Elspeth M. Beauchamp

The evolution of the TOR pathway and its role in cancer.

The target of rapamycin (TOR) pathway is highly conserved among eukaryotes and has evolved to couple nutrient sensing to cellular growth. TOR is found in two distinct signaling complexes in cells, TOR complex 1 (TORC1) and TOR complex 2 (TORC2). These complexes are differentially regulated and act as effectors for the generation of signals that drive diverse cellular processes such as growth, proliferation, protein synthesis, rearrangement of the cytoskeleton, autophagy, metabolism and survival. Mammalian TOR (mTOR) is very important for development in embryos, while in adult organisms it is linked to aging and lifespan effects. In humans, the mTOR pathway is implicated in the tumorigenesis of multiple cancer types and its deregulation is associated with familial cancer syndromes. Because of its high biological relevance, different therapeutic strategies have been developed to target this signaling cascade, resulting in the emergence of unique pharmacological inhibitors that are either already approved for use in clinical oncology or currently under preclinical or clinical development. Multimodal treatment strategies that simultaneously target multiple nodes of the pathway and/or negative feedback regulatory loops may ultimately provide the best therapeutic advantage in targeting this pathway for the treatment of malignancies.

Autophagy Is a Survival Mechanism of Acute Myelogenous Leukemia Precursors during Dual mTORC2/mTORC1 Targeting

Purpose: To examine whether induction of autophagy is a mechanism of leukemic cell resistance to dual mTORC1/mTORC2 inhibitors in acute myelogenous leukemia (AML) leukemic progenitors. Experimental Design: Combinations of different experimental approaches were used to assess induction of autophagy, including immunoblotting to detect effects on LC3II and p62/SQTM1 expression and on ULK1 phosphorylation, immunofluorescence, and electron microscopy. Functional responses were assessed using cell viability and apoptosis assays, and clonogenic leukemic progenitor assays in methylcellulose. Results: We provide evidence that treatment of AML cells with catalytic mTOR inhibitors results in induction of autophagy, which acts as a regulatory mechanism to promote leukemic cell survival. Such induction of autophagy by dual mTORC1/mTORC2 inhibitors partially protects primitive leukemic precursors from the inhibitory effects of such agents and limits their activities. Simultaneous blockade of the autophagic process using chloroquine or by knockdown of ULK1 results in enhanced antileukemic responses. Conclusions: Dual targeting of mTORC2 and mTORC1 results in induction of autophagy in AML cells. Combinations of catalytic mTOR targeting agents and autophagy inhibitors may provide a unique approach to target primitive leukemic precursors in AML. Clin Cancer Res; 20(9); 2400–9. ©2014 AACR.

Central Role of ULK1 in Type I Interferon Signaling

We provide evidence that the Unc-51-like kinase 1 (ULK1) is activated during engagement of the type I interferon (IFN) receptor (IFNR). Our studies demonstrate that the function of ULK1 is required for gene transcription mediated via IFN-stimulated response elements (ISRE) and IFNγ activation site (GAS) elements and controls expression of key IFN-stimulated genes (ISGs). We identify ULK1 as an upstream regulator of p38α mitogen-activated protein kinase (MAPK) and establish that the regulatory effects of ULK1 on ISG expression are mediated possibly by engagement of the p38 MAPK pathway. Importantly, we demonstrate that ULK1 is essential for antiproliferative responses and type I IFN-induced antineoplastic effects against malignant erythroid precursors from patients with myeloproliferative neoplasms. Together, these data reveal a role for ULK1 as a key mediator of type I IFNR-generated signals that control gene transcription and induction of antineoplastic responses.

Direct binding of arsenic trioxide to AMPK and generation of inhibitory effects on acute myeloid leukemia precursors

Arsenic trioxide (As2O3) exhibits potent antineoplastic effects and is used extensively in clinical oncology for the treatment of a subset of patients with acute myeloid leukemia (AML). Although As2O3 is known to regulate activation of several signaling cascades, the key events, accounting for its antileukemic properties, remain to be defined. We provide evidence that arsenic can directly bind to cysteine 299 in AMPKα and inhibit its activity. This inhibition of AMPK by arsenic is required in part for its cytotoxic effects on primitive leukemic progenitors from patients with AML, while concomitant treatment with an AMPK activator antagonizes in vivo the arsenic-induced antileukemic effects in a xenograft AML mouse model. A consequence of AMPK inhibition is activation of the mTOR pathway as a negative regulatory feedback loop. However, when AMPK expression is lost, arsenic-dependent activation of the kinase RSK downstream of MAPK activity compensates the generation of regulatory feedback signals through phosphorylation of downstream mTOR targets. Thus, therapeutic regimens with As2O3 will need to include inhibitors of both the mTOR and RSK pathways in combination to prevent engagement of negative feedback loops and maximize antineoplastic responses. Mol Cancer Ther; 14(1); 202–12. ©2014 AACR.

Human Schlafen 5 (SLFN5) Is a Regulator of Motility and Invasiveness of Renal Cell Carcinoma Cells

ABSTRACT We provide evidence that human SLFN5, an interferon (IFN)-inducible member of the Schlafen (SLFN) family of proteins, exhibits key roles in controlling motility and invasiveness of renal cell carcinoma (RCC) cells. Our studies define the mechanism by which this occurs, demonstrating that SLFN5 negatively controls expression of the matrix metalloproteinase 1 gene (MMP-1), MMP-13, and several other genes involved in the control of malignant cell motility. Importantly, our data establish that SLFN5 expression correlates with a better overall survival in a large cohort of patients with RCC. The inverse relationship between SLFN5 expression and RCC aggressiveness raises the possibility of developing unique therapeutic approaches in the treatment of RCC, by modulating SLFN5 expression.

