ALKBH5 modulates hematopoietic stem and progenitor cell energy metabolism through m6A modification-mediated RNA stability control.

N6-methyladenosine (m6A) RNA modification controls numerous cellular processes. To what extent these post-transcriptional regulatory mechanisms play a role in hematopoiesis has not been fully elucidated. We here show that the m6A demethylase alkB homolog 5 (ALKBH5) controls mitochondrial ATP production and modulates hematopoietic stem and progenitor cell (HSPC) fitness in an m6A-dependent manner. Loss of ALKBH5 results in increased RNA methylation and instability of oxoglutarate-dehydrogenase (Ogdh) messenger RNA and reduction of OGDH protein levels. Limited OGDH availability slows the tricarboxylic acid (TCA) cycle with accumulation of α-ketoglutarate (α-KG) and conversion of α-KG into L-2-hydroxyglutarate (L-2-HG). L-2-HG inhibits energy production in both murine and human hematopoietic cells in vitro. Impaired mitochondrial energy production confers competitive disadvantage to HSPCs and limits clonogenicity of Mll-AF9-induced leukemia. Our study uncovers a mechanism whereby the RNA m6A demethylase ALKBH5 regulates the stability of metabolic enzyme transcripts, thereby controlling energy metabolism in hematopoiesis and leukemia.

Chemo-free maintenance therapy in adult T-cell acute lymphoblastic leukemia: A case report and literature review.

Maintenance therapy in adult T-cell acute lymphoblastic leukemia (T-ALL) is the longest phase but with limited option. The classic drugs used in the maintenance phase such as 6-mercaptopurine, methotrexate, corticosteroid and vincristine have potentially serious toxicities. Optimizing therapy in the modern age, chemo-free maintenance therapy regimens for patients with T-ALL may dramatically improve the maintenance therapeutic landscape. We report here the combination of Anti-programmed cell death protein 1 antibody and histone deacetylase inhibitor as chemo-free maintenance treatment in a T-ALL patient with literature review, thus providing a unique perspective in addition to valuable information which may inform novel therapeutic approaches.

Helicobacter pylori-induced NAT10 stabilizes MDM2 mRNA via RNA acetylation to facilitate gastric cancer progression.

BACKGROUND: N4-acetylcytidine (ac4C), a widespread modification in human mRNAs that is catalyzed by the N-acetyltransferase 10 (NAT10) enzyme, plays an important role in promoting mRNA stability and translation. However, the biological functions and regulatory mechanisms of NAT10-mediated ac4C were poorly defined. METHODS: ac4C mRNA modification status and NAT10 expression levels were analyzed in gastric cancer (GC) samples and compared with the corresponding normal tissues. The biological role of NAT10-mediated ac4C and its upstream and downstream regulatory mechanisms were determined in vitro and in vivo. The therapeutic potential of targeting NAT10 in GC was further explored. RESULTS: Here, we demonstrated that both ac4C mRNA modification and its acetyltransferase NAT10 were increased in GC, and increased NAT10 expression was associated with disease progression and poor patient prognosis. Functionally, we found that NAT10 promoted cellular G2/M phase progression, proliferation and tumorigenicity of GC in an ac4C-depedent manner. Mechanistic analyses demonstrated that NAT10 mediated ac4C acetylation of MDM2 transcript and subsequently stabilized MDM2 mRNA, leading to its upregulation and p53 downregulation and thereby facilitating gastric carcinogenesis. In addition, Helicobacter pylori (Hp) infection contributed to NAT10 induction, causing MDM2 overexpression and subsequent p53 degradation. Further investigations revealed that targeting NAT10 with Remodelin showed anti-cancer activity in GC and augmented the anti-tumor activity of MDM2 inhibitors in p53 wild-type GC. CONCLUSIONS: These results suggest the critical role of NAT10-mediated ac4C modification in GC oncogenesis and reveal a previously unrecognized signaling cascade involving the Hp-NAT10-MDM2-p53 axis during GC development.

Selective inhibition of MCL1 overcomes venetoclax resistance in a murine model of myelodysplastic syndromes.

