Abstract
The PIM family of serine/threonine kinases (PIM1, PIM2, PIM3) are important regulators of signal transduction that phosphorylate proteins essential for cell proliferation, survival, and apoptosis. The PIM kinases are constitutively active and broadly expressed in multiple tissues and up-regulated in various malignancies. We report the discovery of a novel fusion transcript encoding the kinase domain of PIM3 fused to SCO2, a cytochrome c oxidase assembly protein. In transcript sequencing (RNA Seq) of 68 pediatric AML cases, PIM3-SCO2 fusion transcript was computationally identified and experimentally verified in index cases and studied in an independent cohort of pediatric patients with AML.
RNA-Seq performed on the Illumina HiSeq in 68 diagnostic specimens from children with AML treated on COG clinical trials. Sequence reads were mapped to human genome using Novoalign. Four computational methods including Defuse, TophatFusion, FusionMap, and Snowshoes-FTD, were utilized to identify fusion transcripts and after filtering to eliminate false positives, fusions were selected based on observation in 2 or more fusion methods and presence in chimerDB. PIM3-SCO2 was identified as an in-frame fusion transcript in 3 cases with Inv(16) and subsequently verified by RT-PCR and Sanger sequencing. Frequency validation was performed by semi-quantitative expression analysis of PIM3-SCO2 expression levels in 235 AML diagnostic specimens as well as 6 normal bone marrow (NBM) controls. PIM3-SCO2 fusion protein was assessed by Western blot on whole cell lysates from cases with the fusion transcript.
After verification of the fusion, available whole genome sequencing data in matching cases was interrogated and failed to demonstrate genomic counterpart to this fusion transcript, suggesting that this fusion may be the result of transcriptional read-through; also called transcription-induced chimera (TIC). Such fusion transcripts are generated when genes in proximity on a genome strand are spliced together to generate a chimeric product. Frequency validation studies in 235 diagnostic specimens from COG AAML0531 demonstrated that PIM3-SCO2 fusion transcript was highly prevalent in AML and expressed in 187 of the 235 cases of AML (80%) with variable prevalence across different cytogenetic cohorts, with prevalence of 87% in CBF, 56% in MLL, 79% in normal karyotype, and 70% in those with “other” karyotypes (p<0.001) Further evaluation of the expression level of the fusion product demonstrated significant variability among AML patients. Given the high prevalence of fusion transcript in AML specimens, we evaluated the expression of PIM3-SCO2 transcript in normal marrow as well as in non-hematopoietic tissues. PIM3-SCO2 fusion was detected at low levels in whole normal marrow, but was absent in T cells as well non-hematopoietic tissues including cerebellum, cortex, thymus, skeletal muscle, and tongue. Protein lysates from various tissues and in patients with PIM3-SCO2 fusions was interrogated for the presence of PIM3 protein variant using an antibody directed at the amino terminal end of PIM3 protein. Normal PIM3 protein of 36 kDa was detected in HEK-293 kidney derived cell line and in Jurkat cells; however, in patient specimens with the fusion transcript, presence of a 50 kDa protein, which is the expected protein product of the fusion transcript, was confirmed. Although the appropriate PIM3 protein product was observed in non hematopoietic tissues, only the fusion product was observed in hematopoietic cells, including normal marrow, suggesting that the fusion product may be the only translated product of PIM3 in normal hematopoiesis. Gene expression profiling of 226 Dx and 35 relapse samples was performed. Compared to normal marrow, PIM3 exhibited significantly higher expression at diagnosis (p=0.04) and at relapse (p=0.022). In addition, PIM3 related signaling genes were also overrepresented on pathway analysis in Dx and Relapse samples vs. normal bone marrow.
Our data show that a novel PIM3-SCO2 fusion transcript, which is likely a transcription-induced chimera of the two gene transcripts, may be involved in normal hematopoietic development whose expression is highly dysregulated in AML. Expression level of this chimeric product is highly variable in childhood AML, is associated with cytogenetic and molecular subsets, and may identify a potential target for therapeutic intervention.
Disclosures:
No relevant conflicts of interest to declare.
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