Histone Methyltransferase Inhibitor Research Articles

The epigenetic treatment remodel genome-wide histone H4 hyper-acetylation patterns and affect signaling pathways in acute promyelocytic leukemia cells

Although majority of acute promyelocytic leukemia (APL) patients achieve complete remission after the standard treatment, 5–10% of patients are shown to relapse or develop resistance to treatment. In such cases, medications that target epigenetic processes could become an appealing supplementary approach. In this study, we tested the anti-leukemic activity of histone deacetylase inhibitor Belinostat (PXD101) and histone methyltransferase inhibitor 3-Deazaneplanocin A combined with all-trans retinoic acid in APL cells NB4, promyelocytes resembling HL-60 cells and APL patients' cells. After HL-60 and NB4 cell treatment, ChIP-sequencing was performed using antibodies against hyper-acetylated histone H4. Hyper-acetylated histone H4 distribution peaks were compared in treated vs untreated HL-60 and NB4 cells. Results demonstrated that in treated HL-60 cells, the majority of peaks were distributed within the regions of proximal promoters, whereas in treated NB4 cells, hyper-acetylated histone H4 peaks were mainly localized in gene body regions. Further ChIP-seq data analysis revealed the changes in histone H4 hyper-acetylation in promoter/gene body regions of genes involved in cancer signaling pathways. In addition, quantitative gene expression analysis proved changes in various cellular pathways important for carcinogenesis. Epigenetic treatment down-regulated the expression of MTOR, LAMTOR1, WNT2B, VEGFR3, FGF2, FGFR1, TGFA, TGFB1, TGFBR1, PDGFA, PDGFRA and PDGFRB genes in NB4, HL-60 and APL patients’ cells. In addition, effect of epigenetic treatment on protein expression of aforementioned signaling pathways was confirmed with mass spectrometry analysis. Taken together, these results provide supplementary insights into molecular changes that occur during epigenetic therapy application in in vitro promyelocytic leukemia cell model.

Covalent inhibition of NSD1 histone methyltransferase.

The NSD family of histone methyltransferases is associated with various malignancies, including aggressive acute leukemia with NUP98-NSD1 translocation. While NSD proteins represent attractive drug targets, their catalytic SET domains exist in autoinhibited conformation, presenting significant challenges for inhibitor development. Here, we employed a fragment-based screening strategy followed by chemical optimization, which resulted in development of the first-in-class irreversible small molecule inhibitors of the NSD1 SET domain. The crystal structure of NSD1 in complex with covalently bound ligand reveals conformational change in the autoinhibitory loop of the SET domain and formation of a channel-like pocket suitable for targeting with small molecules. Our covalent lead, compound BT5, demonstrates on-target activity in NUP98-NSD1 leukemia cells, including inhibition of H3K36 dimethylation and downregulation of target genes, and impairs colony formation in NUP98-NSD1 patient sample. This study will facilitate development of the next generation of potent and selective inhibitors of the NSD histone methyltransferases.

Long-Term Effect of Post-traumatic Stress in Adolescence on Dendrite Development and H3K9me2/BDNF Expression in Male Rat Hippocampus and Prefrontal Cortex.

Exposure to a harsh environment in early life increases in the risk of post-traumatic stress disorder (PTSD) of an individual. Brain derived neurotrophic factor (BDNF) plays an important role in neurodevelopment in developmental stages. Both chronic and traumatic stresses induce a decrease in the level of BDNF and reduce neural plasticity, which is linked to the pathogenesis of PTSD. Also, studies have shown that stress alters the epigenetic marker H3K9me2, which can bind to the promoter region of the Bdnf gene and reduce BDNF protein level. However, the long-term effects of traumatic stress during adolescence on H3K9me2, BDNF expression and dendrite development are not well-known. The present study established a model of PTSD in adolescent rats using an inescapable foot shock (IFS) procedure. Anxiety-like behaviors, social interaction behavior and memory function were assessed by the open field test, elevated plus maze test, three-chamber sociability test and Morris water maze test. In addition, neuronal development and H3K9me2/BDNF expression in hippocampus (HIP) and prefrontal cortex (PFC) were evaluated by Golgi staining, western blotting, qRT-PCR analysis and CHIP-qPCR analysis. Additionally, the Unc0642, a small molecule inhibitor of histone methyltransferase (EHMT2) was used for intervention. The results showed that the IFS procedure induced the PTSD-like behaviors in rats, resulted in fewer dendrite branches and shorter dendrite length in CA1 of HIP and PFC, increased H3K9me2 level and decreased BDNF expression in HIP and PFC. Also, although all the changes can persist to adulthood, Unc0642 administration relieved most of alterations. Our study suggests that traumatic stress in adolescence leads to immediate and long-term mental disorders, neuronal morphological changes, lower BDNF level and increased H3K9me2 level in the HIP and PFC, indicating that H3K9me2/BDNF dysfunction plays a key role in pathogenesis of PTSD.

