👤 John K Cowell

A truncated derivative of Fibroblast growth factor receptor-1 (FGFR1) (tnFGFR1) independently transforms haematopoietic stem cells leading to a stem cell-like leukaemia/lymphoma syndrome (SCLL). In mouse models, these transformed cells show extensive genetic reprogramming that is distinct from cells transformed with full-length chimeric FGFR1 kinases. We now show that the truncated derivative is insensitive to kinase inhibitors, and its increased expression is related to resistance to kinase inhibitors. Chromatin immunoprecipitation (ChIP) analysis shows that tnFGFR1 can occupy the promoters of the Myc, Flt3 and Kit genes suggesting a transcription regulatory function. However, without a deoxyribonucleic acid-binding motif, tnFGFR1 must interact with binding partners that supply this function. To define this potential regulatory complex, we performed immunoprecipitation-mass spectroscopy (IP-MS) and demonstrate that tnFGFR1 is present in a complex with the splicing factor proline- and glutamine-rich (SFPQ) protein and non-POU domain-containing octamer-binding protein (NONO) protein. In addition, this protein complex is present on the promoters of target genes Flt3 and Kit, and knockdown of either SFPQ or NONO prevents activation of their target genes. In addition, treatment with the NONO inhibitor auranofin suppresses cell proliferation of tnFGFR1-transformed cells in vitro mitigating leukaemia progression in vivo in a mouse model. Thus, future targeting of this tnFGFR1 transcription complex may provide a means for treating tnFGFR1-driven leukaemia. Show less

The development of FGFR1-driven stem cell leukemia and lymphoma syndrome (SCLL) in mouse models is accompanied by an increase in highly heterogenous myeloid derived suppressor cells (MDSCs), which promote immune evasion. To dissect this heterogeneity, we used a combination of CyTOF and scRNA-Seq to define the phenotypes and genotypes of these MDSCs. CyTOF demonstrated increased levels of circulating macrophages in the peripheral blood of leukemic mice, and flow cytometry demonstrated that these macrophages were derived from Ly6C Show less

Leukemias driven by activated, chimeric FGFR1 kinases typically progress to AML which have poor prognosis. Mouse models of this syndrome allow detailed analysis of cellular and molecular changes occurring during leukemogenesis. We have used these models to determine the effects of leukemia development on the immune cell composition in the leukemia microenvironment during leukemia development and progression. Single cell RNA sequencing (scRNA-Seq) was used to characterize leukemia associated neutrophils and define gene expression changes in these cells during leukemia progression. scRNA-Seq revealed six distinct subgroups of neutrophils based on their specific differential gene expression. In response to leukemia development, there is a dramatic increase in only two of the neutrophil subgroups. These two subgroups show specific gene expression signatures consistent with neutrophil precursors which give rise to immature polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). Analysis of gene expression in these precursor cells identified pathways that were specifically upregulated, the most pronounced of which involved matrix metalloproteinases Mmp8 and Mmp9, during leukemia progression. Pharmacological inhibition of MMPs using Ilomastat preferentially restricted in vitro migration of neutrophils from leukemic mice and led to a significantly improved survival in vivo, accompanied by impaired PMN-MDSC recruitment. As a result, levels of T-cells were proportionally increased. In clinically annotated TCGA databases, MMP8 was shown to act as an independent indicator for poor prognosis and correlated with higher neutrophil infiltration and poor pan-cancer prognosis. We have defined specific leukemia responsive neutrophil subgroups based on their unique gene expression profile, which appear to be the precursors of neutrophils specifically associated with leukemia progression. An important event during development of these neutrophils is upregulation MMP genes which facilitated mobilization of these precursors from the BM in response to cancer progression, suggesting a possible therapeutic approach to suppress the development of immune tolerance. Show less

The genome is organized into large scale structures in the interphase nucleus. Pericentromeric heterochromatin represents one such compartment characterized by histones H3 and H4 tri-methylated at K9 and K20 respectively and with a correspondingly low level of histone acetylation. HP1 proteins are concentrated in pericentric heterochromatin and histone deacetylase inhibitors such as trichostatin A (TSA) promote hyperacetylation of heterochromatic nucleosomes and the dispersal of HP1 proteins. We observed that in mouse cells, which contain prominent heterochromatin, DNA topoisomerase IIβ (topoIIβ) is also concentrated in heterochromatic regions. Similarly, a detergent-resistant fraction of topoIIβ is associated with heterochromatin in human cell lines. Treatment with TSA displaced topoIIβ from the heterochromatin with similar kinetics to the displacement of HP1β. Topoisomerase II is the cellular target for a number of clinically important cytotoxic anti-cancer agents known collectively as topoisomerase poisons, and it has been previously reported that histone deacetylase inhibitors can sensitize cells to these drugs. While topoIIα appears to be the major target for most topoisomerase poisons, histone deacetylase-mediated potentiation of these drugs is dependent on topoIIβ. We find that while prior treatment with TSA did not increase the quantity of etoposide-mediated topoIIβ-DNA covalent complexes, it did result in a shift in their distribution from a largely heterochromatin-associated to a pannuclear pattern. We suggest that this redistribution of topoIIβ converts this isoform of topoII to a effective relevant target for topoisomerase poisons. Show less

The HP1 class of chromobox (Cbx) genes encode an evolutionarily conserved family of proteins involved in the packaging of chromosomal domains into a repressive heterochromatic state. The murine Cbx5, Cbx1 and Cbx3 genes encode the three mouse HP1 proteins, mHP1 alpha, -beta and -gamma respectively. Here, we report the cloning of the mouse Cbx3/HP1 gamma gene and the chromosomal localisation of Cbx3 and three Cbx3-related pseudogenes. The Cbx3 structural gene is located on mouse Chromosome 6, close to the Hoxa cluster. Two Cbx3 processed pseudogenes are separated by just 300 bp and are arranged in a head-to-tail configuration on Chromosome 13 while a third pseudogene is found on mouse Chromosome 4. The genomic intron-exon arrangement of Cbx3 is different from the conserved organisation of three other mammalian HP1 genes, Cbx1 (mHP1 beta), CBX3 (hHP1 gamma), and Cbx5 (mHP1 alpha) in that Cbx3 lacks an intron that is present in the others. Show less

Heterochromatin represents a cytologically visible state of heritable gene repression. In the yeast, Schizosaccharomyces pombe, the swi6 gene encodes a heterochromatin protein 1 (HP1)-like chromodomain protein that localizes to heterochromatin domains, including the centromeres, telomeres, and the donor mating-type loci, and is involved in silencing at these loci. We identify here the functional domains of swi6p and demonstrate that the chromodomain from a mammalian HP1-like protein, M31, can functionally replace that of swi6p, showing that chromodomain function is conserved from yeasts to humans. Site-directed mutagenesis, based on a modeled three-dimensional structure of the swi6p chromodomain, shows that the hydrophobic amino acids which lie in the core of the structure are critical for biological function. Gel filtration, gel overlay experiments, and mass spectroscopy show that HP1 proteins can self-associate, and we suggest that it is as oligomers that HP1 proteins are incorporated into heterochromatin complexes that silence gene activity. Show less