Pharmacological Inhibitors of LSD1 Promote Differentiation of Myeloid Leukemia Cells through a Mechanism Independent of Histone Demethylation

Abstract

Lysine Specific Demethylase 1 (LSD1 or KDM1A) is one of a number of epigenetic regulators which have recently emerged as candidate therapeutic targets in acute myeloid leukaemia (AML). It is a flavin adenine dinucleotide (FAD) dependent homolog of the amine oxidase family with an ability to demethylate monomethyl or dimethyl lysine 4 (K4) of histone H3, in addition to other substrates. Pharmacological inhibitors of LSD1 such as the tranylcypromine derivatives have already commenced evaluation in early phase clinical trials. While it has been widely assumed that these compounds promote differentiation of AML cells through inhibition of the demethylase activity of LSD1, the precise mechanisms by which LSD1 inhibitors function has not yet been determined. If changes in histone methylation are a central and critical mediator of the effects of LSD1 inhibitors in promoting AML cell differentiation, it would be expected that global changes in transcription would be tightly linked temporally to changes in histone methylation following drug treatment of cells. Through RNA sequencing and ChIP sequencing experiments performed in human THP1 AML cells treated for 24 hours with a potent and specific tranylcypromine-derivative LSD1 inhibitor (ORY86, trans-N-((2-methoxypyridin-3-yl)methyl)-2-phenylcyclopropan-1-amine), we have established that wholescale up regulation of a myeloid differentiation transcription programme occurs in the absence of any significant genome-wide changes in mono- and di-methyl H3K4 and H3K9 (which are key enzymatic targets of LSD1). Thus LSD1 inhibitor-induced up regulation of myeloid differentiation gene expression is not downstream of changes in histone methylation. We further demonstrated that non-enzymatic functions of LSD1 are essential in AML cells by expressing either wild-type (WT) or catalytically inactive LSD1 (K661A) in LSD1 knockdown (KD) THP1 cells. While LSD1 KD cells exhibit myeloid differentiation and loss of clonogenic potential, both the WT and mutant versions of LSD1 were able to rescue the in vitro clonogenic potential of KD cells to an equivalent extent. Thus the histone demethylase activity of LSD1 is not required to sustain AML blasts in an undifferentiated state. Comparison of the transcriptional consequences of LSD1 inhibition with the transcriptional consequences of transcription factor knockdown in THP1 AML cells using GSEA revealed that pharmacological inhibition of LSD1 mimics depletion of GFI1. Immunoprecipitation experiments confirmed the previously described physical association of GFI1 with LSD1. Critically, the physical interactions of LSD1 with GFI1 was reversed by pharmacological inhibition of LSD1 with ORY86. Furthermore, in ChIP sequencing experiments drug treatment led to dissociation of LSD1 from promoters and enhancers. By contrast, there was no disruption of the endogenous level interaction of LSD1 with RCOR1, HDAC1 and HDAC2 (i.e. the CoREST complex) following drug treatment. To determine whether the inhibitor-induced separation of LSD1 from GFI1 is required for induction of myeloid differentiation by ORY86, we performed experiments using a GFI1-LSD1 fusion construct expressed in THP1 cells under the control of a doxycycline-regulated promoter. This construct tethers LSD1 directly to the transcription factor and circumvents any drug induced physical separation. THP1 cells expressing GFI1-LSD1 were drug resistant (as determined by immunophenotyping and clonogenic potential), in contrast to control cells expressing GFI1, LSD1 or an empty vector in the same inducible system. Thus, drug-induced physical separation of GFI1 from LSD1 is required for THP1 AML cells to undergo differentiation. Our data support a model whereby the physical association of LSD1 with transcription factors such as GFI1 is essential to maintain the differentiation block in AML. Unexpectedly, tranylcypromine-derivative inhibitors target this novel scaffolding function of LSD1, rather than its histone demethylase activity, to promote differentiation of AML cells. Disclosures Lynch: Astra Zeneca: Employment. Ciceri:Oryzon Genomics: Employment. Somervaille:Oryzon Genomics: Research Funding; Imago Biosciences: Consultancy.

ORY-1001, a Potent and Selective Covalent KDM1A Inhibitor, for the Treatment of Acute Leukemia

“…This is in line with the reported observation that reversible and irreversible KDM1A inhibitors provoke the disruption of the KDM1A:GFI1 interaction (Lynch et al, 2014) and eviction of the protein from part of its genomic target sites (McGrath et al, 2016). More specifically, Lynch et al (2014) reported that the differentiation block of THP1 AML cells does not depend on the H3K4me2 demethylase activity of KDM1A but that drug-induced physical separation of GFI1 from KDM1A causes THP1 AML cells to undergo differentiation. Fast inhibitor-mediated dissociation of KDM1A from the SNAG domain of GFI1 may explain the rapid transcriptional responses that precede the changes in the H3K4 methylation levels described in the previous section.…”

“…CTBP1 does not itself contain a SNAG domain, but has been described to interact with proteins that do (Kuppuswamy et al, 2008). This is in line with the reported observation that reversible and irreversible KDM1A inhibitors provoke the disruption of the KDM1A:GFI1 interaction (Lynch et al, 2014) and eviction of the protein from part of its genomic target sites (McGrath et al, 2016). More specifically, Lynch et al (2014) reported that the differentiation block of THP1 AML cells does not depend on the H3K4me2 demethylase activity of KDM1A but that drug-induced physical separation of GFI1 from KDM1A causes THP1 AML cells to undergo differentiation.…”

Upregulation of CD11b and CD86 through LSD1 inhibition promotes myeloid differentiation and suppresses cell proliferation in human monocytic leukemia cells

“…Multiple oral LSD1 inhibitors have already been undergoing phase I or IIa clinical trial in patients with AML ( https://clinicaltrials.gov/ ; ORY-1001 in phase I/IIa study; GSK2879552 in phase I study). In addition, we noticed that Lynch's group reported that physical association of LSD1 with transcription factors such as GFI1 is essential to maintain the differentiation block in AML and tranylcypromine-derivative inhibitors target this novel scaffolding function of LSD1, rather than its histone demethylase activity, to promote differentiation of AML cells [ 76 ]. It suggests that LSD1 might regulate myeloid differentiation and oncogenic proliferation on at least three dimensions: the first one is histone modification, such as H3K4 methylation; the second is non-histone protein methylation, such as p53-K372 methylation; and the last one is non-enzymatic scaffolding function, such as LSD1-GFI1 interaction in MLL-AF9 translocated AML, indicating the complexity of LSD1 function in the different cell contexts.…”

Clinical developments in epigenetic-directed therapies in acute myeloid leukemia

“…105 In vitro studies have found that LSD1 inhibitors do not significantly increase lysine methylation, and inhibition of LSD1-mediated transcriptional repression may contribute to its therapeutic effect. 106 Finally, emerging evidence supports the ability of LSD1 inhibitors, including tranylcypromine, to induce all-trans retinoic acid susceptibility in nonacute promyelocytic leukemia AML. 107 Phase 1 studies of multiple LSD1 inhibitors (INCB059872, IMG-7289, tranylcypromine) in AML are currently underway as monotherapy and combination therapy with all-trans retinoic acid or HMAs (Table 3).…”