By Gilang Cahya 
Cell division, the fundamental process that underpins life, can paradoxically become a harbinger of destruction when it spirals out of control. This unchecked proliferation is the hallmark of cancer, where mutated genes, known as oncogenes, hijack the cellular machinery, leading to the relentless formation of tumors. A significant challenge in cancer therapy has been the genetic evolution of tumors, which often become "independent" from their original oncogenic driver, making it difficult to target the root cause of their replication. However, a recent breakthrough by researchers at Rockefeller University offers a novel strategy to combat this persistent threat, demonstrating a potent new approach against a rare and aggressive form of liver cancer, with implications that could extend to more common malignancies.
Targeting the Genesis of Fibrolamellar Hepatocellular Carcinoma (FLC)
The research, published in the esteemed journal Molecular Therapy, details a pioneering method to silence the specific oncogene responsible for fibrolamellar hepatocellular carcinoma (FLC). FLC is a particularly aggressive and often fatal liver cancer that primarily affects adolescents and young adults, presenting a stark contrast to more common adult liver cancers. The Rockefeller team has successfully employed small interfering RNAs (siRNAs) to disrupt the production of illness-inducing proteins that drive tumor formation in FLC. This innovative therapy was delivered into FLC cells by targeting a specific surface receptor, effectively blocking the oncogene’s relentless activity at its source.
This marks a significant milestone, being the first instance where siRNAs have been utilized to impede the progression of FLC. According to first author Christoph Neumayer, a Ph.D. student in Rockefeller’s Laboratory of Cellular Biophysics, led by Sanford M. Simon, the findings represent a crucial proof of concept. "That’s the really exciting part," Neumayer stated. "This is proof of concept that siRNAs can be used for FLC, as well as adult liver cancers, which are much more common, and other tumor types elsewhere in the body." This broader applicability suggests that the developed therapeutic platform could have far-reaching consequences in the fight against various cancers.
Unraveling the Genetic Anomaly Behind FLC
The genesis of FLC lies in a unique genetic fusion event occurring on chromosome 19. This fusion involves two genes: DNAJB1, which typically produces heat-shock proteins crucial for maintaining cellular stability and homeostasis, and PRKACA, which encodes for protein kinase A (PKA), an enzyme vital for cellular metabolic functions. The resulting chimeric gene, known as DNAJB1::PRKACA, creates a hybrid protein that dysregulates cellular processes, leading to FLC. While the precise molecular mechanisms by which this fusion oncogene drives cancer remain an active area of investigation, its role in FLC development is unequivocally established.
The discovery of this fusion oncogene as the culprit behind FLC was a significant breakthrough in itself, made by researchers in Dr. Simon’s lab in 2014. This pivotal research was deeply personal for Dr. Simon, stemming from the diagnosis of his teenage daughter, Elana, with liver disease. Following the surgical removal of her tumor – the current standard of care for FLC – Elana actively collaborated with her father, contributing to the groundbreaking research that elucidated the disease’s genetic underpinnings, culminating in a publication in the prestigious journal Science. This familial connection has undoubtedly fueled the ongoing dedication of the Simon lab to understanding and treating FLC.
A Multifaceted Therapeutic Strategy
The current siRNA research is part of a comprehensive, three-pronged therapeutic strategy developed by Dr. Simon’s laboratory. This strategy encompasses repurposing existing drugs for FLC treatment, developing novel small molecules known as PROTACs (proteolysis-targeting chimeras) to selectively degrade the DNAJB1::PRKACA oncoprotein, and the innovative siRNA approach. The drug repurposing arm of this strategy is nearing a crucial phase, with advanced preparations underway for a clinical trial. Concurrently, a significant portion of the PROTAC research is integrated into an international collaboration that recently secured a substantial $25 million Cancer Grand Challenges grant. This grant is dedicated to accelerating the development of treatments for high-risk, oncogene-driven pediatric cancers, underscoring the urgency and potential impact of this work.
Prior to the siRNA development, the Simon lab achieved a remarkable success using short hairpin RNAs (shRNAs) in preclinical models. In studies involving mice, they demonstrated that shRNAs, engineered to disrupt messenger RNA (mRNA) transcripts, could completely halt FLC tumor growth. Furthermore, this intervention led to a significant reduction in the size of many tumors, with some even disappearing entirely. This compelling evidence that targeting the fused oncogene could directly impact tumor viability paved the way for further exploration, specifically focusing on derailing the activity of the aberrant kinase A, which was strongly implicated in driving tumor proliferation.
