ALBERT

Description: Alteration of lineage-specific transcriptional programs for hematopoiesis causes differentiation block and promotes leukemia development. Here, we show that AML1/ETO, the most common translocation fusion product in acute myeloid leukemia (AML), counteracts the activity of retinoic acid (RA), a transcriptional regulator of myelopoiesis. AML1/ETO participates in a protein complex with the RA receptor alpha (RARα) at RA regulatory regions on RARβ2, which is a key RA target gene mediating RA activity/resistance in cells. At these sites, AML1/ETO recruits histone deacetylase, DNA methyltransferase, and DNA-methyl-CpG binding activities that promote a repressed chromatin conformation. The link among AML1/ETO, heterochromatic RARβ2 repression, RA resistance, and myeloid differentiation block is indicated by the ability of either siRNA-AML1/ETO or the DNA methylation inhibitor 5-azacytidine to revert these epigenetic alterations and to restore RA differentiation response in AML1/ETO blasts. Finally, RARβ2 is commonly silenced by hypermethylation in primary AML blasts but not in normal hematopoietic precursors, thus suggesting a role for the epigenetic repression of the RA signaling pathway in myeloid leukemogenesis.

Description: As a result of the t(9;22), more than 95% of CMLs and 20–25% of adult ALLs express the p210(BCR-ABL) or the p185(BCR-ABL) fusion protein respectively. The BCR portion of the fusion protein harbors an N-terminal coiled-coil (CC) domain. The CC contains two alpha helical motifs and assembles to dimers with antiparallel orientation. Two of these dimers associate and lead to the tetramerization of BCR. The second alpha-Helix (Helix-2) contributes the majority of the dimer and tetramer interface. In the case of the BCR/ABL fusion protein, the BCR mediated tetramerization of ABL leads to the constitutive activation of the ABL kinase domain. The subsequent permanent activation of multiple downstream signaling pathways induces the leukemic phenotype. Targeted inhibition of BCR/ABL by the ABL kinase inhibitor Imatinib leads to complete remission in CML and ALL patients. However, a large portion of ALL and CML patients in blast crisis become resistant to Imatinib mainly by the acquisition of point mutations in BCR/ABL. Imatinib binds to the inactive conformation of the ABL-kinase which in case of the BCR/ABL fusion protein is present in monomers. We have previously shown that CC-derived peptides that interfere with BCR/ABL tetramer formation reduce the kinase activity of BCR/ABL in vivo and increase the sensitivity of BCR/ABL expressing cells towards Imatinib. Here we investigated the effects of peptides derived from the CC subdomain Helix-2 on BCR/ABL oligomerization and on the biology of cells expressing Wt BCR/ABL and Imatinib-resistant mutants. First we confirmed the interaction between Helix-2 and BCR/ABL by GST-pull-down and co-immunoprecipitation assays. The influence of Helix 2 peptides on the BCR/ABL oligomerization and its capacity to form high-molecular weight (HMW) complexes in vivo was analyzed by size-exclusion HPLC. The effect of Helix 2 on the leukemogenic potential Helix of BCR/ABL was investigated in retrovirally transduced murine IL-3 dependent Ba/F3 cells and human Philadelphia Chromosome (Ph) negative ALL-cell line Nalm-6 and Ph-positve SupB15 cells. Ba/F3 cells transduced with Imatinib-resistant BCR/ABL point mutations were used to investigate the inhibitory potential of CC specific peptides on mutant BCR/ABL in presence and absence of Imatinib. Here we report that CC-specific Helix-2 peptides interact with Wt and mutant BCR/ABL; decrease the autophosphorylation of BCR/ABL in transduced Ba/F3 cells; disrupt the HMW-complexes of Wt and mutant BCR/ABL; abrogate the growth of human Ph-positive expressing SupB15 cells without affecting Ph-negative Nalm-6 cells; v) increase the Imatinib sensitivity of IL-3 independent Ba/F3 cells expressing BCR/ABL; reduce the Imatinib resistance of Ba/F3-cells expressing BCR/ABL P-loop mutants Y253F and E255K. Taken together these results show that the disruption of BCR/ABL tetramerization has strong inhibitory effects on BCR/ABL transformed cells. Furthermore our results indicate that disruption of BCR/ABL tetramerization is a therapeutic option for the treatment of patients harboring Imatinib-resistant BCR/ABL mutations.

Description: Epigenetic modifications regulate developmental genes involved in stem cell identity and lineage choice. NFI-A is a posttranscriptional microRNA-223 (miR-223) target directing human hematopoietic progenitor lineage decision: NFI-A induction or silencing boosts erythropoiesis or granulopoiesis, respectively. Here we show that NFI-A promoter silencing, which allows granulopoiesis, is guaranteed by epigenetic events, including the resolution of opposing chromatin “bivalent domains,” hypermethylation, recruitment of polycomb (PcG)–RNAi complexes, and miR-223 promoter targeting activity. During granulopoiesis, miR-223 localizes inside the nucleus and targets the NFI-A promoter region containing PcGs binding sites and miR-223 complementary DNA sequences, evolutionarily conserved in mammalians. Remarkably, both the integrity of the PcGs-RNAi complex and DNA sequences matching the seed region of miR-223 are required to induce NFI-A transcriptional silencing. Moreover, ectopic miR-223 expression in human myeloid progenitors causes heterochromatic repression of NFI-A gene and channels granulopoiesis, whereas its stable knockdown produces the opposite effects. Our findings indicate that, besides the regulation of translation of mRNA targets, endogenous miRs can affect gene expression at the transcriptional level, functioning in a critical interface between chromatin remodeling complexes and the genome to direct fate lineage determination of hematopoietic progenitors.