Cytokine Leukemia Inhibitory Factor Research Articles

A mechanistic PK/PD model of AZD0171 (anti-LIF) to support Phase II dose selection.

AZD0171 (INN: Falbikitug) is being developed as a humanized monoclonal antibody (mAb), immunoglobulin G subclass 1 (IgG1), which binds specifically to the immunosuppressive human cytokine leukemia inhibitory factor (LIF) and inhibits downstream signaling by blocking recruitment of glycoprotein 130 (gp130) to the LIF receptor (LIFR) subunit (gp190) and the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and is intended to treat adult participants with advanced solid tumors. LIF is a pleiotropic cytokine (and a member of the IL-6 family of cytokines) involved in many physiological and pathological processes and is highly expressed in a subset of solid tumors, including non-small cell lung cancer (NSCLC), colon, ovarian, prostate, and pancreatic cancer. The aim of this work was to develop a mechanistic PK/PD model to investigate the effect of AZD0171 on tumor LIF levels, predict the level of downstream signaling complex (LIF:LIFR:gp130) inhibition, and examine the dose-response relationship to support dose selection for a Phase II clinical study. Modeling results show that tumor LIF is inhibited in a dose-dependent manner with >90% inhibition for 95% of patients at the Phase II clinical dose of 1500 mg Q2W.

Abstract 7465: Leukemia inhibitory factor creates an immune suppressive tumor microenvironment and fosters tumor growth in KRAS-driven pancreatic ductal adenocarcinoma

Abstract Immune checkpoint blockade revolutionized cancer treatments by activating anti-tumor immunity. Pancreatic ductal adenocarcinoma (PDA) patients have not benefited from these advances due to an immune suppressive tumor microenvironment. Identifying PDA cell-encoded factors that drive immune evasion is necessary to unlock new immunologically active treatment options in PDA. We have recently demonstrated that the cytokine Leukemia inhibitory factor (LIF) is highly upregulated in PDA compared to the normal healthy pancreas. LIF is known for its immune suppressive qualities, but data on LIF in PDA is currently limited. We generated a conditional Lif flox (L) allele and crossed it into the well-established KrasLSL-G12D/+, Trp53flox/flox, Pdx1-Cre (KPC) mouse model. LKPC mice (n=29) had longer overall survival (69 days) than control KPC mice (n=29) (59 days), (p<0.05). LKPC mice harbored significantly smaller tumors with an average tumor weight of 0.6g compared to KPC control animals with an average tumor weight of 1.0g (p<0.005). Immunohistochemical evaluation of tumor proliferation measured by Ki-67 positivity in LKPC and KPC tumors at endpoint showed comparable results. Tumor-derived cell lines from KPC and LKPC mice showed no growth differences in vitro nor when reimplanted subcutaneously (sc) in immunodeficient mice. In contrast, we observed a significant decrease in tumor growth and weight in LKPC sc tumors (p<0.05) compared to the KPC sc tumors in syngeneic mice. LKPC sc tumors had significantly fewer neutrophils and more macrophages, particularly MHC II-high macrophages, known for their anti-tumor qualities when compared to KPC sc tumors using flow cytometry. A trend towards more CD8+ T-cells in sc LKPC tumors was also observed. Our genetically engineered mouse model confirmed this finding by immunohistochemical evaluation of tumors at endpoint, which demonstrated a significant increase of CD8+ T-cells in the LKPC animals compared to the KPC controls. In summary, we find that cancer-cell-expressed LIF augments tumor growth and shortens overall survival by changing the immune tumor microenvironment, nominating leukemia inhibitory factor depletion as a promising therapeutic immune target in KRAS-driven pancreatic cancer. Citation Format: Lisa Miller-Phillips, Imene Boukhalfa Hanafi, Alexis J. Combes, Shabana Bano, Lisiena Hysenaj, Jeroen P. Roose, Adam Olshen, Ashley Kiemen, Sarah Umetsu, Man-Tzu Wang, Eric A. Collisson. Leukemia inhibitory factor creates an immune suppressive tumor microenvironment and fosters tumor growth in KRAS-driven pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7465.