Overcoming Cellular Barriers: The siRNA Delivery Challenge
A significant hurdle in targeting the DNAJB1::PRKACA oncogene, particularly its kinase A component, lies in its similarity to the normal, functional kinase A found in healthy cells. As Neumayer explained, "The problem was, fused kinase A and its ‘wild type’ form are nearly identical, so ‘any drug you developed that blocks fused kinase A activity would affect all kinase A, including in normal cells.’ In other words, you would incur a host of problematic side effects." This inherent challenge necessitated a highly specific approach to disable the toxic kinase without compromising essential cellular functions in healthy tissues.
The solution lay in designing siRNAs with exquisite specificity, engineered to target only the aberrant fusion transcript. However, siRNAs, being relatively large RNA molecules, face a significant barrier in crossing the cell membrane, the protective outer layer of cells. This inherent impermeability meant that simply introducing siRNAs into the body would not effectively deliver them to their intracellular targets. The researchers therefore needed to identify a mechanism for efficient and targeted delivery into the FLC cells.
Leveraging the Liver’s Natural Delivery System
Fortuitously, the liver, the organ affected by FLC, possesses a natural pathway that could be exploited for therapeutic delivery. Researchers had previously identified a specific receptor on the surface of liver cells, known as ASGR1 (asialoglycoprotein receptor 1). This receptor acts as a critical component of the liver’s waste disposal system, recognizing and internalizing specific molecules for clearance. The key to unlocking this cellular door was a small molecule, a binding ligand known as GalNAc (N-acetylgalactosamine). When GalNAc molecules bind to ASGR1, they trigger the receptor to internalize the bound molecule, effectively ushering it into the liver cell.
The scientific community had already established the efficacy of GalNAc conjugate therapies, where siRNA molecules are chemically attached to the GalNAc ligand. This strategy has proven successful in delivering therapeutic agents into cells for treating conditions such as hereditary transthyretin-mediated amyloidosis and atherosclerotic cardiovascular disease. Inspired by these existing successes, the Rockefeller team hypothesized that this same delivery mechanism could be adapted to transport their specifically designed siRNAs into FLC cells, thereby targeting the kinase driving the tumors.
Precise Targeting and Promising Outcomes in Preclinical Models
To validate their hypothesis, the researchers meticulously crafted a custom siRNA molecule and chemically conjugated it to the ASGR1 ligand, GalNAc. This novel construct was then tested across a range of FLC tumor models in mice and in human cell cultures. The results were highly encouraging: the GalNAc-conjugated siRNA therapy was efficiently delivered into the target cells in all tested contexts. Crucially, this delivery resulted in a significant reduction in the mRNA levels of the oncogene, effectively halting the production of the disease-driving protein.
The therapeutic intervention led to a tangible impact on tumor progression, with observed stagnation and shrinkage of tumors, indicating their inability to grow. A critical aspect of this research was the assessment of potential toxicity. In the mouse models, no signs of liver toxicity were detected, suggesting that the animals tolerated the siRNA therapy well. "What we saw was tumor inhibition, when we had hoped to outright kill the tumor," Neumayer commented, acknowledging the next steps in optimizing the therapy. "Our future direction will be to try to figure out how to improve that."
Further bolstering the therapeutic potential of this approach, the researchers conducted rigorous tests to ascertain the specificity of their siRNA. They injected the siRNA into tumor cells of a different type of liver cancer, distinct from FLC. As anticipated, the siRNA exhibited no toxic impact on these unrelated cancer cells. This observation was a key validation of the targeted nature of their therapy. "We wanted to show that our siRNA is so targeted to the FLC fusion oncogene that it has no side effects on other cells — even on other cancer cells," Neumayer emphasized. This high degree of specificity is a hallmark of an ideal cancer therapeutic, minimizing off-target effects and maximizing efficacy against the intended target.
Broader Implications and the Future of Genetic Therapies
The implications of this research extend far beyond the treatment of FLC. The successful demonstration of siRNA’s efficacy and targeted delivery in this rare liver cancer opens doors for its application in a much wider spectrum of oncological diseases. Neumayer posited that siRNAs could prove effective in treating tumors throughout the body, provided that a suitable cell-surface receptor and delivery ligand can be identified for each specific cancer type. "I think siRNA medicines as a class will have a big impact over time as a new kind of genetic therapy," he concluded, highlighting the transformative potential of this class of therapeutics.
The ability to precisely target and silence specific oncogenes, without causing undue harm to healthy cells, represents a significant leap forward in cancer treatment. As our understanding of the genetic underpinnings of various cancers continues to expand, therapies like siRNA hold the promise of ushering in an era of personalized and highly effective genetic interventions. The ongoing work by the Simon lab and their collaborators underscores a relentless pursuit of innovative solutions to combat devastating diseases, offering a beacon of hope for patients and a testament to the power of dedicated scientific inquiry. The future of cancer therapy may well be written in the language of RNA, with precise genetic instructions delivered directly to the cells that need them most.
