Benavides-Serrato, A. | bioRxiv Science

bioRxiv2019

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.

cancer biology↗

bioRxiv2017

mTORC2/AKT/HSF1/HuR Constitute A Feed-Forward Loop Regulating Rictor Expression And Tumor Growth In Glioblastoma

Overexpression of Rictor has been demonstrated to result in increased mTORC2 nucleation and activity leading to tumor growth and increased invasive characteristics in glioblastoma multiforme (GBM). However the mechanisms regulating Rictor expression in these tumors is not clearly understood. In this report, we demonstrate that Rictor is regulated at the level of mRNA translation via HSF1-induced HuR activity. HuR is shown to directly bind the 3' UTR of the Rictor transcript and enhance translational efficiency. Moreover, we demonstrate that mTORC2/AKT signaling activates HSF1 resulting in a feed-forward cascade in which continued mTORC2 activity is able to drive Rictor expression. RNAi-mediated blockade of AKT, HSF1 or HuR is sufficient to downregulate Rictor and inhibit GBM growth and invasive characteristics in vitro and suppresses xenograft growth in mice. We further demonstrate that constitutive overexpression of HuR is able to maintain Rictor expression under conditions of AKT or HSF1 loss. In an additional level of regulation, miR-218, a known Rictor targeting miRNA is shown to be subject to mTORC2/STAT3-mediated repression. The expression of these components is also examined in patient GBM samples and correlative associations between the relative expression of these factors support the presence of these signaling relationships in GBM. These data support a role for a feed-forward loop mechanism by which mTORC2 activity stimulates Rictor translational efficiency and suppresses miR-218 resulting in enhanced mTORC2 activity in these tumors.

cancer biology↗