Proteomic analysis reveals a potential role for extracellular vesicles within the erythroblastic island niche.

Introduction: Erythroblastic island (EBI) macrophages play an essential role in the production and maturation of the vast numbers of red blood cells (RBCs) that are produced throughout life. Their location within the bone marrow makes it difficult to study the cellular and molecular interactions associated with their action so we have used an in vitro model of the EBI niche using macrophages derived from human induced pluripotent stem cells (hiPSCs). We previously demonstrated that the activation of the transcription factor KLF1 enhanced the activity of hiPSC-derived EBI macrophages. Methods: To elucidate the mechanisms associated with EBI-like activity we carried out a quantitative proteomic analysis and assessed the role of extracellular vesicles using Nanosight Tracking analyses and media filtration. Results and Discussion: Gene ontology analysis showed that many of the proteins upregulated by KLF1 were protein-binding factors, some of which were associated with the cell membrane or extracellular vesicles We demonstrated that filtration of macrophage-conditioned media resulted in a reduction in the supportive effects on erythroid cell viability and maturation implying a role for extracellular vesicles but this was not KLF1 dependent. Pathway analyses of the proteomic data revealed that proteins upregulated by KLF1 were associated with the citric acid cycle, pyruvate metabolism and ATP synthesis indicating that KLF1-activated macrophages had a metabolic profile comparable to a pro-reparative phenotype. This study has generated a proteomic dataset that could provide new insights into the role of macrophages within the EBI niche and has indicated a potential role for extracellular vesicles in the differentiation and maturation of RBCs in vitro. Further research will aid in the production of RBCs in vitro for use in disease modelling and cell therapy.

Investigation of inherited noncoding genetic variation impacting the pharmacogenomics of childhood acute lymphoblastic leukemia treatment.

Defining genetic factors impacting chemotherapy failure can help to better predict response and identify drug resistance mechanisms. However, there is limited understanding of the contribution of inherited noncoding genetic variation on inter-individual differences in chemotherapy response in childhood acute lymphoblastic leukemia (ALL). Here we map inherited noncoding variants associated with treatment outcome and/or chemotherapeutic drug resistance to ALL cis-regulatory elements and investigate their gene regulatory potential and target gene connectivity using massively parallel reporter assays and three-dimensional chromatin looping assays, respectively. We identify 54 variants with transcriptional effects and high-confidence gene connectivity. Additionally, functional interrogation of the top variant, rs1247117, reveals changes in chromatin accessibility, PU.1 binding affinity and gene expression, and deletion of the genomic interval containing rs1247117 sensitizes cells to vincristine. Together, these data demonstrate that noncoding regulatory variants associated with diverse pharmacological traits harbor significant effects on allele-specific transcriptional activity and impact sensitivity to antileukemic agents.

Human cellular model systems of ß-thalassemia enable in-depth analysis of disease phenotype.

ß-thalassemia is a prevalent genetic disorder causing severe anemia due to defective erythropoiesis, with few treatment options. Studying the underlying molecular defects is impeded by paucity of suitable patient material. In this study we create human disease cellular model systems for ß-thalassemia by gene editing the erythroid line BEL-A, which accurately recapitulate the phenotype of patient erythroid cells. We also develop a high throughput compatible fluorometric-based assay for evaluating severity of disease phenotype and utilize the assay to demonstrate that the lines respond appropriately to verified reagents. We next use the lines to perform extensive analysis of the altered molecular mechanisms in ß-thalassemia erythroid cells, revealing upregulation of a wide range of biological pathways and processes along with potential novel targets for therapeutic investigation. Overall, the lines provide a sustainable supply of disease cells as research tools for identifying therapeutic targets and as screening platforms for new drugs and reagents.

A novel human cellular model of CDA IV enables comprehensive analysis revealing the molecular basis of the disease phenotype.