Treatment for myelodysplastic syndromes (MDS) remains insufficient due to clonal heterogeneity and lack of effective clinical therapies. Dysregulation of apoptosis is observed across MDS subtypes regardless of mutations and represents an attractive therapeutic opportunity. Venetoclax (VEN), a selective inhibitor of anti-apoptotic protein B-cell lymphoma- 2 (BCL2), has yielded impressive responses in older patients with acute myeloid leukemia (AML) and high risk MDS. BCL2 family anti-apoptotic proteins BCL-XL and induced myeloid cell leukemia 1 (MCL1) are implicated in leukemia survival, and upregulation of MCL1 is seen in VEN-resistant AML and MDS. We determined in vitro sensitivity of MDS patient samples to selective inhibitors of BCL2, BCL-XL and MCL1. While VEN response positively correlated with MDS with excess blasts, all MDS subtypes responded to MCL1 inhibition. Treatment with combined VEN + MCL1 inhibtion was synergistic in all MDS subtypes without significant injury to normal hematopoiesis and reduced MDS engraftment in MISTRG6 mice, supporting the pursuit of clinical trials with combined BCL2 + MCL1 inhibition in MDS.

Degree of stemness predicts micro-environmental response and clinical outcomes of diffuse large B-cell lymphoma and identifies a potential targeted therapy.

Some cells within a diffuse large B-cell lymphoma (DLBCL) have the genotype of a stem cell, the proportion of which is termed degree of stemness. We interrogated correlations between the degree of stemness with immune and stromal cell scores and clinical outcomes in persons with DLBCL. We evaluated gene expression data on 1,398 subjects from Gene Expression Omnibus to calculate the degree of stemness. Subjects were classified into low- and high-stemness cohorts based on restricted cubic spline plots. Weighted gene co-expression network analysis (WGCNA) was used to screen for stemness-related genes. Immune and stromal scores correlated with the degree of stemness (both P < 0.001). A high degree of stemness correlated with a shorter progression-free survival (PFS; Hazard Ratio [HR; 95% Confidence Interval [CI] =1.90 (1.37, 2.64; P < 0.001) and a shorter survival (HR = 2.29 (1.53, 3.44; P < 0.001). CDC7 expression correlated with the degree of stemness, and CDC7-inhibitors significantly increased apoptosis (P < 0.01), the proportion of cells in G1 phase (P < 0.01), and inhibited lymphoma growth in a mice xenograft model (P = 0.04). Our data indicate correlations between the degree of stemness, immune and stromal scores, PFS, and survival. These data will improve the prediction of therapy outcomes in DLBCL and suggest potential new therapies.

Targeting the EIF2AK1 Signaling Pathway Rescues Red Blood Cell Production in SF3B1-Mutant Myelodysplastic Syndromes With Ringed Sideroblasts.

SF3B1 mutations, which occur in 20% of patients with myelodysplastic syndromes (MDS), are the hallmarks of a specific MDS subtype, MDS with ringed sideroblasts (MDS-RS), which is characterized by the accumulation of erythroid precursors in the bone marrow and primarily affects the elderly population. Here, using single-cell technologies and functional validation studies of primary SF3B1-mutant MDS-RS samples, we show that SF3B1 mutations lead to the activation of the EIF2AK1 pathway in response to heme deficiency and that targeting this pathway rescues aberrant erythroid differentiation and enables the red blood cell maturation of MDS-RS erythroblasts. These data support the development of EIF2AK1 inhibitors to overcome transfusion dependency in patients with SF3B1-mutant MDS-RS with impaired red blood cell production. SIGNIFICANCE: MDS-RS are characterized by significant anemia. Patients with MDS-RS die from a shortage of red blood cells and the side effects of iron overload due to their constant need for transfusions. Our study has implications for the development of therapies to achieve long-lasting hematologic responses. This article is highlighted in the In This Issue feature, p. 476.

In vivo anti-tumor effect of PARP inhibition in IDH1/2 mutant MDS/AML resistant to targeted inhibitors of mutant IDH1/2.

