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Rhabdomyosarcoma is the most common paediatric soft-tissue sarcoma, and for tumour recurrence, the prognosis is still unfavourable. The current standard therapy consisting of surgery, radiation and combined chemotherapy does not consider the specific biology of this tumour.
Histone deacetylases (HDACs) and the Lysine-specific demethylase-1 (LSD1) are two epigenetic modifiers which are both part of repressor complexes leading to transcriptional silencing of target genes. Whereas HDACs lead to deacetylation of several lysine-residues within the histone tail, LSD1 is specific for demethylation of H3K4me2 and H3K4me1, as well as in a different context for H3K9me2. Rhabdomyosarcoma is reported to harbour high levels of LSD1, but the functional relevance is yet unclear. HDAC inhibition proved to be effective as single agent treatment, however, the proximity of HDAC1/2 and LSD1 in repressor complexes at the DNA implies a suitable rationale for a combination therapy potentially leading to cooperative effects on target gene transcription. In this study, we aimed to evaluate the potential of a combined LSD1 and HDAC inhibition for cell death induction in rhabdomyosarcoma cell lines. Whereas LSD1 inhibitors failed to induce cell death on their own, the combined inhibition of HDACs and LSD1 resulted in highly synergistic cell death induction. This effect extended to several combinations of LSD1 and HDAC inhibitors as well as to four different rhabdomyosarcoma cell lines, two of embryonal and two of alveolar histology.
With the use of the HDAC inhibitor JNJ-26481585 and the reversible LSD1 inhibitor GSK690, we demonstrated that the cell death induced by the combination matches with the details of intrinsic mitochondrial apoptosis. JNJ-26481585/GSK690-induced cell death is partially caspase-dependent and leads to caspase cleavage, followed by substrate cleavage as shown for PARP, as well as loss of the mitochondrial membrane potential.
Furthermore, JNJ-26481585 and GSK690 acted together to transcriptionally upregulate the proapoptotic proteins NOXA, BIM and BMF, which resulted in respective changes on protein level for both cell lines. However, the antiapoptotic BCL-2 family proteins BCL-2, MCL-1 and BCL-xL displayed only minor changes in protein levels upon treatment with GSK690 and JNJ-26481585, which did not rely on transcriptional activity. Therefore, the increase in proapoptotic proteins induces a shift towards proapoptotic signalling at the mitochondrial membrane. This shift is functionally relevant since knockdown of a proapoptotic protein or overexpression of one of the antiapoptotic proteins BCL-2 and MCL-1, as well as a stabilized mutant MCL-1, can significantly protect from GSK690/JNJ-26481585-induced cell death.
Knockdown of the mitochondrial membrane protein BAK, which is directly guarding the mitochondrial membrane integrity, potently protected from GSK690/JNJ-26481585- induced cell death, directly linking the shift in the BCL-2 family proteins to the observed loss of mitochondrial membrane potential and the further downstream activation of caspases. Furthermore, treatment with JNJ-26481585 and GSK690 resulted in a cell cycle arrest in G2/M phase, indicating additional effects on the tumour cells beside apoptosis induction. Taken together, the combined inhibition of LSD1 and HDACs is a promising strategy for rhabdomyosarcoma treatment.