Isoxazole compound ML327 blocks MYC expression and tumor formation in neuroblastoma

Abstract

Neuroblastomas are the most common extracranial solid tumors in children and arise from the embryonic neural crest. MYCN-amplification is a feature of ~30% of neuroblastoma tumors and portends a poor prognosis. Neural crest precursors undergo epithelial-to-mesenchymal transition (EMT) to gain migratory potential and populate the sympathoadrenal axis. Neuroblastomas are posited to arise due to a blockade of neural crest differentiation. We have recently reported effects of a novel MET inducing compound ML327 (N-(3-(2-hydroxynicotinamido) propyl)-5-phenylisoxazole-3-carboxamide) in colon cancer cells. Herein, we hypothesized that forced epithelial differentiation using ML327 would promote neuroblastoma differentiation. In this study, we demonstrate that ML327 in neuroblastoma cells induces a gene signature consistent with both epithelial and neuronal differentiation features with adaptation of an elongated phenotype. These features accompany induction of cell death and G1 cell cycle arrest with blockage of anchorage-independent growth and neurosphere formation. Furthermore, pretreatment with ML327 results in persistent defects in proliferative potential and tumor-initiating capacity, validating the pro-differentiating effects of our compound. Intriguingly, we have identified destabilization of MYC signaling as an early and consistent feature of ML327 treatment that is observed in both MYCN-amplified and MYCN-single copy neuroblastoma cell lines. Moreover, ML327 blocked MYCN mRNA levels and tumor progression in established MYCN-amplified xenografts. As such, ML327 may have potential efficacy, alone or in conjunction with existing therapeutic strategies against neuroblastoma. Future identification of the specific intracellular target of ML327 may inform future drug discovery efforts and enhance our understanding of MYC regulation.

ML327 induces apoptosis and sensitizes Ewing sarcoma cells to TNF-related apoptosis-inducing ligand

“…Similar to our prior findings in epithelial and neural-crest derived cells, protein expression of p53 diminished during ML327-induced apoptosis (Suppl Fig. 1) [11]. These findings, together with our prior observations, suggest that ML327 likely induces apoptosis in a p53-independent manner.…”

“…Intriguingly, the predominant cellular response observed within colon and lung carcinomas is blockade of cellular migration with no observed changes in cellular viability appreciated in vitro and in vivo [10]. The predominant feature of our trials in neuroblastoma is growth arrest and necrosis with minimal induction of apoptotic markers [11]. Herein, we report a marked induction of apoptotic markers, Caspase 3 and PARP cleavage, in association with MET induction in ES cells.…”

“…We originally identified ML327 as a small molecule that is capable of inducing the expression of E-cadherin, a hallmark of epithelial cell fate, in advanced carcinoma cells of the lung and colon [10]. We have systemically expanded our observations to encompass tumors of neural crest origin, neuroblastoma, and now cells of mesenchymal origin (ES) [11]. E-cadherin is consistently upregulated by ML327 in all tested cell lines (colon, lung, breast, neuroblastoma, and ES cells) with the exception of RKO (colon) and MDA-MB-231 (breast) cancer cells, whose E-cadherin promoters are silenced by DNA hypermethylation [10,18].…”

“…ML327 does not alter the cellular viability of colon and lung cancer cells, but is capable of blocking carcinoma cell invasiveness in vivo [10]. Intriguingly, we have recently reported that induction of partial mesenchymal-to-epithelial transition (MET) in neural crest-derived neuroblastomas blocks growth both in vitro and in vivo by inducing cell cycle arrest and necrosis, highlighting the therapeutic potential of this small molecule in cancers of non-epithelial origin [11]. …”