Novel AML specific splice junction evaluation identifies ubc and DMBT1 as top proteins of clinical and therapeutic relevance in pediatric Acute Myeloid Leukemia Free

Acute Myeloid leukemia (AML) is a hematologic malignancy characterized by uncontrollable proliferation of myeloid stem cells. Despite therapeutic advances, a significant proportion of patients experience relapse thus underlying the urgent need for identification of novel biomarkers, enhancement of our understanding of disease progression, and development of novel targeted therapeutic agents for improvement patient outcomes. Alternative splicing is often altered in AML due to mutations in splicing factors and spliceosome components. While each protein-coding gene is predicted to have an average of 7.4 isoforms, most remain unidentified, suggesting many isoforms have yet to be characterized. Discovering novel splice isoforms in AML that are associated with patient outcomes may give insight into how splicing influences disease pathology as well as provide insight into potential novel AML specific isoforms that can be targeted. This study defines the AML-specific splicing landscape and evaluates its association survival outcomes in the 841 patients treated on the AAML1031 trial.

Publicly available RNA seq data from AAML1031 cohort and healthy individuals from GTEx was evaluated to identify splice junctions using the STAR Alignment tool. Junctions were normalized to counts per million and those found in GTEx were removed. Further, uniquely mapped junctions were included by presence in 50 or more samples and multi-mapped junctions were filtered to junctions with a count of 1 or greater in 10 or more samples. Following these filtration steps, 2147 novel AML specific junctions moved to downstream analysis. Cox proportional hazard models were used to test for association of splice junctions with the clinical outcome endpoints overall survival (OS) and event free survival (EFS). The Benjamin Hochberg procedure was used to adjust for false discovery rate. Top significant hits were evaluated for pathway enrichment.

Overall, in 532 patients with specimens from diagnostic bone marrow aspirates and availability of clinical data, 40 gene-annotated splice junctions were significantly associated with OS and 28 were associated with EFS (FDR adjusted P<0.05). Presence of 3 splice junctions associated with better survival which mapped to loci UBC, U2AF and PDK2P. UBC encodes Polyubiquitin C, a component of the ubiquitin-proteasome system that is involved in several signaling pathways including NOTCH2, TLR3 and CSFI (in myeloid cells). It is also often dysregulated in AML. The UBC junction covers a large portion of exon 2 and may represent a biologically relevant aberrant transcript. U2AF1 is commonly mutated as gain-of-function in AML with higher expression of this mutant associated with poorer survival outcomes. This junction may disrupt the canonical functioning of U2AF1. Splice junctions associated with both poor EFS and OS included DMBT1, multiple members of GOLGA8 family of genes, NPIPB8, PDCD6IP, RGPD5, TBC1D3D, and TBC1D3K, ZNG1F and 5 pseudogenes ANAPC13, NEPPSP1, SMG1P5, ULK4P1, and WASH6P. Of significant interest is DMBT1 (deleted in malignant brain tumors) that has been associated with progression of many cancers and has been shown to be involved in immune defense. Three DMBT1 splice gap junctions significant for survival have a common breakpoint close to Serine 1222 at the end of exon 29 resulting in 978bp (intronic region), 5037bp (exons 30-32) and 9096bp (exons 30-35) deletions with subsequent protein coding isoforms being 2542 amino acids (reference) and variant isoforms with 2413 and 1785 amino acids. Given DMBT1 is a transmembrane protein and if the isoforms localizes to the plasma membrane this raises significant relevance to develop therapeutic agents targeting the AML specific isoforms of DMBT1. Within leukemias, copy number variation in DMBT1 has been shown in patients with acute lymphoblastic leukemia.

This study identified novel splice junctions in the pediatric AAML1031 that were predictive of survival outcomes. Identification of survival-associated junctions in genes connected to cancer and cell cycle networks highlights their potential biological relevance in disease pathology and progression, clinical relevance as a predictive biomarkers, and therapeutic relevance for development of novel targeted therapeutic agents.