Activation of endogenous retroviruses and induction of viral mimicry by MEK1/2 inhibition in pancreatic cancer.

While pancreatic ductal adenocarcinomas (PDACs) are addicted to KRAS-activating mutations, inhibitors of downstream KRAS effectors, such as the MEK1/2 kinase inhibitor trametinib, are devoid of therapeutic effects. However, the extensive rewiring of regulatory circuits driven by the attenuation of the KRAS pathway may induce vulnerabilities of therapeutic relevance. An in-depth molecular analysis of the transcriptional and epigenomic alterations occurring in PDAC cells in the initial hours after MEK1/2 inhibition by trametinib unveiled the induction of endogenous retroviruses (ERVs) escaping epigenetic silencing, leading to the production of double-stranded RNAs and the increased expression of interferon (IFN) genes. We tracked ERV activation to the early induction of the transcription factor ELF3, which extensively bound and activated nonsilenced retroelements and synergized with IRF1 (interferon regulatory factor 1) in the activation of IFNs and IFN-stimulated genes. Trametinib-induced viral mimicry in PDAC may be exploited in the rational design of combination therapies in immuno-oncology.

Clonal dominance defines metastatic dissemination in pancreatic cancer.

Tumors represent ecosystems where subclones compete during tumor growth. While extensively investigated, a comprehensive picture of the interplay of clonal lineages during dissemination is still lacking. Using patient-derived pancreatic cancer cells, we created orthotopically implanted clonal replica tumors to trace clonal dynamics of unperturbed tumor expansion and dissemination. This model revealed the multifaceted nature of tumor growth, with rapid changes in clonal fitness leading to continuous reshuffling of tumor architecture and alternating clonal dominance as a distinct feature of cancer growth. Regarding dissemination, a large fraction of tumor lineages could be found at secondary sites each having distinctive organ growth patterns as well as numerous undescribed behaviors such as abortive colonization. Paired analysis of primary and secondary sites revealed fitness as major contributor to dissemination. From the analysis of pro- and nonmetastatic isogenic subclones, we identified a transcriptomic signature able to identify metastatic cells in human tumors and predict patients' survival.

Ether phospholipids are required for mitochondrial reactive oxygen species homeostasis.

Mitochondria are hubs where bioenergetics, redox homeostasis, and anabolic metabolism pathways integrate through a tightly coordinated flux of metabolites. The contributions of mitochondrial metabolism to tumor growth and therapy resistance are evident, but drugs targeting mitochondrial metabolism have repeatedly failed in the clinic. Our study in pancreatic ductal adenocarcinoma (PDAC) finds that cellular and mitochondrial lipid composition influence cancer cell sensitivity to pharmacological inhibition of electron transport chain complex I. Profiling of patient-derived PDAC models revealed that monounsaturated fatty acids (MUFAs) and MUFA-linked ether phospholipids play a critical role in maintaining ROS homeostasis. We show that ether phospholipids support mitochondrial supercomplex assembly and ROS production; accordingly, blocking de novo ether phospholipid biosynthesis sensitized PDAC cells to complex I inhibition by inducing mitochondrial ROS and lipid peroxidation. These data identify ether phospholipids as a regulator of mitochondrial redox control that contributes to the sensitivity of PDAC cells to complex I inhibition.

KRAS inhibition activates an actionable CD24 'don't eat me' signal in pancreas cancer.

