Craig R. Stumpf

The translational landscape of mTOR signalling steers cancer initiation and metastasis

The mammalian target of rapamycin (mTOR) kinase is a master regulator of protein synthesis that couples nutrient sensing to cell growth and cancer. However, the downstream translationally regulated nodes of gene expression that may direct cancer development are poorly characterized. Using ribosome profiling, we uncover specialized translation of the prostate cancer genome by oncogenic mTOR signalling, revealing a remarkably specific repertoire of genes involved in cell proliferation, metabolism and invasion. We extend these findings by functionally characterizing a class of translationally controlled pro-invasion messenger RNAs that we show direct prostate cancer invasion and metastasis downstream of oncogenic mTOR signalling. Furthermore, we develop a clinically relevant ATP site inhibitor of mTOR, INK128, which reprograms this gene expression signature with therapeutic benefit for prostate cancer metastasis, for which there is presently no cure. Together, these findings extend our understanding of how the ‘cancerous’ translation machinery steers specific cancer cell behaviours, including metastasis, and may be therapeutically targeted.

Ribosome-mediated specificity in Hox mRNA translation and vertebrate tissue patterning

Historically, the ribosome has been viewed as a complex ribozyme with constitutive rather than regulatory capacity in mRNA translation. Here we identify mutations of the Ribosomal Protein L38 (Rpl38) gene in mice exhibiting surprising tissue-specific patterning defects, including pronounced homeotic transformations of the axial skeleton. In Rpl38 mutant embryos, global protein synthesis is unchanged; however the translation of a select subset of Homeobox mRNAs is perturbed. Our data reveal that RPL38 facilitates 80S complex formation on these mRNAs as a regulatory component of the ribosome to confer transcript-specific translational control. We further show that Rpl38 expression is markedly enriched in regions of the embryo where loss-of-function phenotypes occur. Unexpectedly, a ribosomal protein (RP) expression screen reveals dynamic regulation of individual RPs within the vertebrate embryo. Collectively, these findings suggest that RP activity may be highly regulated to impart a new layer of specificity in the control of gene expression and mammalian development.

The cancerous translation apparatus

Deregulations in translational control are critical features of cancer initiation and progression. Activation of key oncogenic pathways promotes rapid and dramatic translational reprogramming, not simply by increasing overall protein synthesis, but also by modulating specific mRNA networks that promote cellular transformation. Additionally, ribosomopathies caused by mutations in ribosome components alter translational regulation leading to specific pathological features, including cancer susceptibility. Exciting advances in our understanding of translational control in cancer have illuminated a striking specificity innate to the translational apparatus. Characterizing this specificity will provide novel insights into how cells normally utilize translational control to modulate gene expression, how it is deregulated in cancer, and how these processes can be targeted to develop new cancer therapies.

Assessing gene-level translational control from ribosome profiling

MOTIVATION The translational landscape of diverse cellular systems remains largely uncharacterized. A detailed understanding of the control of gene expression at the level of messenger RNA translation is vital to elucidating a systems-level view of complex molecular programs in the cell. Establishing the degree to which such post-transcriptional regulation can mediate specific phenotypes is similarly critical to elucidating the molecular pathogenesis of diseases such as cancer. Recently, methods for massively parallel sequencing of ribosome-bound fragments of messenger RNA have begun to uncover genome-wide translational control at codon resolution. Despite its promise for deeply characterizing mammalian proteomes, few analytical methods exist for the comprehensive analysis of this paired RNA and ribosome data. RESULTS We describe the Babel framework, an analytical methodology for assessing the significance of changes in translational regulation within cells and between conditions. This approach facilitates the analysis of translation genome-wide while allowing statistically principled gene-level inference. Babel is based on an errors-in-variables regression model that uses the negative binomial distribution and draws inference using a parametric bootstrap approach. We demonstrate the operating characteristics of Babel on simulated data and use its gene-level inference to extend prior analyses significantly, discovering new translationally regulated modules under mammalian target of rapamycin (mTOR) pathway signaling control.

