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PubMed: 31209238, The effectiveness of most cancer targeted therapies is short-lived. Tumors often develop resistance that might be overcome with drug combinations. However, the number of possible combinations is vast, necessitating data-driven approaches to find optimal patient-specific treatments. Here we report AstraZeneca’s large drug combination dataset, consisting of 11,576 experiments from 910 combinations across 85 molecularly characterized cancer cell lines, and results of a DREAM Challenge to evaluate computational strategies for predicting synergistic drug pairs and biomarkers. 160 teams participated to provide a comprehensive methodological development and benchmarking. Winning methods incorporate prior knowledge of drug-target interactions. Synergy is predicted with an accuracy matching biological replicates for >60% of combinations. However, 20% of drug combinations are poorly predicted by all methods. Genomic rationale for synergy predictions are identified, including ADAM17 inhibitor antagonism when combined with PIK3CB/D inhibition contrasting to synergy when combined with other PI3K-pathway inhibitors in PIK3CA mutant cells. © 2019, The Author(s)., National Institute for Health Research, NIHR Wellcome Trust, WT: 102696, 206194 Magyar Tudományos Akadémia, MTA Bayer 668858 PrECISE AstraZeneca, We thank the Genomics of Drug Sensitivity in Cancer and COSMIC teams at the Wellcome Trust Sanger Institute for help with the preparation of the molecular data, Denes Turei for help with Omnipath, and Katjusa Koler for help with matching drug names across combination screens. We thank AstraZeneca for funding and provision of data to the DREAM Consortium to run the challenge, and funding from the European Union Horizon 2020 research (under grant agreement No 668858 PrECISE to J.S.R.), the Joint Research Center for Computational Biomedicine (which is partially funded by Bayer AG) to J.S.R., National Institute for Health Research (NIHR) Sheffield Biomedical Research Center, Premium Postdoctoral Fellowship Program of the Hungarian Academy of Sciences. M.G lab is supported by Wellcome Trust (102696 and 206194)., Competing interests: K.C.B., Z.G., G.Y.D., E.K.Y.T., S.F., and J.R.D. are AstraZeneca employees. K.C.B., Z.G., E.K.Y.T., S.F., and J.R.D. are AstraZeneca shareholders. Y.G. receives personal compensation from Eli Lilly and Company, is a shareholder of Cleerly, Inc., and Ann Arbor Algorithms, Inc. M.G. receives research funding from AstraZeneca and has performed consultancy for Sanofi. The remaining authors declare no competing interests.

Funder: nihr oxford biomedical research centre; funder-id: http://dx.doi.org/10.13039/501100013373, Funder: national institute for health research; funder-id: http://dx.doi.org/10.13039/501100000272, OBJECTIVES: The aim was to investigate the impact of a group-based weight management programme on symptoms of depression and anxiety compared with self-help in a randomised controlled trial (RCT). METHOD: People with overweight (Body Mass Index [BMI]���28kg/m2) were randomly allocated self-help (n = 211) or a group-based weight management programme for 12 weeks (n = 528) or 52 weeks (n = 528) between 18/10/2012 and 10/02/2014. Symptoms were assessed using the Hospital Anxiety and Depression Scale, at baseline, 3, 12 and 24 months. Linear regression modelling examined changes in Hospital Anxiety and Depression Scale between trial arms. RESULTS: At 3 months, there was a -0.6 point difference (95% confidence interval [CI], -1.1, -0.1) in depression score and -0.1 difference (95% CI, -0.7, 0.4) in anxiety score between group-based weight management programme and self-help. At subsequent time points there was no consistent evidence of a difference in depression or anxiety scores between trial arms. There was no evidence that depression or anxiety worsened at any time point. CONCLUSIONS: There was no evidence of harm to depression or anxiety symptoms as a result of attending a group-based weight loss programme. There was a transient reduction in symptoms of depression, but not anxiety, compared to self-help. This effect equates to less than 1 point out of 21 on the Hospital Anxiety and Depression Scale and is not clinically significant.

To provide an accessible and inexpensive method to surveil for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutations, we developed a multiplex real-time reverse transcription-PCR (rRT-PCR) assay, the Spike single-nucleotide polymorphism (SNP) assay, to detect specific mutations in the spike receptor binding domain. A single primer pair was designed to amplify a 348-bp region of spike, and probes were initially designed to detect K417, E484K, and N501Y. The assay was evaluated using characterized variant sample pools and residual nasopharyngeal samples. Variant calls were confirmed by SARS-CoV-2 genome sequencing in a subset of samples. Subsequently, a fourth probe was designed to detect L452R. The lower limit of 95% detection was 2.46 to 2.48 log10 genome equivalents (GE)/ml for the three initial targets (∼1 to 2 GE/reaction). Among 253 residual nasopharyngeal swabs with detectable SARS-CoV-2 RNA, the Spike SNP assay was positive in 238 (94.1%) samples. All 220 samples with threshold cycle (CT) values of

The last few weeks have been filled with despair at the lack of understanding of everyday pressures in UK general practice by NHS England and the media. Despite being responsible for delivering a world-leading vaccination programme, managing record consultation figures,1 adapting to large-scale roll-out of remote consultations, and …

Acute myeloid leukemia (AML) is a heterogeneous disease with a high relapse rate. Cytokine receptor targeted therapies are therapeutically attractive but are subject to resistance-conferring mutations. Likewise, targeting downstream signaling pathways has been difficult. Recent success in the development of synergistic combinations has provided new hope for refractory AML patients. While generally not efficacious as monotherapy, BH3 mimetics are very effective in combination with chemotherapy agents. With this in mind, we further explored novel BH3 mimetic drug combinations and showed that pimozide cooperates with mTOR inhibitors and BH3 mimetics in AML cells. The three-drug combination was able to reach cells that were not as responsive to single or double drug combinations. In Flt3-internal tandem duplication (ITD)-positive cells, we previously showed pimozide to be highly effective when combined with imipramine blue (IB). Here, we show that Flt3-ITD+ cells are sensitive to an IB-induced dynamin 1-like (Drp1)-p38-ROS pathway. Pimozide contributes important calcium channel blocker activity converging with IB on mitochondrial oxidative metabolism. Overall, these data support the concept that antioxidants are a double-edged sword. Rationally designed combination therapies have significant promise for further pre-clinical development and may ultimately lead to improved responses.

