Histone H3 trimethylation at lysine 36 guides m6A RNA modification co-transcriptionally.

DNA and histone modifications have notable effects on gene expression1. Being the most prevalent internal modification in mRNA, the N6-methyladenosine (m6A) mRNA modification is as an important post-transcriptional mechanism of gene regulation2-4 and has crucial roles in various normal and pathological processes5-12. However, it is unclear how m6A is specifically and dynamically deposited in the transcriptome. Here we report that histone H3 trimethylation at Lys36 (H3K36me3), a marker for transcription elongation, guides m6A deposition globally. We show that m6A modifications are enriched in the vicinity of H3K36me3 peaks, and are reduced globally when cellular H3K36me3 is depleted. Mechanistically, H3K36me3 is recognized and bound directly by METTL14, a crucial component of the m6A methyltransferase complex (MTC), which in turn facilitates the binding of the m6A MTC to adjacent RNA polymerase II, thereby delivering the m6A MTC to actively transcribed nascent RNAs to deposit m6A co-transcriptionally. In mouse embryonic stem cells, phenocopying METTL14 knockdown, H3K36me3 depletion also markedly reduces m6A abundance transcriptome-wide and in pluripotency transcripts, resulting in increased cell stemness. Collectively, our studies reveal the important roles of H3K36me3 and METTL14 in determining specific and dynamic deposition of m6A in mRNA, and uncover another layer of gene expression regulation that involves crosstalk between histone modification and RNA methylation.

Gilteritinib-based combination therapy in adult relapsed/refractory FLT3-mutated acute myeloid leukaemia.

Gilteritinib, a potent FMS-like tyrosine kinase 3 (FLT3) inhibitor, was approved for relapsed/refractory (R/R) FLT3-mutated acute myeloid leukaemia (AML) patients but still showed limited efficacy. Here, we retrospectively analysed the efficacy and safety of different gilteritinib-based combination therapies (gilteritinib plus hypomethylating agent and venetoclax, G + HMA + VEN; gilteritinib plus HMA, G + HMA; gilteritinib plus venetoclax, G + VEN) in 33 R/R FLT3-mutated AML patients. The composite complete response (CRc) and modified CRc (mCRc) rates were 66.7% (12/18) and 88.9% (16/18) in patients received G + HMA + VEN, which was higher compared with that in G + HMA (CRc: 18.2%, 2/11; mCRc: 45.5%, 5/11) or G + VEN (CRc: 50.0%, 2/4; mCRc: 50.0%, 2/4). The median overall survival (OS) for G + HMA + VEN, G + HMA and G + VEN treatment was not reached, 160.0 days and 231.0 days. The median duration of remission (DOR) for G + HMA + VEN, G + HMA and G + VEN treatment was not reached, 82.0 days and 77.0 days. Four patients in the G + HMA + VEN group received alloHSCT after remission exhibited prolonged median DOR. The most common grade 3/4 adverse events were cytopenia, febrile neutropenia and pulmonary infection; there were no differences among the three groups. In conclusion, our data demonstrated promising response of G + HMA + VEN combination therapy in R/R FLT3-mutated AML, and it may be considered an effective therapy bridge to transplantation.

Inactivated vaccine dosage and serum IgG levels correlate with persistent COVID-19 infections in hematologic malignancy patients during the Omicron Surge in China.

