BRD9 degraders as chemosensitizers in acute leukemia and multiple myeloma.

Bromodomain-containing protein 9 (BRD9), an essential component of the SWI/SNF chromatin remodeling complex termed ncBAF, has been established as a therapeutic target in a subset of sarcomas and leukemias. Here, we used novel small molecule inhibitors and degraders along with RNA interference to assess the dependency on BRD9 in the context of diverse hematological malignancies, including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and multiple myeloma (MM) model systems. Following depletion of BRD9 protein, AML cells undergo terminal differentiation, whereas apoptosis was more prominent in ALL and MM. RNA-seq analysis of acute leukemia and MM cells revealed both unique and common signaling pathways affected by BRD9 degradation, with common pathways including those associated with regulation of inflammation, cell adhesion, DNA repair and cell cycle progression. Degradation of BRD9 potentiated the effects of several chemotherapeutic agents and targeted therapies against AML, ALL, and MM. Our findings support further development of therapeutic targeting of BRD9, alone or combined with other agents, as a novel strategy for acute leukemias and MM.

Chemoproteomics Enabled Discovery of Selective Probes for NuA4 Factor BRD8.

Bromodomain-containing proteins frequently reside in multisubunit chromatin complexes with tissue or cell state-specific compositions. Recent studies have revealed tumor-specific dependencies on the BAF complex bromodomain subunit BRD9 that are a result of recurrent mutations afflicting the structure and composition of associated complex members. To enable the study of ligand engaged complex assemblies, we established a chemoproteomics approach using a functionalized derivative of the BRD9 ligand BI-9564 as an affinity matrix. Unexpectedly, in addition to known interactions with BRD9 and associated BAF complex proteins, we identify a previously unreported interaction with members of the NuA4 complex through the bromodomain-containing subunit BRD8. We apply this finding, alongside a homology-model-guided design, to develop chemical biology approaches for the study of BRD8 inhibition and to arrive at first-in-class selective and cellularly active probes for BRD8. These tools will empower further pharmacological studies of BRD9 and BRD8 within respective BAF and NuA4 complexes.

Recurrent ubiquitin B silencing in gynecological cancers establishes dependence on ubiquitin C.

Transcriptional repression of ubiquitin B (UBB) is a cancer-subtype-specific alteration that occurs in a substantial population of patients with cancers of the female reproductive tract. UBB is 1 of 2 genes encoding for ubiquitin as a polyprotein consisting of multiple copies of ubiquitin monomers. Silencing of UBB reduces cellular UBB levels and results in an exquisite dependence on ubiquitin C (UBC), the second polyubiquitin gene. UBB is repressed in approximately 30% of high-grade serous ovarian cancer (HGSOC) patients and is a recurrent lesion in uterine carcinosarcoma and endometrial carcinoma. We identified ovarian tumor cell lines that retain UBB in a repressed state, used these cell lines to establish orthotopic ovarian tumors, and found that inducible expression of a UBC-targeting shRNA led to tumor regression, and substantial long-term survival benefit. Thus, we describe a recurrent cancer-specific lesion at the level of ubiquitin production. Moreover, these observations reveal the prognostic value of UBB repression and establish UBC as a promising therapeutic target for ovarian cancer patients with recurrent UBB silencing.

A history of corticosterone exposure regulates fear extinction and cortical NR2B, GluR2/3, and BDNF.

