Pharmacological Induction Research Articles

Modulation of the unfolded protein response pathway as an antiviral approach in airway epithelial cells

IntroductionRhinovirus (RV) infection is a major cause of cystic fibrosis (CF) lung morbidity with limited therapeutic options. Various diseases involving chronic inflammatory response and infection are associated with endoplasmic reticulum (ER) stress and subsequent activation of the unfolded protein response (UPR), an adaptive response to maintain cellular homeostasis. Recent evidence suggests impaired ER stress response in CF airway epithelial cells, this might be a reason for recurrent viral infection in CF. Therefore, assuming that ER stress inducing drugs have antiviral properties, we evaluated the activation of the UPR by selected ER stress inducers as an approach to control virus replication in the CF bronchial epithelium. MethodsWe assessed the levels of UPR markers, namely the glucose-regulated protein 78 (Grp78) and the C/EBP homologous protein (CHOP), in primary CF and control bronchial epithelial cells and in a CF and control bronchial epithelial cell line before and after infection with RV. The cells were also pretreated with ER stress-inducing drugs and RV replication and shedding was measured by quantitative RT-PCR and by a TCID50 assay, respectively. Cell death was assessed by a lactate dehydrogenate (LDH) activity test in supernatants. ResultsWe observed a significantly impaired induction of Grp78 and CHOP in CF compare to control cells following RV infection. The ER stress response could be significantly induced in CF cells by pharmacological ER stress inducers Brefeldin A, Tunicamycin, and Thapsigargin. The chemical induction of the UPR pathway prior to RV infection of CF and control cells reduced viral replication and shedding by up to two orders of magnitude and protected cells from RV-induced cell death. ConclusionRV infection causes an impaired activation of the UPR in CF cells. Rescue of the ER stress response by chemical ER stress inducers reduced significantly RV replication in CF cells. Thus, pharmacological modulation of the UPR might represent a strategy to control respiratory virus replication in the CF bronchial epithelium.

BET proteins regulate homologous recombination-mediated DNA repair: BRCAness and implications for cancer therapy.

Bromodomain and Extra-Terminal (BET) proteins are historically involved in regulating gene expression and BRD4 was recently found to be involved in DNA damage regulation. Aims of our study were to assess BRD4 regulation in homologous recombination-mediated DNA repair and to explore novel clinical strategies through the combinations of the pharmacological induction of epigenetic BRCAness in BRCA1 wild-type triple negative breast cancer (TNBC) cells by means of BET inhibitors and compounds already available in clinic. Performing a dual approach (chromatin immunoprecipitation and RNA interference), the direct relationship between BRD4 and BRCA1/RAD51 expression was confirmed in TNBC cells. Moreover, BRD4 pharmacological inhibition using two BET inhibitors (JQ1 and GSK525762A) induced a dose-dependent reduction in BRCA1 and RAD51 levels and is able to hinder homologous recombination-mediated DNA damage repair, generating a BRCAness phenotype in TNBC cells. Furthermore, BET inhibition impaired the ability of TNBC cells to overcome the increase in DNA damage after platinum salts (i.e., CDDP) exposure, leading to massive cell death, and triggered synthetic lethality when combined with PARP inhibitors (i.e., AZD2281). Altogether, the present study confirms that BET proteins directly regulate the homologous recombination pathway and their inhibition induced a BRCAness phenotype in BRCA1 wild-type TNBC cells. Noteworthy, being this strategy based on drugs already available for human use, it is rapidly transferable and could potentially enable clinicians to exploit platinum salts and PARP inhibitors-based treatments in a wider population of TNBC patients and not just in a specific subgroup, after validating clinical trials.

The DNA Methylation Maintenance Protein UHRF1 Regulates Fetal Globin Expression Independent of HBG Promoter DNA Methylation

