Rosemary Finnon

Flt3 -ITD mutations in a mouse model of radiation-induced acute myeloid leukaemia

Acute myeloid leukaemia (AML) is one of the most common malignancies seen to occur in human populations exposed to ionising radiation.1 Mouse models have been widely used for quantitative and mechanistic studies of radiation leukaemogenesis; further, there is a similarity in the histopathological changes found in human AML and mouse AML. The majority of mouse radiation-induced AMLs (rAMLs) carry deletions of chromosome 2, and the Sfpi1/PU.1 haematopoietic transcription factor is located within the commonly deleted chromosome 2 region.2 In most rAMLs and rAML cell lines (70%), Sfpi1/PU.1 suffers hemizygous loss accompanied by point mutations in the region encoding the ETS DNA-binding domain, leading to base substitutions at the R235 residue of the protein.2, 3, 4 Sfpi1/PU.1 mutations have been identified in human AML, but these are rare; in contrast, the most common mutations in human AML involve the Flt3 receptor tyrosine kinase. Most of these mutations are internal tandem duplication (Flt3-ITD) of 3–400 bp5 that result in ligand-dependent dimerisation and receptor phosphorylation. The presence of a Flt3-ITD appears to confer a more severe phenotype and a poor prognosis for AML sufferers. The majority of the remaining non-ITD mutations in Flt3 occur in the second tyrosine kinase domain, being mostly point mutations within codon 835 or the deletion of 836.6

A major breakpoint cluster domain in murine radiation-induced acute myeloid leukemia*

Cytogenetic and molecular studies have provided evidence of the clustering of chromosome 2 deletion breakpoints in radiation‐induced murine acute myeloid leukemia (AML). Moreover, clustering occurs in at least two fragile domains rich in telomere‐like arrays. Here we describe a physical map of the distal breakpoint cluster and confirm the presence of inverted head‐to‐head telomeric sequence arrays. These potentially recombinogenic sequences were not, however, the direct focus for post‐irradiation chromosome breakage in AML. Instead, the two arrays bordered a 2.5‐kb sequence with properties expected of a nuclear matrix attachment region (MAR). The putative MAR co‐localized in the fragile domain with genes important to the hemopoietic system (leukocyte tyrosine kinase, zinc finger protein 106, erythrocyte protein band 4.2, and β2‐microglobulin (β2m)); the β2m subdomain was a particular focus of breakage. On the basis of these and other data, we suggest that AML‐associated chromosome 2 fragility in the mouse is a consequence of domain‐specific fragility in genomic domains containing numerous genes critical to the hemopoietic system. Copyright

Live cell detection of chromosome 2 deletion and Sfpi1/PU1 loss in radiation-induced mouse acute myeloid leukaemia.

The CBA/H mouse model of radiation-induced acute myeloid leukaemia (rAML) has been studied for decades to bring to light the molecular mechanisms associated with multistage carcinogenesis. A specific interstitial deletion of chromosome 2 found in a high proportion of rAML is recognised as the initiating event. The deletion leads to the loss of Sfpi, a gene essential for haematopoietic development. Its product, the transcription factor PU.1 acts as a tumour suppressor in this model. Although the deletion can be detected early following ionising radiation exposure by cytogenetic techniques, precise characterisation of the haematopoietic cells carrying the deletion and the study of their fate in vivo cannot be achieved. Here, using a genetically engineered C57BL/6 mouse model expressing the GFP fluorescent molecule under the control of the Sfpi1 promoter, which we have bred onto the rAML-susceptible CBA/H strain, we demonstrate that GFP expression did not interfere with X-ray induced leukaemia incidence and that GFP fluorescence in live leukaemic cells is a surrogate marker of radiation-induced chromosome 2 deletions with or without point mutations on the remaining allele of the Sfpi1 gene. This study presents the first experimental evidence for the detection of this leukaemia initiating event in live leukemic cells.

Frequency of acute myeloid leukaemia-associated mouse chromosome 2 deletions in X-ray exposed immature haematopoietic progenitors and stem cells.

