BRD4 inhibition sensitizes diffuse large B-cell lymphoma cells to ferroptosis.

Diffuse large B-cell lymphoma (DLBCL), the most common form of non-Hodgkin lymphoma, is characterized by an aggressive clinical course. In approximately one-third of patients with DLBCL, first-line multiagent immunochemotherapy fails to produce a durable response. Molecular heterogeneity and apoptosis resistance pose major therapeutic challenges in DLBCL treatment. To circumvent apoptosis resistance, the induction of ferroptosis might represent a promising strategy for lymphoma therapy. In this study, a compound library, targeting epigenetic modulators, was screened to identify ferroptosis-sensitizing drugs. Strikingly, bromodomain and extra-terminal domain (BET) inhibitors sensitized cells of the germinal center B-cell-like (GCB) subtype of DLBCL to ferroptosis induction and the combination of BET inhibitors with ferroptosis inducers, such as dimethyl fumarate or RSL3, synergized in the killing of DLBCL cells in vitro and in vivo. On the molecular level, the BET protein BRD4 was found to be an essential regulator of ferroptosis suppressor protein 1 expression and thus to protect GCB-DLBCL cells from ferroptosis. Collectively, we identified and characterized BRD4 as an important player in ferroptosis suppression in GCB-DLBCL and provide a rationale for the combination of BET inhibitors with ferroptosis-inducing agents as a novel therapeutic approach for DLBCL treatment.

Inhibition of glutaminase-1 in DLBCL potentiates venetoclax-induced antitumor activity by promoting oxidative stress.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma in adults, but first-line immunochemotherapy fails to produce a durable response in about one-third of the patients. Because tumor cells often reprogram their metabolism, we investigated the importance of glutaminolysis, a pathway converting glutamine to generate energy and various metabolites, for the growth of DLBCL cells. Glutaminase-1 (GLS1) expression was robustly detected in DLBCL biopsy samples and cell lines. Both pharmacological inhibition and genetic knockdown of GLS1 induced cell death in DLBCL cells regardless of their subtype classification, whereas primary B cells remained unaffected. Interestingly, GLS1 inhibition resulted not only in reduced levels of intermediates of the tricarboxylic acid cycle but also in a strong mitochondrial accumulation of reactive oxygen species. Supplementation of DLBCL cells with α-ketoglutarate or with the antioxidant α-tocopherol mitigated oxidative stress and abrogated cell death upon GLS1 inhibition, indicating an essential role of glutaminolysis in the protection from oxidative stress. Furthermore, the combination of the GLS1 inhibitor CB-839 with the therapeutic BCL2 inhibitor ABT-199 not only induced massive reactive oxygen species (ROS) production but also exhibited highly synergistic cytotoxicity, suggesting that simultaneous targeting of GLS1 and BCL2 could represent a novel therapeutic strategy for patients with DLBCL.

Regional, age-dependent, and genotype-dependent differences in ventricular action potential duration and activation time in 410 Langendorff-perfused mouse hearts.

Although numerous studies have reported the effects of genetic alterations on murine electrophysiology, the range of normal values for ventricular activation, repolarization, and arrhythmias in mouse hearts is not known. We analyzed right ventricular (RV), left ventricular (LV), and septal activation times, monophasic action potential durations (APD), and right ventricular effective refractory periods during spontaneous rhythm, induced AV nodal block, right ventricular pacing (100-300 ms paced cycle length), and programmed stimulation in 410 beating, Langendorff-perfused, wild-type mouse hearts of CD1, DBAC3H, FVBN, C57/Bl6, and hybrid backgrounds (age 203 +/- 132 days). Action potential duration was longer at longer cycle lengths. LV-APD prolonged more than RV-APD, resulting in an increased heterogeneity of APD at longer pacing cycle lengths. Higher heart weight/body weight ratio and DBAC3H and FVB/N backgrounds were associated with long APD, C57Bl/6 background was associated with short APD. Activation times were longer in older hearts. There were no clear-cut sex-dependent APD differences. Sustained spontaneous arrhythmias occurred in 1% of hearts, non-sustained arrhythmias in 18%. Induction of AV block and C57Bl/6 genetic background were associated with spontaneous arrhythmias. Programmed stimulation induced arrhythmias in 51% of hearts. Inducible arrhythmias were associated with advanced age and shorter refractory periods. Ventricular APD in beating mouse hearts show rate- and site-dependent changes comparable to man and large animals. Bradycardia provokes spontaneous arrhythmias in mouse heart, while age-dependent conduction slowing and short refractory periods predispose to induced arrhythmias. Genetic background influences repolarization and arrhythmogenesis. These findings provide systematic data for the design and interpretation of arrhythmia studies in murine disease models.

Load-reducing therapy prevents development of arrhythmogenic right ventricular cardiomyopathy in plakoglobin-deficient mice.

OBJECTIVES: We used a murine model of arrhythmogenic right ventricular cardiomyopathy (ARVC) to test whether reducing ventricular load prevents or slows development of this cardiomyopathy. BACKGROUND: At present, no therapy exists to slow progression of ARVC. Genetically conferred dysfunction of the mechanical cell-cell connections, often associated with reduced expression of plakoglobin, is thought to cause ARVC. METHODS: Littermate pairs of heterozygous plakoglobin-deficient mice (plako(+/-)) and wild-type (WT) littermates underwent 7 weeks of endurance training (daily swimming). Mice were randomized to blinded load-reducing therapy (furosemide and nitrates) or placebo. RESULTS: Therapy prevented training-induced right ventricular (RV) enlargement in plako(+/-) mice (RV volume: untreated plako(+/-) 136 ± 5 µl; treated plako(+/-) 78 ± 5 µl; WT 81 ± 5 µl; p < 0.01 for untreated vs. WT and untreated vs. treated; mean ± SEM). In isolated, Langendorff-perfused hearts, ventricular tachycardias (VTs) were more often induced in untreated plako(+/-) hearts (15 of 25), than in treated plako(+/-) hearts (5 of 19) or in WT hearts (6 of 21, both p < 0.05). Epicardial mapping of the RV identified macro-re-entry as the mechanism of ventricular tachycardia. The RV longitudinal conduction velocity was reduced in untreated but not in treated plako(+/-) mice (p < 0.01 for untreated vs. WT and untreated vs. treated). Myocardial concentration of phosphorylated connexin43 was lower in plako(+/-) hearts with VTs compared with hearts without VTs and was reduced in untreated plako(+/-) compared with WT (both p < 0.05). Plako(+/-) hearts showed reduced myocardial plakoglobin concentration, whereas ß-catenin and N-cadherin concentration was not changed. CONCLUSIONS: Load-reducing therapy prevents training-induced development of ARVC in plako(+/-) mice.