Dopamine (DA) neurons in the ventral tegmental area (VTA) are critical to coping with stress. However, molecular mechanisms regulating their activity and stress-induced depression were not well understood. We found that the receptor tyrosine kinase ErbB4 in VTA was activated in stress-susceptible mice. Deleting ErbB4 in VTA or in DA neurons, or chemical genetic inhibition of ErbB4 kinase activity in VTA suppressed the development of chronic social defeat stress (CSDS)-induced depression-like behaviors. ErbB4 activation required the expression of NRG1 in the laterodorsal tegmentum (LDTg); LDTg-specific deletion of NRG1 inhibited depression-like behaviors. NRG1 and ErbB4 suppressed potassium currents of VTA DA neurons and increased their firing activity. Finally, we showed that acute inhibition of ErbB4 after stress attenuated DA neuron hyperactivity and expression of depression-like behaviors. Together, these observations demonstrate a critical role of NRG1-ErbB4 signaling in regulating depression-like behaviors and identify an unexpected mechanism by which the LDTg-VTA circuit regulates the activity of DA neurons.

Summary Dopamine (DA) transmission is critical to motivation, movement, and emotion. Unlike glutamatergic and GABAergic synapses, the development of DA synapses is less understood. We show that bassoon (BSN) clusters along DA axons in the core of nucleus accumbens (NAcc) were increased in neonatal stages and reduced afterward, suggesting DA synapse elimination. Remarkably, DA neuron-specific ablating neuregulin 3 (NRG3), a protein whose levels correlate with BSN clusters, increased the clusters and impaired DA release and behaviors related to DA transmission. An unbiased screen of transmembrane proteins with the extracellular domain (ECD) of NRG3 identified Caspr3 (contactin associate-like protein 3) as a binding partner. Caspr3 was enriched in striatal medium spiny neurons (MSNs). NRG3 and Caspr3 interact in trans, which was blocked by Caspr3-ECD. Caspr3 null mice displayed phenotypes similar to those in DAT-Nrg3f/f mice in DA axonal BSN clusters and DA transmission. Finally, in vivo disruption of the NRG3-Caspr3 interaction increased BSN clusters. Together, these results demonstrate that DA synapse development is controlled by trans interaction between NRG3 in DA neurons and Caspr3 in MSNs, identifying a novel pair of cell adhesion molecules for brain circuit wiring.

Background Broad testing for respiratory viruses among persons under investigation (PUI) for SARS-CoV-2 is performed inconsistently, limiting our understanding of alternative infections and co-infections in these patients. Here, we used unbiased metagenomic next-generation sequencing (mNGS) to assess the frequencies of 1) alternative viral infections in SARS-CoV-2 RT-PCR negative PUIs and 2) viral co-infections in SARS-CoV-2 RT-PCR positive PUIs. Methods A convenience sample set was selected from PUIs who were tested for SARS-CoV-2 in the Emory Healthcare system during the first 2 months of the pandemic from 02/26-04/23/20. Laboratory results were extracted by chart review; Flu/RSV and multiplex respiratory pathogen PCRs had been performed at the discretion of treating physicians. Excess nasopharyngeal swab samples were retrieved within 72 hours of collection and underwent RNA extraction and SARS-CoV-2 testing by triplex RT-PCR. mNGS was performed by DNAse treatment, random primer cDNA synthesis, Nextera XT tagmentation, and high-depth Illumina sequencing. Reads underwent taxonomic classification by KrakenUniq, as implemented in viral-ngs. Results 53 PUIs were included, 30 negative and 23 positive for SARS-CoV-2 by RT-PCR. Among SARS-CoV-2 negative PUIs, 28 (93%) underwent clinical testing for alternative infections, and 8 (29%) tested positive for another respiratory virus. In all cases, mNGS identified the same virus (Table 1). In another 3 PUIs, mNGS identified two viruses that were not tested for and one that was missed by routine testing. No SARS-CoV-2 was detected by mNGS among RT-PCR negative PUIs. Among SARS-CoV-2 RT-PCR positive PUIs, 18 (69%) underwent clinical testing for co-infections, and none were detected. mNGS did not identify any viral co-infections but did detect SARS-CoV-2 in all 23 PUIs. Table 1: Molecular and Metagenomic Testing of Persons Under Investigation Conclusion Unbiased mNGS offers the powerful opportunity to streamline testing for PUIs by assessing for SARS-CoV-2 and alternative infections simultaneously; this technique can also be used to identify co-infections, but none were observed in our study population. Interestingly, many PUIs had no infection identified on routine testing or mNGS, which may reflect inadequate sampling, rapid virus clearance, or a non-viral process. Disclosures All Authors: No reported disclosures

Summary Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders with symptoms including social deficits, anxiety, and communication difficulties. However, ASD pathogenic mechanisms are poorly understood. Mutations of CUL3, which encodes Cullin 3 (CUL3), a component of an E3 ligase complex, are thought of as risk factors for ASD and schizophrenia (SCZ). CUL3 is abundant in the brain, yet little is known of its function. Here, we show that CUL3 is critical for neurodevelopment. CUL3-deficient mice exhibited social deficits and anxiety-like behaviors with enhanced glutamatergic transmission and neuronal excitability. Proteomic analysis revealed eIF4G1, a protein for Cap-dependent translation, as a potential target of CUL3. ASD-associated cellular and behavioral deficits could be rescued by pharmacological inhibition of the eIF4G1 function and chemogenetic inhibition of neuronal activity. Thus, CUL3 is critical to neural development, neurotransmission, and excitation-inhibition (E-I) balance. Our study provides novel insight into the pathophysiological mechanisms of ASD and SCZ.

