ALBERT

Beschreibung: Cancer is caused by accumulated genomic and epigenetic abnormalities during the development of an individual, particularly during the neonatal period, when developmental plasticity is actively occurring. Myeloid-specific deletion of pten in embryos or after 3 weeks of age causes acute monocytic or myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) following a transient myeloproliferative neoplasm (MPN) in adult mice, which can mimic the human diseases to varying degrees. However, it is not clear how the timing of genomic and epigenetic abnormalities contributes to the disease phenotype in a mouse that is of an age comparable to human children. We hypothesized that during the development/aging process, the timing of when the genomic abnormality or “hit” occurs, such as loss of Pten or Nf1, is a critical determinant of the disease phenotype. We tested this by investigating the effect of somatic deletion of Pten at an age of 8 days, one of the most vulnerable stages for malignancy development in mice with or without a germline mutant Nf1. Through crossbreeding, we generated mice with Ptenfl/flNf1Fcr/+Mx1-Cre+ on a C57BL6/129 genetic background, and conditionally deleted Pten in a myeloid-specific manner by intraperitoneal injection of Poly(I:C). Mice with a pten deletion and mutant Nf1 (ptenkoNf1mut, hereafter referred as double mutant) showed signs of sickness at the end of the 2nd week of life, and all died by age 3-5 weeks (equivalent to 1-3 years old in humans). The natural survival in double mutant mice (n=10) was significantly shorter than those with wild type pten and Nf1 (ptenwt; Nf1wt, hereafter referred as WT, n=6, median 0.9 vs 〉14 months, p

Beschreibung: Juvenile myelomonocytic leukemia (JMML) and chronic myelomonocytic leukemia (CMML) are mixed myelodysplastic syndromes (MDS)/myeloproliferative neoplasms (MPNs) that share similar clinical manifestations, but differ in prognosis and ages at diagnosis. Both are characterized by monocytosis and monocytic infiltration in vital organs. JMML is extremely aggressive, with death occurring within one year after diagnosis in children ( Pten wt/ΔNf1LOH〉 WT (median spleen weight: 228〉180〉134〉56mg, respectively, p

Beschreibung: Acute myeloid leukemia (AML) is a hematologic malignancy that continues to have high relapse and treatment-related mortality rates, despite recent advances in clinical management and therapy. Janus kinase (JAK) inhibitors inhibit the activity of the JAK/STAT pathway and have demonstrated some clinical responses in AML patients. However, survival analysis suggests that more than half of AML patients do not benefit from treatment with JAK inhibitors. Furthermore, PTEN deficiency is frequently found in patients in the late stages of cancer, which causes hyperactivated AKT and MAPK pathways. However, emerging data suggests that leukemia cells with PTEN deficiency are resistant to MAPK inhibitors. Over the past decade, it has been demonstrated that dysregulated epigenetics play an important role in myeloid leukemogenesis. The bromodomain and extraterminal domain (BET) family includes adaptor proteins Brd2, Brd3, Brd4, and Brdt that regulate gene expression via binding to acetylated chromatin and subsequently activating RNA Polymerase II driven transcriptional elongation, resulting in the promotion of gene expression. BRD4 is a BET protein required for disease maintenance in AML. JQ1 is a small molecule that interferes with transcriptional regulators, such as BRD4, by preventing them from interacting with acetylated regions of the genome and thus inhibiting the transcriptional activation of BRD4 target genes. Prior research in lymphocytic leukemia cell lines suggests that JQ1 also decreases STAT5-dependent gene transcriptional activities. We hypothesize that the inhibition of BET proteins may correct the over-activated transcriptional activities in myeloid leukemia cells and induce disease regression. We tested our hypothesis in PTEN deficient myeloid leukemia cell lines, TF-1a and K562, and used human cord blood mononuclear cells (CB) for normal cell comparison. Methods: 1) To test whether JQ1 can inhibit colony formation, we seeded cells on 0.3% agar and McCoys' 5A medium supplemented with nutrients and 15% fetal bovine serum, without cytokines, and added JQ1 diluents to the cultures at concentrations of 32.5-1000nM overnight after the cultures were established. 2) To test whether JQ1 can inhibit leukemia cell proliferation, we cultured cells in liquid medium with JQ1 for 48-72 hours, and quantified the viable cells using alamarBlue® assay. 3) To investigate whether JAK/STAT5 activity is altered by JQ1 in leukemia cells, we quantified phosphorylated STAT5 (pSTAT5) in cells via flow cytometry and western blot. We treated the cells with JQ1 at various concentrations for 2 hours and then stimulated the cells for 15 minutes in medium with 0.5% BSA and 10ng/mL GM-CSF prior to staining the cells with anti-pStat5 (pY694) antibody conjugated with Alexa Fluor® 647 for FACS analysis or lysing the cells for western blot analysis. Results: In the colony formation assay, we found that TF-1a cells were more sensitive to JQ1 than the CB cells and K562, with an IC50 of 62.5-125 nM for TF-1a cells (p