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Although inflammatory mechanisms driving hepatocellular carcinoma (HCC) have been proposed, the regulators of anticancer immunity in HCC remain poorly understood. We found that IL27 receptor (IL27R) signaling promotes HCC development in vivo. High IL27EBI3 cytokine or IL27RA expression correlated with poor prognosis for patients with HCC. Loss of IL27R suppressed HCC in vivo in two different models of hepatocarcinogenesis. Mechanistically, IL27R sig­naling within the tumor microenvironment restrains the cytotoxicity of innate cytotoxic lymphocytes. IL27R ablation enhanced their accumulation and activation, whereas depletion or functional impairment of innate cytotoxic cells abrogated the effect of IL27R disruption. Pharmacologic neutralization of IL27 signaling increased infiltration of innate cytotoxic lymphocytes with upregulated cytotoxic molecules and reduced HCC development. Our data reveal an unexpected role of IL27R signaling as an immunologic checkpoint regulating innate cytotoxic lymphocytes and promoting HCC of different etiologies, thus indicating a therapeutic potential for IL27 pathway blockade in HCC. Significance: HCC, the most common form of liver cancer, is characterized by a poor survival rate and limited treatment options. The discovery of a novel IL27-dependent mechanism controlling anticancer cytotoxic immune response will pave the road for new treatment options for this devastating disease. This article is highlighted in the In This Issue feature, p. 1825

Although inflammatory mechanisms driving hepatocellular carcinoma (HCC) have been proposed, the regulators of anticancer immunity in HCC remain poorly understood. We found that IL27 receptor (IL27R) signaling promotes HCC development in vivo. High IL27EBI3 cytokine or IL27RA expression correlated with poor prognosis for patients with HCC. Loss of IL27R suppressed HCC in vivo in two different models of hepatocarcinogenesis. Mechanistically, IL27R sig­naling within the tumor microenvironment restrains the cytotoxicity of innate cytotoxic lymphocytes. IL27R ablation enhanced their accumulation and activation, whereas depletion or functional impairment of innate cytotoxic cells abrogated the effect of IL27R disruption. Pharmacologic neutralization of IL27 signaling increased infiltration of innate cytotoxic lymphocytes with upregulated cytotoxic molecules and reduced HCC development. Our data reveal an unexpected role of IL27R signaling as an immunologic checkpoint regulating innate cytotoxic lymphocytes and promoting HCC of different etiologies, thus indicating a therapeutic potential for IL27 pathway blockade in HCC.Significance:HCC, the most common form of liver cancer, is characterized by a poor survival rate and limited treatment options. The discovery of a novel IL27-dependent mechanism controlling anticancer cytotoxic immune response will pave the road for new treatment options for this devastating disease.This article is highlighted in the In This Issue feature, p. 1825

Supplementary Data from IL27 Signaling Serves as an Immunologic Checkpoint for Innate Cytotoxic Cells to Promote Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the most common form of liver cancer with poor survival and limited therapeutic options. HCC development is accompanied by underlying chronic inflammation, which represents a major unifying mechanism for tumor promotion. While some tumor-promoting inflammatory mechanisms had been proposed, the identity of immune mechanisms controlling anti-cancer immunity in HCC remain poorly understood. Interleukin (IL)-27 receptor signaling plays an anti-inflammatory role in a variety of infectious and chronic inflammatory diseases. Here, using genetic and pharmacological approaches we found that IL-27 receptor (IL-27R) signaling promotes HCC development in vivo. Genetic loss of IL-27R suppressed HCC in both carcinogen-induced and non-alcoholic steatohepatitis (NASH)-driven models. Mechanistically, the pro-tumorigenic effect was mediated by an immunoregulatory role of IL-27R within the tumor microenvironment, particularly the suppression of cytotoxic Natural killer (NK) cells. Single-cell RNA analysis further established the role of IL-27R signaling in restraining cytotoxic populations of NK cells. IL-27R ablation enhanced the accumulation and activation of cytotoxic NK cells during acute liver injury and in HCC tumors, while depletion or functional impairment of NK cells abrogated the effect of genetic IL-27R disruption. Taken together, our data suggest an unexpected role of IL-27R signaling as a novel immunological checkpoint regulating innate cytotoxic cell activity and promoting development of HCC of different etiologies. Citation Format: Aleksandra M. Mazitova, Turan Aghayev, Jennifer Fang, Amiran Dzutsev, Giorgio Trinchieri, Kerry S. Campbell, Sergei I. Grivennikov, Ekaterina K. Koltsova. IL-27 signaling regulates anti-cancer immune response in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3130.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer with poor survival and limited therapeutic options. HCC has different etiologies, typically associated with viral or carcinogenic insults or fatty liver disease and underlying chronic inflammation presents as a major unifying mechanism for tumor promotion. On the other hand, mechanisms of how inflammatory response can regulate anti-cancer immunity in HCC remain incompletely understood.Interleukin (IL)-27 receptor signaling plays an anti-inflammatory role in a variety of infectious and chronic inflammatory diseases. Here, using genetic and pharmacological approaches we found that IL-27 receptor (IL-27R) signaling promotes HCC development in vivo. Genetic loss of IL-27R suppressed HCC development in both toxin/carcinogen-induced diethylnitrosamine (DEN) and non-alcoholic steatohepatitis (NASH)-driven models. Elevated expression of IL-27RA rendered poor prognosis to HCC patients. Mechanistically, the pro-tumorigenic effect was mediated by immunoregulatory role of IL-27R signaling within the tumor microenvironment, particularly the suppression of Natural killer (NK) cells. IL-27R ablation enhanced the accumulation and activation of cytotoxic NK cells during acute liver injury and in HCC tumors, while depletion of NK cells abrogated the effect of genetic IL-27R disruption.Taken together, our data suggest an unexpected role of IL-27R signaling as a novel immunological checkpoint regulating NK cell activity and promoting development of HCC of different etiologies.

