Novel histone deacetylase inhibitor CT-101 induces γ-globin gene expression in sickle erythroid progenitors with targeted epigenetic effects.

Induction of fetal hemoglobin (HbF) expression ameliorates the clinical severity and prolong survival in persons with sickle cell disease (SCD). Hydroxyurea (HU) is the only FDA-approved HbF inducer however, additional therapeutics that produce an additive effect in SCD are needed. To this end, development of potent Class I histone deacetylase inhibitors (HDACi) for HbF induction represents a rational molecularly targeted approach. In studies here, we evaluated CT-101, a novel Class I-restricted HDACi, a Largazole derivative, for pharmacodynamics, cytotoxicity, and targeted epigenetic effects. In SCD-derived erythroid progenitors, CT-101 induced HbF expression with additive activity in combination with HU. CT-101 preferentially activated γ-globin gene transcription, increased acetylated histone H3 levels, and conferred an open chromatin conformation in the γ-globin promoter. These data indicate CT-101 represents a strong potential candidate as a molecularly targeted inducer of HbF.

Benserazide racemate and enantiomers induce fetal globin gene expression in vivo: Studies to guide clinical development for beta thalassemia and sickle cell disease.

Increased expression of developmentally silenced fetal globin (HBG) reduces the clinical severity of ß-hemoglobinopathies. Benserazide has a relatively benign safety profile having been approved for 50 years in Europe and Canada for Parkinson's disease treatment. Benserazide was shown to activate HBG gene transcription in a high throughput screen, and subsequent studies confirmed fetal hemoglobin (HbF) induction in erythroid progenitors from hemoglobinopathy patients, transgenic mice containing the entire human ß-globin gene (ß-YAC) and anemic baboons. The goal of this study is to evaluate efficacies and plasma exposure profiles of benserazide racemate and its enantiomers to select the chemical form for clinical development. Intermittent treatment with all forms of benserazide in ß-YAC mice significantly increased proportions of red blood cells expressing HbF and HbF protein per cell with similar pharmacokinetic profiles and with no cytopenia. These data contribute to the regulatory justification for development of the benserazide racemate. Additionally, dose ranges and frequencies required for HbF induction using racemic benserazide were explored. Orally administered escalating doses of benserazide in an anemic baboon induced γ-globin mRNA up to 13-fold and establish an intermittent dose regimen for clinical studies as a therapeutic candidate for potential treatment of ß-hemoglobinopathies.

MIR29B mediates epigenetic mechanisms of HBG gene activation.

Sickle cell disease (SCD) affects over 2 million people worldwide with high morbidity and mortality in underdeveloped countries. Therapeutic interventions aimed at reactivating fetal haemoglobin (HbF) is an effective approach for improving survival and ameliorating the clinical severity of SCD. A class of agents that inhibit DNA methyltransferase (DNMT) activity show promise as HbF inducers because off-target effects are not observed at low concentrations. However, these compounds are rapidly degraded by cytidine deaminase when taken by oral administration, creating a critical barrier to clinical development for SCD. We previously demonstrated that microRNA29B (MIR29B) inhibits de novo DNMT synthesis, therefore, the goal of our study was to determine if MIR29 mediates HbF induction. Overexpression of MIR29B in human KU812 cells and primary erythroid progenitors significantly increased the percentage of HbF positive cells, while decreasing the expression of DNMT3A and the HBG repressor MYB. Furthermore, HBG promoter methylation levels decreased significantly following MIR29B overexpression in human erythroid progenitors. We subsequently, observed higher MIR29B expression in SCD patients with higher HbF levels compared to those with low HbF. Our findings provide evidence for the ability of MIR29B to induce HbF and supports further investigation to expand treatment options for SCD.

Conjugate prodrug AN-233 induces fetal hemoglobin expression in sickle erythroid progenitors and ß-YAC transgenic mice.

Pharmacologic induction of fetal hemoglobin (HbF) is an effective strategy for treating sickle cell disease (SCD) by ameliorating disease severity. Hydroxyurea is the only FDA-approved agent that induces HbF, but significant non-responders and requirement for frequent monitoring of blood counts for drug toxicity limit clinical usefulness. Therefore, we investigated a novel prodrug conjugate of butyric acid (BA) and δ-aminolevulinate (ALA) as a potential HbF inducing agent, using erythroid precursors and a preclinical ß-YAC mouse model. We observed significantly increased γ-globin gene transcription and HbF expression mediated by AN-233 in K562 cells. Moreover, AN-233 stimulated mild heme biosynthesis and inhibited expression of heme-regulated eIF2α kinase involved in silencing γ-globin expression. Studies using primary erythroid precursors generated from sickle peripheral blood mononuclear cells verified the ability of AN-233 to induce HbF, increase histone H3 and H4 acetylation levels at the γ-globin promoter and reduce erythroid precursor sickling by 50%. Subsequent drug treatment of ß-YAC transgenic mice confirmed HbF induction in vivo by AN-233 through an increase in the percentage of HbF positive red blood cells and HbF levels measured by flow cytometry. These data support the potential development of AN-233 for the treatment of SCD.

