Sorafenib is a multikinase inhibitor that induces apoptosis in human leukemia and other malignant cells.Recently, we demonstrated that sorafenib diminishes Mcl-1 protein expression by inhibiting translation through a MEK1/2-ERK1/2 signaling-independent mechanism and that this phenomenon plays a key functional role in sorafenib-mediated lethality. Here, we report that inducible expression of constitutively active MEK1 fails to protect cells from sorafenib-mediated lethality, indicating that sorafenib-induced cell death is unrelated to MEK1/2-ERK1/2 pathway inactivation. Notably, treatment with sorafenib induced endoplasmic reticulum (ER) stress in human leukemia cells (U937) manifested by immediate cytosolic-calcium mobilization, GADD153 and GADD34 protein induction, PKR-like ER kinase (PERK) and eukaryotic initiation factor 2␣ (eIF2␣) phosphorylation, XBP1 splicing, and a general reduction in protein synthesis as assessed by [35 S]methionine incorporation. These events were accompanied by pronounced generation of reactive oxygen species through a mechanism dependent upon cytosolic-calcium mobilization and a significant decline in GRP78/Bip protein levels. Interestingly, enforced expression of IRE1␣ markedly reduced sorafenib-mediated apoptosis, whereas knockdown of IRE1␣ or XBP1, disruption of PERK activity, or inhibition of eIF2␣ phosphorylation enhanced sorafenib-mediated lethality. Finally, downregulation of caspase-2 or caspase-4 by small interfering RNA significantly diminished apoptosis induced by sorafenib. Together, these findings demonstrate that ER stress represents a central component of a MEK1/2-ERK1/2-independent cell death program triggered by sorafenib.The discovery that aberrant activation of the Ras-Raf-MEK1/2-ERK1/2 module occurs in a high percentage (30%) of human cancers (12, 42, 48, 57) provided a rationale for attempting to disrupt this pathway as a candidate anticancer strategy. Recently, a number of specific inhibitors designed to interrupt this pathway at the level of Ras, Raf, or MEK1/2 have been developed. Among these, sorafenib, a biaryl urea, also known as BAY 43-9006 or Nexavar, was initially developed as a specific inhibitor of C-Raf and B-Raf. However, subsequent studies revealed that it also inhibits several other tyrosine kinases involved in tumor progression, including VEGFR-2, VEGFR-3, PDGFR-␤, Flt3, c-Kit, and Ret (6, 60). Interestingly, sorafenib has been shown to inhibit mutant (V600E) B-Raf kinase activities in vitro and to diminish MEK/extracellular signal-regulated kinase (ERK) activation in various tumor cell lines, including those harboring mutant Ras or B-Raf (25,56,60). Although this compound has potent activity in preclinical tumor xenograft models against a variety of tumor cell types (60), has shown promising activity in a number of clinical trials, and has recently been approved for the treatment of advanced renal cell carcinoma (51), the mechanism(s) by which it exerts its antitumor activity has not been fully elucidated and is currently the subject of ongoing i...

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Imaging neuronal networks provides a foundation for understanding the nervous system, but resolving dense nanometer-scale structures over large volumes remains challenging for light (LM) and electron microscopy (EM). Here, we show that X-ray holographic nano-tomography (XNH) can image millimeter-scale volumes with sub-100 nm resolution, enabling reconstruction of dense wiring in Drosophila melanogaster and mouse nervous tissue. We performed correlative XNH and EM to reconstruct hundreds of cortical pyramidal cells, and show that more superficial cells receive stronger synaptic inhibition on their apical dendrites. By combining multiple XNH scans, we imaged an adult Drosophila leg with sufficient resolution to comprehensively catalog mechanosensory neurons and trace individual motor axons from muscles to the central nervous system. To accelerate neuronal reconstructions, we trained a convolutional neural network to automatically segment neurons from XNH volumes. Thus, XNH bridges a key gap between LM and EM, providing a new avenue for neural circuit discovery.