Current therapy for acute myeloid leukemia (AML) primarily includes high-dose cytotoxic chemotherapy with or without allogeneic stem cell transplantation. Targeting unique cellular metabolism of cancer cells is a potentially less toxic approach. Monotherapy with mitochondrial inhibitors like metformin have met with limited success since escape mechanisms such as increased glycolytic ATP production, especially in hyperglycemia, can overcome the metabolic blockade. As an alternative strategy for metformin therapy, we hypothesized that the combination of 6-benzylthioinosine (6-BT), a broad-spectrum metabolic inhibitor, and metformin could block this drug resistance mechanism. Metformin treatment alone resulted in significant suppression of ROS and mitochondrial respiration with increased glycolysis accompanied by modest cytotoxicity (10–25%). In contrast, 6-BT monotherapy resulted in inhibition of glucose uptake, decreased glycolysis, and decreased ATP with minimal changes in ROS and mitochondrial respiration. The combination of 6-BT with metformin resulted in significant cytotoxicity (60–70%) in monocytic AML cell lines and was associated with inhibition of FLT3-ITD activated STAT5 and reduced c-Myc and GLUT-1 expression. Therefore, although the anti-tumor and metabolic effects of metformin have been limited by the metabolic reprogramming within cells, the novel combination of 6-BT and metformin targets this bypass mechanism resulting in reduced glycolysis, STAT5 inhibition, and increased cell death.

Neuregulin3 (NRG3) is a growth factor of the neuregulin (NRG) family and a risk gene of various severe mental illnesses including schizophrenia, bipolar disorders, and major depression. However, the physiological function of NRG3 remains poorly understood. Here we show that loss of Nrg3 in GFAP-Nrg3f/f mice increased glutamatergic transmission, but had no effect on GABAergic transmission. These phenotypes were observed in Nex-Nrg3f/f mice, where Nrg3 was specifically knocked out in pyramidal neurons, indicating that Nrg3 regulates glutamatergic transmission by a cell-autonomous mechanism. Consequently, in the absence of Nrg3 in pyramidal neurons, mutant mice displayed various behavioral deficits related to mental illnesses. We show that the Nrg3 mutation decreased paired-pulse facilitation, increased decay of NMDAR currents when treated with MK801, and increased minimal stimulation-elicited response, providing evidence that the Nrg3 mutation increases glutamate release probability. Notably, Nrg3 is a presynaptic protein that regulates the SNARE-complex assembly. Finally, increased Nrg3 levels, as observed in patients with severe mental illnesses, suppressed glutamatergic transmission. Together, these observations indicate that, unlike the prototype Nrg1, the effect of which is mediated by activating ErbB4 in interneurons, Nrg3 is critical in controlling glutamatergic transmission by regulating the SNARE complex at the presynaptic terminals, identifying a function of Nrg3 and revealing a pathophysiological mechanism for hypofunction of the glutamatergic pathway in Nrg3-related severe mental illnesses.

Aberrant cytokine signaling initiated from mutant receptor tyrosine kinases (RTKs) provides critical growth and survival signals in high risk acute myeloid leukemia (AML). Inhibitors to FLT3 have already been tested in clinical trials, however, drug resistance limits clinical efficacy. Mutant receptor tyrosine kinases are mislocalized in the endoplasmic reticulum (ER) of AML and play an important role in the non-canonical activation of signal transducer and activator of transcription 5 (STAT5). Here, we have tested a potent new drug called imipramine blue (IB), which is a chimeric molecule with a dual mechanism of action. At 200–300 nM concentrations, IB is a potent inhibitor of STAT5 through liberation of endogenous phosphatase activity following NADPH oxidase (NOX) inhibition. However, at 75–150 nM concentrations, IB was highly effective at killing mutant FLT3-driven AML cells through a similar mechanism as thapsigargin (TG), involving increased cytosolic calcium. IB also potently inhibited survival of primary human FLT3/ITD+ AML cells compared to FLT3/ITDneg cells and spared normal umbilical cord blood cells. Therefore, IB functions through a mechanism involving vulnerability to dysregulated calcium metabolism and the combination of fusing a lipophilic amine to a NOX inhibiting dye shows promise for further pre-clinical development for targeting high risk AML.

Ninety cannabis-dependent adults seeking treatment were randomly assigned to receive cognitive-behavioral therapy, abstinence-based voucher incentives, or their combination. Treatment duration was 14 weeks, and outcomes were assessed for 12 months posttreatment. Findings suggest that (a) abstinence-based vouchers were effective for engendering extended periods of continuous marijuana abstinence during treatment, (b) cognitive-behavioral therapy did not add to this during-treatment effect, and (c) cognitive-behavioral therapy enhanced the posttreatment maintenance of the initial positive effect of vouchers on abstinence. This study extends the literature on cannabis dependence, indicating that a program of abstinence-based vouchers is a potent treatment option. Discussion focuses on the strengths of each intervention, the clinical significance of the findings, and the need to continue efforts toward development of effective interventions.

The balance between matrix metalloproteinase (MMP) and tissue inhibitor of matrix metalloproteinase (TIMP) is important for extracellular matrix interactions of hematopoietic cells. MMP-independent growth modulating activity for TIMP-1 on B lymphocytes and erythroid progenitors has also been described, but a role for TIMP-1 in myelomonocytic differentiation has not been previously reported. In this study, we demonstrate that TIMP-1 overexpression impairs differentiation of the myeloblastic M1 cell line following interleukin (IL)-6 stimulation. We generated retroviral vectors coexpressing human TIMP-1 and the green fluorescent protein (GFP) and stably transduced murine M1 myeloid cells. TIMP-1 expressing cells showed a large reduction in IL-6-induced macrophage differentiation in vitro that was reversible with a specific monoclonal antibody. The differentiation delay in M1/TIMP-1 cells was also specifically reversible by pharmacologic phosphatidylinositol-3 kinase (PI3-K) inhibition. Additionally, overexpression of a TIMP-1/GFP fusion protein also impaired M1 differentiation and this protein was localized to the cell surface, consistent with an autocrine receptor-mediated mechanism. Surprisingly, TIMP-1 transduced cells had a selective advantage for growth in IL-6, indicating that functional effects on growth and differentiation of M1 cells were primarily through an autocrine mechanism. Intrinsic TIMP-1 expression in myeloid leukemia cells might thus impact upon survival or differentiation.

