Metabolic dependencies of acute myeloid leukemia stem cells.

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy primarily driven by an immature population of AML cells termed leukemia stem cells (LSCs) that are implicated in AML development, chemoresistance, and relapse. An emerging area of research in AML focuses on identifying and targeting the aberrant metabolism in LSCs. Dysregulated metabolism is involved in sustaining functional properties of LSCs, impeding myeloid differentiation, and evading programmed cell death, both in the process of leukemogenesis and in response to chemotherapy. This review discusses recent discoveries regarding the aberrant metabolic processes of AML LSCs that have begun to change the therapeutic landscape of AML.

An oncogenic enhancer encodes selective selenium dependency in AML.

Deregulation of transcription is a hallmark of acute myeloid leukemia (AML) that drives oncogenic expression programs and presents opportunities for therapeutic targeting. By integrating comprehensive pan-cancer enhancer landscapes with genetic dependency mapping, we find that AML-enriched enhancers encode for more selective tumor dependencies. We hypothesized that this approach could identify actionable dependencies downstream of oncogenic driver events and discovered a MYB-regulated AML-enriched enhancer regulating SEPHS2, a key component of the selenoprotein production pathway. Using a combination of patient samples and mouse models, we show that this enhancer upregulates SEPHS2, promoting selenoprotein production and antioxidant function required for AML survival. SEPHS2 and other selenoprotein pathway genes are required for AML growth in vitro. SEPHS2 knockout and selenium dietary restriction significantly delay leukemogenesis in vivo with little effect on normal hematopoiesis. These data validate the utility of enhancer mapping in target identification and suggest that selenoprotein production is an actionable target in AML.

The histone H3.3 chaperone HIRA restrains erythroid-biased differentiation of adult hematopoietic stem cells.

Histone variants contribute to the complexity of the chromatin landscape and play an integral role in defining DNA domains and regulating gene expression. The histone H3 variant H3.3 is incorporated into genic elements independent of DNA replication by its chaperone HIRA. Here we demonstrate that Hira is required for the self-renewal of adult hematopoietic stem cells (HSCs) and to restrain erythroid differentiation. Deletion of Hira led to rapid depletion of HSCs while differentiated hematopoietic cells remained largely unaffected. Depletion of HSCs after Hira deletion was accompanied by increased expression of bivalent and erythroid genes, which was exacerbated upon cell division and paralleled increased erythroid differentiation. Assessing H3.3 occupancy identified a subset of polycomb-repressed chromatin in HSCs that depends on HIRA to maintain the inaccessible, H3.3-occupied state for gene repression. HIRA-dependent H3.3 incorporation thus defines distinct repressive chromatin that represses erythroid differentiation of HSCs.

Nuclear NAD+ homeostasis governed by NMNAT1 prevents apoptosis of acute myeloid leukemia stem cells.

Metabolic dysregulation underlies malignant phenotypes attributed to cancer stem cells, such as unlimited proliferation and differentiation blockade. Here, we demonstrate that NAD+ metabolism enables acute myeloid leukemia (AML) to evade apoptosis, another hallmark of cancer stem cells. We integrated whole-genome CRISPR screening and pan-cancer genetic dependency mapping to identify NAMPT and NMNAT1 as AML dependencies governing NAD+ biosynthesis. While both NAMPT and NMNAT1 were required for AML, the presence of NAD+ precursors bypassed the dependence of AML on NAMPT but not NMNAT1, pointing to NMNAT1 as a gatekeeper of NAD+ biosynthesis. Deletion of NMNAT1 reduced nuclear NAD+, activated p53, and increased venetoclax sensitivity. Conversely, increased NAD+ biosynthesis promoted venetoclax resistance. Unlike leukemia stem cells (LSCs) in both murine and human AML xenograft models, NMNAT1 was dispensable for hematopoietic stem cells and hematopoiesis. Our findings identify NMNAT1 as a previously unidentified therapeutic target that maintains NAD+ for AML progression and chemoresistance.

Protein quality control through endoplasmic reticulum-associated degradation maintains haematopoietic stem cell identity and niche interactions.