PURPOSE: The essence of this scholarly work was to carefully outline the key factors intensifying the virulence and protracted contagion of COVID-19, particularly among individuals afflicted with hematologic malignancies (HM), in an epoch predominantly governed by the Omicron variant. METHODS: Adults with HM diagnosed with COVID-19 from November 2022 to February 2023 were monitored in this retrospective study. Patient blood samples yielded biochemical data, and COVID-19 was confirmed through RNA or antigen testing. The factors affecting severity and infection duration were examined using both univariate and multivariate logistic regression analyses. For calculating the overall survival probabilities, the Kaplan-Meier product limit approach was employed. RESULTS: In the examined cohort, 133 individuals diagnosed with HM and concomitantly infected with COVID-19 were scrutinized. Of the participants, 29.3% (39 patients) were classified as Severe/Critical, while the other 70.7% (94 patients) were categorized as Non-severe. A significant difference was observed in vaccination status: 61.7% of patients in the Non-severe group had received at least a two-dose vaccine regimen, whereas 61.5% of the Severe/Critical group had either minimal or only one dose of vaccination. The data analysis revealed that elevated C-reactive protein levels (≥ 100 mg/L) significantly raised the risk of severe/critical conditions in HM patients with COVID-19, as determined by advanced multivariate logistic regression. The odds ratio was 3.415 with a 95% confidence interval of 1.294-9.012 (p = 0.013). Patients who continued to have positive nucleic acid tests and ongoing symptoms beyond 30 days were categorized as having a persistent infection, whereas those who achieved infection control within this timeframe were categorized as having infection recovery. Of the HM cohort, 11 did not survive beyond 30 days after diagnosis. The results from a competing risk model revealed that increased interleukin-6 levels (HR: 2.626, 95% CI: 1.361-5.075; p = 0.004) was significantly associated with persistent infection. Conversely, receiving more than two vaccine doses (HR: 0.366, 95% CI: 0.158-0.846; p = 0.019), and having high IgG levels (≥ 1000 mg/dl) (HR: 0.364, 95% CI: 0.167-0.791; p = 0.011), were associated with infection recovery. There was a notable disparity in survival rates between patients with persistent infections and infection recovery, with those in the non-persistent group demonstrating superior survival outcomes (P < 0.001). CONCLUSIONS: In conclusion, the study determined that HM patients with COVID-19 and increased C-reactive protein levels had a higher likelihood of severe health outcomes. Persistent infection tended to be more prevalent in those with vaccine dosages (< 2 doses), lower IgG levels, and higher interleukin-6 levels.

Influenza A (H1N1) virus induced long-term remission in a refractory acute myeloid leukaemia.

There have been reports of haematological cancer patients achieving spontaneous remission after being infected with the influenza A or SARS-COV-2 virus. Here, we present the first case of long-term complete remission (CR) induced by influenza A (IAV, H1N1 subtype) in a refractory AML patient and have functionally validated this finding in two different animal disease models. We observed a significant increase in the proportion of helper T cells in the patient after IAV infection. The levels of cytokines, including IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ and TNF-α, were higher in IAV-infected patients compared with control groups. These findings indicate that the anti-tumour effects induced by IAV are closely related to the modification of the immune response. Our study provides new evidence of the anti-tumour effects of IAV from a clinical practice perspective.

Glutamate dehydrogenase 1: A novel metabolic target in inhibiting acute myeloid leukaemia progression.

Glutamine metabolic reprogramming in acute myeloid leukaemia (AML) cells contributes to the decreased sensitivity to antileukemic drugs. Leukaemic cells, but not their myeloid counterparts, largely depend on glutamine. Glutamate dehydrogenase 1 (GDH1) is a regulation enzyme in glutaminolysis. However, its role in AML remains unknown. Here, we reported that GDH1 was highly expressed in AML: high GDH1 was one of the independent negative prognostic factors in AML cohort. The dependence of leukaemic cells on GDH1 was proved both in vitro and in vivo. High GDH1 promoted cell proliferation and reduced survival time of leukaemic mice. Targeting GDH1 eliminated the blast cells and delayed AML progression. Mechanistically, GDH1 knockdown inhibited glutamine uptake by downregulating SLC1A5. Moreover, GDH1 invalidation also inhibited SLC3A2 and abrogated the cystine-glutamate antiporter system Xc- . The reduced cystine and glutamine disrupted the synthesis of glutathione (GSH) and led to the dysfunction of glutathione peroxidase-4 (GPX4), which maintains the lipid peroxidation homeostasis by using GSH as a co-factor. Collectively, triggering ferroptosis in AML cells in a GSH depletion manner, GDH1 inhibition was synthetically lethal with the chemotherapy drug cytarabine. Ferroptosis induced by inhibiting GDH1 provides an actionable therapeutic opportunity and a unique target for synthetic lethality to facilitate the elimination of malignant AML cells.

