A stemness screen reveals C3orf54/INKA1 as a promoter of human leukemia stem cell latency.

There is a growing body of evidence that the molecular properties of leukemia stem cells (LSCs) are associated with clinical outcomes in acute myeloid leukemia (AML), and LSCs have been linked to therapy failure and relapse. Thus, a better understanding of the molecular mechanisms that contribute to the persistence and regenerative potential of LSCs is expected to result in the development of more effective therapies. We therefore interrogated functionally validated data sets of LSC-specific genes together with their known protein interactors and selected 64 candidates for a competitive in vivo gain-of-function screen to identify genes that enhanced stemness in human cord blood hematopoietic stem and progenitor cells. A consistent effect observed for the top hits was the ability to restrain early repopulation kinetics while preserving regenerative potential. Overexpression (OE) of the most promising candidate, the orphan gene C3orf54/INKA1, in a patient-derived AML model (8227) promoted the retention of LSCs in a primitive state manifested by relative expansion of CD34+ cells, accumulation of cells in G0, and reduced output of differentiated progeny. Despite delayed early repopulation, at later times, INKA1-OE resulted in the expansion of self-renewing LSCs. In contrast, INKA1 silencing in primary AML reduced regenerative potential. Mechanistically, our multidimensional confocal analysis found that INKA1 regulates G0 exit by interfering with nuclear localization of its target PAK4, with concomitant reduction of global H4K16ac levels. These data identify INKA1 as a novel regulator of LSC latency and reveal a link between the regulation of stem cell kinetics and pool size during regeneration.

The unfolded protein response governs integrity of the haematopoietic stem-cell pool during stress.

The blood system is sustained by a pool of haematopoietic stem cells (HSCs) that are long-lived due to their capacity for self-renewal. A consequence of longevity is exposure to stress stimuli including reactive oxygen species (ROS), nutrient fluctuation and DNA damage. Damage that occurs within stressed HSCs must be tightly controlled to prevent either loss of function or the clonal persistence of oncogenic mutations that increase the risk of leukaemogenesis. Despite the importance of maintaining cell integrity throughout life, how the HSC pool achieves this and how individual HSCs respond to stress remain poorly understood. Many sources of stress cause misfolded protein accumulation in the endoplasmic reticulum (ER), and subsequent activation of the unfolded protein response (UPR) enables the cell to either resolve stress or initiate apoptosis. Here we show that human HSCs are predisposed to apoptosis through strong activation of the PERK branch of the UPR after ER stress, whereas closely related progenitors exhibit an adaptive response leading to their survival. Enhanced ER protein folding by overexpression of the co-chaperone ERDJ4 (also called DNAJB9) increases HSC repopulation capacity in xenograft assays, linking the UPR to HSC function. Because the UPR is a focal point where different sources of stress converge, our study provides a framework for understanding how stress signalling is coordinated within tissue hierarchies and integrated with stemness. Broadly, these findings reveal that the HSC pool maintains clonal integrity by clearance of individual HSCs after stress to prevent propagation of damaged stem cells.

The microRNA-producing enzyme Dicer1 is essential for zebrafish development.

MicroRNAs (miRNAs) are produced by the Dicer1 enzyme; the role of Dicer1 in vertebrate development is unknown. Here we report target-selected inactivation of the dicer1 gene in zebrafish. We observed an initial build-up of miRNA levels, produced by maternal Dicer1, in homozygous dicer1 mutants, but miRNA accumulation stopped after a few days. This resulted in developmental arrest around day 10. These results indicate that miRNA-producing Dicer1 is essential for vertebrate development.

The Wnt/beta-catenin pathway regulates cardiac valve formation.

