HONG KONG – An Australian study has identified a critical molecular feedback loop that helps initiate and drive neuroblastoma, which may result in a badly needed new treatment strategy for the disease, Sydney Children’s Hospital and University of New South Wales (UNSW) researchers reported in the Nov. 4, 2015, issue of Science Translational Medicine.

The most common solid tumor in early childhood with an incidence of around 1:7500, “Neuroblastoma is a malignant tumor of neural crest origin, which may arise from the adrenal gland and anywhere along the sympathetic [nervous system] trunk,” said Kenneth Wong, a clinical associate professor with the Department of Surgery, Li Ka Shing Faculty of Medicine, the University of Hong Kong (HKU).

The prognosis obviously depends on the stage of disease at presentation, but neuroblastoma frequently presents at a clinically advanced stage of the disease, often with primary or secondary resistance to conventional chemotherapy regimens.

“Approximately 50 percent of children will present with advanced stage 4 disease because very often the tumor grows inside without any overt symptoms and signs of the malignancy,” explained Wong.

However, patients with advanced disease tend not to respond well to conventional treatments. “Approximately 50 percent of patients have advanced disease,” said study corresponding author Glenn Marshall, a professor and director of the Kids Cancer Centre at Sydney Children’s Hospital and head of the Molecular Carcinogenesis Program at the Children’s Cancer Institute, UNSW.

Despite receiving intensive treatments including “cytotoxic chemotherapy, autologous bone marrow transplant, radiotherapy, surgery, retinoic acid,” and monoclonal antibodies targeting diasialoganglioside (anti-GD2 antibody) on tumor cells, “with all this, the cure rate is still only 50 percent,” said Marshall.

He noted that in around 25 percent of cases, a poor clinical outcome correlates strongly with amplification of the c-MYC homolog MYCN, which has been validated as an oncogene in a tyrosine hydroxylase (TH-MYCN) transgenic mouse model, which reflects many of the features of human neuroblastoma.

However, in addition to amplification of the MYCN oncogene acting as a biomarker for a poor treatment response in high-risk neuroblastoma patients, increased activation of MYC transcriptional target genes has also been reported to predict poor prognosis in patients with metastatic clinical stage 4 disease, independent of MYCN amplification.

“MYCN overexpression is a key driver of neuroblastoma,” said Marshall, noting that targeting MYCN protein directly in cancer has been difficult, due to the lack of a crystal structure and the diverse role played by MYC proteins in cancer and the important role of MYC proteins in normal cells.

ACHILLE’S HEEL

“We hypothesized that among genes regulated by MYCN, there would be an Achilles heel,” Marshall told BioWorld Today. “The relationship between our MYC [gene] signature and patient prognosis is one of the first [to be identified] in the disease.”

Some patients with stage 4 disease had very low expression of MYCN, but high levels of homolog c-MYC, suggesting that c-MYC acts in a functionally redundant manner regarding MYC-directed transcription, given that c-MYC expression is inversely correlated to MYCN.

This suggests that targeting genes downstream from the c-MYC proteins may represent a potential new therapeutic strategy for the treatment of high-risk neuroblastoma patients.

“As cancer driver genes, c-MYC and MYCN seem to be interchangeable in the disease, but MYCN is far more common,” explained Marshall. “Moreover, our findings, which can be generalized to c-MYC, may be applied to other c-MYC-driven cancers, [which account for] 50 percent of all human cancers.”

The UNSW researchers used this prognostic group of MYC target genes or gene signature to identify histone chaperone facilitates chromatin transcription (FACT), a protein complex involved in transcription and DNA replication, as a promising therapeutic target for neuroblastoma.

“We found that FACT and MYCN expression act in a positive feedback regulatory loop in neuroblastoma cells at the point of tumor initiation and later during tumor progression. This relationship results in a remarkable susceptibility of neuroblastoma cells to the FACT inhibitor, CBL0137,” said Marshall.

