Increased Efficacy of Histone Methyltransferase G9a Inhibitors Against MYCN-Amplified Neuroblastoma.

Jacob Bellamy,Karim Malik,Zsombor Melegh,Daniel Catchpoole,Madhu Kollareddy,Anthony Dallosso,Marianna Szemes

doi:10.3389/fonc.2020.00818

Jacob Bellamy, Karim Malik + Show 5 more

Open Access

https://doi.org/10.3389/fonc.2020.00818

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Abstract

Targeted inhibition of proteins modulating epigenetic changes is an increasingly important priority in cancer therapeutics, and many small molecule inhibitors are currently being developed. In the case of neuroblastoma (NB), a pediatric solid tumor with a paucity of intragenic mutations, epigenetic deregulation may be especially important. In this study we validate the histone methyltransferase G9a/EHMT2 as being associated with indicators of poor prognosis in NB. Immunological analysis of G9a protein shows it to be more highly expressed in NB cell-lines with MYCN amplification, which is a primary determinant of dismal outcome in NB patients. Furthermore, G9a protein in primary tumors is expressed at higher levels in poorly differentiated/undifferentiated NB, and correlates with high EZH2 expression, a known co-operative oncoprotein in NB. Our functional analyses demonstrate that siRNA-mediated G9a depletion inhibits cell growth in all NB cell lines, but, strikingly, only triggers apoptosis in NB cells with MYCN amplification, suggesting a synthetic lethal relationship between G9a and MYCN. This pattern of sensitivity is also evident when using small molecule inhibitors of G9a, UNC0638, and UNC0642. The increased efficacy of G9a inhibition in the presence of MYCN-overexpression is also demonstrated in the SHEP-21N isogenic model with tet-regulatable MYCN. Finally, using RNA sequencing, we identify several potential tumor suppressor genes that are reactivated by G9a inhibition in NB, including the CLU, FLCN, AMHR2, and AKR1C1-3. Together, our study underlines the under-appreciated role of G9a in NB, especially in MYCN-amplified tumors.

Highlights

Neuroblastoma (NB) is a biologically and clinically heterogeneous cancer arising from the developing sympathetic nervous system
Kaplan–Meier analysis of overall survival showed that high G9a/EHMT2 expression was significantly associated with poor survival (Figure 1A)
In order to assess whether this relationship at the RNA level was apparent at the protein level, we conducted immunoblotting of G9a protein expression in NB cell lines with and without MNA, and confirmed that increased G9a protein expression was apparent in MNA NB cell lines (Figures 1C,D)

Summary

Introduction

Neuroblastoma (NB) is a biologically and clinically heterogeneous cancer arising from the developing sympathetic nervous system. Change of function gene mutations are relatively scarce in NB, but include the oncogene ALK, which is frequently mutated in familial NB and in up to 10% of sporadic cases [4]. This has prompted the notion that epigenetic aberrations are likely to contribute to NB pathogenesis. Consistent with this hypothesis, evidence has accrued for the involvement of epigenetic modifiers, including histone methyltransferases (HMTs) in NB tumorigenesis. EZH2 has been independently shown to repress tumor suppressor genes in neuroblastoma, including CASZ1, RUNX3, NGFR, and CLU [10]. The widespread involvement of HMTs in tumorigenesis has led to concerted pharmaceutical interest in developing selective inhibitors for HMTs [12]

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