SHIP1 and PTEN Co-Inactivation and Akt Pathway Targeting in ALL.

Abstract 2386

Poster Board II-363

Despite success in the treatment of Acute lymphoblastic leukemia (ALL), relapse and death continue to occur and in survivors, secondary malignancies due to the impact of aggressive chemotherapy negatively impact the quality and duration of life. Akt plays a central role in signal transduction and is negatively regulated by PTEN and, in cells of hematopoietic lineage, SHIP1; it is positively regulated by PIK3CA. Despite multiple levels of regulation, Akt is aberrantly activated in many cancers including ALL. To understand the mechanism behind Akt activation in ALL, PIK3CA, PTEN and SHIP1 genes were assessed in leukemia cell lines and primary samples. No sample harbored PIK3CA mutation. PTEN was expressed in just one-third of the cell lines analyzed, but in two-thirds of the primary ALL of both T- and B-lineage, though all were in the phosphorylated (inactivated) form. SHIP1 was expressed in all leukemia cell lines, except for Jurkat and K562 cell lines. In general, expression of SHIP1 protein was much higher in cell lines of the B-lineage than of the T-lineage. The Jurkat T-ALL cell line, long observed to lack SHIP1 protein, harbored biallelic inactivation by null mutation and a frame-shift deletion; no SHIP1 mutations were identified in the K562 cell line. In contrast to leukemia cell lines, no full length SHIP1 was detected in primary T-ALL, and only ∼20% of the primary B-precursor ALL expressed full length SHIP despite the expression of full length transcript. Both ALL lineages expressed truncated isoforms of SHIP, which cloning and sequence analysis revealed was due to premature termination resulting from the frame-shift and other translationally-inactivating alternative splicing. The co-inactivation of SHIP and PTEN may create therapeutic opportunities. The PI3KCA inhibitor LY294002 has been shown to sensitize glioblastoma and colon carcinoma to doxorubicin. To determine if these agents acted synergistically in T-ALL, cell lines Molt16 (SHIP1+/PTEN-), Jurkat (SHIP1-/PTEN-) and normal MNCs (SHIP1+/PTEN+) were subjected to various combinations of the PIK3CA inhibitor LY294002 and doxorubicin and cell proliferation and viability assessed. Normal cells were generally insensitive to both agents except at high concentrations. LY294002 and doxorubicin inhibited viability in Molt16 with IC50s of 10 uM and 0.1 ug/ml, respectively, with no evidence of synergy. Jurkat cells exhibited a similar IC50 to LY294002, but were more sensitive to doxorubicin (IC50s of 10 uM and 0.025 ug/ml, respectively), with the two agents demonstrating some synergy when added simultaneously at low concentrations. In summary, we present evidence of Akt pathway deregulation in T-ALL due to SHIP1/PTEN inactivation and suggest that agents targeting this pathway may provide new directions into the therapeutic treatment of T-ALL.