Personalized Immunotherapy of Patients: Defining by Single-cell RNA-seq with Artificial Intelligence.

Immunotherapy, including immune cell therapy and targeted therapy, is gradually developed through the ongoing discovery of molecular compounds or immune cells. Choosing the best one or the best combination of target compounds and immune-cell therapy is a challenge for clinical scientists and clinicians. We have found variable efficacy individually after tumor-infiltrating lymphocyte (TIL) therapy, and now TILs have been discovered in a group of heterogeneous immune cells. To select the best immunotherapy for each patient, we started to study TIL genomics, including single-cell mRNA differential display from TIL published in 2007 and single-cell RNA-seq from TIL published in 2013, set up TIL quantitative network in 2015, researched machine-learning model for immune therapy in 2022. These manual reports single-cell RNA-seq data combined with machine learning to evaluate the optimal compounds and immune cells for individual patients. The machine-learning model, one of artificial intelligence, can estimate targeting genomic variance from single-cell RNA-seq so that they can cover thirteen kinds of immune cell therapies and ongoing FDA-approved targeted therapies such as PD1 inhibitors, PDL1 inhibitors, and CTLA4 inhibitors, as well as other different treatments such as HDACI or DNMT1 inhibitors, FDA-approved drugs. Moreover, also cover Phase-1, Phase-2, Phase-3, and Phase-4 of clinical trials, such as TIL, CAR T-cells, TCR T-cells. Single-cell RNA-seq with an Artificial intelligence estimation system is much better than our published models from microarrays or just cell therapy. The medical goal is to address three issues in clinical immunotherapy: the increase of efficacy; the decrease of adverse effects and the decrease of the cost in clinical applications.

Characterization of the transcriptome profiles related to globin gene switching during in vitro erythroid maturation.

BACKGROUND: The fetal and adult globin genes in the human ß-globin cluster on chromosome 11 are sequentially expressed to achieve normal hemoglobin switching during human development. The pharmacological induction of fetal γ-globin (HBG) to replace abnormal adult sickle ßS-globin is a successful strategy to treat sickle cell disease; however the molecular mechanism of γ-gene silencing after birth is not fully understood. Therefore, we performed global gene expression profiling using primary erythroid progenitors grown from human peripheral blood mononuclear cells to characterize gene expression patterns during the γ-globin to ß-globin (γ/ß) switch observed throughout in vitro erythroid differentiation. RESULTS: We confirmed erythroid maturation in our culture system using cell morphologic features defined by Giemsa staining and the γ/ß-globin switch by reverse transcription-quantitative PCR (RT-qPCR) analysis. We observed maximal γ-globin expression at day 7 with a switch to a predominance of ß-globin expression by day 28 and the γ/ß-globin switch occurred around day 21. Expression patterns for transcription factors including GATA1, GATA2, KLF1 and NFE2 confirmed our system produced the expected pattern of expression based on the known function of these factors in globin gene regulation. Subsequent gene expression profiling was performed with RNA isolated from progenitors harvested at day 7, 14, 21, and 28 in culture. Three major gene profiles were generated by Principal Component Analysis (PCA). For profile-1 genes, where expression decreased from day 7 to day 28, we identified 2,102 genes down-regulated > 1.5-fold. Ingenuity pathway analysis (IPA) for profile-1 genes demonstrated involvement of the Cdc42, phospholipase C, NF-Kß, Interleukin-4, and p38 mitogen activated protein kinase (MAPK) signaling pathways. Transcription factors known to be involved in γ-and ß-globin regulation were identified.The same approach was used to generate profile-2 genes where expression was up-regulated over 28 days in culture. IPA for the 2,437 genes with > 1.5-fold induction identified the mitotic roles of polo-like kinase, aryl hydrocarbon receptor, cell cycle control, and ATM (Ataxia Telangiectasia Mutated Protein) signaling pathways; transcription factors identified included KLF1, GATA1 and NFE2 among others. Finally, profile-3 was generated from 1,579 genes with maximal expression at day 21, around the time of the γ/ß-globin switch. IPA identified associations with cell cycle control, ATM, and aryl hydrocarbon receptor signaling pathways. CONCLUSIONS: The transcriptome analysis completed with erythroid progenitors grown in vitro identified groups of genes with distinct expression profiles, which function in metabolic pathways associated with cell survival, hematopoiesis, blood cells activation, and inflammatory responses. This study represents the first report of a transcriptome analysis in human primary erythroid progenitors to identify transcription factors involved in hemoglobin switching. Our results also demonstrate that the in vitro liquid culture system is an excellent model to define mechanisms of global gene expression and the DNA-binding protein and signaling pathways involved in globin gene regulation.

