Gabriela Galicia-Vázquez

The Energy Sensor AMPK Regulates T Cell Metabolic Adaptation and Effector Responses In Vivo

Naive T cells undergo metabolic reprogramming to support the increased energetic and biosynthetic demands of effector T cell function. However, how nutrient availability influences T cell metabolism and function remains poorly understood. Here we report plasticity in effector T cell metabolism in response to changing nutrient availability. Activated T cells were found to possess a glucose-sensitive metabolic checkpoint controlled by the energy sensor AMP-activated protein kinase (AMPK) that regulated mRNA translation and glutamine-dependent mitochondrial metabolism to maintain T cell bioenergetics and viability. T cells lacking AMPKα1 displayed reduced mitochondrial bioenergetics and cellular ATP in response to glucose limitation in vitro or pathogenic challenge in vivo. Finally, we demonstrated that AMPKα1 is essential for T helper 1 (Th1) and Th17 cell development and primary T cell responses to viral and bacterial infections in vivo. Our data highlight AMPK-dependent regulation of metabolic homeostasis as a key regulator of T cell-mediated adaptive immunity.

Antitumor Activity and Mechanism of Action of the Cyclopenta[b]benzofuran, Silvestrol

Background Flavaglines are a family of natural products from the genus Aglaia that exhibit anti-cancer activity in vitro and in vivo and inhibit translation initiation. They have been shown to modulate the activity of eIF4A, the DEAD-box RNA helicase subunit of the eukaryotic initiation factor (eIF) 4F complex, a complex that stimulates ribosome recruitment during translation initiation. One flavagline, silvestrol, is capable of modulating chemosensitivity in a mechanism-based mouse model. Methodology/Principal Findings Among a number of flavagline family members tested herein, we find that silvestrol is the more potent translation inhibitor among these. We find that silvestrol impairs the ribosome recruitment step of translation initiation by affecting the composition of the eukaryotic initiation factor (eIF) 4F complex. We show that silvestrol exhibits significant anticancer activity in human breast and prostate cancer xenograft models, and that this is associated with increased apoptosis, decreased proliferation, and inhibition of angiogenesis. We demonstrate that targeting translation by silvestrol results in preferential inhibition of weakly initiating mRNAs. Conclusions/Significance Our results indicate that silvestrol is a potent anti-cancer compound in vivo that exerts its activity by affecting survival pathways as well as angiogenesis. We propose that silvestrol mediates its effects by preferentially inhibiting translation of malignancy-related mRNAs. Silvestrol appears to be well tolerated in animals.

Evidence for a Functionally Relevant Rocaglamide Binding Site on the eIF4A–RNA Complex

Translation initiation is an emerging target in oncology and neurobiology indications. Naturally derived and synthetic rocaglamide scaffolds have been used to interrogate this pathway; however, there is uncertainty regarding their precise mechanism(s) of action. We exploited the genetic tractability of yeast to define the primary effect of both a natural and a synthetic rocaglamide in a cellular context and characterized the molecular target using biochemical studies and in silico modeling. Chemogenomic profiling and mutagenesis in yeast identified the eIF (eukaryotic Initiation Factor) 4A helicase homologue as the primary molecular target of rocaglamides and defined a discrete set of residues near the RNA binding motif that confer resistance to both compounds. Three of the eIF4A mutations were characterized regarding their functional consequences on activity and response to rocaglamide inhibition. These data support a model whereby rocaglamides stabilize an eIF4A-RNA interaction to either alter the level and/or impair the activity of the eIF4F complex. Furthermore, in silico modeling supports the annotation of a binding pocket delineated by the RNA substrate and the residues identified from our mutagenesis screen. As expected from the high degree of conservation of the eukaryotic translation pathway, these observations are consistent with previous observations in mammalian model systems. Importantly, we demonstrate that the chemically distinct silvestrol and synthetic rocaglamides share a common mechanism of action, which will be critical for optimization of physiologically stable derivatives. Finally, these data confirm the value of the rocaglamide scaffold for exploring the impact of translational modulation on disease.

