Bromodomain and extraterminal (BET) bromodomain (BRD) proteins are epigenetic readers that bind to acetylated lysine residues on chromatin, acting as co-activators or co-repressors of gene expression. BRD2 and BRD4, members of the BET family, are significantly increased in glioblastoma multiforme (GBM), the most common primary adult brain cancer. OTX015 (MK-8628), a novel BRD2/3/4 inhibitor, is under evaluation in dose-finding studies in solid tumors, including GBM. We investigated the pharmacologic characteristics of OTX015 as a single agent and combined with targeted therapy or conventional chemotherapies in glioblastoma cell lines. OTX015 displayed higher antiproliferative effects compared to its analog JQ1, with GI 50 values of approximately 0.2 mM. In addition, C-MYC and CDKN1A mRNA levels increased transiently after 4 h-exposure to OTX015, while BRD2, SESN3, HEXIM-1, HIST2H2BE, and HIST1H2BK were rapidly upregulated and sustained after 24 h. Studies in three additional GBM cell lines supported the antiproliferative effects of OTX015. In U87MG cells, OTX015 showed synergistic to additive activity when administered concomitant to or before SN38, temozolomide or everolimus. Single agent oral OTX015 significantly increased survival in mice bearing orthotopic or heterotopic U87MG xenografts. OTX015 combined simultaneously with temozolomide improved mice survival over either single agent. The passage of OTX015 across the blood-brain barrier was demonstrated with OTX015 tumor levels 7 to 15-fold higher than in normal tissues, along with preferential binding of OTX015 to tumor tissue. The significant antitumor effects seen with OTX015 in GBM xenograft models highlight its therapeutic potential in GBM patients, alone or combined with conventional chemotherapies.Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary brain tumor in humans, accounting for 52% of all functional tissue brain tumors and 20% of all intracranial tumors. 1,2 Standard of care is typically surgery followed by radiotherapy with the DNA alkylating agent temozolomide. 3 Median survival after diagnosis is only 15 months, leaving a large unmet medical need. 4 Alternative therapeutic approaches have focused on targeted therapies against processes known to play a major role in GBM development, such as blocking angiogenesis with the anti-VEGF agent, bevacizumab. 5 The topoisomerase inhibitor irinotecan which blocks DNA replication causing cell death has also shown hints of activity against malignant glioma in the clinical setting. 6,7 Key words: BET inhibitor, OTX015 (MK-8628), glioblastoma, blood brain barrier, combination studies. Abbreviations: 95% CI: 95% confidence interval; BET: bromodomain and extraterminal; BID: bi-daily; BRD: bromodomain; CI: combination index; E max : drug efficacy as percent cell proliferation inhibition at the highest dose; GBM: glioblastoma multiforme; GI 50 : drug concentration at which cell proliferation is reduced by half; IP: intraperitoneal; MTX: methotrexate; po: per orally; VEGF: vascula...

Controlled cell death, or apoptosis, occurs in response to many different environmental stimuli. The apoptotic cascade that occurs within the cell in response to these cues leads to morphological and biochemical changes that trigger the dismantling and packaging of the cell. Caspases are a family of cysteine-dependent aspartate-directed proteases that play an integral role in the cascade that leads to apoptosis. Caspases are grouped as either initiators or effectors of apoptosis, depending on where they enter the cell death process. Prior to activation, initiator caspases are present as monomers that must dimerize for full activation whereas effector caspases are present as dimeric zymogens that must be processed for full activation. The stability of the dimer may be due predominately to the interactions in the dimer interface as each caspase has unique properties in this region that lend to its specific mode of activation. Moreover, dimerization is responsible for active site formation because both monomers contribute residues that enable the formation of a fully functional active site. Overall, dimerization plays a key role in the ability of caspases to form fully functional proteases.

Highlights d Distinct transcriptional programs characterize prostate CSC and bulk tumor cells d BRD4 promotes mitochondrial biogenesis and metabolic plasticity in prostate CSCs d Mitochondrial fission enables asymmetric division and prostate CSC self-renewal d BRD4 inhibitors block mitochondrial fission and hinder selfrenewal of prostate CSCs

Cytotoxic approaches to killing tumor cells, such as chemotherapeutic agents, γ-irradiation, suicide genes or immunotherapy, have been shown to induce cell death through apoptosis. The intrinsic apoptotic pathway is activated following treatment with cytotoxic drugs, and these reactions ultimately lead to the activation of caspases, which promote cell death in tumor cells. In addition, activation of the extrinsic apoptotic pathway with death-inducing ligands leads to an increased sensitivity of tumor cells toward cytotoxic stimuli, illustrating the interplay between the two cell death pathways. In contrast, tumor resistance to cytotoxic stimuli may be due to defects in apoptotic signaling. As a result of their importance in killing cancer cells, a number of apoptotic molecules are implicated in cancer therapy. The knowledge gleaned from basic research into apoptotic pathways from cell biological, structural, biochemical, and biophysical approaches can be used in strategies to develop novel compounds that eradicate tumor cells. In addition to current drug targets, research into molecules that activate procaspase-3 directly may show the direct activation of the executioner caspase to be a powerful therapeutic strategy in the treatment of many cancers.

The native ensemble of caspases is described globally by a complex energy landscape where the binding of substrate selects for the active conformation, whereas targeting an allosteric site in the dimer interface selects an inactive conformation that contains disordered active-site loops. Mutations and posttranslational modifications stabilize high-energy inactive conformations, with mostly formed, but distorted, active sites. To examine the interconversion of active and inactive states in the ensemble, we used detection of related solvent positions to analyze 4,995 waters in 15 highresolution (<2.0 Å) structures of wild-type caspase-3, resulting in 450 clusters with the most highly conserved set containing 145 water molecules. The data show that regions of the protein that contact the conserved waters also correspond to sites of posttranslational modifications, suggesting that the conserved waters are an integral part of allosteric mechanisms. To test this hypothesis, we created a library of 19 caspase-3 variants through saturation mutagenesis in a single position of the allosteric site of the dimer interface, and we show that the enzyme activity varies by more than four orders of magnitude. Altogether, our database consists of 37 high-resolution structures of caspase-3 variants, and we demonstrate that the decrease in activity correlates with a loss of conserved water molecules. The data show that the activity of caspase-3 can be fine-tuned through globally desolvating the active conformation within the native ensemble, providing a mechanism for cells to repartition the ensemble and thus fine-tune activity through conformational selection.C aspase function in cell development and cell death results from a continuum of enzyme activity, in which an as-yetundefined activity threshold is required for cell death. At subthreshold levels, caspase activity is important for a variety of physiological reactions (referred to as adaptive responses), including remodeling the cytoplasm (1), cell differentiation (2), neuron pruning (3), receptor endocytosis (4), macrophage function (5), and development of the eye lens (6) and inner ear (7). The roles of caspases in apoptosis are well known, but their roles in adaptive responses are less clear, particularly in regard to how cells set the threshold of caspase activity to limit apoptosis while ensuring sufficient activity for signaling and differentiation.Cells use two general mechanisms to modify caspase activity, through modulating levels of active caspase or through allosteric mechanisms that change the distribution of conformations in the native ensemble, although the two are not mutually exclusive. Levels of caspase-3 are controlled by cleavage of the inactive zymogen to yield a dimer of protomers (Fig. 1A) (8,9), and this process is responsive to several signaling pathways, such as transient expression of the Bad-Bax cascade (10) or phosphorylation of the zymogen (Fig. 1B) (11). Alternatively, inhibitor of apoptosis proteins (IAPs) affect levels of active caspase-3 by dire...