Treatment options for patients with relapsed/refractory acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are scarce. Recurring mutations, such as mutations in isocitrate dehydrogenase-1 and -2 (IDH1/2) are found in subsets of AML and MDS, are therapeutically targeted by mutant enzyme-specific small molecule inhibitors (IDHmi). IDH mutations induce diverse metabolic and epigenetic changes that drive malignant transformation. IDHmi alone are not curative and resistance commonly develops, underscoring the importance of alternate therapeutic options. We were first to report that IDH1/2 mutations induce a homologous recombination (HR) defect, which confers sensitivity to poly (ADP)-ribose polymerase inhibitors (PARPi). Here, we show that the PARPi olaparib is effective against primary patient-derived IDH1/2-mutant AML/ MDS xeno-grafts (PDXs). Olaparib efficiently reduced overall engraftment and leukemia-initiating cell frequency as evident in serial transplantation assays in IDH1/2-mutant but not -wildtype AML/MDS PDXs. Importantly, we show that olaparib is effective in both IDHmi-naïve and -resistant AML PDXs, critical given the high relapse and refractoriness rates to IDHmi. Our pre-clinical studies provide a strong rationale for the translation of PARP inhibition to patients with IDH1/2-mutant AML/ MDS, providing an additional line of therapy for patients who do not respond to or relapse after targeted mutant IDH inhibition.

Precision analysis of mutant U2AF1 activity reveals deployment of stress granules in myeloid malignancies.

Splicing factor mutations are common among cancers, recently emerging as drivers of myeloid malignancies. U2AF1 carries hotspot mutations in its RNA-binding motifs; however, how they affect splicing and promote cancer remain unclear. The U2AF1/U2AF2 heterodimer is critical for 3' splice site (3'SS) definition. To specifically unmask changes in U2AF1 function in vivo, we developed a crosslinking and immunoprecipitation procedure that detects contacts between U2AF1 and the 3'SS AG at single-nucleotide resolution. Our data reveal that the U2AF1 S34F and Q157R mutants establish new 3'SS contacts at -3 and +1 nucleotides, respectively. These effects compromise U2AF2-RNA interactions, resulting predominantly in intron retention and exon exclusion. Integrating RNA binding, splicing, and turnover data, we predicted that U2AF1 mutations directly affect stress granule components, which was corroborated by single-cell RNA-seq. Remarkably, U2AF1-mutant cell lines and patient-derived MDS/AML blasts displayed a heightened stress granule response, pointing to a novel role for biomolecular condensates in adaptive oncogenic strategies.

Effects of NRAS Mutations on Leukemogenesis and Targeting of Children With Acute Lymphoblastic Leukemia.

Through the advancements in recent decades, childhood acute lymphoblastic leukemia (ALL) is gradually becoming a highly curable disease. However, the truth is there remaining relapse in ∼15% of ALL cases with dismal outcomes. RAS mutations, in particular NRAS mutations, were predominant mutations affecting relapse susceptibility. KRAS mutations targeting has been successfully exploited, while NRAS mutation targeting remains to be explored due to its complicated and compensatory mechanisms. Using targeted sequencing, we profiled RAS mutations in 333 primary and 18 relapsed ALL patients and examined their impact on ALL leukemogenesis, therapeutic potential, and treatment outcome. Cumulative analysis showed that RAS mutations were associated with a higher relapse incidence in children with ALL. In vitro cellular assays revealed that about one-third of the NRAS mutations significantly transformed Ba/F3 cells as measured by IL3-independent growth. Meanwhile, we applied a high-throughput drug screening method to characterize variable mutation-related candidate targeted agents and uncovered that leukemogenic-NRAS mutations might respond to MEK, autophagy, Akt, EGFR signaling, Polo-like Kinase, Src signaling, and TGF-ß receptor inhibition depending on the mutation profile.

Association Between NR3C1 Mutations and Glucocorticoid Resistance in Children With Acute Lymphoblastic Leukemia.

Treatment outcomes in children with acute lymphoblastic leukemia (ALL) have been improved substantially, with a cure rate exceeding 80% using conventional therapy. However, the outcome for patients with relapsed/refractory ALL remains unsatisfactory, despite the fact that these patients generally receive more intense therapy. Glucocorticoid (GC) resistance is a leading cause of treatment failure and relapse in ALL. Abnormal NR3C1 transcription and/or translation is strongly associated with GC resistance, but the underlying molecular mechanism and the clinical value of NR3C1 alterations with GC resistance in ALL treatment remain unclear. This study applied panel sequencing to 333 newly diagnosed and 18 relapsed ALL samples to characterize the link between NR3C1 and ALL further. We identified NR3C1 mutations in three patients with newly diagnosed ALL (0.9%) and two patients with relapsed ALL (11.1%). Functional analyses revealed that four of these five NR3C1 mutations (p. R477H, p. Y478C, p. P530fs, and p. H726P) were loss-of-function (LoF) mutations. A drug sensitivity test further showed that LoF NR3C1 mutations influence GC resistance. Saturated mutagenesis of hotspot R477 demonstrated the importance of this residue for NR3C1 function. The dominant-negative effect of p. R477C and p. R477S and the non-dominant negative effect of p. R477H and p. Y478C suggests multiple mechanisms underlying GC resistance. Thus, primary or acquired genomic lesions in NR3C1 may play a critical role in GC resistance and contribute to ALL treatment failure and/or relapse.

