CHOP Researchers Elucidate Mechanism Behind Immunotherapy Resistance in B-ALL

CHOP researchers discovered aberrant splicing may be behind treatment resistance in B-ALL

Certain cancer treatments harness the power of the patient's own immune system to kill cancer cells. These immunotherapies, particularly chimeric antigen receptor (CAR) T cell therapy that targets a protein called CD19 protein on the surfaces of B cells, are considered a breakthrough in the treatment of pediatric B-lymphoblastic leukemia (B-ALL).

Despite their effectiveness and success, approximately 50 percent of patients with B-ALL experience relapse and many move on to an immunotherapy that targets a different surface protein — CD22. However, many patients treated using this approach also go on to relapse.

Researchers at Children's Hospital of Philadelphia identified a mechanism that may be the culprit behind resistance to CD22-directed immunotherapy: aberrant splicing of the messenger RNA encoding CD22. This mis-splicing leads to downregulation of the CD22 antigen protein and makes malignant cells resistant to the CD22-directed immunotherapy. The findings appeared in Blood Cancer Discovery.

Andrei Thomas-Tikhonenko, PhD

"Previous work demonstrated that aberrant splicing is involved in resistance to CD19-directed therapies, and our foundational finding here is that in B-ALL CD22 is mis-spliced just as profoundly, if not more profoundly than CD19," said senior co-author Andrei Thomas-Tikhonenko, PhD, chief of the Division of Cancer Pathobiology at CHOP, and professor of Pathology and Laboratory Medicine at the University of Pennsylvania Perelman School of Medicine. "As CD22-directed immunotherapies emerge as potential frontline treatments for patients with B-ALL, we will have the opportunity to study the molecular mechanisms of CD22 aberrant splicing and its abnormal protein isoforms, which could possibly be used as predictive biomarkers - and possibly even treatment targets - for new CD22-directed immunotherapies."

Cancer cells often evade immunotherapy through a process known as antigen escape — when targets, or antigens, on the cancer cell are no longer visible to the immune system. The antigen in this case is CD22. Despite the downregulation of the surface CD22, the production of CD22 mRNA continued, leading the researchers to suspect that aberrant splicing may be a culprit.

Messenger RNAs, which contain codes used to create proteins, are pieced together from building blocks called exons through a process termed splicing. In aberrant splicing, RNA sequences are cut and re-connected in a way that may include or skip distinct exons, resulting in an altered messenger RNA code.

"Antigen escape is becoming increasingly acknowledged as a mechanism of resistance to many immunotherapies," said first author Sisi Zheng, MD, a former CHOP pediatric hematology/oncology fellow who is now an instructor at the University of Texas Southwestern Medical Center. "CD22 is a particularly relevant gene to study because CD22-directed immunotherapy is becoming a therapy option for many B-ALL patients, not just in the relapsed setting but in the front-line as well. Thus, it behooves us to be able to differentiate the patients who are more or less likely to develop these splicing-mediated mechanisms of resistance."

The researchers, who also included co-senior author Sarah K. Tasian, MD, chief of the Hematologic Malignances Program at CHOP and associate professor of Pediatrics at the University of Pennsylvania Perelman School of Medicine, analyzed RNA sequencing data of more than 200 B-ALL samples and compared it with data from multiple healthy bone marrow donors. They identified numerous splicing variations in the B-ALL samples, including a novel isoform that involves skipping CD22 exons 5 and 6, and several variants that involved skipping exon 2.

They evaluated these variants' possible role in immunotherapy resistance. They found that skipping exons 5 and 6 results in the loss of part of the protein to which several antibodies and CARs attach, which reduced the efficacy of certain immunotherapies. When analyzing the effect of skipping exon 2, they found that this molecular event decreased expression of all CD22 protein isoforms, and hindered recognition by antibodies, including inotuzumab, the antibody-drug conjugate that targets CD22.

When they studied relapsed B-ALL samples from children treated with inotuzumab in a Children's Oncology Group trial, they noted that the CD22 isoform skipping exons 2 through 6 was prevalent in this population. In one patient, it was the only CD22 isoform, and that patient did not respond to inotuzumab.

"Inotuzumab is an important drug in our ALL immunotherapy armamentarium for children and adolescents with relapsed leukemia and is now under evaluation in newly diagnosed patients via frontline clinical trials," Dr. Tasian said. "The CD22 variants identified in our study may be used as predictive biomarkers of inotuzumab treatment response versus failure, as well as provide rationale for investigation of alternative antibody-based and cellular immunotherapeutic strategies."

Other key researchers who contributed their expertise to this project are Ammar S. Naqvi, PhD, Katharina E. Hayer, Zhiwei Ang, PhD, and Manuel Torres-Diz, PhD, from CHOP and Kristen Lynch, PhD, Marco Ruella, MD, and Yoseph Barash, PhD, from Penn.