Targeting the mTORC2 signaling complex in B cell malignancies

Hyperactive PI3 kinase-Akt (PI3K-Akt) signaling has an important role in cell growth and resistance to apoptosis in B cell malignancies. Inhibition of this pathway by blocking PI3K activity, and or inhibiting mTORC1/2 signaling complexes is an active area of research in B cell leukemia/lymphoma such as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). With a tissue-scan array, the expression of Rictor is a component of the mTORC2 complex was determined by quantitative PCR in a number of B cell malignancies. Rictor was found to be over-expressed in CLL and MCL cells as compared to normal B cells with no over-expression in Hodgkins and non-Hodgkins lymphomas. Inactivation of Rictor was performed by shRNA in two Mantle cell lines and these stable Rictor knockdown cell lines demonstrated a slower growth of cells as compared to scrambled shRNA control. In addition, there was a decrease of mTORC2 signaling and B cell receptor (BCR) cross-linking mediated Akt (Ser473) and NDRG1 (Thr 346) phosphorylation. To specifically disrupt the mTORC2 signaling complex and target Rictor overexpression, previously identified inhibitors that block Rictor and MTOR interaction in a yeast two-hybrid system were analyzed. Treatment of primary CLL specimens with these inhibitors followed by immunoprecipitation experiments confirmed the disruption of the mTORC2 complex. These inhibitors also induced apoptosis in CLL specimens and were more effective than rapamycin, an MTOR inhibitor and pp242, an mTORC1 and 2 inhibitors, at equimolar concentrations. Treatment of CLL specimens with the lead inhibitor, compound#6, resulted in inhibition of p-Akt, p-GSK 3 beta, p-PKC alpha, p-Foxo1, and p-Foxo3, with minimal effect on the phosphorylation of an mTORC1 target gene, S6 kinase. In comparison with Idelalisib (CAL-101), a clinically approved PI3Kinase p110 delta inhibitor in CLL, comp#6 is more effective in inducing apoptosis in primary CLL specimens at equimolar concentrations (mean 51.2, SD 21.7 as compared to mean 26.9, SD 17.2). The data support the effectiveness of these novel inhibitors that specifically disrupt the mTORC2 complex in primary CLL specimens.

A central role for a region in the middle

A domain called the 'Conserved region in the middle' is responsible for target recognition in the TORC2 complex in fission yeast and the mTORC2 complex in mammals.

The Critical Roles of SIN1 in Platelet Activation and Myocardial Infarction

Mammalian stress-activated protein kinase interacting protein 1 (SIN1) is an essential subunit of the mTORC2 complex, which regulates Akt activation by phosphorylation of Akt at Ser473 residue. Despite the function of Akt in platelet activation and thrombosis was well studied, the role of SIN1 in platelet activation and thrombosis remains unknown. In this study, we observed that megakaryocyte/platelet specific SIN1 deficiency caused 30% reduction of platelet counts in peripheral blood probably by blockage of megakaryocyte differentiation and enhancing platelet apoptosis, suggesting that SIN1 had an important role in thrombopoiesis. More importantly, SIN1 deficiency caused a defect in platelet aggregation in response to low level of thrombin, U46619, ADP and collagen. SIN1 deficiency also exhibited diminished ability of platelet to spread on immobilized fibrinogen and the decreased rate of clot retraction in platelet-rich plasma containing SIN1 deficient platelets. mTORC2 complex analysis revealed that the expression levels of Rictor, another mTORC2 component, were significantly diminished in SIN1 deficient platelet. And SIN1 deficiency attenuated agonist-induced phosphorylation of Akt at Ser473, Thr308 and Thr450, and Gsk3β at Ser9 in platelet. SIN1 could be phosphorylated at Thr86, which correlated with the phosphorylation of Akt at Ser473 in activated platelets. Further study demonstrated that the phosphorylation levels of SIN1 at Thr86 and Akt at Ser473 and Thr450, but not at Thr308 were enhanced in the platelets collected from ST-segment elevation myocardial infarction (STEMI) patients, indicating that SIN1 activation correlated with myocardial infarction process. A mouse model of chronic myocardial infarction (MI) was performed and the results demonstrated that platelet-specific SIN1 deficient mice had less platelet activation, reduced MI size, and improved post-MI heart function. In conclusion, SIN1 plays critical roles in platelet activation, MI and post-MI heart failure, therefore serves as a target for therapeutic intervention in the thrombosis and myocardial infarction. Disclosures No relevant conflicts of interest to declare.