Pharmacological or genetic induction of fetal hemoglobin (HbF, α2γ2) in adult red blood cells is a proven strategy to ameliorate the clinical symptoms of sickle cell disease (SCD) and β-thalassemia. Therefore, efforts are underway to better understand mechanisms that mediate the perinatal switch from HbF to adult hemoglobin (HbA, α2β2). We performed a CRISPR-Cas9/guide (g) RNA screen to identify novel proteins that regulate HbF production in HUDEP-2 cells, a human erythroid line that normally expresses HbA. We identified UHRF1 (ubiquitin-like with PHD and RING finger domains 1) as a repressor of HbF production. UHRF1 binds hemi-methylated DNA and recruit DNA methyltransferase 1 (DNMT1) to ensure faithful maintenance of DNA methylation during DNA replication. Numerous UHRF1-interacting proteins, including DNMT1, EHMT1/2 and HDAC2 are associated with γ-globin repression. We used CRISPR/Cas9 and RNA interference to validate UHRF1 as a HbF regulator. Compared to non-targeting gRNA UHRF1 disruption using Cas9 + 2 separate gRNAs increased the γ-globin/γ+β-globin RNA ratio from 1.9 to 25.8/27.1% (P<0.01), increased the fraction of HbF immunostaining cells (“F-cells”) from 7.5 to 25.1/35.4% and increased HbF protein from 2.10 to 16.3/15.0% (P<0.01) in HUDEP-2 cells. Compared to a control luciferase shRNA, 2 different UHRF1 shRNAs increased theγ-globin/γ+β-globin RNA ratio from 9.68% to 21.59/28.93% (P<0.01), increased the F-cell fraction from 37.9 to 49.8/55.6% and increased HbF protein from 9.1 to 16.18/18.5% (P<0.05) in erythroid cells derived from normal adult peripheral blood CD34+ cells. UHRF1 deficiency did not alter erythroid maturation or expression of key transcription factor genes that regulate HbF expression in HUDEP-2 or CD34+ cells (BCL11A, ZBTB7A, MYB and KLF1). UHRF1 mutant proteins defective in recognizing H3K9me2 (FW237/238AA), binding to hemi-methylated DNA (R491A) or ubiquitination of H3K23 to enhance DNMT1 recruitment (C741A), were unable to repress HBG1/HBG2. These mutations have the most profound effects on maintaining DNA methylation, indicating that UHRF1 represses HBG1/HBG2 in HUDEP-2 cells through this mechanism.UHRF1 knockout induced genome-wide demethylation including 6 CpG sites located at positions -162, -53, -50, +6, +17, +50 positions relative to the γ-globin (HBG1 and HBG2) transcription start site. Demethylation of these sites is thought to be required for γ-globin de-repression. However, forced demethylation of these cytosines in HUDEP-2 cells using specific gRNAs + dead (d) Cas9-TET1 was not sufficient to activate γ-globin expression when UHRF1 was present. Additionally, dCas9-DNMT3a-mediated methylation of the HBG promoter CpG residues in UHRF1 knockdown HUDEP-2 cells did not inhibit γ-globin expression in UHRF1 knockout HUDEP-2 cells. Based on these studies, we conclude that: 1) UHRF1 regulates γ-globin transcription; 2) demethylation of CpG sites at the HBG gene promoters is neither necessary or sufficient for γ-globin induction; 3) UHRF1 regulates γ-to-β globin gene switching either by methylating DNA regions other than those present around the HBG promoter or through non-canonical activities. Distinguishing these mechanisms will elucidate further our understanding of globin gene switching and could identify new pathways for pharmacological induction of HbF. DisclosuresNo relevant conflicts of interest to declare.

Domain-Focused CRISPR-Cas9 Screen Identifies the E3 Ubiquitin Ligase Substrate Adaptor Protein SPOP as a Novel Repressor of Fetal Hemoglobin

Elevated levels of fetal hemoglobin (HbF) are beneficial to patients with sickle cell disease (SCD) and b-thalassemia. The identification of pharmacologically controllable pathways that regulate HbF levels remains an important goal in the treatment of these disorders. BCL11A and LRF (ZBTB7A) are the two major transcriptional repressors of g-globin (fetal beta-type globin). However, since transcription factors are challenging to inhibit with small molecules, we aimed to identify novel HbF regulators that might be amenable to control by pharmacologic means.Using an improved protein-domain based CRISPR-Cas9 screen recently developed in our laboratory (Grevet et al., Science 361:6399, 285-290), we screened for new HbF regulators using a gRNAs library targeting chromatin associated proteins including BTB domain containing factors. We identified SPOP, a substrate adaptor of the CUL3 ubiquitin ligase complex, as a novel inhibitor of g-globin expression. Depletion of SPOP strongly elevated the mRNA and protein levels of g-globin in both HUDEP-2 and primary human CD34+ derived erythroid cells. The effects of SPOP loss were remarkably selective as evidenced by RNA-seq and whole cell mass spectrometry. Importantly, SPOP depletion did not appear to impair cell growth or differentiation. Mechanistically, we demonstrated that the repressive role of SPOP on g-globin levels is dependent on the CUL3 ubiquitin ligase complex. Additionally, forced expression of a dominant negative form of SPOP (Y87N) unable to bind to its substrates strongly induced HbF production. We also found that SPOP depletion did not measurably alter LRF levels and modestly reduced BCL11A levels. Overexpression of BCL11A did not restore g-globin silencing in SPOP depleted cells, indicating that SPOP works largely independently of these two factors. Finally, we tested whether the effects of SPOP depletion can be augmented through the use of experimental pharmacologic HbF inducers, such as pomalidomide. In proof of principle experiments we found that SPOP depletion and pomalidomide treatment induced HbF production to levels higher than either treatment alone.In sum, our work uncovered SPOP as a new regulator of HbF and suggests targeting SPOP alone or in combination with other HbF inducers may provide a novel therapeutic strategy for the treatment of hemoglobinopathies such as sickle cell disease and b-thalassemia. DisclosuresNo relevant conflicts of interest to declare.

Heme-Regulated Inhibitor (HRI) Loss and Pharmacologic Treatments Cooperate to Strongly Elevate Fetal Hemoglobin and Reduce Sickle Cell Formation

