Faculty Bibliography

Muscle function and mass begin declining in adults long before evidence of sarcopenia and include reduced mitochondrial function, although much remains to be characterized. We found that mRNA decay factor AU-rich mRNA binding factor 1 (AUF1), which stimulates myogenesis, is strongly reduced in skeletal muscle of adult and older mice in the absence of evidence of sarcopenia. Muscle-specific adeno-associated virus (AAV)8-AUF1 gene therapy increased expression of AUF1, muscle function, and mass. AAV8 AUF1 muscle gene transfer in 12-month-old mice increased the levels of activated muscle stem (satellite) cells, increased muscle mass, reduced markers of muscle atrophy, increased markers of mitochondrial content and muscle fiber oxidative capacity, and enhanced exercise performance to levels of 3-month-old mice. With wild-type and AUF1 knockout mice and cultured myoblasts, AUF1 supplementation of muscle fibers was found to increase expression of Peroxisome Proliferator-activated Receptor Gamma Co-activator 1-alpha (PGC1α), a major effector of skeletal muscle mitochondrial oxidative metabolism. AUF1 stabilized and increased translation of the pgc1α mRNA, which is strongly reduced in adult muscle in the absence of AUF1 supplementation. Skeletal muscle-specific gene transfer of AUF1 therefore restores muscle mass, increases exercise endurance, and may provide a therapeutic strategy for age-related muscle loss.

Regulatory T cells (Treg cells) inhibit effector T cells and maintain immune system homeostasis. Treg cell maturation in peripheral sites requires inhibition of protein kinase mTORC1 and TGF-beta-1 (TGF-beta). While Treg cell maturation requires protein synthesis, mTORC1 inhibition downregulates it, leaving unanswered how Treg cells achieve essential mRNA translation for development and immune suppression activity. Using human CD4⁺ T cells differentiated in culture and genome-wide transcription and translation profiling, here we report that TGF-beta transcriptionally reprograms naive T cells to express Treg cell differentiation and immune suppression mRNAs, while mTORC1 inhibition impairs translation of T cell mRNAs but not those induced by TGF-beta. Rather than canonical mTORC1/eIF4E/eIF4G translation, Treg cell mRNAs utilize the eIF4G homolog DAP5 and initiation factor eIF3d in a non-canonical translation mechanism that requires cap-dependent binding by eIF3d directed by Treg cell mRNA 5' noncoding regions. Silencing DAP5 in isolated human naive CD4⁺ T cells impairs their differentiation into Treg cells. Treg cell differentiation is mediated by mTORC1 downregulation and TGF-beta transcriptional reprogramming that establishes a DAP5/eIF3d-selective mechanism of mRNA translation.

Orellana et al. (2021) and Dai et al. (2021) demonstrate that increased m⁷G modification of a subset of tRNAs by the METTL1/WDR4 complex stabilizes these mRNAs against decay, increases translation efficiency, reduces ribosome pausing, is associated with poor survival in human cancers, and is directly transforming.

Inhibition of mTORC1 signaling has been shown to diminish growth of meningiomas and schwannomas in preclinical studies, and clinical data suggest that everolimus, an orally administered mTORC1 inhibitor, may slow tumor progression in a subset of NF2 patients with vestibular schwannoma (VS). To assess the pharmacokinetics, pharmacodynamics and potential mechanisms of treatment resistance, we performed a pre-surgical (phase 0) clinical trial of everolimus in patients undergoing elective surgery for VS or meningiomas. Eligible patients with meningioma or VS requiring tumor resection enrolled on study received everolimus 10 mg daily for 10 days immediately prior to surgery. Everolimus blood levels were determined immediately prior to and after surgery. Tumor samples were collected intraoperatively. Ten patients completed protocol therapy. Median pre- and post-operative blood levels of everolimus were found to be in a high therapeutic range (17.4 ng/ml and 9.4 ng/ml, respectively). Median tumor tissue drug concentration determined by mass spectrometry was 24.3 pg/mg (range 9.2-169.2). We observed only partial inhibition of phospho-S6 in the treated tumors, indicating incomplete target inhibition compared to control tissues from untreated patients (p=0.025). Everolimus led to incomplete inhibition of mTORC1 and downstream signaling. These data may explain the limited anti-tumor effect of everolimus observed in clinical studies for NF2 patients and will inform the design of future pre-clinical and clinical studies targeting mTORC1 in meningiomas and schwannomas.

BACKGROUND:Circulating tumor DNAs (ctDNAs) are highly promising cancer biomarkers, potentially applicable for noninvasive liquid biopsy and disease monitoring. However, to date, sequencing of ctDNAs has proven to be challenging primarily due to small sample size and high background of fragmented cell-free DNAs (cfDNAs) derived from normal cells in the circulation, specifically in early stage cancer. METHODS:Solid-state nanopores (ssNPs) have recently emerged as a highly efficient tool for single-DNA sensing and analysis. Herein, we present a rapid nanopore genotyping strategy to enable an amplification-free identification and classification of ctDNA mutations. A biochemical ligation detection assay was used for the creation of specific fluorescently-labelled short DNA reporter molecules. Color conjugation with multiple fluorophores enabled a unique multi-color signature for different mutations, offering multiplexing potency. Single-molecule readout of the fluorescent labels was carried out by electro-optical sensing via solid-state nanopores drilled in titanium oxide membranes. RESULTS:As proof of concept, we utilized our method to detect the presence of low-quantity ERBB2 F310S and PIK3Ca H1047R breast cancer mutations from both plasmids and xenograft mice blood samples. We demonstrated an ability to distinguish between a wild type and a mutated sample, and between the different mutations in the same sample. CONCLUSIONS:Our method can potentially enable rapid and low cost ctDNA analysis that completely circumvents PCR amplification and library preparation. This approach will thus meet a currently unmet demand in terms of sensitivity, multiplexing and cost, opening new avenues for early diagnosis of cancer.