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Purpose The purpose of this study was to determine whether HDAC inhibitors (HDACIs) such as vorinostat or entinostat (SNDX-275) could increase the lethality of the dual Bcr/Abl-aurora kinase inhibitor KW-2449 in various Bcr/Abl+ human leukemia cells, including those resistant to imatinib mesylate (IM). Experimental Design Bcr/Abl+ CML and ALL cells, including those resistant to IM (T315I, E255K) were exposed to KW-2449 in the presence or absence of vorinostat or SNDX-275, after which apoptosis and effects on signaling pathways were examined. In vivo studies combining HDACIs and KW2449 were performed using a systemic IM-resistant ALL xenograft model. Results Co-administration of HDACIs synergistically increased KW-2449 lethality in vitro in multiple CML and Ph+ ALL cell types including human IM resistant cells (e.g. BV-173/E255K, Adult/T315I). Combined treatment resulted in inactivation of Bcr/Abl and downstream targets (e.g. STAT5 and CRKL), as well as increased ROS generation and DNA damage (γH2A.X). The latter events and cell death were significantly attenuated by free radical scavengers (TBAP). Increased lethality was also observed in primary CD34+ cells from patients with CML, but not in normal CD34+ cells. Finally, minimally active vorinostat or SNDX275 doses markedly increased KW2449 anti-tumor effects and significantly prolonged the survival of murine xenografts bearing IM-resistant ALL cells (BV173/E255K). Conclusions HDACIs increase KW-2449 lethality in Bcr/Abl+ cells in association with inhibition of Bcr/Abl, generation of ROS, and induction of DNA damage. This strategy preferentially targets primary Bcr/Abl+ hematopoietic cells and exhibits enhanced in vivo activity. Combining KW-2449 with HDACIs warrants attention in IM-resistant Bcr/Abl+ leukemias.

Interactions between the dual Bcr/Abl and aurora kinase inhibitor MK-0457 and the histone deacetylase inhibitor vorinostat were examined in Bcr/Abl ؉ leukemia cells, including those resistant to imatinib mesylate (IM), particularly those with the T315I mutation. Coadministration of vorinostat dramatically increased MK-0457 lethality in K562 and LAMA84 cells. Notably, the MK-0457/vorinostat regimen was highly active against primary CD34 ؉ chronic myelogenous leukemia ( IntroductionChronic myelogenous leukemia (CML) is characterized by the Philadelphia chromosome (Ph; 22q), which is responsible for the chimeric fusion oncoprotein Bcr/Abl. The Bcr/Abl kinase is constitutively active and signals downstream to multiple survival pathways, 1 providing CML cells with a survival advantage over their normal counterparts and conferring resistance against cytotoxic agents. 2 The treatment of CML has been revolutionized by the introduction of the kinase inhibitor imatinib mesylate (IM; Gleevec, Novartis, Basel, Switzerland), which is highly active in patients with chronic-phase CML 3 but less active in patients with accelerated or blast-phase disease. 4 However, almost all patients who initially respond eventually develop resistance to this agent. Mechanisms of resistance include bcr/abl gene amplification, increased expression of the Bcr/Abl protein, and most commonly, point mutations in various domains of the Bcr/Abl kinase, including the activation loop, the phosphorylation loop, or the gatekeeper region. 5 This phenomenon stimulated the development of secondgeneration Bcr/Abl kinase inhibitors (eg, dasatinib and nilotinib), which are active against proteins bearing most mutations. 6,7 However, these agents are inactive against cells with gatekeeper region mutations, most notably T315I, 8 prompting the search for newer Bcr/Abl kinase inhibitors active against such mutants.The aurora kinases (A, B, and C) represent a family of serine/threonine kinases involved in the control of mitosis. 9 Deregulation of aurora kinase activity leads to disruption of cell-cycle progression, mitotic abnormalities, and genetic instability. 10 Importantly, aurora kinases are overexpressed and/or activated in a variety of tumor cells, suggesting a role for this family in tumorigenesis. 9,10 MK-0457 is a small-molecule, novel pan-aurora kinase inhibitor 9 with demonstrated activity against wild-type (wt) and mutated Bcr/Abl, [11][12][13] including the T315I mutation, as well as FLT3 and JAK2. MK-0457 delays entry into mitosis, leads to aberrant cytokinesis, induces apoptosis in several human tumor types, and is being evaluated in patients with a variety of malignant diseases. 9 MK-0457 potently inhibits aurora kinases (particularly aurora A and B) in tumor cells, manifested by down-regulation of phosphorylated histone H3 at Ser10. 9 This results in multiple events, including aberrant cell-cycle progression and accumulation of polyploid cells with DNA content of 4N or more, which collectively trigger cell death. 14,15 Very recently, it was repor...