Stem cells need to be protected from genotoxic and proteotoxic stress to maintain a healthy pool throughout life1-3. Little is known about the proteostasis mechanism that safeguards stem cells. Here we report endoplasmic reticulum-associated degradation (ERAD) as a protein quality checkpoint that controls the haematopoietic stem cell (HSC)-niche interaction and determines the fate of HSCs. The SEL1L-HRD1 complex, the most conserved branch of ERAD4, is highly expressed in HSCs. Deletion of Sel1l led to niche displacement of HSCs and a complete loss of HSC identity, and allowed highly efficient donor-HSC engraftment without irradiation. Mechanistic studies identified MPL, the master regulator of HSC identity5, as a bona fide ERAD substrate that became aggregated in the endoplasmic reticulum following ERAD deficiency. Restoration of MPL signalling with an agonist partially rescued the number and reconstitution capacity of Sel1l-deficient HSCs. Our study defines ERAD as an essential proteostasis mechanism to safeguard a healthy stem cell pool by regulating the stem cell-niche interaction.

Function of Arl4aa in the Initiation of Hematopoiesis in Zebrafish by Maintaining Golgi Complex Integrity in Hemogenic Endothelium.

ADP-ribosylation factor-like 4aa (Arl4aa) is a member of the ADP-ribosylation factor family. It is expressed in hematopoietic tissue during embryonic development, but its function was unknown. Zebrafish arl4aa is preferentially expressed in the ventral wall of the dorsal aorta (VDA) at 24 and 36 hpf and in caudal hematopoietic tissue at 48 hpf. Morpholino knockdown and transcription activator-like effector nuclease (TALEN) knockout of arl4aa significantly reduced expression of genes associated with definitive hematopoietic stem cells (HSCs). Golgi complex integrity in VDA was disrupted as shown by transmission electron microscopy and immunostaining of Golgi membrane Giantin. Mechanistically, arl4aa knockdown reduced Notch signaling in the VDA and its target gene expression. Protein expression of NICD was also reduced. Effects of arl4aa knockdown on definitive hematopoiesis could be restored by NICD expression. This study identified arl4aa as a factor regulating initiation of definitive HSCs by maintaining the integrity of Golgi complex and, secondarily, maturation of the Notch receptor.

AMP-activated protein kinase links acetyl-CoA homeostasis to BRD4 recruitment in acute myeloid leukemia.

Altered metabolism fuels 2 hallmark properties of cancer cells: unlimited proliferation and differentiation blockade. Adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of bioenergetics crucial for glucose metabolism in acute myeloid leukemia (AML), and its inhibition delays leukemogenesis, but whether the metabolic function of AMPK alters the AML epigenome remains unknown. Here, we demonstrate that AMPK maintains the epigenome of MLL-rearranged AML by linking acetyl-coenzyme A (CoA) homeostasis to Bromodomain and Extra-Terminal domain (BET) protein recruitment to chromatin. AMPK deletion reduced acetyl-CoA and histone acetylation, displacing BET proteins from chromatin in leukemia-initiating cells. In both mouse and patient-derived xenograft AML models, treating with AMPK and BET inhibitors synergistically suppressed AML. Our results provide a therapeutic rationale to target AMPK and BET for AML therapy.

Clonal expansion and myeloid leukemia progression modeled by multiplex gene editing of murine hematopoietic progenitor cells.

Recent advances in next-generation sequencing have identified novel mutations and revealed complex genetic architectures in human hematological malignancies. Moving forward, new methods to quickly generate animal models that recapitulate the complex genetics of human hematological disorders are needed to transform the genetic information to new therapies. Here, we used a ribonucleoprotein-based CRISPR/Cas9 system to model human clonal hematopoiesis of indeterminate potential and acute myeloid leukemia (AML). We edited multiple genes recurrently mutated in hematological disorders, including those encoding epigenetic regulators, transcriptional regulators, and signaling components in murine hematopoietic stem/progenitor cells. Tracking the clonal dynamics by sequencing the indels induced by CRISPR/Cas9 revealed clonal expansion in some recipient mice that progressed to AML initiated by leukemia-initiating cells. Our results establish that the CRISPR/Cas9-mediated multiplex mutagenesis can be used to engineer a variety of murine models of hematological malignancies with complex genetic architectures seen in human disease.

Design and Application of a DNA-Encoded Macrocyclic Peptide Library.

