Celebrating the 10th Anniversary
A decade of discoveries in Cancer Discovery. For the community. By the Community.
Their impactful studies. In their words.
Our original study of the epitope loss taught us a valuable lesson that genomic sequencing often does not tell the whole story, and that post-transcriptional regulation such as alternative splicing can be a potent driver of resistance to immunotherapy.
Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy
Q: What unanswered questions in the field was this study addressing?
A: (Elena Sotillo) We engaged in this study as the world’s first pediatric patients with relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL) were receiving lifesaving CD19-directed chimeric antigen receptor (CAR)-armed autologous T-cells. Sadly, in some of them responses proved to be short-lived and their leukemias were rapidly reemerging. About half of these post–CART-19 relapses were due to the lack of persistence or activity of the CAR T cells. However, in the other half, CAR T cells were perfectly functional, but the leukemias kept returning as CD19-negative diseases. We thought that understanding the underlying molecular mechanism would be essential to the development of novel therapeutic approaches to predict, prevent, reverse, or circumvent antigen loss.
A: (Andrei Thomas-Tikhonenko) Prior work from our laboratory had indicated that CD19 is essential for the survival and proliferation of malignant B cells in lymphomas originating in lymph nodes and secondary lymphoid tissues. Whether CD19 is an essential gene in leukemias derived from bone marrow–resident immature B-cell precursors remained an open question. Spoiler alert: it isn’t!
Q: What surprised or excited you most about the findings?
A: (Andrei Thomas-Tikhonenko) Going in, we did not really know what to expect, and the experiments were done literally in real time. My gut feeling (completely wrong, in retrospect) was that the mechanism of epitope loss would be predictable and "boring," say gene deletion or promoter hypermethylation. Then a rotating graduate student performed some simple RT-PCR experiments on the mRNAs extracted from post–CART-19 "CD19-negative" samples and discovered that CD19 transcripts were still there. On top of that, whole-exome sequencing revealed that not every relapsed leukemia had inactivating mutations in the CD19 gene and in those that had, such mutations were often subclonal. Yet, according to clinical flow cytometry, every single cell lacked CD19 on the cell surface. How was that possible? We decided to take a step back and reanalyze our RNA samples and figure out what CD19 transcripts actually looked like.
A: (Elena Sotillo) The first surprising thing we discovered was that post-treatment CD19 transcripts often missed exons 5 and 6, which correspond to the transmembrane domain of the protein. Without that domain, CD19 won’t be able to anchor itself in the plasma membrane and serve as the target for immunotherapy. The second splice variant was even more interesting because it lacked exon 2, which encodes the IgG-like loop necessary for the recognition by CAR T cells. Moreover, exon 2 skipping affects the overall protein structure, making it nearly impossible to reach the cell surface. This led us to propose a model where genetic mutations and RNA splicing work hand-in-hand to ensure that no CD19 epitopes are visible to CART-19 cells.
Q: What has been the paper’s greatest impact in the years that followed?
A: (Elena Sotillo) Once it became clear that mutant and the newly discovered misspliced CD19 isoforms fail to reach the plasma membrane, the search for alternative targets to treat surface CD19-negative leukemias began in earnest in several labs across the country. CD22 has quickly emerged as the front-runner, but other promising immunotherapeutics are now in preclinical and clinical development.
A: (Andrei Thomas-Tikhonenko) Our original study of the epitope loss taught us a valuable lesson that genomic sequencing often does not tell the whole story, and that post-transcriptional regulation such as alternative splicing can be a potent driver of resistance to immunotherapy. On the other hand, there is a growing realization that it can be a rich source of tumor-specific neoantigens for both conventional T-cell receptors and CARs. We are now actively engaged in this research through the Pediatric Immunotherapy Discovery and Development Network (PI-DDN) funded by the Beau Biden Cancer Moonshot Initiative and hope to move this work into the preclinical space before long.