Molecular characterization of ESR1 variants in breast cancer.

PURPOSE: Estrogen receptor 1 (ESR1) mutations and fusions typically arise in patients with hormone receptor-positive breast cancer after aromatase inhibitor therapy, whereby ESR1 is constitutively activated in a ligand-independent manner. These variants can impact treatment response. Herein, we characterize ESR1 variants among molecularly profiled advanced breast cancers. METHODS: DNA next-generation sequencing (592-gene panel) data from 9860 breast cancer samples were retrospectively reviewed. Gene fusions were detected using the ArcherDx fusion assay or whole transcriptome sequencing (n = 344 and n = 4305, respectively). Statistical analyses included Chi-square and Fisher's exact tests. RESULTS: An ESR1 ligand-binding domain (LBD) mutation was detected in 8.6% of tumors evaluated and a pathogenic ESR1 fusion was detected in 1.6%. Most ESR1 LBD mutations/fusions were from estrogen receptor (ER)-positive samples (20.1% and 4.9%, respectively). The most common ESR1 LBD mutations included D538G (3.3%), Y537S (2.3%), and E380Q (1.1%) mutations. Among biopsy sites, ESR1 LBD mutations were most observed in liver metastases. Pathogenic ESR1 fusions were identified in 76 samples (1.6%) with 40 unique fusion partners. Evaluating co-alterations, ESR1 variant (mutation/fusion) samples more frequently expressed androgen receptor (78.0% vs 58.6, P < 0.0001) and less frequently immune checkpoint proteins than ESR1 wild-type (PD-1 20.0% vs 53.4, P < 0.05; immune cell PD-L1 10.0% vs 30.2, P < 0.0001). CONCLUSION: We have described one of the largest series of ESR1 fusions reported. ESR1 LBD mutations were commonly identified in ER-positive disease. Limited data exists regarding the clinical impact of ESR1 fusions, which could be an area for future therapeutic exploration.

Clinical outcomes associated with expression of aurora kinase and p53 family members in muscle-invasive bladder cancer.

Biomarkers are needed in muscle-invasive bladder cancer (MIBC). We previously reported that high tumor aurora kinase (AURK) A expression identifies patients with MIBC with poor prognosis. Aberrant p53 expression has also been associated with poor outcomes in MIBC, though to the best of our knowledge, co-expression rates of p53 and aurora kinases have not been previously described in MIBC. As aurora kinase and p53 family members may co-regulate each other, the present study investigated whether tumor p53 or p63 protein expression influenced the prognostic value of AURKA in a pilot study of 50 patients with MIBC treated with curative intent. Immunohistochemistry for AURKA, AURKB, p53 and p63 were performed on archival pre-treatment tumor specimens and correlated with clinical outcomes in patients with MIBC who received neoadjuvant chemotherapy (NAC) prior to cystectomy. Baseline p53 [hazard ratio (HR) 1.46; 95% confidence interval (CI)=0.55-3.9; P=0.448) and p63 (HR 2.02; 95% CI=0.51-8.1; P=0.313) protein expression did not predict for overall survival (OS). Low p53 protein expression did not correlate with high AURKA (φ=0.190) or AURKB (φ=0.075) expression. However, in tumors with low p53 expression (n=17), the presence of either high AURKA or AURKB expression levels predicted an increased risk for relapse (HR 27.1; 95% CI=2.7-270.1; P=0.005) and mortality (HR 14.9; 95% CI=2.3-95.6; P=0.004) compared to tumors with both low AURKA and AURKB levels. The relationship between p63 and AURKA/B expression levels was not tested due to the prevalence (80%) of high p63 expression in the present cohort. In tumors with low AURKA expression, p53 status did not predict for OS (HR 0.62; 95% CI 0.2-3.2; P=0.572). In multivariable analysis, only high baseline AURKA expression predicted for inferior OS (HR 4.9; 95% CI 1.7-14.1; P=0.003). To the best of our knowledge, the present study was the first to report co-expression of p53 and aurora kinase family members in MIBC, and although wild-type p53 may regulate the aurora kinases in preclinical models, the adverse prognostic value of tumor AURKA overexpression was independent from baseline tumor p53 protein expression in the present cohort. AURKA remains an important prognostic biomarker in patients with MIBC and warrants further evaluation in prospective studies to validate whether baseline AURKA can identify patients that are unlikely to benefit from standard of care with NAC.