An Integrative Approach to the Current Treatment of HIV-Associated Neurocognitive Disorders and the Implementation of Leukemia Inhibitor Factor as a Mediator of Neurocognitive Preservation.

HIV-associated neurocognitive disorders (HANDs) continue to impact patients despite antiretroviral therapy. A combination of antiretroviral therapies can diminish the HIV viral load to near undetectable levels, but fails to preserve neurocognitive integrity. The cytokine leukemia inhibitory factor (LIF) has shown neuroprotective properties that could mitigate neurodegeneration in HANDs. The LIF promotes neurogenesis, neural cell differentiation, and survival. Combination antiretroviral therapy reduces severe forms of HANDs, but neurocognitive impairment persists; additionally, some antiretrovirals have additional adverse neurotoxic effects. The LIF counteracts neurotoxic viral proteins and limits neural cell damage in models of neuroinflammation. Adding the LIF as an adjuvant therapy to enhance neuroprotection merits further research for managing HANDs. The successful implementation of the LIF to current therapies would contribute to achieving a better quality of life for the affected population.

Leukemia inhibitory factor protects against graft-versus-host disease while preserving graft-versus-leukemia activity

AbstractGraft-versus-host disease (GVHD) remains a major complication after allogeneic hematopoietic stem cell transplantation, a widely used therapy for hematologic malignancies and blood disorders. Here, we report an unexpected role of cytokine leukemia inhibitory factor (LIF) in protecting against GVHD development. Administrating recombinant LIF protein (rLIF) protects mice from GVHD-induced tissue damage and lethality without compromising the graft-versus-leukemia activity, which is crucial to prevent tumor relapse. We found that rLIF decreases the infiltration and activation of donor immune cells and protects intestinal stem cells to ameliorate GVHD. Mechanistically, rLIF downregulates IL-12–p40 expression in recipient dendritic cells after irradiation through activating STAT1 signaling, which results in decreased major histocompatibility complex II levels on intestinal epithelial cells and decreased donor T-cell activation and infiltration. This study reveals a previously unidentified protective role of LIF for GVHD-induced tissue pathology and provides a potential effective therapeutic strategy to limit tissue pathology without compromising antileukemic efficacy.

Abstract 1062: Antiproliferative activity of EC359, an inhibitor of leukemia inhibitory factor receptor (LIF-R), singly and in combination with chemotherapy drugs against pancreatic cancer cell lines

Abstract Although combination chemotherapy treatments such as FOLFIRINOX (5-fluorouracil, oxaliplatin, irinotecan), gem-abraxane (gemcitabine and nab-paclitaxel) and GTX (gemcitabine, docetaxel and capecitabine) have improved survival of pancreatic cancer patients incrementally, 5-year-survival for this disease remains dismal. New and more effective treatments are needed. Pancreatic stellate cells (PSCs), a form of fibroblasts that comprise much of the pancreatic tumor microenvironment, interact with the cancer cells via the cytokine leukemia inhibitory factor (LIF) and its receptor, LIF-R. This interaction contributes to tumor progression, survival, and resistance to various therapies, and is an important target for novel therapy. A small molecule inhibitor of LIF-R, EC359, inhibits growth of breast cancer cell line tumor explants in mice (Mol Cancer Ther 18: 1341-1354, 2019) and potentially modulates the activity of gemcitabine against such explants (Genes Cancer 10: 1-10, 2019). Given the involvement of the LIF axis in pancreatic cancer, EC359 was tested for its ability to inhibit proliferation of the human pancreatic cancer cell lines PANC-1, Capan-1, and Capan-2, and its possible influence on activity of chemotherapy drugs against these cell lines. Five-day drug exposures were conducted in 96-well plates. Relative cell density determined using vital stains (alamarBlue©, neutral red) was reported as a percent of the fluorescence/absorbance of control cultures. The 3 cell lines exhibited different sensitivities to EC359, with IC50 values ranging from 5 nM (Capan-1) to 150 nM (Capan-2). When combined with anti-pancreatic cancer standard-of-care drugs, EC359 yielded synergistic interactions in a concentration-dependent manner. Depending on the cell line, EC359 decreased the IC50 value for oxaliplatin by as much as 83%. Such enhancements of cytotoxicity for other drugs were up to 84% for docetaxel, 67% for paclitaxel, and 50% for irinotecan. EC359 was also synergistic with IBR120 (a novel small molecule inhibitor of RAD51) against PANC-1 cells. EC359 additively inhibited proliferation in combination with irinotecan or 5-FUdR. The nanomolar activity of EC359 against pancreatic cancer cell lines makes it a good candidate for potential treatment of this generally refractory disease. The synergistic interaction of EC359 with standard-of-care drugs provides an opportunity for increasing the therapeutic index for these agents, and ultimately improving clinical outcomes for pancreatic cancer and other cancers against which EC359 is currently in clinical trial. Citation Format: Peter J. Ferguson, Hareesh Nair, Mark D. Vincent, James Koropatnick. Antiproliferative activity of EC359, an inhibitor of leukemia inhibitory factor receptor (LIF-R), singly and in combination with chemotherapy drugs against pancreatic cancer cell lines [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 1062.