Signal transducer and activator of transcription-6 (STAT6) plays important roles in cytokine signaling via interleukin-4 and -13 receptors (IL-4R and IL-13R). Mice in which STAT6 has been disrupted by homologous recombination show defects in T helper cell type 2 (Th2) lymphocyte production, resulting in an accumulation of Th1 cells. In addition to defects in differentiation and proliferation of T lymphocytes, STAT6-deficient mice show increased cell-cycle activation and frequency of myeloid progenitors. Although this has been shown to be mediated through Oncostatin M production by T cells, IL-4Rα and STAT6 have also recently been found to be enriched for expression in primitive hematopoietic stem cells (HSCs) in gene expression-profiling studies. Therefore, we have investigated whether defects in hematopoietic function in mice lacking STAT6 expression extended into the primitive hematopoietic compartments of the bone marrow. Here, we report that STAT6 deficiency increased bone marrow-committed myeloid progenitors but did not alter the number of cells enriched for HSC/multipotent progenitors, primitive cobblestone area-forming cells assayed in vitro, or bone marrow short-term or long-term repopulating cells assayed in vivo. Therefore, the requirement for STAT6 activation during hematopoiesis is limited, and primitive hematopoietic cell types are insulated against possible effects of cytokine stimulation by Th1 cells.

Signal transducer and activator of transcription-5 (STAT5) plays an important role in repopulating activity of hematopoietic stem cells (HSCs). However, the relationship of STAT5 activation with early acting cytokine receptors is not well established. We have directly compared bone marrow (BM) from mice mutant for STAT5a and STAT5b (STAT5ab-/-) with that from mice lacking c-Mpl (c-Mpl-/-), the thrombopoietin receptor. Both STAT5 and c-Mpl deficiency only mildly affected committed myeloid progenitors assayed in vitro, but STAT5ab-/- BM showed lower Gr-1+ (4.4-fold), B220+ (23-fold), CD4+ (20-fold), and Ter119+ (17-fold) peripheral blood repopulating activity than c-Mpl-/- BM against wild-type competitor in long-term repopulating assays in vivo. Direct head-to-head competitions of STAT5ab-/- BM and c-Mpl-/- BM showed up to a 25-fold reduction in STAT5ab-/- contribution. Differences affecting reconstitution of primitive c-Kit+Lin-Sca-1+ multipotent progenitor (MPP)/HSC (1.8-fold) and c-Kit+Lin-Sca-1- oligopotent progenitor BM fractions (3.3-fold) were more modest. In serial transplantation experiments, STAT5ab-/- and c-Mpl-/- BM both failed to provide consistent engraftment in tertiary hosts and could not radioprotect lethally irradiated quaternary recipients. These results indicate substantial overlap in c-Mpl-STAT5 signaling defects at the MPP/HSC level but indicate that STAT5 is activated independent of c-Mpl to promote multilineage hematopoietic differentiation. (Blood. 2004;103:2965-2972)

Secreted growth factors are integral components of the bone marrow (BM) niche and can regulate survival, proliferation, and differentiation of committed hematopoietic stem cells (HSCs). However, downstream genes activated in HSCs by early-acting cytokines are not well characterized. To better define intracellular cytokine signaling in HSC function, we have analyzed mice lacking expression of both signal transducer and activator of transcription 5a (STAT5a) and STAT5b (STAT5ab−/−). These studies specifically avoided possible autoimmune and/or splenomegaly disease-mediated indirect effects on HSC function by using 2 independent approaches: (1) by crossing onto the C57Bl/6 RAG2−/− background, and (2) by generation of wild-type chimeric mice reconstituted with transplanted STAT5ab−/− BM cells. These experiments demonstrated that STAT5-deficient HSCs have cell autonomous defects in competitive long-term repopulating activity. Furthermore, in the chimeric mice, injected wild-type BM cells showed a progressive multilineage competitive repopulating advantage in vivo, demonstrating that steady-state hematopoiesis was also highly STAT5-dependent. Consistent with the in vivo repopulating deficiency, when Sca-1+c-kit+lin− (KLS) cells were isolated and stimulated with growth factors in vitro, up to a 13-fold reduced expansion of total nucleated cells was observed in response to cocktails containing interleukin 3 (IL-3), IL-6, stem cell factor (SCF), Flt3 ligand, and thrombopoietin. Notably, a 10-fold reduction in expansion was observed with IL-3 and SCF. However, STAT5 activation was not required for regeneration of the KLS pool in vivo following transplant or for secondary repopulating ability. These studies support a major role for STAT5 activation as a cellular determinant of cytokine-mediated HSC repopulating potential but not self-renewal capacity.

Dysregulation of cytokine signaling pathways is associated with benign and malignant hematologic disorders. Improvements in therapy rely on understanding the biology of the pathways and the proteins involved. Studying these pathways in patient samples is challenging as samples are difficult to obtain, contain fewer cells, and are heterogeneous in nature. To address some of these difficulties, we have utilized the technique of microcapillary electrophoresis. Using the NanoPro 1000 system (ProteinSimple) which is built on an automated, capillary-based immunoassay platform, we have developed rapid and quantitative assays for specific proteins from relatively small sample sizes. The NanoPro provides precise and quantitative data of the phosphorylation states of a specific protein of interest. We describe our experience with NanoPro assay development and optimization with specific application toward understanding aberrant cytokine signaling in human leukemia cells.