KRAS G12C inhibitor (G12Ci) has produced encouraging, albeit modest and transient, clinical benefit in pancreatic ductal adenocarcinoma (PDAC). Identifying and targeting resistance mechanisms to G12Ci treatment is therefore crucial. To better understand the tumor biology of the KRAS G12C allele and possible bypass mechanisms, we developed a novel autochthonous KRAS G12C -driven PDAC model. Compared to the classical KRAS G12D PDAC model, the G12C model exhibit slower tumor growth, yet similar histopathological and molecular features. Aligned with clinical experience, G12Ci treatment of KRAS G12C tumors produced modest impact despite stimulating a 'hot' tumor immune microenvironment. Immunoprofiling revealed that CD24, a 'do-not-eat-me' signal, is significantly upregulated on cancer cells upon G12Ci treatment. Blocking CD24 enhanced macrophage phagocytosis of cancer cells and significantly sensitized tumors to G12Ci treatment. Similar findings were observed in KRAS G12D -driven PDAC. Our study reveals common and distinct oncogenic KRAS allele-specific biology and identifies a clinically actionable adaptive mechanism that may improve the efficacy of oncogenic KRAS inhibitor therapy in PDAC. Significance: Lack of faithful preclinical models limits the exploration of resistance mechanisms to KRAS G12C inhibitor in PDAC. We generated an autochthonous KRAS G12C -driven PDAC model, which revealed allele-specific biology of the KRAS G12C during PDAC development. We identified CD24 as an actionable adaptive mechanisms in cancer cells induced upon KRAS G12C inhibition and blocking CD24 sensitizes PDAC to KRAS inhibitors in preclinical models.

Fibroblast Growth Factor Receptor Inhibitors Reduce Adipogenesis of Orbital Fibroblasts and Enhance Myofibroblastic Differentiation in Graves' Orbitopathy.

Purpose: Orbital fibroblasts are involved in pathogenesis of Graves' orbitopathy (GO). Fibroblast growth factor (FGF) affects fibroblasts of GO. This study aims to investigate the roles of FGF and FGF receptor (FGFR) in GO.Methods: Serum FGF proteins and orbital fibroblast FGFR proteins and mRNAs were measured in GO patients and controls. Orbital fibroblasts of GO were cultured and accessed for changes in proliferation (by nuclei number and MTT), myofibroblastic differentiation (by α-SMA), and adipogenesis (by oil droplets using Oil Red O stain) under FGF1 with or without FGFR inhibitors (FGFRi).Results: Serum FGF1 and FGF2 were increased in GO patients. FGFR1 was the most abundantly expressed FGFR in GO orbital fibroblasts. FGF1 increased GO fibroblast proliferation/adipogenesis and suppressed myofibroblastic differentiation, while FGFRi reversed these effects.Conclusion: FGF signaling may be involved in GO pathogenesis. Manipulation of FGF-FGFR pathway for GO treatment is worthy of further investigation.Registration number on Clinicaltrials.gov: NCT03324022.

Immune Phenotype and Response to Neoadjuvant Therapy in Triple-Negative Breast Cancer.

PURPOSE: Increasing tumor-infiltrating lymphocytes (TIL) is associated with higher rates of pathologic complete response (pCR) to neoadjuvant therapy (NAT) in patients with triple-negative breast cancer (TNBC). However, the presence of TILs does not consistently predict pCR, therefore, the current study was undertaken to more fully characterize the immune cell response and its association with pCR. EXPERIMENTAL DESIGN: We obtained pretreatment core-needle biopsies from 105 patients with stage I-III TNBC enrolled in ARTEMIS (NCT02276443) who received NAT from Oct 22, 2015 through July 24, 2018. The tumor-immune microenvironment was comprehensively profiled by performing T-cell receptor (TCR) sequencing, programmed death-ligand 1 (PD-L1) IHC, multiplex immunofluorescence, and RNA sequencing on pretreatment tumor samples. The primary endpoint was pathologic response to NAT. RESULTS: The pCR rate was 40% (42/105). Higher TCR clonality (median = 0.2 vs. 0.1, P = 0.03), PD-L1 positivity (OR: 2.91, P = 0.020), higher CD3+:CD68+ ratio (median = 14.70 vs. 8.20, P = 0.0128), and closer spatial proximity of T cells to tumor cells (median = 19.26 vs. 21.94 µm, P = 0.0169) were associated with pCR. In a multivariable model, closer spatial proximity of T cells to tumor cells and PD-L1 expression enhanced prediction of pCR when considered in conjunction with clinical stage. CONCLUSIONS: In patients receiving NAT for TNBC, deep immune profiling through detailed phenotypic characterization and spatial analysis can improve prediction of pCR in patients receiving NAT for TNBC when considered with traditional clinical parameters.