Combined liver-cytokine humanization comes to the rescue of circulating human red blood cells.

In vivo models that recapitulate human erythropoiesis with persistence of circulating red blood cells (RBCs) have remained elusive. We report an immunodeficient murine model in which combined human liver and cytokine humanization confer enhanced human erythropoiesis and RBC survival in the circulation. We deleted the fumarylacetoacetate hydrolase (Fah) gene in MISTRG mice expressing several human cytokines in place of their murine counterparts. Liver humanization by intrasplenic injection of human hepatocytes (huHep) eliminated murine complement C3 and reduced murine Kupffer cell density. Engraftment of human sickle cell disease (SCD)-derived hematopoietic stem cells in huHepMISTRGFah -/- mice resulted in vaso-occlusion that replicated acute SCD pathology. Combined liver-cytokine-humanized mice will facilitate the study of diseases afflicting RBCs, including bone marrow failure, hemoglobinopathies, and malaria, and also preclinical testing of therapies.

m6A Modification Prevents Formation of Endogenous Double-Stranded RNAs and Deleterious Innate Immune Responses during Hematopoietic Development.

N6-methyladenosine (m6A) is the most abundant RNA modification, but little is known about its role in mammalian hematopoietic development. Here, we show that conditional deletion of the m6A writer METTL3 in murine fetal liver resulted in hematopoietic failure and perinatal lethality. Loss of METTL3 and m6A activated an aberrant innate immune response, mediated by the formation of endogenous double-stranded RNAs (dsRNAs). The aberrantly formed dsRNAs were long, highly m6A modified in their native state, characterized by low folding energies, and predominantly protein coding. We identified coinciding activation of pattern recognition receptor pathways normally tasked with the detection of foreign dsRNAs. Disruption of the aberrant immune response via abrogation of downstream Mavs or Rnasel signaling partially rescued the observed hematopoietic defects in METTL3-deficient cells in vitro and in vivo. Our results suggest that m6A modification protects against endogenous dsRNA formation and a deleterious innate immune response during mammalian hematopoietic development.

Humanized mice as preclinical models for myeloid malignancies.

Humanized mice have proven to be invaluable for human hematological translational research since they offer essential tools to dissect disease biology and to bridge the gap between pre-clinical testing of novel therapeutics and their clinical applications. Many efforts have been placed to advance and optimize humanized mice to support the engraftment, differentiation, and maintenance of hematopoietic stem cells (HSCs) and the human hematological system in order to broaden the scope of applications of such models. This review covers the background of humanized mice, how they are used as platforms to model myeloid malignancies, and the various current and potential approaches to further enhance their utilization in biomedical research.

Functional Analysis of Human Hematopoietic Stem Cells In Vivo in Humanized Mice.

Ex vivo generation and expansion of functional hematopoietic stem cells represents the holy grail of reprogramming and would constitute a major advance in stem cell therapies and generation of blood cellular products. In vivo testing is critical to assure proper cell intrinsic function in an organismal context. Here we describe methods for the generation of human hematopoiesis chimeric mice and evaluation of hematopoietic stem cell function. The choice of mouse model, stem cell source, and transplantation route can be adjusted to suit the desired application.

A highly efficient and faithful MDS patient-derived xenotransplantation model for pre-clinical studies.