Tregs dysfunction aggravates postoperative cognitive impairment in aged mice.

OBJECTIVES: Enhanced neuroinflammation is an important mechanism underlying perioperative neurocognitive disorders. Regulatory T cells (Tregs) play a crucial role in regulating systemic immune responses. The present study was aimed to investigate the participation of Tregs in the development of postoperative cognitive dysfunction (POCD). METHODS: Surgery-associated neurocognitive disorder was induced in 18-month-old mice subjected to internal fixation of tibial fracture. Morris water maze was used to examine mice cognitive function. Splenic Tregs were collected for RNA sequencing and flow cytometry. Levels of inflammatory factors in the circulation and hippocampus were measured by enzyme-linked immunosorbent assay. Protein presences of tight junction proteins were detected by immunofluorescence. RESULTS: Surgery of internal fixation of tibial fracture induced cognitive impairment in aged mice, accompanied by elevated plasma levels of inflammatory factors and increased circulating Tregs. Transfusion of Tregs from young mice partially restored the structure of the blood-brain barrier and alleviated POCD in aged mice. Compared with young Tregs, differentially expressed genes in aged Tregs were enriched in tumor necrosis factor (TNF) signaling pathway and cytokine-cytokine receptor interaction. Flow cytometry revealed that aged Tregs had blunted functions under basal and stimulated conditions. Blockade of the CD25 epitope protected the blood-brain barrier structure, reduced TNF-α levels in the hippocampus, and improved surgery-associated cognition in aged mice. CONCLUSIONS: Blocking peripheral regulatory T cells improves surgery-induced cognitive function in aged mice. Therefore, aged Tregs play an essential role in the occurrence of POCD.

Telocytes reduce oxidative stress by downregulating DUOX2 expression in inflamed lungs of mice.

Telocytes (TCs), a novel type of interstitial cells, have been found to participate in tissue protection and repair. In this study, we investigated the antioxidative effects of TCs in inflamed lungs of mice. Acute respiratory distress syndrome (ARDS) mice were used as models of inflamed lungs of mice. Gene sequencing was used to screen the differentially expressed miRNAs in TCs after lipopolysaccharide (LPS) stimulation. AntagomiR-146a-5p-pretreated TCs were first injected into mice, and antioxidant activity of TCs was estimated. TCs, RAW264.7 cells, and MLE-12 cells were collected for the detection of expressions of NOX1-4, DUOX1-2, SOD1-3, GPX1-2, CAT, Nrf2, miR-146a-5p, and miR-21a-3p after LPS stimulation. Silencing miRNAs were delivered to examine the involved signaling pathways. Oxidative stress was examined by measuring malondialdehyde (MDA) levels. We found that microRNA-146a-5p and microRNA-21a-3p were upregulated in TCs after LPS stimulation. ARDS mice that were preinfused with TCs had lower lung tissue injury scores, lung wet-dry ratios, white blood cell counts in alveolar lavage fluid and lower MDA concentrations in lung tissue. However, in antagomiR-146a-5p-pretreated ARDS mice, the infusion of TCs caused no corresponding changes. After LPS stimulation, DUOX2 and MDA concentrations were downregulated in TCs, while DUOX2 was restored by antagomiR-146a-5p in TCs. Dual-luciferase reporter assay confirmed that CREB1 was downregulated by miR-146a-5p, while DUOX2 was downregulated by CREB1, which was confirmed by treating TCs with a specific CREB1 inhibitor. This study demonstrates that LPS stimulation upregulates miR-146a-5p in TCs, which downregulates the CREB1/DUOX2 pathway, resulting in a decrease in oxidative stress in cultured TCs. TCs reduce LPS-induced oxidative stress by decreasing DUOX2 in inflamed lungs of mice.

Phytochemical library screening reveals betulinic acid as a novel Skp2-SCF E3 ligase inhibitor in non-small cell lung cancer.

