Long-term follow-up of levonorgestrel intrauterine device for atypical hyperplasia and early endometrial cancer reveals relapse characterized by immune exhaustion.

BACKGROUND: Nonsurgical treatment options are increasingly needed for endometrial atypical hyperplasia (AH) and endometrioid endometrial cancer (EEC). Despite promising initial response rates, prospective long-term data and determinants for relapse are limited. METHODS: Follow-up data from patients in our prospective phase II trial of LIUD for AH/G1EEC were collected from medical records. Spatial transcriptomics (Nanostring GeoMX digital spatial profiling) with in silico cell type deconvolution and pathway analyses were employed on longitudinal biopsy samples from five patients across pre-treatment, on-treatment, and relapse. RESULTS: Of 43 participants exhibiting initial response to LIUD, 41 had follow-up data. Sixteen (39%) experienced relapse. Clinical factors associated with shorter response duration included younger age, initial diagnosis of G1EEC, lack of response at six months, premenopausal status, and Hispanic ethnicity (p<0.05), but only six-month response status remained a significant predictor in a multivariate model (p=0.023). LIUD increased abundance of NK cells (DMCP-counter score=46.13, FDR=0.004) and cytotoxic lymphocytes (DMCP-counter score=277.67, FDR=0.004), as well as lymphocyte cytotoxicity markers PRF1 (log2FC=1.62, FDR=0.025) and GZMA (log2FC=2.47, FDR=0.008). NK cells were reduced at relapse (DMCP-counter score=-55.96, FDR=0.02). Immune-related pathways (IFNα-response and TGFß-signaling) were enriched at relapse (FDR<0.05). IDO1 expression, reflecting immune exhaustion, was upregulated at relapse (FDR<0.05). CONCLUSIONS: Upfront resistance and relapse after initial response to LIUD for AH/G1EEC impacts nearly half of patients, remaining a major hurdle for non-surgical treatment of AH/G1EEC. Molecular studies evaluating longitudinal biopsies from a small cohort implicate immune mechanisms at relapse, including reversal of progestin-related immunomodulation and increased immune exhaustion.

Mitochondrial defects and metabolic vulnerabilities in Lynch syndrome-associated MSH2-deficient endometrial cancer.

Lynch syndrome (LS) is defined by inherited mutations in DNA mismatch repair genes, including MSH2, and carries 60% lifetime risk of developing endometrial cancer (EC). Beyond hypermutability, specific mechanisms for LS-associated endometrial carcinogenesis are not well understood. Here, we assessed the effects of MSH2 loss on EC pathogenesis using a novel mouse model (PR-Cre Msh2 flox/flox , abbreviated Msh2KO), primary cell lines established from this model, human tissues, and human EC cell lines with isogenic MSH2 knockdown. Beginning at eight months of age, 30% of Msh2KO mice exhibited endometrial atypical hyperplasia (AH), a precancerous lesion. At 12 to 16 months of age, 47% of Msh2KO mice exhibited either AH or ECs with histologic features similar to human LS-related ECs. Transcriptomic profiling of EC from Msh2KO mice revealed a transcriptomic signature for mitochondrial dysfunction. Studies in vitro and in vivo revealed mitochondrial dysfunction based upon two mechanisms: marked mitochondrial content reduction, along with pronounced disruptions to the integrity of retained mitochondria. Human LS-related ECs also exhibited mitochondrial content reduction compared with non-LS-related ECs. Functional studies revealed metabolic reprogramming of MSH2-deficient EC cells in vitro , including reduced oxidative phosphorylation and increased susceptibility to glycolysis suppression. We are the first to identify mitochondrial dysfunction and metabolic disruption as a consequence of MSH2 deficiency-related EC. Mitochondrial and metabolic aberrations should be evaluated as novel biomarkers for endometrial carcinogenesis or risk stratification and could serve as targets for cancer interception in women with LS. Significance: This is the first study to report mitochondrial dysfunction contributing to MSH2-deficient endometrial cancer development, identifying a noncanonical pathway for MSH2 deficient carcinogenesis, which also imparts vulnerability to metabolic targeting.

Charge-based interactions through peptide position 4 drive diversity of antigen presentation by human leukocyte antigen class I molecules.

