Rationale/Hypothesis: The goal of my research is to determine whether c- Abl's regulation of alternative splicing is responsible not just for the initiation and progression of cancer, but also for cellular and tissue response to chemotherapy. My preliminary data has shown that c-Abl regulates the balance between the two different splice variants of caspase-2, a key effector in the cellular apoptotic machinery that responds to chemotherapeutic signals. Artificial overexpression of c-Abl promotes a rapid upregulation of caspase-2-short, a truncated version of caspase-2. This splice variant has been demonstrated to be anti-apoptotic in cell culture systems and is present only in neural tissue, which is highly resistant to many chemotherapeutic strategies.

Background: Previously published research has shown that when the normal function of c-Abl is compromised by the chromosomal fusion of the abl gene with the bcr gene, as is the case in chronic myelogenous leukemia (CML), there is a significant change in the alternative splicing of pre-mRNA transcripts. Separate studies have provided evidence that this differential splicing has a causative role in leukemic progression. BCR-ABL+ pre-B lymphoblastic leukemia cells express a splice variant of the ikaros gene. This alternative transcript produces a dominant negative version of Ikaros (IK6) that is not present in non-leukemic pre-B cells. Application of the BCRABL inhibitor Imatinib to the BCR-ABL+ pre-B lymphoblastic leukemia cells dramatically decreases the presence of the splice variant and RNAi directed against the splice variant partially restores lineage fidelity and proper differentiation.

Specific Aims: I am currently examining two possible mechanisms by which c- Abl could control alternative splicing of caspase-2. One possibility to be considered is whether or not c-Abl regulates the production of microRNAs that regulate the expression and/or translation of certain splicing factors. Our microarray experiments indicate that overexpression of c-Abl causes an upregulation of miR-124, a microRNA known to regulate PTB and nPTB. These two splicing factors are responsible for an alternative splicing program that produces neurally-specific transcripts such as caspase-2-short. The connection between c-Abl and miR-124 is supported by the fact that both miR-124 and c-Abl initiate their expression at similar times and places during early neural development.

The other possible mechanism for c-Abl's effect on alternative splicing involves its capacity to phosphorylate the CTD tail of RNA Pol II. Our lab has shown that c-Abl alters the alternative splicing of CD44, a gene whose different transcripts may regulate certain aspects of tumorigenesis. Our work on c-Abl's role in the splicing of CD44 has generated strong evidence that phosphorylation of the CTD tail by c-Abl may alter the binding affinity of several splicing factors for the RNA Pol II complex. This, in turn, promotes the production of different CD44 splice variants. This phosphorylation of the CTD tail by c-Abl may be responsible controlling the production of the two splice variants of caspase-2.

Relevance to Cancer: In addition to the tumorigenic splicing changes that occur in BCR-ABL+ leukemias, there is evidence that splicing changes directly alter chemotherapeutic response. Many cancers demonstrate high expression levels of prosurvival splice variants and in vivo mouse models have shown that chemotherapeutic outcome can be determined by these expression levels. Anti-apoptotic splice variants of members of the Bcl-2, p53, and Caspase families have been shown to dramatically decrease the levels of apoptotic response to a variety of drugs. There is a distinct possibility c-Abl may regulate pro-survival splicing of many apoptotic genes and that aberrant levels of caspase-2 may be a key player in chemo-resistance. The capacity to regulate expression of both these cellular entities with drugs or techniques like RNAi allows for the possibility of chemoresistance to be potentially alleviated. This would result in immediate benefits for cancer therapeutics as it would increase the efficiency and safety of many drugs already in use without requiring the massive investment in time and resources required for the creation of new drugs.

PUBLICATIONS (resulting from this training)

Information pending - trainee just commenced training