Abdominal Aortic Aneurysm (AAA) is a vascular disease, where aortic wall degradation is mediated by accumulated immune cells. Though cytokines regulate the inflammatory milieu within the aortic wall, their contribution to AAA through distant alterations, particularly in the control of hematopoietic stem cells proliferation and myeloid cell differentiation remains poorly defined. Here we report an unexpected pathogenic role for interleukin-27 receptor (IL-27R) in AAA development as genetic inactivation of IL-27R protected mice from AAA induced by Angiotensin (Ang) II. The mitigation of AAA in IL-27R deficient mice is associated with a blunted accumulation of myeloid cells in suprarenal aortas due to the surprising attenuation of Ang II-induced expansion of HSCs. The loss of IL-27R engages transcriptional programs that promote HSCs quiescence and suppresses myeloid lineage differentiation, decreasing mature cell production and myeloid cell accumulation in the aorta.We, therefore, illuminate how a prominent vascular disease can be distantly driven by cytokine dependent regulation of the bone marrow precursors.

The transcription factor JUN is frequently overexpressed in multiple genetic subtypes of acute myeloid leukemia (AML); however, the functional role of JUN in AML is not well defined. Here we report that short hairpin RNA (shRNA)-mediated inhibition of JUN decreases AML cell survival and propagation in vivo. By performing RNA sequencing analysis, we discovered that JUN inhibition reduces the transcriptional output of the unfolded protein response (UPR), an intracellular signaling transduction network activated by endoplasmic reticulum (ER) stress. Specifically, we found that JUN is activated by MEK signaling in response to ER stress, and that JUN binds to the promoters of several key UPR effectors, such as XBP1 and ATF4, to activate their transcription and allow AML cells to properly negotiate ER stress. In addition, we observed that shRNA-mediated inhibition of XBP1 or ATF4 induces AML cell apoptosis and significantly extends disease latency in vivo tying the reduced survival mediated by JUN inhibition to the loss of pro-survival UPR signaling. These data uncover a previously unrecognized role of JUN as a regulator of the UPR as well as provide key new insights into the how ER stress responses contribute to AML and identify JUN and the UPR as promising therapeutic targets in this disease.

Abdominal Aortic Aneurysm (AAA) is a vascular disease, where aortic wall degradation is mediated by accumulated immune cells. Several cytokines have been suggested to play role in AAA, however the role of cytokines in immune cell accumulation and AAA progression remains poorly defined. Here we report an unexpected role of IL-27R signaling in the development of AAA. We found that in an animal model of AAA, prolonged infusion of Angiotensin (Ang) II robustly induced AAA formation in hyperlipidemic Apoe -/- and Apoe -/- Il27ra +/- mice but failed to do so in Apoe -/- Il27ra -/- mice. This mitigation of AAA formation in Apoe -/- Il27ra -/- mice was associated with a blunted accumulation of pathogenic myeloid cells in suprarenal aortas resulting from a reduction in Ang II-induced hematopoietic stem and progenitor cell (HSPCs) expansion. We found that in the absence of IL-27R signaling HSPCs were unable to fully downregulate p21 expression in response to Ang II, which impeded the ability of Apoe -/- Il27ra -/- HSPCs to proliferate. Collectively, these data demonstrate that IL-27R signaling influences AAA development by activating emergency hematopoiesis in response to elevated Ang II. Further, they provide new insights into how the immunoregulatory cytokine IL-27 drives a prominent lethal vascular disease by distant regulation of stress/emergency hematopoiesis.