Hypomethylating Agents in Lymphoma.

Epigenetic mutations are frequent and pathogenic in select subtypes of lymphoma, and agents modulating DNA and histone methylation-such as inhibitors of DNMT and EZH2, respectively-have demonstrated promise in treating these diseases. In particular, lymphomas derived from the germinal center-GC-DLBCL, FL, and AITL-are all characterized by epigenetic derangements. In an effort to target these derangements, DNMT inhibitors have been investigated as a means of improving responsiveness to chemotherapy in DLBCL patients, or as monotherapy or in combination with other epigenetic agents in the treatment of TCL. Histone methyltransferase inhibitors have demonstrated effectiveness in R/R FL patients with EZH2-activating mutations. New treatment options that target the pathogenesis of disease are needed. HDAC inhibitors have been in the clinic for over a decade for the treatment of lymphoma, and now methyltransferase inhibitors are finding their niche for this disease.

HMTase Inhibitors as a Potential Epigenetic-Based Therapeutic Approach for Friedreich's Ataxia.

Friedreich’s ataxia (FRDA) is a progressive neurodegenerative disorder caused by a homozygous GAA repeat expansion mutation in intron 1 of the frataxin gene (FXN), which instigates reduced transcription. As a consequence, reduced levels of frataxin protein lead to mitochondrial iron accumulation, oxidative stress, and ultimately cell death; particularly in dorsal root ganglia (DRG) sensory neurons and the dentate nucleus of the cerebellum. In addition to neurological disability, FRDA is associated with cardiomyopathy, diabetes mellitus, and skeletal deformities. Currently there is no effective treatment for FRDA and patients die prematurely. Recent findings suggest that abnormal GAA expansion plays a role in histone modification, subjecting the FXN gene to heterochromatin silencing. Therefore, as an epigenetic-based therapy, we investigated the efficacy and tolerability of two histone methyltransferase (HMTase) inhibitor compounds, BIX0194 (G9a-inhibitor) and GSK126 (EZH2-inhibitor), to specifically target and reduce H3K9me2/3 and H3K27me3 levels, respectively, in FRDA fibroblasts. We show that a combination treatment of BIX0194 and GSK126, significantly increased FXN gene expression levels and reduced the repressive histone marks. However, no increase in frataxin protein levels was observed. Nevertheless, our results are still promising and may encourage to investigate HMTase inhibitors with other synergistic epigenetic-based therapies for further preliminary studies.

The Promise for Histone Methyltransferase Inhibitors for Epigenetic Therapy in Clinical Oncology: A Narrative Review.

Epigenetic processes are essential for normal development and the maintenance of tissue-specific gene expression in mammals. Changes in gene expression and malignant cellular transformation can result from disruption of epigenetic mechanisms, and global disruption in the epigenetic landscape is a key feature of cancer. The study of epigenetics in cancer has revealed that human cancer cells harbor both genetic alterations and epigenetic abnormalities that interplay at all stages of cancer development. Unlike genetic mutations, epigenetic aberrations are potentially reversible through epigenetic therapy, providing a therapeutically relevant treatment option. Histone methyltransferase inhibitors are emerging as an epigenetic therapy approach with great promise in the field of clinical oncology. The recent accelerated approval of the enhancer of zeste homolog 2 (EZH2; also known as histone-lysine N-methyltransferase EZH2) inhibitor tazemetostat for metastatic or locally advanced epithelioid sarcoma marks the first approval of such a compound for the treatment of cancer. Many other histone methyltransferase inhibitors are currently in development, some of which are being tested in clinical studies. This review focuses on histone methyltransferase inhibitors, highlighting their potential in the treatment of cancer. We also discuss the role for such epigenetic drugs in overcoming epigenetically driven drug resistance mechanisms, and their value in combination with other therapeutic approaches such as immunotherapy.

