoxy-1-methyl-1H-pyrazol-4-yl)amino)-9-methyl-9H-purin-2-yl)pyrrolidine-3-yl)acrylamide (PF-06747775) through Structure-Based Drug Design: A High Affinity Irreversible Inhibitor Targeting Oncogenic EGFR Mutants with Selectivity over Wild-Type EGFR.

Mutant epidermal growth factor receptor (EGFR) is a major driver of non-small-cell lung cancer (NSCLC). Marketed first generation inhibitors, such as erlotinib, effect a transient beneficial response in EGFR mutant NSCLC patients before resistance mechanisms render these inhibitors ineffective. Secondary oncogenic EGFR mutations account for approximately 50% of relapses, the most common being the gatekeeper T790M substitution that renders existing therapies ineffective. The discovery of PF-06459988 (1), an irreversible pyrrolopyrimidine inhibitor of EGFR T790M mutants, was recently disclosed.1 Herein, we describe our continued efforts to achieve potency across EGFR oncogenic mutations and improved kinome selectivity, resulting in the discovery of clinical candidate PF-06747775 (21), which provides potent EGFR activity against the four common mutants (exon 19 deletion (Del), L858R, and double mutants T790M/L858R and T790M/Del), selectivity over wild-type EGFR, and desirable ADME properties. Compound 21 is currently being evaluated in phase-I clinical trials of mutant EGFR driven NSCLC.

Diabetes management in San Diego's Chamorro community.

PURPOSE: The purpose of this study was to assess the diabetes risk status, incidence, and morbidity within San Diego's Chamorro community as a foundation to help community leaders and health care providers create culturally customized health promotion interventions. METHODS: The Behavioral Risk Factor Surveillance Survey was used to query a randomly selected, convenience sample of San Diego Chamorros (N = 228) drawn from the Chamorro Directory International. Based on individual survey responses, participants were mailed personalized health-promoting information. Subsequently, they received information that addressed the most commonly observed overall threats to the Chamorro community's health. RESULTS: A higher than average prevalence of diabetes and gestational diabetes was reported by study participants along with a high prevalence of the risk factors associated with the premature onset of diabetes and its consequences. CONCLUSION: Collaborative partnerships between health professionals and community leaders can help identify opportunities and strategies for improving the health of the nation's population subgroups. San Diego's Chamorro community leaders now have a clearer understanding of the prevalence of diabetes risk factors within their community and can begin working with public health educators to create culturally aligned diabetes prevention and management programs. Given the willingness of Chamorro leaders to get involved in the development of a diabetes awareness campaign and the community's closely knit social network, it should be possible to promote (1) community participation in the intervention program, (2) an increase in the community's adherence to recommended behavioral changes, and (3) identification of additional program modifications that will further enhance the program's cultural relevance.

Effects of Activating Mutations on EGFR Cellular Protein Turnover and Amino Acid Recycling Determined Using SILAC Mass Spectrometry.

Rapid mutations of proteins that are targeted in cancer therapy often lead to drug resistance. Often, the mutation directly affects a drug's binding site, effectively blocking binding of the drug, but these mutations can have other effects such as changing the protein turnover half-life. Utilizing SILAC MS, we measured the cellular turnover rates of an important non-small cell lung cancer target, epidermal growth factor receptor (EGFR). Wild-type (WT) EGFR, EGFR with a single activating mutant (Del 746-750 or L858R), and the drug-resistant double mutant (L858R/T790M) EGFR were analyzed. In non-small cell lung cancer cell lines, EGFR turnover rates ranged from 28 hours in A431 cells (WT) to 7.5 hours in the PC-9 cells (Del 746-750 mutant). The measurement of EGFR turnover rate in PC-9 cells dosed with irreversible inhibitors has additional complexity due to inhibitor effects on cell viability and results were reported as a range. Finally, essential amino acid recycling (K and R) was measured in different cell lines. The recycling was different in each cell line, but the overall inclusion of the effect of amino acid recycling on calculating EGFR turnover rates resulted in a 10-20% reduction in rates.

Tracheal stenting for primary tracheal mucosa-associated lymphoid tissue lymphoma.

