SHIP1 Is Present but Strongly Downregulated in T-ALL, and after Restoration Suppresses Leukemia Growth in a T-ALL Xenotransplantation Mouse Model.

Acute lymphoblastic leukemia (ALL) is the most common cause of cancer-related death in children. Despite significantly increased chances of cure, especially for high-risk ALL patients, it still represents a poor prognosis for a substantial fraction of patients. Misregulated proteins in central switching points of the cellular signaling pathways represent potentially important therapeutic targets. Recently, the inositol phosphatase SHIP1 (SH2-containing inositol 5-phosphatase) has been considered as a tumor suppressor in leukemia. SHIP1 serves as an important negative regulator of the PI3K/AKT signaling pathway, which is frequently constitutively activated in primary T-ALL. In contrast to other reports, we show for the first time that SHIP1 has not been lost in T-ALL cells, but is strongly downregulated. Reduced expression of SHIP1 leads to an increased activation of the PI3K/AKT signaling pathway. SHIP1-mRNA expression is frequently reduced in primary T-ALL samples, which is recapitulated by the decrease in SHIP1 expression at the protein level in seven out of eight available T-ALL patient samples. In addition, we investigated the change in the activity profile of tyrosine and serine/threonine kinases after the restoration of SHIP1 expression in Jurkat T-ALL cells. The tyrosine kinase receptor subfamilies of NTRK and PDGFR, which are upregulated in T-ALL subgroups with low SHIP1 expression, are significantly disabled after SHIP1 reconstitution. Lentiviral-mediated reconstitution of SHIP1 expression in Jurkat cells points to a decreased cellular proliferation upon transplantation into NSG mice in comparison to the control cohort. Together, our findings will help to elucidate the complex network of cell signaling proteins, further support a functional role for SHIP1 as tumor suppressor in T-ALL and, much more importantly, show that full-length SHIP1 is expressed in T-ALL samples.

Detection of N-RAS and K-RAS in their active GTP-bound form in acute myeloid leukemia without activating RAS mutations.

RAS genes, predominantly N-RAS and K-RAS, have been implicated in the pathogenesis of acute myeloid leukemia (AML), due to activating RAS mutations detectable in approximately 20% of AML patients. In the present study, RAS proteins were detected in their activated, GTP-bound form, in AML patients (n = 10) not expressing mutated forms of H-RAS, K-RAS and N-RAS. Further analysis revealed the simultaneous presence of N-RAS and K-RAS proteins in the GTP-bound state in seven out of 10 AML samples. In four out of 10 samples the levels of RAS-GTP were comparable to an AML cell line (TF-1) with an activating N-RAS mutation (Q61P). The detection of RAS-GTP in AML patients without RAS mutations further supports a functional role of RAS proteins in the pathogenesis of AML and may explain the observed effects of RAS inhibitors in some AML patients in the absence of activating RAS mutations.

Leukemia-associated mutations in SHIP1 inhibit its enzymatic activity, interaction with the GM-CSF receptor and Grb2, and its ability to inactivate PI3K/AKT signaling.

The inositol 5-phosphatase SHIP1 is a negative regulator of the PI3K/AKT pathway, which is constitutively activated in 50-70% of acute myeloid leukemias (AML). Ten different missense mutations in SHIP1 have been described in 3% of AML patients suggesting a functional role of SHIP1 in AML. Here, we report the identification of two new SHIP1 mutations T162P and R225W that were detected in 2 and 1 out of 96 AML patients, respectively. The functional analysis of all 12 AML-associated SHIP1 mutations, one ALL-associated SHIP1 mutation (Q1076X) and a missense SNP (H1168Y) revealed that two mutations i.e. Y643H and P1039S abrogated the ability of SHIP1 to reduce constitutive PI3K/AKT signaling in Jurkat cells. The loss of function of SHIP1 mutant Y643H which is localized in the inositol phosphatase domain was due to a reduction of the specific activity by 84%. Because all other SHIP1 mutants had a normal enzymatic activity, we assumed that these SHIP1 mutants may be functionally impaired due to a loss of interaction with plasma membrane receptors or adapter proteins. In agreement with this model, we found that the SHIP1 mutant F28L located in the FLVR motif of the SH2 domain was incapable of binding tyrosine-phosphorylated proteins including the GM-CSF receptor and that the SHIP1 mutant Q1076X lost its ability to bind to the C-terminal SH3 domain of the adapter protein Grb2. In addition, SHIP1 mutant P1039S which does not reduce PI3K/AKT signaling anymore is located in a PXXP SH3 domain consensus binding motif suggesting that mutation of the conserved proline residue interferes with binding of SHIP1 to a so far unidentified SH3 domain containing protein. In summary, our data indicate that SHIP1 mutations detected in human leukemia patients impair the negative regulatory function of SHIP1 on PI3K/AKT signaling in leukemia cells either directly by reduced enzymatic activity or indirectly by disturbed protein interaction with tyrosine-phosphorylated membrane receptors or adapter proteins. These results further support a functional role of SHIP1 as tumor suppressor protein in the pathogenesis of AML.