A Histone Methyltransferase Inhibitor Can Reverse Epigenetically Acquired Drug Resistance in the Malaria Parasite Plasmodium falciparum.

Malaria parasites invade and replicate within red blood cells (RBCs), extensively modifying their structure and gaining access to the extracellular environment by placing the plasmodial surface anion channel (PSAC) into the RBC membrane. Expression of members of the cytoadherence linked antigen gene 3 (clag3) family is required for PSAC activity, a process that is regulated epigenetically. PSAC is a well-established route of uptake for large, hydrophilic antimalarial compounds, and parasites can acquire resistance by silencing clag3 gene expression, thereby reducing drug uptake. We found that exposure to sub-IC50 concentrations of the histone methyltransferase inhibitor chaetocin caused substantial changes in both clag3 gene expression and RBC permeability, and reversed acquired resistance to the antimalarial compound blasticidin S that is transported through PSACs. Chaetocin treatment also altered progression of parasites through their replicative cycle, presumably by changing their ability to modify chromatin appropriately to enable DNA replication. These results indicate that targeting histone modifiers could represent a novel tool for reversing epigenetically acquired drug resistance in P. falciparum.

Exon Skipping in Directly Reprogrammed Myotubes Obtained from Human Urine-Derived Cells.

Duchenne muscular dystrophy (DMD), a progressive and fatal muscle disease, is caused by mutations in the DMD gene that result in the absence of dystrophin protein. To date, we have completed an investigator-initiated first-in-human study at the National Center of Neurology and Psychiatry based on the systemic injection of the morpholino oligonucleotides which are prone to exon-53 skipping. For the effective treatment of DMD, in vitro testing with myoblasts derived from DMD patients to screen drugs and assess patient eligibility before undertaking clinical trials is thought to be essential. Very recently, we reported a new MYOD1-converted urine-derived cell (UDC) treated with the histone methyltransferase inhibitor (3-deazaneplanocin A hydrochloride), as a cellular model of DMD. The new autologous UDC might show phenocopy of the disease-specific phenotypes of DMD, leading to the application of precision medicine in a variety of muscle-related diseases. In this article, we describe a detailed protocol for efficient modelling of DMD muscle cells using MYOD1-converted UDCs along with reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry to evaluate the restoration of dystrophin mRNA and protein levels after exon skipping.

Epigenetically modifying the Foxp3 locus for generation of stable antigen-specific Tregs as cellular therapeutics.

Foxp3+ regulatory T cells (Tregs) are potent immunoregulatory cells, prompting strong interests in manipulating them for therapeutic purposes. However, significant challenges remain, including their heterogeneity and functional instability. Here we focused on the inducible Tregs (iTregs) and studied whether the Foxp3 locus can be epigenetically edited ex vivo to produce stable therapeutic iTregs. Under iTreg-inducing condition where activated CD4+ T effector cells were converted to Foxp3+ Tregs, we tested approximately 30 compounds and identified 3 chromatin-modifying chemical compounds (3C) consisting of sodium butyrate (a broad histone deacetylase inhibitor), UNC0646 (a histone methyltransferase inhibitor), and vitamin C (a TET dioxygenase co-activator), that together produced complete demethylation at the conserved noncoding sequence 2 (CNS2) region of Foxp3 locus. We found that iTregs induced in the presence of 3C (3C-iTregs) are stable, even after exposure to inflammatory cytokines. They expressed high levels of Foxp3 and exhibited potent suppressive activities both in vitro and in vivo. We showed that in models of autoimmunity and transplant rejection, adoptive transfer of antigen-specific 3C-iTregs prevented the induction of experimental autoimmune encephalitis and enabled long-term skin allograft survival. Our data demonstrate that the Foxp3 locus can be epigenetically edited ex vivo to generate stable therapeutic iTregs.

Small Molecules Targeting H3K9 Methylation Prevent Silencing of Reactivated FMR1 Alleles in Fragile X Syndrome Patient Derived Cells.