Epithelial memory of inflammation limits tissue damage while promoting pancreatic tumorigenesis.

Inflammation is a major risk factor for pancreatic ductal adenocarcinoma (PDAC). When occurring in the context of pancreatitis, KRAS mutations accelerate tumor development in mouse models. We report that long after its complete resolution, a transient inflammatory event primes pancreatic epithelial cells to subsequent transformation by oncogenic KRAS. Upon recovery from acute inflammation, pancreatic epithelial cells display an enduring adaptive response associated with sustained transcriptional and epigenetic reprogramming. Such adaptation enables the reactivation of acinar-to-ductal metaplasia (ADM) upon subsequent inflammatory events, thereby limiting tissue damage through a rapid decrease of zymogen production. We propose that because activating mutations of KRAS maintain an irreversible ADM, they may be beneficial and under strong positive selection in the context of recurrent pancreatitis.

Oncogenic KRAS-Driven Metabolic Reprogramming in Pancreatic Cancer Cells Utilizes Cytokines from the Tumor Microenvironment.

A hallmark of pancreatic ductal adenocarcinoma (PDAC) is an exuberant stroma comprised of diverse cell types that enable or suppress tumor progression. Here, we explored the role of oncogenic KRAS in protumorigenic signaling interactions between cancer cells and host cells. We show that KRAS mutation (KRAS*) drives cell-autonomous expression of type I cytokine receptor complexes (IL2rγ-IL4rα and IL2rγ-IL13rα1) in cancer cells that in turn are capable of receiving cytokine growth signals (IL4 or IL13) provided by invading Th2 cells in the microenvironment. Early neoplastic lesions show close proximity of cancer cells harboring KRAS* and Th2 cells producing IL4 and IL13. Activated IL2rγ-IL4rα and IL2rγ-IL13rα1 receptors signal primarily via JAK1-STAT6. Integrated transcriptomic, chromatin occupancy, and metabolomic studies identified MYC as a direct target of activated STAT6 and that MYC drives glycolysis. Thus, paracrine signaling in the tumor microenvironment plays a key role in the KRAS*-driven metabolic reprogramming of PDAC. SIGNIFICANCE: Type II cytokines, secreted by Th2 cells in the tumor microenvironment, can stimulate cancer cell-intrinsic MYC transcriptional upregulation to drive glycolysis. This KRAS*-driven heterotypic signaling circuit in the early and advanced tumor microenvironment enables cooperative protumorigenic interactions, providing candidate therapeutic targets in the KRAS* pathway for this intractable disease.

Pre-existing Functional Heterogeneity of Tumorigenic Compartment as the Origin of Chemoresistance in Pancreatic Tumors.

Adaptive drug-resistance mechanisms allow human tumors to evade treatment through selection and expansion of treatment-resistant clones. Here, studying clonal evolution of tumor cells derived from human pancreatic tumors, we demonstrate that in vitro cultures and in vivo tumors are maintained by a common set of tumorigenic cells that can be used to establish clonal replica tumors (CRTs), large cohorts of animals bearing human tumors with identical clonal composition. Using CRTs to conduct quantitative assessments of adaptive responses to therapeutics, we uncovered a multitude of functionally heterogeneous subpopulations of cells with differential degrees of drug sensitivity. High-throughput isolation and deep characterization of unique clonal lineages showed genetic and transcriptomic diversity underlying functionally diverse subpopulations. Molecular annotation of gemcitabine-naive clonal lineages with distinct responses to treatment in the context of CRTs generated signatures that can predict the response to chemotherapy, representing a potential biomarker to stratify patients with pancreatic cancer.

IL-6/JAK1 pathway drives PD-L1 Y112 phosphorylation to promote cancer immune evasion.

