Depletion of tet2 results in age-dependent changes in DNA methylation and gene expression in a zebrafish model of myelodysplastic syndrome.

Introduction: Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic disorders characterized by ineffective hematopoiesis, cytopenias, and dysplasia. The gene encoding ten-eleven translocation 2 (tet2), a dioxygenase enzyme that catalyzes the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine, is a recurrently mutated tumor suppressor gene in MDS and other myeloid malignancies. Previously, we reported a stable zebrafish line with a loss-of-function mutation in the tet2 gene. The tet2m/m-mutant zebrafish developed a pre-MDS state with kidney marrow dysplasia, but normal circulating blood counts by 11 months of age and accompanying anemia, signifying the onset of MDS, by 24 months of age. Methods: In the current study, we collected progenitor cells from the kidney marrows of the adult tet2m/m and tet2wt/wt fish at 4 and 15 months of age and conducted enhanced reduced representation of bisulfite sequencing (ERRBS) and bulk RNA-seq to measure changes in DNA methylation and gene expression of hematopoietic stem and progenitor cells (HSPCs). Results and discussion: A global increase in DNA methylation of gene promoter regions and CpG islands was observed in tet2m/m HSPCs at 4 months of age when compared with the wild type. Furthermore, hypermethylated genes were significantly enriched for targets of SUZ12 and the metal-response-element-binding transcription factor 2 (MTF2)-involved in the polycomb repressive complex 2 (PRC2). However, between 4 and 15 months of age, we observed a paradoxical global decrease in DNA methylation in tet2m/m HSPCs. Gene expression analyses identified upregulation of genes associated with mTORC1 signaling and interferon gamma and alpha responses in tet2m/m HSPCs at 4 months of age when compared with the wild type. Downregulated genes in HSPCs of tet2-mutant fish at 4 months of age were enriched for cell cycle regulation, heme metabolism, and interleukin 2 (IL2)/signal transducer and activator of transcription 5 (STAT5) signaling, possibly related to increased self-renewal and clonal advantage in HSPCs with tet2 loss of function. Finally, there was an overall inverse correlation between overall increased promoter methylation and gene expression.

Synthetic lethal targeting of TET2-mutant haematopoietic stem and progenitor cells by XPO1 inhibitors.

TET2 inactivating mutations serve as initiating genetic lesions in the transformation of haematopoietic stem and progenitor cells (HSPCs). In this study, we analysed known drugs in zebrafish embryos for their ability to selectively kill tet2-mutant HSPCs in vivo. We found that the exportin 1 (XPO1) inhibitors, selinexor and eltanexor, selectively kill tet2-mutant HSPCs. In serial replating colony assays, these small molecules were selectively active in killing murine Tet2-deficient Lineage-, Sca1+, Kit+ (LSK) cells, and also TET2-inactivated human acute myeloid leukaemia (AML) cells. Selective killing of TET2-mutant HSPCs and human AML cells by these inhibitors was due to increased levels of apoptosis, without evidence of DNA damage based on increased γH2AX expression. The finding that TET2 loss renders HSPCs and AML cells selectively susceptible to cell death induced by XPO1 inhibitors provides preclinical evidence of the selective activity of these drugs, justifying further clinical studies of these small molecules for the treatment of TET2-mutant haematopoietic malignancies, and to suppress clonal expansion in age-related TET2-mutant clonal haematopoiesis.

Synergistic melanoma cell death mediated by inhibition of both MCL1 and BCL2 in high-risk tumors driven by NF1/PTEN loss.

Melanomas driven by loss of the NF1 tumor suppressor have a high risk of treatment failure and effective therapies have not been developed. Here we show that loss-of-function mutations of nf1 and pten result in aggressive melanomas in zebrafish, representing the first animal model of NF1-mutant melanomas harboring PTEN loss. MEK or PI3K inhibitors show little activity when given alone due to cross-talk between the pathways, and high toxicity when given together. The mTOR inhibitors, sirolimus, everolimus, and temsirolimus, were the most active single agents tested, potently induced tumor-suppressive autophagy, but not apoptosis. Because addition of the BCL2 inhibitor venetoclax resulted in compensatory upregulation of MCL1, we established a three-drug combination composed of sirolimus, venetoclax, and the MCL1 inhibitor S63845. This well-tolerated drug combination potently and synergistically induces apoptosis in both zebrafish and human NF1/PTEN-deficient melanoma cells, providing preclinical evidence justifying an early-stage clinical trial in patients with NF1/PTEN-deficient melanoma.

Synthetic lethal targeting of TET2-mutant hematopoietic stem and progenitor cells (HSPCs) with TOP1-targeted drugs and PARP1 inhibitors.