Truncation of the tumour suppressor adenomatous polyposis coli (Apc) constitutively activates the Wnt/beta-catenin signalling pathway. Apc has a role in development: for example, embryos of mice with truncated Apc do not complete gastrulation. To understand this role more fully, we examined the effect of truncated Apc on zebrafish development. Here we show that, in contrast to mice, zebrafish do complete gastrulation. However, mutant hearts fail to loop and form excessive endocardial cushions. Conversely, overexpression of Apc or Dickkopf 1 (Dkk1), a secreted Wnt inhibitor, blocks cushion formation. In wild-type hearts, nuclear beta-catenin, the hallmark of activated canonical Wnt signalling, accumulates only in valve-forming cells, where it can activate a Tcf reporter. In mutant hearts, all cells display nuclear beta-catenin and Tcf reporter activity, while valve markers are markedly upregulated. Concomitantly, proliferation and epithelial-mesenchymal transition, normally restricted to endocardial cushions, occur throughout the endocardium. Our findings identify a novel role for Wnt/beta-catenin signalling in determining endocardial cell fate.

ATP7B variant c.1934T > G p.Met645Arg causes Wilson disease by promoting exon 6 skipping.

Wilson disease is a recessive genetic disorder caused by pathogenic loss-of-function variants in the ATP7B gene. It is characterized by disrupted copper homeostasis resulting in liver disease and/or neurological abnormalities. The variant NM_000053.3:c.1934T > G (Met645Arg) has been reported as compound heterozygous, and is highly prevalent among Wilson disease patients of Spanish descent. Accordingly, it is classified as pathogenic by leading molecular diagnostic centers. However, functional studies suggest that the amino acid change does not alter protein function, leading one ClinVar submitter to question its pathogenicity. Here, we used a minigene system and gene-edited HepG2 cells to demonstrate that c.1934T > G causes ~70% skipping of exon 6. Exon 6 skipping results in frameshift and stop-gain, leading to loss of ATP7B function. The elucidation of the mechanistic effect for this variant resolves any doubt about its pathogenicity and enables the development of genetic medicines for restoring correct splicing.

A novel method for detecting the cellular stemness state in normal and leukemic human hematopoietic cells can predict disease outcome and drug sensitivity.

Acute leukemia is an aggressive blood malignancy with low survival rates. A high expression of stem-like programs in leukemias predicts poor prognosis and is assumed to act in an aberrant fashion in the phenotypically heterogeneous leukemia stem cell (LSC) population. A lack of suitable genome engineering tools that can isolate LSCs based on their stemness precludes their comprehensive examination and full characterization. We hypothesized that tagging endogenous stemness-regulatory regions could generate a genome reporter for the putative leukemia stemness-state. Our analysis revealed that the ERG + 85 enhancer region can serve as a marker for stemness-state and a fluorescent lentiviral reporter was developed that can accurately recapitulate the endogenous activity. Using our novel reporter, we revealed cellular heterogeneity in several leukemia cell lines and patient-derived samples. Alterations in reporter activity were associated with transcriptomic and functional changes that were closely related to the hematopoietic stem cell (HSC) identity. Notably, the differentiation potential was skewed towards the erythro-megakaryocytic lineage. Moreover, an ERG + 85High fraction of AML cells could regenerate the original cellular heterogeneity and was enriched for LSCs. RNA-seq analysis coupled with in silico drug-screen analysis identified 4HPR as an effective inhibitor of ERG + 85High leukemia growth. We propose that further utilization of our novel molecular tool will identify crucial determinants of LSCs, thus providing a rationale for their therapeutic targeting.

Substrate requirements for let-7 function in the developing zebrafish embryo.

MicroRNAs (miRNAs) are involved in the regulation of gene expression at the post-transcriptional level by base pairing to the 3'-UTR (untranslated region) of mRNAs. The let-7 miRNA was first discovered in Caenorhabditis elegans and is evolutionarily conserved. We used zebrafish embryos as a vertebrate in vivo system to study substrate requirements for function of let-7. Injection of a double-stranded let-7 miRNA into the zygotes of zebrafish and frogs causes specific phenotypic defects. Only the antisense strand of the let-7 duplex has biological activity. In addition, co-injected mRNA of gfp fused to the 3'-UTR of a zebrafish lin-41 ortholog (a presumed target of let-7) is silenced by let-7. Point mutant studies revealed that the two let-7 target sites in the lin-41 3'-UTR are both essential and sufficient for silencing. let-7 and mir221 together, but not either of them alone, can silence a construct with one of the let-7 target sites replaced by a target site for mir221, showing that two different miRNAs can provide the required cooperative effect. let-7 target sites can be moved around: they are also functional when positioned in the coding sequence or even in the 5'-UTR of gfp. We took advantage of reporter and phenotypic assays to analyze the activity of all possible point mutant derivatives of let-7 and found that only the 5' region is critical for function of let-7.