In fragile X syndrome (FXS), expansion of a CGG repeat tract in the 5′-untranslated region of the FMR1 gene to >200 repeats causes transcriptional silencing by inducing heterochromatin formation. Understanding the mechanism of FMR1 silencing is important as gene reactivation is a potential treatment approach for FXS. To date, only the DNA demethylating drug 5-azadeoxycytidine (AZA) has proved effective at gene reactivation; however, this drug is toxic. The repressive H3K9 methylation mark is enriched on the FMR1 gene in FXS patient cells and is thus a potential druggable target. However, its contribution to the silencing process is unclear. Here, we studied the effect of small molecule inhibitors of H3K9 methylation on FMR1 expression in FXS patient cells. Chaetocin showed a small effect on FMR1 gene reactivation and a synergistic effect on FMR1 mRNA levels when used in combination with AZA. Additionally, chaetocin, BIX01294 and 3-Deazaneplanocin A (DZNep) were able to significantly delay the re-silencing of AZA-reactivated FMR1 alleles. These data are consistent with the idea that H3K9 methylation precedes DNA methylation and that removal of DNA methylation is necessary to see the optimal effect of histone methyl-transferase (HMT) inhibitors on FMR1 gene expression. Nonetheless, our data also show that drugs targeting repressive H3K9 methylation marks are able to produce sustained reactivation of the FMR1 gene after a single dose of AZA.

Targeting p53 and histone methyltransferases restores exhausted CD8+ T cells in HCV infection

Hepatitis C virus infection (HCV) represents a unique model to characterize, from early to late stages of infection, the T cell differentiation process leading to exhaustion of human CD8+ T cells. Here we show that in early HCV infection, exhaustion-committed virus-specific CD8+ T cells display a marked upregulation of transcription associated with impaired glycolytic and mitochondrial functions, that are linked to enhanced ataxia-telangiectasia mutated (ATM) and p53 signaling. After evolution to chronic infection, exhaustion of HCV-specific T cell responses is instead characterized by a broad gene downregulation associated with a wide metabolic and anti-viral function impairment, which can be rescued by histone methyltransferase inhibitors. These results have implications not only for treatment of HCV-positive patients not responding to last-generation antivirals, but also for other chronic pathologies associated with T cell dysfunction, including cancer.

Chaetocin Abrogates the Self-Renewal of Bladder Cancer Stem Cells <i>via</i> the Suppression of KMT1A-GATA3-STAT3 Circuit

Background: Bladder cancer stem cells (BCSCs) have the abilities of self-renewal, differentiation, metastasis, conferring drug resistance and exhibiting high tumorigenicity. KMT1A-GATA3-STAT3 axis was proved to maintain the stemness of BCSCs, in which KMT1A was highly expressed in BCSCs rather than bladder cancer non-stem cells (BCNSCs) and normal bladder epithelial cells. However, the therapeutic effect of targeting KMT1A in bladder cancer (BC) or BCSCs remains unknown. Methods: In this study, we confirmed that the expression of KMT1A was remarkably higher in BCSCs than those in BCNSCs. Six histone methyltransferase inhibitors AMI-1, BIX-01294, chaetocin, DZNeP, GSK343 and UNC0631 were investigated targeting KMT1A through cell proliferation assay. Findings: Among those, chaetocin suppressed the cell propagation, induced apoptosis and caused G1 phase cell cycle arrest of BC cells, without influencing normal bladder epithelial cells. More importantly, chaetocin abrogated the self-renewal of BCSCs via the suppression of KMT1A-GATA3-STAT3 circuit and other stemness-related pathways. Finally, intravesical instillation of chaetocin inhibited the formation of BCSCs tumor spheres and xenograft tumors. Chaetocin is an effective histone methyltransferase inhibitor targeting KMT1A in BCSCs. Furthermore, chaetocin abrogated the stemness maintenance and tumor growth of BCSCs via the suppression of KMT1A-GATA3-STAT3 circuit. In future, chaetocin can be a potential therapeutic strategy for BC. Funding Statement: This work was supported by the Basic Scientific Research Operating Expenses for Central Universities (No. buctrc201910), National Natural Science Foundation of China (81602644), the project of Basic Research Cooperation of Beijing-Tianjin-Hebei (H2019104018) and the grant from the Ministry of Science and Technology of China (2010ZX09401-403). Declaration of Interests: The authors have declared that no competing interest exists. Ethics Approval Statement: Human tissues were obtained from The Second Affiliated Hospital of Kunming Medical University College (Kunming, China) with informed consent and approved by the Research Ethics Board at The Second Affiliated Hospital of Kunming Medical University. The mice were maintained under standard conditions according to the institutional guidelines for animal care. All animal experiments were approved by the Animal Ethics Committee of The Second Affiliated Hospital of Kunming Medical University College (Kunming, China) and were carried out in compliance with the Animal Management Rules of the Ministry of Health of the People's Republic of China.