CRISPR-Mediated Drug-Target Validation Reveals Selective Pharmacological Inhibition of the RNA Helicase, eIF4A

SUMMARY Targeting translation initiation is an emerging anti-neoplastic strategy that capitalizes on de-regulated upstream MAPK and PI3K-mTOR signaling pathways in cancers. A key regulator of translation that controls ribosome recruitment flux is eukaryotic initiation factor (eIF) 4F, a hetero-trimeric complex composed of the cap binding protein eIF4E, the scaffolding protein eIF4G, and the RNA helicase eIF4A. Small molecule inhibitors targeting eIF4F display promising anti-neoplastic activity in preclinical settings. Among these are some rocaglate family members that are well tolerated in vivo, deplete eIF4F of its eIF4A helicase subunit, have shown activity as single agents in several xenograft models, and can reverse acquired resistance to MAPK and PI3K-mTOR targeted therapies. Herein, we highlight the power of using genetic complementation approaches and CRISPR/Cas9-mediated editing for drug-target validation ex vivo and in vivo, linking the anti-tumor properties of rocaglates to eIF4A inhibition.

Inhibitors of Translation Targeting Eukaryotic Translation Initiation Factor 4A

The RNA helicases eIF4AI and eIF4AII play key roles in recruiting ribosomes to mRNA templates during eukaryotic translation initiation. Small molecule inhibitors of eIF4AI and eIF4AII have been useful for chemically dissecting their role in translation in vitro and in vivo. Here, we describe a screen performed on a small focused library of kinase inhibitors to identify a novel helicase inhibitor. We describe assays that have been critical for characterizing novel RNA helicase inhibitors.

High-throughput assays probing protein-RNA interactions of eukaryotic translation initiation factors.

Protein-RNA interactions are involved in all facets of RNA biology. The identification of small molecules that selectively block such bimolecular interactions could provide insight into previously unexplored steps of gene regulation. Such is the case for regulation of eukaryotic protein synthesis where interactions between messenger RNA (mRNA) and several eukaryotic initiation factors govern the recruitment of 40S ribosomes (and associated factors) to mRNA templates during the initiation phase. We have designed simple fluorescence polarization-based high-throughput screening assays that query the binding of several translation factors to RNA and found that the mixed inhibitor p-chloromercuribenzoate interferes with poly(A) binding protein-RNA interaction.

Regulation of Eukaryotic Initiation Factor 4AII by MyoD during Murine Myogenic Cell Differentiation

Gene expression during muscle cell differentiation is tightly regulated at multiple levels, including translation initiation. The PI3K/mTOR signalling pathway exerts control over protein synthesis by regulating assembly of eukaryotic initiation factor (eIF) 4F, a heterotrimeric complex that stimulates recruitment of ribosomes to mRNA templates. One of the subunits of eIF4F, eIF4A, supplies essential helicase function during this phase of translation. The presence of two cellular eIF4A isoforms, eIF4AI and eIF4AII, has long thought to impart equivalent functions to eIF4F. However, recent experiments have alluded to distinct activities between them. Herein, we characterize distinct regulatory mechanisms between the eIF4A isoforms during muscle cell differentiation. We find that eIF4AI levels decrease during differentiation whereas eIF4AII levels increase during myofiber formation in a MyoD-dependent manner. This study characterizes a previously undefined mechanism for eIF4AII regulation in differentiation and highlights functional differences between eIF4AI and eIF4AII. Finally, RNAi-mediated alterations in eIF4AI and eIF4AII levels indicate that the myogenic process can tolerate short term reductions in eIF4AI or eIF4AII levels, but not both.

Del11q-positive CLL lymphocytes exhibit altered glutamine metabolism and differential response to GLS1 and glucose metabolism inhibition

Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of malignant B cells, and their accumulation in the blood stream and homing tissues. In the CLL context, the chromosomal aberrations del17p and del11q, spanning TP53 and ATM locus, respectively, are considered poor outcome predictors. As well, CD38, ZAP70, and unmutated status of the IgVH locus represent bad prognosis markers. Despite the outstanding clinical results yielded by ibrutinib (mainly used in relapsed or refractory settings), no complete remission is guaranteed due to the arising of resistance and relapse upon treatment discontinuation. The lack of tailored therapies upon ibrutinib failure represents a major challenge, particularly in del11q and del17p cases refractory to conventional therapies. Recently, the analysis of metabolic reprogramming has uncovered tumor cell vulnerabilities, leading to the development of novel therapeutic approaches; such as the use of ritonavir (glucose uptake inhibitor) and metformin (OxPhos inhibitor) for multiple myeloma treatment. The aim of this work was to define targetable metabolic features in CLL lymphocytes with respect to their del11q status, since ATM—a reactive oxygen species (ROS) sensor and metabolic regulator—and miR125—metabolic regulator—genes are comprised within del11q. Also, del11q has been linked to increased insulin receptor expression. We used a panel of 26 CLL primary samples donated from affected patients, 23% of which were del11q-positive, which represent occurrence of del11q cases in the clinical setting (~20%), while del17p cases were excluded (Supplementary Table 1). CLL lymphocytes were maintained in a physiological glucose concentration, since our group noticed that CLL cells exposed to high or limited glucose levels display different metabolic responses. Under basal conditions, no significant difference was found in glucose or glutamine uptake, total or reduced glutathione, and ROS levels (Supplementary Fig. 1a–d) between del11q-positive (hereafter del11q) and del11q-negative (hereafter wildtype) CLL lymphocytes, suggesting that del11q CLL lymphocytes are adapted to reduced ATM expression. CLL lymphocyte metabolism was explored by challenging these lymphocytes with a panel of metabolic inhibitors (Supplementary Table 2). The disturbance of glycolysis (2DG), glucose uptake (ritonavir) or OxPhos (oligomycin) decreased all CLL cell viability. Del11q CLL cells displayed enhanced sensitivity for the three treatments. Inhibiting fatty acid oxidation (etomoxir) was equally cytotoxic to all CLL lymphocytes; conversely, the inhibition of the pentose phosphate pathway (PPP) (DHEA) or the one-carbon metabolism (AMPA) was not cytotoxic to CLL cells (Fig. 1a). Curtailing the first step of glutamine metabolism, via glutaminase (GLS1) inhibition (compound 968), was cytotoxic only to del11q CLL cells (Fig. 1a). Since glutamine and glutamate uptake were similar between del11q and wild-type CLL cells (Supplementary Fig. 1e, f), it is likely that differential utilization of these metabolites is associated with del11q status. A distinctive characteristic of del11q CLL cells was the consumption of ammonia under basal conditions, while wild-type CLL lymphocytes

Ibrutinib Resistance Is Reduced by an Inhibitor of Fatty Acid Oxidation in Primary CLL Lymphocytes

Chronic Lymphocytic Leukemia (CLL) is an incurable disease, characterized by the accumulation of malignant B-lymphocytes in the blood stream (quiescent state) and homing tissues (where they can proliferate). In CLL, the targeting of B-cell receptor signaling through a Burtons tyrosine kinase inhibitor (ibrutinib) has rendered outstanding clinical results. However, complete remission is not guaranteed due to drug resistance or relapse, revealing the need for novel approaches for CLL treatment. The characterization of metabolic rewiring in proliferative cancer cells is already being applied for diagnostic and therapeutic purposes, but our knowledge of quiescent cell metabolism—relevant for CLL cells—is still fragmentary. Recently, we reported that glutamine metabolism in primary CLL cells bearing the del11q deletion is different from their del11q negative counterparts, making del11q cells especially sensitive to glutaminase and glycolysis inhibitors. In this work, we used our primary CLL lymphocyte bank and compounds interfering with central carbon metabolism to define metabolic traits associated with ibrutinib resistance. We observe a differential basal metabolite uptake linked to ibrutinib resistance, favoring glutamine uptake and catabolism. Upon ibrutinib treatment, the redox balance in ibrutinib resistant cells is shifted toward NADPH accumulation, without an increase in glutamine uptake, suggesting alternative metabolic rewiring such as the activation of fatty acid oxidation. In accordance to this idea, the curtailing of fatty acid oxidation by CPT1 inhibition (etomoxir) re-sensitized resistant cells to ibrutinib. Our results suggest that fatty acid oxidation could be explored as a target to overcome ibrutinib resistance.

Current and Emerging Therapies Targeting Translation

The highly regulated process of protein synthesis is frequently dysregulated in cancers. In nontransformed cells, one of the primary control points occurs when ribosomal subunits are recruited to mRNA templates. This process is limited by the availability of the trans-acting factor, eIF4F, a heterotrimeric complex composed of the eIF4E cap-binding protein; the scaffold protein, eIF4G; and the ATP-dependent DEAD box helicase eIF4A. As the eIF4F checkpoint is under regulation of the PI3K/AKT/mTOR cascade and its subunits are transcriptional targets of MYC, this step is often dysregulated in human cancers. In this review, we discuss the development, mode of action and biological activity of small-molecule inhibitors that interfere with eIF4F activity and their potential for clinical development.