Optimizing the management of chronic pain in sickle cell disease.

Chronic pain in sickle cell disease (SCD) refers to pain present on most days lasting over six months. It can start during childhood and the prevalence increases with age. By adulthood, over 55% of patients experience pain on over 50% of days; 29% reporting pain on 95% of days. The true prevalence of chronic pain in SCD is likely underappreciated as it is mostly managed at home. Patients with chronic pain and SCD frequently seek acute care for exacerbation of underlying chronic pain difficult to distinguish from their usual acute vaso-occlusive crises. When treating chronic pain in SCD, the challenge is distinguishing between non-SCD related etiologies versus chronic pain resulting from SCD pathophysiological processes. This distinction is important to delineate as it will drive appropriate management strategies. Chronic pain in SCD has profound consequences for the patient; is often associated with comorbid psychiatric illnesses (depression and anxiety), not dissimilar from other chronic pain syndromes. They may also experience challenges with sleep hygiene, various somatic symptoms, and chronic fatigue that impair quality of life. How best to treat chronic pain in SCD is not definitively established. Both acute and chronic pain in SCD is typically treated with opioids. Emerging data suggests that chronic opioid therapy (COT) is a suboptimal treatment strategy for chronic pain. This review will discuss the complexity of managing chronic pain in SCD; pain that may be dependent or independent of the underlying SCD diagnosis. We will also describe alternative treatment approaches to high-dose COT.

Ubiquitin-Activated Interaction Traps (UBAITs): Tools for Capturing Protein-Protein Interactions.

UBAITs (Ubiquitin-Activated Interaction Traps) are reagents that capitalize on the biochemistry of the ubiquitin system to covalently trap transient protein-protein interactions. UBAITs consist of an affinity-tagged protein of interest fused to a short linker followed by a C-terminal ubiquitin moiety. When charged in an E1- and E2-dependent manner, the C-terminal ubiquitin moiety of the UBAIT has the potential to form an amide linkage with lysine side chains of a protein that interacts transiently with the protein of interest, thereby covalently trapping the protein-protein interaction. The partner protein can then be identified by affinity-based purification of the UBAIT coupled with mass spectroscopy methods. While originally designed to identify substrates of ubiquitin ligases, UBAITs can, in principle, be used for identifying interaction partners of virtually any protein of interest. Here we describe methods for utilizing UBAITs in both cell-based and in vitro experiments.

Enzyme-substrate relationships in the ubiquitin system: approaches for identifying substrates of ubiquitin ligases.

Protein ubiquitylation is an important post-translational modification, regulating aspects of virtually every biochemical pathway in eukaryotic cells. Hundreds of enzymes participate in the conjugation and deconjugation of ubiquitin, as well as the recognition, signaling functions, and degradation of ubiquitylated proteins. Regulation of ubiquitylation is most commonly at the level of recognition of substrates by E3 ubiquitin ligases. Characterization of the network of E3-substrate relationships is a major goal and challenge in the field, as this expected to yield fundamental biological insights and opportunities for drug development. There has been remarkable success in identifying substrates for some E3 ligases, in many instances using the standard protein-protein interaction techniques (e.g., two-hybrid screens and co-immunoprecipitations paired with mass spectrometry). However, some E3s have remained refractory to characterization, while others have simply not yet been studied due to the sheer number and diversity of E3s. This review will discuss the range of tools and techniques that can be used for substrate profiling of E3 ligases.

Ubiquitin-Activated Interaction Traps (UBAITs) identify E3 ligase binding partners.