Increasing fetal hemoglobin (HbF) provides significant clinical benefit for patients with sickle cell disease (SCD) and is a critical goal that is being pursued with a variety of pharmacologic strategies. Hydroxyurea (HU) is currently the only FDA-approved drug aimed at raising HbF for SCD patients but is limited in its efficacy. Recent work in our laboratory utilizing a protein kinase-domain focused CRISPR-Cas9 based genetic screen identified heme-regulated inhibitor (HRI), an erythroid-specific protein kinase, as a novel HbF regulator (Grevet et al., Science 361:6399, 285-290). Depleting HRI in a human erythroid cell line and primary human erythroid cultures significantly raised HbF levels. Besides HU, several compounds have been recently identified as in vitro HbF inducers, including pomalidomide, a third-generation imide, and UNC0638, an EHMT 1/2 histone methyltransferase inhibitor. While all these approaches induce HbF in vitro, it remains to be seen whether these would achieve sufficient levels of HbF induction in patients when used as single agents. Although the specific mechanisms by which HRI, HU, pomalidomide and UNC0638 regulate HbF remain to be fully resolved, we hypothesized that they might work in distinct pathways, and that by combining them might improve effect size and diminish potential adverse effects on erythroid cell maturation and viability.We utilized a three-stage in vitro culture system that recapitulates normal erythropoiesis by terminal differentiation of adult CD34+ hematopoietic cells. Primary human CD34+ cells were treated with select pharmacologic inducers of HbF, including hydroxyurea, pomalidomide, or UNC0638, in combination with shRNA lentiviral knockdown of HRI. HbF levels were assessed by RT-qPCR, Western blot, flow cytometry, and cation-exchange HPLC. We find that knockdown of HRI leads to significant HbF induction as expected. We further observed an inverse correlation between remaining HRI levels and HbF induction with maximal effect size requiring 85%-90% HRI depletion. HU displayed comparatively little activity and failed to increase the effects of HRI knockdown. However, treatment with pharmacologic levels of pomalidomide or UNC0638 combined with HRI depletion each showed greater than additive effects in HbF with levels reaching 25-30% HbF for UNC0638-treated and 30-40% HbF for pomalidomide-treated combinations, suggesting underlying cooperativity of HbF induction. RT-qPCR and Western blot analyses suggest that the diminished expression of the HbF repressor BCL11A accounts in large part for HbF induction in HRI knockdown samples, particularly when combined with pomalidomide treatment in which Bcl11A depletion was greater than 90%, while other HbF repressors such as LRF were unchanged. To determine whether HbF induction results in reduced sickle cell formation, we performed combination HRI depletion and HbF pharmacologic induction in primary CD34+ cells derived from sickle cell patients. HRI depletion in primary SCD cells showed a marked increase in HbF from baseline levels; in addition, significant cooperativity with pomalidomide and UNC0638 was observed, achieving 45-50% HbF for UNC0638-treated and 50-60% HbF for pomalidomide-treated drug combinations with no apparent detrimental effects on erythroid differentiation or maturation. Importantly, combination of HRI knockdown and HbF pharmacologic treatments markedly reduced in vitro sickling as measured by low-oxygen sickling assays, suggesting significant amelioration of the sickle cell phenotype in vitro.Overall, we find that the combination of HbF pharmacologic induction and shRNA-mediated HRI inhibition results in significant cooperative upregulation of HbF levels in both normal and sickle cell-derived primary human cells without impairing red cell maturation. We are currently exploring additional potential synergies with HbF regulators to determine the optimal modalities to maximize HbF induction. While no effective and specific HRI inhibitors are currently available, our work suggests that future small molecule inhibition of HRI may be combined with other pharmacotherapies to achieve significant, clinically meaningful HbF induction for the treatment of SCD and other hemoglobinopathies. DisclosuresNo relevant conflicts of interest to declare.

Engineered Human Umbilical Cord Derived Erythroid Progenitor Cells (HUDEP2) with Sickle or β-Thalassemia Mutation: An in-Vitro System for Testing Pharmacological Induction of Fetal Hemoglobin