Red blood cell disorders can result in severe anemia. One such disease congenital dyserythropoietic anemia IV (CDA IV) is caused by the heterozygous mutation E325K in the transcription factor KLF1. However, studying the molecular basis of CDA IV is severely impeded by the paucity of suitable and adequate quantities of material from patients with anemia and the rarity of the disease. We, therefore, took a novel approach, creating a human cellular disease model system for CDA IV that accurately recapitulates the disease phenotype. Next, using comparative proteomics, we reveal extensive distortion of the proteome and a wide range of disordered biological processes in CDA IV erythroid cells. These include downregulated pathways the governing cell cycle, chromatin separation, DNA repair, cytokinesis, membrane trafficking, and global transcription, and upregulated networks governing mitochondrial biogenesis. The diversity of such pathways elucidates the spectrum of phenotypic abnormalities that occur with CDA IV and impairment to erythroid cell development and survival, collectively explaining the CDA IV disease phenotype. The data also reveal far more extensive involvement of KLF1 in previously assigned biological processes, along with novel roles in the regulation of intracellular processes not previously attributed to this transcription factor. Overall, the data demonstrate the power of such a model cellular system to unravel the molecular basis of disease and how studying the effects of a rare mutation can reveal fundamental biology.

Functional investigation of inherited noncoding genetic variation impacting the pharmacogenomics of childhood acute lymphoblastic leukemia treatment.

Although acute lymphoblastic leukemia (ALL) is the most common childhood cancer, there is limited understanding of the contribution of inherited genetic variation on inter-individual differences in chemotherapy response. Defining genetic factors impacting therapy failure can help better predict response and identify drug resistance mechanisms. We therefore mapped inherited noncoding variants associated with chemotherapeutic drug resistance and/or treatment outcome to ALL cis-regulatory elements and investigated their gene regulatory potential and genomic connectivity using massively parallel reporter assays and promoter capture Hi-C, respectively. We identified 53 variants with reproducible allele-specific effects on transcription and high-confidence gene targets. Subsequent functional interrogation of the top variant (rs1247117) determined that it disrupted a PU.1 consensus motif and PU.1 binding affinity. Importantly, deletion of the genomic interval containing rs1247117 sensitized ALL cells to vincristine. Together, these data demonstrate that noncoding regulatory variation associated with diverse pharmacological traits harbor significant effects on allele-specific transcriptional activity and impact sensitivity to chemotherapeutic agents in ALL.

Epigenomic profiling of glucocorticoid responses identifies cis-regulatory disruptions impacting steroid resistance in childhood acute lymphoblastic leukemia.

Glucocorticoids (GCs) are a mainstay of contemporary, multidrug chemotherapy in the treatment of childhood acute lymphoblastic leukemia (ALL), and resistance to GCs remains a major clinical concern. Resistance to GCs is predictive of ALL relapse and poor clinical outcome, and therefore represents a major hurdle limiting further improvements in survival rates. While advances have been made in identifying genes implicated in GC resistance, there remains an insufficient understanding of the impact of cis-regulatory disruptions in resistance. To address this, we mapped the gene regulatory response to GCs in two ALL cell lines using functional genomics and high-throughput reporter assays and identified thousands of GC-responsive changes to chromatin state, including the formation of over 250 GC-responsive super-enhancers and a depletion of AP-1 bound cis-regulatory elements implicated in cell proliferation and anti-apoptotic processes. By integrating our GC response maps with genetic and epigenetic datasets in primary ALL cells from patients, we further uncovered cis-regulatory disruptions at GC-responsive genes that impact GC resistance in childhood ALL. Overall, these data indicate that GCs initiate pervasive effects on the leukemia epigenome, and that alterations to the GC gene regulatory network contribute to GC resistance.

Amino acid stress response genes promote L-asparaginase resistance in pediatric acute lymphoblastic leukemia.