Why do tumor-infiltrating lymphocytes have variable efficacy in the treatment of solid tumors?

Lymphocytes in tumor tissue are called tumor-infiltrating lymphocytes (TILs), and they play a key role in the control and treatment of tumor diseases. Since the discovery in 1987 that cultured TILs can kill tumor cells more than 100 times more effectively than T-cells cultured from peripheral blood in melanoma, it has been confirmed that cultured TILs can successfully cure clinical patients with melanoma. Since 1989, after we investigated TIL isolation performance from solid tumors, we modified some procedures to increase efficacy, and thus successfully established new TIL isolation and culture methods in 1994. Moreover, our laboratory and clinicians using our cultured TILs have published more than 30 papers. To improve the efficacy of TILs, we have been carrying out studies of TIL efficacy to treat solid tumor diseases for approximately 30 years. The three main questions of TIL study have been "How do TILs remain silent in solid tumor tissue?", "How do TILs attack homologous and heterologous antigens from tumor cells of solid tumors?", and "How do TILs infiltrate solid tumor tissue from a distance into tumor sites to kill tumor cells?". Research on these three issues has increasingly answered these questions. In this review I summarize the main issues surrounding TILs in treating solid tumors. This review aims to study the killing function of TILs from solid tumor tissues, thereby ultimately introducing the optimal strategy for patients suffering from solid tumors through personalized immunotherapy in the near future.

Machine-learning Modeling for Personalized Immunotherapy- An Evaluation Module.

Immune-cell therapy and targeting therapy are in rapid development to treat tumor diseases. However, current immune-cell therapy and targeting immunotherapy often face three challenges (three Ss): safety challenges such as cytokine releasing syndrome (C.R.S.); specificity targeting problems such as low efficacy caused by off-targeting tumor cells; unsatisfying payment are confounded to clinical patients and physicians. We have been studying immunotherapy for more than thirty years, and recently, personalized immunotherapy to treat tumor disease has been proposed. After we discovered quiescent genes from immune cells within the tumor microenvironment, we set up single-cell genomics analysis, studying heterogeneous immune responses from multiple tumor antigens (neo-antigen); here, we further introduce a new generation of immunotherapy module by using a machine-learning model to assess optimal immunotherapy. The machine-learning model combined with single-cell genomic analysis can predict optimal immune-cell (such as T-cells) and other optimal targeting drugs such as PD1 and CTLA4 inhibitors for the patient to use.

Salubrinal induces fetal hemoglobin expression via the stress-signaling pathway in human sickle erythroid progenitors and sickle cell disease mice.

Sickle cell disease (SCD) is an inherited blood disorder caused by a mutation in the HBB gene leading to hemoglobin S production and polymerization under hypoxia conditions leading to vaso-occlusion, chronic hemolysis, and progressive organ damage. This disease affects ~100,000 people in the United States and millions worldwide. An effective therapy for SCD is fetal hemoglobin (HbF) induction by pharmacologic agents such as hydroxyurea, the only Food and Drug Administration-approved drug for this purpose. Therefore, the goal of our study was to determine whether salubrinal (SAL), a selective protein phosphatase 1 inhibitor, induces HbF expression through the stress-signaling pathway by activation of p-eIF2α and ATF4 trans-activation in the γ-globin gene promoter. Sickle erythroid progenitors treated with 24µM SAL increased F-cells levels 1.4-fold (p = 0.021) and produced an 80% decrease in reactive oxygen species. Western blot analysis showed SAL enhanced HbF protein by 1.6-fold (p = 0.0441), along with dose-dependent increases of p-eIF2α and ATF4 levels. Subsequent treatment of SCD mice by a single intraperitoneal injection of SAL (5mg/kg) produced peak plasma concentrations at 6 hours. Chronic treatments of SCD mice with SAL mediated a 2.3-fold increase in F-cells (p = 0.0013) and decreased sickle erythrocytes supporting in vivo HbF induction.

