Increased adipose tissue lipogenesis is associated with enhanced insulin sensitivity. Mice overexpressing the Glut4 glucose transporter in adipocytes have elevated lipogenesis and increased glucose ...tolerance despite being obese with elevated circulating fatty acids. Lipidomic analysis of adipose tissue revealed the existence of branched fatty acid esters of hydroxy fatty acids (FAHFAs) that were elevated 16- to 18-fold in these mice. FAHFA isomers differ by the branched ester position on the hydroxy fatty acid (e.g., palmitic-acid-9-hydroxy-stearic-acid, 9-PAHSA). PAHSAs are synthesized in vivo and regulated by fasting and high-fat feeding. PAHSA levels correlate highly with insulin sensitivity and are reduced in adipose tissue and serum of insulin-resistant humans. PAHSA administration in mice lowers ambient glycemia and improves glucose tolerance while stimulating GLP-1 and insulin secretion. PAHSAs also reduce adipose tissue inflammation. In adipocytes, PAHSAs signal through GPR120 to enhance insulin-stimulated glucose uptake. Thus, FAHFAs are endogenous lipids with the potential to treat type 2 diabetes.
Display omitted
•Lipidomics identifies bioactive fatty acid esters of hydroxyl-fatty acids (FAHFAs)•FAHFAs are low in insulin-resistant humans and correlate with insulin sensitivity•These lipids improve insulin secretion and glucose tolerance in mice•FAHFAs block inflammatory cytokine production and reduce adipose inflammation
A new class of lipids that are low in insulin-resistant humans promotes glucose tolerance and ameliorates inflammation associated with obesity in mouse models.
The success of targeted or immune therapies is often hampered by the emergence of resistance and/or clinical benefit in only a subset of patients. We hypothesized that combining targeted therapy with ...immune modulation would show enhanced antitumor responses. Here, we explored the combination potential of erdafitinib, a fibroblast growth factor receptor (FGFR) inhibitor under clinical development, with PD-1 blockade in an autochthonous FGFR2
/p53
lung cancer mouse model. Erdafitinib monotherapy treatment resulted in substantial tumor control but no significant survival benefit. Although anti-PD-1 alone was ineffective, the erdafitinib and anti-PD-1 combination induced significant tumor regression and improved survival. For both erdafitinib monotherapy and combination treatments, tumor control was accompanied by tumor-intrinsic, FGFR pathway inhibition, increased T-cell infiltration, decreased regulatory T cells, and downregulation of PD-L1 expression on tumor cells. These effects were not observed in a KRAS
-mutant genetically engineered mouse model, which is insensitive to FGFR inhibition, indicating that the immune changes mediated by erdafitinib may be initiated as a consequence of tumor cell killing. A decreased fraction of tumor-associated macrophages also occurred but only in combination-treated tumors. Treatment with erdafitinib decreased T-cell receptor (TCR) clonality, reflecting a broadening of the TCR repertoire induced by tumor cell death, whereas combination with anti-PD-1 led to increased TCR clonality, suggesting a more focused antitumor T-cell response. Our results showed that the combination of erdafitinib and anti-PD-1 drives expansion of T-cell clones and immunologic changes in the tumor microenvironment to support enhanced antitumor immunity and survival.
