LSD1-mediated Grob-like fragmentation as a novel drug resistance mechanism

Abstract

Small molecules are powerful tools to illuminate biological functions and have played an instrumental role in making seminal discoveries in chromatin biology. Dysregulation of epigenetic states has been directly implicated in cancers such as acute myeloid leukemia (AML), highlighting the utility in developing therapies that target epigenetic regulators and in characterizing and mitigating resultant drug resistance mechanisms. Herein we present our strategy to elucidate a novel Grob-like fragmentation mechanism involving small molecule epigenetic inhibitors of lysine-specific demethylase 1 (LSD1), a chromatin modifier that regulates histone methylation and exerts dynamic control over numerous transcriptional programs. LSD1 plays a critical role in hematopoiesis through formation of a corepressor complex with growth factor independence 1(B) (GFI1(B)), and previous work from our group indicates that LSD1 complex formation with GFI1(B) is essential for AML proliferation, while its demethylase activity is dispensable. Our characterization of an unprecedented fragmentation mechanism illuminates the demethylase-specific activity of LSD1 inhibitors that rescue binding activity to GFI1(B), reversing inhibitor-related hematotoxicity and antiproliferative effects in AML. We interrogated the requirements for Grob-like fragmentation of covalent LSD1-inhibitor adducts through kinetic and structure-activity relationship studies on T 448, a lead compound for treatment of neuropsychiatric illnesses associated with epigenetic dysregulation. We observed that N-aryl benzamide substitution was essential in facilitating covalent LSD1-inhibitor adduct cleavage and that substitution meta to the tranylcypromine warhead directed Grob like fragmentation in wild-type LSD1. We conversely identified an allosteric mutant of LSD1, called TTASdel, that disrupts distal D helical hydrogen-bonding interactions to stimulate fragmentation of a para-substituted inhibitor adduct and confer drug resistance in AML. Overall, our efforts elucidate new reactivity of LSD1 and detail the mechanism responsible for early clinical success of demethylase-specific inhibitors as therapeutics for neurological disease indications.