Inactivating mutations in TET2 serve as an initiating genetic lesion in the transformation of hematopoietic stem and progenitor cells (HSPCs). Thus, effective therapy for this subset of patients would ideally include drugs that are selectively lethal in TET2-mutant HSPCs, at dosages that spare normal HSPCs. In this study, we tested 129 FDA-approved anticancer drugs in a tet2-deficient zebrafish model and showed that topoisomerase 1 (TOP1)-targeted drugs and PARP1 inhibitors selectively kill tet2-mutant HSPCs. We found that Tet2-deficient murine bone marrow progenitors and CRISPR-Cas9-induced TET2-mutant human AML cells were more sensitive to both classes of drugs compared with matched control cells. The mechanism underlying the selective killing of TET2-mutant blood cells by these drugs was due to aberrantly low levels of tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme that is important for removing TOP1 cleavage complexes (TOP1cc). Low TDP1 levels yield sensitivity to TOP1-targeted drugs or PARP1 inhibitors and an inability to remove TOP1 cleavage complexes, leading to DNA double-strand breaks and cell death. The finding that TET2 mutations render HSPCs uniquely vulnerable to disruption of TOP1 and PARP1 activity may therefore represent a unique opportunity to use relatively low dosages of these drugs for the "precision therapy" of TET2-mutant myeloid malignancies.

Disruption of asxl1 results in myeloproliferative neoplasms in zebrafish.

Somatic loss-of-function mutations of the additional sex combs-like transcriptional regulator 1 (ASXL1) gene are common genetic abnormalities in human myeloid malignancies and induce clonal expansion of mutated hematopoietic stem cells (HSCs). To understand how ASXL1 disruption leads to myeloid cell transformation, we generated asxl1 haploinsufficient and null zebrafish lines using genome-editing technology. Here, we show that homozygous loss of asxl1 leads to apoptosis of newly formed HSCs. Apoptosis occurred via the mitochondrial apoptotic pathway mediated by upregulation of bim and bid Half of the asxl1+/- zebrafish had myeloproliferative neoplasms (MPNs) by 5 months of age. Heterozygous loss of asxl1 combined with heterozygous loss of tet2 led to a more penetrant MPN phenotype, while heterozygous loss of asxl1 combined with complete loss of tet2 led to acute myeloid leukemia (AML). These findings support the use of asxl1+/- zebrafish as a strategy to identify small-molecule drugs to suppress the growth of asxl1 mutant but not wild-type HSCs in individuals with somatically acquired inactivating mutations of ASXL1.

Phospholipase C gamma-1 is required for granulocyte maturation in zebrafish.

The regulation of hematopoiesis is generally evolutionarily conserved from zebrafish to mammals, including hematopoietic stem cell formation and blood cell lineage differentiation. In zebrafish, primitive granulocytes originate at two distinct regions, the anterior lateral plate mesoderm (A-LPM) and the intermediate cell mass (ICM). Few studies in the zebrafish have examined genes specifically required for the granulocytic lineage. In this study, we identified the responsible gene for a zebrafish mutant that has relatively normal hematopoiesis, except decreased expression of the granulocyte-specific gene mpx. Positional cloning revealed that phospholipase C gamma-1 (plcg1) was mutated. Deficiency of plcg1 function specifically affected development of granulocytes, especially the maturation process. These results suggested that plcg1 functioned specifically in zebrafish ICM granulopoiesis for the first time. Our studies suggest that specific pathways regulate the differentiation of the hematopoietic lineages.

Heritable and lineage-specific gene knockdown in zebrafish embryo.

BACKGROUND: Reduced expression of developmentally important genes and tumor suppressors due to haploinsufficiency or epigenetic suppression has been shown to contribute to the pathogenesis of various malignancies. However, methodology that allows spatio-temporally knockdown of gene expression in various model organisms such as zebrafish has not been well established, which largely limits the potential of zebrafish as a vertebrate model of human malignant disorders. PRINCIPAL FINDING: Here, we report that multiple copies of small hairpin RNA (shRNA) are expressed from a single transcript that mimics the natural microRNA-30e precursor (mir-shRNA). The mir-shRNA, when microinjected into zebrafish embryos, induced an efficient knockdown of two developmentally essential genes chordin and alpha-catenin in a dose-controllable fashion. Furthermore, we designed a novel cassette vector to simultaneously express an intronic mir-shRNA and a chimeric red fluorescent protein driven by lineage-specific promoter, which efficiently reduced the expression of a chromosomally integrated reporter gene and an endogenously expressed gata-1 gene in the developing erythroid progenitors and hemangioblasts, respectively. SIGNIFICANCE: This methodology provides an invaluable tool to knockdown developmental important genes in a tissue-specific manner or to establish animal models, in which the gene dosage is critically important in the pathogenesis of human disorders. The strategy should be also applicable to other model organisms.

Mir-144 selectively regulates embryonic alpha-hemoglobin synthesis during primitive erythropoiesis.

Precise transcriptional control of developmental stage-specific expression and switching of alpha- and beta-globin genes is significantly important to understand the general principles controlling gene expression and the pathogenesis of thalassemia. Although transcription factors regulating beta-globin genes have been identified, little is known about the microRNAs and trans-acting mechanism controlling alpha-globin genes transcription. Here, we show that an erythroid lineage-specific microRNA gene, miR-144, expressed at specific developmental stages during zebrafish embryogenesis, negatively regulates the embryonic alpha-globin, but not embryonic beta-globin, gene expression, through physiologically targeting klfd, an erythroid-specific Krüppel-like transcription factor. Klfd selectively binds to the CACCC boxes in the promoters of both alpha-globin and miR-144 genes to activate their transcriptions, thus forming a negative feedback circuitry to fine-tune the expression of embryonic alpha-globin gene. The selective effect of the miR-144-Klfd pathway on globin gene regulation may thereby constitute a novel therapeutic target for improving the clinical outcome of patients with thalassemia.