Researchers Reveal How B-ALL Leukemia Tumors Resist Immunotherapy

Leukemia tumors are cleverly deceptive, but researchers at Children’s Hospital of Philadelphia (CHOP) have figured out one of their tricks that allows cancer cells to be invisible to chimeric antigen receptor-armed T cells (CTL019), a new form of investigational immunotherapy.

In recent years, CTL019 therapy has gained attention as an investigational therapy to treat high-risk B cell acute lymphoblastic leukemia (B-ALL) that resists standard chemotherapy, radiation, and stem cell transplants. Researchers at CHOP and the University of Pennsylvania genetically reprogrammed T cells to potentially seek and destroy B-cells, including tumor cells, that express the antigen CD19, a protein essential to cell processes that appears on the cells’ surface.

Earlier this year, the CHOP/Penn team presented data at the annual meeting of the American Society of Pediatric Hematology/Oncology, showing that 78 percent of children who received CTL019 treatment were still surviving at one year, yet there are relapses after CTL019 therapy, according to Stephan Grupp, MD, PhD, a Pediatric Oncologist at CHOP and Professor of Pediatrics at the Perelman School of Medicine at the University of Pennsylvania.

Andrei Thomas-Tikhonenko, PhD, a Cancer Cell Biologist, and Elena Sotillo, PhD, a Senior Scientist in his lab at CHOP, have revealed one of the reasons why these treatment failures may occur: alternative splicing.

During gene expression, splicing assembles building blocks called exons to create a mature template for protein synthesis known as messenger RNA. However, sometimes exons are left out. The result is the creation of splicing variants that can cause tumor growth, or in this case, resistance to CTL019 therapy.

“Our findings were extremely surprising, and they happened very quickly,” Dr. Sotillo said. “We found that CD19 is expressed, but the cancer cell is hiding exon 2 — the region that is required for CTL019 to find it and kill it.”

The way alternative splicing makes CD19 invisible to the immune system is like a magician who slides a card up his sleeve, Dr. Thomas-Tikhonenko explained. The card is still there without the audience realizing it. In the same way, the CD19 protein is made, but it is an isoform that is lacking a piece (exon 2), and it is not fully present on the tumors’ surface. To a CTL019 reprogrammed T cell, it would seem like CD19 had vanished.

The research team described how these mechanisms work in a paper published in Cancer Discovery. With the help of Kristen Lynch, PhD, a Professor of Biochemistry and Biophysics at the University of Pennsylvania, they discovered a splicing factor called SRSF3 that binds to exon 2 and promotes its inclusion. Levels of SRSF3 were lower in the relapsed leukemia cases that the researchers studied, Dr. Sotillo said.

Understanding the nuances of this splicing machinery will be the focus of the team’s future research. Scientists in Dr. Thomas-Tikhonenko’s lab aim to identify drugs that could restore splicing and make the full length of the entire CD19 protein. In other words, perhaps they will find a way to bring the missing card back from the magician’s sleeve.

“Really good epigenetic drugs are in clinical trials or at least available for research use that we think might work by regulating splicing,” Dr. Thomas-Tikhonenko said. “That is a new concept we plan to pursue. Splicing can be manipulated, and this gives us an opportunity to come up with something that would force exon 2 to be included.”

Another approach is for researchers to design new chimeric antibodies that do not depend solely on exon 2 to work. Instead, they could target multiple building blocks of the CD19 protein, such as exons 3 or 4.

“That would prevent resistance from occurring,” Dr. Sotillo said. “The antibodies would recognize other regions on the same protein to function.”

In addition to suggesting new research and treatment avenues, greater appreciation of alternative splicing has broader implications for the development of immunotherapeutics, Dr. Thomas-Tikhonenko said. He pointed out that CD19 isoforms without exon 2 had been reported previously in research databases, but until now they have not received much attention.

“Down the road, people will do their research a little differently and consider what part of the protein is being targeted by a new therapy and whether it is prone to alternative splicing or skipping of exons,” said Dr. Thomas-Tikhonenko, who also is a professor of Pathology and Laboratory Medicine at UPenn. “They can go to the databases and figure out what gene this really is and its pre-existing isoforms because there could be a built-in resistance mechanism.”

When researchers identify these isoforms in patients’ tumors, they also could be used as a way to predict treatment success with CT019. Unfortunately, resistance based on the disappearance of antigen is known to be a problem with other types of cancer therapies, so the researchers are interested to see whether alternative splicing plays a major role in curtailing the effectiveness of those methods as well.

The entire research team is excited about their accomplishment and the new directions that it opens. Some key people who contributed their expertise to the project from CHOP included Dr. Grupp, David Barrett, MD, PhD; Kathryn Black, PhD; Asen Bagashev, PhD; and John Maris, MD. In addition to Dr. Lynch, at UPenn the investigators worked closely with Marco Ruella, MD, and Yoseph Barash, PhD.

“At the end of the day, it was a huge collaborative effort that turned out great,” Dr. Sotillo said.