Protein-tyrosine phosphatase 1B (PTP1B) regulates food intake (FI) and energy expenditure (EE) by inhibiting leptin signaling in the hypothalamus. In peripheral tissues, PTP1B regulates insulin ...signaling, but its effects on CNS insulin action are largely unknown. Mice harboring a whole-brain deletion of the gene encoding PTP1B (Ptpn1) are lean, leptin-hypersensitive, and resistant to high fat diet-induced (HFD-induced) obesity. Arcuate proopiomelanocortin (POMC) neuron-specific deletion of Ptpn1 causes a similar, but much milder, phenotype, suggesting that PTP1B also acts in other neurons to regulate metabolism. Steroidogenic factor-1-expressing (SF-1-expressing) neurons in the ventromedial hypothalamus (VMH) play an important role in regulating body weight, FI, and EE. Surprisingly, Ptpn1 deletion in SF-1 neurons caused an age-dependent increase in adiposity in HFD-fed female mice. Although leptin sensitivity was increased and FI was reduced in these mice, they had impaired sympathetic output and decreased EE. Immunohistochemical analysis showed enhanced leptin and insulin signaling in VMH neurons from mice lacking PTP1B in SF-1 neurons. Thus, in the VMH, leptin negatively regulates FI, promoting weight loss, whereas insulin suppresses EE, leading to weight gain. Our results establish a novel role for PTP1B in regulating insulin action in the VMH and suggest that increased insulin responsiveness in SF-1 neurons can overcome leptin hypersensitivity and enhance adiposity.
Tumor-associated macrophages (TAM) play an important role in maintaining the immunosuppressive state of the tumor microenvironment (TME). High levels of CD163+ TAMs specifically are associated with ...poor prognosis in many solid tumor types. Targeting TAMs may represent a key approach in development of the next generation of cancer immune therapeutics. Members of the leukocyte immunoglobulin-like receptor B (LILRB) family, including LILRB2 (ILT4), are known to transmit inhibitory signals in macrophages and other myeloid cells. Leveraging bulk and single cell RNA-sequencing datasets, as well as extensive immunophenotyping of human tumors, we found that LILRB2 is highly expressed on CD163+ CD11b+ cells in the TME and that LILRB2 expression correlates with CD163 expression across many tumor types. To target LILRB2, we have developed JTX-8064, a highly potent and selective antagonistic mAb. JTX-8064 blocks LILRB2 binding to its cognate ligands, including classical and nonclassical MHC molecules. In vitro, JTX-8064 drives the polarization of human macrophages and dendritic cells toward an immunostimulatory phenotype. As a result, human macrophages treated with a LILRB2 blocker are reprogrammed to increase the activation of autologous T cells in co-culture systems. Furthermore, JTX-8064 significantly potentiates the activity of anti-PD-1 in allogeneic mixed lymphocyte reaction. In a human tumor explant culture, pharmacodynamic activity of JTX-8064 was observed in monotherapy and in combination with anti-PD-1. Collectively, our work provides strong translational and preclinical rationale to target LILRB2 in cancer.
We developed a screening assay in which luciferized ID8 expressing OVA was cocultured with transgenic CD8
T cells specifically recognizing the model antigen in an H-2b-restricted manner. The assay ...was screened with a small-molecule library to identify compounds that inhibit or enhance T cell-mediated killing of tumor cells. Erlotinib, an EGFR inhibitor, was the top compound that enhanced T-cell killing of tumor cells. Subsequent experiments with erlotinib and additional EGFR inhibitors validated the screen results. EGFR inhibitors increased both basal and IFNγ-induced MHC class-I presentation, which enhanced recognition and lysis of tumor cell targets by CD8
cytotoxic T lymphocytes. The ID8 cell line was also transduced to constitutively express Cas9, and a pooled CRISPR screen, utilizing the same target tumor cell/T-cell assay, identified single-guide (sg)RNAs targeting
that sensitized tumor cells to T cell-mediated killing. Combination of PD-1 blockade with EGFR inhibition showed significant synergistic efficacy in a syngeneic model, further validating EGFR inhibitors as immunomodulatory agents that enhance checkpoint blockade. This assay can be screened in high-throughput with small-molecule libraries and genome-wide CRISPR/Cas9 libraries to identify both compounds and target genes, respectively, that enhance or inhibit T-cell recognition and killing of tumor cells. Retrospective analyses of squamous-cell head and neck cancer (SCCHN) patients treated with the combination of afatinib and pembrolizumab demonstrated a rate of clinical activity exceeding that of each single agent. Prospective clinical trials evaluating the combination of an EGFR inhibitor and PD-1 blockade should be conducted.
