More than six years after Emily Whitehead became the first child to receive chimeric antigen receptor (CAR) T-cell therapy, doctors have had remarkable success in turning the immune systems of even more children with acute lymphoblastic leukemia (ALL) into top-notch fighters against the disease. For some patients, however, these superhero T-cells still fail in their mission to find and fight their cancer targets.
At Children’s Hospital of Philadelphia Research Institute, basic and bioinformatics scientists are working hard to unravel the mechanisms by which leukemia cells resist CAR T-cell therapy and find useful workarounds. With the support of a new grant from the National Cancer Institute (NCI), Andrei Thomas-Tikhonenko, PhD, chief of the Division of Cancer Pathobiology, and his fellow Principal Investigator Yoseph Barash, PhD, associate professor of Genetics at the University of Pennsylvania, are leading a novel investigation into one such mechanism known as alternative splicing.
Building on previous innovative findings, the project seeks to lay the groundwork for the development of new immunotherapies that target pediatric cancers with increased specificity. Of note, the grant uses the so-called U01 funding mechanism, or Research Project Cooperative Agreement. In the true spirit of collaborative science, the grant complements and informs other projects funded by the NCI’s Pediatric Immunotherapy Discovery and Development Network (PI-DDN), a multi-institute group whose aim is to better understand how to harness the power of the immune system to treat childhood cancers.
“Our original desire was to understand how resistance to CAR T-cell therapy develops,” Dr. Thomas-Tikhonenko said. “Working with Stephan Grupp here at CHOP, we saw how the clinical trials yielded amazing responses to CAR T-cells targeting CD19, but unfortunately, also relapses due to CD19 loss. And as time goes by, those relapses emerge as major obstacles to therapy. So, our first aim was to understand the mechanisms by which leukemias become CD19-negative.”
Placing Alternative Splicing Under the Microscope
CAR T-cells can fail for a slew of reasons Dr. Thomas-Tikhonenko explained. Some mechanisms are more traditional, like when a patient acquires mutations in the CD19 gene, resulting in gene silencing or a mutated protein. But because not all resistant tumors have such mutations, Dr. Thomas-Tikhonenko’s team believed it was important to investigate other possible alterations.
One that increasingly caught their attention was alternative splicing, a regulated process by which different protein variants (known as isoforms) can be encoded by a single gene.
As DNA — the rough blueprint of our genes — is transcribed into messenger RNA (mRNA) to direct protein production, those mRNA molecules must be edited, or “spliced” into continuous pieces. Splicing involves the scanning of the gene’s “text,” the stitching together of sensible pieces (exons), and the disposal of everything else.
But through what is known as alternative splicing, this editing process can take many surprising turns: Parts of the gene text can be skipped, nonsensical sequences can be retained, and non-neighboring exons can be joined together. The jumbled code can then result in proteins that have abnormal functions, new functions, or no function whatsoever.
If a protein like CD19 is assembled abnormally as a result of mis-splicing, it could appear “invisible” to the immune system and to the hunter T-cells seeking it out. This is exactly what Dr. Thomas-Tikhonenko and his team reported in a 2015 study published in Cancer Discovery.
“What we found is that the piece needed for the CD19 protein to be recognized is missing from some of the assembly lines,” said Dr. Thomas-Tikhonenko, who described the phenomenon as a clever disappearing act perfected by leukemia cells.
More Alterations, More Ways to Target B-Cell ALL?
Now, with the support of the NCI grant, Dr. Thomas-Tikhonenko and a multidisciplinary team can dig deeper into alternative splicing by exploring myriad potential alterations that can occur in leukemia cells. Their mission is fueled by a recent discovery that CD19 is not the only gene affected by splicing. In a paper published in November in Nucleic Acids Research, the researchers reported leukemia cells actually had thousands of alterations in splicing, resulting in proteins that either don’t assemble at all or assemble in a different way. Postdoctoral fellow Ammar Naqvi, PhD, the lead author on that paper, and Katharina Hayer, bioinformatics scientist, pointed out that some of them acted deep within the cell nucleus and helped convert normal blood cells into leukemia, while others appeared on the cell surface to define their identity.
One such protein is CD22; like CD19, CD22 is expressed on the surface of B-ALL cancer cells and thus, is an attractive target for immunotherapy. (A clinical trial at CHOP is currently evaluating the use of CAR T-cells targeted to CD22.)
“What we saw was that CD22 was also mis-spliced,” Dr. Thomas-Tikhonenko said. “There are isoforms, or variants of the CD22 protein, that are missing certain pieces. And when you miss those pieces, you could develop resistance to immunotherapy.”