Leukaemia inhibitory factor in gastric cancer: friend or foe?

IL-6 family cytokine leukaemia inhibitory factor (LIF) study has deciphered a variety of effects, in physiology as well as pathology. Despite the sudden arousal in LIF interest in cancers, its study in the gastric cancer (GC) context has been put aside. Only few related studies can be found in literature, most of them investigating IL-6/STAT3 signalling in GC, and not the particular LIF/LIFRβ signalisation. LIF/LIFR has opposing effects depending on the signalling pathways involved. This review relates the pro- and anti-tumorigenic aspects of LIF/LIFR in GC, taking also into account facts from other types of cancer. A better understanding of these issues would undoubtedly help postulate interesting hypotheses and perspectives for future LIF/LIFR study and its use in GC therapies, where options tend to be limited in number and efficiency.

A muscle cell-macrophage axis involving matrix metalloproteinase 14 facilitates extracellular matrix remodeling with mechanical loading.

The extracellular matrix (ECM) in skeletal muscle plays an integral role in tissue development, structural support, and force transmission. For successful adaptation to mechanical loading, remodeling processes must occur. In a large cohort of older adults, transcriptomics revealed that genes involved in ECM remodeling, including matrix metalloproteinase 14 (MMP14), were the most upregulated following 14 weeks of progressive resistance exercise training (PRT). Using single‐cell RNA‐seq, we identified macrophages as a source of Mmp14 in muscle following a hypertrophic exercise stimulus in mice. In vitro contractile activity in myotubes revealed that the gene encoding cytokine leukemia inhibitory factor (LIF) is robustly upregulated and can stimulate Mmp14 expression in macrophages. Functional experiments confirmed that modulation of this muscle cell‐macrophage axis facilitated Type I collagen turnover. Finally, changes in LIF expression were significantly correlated with MMP14 expression in humans following 14 weeks of PRT. Our experiments reveal a mechanism whereby muscle fibers influence macrophage behavior to promote ECM remodeling in response to mechanical loading.

Cancer-associated adipocytes promote the invasion and metastasis in breast cancer through LIF/CXCLs positive feedback loop.

Cancer-associated adipocytes (CAAs), which are adipocytes transformed by cancer cells, are of great importance in promoting the progression of breast cancer. However, the underlying mechanisms involved in the crosstalk between cancer cells and adipocytes are still unknown. Here we report that CAAs and breast cancer cells communicate with each other by secreting the cytokines leukemia inhibitory factor (LIF) and C-X-C subfamily chemokines (CXCLs), respectively. LIF is a pro-inflammatory cytokine secreted by CAAs, which promotes migration and invasion of breast cancer cells via the Stat3 signaling pathway. The activation of Stat3 induced the secretion of glutamic acid-leucine-arginine (ELR) motif CXCLs (CXCL1, CXCL2, CXCL3 and CXCL8) in tumor cells. Interestingly, CXCLs in turn activated the ERK1/2/NF-κB/Stat3 signaling cascade to promote the expression of LIF in CAAs. In clinical breast cancer pathology samples, the up-regulation of LIF in paracancerous adipose tissue was positively correlated with the activation of Stat3 in breast cancer. Furthermore, we verified that adipocytes enhanced lung metastasis of breast cancer cells, and the combination of EC330 (targeting LIF) and SB225002 (targeting C-X-C motility chemokine receptor 2 (CXCR2)) significantly reduced lung metastasis of breast cancer cells in vivo. Our findings reveal that the interaction of adipocytes with breast cancer cells depends on a positive feedback loop between the cytokines LIF and CXCLs, which promotes breast cancer invasion and metastasis.

