Abstract LB-49: Identification of C-terminal truncated splice variants of the apoptosis-stimulating protein of p53-2 (ASPP2) in acute leukemia.
Authors: Kampa-Schittenhelm Kerstin M. , Lopez Charles D. , Illing Barbara , Walter Michael , Schittenhelm Marcus M. 
Published: August 2014

ASPP2 is an independent haploinsufficient tumor suppressor (Kampa et al., PNAS2009) and studies in tumors including breast cancer and aggressive lymphoma have shown that attenuation/loss of ASPP2 is a common occurrence. In acute leukemia, we have evidence that attenuation of ASPP2 significantly associates with higher-risk disease and poor clinical outcome (Schittenhelm et al.,ASH2012).

In an attempt to screen for ASPP2 gene/transcript aberrations we identified two novel splicing variants of ASPP2 in acute myeloid and lymphoid leukemia blasts from 80 consented patients: The more prevalent isoform lacking exon 17, named ASPP2κ, was detected in &gt;30% of patients. ASPP2μ, lacking ex16/17, was identified in one patient. A quantitative (q)PCR assay on genomic DNA to compare exon 15 versus exon 17 expression levels did not reveal any significant differences arguing for a splicing event rather than mutation. Isolating CD34(+) leukemia blasts using cell sorting technology demonstrated ASPP2κ-expression in leukemia stem/progenitor cells.

Lack of exon 16 (resp. exon 16/17) results in a reading frame-shift with a premature translation stop, omitting most of the C-terminus - which harbors the p53-binding sites. We generated isoform-specific antibodies directed against the hypothetical fusion site. Sequence-specificity was confirmed by BlastN search. Co-immunoprecipitation using antibodies targeting the ASPP2 N-terminal and probing for ASPP2κ-specific antibodies revealed genuine translation into two protein isoforms (ASPP2κ-1/2, corresponding to ASPP2-1/2) with the predicted sizes of 97 and 111 kDa. Western immunoblotting and intracellular semi-quantitative immunophenotyping of freshly isolated native leukemia as well as bone marrow donor samples demonstrated ASPP2κ-1/2 to be specifically expressed in leukemia.

Additionally we developed a highly isoform—specific qRT-PCR assay running on a light-cycler platform: A restriction motif-specific ASPP2 exon 17 digest was performed to deplete “wildtype” ASPP2 and ASPP2κ-specific primer were generated. Although ASPP2κ amplicons were detectable in physiologic cells, high expression levels were exclusively found in leukemic blasts. Tantalizingly ASPP2κ as well as ASPP2μ dramatically decreased in patients achieving complete remission (CR) - whereas patients failing remission retained high transcript levels.

Preliminary data suggest, that transfection of ASPP2κ in murine pro-B Ba/F3 cells renders cells to more aggressive biology with mitotic failure and perturbed cellular proliferation. Systematic analysis of other tumor entities and functional analysis including other models are ongoing.

In summary, we identified two novel ASPP2 splice variants lacking the p53-binding site - which are predominantly detected in acute leukemia and may link to early tumorigenesis.

Citation Format: Kerstin M. Kampa-Schittenhelm, Charles D. Lopez, Barbara Illing, Michael Walter, Marcus M. Schittenhelm. Identification of C-terminal truncated splice variants of the apoptosis-stimulating protein of p53-2 (ASPP2) in acute leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-49. doi:10.1158/1538-7445.AM2013-LB-49