Human leukocyte antigen class I (HLA-I) molecules bind and present peptides at the cell surface to facilitate the induction of appropriate CD8+ T cell-mediated immune responses to pathogen- and self-derived proteins. The HLA-I peptide-binding cleft contains dominant anchor sites in the B and F pockets that interact primarily with amino acids at peptide position 2 and the C-terminus, respectively. Nonpocket peptide-HLA interactions also contribute to peptide binding and stability, but these secondary interactions are thought to be unique to individual HLA allotypes or to specific peptide antigens. Here, we show that two positively charged residues located near the top of peptide-binding cleft facilitate interactions with negatively charged residues at position 4 of presented peptides, which occur at elevated frequencies across most HLA-I allotypes. Loss of these interactions was shown to impair HLA-I/peptide binding and complex stability, as demonstrated by both in vitro and in silico experiments. Furthermore, mutation of these Arginine-65 (R65) and/or Lysine-66 (K66) residues in HLA-A*02:01 and A*24:02 significantly reduced HLA-I cell surface expression while also reducing the diversity of the presented peptide repertoire by up to 5-fold. The impact of the R65 mutation demonstrates that nonpocket HLA-I/peptide interactions can constitute anchor motifs that exert an unexpectedly broad influence on HLA-I-mediated antigen presentation. These findings provide fundamental insights into peptide antigen binding that could broadly inform epitope discovery in the context of viral vaccine development and cancer immunotherapy.

Interleukin-6 blockade abrogates immunotherapy toxicity and promotes tumor immunity.

Immune checkpoint blockade (ICB) therapy frequently induces immune-related adverse events. To elucidate the underlying immunobiology, we performed a deep immune analysis of intestinal, colitis, and tumor tissue from ICB-treated patients with parallel studies in preclinical models. Expression of interleukin-6 (IL-6), neutrophil, and chemotactic markers was higher in colitis than in normal intestinal tissue; T helper 17 (Th17) cells were more prevalent in immune-related enterocolitis (irEC) than T helper 1 (Th1). Anti-cytotoxic T-lymphocyte-associated antigen 4 (anti-CTLA-4) induced stronger Th17 memory in colitis than anti-program death 1 (anti-PD-1). In murine models, IL-6 blockade associated with improved tumor control and a higher density of CD4+/CD8+ effector T cells, with reduced Th17, macrophages, and myeloid cells. In an experimental autoimmune encephalomyelitis (EAE) model with tumors, combined IL-6 blockade and ICB enhanced tumor rejection while simultaneously mitigating EAE symptoms versus ICB alone. IL-6 blockade with ICB could de-couple autoimmunity from antitumor immunity.

Novel polymer-based system for intrauterine delivery of everolimus for anti-cancer applications.

Non-surgical treatment options for low-grade endometrial cancer and precancerous lesions are a critical unmet need for women who wish to preserve fertility or are unable to undergo hysterectomy. The PI3K/AKT/mTOR pathway is frequently activated in endometrial cancers and has been associated with resistance to endocrine therapy, making it a compelling target for early stage disease. Oral everolimus, an inhibitor against mTORC1, has shown clinical benefit in advanced or recurrent disease but has severe adverse effects that may lead to treatment interruption or dose reduction. To overcome this, we developed a polymer-based intrauterine delivery system to achieve persistent, local delivery of everolimus without systemic exposure. In vivo studies, using a rat model, showed that a poly(propylene fumarate)-based rod loaded with everolimus achieved everolimus delivery to the endometrium with levels similar to oral administration, but with limited systemic exposure and up to 84 days of release. Biological activity of everolimus delivered with this system was confirmed, measured by reduced lumen epithelial cell height and PI3K pathway biomarkers. This study shows a promising new delivery approach for anti-cancer drugs for non-surgical treatment of low-grade endometrial cancer.

IL-1α Mediates Innate and Acquired Resistance to Immunotherapy in Melanoma.

Inflammation has long been associated with cancer initiation and progression; however, how inflammation causes immune suppression in the tumor microenvironment and resistance to immunotherapy is not well understood. In this study, we show that both innate proinflammatory cytokine IL-1α and immunotherapy-induced IL-1α make melanoma resistant to immunotherapy. In a mouse melanoma model, we found that tumor size was inversely correlated with response to immunotherapy. Large tumors had higher levels of IL-1α, Th2 cytokines, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), and regulatory T cells but lower levels of IL-12, Th1 cytokines, and activated T cells. We found that therapy with adenovirus-encoded CD40L (rAd.CD40L) increased tumor levels of IL-1α and PMN-MDSCs. Blocking the IL-1 signaling pathway significantly decreased rAd.CD40L-induced PMN-MDSCs and their associated PD-L1 expression in the tumor microenvironment and enhanced tumor-specific immunity. Similarly, blocking the IL-1 signaling pathway improved the antimelanoma activity of anti-PD-L1 Ab therapy. Our study suggests that blocking the IL-1α signaling pathway may increase the efficacy of immunotherapies against melanoma.