Combining epigenetic and immune therapy to overcome cancer resistance.

Cancer undergoes "immune editing" to evade destruction by cells of the host immune system including natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Current adoptive cellular immune therapies include CAR T cells and dendritic cell vaccines, strategies that have yet to show success for a wide range of tumors. Cancer resistance to immune therapy is driven by extrinsic factors and tumor cell intrinsic factors that contribute to immune evasion. These extrinsic factors include immunosuppressive cell populations such as regulatory T cells (Tregs), tumor-associated macrophages (TAMS), and myeloid-derived suppressor cells (MDSCs). These cells produce and secrete immunosuppressive factors and express inhibitory ligands that interact with receptors on T cells including PD-1 and CTLA-4. Immune checkpoint blockade (ICB) therapies such as anti-PD-1 and anti-CTLA-4 have shown success by increasing immune activation to eradicate cancer, though both primary and acquired resistance remain a problem. Tumor cell intrinsic factors driving primary and acquired resistance to these immune therapies include genetic and epigenetic mechanisms. Epigenetic therapies for cancer including DNA methyltransferase inhibitors (DNMTi), histone deacetylase inhibitors (HDACi), and histone methyltransferase inhibitors (HMTi) can stimulate anti-tumor immunity in both tumor cells and host immune cells. Here we discuss in detail tumor mechanisms of immune evasion and how common epigenetic therapies for cancer may be used to reverse immune evasion. Lastly, we summarize current clinical trials combining epigenetic therapies with immune therapies to reverse cancer immune resistance mechanisms.

Histone Methyltransferases as Therapeutic Targets for Kidney Diseases.

Emerging evidence has demonstrated that epigenetic regulation plays a vital role in gene expression under normal and pathological conditions. Alterations in the expression and activation of histone methyltransferases (HMTs) have been reported in preclinical models of multiple kidney diseases, including acute kidney injury, chronic kidney disease, diabetic nephropathy, polycystic kidney disease, and renal cell carcinoma. Pharmacological inhibition of these enzymes has shown promise in preclinical models of those renal diseases. In this review, we summarize recent knowledge regarding expression and activation of various HMTs and their functional roles in some kidney diseases. The preclinical activity of currently available HMT inhibitors and the mechanisms of their actions are highlighted.

16 Improvement of porcine cloned embryo developmental competence via KDM4A overexpression and H3K9me3 methyltransferase inhibitor treatment

Aberrant epigenetic reprogramming is a major reason for the developmental failure of somatic cell nuclear transfer (SCNT) embryos. Histone H3 lysine 9 trimethylation (H3K9me3), a histone marker for transcriptional repression, is considered a key barrier to the development of cloned embryos. In the present study, we found that H3K9me3 was much higher in SCNT embryos than in IVF embryos at the 4-cell and 2-cell stages; H3K9me3 demethylase KDM4A mRNA was injected into cloned embryos 5h after activation. The intensity of H3K9me3 modification decreased obviously after microinjection of KDM4A mRNA, and the developmental efficiency of porcine cloned embryos was enhanced significantly compared with control (32.2±4.2 vs. 21.0±3.5; P<0.05). Moreover, we evaluated the effect of chaetocin, an inhibitor of histone methyltransferase suv39h1/2, on SCNT embryo development. The results showed that 10 nM chaetocin not only suppressed the H3K9me3 modification in porcine embryonic fibroblast but also downregulated the expression of SUV39H1, SUV39H2, and KDM4D. However, treatment of cloned embryos with 10 nM chaetocin efficiently decreased the H3K9me3 level. Importantly, chaetocin treatment at the 4-cell stage for 6h significantly increased the blastocyst rate (57.8±4.3 vs. 43.5±1.8; P<0.05) compared with the control group. Furthermore, because a recent study showed that a high level of H3K9me3 appears in the reprogramming resistance region of nuclear transfer embryos during zygogene activation (ZGA), we examined the expression levels of ZGA-related genes. The qPCR results showed that the expression of ZGA-related genes increased significantly in SCNT embryos with chaetocin treatment compared with the control. These results suggested that chaetocin treatment can improve the efficiency of SCNT reprogramming during ZGA. In summary, our results suggested that H3K9me3 acts as an epigenetic barrier in porcine SCNT reprogramming and that a suv39h1/2 inhibitor can effectively reduce the H3K9me3 level in the early reprogramming phase and further improve the invitro developmental competence. Due to the existence of a variety of abnormal epigenetic mechanisms during somatic cell reprogramming, the combined use of small-molecule inhibitors is required in future studies. Data analyses were performed using SPSS software. Significance was set at P<0.05 unless otherwise specified. The results are expressed as the mean±standard deviation. This work was supported by the National Natural Science Foundation of China (grant number 31601942); the Postdoctoral Science Foundation of Heilongjiang Province (grant number LBH-Z17010); and the Fund for the National Key Research and Development program of China-Stem Cell and Translational Research (2016YFA0100200).

