Disruption of DEPTOR/mTORC1/mTORC2 Signaling Cascade Using a Novel Selective mTOR Kinase Inhibitor AZD8055 Results In Growth Arrest and Apoptosis In Multiple Myeloma Cells
Authors: Cirstea Diana , Hideshima Teru , Santo Loredana , Pozzi Samantha , Vallet Sonia , Patel Kishan , Gorgun Gullu , Hu Yiguo , Guichard Sylvie M , Mahindra Anuj , Anderson Kenneth C. , Raje Noopur 
Published: October 2019

Abstract
Abstract 791
Targeting PI3K/Akt/mTOR signaling is among one of the promising therapeutic strategies in multiple myeloma (MM), since it facilitates MM cell survival and development of drug resistance in the context of the bone marrow microenvironment. Specifically, regulation of PI3K activity, which mediates MM cell growth and drug resistance, by mTOR complex 1 (mTORC1) provides the rationale for use of rapamycin analogs for MM treatment. However, rapamycin alone fails to overcome bone marrow-induced proliferation of MM cells, at least in part, because of the mTORC1-dependent feedback loops which activate PI3K/Akt. More recently, extensive studies of the mTOR network have identified mTORC2 as a “rapamycin-insensitive” complex. Sharing mTOR kinase as a common catalytic subunit, mTORC1 and mTORC2 mediate two distinct pathways: mTORC1 controls cell growth by phosphorylating key regulators of protein synthesis S6 kinase 1 (P70S6K) and the eIF-4E-binding protein 1 (4E-BP1); mTORC2 modulates cell survival and drug resistance by phosphorylating target proteins including Akt and serum/glucocorticoid regulated kinase 1(SGK1)/N-myc downstream regulated 1 (NDRG1). Moreover, studies have also revealed overexpression of a novel mTOR-interacting protein DEP domain containing 6 (DEPTOR), which can modulate mTOR activity and promote PI3K/mTORC2 signaling in primary MM tumor cells and in MM cell lines while mTORC1 remains silenced. We therefore hypothesized that targeting mTOR may disrupt DEPTOR/mTOR interaction and silence mTORC1/mTORC2 signaling, thereby overcoming mTOR resistance in MM cells. To confirm this idea, we used AZD8055, an orally bioavailable selective ATP-competitive mTOR kinase inhibitor, in our MM preclinical models. AZD8055- treatment of MM.1S inhibited phosphorylation of both mTORC1 and mTORC2 substrates: P70S6K; 4E-BP1 including the rapamycin-resistant T37/46 – downstream targets of mTORC1; as well as Akt and NDRG1 – effectors of mTORC2 refractory to rapamycin. Interestingly, AZD8055-mediated mTORC1/mTORC2 downregulation was associated with DEPTOR upregulation, which is consistent with the finding that DEPTOR expression is negatively regulated by mTORC1 and mTORC2. Moreover, inhibition of mTORC1 alone by rapamycin resulted in reduction of DEPTOR, associated with Akt activation. Furthermore, we observed that DEPTOR expression was decreased in MM.1S cells cultured with IL-6, IGF-1 or bone marrow stromal cells (BMSCs), which stimulate PI3K/Akt/mTOR signaling, evidenced by enhanced P70S6K and Akt phosphorylation. Unlike rapamycin, AZD8055 reversed those effects and inhibited MM.1S proliferation, even in the presence of these cytokines or BMSCs. AZD8055-induced growth inhibition was associated with apoptosis, evidenced by caspase-9, -3 and PARP cleavage in a time-dependent fashion (80% apoptotic cells at 72 hour culture as detected by Annexin V/PI staining). Moreover, AZD8055 induced cytotoxicity even in rapamycin resistant MM cell lines and primary patient MM cells. Finally, AZD8055 demonstrated significant anti-MM activity in an in vivo human MM cell xenograft SCID mouse model. Taken together, our data show that disruption of DEPTOR/mTORC1/mTORC2 cascade in MM cells results in significant anti-tumor effects, providing the framework for future clinical trials of AZD8055 to improve patient outcome in MM.

Disclosures:
Guichard: AstraZeneca: Employment, Shareholder AstraZeneca. Anderson:Millenium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; BMS: Consultancy; Acetylon: Membership on an entity's Board of Directors or advisory committees, Ownership interest (inc stock options) in a Start up company. Raje:AstraZeneca: Research Funding; Acetylon: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.