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Supplementary Table 1 from Antitumor Effects of a Combined 5-Aza-2′Deoxycytidine and Valproic Acid Treatment on Rhabdomyosarcoma and Medulloblastoma in Ptch Mutant Mice

Patched (Ptch) heterozygous mice develop medulloblastoma (MB) and rhabdomyosarcoma (RMS) resembling the corresponding human tumors. We have previously shown that epigenetic silencing of the intact Ptch allele contributes to tumor formation in this model. Here, we investigated whether targeting of epigenetic silencing mechanisms could be useful in the treatment of Ptch-associated cancers. A reduction of endogenous DNA methyltransferase1 (Dnmt1) activity significantly reduced tumor incidence in heterozygous Ptch knockout mice. A combined treatment with the Dnmt inhibitor 5-aza-2′deoxycytidine (5-aza-dC) and the histone deacetlyase (HDAC) inhibitor valproic acid (VPA) efficiently prevented MB and RMS formation, whereas monotherapies with either drug were less effective. Wild-type Ptch expression was efficiently reactivated in tumors by 5-aza-dC/VPA combination therapy. This was associated with reduced methylation of the Ptch promoter and induction of histone hyperacetylation suggesting inhibition of HDACs in vivo. However, the treatment was not effective in clinically overt, advanced stage tumors. This is a first in vivo demonstration that targeting of Dnmt and HDAC activities is highly effective in preventing formation of Ptch-associated tumors. The results suggest a novel clinical strategy for consolidation therapy of corresponding tumors in humans after completion of conventional treatment. Our data also suggest that epigenetic therapy may be less effective in treating advanced stages of tumors, at least in this tumor model. [Cancer Res 2009;69(3):887–95]

Patched (Ptch) heterozygous mice develop medulloblastoma (MB) and rhabdomyosarcoma (RMS) resembling the corresponding human tumors. We have previously shown that epigenetic silencing of the intact Ptch allele contributes to tumor formation in this model. Here, we investigated whether targeting of epigenetic silencing mechanisms could be useful in the treatment of Ptch-associated cancers. A reduction of endogenous DNA methyltransferase1 (Dnmt1) activity significantly reduced tumor incidence in heterozygous Ptch knockout mice. A combined treatment with the Dnmt inhibitor 5-aza-2′deoxycytidine (5-aza-dC) and the histone deacetlyase (HDAC) inhibitor valproic acid (VPA) efficiently prevented MB and RMS formation, whereas monotherapies with either drug were less effective. Wild-type Ptch expression was efficiently reactivated in tumors by 5-aza-dC/VPA combination therapy. This was associated with reduced methylation of the Ptch promoter and induction of histone hyperacetylation suggesting inhibition of HDACs in vivo. However, the treatment was not effective in clinically overt, advanced stage tumors. This is a first in vivo demonstration that targeting of Dnmt and HDAC activities is highly effective in preventing formation of Ptch-associated tumors. The results suggest a novel clinical strategy for consolidation therapy of corresponding tumors in humans after completion of conventional treatment. Our data also suggest that epigenetic therapy may be less effective in treating advanced stages of tumors, at least in this tumor model. [Cancer Res 2009;69(3):887–95]

Pharmacological induction of hemoglobin F expression may be a promising approach for the treatment of beta-thalassemia and sickle cell disease. Valproic acid, a drug frequently used for the treatment of seizure disorders, has been shown to enhance fetal hemoglobin synthesis in erythroid cells. However, this effect is only modest and requires relative high concentrations. Therefore, the drug appears not to be applicable for the treatment of beta-globin chain disorders. Here, we describe the identification of novel valproic acid derivatives with potent hemoglobin F inducing activities at concentrations that presumably can be obtained in vivo.

Pharmacologic stimulation of fetal hemoglobin (HbF) expression may be a promising approach for the treatment of β-thalassemia. In this study, we have investigated the HbF-inducing activity and molecular mechanisms of specific histone deacetylase (HDAC) inhibitors in human K562 erythroleukemia cells. Apicidin was the most potent agent compared with other HDAC inhibitors (trichostatin A, MS-275, HC-toxin, suberoylanilide hydroxamic acid [SAHA]) and previously tested compounds (butyrate, phenylbutyrate, isobutyramide, hydroxyurea, 5-aza-cytidine), leading to a 10-fold stimulation of HbF expression at nanomolar to micromolar concentrations. Hyperacetylation of histones correlated with the ability of HDAC inhibitors to stimulate HbF synthesis. Furthermore, analysis of different mitogen-activated protein (MAP) kinase signaling pathways revealed that p38 signaling was activated following apicidin treatment of cells and that inhibition of this pathway abolished the HbF-inducing effect of apicidin. Additionally, activation of the Aγ-globin promoter by apicidin could be inhibited by p38 inhibitor SB203580. In summary, the novel HDAC inhibitor apicidin was found to be a potent inducer of HbF synthesis in K562 cells. The present data outline the role of histone hyperacetylation and p38 MAP kinase signaling as molecular targets for pharmacologic stimulation of HbF production in erythroid cells.

Valproate has been found to stimulate fetal hemoglobin (HbF) synthesis in patients with sickle cell disease. In accordance with these clinical observations, we found a moderate induction of HbF synthesis in K562 erythroid cells in vitro. Investigation of the role of the mitogen-activated protein kinase (MAPK) pathways by Western blot analysis and use of specific kinase inhibitors suggests that inhibition of ERK pathway and activation of the p38 pathway may contribute to the HbF-inducing activity of valproate.