Atherosclerosis and obesity share pathological features including inflammation mediated by innate and adaptive immune cells. LXRα plays a central role in the transcription of inflammatory and metabolic genes. LXRα is modulated by phosphorylation at serine 196 (LXRα pS196), however, the consequences of LXRα pS196 in hematopoietic cell precursors in atherosclerosis and obesity have not been investigated. To assess the importance of LXRα phosphorylation, bone marrow from LXRα WT and S196A mice was transplanted into Ldlr^-/- mice, which were fed a western diet prior to evaluation of atherosclerosis and obesity. Plaques from S196A mice showed reduced inflammatory monocyte recruitment, lipid accumulation, and macrophage proliferation. Expression profiling of CD68⁺ and T cells from S196A mouse plaques revealed downregulation of pro-inflammatory genes and in the case of CD68⁺ upregulation of mitochondrial genes characteristic of anti-inflammatory macrophages. Furthermore, S196A mice had lower body weight and less visceral adipose tissue; this was associated with transcriptional reprograming of the adipose tissue macrophages and T cells, and resolution of inflammation resulting in less fat accumulation within adipocytes. Thus, reducing LXRα pS196 in hematopoietic cells attenuates atherosclerosis and obesity by reprogramming the transcriptional activity of LXRα in macrophages and T cells to promote an anti-inflammatory phenotype.

BACKGROUND:The BCL2 inhibitor venetoclax has shown efficacy in several hematologic malignancies, with the greatest response rates in indolent blood cancers such as chronic lymphocytic leukaemia. There is a lower response rate to venetoclax monotherapy in diffuse large B-cell lymphoma (DLBCL). METHODS:We tested inhibitors of cap-dependent mRNA translation for the ability to sensitise DLBCL and mantle cell lymphoma (MCL) cells to apoptosis by venetoclax. We compared the mTOR kinase inhibitor (TOR-KI) MLN0128 with SBI-756, a compound targeting eukaryotic translation initiation factor 4G1 (eIF4G1), a scaffolding protein in the eIF4F complex. RESULTS:Treatment of DLBCL and MCL cells with SBI-756 synergised with venetoclax to induce apoptosis in vitro, and enhanced venetoclax efficacy in vivo. SBI-756 prevented eIF4E-eIF4G1 association and cap-dependent translation without affecting mTOR substrate phosphorylation. In TOR-KI-resistant DLBCL cells lacking eIF4E binding protein-1, SBI-756 still sensitised to venetoclax. SBI-756 selectively reduced translation of mRNAs encoding ribosomal proteins and translation factors, leading to a reduction in protein synthesis rates in sensitive cells. When normal lymphocytes were treated with SBI-756, only B cells had reduced viability, and this correlated with reduced protein synthesis. CONCLUSIONS:Our data highlight a novel combination for treatment of aggressive lymphomas, and establishes its efficacy and selectivity using preclinical models.

Pancreatic ductal adenocarcinoma (PDAC) tumors have a nutrient-poor, desmoplastic, and highly innervated tumor microenvironment. Although neurons can release stimulatory factors to accelerate PDAC tumorigenesis, the metabolic contribution of peripheral axons has not been explored. We found that peripheral axons release serine (Ser) to support the growth of exogenous Ser (exSer)-dependent PDAC cells during Ser/Gly (glycine) deprivation. Ser deprivation resulted in ribosomal stalling on two of the six Ser codons, TCC and TCT, and allowed the selective translation and secretion of nerve growth factor (NGF) by PDAC cells to promote tumor innervation. Consistent with this, exSer-dependent PDAC tumors grew slower and displayed enhanced innervation in mice on a Ser/Gly-free diet. Blockade of compensatory neuronal innervation using LOXO-101, a Trk-NGF inhibitor, further decreased PDAC tumor growth. Our data indicate that axonal-cancer metabolic crosstalk is a critical adaptation to support PDAC growth in nutrient poor environments.

Maturation of regulatory T cells (Tregs) in peripheral sites is known to require TGFbeta exposure and inhibition of protein kinase mTORC1. It is well known that mTOR inhibition is associated with repression of cap-dependent mRNA translation, which represents the major mechanism for protein synthesis, leaving unanswered how Tregs carry-out essential translation for development and immune suppression activity. To answer this question, we performed genome-wide transcription and translation profiling in CD4⁺ CD127^dim/+ CD25⁺ Tregs derived from anti-CD3/CD28-activated human naive CD4 T cells, treated with the mTORC1 inhibitor RAD001 and/or TGFbeta. We found that TGFbeta activated both Treg differentiation and immune suppression genes, while mTORC1 inhibition selectively blocked translation of most T cell mRNAs except those induced by TGFbeta, including FOXP3, CTLA-4, CD101 or CD103, locking in Treg lineage commitment and immune suppression function. These canonical Treg fate-determining mRNAs were resistant to mTORC1 inhibition, an effect mediated in part by their 5'-untranslated regions through an alternate form of appears to be cap-dependent, eIF4E-independent mRNA translation. In conclusion, TGFbeta transcriptional reprogramming together with mTORC1-independent translational reprogramming enable a privileged translation mechanism by which activated CD4 T cells become Tregs