Genomic expression analysis by single-cell mRNA differential display of quiescent CD8 T cells from tumour-infiltrating lymphocytes obtained from in vivo liver tumours.

We performed a genomic study combining single-cell mRNA differential display and RNA subtractive hybridization to elucidate CD8 T-cell quiescence/ignorance. By comparing actively maintained quiescent CD8 T cells from liver tumour tumour-infiltrating lymphocytes (TILs) with quiescent T cells at the single-cell level, we identified differentially expressed candidate genes by high-throughput screening and comparative analysis of expressed sequence tags (ESTs). While genes for the T-cell receptor, tumour necrosis factor (TNF) receptor, TNF-related apoptosis inducing ligand (TRAIL) and perforin were down-regulated, key genes such as Tob, transforming growth factor (TGF)-beta, lung Krüpple-like factor (LKLF), Sno-A, Ski, Myc, Ets-2 repressor factor (ERF) and RE1-silencing transcription factor (REST/NRSF) complex were highly expressed in the quiescent TIL CD8 cells. Real-time polymerase chain reaction (PCR) further confirmed these results. A regulation model is proposed for actively maintained quiescence in CD8 T cells, including three components: up-regulation of the TGF-beta pathway, a shift in the MYC web and inhibition of the cell cycle.

Regulation of sexual dimorphism: mutational and chemogenetic analysis of the doublesex DM domain.

Doublesex (dsx) is a transcription factor in Drosophila that regulates somatic sexual differentiation. Male- and female-specific splicing isoforms of DSX share a novel DNA-binding domain, designated the DM motif. Broadly conserved among metazoan sex-determining factors, the DM domain contains a nonclassical zinc module and binds in the DNA minor groove. Here, we characterize the DM motif by site-directed and random mutagenesis using a yeast one-hybrid (Y1H) system and extend this analysis by chemogenetic complementation in vitro. The Y1H system is based on a sex-specific Drosophila enhancer element and validated through studies of intersexual dsx mutations. We demonstrate that the eight motif-specific histidines and cysteines engaged in zinc coordination are each critical and cannot be interchanged; folding also requires conserved aliphatic side chains in the hydrophobic core. Mutations that impair DNA binding tend to occur at conserved positions, whereas neutral substitutions occur at nonconserved sites. Evidence for a specific salt bridge between a conserved lysine and the DNA backbone is obtained through the synthesis of nonstandard protein and DNA analogs. Together, these results provide molecular links between the structure of the DM domain and its function in the regulation of sexual dimorphism.

Mechanisms of NRF2 activation to mediate fetal hemoglobin induction and protection against oxidative stress in sickle cell disease.

IMPACT STATEMENT: Sickle cell disease (SCD) is a group of inherited blood disorders caused by mutations in the human ß-globin gene, leading to the synthesis of abnormal hemoglobin S, chronic hemolysis, and oxidative stress. Inhibition of hemoglobin S polymerization by fetal hemoglobin holds the greatest promise for treating SCD. The transcription factor NRF2, is the master regulator of the cellular oxidative stress response and activator of fetal hemoglobin expression. In animal models, various small chemical molecules activate NRF2 and ameliorate the pathophysiology of SCD. This review discusses the mechanisms of NRF2 regulation and therapeutic strategies of NRF2 activation to design the treatment options for individuals with SCD.

MIR-144-mediated NRF2 gene silencing inhibits fetal hemoglobin expression in sickle cell disease.

