PF206 DUAL TARGETING OF LYSOSOMES AND MITOCHONDRIA BY ANTIHISTAMINES TO ERADICATE LEUKEMIC CELLS (2019)

Background: A number of different genetic alterations are known to be involved in leukemogenesis but, to date, only MLL fusion genes have been demonstrated capable of independently inducing the transformation of hematopoietic progenitors and mature cells into AML cells in vivo . Early transformation‐related events are considered to induce general alterations in cell physiology that lay the foundations for cancer cell progression. We then hypothesized that reverting those early leukemogenic changes would potentially undermine pathways that ultimately sustain AML, and performed an in silico screening to identify drugs of interest. Aims: The main objective of the study was to reposition drugs against AML based on a transformation‐associated gene signature and characterize its mechanism of action. Methods: For the in silico screening, an early MLL‐AF9 gene signature was queried in connectivity maps, and drugs were ranked according to their potential to revert the gene signature. To study the antileukemic effects of selected drugs, cell viability and clonogenicity studies were performed in 4 human AML cell lines, cells from 9 AML patients, cells from 5 healthy‐donor buffy coats and 4 cord blood samples. In vivo studies were performed with conditioned NOD.Cg‐Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice xenotransplanted with AML or cord blood cells treated with antihistamines for 18 h and engrafted for 8 weeks. Effects on lysosomal compartment were assessed by Lysotracker staining, galectin puncta formation and cathepsin B immunofluorescence studies. Effects on mitochondria were studied by rhodamine‐123 and MitoSOX staining, and with caspaseGlo 3/7 assay. Results: Antihistamine drug family was identified in the screening as reverting the MLL‐AF9‐associated gene signature, and, indeed, 4 out of 10 tested antihistamines (ebastine, loratadine, terfenadine, rupatadine) proved effective against AML in vitro and ex vivo in the low micromolar range, while mostly sparing healthy cells. Antileukemic effects were observed irrespective of specific AML subtypes and mutations. In vivo studies further corroborated the results, with ebastine treatment practically abolishing AML engraftment while sparing healthy hematopoiesis. When the mechanism of action was interrogated, effects were shown to be histamine receptor 1‐independent, as no reversion was observed with the agonists for the receptor and several highly‐specific antihistamines had no effect. Moreover, cytotoxic effects were observed in cell lines with no expression of histamine receptor 1. Further studies concerning the mechanism of action found an expansion in lysosomes followed by lysosomal membrane permeabilization and cathepsin release to cytoplasm, described to lead to cell death. Concomitantly to lysosomal expansion, we detected dissipation of mitochondrial membrane potential, mitochondrial reactive oxygen species generation and caspase activation upon antihistamine treatment. The 4 antileukemic antihistamines shared cationic amphiphilic properties, known to induce tropism to both targeted organelles thus providing an explanation for the observed effects. Summary/Conclusion: A group of antihistamines possessing cationic amphiphilic structure selectively eradicate AML cells independently of histamine receptors by targeting both lysosomes and mitochondria, known to be altered and more fragile in AML compared to their healthy counterpart. This dual mechanism is based on physicochemical properties of drugs and emerges as a novel therapeutic approach for AML eradication.