The effect of cytokine leukemia-inhibitory factor (LIF) and interleukin-11 (IL-11) gene expression on the primary infertility related to polycystic ovary syndrome, Tubal factor, and Unexplained infertility in Turkish women

BackgroundSuccessful implantation of blastocysts is indeed an important path in mammalian reproduction that is governed by a complicated web of cytokines interactions. Leukemia inhibitory factor (LIF) and interleukin-11 (IL-11) part of the interleukin (IL)-6 groups are cytokines that are needed for effective implantation and prevent infertility symptoms. This study aimed to determine the expression level (LIF, IL-11) genes in patients with primary infertility related to polycystic ovary syndrome (PCOS), tubal factor infertility (TFI), and unexplained infertility (UI).ResultsIn this study, 75 infertility women and 40 controls were involved. The expressions of LIF and IL-11 genes were evaluated by quantitative real-time polymerase chain reaction qRT–PCR Light Cycler in patients and healthy controls. PCOS, TFI, and UI groups showed promising results regarding LIF gene, which appeared at very small levels compared to the control (p < 0.0001). Regarding IL-11, the two groups TFI and UI were significantly linked to the lower level of gene expression, while the PCOS group has no significant difference when it is compared to the control group (p < 0.0001, < 0.05, 0.19), respectively.ConclusionThe current findings show that low levels of LIF and IL-11 gene expression are linked to various primary infertility conditions, including PCOS, tubal factor, and unexplained infertility since they play a fundamental role in embryo implantation.

LIF and bFGF enhanced chicken primordial follicle activation by Wnt/β-catenin pathway

The cytokines leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) are closely related to the development of primordial follicles. In this study, the functions and correlation of LIF and bFGF in the development of chicken primordial follicles were examined, along with the signaling pathways including protein kinase B (AKT), extracellular regulated protein kinase (ERK) and Wnt/β-catenin signaling pathways. Ovarian tissues were collected from four-day-old chicks and incubated with LIF and bFGF alone or in combination for three days to observe the changes in follicular development. Results showed that there was a time-dependent correlation between the changes in expression of LIF/its receptor (LIFR) and the developmental process of primordial follicles. LIF and bFGF exerted a synergistic effect on the activation of primordial follicles. However, SC144 (an antagonist of LIFR) inhibited this stimulating action. The effect by LIF and bFGF were shown to operate at AKT and ERK signaling pathways to suppress cell apoptosis and promote proliferation (P < 0.05) via the Wnt/β-catenin signaling (P < 0.05). In conclusion, local cytokines LIF and bFGF functioned to enhance the activation of chicken primordial follicles by increasing cell proliferation and decreasing apoptosis in the ovary involving AKT, ERK and Wnt/β-catenin signaling.

TCF/LEF regulation of the topologically associated domain ADI promotes mESCs to exit the pluripotent ground state.

Mouse embryonic stem cells (mESCs) can be maintained invitro in defined N2B27 medium supplemented with two chemical inhibitors for GSK3 and MEK (2i) and the cytokine leukemia inhibitory factor (LIF), which act synergistically to promote self-renewal and pluripotency. Here, we find that genetic deletion of the four genes encoding the TCF/LEF transcription factors confers mESCs with the ability to self-renew in N2B27 medium alone. TCF/LEF quadruple knockout (qKO) mESCs display dysregulation of several genes, including Aire, Dnmt3l, and IcosL, located adjacent to each other within a topologically associated domain (TAD). Aire, Dnmt3l, and IcosL appear to be regulated by TCF/LEF in a β-catenin independent manner. Moreover, downregulation of Aire and Dnmt3l in wild-type mESCs mimics the loss of TCF/LEF and increases mESC survival in the absence of 2iL. Hence, this study identifies TCF/LEF effectors that mediate exit from the pluripotent state.

