Researchers Identify Promising Drug Target in Pediatric Neuroblastoma

Investigators had culled the list of suspects down to two. But which one was the guilty party, or were both? The pair worked together so seamlessly, it was difficult to tell where one’s role began and the other’s ended. In some respects, they even looked alike.

Dana-Farber scientists have now teased apart the relationship between the two suspects — the proteins EP300 and CBP — to discover that EP300 is critical for a high-risk form of pediatric neuroblastoma. The finding, reported recently in the journal Cancer Discovery, led to the development of a compound that earmarks EP300 for destruction. When the compound was tested in neuroblastoma cells and animal models of the disease, it led to the rapid death of cancerous cells while having little effect on normal cells.

The work not only provides scientists with a new tool for studying EP300 in other types of cancer but may also serve as the basis for drugs that target the protein in patients with neuroblastoma — potentially providing a targeted therapy for the disease, which strikes about 650 children a year in the United States, mostly before age five.

“We want to identify the unique dependencies of neuroblastoma — the proteins that neuroblastoma cells rely on but are less essential to normal cells,” says Dana-Farber’s Jun Qi, PhD, of Cancer Biology, the study’s senior author. “Drugs that target those specific proteins have the potential to disable or kill neuroblastoma cells while producing fewer side effects than current therapies.”

Qi and his colleagues began their search with a map produced by the Broad Institute of MIT and Harvard that lists genes especially important for cancer cell survival.

“We saw that for most neuroblastoma cells, CBP and EP300 appeared to be critical,” Qi says.

CBP and EP300 are like cousins in a family business. They’re both enzymes that provide a spark for gene activity. They do this by activating specific sites on histones, proteins that hold DNA in place. Activation occurs when a chemical reaction causes an acetyl group — part of a molecule in acetic acid – to be added to these sites, a process known as acetylation. That sets in motion a series of events by which the DNA code is mistakenly activated in cancer cells. CBP and EP300 collaborate so closely that their roles in controlling gene activity sometimes overlap. They also share structural similarities. Within the scientific literature, they’re sometimes referred to as a single unit — EP300/CBP.

Although cells may benefit from having a pair of look-alike, act-alike proteins, the similarities of EP300 and CBP have hindered scientists from studying each one’s specific role – and determining whether one or both are involved in neuroblastoma.

Jun Qi, PhD
Jun Qi, PhD.

Honing in on a clue

For Qi and his associates, the Broad Institute’s gene dependency map provided a key clue.

“It indicated that neuroblastomas with an amplified MYCN gene were especially reliant on EP300 but not CBP,” Qi remarks.

To check if that is the case, the researchers used CRISPR-Cas9 gene-editing technology to shut down EP300 and CBP in MYCN-amplified neuroblastoma cells in the lab. The cells in which EP300 was targeted began dying. The cells in which CBP was targeted showed few changes.

A series of follow-up experiments confirmed the dependency of MYCN-amplified neuroblastoma on EP300 rather than CBP.

“We showed that if you knock out [deactivate] EP300, acetylation levels drop drastically, causing fewer genes to be switched on, Qi explains, “whereas when you knock out CBP, acetylation levels don’t change. CBP is unable to compensate for the loss of EP300. This is unique among the cancers linked to EP300 and CBP.”

The structural resemblance between the proteins has impeded efforts to design drug molecules that target one of the proteins but not the other. Qi and his colleagues dealt with that challenge by creating a compound that connects EP300 directly to an enzyme known as an E3 ubiquitin ligase. The ligase applies a protein tag that destines EP300 for disposal by the cell’s waste-processing machinery. In contrast to conventional drug molecules that disable cancer-related proteins, the compound developed by Qi’s team, known as a PROTAC, removes them entirely.

Members of Qi’s lab and the labs of Kimberly Stegmaier, MD, and A. Thomas Look, MD, were on the research team. They used the new compound, called JQAD1 (the “JQ” standing for Jun Qi), in laboratory lines of MYCN-amplified neuroblastoma cells and in animal models of the disease and found it rapidly caused cancerous cells to die while producing minimal toxicity in normal cells.

“One of the exciting things about this work is that JQAD1, as a first-generation compound, shows efficacy in killing neuroblastoma cells while sparing CBP protein in other, normal, cells,” says the new study’s co-first author Adam Durbin MD, PhD, a former member of Look’s lab and now at St. Jude’s Children’s Research Hospital. “As we work to improve and refine JQAD1, we hope this work sets the groundwork to develop compounds we can move forward to help children with advanced cancers like neuroblastoma, and perhaps other types of cancer as well, while limiting the usual toxicities of therapy.”

Other Dana-Farber contributors to the study include co-first author Tingjian Wang, MD, PhD; along with Virangika Wimalasena; Mark W. Zimmerman, PhD; Deyao Li, PhD; Neekesh V. Dharia, MD, PhD; Paul M.C. Park; Logan H. Sigua; Ken Morita, MD, PhD; Amy Saur Conway; and Amanda L. Robichaud.