A DNA-encoded macrocyclic peptide library was designed and synthesized with 2.4 × 1012 members composed of 4-20 natural and non-natural amino acids. Affinity-based selection was performed against two therapeutic targets, VHL and RSV N protein. On the basis of selection data, some peptides were selected for resynthesis without a DNA tag, and their activity was confirmed.

A Zebrafish Model for Evaluating the Function of Human Leukemic Gene IDH1 and Its Mutation.

The recent advent of next-generation sequencing (NGS) has greatly accelerated identification of gene mutations in myeloid malignancies at unprecedented speed that will soon outpace their functional validation by conventional laboratory techniques and animal models. A high-throughput whole-organism model is useful for the functional validation of new mutations. We recently reported the use of zebrafish to evaluate the hematopoietic function of isocitrate dehydrogenase 1 (IDH1) and the effects of expressing human IDH1-R132H that is frequently identified in human acute myeloid leukemia (AML), in myelopoiesis, with a view to develop zebrafish as a model of AML. Here, we use IDH1 as an example to describe a comprehensive approach to evaluate hematopoietic gene function and the effects of mutations using zebrafish as a model.

Fishing the targets of myeloid malignancies in the era of next generation sequencing.

Recent advent in next generation sequencing (NGS) and bioinformatics has generated an unprecedented amount of genetic information in myeloidmalignancies. This information may shed lights to the pathogenesis, diagnosis and prognostication of these diseases and provide potential targets for therapeutic intervention. However, the rapid emergence of genetic information will quickly outpace their functional validation by conventional laboratory platforms. Foundational knowledge about zebrafish hematopoiesis accumulated over the past two decades and novel genomeediting technologies and research strategies in thismodel organismhavemade it a unique and timely research tool for the study of human blood diseases. Recent studies modeling human myeloid malignancies in zebrafish have also highlighted the technical feasibility and clinical relevance of thesemodels. Careful validation of experimental protocols and standardization among laboratorieswill further enhance the application of zebrafish in the scientific communities and provide important insights to the personalized treatment ofmyeloid malignancies.

Functions of idh1 and its mutation in the regulation of developmental hematopoiesis in zebrafish.

Isocitrate dehydrogenase 1 mutation (IDH1-R132H) was recently identified in acute myeloid leukemia with normal cytogenetics. The mutant enzyme is thought to convert α-ketoglutarate to the pathogenic 2-hydroxyglutarate (2-HG) that affects DNA methylation via inhibition of ten-eleven translocation 2. However, the role of wild-type IDH1 in normal hematopoiesis and its relevance to acute myeloid leukemia is unknown. Here we showed that zebrafish idh1 (zidh1) knockdown by morpholino and targeted mutagenesis by transcription activator-like effector nuclease might induce blockade in myeloid differentiation, as evident by an increase in pu.1 and decrease in mpo, l-plastin, and mpeg1 expression, and significantly reduce definitive hematopoiesis. Morpholino knockdown of zidh2 also induced a blockade in myeloid differentiation but definitive hematopoiesis was not affected. The hematopoietic phenotype of zidh1 knockdown was not rescuable by zidh2 messenger RNA, suggesting nonredundant functions. Overexpression of human IDH1-R132H or its zebrafish ortholog resulted in 2-HG elevation and expansion of myelopoiesis in zebrafish embryos. A human IDH1-R132H-specific inhibitor (AGI-5198) significantly ameliorated both hematopoietic and 2-HG responses in human but not zebrafish IDH1 mutant expression. The results provided important insights to the role of zidh1 in myelopoiesis and definitive hematopoiesis and of IDH1-R132H in leukemogenesis.

Functions of flt3 in zebrafish hematopoiesis and its relevance to human acute myeloid leukemia.

