No abstract

Aberrant activation of mTOR signaling in acute myeloid leukemia (AML) results in a survival advantage that promotes the malignant phenotype. To improve our understanding of factors that contribute to mammalian target of rapamycin (mTOR) signaling activation and identify novel therapeutic targets, we searched for unique interactors of mTOR complexes through proteomics analyses. We identify cyclin dependent kinase 9 (CDK9) as a novel binding partner of the mTOR complex scaffold protein, mLST8. Our studies demonstrate that CDK9 is present in distinct mTOR-like (CTOR) complexes in the cytoplasm and nucleus. In the nucleus, CDK9 binds to RAPTOR and mLST8, forming CTORC1, to promote transcription of genes important for leukemogenesis. In the cytoplasm, CDK9 binds to RICTOR, SIN1, and mLST8, forming CTORC2, and controls messenger RNA (mRNA) translation through phosphorylation of LARP1 and rpS6. Pharmacological targeting of CTORC complexes results in suppression of growth of primitive human AML progenitors in vitro and elicits strong antileukemic responses in AML xenografts in vivo.

We provide evidence for a unique pathway engaged by the type II IFN receptor, involving mTORC2/AKT-mediated downstream regulation of mTORC1 and effectors. These events are required for formation of the eukaryotic translation initiation factor 4F complex (eIF4F) and initiation of mRNA translation of type II interferon-stimulated genes. Our studies establish that Rictor is essential for the generation of type II IFN-dependent antiviral and antiproliferative responses and that it controls the generation of type II IFN-suppressive effects on normal and malignant hematopoiesis. Together, our findings establish a central role for mTORC2 in IFN␥ signaling and type II IFN responses.IFNs are cytokines that exhibit antiviral, immunomodulatory, growth-inhibitory, and cytotoxic properties (1-12). The critical roles of these cytokines in the innate immune system have provoked clinical interest and extensive studies to explore their therapeutic potential. These studies, spanning several decades, have definitively established their utility in the treatment of viral syndromes, many malignancies, and some autoimmune disorders (1-12).IFN␥, the sole type II IFN, binds to the IFNGR1 and IFNGR2 subunits of the type II IFN receptor with high affinity and activates the Janus kinases Jak1 and Jak2, leading to engagement of Jak-Stat pathways and transcriptional activation of IFN␥-regulated genes (13-16). Activation of the Jak-Stat pathway is critical for the IFN␥ transcriptional control of IFN-stimulated genes (ISGs) 3 and, subsequently, for the generation of IFN␥-induced biological responses (13-16). Beyond the classical JakStat pathways, several other signaling pathways have been shown to be activated by the type II IFN receptor, and their function appears to be critical for IFN␥ responses. These include PKC (17), MAP kinase (18,19), and Mnk kinase cascades (20). There is evidence that the AKT/mTOR pathway is engaged in IFN␥ signaling, controlling the initiation of mRNA translation for ISGs (21,22). However, the precise contribution of different mTOR complexes in this process and the sequence of events leading to ISG mRNA translation remain to be determined.The mTOR kinase forms the catalytic core of two known complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (24 -35). mTORC1 is a protein complex consisting of mTOR, mammalian lethal with Sec 13 protein 8/G-protein ␤-protein subunit like (mLST8/G␤L), rapamycin-sensitive companion of mTOR (Raptor), Akt/PKB substrate 40 kDa (Pras40), and DEP domain-containing mTOR-interacting protein (Deptor) (24, 25). mTORC1 is known as a key regulator of pathways involved in the initiation of mRNA translation and is inhibited by allosteric inhibitors such as rapamycin, everolimus, temsirolimus, and other rapalogs (24,25). mTORC2 is comprised of mTOR, mLST8, rapamycin-insensitive companion of mTOR (Rictor), mammalian stress-activated protein kinase interacting protein 1 (Sin1), protein observed with rictor 1/2 (protor 1/2), and deptor (26 -32). Although the two mTOR complexes have different...

The mammalian target of rapamycin (mTOR) and phosphoinositide-3-kinase (PI3K) pathways are often aberrantly activated in acute myeloid leukemia (AML) and play critical roles in proliferation and survival of leukemia cells. We provide evidence that simultaneous targeting of mTOR complexes with the catalytic mTOR inhibitor OSI-027 and of the p110α subunit of PI3K with the specific inhibitor BYL-719 results in efficient suppression of effector pathways and enhanced induction of apoptosis of leukemia cells. Importantly, such a combined targeting approach results in enhanced suppression of primitive leukemic progenitors from patients with AML. Taken together, these findings raise the possibility of combination treatments of mTOR and p110α inhibitors as a unique approach to enhance responses in refractory AML.

Interferons (IFNs) are key initiators and effectors of the immune response against malignant cells and also directly inhibit tumor growth. IFNα is highly effective in the treatment of myeloproliferative neoplasms (MPNs), but the mechanisms of action are unclear and it remains unknown why some patients respond to IFNα and others do not. Here, we identify and characterize a pathway involving PKCδ-dependent phosphorylation of ULK1 on serine residues 341 and 495, required for subsequent activation of p38 MAPK. We show that this pathway is essential for IFN-suppressive effects on primary malignant erythroid precursors from MPN patients, and that increased levels of ULK1 and p38 MAPK correlate with clinical response to IFNα therapy in these patients. We also demonstrate that IFNα treatment induces cleavage/activation of the ULK1-interacting ROCK1/2 proteins in vitro and in vivo, triggering a negative feedback loop that suppresses IFN responses. Overexpression of ROCK1/2 is seen in MPN patients and their genetic or pharmacological inhibition enhances IFN-anti-neoplastic responses in malignant erythroid precursors from MPN patients. These findings suggest the clinical potential of pharmacological inhibition of ROCK1/2 in combination with IFN-therapy for the treatment of MPNs.