Inherited genetic modifiers and pharmacologic agents that enhance fetal hemoglobin (HbF) expression reverse the clinical severity of sickle cell disease (SCD). Recent efforts to develop novel strategies of HbF induction include discovery of molecular targets that regulate γ-globin gene transcription and translation. The purpose of this study was to perform genome-wide microRNA (miRNA) analysis to identify genes associated with HbF expression in patients with SCD. We isolated RNA from purified reticulocytes for microarray-based miRNA expression profiling. Using samples from patients with contrasting HbF levels, we observed an eightfold upregulation of miR-144-3p (miR-144) and miR-144-5p in the low-HbF group compared with those with high HbF. Additional analysis by reverse transcription quantitative polymerase chain reaction confirmed individual miR-144 expression levels of subjects in the two groups. Subsequent functional studies in normal and sickle erythroid progenitors showed NRF2 gene silencing by miR-144 and concomitant repression of γ-globin transcription; by contrast, treatment with miR-144 antagomir reversed its silencing effects in a dose-dependent manner. Because NRF2 regulates reactive oxygen species levels, additional studies investigated mechanisms of HbF regulation using a hemin-induced oxidative stress model. Treatment of KU812 cells with hemin produced an increase in NRF2 expression and HbF induction that reversed with miR-144 pretreatment. Chromatin immunoprecipitation assay confirmed NRF2 binding to the γ-globin antioxidant response element, which was inhibited by miR-144 mimic treatment. The genome-wide miRNA microarray and primary erythroid progenitor data support a miR-144/NRF2-mediated mechanism of γ-globin gene regulation in SCD.

SRY-directed DNA bending and human sex reversal: reassessment of a clinical mutation uncovers a global coupling between the HMG box and its tail.

Sex-reversal mutations in human SRY cluster within its high-mobility group box, a conserved motif of DNA bending. A classical substitution at the crux of this angular domain (M64I) has been reported to impair DNA bending but not DNA binding, implying that sharp bending is required for transcriptional activation and testis determination. Surprisingly, we report that this defect was an inadvertent consequence of protein truncation: in the intact protein, sharp DNA bending is restored by the basic tail of the high-mobility group box. Structural coupling between box and tail is tuned to the native DNA bend angle, damping conformational fluctuations and enabling bidirectional induced fit within the bent complex. M64I-associated sex reversal is instead caused by the impaired function of a flanking non-classical nuclear localization signal (NLS). Similar impairment is caused by M64A, suggesting that mislocalization is due to loss of an M64-specific function and not gain of a non-native I64-specific function. Transcriptional activity, attenuated by mislocalization, is rescued by fusion of a heterologous NLS. In a male embryonic gonadal cell line, M64I and M64A SRY-NLS fusion proteins exhibit native transcriptional activation of Sox9, a key step in testicular differentiation. Our results suggest that male development is robust to subtle alterations in SRY-DNA architecture but depends critically on nuclear localization. The previously unsuspected role of M64 within a non-classical NLS may contribute to its invariance among SOX-related and LEF-1-related transcription factors.

Dimethyl fumarate increases fetal hemoglobin, provides heme detoxification, and corrects anemia in sickle cell disease.

Sickle cell disease (SCD) results from a point mutation in the ß-globin gene forming hemoglobin S (HbS), which polymerizes in deoxygenated erythrocytes, triggering recurrent painful vaso-occlusive crises and chronic hemolytic anemia. Reactivation of fetal Hb (HbF) expression ameliorates these symptoms of SCD. Nuclear factor (erythroid derived-2)-like 2 (Nrf2) is a transcription factor that triggers cytoprotective and antioxidant pathways to limit oxidative damage and inflammation and increases HbF synthesis in CD34+ stem cell-derived erythroid progenitors. We investigated the ability of dimethyl fumarate (DMF), a small-molecule Nrf2 agonist, to activate γ-globin transcription and enhance HbF in tissue culture and in murine and primate models. DMF recruited Nrf2 to the γ-globin promoters and the locus control region of the ß-globin locus in erythroleukemia cells, elevated HbF in SCD donor-derived erythroid progenitors, and reduced hypoxia-induced sickling. Chronic DMF administration in SCD mice induced HbF and increased Nrf2-dependent genes to detoxify heme and limit inflammation. This improved hematological parameters, reduced plasma-free Hb, and attenuated inflammatory markers. Chronic DMF administration to nonanemic primates increased γ-globin mRNA in BM and HbF protein in rbc. DMF represents a potential therapy for SCD to induce HbF and augment vasoprotection and heme detoxification.

DNA alterations in human aberrant crypt foci and colon cancers by random primed polymerase chain reaction.

Colon cancers are the result of the accumulation of multiple genetic alterations. To evaluate the role genomic instability plays during tumor development, we compared DNA fingerprints of 44 aberrant crypt foci (ACF; the earliest identified neoplastic lesion in the colon), 23 cancers, and normal crypts generated by random primers with PCR. The PCR products, separated by PAGE and viewed after silver staining, demonstrate altered fingerprints for 23.3% of the ACF and 95.7% of the cancers. In this first study of human ACF with this approach, the finding of altered DNA fingerprints in these microscopic lesions suggests that genomic instability can occur very early in human colon tumorigenesis.