Skp2 is overexpressed in multiple cancers and plays a critical role in tumor development through ubiquitin/proteasome-dependent degradation of its substrate proteins. Drugs targeting Skp2 have exhibited promising anticancer activity. Here, we identified a plant-derived Skp2 inhibitor, betulinic acid (BA), via high-throughput structure-based virtual screening of a phytochemical library. BA significantly inhibited the proliferation and migration of non-small cell lung cancer (NSCLC) through targeting Skp2-SCF E3 ligase both in vitro and in vivo. Mechanistically, BA binding to Skp2, especially forming H-bonds with residue Lys145, decreases its stability by disrupting Skp1-Skp2 interactions, thereby inhibiting the Skp2-SCF E3 ligase and promoting the accumulation of its substrates; that is, E-cadherin and p27. In both subcutaneous and orthotopic xenografts, BA significantly inhibited the proliferation and metastasis of NSCLC through targeting Skp2-SCF E3 ligase and upregulating p27 and E-cadherin protein levels. Taken together, BA can be considered a valuable therapeutic candidate to inhibit metastasis of NSCLC.

Minimal residual disease level determined by flow cytometry provides reliable risk stratification in adults with T-cell acute lymphoblastic leukaemia.

Minimal residual disease (MRD) is an important independent prognostic factor for relapse and survival in acute lymphoblastic leukaemia (ALL). Compared with adult B-cell ALL, reports of adult T-cell ALL (T-ALL) MRD have been scarce and mostly based on molecular methods. We evaluated the prognostic value of multiparameter flow cytometry (FCM)-based MRD at the end of induction (EOI-MRD). The present retrospective study included 94 adult patients with T-ALL. MRD was detected by six- to eight-colour FCM. Patients who were EOI-MRD positive had a higher cumulative incidence of relapse (CIR) (87·6% vs. 38·8%, P = 0·0020), and a lower relapse-free survival (RFS) (5·4% vs. 61·0%, P = 0·0005) and overall survival (OS) (32·7% vs. 69·7%, P < 0·0001) than those who were EOI-MRD negative. Moreover, for patients who received allogeneic haematopoietic stem cell transplantation (allo-HSCT) at their first remission, EOI-MRD positivity was predictive of post-transplant relapse (2-year CIR: 68·2% vs. 4·0%, P = 0·0003). Multivariate analysis showed that EOI-MRD was an independent prognostic factor for CIR [hazard ratio (HR) 2·139, P = 0·046], RFS (HR 2·125, P = 0·048) and OS (HR 2·987, P = 0·017). In conclusion, EOI-MRD based on FCM was an independent prognostic factor for relapse and survival in adult T-ALL. For patients who underwent HSCT, EOI-MRD could be used to identify patients with a high risk of relapse after allo-HSCT.

CDKN2A deletions are associated with poor outcomes in 101 adults with T-cell acute lymphoblastic leukemia.

The identification of genetic risk subgroups of T-cell acute lymphoblastic leukemia (T-ALL) may provide evidence for risk stratification and individualized treatment. We investigated the characteristics and prognostic value of tumor suppressor gene CDKN2A deletions in 101 patients with T-ALL. The CDKN2A deletion was present in 23% (23/101) of T-ALL by fluorescence in situ hybridization (FISH). The most common type of CDKN2A deletion was homozygous deletion (70%, 16/23). A lower frequency of CDKN2A deletion was found in patients with early T-cell precursor (ETP) ALL than in patients with non-ETP-ALL (10.4% vs 34.0%; P = .008). Deletion of CDKN2A was significantly associated with younger age (P = .001), higher white blood cell (WBC) count (P < .001) and higher lactate dehydrogenase (LDH) level (P = .002). Patients with CDKN2A deletion had lower 2-year overall survival (OS) and event-free survival (EFS) rates than patients without CDKN2A deletion (2-year OS: 18.6% ± 8.9% vs 47.4% ± 6.2%, P = .032; EFS: 16.4 ± 8.3 vs 38.6 ± 5.9%, P = .022). In multivariable analysis, CDKN2A deletion was an independent adverse prognostic factor for OS (P = .016). In conclusion, adult T-ALL patients with CDKN2A deletion had a poor prognosis, and these patients might benefit from intensive chemotherapy or allogeneic hematopoietic stem-cell transplantation.