Primary tracheal mucosa-associated lymphoid tissue (MALT) lymphoma is extremely rare. We report a 49-year-old female patient with the complaint of dyspnea. Fiberoptic bronchoscopy showed polypoid, variable-sized and irregular nodules causing narrowing of the tracheal lumen from the proximal trachea to the left main bronchus. Because of severe stenosis in the airway and the severity of symptoms, this case was unresectable. The patient was then treated successfully with placement of an endobronchial stent through bronchofibroscopy. After the placement of the stent, bronchoscopic biopsy was performed. Pathological analysis confirms a diagnosis of MALT-associated malignant lymphoma. We performed systemic chemotherapy on the patient. The temporary stent was removed after the reduction of the stenosis. This is the first case in which tracheal MALT lymphoma was treated successfully following tracheal stent insertion guided by bronchofibroscopy. Temporary tracheal stenting can be a favorable choice for a patient with tracheal stenosis caused by primary tracheal MALT lymphoma.

Discovery of aryloxy tetramethylcyclobutanes as novel androgen receptor antagonists.

An aryloxy tetramethylcyclobutane was identified as a novel template for androgen receptor (AR) antagonists via cell-based high-throughput screening. Follow-up to the initial "hit" established 5 as a viable lead. Further optimization to achieve full AR antagonism led to the discovery of 26 and 30, both of which demonstrated excellent in vivo tumor growth inhibition upon oral administration in a castration-resistant prostate cancer (CRPC) animal model.

Pharmacological characterization of a small molecule inhibitor of c-Jun kinase.

c-Jun NH(2)-terminal kinase (JNK) plays an important role in insulin resistance; however, identification of pharmacologically potent and selective small molecule JNK inhibitors has been limited. Compound A has a cell IC(50) of 102 nM and is at least 100-fold selective against related kinases and 27-fold selective against glycogen synthase kinase-3beta and cyclin-dependent kinase-2. In C57BL/6 mice, compound A reduced LPS-mediated increases in both plasma cytokine levels and phosphorylated c-Jun in adipose tissue. Treatment of mice fed a high-fat diet with compound A for 3 wk resulted in a 13.1 +/- 1% decrease in body weight and a 9.3 +/- 1.5% decrease in body fat, compared with a 6.6 +/- 2.1% increase in body weight and a 6.7 +/- 2.1% increase in body fat in vehicle-treated mice. Mice pair fed to those that received compound A exhibited a body weight decrease of 7 +/- 1% and a decrease in body fat of 1.6 +/- 1.3%, suggesting that reductions in food intake could not account solely for the reductions in adiposity observed. Compound A dosed at 30 mg/kg for 13 days in high-fat fed mice resulted in a significant decrease in phosphorylated c-Jun in adipose tissue accompanied by a decrease in weight and reductions in glucose and triglycerides and increases in insulin sensitivity to levels comparable with those in lean control mice. The ability of compound A to reduce the insulin-stimulated phosphorylation of insulin receptor substrate-1 (IRS-1) von Ser307 and partially reverse the free fatty acid inhibition of glucose uptake in 3T3L1 adipocytes, suggests that enhancement of insulin signaling in addition to weight loss may contribute to the effects of compound A on insulin sensitization in vivo. Pharmacological inhibition of JNK using compound A may therefore offer an effective therapy for type 2 diabetes mediated at least in part via weight reduction.

Cancer risk factor assessment among Chamorro men in San Diego.

BACKGROUND: Little is known about the health of Chamorros residing in the United States. METHOD: The CDC's Behavioral Risk Factor Surveillance System was used to assess cancer-related behavioral risk factors in San Diego's Chamorro men, and the Chamorro Directory International was used to recruit them. RESULTS: Of the 100 Chamorro men, 19% smoked, 38.4% reported eating 5 or more fruits and vegetables daily, 48.8% reported being current on their prostate cancer screening, and 74.4% reported being current on their colorectal cancer screening. CONCLUSIONS: This sample of Chamorro men reported higher rates of health-promoting practices than nationwide averages.

Cancer risk factor assessment among Chamorro women.

BACKGROUND: Little is known about the health of Chamorros residing in the United States. METHODS: The Chamorro Directory International and CDC's Behavioral Risk Factor Surveillance System were used to assess behavioral risk factors and health needs of San Diego's Chamorro women. RESULTS: Of 128 Chamorro women, 42.2% reported having a total frequency of five or more fruits and vegetables daily, 54.5% reported being current on their colorectal cancer screening, and 9.4% smoke. CONCLUSIONS: While this sample of Chamorro women reported higher rates of healthpromoting practices than nationwide averages, significant health improvement opportunities remain.