Abstract
Introduction: Jounce has generated cell type-specific gene signatures as a means of probing The Cancer Genome Atlas and other large datasets to identify targets that may be important immune ...checkpoints. Using a tumor-associated macrophage (TAM) gene signature, we have found a strong correlation and coherence between TAMs and LILRB2 (leukocyte immunoglobulin like receptor B2; ILT4) across multiple tumors types. LILRB2 is a myeloid cell surface receptor containing four extracellular immunoglobulin domains, a transmembrane domain, and three cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Ligation of LILRB2 on myeloid cells, via its endogenous ligands (classical MHC I molecules e.g. HLA-A, HLA-B and non-classical MHC I molecules e.g. HLA-G), provides a negative signal that inhibits stimulation of an immune response. HLA-G is recognized as an important immunosuppressive molecule playing a role in maternal-fetal tolerance and being overexpressed in cancer - often associated with advanced disease stage and poor prognosis. As tumor-associated macrophages are known to suppress the anti-cancer immune response, these findings provide rationale for targeting LILRB2.
Methods and Results: We have generated a panel of monoclonal antibodies that bind specifically to LILRB2, but not other LILR family members, and can block binding of LILRB2 to MHC I molecules (i.e. HLA-A and HLA-G). In vitro differentiated monocyte-derived macrophages (MDMs) cultured for 24h in the presence of anti-LILRB2 antibodies and lipopolysaccharide (LPS) show polarization toward a more inflammatory phenotype - secreting higher levels of TNF-α and IL-6 with decreased amounts of IL-10 and CCL2 as compared to an isotype control antibody. NanoString mRNA analysis revealed that, in the absence of LPS or any additional stimuli, MDMs cultured with anti-LILRB2 antibodies showed gene changes consistent with inflammatory or M1-like polarization of macrophages. Anti-LILRB2 antibodies were also evaluated in human tumor histoculture and induced pharmacodynamic responses consistent with macrophage and T cell activation in a variety of tumor types. While mice do not express LILRB2 specifically, they do express a LILRB-like molecule known as Pirb. Mice that are deficient in Pirb display resistance to mouse colon 38 (MC-38) tumor growth suggesting this pathway functions as immune checkpoint in cancer.
Conclusions: Based on these preclinical data, JTX-8064, a high affinity LILRB2-specific humanized antagonist monoclonal antibody, is being developed as an immunotherapeutic to reprogram suppressive macrophages within the tumor microenvironment.
Citation Format: Heather Cohen, Yasmin Hashambhoy-Ramsay, Lauren R. Pepper, Jeffrey Y. Smith, Margaret Willer, Kevin Guay, Vikki Spaulding, Kristin O'Malley, Monica Gostissa, Abha Dhaneshwar, Edward C. Stack, Alessandro Mora, Donald R. Shaffer. Preclinical evaluation of JTX-8064, an anti-LILRB2 antagonist antibody, for reprogramming tumor-associated macrophages abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5007.
