Inhibiting stromal Class I HDACs curbs pancreatic cancer progression.

Oncogenic lesions in pancreatic ductal adenocarcinoma (PDAC) hijack the epigenetic machinery in stromal components to establish a desmoplastic and therapeutic resistant tumor microenvironment (TME). Here we identify Class I histone deacetylases (HDACs) as key epigenetic factors facilitating the induction of pro-desmoplastic and pro-tumorigenic transcriptional programs in pancreatic stromal fibroblasts. Mechanistically, HDAC-mediated changes in chromatin architecture enable the activation of pro-desmoplastic programs directed by serum response factor (SRF) and forkhead box M1 (FOXM1). HDACs also coordinate fibroblast pro-inflammatory programs inducing leukemia inhibitory factor (LIF) expression, supporting paracrine pro-tumorigenic crosstalk. HDAC depletion in cancer-associated fibroblasts (CAFs) and treatment with the HDAC inhibitor entinostat (Ent) in PDAC mouse models reduce stromal activation and curb tumor progression. Notably, HDAC inhibition (HDACi) enriches a lipogenic fibroblast subpopulation, a potential precursor for myofibroblasts in the PDAC stroma. Overall, our study reveals the stromal targeting potential of HDACi, highlighting the utility of this epigenetic modulating approach in PDAC therapeutics.

A super-enhancer-regulated RNA-binding protein cascade drives pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy in need of new therapeutic options. Using unbiased analyses of super-enhancers (SEs) as sentinels of core genes involved in cell-specific function, here we uncover a druggable SE-mediated RNA-binding protein (RBP) cascade that supports PDAC growth through enhanced mRNA translation. This cascade is driven by a SE associated with the RBP heterogeneous nuclear ribonucleoprotein F, which stabilizes protein arginine methyltransferase 1 (PRMT1) to, in turn, control the translational mediator ubiquitin-associated protein 2-like. All three of these genes and the regulatory SE are essential for PDAC growth and coordinately regulated by the Myc oncogene. In line with this, modulation of the RBP network by PRMT1 inhibition reveals a unique vulnerability in Myc-high PDAC patient organoids and markedly reduces tumor growth in male mice. Our study highlights a functional link between epigenetic regulation and mRNA translation and identifies components that comprise unexpected therapeutic targets for PDAC.

Inhibiting Stromal Class I HDACs Curbs Pancreatic Cancer Progression.

Oncogenic lesions in pancreatic ductal adenocarcinoma (PDAC) hijack the epigenetic machinery in stromal components to establish a desmoplastic and therapeutic resistant tumor microenvironment (TME). Here we identify Class I histone deacetylases (HDACs) as key epigenetic factors facilitating the induction of pro-desmoplastic and pro-tumorigenic transcriptional programs in pancreatic stromal fibroblasts. Mechanistically, HDAC-mediated changes in chromatin architecture enable the activation of pro-desmoplastic programs directed by serum response factor (SRF) and forkhead box M1 (FOXM1). HDACs also coordinate fibroblast pro-inflammatory programs inducing leukemia inhibitory factor (LIF) expression, supporting paracrine pro-tumorigenic crosstalk. HDAC depletion in cancer-associated fibroblasts (CAFs) and treatment with the HDAC inhibitor entinostat (Ent) in PDAC mouse models reduce stromal activation and curb tumor progression. Notably, HDAC inhibition (HDACi) enriches a lipogenic fibroblast subpopulation, a potential precursor for myofibroblasts in the PDAC stroma. Overall, our study reveals the stromal targeting potential of HDACi, highlighting the utility of this epigenetic modulating approach in PDAC therapeutics.

Stromal cues regulate the pancreatic cancer epigenome and metabolome.

A fibroinflammatory stromal reaction cooperates with oncogenic signaling to influence pancreatic ductal adenocarcinoma (PDAC) initiation, progression, and therapeutic outcome, yet the mechanistic underpinning of this crosstalk remains poorly understood. Here we show that stromal cues elicit an adaptive response in the cancer cell including the rapid mobilization of a transcriptional network implicated in accelerated growth, along with anabolic changes of an altered metabolome. The close overlap of stroma-induced changes in vitro with those previously shown to be regulated by oncogenic Kras in vivo suggests that oncogenic Kras signaling-a hallmark and key driver of PDAC-is contingent on stromal inputs. Mechanistically, stroma-activated cancer cells show widespread increases in histone acetylation at transcriptionally enhanced genes, implicating the PDAC epigenome as a presumptive point of convergence between these pathways and a potential therapeutic target. Notably, inhibition of the bromodomain and extraterminal (BET) family of epigenetic readers, and of Bromodomain-containing protein 2 (BRD2) in particular, blocks stroma-inducible transcriptional regulation in vitro and tumor progression in vivo. Our work suggests the existence of a molecular "AND-gate" such that tumor activation is the consequence of mutant Kras and stromal cues, providing insight into the role of the tumor microenvironment in the origin and treatment of Ras-driven tumors.

