Combined Approach to Leukemic Differentiation Using Transcription Factor PU.1-Enhancing Agents.

The transcription factor PU.1 (Purine-rich DNA binding, SPI1) is a key regulator of hematopoiesis, whose level is influenced by transcription through its enhancers and its post-transcriptional degradation via microRNA-155 (miR-155). The degree of transcriptional regulation of the PU.1 gene is influenced by repression via DNA methylation, as well as other epigenetic factors, such as those related to progenitor maturation status, which is modulated by the transcription factor Myeloblastosis oncogene (MYB). In this work, we show that combinatorial treatment of acute myeloid leukemia (AML) cells with DNA methylation inhibitors (5-Azacytidine), MYB inhibitors (Celastrol), and anti-miR-155 (AM155) ideally leads to overproduction of PU.1. We also show that PU.1 reactivation can be compensated by miR-155 and that only a combined approach leads to sustained PU.1 derepression, even at the protein level. The triple effect on increasing PU.1 levels in myeloblasts stimulates the myeloid transcriptional program while inhibiting cell survival and proliferation, leading to partial leukemic differentiation.

Epigenetic control of SPI1 gene by CTCF and ISWI ATPase SMARCA5.

CCCTC-binding factor (CTCF) can both activate as well as inhibit transcription by forming chromatin loops between regulatory regions and promoters. In this regard, Ctcf binding on non-methylated DNA and its interaction with the Cohesin complex results in differential regulation of the H19/Igf2 locus. Similarly, a role for CTCF has been established in normal hematopoietic development; however its involvement in leukemia remains elusive. Here, we show that Ctcf binds to the imprinting control region of H19/Igf2 in AML blasts. We also demonstrate that Smarca5, which also associates with the Cohesin complex, facilitates Ctcf binding to its target sites on DNA. Furthermore, Smarca5 supports Ctcf functionally and is needed for enhancer-blocking effect at ICR. We next asked whether CTCF and SMARCA5 control the expression of key hematopoiesis regulators. In normally differentiating myeloid cells both CTCF and SMARCA5 together with members of the Cohesin complex are recruited to the SPI1 gene, a key hematopoiesis regulator and leukemia suppressor. Due to DNA methylation, CTCF binding to the SPI1 gene is blocked in AML blasts. Upon AZA-mediated DNA demethylation of human AML blasts, CTCF and SMARCA5 are recruited to the -14.4 Enhancer of SPI1 gene and block its expression. Our data provide new insight into complex SPI1 gene regulation now involving additional key epigenetic factors, CTCF and SMARCA5 that control PU.1 expression at the -14.4 Enhancer.