From predicted neoantigen to T cell therapy for acute myeloid leukemia

The spliceosome is a cluster of proteins that process precursor RNAs into mature messenger RNAs before they are made into proteins. Mutations in the genes encoding these splicing factors frequently occur in myelodysplastic syndromes (MDS), for which blood precursor stem cells fail to generate new blood cells, and in secondary acute myeloid leukemia (sAML), a cancer to which MDS can progress. Professor Dr. Marie Bleakley and Research Associate Dr. Melinda Biernacki in the Translational Science and Therapeutics Division at Fred Hutchinson Cancer Center reasoned that these splicing factor mutations may provide an opportunity for targeted T cell mediated therapy. The Bleakley lab’s recent publication in the Journal for ImmunoTherapy of Cancer details their identification and characterization of neoantigens, protein fragments that activate the immune system, from cancer cells with U2AF1 splicing factor mutations.

Since mutations in splicing factor genes SRSF2 and U2AF1 correlate with poor disease outcomes for MDS or sAML, the researchers selected these genes to lead their investigations of identifying new neoantigens and of testing these as targets for T cell-mediated MDS and AML cancer cell killing. While this treatment approach would be limited to cells with a specific mutation, this approach provides a road map for tailored treatments to attack patient-specific alterations, the holy grail of precision medicine.

The researchers first performed computer simulations to predict if wildtype or mutant forms of SRSF2 or U2AF1 would be processed into neoantigens that could bind to the cellular machinery that present these peptides as beacons for cancer killing T cell recruitment. Excitingly, a neoantigen that included the single amino acid change from glutamine (Q) to arginine (R) at site 157 in the U2AF1^Q157R mutant protein performed well in their simulations while the wildtype protein fragment did not. Therefore, these simulations predict that presentation of one U2AF1^Q157R fragment that includes the R amino acid mutation at site 157 will activate T cell-mediated killing of the cancer cell, but the equivalent peptide generated from the wildtype U2AF1 protein will not.

Moving from simulations to cell-based assays, the researchers next used cell culture systems to test neoantigen processing, presentation, and T cell activation. In these assays, T cells from healthy donors recognized and killed blood cancer cells that expressed U2AF1^Q157R in the context of the correct antigen presenting machinery confirming neoantigen processing and presentation, and T cell activation. AML cells expressing wildtype U2AF1 were not recognized by the mutation specific T cells. “To the best of our knowledge, this is the first discovery of neoantigens arising from spliceosome mutations in myeloid malignancies,” shared Dr. Biernacki.

Carrying this discovery of U2AF1^Q157R neoantigens from observation to therapy, the researchers generated T cells expressing a U2AF1^Q157R specific T cell receptor. For mouse experiments, transfer of these engineered T cells to the mouse should target only the cancer cells presenting the U2AF1^Q157R neoantigens. The researchers evaluated their engineered T cells using cells in culture and in vivo mouse experiments and were able to demonstrate specific U2AF1^Q157R mutant AML cancer cell killing. “The neoantigens we identified are potential therapeutic targets since they should be present only on aberrant neoplastic cells, providing for exquisite specificity of T cell therapies directed against them,” stated Dr. Biernacki.

These findings provide a glimpse beyond the reef into the unknowns that limit our understanding of the aggressive myeloid leukemia, but one thought in particular is on Dr. Biernacki’s mind. “I think one key question is how best to translate these findings into new therapies for patients,” stated Dr. Biernacki. “Acute myeloid leukemia and myelodysplastic neoplasms are heterogeneous diseases, so there's not likely to be a one-size-fits-all target. The U2AF1 neoantigens we identified in this paper are important tools in the toolbox for developing T cell therapies for myeloid malignancies, but a big part of our future research will be continuing to grow our toolbox of antigens.” This work to expand the toolbox of new targets for immune based therapies to treat myeloid malignancies was supported by the Fred Hutch/University of Washington/Seattle Children's Cancer Consortium.

The spotlighted research was funded by the MPN Research Foundation, National Institutes of Health, Amy Strelzer Manasevit Research Program, Gerdin Family Endowed Chair for Leukemia Research, and Fred Hutchinson Cancer Center.

Fred Hutch/University of Washington/Seattle Children's Cancer Consortium members Vivian Oehler, Derek Stirewalt, David Wu, Anthony Rongvaux, and Marie Bleakley contributed to this work.

Biernacki MA, Lok J, Black RG, Foster KA, Cummings C, Woodward KB, Monahan T, Oehler VG, Stirewalt DL, Wu D, Rongvaux A, Deeg HJ, Bleakley M. 2023. Discovery of U2AF1 neoantigens in myeloid neoplasms. J Immunother Cancer. 11(12):e007490.