Mutant KRAS Downregulates the Receptor for Leukemia Inhibitory Factor (LIF) to Enhance a Signature of Glycolysis in Pancreatic Cancer and Lung Cancer.

Pancreatic cancer is characterized by aberrant activity of oncogenic KRAS, which is mutated in 90% of pancreatic adenocarcinomas. Because KRAS itself is a challenging therapeutic target, we focused on understanding key signaling pathways driven by KRAS as a way to reveal dependencies that are amenable to therapeutic intervention. Analyses in primary human pancreatic cancers and model systems revealed that the receptor for the cytokine leukemia inhibitory factor (LIF) is downregulated by mutant KRAS. Furthermore, downregulation of the LIF receptor (LIFR) is necessary for KRAS-mediated neoplastic transformation. We found LIFR exerts inhibitory effects on KRAS-mediated transformation by inhibiting expression of the glucose transporter GLUT1, a key mediator of the enhanced glycolysis found in KRAS-driven malignancies. Decreased LIFR expression leads to increased GLUT1 as well as increases in glycolysis and mitochondrial respiration. The repression of GLUT1 by LIFR is mediated by the transcription factor STAT3, indicating a tumor-suppressive role for STAT3 within cancer cells with mutated KRAS. Finally, reflecting a clinically important tumor-suppressive role of LIFR, decreased LIFR expression correlates with shorter survival in pancreatic cancer patients with mutated KRAS. Similar findings were found in non-small cell lung cancers driven by mutated KRAS, suggesting that silencing LIFR is a generalized mechanism of KRAS-mediated cellular transformation. These results indicate that the LIFR/STAT3 pathway may mediate either tumor-promoting or tumor-suppressive signaling pathways depending on the genetic background of tumor cells, and may play diverse roles within other cells in the tumor microenvironment. IMPLICATIONS: Mutant KRAS drives downregulation of the receptor for LIF, thereby allowing an increase in expression of the glucose transporter GLUT1 and increases in glycolysis and mitochondrial respiration.

Cross-reactivity of two human IL-6 family cytokines OSM and LIF explored by protein-protein docking and molecular dynamics simulation

BackgroundOncostatin M (OSM) and leukemia inhibitory factor (LIF) are two important pro-inflammatory cytokines of the interleukin-6 (IL-6) family. The two cytokines mediated signaling was recently found to be closely associated with cancer and chronic inflammation, which represent promising therapeutic targets for the treatment of many solid tumors and inflammatory disease. As the most closely related members, cross-reactivity of them may result in undesired activation of off-target cells, leading to toxicity or lack of efficacy of the therapeutic effects. However, the mechanism of the cross-reactivity of OSM and LIF is not well understood. MethodsIn this work, protein-protein docking, molecular dynamics (MD) simulations with explicit solvent and post endpoints binding free energy (BFE) analysis were carried out to further understand the structural and energetic principles of interactions between the two cytokines and the shared receptor LIFR. ResultsFor the first time, the simulation given a computational model of OSM-LIFR interaction, and provided significant insights into the mechanism of OSM and LIF cross-react with LIFR. The identified common features shared by OSM and LIF bind to LIFR involving 10 “conserved” residues (90% similarity) distributed at the binding site III comprised of AB loop, BC loop and D helix. In addition, 11 shared residues were identified in LIFR contribute 77.85% and 84.63% energies for OSM and LIF binding, which play a critical role in the formation of the two cytokine-receptor complexes. Moreover, the “nonconserved” residues at the same position of cytokines such as Asp41 in OSM and Pro51 in LIF as well as the three residues (Glu338, Asn201 and Glu260) in LIFR were also discovered. ConclusionsThese important information may facilitate the rational design of novel chemical or biological agents with less toxicity and improved efficacy.