Glycosylation of immune receptors and ligands, such as T cell receptor and coinhibitory molecules, regulates immune signaling activation and immune surveillance. However, how oncogenic signaling initiates glycosylation of coinhibitory molecules to induce immunosuppression remains unclear. Here we show that IL-6-activated JAK1 phosphorylates programmed death-ligand 1 (PD-L1) Tyr112, which recruits the endoplasmic reticulum-associated N-glycosyltransferase STT3A to catalyze PD-L1 glycosylation and maintain PD-L1 stability. Targeting of IL-6 by IL-6 antibody induced synergistic T cell killing effects when combined with anti-T cell immunoglobulin mucin-3 (anti-Tim-3) therapy in animal models. A positive correlation between IL-6 and PD-L1 expression was also observed in hepatocellular carcinoma patient tumor tissues. These results identify a mechanism regulating PD-L1 glycosylation initiation and suggest the combination of anti-IL-6 and anti-Tim-3 as an effective marker-guided therapeutic strategy.

Exosomal TGF-ß1 is correlated with lymphatic metastasis of gastric cancers.

Exosomes are nano-vesicles transporting bioactive material between cells. This study explored the prognostic association of exosomal TGF-ß1 with lymph node (LN) metastasis of gastric cancer (GC). TGF-ß1 expressions in the exosomes isolated from the gastroepiploic veins of 61 GC patients analyzed by ELISA. The regulatory T (Treg) cells in celiac LNs of gastric cancer analyzed by immunohistochemistry. Exosomal TGF-ß1 expression and the ratio of Treg cells in draining LNs were both significantly associated with pathological stages and LN metastasis of gastric cancer. Besides, the exosomal TGF-ß1 expression and Treg proportion in LN were also significantly correlated in gastric cancer patients. Recombinant TGF-ß1 and exosomes isolated from GC patients were used to induce FOXP3+ Treg cells from naïve T cells in vitro. Compared to the control, recombinant TGF-ß1 induced more CD25 (41%), FOXP3 (19%) and CTLA-4 (47%), while reduced CD45RA expression by 38% in primary naïve T cell cultures (p<0.01). Exosomes treatment induced more CD25 and 45% higher CTLA-4 expression, and increased 29% higher of CD45RA-negative cells than recombinant TGF-ß1 did (p<0.01). Adding TGF-ß1 neutralizing antibody partially abrogated the effects of exosomes on Treg induction. Our study showed exosomal TGF-ß1 related to lymph node metastasis and the ratio of Treg cells in lymph nodes of gastric cancers. Exosomes from gastric cancer patients could induce Treg cells formation through the effect of TGF-ß1.

microRNA-183 Mediates Protective Postconditioning of the Liver by Repressing Apaf-1.

AIMS: Ischemic postconditioning (iPoC) is known to mitigate ischemia-reperfusion (IR) injury of the liver, the mechanisms of which remain to be elucidated. This study explored the role of microRNA-183 (miR-183) in the protective mechanism of iPoC. RESULTS: Microarray analysis showed miR-183 was robustly expressed in rats' livers with iPoC. miR-183 repressed the mRNA expression of Apaf-1, which is an apoptosis promoting factor. Using an oxygen-glucose deprivation (OGD) injury model in Clone 9 cells, hypoxic postconditioning (HPoC) and an miR-183 mimetic significantly decreased cell death after OGD, but miR-183 inhibitors eliminated the protection of HPoC. The increased expression of Apaf-1 and the downstream activation of capsase-3/9 after OGD were mitigated by HPoC or the addition of miR-183 mimetics, whereas miR-183 inhibitor diminished the effect of HPoC on Apaf-1-caspase signaling. In the in vivo experiment, iPoC and agomiR-183 decreased the expression of serum ALT after liver IR in the mice, but antagomiR-183 mitigated the effect of iPoC. The results of hematoxylin and eosin and TUNEL staining were compatible with the biochemical assay. Moreover, iPoC and agomiR-183 decreased the expression of Apaf-1 and 4-HNE after IR injury in mouse livers, whereas the antagomiR-mediated prevention of miR-183 expression led to increased protein expression of Apaf-1 and 4-HNE in the postischemic livers. INNOVATION: Our experiment showed the first time that miR-183 was induced in protective postconditioning and reduced reperfusion injury of the livers via the targeting of apoptotic signaling. CONCLUSION: miR-183 mediated the tolerance induced by iPoC in livers via Apaf-1 repressing. Antioxid. Redox Signal. 26, 583-597.