DISSECTING THE CONTRIBUTION OF THE HISTONE DEMETHYLASE LSD1 IN RETINOIC ACID-INDUCED DIFFERENTIATION OF APL CELLS
Creators: name: CECCACCI, ELENA
Publisher: name: Universit� degli Studi di Milano
Year: 2017

description: Acute promyelocytic leukemia (APL) is a cytogenetically distinct subtype of acute myeloid leukemia, characterized by the chromosomal translocation t(15;17) that involves the retinoic acid receptor (RAR) gene and leads to the production of the fusion protein PML-RAR?. In the past it has been successfully treated with all- trans retinoic acid at high doses to differentiate the leukemic blast. The fusion protein indeed retains the capability to binds DNA with an even stronger affinity and recruits repressive co-factors, making the cells insensitive to physiological concentrations of retinoic acid. In the last few years, also the lysine-specific demethylase (LSD1) protein has emerged as important target for the epigenetic therapy of cancer. We found that both pharmacological inhibition and knock down of LSD1 are able to sensitize NB4 cells - a cells line derived from an APL patient - to lower (physiological) doses of retinoic acid (RA) causing growth arrest and differentiation without degradation of the fusion protein. In order to elucidate the role of LSD1 in this mechanism, we characterized the LSD1 genomic distribution in acute myeloid leukemia by ChIP-seq experiment and performed RNA-seq and ChIP-seq for H3K4me1/me2/me3 and H3K27ac in all the four treatments (DMSO as control, RA low, RA high, LSD1 inhibition and cotreatment of LSD1 inhibitor and RA low). Results of RNA-seq analyses show significant changes in gene expression only after co-treatment (RAlow + LSD1i) and RA high, in line with experimental evidence of phenotype. Moreover, in addition to an high overlap between genes expressed in both co-treatment and RAhigh, we observe a significative number of cotreatment-specific expressed genes that suggest a putative synergistic and stronger effect of the cotreatment compare to the retinoic acid high alone. In parallel, the data on histone modifications obtained through ChIP-seq experiments show a significant increase of the di-metylation of H3K4 after treatment with inhibitor. This mainly occurs in regions marked with peaks of LSD1 and associated with genes involved in cell differentiation (genes over- expressed in co-treatment, but not in other single treatments). In the same regions we observe the presence of H3K27 acetylation after treatment with retinoic acid but not after LSD1 inhibition. Only after treatment with both drugs the regions acquire the two histone marks, and we can observe an effective phenotype of differentiation, correlating with the observed change in gene expression. An hypothesis might be that there are regulatory regions linked by LSD1, which undergo a kind of ?pre-mark? in histone modifications after treatment with the inhibitor (gain of H3K4me2) or with retinoic acid (gain of H3K27ac), which is necessary but not sufficient to determine a change in expression, found only after co-treatment in the simultaneous presence of both epigenetic modifications. Overall the combination of the LSD1 inhibition and RA low bypasses the block of PML-RAR fusion protein activating a different pathway of genes compared to RA high with stronger effects on the differentiation of the cells. Taken together our results contribute to understand the role of LSD1 in the RA- induced differentiation of leukemic cells, suggest new therapeutic strategies for the intervention in APL and potentially other leukemias, and highlight the importance of combination therapies as new potent weapon in the cancer treatments.