Acute myeloid leukemia (AML) is a malignant disease characterized by uncontrolled proliferation, differentiation arrest and accumulation of immature myeloid progenitors. Despite recent developments and the approval of new therapeutic agents in the last few years, long term survival of AML, particularly in elderly patients remains an unmet medical need.The use of all-trans retinoic acid (ATRA) in Acute Promyelocytic Leukemia has proven that differentiation therapy may significantly change the survival of AML patients, however the success in APL has not been translated to other groups of AML. Therefore, the identification of new therapeutic agents that may induce the differentiation of AML blasts represents an attractive new target. Furthermore, it is well known that epigenetic alterations have an important role in the development and maintenance of cancer and AML in particular. Thus, our aim was to develop new small molecules targeting epigenetic modifying enzymes like DNA methyltransferases (DNMT), histone methyltransferases or histone deacetylase (HDAC) with the aim of inducing differentiation in AML.
We performed a screening of over 50 small molecules synthesized by our group. The design was performed in-house using a knowledge and structure based strategy and the read out of the screening was based on changes in expression of CD11b (a well described marker of myeloid differentiation) after in vitro treatment of AML cells lines. Interestingly, we found several compounds with high capacity to promote the differentiation of leukemic cells in AML cells lines at low non-cytotoxic doses, selecting CM-444 and CM-1758 as our lead compounds (Figure 1a).A complete biochemical characterization showed that both compounds are specific pan-HDACs inhibitors (HDACi). CM-444 and CM-1758 induced in vitro cell differentiation in all subtypes of AML, independently of the AML genetic subgroups or the presence of mutations, which was significantly more pronounced that differentiation induced by reference compounds such as Panobinostat, Vorinostat, Entinostat, Tubastatin or Quisinostat, previously described HDACi. CM-444 and CM-1758 also induced in vivo differentiation in xenogeneic models of AML. AML differentiation was associated with induction of CD11b, downregulation of c-MYC, overexpression of transcription factors that govern the myeloid differentiation and morphologic changes. In addition, these compounds promoted in vitro differentiation of patient-derived AML blasts. The complete transcriptome analysis by RNA-Seq carried out in AML cell lines after CM-444, CM-1758, Panobinostat or Vorinostat treatment showed changes in genes implicated in differentiation, but without explaining the differences among the different HDACi. Analysis of the complete acetylome and proteome before and after treatment with CM-444 and CM-1758 in comparison with other HDACi showed differential acetylation of non-histone proteins included in the GO categories of Zn binding proteins and nucleic acid binding proteins (Figure 1b). Most of these proteins are epigenetic enzymes and have been related to AML and myeloid differentiation, such as MLL2, EP300 or BRD4.
In summary, we have developed and characterized novel epigenetic small molecules with a high in vitro and in vivo capacity of differentiating AML cells. These compounds might be an effective differentiation-based therapy to be tested in AML. Besides, the mechanism of differentiation of these compounds is due, at least in part, to the acetylation of non-histone epigenetic proteins, which are key in the myeloid differentiation.
Disclosures
Paiva: Celgene, Janssen, Sanofi and Takeda: Consultancy; Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche and Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria.
Small-molecule targeting of MUSASHI RNA-binding activity in acute myeloid leukemia