Endometrial biomarkers in premenopausal women with obesity: an at-risk cohort.

BACKGROUND: Obesity is a well-known risk factor for endometrial cancer, but the mechanisms of obesity-related carcinogenesis are not well defined, particularly for premenopausal women. With the continuing obesity epidemic, increases in the incidence of endometrial cancer and a younger age of diagnosis are often attributed to a hyperestrogenic state created by hormone production in adipose tissue, but significant knowledge gaps remain. The balance of estrogen-responsive signals has not been defined in the endometrium of premenopausal women with obesity, where obesity may not create hyperestrogenism in the context of ovaries being the primary source of estrogen production. Obesity is associated with a state of low-grade, chronic inflammation that can promote tumorigenesis, and it is also known that hormonal changes alter the immune microenvironment of the endometrium. However, limited research has been conducted on endometrial immune-response changes in women who have an increased risk for cancer due to obesity. OBJECTIVE: Endometrial estrogen-regulated biomarkers, previously shown to be dysregulated in endometrial cancer, were evaluated in a cohort of premenopausal women to determine if obesity is associated with differences in the biomarker expression levels, which might reflect an altered risk of developing cancer. The expression of a multiplexed panel of immune-related genes was also evaluated for expression differences related to obesity. STUDY DESIGN: Premenopausal women with a body mass index of ≥30 kg/m2 (n=97) or a body mass index of ≤25 kg/m2 (n=33) were prospectively enrolled in this cross-sectional study, which included the assessment of serum metabolic markers and a timed endometrial biopsy for pathologic evaluation, hormone-regulated biomarker analysis, and immune response gene expression analysis. Medical and gynecologic histories were obtained. Endometrial gene expression markers were also compared across the body mass index groups in a previous cohort of premenopausal women with an inherited cancer risk (Lynch syndrome). RESULTS: In addition to known systemic metabolic differences, histologically normal endometria from women with obesity showed a decrease in gene expression of progesterone receptor (P=.0027) and the estrogen-induced genes retinaldehyde dehydrogenase 2 (P=.008), insulin-like growth factor 1 (P=.016), and survivin (P=.042) when compared with women without obesity. The endometrial biomarkers insulin-like growth factor 1, survivin, and progesterone receptor remained statistically significant in multivariate linear regression models. In contrast, women with obesity and Lynch syndrome had an increased expression of insulin-like growth factor 1 (P=.017). There were no differences in endometrial proliferation, and limited endometrial immune differences were observed. CONCLUSION: When comparing premenopausal women with and without obesity in the absence of endometrial pathology or an inherited cancer risk, the expression of the endometrial biomarkers does not reflect a local hyperestrogenic environment, but it instead reflects a decreased cancer risk profile that may be indicative of a compensated state. In describing premenopausal endometrial cancer risk, it may be insufficient to attribute a high-risk state to obesity alone; further studies are warranted to evaluate individualized biomarker profiles for differences in the hormone-responsive signals or immune response. In patients with Lynch syndrome, the endometrial biomarker profile suggests that obesity further increases the risk of developing cancer.

Vestigial-like 1 is a shared targetable cancer-placenta antigen expressed by pancreatic and basal-like breast cancers.

Cytotoxic T lymphocyte (CTL)-based cancer immunotherapies have shown great promise for inducing clinical regressions by targeting tumor-associated antigens (TAA). To expand the TAA landscape of pancreatic ductal adenocarcinoma (PDAC), we performed tandem mass spectrometry analysis of HLA class I-bound peptides from 35 PDAC patient tumors. This identified a shared HLA-A*0101 restricted peptide derived from co-transcriptional activator Vestigial-like 1 (VGLL1) as a putative TAA demonstrating overexpression in multiple tumor types and low or absent expression in essential normal tissues. Here we show that VGLL1-specific CTLs expanded from the blood of a PDAC patient could recognize and kill in an antigen-specific manner a majority of HLA-A*0101 allogeneic tumor cell lines derived not only from PDAC, but also bladder, ovarian, gastric, lung, and basal-like breast cancers. Gene expression profiling reveals VGLL1 as a member of a unique group of cancer-placenta antigens (CPA) that may constitute immunotherapeutic targets for patients with multiple cancer types.