To describe an alternative, novel surgical approach to the repair of a ruptured globe in the second reported patient with keratoglobus, Ehlers-Danlos type VI, and normal lysyl hydroxylase.Under general anesthesia, a 360 degrees conjunctival peritomy was performed and the epithelium was removed from the keratoglobus cornea. Descemet's membrane and endothelium were removed from a fresh donor corneoscleral ring. An onlay epikeratoplasty was then performed and the entire donor corneoscleral button was secured to the sclera with 9-0 nylon suture. The conjunctiva was brought into position and tacked down over the edge of the donor graft. After 4 months, a full-thickness penetrating keratoplasty was then performed.The patient ultimately had a ruptured globe in the fellow eye and thus required the identical procedure OU. Both eyes were successfully repaired with a final visual acuity OU of 20/100.The described surgical approach allowed successful surgical repair of bilateral ruptured globes in a patient with keratoglobus, Ehlers-Danlos type VI, and normal lysyl hydroxylase levels. In the previous report of such a patient by Judisch et al. (1), the attempt at surgical repair was unsuccessful and ended in enucleation. The described surgical technique may be used to treat advanced keratoglobus or oculus fragilis.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults and the 2nd most common in children. Despite advances in our understanding of AML biology, long term survival remains suboptimal. Current therapy primarily involves high dose cytotoxic chemotherapy and possible allogeneic stem cell transplant, both of which are heavily myelosuppressive and carry some treatment related mortality. The FLT3/ITD mutation is found in 20-30% of adults and 10-20% of children with AML, and despite intensive therapy survival outcomes are dismal. Recent strategies using targeted therapies (e.g. FLT3 tyrosine kinase inhibitors-TKIs) have so far yielded modest responses, and most relapsed/refractory patients will still die of their leukemia. Thus selective agents targeting FLT3 mutated AML but not benign hematopoiesis are needed. A large portion of AML, including those with FLT3/ITD, has elevated levels of reactive oxygen species (ROS), and AML with high ROS is highly proliferative. Interestingly, mislocalization of mutant type III receptor tyrosine kinases (RTKs, e.g. FLT3 and c-KIT) occurs in leukemia. In FLT3/ITD, a portion of underglycosylated RTK accumulates on the endoplasmic reticulum (ER) in a microenvironment with high levels of localized ROS generated from ER-bound NADPH Oxidase 4 (Nox4) and through tight connections with mitochondria. ROS has been demonstrated in recent years to be an integral part of cell signaling, typically through suppression of protein tyrosine phosphatases. Therefore, we hypothesized that elevated ROS plays an important role in leukemic cell signaling in FLT3/ITD+ AML. To this end, we have investigated the effects of the Nox4 inhibitor imipramine blue (IB), a novel potent agent from the class of triphenylmethane dyes (Munson et al, Sci Transl Med, Vol. 4, 2012). We used FLT3/ITD+ AML cell lines (MV4-11, MOLM-14), cell lines without FLT3/ITD (OCI-AML3, K562, HEL, HL-60), and Ba/F3 and 32D cells transduced with FLT3 and c-KIT mutants, respectively. We screened inhibitors of mitochondrial complex I and NOX inhibitors to determine their effects on ROS levels by H2-DCF-DA staining/flow cytometry and on cell viability by trypan blue exclusion assay. While all mitochondrial and NOX4 inhibitors tested effectively reduced total cellular ROS levels, only Nox4 inhibition with IB achieved selective cytotoxicity for FLT3/ITD+ cell lines with high potency (IC50 =125 nM in MV4-11). The Annexin V assay showed that IB causes cell death through an apoptotic mechanism in MV4-11 cells. To determine effects of Nox4 inhibition on leukemic cell signaling, specifically on protein tyrosine phosphatases (PTPs), we studied the signal transducer and activator of transcription 5 (STAT5). We observed phosphorylated STAT5 (pSTAT5) levels by Western blot, and sodium orthovanadate (Na3VO4) was used for PTP inhibition. IB caused dose dependent decreases in pSTAT5 levels after 4 hours of exposure, and this effect was reversed when Na3VO4 was added, implicating reactivated PTPs as the cause of pSTAT5 suppression. To determine the downstream effects of STAT5 inhibition by IB, STAT5 target genes were evaluated by qRT-PCR. Four hour IB treatment caused decreased expression of common STAT5 target genes (Pim1, c-Myc, Cish) indicating effective suppression of STAT5 function. To optimize cytotoxicity, we then combined IB with the STAT5 inhibitor pimozide. A combination of 75 nM IB/5µM pimozide was highly synergistic in MV4-11 (5 fold higher cytotoxicity over individual drugs, 3 fold higher than their additive effect). IB/pimozide selectively killed FLT3/ITD+ AML cell lines while sparing AML cells without FLT3/ITD and benign CD34+ cord blood cells. IB and IB/pimozide were also equally effective in killing Ba/F3 FLT3/ITD cells with point mutations causing resistance to TKIs and selectively killed 32D cells with c-KIT (D814V) mutation over those with wild type c-KIT. Thus we have shown that the Nox4 inhibitor IB is a potent and selective inducer of apoptosis for FLT3/ITD+ AML in vitro, and the novel combination of IB and pimozide selectively targets FLT3/ITD and c-KIT positive mutants, including TKI-resistant FLT3/ITD mutants. We propose that IB alone or in combination with pimozide represents a novel therapeutic strategy, and testing in primary patient samples and in vivo models are currently underway. Disclosures Shah: Plexxikon Inc.: Research Funding; Pfizer: Research Funding; Bristol-Myers Squibb: Research Funding. Arbiser:ABBY Therapeutics: Other: Jack L Arbiser is listed as inventor on a US Patent for imipramine blue. He is cofounder of ABBY Therapeutics, which has licensed imipramine blue from Emory University.

Homologous disruption of expression of signal transducer and activator of transcription 5a (STAT5a) and STAT5b (STAT5ab–/–) in mice results in hematopoietic stem cells (HSCs) that can engraft irradiated hosts alone but are noncompetitive against wild-type HSCs. To explore mechanisms for this phenotype, we crossed the STAT5 mutations onto an HW80 background congenic to the original C57BL/6 that differs in a small chromosome 7 genomic locus. We previously demonstrated that C57BL/6 or HW80 background STAT5ab–/– bone marrow (BM) cells showed equal repopulating function either competitively or noncompetitively in irradiated hosts. However, one intraperitoneal injection of wild-type green fluorescent protein (GFP) transgenic BM cells into unconditioned newborn STAT5ab–/– recipients of either background was sufficient for high-level donor engraftment. Furthermore, haploinsufficiency of STAT5 (STAT5ab+/–) allowed improved engraftment over wild-type recipients, indicating a dose-dependent requirement for STAT5 activation. In reciprocal experiments, STAT5ab–/– BM was transplanted into nonirradiated W/Wv hosts. In these mice, C57BL/6 STAT5ab–/– BM cells were 10-fold more defective in long-term engraftment than control wild-type BM cells and HW80 STAT5ab–/– BM cells were 5- to 10-fold more defective than C57BL/6 STAT5ab–/– BM cells. Therefore, we conclude that STAT5 plays a critical role during steady-state HSC engraftment and a chromosome 7 modifier locus regulates this activity.

