KDM3A/Ets1/MCAM axis promotes growth and metastatic properties in Rhabdomyosarcoma

Abstract

Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy of childhood. RMS exists as two major disease subtypes, with oncofusion-positive RMS (FP-RMS) typically carrying a worse prognosis than oncofusion-negative RMS (FN-RMS), in part due to higher propensity for metastasis. Epigenetic mechanisms have recently emerged as critical players in the pathogenesis of pediatric cancers, as well as potential new therapeutic vulnerabilities. Herein, we show that the epigenetic regulator KDM3A, a member of the Jumonji-domain histone demethylase (JHDM) family, is overexpressed, potently promotes colony formation and transendothelial invasion, and activates the expression of genes involved in cell growth, migration and metastasis, in both FN-RMS and FP-RMS. In mechanistic studies, we demonstrate that both RMS subtypes utilize a KDM3A/Ets1/MCAM disease-promoting axis recently discovered in Ewing Sarcoma, another aggressive pediatric cancer of distinct cellular and molecular origin. We further show that KDM3A depletion in FP-RMS cells inhibits both tumor growth and metastasis in vivo, and that RMS cells are highly sensitive to colony growth inhibition by the pan-JHDM inhibitor JIB-04. Together, our studies reveal an important role for the KDM3A/Ets1/MCAM axis in pediatric sarcomas of distinct cellular and molecular ontogeny, and identify new targetable vulnerabilities in RMS.

KDM3A/Ets1 epigenetic axis contributes to PAX3/FOXO1‐driven and independent disease‐promoting gene expression in fusion‐positive Rhabdomyosarcoma

“…The transcriptome data are summarized in Figs 2A and . Consistent with the known functions of KDM3A and Ets1 as activators of gene expression [11,19], and with Ets1 downstream of KDM3A [12], approximately one third of genes positively controlled by KDM3A were also positively regulated by Ets1, while roughly one half of genes positively controlled by Ets1 were also positively regulated by KDM3A, in each cell line (Figs 2A and ). The KDM3A and Ets1 regulated transcriptomes showed substantial conservation between the two different FP‐RMS cell lines, with approximately one third of all genes positively controlled by each factor in Rh41 cells also similarly regulated in Rh30 cells, and, conversely, nearly two thirds of KDM3A positively controlled genes and one half of Ets1 positively controlled genes in Rh30 cells showing similar regulation in Rh41 cells (Figs 2A and ).…”

“…Our previous studies identified the epigenetic regulator KDM3A as a potent novel disease-promoting factor in ES and RMS, and the Ets1 transcription factor as a downstream mechanism contributing to KDM3A effects [12,14,40]. In the present study, we show that, in FP-RMS, Ets1 itself is an important promoter of disease-relevant phenotypes, namely growth, invasion, and metastasis.…”

“…KDM3A and Ets1 phenotypes and transcriptomes, defined in this and our previous studies [12], provide a new perspective for understanding and dissecting disease‐promoting gene expression in FP‐RMS. As an initial insight into such mechanisms, our H3K27Ac ChIP studies suggest that KDM3A and Ets1 may control the activation of the disease‐promoting gene MEST through both promoter and PAX3/FOXO1‐associated SE regulatory elements.…”

“…We therefore examined the phenotypic effects of Ets1 shRNA‐mediated depletion in patient‐derived FN‐RMS (RD and SMS‐CTR) and FP‐RMS (Rh30 and Rh41) cell lines. Similar to our prior studies [12], we employed the clonogenic assay and the transendothelial invasion assay to evaluate properties important for sarcoma growth and dissemination, respectively. In all four cell lines, depletion of Ets1 resulted in potent inhibition of clonogenic growth (Fig.…”

“…Scrambled (nontargeting control) shRNA (Addgene plasmid 1864), and KDM3A and Ets1 targeting shRNAs are described in Ref. [14] and have been previously validated in RMS cell lines used here [12]; shRNAs 1 and 2 for MEST correspond to TRCN0000075318 and TRCN0000075320 (Sigma Mission shRNAs, distributed via the University of Colorado Cancer Center Functional Genomics Core Facility). Following transduction, cells were selected with 1 µg·mL −1 of puromycin for 3–4 days, and depletion of gene expression was verified using protein immunoblotting, or quantitative reverse transcription–polymerase chain reaction (qRT‐PCR) (for MEST, for which reliable antibodies were not identified).…”