Anti-OX40 Antibody Directly Enhances The Function of Tumor-Reactive CD8+ T Cells and Synergizes with PI3Kß Inhibition in PTEN Loss Melanoma.

PURPOSE: OX40 agonist-based combinations are emerging as a novel avenue to improve the effectiveness of cancer immunotherapy. To better guide its clinical development, we characterized the role of the OX40 pathway in tumor-reactive immune cells. We also evaluated combining OX40 agonists with targeted therapy to combat resistance to cancer immunotherapy.Experimental Design: We utilized patient-derived tumor-infiltrating lymphocytes (TILs) and multiple preclinical models to determine the direct effect of anti-OX40 agonistic antibodies on tumor-reactive CD8+ T cells. We also evaluated the antitumor activity of an anti-OX40 antibody plus PI3Kß inhibition in a transgenic murine melanoma model (Braf mutant, PTEN null), which spontaneously develops immunotherapy-resistant melanomas. RESULTS: We observed elevated expression of OX40 in tumor-reactive CD8+ TILs upon encountering tumors; activation of OX40 signaling enhanced their cytotoxic function. OX40 agonist antibody improved the antitumor activity of CD8+ T cells and the generation of tumor-specific T-cell memory in vivo. Furthermore, combining anti-OX40 with GSK2636771, a PI3Kß-selective inhibitor, delayed tumor growth and extended the survival of mice with PTEN-null melanomas. This combination treatment did not increase the number of TILs, but it instead significantly enhanced proliferation of CD8+ TILs and elevated the serum levels of CCL4, CXCL10, and IFNγ, which are mainly produced by memory and/or effector T cells. CONCLUSIONS: These results highlight a critical role of OX40 activation in potentiating the effector function of tumor-reactive CD8+ T cells and suggest further evaluation of OX40 agonist-based combinations in patients with immune-resistant tumors.

Macrophage depletion through colony stimulating factor 1 receptor pathway blockade overcomes adaptive resistance to anti-VEGF therapy.

Anti-angiogenesis therapy has shown clinical benefit in patients with high-grade serous ovarian cancer (HGSC), but adaptive resistance rapidly emerges. Thus, approaches to overcome such resistance are needed. We developed the setting of adaptive resistance to anti-VEGF therapy, and performed a series of in vivo experiments in both immune competent and nude mouse models. Given the pro-angiogenic properties of tumor-associated macrophages (TAMs) and the dominant role of CSF1R in macrophage function, we added CSF1R inhibitors following emergence of adaptive resistance to anti-VEGF antibody. Mice treated with a CSF1R inhibitor (AC708) after anti-VEGF antibody resistance had little to no measurable tumor burden upon completion of the experiment while those that did not receive a CSF1R inhibitor still had abundant tumor. To mimic clinically used regimens, mice were also treated with anti-VEGF antibody and paclitaxel until resistance emerged, and then a CSF1R inhibitor was added. The addition of a CSF1R inhibitor restored response to anti-angiogenesis therapy, resulting in 83% lower tumor burden compared to treatment with anti-VEGF antibody and paclitaxel alone. Collectively, our data demonstrate that the addition of a CSF1R inhibitor to anti-VEGF therapy and taxane chemotherapy results in robust anti-tumor effects.

SLC45A2: A Melanoma Antigen with High Tumor Selectivity and Reduced Potential for Autoimmune Toxicity.

Cytotoxic T lymphocyte (CTL)-based immunotherapies have had remarkable success at generating objective clinical responses in patients with advanced metastatic melanoma. Although the melanocyte differentiation antigens (MDA) MART-1, PMEL, and tyrosinase were among the first melanoma tumor-associated antigens identified and targeted with immunotherapy, expression within normal melanocytes of the eye and inner ear can elicit serious autoimmune side effects, thus limiting their clinical potential as CTL targets. Using a tandem mass spectrometry (MS) approach to analyze the immunopeptidomes of 55 melanoma patient-derived cell lines, we identified a number of shared HLA class I-bound peptides derived from the melanocyte-specific transporter protein SLC45A2. Antigen-specific CTLs generated against HLA-A*0201- and HLA-A*2402-restricted SLC45A2 peptides effectively killed a majority of HLA-matched cutaneous, uveal, and mucosal melanoma cell lines tested (18/25). CTLs specific for SLC45A2 showed significantly reduced recognition of HLA-matched primary melanocytes that were, conversely, robustly killed by MART1- and PMEL-specific T cells. Transcriptome analysis revealed that SLC45A2 mRNA expression in normal melanocytes was less than 2% that of other MDAs, therefore providing a more favorable melanoma-to-melanocyte expression ratio. Expression of SLC45A2 and CTL sensitivity could be further upregulated in BRAF(V600E)-mutant melanoma cells upon treatment with BRAF or MEK inhibitors, similarly to other MDAs. Taken together, our study demonstrates the feasibility of using tandem MS as a means of discovering shared immunogenic tumor-associated epitopes and identifies SLC45A2 as a promising immunotherapeutic target for melanoma with high tumor selectivity and reduced potential for autoimmune toxicity. Cancer Immunol Res; 5(8); 618-29. ©2017 AACR.