Understanding the genomic and epigenetic mechanisms of drug resistance in pediatric acute lymphoblastic leukemia (ALL) is critical for further improvements in treatment outcomes. The role of transcriptomic response in conferring resistance to l-asparaginase (LASP) is poorly understood beyond asparagine synthetase (ASNS). We defined reproducible LASP response genes in LASP-resistant and LASP-sensitive ALL cell lines as well as primary leukemia samples from newly diagnosed patients. Defining target genes of the amino acid stress response-related transcription factor activating transcription factor 4 (ATF4) in ALL cell lines using chromatin immunoprecipitation sequencing (ChIP-seq) revealed 45% of genes that changed expression after LASP treatment were direct targets of the ATF4 transcription factor, and 34% of these genes harbored LASP-responsive ATF4 promoter binding events. SLC7A11 was found to be a response gene in cell lines and patient samples as well as a direct target of ATF4. SLC7A11 was also one of only 2.4% of LASP response genes with basal level gene expression that also correlated with LASP ex vivo resistance in primary leukemia cells. Experiments using chemical inhibition of SLC7A11 with sulfasalazine, gene overexpression, and partial gene knockout recapitulated LASP resistance or sensitivity in ALL cell lines. These findings show the importance of assessing changes in gene expression following treatment with an antileukemic agent for its association with drug resistance and highlight that many response genes may not differ in their basal expression in drug-resistant leukemia cells.

Reproducible immortalization of erythroblasts from multiple stem cell sources provides approach for sustainable RBC therapeutics.

Developing robust methodology for the sustainable production of red blood cells in vitro is essential for providing an alternative source of clinical-quality blood, particularly for individuals with rare blood group phenotypes. Immortalized erythroid progenitor cell lines are the most promising emergent technology for achieving this goal. We previously created the erythroid cell line BEL-A from bone marrow CD34+ cells that had improved differentiation and enucleation potential compared to other lines reported. In this study we show that our immortalization approach is reproducible for erythroid cells differentiated from bone marrow and also from far more accessible peripheral and cord blood CD34+ cells, consistently generating lines with similar improved erythroid performance. Extensive characterization of the lines shows them to accurately recapitulate their primary cell equivalents and provides a molecular signature for immortalization. In addition, we show that only cells at a specific stage of erythropoiesis, predominantly proerythroblasts, are amenable to immortalization. Our methodology provides a step forward in the drive for a sustainable supply of red cells for clinical use and for the generation of model cellular systems for the study of erythropoiesis in health and disease, with the added benefit of an indefinite expansion window for manipulation of molecular targets.

Identification of the transcription factor MAZ as a regulator of erythropoiesis.

Erythropoiesis requires a combination of ubiquitous and tissue-specific transcription factors (TFs). Here, through DNA affinity purification followed by mass spectrometry, we have identified the widely expressed protein MAZ (Myc-associated zinc finger) as a TF that binds to the promoter of the erythroid-specific human α-globin gene. Genome-wide mapping in primary human erythroid cells revealed that MAZ also occupies active promoters as well as GATA1-bound enhancer elements of key erythroid genes. Consistent with an important role during erythropoiesis, knockdown of MAZ reduces α-globin expression in K562 cells and impairs differentiation in primary human erythroid cells. Genetic variants in the MAZ locus are associated with changes in clinically important human erythroid traits. Taken together, these findings reveal the zinc-finger TF MAZ to be a previously unrecognized regulator of the erythroid differentiation program.

Identification of small molecules that mitigate vincristine-induced neurotoxicity while sensitizing leukemia cells to vincristine.

Vincristine (VCR) is one of the most widely prescribed medications for treating solid tumors and acute lymphoblastic leukemia (ALL) in children and adults. However, its major dose-limiting toxicity is peripheral neuropathy that can disrupt curative therapy. Peripheral neuropathy can also persist into adulthood, compromising quality of life of childhood cancer survivors. Reducing VCR-induced neurotoxicity without compromising its anticancer effects would be ideal. Here, we show that low expression of NHP2L1 is associated with increased sensitivity of primary leukemia cells to VCR, and that concomitant administration of VCR with inhibitors of NHP2L1 increases VCR cytotoxicity in leukemia cells, prolongs survival of ALL xenograft mice, but decreases VCR effects on human-induced pluripotent stem cell-derived neurons and mitigates neurotoxicity in mice. These findings offer a strategy for increasing VCR's antileukemic effects while reducing peripheral neuropathy in patients treated with this widely prescribed medication.