Introduction: Sickle cell disease (SCD) and β-thalassemia are inherited blood disorders caused by mutations in the β-globin gene (HBB). Elucidation of the multiple pathophysiologic mechanisms in SCD and β-thalassemia has resulted in an increasing efforts to identify new treatment modalities to ameliorate the consequences of the disease. However, no consistent in vitro system exists for studies of pharmacological therapies for the diseases. Human umbilical cord-derived erythroid progenitor cells (HUDEP2) are an immortalized CD34+ hematopoietic stem cell-derived erythroid precursor cell line that can differentiate into red blood cells. Here, we engineered sickle HUDEP2 and β-thalassemia HUDEP2 clonal lines through CRISPR/Cas9-mediated editing of the human HBB. We sought to establish if these engineered cell lines exhibit disease phenotypes, and if upon in vitro erythroid differentiation they produce fetal hemoglobin (HbF) in response to hydroxyurea, the only FDA-approved drug for HbF induction. Our goal is to create an in vitro system to test new HbF inducers for treating SCD or β-thalassemia.Materials and Methods: We delivered Hi-Fidelity Streptococcus pyogenes (Sp) Cas9 protein and CRISPR guide RNA as a ribonucleoprotein complex in conjunction with a single-stranded DNA donor (ssODN) template to introduce the sickle or K17X (A<T) or codon 6 [-G] β-thalassemia mutation into the HBB locus of HUDEP2 cells. Edited HUDEP2 cells were single-cell sorted into multiple 96-well plates and expanded. The genotype of the clones was determined using a probe-based droplet digital PCR assay and confirmed through Sanger sequencing. Native polyacrylamide gel electrophoresis and high-performance liquid chromatography (HPLC) were used to confirm the hemoglobin phenotype. Normal parental cell line, sickle clone, and two individual β-thalassemia clones were used to test the pharmacological induction of HbF. We initiated drug treatment in the expansion phase with 30 µM hydroxyurea. Trypan Blue staining and CD71/CD233/CD235 staining determined the effect of the drugs on the viability, growth rate and erythroid development of HUDEP2 lines. After 10 days of drug treatment, differentiated HUDEP2 were analyzed for globin expression through RT-qPCR and HPLC, and HbF positive cells (F-cells) were quantified via flow cytometry. Cells were placed at 2% O2 for four hours, fixed in glutaraldehyde, stained, and viewed under magnification to assess sickling potential.Results and Discussion: We generated multiple clones with biallelic sickle or β-thalassemia mutations. Sickle HUDEP2 clones almost exclusively expressed sickle hemoglobin with low level of HbF and hemoglobin A2 (HbA2), and β-thalassemia HUDEP2 clones produced no normal adult hemoglobin, 8-10% HbF, and 26-28% HbA2. On HPLC analysis, β-Thalassemia HUDEP2 clones had an unknown tall peak (39-45%) between HbF and HbA consistent with an α-globin homotetramer (α4). When subjected to hypoxic conditions for 4 hours, sickle HUDEP2 produced sickle cells. HUDEP2 parent cells did not sickle under hypoxic conditions. Hydroxyurea induced 3.8-fold, 1.8-fold, and 1.6-fold increases in γ-globin gene (HBG) expression; 2.9-fold, 1.4-fold, and 1.4-fold increases in the percentages of F-cells; 1.4-fold, 1.2-fold, and 1.6-fold increase in the percentages of HbF in sickle, K17X(A<T) and codon 6[-G] β-thalassemia HUDEP2 clones, respectively. No change was observed in CD71/CD235 positive HUDEP2 cells in the presence hydroxyurea. This finding demonstrated that hydroxyurea treatment induces HBG expression as well as HbF and F-cells in engineered sickle and β-thalassemia HUDEP2 clones. Future work will include screening other pharmacological compounds as well as studying the mechanism of HbF induction by using HUDEP2 clones.Conclusions: Our engineered sickle and β-thalassemia HUDEP2 cell lines have properties similar to those of patient erythroid cells and respond to the known HbF inducer hydroxyurea. This in vitro model system may facilitate the drug-discovery process by enabling multimodal drug screening on a large scale with consistent and reproducible results.Acknowledgments: This work was supported by the Cancer Prevention and Research Institute of Texas grants RR140081 and RP170721 (to G.B.) and the National Heart, Lung and Blood Institute of NIH (1K08DK110448 to V.S.) DisclosuresNo relevant conflicts of interest to declare.

How KEAP1 Deletion in Single Cell Mitigates Sickle Cell Pathology

Sickle cell disease (SCD) is one of the most frequent blood disorders caused by a point mutation of β-globin gene. In the pathology of SCD, we often encounter contributions of vascular and cellular components. The activation of vascular endothelial cells promotes adhesion of inflammatory cells and vaso-occlusion responsible for low blood flow, hypoxemia and organ damages. Hemolysis triggers activation and recruitment of myeloid cells to the inflammation site, further sustaining the inflammatory process. Recent progress has created the need to examine targeted induction of the KEAP1-NRF2 system as a potential therapeutic tool for the SCD. We have demonstrated that genetic as well as pharmacological induction of NRF2 in SCD prevent organ damages and suppress inflammation, although it still remains unclear how the NRF2 induction modifies the pathology or which type of cells contribute to the NRF2-mediated improvement of SCD. To address these questions, we conducted targeted deletion of Keap1 gene and induced expression of Nrf2 gene in endothelial cells and macrophages/monocytes using Tie1-Cre and LysM-Cre system, respectively. We found that SCD::Keap1F/F::Tie1-Cre mice (NRF2-increase in endothelial cells) showed low hematocrit and low platelet counts. While severe infiltration of monocytes, lymphocytes and macrophages was observed in the lungs of SCD mice, such phenotype was diminished in the SCD::Keap1F/F::Tie1-Cre mice. In the compound mutant mice, livers showed fewer necrotic zones and less to none accumulation of free iron. On the contrary, imaging MS (iMScope) analyses revealed decrease of free heme in livers and spleens in SCD::Keap1F/F::LysM-Cre mice (NRF2-increase in macrophages/monocytes). Furthermore, upregulations of most known Nrf2-dependent pathways and other yet-to-be-defined pathways are found in an RNA-sequencing analysis of the mice. Importantly, NRF2 induction enhanced heme catabolism and decreases the plasma level of free heme and the quantity of accumulated free heme in the SCD::Keap1F/F::LysM-Cre mouse tissues, whereas the change is not obvious in SCD::Keap1F/F::Tie1-Cre mice. On the contrary, vascular leakage of fluids was decreased in the SCD::Keap1F/F::Tie1-Cre mice. Thus, our results demonstrate that cell-lineage-specific activation of Nrf2 does inhibit the inflammation and organ injury with some particularities to each genotype. DisclosuresNo relevant conflicts of interest to declare.

