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1416 Non-Canonical Exon Usage in Pediatric Leukemia: Implications for Chemoresistance

Program: Oral and Poster Abstracts
Session: 614. Acute Lymphoblastic Leukemias: Therapies, Excluding Transplantation and Cellular Immunotherapies: Poster I
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Non-Biological therapies, Chemotherapy, Therapies
Saturday, December 10, 2022, 5:30 PM-7:30 PM

Manuel Torres-Diz, PhD1, Clara Reglero, PhD2*, Catherine D Falkenstein, BS3*, Zhiwei Ang, PhD1*, Priyanka Sehgal, PhD1*, Caleb Radens, PhD4*, Yoseph Barash, PhD4*, Sarah K Tasian, MD3, Adolfo Ferrando, M.D., PhD2,5,6,7* and Andrei Thomas-Tikhonenko, PhD8,9,10

1Cancer Pathobiology, The Children's Hospital of Philadelphia, Philadelphia, PA
2Institute for Cancer Genetics, Columbia University, New York, NY
3Division of Oncology and Center for Childhood Cancer Research, Children’s Hospital of Philadelphia, Philadelphia, PA
4Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA
5Department of Pediatrics, Columbia University Medical Center, New York, NY
6Department of Systems Biology, Columbia University, New York, NY
7Department of Pathology, Columbia University Medical Center, New York, NY
8Children's Hospital of Philadelphia and Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA
9Division of Experimental Pathology, Cancer Pathobiology, The Children's Hospital of Philadelphia, Philadelphia, PA
10Professor of Pathology & Laboratory Medicine and Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia

B-lymphoblastic leukemia (B-ALL) is a heterogeneous, chromosome translocation-driven disease where the prevalence of somatic mutations and copy number variations is relatively low. Previous B-ALL whole exome sequencing efforts by other groups have focused upon mutations acquired under therapeutic pressure, but they have not identified universal resistance gene(s). Instead, relapse-specific mutations occurred in multiple genetic loci often involved in resistance to either glucocorticoids or purine analogs (e.g., 6-mercaptopurine, or 6-MP). The most prevalent target, NT5C2, encodes the enzyme 5'-nucleotidase/cytosolic II involved in 6-MP catabolism, but these gain-of-function mutations were found in a minority of relapsed/refractory (r/r) B-ALL samples (~25%). This lack of concordance between the genotype and the phenotype suggested a possibility that instead of mutations, NT5C2 is predominantly affected by post-transcriptional events, such as aberrant mRNA splicing (AS).

To address this possibility, we have analyzed several richly annotated RNA-Seq datasets, including NCI TARGET, which presently includes several hundred baseline childhood B-ALL samples as well as 48 paired diagnostic and relapse samples. In baseline B-ALL samples, we indeed discovered an abnormally spliced NT5C2 mRNA isoform containing the cryptic in-frame exon 4a (NT5ex4a). Of note, NT5ex4a levels were further increased in relapses compared to diagnostic samples, consistent with its putative role in chemoresistance. Furthermore, NT5ex4a mapped to full-length protein-coding transcripts and resulted in inclusion of 8 extra amino acids near the ATP-binding effector site 2.

Using bacterially produced protein, we demonstrated that at low concentrations of ATP NT5ex4a exhibited elevated enzymatic activity compared to the canonical isoform. Consistent with this biochemical finding, in reconstituted NT5C2low B-ALL cells NT5ex4a conferred the same level of resistance to 6-MP as the variant with the R238W hotspot mutation (2-log difference in IC50). Conversely, NT5ex4a-KO CRISPR/Cas9 engineered cells reduced cell survival in the presence of 6-MP. The role of NT5ex4a in chemoresistance in vivo was further confirmed in B-ALL cells expressing this alternative isoform. Therefore, inclusion of NT5C2 exon 4a phenocopies relapse-specific mutations and could serve as both a valuable predictive biomarker in B-ALL and potentially chronic myelogenous (CML) and acute myeloid leukemia (AML). Additionally, at least in vitro, expression of this non-canonical isoform conferred collateral sensitivity to the purine biosynthesis inhibitor Mizoribine, suggesting the existence of a therapeutic window to treat leukemias with dysregulated splicing.

Disclosures: Radens: GSK: Ended employment in the past 24 months. Ferrando: Regeneron Therapeutics: Current Employment.

*signifies non-member of ASH