BioProject

Histone lysine demethylases (KDMs) are emerging as therapeutic targets in cancer. Development of potent KDM inhibitors may provide additional options for epigenomics-oriented therapies. Using a Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) functional demethylation assay, in combination with a high-content immunofluorescence imaging phenotypic screen, Matrix-Assisted Laser Desorption/Ionization- Fourier Transform Ion Cyclotron Resonance mass spectrometry (MALDI-FTICR MS) and Amplified Luminescent Proximity Homogeneous Assay (ALPHA), we identified geldanamycin, an inhibitor of heat shock protein 90 (Hsp90), as a novel inhibitor of JmjC-domain containing demethylases such as KDM4B. We further found that geldanamycin can destabilize the PAX3-FOXO1 fusion oncoprotein, an Hsp90 client, which is a driver of clinically unfavorable alveolar rhabdomyosarcoma (aRMS). We then hypothesized that dual inhibition of PAX3-FOXO1 and epigenetic modifiers of aRMS would have synergistic antitumor activity. We repurposed the geldanamycin analog 17-DMAG to target aRMS and found that 17-DMAG significantly delays tumor growth , extends survival in xenograft mouse models, and inhibits expression of PAX3-FOXO1 targets and multiple oncogenic pathways including MYC, E2F and NOTCH. In addition, the combination of 17-DMAG with conventional chemotherapy or the bromodomain inhibitor JQ1 significantly enhances therapeutic efficacy. In summary, we have identified geldanamycin and 17-DMAG as dual KDM/Hsp90 inhibitors and 17-DMAG is efficacious against PAX3-FOXO1-driven rhabdomyosarcoma. Overall design: Rh30 cells were subcutaneously implanted in CB17 SCID mice. When tumor volume reached about 200 mm3, mice were treated with vehicle, 17-DMAG(25mg/kg), 17-AAG (50mg/kg), twice daily, via IP injection, on every Monday and Thursady. Total RNA was extracted from xenograft tissues by RNeasy Mini Kit (cat. # 74104) from QIAGEN. Paired-end sequencing was performed using the High-Seq platform with 100bp read length. Reads were aligned to the human GRCh37-lite using SJCRH’s Strongarm pipeline. Counts per gene were obtained using htseq-count version 0.6.1 with Gencode vM5 level 1and 2 gene annotations. Counts were normalized with VOOM and analyzed with LIMMA within the R statistical environment. Significance was defined as having a false discovery rate (FDR) <0.05.