Heterogeneity in Multi-Organ Expression of HO-1 and PlGF in Sickle Mice Mimic Exposure of Non-Sickle Mice to Extracellular Heme Via Nrf2-Dependent Pathways

Heme is a byproduct of hemolysis released into the circulation. Heme oxygenase-1 (HO-1), the rate limiting enzyme breaks down heme from damaged or senescent red blood cells, exerting antioxidant and anti-inflammatory cytoprotective effect, especially in sickle cell disease (SCD). HO-1 is regulated by heme through the NRF2 transcription factor. Placental Growth Factor (PlGF) is an angiogenic growth factor secreted by proliferating erythroblasts during normal development, which we previously have found is also activated by heme, secondarily regulating expression of the vasoconstrictor endothelin-1 that promotes pulmonary hypertension. Because hemolysis contributes to organ damage in SCD, our objective was to assess the multi-organ expression of HO-1 and PlGF in adult sickle (SS) mice as a marker of heme uptake and vascular injury respectively, and to identify most at risk organs during acute elevation of circulating extracellular heme. Furthermore, we investigated the transcriptional response of HO-1 and PlGF in different organs in C57BL/6J wild type (WT) and Nrf2 null mice following acute exposure to heme to understand the molecular pathways involved.HO-1 expression varied dramatically by organ in untreated SS mice, by real time PCR. Using Delta Ct (most negative=higher expression), there was heterogeneity in expression of HO-1 among different organs (p<0.0001, One-way ANOVA). Spleen (-5.1±0.1), liver (-4.3±0.0) and kidney (-2.5±0.6) showed higher expression compared to other organs (Fig. 1a). PlGF expression on the other hand, was higher in the heart (2.9±0.3), lung (3.1±0.2) and kidney (5.0±0.6) (p<0.0001, One-way ANOVA). To mimic acute hemolysis, 50µmoles/kg bodyweight of heme was infused into SS mice, and organs were harvested 3 hours later. The expression of HO-1 and PlGF was strongly induced (p<0.0001, One-way ANOVA), augmenting the previous pattern observed in untreated SS mice. Surprisingly, we observed a submaximal response to HO-1 induction in the liver of SS mice injected with hemin compared to control. We confirmed the variability in organ response to heme in WT mice injected intravenously with saline or hemin (120µmoles/kg body weight), followed by harvest of the spleen, liver, kidney, lung and heart 3 hours after injection. Heme oxygenase-1 mRNA levels increased sharply in organs from WT mice that received hemin compared with saline control: liver (207-fold), heart (201-fold), kidney (101-fold), lung (69-fold) and spleen (7-fold). This induction was largely Nrf2-dependent, with 59-78% lower response in Nrf2 null mice in every organ except kidney. The 101-fold induction of HO-1 in the kidney was essentially Nrf2 independent. Next, we measured heme content of each organ 3 hours after injection of heme. The organs with highest heme induction of HO-1 appeared to clear heme more rapidly (R2=0.82, p=0.03) (Fig. 1b) and interestingly, the expression of the heme importer Hrg1 was also induced significantly in these organs (R2=0.82, p=0.004). PlGF expression in WT mice was induced by heme more strongly in the heart, lung and kidney (parallel to SS mice) and was approximately 50% Nrf2-dependent in all organs sampled.The reticulo-endothelial organs have been shown by others to take up free heme avidly, but these are the first data to demonstrate widely ranging differences in heme response among other organs, and the degree of HO-1 induction in the heart is surprisingly comparable to liver and kidney. Also, pharmacological induction of HO-I has been proposed as a therapy in SCD. Our result showing submaximal induction of HO-1 in SS mice suggests suboptimal expression of HO-1 in SCD. This is consistent with published benefit in sickle cell mice of HO-1 augmentation by gene transfer or Nrf2-inducing agents. Understanding the mechanism of organ-specific response to heme and HO-1/PGF induction could justify the introduction of early therapy to mitigate heme-mediated injury to organs and tissues in SCD and slow progression to organ failure, especially in the aging population of adults with SCD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Role of Adenosine Monophosphate Activated Protein Kinase Alpha 1 (AMPK) in Gamma-Globin Regulation

Background: Fetal hemoglobin (HbF, α2g2) induction has long been an area of investigation, as it is known to reduce the clinical complications of sickle cell disease (SCD) and beta thalassemia. Progress in identifying novel HbF inducing strategies has been stymied by an incomplete understanding of gamma-globin regulation. We used natural genetic variation to identify novel genes and pathways associated with HbF levels in patients with SCD. Our whole exome sequencing analysis of 1290 samples from patients with SCD identified the insulin signaling pathway to be related to HbF regulation. Functional studies performed in hematopoietic stem and progenitor cells (HSPCs) from patients with SCD established that FOXO3 is a positive regulator of HbF, and that metformin, a FOXO3 and AMPK activator, can induce HbF (Zhang et al, Blood 2018). We hypothesized that other proteins in the insulin signaling pathway, particularly AMPK, a direct activator of FOXO3, may contribute to HbF regulation and be a potential target for pharmacologic induction of HbF.Objectives: We now seek to determine the role of AMPK and AMPK activators such as piceatannol in HbF regulation through functional studies in HSPCs from patients with SCD.Methods: HSPCs from 3 unique patients with SCD were transduced with AMPK shRNA on day 5 of two phase primary erythroid culture. AMPK, FOXO3, gamma and beta globin gene expression were measured by RT-qPCR and HbF by HPLC respectively on day 14 of culture. HSPCs from 3 unique patients with SCD were treated with AICAR, piceatannol at 12.5µM and metformin at 100 µM on day 7 of erythroid culture. Cell lysate was collected on day 14, and AMPK, FOXO3, gamma and beta globin gene expression and protein levels measured by RT-qPCR and western blot respectively. Levels of pAMPK, at Thr172, were quantified by western blot. 1 µM Compound C was added with piceatannol and with metformin in separate erythroid cultures on day 7, and the effect on gamma globin and phosphorylation of AMPK at Thr172 was measured on day 14 by RT-qPCR and western blot respectively.Results: 70% knockdown of AMPK resulted in a 50% decrease in HbF (p<0.01) and a three-fold reduction in gamma-globin expression (p<0.001). HSPCs treated with metformin or piceatannol exhibited a 2-3 fold rise in AMPK, FOXO3 and gamma globin gene expression (p<0.001). HSPCs treated with piceatannol and metformin showed an increase in pAMPK at Thr172, the activated form of AMPK. In the presence of a specific AMPK inhibitor, Compound C, metformin and piceatannol, no induction of gamma globin was observed (Figure 1), and pAMPK was reduced to untreated levels.Conclusions: Knockdown of AMPK in HSPCs reduces gamma globin expression and %HbF, supporting the role of AMPK in gamma globin regulation. Drugs known to activate AMPK, metformin and piceatannol, increase gamma globin in SCD patient derived HSPCs. Pharmacologic blockage of AMPK activity with Compound C results in reduction of HbF induction, and reduces the gamma globin induction of metformin and piceatannol to untreated levels. We therefore conclude that AMPK is a positive regulator of HbF, and that pharmacologic induction of HbF with metformin and piceatannol requires AMPK activity. Further work is needed to establish if FOXO3 and AMPK alone are instrumental in HbF regulation, or if other proteins in the insulin signaling pathway may play a role in HbF regulation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pharmacological induction of Heat Shock Protein 70 by celastrol protects motoneurons from excitotoxicity in rat spinal cord in vitro