Abstract
Neoantigens are emerging as attractive vaccine targets for personalized cancer immunotherapy. As opposed to tumor-associated antigens, neoantigens contain non-synonymous mutations that ...enable their identification as foreign targets not subject to central tolerance in the thymus. Personalized cancer vaccines leverage neoantigens to specifically direct the immune system to recognize cancer cells for the coordinated attack and destruction of tumors. While in silico methods are commonly used to predict immunogenic neoantigens primarily via putative binding to major histocompatibility complexes (MHC), the positive predictive value of these approaches is low as they cannot account for the complexity of antigen processing, the diversity of MHC class I and class II alleles, and the additional steps of T cell activation. Ex vivo technologies have the potential to overcome the limitations of neoantigen identification by utilizing biologically-relevant testing. ATLAS™ is an unbiased immune response profiling platform that enables comprehensive screening of a tumor mutanome by using a patient's own autologous immune cells, specifically monocyte-derived dendritic cells (MDDC) as antigen presenting cells (APCs) and sorted CD8+ and CD4+ T cells. By utilizing autologous APCs and T cells, ATLAS is agnostic to MHC diversity and assesses preexisting T cell responses to any given mutation. Patient MDDC are pulsed with an ordered array of Escherichia coli expressing patient-specific mutations as short polypeptides. CD8+ and CD4+ T cell response screening is performed using APCs and E. coli with and without pore-forming lysteriolysin O (cLLO) facilitating MHC class I or class II presentation, respectively. Thus, preexisting patient T cell responses to cancer antigens can be characterized by inflammatory cytokine secretion. We utilized a mouse melanoma model to demonstrate the capability of the ATLAS platform for identification of vaccine neoantigens. Whole exome sequencing was performed on B16F10 melanomas resected from C57BL/6 mice, identifying >1600 non-somatic, non-silent mutations. E. coli libraries individually expressing all mutations were constructed and used to screen APCs and T cells from the spleens of B16F10 tumor-bearing mice. Biologically relevant neoantigens were identified by their ability to modulate the secretion of inflammatory cytokines by CD4+ and CD8+ T cells. The significance of the identified neoantigens in comparison to predicted and previously reported B16F10 antigens is described. Top neoantigen candidates were selected and manufactured as synthetic long peptides. Therapeutic vaccination with ATLAS-identified neoantigens in tumor challenge studies is planned and progress will be reported. These studies demonstrate a biologically-relevant approach to improve neoantigen selection for personalized cancer vaccine design enabling improved therapeutic efficacy.
Citation Format: Hanna Starobinets, Catarina Nogueira, Kyle Ferber, Huilei Xu, Abha Dhaneshwar, Jason R. Dobson, James Loizeaux, James Foti, Michael O'Keefe, Erick Donis, Wendy Broom, Pamela Carroll, Paul Kirschmeier, Jessica B. Flechtner, Hubert Lam. Ex vivo ATLAS-identification of neoantigens for personalized cancer immunotherapy in mouse melanoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5718.
Abstract
As immune checkpoint blocking antibodies increasing become foundational therapies for the treatment of cancer, there is a pressing need to identify compounds that synergize with checkpoint ...blockade as the basis of combinatorial treatment regimens. We have developed a screening assay in which a luciferized tumor cell line expressing a model antigen is co-cultured with a transgenic CD8+ T cell specifically recognizing the model antigen in a H-2b-restricted manner. The target tumor cell/T cell assay was screened with a small molecule library to identify compounds that inhibit or enhance T cell-mediated killing of tumor cells in an antigen-dependent manner. The EGFR inhibitor Erlotinib was the top hit that enhanced T cell killing of tumor cells. Subsequent experiments with Erlotinib and additional EGFR inhibitors validated the screen result. EGFR inhibitors increase both basal and IFN-γ-induced antigen processing and presentation of MHC class-I, which enhanced recognition and lysis by CD8+ cytotoxic T lymphocytes. The tumor cell line was also transduced to constitutively express Cas9, and a pooled CRISPR screen utilizing the same target tumor cell/T cell assay identified sgRNAs targeting EGFR as sensitizing tumor cells to T cell-mediated killing. Combination of PD-1 blockade with EGFR inhibition showed significant synergistic efficacy in the MC38 syngeneic colon cancer model that was superior to PD-1 blockade or EGFR inhibition alone, further validating EGFR inhibitors as immunomodulatory agents that enhance PD-1 checkpoint blockade. This novel target tumor cell/T cell assay can be screened in high-throughput with small molecule libraries and genome-wide CRISPR/Cas9 libraries to identify both compounds AND target genes, respectively, that enhance or inhibit T cell recognition and killing of tumor cells.
Citation Format: Patrick H. Lizotte, Troy Luster, Megan E. Cavanaugh, Luke J. Taus, Abha Dhaneshwar, Naomi Mayman, Aaron Yang, Mark Bittinger, Paul Kirschmeier, Nathanael S. Gray, David A. Barbie, Pasi A. Janne. High-throughput immune-oncology screen identifies EGFR inhibitors as potent enhancers of CTL antigen-specific tumor cell killing abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4935.
