ONC201 Suppresses Neuroblastoma Growth by Interrupting Mitochondrial Function and Reactivating Nuclear ATRX Expression While Decreasing MYCN

Abstract

Neuroblastoma (NB) is characterized by several malignant phenotypes that are difficult to treat effectively without combination therapy. The therapeutic implication of mitochondrial ClpXP protease ClpP and ClpX has been verified in several malignancies, but is unknown in NB. Firstly, we observed a significant increase in ClpP and ClpX expression in immature and mature ganglion cells as compared to more malignant neuroblasts and less malignant Schwannian-stroma-dominant cell types in human neuroblastoma tissues. We used ONC201 targeting ClpXP to treat NB cells, and found a significant suppression of mitochondrial protease, i.e., ClpP and ClpX, expression and downregulation of mitochondrial respiratory chain subunits SDHB and NDUFS1. The latter was associated with a state of energy depletion, increased reactive oxygen species, and decreased mitochondrial membrane potential, consequently promoting apoptosis and suppressing cell growth of NB. Treatment of NB cells with ONC201 as well as the genetic attenuation of ClpP and ClpX through specific short interfering RNA (siRNA) resulted in the significant upregulation of the tumor suppressor alpha thalassemia/mental retardation X-linked (ATRX) and promotion of neurite outgrowth, implicating mitochondrial ClpXP proteases in MYCN-amplified NB cell differentiation. Furthermore, ONC201 treatment significantly decreased MYCN protein expression and suppressed tumor formation with the reactivation of ATRX expression in MYCN-amplified NB-cell-derived xenograft tumors. Taken together, ONC201 could be the potential agent to provide diversified therapeutic application in NB, particularly in NB with MYCN amplification.

ONC201 Exerts Oncogenic Effects Beyond its Mitochondria-Disturbing Role in Neuroblastoma Subsets

“…ATRX has been implicated in neuronal differentiation and functions as a tumor suppressor, protecting cells from DNA replication stress by resolving di cult-to-replicate G-quadruplex DNA structures [34]. In our previous study, both ONC201 treatment and the genetic attenuation of ClpP and ClpX expression resulted in signi cant upregulation of the tumor suppressor ATRX, promoting neurite outgrowth in MYCN-ampli ed NB cells [5]. In the present study, we showed the association of ATRX expression with maturation of human NB tissues.…”

“…Purhonen et al reported that complex III de ciency triggers upregulation of c-Myc expression, followed by excessive anabolic metabolism and increased cell proliferation, under conditions of energy and biosynthetic precursor de ciency [26]. Our previous study showed that ONC201 downregulated the mitochondrial respiratory chain subunits SDHB and NDUFS1, resulting in energy de ciency and further trigger c-Myc expression [5], similar to the results reported by Purhonen et al[26], although the precise mechanism remains unclear.…”

“…ONC201 treatment previously was able to signi cantly reduce MYCN protein expression, inhibit tumor formation, and reactivate alpha thalassemia mental retardation X-linked (ATRX) expression in MYCNampli ed NB cell-derived xenograft tumors [5]. However, there have been no studies of ATRX expression in non-MYCN-ampli ed NB xenografts and cell lines.…”

“…In our previous study [5], ONC201 suppressed the expression of ClpP and caseinolytic mitochondrial matrix peptidase proteolytic subunit X (ClpX), while downregulating the mitochondrial respiratory chain subunits succinate dehydrogenase complex iron sulfur subunit B (SDHB) and NADH:ubiquinone oxidoreductase core subunit S1 (NDUFS1); these effects resulted in energy depletion and increased reactive oxygen species (ROS) levels, along with suppression of NB cell growth, regardless of MYCN ampli cation status. However, ONC201 only inhibited tumor development in xenograft tumors derived from NB cells with ampli ed MYCN; this inhibition correlated with a substantial reduction in MYCN protein levels and the reactivation of ATRX expression.…”

BMP2 and BMP7 cooperate with H3.3K27M to promote quiescence and invasiveness in pediatric diffuse midline gliomas

“…BMP2 has been shown to be induced by hypoxia 51 or reactive oxygen species (ROS) 52 . Accordingly, exposure of ACVR1 WT H3.3K27M DIPG cells to hypoxia or ONC201, which is known to significantly increase ROS production 53,54 , are both sufficient to induce a significant increase in BMP2 expression (Figure 3A). Thus, BMP2 can be produced autonomously by tumor cells in response to specific stresses.…”