The secondary phase of spinal cord injury arising after the primary lesion largely extends the damage severity with delayed negative consequences for sensory-motor pathways. It is, therefore, important to find out if enhancing intrinsic mechanisms of neuroprotection can spare motoneurons that are very vulnerable cells. This issue was investigated with an in vitro model of rat spinal cord excitotoxicity monitored for up to 24hr after the primary injury evoked by kainate. This study sought to pharmacologically boost the expression of heat shock proteins (HSP) to protect spinal motoneurons using celastrol to investigate if the rat spinal cord can upregulate HSP as neuroprotective mechanism. Despite its narrow range of drug safety in vitro, celastrol was not toxic to the rat spinal cord at 0.75μM concentration and enhanced the expression of HSP70 by motoneurons. When celastrol was applied either before or after kainate, the number of dead motoneurons was significantly decreased and the nuclear localization of the cell death biomarker AIF strongly inhibited. Nevertheless, electrophysiological recording showed that protection of lumbar motor networks by celastrol was rather limited as reflex activity was impaired and fictive locomotion largely depressed, suggesting that functional deficit persisted, though the networks could express slow rhythmic oscillations. While our data do not exclude further recovery at later times beyond the experimental observations, the present results indicate that the upregulated expression of HSP in the aftermath of acute injury may be an interesting avenue for early protection of spinal motoneurons.

NOXA genetic amplification or pharmacologic induction primes lymphoma cells to BCL2 inhibitor-induced cell death

Although diffuse large B cell lymphoma (DLBCL) cells widely express the BCL2 protein, they rarely respond to treatment with BCL2-selective inhibitors. Here we show that DLBCL cells harboring PMAIP1/NOXA gene amplification were highly sensitive to BCL2 small-molecule inhibitors. In these cells, BCL2 inhibition induced cell death by activating caspase 9, which was further amplified by caspase-dependent cleavage and depletion of MCL1. In DLBCL cells lacking NOXA amplification, BCL2 inhibition was associated with an increase in MCL1 protein abundance in a BIM-dependent manner, causing a decreased antilymphoma efficacy. In these cells, dual inhibition of MCL1 and BCL2 was required for enhanced killing. Pharmacologic induction of NOXA, using the histone deacetylase inhibitor panobinostat, decreased MCL1 protein abundance and increased lymphoma cell vulnerability to BCL2 inhibitors in vitro and in vivo. Our data provide a mechanistic rationale for combination strategies to disrupt lymphoma cell codependency on BCL2 and MCL1 proteins in DLBCL.

Intravenous Heme Arginate Induces HO-1 (Heme Oxygenase-1) in the Human Heart.

Objective- HO-1 (heme oxygenase-1) induction may prevent or reduce ischemia-reperfusion injury. We previously evaluated its in vivo induction after a single systemic administration of heme arginate in peripheral blood mononuclear cells. The current trial was designed to assess the pharmacological tissue induction of HO-1 in the human heart with heme arginate in vivo. Approach and Results- Patients planned for conventional aortic valve replacement received placebo (n=8), 1 mg/kg (n=7) or 3 mg/kg (n=9) heme arginate infused intravenously 24 hours before surgery. A biopsy of the right ventricle was performed directly before aortic cross-clamping and after cross-clamp release. In addition, the right atrial appendage was partially removed for analysis. HO-1 protein and mRNA concentrations were measured in tissue samples and in peripheral blood mononuclear cells before to and up to 72 hours after surgery. No study medication-related adverse events occurred. A strong, dose-dependent effect on myocardial HO-1 mRNA levels was observed (right ventricle: 7.9±5.0 versus 88.6±49.1 versus 203.6±148.7; P=0.002 and right atrium: 10.8±8.8 versus 229.8±173.1 versus 392.7±195.7; P=0.001). This was paralleled by a profound increase of HO-1 protein concentration in atrial tissue (8401±3889 versus 28 585±10 692 versus 29 022±8583; P<0.001). Surgery and heme arginate infusion significantly increased HO-1 mRNA concentration in peripheral blood mononuclear cells ( P<0.001). HO-1 induction led to a significant increase of postoperative carboxyhemoglobin (1.7% versus 1.4%; P=0.041). No effect on plasma HO-1 protein levels could be detected. Conclusions- Myocardial HO-1 mRNA and protein can be dose-dependently induced by heme arginate. Protective effects of this therapeutic strategy should be evaluated in upcoming clinical trials. Clinical Trial Registration- URL: http://www.clinicaltrials.gov . Unique identifier: NCT02314780.