Summary Although commensal flora is involved in the regulation of immunity, the interplay between cytokine signaling and microbiota in atherosclerosis remains unknown. We found that interleukin (IL)-23 and its downstream target IL-22 restricted atherosclerosis by repressing pro-atherogenic microbiota. Inactivation of IL-23-IL-22 signaling led to deterioration of the intestinal barrier, dysbiosis, and expansion of pathogenic bacteria with distinct biosynthetic and metabolic properties, causing systemic increase in pro-atherogenic metabolites such as lipopolysaccharide (LPS) and trimethylamine N-oxide (TMAO). Augmented disease in the absence of the IL-23-IL-22 pathway was mediated in part by pro-atherogenic osteopontin, controlled by microbial metabolites. Microbiota transfer from IL-23-deficient mice accelerated atherosclerosis, whereas microbial depletion or IL-22 supplementation reduced inflammation and ameliorated disease. Our work uncovers the IL-23-IL-22 signaling as a regulator of atherosclerosis that restrains expansion of pro-atherogenic microbiota and argues for informed use of cytokine blockers to avoid cardiovascular side effects driven by microbiota and inflammation.

Abdominal Aortic Aneurysm (AAA) is a vascular disease, where aortic wall degradation is in part mediated by the accumulation of immune cells leading to aortic wall rupture and bleeding which is often fatal for the patient. Smoking, age, male gender, hypertension and atherosclerosis are major risk factors, however the mechanism of AAA development is still elusive. Though cytokines regulate the inflammatory milieu within the aortic wall, their possible role in systemic regulation of this disease, particularly in the control of hematopoietic stem cell proliferation, myeloid cell differentiation and their subsequent accumulation in AAA remains poorly defined. Here we report an unexpected pathogenic role of IL-27R signaling in the development of AAA. We found that in an animal model of AAA, prolonged infusion of Angiotensin (Ang) II robustly induces AAA formation in hyperlipidemic Apoe−/− and Apoe−/−Il27ra+/− mice but not in IL-27R deficient Apoe−/− mice. This mitigation of AAA formation in Apoe−/−Il27ra−/− mice is associated with a blunted accumulation of myeloid cells in suprarenal aortas due to a reduction of Ang II-induced hematopoietic stem and progenitor cell (HSPCs) expansion. We found that the absence of IL-27R signaling engages transcriptional programs that promote HSCs self-renewal program and suppress myeloid lineage differentiation, resulting in decreased mature myeloid cell production and thus a concomitant lack of Ang II-induced myeloid cell accumulation in the suprarenal aortas. Collectively, these data demonstrate that IL-27R signaling influences AAA development by potentiating Ang II induced myelopoiesis and may represent a molecular point of therapeutic intervention in AAA.

Over 30% of acute myeloid leukemia (AML) patients do not respond to first-line chemotherapy, and a significant portion of patients that do initially respond subsequently relapse with resistant disease. These unsatisfactory outcomes indicate that AML cells either rapidly evolve or inherently possess mechanisms for evading standard chemotherapeutic approaches. Several studies have suggested that AML cells utilize pathways that regulate intracellular redox biology to promote chemotherapy resistance; however, the precise pathways governing resistance and redox biology in AML have yet to be fully determined. We recently discovered that the FOXO family of transcription factors, which have been traditionally considered tumor suppressor genes, actually support AML cell survival and the differentiation blockade. Specifically, we observed that the expression of FOXO1 and FOXO3 are significantly increased in approximately 40% of primary human AML samples (p Previous studies have shown that, in variety of cell types, FOXOs influence the intracellular redox environment by suppressing the production of reactive oxygen species (ROS). Therefore, to determine the molecular role of FOXOs in AML, we initially focused on the impact of FOXO inhibition on AML cell redox biology. Using fluorogenic probes that detect either total intracellular ROS content (CellRox) or superoxide production (MitoSox), we found that shRNA-mediated inhibition of FOXO3 did not affect total levels of intracellular ROS or superoxides. However, using a lipid peroxidation sensor (BODIPY¨ 581/591 C11), we did observe that two distinct FOXO3-targeting shRNAs increased both homeostatic and stress-induced levels of lipid peroxides in AML cells (shFOXO3-1, p=0.0004; shFOXO3-2, p=0.0023). Consistent with this, we also found that AML cells treated with a chemical inhibitor of FOXOs (AS1842856) display increased steady-state levels of intracellular lipid peroxides (p=0.0076) as well as increased signs of differentiation (CD11b and morphological changes) and death (Annexin V staining). To elucidate the importance of lipid peroxidation in AML, we evaluated how two chemical anti-oxidants, N-acetyl-L-cysteine (NAC) and butylated hydroxyanisole (BHA), impact the anti-leukemia effects and increased lipid peroxidation mediated by FOXO inhibition. From these analyses, we have observed that BHA treatment suppresses lipid peroxide production and partly blocks AML cell death induced by shRNA-mediated FOXO3 inhibition (shFOXO3-1, p=0.0001; shFOXO3-2, p Both basic and clinical studies have shown that anthracyclines such as daunorubicin (DNR) induce lipid peroxidation; however, the role of lipid peroxidation in chemotherapy effectiveness is largely unknown. We have discovered that co-treatment of AML cells with DNR and BHA (but not NAC) completely blocks the cytotoxic effects of DNR (p Collectively, these results suggest that FOXOs are critical mediators of AML progression and chemotherapy resistance by directly regulating intracellular lipid peroxide levels. Disclosures No relevant conflicts of interest to declare.