The murine myelomonocytic cell line WEHI‐3B exhibits ectopic expression of the genes encoding the homeobox protein, Hox‐2.4, and the myeloid growth factor, interleukin‐3 (IL‐3). We showed previously that concomitant expression of IL‐3 and Hox‐2.4 in bone marrow cells induced the development of transplantable growth factor‐independent tumours resembling the WEHI‐3B tumour. We have now investigated the effect of enforced expression of Hox‐2.4 alone. Bone marrow cells were infected with Hox‐2.4 retrovirus and then either cultured in agar or transplanted into irradiated mice. In vitro, colonies derived from virus‐infected cells readily yielded IL‐3‐dependent, non‐tumorigenic cell lines of the myelomonocytic, megakaryocytic and mast cell lineages. Surprisingly, both the establishment and maintenance of these lines required very high concentrations of IL‐3 and reduced levels promoted differentiation. Transplanted mice analysed after 3 months appeared normal but their spleen and bone marrow contained abundant provirus‐bearing progenitor cells, from which IL‐3‐dependent long‐term cell lines could readily be established in vitro. Four of 18 animals monitored for up to 12 months eventually developed clonal leukaemia, associated in three cases with IL‐3 production. Thus ectopic expression of Hox‐2.4 enhances self‐renewal of immature myeloid progenitors and progression to a fully malignant state is favoured by somatic mutations conferring autocrine production of IL‐3.
Highlights d Genuine macrophages are absent from hematopoietic tissue single-cell preparations d Fragmented remnants of macrophages containing cytoplasm adhere to other cells d Macrophage remnants convolute hematopoietic tissue flow cytometry analysis d Remnant-derived macrophage-RNA is misattributed to other cells in RNA-seq data
The homeobox transcription factor Mtx2 is essential for epiboly, the first morphogenetic movement of gastrulation in zebrafish. Morpholino knockdown of Mtx2 results in stalling of epiboly and lysis due to yolk rupture. However, the mechanism of Mtx2 action is unknown. The role of mtx2 is surprising as most mix/bix family genes are thought to have roles in mesendoderm specification. Using a transgenic sox17-promoter driven EGFP line, we show that Mtx2 is not required for endoderm specification but is required for correct morphogenetic movements of endoderm and axial mesoderm. During normal zebrafish development, mtx2 is expressed at both the blastoderm margin and in the zebrafish equivalent of visceral endoderm, the extra-embryonic yolk syncytial layer (YSL). We show that formation of the YSL is not Mtx2 dependent, but that Mtx2 directs spatial arrangement of YSL nuclei. Furthermore, we demonstrate that Mtx2 knockdown results in loss of the YSL F-actin ring, a microfilament structure previously shown to be necessary for epiboly progression. In summary, we propose that Mtx2 acts within the YSL to regulate morphogenetic movements of both embryonic and extra-embryonic tissues, independently of cell fate specification.
The caudal-related homeobox transcription factor CDX2 is expressed in leukemic cells but not during normal blood formation. Retroviral overexpression of Cdx2 induces AML in mice, however the developmental stage at which CDX2 exerts its effect is unknown. We developed a conditionally inducible Cdx2 mouse model to determine the effects of in vivo, inducible Cdx2 expression in hematopoietic stem and progenitor cells (HSPCs). Cdx2-transgenic mice develop myelodysplastic syndrome with progression to acute leukemia associated with acquisition of additional driver mutations. Cdx2-expressing HSPCs demonstrate enrichment of hematopoietic-specific enhancers associated with pro-differentiation transcription factors. Furthermore, treatment of Cdx2 AML with azacitidine decreases leukemic burden. Extended scheduling of low-dose azacitidine shows greater efficacy in comparison to intermittent higher-dose azacitidine, linked to more specific epigenetic modulation. Conditional Cdx2 expression in HSPCs is an inducible model of de novo leukemic transformation and can be used to optimize treatment in high-risk AML.
Mice lacking the erythroid Kruppel-like factor (EKLF) die in utero at embryonic day 15 (E15) from severe anemia. EKLF−/− embryos display a marked deficit in β-globin gene expression. To test whether β-globin deficiency was solely responsible for the anemia and intrauterine death, we corrected the globin chain imbalance in EKLF−/− embryos by breeding with a strain of mice that express high levels of human γ-globin. Despite efficient production of hybrid m2-hγ2 hemoglobin in the fetal livers of EKLF−/− animals, hemolysis was not corrected and survival was not prolonged. We concluded that deficiency of nonglobin EKLF target genes is a major contributor to the definitive red blood cell abnormalities and prenatal death in EKLF−/−embryos. These results suggest that strategies designed to antagonize EKLF function in adults with hemoglobinopathy, in an attempt to reactivate γ-globin gene expression, may adversely affect other essential aspects of red blood cell physiology.
LBA7006 Background: PAC is a potent JAK2 inhibitor without significant JAK1 inhibition with minimal myelosuppression in early-phase studies in MF. Methods: The efficacy and safety of daily oral PAC was compared to BAT (2:1 randomization stratified for risk and platelet count). The 10 endpoint was the proportion of ITT patients (pts) achieving ≥ 35% spleen volume reduction (SVR) at week (wk) 24 by centrally reviewed MRI or CT. Secondary endpoints included the proportion achieving ≥ 50% reduction in total symptom score (TSS) at wk 24 using the MPN Symptom Assessment Form. Results: Patients:327 were enrolled (PAC:220, BAT:107), 62% with 10 MF. Median time from diagnosis was 1.12 years (PAC 0.99, BAT 1.60): 32% and 15% had a platelet counts < 100,000/µL or <50,000/ µL; 75% were JAK2V617F positive. Efficacy: The median duration of treatment was 16.2 months PAC and 5.9 months BAT. Sixty-two percent of BAT patients received active disease directed therapy. The SVR rates at week 24 were 19.1% for PAC vs. 4.7% for BAT (p=0.0003) in ITT and 25% vs. 5.9% (p=0.0001) in the evaluable population. 79% of BAT patients crossed over to PAC; 21% had achieved a >35% reduction in spleen volume at data cutoff. TSS composite V1 + V2 response rates were 24.5%for PAC vs. 6.5% for BAT (p<0.0001) by ITT, and were 40.9% vs. 9.9% in evaluable pts (p<0.0001). Efficacy with baseline cytopenias: In pts with <100,000 and <50,000 platelets/μ/L, the SVR rates were 16.7% for PAC vs. 0% for BAT (p=0.009), and 22.9% vs. 0% (p=0.045) by ITT and 23.5% vs. 0% (p=0.007) and 33.3% vs. 0% (p=0.037) in evaluable pts. In RBC transfusion dependent pts, 25.7% of PAC pts became RBC independent vs. 0% of BAT pts (p=0.043). Safety: The most common adverse events (AE) for PAC were diarrhea, nausea, and vomiting; (grade 3 were <5%, <1%, <1% respectively). Hematologic AEs were similar between PAC and BAT. Conclusions: This study demonstrated PAC was well tolerated and induced significant and sustained SVR and symptom control even in patients with severe thrombocytopenia. PAC therapy resulted in RBC transfusion independence in a significant proportion of pts. Clinical trial information: NCT01773187.
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