Comprehensive preclinical studies of Myelodysplastic Syndromes (MDS) have been elusive due to limited ability of MDS stem cells to engraft current immunodeficient murine hosts. Here we report a MDS patient-derived xenotransplantation model in cytokine-humanized immunodeficient "MISTRG" mice that provides efficient and faithful disease representation across all MDS subtypes. MISTRG MDS patient-derived xenografts (PDX) reproduce patients' dysplastic morphology with multi-lineage representation, including erythro- and megakaryopoiesis. MISTRG MDS-PDX replicate the original sample's genetic complexity and can be propagated via serial transplantation. MISTRG MDS-PDX demonstrate the cytotoxic and differentiation potential of targeted therapeutics providing superior readouts of drug mechanism of action and therapeutic efficacy. Physiologic humanization of the hematopoietic stem cell niche proves critical to MDS stem cell propagation and function in vivo. The MISTRG MDS-PDX model opens novel avenues of research and long-awaited opportunities in MDS research.

SRSF2 mutations drive oncogenesis by activating a global program of aberrant alternative splicing in hematopoietic cells.

Recurrent mutations in the splicing factor SRSF2 are associated with poor clinical outcomes in myelodysplastic syndromes (MDS). Their high frequency suggests these mutations drive oncogenesis, yet the molecular explanation for this process is unclear. SRSF2 mutations could directly affect pre-mRNA splicing of a vital gene product; alternatively, a whole network of gene products could be affected. Here we determine how SRSF2 mutations globally affect RNA binding and splicing in vivo using HITS-CLIP. Remarkably, the majority of differential binding events do not translate into alternative splicing of exons with SRSF2P95H binding sites. Alternative splice alterations appear to be dominated by indirect effects. Importantly, SRSF2P95H targets are enriched in RNA processing and splicing genes, including several members of the hnRNP and SR families of proteins, suggesting a "splicing-cascade" phenotype wherein mutation of a single splicing factor leads to widespread modifications in multiple RNA processing and splicing proteins. We show that splice alteration of HNRNPA2B1, a splicing factor differentially bound and spliced by SRSF2P95H, impairs hematopoietic differentiation in vivo. Our data suggests a model whereby the recurrent mutations in splicing factors set off a cascade of gene regulatory events that together affect hematopoiesis and drive cancer.

2-Hydroxyglutarate produced by neomorphic IDH mutations suppresses homologous recombination and induces PARP inhibitor sensitivity.

2-Hydroxyglutarate (2HG) exists as two enantiomers, (R)-2HG and (S)-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations, whereas the latter is produced under pathologic processes such as hypoxia. We report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitors. This "BRCAness" phenotype of IDH mutant cells can be completely reversed by treatment with small-molecule inhibitors of the mutant IDH1 enzyme, and conversely, it can be entirely recapitulated by treatment with either of the 2HG enantiomers in cells with intact IDH1/2 proteins. We demonstrate mutant IDH1-dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH, rather than attempting to block 2HG production, by targeting the 2HG-dependent HR deficiency with PARP inhibition. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair, and genetic instability.

Small interfering RNA targeting NF-κB attenuates lipopolysaccharide-induced acute lung injury in rats.

BACKGROUND: To investigate the anti-inflammatory effects of specific small interfering RNA targeting NF-κB on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in rats. METHOD: Acute lung injury was induced in Sprague-Dawley rats by intraperitoneal injection with LPS (5 mg/kg), followed by immediate intratracheal instillation of siRNA targeting NF-κB p65 (40 µg/ml). Animals in each group were sacrificed at 1 h or 8 h after the instillation. Pulmonary histological changes were evaluated by hematoxylin-eosin staining. The levels of NF-κB and TNF-α were measured by qRT-PCR. Expressions of NF-κB in lung cells and TNF-α in bronchoalveolar lavage fluid (BALF) were determined by western blot analysis and enzyme-linked immunosorbent assay (ELISA) respectively. RESULTS: LPS administration reduced the rectal temperature and white blood cell counts at 1 h, increased lung wet/dry weight ratios, caused evident lung histopathological injury, and increased the detectable transcript and cytokine levels of TNF-α in lung tissue in BALF. siRNA targeting of NF-κB p65 effectively abrogated the expression of NF-κB p65 in lung cells and, aside from rectal temperatures, ameliorated all changes induced by LPS. CONCLUSIONS: NF-κB knockdown exerts anti-inflammatory effects on LPS-induced ALI especially in the initial phase, which may be due in part to reduced levels of the proinflammatory cytokine TNF-α. NF-κB siRNA's rapidity and effectiveness to abrogate ALI development may provide an effective therapeutic method with future clinical applications.