Deregulation of DUX4 and ERG in acute lymphoblastic leukemia.

Chromosomal rearrangements deregulating hematopoietic transcription factors are common in acute lymphoblastic leukemia (ALL). Here we show that deregulation of the homeobox transcription factor gene DUX4 and the ETS transcription factor gene ERG is a hallmark of a subtype of B-progenitor ALL that comprises up to 7% of B-ALL. DUX4 rearrangement and overexpression was present in all cases and was accompanied by transcriptional deregulation of ERG, expression of a novel ERG isoform, ERGalt, and frequent ERG deletion. ERGalt uses a non-canonical first exon whose transcription was initiated by DUX4 binding. ERGalt retains the DNA-binding and transactivation domains of ERG, but it inhibits wild-type ERG transcriptional activity and is transforming. These results illustrate a unique paradigm of transcription factor deregulation in leukemia in which DUX4 deregulation results in loss of function of ERG, either by deletion or induced expression of an isoform that is a dominant-negative inhibitor of wild-type ERG function.

Ectopic miR-125a Expression Induces Long-Term Repopulating Stem Cell Capacity in Mouse and Human Hematopoietic Progenitors.

Umbilical cord blood (CB) is a convenient and broadly used source of hematopoietic stem cells (HSCs) for allogeneic stem cell transplantation. However, limiting numbers of HSCs remain a major constraint for its clinical application. Although one feasible option would be to expand HSCs to improve therapeutic outcome, available protocols and the molecular mechanisms governing the self-renewal of HSCs are unclear. Here, we show that ectopic expression of a single microRNA (miRNA), miR-125a, in purified murine and human multipotent progenitors (MPPs) resulted in increased self-renewal and robust long-term multi-lineage repopulation in transplanted recipient mice. Using quantitative proteomics and western blot analysis, we identified a restricted set of miR-125a targets involved in conferring long-term repopulating capacity to MPPs in humans and mice. Our findings offer the innovative potential to use MPPs with enhanced self-renewal activity to augment limited sources of HSCs to improve clinical protocols.

MicroRNA function in animal development.

MicroRNAs (miRNAs) are small non-coding RNA molecules that post-transcriptionally regulate gene expression by base-pairing to mRNAs. Hundreds of miRNAs have been identified in various multicellular organisms and many miRNAs are evolutionarily conserved. Although the biological functions of most miRNAs are unknown, miRNAs are predicted to regulate up to 30% of the genes within the human genome. Gradually, we are beginning to understand the functions of individual miRNAs and the general function of miRNA action. Here, we review the recent advances in miRNA biology in animals. Particularly, we focus on the roles of miRNAs in vertebrate development and disease.

Continuous exchange of sequence information between dispersed Tc1 transposons in the Caenorhabditis elegans genome.

In a genome-wide analysis of the active transposons in Caenorhabditis elegans we determined the localization and sequence of all copies of each of the six active transposon families. Most copies of the most active transposons, Tc1 and Tc3, are intact but individually have a unique sequence, because of unique patterns of single-nucleotide polymorphisms. The sequence of each of the 32 Tc1 elements is invariant in the C. elegans strain N2, which has no germline transposition. However, at the same 32 Tc1 loci in strains with germline transposition, Tc1 elements can acquire the sequence of Tc1 elements elsewhere in the N2 genome or a chimeric sequence derived from two dispersed Tc1 elements. We hypothesize that during double-strand-break repair after Tc1 excision, the template for repair can switch from the Tc1 element on the sister chromatid or homologous chromosome to a Tc1 copy elsewhere in the genome. Thus, the population of active transposable elements in C. elegans is highly dynamic because of a continuous exchange of sequence information between individual copies, potentially allowing a higher evolution rate than that found in endogenous genes.

