FT-6876, a Potent and Selective Inhibitor of CBP/p300, is Active in Preclinical Models of Androgen Receptor-Positive Breast Cancer.

BACKGROUND: Patients with triple-negative breast cancer (TNBC) expressing the androgen receptor (AR) respond poorly to neoadjuvant chemotherapy, although AR antagonists have shown promising clinical activity, suggesting these tumors are AR-dependent. cAMP responsive element binding protein (CREB)-binding protein (CBP) and p300 are transcriptional co-activators for the AR, a key driver of AR+ breast and prostate cancer, and may provide a novel therapeutic target in AR+ TNBC. OBJECTIVES: The aim of this study was to determine the therapeutic potential of FT-6876, a new CBP/p300 bromodomain inhibitor, in breast cancer models with a range of AR levels in vitro and in vivo. METHODS: Effects of FT-6876 on the CBP/p300 pathway were determined by combining chromatin immunoprecipitation (ChIP) with precision run-on sequencing (PRO-seq) complemented with H3K27 acetylation (Ac) and transcriptional profiling. The antiproliferative effect of FT-6876 was also measured in vitro and in vivo. RESULTS: We describe the discovery of FT-6876, a potent and selective CBP/p300 bromodomain inhibitor. The combination of ChIP and PRO-seq confirmed the reduction in H3K27Ac at specific promoter sites concurrent with a decrease in CBP/p300 on the chromatin and a reduction in nascent RNA and enhancer RNA. This was associated with a time- and concentration-dependent reduction in H3K37Ac associated with a decrease in AR and estrogen receptor (ER) target gene expression. This led to a time-dependent growth inhibition in AR+ models, correlated with AR expression. Tumor growth inhibition was also observed in AR+ tumor models of TNBC and ER+ breast cancer subtypes with consistent pharmacokinetics and pharmacodynamics. CONCLUSION: Our findings demonstrate FT-6876 as a promising new CBP/p300 bromodomain inhibitor, with efficacy in preclinical models of AR+ breast cancer.

Intermittent treatment of BRAFV600E melanoma cells delays resistance by adaptive resensitization to drug rechallenge.

Patients with melanoma receiving drugs targeting BRAFV600E and mitogen-activated protein (MAP) kinase kinases 1 and 2 (MEK1/2) invariably develop resistance and face continued progression. Based on preclinical studies, intermittent treatment involving alternating periods of drug withdrawal and rechallenge has been proposed as a method to delay the onset of resistance. The beneficial effect of intermittent treatment has been attributed to drug addiction, where drug withdrawal reduces the viability of resistant cells due to MAP kinase pathway hyperactivation. However, the mechanistic basis of the intermittent effect is incompletely understood. We show that intermittent treatment with the BRAFV600E inhibitor, LGX818/encorafenib, suppresses growth compared with continuous treatment in human melanoma cells engineered to express BRAFV600E, p61-BRAFV600E, or MEK2C125 oncogenes. Analysis of the BRAFV600E-overexpressing cells shows that, while drug addiction clearly occurs, it fails to account for the advantageous effect of intermittent treatment. Instead, growth suppression is best explained by resensitization during periods of drug removal, followed by cell death after drug readdition. Continuous treatment leads to transcriptional responses prominently associated with chemoresistance in melanoma. By contrast, cells treated intermittently reveal a subset of transcripts that reverse expression between successive cycles of drug removal and rechallenge and include mediators of cell invasiveness and the epithelial-to-mesenchymal transition. These transcripts change during periods of drug removal by adaptive switching, rather than selection pressure. Resensitization occurs against a background of sustained expression of melanoma resistance genes, producing a transcriptome distinct from that of the initial drug-naive cell state. We conclude that phenotypic plasticity leading to drug resensitization can underlie the beneficial effect of intermittent treatment.

Genome-wide binding analysis of 195 DNA binding proteins reveals "reservoir" promoters and human specific SVA-repeat family regulation.

A key aspect in defining cell state is the complex choreography of DNA binding events in a given cell type, which in turn establishes a cell-specific gene-expression program. Here we wanted to take a deep analysis of DNA binding events and transcriptional output of a single cell state (K562 cells). To this end we re-analyzed 195 DNA binding proteins contained in ENCODE data. We used standardized analysis pipelines, containerization, and literate programming with R Markdown for reproducibility and rigor. Our approach validated many findings from previous independent studies, underscoring the importance of ENCODE's goals in providing these reproducible data resources. We also had several new findings including: (i) 1,362 promoters, which we refer to as 'reservoirs,' that are defined by having up to 111 different DNA binding-proteins localized on one promoter, yet do not have any expression of steady-state RNA (ii) Reservoirs do not overlap super-enhancer annotations and distinct have distinct properties from super-enhancers. (iii) The human specific SVA repeat element may have been co-opted for enhancer regulation and is highly transcribed in PRO-seq and RNA-seq. Collectively, this study performed by the students of a CU Boulder computational biology class (BCHM 5631 -Spring 2020) demonstrates the value of reproducible findings and how resources like ENCODE that prioritize data standards can foster new findings with existing data in a didactic environment.

Specificity of Phosphorylation Responses to Mitogen Activated Protein (MAP) Kinase Pathway Inhibitors in Melanoma Cells.