We describe a new class of reagents for identifying substrates, adaptors, and regulators of HECT and RING E3s. UBAITs (Ubiquitin-Activated Interaction Traps) are E3-ubiquitin fusion proteins and, in an E1- and E2-dependent manner, the C-terminal ubiquitin moiety forms an amide linkage to proteins that interact with the E3, enabling covalent co-purification of the E3 with partner proteins. We designed UBAITs for both HECT (Rsp5, Itch) and RING (Psh1, RNF126, RNF168) E3s. For HECT E3s, trapping of interacting proteins occurred in vitro either through an E3 thioester-linked lariat intermediate or through an E2 thioester intermediate, and both WT and active-site mutant UBAITs trapped known interacting proteins in yeast and human cells. Yeast Psh1 and human RNF126 and RNF168 UBAITs also trapped known interacting proteins when expressed in cells. Human RNF168 is a key mediator of ubiquitin signaling that promotes DNA double-strand break repair. Using the RNF168 UBAIT, we identify H2AZ--a histone protein involved in DNA repair--as a new target of this E3 ligase. These results demonstrate that UBAITs represent powerful tools for profiling a wide range of ubiquitin ligases.

Bacterial persistence increases as environmental fitness decreases.

Since persister cells cause chronic infections and since Escherichia coli toxin MqsR increases persisters, we used protein engineering to increase the toxicity of MqsR to gain insights into persister cell formation. Through two amino acid replacements that increased the stability of MqsR, toxicity and persistence were increased. A whole-transcriptome study revealed that the MqsR variant increased persistence by repressing genes for acid resistance, multidrug resistance and osmotic resistance. Corroborating these microarray results, deletion of rpoS, as well as the genes that the master stress response regulator RpoS controls, increased persister formation dramatically to the extent that nearly the whole population became persistent. Furthermore, wild-type cells stressed by prior treatment to acid or hydrogen peroxide increased persistence 12 000-fold. Whole-transcriptome analyses of persister cells generated by two different methods (wild-type cells pretreated with hydrogen peroxide and the rpoS deletion) corroborated the importance of suppressing RpoS in persister cell formation. Therefore, the more toxic MqsR increases persistence by decreasing the ability of the cell to respond to antibiotic stress through its RpoS-based regulation of acid resistance, multidrug resistance and osmotic resistance systems.

Antibiotic selection leads to inadvertent selection of nfxC-type phenotypic mutants in Pseudomonas aeruginosa.

Spontaneous nfxC-type phenotypic mutants of Pseudomonas aeruginosa are characterized by chloramphenicol resistance and increased expression of the multidrug efflux pump, MexEF-OprN. These mutants frequently arise during the propagation of bacteria in laboratory conditions. This study shows that some of the discrepancies in transcriptomic/phenotypic profiles of two rsmA mutants, PAZH13 (rsmA mutant in strain PAO1) and PAKΔrsmA, are due to the nfxC-type nature of PAZH13. This indicates that awareness of the possible occurrence of these spontaneous nfxC-type phenotypes is necessary. Screening of mexE levels could provide clarity when an nfxC-type phenotype is suspected.

MexT modulates virulence determinants in Pseudomonas aeruginosa independent of the MexEF-OprN efflux pump.

In the human pathogen Pseudomonas aeruginosa, the LysR-family regulator MexT modulates the induction of the tripartite MexEF-OprN resistance nodulation-division multi-drug efflux system resulting in increased resistance to diverse antibiotics. The MexEF-OprN system is normally quiescent in wild-type cells, but is highly induced in nfxC-type phenotypic mutants in a MexT dependent manner. In addition to antibiotic resistance, induction of mexEF-oprN in nfxC-type mutants has been linked to reduced levels of homoserine lactone-dependent virulence traits, including pyocyanin, elastase, rhamnolipids and PQS and to reduced expression of type three secretion effector proteins. In this study, MexT is overexpressed in wild-type PAO1 and an isogenic mexEF deletion mutant to determine if MexT regulates diverse virulence phenotypes dependent or independent of MexEF-OprN. It is shown that MexT regulates type three secretion, pyocyanin production and early surface attachment independent of MexEF-OprN. In contrast, MexT modulation of the expression of the virulence genes rhlA, lasB and hcnB is dependent on MexEF-OprN, which apparently mediates these effects via efflux of cell-signaling intermediates. The data presented demonstrates that MexT may play a more global role in modulating P. aeruginosa virulence than previously reported and suggests that MexT regulates diverse targets that mediate phenotypic alterations independent of MexEF-OprN.