IntroductionChronic myelogenous leukemia (CML) is a stem-cell disease characterized in 95% of cases by the reciprocal translocation of the long arms of chromosomes 9 and 22, resulting in a chimeric fusion protein with constitutively active tyrosine kinase activity (Bcr/ Abl). 1,2 Bcr/Abl signals downstream to multiple survival pathways, including STAT5, Bcl-x L , ERK1/2 (extracellular signal regulated kinase 1/2), and NF-B, among others, which collectively confer a survival advantage on CML cells compared with their normal counterparts. 2,3 The therapy of CML has changed dramatically with the introduction of imatinib mesylate (Gleevec), a tyrosine kinase inhibitor that inhibits Bcr/Abl as well as other kinases including c-Kit. 4,5 Despite the success of imatinib mesylate in CML patients, it is less effective in patients with more advanced disease (eg, accelerated or blast phase). [6][7][8] In addition, patients who initially respond eventually become refractory to imatinib due to the development of increased expression of Bcr/Abl, or more commonly, the appearance of mutations in the kinase domain that prevent drug binding and inhibitory activity. [9][10][11] For these reasons, attempts to circumvent or overcome imatinib mesylate resistance represent the focus of intense interest.One approach to this problem involves combining imatinib mesylate with other signaling inhibitors, and combination studies involving agents such as flavopiridol, 12 farnesyltransferase inhibitors, 13,14 histone deacetylase inhibitors, 15,16 and Akt inhibitors 17 have been described. Another strategy involves the design of second-generation Bcr/Abl kinase inhibitors that are more active than imatinib mesylate and/or able to kill Bcr/Abl ϩ cells that have become resistant to imatinib mesylate. An example of such agents is BMS-354825 (dasatinib), a dual Bcr/Abl and Src kinase inhibitor that is active against Bcr/Abl ϩ cells when administered at nanomolar concentrations. 18,19 Notably, dasatinib is active against cells exhibiting certain Bcr/Abl mutations (eg, E255K, M351T), but is relatively ineffective against cells with T315I mutation, which occupies a "gatekeeper" position in the Bcr/Abl kinase region. 18,20 The relative contribution of Bcr/Abl and Src kinase inhibition in the lethality of dasatinib remains to be fully elucidated. Recent preclinical studies suggest potential benefit for combining imatinib mesylate with Bcr/Abl kinase inhibitors such as dasatinib. 21 The Raf1/MEK1/2/ERK1/2 pathway is an important survival signaling cascade involved in cell proliferation, differentiation, and transformation. [22][23][24] It has also been implicated in the antiapoptotic actions of Bcr/Abl. 2 While MEK activity appears restricted to only one class of substrates, ERK activates more than 70 substrates including nuclear transcription factors. [22][23][24][25] For this reason, several pharmacologic MEK1/2 inhibitors have recently entered the clinic, and have been shown to inhibit their targets (ie, ERK1/2 phosphorylation) when administered at we...

Many animals use coordinated limb movements to interact with and navigate through the environment. To investigate circuit mechanisms underlying locomotor behavior, we used serial-section electron microscopy (EM) to map synaptic connectivity within a neuronal network that controls limb movements. We present a synapse-resolution EM dataset containing the ventral nerve cord (VNC) of an adult female Drosophila melanogaster. To generate this dataset, we developed GridTape, a technology that combines automated serial-section collection with automated high-throughput transmission EM. Using this dataset, we reconstructed 507 motor neurons, including all those that control the legs and wings. We show that a specific class of leg sensory neurons directly synapse onto the largest-caliber motor neuron axons on both sides of the body, representing a unique feedback pathway for fast limb control. We provide open access to the dataset and reconstructions registered to a standard atlas to permit matching of cells between EM and light microscopy data. We also provide GridTape instrumentation designs and software to make large-scale EM data acquisition more accessible and affordable to the scientific community.