Two broad goals have now emerged for the new grant. First, the Penn team led by Dr. Barash hopes to learn more about what drives alternative splicing with the help of large amounts of data — some generated at CHOP and some taken from a variety of databases around the country. The hope is to discover what is responsible for the alterations in splicing, such as whether it’s encoded in a patient’s DNA, so as to have the power to potentially correlate changes in DNA with changes in splicing.
Down the road, clinicians might in fact query a patient’s genes to identify abnormally spliced proteins even before beginning treatment.
“I think the most impactful finding would be to be able to analyze the DNA of a patient and say: Look, this patient has a certain variant in, for example, gene X, and this variant correlates with an abnormal protein isoform, while gene Y is expressed normally,” Dr. Thomas-Tikhonenko said. “Therefore, this patient could benefit from a therapy directed at protein Y, but not protein X.”
The second goal is to further interrogate splicing as a mechanism of resistance using CD22 as just one example. That involves learning more about what is responsible for generating various CD22 isoforms and testing different immunotherapy approaches to see how they behave.
“Perhaps we can say: Well, this therapeutic doesn’t care about alternative splicing, but this one does,” Dr. Thomas-Tikhonenko said. “Then can we pick and choose immunotherapies based on what protein isoform they target.”
Two postdoctoral fellows in the Thomas-Tikhonenko lab, Asen Bagashev, PhD, and Mukta Asnani, PhD, are currently working to find answers to this question in close collaboration with Kristen Lynch, PhD, Chair of the Department of Biochemistry and Biophysics at Penn.
What excites Dr. Thomas-Tikhonenko the most is turning alternative splicing on its head.
“Saying that alternative splicing always gets in the way is not particularly helpful,” he explained. “So, can we actually use information about alternative splicing to some good ends.”
The idea is to develop new immunotherapies that would target abnormally spliced proteins. One example would be an antibody that recognizes a novel junction caused by splicing together two non-neighboring exons. Sisi Zheng, MD, a second-year Pediatric Hematology/Oncology Fellow, took on this project last summer and successfully generated the first antibody of that kind. Such an approach could avoid some of the collateral damages in current CAR T-cell treatments, mainly the ablation of all healthy, non-cancerous antibody-producing B-cells, which also have CD19 and CD22 on their surfaces.
“Even when CAR T-cell therapy works well, the patient won’t have any antibodies left,” Dr. Thomas-Tikhonenko said. “But perhaps we could develop better immunotherapies that target not just B-cell lineage-specific markers but their tumor-specific isoforms The thought is, if you target some of these isoforms that don’t exist in normal cells or exist in very low levels, you can not only develop new CAR T-cell therapies, but these therapies would presumably spare normal cells.”
While much of this research is work-in-progress, CD19 and CD22 are just two examples of mis-spliced proteins that the team can target through this research project. In other words, if their findings are not what they hoped for in one particular set of experiments, there are dozens of other interesting isoforms they would move on to investigate.
“I’m hoping that our grant will be successful in that we will be able to develop a drug or an antibody,” he said. “But I’m also excited about the collaborative nature of this science. I’m thrilled about sharing the data we generate with the broad community with the understanding that people will follow up on some of our observations.”
Expanding Our Idea of Precision Medicine
As if that isn’t exciting enough, Dr. Thomas-Tikhonenko believes that a focus on abnormally spliced proteins in leukemia cells could have the potential to expand how we currently think of precision medicine in pediatrics.
Currently, research through the NCI-COG Pediatric MATCH program seeks to “match” patients with common mutations to treatments that target the genetic change found in their tumors rather than their type of cancer or cancer site. One shining example for this approach is successful targeting of mutations in the anaplastic lymphoma kinase (ALK) gene, Dr. Thomas-Tikhonenko said. The ALK mutation is commonly involved in lung cancer and is also found in pediatric neuroblastoma. Thus, scientists could use drugs typically prescribed for lung cancer as a springboard to improve and develop effective neuroblastoma treatments. The tricky part, however, is that while many mutations exist in adult cancers, they simply don’t exist in as large a number of pediatric cancers.
“The challenge is that the rate of mutations is low, and the rate of recurrent mutations is even lower,” Dr. Thomas-Tikhonenko said. “MATCH is a great idea, but when it comes down to actually finding pediatric patients with actionable mutations, there is a paucity of good options.”
That’s where alternative splicing could come in, he continued: “We can say that there may not be as many mutations in the genome of this cancer, but there are other alterations, such as mis-spliced proteins, that can be exploited.”
Learn more about Dr. Thomas-Tikhonenko’s research into alternative splicing.