Activated Src kinases downstream of BCR-ABL and Flt3 induces proteasomal degradation of SHIP1 by phosphorylation of tyrosine 1021.

Within the various subtypes of ALL, patients with a BCR-ABL-positive background as well as with a genetic change in the KMT2A gene have by far the worst survival probabilities. Interestingly, both subtypes are characterized by highly activated tyrosine kinases. SHIP1 serves as an important negative regulator of the PI3K/AKT signaling pathway, which is often constitutively activated in ALL. The protein expression of SHIP1 is decreased in most T-ALL and in some subgroups of B-ALL. In this study, we analyzed the expression of SHIP1 protein in detail in the context of groups with aberrant activated tyrosine kinases, namely BCR-ABL (Ph+) and Flt3 (KMT2A translocations). We demonstrate that constitutively activated Src kinases downstream of BCR-ABL and receptor tyrosine kinases reduce the SHIP1 expression in a SHIP1-Y1021 phosphorylated-dependent manner with subsequent ubiquitin marked proteasomal degradation. Inhibition of BCR-ABL (Imatinib), Flt3 (Quizartinib) or Src-Kinase-Family (Saracatinib) leads to significant reconstitution of SHIP1 protein expression. These results further support a functional role of SHIP1 as tumor suppressor protein and could be the basis for the establishment of a targeted therapy form.

Reduced expression and activity of patient-derived SHIP1 phosphatase domain mutants.

The characterization of dysregulated proteins in cell signaling pathways is important for the development of therapeutic approaches. The PI3K/AKT/mTOR pathway is frequently upregulated in cancer cells and the SH2-containing inositol 5-phosphatase SHIP1 can act as a negative regulator of the PI3K/AKT pathway. In this study, we investigated different patient-derived mutations within the conserved phosphatase domain of SHIP1. We could demonstrate that 2 out of 7 SHIP1-phosphatase domain mutations (G585K and R673Q) possessed reduced protein expression and reduced enzymatic activity in comparison to SHIP1 wild type (WT) protein and two additional mutations (E452K, R551Q) possessed reduced enzymatic activity at a comparable expression level compared to SHIP1 WT in the cell line H1299. The investigated mutations resulted in protein expression levels that were up to 93% lower than those of the SHIP1 WT for SHIP1 mutant R673Q and the enzymatic activity was below the detection limit of the performed phosphatase assay. Whereas the protein level of the R673Q mutant was reduced in comparison to SHIP1 WT the mRNA level was comparable indicating a post-transcriptional regulation. SHIP1 R673Q was rapidly degraded, with a calculated half-life of l.5 h. In addition, SHIP1 R673Q levels were significantly increased by the treatment with the proteasome inhibitor MG-132 in comparison to the DMSO control. Therefore, SHIP1 was confirmed as the target of enhanced proteasomal degradation. Computational analysis of the wild type and mutant protein structures revealed that the loss of the positively charged arginine residue R673 is associated with the loss of two salt bridges to the negatively charged amino acids D617 and E634 leading to an intramolecular instability of the mutated SHIP1 R673Q protein. Six out of seven SHIP1 mutants significantly affected the PI3K/AKT/mTOR pathway in the three cancer cell lines H1299, Reh and Sem. Four out of seven SHIP1 mutants affected phosphorylation of AKT and its target GSK3ß positively compared to SHIP1 WT, whereas a negative effect on the phosphorylation of S6 was found in five out of seven mutants. In general, SHIP1 mutants impacting signal transduction were either associated with decreased SHIP1 activity or SHIP1 expression or both. Overall, the presented results indicate a regulation of the protein expression and activity of SHIP1 by patient-derived mutations in its phosphatase domain.

Investigation of the function of the PI3-Kinase / AKT signaling pathway for leukemogenesis and therapy of acute childhood lymphoblastic leukemia (ALL).