TCF/LEF Regulation of the Topologically Associated Domain &lt;i&gt;ADI&lt;/i&gt; Promotes mESCs to Exit the Pluripotent Ground State

Mouse embryonic stem cells (mESCs) can be maintained in vitro in defined N2B27 medium supplemented with two chemical inhibitors for GSK3 and MEK (2i) and the cytokine leukemia inhibitory factor (LIF), which act synergistically to promote self-renewal and pluripotency. Here, we find that genetic deletion of the four genes encoding the TCF/LEF transcription factors confers mESCs with the ability to self-renew in N2B27 medium alone. TCF/LEF quadruple knockout (qKO) mESCs display dysregulation of several genes, including Aire, Dnmt3l and IcosL, located adjacent to each other within a topologically associated domain (TAD). Aire, Dnmt3l and IcosL appear to be regulated by TCF/LEF in a s-catenin independent manner. Moreover, downregulation of Aire and Dnmt3l in wild-type mESCs mimics the loss of TCF/LEF and increases mESC survival in the absence of 2iL. Hence, this study identifies novel TCF/LEF effectors that mediate exit from the pluripotent state.

Transient Receptor Potential Ankyrin 1 (TRPA1) Is Involved in Upregulating Interleukin-6 Expression in Osteoarthritic Chondrocyte Models.

Transient receptor potential ankyrin 1 (TRPA1) is a membrane-bound ion channel found in neurons, where it mediates nociception and neurogenic inflammation. Recently, we have discovered that TRPA1 is also expressed in human osteoarthritic (OA) chondrocytes and downregulated by the anti-inflammatory drugs aurothiomalate and dexamethasone. We have also shown TRPA1 to mediate inflammation, pain, and cartilage degeneration in experimental osteoarthritis. In this study, we investigated the role of TRPA1 in joint inflammation, focusing on the pro-inflammatory cytokine interleukin-6 (IL-6). We utilized cartilage/chondrocytes from wild-type (WT) and TRPA1 knockout (KO) mice, along with primary chondrocytes from OA patients. The results show that TRPA1 regulates the synthesis of the OA-driving inflammatory cytokine IL-6 in chondrocytes. IL-6 was highly expressed in WT chondrocytes, and its expression, along with the expression of IL-6 family cytokines leukemia inhibitory factor (LIF) and IL-11, were significantly downregulated by TRPA1 deficiency. Furthermore, treatment with the TRPA1 antagonist significantly downregulated the expression of IL-6 in chondrocytes from WT mice and OA patients. The results suggest that TRPA1 is involved in the upregulation of IL-6 production in chondrocytes. These findings together with previous results on the expression and functions of TRPA1 in cellular and animal models point to the role of TRPA1 as a potential mediator and novel drug target in osteoarthritis.

Effects of Dimethyl Sulfoxide on the Pluripotency and Differentiation Capacity of Mouse Embryonic Stem Cells.

Mouse embryonic stem cells (mESCs) go through self-renewal in the existence of the cytokine leukemia inhibitory factor (LIF). LIF is added to the mouse stem cells culture medium, and its removal results in fast differentiation. Dimethyl sulfoxide (DMSO) is one of the most used solvents in drug test. We exposed 4-day mESC cultures to different concentrations of DMSO (0.1%, 0.5%, 1.0%, and 2.0%) to identify the safest dose exhibiting efficacy as a solvent. mESCs grown under general pluripotency conditions in the absence of LIF were treated with DMSO. In addition, as a control for differentiation, mESCs were grown in the absence of LIF. DMSO upregulated the mRNA expression level of pluripotency markers. Moreover, DMSO reduced the mRNA expression levels of ectodermal marker (β-tubulin3), mesodermal marker (Hand1), and endodermal markers (Foxa2 and Sox17) in mESCs. These results indicate that DMSO treatment enhances the pluripotency and disrupts the differentiation of mESCs. We also show that members of the Tet oncogene family are critical to inhibiting the differentiation and methylation of mESCs. DMSO is appropriate to sustain the pluripotency of mESCs in the absence of LIF, and that mESCs can be sustained in an undifferentiated state using DMSO. Therefore, DMSO may, in part, function as a substitute for LIF.

JAK Inhibitors Suppress Cancer Cachexia-Associated Anorexia and Adipose Wasting in Mice.