FMS-like tyrosine kinase 3 (FLT3) is expressed in human hematopoietic stem and progenitor cells (HSPCs) but its role during embryogenesis is unclear. In acute myeloid leukemia (AML), internal tandem duplication (ITD) of FLT3 at the juxtamembrane (JMD) and tyrosine kinase (TKD) domains (FLT3-ITD(+)) occurs in 30% of patients and is associated with inferior clinical prognosis. TKD mutations (FLT3-TKD(+)) occur in 5% of cases. We made use of zebrafish to examine the role of flt3 in developmental hematopoiesis and model human FLT3-ITD(+) and FLT3-TKD(+) AML. Zebrafish flt3 JMD and TKD were remarkably similar to their mammalian orthologs. Morpholino knockdown significantly reduced the expression of l-plastin (pan-leukocyte), csf1r, and mpeg1 (macrophage) as well as that of c-myb (definitive HSPCs), lck, and rag1 (T-lymphocyte). Expressing human FLT3-ITD in zebrafish embryos resulted in expansion and clustering of myeloid cells (pu.1(+), mpo(+), and cebpα(+)) which were ameliorated by AC220 and associated with stat5, erk1/2, and akt phosphorylation. Human FLT3-TKD (D835Y) induced significant, albeit modest, myeloid expansion resistant to AC220. This study provides novel insight into the role of flt3 during hematopoiesis and establishes a zebrafish model of FLT3-ITD(+) and FLT3-TKD(+) AML that may facilitate high-throughput screening of novel and personalized agents.

Hydrogen exchange-mass spectrometry measures stapled peptide conformational dynamics and predicts pharmacokinetic properties.

Peptide drugs have traditionally suffered from poor pharmacokinetic properties due to their conformational flexibility and the interaction of proteases with backbone amide bonds. "Stapled Peptides" are cyclized using an all-hydrocarbon cross-linking strategy to reinforce their α-helical conformation, yielding improved protease resistance and drug-like properties. Here we demonstrate that hydrogen exchange-mass spectrometry (HX-MS) effectively probes the conformational dynamics of Stapled Peptides derived from the survivin-borealin protein-protein interface and predicts their susceptibility to proteolytic degradation. In Stapled Peptides, amide exchange was reduced by over five orders-of-magnitude versus the native peptide sequence depending on staple placement. Furthermore, deuteration kinetics correlated directly with rates of proteolysis to reveal the optimal staple placement for improved drug properties.

Stapled α-helical peptide drug development: a potent dual inhibitor of MDM2 and MDMX for p53-dependent cancer therapy.

Stapled α-helical peptides have emerged as a promising new modality for a wide range of therapeutic targets. Here, we report a potent and selective dual inhibitor of MDM2 and MDMX, ATSP-7041, which effectively activates the p53 pathway in tumors in vitro and in vivo. Specifically, ATSP-7041 binds both MDM2 and MDMX with nanomolar affinities, shows submicromolar cellular activities in cancer cell lines in the presence of serum, and demonstrates highly specific, on-target mechanism of action. A high resolution (1.7-Å) X-ray crystal structure reveals its molecular interactions with the target protein MDMX, including multiple contacts with key amino acids as well as a role for the hydrocarbon staple itself in target engagement. Most importantly, ATSP-7041 demonstrates robust p53-dependent tumor growth suppression in MDM2/MDMX-overexpressing xenograft cancer models, with a high correlation to on-target pharmacodynamic activity, and possesses favorable pharmacokinetic and tissue distribution properties. Overall, ATSP-7041 demonstrates in vitro and in vivo proof-of-concept that stapled peptides can be developed as therapeutically relevant inhibitors of protein-protein interaction and may offer a viable modality for cancer therapy.

Aldehyde complexes with protonated peptides in the gas phase.

This Article presents a study of aldehyde complexes with peptide ions formed by bimolecular collisions in the gas phase. Desolvated ions generated by electrospray ionization are stored within a radio frequency (RF) ion trap and exposed to aldehyde vapor. Mass spectrometry measurements were performed on the resulting aldehyde complexes formed with single amino acids (LysH(+), HisH(+), and ArgH(+)) and polypeptides [Pro(n)-Lys+2H](2+) and [(Gly-Ser)(m)-Lys+2H](2+). These data identify several interesting and unexpected aspects of the aldehyde complex kinetics. It is observed that the formation of stable complexes requires the presence of water vapor. The formation kinetics of aldehyde-peptide complexes exhibits multiexponential time dependence that is modeled by interactions in the presence of structural heterogeneity. Aldehyde binding appears to involve a competition between conformers with unhindered access to protonation sites and conformers with intramolecular solvation of these sites. Proton transfer to the aldehyde ligand is responsible for the loss of the complexes. This is supported by proton affinity calculations and identified by reaction products exhibiting loss of protonation by the parent ion accompanied by the appearance of aldehyde cations.

Developmental toxicity of cypermethrin in embryo-larval stages of zebrafish.