Homoharringtonine exhibits potent anti-tumor effect and modulates DNA epigenome in acute myeloid leukemia by targeting SP1/TET1/5hmC.

Homoharringtonine, a plant alkaloid, has been reported to suppress protein synthesis and has been approved by the US Food and Drug Administration for the treatment of chronic myeloid leukemia. Here we show that in acute myeloid leukemia (AML), homoharringtonine potently inhibits cell growth/viability and induces cell cycle arrest and apoptosis, significantly inhibits disease progression in vivo, and substantially prolongs survival of mice bearing murine or human AML. Strikingly, homoharringtonine treatment dramatically decreases global DNA 5-hydroxymethylcytosine abundance through targeting the SP1/TET1 axis, and TET1 depletion mimics homoharringtonine's therapeutic effects in AML. Our further 5hmC-seq and RNA-seq analyses, followed by a series of validation and functional studies, suggest that FLT3 is a critical down-stream target of homoharringtonine/SP1/TET1/5hmC signaling, and suppression of FLT3 and its downstream targets (e.g. MYC) contributes to the high sensitivity of FLT3-mutated AML cells to homoharringtonine. Collectively, our studies uncover a previously unappreciated DNA epigenome-related mechanism underlying the potent antileukemic effect of homoharringtonine, which involves suppression of the SP1/TET1/5hmC/FLT3/MYC signaling pathways in AML. Our work also highlights the particular promise of clinical application of homoharringtonine to treat human AML with FLT3 mutations, which accounts for more than 30% of total cases of AML.

6-O-angeloylplenolin exerts neuroprotection against lipopolysaccharide-induced neuroinflammation in vitro and in vivo.

Neuroinflammation is one of the critical events in neurodegenerative diseases, whereas microglia play an important role in the pathogenesis of neuroinflammation. In this study, we investigated the effects of a natural sesquiterpene lactone, 6-O-angeloylplenolin (6-OAP), isolated from the traditional Chinese medicine Centipeda minima (L.) A.Br., on neuroinflammation and the underlying mechanisms. We showed that treatment with lipopolysaccharide (LPS) caused activation of BV2 and primary microglial cells and development of neuroinflammation in vitro, evidenced by increased production of inflammatory cytokines TNF-α and IL-1ß, the phosphorylation and nuclear translocation of NF-κB, and the transcriptional upregulation of COX-2 and iNOS, leading to increased production of proinflammatory factors NO and PGE2. Moreover, LPS treatment induced oxidative stress through increasing the expression levels of NOX2 and NOX4. Pretreatment with 6-OAP (0.5-4 µM) dose-dependently attenuated LPS-induced NF-κB activation and oxidative stress, thus suppressed neuroinflammation in the cells. In a mouse model of LPS-induced neuroinflammation, 6-OAP (5-20 mg·kg-1·d-1, ip, for 7 days before LPS injection) dose-dependently inhibited the production of inflammatory cytokines, the activation of the NF-κB signaling pathway, and the expression of inflammatory enzymes in brain tissues. 6-OAP pretreatment significantly ameliorated the activation of microglia and astrocytes in the brains. 6-OAP at a high dose caused a much stronger antineuroinflammatory effect than dexamethansone (DEX). Furthermore, we demonstrated that 6-OAP pretreatment could inhibit LPS-induced neurite and synaptic loss in vitro and in vivo. In conclusion, our results demonstrate that 6-OAP exerts antineuroinflammatory effects and can be considered a novel drug candidate for the treatment of neuroinflammatory diseases.

Inhibition of glycogen synthase kinase 3ß improves cognitive function in aged mice by upregulating claudin presences in cerebral endothelial cells.