Signal transducers and activators of transcription (STATs) are intracellular mediators of cytokine receptor signals. Because many early-acting growth factors have been implicated in STAT5 activation, this study sought to investigate whether STAT5 may be a transcriptional regulator of hematopoietic stem cell (HSC) long-term repopulating activity. To test this possibility, bone marrow (BM) and fetal liver (FL) cells from mice containing homozygous deletions of both STAT5a and STAT5b genes (STAT5ab−/−) were characterized for hematopoietic repopulating activities. BM and FL grafts were capable of repopulating lymphoid and myeloid lineages of lethally irradiated primary and secondary hosts, with defects observed primarily in T-lymphocyte engraftment. Because only a fraction of normal HSC function is required to reconstitute hematopoiesis, competitive repopulation assays of adult BM or FL cells were used against wild type adult BM or FL cells to quantitate stem cell function. In these analyses, average 25-, 28-, 45-, and 68-fold decreases in normal repopulating activity were evident in granulocyte (Gr-1+), macrophage (Mac-1+), erythroid progenitor (Ter119+), and B-lymphocyte (B220+) populations, respectively, with T lymphocytes (CD4+) always undetectable from the STAT5ab−/− graft. Consistent with previous reports of divergence between stem cell phenotype and function in cases of perturbed hematopoiesis, the absolute number of cells within Sca-1+c-kit+lin− or lin− Hoechst 33342 side population fractions was not significantly different between wild type and STAT5ab−/−BM or FL cells. These results demonstrate that a significant proportion of the growth factor signals required for multilineage reconstitution potential of HSCs is STAT5 dependent.

Background: Cytogenetic and molecular aberrations have improved risk stratification of acute myeloid leukemia (AML) patients, however survival continues to be low (40-50%) and current therapy primarily includes high dose cytotoxic chemotherapy with or without allogeneic stem cell transplant. In AML, metabolomic analysis has identified key differences in glycolysis, gluconeogenesis and tri-carboxylic acid (TCA) pathways between normal and AML cells. Monotherapy with mitochondrial inhibitors like Metformin have met with little success since cancer cells use glycolytic escape mechanisms to overcome the decreased mitochondrial oxidative phosphorylation. Here we demonstrate a novel therapeutic approach for overcoming this route of Metformin resistance. Methods: Metformin, a well-established anti-diabetic drug, is a strong inhibitor of mitochondrial respiration and is also considered an inhibitor of mTOR signaling acting via AMPK activation. Despite its therapeutic potential, cytotoxicity of AML cells with Metformin alone at achievable plasma levels (10-20 mM) is modest due in part to increased glycolytic ATP production as a bypass mechanism. This has limited the effectiveness of Metformin in AML and led us to investigate mechanisms for reversal of this resistance mechanism to improve the pre-clinical efficacy. 6-Benzylthioinosine (6-BT) is a small molecule that we have shown depletes ATP and can cause differentiation in myeloid leukemia cells (at 10µM concentration) with minimal toxicity to normal cells (LD50 >100µM) (Wald et al. Cancer Res, 2008). To improve on cytotoxicity seen with Metformin, we hypothesized that the combination of 6-BT and Metformin would enhance cytotoxicity in AML. The combination of Metformin (10mM) and 6-BT (10 µM) were tested alone or combined. We compared the effects of the combination treatment on mTOR signaling and caspase-3 activation. We also measured changes in reactive oxygen species (ROS), intracellular ATP, extracellular glucose levels and GLUT-1 expression. Glycolytic flux was measured by Extracellular Acidification Rate (ECAR) and oxidative phosphorylation was measured by Oxygen Consumption Rate (OCR) using Seahorse XF Analyzer. All experiments were performed in triplicate and Student’s two tailed t-test was used to calculate p-values (less than 0.05 were considered to be statistically significant). Results: Metformin and 6-BT alone caused inhibition of cell growth (< 3-fold) and modest cytotoxicity (10-40%, SD ± 6.3%) in all AML cell lines tested (MV4-11, MOLM-14, OCI-AML3, Nomo-1 and THP-1). However the combination of 6-BT with Metformin resulted in marked growth inhibition (30-50 fold) and cytotoxicity (90-100%, SD ± 8.3%) in monocytic AML cell lines (MV4-11 and MOLM-14) within 48 hours of exposure, which was partially dependent on caspase-3 induction. Apoptosis induction was also detected by Annexin V assay. Cytotoxic effects were independent of mTOR inhibition since AZD8055 showed no synergy with the 6-BT or the combination. 6-BT has structural similarity with 6-Mercaptopurine (6-MP), however the combination of Metformin and 6-MP also showed no synergy. 6-BT combined with Metformin caused significant (50-70%) reduction in intracellular ATP levels in the monocytic AML lines without significant extracellular glucose depletion suggestive of glycolytic inhibition. As expected, Metformin significantly suppressed oxidative phosphorylation as evident by near complete inhibition of OCR ( Conclusions: The anti-tumor and metabolic effects of Metformin have been limited by the metabolic reprogramming within cells that result in low cytotoxicity. Here we show that the novel combination of 6-BT and Metformin targets this metabolic bypass mechanism resulting in increased cell death. This is the first demonstration that 6-BT, a drug that is selectively taken up and phosphorylated by AML cells, can exert cytotoxicity involving down modulation of glycolysis. We propose that the combination of 6-BT and Metformin represents a new therapeutic strategy that warrants further testing in primary patient samples. Disclosures No relevant conflicts of interest to declare.

PURPOSE: A patient developed significant corneal complications from air bag deployment, 17 months after laser in situ keratomileusis (LASIK). METHODS: Case report, slit-lamp microscopy, and review of the medical literature. RESULTS: A 37-year-old woman underwent bilateral LASIK with resultant 20/20 uncorrected visual acuity. Seventeen months later, she sustained facial and ocular injuries from air bag deployment during a motor vehicle accident. Examination revealed bilateral corneal abrasions, partial dislocation of the right corneal LASIK flap, and a hyphema in the right eye. The LASIK flap was realigned, but recovery was complicated by a slowly healing epithelial defect and flap edema. One month following the injury, epithelial ingrowth beneath the LASIK flap was noted. Surgical elevation of the flap and removal of the epithelial ingrowth was performed. Eight months later, epithelial ingrowth was absent and the visual acuity was 20/40. Residual irregular astigmatism necessitated rigid gas permeable contact lens fitting to achieve 20/20 visual acuity. CONCLUSIONS: Air bags may cause significant ocular trauma. The wound healing response of LASIK allows corneal flap separation from its stromal bed for an indeterminate time after surgery. Discussion of the possible risk of corneal trauma as part of informed consent prior to LASIK may be appropriate. [J Refract Surg 2000;16:373-374]