Investigating Myc-Dependent Translational Regulation in Normal and Cancer Cells

There is an increasing realization that a primary role for Myc in driving cellular growth and cell cycle progression relies on Mycs ability to increase the rate of protein synthesis. Myc induces myriad changes in both global and specific mRNA translation. Herein, we present three assays that allow researchers to measure changes in protein synthesis at the global level as well as alterations in the translation of specific mRNAs. Metabolic labeling of cells with (35)S-containing methionine and cysteine is presented as a method to measure the overall rate of global protein synthesis. The bicistronic reporter assay is employed to determine levels of cap-dependent and cap-independent translation initiation in the cell. Finally, isolation of polysome-associated mRNAs followed by next-generation sequencing, microarray or quantitative real-time PCR (qRT-PCR) analysis is utilized to detect changes in the abundance of specific mRNAs that are regulated upon Myc hyperactivation. The protocols described in this chapter can be used to understand how and to what extent Myc-dependent regulation of translation influences normal cellular functions as well as tumorigenesis.

Structure-based Design of Pyridone-Aminal eFT508 Targeting Dysregulated Translation by Selective Mitogen-activated Protein Kinase Interacting Kinases 1 and 2 (MNK1/2) Inhibition.

Dysregulated translation of mRNA plays a major role in tumorigenesis. Mitogen-activated protein kinase interacting kinases (MNK)1/2 are key regulators of mRNA translation integrating signals from oncogenic and immune signaling pathways through phosphorylation of eIF4E and other mRNA binding proteins. Modulation of these key effector proteins regulates mRNA, which controls tumor/stromal cell signaling. Compound 23 (eFT508), an exquisitely selective, potent dual MNK1/2 inhibitor, was designed to assess the potential for control of oncogene signaling at the level of mRNA translation. The crystal structure-guided design leverages stereoelectronic interactions unique to MNK culminating in a novel pyridone-aminal structure described for the first time in the kinase literature. Compound 23 has potent in vivo antitumor activity in models of diffuse large cell B-cell lymphoma and solid tumors, suggesting that controlling dysregulated translation has real therapeutic potential. Compound 23 is currently being evaluated in Phase 2 clinical trials in solid tumors and lymphoma. Compound 23 is the first highly selective dual MNK inhibitor targeting dysregulated translation being assessed clinically.

Abstract 596: eFT508, a potent and highly selective inhibitor of MNK1/2 regulates immune checkpoint and cytokine expression promoting anti-tumor immunity