Merestinib blocks Mnk kinase activity in acute myeloid leukemia progenitors and exhibits antileukemic effects in vitro and in vivo

Mitogen-activated protein kinase interacting protein kinases (Mnks) play important roles in the development and progression of acute myeloid leukemia (AML) by regulating eukaryotic translation initiation factor 4E (eIF4E) activation. Inhibiting Mnk1/2-induced phosphorylation of eIF4E may represent a unique approach for the treatment of AML. We provide evidence for antileukemic effects of merestinib, an orally bioavailable multikinase inhibitor with suppressive effects on Mnk activity. Our studies show that merestinib effectively blocks eIF4E phosphorylation in AML cells and suppresses primitive leukemic progenitors from AML patients in vitro and in an AML xenograft model in vivo. Our findings provide evidence for potent preclinical antileukemic properties of merestinib and support its clinical development for the treatment of patients with AML.

Resveratrol enhances the suppressive effects of arsenic trioxide on primitive leukemic progenitors

Efforts to enhance the antileukemic properties of arsenic trioxide are clinically relevant and may lead to the development of new therapeutic approaches for the management of certain hematological malignancies. We provide evidence that concomitant treatment of acute myeloid leukemia (AML) cells or chronic myeloid leukemia (CML) cells with resveratrol potentiates arsenic trioxide-dependent induction of apoptosis. Importantly, clonogenic assays in methylcellulose demonstrate potent suppressive effects of the combination of these agents on primitive leukemic progenitors derived from patients with AML or CML. Taken together, these findings suggest that combinations of arsenic trioxide with resveratrol may provide an approach for targeting of early leukemic precursors and, possibly, leukemia initiating stem cells.

Pre-clinical evidence of PIM kinase inhibitor activity in BCR-ABL1 unmutated and mutated Philadelphia chromosome-positive (Ph+) leukemias.

We investigated the efficacy of targeting the PIM kinase pathway in Philadelphia chromosome-positive (Ph+) leukemias. We provide evidence that inhibition of PIM, with the pan-PIM inhibitor SGI-1776, results in suppression of classic PIM effectors and also elements of the mTOR pathway, suggesting interplay between PIM and mTOR signals. Our data demonstrate that PIM inhibition enhances the effects of imatinib mesylate on Ph+ leukemia cells. We also found that PIM inhibition results in suppression of leukemic cell proliferation and induction of apoptosis of Ph+ leukemia cells, including those resistant to imatinib mesylate. Importantly, inhibition of PIM results in enhanced suppression of primary leukemic progenitors from patients with CML. Altogether these findings suggest that pharmacological PIM targeting may provide a unique therapeutic approach for the treatment of Ph+ leukemias.

BH3 mimetics and multi-kinase inhibition in AML

In this issue of Blood, Rahmani et al show in preclinical studies that the combination of the multi-kinase inhibitor sorafenib with the BH3 mimetic obatoclax results in enhanced antileukemic effects compared with the effects of each agent alone. This work has important clinical implications because it describes a novel approach to overcome acute myeloid leukemia (AML) cell resistance by combining agents that are currently being investigated in trials as single agents.

Differential Response of Glioma Stem Cells to Arsenic Trioxide Therapy Is Regulated by MNK1 and mRNA Translation

Mesenchymal (MES) and proneural (PN) are two distinct glioma stem cell (GSC) populations that drive therapeutic resistance in glioblastoma (GBM). We screened a panel of 650 small molecules against patient-derived GBM cells to discover compounds targeting specific GBM subtypes. Arsenic trioxide (ATO), an FDA-approved drug that crosses the blood–brain barrier, was identified as a potent PN-specific compound in the initial screen and follow-up validation studies. Furthermore, MES and PN GSCs exhibited differential sensitivity to ATO. As ATO has been shown to activate the MAPK-interacting kinase 1 (MNK1)-eukaryotic translation initiation factor 4E (eIF4E) pathway and subsequent mRNA translation in a negative regulatory feedback manner, the mechanistic role of ATO resistance in MES GBM was explored. In GBM cells, ATO-activated translation initiation cellular events via the MNK1–eIF4E signaling axis. Furthermore, resistance to ATO in intracranial PDX tumors correlated with high eIF4E phosphorylation. Polysomal fractionation and microarray analysis of GBM cells were performed to identify ATOs effect on mRNA translation and enrichment of anti-apoptotic mRNAs in the ATO-induced translatome was found. Additionally, it was determined that MNK inhibition sensitized MES GSCs to ATO in neurosphere and apoptosis assays. Finally, examination of the effect of ATO on patients from a phase I/II clinical trial of ATO revealed that PN GBM patients responded better to ATO than other subtypes as demonstrated by longer overall and progression-free survival. Implications: These findings raise the possibility of a unique therapeutic approach for GBM, involving MNK1 targeting to sensitize MES GSCs to drugs like arsenic trioxide. Mol Cancer Res; 16(1); 32–46. ©2017 AACR.