Drug screening approach combines epigenetic sensitization with immunochemotherapy in cancer

BackgroundThe epigenome plays a key role in cancer heterogeneity and drug resistance. Hence, a number of epigenetic inhibitors have been developed and tested in cancers. The major focus of most studies so far has been on the cytotoxic effect of these compounds, and only few have investigated the ability to revert the resistant phenotype in cancer cells. Hence, there is a need for a systematic methodology to unravel the mechanisms behind epigenetic sensitization.ResultsWe have developed a high-throughput protocol to screen non-simultaneous drug combinations, and used it to investigate the reprogramming potential of epigenetic inhibitors. We demonstrated the effectiveness of our protocol by screening 60 epigenetic compounds on diffuse large B-cell lymphoma (DLBCL) cells. We identified several histone deacetylase (HDAC) and histone methyltransferase (HMT) inhibitors that acted synergistically with doxorubicin and rituximab. These two classes of epigenetic inhibitors achieved sensitization by disrupting DNA repair, cell cycle, and apoptotic signaling. The data used to perform these analyses are easily browsable through our Results Explorer. Additionally, we showed that these inhibitors achieve sensitization at lower doses than those required to induce cytotoxicity.ConclusionsOur drug screening approach provides a systematic framework to test non-simultaneous drug combinations. This methodology identified HDAC and HMT inhibitors as successful sensitizing compounds in treatment-resistant DLBCL. Further investigation into the mechanisms behind successful epigenetic sensitization highlighted DNA repair, cell cycle, and apoptosis as the most dysregulated pathways. Altogether, our method adds supporting evidence in the use of epigenetic inhibitors as sensitizing agents in clinical settings.

The repositioning of epigenetic probes/inhibitors identifies new anti-schistosomal lead compounds and chemotherapeutic targets.

Praziquantel represents the frontline chemotherapy used to treat schistosomiasis, a neglected tropical disease (NTD) caused by infection with macro-parasitic blood fluke schistosomes. While this drug is safe, its inability to kill all schistosome lifecycle stages within the human host often requires repeat treatments. This limitation, amongst others, has led to the search for novel anti-schistosome replacement or combinatorial chemotherapies. Here, we describe a repositioning strategy to assess the anthelmintic activity of epigenetic probes/inhibitors obtained from the Structural Genomics Consortium. Thirty-seven epigenetic probes/inhibitors targeting histone readers, writers and erasers were initially screened against Schistosoma mansoni schistosomula using the high-throughput Roboworm platform. At 10 μM, 14 of these 37 compounds (38%) negatively affected schistosomula motility and phenotype after 72 hours of continuous co-incubation. Subsequent dose-response titrations against schistosomula and adult worms revealed epigenetic probes targeting one reader (NVS-CECR2-1), one writer (LLY-507 and BAY-598) and one eraser (GSK-J4) to be particularly active. As LLY-507/BAY-598 (SMYD2 histone methyltransferase inhibitors) and GSK-J4 (a JMJD3 histone demethylase inhibitor) regulate an epigenetic process (protein methylation) known to be critical for schistosome development, further characterisation of these compounds/putative targets was performed. RNA interference (RNAi) of one putative LLY-507/BAY-598 S. mansoni target (Smp_000700) in adult worms replicated the compound-mediated motility and egg production defects. Furthermore, H3K36me2, a known product catalysed by SMYD2 activity, was also reduced by LLY-507 (25%), BAY-598 (23%) and siSmp_000700 (15%) treatment of adult worms. Oviposition and packaging of vitelline cells into in vitro laid eggs was also significantly affected by GSK-J4 (putative cell permeable prodrug inhibitor of Smp_034000), but not by the related structural analogue GSK-J1 (cell impermeable inhibitor). Collectively, these results provide further support for the development of next-generation drugs targeting schistosome epigenetic pathway components. In particular, the progression of histone methylation/demethylation modulators presents a tractable strategy for anti-schistosomal control.