Dysregulated translation of messenger RNA (mRNA) plays a role in the pathogenesis of multiple solid tumors and hematological malignancies. MNK1 and MNK2 integrate signals from several oncogenic and immune signaling pathways (including RAS, Toll-like receptors and T cell receptor) by phosphorylating eukaryotic initiation factor 4E (eIF4E) and other key effector proteins including hnRNPA1 and PSF. Phosphorylation of these RNA-binding proteins by MNK1 and MNK2 selectively regulates the stability and translation of a subset of cellular mRNA that control tumor/stromal cell signaling, the tumor microenvironment and immune cell function. eFT508 is a potent and highly selective inhibitor of both MNK1 and MNK2. Ribosome profiling has demonstrated that inhibition of MNK1 and MNK2 by eFT508 selectively regulates the translational efficiency and mRNA stability of a subset of genes that include inflammatory cytokines/chemokines, regulators of stress response, and effectors of anti-tumor immune response. Given the importance of MAPK signaling and translational control to immune cell activation and differentiation, the immunological effect of eFT508 was further evaluated in both normal human immune cells in vitro and immunocompetent syngeneic cancer models in vivo. eFT508 treatment of normal donor T cells has no deleterious effect on αCD3/αCD28 stimulated IL-2 production, T cell proliferation or T cell viability. However, eFT508 selectively down regulates the induction of IL-10 and specific immune checkpoint receptors, including PD-1 and LAG3. Further evaluation of the mechanism of translational regulation has shown LAG3 mRNA contains specific sequence elements in the 5’-untranslated region (UTR) that confer sensitivity to eFT508. In addition, IL-10 mRNA is destabilized upon treatment with eFT508 leading to significant inhibition of IL-10 production in activated T cells. Furthermore, eFT508 treatment results in upregulation of MHC class II molecules on tumor cells, macrophage and dendritic cells through an IL-10/MARCH1 dependent mechanism. The in vivo antitumor effect of eFT508 was assessed in the CT26 BALB/C syngeneic tumor model. CT26 mouse tumor cell proliferation and survival are insensitive to eFT508 in vitro. In vivo, daily oral treatment with 1 mg/kg eFT508 results in significant anti-tumor activity, modulation of tumor infiltrating lymphocytes and establishment of immune memory. In addition, combination of eFT508 with either anti-PD-1 or anti-PD-L1 monoclonal antibodies results in marked efficacy, significantly increasing the percentage of responder animals. eFT508 is currently under evaluation in two phase I/II clinical trials for patients with advanced solid tumors and patients with advanced lymphoma respectively. These findings support further clinical evaluation of eFT508 in combination with checkpoint blockade. Citation Format: Kevin R. Webster, Vikas K. Goel, Jocelyn Staunton, Craig R. Stumpf, Rajesh Sharma, Ivy N. Hung, Gregory S. Parker, Jolene Molter, Gary G. Chiang, Christopher J. Wegerski, Samuel Sperry, Vera Huang, Joan Chen, Peggy A. Thompson, Chinh Tran, Justin T. Ernst, Paul A. Sprengeler, Siegfried H. Reich. eFT508, a potent and highly selective inhibitor of MNK1/2 regulates immune checkpoint and cytokine expression promoting anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 596. doi:10.1158/1538-7445.AM2017-596

Abstract PR07: Myc-dependent translation makes an impact: Tailor-made protein expression for cancer development

Our research delineates how the “cancer translatome” can reprogram gene expression at the post-transcriptional level, by controlling the translation of specific subsets of mRNAs during distinct steps of cancer progression. Here, we have genetically engineered a novel mouse model in which two of the most commonly perturbed oncogenes, MYC and KRAS, are stochastically activated in a small number of hepatocytes resulting in a dramatic increase in metastatic tumors, recapitulating the process of liver tumor development in human cancers. Therefore, we have used this model to detail the evolution of gene expression from primary tumor initiation to metastasis at both the transcriptional and translational levels. We used a genome-wide ribosome profiling approach to assess changes in gene expression at the earliest possible time point after oncogenic activation to metastasis formation in order to develop the first functional comprehensive network of widespread changes in translational regulation that occur during the course of tumor development. We characterized networks of translational regulation unique to each individual oncogene, as well as networks that are synergistically regulated upon activation of both oncogenes. For example, we have identified common networks that are translationally induced upon activation of either MYC or KRAS (e.g. protein synthesis, energy metabolism). We discovered that each oncogene also regulates the translation of unique mRNA networks. MYC induces the translation of genes involved in cell proliferation, mRNA splicing, and lipid metabolism, while KRAS specifically induces the translation of stress response genes, especially the pathway that responds to hypoxia. In addition to the additive effects of regulating their cognate mRNA networks, the combination of MYC and KRAS activation in the same cells can also induce a synergistic effect on the translation of new networks including genes involved in integrin signaling, which may partially explain the increased metastasis observed in this mouse model. Additionally, we uncovered independent regulatory mechanisms that may allow for temporal regulation of discrete oncogenic processes, either at the level of transcription or translation, required at different times during the course of tumor development. Understanding the mechanisms and specific targets of translational regulation downstream of MYC and KRAS activation will provide a novel platform from which to develop therapeutic strategies targeting cancers possessing two of the most common and currently “undruggable” oncogenic lesions. Citation Format: Craig Stumpf, Tom Cunningham, Davide Ruggero. Myc-dependent translation makes an impact: Tailor-made protein expression for cancer development. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr PR07.