Overall cure rates in acute myeloid leukemia (AML) continue to range between 60-65% with disease relapse in AML being a major cause of mortality. Risk stratification by cytogenetic and molecular aberrations has changed the prognosis for some patients, but for a majority of the patients (especially with normal cytogenetics) improvement in therapy is needed. Understanding key differences in survival pathways such as the mTOR pathway within leukemic cells could be potentially exploited to develop new drug therapy and design relapse therapy. Tracking pathway activation in patient samples is challenging in AML since samples are difficult to obtain, contain fewer cells, and are heterogeneous in nature. The NanoPro 1000 system (ProteinSimple) is built on an automated, capillary-based immunoassay platform and enables a rapid and quantitative analysis of specific proteins and their phosphorylation states. We have used this novel nano-technology based system to study the mTOR pathway in AML samples (n=8) obtained from the Children's Oncology Group (COG) Myeloid Disease Reference Laboratory. Assays for downstream mTOR target protein-eukaryotic initiation factor 4E binding protein 1 (4EBP1) were standardized using AML cell lines (MV4-11, MOLM-14, OCI-AML3 and HL-60) prior to testing in patient samples. The assay was able to detect as little as 80 ng of protein which corresponded to 1000 cells per assay. MV4-11 cells were treated with the dual mTOR 1/2 inhibitor AZD-8055 for 24 hours and reduced phosphorylation of 4EBP1 was confirmed by Western Blotting and corroborated by the NanoPro assay. This nano-immunoassay was able to reliably quantify amounts of 4EBP1 (total and phosphorylated forms) in primary pediatric AML samples (n=8) thawed following cryopreservation. The relative luminescence units (measure of chemiluminescence and protein signal) were plotted against exposure time and the linearity of the graph provided evidence that the signal is sensitive over a range of three logs. Further, these samples were treated with AZD-8055 for 24 hours followed by analysis of changes in signaling profiles for total and phosphorylated Ser65 and Thr37/46. As expected, complete reduction of phosphorylation was noted in the treated samples (n=4). The assays obtained were quantifiable, consistent, and reproducible and enabled us to delineate activation patterns of the mTOR pathway in primary AML samples and evaluate drug efficacy for targeted agents. Our data provides a strong basis for testing this platform on a larger scale and our long term aim is to utilize this novel nano-immunoassay based platform prospectively in de-novo AML to be able to identify poor responders who might benefit from early introduction of targeted therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C200. Citation Format: Himalee Sabnis, Heath Bradley, Todd Cooper, Kevin Bunting. Rapid nano-immunoassay for evaluation of mTOR pathway activation in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C200.

Objective To determine whether outer retinal changes occur in chronic, presumed primary open-angle glaucoma (POAG). Methods The outer retinas from 128 human eyes with a diagnosis of chronic glaucoma (presumably POAG in most cases) and 90 control eyes were examined histologically by 3 masked observers for photoreceptor swelling and loss. Retinas from 9 rhesus monkeys with glaucoma induced experimentally by laser trabecular destruction were compared with 7 fellow (control) eyes. The mean pressure elevations in the eyes with laser trabecular destruction ranged from 26.6 to 53.6 mm Hg with durations varying from 7 to 33 weeks. Results Swelling of the red- and green-sensitive cones was observed in a statistically significantly greater proportion of human eyes with presumed POAG compared with the control eyes. Patchy loss of red/green cones and rods was also found in some of the glaucomatous retinas. In a subset of the human eyes with end-stage disease, cone swelling was a variable finding. Although no photoreceptor loss was found in the 9 monkey eyes with experimental glaucoma, 8 had swelling of their red/green cones that was remarkably similar to that seen in the human eyes. Swelling was not present in any of the control monkey eyes. Conclusions The photoreceptors are affected by chronically elevated intraocular pressure. Clinical Relevance These findings may explain some of the abnormalities of color vision and the electrophysiological effects that have been observed in patients with POAG.

Improvements to the original procedure of using a panel of monoclonal antibodies and magnetic microspheres for the depletion of tumour cells from bone marrow are described. These include a completely disposable system for the magnetic depletion of tumour cells coated with magnetic microspheres. Properties of a new series of microspheres are compared with the old M330 beads in their ability to deplete neuroblasts from both model systems and 50 bone marrows harvested from Stage IV neuroblastoma patients. Using human neuroblastoma cell lines labelled with the DNA intercalating, Hoechst dye 33342 a 5% tumour contamination can routinely be removed from 5 X 10(6) - 5 X 10(7) nucleated cells. Analysis of the 50 purged marrows revealed that 10 were visibly contaminated with tumour (by conventional cytology and immunological procedures). In all but one case, tumour cells were removed. In this instance the tumour:bead ratio fell to 1:4 indicating the importance of maintaining a sufficient number of beads in the system. Red cell contamination of marrow was also kept extremely low so preventing possible physical blockade of bead:tumour cell interaction. Marrow engraftment was rapid in this group, apart from patients who had been exposed to high doses of alkylating agents prior to autografting. Images Figure 2