Profiling chromatin accessibility in pediatric acute lymphoblastic leukemia identifies subtype-specific chromatin landscapes and gene regulatory networks.

Acute lymphoblastic leukemia (ALL) is a hematopoietic malignancy comprised of molecular subtypes largely characterized by aneuploidy or recurring chromosomal rearrangements. Despite extensive information on the ALL transcriptome and methylome, there is limited understanding of the ALL chromatin landscape. We therefore mapped accessible chromatin in 24 primary ALL cell biospecimens comprising three common molecular subtypes (DUX4/ERG, ETV6-RUNX1 and hyperdiploid) from patients treated at St. Jude Children's Research Hospital. Our findings highlight extensive chromatin reprogramming in ALL, including the identification ALL subtype-specific chromatin landscapes that are additionally modulated by genetic variation. Chromatin accessibility differences between ALL and normal B-cells implicate the activation of B-cell repressed chromatin domains and detail the disruption of normal B-cell development in ALL. Among ALL subtypes, we uncovered roles for basic helix-loop-helix, homeodomain and activator protein 1 transcription factors in promoting subtype-specific chromatin accessibility and distinct gene regulatory networks. In addition to chromatin subtype-specificity, we further identified over 3500 DNA sequence variants that alter the ALL chromatin landscape and contribute to inter-individual variability in chromatin accessibility. Collectively, our data suggest that subtype-specific chromatin landscapes and gene regulatory networks impact ALL biology and contribute to transcriptomic differences among ALL subtypes.

Characterization and evolutionary origin of novel C2H2 zinc finger protein (ZNF648) required for both erythroid and megakaryocyte differentiation in humans.

Human ZNF648 is a novel poly C-terminal C2H2 zinc finger protein identified amongst the most dysregulated proteins in erythroid cells differentiated from iPSC. Its nuclear localisation and structure indicate it is likely a DNA-binding protein. Using a combination of ZNF648 overexpression in an iPSC line and primary adult erythroid cells, ZNF648 knockdown in primary adult erythroid cells and megakaryocytes, comparative proteomics and transcriptomics we show that ZNF648 is required for both erythroid and megakaryocyte differentiation. Orthologues of ZNF648 were detected across Mammals, Reptilia, Actinopterygii, in some Aves, Amphibia and Coelacanthiformes suggesting the gene originated in the common ancestor of Osteichthyes (Euteleostomi or bony fish). Conservation of the C-terminal zinc finger domain is higher, with some variation in zinc finger number but a core of at least six zinc fingers conserved across all groups, with the N-terminus recognisably similar within but not between major lineages. This suggests the N-terminus of ZNF648 evolves faster than the C-terminus, however this is not due to exon-shuffling as the entire coding region of ZNF648 is within a single exon. As for other such transcription factors, the N-terminus likely carries out regulatory functions, but showed no sequence similarity to any known domains. The greater functional constraint on the zinc finger domain suggests ZNF648 binds at least some similar regions of DNA in the different organisms. However, divergence of the N-terminal region may enable differential expression, allowing adaptation of function in the different organisms.

Integrative genomic analyses reveal mechanisms of glucocorticoid resistance in acute lymphoblastic leukemia.

Identification of genomic and epigenomic determinants of drug resistance provides important insights for improving cancer treatment. Using agnostic genome-wide interrogation of mRNA and miRNA expression, DNA methylation, SNPs, CNAs and SNVs/Indels in primary human acute lymphoblastic leukemia cells, we identified 463 genomic features associated with glucocorticoid resistance. Gene-level aggregation identified 118 overlapping genes, 15 of which were confirmed by genome-wide CRISPR screen. Collectively, this identified 30 of 38 (79%) known glucocorticoid-resistance genes/miRNAs and all 38 known resistance pathways, while revealing 14 genes not previously associated with glucocorticoid-resistance. Single cell RNAseq and network-based transcriptomic modelling corroborated the top previously undiscovered gene, CELSR2. Manipulation of CELSR2 recapitulated glucocorticoid resistance in human leukemia cell lines and revealed a synergistic drug combination (prednisolone and venetoclax) that mitigated resistance in mouse xenograft models. These findings illustrate the power of an integrative genomic strategy for elucidating genes and pathways conferring drug resistance in cancer cells.

Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia.

In vivo, mammalian cells reside in an environment of 0.5-10% O2 (depending on the tissue location within the body), whilst standard in vitro cell culture is carried out under room air. Little is known about the effects of this hyperoxic environment on treatment-induced oxidative stress, relative to a physiological oxygen environment. In the present study we investigated the effects of long-term culture under hyperoxia (air) on photodynamic treatment. Upon photodynamic irradiation, cells which had been cultured long-term under hyperoxia generated higher concentrations of mitochondrial reactive oxygen species, compared with cells in a physioxic (2% O2) environment. However, there was no significant difference in viability between hyperoxic and physioxic cells. The expression of genes encoding key redox homeostasis proteins and the activity of key antioxidant enzymes was significantly higher after the long-term culture of hyperoxic cells compared with physioxic cells. The induction of antioxidant genes and increased antioxidant enzyme activity appear to contribute to the development of a phenotype that is resistant to oxidative stress-induced cellular damage and death when using standard cell culture conditions. The results from experiments using selective inhibitors suggested that the thioredoxin antioxidant system contributes to this phenotype. To avoid artefactual results, in vitro cellular responses should be studied in mammalian cells that have been cultured under physioxia. This investigation provides new insights into the effects of physioxic cell culture on a model of a clinically relevant photodynamic treatment and the associated cellular pathways.

Regulation of the Human Fc-Neonatal Receptor alpha-Chain Gene FCGRT by MicroRNA-3181.

PURPOSE: FCGRT encodes the alpha-chain component of the neonatal Fc receptor (FcRn). FcRn is critical for the trafficking of endogenous and exogenous IgG molecules and albumin in various tissues. Few regulators of FcRn expression have been identified. We investigated the epigenetic regulation of FcRn by two microRNAs (hsa-miR-3181 and hsa-miR-3136-3p) acting on FCGRT. METHODS: The binding of candidate microRNAs to the 3'-untranslated region of FCGRT was evaluated using luciferase reporter constructs in CHO cells. The effect of microRNAs on FCGRT mRNA and FcRn protein expression was evaluated using specific microRNA mimics and inhibitor transfections in A549, HEK293 and HepG2 cells. RESULTS: Hsa-miR-3181 mimic reduced luciferase reporter activity by 70.1% (10 nM, P < 0.0001). In A549, HEK293 and HepG2 cells, hsa-miR-3181 decreased FCGRT mRNA expression (48.6%, 51.3% and 43.5% respectively, 25 nM, P < 0.05). The hsa-miR-3181 mimic decreased the expression of FcRn protein by 40% after 48 h (25 nM, P < 0.001). The mature form of hsa-miR-3181 was detected in samples of human liver. CONCLUSIONS: These data suggest that hsa-miR-3181 is an epigenetic regulator of FCGRT expression. The identification of this regulator of FCGRT may provide insights into a potential determinant of interindividual variability in FcRn expression.

Transcriptional regulation of the canine carbonyl reductase 1 gene (cbr1) by the specificity protein 1 (Sp1).

The clinical use of anthracyclines to treat various canine cancers is limited by the development of cardiotoxicity. The intra-cardiac synthesis of anthracycline C-13 alcohol metabolites (e.g. daunorubicinol) contributes to the development of cardiotoxicity. Canine carbonyl reductase 1 (cbr1) catalyzes the reduction of daunorubicin into daunorubicinol. Recent mapping of the cbr1 locus by sequencing DNA samples from dogs from various breeds revealed a cluster of conserved motifs for the transcription factor Sp1 in the putative promoter region of cbr1. We hypothesized that the variable number of Sp1 motifs could impact the transcription of canine cbr1. In this study, we report the functional characterization of the canine cbr1 promoter. Experiments with reporter constructs and chromatin immunoprecipitation show that cbr1 transcription depends on the binding of Sp1 to the proximal promoter. Site-directed mutagenesis experiments suggest that the variable number of Sp1 motifs impacts the transcription of canine cbr1. Inhibition of Sp1-DNA binding decreased canine cbr1 mRNA levels by 54% in comparison to controls, and also decreased enzymatic carbonyl reductase activity for the substrates daunorubicin (16%) and menadione (23%). The transactivation of Sp1 increased the expression of cbr1 mRNA (67%), and increased carbonyl reductase activity for daunorubicin (35%) and menadione (27%). These data suggest that the variable number of Sp1 motifs in the canine cbr1 promoter may impact the pharmacodynamics of anthracyclines in canine cancer patients.

