Identification of a CARM1 Inhibitor with Potent In Vitro and In Vivo Activity in Preclinical Models of Multiple Myeloma

Abstract

CARM1 is an arginine methyltransferase with diverse histone and non-histone substrates implicated in the regulation of cellular processes including transcriptional co-activation and RNA processing. CARM1 overexpression has been reported in multiple cancer types and has been shown to modulate oncogenic pathways in in vitro studies. Detailed understanding of the mechanism of action of CARM1 in oncogenesis has been limited by a lack of selective tool compounds, particularly for in vivo studies. We describe the identification and characterization of, to our knowledge, the first potent and selective inhibitor of CARM1 that exhibits anti-proliferative effects both in vitro and in vivo and, to our knowledge, the first demonstration of a role for CARM1 in multiple myeloma (MM). EZM2302 (GSK3359088) is an inhibitor of CARM1 enzymatic activity in biochemical assays (IC50 = 6 nM) with broad selectivity against other histone methyltransferases. Treatment of MM cell lines with EZM2302 leads to inhibition of PABP1 and SMB methylation and cell stasis with IC50 values in the nanomolar range. Oral dosing of EZM2302 demonstrates dose-dependent in vivo CARM1 inhibition and anti-tumor activity in an MM xenograft model. EZM2302 is a validated chemical probe suitable for further understanding the biological role CARM1 plays in cancer and other diseases.

A Chemical Probe of CARM1 Alters Epigenetic Plasticity against Breast Cancer Cell Invasion

“…Competitive assays with SAM cofactor and peptide substrate showed that 2a and 5a act To the best of our knowledge, EZM2302, TP-064, SKI-73 (www.thesgc.org/chemicalprobes/SKI-73) and their derivatives are the only selective and cell-active CARM1 inhibitors. (Drew et al, 2017;Nakayama et al, 2018) While the potency, selectivity, on-target engagement and potential off-target effects associated with these compounds have been examined in vitro and in cellular contexts as chemical probes, EZM2302, TP-064, SKI-73 are distinct by their molecular scaffolds and modes of interaction with CARM1 (www.thesgc.org/chemical-probes/SKI-73). (Drew et al, 2017;Nakayama et al, 2018) SKI-73 is a cofactor analog inhibitor embedding a N6'homosinefungin moiety to engage the SAM binding site of CARM1 in a cofactor-competitive, substrate-noncompetitive manner; EZM2302 and TP-064 occupy the substrate-binding pocket of CARM1 in a SAH-uncompetitive or SAM-noncompetitive manner.…”

“…(Drew et al, 2017;Nakayama et al, 2018) While the potency, selectivity, on-target engagement and potential off-target effects associated with these compounds have been examined in vitro and in cellular contexts as chemical probes, EZM2302, TP-064, SKI-73 are distinct by their molecular scaffolds and modes of interaction with CARM1 (www.thesgc.org/chemical-probes/SKI-73). (Drew et al, 2017;Nakayama et al, 2018) SKI-73 is a cofactor analog inhibitor embedding a N6'homosinefungin moiety to engage the SAM binding site of CARM1 in a cofactor-competitive, substrate-noncompetitive manner; EZM2302 and TP-064 occupy the substrate-binding pocket of CARM1 in a SAH-uncompetitive or SAM-noncompetitive manner. (Drew et al, 2017;Nakayama et al, 2018) In particular, the prodrug property of SKI-73 allows its ready cellular uptake, followed by rapid conversion into its active forms inside cells.…”

“…(Blanc and Richard, 2017;Yang and Bedford, 2013) The requirement of CARM1 is implicated in multiple cancers with its methyltransferase activity particularly addicted by hematopoietic malignancies and metastatic breast cancer. (Drew et al, 2017;Greenblatt et al, 2018;Nakayama et al, 2018;Wang et al, 2014) Our prior efforts using in vivo mouse and in vitro cell models uncovered the role of CARM1 in promoting breast cancer metastasis. (Wang et al, 2014) Mechanistically, CARM1 methylates Arg1064 of BAF155 and thus facilitates the recruitment of the BAF155-containing SWI/SNF complex to a specific subset of gene loci essential for breast cancer metastasis.…”

“…(Wang et al, 2014) While this cancer relevance inspired the development of CARM1 inhibitors, (Kaniskan et al, 2018;Scheer et al, 2019) many small-molecule CARM1 inhibitors lack target selectivity or cellular activity (Kaniskan et al, 2018)---two essential criteria of chemical probes. (Frye, 2010) To the best of our knowledge, EZM2302, (Drew et al, 2017;Greenblatt et al, 2018) TP-064 (Nakayama et al, 2018) and SKI-73 (www.thesgc.org/chemical-probes/SKI-73) are the only selective and cell-active CARM1 chemical probes, which were developed by Epizyme, Takeda/SGC(Structural Genomic Consortium), and our team, respectively. EZM2302 and TP-064 were developed through conventional small-molecule scaffolds occupying the substrate-binding pocket of CARM1.…”

“…(Drew et al, 2017;Greenblatt et al, 2018;Nakayama et al, 2018) The potential utility of EZM2302 and TP-064 is implicated by their selective anti-proliferative effects on hematopoietic cancer cells, in particular multiple myeloma cells. (Drew et al, 2017;Greenblatt et al, 2018;Nakayama et al, 2018) However, definitive molecular mechanisms of the CARM1 addiction in these contexts remain elusive. (Greenblatt et al, 2018) Here we report the characterization and novel utility of SKI-73---a chemical probe of CARM1 with pro-drug properties.…”