Exposure to ionising radiation can lead to an increased risk of cancer, particularly leukaemia. In radiation-induced acute myeloid leukaemia (rAML), a partial hemizygous deletion of mouse chromosome 2 is a common feature in several susceptible strains. The deletion is an early event detectable 24 h after exposure in bone marrow cells using cytogenetic techniques. Expanding clones of bone marrow cells with chromosome 2 deletions can be detected less than a year after exposure to ionising radiation in around half of the irradiated mice. Ultimately, 15–25% of exposed animals develop AML. It is generally assumed that leukaemia originates in an early progenitor cell or haematopoietic stem cell, but it is unknown whether the original chromosome damage occurs at a similar frequency in committed progenitors and stem cells. In this study, we monitored the frequency of chromosome 2 deletions in immature bone marrow cells (Lin−) and haematopoietic stem cells/multipotent progenitor cells (LSK) by several techniques, fluorescent in situ hybridisation (FISH) and through use of a reporter gene model, flow cytometry and colony forming units in spleen (CFU-S) following ex vivo or in vivo exposure. We showed that partial chromosome 2 deletions are present in the LSK subpopulation, but cannot be detected in Lin− cells and CFU-S12 cells. Furthermore, we transplanted irradiated Lin− or LSK cells into host animals to determine whether specific irradiated cell populations acquire an increased proliferative advantage compared to unirradiated cells. Interestingly, the irradiated LSK subpopulation containing cells carrying chromosome 2 deletions does not appear to repopulate as well as the unirradiated population, suggesting that the chromosomal deletion does not provide an advantage for growth and in vivo repopulation, at least at early stages following occurrence.

Analysis of loss of heterozygosity in lymphoma and leukaemia arising in F1 hybrid mice locates a common region of chromosome 4 loss.

Previous studies have identified five lymphoma‐related tumour suppressor gene regions on murine chromosome 4. Using detailed allelotype analysis on a range of lympho‐haematopoietic tumour types arising in F1 hybrid mice, we now show a consistent pattern of loss of heterozygosity (LOH) which identifies a common region of loss delineated by microsatellites D4Mit21 and D4Mit53 on proximal chromosome 4. This critical segment corresponds to the thymic lymphoma tumour suppressor region 5 (TLSR5) identified in an earlier study. Tumours of this type have also been reported as showing allelic loss from the Trp53 and Ikaros regions on chromosome 11. In the present study, only a small fraction of tumours showed LOH in the Ikaros region, while a minority of lymphomas, but not acute myeloid leukaemias, showed allelic loss of the chromosome 11 segment encoding Trp53. These and other data indicate strongly that the genomic regions identified as showing recurrent LOH depend on the genetic background of the mice. Overall, the results indicate a key role for a tumour suppressor gene(s) encoded in an ∼3 cM segment on proximal chromosome 4 and provide an experimental basis for the further investigation of the functional role of candidate genes which include Pax5 and Tgfbr1.

Transcriptomic and proteomic analysis of mouse radiation-induced acute myeloid leukaemia (AML)

A combined transcriptome and proteome analysis of mouse radiation-induced AMLs using two primary AMLs, cell lines from these primaries, another cell line and its in vivo passage is reported. Compared to haematopoietic progenitor and stem cells (HPSC), over 5000 transcriptome alterations were identified, 2600 present in all materials. 55 and 3 alterations were detected in the proteomes of the cell lines and primary/in vivo passage material respectively, with one common to all materials. In cell lines, approximately 50% of the transcriptome changes are related to adaptation to cell culture, and in the proteome this proportion was higher. An AML ‘signature’ of 17 genes/proteins commonly deregulated in primary AMLs and cell lines compared to HPSCs was identified and validated using human AML transcriptome data. This also distinguishes primary AMLs from cell lines and includes proteins such as Coronin 1, pontin/RUVBL1 and Myeloperoxidase commonly implicated in human AML. C-Myc was identified as having a key role in radiation leukaemogenesis. These data identify novel candidates relevant to mouse radiation AML pathogenesis, and confirm that pathways of leukaemogenesis in the mouse and human share substantial commonality.

NOD Scid Gamma Mice Are Permissive to Allogeneic HSC Transplantation without Prior Conditioning

Scid hematopoietic stem cells (HSCs) have an intrinsic defect in their maintenance within the bone marrow (BM) niche which facilitates HSC transplantation without the absolute requirement of prior conditioning. Nevertheless, NOD scid mice have a significantly altered life span due to early development of thymic lymphomas, which compromises the ability to study the long-term fate of exogenous HSCs and their progeny. Here, we present data on the transplantation of HSCs into NOD scid gamma (NSG) mice to achieve long-term engraftment without prior conditioning. We transplanted allogeneic HSCs constitutively expressing the mCherry fluorescent marker into age-matched NSG mice and assessed donor chimerism 6 months post-transplantation. All transplanted NSG mice showed long-term myeloid and lymphoid cell chimerism. Also, in vivo irradiated HSCs showed long-term engraftment, although overall white blood cell (WBC) donor chimerism was lower compared with non-irradiated HSCs. Using this novel NSG transplantation model, we will be able to study the effects of low dose in vivo X-ray exposure on the long-term fate of HSCs, without the requirement of prior radio-ablation of the recipient, and thus leaving the recipient’s BM microenvironment uncompromised. In conclusion, we demonstrated for the first time that allogeneic HSCs from a different inbred strain can compete for niches in the BM compartment of NSG mice.

Influence of radiation quality on mouse chromosome 2 deletions in radiation-induced acute myeloid leukaemia.