Cachexia, a syndrome of muscle atrophy, adipose loss, and anorexia, is associated with reduced survival in cancer patients. The colon adenocarcinoma C26c20 cell line secretes the cytokine leukemia inhibitory factor (LIF) which induces cachexia. We characterized how LIF promotes cachexia-associated weight loss and anorexia in mice through JAK-dependent changes in adipose and hypothalamic tissues. Cachexia was induced in vivo with the heterotopic allotransplanted administration of C26c20 colon adenocarcinoma cells or the intraperitoneal administration of recombinant LIF in the absence or presence of JAK inhibitors. Blood, adipose, and hypothalamic tissues were collected and processed for cyto/adipokine ELISAs, immunoblot analysis, and quantitative RT-PCR. Cachexia-associated lipolysis was induced in vitro by stimulating differentiated adipocytes with recombinant LIF or IL-6 in the absence or presence of lipase or JAK inhibitors. These adipocytes were processed for glycerol release into the media, immunoblot analysis, and RT-PCR. Tumor-secreted LIF induced changes in adipose tissue expression and serum levels of IL-6 and leptin in a JAK-dependent manner influencing cachexia-associated adipose wasting and anorexia. We identified two JAK inhibitors that block IL-6 family-mediated adipocyte lipolysis and IL-6 induction using an in vitro cachexia lipolysis assay. JAK inhibitors administered to the in vivo C26c20 cancer cachexia mouse models led to 1) a decrease in STAT3 phosphorylation in hypothalamic and adipose tissues, 2) a reverse in the cachexia serum cyto/adipokine signature, 3) a delay in cancer cachexia-associated anorexia and adipose loss, and 4) an improvement in overall survival. JAK inhibitors suppress LIF-associated adipose loss and anorexia in both in vitro and in vivo models of cancer cachexia.

Binary Colloidal Crystal (BCC) Substrates for Controlling the Fate of Mouse Embryonic Stem Cells

Understanding the interactions of stem cells with surface topography can give us an invaluable tool in controlling stemness and fate of stem cells for further use in biomedical applications. In this study, we have fabricated topographical features using a class of cell culture substrates called binary colloidal crystals (BCCs), that are made by self-assembly of mixtures of spherical micron sized silica (Si) and nanometer sized polystyrene (PS) or poly (methyl methacrylate) (PMMA) particles. The substrates formed are arrays of ordered, hexagonally packed large Si particles inter-dispersed with the PS particles that are stabilized by gentle heating, which melts the PS or PMMA forming substrates suitable for cell culture. BCC substrates were used for culture of mouse embryonic stem cells (mESCs). Compared to tissue culture plates, COM1 (Si5-PMMA0.4), COM2 (Si5-PS0.4) and COM4 (Si2-PSC0.22) have shown to provide a better support for mESC proliferation in the presence of the cytokine leukemia inhibitory factor (LIF). The behavior of mESCs with the BCCs in presence and absence of LIF, was further explored and it was found that interaction of mESCs with the culture substrate can be controlled by tuning surface topography and roughness, which is determined by the size and type of particles used in making BCCs. Furthermore, it was shown that limiting cell-surface interactions and controlling colony shape can promote stemness maintenance on COM1 and COM2 substrates as indicated by better proliferation and higher expression of pluripotency genes including Nanog both in presence and in absence of LIF. Together with higher expression of GATA6 gene, it can be stated that these surfaces can be used for endodermic priming of mESCs. Therefore, we believe that these surfaces, especially COM1 and COM2 surfaces can be beneficial as stem cell culture systems for further use in biomedical research.

