The clinical development of drug combinations is typically achieved through trial-and-error or via insight gained through a detailed molecular understanding of dysregulated signaling pathways in a specific cancer type. Unbiased small-molecule combination (matrix) screening represents a high-throughput means to explore hundreds and even thousands of drug-drug pairs for potential investigation and translation. Here, we describe a high-throughput screening platform capable of testing compounds in pairwise matrix blocks for the rapid and systematic identification of synergistic, additive, and antagonistic drug combinations. We use this platform to define potential therapeutic combinations for the activated B-cell-like subtype (ABC) of diffuse large B-cell lymphoma (DLBCL). We identify drugs with synergy, additivity, and antagonism with the Bruton's tyrosine kinase inhibitor ibrutinib, which targets the chronic active B-cell receptor signaling that characterizes ABC DLBCL. Ibrutinib interacted favorably with a wide range of compounds, including inhibitors of the PI3K-AKT-mammalian target of rapamycin signaling cascade, other B-cell receptor pathway inhibitors, Bcl-2 family inhibitors, and several components of chemotherapy that is the standard of care for DLBCL.translational research | PCI-32765 | Imbruvica

Prolonged exposure of rodent ␤-cells to combinations of cytokines induces the inducible form of nitric oxide synthase (iNOS) expression and Fas expression, nitric oxide (NO) production, and cell death. It also induces the expression of potential "defense" genes, such as manganese superoxide dismutase (MnSOD) and heat shock protein (hsp) 70. NO is a radical with multifaceted actions. Recent studies have shown that NO, in addition to having cytotoxic actions, may regulate gene transcription. It remains unclear whether NO mediates cytokine-induced gene expression and subsequent ␤-cell death. Previous studies using NO synthase blockers yielded conflicting results, which may be due to nonspecific effects of these agents. In this study, we examined the effects of cytokines on gene expression, determined by reverse transcriptase-polymerase chain reaction (RT-PCR), and viability, determined by nuclear dyes, of pancreatic islets or fluorescence-activated cell sorter (FACS)-purified ␤-cells isolated from iNOS knockout mice (iNOS -/ -, background C57BL/6x129SvEv) or their respective controls (C57BL/6x129SvEv). The combination of cytokines used was interleukin-1␤ (50 U/ml) plus ␥-interferon (1,000 U/ml) plus tumor necrosis factor-␣ (1,000 U/ml). The lack of cytokine-induced iNOS activity in the iNOS -/-islet cells was confirmed by RT-PCR and nitrite determination. Cytokines induced a >3-fold increase in Fas and MnSOD mRNA expression in wild-type (WT) and iNOS -/-islets. On the other hand, hsp 70 was induced in WT but not in iNOS -/-islets. Prolonged (6-9 days) exposure of WT islets to cytokines leads to an 80-90% decrease in islet cell viability, whereas viability decreased by only 10-30% in iNOS -/-islet cells. To determine the mode of cytokine-induced cell death, FACS-purified ␤-cells were exposed to the same cytokines. After 9 days, the apoptosis index was similarly increased in WT (39 ± 3%) and iNOS -/-(33 ± 4%) ␤-cells. On the other hand, cytokines increased necrosis in WT (20 ± 4%) but not in iNOS -/-(7 ± 3%) ␤-cells. From these data, we concluded that 1) NO is required for cytokine-induced hsp 70 mRNA expression but not for Fas and MnSOD expression, 2) cytokines induce both apoptosis and necrosis in mouse ␤-cells, and 3) cytokine-induced apoptosis is mostly NO-independent, whereas necrosis requires NO formation. Diabetes

In the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL), NF-κB activity is essential for viability of the malignant cells and is sustained by constitutive activity of IκB kinase (IKK) in the cytoplasm. Here, we report an unexpected role for the bromodomain and extraterminal domain (BET) proteins BRD2 and BRD4 in maintaining oncogenic IKK activity in ABC DLBCL. IKK activity was reduced by small molecules targeting BET proteins as well as by genetic knockdown of BRD2 and BRD4 expression, thereby inhibiting downstream NF-κB-driven transcriptional programs and killing ABC DLBCL cells. Using a high-throughput platform to screen for drug-drug synergy, we observed that the BET inhibitor JQ1 combined favorably with multiple drugs targeting B-cell receptor signaling, one pathway that activates IKK in ABC DLBCL. The BTK kinase inhibitor ibrutinib, which is in clinical development for the treatment of ABC DLBCL, synergized strongly with BET inhibitors in killing ABC DLBCL cells in vitro and in a xenograft mouse model. These findings provide a mechanistic basis for the clinical development of BET protein inhibitors in ABC DLBCL, particularly in combination with other modulators of oncogenic IKK signaling.cancer therapy | drug synergism T he activated B-cell like subtype (ABC) of diffuse large B-cell lymphoma (DLBCL) has an aggressive clinical course compared with other DLBCL subtypes, with an overall survival of only 40% with current multidrug chemotherapies (1, 2). In recent years, detailed genetic and functional genomic analyses unveiled the key oncogenic mechanisms that sustain the aggressiveness of this subtype. Notably, all ABC DLBCLs rely on constitutive NF-κB activation for survival (3). Various oncogenic events converge on NF-κB to promote lymphomagenesis. About 10% of ABC DLBCL tumors have activating mutations affecting CARD11, a scaffolding protein required for the assembly of the CARD11-BCL10-MALT1 (CBM) complex. Mutant CARD11 proteins spontaneously generate cytoplasmic CBM aggregates that drive constitutive NF-κB activity (4). ABC DLBCL tumors with wild-type CARD11 use other mechanisms to activate NF-κB. In 20% of cases, signals emanating from the B-cell receptor (BCR) are augmented by somatically acquired mutations targeting the BCR subunits CD79A and CD79B (5). In 39% of ABC DLBCLs, NF-κB is activated by somatic mutations targeting MyD88, an adaptor protein in the Toll-like receptor (TLR) pathway (6). In normal B cells, stimulus-dependent engagement of the BCR and MyD88 pathways activates IκB kinase (IKK), which phosphorylates ΙκBα, thereby promoting its degradation and allowing NF-κB transcription factors to enter the nucleus and activate a distinctive set of target genes. By contrast, ABC DLBCL cells become addicted to constitutive activity of IKK such that its inhibition is lethal (7). Recent therapeutic efforts to target oncogenic signaling in ABC DLBCL have focused on ibrutinib, a selective inhibitor of the kinase BTK that transmits signals from the BCR to the NF-κB pathway (5)...