Inhibition of the Mitochondrial Protease ClpP as a Therapeutic Strategy for Human Acute Myeloid Leukemia.

From an shRNA screen, we identified ClpP as a member of the mitochondrial proteome whose knockdown reduced the viability of K562 leukemic cells. Expression of this mitochondrial protease that has structural similarity to the cytoplasmic proteosome is increased in leukemic cells from approximately half of all patients with AML. Genetic or chemical inhibition of ClpP killed cells from both human AML cell lines and primary samples in which the cells showed elevated ClpP expression but did not affect their normal counterparts. Importantly, Clpp knockout mice were viable with normal hematopoiesis. Mechanistically, we found that ClpP interacts with mitochondrial respiratory chain proteins and metabolic enzymes, and knockdown of ClpP in leukemic cells inhibited oxidative phosphorylation and mitochondrial metabolism.

Reduced lymphoid lineage priming promotes human hematopoietic stem cell expansion.

The hematopoietic system sustains regeneration throughout life by balancing self-renewal and differentiation. To stay poised for mature blood production, hematopoietic stem cells (HSCs) maintain low-level expression of lineage-associated genes, a process termed lineage priming. Here, we modulated expression levels of Inhibitor of DNA binding (ID) proteins to ask whether lineage priming affects self-renewal of human HSCs. We found that lentiviral overexpression of ID proteins in cord blood HSCs biases myeloerythroid commitment at the expense of lymphoid differentiation. Conversely, reducing ID2 expression levels increases lymphoid potential. Mechanistically, ID2 inhibits the transcription factor E47 to attenuate B-lymphoid priming in HSCs and progenitors. Strikingly, ID2 overexpression also results in a 10-fold expansion of HSCs in serial limiting dilution assays, indicating that early lymphoid transcription factors antagonize human HSC self-renewal. The relationship between lineage priming and self-renewal can be exploited to increase expansion of transplantable human HSCs and points to broader implications for other stem cell populations.

Variable clonal repopulation dynamics influence chemotherapy response in colorectal cancer.

Intratumoral heterogeneity arises through the evolution of genetically diverse subclones during tumor progression. However, it remains unknown whether cells within single genetic clones are functionally equivalent. By combining DNA copy number alteration (CNA) profiling, sequencing, and lentiviral lineage tracking, we followed the repopulation dynamics of 150 single lentivirus-marked lineages from 10 human colorectal cancers through serial xenograft passages in mice. CNA and mutational analysis distinguished individual clones and showed that clones remained stable upon serial transplantation. Despite this stability, the proliferation, persistence, and chemotherapy tolerance of lentivirally marked lineages were variable within each clone. Chemotherapy promoted the dominance of previously minor or dormant lineages. Thus, apart from genetic diversity, tumor cells display inherent functional variability in tumor propagation potential, which contributes to both cancer growth and therapy tolerance.

Catalytic site remodelling of the DOT1L methyltransferase by selective inhibitors.

Selective inhibition of protein methyltransferases is a promising new approach to drug discovery. An attractive strategy towards this goal is the development of compounds that selectively inhibit binding of the cofactor, S-adenosylmethionine, within specific protein methyltransferases. Here we report the three-dimensional structure of the protein methyltransferase DOT1L bound to EPZ004777, the first S-adenosylmethionine-competitive inhibitor of a protein methyltransferase with in vivo efficacy. This structure and those of four new analogues reveal remodelling of the catalytic site. EPZ004777 and a brominated analogue, SGC0946, inhibit DOT1L in vitro and selectively kill mixed lineage leukaemia cells, in which DOT1L is aberrantly localized via interaction with an oncogenic MLL fusion protein. These data provide important new insight into mechanisms of cell-active S-adenosylmethionine-competitive protein methyltransferase inhibitors, and establish a foundation for the further development of drug-like inhibitors of DOT1L for cancer therapy.

Zebrafish Tie-2 shares a redundant role with Tie-1 in heart development and regulates vessel integrity.