“Considerable evidence shows that MYCN is a major driver and initiator of neuroblastoma,” he said, noting the UNSW researchers had previously shown that MYCN-initiated embryonal pre-cancer cells required much higher levels of MYCN to go to a fully transformed malignant state.

“Here we show that another mechanism driving even higher levels of MYCN is a forward feedback loop between FACT and MYCN expression. High MYCN levels stimulate FACT transcription, then high FACT levels stimulate MYCN transcription and stabilize the MYCN protein to rapidly increase MYCN levels, fueling the fire of cancer,” Marshall explained.

“Because embryonal cancers occur early in life, there must be a mechanism of high-speed carcinogenesis which, unlike adult cancers that which are dependent on slow accumulation of gene mutations, are in some way dependent on an initiating lesion such as increased MYCN levels,” he suggested. “A forward feedback loop between FACT and MYCN is just such a mechanism which is not dependent on a new mutation.”

CBL0137

A small molecule curaxin compound, CBL0137 was originally developed at the Roswell Park Cancer Institute in Buffalo, N.Y., by Andrei Gudkov and Katerina Gurova, who are long-term collaborators with the UNSW researchers and were coauthors of the present study.

“We began working on CBL because it was discovered as a [tumor phosphoprotein] p53 activator and we had previously shown that embryonal pre-cancer cells were susceptible to transformation by MYCN overexpression, because of transient p53 repression,” said Marshall.

“This appears to be a feature of several embryonal pre-cancer cell types. Importantly, we have shown that CBL can block neuroblastoma initiation in mouse models. We then looked at its putative target FACT and were surprised to find it had all the characteristics of an oncogene and had this very unique interaction with MYCN.”

CBL0137 markedly reduced tumor initiation and progression in vivo. “All of the in vivo CBL0137 testing was done in mouse models of neuroblastoma, while the initial gene expression arrays were performed using RNA from human neuroblastoma tumor tissue,” said Marshall

In addition, because of the role played by FACT in repairing DNA damage caused by genotoxic drugs, “CBL0137 created a synthetic lethal environment when combined with conventional genotoxic chemotherapy, suggesting a promising treatment approach for this aggressive childhood malignancy.”

Marshall is optimistic regarding further development of CBL0137 as a candidate molecule for neuroblastoma. “We hope to have an international trial in the U.S. and Sydney within the next 18 months for relapsed solid tumors in children,” he said, adding that CBL0137 is currently in early phase clinical trials in the US and Russia for a variety of cancer types.

“These new research findings are very exciting and could potentially confer improved survival in patients with MYCN amplification, who are those patients with the worst prognosis,” commented HKU’s Wong. “However, clinical trials are needed to see if the mouse data can be translated into humans.”

CLINICAL TRIALS

“Our laboratory tests told us that CBL0137 was likely to be very effective against the most aggressive neuroblastomas, and indeed the most aggressive forms of other childhood cancers,” said study co-author Michelle Haber, a professor and executive director of Children’s Cancer Institute and head of its Experimental Therapeutics Program.

“But what is particularly exciting is that, in contrast to many other chemotherapeutic agents, CBL0137 does not damage DNA, and it is DNA damage that is responsible for the many unpleasant and serious side-effects that frequently affect children after they are cured of their cancer.

“The drug is currently in phase I clinical trials for adults, which means that safe dosage levels are being tested. Once the adult trials are completed, a phase I trial for children with refractory neuroblastoma and other aggressive childhood cancers will open in the U.S. and Australia,” she said.

“The early phase trial, in particular in conjunction with conventional chemotherapy, as well as our results in mice, appeared to show a synergy between chemotherapy and CBL0137, due to it blocking DNA repair,” said Marshall.

“We are currently investigating combination therapies with other chromatin modifier agents and continue to investigate the possibility that CBL0137 and/or other agents might be effective in killing embryonal pre-cancer cells and we are considering testing in animal models of embryonal cancer prevention.”