Cypermethrin, a type II pyrethroid insecticide, is widely used throughout the world in agriculture, forestry, horticulture and homes. Though the neurotoxicity of cypermethrin has been thoroughly studied in adult rodents, little is so far available regarding the developmental toxicity of cypermethrin to fish in early life stages. To explore the potential developmental toxicity of cypermethrin, 4-h post-fertilization (hpf) zebrafish embryos were exposed to various concentrations of cypermethrin (0, 25, 50, 100, 200 and 400 µg L⁻¹) until 96 h. Among a suite of morphological abnormalities, the unique phenotype curvature was observed at concentrations as low as 25 µg L⁻¹. Studies revealed that 400 µg L⁻¹ cypermethrin significantly increased malondialdehyde production. In addition, activity of antioxidative enzymes including superoxide dismutase and catalase were significantly induced in zebrafish larvae in a concentration-dependent manner. To further investigate the toxic effects of cypermethrin on fish, acridine orange (AO) staining was performed at 400 µg L⁻¹ cypermethrin and the result showed notable signs of apoptosis mainly in the nervous system. Cypermethrin also down-regulated ogg1 and increased p53 gene expression as well as the caspase-3 activity. Our results demonstrate that cypermethrin was able to induce oxidative stress and produce apoptosis through the involvement of caspases in zebrafish embryos. In this study, we investigated the developmental toxicity of cypermethrin using zebrafish embryos, which could be helpful in fully understanding the potential mechanisms of cypermethrin exposure during embryogenesis and also suggested that zebrafish could serve as an ideal model for studying developmental toxicity of environmental contaminants.

Assessment of an association between an aryl hydrocarbon receptor gene (AHR) polymorphism and risk of male infertility.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxicity of a variety of environmental chemicals, such as polycyclic aromatic hydrocarbons (PAHs) and dioxins. We hypothesized that polymorphisms of AHR may result in significant differences in sensitivity to toxic effects of PAHs or dioxins and contribute to susceptibility to male infertility. To address this possibility, we conducted a study including 580 idiopathic infertile subjects and 580 fertile controls to assess associations between the male infertility risk and six tagging single nucleotide polymorphisms of AHR gene. Additionally, correlations between AHR polymorphisms and sperm concentration, levels of DNA fragmentation, and benzo(a)pyrene diolepoxide (BPDE)-DNA adducts in sperm were determined in 420 patients. Genotypes were determined using the ABI OpenArray platform. Sperm DNA fragmentation was evaluated by terminal deoxyribonucleotidyl transferase (TdT)-mediated dUDP nick-end labelling (TUNEL) assay, and sperm BPDE-DNA adducts were measured by immunofluorescent assay using flow cytometry. We found that the G variant of rs2158041 was associated with significantly increased risk of male infertility (adjusted odds ratio 1.53, 95% confidence interval 1.21-1.93; p = 6.0 x 10⁻6; GA/AA vs. GG genotypes). Furthermore, patients with rs2158041 AA genotype showed a reduced sperm concentration. In addition, a gradual increase of sperm DNA fragmentation and sperm BPDE-DNA adducts was found among the three rs2158041 subgroups (GG → GA → AA), though the differences were not statistically significant. These results suggested that the AHR polymorphism might be associated with individual risk of male infertility in the Chinese population study.

Effects of non-occupational environmental exposure to pyrethroids on semen quality and sperm DNA integrity in Chinese men.

Observations in several western and Asiatic countries point toward a decline in semen quality which may be associated with environmental exposures. To investigate the effect of environmental exposure to pyrethroids on sperm DNA integrity and semen quality, 240 men were recruited from an infertility clinic through the clinic following strict eligibility screening. Urinary 3-phenoxybenzoic acid (3-PBA) concentration, semen quality, and sperm DNA integrity were evaluated. After adjustment for potential confounders, a significant inverse correlation was observed between the urinary 3-PBA level and the sperm concentration (ß=-0.27, 95%CI: -0.41 to -0.12, P<0.001). Moreover, we also found a significant positive correlation between urinary 3-PBA level and sperm DNA fragmentation (ß=0.27, 95%CI: 0.15-0.39, P<0.001). Our results suggest that non-occupational environmental pyrethroids exposure may have a negative impact on sperm DNA integrity and semen quality in Chinese males.