The hydroxypyridinone iron chelator CP94 increases methyl-aminolevulinate-based photodynamic cell killing by increasing the generation of reactive oxygen species.

Methyl-aminolevulinate-based photodynamic therapy (MAL-PDT) is utilised clinically for the treatment of non-melanoma skin cancers and pre-cancers and the hydroxypyridinone iron chelator, CP94, has successfully been demonstrated to increase MAL-PDT efficacy in an initial clinical pilot study. However, the biochemical and photochemical processes leading to CP94-enhanced photodynamic cell death, beyond the well-documented increases in accumulation of the photosensitiser protoporphyrin IX (PpIX), have not yet been fully elucidated. This investigation demonstrated that MAL-based photodynamic cell killing of cultured human squamous carcinoma cells (A431) occurred in a predominantly necrotic manner following the generation of singlet oxygen and ROS. Augmenting MAL-based photodynamic cell killing with CP94 co-treatment resulted in increased PpIX accumulation, MitoSOX-detectable ROS generation (probably of mitochondrial origin) and necrotic cell death, but did not affect singlet oxygen generation. We also report (to our knowledge, for the first time) the detection of intracellular PpIX-generated singlet oxygen in whole cells via electron paramagnetic resonance spectroscopy in conjunction with a spin trap.

Characterization of the Canine Anthracycline-Metabolizing Enzyme Carbonyl Reductase 1 (cbr1) and the Functional Isoform cbr1 V218.

The anthracyclines doxorubicin and daunorubicin are used in the treatment of various human and canine cancers, but anthracycline-related cardiotoxicity limits their clinical utility. The formation of anthracycline C-13 alcohol metabolites (e.g., doxorubicinol and daunorubicinol) contributes to the development of anthracycline-related cardiotoxicity. The enzymes responsible for the synthesis of anthracycline C-13 alcohol metabolites in canines remain to be elucidated. We hypothesized that canine carbonyl reductase 1 (cbr1), the homolog of the prominent anthracycline reductase human CBR1, would have anthracycline reductase activity. Recombinant canine cbr1 (molecular weight: 32.8 kDa) was purified from Escherichia coli. The enzyme kinetics of "wild-type" canine cbr1 (cbr1 D218) and a variant isoform (cbr1 V218) were characterized with the substrates daunorubicin and menadione, as well as the flavonoid inhibitor rutin. Canine cbr1 catalyzes the reduction of daunorubicin to daunorubicinol, with cbr1 D218 and cbr1 V218 displaying different kinetic parameters (cbr1 D218 Km: 188 ± 144 µM versus cbr1 V218 Km: 527 ± 136 µM, P < 0.05, and cbr1 D218 Vmax: 6446 ± 3615 nmol/min per milligram versus cbr1 V218 Vmax: 15539 ± 2623 nmol/min per milligram, P < 0.01). Canine cbr1 also metabolized menadione (cbr1 D218 Km: 104 ± 50 µM, Vmax: 2034 ± 307 nmol/min per milligram). Rutin acted as a competitive inhibitor for the reduction of daunorubicin (cbr1 D218 Ki: 1.84 ± 1.02 µM, cbr1 V218 Ki: 1.38 ± 0.47 µM). These studies show that canine cbr1 metabolizes daunorubicin and provide the necessary foundation to characterize the role of cbr1 in the variable pharmacodynamics of anthracyclines in canine cancer patients.