A history of exposure to stressors may be a predisposing factor for developing posttraumatic stress disorder (PTSD) after trauma. Extinction of conditioned fear appears to be impaired in PTSD, but the consequences of prior stress or excess glucocorticoid exposure for extinction learning are not known. We report that prior chronic exposure to the stress hormone, corticosterone (CORT), decreases endogenous CORT secretion upon context reexposure and impairs extinction after contextual fear conditioning in rats, while leaving fear memory acquisition and expression intact. Posttraining administration of the glucocorticoid receptor (GR) antagonist, RU38486, partially mimicked prior CORT exposure effects on freezing during fear extinction training. Extinction of conditioned fear is an active learning process thought to involve glutamatergic targets--including specific NMDA and AMPA receptor subunits--in the ventromedial prefrontal cortex (vmPFC), which includes the prelimbic, infralimbic, and medial orbitofrontal cortices. After CORT exposure, decreases in the NMDA receptor NR2B subunit and AMPA receptor subunits, GluR2/3, as well as brain-derived neurotrophic factor, were detected in cortical regions, but not dorsal hippocampus (CA1). Receptor subunit expression levels in the vmPFC correlated with freezing during training. In addition, prior CORT selectively decreased sucrose preference, consistent with established models of anhedonia and with blunted affect in PTSD. Together, these data suggest a cellular mechanism by which chronically elevated glucocorticoid exposure--as may be experienced during repeated exposure to stressors--interferes with the neural systems that modulate behavioral flexibility and may thereby contribute to psychopathological fear states.

Regionally specific regulation of ERK MAP kinase in a model of antidepressant-sensitive chronic depression.

BACKGROUND: Elevated phosphorylation of neurotrophin-regulated transcription factors, such as cyclic adenosine monophosphate (cAMP)-response element binding protein (CREB), in the hippocampus has been proposed as a common mediator of antidepressant (ADT) efficacy in otherwise naive rodents. The intracellular factors by which ADTs and glucocorticoids, causal factors in depression, regulate depression-like behavior remain unclear, but extracellular signal-regulated kinase 1/2 (ERK1/2), upstream of CREB, is a likely candidate. METHODS: We explored the long-term consequences of glucocorticoid exposure and subsequent ADT treatment in a novel model of chronic depression. Motivated behaviors, immobility during tail suspension, and ERK1/2, known to be required for behavioral response to ADTs, were quantified. RESULTS: Chronic corticosterone (CORT) increased immobility, decreased responding in an operant conditioning task of motivation, and selectively reduced phosphorylated ERK1/2 (pERK1/2) in the dentate gyrus. Behavioral and biochemical measures were restored to baseline by amitriptyline (AMI) treatment. Corticosterone regulated pERK1/2 on a time course that paralleled increases in heat shock proteins associated with depression and decreased tyrosine kinase receptor B (trkB) phosphorylation. Chronic AMI also produced regionally dissociable effects on pERK1/2 in CA1/CA3, amygdala, and striatum, but not prefrontal cortex. CONCLUSIONS: Antidepressant efficacy in a motivational task and behavioral despair assay are associated with altered limbic pERK1/2, including restored pERK1/2 in the dentate gyrus after stress-related insult.

"Waste not, want not": determining the optimal priming volume for intravenous insulin infusions.

BACKGROUND: Insulin adsorbs to plastics used for intravenous (IV) tubing. As a result, clinical IV insulin infusion procotols advise an initial priming volume of up to 50 mL, which may be wasteful-especially since most institutions use 100-mL IV solution bags. In this brief report, we sought to determine the optimal priming volume required for clinical IV insulin infusions. METHODS: One hundred units of regular human insulin was dissolved into 100 mL of 0.9% NaCl. Employing a standard polypropylene infusion set, a priming infusion was started. At 10- mL intervals, from 0 to 50 mL, effluent was collected directly into glass vials. After dilution (1:10,000) using a proprietary buffer, insulin concentrations were then measured using a double antibody radioimmunoassay. Twenty individually prepared insulin bags were tested in this manner. RESULTS: Insulin levels without prime were 15.8% [95% confidence interval (CI), 9.1-22.6%] lower than insulin levels following 50 mL of prime (designated as "maximal values"). After a priming volume of 10 mL, insulin adsorption losses fell to a marginally significant 6.6% (95% CI, 0.1-13.1%). Following 20 mL of prime, insulin concentrations were indistinguishable from maximal values (3.4% loss, 95% CI, -0.2% to 7.1%). CONCLUSIONS: For standard IV insulin infusions, a priming volume of 20 mL is sufficient to minimize the effect of insulin adsorption losses to IV lines. Priming volumes exceeding 20 mL are wasteful, increase costs, and generate unnecessary work for nurses and pharmacists.