Acute myeloid leukemia with complex karyotype (CK-AML) is a distinct biological entity associated with a very poor outcome. Since complex karyotypes frequently contain deletions of the chromosomal region 12p13 encompassing the tumor suppressor genes ETV6 and CDKN1B, we aimed to unravel their modes of inactivation in CK-AML. To decipher deletions, mutations and methylation of ETV6 and CDKN1B, arrayCGH, SNP arrays, direct sequencing of all coding exons and pyrosequencing of the 5′UTR CpG islands of ETV6 and CDKN1B were performed. In total, 39 of 79 patients (49%) showed monoallelic deletions of 12p13 according to karyotypic data and 20 of 43 patients (47%) according to genomic profiling. Genomic profiling led to the minimal deleted region covering the 3′-UTR of ETV6 and CDKN1B. Direct sequencing revealed one novel monoallelic frameshift mutation in ETV6 while no mutations in CDKN1B were identified. Furthermore, methylation levels of ETV6 and CDKN1B did not indicate transcriptional silencing of any of these genes. ETV6 and CDKN1B had reduced expression levels in CK-AML patients with deletion in 12p13 as compared to CK-AML without deletion in 12p13, while the other genes (BCL2L14, LRP6, DUSP16 and GPRC5D) located within the minimal deleted region in 12p13 had very low or missing expression in CK-AML irrespective of their copy number status. ETV6 and CDKN1B are mainly affected by small monoallelic deletions, whereas mutations and hypermethylation play a minor role in CK-AML. Reduced gene dosage led to reduced gene expression levels, pointing to haploinsufficiency as the relevant mechanism of inactivation of ETV6 and CDKN1B in CK-AML.
Megakaryocytes (MKs), the precursors of blood platelets, are large, polyploid cells residing mainly in the bone marrow. We have previously shown that balanced signaling of the Rho GTPases RhoA and Cdc42 is critical for correct MK localization at bone marrow sinusoids in vivo. Using conditional RhoA/Cdc42 double-knockout (DKO) mice, we reveal here that RhoA/Cdc42 signaling is dispensable for the process of polyploidization in MKs but essential for cytoplasmic MK maturation. Proplatelet formation is virtually abrogated in the absence of RhoA/Cdc42 and leads to severe macrothrombocytopenia in DKO animals. The MK maturation defect is associated with downregulation of myosin light chain 2 (MLC2) and β1-tubulin, as well as an upregulation of LIM kinase 1 and cofilin-1 at both the mRNA and protein level and can be linked to impaired MKL1/SRF signaling. Our findings demonstrate that MK endomitosis and cytoplasmic maturation are separately regulated processes, and the latter is critically controlled by RhoA/Cdc42.
Failure in airway regeneration after injury may result in profound consequences like a disease with bronchiolitis obliterans. Re-building of ciliated cells is a good marker for resumed airway activity. We decided to investigate whether M3E3C3,a fetal hamster stem equivalent cell line, is capable of in vitro ciliogenesis. Immunoreactivity and qRT-PCR have revealed that this cell line expresses a stem cell marker Sox2 and another marker, Clara cell secretory protein (CCSP) specific for the stem cells in the bronchiolar region. An embryonic stem cell factor Oct4 was negative.
Platelets are small anucleate cells that circulate in the blood in a resting state but can be activated by external cues. In case of need, platelets from blood donors can be transfused. As an alternative source, platelets can be produced from induced pluripotent stem cells (iPSCs); however, recovered numbers are low.To optimize megakaryocyte (MK) and platelet output from murine iPSCs, we investigated overexpression of the transcription factors GATA-binding factor 1 (GATA1); nuclear factor, erythroid 2; and pre-B-cell leukemia transcription factor 1 (Pbx1) and a hyperactive variant of the small guanosine triphosphatase RhoA (RhoAhc).To avoid off-target effects, we generated iPSCs carrying the reverse tetracycline-responsive transactivator M2 (rtTA-M2) in the Rosa26 locus and expressed the factors from Tet-inducible gammaretroviral vectors. Differentiation of iPSCs was initiated by embryoid body (EB) formation. After EB dissociation, early hematopoietic progenitors were enriched and cocultivated on OP9 feeder cells with thrombopoietin and stem cell factor to induce megakaryocyte (MK) differentiation.Overexpression of GATA1 and Pbx1 increased MK output 2- to 2.5-fold and allowed prolonged collection of MK. Cytologic and ultrastructural analyses identified typical MK with enlarged cells, multilobulated nuclei, granule structures, and an internal membrane system. However, GATA1 and Pbx1 expression did not improve MK maturation or platelet release, although in vitro-generated platelets were functional in spreading on fibrinogen or collagen-related peptide.We demonstrate that the use of rtTA-M2 transgenic iPSCs transduced with Tet-inducible retroviral vectors allowed for gene expression at later time points during differentiation. With this strategy we could identify factors that increased in vitro MK production.