The BRAF-MKK1/2-ERK1/2 pathway is constitutively activated in response to oncogenic mutations of BRAF in many cancer types, including melanoma. Although small molecules that inhibit oncogenic BRAF and MAP kinase kinase (MKK)1/2 have been successful in clinical settings, resistance invariably develops. High affinity inhibitors of ERK1/2 have been shown in preclinical studies to bypass the resistance of melanoma and colon cancer cells to BRAF and MKK1/2 inhibitors, and are thus promising additions to current treatment protocols. But still unknown is how molecular responses to ERK1/2 inhibitors compare with inhibitors currently in clinical use. Here, we employ quantitative phosphoproteomics to evaluate changes in phosphorylation in response to the ERK inhibitors, SCH772984 and GDC0994, and compare these to the clinically used MKK1/2 inhibitor, trametinib. Combined with previous studies measuring phosphoproteomic responses to the MKK1/2 inhibitor, selumetinib, and the BRAF inhibitor, vemurafenib, the outcomes reveal key insights into pathway organization, phosphorylation specificity and off-target effects of these inhibitors. The results demonstrate linearity in signaling from BRAF to MKK1/2 and from MKK1/2 to ERK1/2. They identify likely targets of direct phosphorylation by ERK1/2, as well as inhibitor off-targets, including an off-target regulation of the p38α mitogen activated protein kinase (MAPK) pathway by the MKK1/2 inhibitor, trametinib, at concentrations used in the literature but higher than in vivo drug concentrations. In addition, several known phosphorylation targets of ERK1/2 are insensitive to MKK or ERK inhibitors, revealing variability in canonical pathway responses between different cell systems. By comparing multiple inhibitors targeted to multiple tiers of protein kinases in the MAPK pathway, we gain insight into regulation and new targets of the oncogenic BRAF driver pathway in cancer cells, and a useful approach for evaluating the specificity of drugs and drug candidates.

Identification of a serum-induced transcriptional signature associated with type 1 diabetes in the BioBreeding rat.

OBJECTIVE: Inflammatory mediators associated with type 1 diabetes are dilute and difficult to measure in the periphery, necessitating development of more sensitive and informative biomarkers for studying diabetogenic mechanisms, assessing preonset risk, and monitoring therapeutic interventions. RESEARCH DESIGN AND METHODS: We previously utilized a novel bioassay in which human type 1 diabetes sera were used to induce a disease-specific transcriptional signature in unrelated, healthy peripheral blood mononuclear cells (PBMCs). Here, we apply this strategy to investigate the inflammatory state associated with type 1 diabetes in biobreeding (BB) rats. RESULTS: Consistent with their common susceptibility, sera of both spontaneously diabetic BB DRlyp/lyp and diabetes inducible BB DR+/+ rats induced transcription of cytokines, immune receptors, and signaling molecules in PBMCs of healthy donor rats compared with control sera. Like the human type 1 diabetes signature, the DRlyp/lyp signature, which is associated with progression to diabetes, was differentiated from that of the DR+/+ by induction of many interleukin (IL)-1-regulated genes. Supplementing cultures with an IL-1 receptor antagonist (IL-1Ra) modulated the DRlyp/lyp signature (P < 10(-6)), while administration of IL-1Ra to DRlyp/lyp rats delayed onset (P = 0.007), and sera of treated animals did not induce the characteristic signature. Consistent with the presence of immunoregulatory cells in DR+/+ rats was induction of a signature possessing negative regulators of transcription and inflammation. CONCLUSIONS: Paralleling our human studies, serum signatures in BB rats reflect processes associated with progression to type 1 diabetes. Furthermore, these studies support the potential utility of this approach to detect changes in the inflammatory state during therapeutic intervention.

Single-nucleotide polymorphisms in the IL2RA gene are associated with age at diagnosis in late-onset Finnish type 1 diabetes subjects.

The onset of type 1 diabetes can occur at any age, with as many as half of all cases diagnosed after age 15. Despite this wide distribution in age at diagnosis, most genetic studies focus on cases diagnosed in childhood or during early adulthood. To better understand the genetics of late-onset type 1 diabetes, we collected a Finnish case/control cohort with all cases diagnosed between ages 15 and 40. We genotyped 591 probands and 1,538 control subjects at regions well established as susceptibility loci in early onset type 1 diabetes. These loci were then tested for disease association and age-at-diagnosis effects. Using logistic regression, we found that single-nucleotide polymorphisms (SNPs) at the INS, PTPN22, and IFIH1 loci were associated with late-onset disease (OR (95%CI) = 0.57(0.47-0.69), p = 2.77 x 10(-9); OR (95%CI) = 1.50 (1.27-1.78), p = 3.98 x 10(-6); and OR (95%CI) = 0.81(0.71-0.93), p = 0.0028, respectively). In contrast, a disease association was not detected for two SNPs at the IL2RA locus (rs11594656 and rs41295061). Despite this, we did find an independent age-at-diagnosis effect for each IL2RA SNP using a multivariate Cox proportional hazards model (p = 0.003, 0.002, respectively). Taken together, polymorphisms at the IL2RA locus were a major determinant of age at diagnosis in our cohort with an effect at par with the HLA-DQ2/DQ8 genotype as measured by hazard ratios. These findings suggest that the IL2RA locus controls both the susceptibility to disease and its time of occurrence. Thus, we believe the IL2/IL2R axis represents a potential therapeutic target for delaying the onset of disease.