Glycogen synthase kinase-3ß (GSK-3ß), a serine/threonine protein kinase, is widely distributed in mammalian brains. Since GSK-3ß plays a vital role in the development of neurodegenerative disorders, the present study was designed to investigate the role of GSK-3ß in the blood-brain barrier (BBB) permeability in aged mice. Morris water maze test was used to examine mouse cognitive function. BBB permeability was examined by the leakage of fluorescence signals of low-molecular weight dextran. GSK-3ß inhibitor, 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), was administrated in aged mice and in cultured mouse brain microvascular endothelial cells (bEnd.3). Compared with young mice, aged mice had increased leftover signals of dextran in the hippocampus and a lower score in the maze test, suggesting that aged mice have abnormal leakage of BBB and cognitive dysfunction. The protein expression of Toll-like receptor 4 (TLR4) was increased, whereas the protein expressions of junction proteins (claudin1 and claudin5) were reduced in endothelial cells of BBB in aged mice. Phosphorylated level of serine 9, an inhibitory residue in GSK-3ß protein, was decreased. TDZD-8 treatment downregulated TLR4 protein expression, upregulated claudin1 and claudin5 protein expressions, and significantly improved cognitive function in aged mice. In bEnd.3 cells, TDZD-8 treatment reduced TLR4 expression and increased claudin5 expression in cells stimulated with lipopolysaccharides. In conclusion, the inhibition of GSK-3ß activity downregulates aging-induced TLR4 expression and restores the BBB integrity, resulting in the improvement of cognitive function in aged mice.

The regulatory effect of microRNA-21a-3p on the promotion of telocyte angiogenesis mediated by PI3K (p110α)/AKT/mTOR in LPS induced mice ARDS.

BACKGROUND: Telocytes (TCs) are newly identified interstitial cells that participate in tissue protection and repair. The present study investigated the mechanisms underlying the protective effect of TCs in a mouse model of respiratory distress. METHODS: The mouse model of acute respiratory distress syndrome (ARDS) was established by intratracheal instillation of lipopolysaccharide (LPS). After instillation of TCs culture medium, lung injury was assessed, and angiogenesis markers, including CD31 and endothelial nitric oxide synthase (eNOS), were detected by immunofluorescence. Bioinformatics analysis was used to screen significantly differentially expressed microRNAs (miRNAs) in cultured TCs stimulated with LPS, and the regulation of downstream angiogenesis genes by these miRNAs was analysed and verified. PI3K subunits and pathways were evaluated by using a PI3K p110α inhibitor to study the involved mechanisms. RESULTS: In ARDS mice, instillation of TCs culture medium ameliorated LPS-induced inflammation and lung injury and increased the protein levels of CD31 and eNOS in the injured lungs. A total of 7 miRNAs and 1899 mRNAs were differentially regulated in TCs stimulated with LPS. Functional prediction analysis showed that the differentially expressed mRNAs were enriched in angiogenesis-related processes, which were highly correlated with miR-21a-3p. Culture medium from TCs with miR-21a-3p inhibition failed to promote angiogenesis in mouse models of LPS-induced ARDS. In cultured TCs, LPS stimulation upregulated the expression of miR-21a-3p, which further targeted the transcription factor E2F8 and decreased Notch2 protein expression. TCs culture medium enhanced hemangioendothelioma endothelial cells (EOMA cells) proliferation, which was blocked by the miR-21a-3p inhibitor. The PI3K p110α inhibitor decreased vascular endothelial growth factor levels in LPS-stimulated TCs and reversed the enhancing effect of TCs culture medium on EOMA cells proliferation. CONCLUSIONS: TCs exerted protective effects under inflammatory conditions by promoting angiogenesis via miR-21a-3p. The PI3K p110α subunit and transcriptional factor E2F8 could be involved in this process.

Influence of gene modification in biological behaviors and responses of mouse lung telocytes to inflammation.