The 4E-BP-eIF4E axis promotes rapamycin-sensitive growth and proliferation in lymphocytes.

Rapamycin has been used as a clinical immunosuppressant for many years; however, the molecular basis for its selective effects on lymphocytes remains unclear. We investigated the role of two canonical effectors of the mammalian target of rapamycin (mTOR): ribosomal S6 kinases (S6Ks) and eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs). S6Ks are thought to regulate cell growth (increase in cell size), and 4E-BPs are thought to control proliferation (increase in cell number), with mTORC1 signaling serving to integrate these processes. However, we found that the 4E-BP-eIF4E signaling axis controlled both the growth and proliferation of lymphocytes, processes for which the S6Ks were dispensable. Furthermore, rapamycin disrupted eIF4E function selectively in lymphocytes, which was due to the increased abundance of 4E-BP2 relative to that of 4E-BP1 in these cells and the greater sensitivity of 4E-BP2 to rapamycin. Together, our findings suggest that the 4E-BP-eIF4E axis is uniquely rapamycin-sensitive in lymphocytes and that this axis promotes clonal expansion of these cells by coordinating growth and proliferation.

New frontiers in translational control of the cancer genome.

The past several years have seen dramatic leaps in our understanding of how gene expression is rewired at the translation level during tumorigenesis to support the transformed phenotype. This work has been driven by an explosion in technological advances and is revealing previously unimagined regulatory mechanisms that dictate functional expression of the cancer genome. In this Review we discuss emerging trends and exciting new discoveries that reveal how this translational circuitry contributes to specific aspects of tumorigenesis and cancer cell function, with a particular focus on recent insights into the role of translational control in the adaptive response to oncogenic stress conditions.

Differential Requirements for eIF4E Dose in Normal Development and Cancer.

eIF4E, the major cap-binding protein, has long been considered limiting for translating the mammalian genome. However, the eIF4E dose requirement at an organismal level remains unexplored. By generating an Eif4e haploinsufficient mouse, we found that a 50% reduction in eIF4E expression, while compatible with normal development and global protein synthesis, significantly impeded cellular transformation. Genome-wide translational profiling uncovered a translational program induced by oncogenic transformation and revealed a critical role for the dose of eIF4E, specifically in translating a network of mRNAs enriched for a unique 5' UTR signature. In particular, we demonstrate that the dose of eIF4E is essential for translating mRNAs that regulate reactive oxygen species, fueling transformation and cancer cell survival in vivo. Our findings indicate eIF4E is maintained at levels in excess for normal development that are hijacked by cancer cells to drive a translational program supporting tumorigenesis.

Myc and mTOR converge on a common node in protein synthesis control that confers synthetic lethality in Myc-driven cancers.

Myc is one of the most commonly deregulated oncogenes in human cancer, yet therapies directly targeting Myc hyperactivation are not presently available in the clinic. The evolutionarily conserved function of Myc in modulating protein synthesis control is critical to the Myc oncogenic program. Indeed, enhancing the protein synthesis capacity of cancer cells directly contributes to their survival, proliferation, and genome instability. Therefore, inhibiting enhanced protein synthesis may represent a highly relevant strategy for the treatment of Myc-dependent human cancers. However, components of the translation machinery that can be exploited as therapeutic targets for Myc-driven cancers remain poorly defined. Here, we uncover a surprising and important functional link between Myc and mammalian target of rapamycin (mTOR)-dependent phosphorylation of eukaryotic translation initiation factor 4E binding protein-1 (4EBP1), a master regulator of protein synthesis control. Using a pharmacogenetic approach, we find that mTOR-dependent phosphorylation of 4EBP1 is required for cancer cell survival in Myc-dependent tumor initiation and maintenance. We further show that a clinical mTOR active site inhibitor, which is capable of blocking mTOR-dependent 4EBP1 phosphorylation, has remarkable therapeutic efficacy in Myc-driven hematological cancers. Additionally, we demonstrate the clinical implications of these results by delineating a significant link between Myc and mTOR-dependent phosphorylation of 4EBP1 and therapeutic response in human lymphomas. Together, these findings reveal that an important mTOR substrate is found hyperactivated downstream of Myc oncogenic activity to promote tumor survival and confers synthetic lethality, thereby revealing a unique therapeutic approach to render Myc druggable in the clinic.