Background: Most people who stop smoking gain weight. This can discourage some people from making a quit attempt and risks offsetting some, but not all, of the health advantages of quitting. Interventions to prevent weight gain could improve health outcomes, but there is a concern that they may undermine quitting. Objectives: To systematically review the effects of: (1) interventions targeting post‐cessation weight gain on weight change and smoking cessation (referred to as 'Part 1') and (2) interventions designed to aid smoking cessation that plausibly affect post‐cessation weight gain (referred to as 'Part 2'). Search methods: Part 1 ‐ We searched the Cochrane Tobacco Addiction Group's Specialized Register and CENTRAL; latest search 16 October 2020. Part 2 ‐ We searched included studies in the following 'parent' Cochrane reviews: nicotine replacement therapy (NRT), antidepressants, nicotine receptor partial agonists, e‐cigarettes, and exercise interventions for smoking cessation published in Issue 10, 2020 of the Cochrane Library. We updated register searches for the review of nicotine receptor partial agonists. Selection criteria Part 1 ‐ trials of interventions that targeted post‐cessation weight gain and had measured weight at any follow‐up point or smoking cessation, or both, six or more months after quit day. Part 2 ‐ trials included in the selected parent Cochrane reviews reporting weight change at any time point. Data collection and analysis: Screening and data extraction followed standard Cochrane methods. Change in weight was expressed as difference in weight change from baseline to follow‐up between trial arms and was reported only in people abstinent from smoking. Abstinence from smoking was expressed as a risk ratio (RR). Where appropriate, we performed meta‐analysis using the inverse variance method for weight, and Mantel‐Haenszel method for smoking. Main results: Part 1: We include 37 completed studies; 21 are new to this update. We judged five studies to be at low risk of bias, 17 to be at unclear risk and the remainder at high risk. An intermittent very low calorie diet (VLCD) comprising full meal replacement provided free of charge and accompanied by intensive dietitian support significantly reduced weight gain at end of treatment compared with education on how to avoid weight gain (mean difference (MD) −3.70 kg, 95% confidence interval (CI) −4.82 to −2.58; 1 study, 121 participants), but there was no evidence of benefit at 12 months (MD −1.30 kg, 95% CI −3.49 to 0.89; 1 study, 62 participants). The VLCD increased the chances of abstinence at 12 months (RR 1.73, 95% CI 1.10 to 2.73; 1 study, 287 participants). However, a second study found that no‐one completed the VLCD intervention or achieved abstinence. Interventions aimed at increasing acceptance of weight gain reported mixed effects at end of treatment, 6 months and 12 months with confidence intervals including both increases and decreases in weight gain compared with no advice or health education. Due to high heterogeneity, we did not combine the data. These interventions increased quit rates at 6 months (RR 1.42, 95% CI 1.03 to 1.96; 4 studies, 619 participants; I2 = 21%), but there was no evidence at 12 months (RR 1.25, 95% CI 0.76 to 2.06; 2 studies, 496 participants; I2 = 26%). Some pharmacological interventions tested for limiting post‐cessation weight gain (PCWG) reduced weight gain at the end of treatment (dexfenfluramine, phenylpropanolamine, naltrexone). The effects of ephedrine and caffeine combined, lorcaserin, and chromium were too imprecise to give useful estimates of treatment effects. There was very low‐certainty evidence that personalized weight management support reduced weight gain at end of treatment (MD −1.11 kg, 95% CI −1.93 to −0.29; 3 studies, 121 participants; I2 = 0%), but no evidence in the longer‐term 12 months (MD −0.44 kg, 95% CI −2.34 to 1.46; 4 studies, 530 participants; I2 = 41%). There was low to very low‐certainty evidence that detailed weight management education without personalized assessment, planning and feedback did not reduce weight gain and may have reduced smoking cessation rates (12 months: MD −0.21 kg, 95% CI −2.28 to 1.86; 2 studies, 61 participants; I2 = 0%; RR for smoking cessation 0.66, 95% CI 0.48 to 0.90; 2 studies, 522 participants; I2 = 0%). Part 2: We include 83 completed studies, 27 of which are new to this update. There was low certainty that exercise interventions led to minimal or no weight reduction compared with standard care at end of treatment (MD −0.25 kg, 95% CI −0.78 to 0.29; 4 studies, 404 participants; I2 = 0%). However, weight was reduced at 12 months (MD −2.07 kg, 95% CI −3.78 to −0.36; 3 studies, 182 participants; I2 = 0%). Both bupropion and fluoxetine limited weight gain at end of treatment (bupropion MD −1.01 kg, 95% CI −1.35 to −0.67; 10 studies, 1098 participants; I2 = 3%); (fluoxetine MD −1.01 kg, 95% CI −1.49 to −0.53; 2 studies, 144 participants; I2 = 38%; low‐ and very low‐certainty evidence, respectively). There was no evidence of benefit at 12 months for bupropion, but estimates were imprecise (bupropion MD −0.26 kg, 95% CI −1.31 to 0.78; 7 studies, 471 participants; I2 = 0%). No studies of fluoxetine provided data at 12 months. There was moderate‐certainty that NRT reduced weight at end of treatment (MD −0.52 kg, 95% CI −0.99 to −0.05; 21 studies, 2784 participants; I2 = 81%) and moderate‐certainty that the effect may be similar at 12 months (MD −0.37 kg, 95% CI −0.86 to 0.11; 17 studies, 1463 participants; I2 = 0%), although the estimates are too imprecise to assess long‐term benefit. There was mixed evidence of the effect of varenicline on weight, with high‐certainty evidence that weight change was very modestly lower at the end of treatment (MD −0.23 kg, 95% CI −0.53 to 0.06; 14 studies, 2566 participants; I2 = 32%); a low‐certainty estimate gave an imprecise estimate of higher weight at 12 months (MD 1.05 kg, 95% CI −0.58 to 2.69; 3 studies, 237 participants; I2 = 0%). Authors' conclusions: Overall, there is no intervention for which there is moderate certainty of a clinically useful effect on long‐term weight gain. There is also no moderate‐ or high‐certainty evidence that interventions designed to limit weight gain reduce the chances of people achieving abstinence from smoking.

Background Overall cure rates in acute myeloid leukemia (AML) continue to range between 60-65% with disease relapse being a major cause of mortality. The PI3K-Akt-mTOR kinase pathway plays a vital role in pro-survival signals within leukemic cells and inhibition of this pathway is being investigated to improve patient outcomes. Tracking activation of multiple signaling proteins simultaneously in patient samples can be challenging especially with limiting cell numbers within rare sub-populations. Methods The NanoPro 1000 system (ProteinSimple) is built on an automated, capillary-based immunoassay platform and enables a rapid and quantitative analysis of specific proteins and their phosphorylation states. We have utilized this nano-immunoassay to examine activation of Akt 1/2/3 and downstream mTOR target - eukaryotic initiation factor 4E-Binding Protein 1 (4EBP1). Results Assays for Akt 1/2/3 and 4EBP1 were standardized using AML cell lines (MV4-11, MOLM-14, OCI-AML3 and HL-60) prior to testing in patient samples. Target inhibition was studied using mTOR 1/2 inhibitor AZD-8055 and results were corroborated by Western blotting. The assay was able to quantify nanogram amounts of 4EBP1 and Akt 1/2/3 in AML cell lines and primary pediatric AML samples and results were quantifiable, consistent and reproducible. Conclusion Our data provides a strong basis for testing this platform on a larger scale and our long term aim is to utilize this nano-immunoassay prospectively in de-novo AML to be able to identify poor responders who might benefit from early introduction of targeted therapy.