Acute lymphoblastic leukemia is the most common cause of cancer-related death in children and, especially for patients in a high-risk group, still represents a poor prognosis. The PI3K/AKT/mTOR signaling pathway has been identified as a frequently constitutively activated switching point in the disease of ALL. Despite the knowledge of the therapeutic importance of the signaling pathway, the results of clinically effective treatment strategies have so far been extremely sobering. In particular, monotherapy approaches represent a major problem with regard to cell resistance. In this work, the PI3K/AKT/mTOR signaling pathway was examined as a therapeutic target for the treatment of childhood acute lymphoblastic leukemia (ALL) with a new therapeutic approach to avoid cell resistance. Therefore, we used a combined therapeutic approach with inhibitors directed against AKT (MK2206), mTOR (RAD001) and the most prominent and aberrantly activated tyrosine kinase. In case of BCR-ABL-positive B-ALL cells we used a combination with the classic inhibitor Imatinib and in case of MLL-AF4-positive B-ALL cells we used a combination with Quizartinib (directed against FLT3). We show, in particular compared to the monotherapies, a highly significant inhibition of the growth of these cells after this new specific triple combination therapy. Furthermore, we show that inhibiting AKT alone leads to a feedback mechanism and an upregulation of the phosphorylation of a number of receptor-tyrosine-kinases. After isoform-specific knockdown of the three AKT isoforms in ALL cells we identified that especially ErbB2/Her2 is most strongly phosphorylated in cells with AKT2 knockdown. AKT isoform 1 and 2 knockdown cells show, in contrast to AKT isoform 3 knockdown cells, a weak proliferation and are presumably kept alive among others by the increased phosphorylation of the receptor-tyrosine-kinase ErbB2. This work provides first indications for a new combination therapy of B-ALL cells, which is directed against AKT, mTOR and a predominantly highly activated kinase.

Analysis of the FLVR motif of SHIP1 and its importance for the protein stability of SH2 containing signaling proteins.

Binding of proteins with SH2 domains to tyrosine-phosphorylated signaling proteins is a key mechanism for transmission of biological signals within the cell. Characterization of dysregulated proteins in cell signaling pathways is important for the development of therapeutic approaches. The AKT pathway is a frequently upregulated pathway in most cancer cells and the SH2-containing inositol 5-phosphatase SHIP1 is a negative regulator of the AKT pathway. In this study we investigated different mutations of the conserved FLVR motif of the SH2 domain and putative phosphorylation sites of SHIP1 which are located in close proximity to its FLVR motif. We demonstrate that patient-derived SHIP1-FLVR motif mutations e.g. F28L, and L29F possess reduced protein expression and increased phospho-AKT-S473 levels in comparison to SHIP1 wildtype. The estimated half-life of SHIP1-F28L protein was reduced from 23.2 h to 0.89 h in TF-1 cells and from 4.7 h to 0.6 h in Jurkat cells. These data indicate that the phenylalanine residue at position 28 of SHIP1 is important for its stability. Replacement of F28 with other aromatic residues like tyrosine and tryptophan preserves protein stability while replacement with non-aromatic amino acids like leucine, isoleucine, valine or alanine severely affects the stability of SHIP1. In consequence, a SHIP1-mutant with an aromatic amino acid at position 28 i.e. F28W can rescue the inhibitory function of wild type SHIP1, whereas SHIP1-mutants with non-aromatic amino acids i.e. F28V do not inhibit cell growth anymore. A detailed structural analysis revealed that F28 forms hydrophobic surface contacts in particular with W5, I83, L97 and P100 which can be maintained by tyrosine and tryptophan residues, but not by non-aromatic residues at position 28. In line with this model of mutation-induced instability of SHIP1-F28L, treatment of cells with proteasomal inhibitor MG132 was able to rescue expression of SHIP1-F28L. In addition, mutation of putative phosphorylation sites S27 and S33 adjacent to the FLVR motif of SHIP1 have an influence on its protein stability. These results further support a functional role of SHIP1 as tumor suppressor protein and indicate a regulation of protein expression of SH2 domain containing proteins via the FLVR motif.