Heme-Regulated Inhibitor (HRI) Depletion Cooperates with Pharmacologic Inducers 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 but is limited in its efficacy. Recent work in our laboratory identified heme-regulated inhibitor (HRI), an erythroid-specific protein kinase, as a novel HbF regulator (Grevet et al., Science 2018). Depleting HRI in primary human erythroid cells significantly raised HbF levels; however, maximal HbF induction by HRI knockdown required at least 80-85% HRI depletion. It is currently unknown whether this degree of HRI inhibition can be achieved pharmacologically. 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. We therefore set out to test whether combining HRI depletion with pharmacologic HbF inducers would cooperatively increase HbF levels and diminish potential adverse effects on erythroid cell maturation and viability. We performed three-stage in vitro culture of human CD34+ cells treated with select pharmacologic inducers of HbF, including HU, pomalidomide, or UNC0638, in combination with shRNA knockdown of HRI. HbF levels were assessed by RT-qPCR, Western blot, flow cytometry, and HPLC. We found that treatment with HU displayed comparatively little activity and failed to increase effects of HRI knockdown. However, treatment with UNC0638 combined with HRI depletion showed greater than additive effects on HbF with levels reaching 25-30% HbF, while HRI depletion combined with pomalidomide treatment showed the highest levels of cooperativity, reaching 30-40% HbF. Combination of HRI depletion and HbF pharmacologic inducers showed minimal effects on the erythroid transcriptome by RNA-Seq and did not significantly impair erythroid maturation. Intriguingly, dose-titration experiments indicated that HRI knockdown sensitizes erythroid cells to low doses of pomalidomide, maintaining HbF levels of greater than 40% in HRI-depleted samples despite a ten-fold decrease in pomalidomide concentration. Diminished expression of the HbF repressor BCL11A accounted in large part for HbF induction in HRI knockdown samples, particularly when combined with pomalidomide treatment in which BCL11A depletion exceeded 90%, while other HbF repressors such as LRF were unchanged. We will present RNASeq analyses aimed at elucidating mechanisms of HRI cooperativity. Finally, we found that HRI depletion in SCD patient-derived cells showed significant cooperativity with pomalidomide and UNC0638, achieving 45-50% HbF for UNC0638-treated and 50-60% HbF for pomalidomide-treated drug combinations. 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 sum, we find that dual targeting of HbF induction via HRI inhibition and pharmacologic inducers results in successful cooperative upregulation of HbF levels in both normal and SCD primary human cells without impairing red cell maturation. Furthermore, our data suggest that dose titration of HbF inducers combined with HRI depletion could maximize HbF induction while potentially reducing off-target effects. Moreover, HRI is an attractive target for HbF induction as it is expressed in an erythroid-specific manner. While no specific HRI inhibitors are currently available, our work suggests that future small molecule inhibitors of HRI may be combined with other pharmacotherapies to achieve significant, clinically meaningful HbF induction for the treatment of SCD and other hemoglobinopathies. Disclosures Abdulmalik: The Children's Hospital of Philadelphia: Patents & Royalties: Provisional Patent. Blobel:Bioverativ: Research Funding; Pfizer: Research Funding.

A Phase Ib/II Study of the Histone Methyltransferase Inhibitor Pinometostat in Combination with Azacitidine in Patients with 11q23-Rearranged Acute Myeloid Leukemia

A Phase Ib/II Study of the Histone Methyltransferase Inhibitor Pinometostat in Combination with Azacitidine in Patients with 11q23-Rearranged Acute Myeloid Leukemia

Respiratory Epithelial Cells Can Remember Infection: A Proof-of-Concept Study.

Human bronchial epithelial cells play a key role in airway immune homeostasis. We hypothesized that these sentinel cells can remember a previous contact with pathogen compounds and respond nonspecifically to reinfection, a phenomenon called innate immune memory. We demonstrated that their preexposure to Pseudomonas aeruginosa flagellin modify their inflammatory response to a second, nonrelated stimulus, including live pathogens or lipopolysaccharide. Using histone acetyltransferase and methyltransferase inhibitors, we showed that this phenomenon relied on epigenetic regulation. This report is a major breakthrough in the field of multimicrobial respiratory tract infections, wherein control of inflammatory exacerbations is a major therapeutic issue.