Identification of Potential Therapeutic Targets Using Genome-Wide Analysis of Alternative Splicing (AS) In Patients with Acute Myeloid Leukemia (AML)

Abstract 177

Current therapy cures only a fraction of adults with AML, and it is clear that new treatments, specifically targeted to the genetic or epigenetic lesions that cause the disease, are needed to improve outcome. While monoclonal antibodies against cell surface antigens have been effective therapeutics in other hematologic malignancies, there has only been limited success in AML, partly due to the sharing of many antigens with normal stem cells. We have previously shown that alternative splicing (AS) of genes is common in AML cells when compared to normal CD34+ cells, and here we report efforts to identify alternatively spliced genes encoding surface proteins that could yield novel antibody targets. In order to identify genes that are differentially spliced between AML and normal progenitor cells (NPC), we extended our original genome-wide AS study of 27 AML patient samples to include 123 AML patient samples, 8 NPC's, and 11 AML cell lines.

For this study, samples were hybridized to the Affymetrix Human Exon 10ST array. Results were analyzed using commercially available Xray software from Biotique Systems. We identified 217 genes that were differentially spliced in > 35% of patients with AML compared to NPC. An average of 30 differentially spliced genes was observed in each individual patient. Splicing events in any given patients ranged from 10 to 50. Of the 217 commonly spliced genes, 33 genes were found to encode trans-membrane proteins.

Three genes, NOTCH2, FLT3 and CD13, were selected for further study. First, the exon array results were validated by RT-PCR, qRT-PCR, and DNA cloning/sequencing from 10 patients. In each case, at least one aberrant splice form was identified that is predicted to encode one or more alternative extracellular regions of the protein. These AS events did not otherwise change the open reading frame of these genes. Two different splice variants of NOTCH2 (NOTCH2-Va and -Vb) were detected in more than 80% (P=0.0001) AML patients, but at undetectable or minimal levels in NPC's.

Similarly, we detected three novel aberrant splice variants of FLT3 and CD13, which we have designated as FLT3-Va, -Vb, and -Vc and CD13-Va, -Vb, -Vc, at least one of which was detected in all AML patient samples tested, alone or with full length transcripts. The breakdown for the frequency of expression for these variants in AML patients are as follows: FLT3 -Va (68/123; 55.3%; P=0.001); -Vb (62/123; 50.4%; P=0.003); -Vc (14/123; 11.4%; P=0.15) and CD13-Va (73/123; 59.3%; P=0.001); -Vb (57/123; 46.3%; P=0.005); -Vc (34/123; 27.6%; P=0.042). None of these variants were observed in BM or PB samples from normal donors.

We evaluated the expression frequency of these splice variants in patient groups with different FAB subtypes, as well as in patients groups with cytogenetically normal or complex AML. This analysis identified frequent expression of NOTCH2-Va splice variant in patients (more than 85% patients) with M0, M1, M2, M5 and M6 AML, while FLT3-Va and -Vb variants were detected in more than 50 % of patients diagnosed with M1, M2 and M5 AML. CD13-Va and -Vb variants were identified in more than 60 % of M6 AML, while CD13-Va is expressed more than 80% of M1 AML. Interestingly, the NOTCH2-Va splice variant is expressed in nearly 90% of patients with cytogenetically complex AML, while NOTCH2-Va, FLT3-Va, -Vb and FLT3-Va were expressed more than 60% of patients with normal karyotype AML. Moderate expression frequency of other splice variants has been observed in AML patients with different subgroups analyzed.

Overall, our results from genome-wide AS analysis suggest that alternative splicing is a common event in AML, with some splice variants being detected in a significant number of different subgroups of patients. The clinical consequences and significance of this finding, as well as frequency of novel variant transcripts in large population of AML is currently the focus of further investigation. Screening and correlation analysis of additional 300 patients samples obtained at time of diagnosis as well as during relapse are in progress. Certain of the more common splice variants may generate new targets for the development of novel therapeutics.