Mithramycin 2′-Oximes with Improved Selectivity, Pharmacokinetics, and Ewing Sarcoma Antitumor Efficacy

Abstract

Mithramycin A (MTM) inhibits the oncogenic transcription factor EWS-FLI1 in Ewing sarcoma, but poor pharmacokinetics (PK) and toxicity limit its clinical use. To address this limitation, we report an efficient MTM 2′-oxime (MTM ox ) conjugation strategy for rapid MTM diversification. Comparative cytotoxicity assays of 41 MTM ox analogues using E-twenty-six (ETS) fusion-dependent and ETS fusion-independent cancer cell lines revealed improved ETS fusionindependent/dependent selectivity indices for select 2′-conjugated analogues as compared to MTM. Luciferase-based reporter assays demonstrated target engagement at low nM concentrations, and molecular assays revealed that analogues inhibit the transcriptional activity of EWS-FLI1. These in vitro screens identified MTM ox 32E (a Phe−Trp dipeptide-based 2′-conjugate) for in vivo testing. Relative to MTM, MTM ox 32E displayed an 11-fold increase in plasma exposure and improved efficacy in an Ewing sarcoma xenograft. Importantly, these studies are the first to point to simple C3 aliphatic side-chain modification of MTM as an effective strategy to improve PK.

Interleukin-10 suppression enhances T-cell antitumor immunity and responses to checkpoint blockade in chronic lymphocytic leukemia

“…Like MTM, MTM ox 32 E interacts with GC rich regions of DNA. MTM ox 32 E had better pharmacokinetics, tolerability, and selectivity than MTM and other analogues(26). MTM ox 32 E showed some selectivity for inhibiting Sp1 binding on the IL-10 promoter in cell culture.…”

“…Concentrations of inhibitors and stimulants are listed in figure legends. Anti-IgM (Jackson ImmunoResearch, West Grove, PA), anti-CD3, anti-CD28 (BioLegend) and MTM (Enzo Life Sciences, Farmingdale, NY) are commercially sourced, while MTM ox 32 E was synthesized in house(26). Secreted cytokines were measured with ELISA MAX Standard Set Mouse or Human kits according to the manufacturer’s protocol (BioLegend).…”

“…We suppressed CLL-derived IL-10 with anti-IL-10 or by inhibiting Sp1, a transcription factor required for CLL IL-10 production(4), with a novel analog of mithramycin (MTM ox 32 E , structure in supplementary methods). MTM ox 32 E exhibits reduced toxicity and increased potency over the parent molecule in other cancer models(25, 26). This treatment significantly decreased CLL burden in the blood, spleen and bone marrow, and when combined with anti-PD-L1, suppressing IL-10 reduced tumor burden and enhanced T-cell functionality over ICB alone.…”

Mithplatins: Mithramycin SA‐Pt(II) Complex Conjugates for the Treatment of Platinum‐Resistant Ovarian Cancers

“…[23] This new semisynthetic approach also stimulated our recent MTM-oxime based derivatization strategy. [24] A similar oxidative cleavage was previously used by Preobrazhenskaya et al for the 3-side-chain cleavage of olivomycin. [25] However, olivomycin -in contrast to mithramycindoes not have any sugar moiety vulnerable to this oxidative cleavage procedure.…”

Insight into mithramycin disruption of ETS transcription leads to improved understanding of more selective analogs

“…As a result, efforts have been focused on identifying MTM analogs with comparable or improved activity but reduced toxicity. In previous reports, the Grohar (Osgood et al, 2016), the Rohr (Mitra et al, 2018), and the Leggas/Thorson labs (Liu et al, 2020) MTM is known to bind X(G/C)(G/C)Xrich regions as an Mg 2+ coordinated dimer in the minor groove of DNA, while many transcription factors including ETS transcription factors bind DNA in the major grove. As a result, MTM is unlikely to compete for binding sites with these transcription factors but could either selectively bind to DNA in the presence of these proteins or alter DNA in such a way as to alter their activity.…”