ALBERT

Description: Myelodysplastic syndrome (MDS) is a disorder arising from hematopoietic stem and progenitor cell (HSPC) dysfunction resulting in ineffective hematopoiesis. Mutations in the spliceosomal component SF3B1 (Splicing Factor 3B, subunit 1) are prevalent in MDS, but how this leads to aberrant hematopoiesis is unclear. We elucidated the in vivo consequences of sf3b1 misregulation on hematopoiesis and splicing using a zebrafish sf3b1 (sf3b1hi3394) loss-of-function mutant. Primitive erythropoiesis initiates normally in sf3b1 mutants, as evidenced by normal expression of scl (stem cell leukemia), gata1 and beta-globin, but maturation is defective as shown by decreased hemoglobinization and an increase in immature erythroblasts at 48 hours post fertilization (hpf). Similarly, primitive myelopoiesis initiates normally as early expression of scl and pu.1 are normal, but expression of the differentiation markers l-plastin and myeloperoxidase are diminished in mutants at 24 and 28 hpf. Hemogenic endothelial and HSPC markers runx1 and gata2b are also diminished, while the pan-endothelial marker kdrl (kinase insert domain receptor like) and aorta-specific markers notch1b and notch3 are normally expressed at 24 hpf, indicating hematopoietic induction is more sensitive to sf3b1 loss than vascular formation. RNA-seq analysis of sf3b1 mutants revealed large-scale splicing defects and alterations, particularly affecting introns with suboptimal splice sites. Comparison of RNA-seq data from zebrafish sf3b1 mutants and MDS patients harboring SF3B1 mutations revealed a significant overlap in misspliced genes and in the corresponding affected pathways, including many pathways implicated in MDS pathology such as TGFβ (Transforming Growth Factor-Beta) and immune signaling. Together, these results show conservation between zebrafish mutants and human cells with SF3B1 mutations. We next took a chemical genetic approach and discovered sf3b1 mutants were more sensitive than wild type siblings to treatment with E7107, a small molecule inhibitor of the spliceosome, indicating a potential therapeutic window for spliceosome modulators in MDS. Motivated by this finding, we performed a large-scale chemical modifier screen and identified many new lead compounds (6 out of 562 screened) that also show synthetic lethality in sf3b1 mutants. These small molecules are enriched in those with anti-inflammatory properties, suggesting aberrant immune signaling could underlie some of the defects in sf3b1 mutants. Together, our data demonstrate the molecular and cellular consequences of sf3b1 loss in vivo and establish sf3b1 zebrafish mutants as a novel model for discovery of therapeutics for MDS. Disclosures Smith: H3 Biomedicine: Employment. Buonamici:H3 Biomedicine: Employment.

Description: Myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML) are clonal hematopoietic disorders resulting from genetic alterations in hematopoietic stem cells. These myeloid disorders are clinically heterogeneous and biologically complex. Despite major advances in understanding the genetic and molecular landscape of MDS/AML, along with the introduction of newer and targeted therapies, the cure rates in AML are still only about 60% in children, and much lower in adults. Exploiting the genetic tractability of the zebrafish (Danio rerio) vertebrate model, we are investigating the role of a novel epigenetic regulator, Chromodomain helicase DNA binding protein-1 (CHD1) in hematopoiesis and its misregulation leading to MDS and AML. CHD1 is located at chromosome 5q21, which lies within the most frequent breakpoints seen with the deletion of the long arm of chromosome 5 [del (5q)] in patients with MDS and AML. In addition to del (5q), we found that CHD1 levels are significantly lower in bone marrow cells of patients with other forms of MDS, relative to normal controls. Using CRISPR/Cas9-mediated targeted mutagenesis in zebrafish, we created chd1 homozygous mutant fish. We confirmed a marked decrease in chd1 gene expression in these mutant fish. Chd1 homozygous mutants are viable and fertile as adults, with no significant developmental or hematopoietic phenotypes observed during embryogenesis. As CHD1 can act as a tumor suppressor and is linked to the DNA damage response, we hypothesized that chd1 mutant zebrafish would be more sensitive to DNA damaging agents. Indeed, we found that chd1 mutants have increased sensitivity to ionizing radiation as evidenced by elevated brain cell death measured by whole mount imaging of live embryos and immunofluorescence for activated Caspase 3, a marker of apoptosis. We also generated chd1het; tp53het zebrafish to test whether chd1 haploinsufficiency could accelerate tumor rates in tp53 mutant fish. Single heterozygotes chd1het or tp53het usually do not form tumors at one year of age, but chd1het; tp53het double heterozygous zebrafish showed substantial tumor growth by one year of age. Taken together, our data suggest that CHD1 may play a key role in protecting genomic integrity, explaining why diminished CHD1 levels could contribute to the pathogenesis of MDS and AML. This genetic interaction may be especially crucial in patients with combined del (5q) and TP53 alterations, and could contribute to the increased severity seen in this group. Our findings suggest these and other CHD1-deficient patients may be resistant to standard therapies due to attenuated DNA damage responses, allowing their AML to survive DNA damage caused by conventional anti-cancer treatments. Disclosures No relevant conflicts of interest to declare.