Assessment after 120?h of exposure to GSK690 and JNJ-26481585 showed profound suppression of cell viability over prolonged time (Supplementary Fig. upregulate mRNA levels of the proapoptotic BH3-only proteins BMF, PUMA, Tolfenamic acid 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 functions 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 Tolfenamic acid LSD1 and HDAC inhibition synergistically induces cell death in RMS cells by shifting the ratio of pro- and Tolfenamic acid 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 encouraging new therapeutic approach in RMS. RMS represents the most frequent soft-tissue sarcoma in children and comprises two major subtypes, that is, embryonal RMS (eRMS) and alveolar RMS (aRMS).1, 2, 3 Despite multimodal therapy consisting of surgery, chemotherapy and radiation, the overall survival for patients with advanced disease is still very poor.4 This highlights the urgent medical need for innovative treatment concepts. The antineoplastic activity of chemo-, immuno-, or radiotherapy largely depends on the induction of programmed cell death in tumor cells.5 Apoptosis is one of the most extensively analyzed forms of programmed cell death that is highly conserved throughout evolution and typically disturbed in cancer cells.6 Two key signaling pathways to apoptotic cell death have been delineated, namely the intrinsic (mitochondrial) and the extrinsic (death-receptor) pathway, which both eventually lead to activation of caspases.5, 7 Within the intrinsic pathway, pro- and antiapoptotic proteins of the BCL-2 family control outer mitochondrial membrane permeabilization (MOMP).7, 8 A shift towards proapoptotic BCL-2 family proteins favors MOMP, followed by the release of cytochrome C and second mitochondria-derived activator of caspases (Smac) from the mitochondrial intermembrane space into the cytosol.7, 8 Cytochrome C initiates formation of the apoptosome and activation of initiator caspase-9 which in turn activates caspase-3, eventually leading to the execution of apoptotic cell death.9 Smac contributes to the activation of caspases as it binds to and thereby antagonizes XIAP, a member of the Inhibitor of Apoptosis family of proteins.10 Post-translational modifications of histone proteins such as acetylation, methylation or phosphorylation create a histone code, which provides the basis for the transcriptional activity of numerous genes.11, 12 Removal of histone acetylation and demethylation of H3K4 reduce transcriptional activity and are conducted by repressor MF1 complexes, like the CoREST complex that contains HDAC1 or HDAC2, as well as LSD1.13, 14, 15, 16 HDACs have been implicated in contributing to oncogenesis by silencing tumor suppressor genes and apoptosis inducers.17, 18 LSD1 is known as a regulator of a wide spectrum of biological processes including pluripotency, differentiation, metabolic processes, as well Tolfenamic acid as cancer development and progression.19, 20, 21 In RMS, HDAC inhibition has been shown to reverse oncogenic features and induce cell death.22, 23, 24 In recent years, a broad range of inhibitors of epigenetic modifiers has been developed. JNJ-26481585 (Quisinostat) is a second-generation HDAC inhibitor that blocks class I and II HDACs with high potency.25 LSD1 inhibition was first described for the antidepressant agent Tranylcypromine, a MAO-A and MAO-B inhibitor that also inhibits LSD1 due to the high similarity of the catalytic sites of LSD1, MAO-A and MAO-B.26 In recent years, more specific LSD1 inhibitors have been developed, some of which have already progressed to clinical trials for the treatment of leukemia or lung cancer.27, 28 High LSD1 levels have been detected in several types of solid tumors or hematological malignancies and have been associated with poor prognosis.19 Recently, LSD1 has also been shown to be overexpressed in primary RMS samples.29, 30 However, little is yet known about whether or not LSD1 may serve as a therapeutic target in RMS. Therefore, the current study aims at investigating the potential of LSD1 inhibition in RMS cells, either alone or in combination with other epigenetic modifiers such as HDAC inhibitors. Results LSD1 and HDAC inhibitors synergize to induce cell death in RMS cells To investigate the therapeutic potential of LSD1 inhibition in RMS, we tested the effects of the reversible LSD1 inhibitor GSK690 alone and in combination with the second-generation HDAC inhibitor JNJ-26481585 in RMS cell lines, which represent eRMS (RD, TE381.T) and aRMS (RH30, RMS13) as the two major histological subtypes. Treatment with GSK690 alone had no or little effect on the induction of cell death, as measured by DNA fragmentation, a typical parameter of apoptotic cell death (Figure 1a), and by propidium iodide (PI) staining (Supplementary Fig. 1A). Importantly, GSK690 acted together with JNJ-26481585 to induce cell death in all.