Loss of Ercc1 Results in a Time- and Dose-Dependent Reduction of Proliferating Early Hematopoietic Progenitors

The endonuclease complex Ercc1/Xpf is involved in interstrand crosslink repair and functions downstream of the Fanconi pathway. Loss of Ercc1 causes hematopoietic defects similar to those seen in Fanconi Anemia. Ercc1−/− mice die 3-4 weeks after birth, which prevents long-term follow up of the hematopoietic compartment. We used alternative Ercc1 mouse models to examine the effect of low or absent Ercc1 activity on hematopoiesis. Tie2-Cre-driven deletion of a floxed Ercc1 allele was efficient (>80%) in fetal liver hematopoietic cells. Hematopoietic stem and progenitor cells (HSPCs) with a deleted allele were maintained in mice up to 1 year of age when harboring a wt allele, but were progressively outcompeted when the deleted allele was combined with a knockout allele. Mice with a minimal Ercc1 activity expressed by 1 or 2 hypomorphic Ercc1 alleles have an extended life expectancy, which allows analysis of HSPCs at 10 and 20 weeks of age. The HSPC compartment was affected in all Ercc1-deficient models. Actively proliferating multipotent progenitors were most affected as were myeloid and erythroid clonogenic progenitors. In conclusion, lack of Ercc1 results in a severe competitive disadvantage of HSPCs and is most deleterious in proliferating progenitor cells

Retroviral Integration Mutagenesis in Mice and Comparative Analysis in Human AML Identify Reduced PTP4A3 Expression as a Prognostic Indicator

Acute myeloid leukemia (AML) results from multiple genetic and epigenetic aberrations, many of which remain unidentified. Frequent loss of large chromosomal regions marks haplo-insufficiency as one of the major mechanisms contributing to leukemogenesis. However, which haplo-insufficient genes (HIGs) are involved in leukemogenesis is largely unknown and powerful experimental strategies aimed at their identification are currently lacking. Here, we present a new approach to discover HIGs, using retroviral integration mutagenesis in mice in which methylated viral integration sites and neighbouring genes were identified. In total we mapped 6 genes which are flanked by methylated viral integration sites (mVIS). Three of these, i.e., Lrmp, Hcls1 and Prkrir, were up regulated and one, i.e., Ptp4a3, was down regulated in the affected tumor. Next, we investigated the role of PTP4A3 in human AML and we show that PTP4A3 expression is a negative prognostic indicator, independent of other prognostic parameters. In conclusion, our novel strategy has identified PTP4A3 to potentially have a role in AML, on one hand as a candidate HIG contributing to leukemogenesis in mice and on the other hand as a prognostic indicator in human AML

The gene encoding the transcriptional regulator Yin Yang 1 (YY1) is a myeloid transforming gene interfering with neutrophilic differentiation

The genetic defects underlying the pathogenesis of acute myeloid leukemia (AML) are still largely unknown. Retroviral insertion mutagenesis in mice has become a powerful tool to identify candidate genes involved in the development of leukemia and lymphoma. We have used this strategy with the 1.4 strain of Graffi murine leukemia virus (MuLV), which predominantly causes myeloid leukemias. Here, we report that Graffi-1.4-induced AML frequently harbors virus integrations in the gene encoding the transcription factor Yin Yang 1 (YY1). These integrations occurred in both orientations, and all were located in the 5' promoter region of the gene, 0.5 to 1.5 kb upstream of the major transcriptional start site. Luciferase reporter assays showed

Deficiency of the ribosome biogenesis gene Sbds in hematopoietic stem and progenitor cells causes neutropenia in mice by attenuating lineage progression in myelocytes