Trouble at the core: BRAF(V600E) drives multiple modes of T-cell suppression in melanoma.

Several studies have demonstrated that oncogenic BRAF(V600E) promotes T-cell suppression in melanoma by upregulating the transcription of a multitude of immunomodulatory chemokine and cytokine genes. BRAF(V600E) has now been shown to act even more directly to evade cytotoxic T-cell recognition, by driving rapid internalization of human leukocyte antigen (HLA) class I from the tumor-cell surface and its intracellular sequestration.

BRAFV600E Co-opts a Conserved MHC Class I Internalization Pathway to Diminish Antigen Presentation and CD8+ T-cell Recognition of Melanoma.

Oncogene activation in tumor cells induces broad and complex cellular changes that contribute significantly to disease initiation and progression. In melanoma, oncogenic BRAF(V600E) has been shown to drive the transcription of a specific gene signature that can promote multiple mechanisms of immune suppression within the tumor microenvironment. We show here that BRAF(V600E) also induces rapid internalization of MHC class I (MHC-I) from the melanoma cell surface and its intracellular sequestration within endolysosomal compartments. Importantly, MAPK inhibitor treatment quickly restored MHC-I surface expression in tumor cells, thereby enhancing melanoma antigen-specific T-cell recognition and effector function. MAPK pathway-driven relocalization of HLA-A*0201 required a highly conserved cytoplasmic serine phosphorylation site previously implicated in rapid MHC-I internalization and recycling by activated immune cells. Collectively, these data suggest that oncogenic activation of BRAF allows tumor cells to co-opt an evolutionarily conserved MHC-I trafficking pathway as a strategy to facilitate immune evasion. This link between MAPK pathway activation and the MHC-I cytoplasmic tail has direct implications for immunologic recognition of tumor cells and provides further evidence to support testing therapeutic strategies combining MAPK pathway inhibition with immunotherapies in the clinical setting.

Activation of the inflammasome and enhanced migration of microparticle-stimulated dendritic cells to the draining lymph node.

Porous silicon microparticles presenting pathogen-associated molecular patterns mimic pathogens, enhancing internalization of the microparticles and activation of antigen presenting dendritic cells. We demonstrate abundant uptake of microparticles bound by the TLR-4 ligands LPS and MPL by murine bone marrow-derived dendritic cells (BMDC). Labeled microparticles induce concentration-dependent production of IL-1ß, with inhibition by the caspase inhibitor Z-VAD-FMK supporting activation of the NLRP3-dependent inflammasome. Inoculation of BALB/c mice with ligand-bound microparticles induces a significant increase in circulating levels of IL-1ß, TNF-α, and IL-6. Stimulation of BMDC with ligand-bound microparticles increases surface expression of costimulatory and MHC molecules, and enhances migration of BMDC to the draining lymph node. LPS-microparticles stimulate in vivo C57BL/6 BMDC and OT-1 transgenic T cell interactions in the presence of OVA SIINFEKL peptide in lymph nodes, with intact nodes imaged using two-photon microscopy. Formation of in vivo and in vitro immunological synapses between BMDC, loaded with OVA peptide and LPS-microparticles, and OT-1 T cells are presented, as well as elevated intracellular interferon gamma levels in CD8(+) T cells stimulated by BMDC carrying peptide-loaded microparticles. In short, ligand-bound microparticles enhance (1) phagocytosis of microparticles; (2) BMDC inflammasome activation and upregulation of costimulatory and MHC molecules; (3) cellular migration of BMDC to lymphatic tissue; and (4) cellular interactions leading to T cell activation in the presence of antigen.