Abstract
Targeted therapies against activated oncogenes, such as receptor tyrosine kinases, have significantly prolonged non-small cell lung cancer (NSCLC) patient survival, but the development of ...resistance limits the durability of clinical response. Genetic alterations which constitutively activate Fibroblast Growth Factor Receptors (FGFR) have been observed in patients with NSCLC. Erdafitinib (JNJ-42756493), an orally bioavailable pan-FGFR inhibitor discovered as part of a collaboration between Janssen and Astex Pharmaceuticals, has been shown to inhibit FGFR signaling pathways resulting in cell death and tumor growth inhibition in both in vitro and in vivo models of FGFR pathway aberration. Further, erdafitinib has been shown to have favorable pharmaceutical properties with manageable side effects in humans and several clinical trials are currently underway. One potential strategy to enhance the durability of response to targeted therapies, such as FGFR inhibitors, is to couple them with immunotherapy. In this setting, T cell responses primed and activated by increased antigen release resulting from the tumor cell targeted therapy could be enhanced and maintained by T-cell directed checkpoint blockade. To test this hypothesis, we evaluated erdafitinib in combination with an anti-programmed death-1 (PD-1) blocking antibody in an autochthonous FGFR2K660N/p53 genetically engineered mouse model (GEMM) of lung cancer, in which tumors develop within the context of an intact immune microenvironment. Cohorts of tumor bearing FGFR2K660N/p53 mutant mice treated with erdafitinib with or without anti-PD-1 showed significant tumor regressions compared to control and anti-PD-1 alone groups. Despite lack of differences in acute tumor responses between erdafitinib monotherapy and combination therapy, we observed significant survival benefit in the combination group erdafitinib alone (median survival 19.7 weeks vs 13.4 weeks, p<0.004). In a separate study, similar tumor regressions were noted in the FGFR-driven GEMM at 1 week of erdafitinib with or without anti-PD-1 treatment, while no such response to these treatments was noted in a KRAS-driven lung cancer GEMM. Immune profiling of tumor specimens revealed high baseline expression of programmed death ligand-1 (PD-L1) expression by IHC and flow cytometry. Following combination treatment, subsequent immunohistochemistry (IHC) analyses showed a significant decrease in Ki67 and PD-L1 positive tumor cells, accompanied by an increase in cluster of differentiation 3 (CD3) positive tumor-infiltrating cells in combination group as compared to control. T cell function is not inhibited by erdafitinib, as measured in vitro by mixed lymphocyte reaction and cytomegalovirus recall assays. Additional changes observed in lung tumors across treatment groups in immune cell infiltration, functionality, and T-cell clonality will be discussed. These data suggest that combination treatment of erdafitinib and PD-1 blockade drives improved survival in FGFR2-driven model of lung cancer by simultaneous inhibition of FGFR pathway in tumor cells and enhancement of anti-tumor immunity. Thus, data here provide rationale for the combined clinical testing of erdafitinib and PD-1 blockade in patients with FGFR-altered lung cancers.
Citation Format: Sangeetha Palakurthi, Mari Kuraguchi, Sima Zacharek, Jeff Liu, Dennis Bonal, Wei Huang, Kristin Depeaux, Abha Dhaneshwar, Sam Regan, Dyane Bailey, Martha Gowaski, Mei Zheng, Roderick Bronson, Catherine Ferrante, Enrique Zudaire, Sylvie Laquerre, Mark Bittinger, Kirschmeier Paul, Kathryn Packman, Raluca I. Verona, Kwok-Kin Wong, Matthew V. Lorenzi. Improved survival with erdafitinib (JNJ-42756493) and PD-1 blockade mediated by enhancement of anti-tumor immunity in an FGFR2-driven genetically engineered mouse model of lung cancer. abstract. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B27.
PDF
