Loss of the KN Motif and AnKyrin Repeat Domain 1 (KANK1) Leads to Lymphoid Compartment Dysregulation in Murine Model.

The KN Motif and AnKyrin Repeat Domain 1 (KANK1) is proposed as a tumour suppressor gene, as its expression is reduced or absent in several types of tumour tissue, and over-expressing the protein inhibited the proliferation of tumour cells in solid cancer models. We report a novel germline loss of heterozygosity mutation encompassing the KANK1 gene in a young patient diagnosed with myelodysplastic neoplasm (MDS) with no additional disease-related genomic aberrations. To study the potential role of KANK1 in haematopoiesis, we generated a new transgenic mouse model with a confirmed loss of KANK1 expression. KANK1 knockout mice did not develop any haematological abnormalities; however, the loss of its expression led to alteration in the colony forming and proliferative potential of bone marrow (BM) cells and a decrease in hematopoietic stem and progenitor cells (HSPCs) population frequency. A comprehensive marker expression analysis of lineage cell populations indicated a role for Kank1 in lymphoid cell development, and total protein analysis suggests the involvement of Kank1 in BM cells' cytoskeleton formation and mobility.

NSD1 in erythroid differentiation and leukemogenesis.

We have uncovered a novel role for the nuclear receptor-binding SET domain protein 1 (NSD1) in human and murine erythroid differentiation. Mechanistically, we found that the histone methyltransferase activity of NSD1 is essential for chromatin binding, protein interactions and target gene activation of the erythroid transcriptional master regulator GATA1.

Nuclear interacting SET domain protein 1 inactivation impairs GATA1-regulated erythroid differentiation and causes erythroleukemia.

The nuclear receptor binding SET domain protein 1 (NSD1) is recurrently mutated in human cancers including acute leukemia. We show that NSD1 knockdown alters erythroid clonogenic growth of human CD34+ hematopoietic cells. Ablation of Nsd1 in the hematopoietic system of mice induces a transplantable erythroleukemia. In vitro differentiation of Nsd1-/- erythroblasts is majorly impaired despite abundant expression of GATA1, the transcriptional master regulator of erythropoiesis, and associated with an impaired activation of GATA1-induced targets. Retroviral expression of wildtype NSD1, but not a catalytically-inactive NSD1N1918Q SET-domain mutant induces terminal maturation of Nsd1-/- erythroblasts. Despite similar GATA1 protein levels, exogenous NSD1 but not NSDN1918Q significantly increases the occupancy of GATA1 at target genes and their expression. Notably, exogenous NSD1 reduces the association of GATA1 with the co-repressor SKI, and knockdown of SKI induces differentiation of Nsd1-/- erythroblasts. Collectively, we identify the NSD1 methyltransferase as a regulator of GATA1-controlled erythroid differentiation and leukemogenesis.

Murine Models of Acute Myeloid Leukaemia.

Acute myeloid leukaemia (AML) is a rare but severe form of human cancer that results from a limited number of functionally cooperating genetic abnormalities leading to uncontrolled proliferation and impaired differentiation of hematopoietic stem and progenitor cells. Before the identification of genetic driver lesions, chemically, irradiation or viral infection-induced mouse leukaemia models provided platforms to test novel chemotherapeutics. Later, transgenic mouse models were established to test the in vivo transforming potential of newly cloned fusion genes and genetic aberrations detected in patients' genomes. Hereby researchers constitutively or conditionally expressed the respective gene in the germline of the mouse or reconstituted the hematopoietic system of lethally irradiated mice with bone marrow virally expressing the mutation of interest. More recently, immune deficient mice have been explored to study patient-derived human AML cells in vivo. Unfortunately, although complementary to each other, none of the currently available strategies faithfully model the initiation and progression of the human disease. Nevertheless, fast advances in the fields of next generation sequencing, molecular technology and bioengineering are continuously contributing to the generation of better mouse models. Here we review the most important AML mouse models of each category, briefly describe their advantages and limitations and show how they have contributed to our understanding of the biology and to the development of novel therapies.

A Novel Inducible Mouse Model of MLL-ENL-driven Mixed-lineage Acute Leukemia.

Previous retroviral and knock-in approaches to model human t(11;19)+ acute mixed-lineage leukemia in mice resulted in myeloproliferation and acute myeloid leukemia not fully recapitulating the human disease. The authors established a doxycycline (DOX)-inducible transgenic mouse model "iMLL-ENL" in which induction in long-term hematopoietic stem cells, lymphoid primed multipotent progenitor cells, multipotent progenitors (MPP4) but not in more committed myeloid granulocyte-macrophage progenitors led to a fully reversible acute leukemia expressing myeloid and B-cell markers. iMLL-ENL leukemic cells generally expressed lower MLL-ENL mRNA than those obtained after retroviral transduction. Disease induction was associated with iMLL-ENL levels exceeding the endogenous Mll1 at mRNA and protein levels. In leukemic cells from t(11;19)+ leukemia patients, MLL-ENL mRNA also exceeded the endogenous MLL1 levels suggesting a critical threshold for transformation. Expression profiling of iMLL-ENL acute leukemia revealed gene signatures that segregated t(11;19)+ leukemia patients from those without an MLL translocation. Importantly, B220+ iMLL-ENL leukemic cells showed a higher in vivo leukemia initiation potential than coexisting B220- cells. Collectively, characterization of a novel transgenic mouse model indicates that the cell-of-origin and the fusion gene expression levels are both critical determinants for MLL-ENL-driven acute leukemia.

JAK2V617F allele burden in patients with myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) are clonal malignant diseases that represent a group of conditions including polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF). The JAK2-V617F mutation is prevalent in almost all patients with MPNs and has become a valuable biomarker for diagnosis of MPNs. A different allele burden in these entities has long been noticed. The aim of our study was to assess the JAK2 allele burden in our JAK2V617F positive cases and its association with phenotype if any and to select a simple, sensitive assay for use in our clinical molecular diagnostic laboratory. Methodologies reported in this literature include amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and real-time quantitative polymerase chain reaction (RQ-PCR). We analyzed 174 cases by RQ-PCR for the quantification of JAK2V617F were initially screened by ARMS-PCR. We found that V617F allele burden in the entire population of patients was 73 % ranging from 0.97 to 95 %. The median V617F allele burden in PV patients was 40 %, MF was 95 %, and ET was 25 %. ARMS-PCR and RQ-PCR were proven to be sensitive since ARMS-PCR is a qualitative method; it can be used to screen JAK2V617F mutation and RQ-PCR was used to quantify the V617F cells. Our study suggests that JAK2V617F positivity is associated with MPNs, and its allele burden is an excellent diagnostic marker for disease subtypes, prognosis, disease phenotype and complication, and evolution. The data indicates that ARMS-PCR is simple and can be easily performed for the primary screening of JAK2V617F mutation, and RQ-PCR is sensitive enough to detect low mutant allele levels (>10 %), specific enough not to produce false positive results, and can be performed for the JAK2V617F allele burden quantification.