GLI1 is regulated through Smoothened-independent mechanisms in neoplastic pancreatic ducts and mediates PDAC cell survival and transformation.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by the deregulation of the hedgehog signaling pathway. The Sonic Hedgehog ligand (Shh), absent in the normal pancreas, is highly expressed in pancreatic tumors and is sufficient to induce neoplastic precursor lesions in mouse models. We investigated the mechanism of Shh signaling in PDAC carcinogenesis by genetically ablating the canonical bottleneck of hedgehog signaling, the transmembrane protein Smoothened (Smo), in the pancreatic epithelium of PDAC-susceptible mice. We report that multistage development of PDAC tumors is not affected by the deletion of Smo in the pancreas, demonstrating that autocrine Shh-Ptch-Smo signaling is not required in pancreatic ductal cells for PDAC progression. However, the expression of Gli target genes is maintained in Smo-negative ducts, implicating alternative means of regulating Gli transcription in the neoplastic ductal epithelium. In PDAC tumor cells, we find that Gli transcription is decoupled from upstream Shh-Ptch-Smo signaling and is regulated by TGF-beta and KRAS, and we show that Gli1 is required both for survival and for the KRAS-mediated transformed phenotype of cultured PDAC cancer cells.

Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy.

The tumor microenvironment consists of stromal cells and extracellular factors that evolve in parallel with carcinoma cells. To gain insights into the activities of stromal cell populations, we developed and applied multicolor imaging techniques to analyze the behavior of these cells within different tumor microenvironments in the same live mouse. We found that regulatory T-lymphocytes (Tregs) migrated in proximity to blood vessels. Dendritic-like cells, myeloid cells and carcinoma-associated fibroblasts all exhibited higher motility in the microenvironment at the tumor periphery than within the tumor mass. Since oxygen levels differ between tumor microenvironments, we tested if acute hypoxia could account for the differences in cell migration. Direct visualization revealed that Tregs ceased migration under acute systemic hypoxia, whereas myeloid cells continued migrating. In the same mouse and microenvironment, we experimentally subdivided the myeloid cell population and revealed that uptake of fluorescent dextran defined a low-motility subpopulation expressing markers of tumor-promoting, alternatively activated macrophages. In contrast, fluorescent anti-Gr1 antibodies marked myeloid cells patrolling inside tumor vessels and in the stroma. Our techniques allow real-time combinatorial analysis of cell populations based on spatial location, gene expression, behavior and cell surface molecules within intact tumors. The techniques are not limited to investigations in cancer, but could give new insights into cell behavior more broadly in development and disease.

Distinct structural requirements of GATA-3 for the regulation of thymocyte and Th2 cell differentiation.

GATA-3, the only T cell-specific member of the GATA family of transcription factors, is essential for the intrathymic development of CD4+ T cells and for the differentiation of Th2 cells. However, whether distinct biochemical features, unique to GATA-3 compared with other GATA family members, are required to drive T cell transcriptional programs or whether the T cell-specific functions of GATA-3 can simply be ascribed to its expression pattern is unclear. Nor do we understand the protein structural requirements for each individual function of GATA-3. In this study, we report that a heterologous GATA factor, GATA-4, was competent in supporting the development of CD4+ T cells but could not fully compensate for GATA-3 in regulating the expression of Th cytokines. Specifically, GATA-3 was more potent than GATA-4 in driving the production of IL-13 due to a mechanism independent of DNA binding or chromatin remodeling of the IL-13 locus. The difference was mapped to a partially conserved region C-terminal to the second zinc finger. Converting a single proline residue located in this region of GATA-4 to its counterpart, a methionine of GATA-3, was sufficient to enhance the IL-13-promoting function of GATA-4 but had no effect on other cytokines. Taken together, our data demonstrate that the unique function of GATA-3 is conferred by both its cell type-specific expression and distinct protein structure.

GATA-3 deficiency abrogates the development and maintenance of T helper type 2 cells.

T helper type 2 (Th2) cells secrete IL-4, IL-5, IL-10, and IL-13 and mediate allergic and asthmatic disease. GATA-3 is a Th2-specific transcription factor that appears in overexpression studies and transgenic systems to function as a Th2 lineage determinant. Because GATA-3 is also crucial for development of the T lineage and throughout thymic development, direct demonstration that GATA-3 is required for Th2 development by targeted deletion has been lacking. Using a conditional knockout approach, we found that GATA-3 is required for optimal Th2 cytokine production in vitro and in vivo. Our data also show that GATA-3 expression must be sustained to maintain the Th2 phenotype.

Critical roles for transcription factor GATA-3 in thymocyte development.

The transcription factor GATA-3 is expressed at every stage of thymic development, but its role in thymocyte differentiation is unknown. The fact that RAG chimeric animals lacking GATA-3 cannot generate early thymocytes from common lymphoid progenitors has thus far precluded investigation of the function of GATA-3 in the thymus. To address this, we generated mice deficient in GATA-3 at early and late stages of thymic differentiation. Our studies revealed that GATA-3 is involved in beta selection and is indispensable for single-positive CD4 thymocyte development. Thus, our data demonstrate that the coordinated and regulated expression of GATA-3 at each stage of thymic development is critical for the generation of mature T cells.