Shwachman-Diamond syndrome is a congenital bone marrow failure disorder characterized by debilitating neutropenia. The disease is associated with loss-of-function mutations in the SBDS gene, implicated in ribosome biogenesis, but the cellular and molecular events driving cell specific phenotypes in ribosomopathies remain poorly defined. Here, we established what is to our knowledge the first mammalian model of neutropenia in Shwachman-Diamond syndrome through targeted downregulation of Sbds in hematopoietic stem and progenitor cells expressing the myeloid transcription factor CCAAT/enhancer binding protein a (Cebpa). Sbds deficiency in the myeloid lineage specifically affected myelocytes and their downstream progeny while, unexpectedly, it was well tolerated by rapidly cycling hematopoietic progenitor cells. Molecular insights provided by massive parallel sequencing supported cellular observations of impaired cell cycle exit and formation of secondary granules associated with the defect of myeloid lineage progression in myelocytes. Mechanistically, Sbds deficiency activated the p53 tumor suppressor pathway and induced apoptosis in these cells. Collectively, the data reveal a previously unanticipated, selective dependency of myelocytes and downstream progeny, but not rapidly cycling progenitors, on this ubiquitous ribosome biogenesis protein, thus providing a cellular basis for the understanding of myeloid lineage biased defects in Shwachman-Diamond syndrome

Sequential gain of mutations in severe congenital neutropenia progressing to acute myeloid leukemia

Severe congenital neutropenia (SCN) is a BM failure syndrome with a high risk of progression to acute myeloid leukemia (AML). The underlying genetic changes involved in SCN evolution to AML are largely unknown. We obtained serial hematopoietic samples from an SCN patient who developed AML 17 years after the initiation of G-CSF treatment. Next-generation sequencing was performed to identify mutations during disease progression. In the AML phase, we found 12 acquired nonsynonymous mutations. Three of these, in CSF3R, LLGL2, and ZC3H18, co-occurred in a subpopulation of progenitor cells already in the early SCN phase. This population expanded over time, whereas clones harboring only CSF3R mutations disappeared from the BM. The other 9 mutations were only apparent in the AML cells and affected known AML-associated genes (RUNX1 and ASXL1) and chromatin remodelers (SUZ12 and EP3

Cooperativity of RUNX1 and CSF3R mutations in severe congenital neutropenia: a unique pathway in myeloid leukemogenesis

Key Points CN/AML patients have a high frequency of CSF3R and RUNX1 mutations. CSF3R and RUNX1 mutations induce elevated proliferation of CD34+ cells.</jats:p

Cooperativity of RUNX1 and CSF3R mutations in the development of leukemia in severe congenital neutropenia: a unique pathway in myeloid leukemogenesis

Severe congenital neutropenia (CN) is a pre-leukemic bone marrow failure syndrome with a 20% risk of evolving into leukemia or MDS. Patterns of acquisition of leukemia-associated mutations were investigated using next-generation deep-sequencing in 31 CN patients who developed leukemia or MDS. Twenty (64.5%) of the 31 patients had mutations in RUNX1. A majority of patients with RUNX1 mutations (80.5%) also had acquired CSF3R mutations. In contrast to their high frequency in CN patients who developed leukemia or MDS, RUNX1 mutations were found in only 9 of 307 (2.9%) patients with de novo pediatric AML. A sequential analysis at stages prior to overt leukemia revealed RUNX1 mutations to be late events in leukemic transformation. Single-cell analyses in two patients showed that RUNX1 and CSF3R mutations were present in the same malignant clone. Functional studies demonstrated elevated G-CSF-induced proliferation with diminished myeloid differentiation of hematopoietic CD34(+) cells co-expressing mutated forms of RUNX1 and CSF3R. The high frequency of cooperating RUNX1 and CSF3R mutations in CN patients suggests a novel molecular pathway of leukemogenesis: mutations in the hematopoietic cytokine receptor (G-CSFR) in combination with the second mutations in the downstream hematopoietic transcription fator (RUNX1). The detection of both RUNX1 and CSF3R mutations could be used as a marker for identifying CN patients with a high risk of progressing to leukemia or MDS.status: publishe