Defective transcription elongation in a subset of cancers confers immunotherapy resistance

The nature and role of global transcriptional deregulations in cancers are not fully understood. We report that a large proportion of cancers have widespread defects in mRNA transcription elongation (TE). Cancers with TE defects (TEdeff) display spurious transcription and defective mRNA processing of genes characterized by long genomic length, poised promoters and inducible expression. Signaling pathways regulated by such genes, such as pro-inflammatory response pathways, are consistently suppressed in TEdeff tumors. Remarkably, TEdeff correlates with the poor response and outcome in immunotherapy, but not chemo- or targeted therapy, -treated renal cell carcinoma and metastatic melanoma patients. Forced pharmacologic or genetic induction of TEdeff in tumor cells impairs pro-inflammatory response signaling, and imposes resistance to the innate and adaptive anti-tumor immune responses and checkpoint inhibitor therapy in vivo. Therefore, defective TE is a previously unknown mechanism of tumor immune resistance, and should be assessed in cancer patients undergoing immunotherapy.

Heme oxygenase-1 induction attenuates senescence in chronic obstructive pulmonary disease lung fibroblasts by protecting against mitochondria dysfunction.

Chronic obstructive pulmonary disease (COPD) is associated with lung fibroblast senescence, a process characterized by an irreversible proliferation arrest associated with secretion of inflammatory mediators. ROS production, known to induce senescence, is increased in COPD fibroblasts and mitochondria dysfunction participates in this process. Among the battery of cellular responses against oxidative stress damage, heme oxygenase (HO)‐1 plays a critical role in defending the lung against oxidative stress and inflammation. Therefore, we investigated whether pharmacological induction of HO‐1 by chronic hemin treatment attenuates senescence and improves dysfunctional mitochondria in COPD fibroblasts. Fibroblasts from smoker controls (S‐C) and COPD patients were isolated from lung biopsies. Fibroblasts were long‐term cultured in the presence or absence of hemin, and/or ZnPP or QC‐15 (HO‐1 inhibitors). Lung fibroblasts from smokers and COPD patients displayed in long‐term culture a senescent phenotype, characterized by a reduced replicative capacity, an increased senescence and inflammatory profile. These parameters were significantly higher in senescent COPD fibroblasts which also exhibited decreased mitochondrial activity (respiration, glycolysis, and ATP levels) which led to an increased production of ROS, and mitochondria biogenesis and impaired mitophagy process. Exposure to hemin increased the gene and protein expression level of HO‐1 in fibroblasts and diminished ROS levels, senescence, the inflammatory profile and simultaneously rescued mitochondria dysfunction by restoring mitophagy in COPD cells. The effects of hemin were abolished by a cotreatment with ZnPP or QC‐15. We conclude that HO‐1 attenuates senescence in COPD fibroblasts by protecting, at least in part, against mitochondria dysfunction and restoring mitophagy.

Heat Shock Proteins as a Potential Therapeutic Target in the Treatment of Gestational Diabetes Mellitus: What We Know so Far.

Gestational diabetes mellitus (GDM) is a complex condition that involves a variety of pathological mechanisms, including pancreatic β-cell failure, insulin resistance, and inflammation. There is an increasing body of literature suggesting that these interrelated phenomena may arise from the common mechanism of endoplasmic reticulum (ER) stress. Both obesity-associated nutrient excess and hyperglycemia disturb ER function in protein folding and transport. This results in the accumulation of polypeptides in the ER lumen and impairs insulin secretion and signaling. Exercise elicits metabolic adaptive responses, which may help to restore normal chaperone expression in insulin-resistant tissues. Pharmacological induction of chaperones, mimicking the metabolic effect of exercise, is a promising therapeutic tool for preventing GDM by maintaining the body’s natural stress response. Metformin, a commonly used diabetes medication, has recently been identified as a modulator of ER-stress-associated inflammation. The results of recent studies suggest the potential use of chemical ER chaperones and antioxidant vitamins as therapeutic interventions that can prevent glucose-induced ER stress in GDM placentas. In this review, we discuss whether chaperones may significantly contribute to the pathogenesis of GDM, as well as whether they can be a potential therapeutic target in GDM treatment.

