Concomitant epigenetic targeting of LSD1 and HDAC synergistically induces mitochondrial apoptosis in rhabdomyosarcoma cells

Abstract

The lysine-specific demethylase 1 (LSD1) is overexpressed in several cancers including rhabdomyosarcoma (RMS). However, little is yet known about whether or not LSD1 may serve as therapeutic target in RMS. We therefore investigated the potential of LSD1 inhibitors alone or in combination with other epigenetic modifiers such as histone deacetylase (HDAC) inhibitors. Here, we identify a synergistic interaction of LSD1 inhibitors (i.e., GSK690, Ex917) and HDAC inhibitors (i.e., JNJ-26481585, SAHA) to induce cell death in RMS cells. By comparison, LSD1 inhibitors as single agents exhibit little cytotoxicity against RMS cells. Mechanistically, GSK690 acts in concert with JNJ-26481585 to upregulate mRNA levels of the proapoptotic BH3-only proteins BMF, PUMA, BIM and NOXA. This increase in mRNA levels is accompanied by a corresponding upregulation of BMF, PUMA, BIM and NOXA protein levels. Importantly, individual knockdown of either BMF, BIM or NOXA significantly reduces GSK690/JNJ-26481585-mediated cell death. Similarly, genetic silencing of BAK significantly rescues cell death upon GSK690/JNJ-26481585 cotreatment. Also, overexpression of antiapoptotic BCL-2 or MCL-1 significantly protects RMS cells from GSK690/JNJ-26481585-induced cell death. Furthermore, GSK690 acts in concert with JNJ-26481585 to increase activation of caspase-9 and -3. Consistently, addition of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) significantly reduces GSK690/JNJ-26481585-mediated cell death. In conclusion, concomitant LSD1 and HDAC inhibition synergistically induces cell death in RMS cells by shifting the ratio of pro- and antiapoptotic BCL-2 proteins in favor of apoptosis, thereby engaging the intrinsic apoptotic pathway. This indicates that combined treatment with LSD1 and HDAC inhibitors is a promising new therapeutic approach in RMS.

Targeting ferroptosis in rhabdomyosarcoma cells

“…Cell death was assessed by Hoechst/propidium iodide (PI) staining, analyzing the plasma membrane permeability using fluorescence microscopy (ImageXpress Mikro XLS), as described previously . To determine cellular GSH levels GSH/GSSG‐Glo assay (Promega, Madison, WI) was used following the instruction manual.…”

“…Cell death was assessed by Hoechst/propidium iodide (PI) staining, analyzing the plasma membrane permeability using fluorescence microscopy (ImageXpress Mikro XLS), as described previously. 18 To determine cellular GSH levels GSH/GSSG-Glo assay (Promega, Madison, WI) was used following the instruction manual. In parallel, cells were stained with 1 μg/μL Hoechst-Dye (Sigma-Aldrich) and measured by fluorescence microscopy (ImageXpress Mikro XLS) with automated analysis using MetaXpress Software (Molecular Devices).…”

Dodging the bullet: therapeutic resistance mechanisms in pediatric cancers

“…This overexpression can be driven by chromosomal translocations, through signal transduction from extracellular signaling [139] , alternative splicing [140] or epigenetic dysregulation [141] . Antiapoptotic BCL2 family expression has been seen in numerous pediatric cancers, including neuroblastomas [142][143][144] , leukemias [141,[145][146][147] , lymphomas [148,149] , brain tumors [150][151][152] , and sarcomas [153][154][155][156] .…”

Targeted Therapy–based Combination Treatment in Rhabdomyosarcoma

“…In addition to HDAC, lysine-specific demethylase 1 (LSD1) is overexpressed in rhabdomyosarcoma. Combination treatment of LSD1 and HDAC inhibitors showed synergism in ARMS and ERMS cell lines (82). HDAC inhibitors in combination with either targeted therapies or chemotherapy are in clinical trials (NCT01106872, NCT00937495, NCT01132911, NCT01294670; Table 2) and a subset of patients with advanced STS responded to monotherapy and combination treatments (NCT00878800; Table 2; refs.…”