Leukaemia is the prevailing neoplastic disorder of the hematopoietic system. Epidemiological analyses of the survivors of the Japanese atomic bombings show that exposure to ionising radiation (IR) can cause leukaemia. Although a clear association between radiation exposure and leukaemia development is acknowledged, the underlying mechanisms remain incompletely understood. A hemizygous deletion on mouse chromosome 2 (del2) is a common feature in several mouse strains susceptible to radiation-induced acute myeloid leukaemia (rAML). The deletion is an early event detectable 24h after exposure in bone marrow cells. Ultimately, 15-25% of exposed animals develop AML with 80-90% of cases carrying del2. Molecular mapping of leukaemic cell genomes identified a minimal deleted region (MDR) on chromosome 2 (chr2) in which a tumour suppressor gene, Sfpi1 is located, encoding the transcription factor PU.1, essential in haematopoiesis. The remaining copy of Sfpi1 has a point mutation in the coding sequence for the DNA-binding domain of the protein in 70% of rAML, which alters a single CpG sequence in the codon for arginine residue R235. In order to identify chr2 deletions and Sfpi.1/PU.1 loss, we performed array comparative genomic hybridization (aCGH) on a unique panel of 79rAMLs. Using a custom made CGH array specifically designed for mouse chr2, we analysed at unprecedentedly high resolution (1.4M array- 148bp resolution) the size of the MDR in low LET and high-LET induced rAMLs (32 X-ray- and 47 neutron-induced). Sequencing of Sfpi1/PU.1DNA binding domain identified the presence of R235 point mutations, showing no influence of radiation quality on R235 type or frequency. We identified for the first time rAML cases with complex del2 in a subset of neutron-induced AMLs. This study allowed us to re-define the MDR to a much smaller 5.5Mb region (still including Sfpi1/PU.1), identical regardless of radiation quality.

Tracking preleukemic cells in vivo to reveal the sequence of molecular events in radiation leukemogenesis

Epidemiological studies have demonstrated an increased leukemia incidence following ionizing radiation exposure, but to date, the target cells and underlying mechanisms of radiation leukemogenesis remain largely unidentified. We engineered a mouse model carrying a different fluorescent marker on each chromosome 2, located inside the minimum deleted region occurring after radiation exposure and recognized as the first leukemogenic event. Using this tailored model, we report that following radiation exposure, more than half of asymptomatic CBA Sfpi1GFP/mCh mice presented with expanding clones of preleukemic hematopoietic cells harboring a hemizygous interstitial deletion of chromosome 2. Moreover, following isolation of preleukemic hematopoietic stem and progenitor cells irradiated in their native microenvironment, we identified the presence of Sfpi1 point mutations within a subpopulation of these preleukemic cells expanding rapidly (increasing from 6% to 55% in 21 days in peripheral blood in one case), hence identifying for the first time the presence of such cells within a living animal. Importantly, we also report a previously undescribed gender difference in the phenotype of the preleukemic cells and leukemia, suggesting a gender imbalance in the radiation-induced leukemic target cell. In conclusion, we provide novel insights into the sequence of molecular events occurring during the (radiation-induced) leukemic clonal evolution.

The Influence of the CTIP Polymorphism, Q418P, on Homologous Recombination and Predisposition to Radiation-Induced Tumorigenesis (mainly rAML) in Mice

Exposure to ionizing radiation increases the incidence of acute myeloid leukemia (AML), which has been diagnosed in Japanese atomic bombing survivors, as well as patients treated with radiotherapy. The genetic basis for susceptibility to radiation-induced AML is not well characterized. We previously identified a candidate murine gene for susceptibility to radiation-induced AML (rAML): C-terminal binding protein (CTBP)-interacting protein (CTIP)/retinoblastoma binding protein 8 (RBBP8). This gene is essential for embryonic development, double-strand break (DSB) resection in homologous recombination (HR) and tumor suppression. In the 129S2/SvHsd mouse strain, a nonsynonymous single nucleotide polymorphism (nsSNP) in Ctip, Q418P, has been identified. We investigated the role of Q418P in radiation-induced carcinogenesis and its effect on CTIP function in HR. After whole-body exposure to 3 Gy of X rays, 11 out of 113 (9.7%) 129S2/SvHsd mice developed rAML. Furthermore, 129S2/SvHsd mouse embryonic fibroblasts (MEFs) showed lower levels of recruitment of HR factors, Rad51 and replication protein A (RPA) to radiation-induced foci, compared to CBA/H and C57BL/6 MEFs, isolated from rAML-sensitive and resistant strains, respectively. Mitomycin C and alpha particles induced lower levels of sister chromatid exchanges in 129S2/SvHsd cells compared to CBA/H and C57BL/6. Our data demonstrate that Q418P nsSNP influences the efficiency of CTIP function in HR repair of DNA DSBs in vitro and in vivo, and appears to affect susceptibility to rAML.