BET Inhibition-Induced GSK3β Feedback Enhances Lymphoma Vulnerability to PI3K Inhibitors

The phosphatidylinositol 3 kinase (PI3K)-glycogen synthase kinase \u3b2 (GSK3\u3b2) axis plays a central role in MYC-driven lymphomagenesis, and MYC targeting with bromodomain and extraterminal protein family inhibitors (BETi) is a promising treatment strategy in lymphoma. In a high-throughput combinatorial drug screening experiment, BETi enhance the antiproliferative effects of PI3K inhibitors in a panel of diffuse large B cell lymphoma (DLBCL) and Burkitt lymphoma cell lines. BETi or MYC silencing upregulates several PI3K pathway genes and induces GSK3\u3b2 S9 inhibitory phosphorylation, resulting in increased \u3b2-catenin protein abundance. Furthermore, BETi or MYC silencing increases GSK3\u3b2 S9 phosphorylation levels and \u3b2-catenin protein abundance through downregulating the E2 ubiquitin conjugating enzymes UBE2C and UBE2T. In a mouse xenograft DLBCL model, BETi decrease MYC, UBE2C, and UBE2T and increase phospho-GSK3\u3b2 S9 levels, enhancing the anti-proliferative effect of PI3K inhibitors. Our study reveals prosurvival feedbacks induced by BETi involving GSK3\u3b2 regulation, providing a mechanistic rationale for combination strategies. In this study, Derenzini et al. demonstrate that BET inhibitors enhance lymphoma vulnerability to PI3K inhibitors by inducing GSK3\u3b2 feedback in a MYC-dependent manner and by downregulating E2-ubiquitin conjugating enzymes, which further enhance the feedback. These data provide the rationale for combining BET and PI3K inhibitors in lymphoma therapy

The quantitation of Trypanosoma congolense in calves. 1. Hematological changes

Anemia due to Trypanosoma congolense infection in neonatal and 6-month-old calves was hemolytic and moderately responsive. The red cells were macrocytic and normochromic in both groups of calves and the anemia was milder in infected neonatal calves compared to the 6-month-old calves. The neonatal calves maintained normal white blood cell counts and developed a lymphocytosis at two weeks of infection. The six-month-old calves developed mild neutropenic leukopenia at one week post-infection without lymphocytosis. Plasma iron turnover rates were not different between infected and control calves in either age group at the second week of infection but both groups of infected calves had higher turnover rates at the sixth week of infection. The mean percent of radioiron uptake into peripheral blood red cells was similar in the controls and infected calves, but the rate of appearance of labelled red cells in the peripheral blood was more rapid in the infected calves. Radioiron uptake was depressed in the infected six-month-old calves during second week of infection but not in the sixth week. Apparent plasma volumes were increased in both groups of calves at both time periods

A cytofluorometric method of counting trypanosomes

A rapid automated method of counting trypanosomes was developed using cytofluorometric methods. Organisms were vitally stained with acridine orange, and DEAE cellulose separated T. congolese were accurately counted at all concentrations. T. congolense in heparinized whole bovine blood were also counted accurately at levels greater than 1,000 per microliter. At lower levels, small fluorescent particles in whole blood interfered with the counts

The quantitation of trypanosoma congolense in calves. III. A quantitative comparison of trypanosomes in jugular vein and microvasculature and tests of dispersing agents

Hemocytometer and cytofluorometric methods were used to count trypanosomes in the blood of neonatal and six-month-old calves infected with T. congolense. The correlation between these methods was highest in the six-month-old calves because their parasitemia was greater and the accuracy of cytofluorometric counting is reduced below 1,000 trypanosomes per microliter of blood. Fine needle aspiration biopsies showed many trypanosomes marginated in cerebral cortical capillaries, but few in muscle or liver. Quantitatively, the cerebral capillaries in the infected six-month-old calves were significantly wider than their controls with fewer endothelial nuclei suggesting capillary dilatation. There were fewer trypanosomes and no significant changes in capillary width in the neonatal calves. Several drugs were tried for activity in dispersing marginated trypanosomes into axial flow. Berenil and dexamethasone had a significant effect on trypanosome counts in blood and tissue whereas, epinephrine caused a fleeting increase in parasitemia. Hetastarch, dextran and cyclophosphamide had no specific effect. Fine needle aspiration bipsies of brain, thirty minutes after Berenil injection showed that the capillaries were cleared of trypanosomes

The quantitation of Trypanosoma congolense in calves. Iv. In vitro culture of myeloid and erythroid marrow cells

Myeloid and erythroid cultures were prepared from bone marrow aspirates taken from control and T. congolense infected neonatal and six-month-old calves. The aspirates obtained from the infected animals were more cellular than the controls and it was found that the removal of adherent cells prior to Ficoll separation improved the sensitivity of the cultures to colony stimulating factors and removed the background connective tissue cells and megakaryocytes from the resulting colonies. There was an apparent decrease in the number of myeloid colonies from the infected six-month-old calves, but an initial increase in myeloid colonies in the infected neonatal calves compared to their controls. There were no significant differences between infected and control calves for the numbers of erythroid colonies produced, but the degree of maturation as measured by hemoglobinization was decreased in two infected six-month-old calves at week 13 of infection

The quantitation of Trypanosoma congolense in calves. II. Biochemical change

Changes in serum electrolytes, osmolality, total proteins and protein fractions, lipid levels and organ function tests were determined in neonatal and 6-month-old calves infected with T. congolense TREU 112 and control calves. There were no consistent changes in electrolytes or osmolality for either age group of calves. There was a decrease in total serum protein in the infected 6-month-old calves, caused by decreases in the albumin and beta 2-globulin fractions. The total serum protein level remained constant in the infected neonatal calves, but there was a significant decrease in the albumin fraction which was compensated for by an increase in alpha-globulin and gamma-globulin fractions. Total serum lipids and cholesterol were decreased and triglyceride levels and erythrocyte phospholipid levels were significantly elevated in both age groups of infected calves. Tests of adrenal and hepatic function were not significantly different in infected calves compared to control calves of the same age group. Thyroid function tests showed that T3 was significantly elevated in infected 6-month-old calves, whereas T4 was significantly decreased in infected neonatal calves