Leukemia inhibitory factor regulates the activation of inflammatory signals in macrophages and trophoblast cells

The pleiotropic cytokine leukemia inhibitory factor (LIF) is a key gestational factor known to establish dynamic cellular and molecular cross talk at the feto–maternal interface. Previously, we described the regulatory role of the LIF–trophoblast–IL10 axis in the process of macrophage deactivation in response to pro-inflammatory cytokines. However, the direct regulatory effects of LIF in macrophage and trophoblast cell function remains elusive. In this study, we aimed to examine whether and how LIF regulates the behavior of macrophages and trophoblast cells in response to pro-inflammatory stress factors. We found that LIF modulated the activating effects of interferon-gamma (IFNγ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in macrophages and trophoblast cells by reducing the phosphorylation levels of signal transducer and activator of transcription-1 (Stat1) and -5 (Stat5). Cell activation with IFNγ inhibited cell invasion and migration but this immobilizing effect was abrogated when macrophages and trophoblast cells were deactivated with LIF; macrophage cell motility restitution could in part be explained by the positive effects of LIF in Stat3 activation and matrix metalloproteinase 9 (MMP-9) expression. Pharmacological inhibition of Stat1 and Stat3 indicated that IFNγ-induced Stat1 activation mediated macrophage motility inhibition, and that cell motility in IFNγ-activated macrophages is restored via LIF-induced Stat3 activation and Stat1 inhibition. Moreover, IFNγ-induced TNFα gene expression was also abrogated by LIF through Stat1 inhibition and Stat3 activation. Finally, we have found that cell invasion of trophoblast cells is inhibited when they were cocultured with GM-CSF-differentiated, IFNγ-stimulated macrophages. This effect, however, was inhibited when macrophages were exposed to LIF. Overall, this in vitro study reveals for the first time the anti-inflammatory and pro-gestational activities of LIF by acting directly on macrophages and trophoblast cells.

Abstract B32: Mutant KRAS downregulates LIFR to enhance glycolysis in pancreatic cancer

Abstract One of the hallmarks of pancreatic cancer is aberrant activity of oncogenic KRAS, which is mutated and constitutively activated in greater than 90% of pancreatic adenocarcinomas. While KRAS itself is a challenging therapeutic target, we have focused on understanding the key signaling pathways driven by KRAS, which may reveal dependencies that are amenable to therapeutic intervention. By performing bioinformatics analysis on primary human pancreatic cancers coupled with validation in cancer cell lines, we found that the receptor for the cytokine leukemia inhibitory factor (LIF) was downregulated by mutant KRAS. Furthermore, downregulation of the LIF receptor (LIFR) was necessary for KRAS-mediated neoplastic transformation. One of the key features of KRAS-driven cancer is enhanced glycolysis. We found LIFR exerts inhibitory effects on KRAS-mediated transformation by inhibiting expression of the glucose transporter GLUT1, a key mediator of the enhanced glycolysis found in KRAS-driven malignancies. Interestingly, this downregulation of GLUT1 by LIFR is mediated by the transcription factor STAT3, indicating a tumor-suppressive role for STAT3 within cancer cells with mutated KRAS. Finally, reflecting a clinically important tumor-suppressive role of LIFR, decreased LIFR expression correlates with shorter survival in pancreatic cancer patients with mutated KRAS. Similar findings were found in non-small cell lung cancers driven by mutated KRAS, suggesting that silencing LIFR is a generalized mechanism of KRAS-mediated cellular transformation. These findings highlight the intriguing concept that the LIFR/STAT3 pathway may mediate either tumor-promoting or tumor-suppressive signaling pathways, depending on cellular genetic background and specific cell types, even within one cancerous tissue. For example, while LIF in the tumor microenvironment may act on pancreatic stellate cells to promote dense stromal formation, activation of LIFR/STAT3 within pancreatic cancer cells limits cell survival. The inhibition of LIFR expression in pancreatic cancer cells upregulates GLUT1 levels in order to support the enhanced glycolysis necessary for malignant transformation. This selective silencing of LIFR within cancer cells allows the pancreatic cancer cells to evade the inhibitory effects of LIF, while other cells in the tumor microenvironment have increased function in the presence of LIF. Therefore, the elucidation of the complex role of the LIF/LIFR/STAT3 axis in pancreatic cancer is essential to allow the design of rational therapeutic interventions for patients with pancreatic cancer. Citation Format: Suhu Liu, Helen I. Gandler, Sarah R. Walker, Darwin Q. Ye, Zachary T. Giaccone, Jonathan A. Nowak, Andrew J. Aguirre, Brian M. Wolpin, David A. Frank. Mutant KRAS downregulates LIFR to enhance glycolysis in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B32.