BACKGROUND: Telocytes play key roles in maintenance of organ/tissue function and prevention of organ injury. However, there are great challenges to investigate telocytes functions using primary telocytes, due to the difficulties of isolation, identification, and stability. The present study aims at constructing continuous cell strain of mouse lung telocyte cell line with stable characters by gene modification and investigating biological behaviors and responses of gene-modified telocytes to inflammation. METHODS: Mouse primary lung telocytes were isolated and identified using immune-labeling markers and immunoelectron microscopy. Primary telocytes were transformed with Simian vacuolating virus 40 small and large T antigen (SV40). Biological characters, behaviors morphology, and proliferation of those gene-modified telocytes were defined and monitored dynamically for 50 generations, as compared with primary lung telocytes. Cell cycle of mouse primary lung telocytes or gene-modified telocytes was detected by flow cytometry. RESULTS: Gene modified telocytes of generations 5, 10, 30 and 50 were observed with telopodes and also showed CD34 and ckit positive. Multiple cellular morphology were also observed on telocyte cell-line under monitor of celliq and enhanced cell proliferation were showed. SV40 transduction was also reduced apoptosis and increased the ratio of S and G2 phases in telocyte cell-line. CONCLUSION: We successfully constructed mouse lung telocyte cell-line which maintained the biological properties and behaviors as primary telocytes and could responses to inflammation induced by LPS. Thus, gene-modified lung telocytes, Telocyte Line, would provide a cell tool for researchers exploring the roles and applications of telocytes involved in physiological and pathological states in future.

The high NRF2 expression confers chemotherapy resistance partly through up-regulated DUSP1 in myelodysplastic syndromes.

Although cytarabine has been widely considered as one of the chemotherapy drugs for high-risk myelodysplastic syndromes (MDS), the overall response rate is only approximately 20-30%. Nuclear factor erythroid 2-related factor 2 (NRF2, also called NFE2L2) has been shown to play a pivotal role in preventing cancer cells from being affected by chemotherapy. However, it is not yet known whether NRF2 can be used as a prognostic biomarker in MDS, or whether elevated NRF2 levels are associated with cytarabine resistance. Here, we found that NRF2 expression levels in bone marrow from high-risk patients exceeded that of low-risk MDS patients. Importantly, high NRF2 levels are correlated with inferior outcomes in MDS patients (n=137). Downregulation of NRF2 by the inhibitor Luteolin, or lentiviral shRNA knockdown, enhanced the chemotherapeutic efficacy of cytarabine, while MDS cells treated by NRF2 agonist Sulforaphane showed increased resistance to cytarabine. More importantly, pharmacological inhibition of NRF2 could sensitize primary high-risk MDS cells to cytarabine treatment. Mechanistically, downregulation of dual specificity protein phosphatase 1, an NRF2 direct target gene, could abrogate cytarabine resistance in NRF2 elevated MDS cells. Silencing NRF2 or dual specificity protein phosphatase 1 also significantly sensitized cytarabine treatment and inhibited tumors in MDS cells transplanted mouse models in vivo Our study suggests that targeting NRF2 in combination with conventional chemotherapy could pave the way for future therapy for high-risk MDS.

Setd2 regulates quiescence and differentiation of adult hematopoietic stem cells by restricting RNA polymerase II elongation.

SET domain containing 2 (Setd2), encoding a histone methyltransferase, is associated with many hematopoietic diseases when mutated. By generating a novel exon 6 conditional knockout mouse model, we describe an essential role of Setd2 in maintaining the adult hematopoietic stem cells. Loss of Setd2 results in leukopenia, anemia, and increased platelets accompanied by hypocellularity, erythroid dysplasia, and mild fibrosis in bone marrow. Setd2 knockout mice show significantly decreased hematopoietic stem and progenitor cells except for erythroid progenitors. Setd2 knockout hematopoietic stem cells fail to establish long-term bone marrow reconstitution after transplantation because of the loss of quiescence, increased apoptosis, and reduced multiple-lineage terminal differentiation potential. Bioinformatic analysis revealed that the hematopoietic stem cells exit from quiescence and commit to differentiation, which lead to hematopoietic stem cell exhaustion. Mechanistically, we attribute an important Setd2 function in murine adult hematopoietic stem cells to the inhibition of the Nsd1/2/3 transcriptional complex, which recruits super elongation complex and controls RNA polymerase II elongation on a subset of target genes, including Myc Our results reveal a critical role of Setd2 in regulating quiescence and differentiation of hematopoietic stem cells through restricting the NSDs/SEC mediated RNA polymerase II elongation.