Hydroxypropyl-β-cyclodextrin protects from kidney disease in experimental Alport syndrome and focal segmental glomerulosclerosis

Studies suggest that altered renal lipid metabolism plays a role in the pathogenesis of diabetic kidney disease and that genetic or pharmacological induction of cholesterol efflux protects from the development of diabetic kidney disease and focal segmental glomerulosclerosis (FSGS). Here we tested whether altered lipid metabolism contributes to renal failure in the Col4a3 knockout mouse model for Alport Syndrome. There was an eight-fold increase in the cholesterol content in renal cortexes of mice with Alport Syndrome. This was associated with increased glomerular lipid droplets and cholesterol crystals. Treatment of mice with Alport Syndrome with hydroxypropyl-β-cyclodextrin (HPβCD) reduced cholesterol content in the kidneys of mice with Alport Syndrome and protected from the development of albuminuria, renal failure, inflammation and tubulointerstitial fibrosis. Cholesterol efflux and trafficking-related genes were primarily affected in mice with Alport Syndrome and were differentially regulated in the kidney cortex and isolated glomeruli. HPβCD also protected from proteinuria and mesangial expansion in a second model of non-metabolic kidney disease, adriamycin-induced nephropathy. Consistent with our experimental findings, microarray analysis confirmed dysregulation of several lipid-related genes in glomeruli isolated from kidney biopsies of patients with primary FSGS enrolled in the NEPTUNE study. Thus, lipid dysmetabolism occurs in non-metabolic glomerular disorders such as Alport Syndrome and FSGS, and HPβCD improves renal function in experimental Alport Syndrome and FSGS.

Reduction in polyamine catabolism leads to spermine-mediated airway epithelial injury and induces asthma features.

Airway epithelial injury is a crucial component of acute and severe asthma pathogenesis and a promising target for treatment of refractory asthma. However, the underlying mechanism of epithelial injury remains poorly explored. Although high levels of polyamines, mainly spermine, have been found in asthma and comorbidity, their role in airway epithelial injury and the cause of their altered levels in asthma have not been explored. We measured key polyamine metabolic enzymes in lung samples from normal and asthmatic subjects and in mice with OVA-induced allergic airway inflammation (AAI). Polyamine metabolism was modulated using pharmacologic/genetic modulators. Epithelial stress and apoptosis were measured by TSLP levels and TUNEL assay, respectively. We found loss of the polyamine catabolic enzymes spermidine/spermine-N (1)-acetyltransferase-1 (SAT1) and spermine oxidase (SMOX) predominantly in bronchial epithelial cells (BECs) of human asthmatic lung samples and mice with AAI. In naïve mice, SAT1 or SMOX knockdown led to airway hyper-responsiveness, remodeling, and BEC apoptosis. Conversely, in mice with AAI, overexpression of either SAT1 or SMOX alleviated asthmatic features and reduced TSLP levels and BEC apoptosis. Similarly, while pharmacological induction of SAT1 and SMOX using the polyamine analogue bis(ethyl)norspermine (BENSPM) alleviated asthmatic features with reduced TSLP levels and BEC apoptosis, pharmacological inhibition of these enzymes using BERENIL or MDL72527, respectively, worsened them. Spermine accumulation in lungs correlated with BEC apoptosis, and spermine treatment caused apoptosis of human BEAS-2B cells invitro. Spermine induces BEC injury. Induction of polyamine catabolism may represent a novel therapeutic approach for asthma via reversing BEC stress.

Heme Oxygenase-1 as a Modulator of Intestinal Inflammation Development and Progression.

Heme Oxygenase 1 (HMOX1) is an enzyme that catalyzes the reaction that degrades the heme group contained in several important proteins, such as hemoglobin, myoglobin, and cytochrome p450. The enzymatic reaction catalyzed by HMOX1 generates Fe2+, biliverdin and CO. It has been shown that HMOX1 activity and the by-product CO can downmodulate the damaging immune response in several models of intestinal inflammation as a result of pharmacological induction of HMOX1 expression and the administration of non-toxic amounts of CO. Inflammatory Bowel Diseases, which includes Crohn's Disease (CD) and Ulcerative Colitis (UC), are one of the most studied ailments associated to HMOX1 effects. However, microbiota imbalances and infections are also important factors influencing the occurrence of acute and chronic intestinal inflammation, where HMOX1 activity may play a major role. As part of this article we discuss the immune modulatory capacity of HMOX1 during IBD, as well during the infections and interactions with the microbiota that contribute to this inflammatory disease.

Avoiding Induction of Labour for Prolonged Pregnancy

Current national guidelines in many countries recommend that otherwise healthy pregnant women be offered induction of labour if the pregnancy exceeds 10 days beyond term. Many women in this situation wish to avoid clinical or pharmacological induction techniques, and seek advice on alternatives from their midwives. The UK northwest midwives research group conducted a survey of midwives based on four sites. The formal evidence base for the most frequently recommended practices was then reviewed. The aim was to identify midwives’ knowledge of techniques to avoid clinical or pharmacological induction of labour for post-maturity in healthy women and babies; the related advice they would give to women; and the formal evidence base for those techniques most frequently recommended.

FoxO Function Is Essential for Maintenance of Autophagic Flux and Neuronal Morphogenesis in Adult Neurogenesis

Autophagy is a conserved catabolic pathway with emerging functions in mammalian neurodevelopment and human neurodevelopmental diseases. The mechanisms controlling autophagy in neuronal development are not fully understood. Here, we found that conditional deletion of the Forkhead BoxO transcription factors FoxO1, FoxO3, and FoxO4 strongly impaired autophagic flux in developing neurons of the adult mouse hippocampus. Moreover, FoxO deficiency led to altered dendritic morphology, increased spine density, and aberrant spine positioning in adult-generated neurons. Strikingly, pharmacological induction of autophagy was sufficient to correct abnormal dendrite and spine development of FoxO-deficient neurons. Collectively, these findings reveal a novel link between FoxO transcription factors, autophagic flux, and maturation of developing neurons.