Tie-2 is a member of the receptor tyrosine kinase family and is required for vascular remodeling and maintenance of mammalian vessel integrity. A number of mutations in the human TIE2 gene have been identified in patients suffering from cutaneomucosal venous malformations and ventricular septal defects. How exactly Tie-2 signaling pathways play different roles in both vascular development and vascular stability is unknown. We have generated a zebrafish line carrying a stop mutation in the kinase domain of the Tie-2 receptor. Mutant embryos lack Tie-2 protein, but do not display any defect in heart and vessel development. Simultaneous loss of Tie-1 and Tie-2, however, leads to a cardiac phenotype. Our study shows that Tie-1 and Tie-2 are not required for early heart development, yet they have redundant roles for the maintenance of endocardial-myocardial connection in later stages. Tie-2 and its ligand Angiopoietin-1 have also been reported to play an important role in vessel stability. We used atorvastatin and simvastatin, drugs that cause bleeding in wild-type zebrafish larvae, to challenge vessel stability in tie-2 mutants. Interestingly, recent clinical studies have reported hemorrhagic stroke as a side effect of atorvastatin treatment. Exposure of embryos to statins revealed that tie-2 mutants are significantly protected from statin-induced bleeding. Furthermore, tie-2 mutants became less resistant to bleeding after VE-cadherin knockdown. Taken together, these data show that atorvastatin affects vessel stability through Tie-2, and that VE-cadherin and Tie-2 act in concert to allow vessel remodeling while playing a role in vessel stability. Our study introduces an additional vertebrate model to study in vivo the function of Tie-2 in development and disease.

T-cell factor 4 (Tcf7l2) maintains proliferative compartments in zebrafish intestine.

Previous studies have shown that Wnt signals, relayed through beta-catenin and T-cell factor 4 (Tcf4), are essential for the induction and maintenance of crypts in mice. We have now generated a tcf4 (tcf7l2) mutant zebrafish by reverse genetics. We first observe a phenotypic defect at 4 weeks post-fertilization (wpf), leading to death at about 6 wpf. The phenotype comprises a loss of proliferation at the base of the intestinal folds of the middle and distal parts of the intestine. The proximal intestine represents an independent compartment, as it expresses sox2 in the epithelium and barx1 in the surrounding mesenchyme, which are early stomach markers in higher vertebrates. Zebrafish are functionally stomach-less, but the proximal intestine might share its ontogeny with the mammalian stomach. Rare adult homozygous tcf4(-/-) 'escapers' show proliferation defects in the gut epithelium, but have no other obvious abnormalities. This study underscores the involvement of Tcf4 in maintaining proliferative self-renewal in the intestine throughout life.

In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes.

MicroRNAs (miRNAs) are 20-23 nucleotide (nt) RNA molecules that regulate gene expression post-transcriptionally. A key step toward understanding the function of the hundreds of miRNAs identified in animals is to determine their expression during development. Here we performed a detailed analysis of conditions for in situ detection of miRNAs in the zebrafish embryo using locked nucleic acid (LNA)-modified DNA probes and report expression patterns for 15 miRNAs in the mouse embryo.

Phylogenetic shadowing and computational identification of human microRNA genes.

We sequenced 122 miRNAs in 10 primate species to reveal conservation characteristics of miRNA genes. Strong conservation is observed in stems of miRNA hairpins and increased variation in loop sequences. Interestingly, a striking drop in conservation was found for sequences immediately flanking the miRNA hairpins. This characteristic profile was employed to predict novel miRNAs using cross-species comparisons. Nine hundred and seventy-six candidate miRNAs were identified by scanning whole-genome human/mouse and human/rat alignments. Most of the novel candidates are conserved also in other vertebrates (dog, cow, chicken, opossum, zebrafish). Northern blot analysis confirmed the expression of mature miRNAs for 16 out of 69 representative candidates. Additional support for the expression of 179 novel candidates can be found in public databases, their presence in gene clusters, and literature that appeared after these predictions were made. Taken together, these results suggest the presence of significantly higher numbers of miRNAs in the human genome than previously estimated.