Supplementary Figure 1 from Nm23-H1 Suppresses Metastasis by Inhibiting Expression of the Lysophosphatidic Acid Receptor <i>EDG2</i>

Supplementary Table 1, Figure Legend from Nm23-H1 Suppresses Metastasis by Inhibiting Expression of the Lysophosphatidic Acid Receptor <i>EDG2</i>

Melanoma is the deadliest form of skin cancer due to the lack of widely effective therapies for advanced disease. Recently FDA-approved immunotherapies, such as immune checkpoint blockade (ICB) by CTLA-4 and PD-1/PD-L1 antibodies, provide unprecedent durable responses but in less than 40% of late stage melanoma patients. While high mutational loads characteristic of responsive tumors has not shown predictive value of patient outcome, accumulating evidences suggest a key role for neoantigens in the response to ICB. Moreover, new T-cell transfer- and vaccine-based therapeutic strategies highlighted the relevance of an efficient identification and prioritization of highly immunogenic neoantigens to improve immunotherapies. However, mechanistic studies are not possible in humans, and the development of adequate predictive methods is still the center of intense debate in the field. Here we use two genetically engineered melanoma mouse models exhibiting distinct response to ICB. Melanomas induced by neonatal ultraviolet radiation (UV) in a HGF-transgenic mouse (HGF-tg) showed high sensitivity to anti-CTLA-4, whereas UV-induced melanomas in HGF-tg; BrafV600E; Cdkn2a+/- mouse (Braf/HGF) did not respond. We hypothesized these models will allow us to identify the neoantigen features required for ICB response including type, expression levels and allele frequency patterns. High tumor immunogenicity, assessed by in vivo vaccination assays, as well as increased T-cell infiltration upon anti-CTLA-4 treatment were correlated to greater response. Moreover, exome and RNA sequencing analyses revealed similar mutational and neoantigen load in both models, albeit with no common expressed mutations. Notably, both models expressed similar levels of antigen presentation related genes (e.g. B2m, H2-Kd, Tap1) suggesting that specific neoantigens in HGF-tg melanoma cells may contribute to their sensitivity to anti-CTLA-4. To test this, we predicted MHC-I/-II binding of HGF-tg melanoma mutated epitopes in silico and generated a “neo-epitope” library. Importantly, the expression and allele frequency of most of selected mutations were decreased in anti-CTLA-4 responder HGF melanomas. The “neo-epitope” library was transduced into Braf/HGF non-responder cells and future studies will identify the neo-epitopes lost upon ICB, representing determinants of therapeutic success. Additionally, in vivo vaccination assays using synthetic mutant peptides will be performed to validate each neoantigen candidate. We anticipate that our studies will provide insight into the role that neoantigens play in melanoma immunotherapy responses. (EPG and CPD contribute to this abstract equally). Citation Format: Eva Perez Guijarro, Chi-Ping Day, Zoe W. Ohler, Rajaa El Meskini, Howard Yang, Suman Vodnala, Cari Graff-Cherry, Sung Chin, Anyen Fon, Helen Michael, Maxwell Lee, Terry Van Dyke, Shyam Sharan, Glenn Merlino. Functional characterization of neoantigens determining immune checkpoint blockade response in mouse models of human 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 5720.

Abstract Melanoma is an aggressive and lethal disease with no efficacious therapies for a broad subset of late stage patients. Current immunotherapies, including immune checkpoint blockade (ICB), may prolong survival in certain patients, but generate responses in less than 40% of the treated cohort. This demands a better understanding of molecular mechanisms underlying the lack of response and acquired resistance to ICB. However, functional studies are limited in patients, and current preclinical studies are handicapped by the absence of appropriate mouse models that recapitulate the pathological and immunological diversity of human melanomas. Here we develop four syngeneic melanoma mouse models with human-relevant genetic modifications and carcinogenic agents, which we hypothesize will mirror the spectrum of responses to ICB and offer a platform for future mechanistic studies in melanoma. The models are: 1) neonatal ultraviolet radiation (UV)-induced melanoma in a HGF-transgenic mouse, in which melanocyte localization at the epidermal-dermal junction mimics human distribution (HU); 2) 7,12-Dimethylbenz(a)anthracene (DMBA)-induced melanoma in a HGF-tg and Cdk4R24C mouse (HC4D); 3) UV-induced melanoma in a BrafCA/+; HGF-tg; Cdkn2aflox/+; Tyr-CreERT2-t mouse (BHCU); and 4) UV-induced melanoma in a BrafCA/+; Ptenflox/+; Cdkn2aflox/+; Tyr-CreERT2-tg mouse (BPCU). Exome sequencing of the four models reveals a high correlation with mutational subtypes previously described in human melanoma. BPCU and BHCU represent different Braf mutant patient populations and HU and HC4D represent triple wildtype melanoma (non-BRAF, -NRAS, -NF1). The four mouse models demonstrate distinct responses to ICB with anti-CTLA-4 treatment. While HU and HC4D melanomas show high or partial sensitivity to anti-CTLA-4, respectively, BPCU and BHCU do not respond to treatment. In vivo vaccination assays demonstrate that the anti-CTLA-4 response in our models is linked to increased tumor immunogenicity. However, the number of non-synonymous mutations and antigen presentation functionality do not correlate with ICB efficacy. Tumor infiltration by T cells was assessed by CD3 and FoxP3 immunostaining and gene expression analysis. Although clear differential gene expression profiles are noted among the four models and in those tumors responding to the treatment, we unexpectedly found that “hot” melanomas (e.g., showing upregulation of inflammatory pathways and high T-cell infiltration) do not necessarily predict ICB efficacy. These results suggest that additional mechanisms could help determine the response or intrinsic resistance to anti-CTLA-4 and open new avenues for future research and treatment. Overall, our study offers four genetically and phenotypically distinct mouse models representing diverse human melanoma subtypes as powerful tools for the mechanistic study of the response to immunotherapies in melanoma. Citation Format: Corinne Rauck, Eva Perez-Guijarro, Zoe W. Ohler, Rajaa El E. Meskini, Howard Yang, Suman Vodnala, Cari Graff-Cherry, Sung Chin, Anyen Fon, Helen Michael, Maxwell Lee, Terry Van Dyke, Shyam Sharan, Glenn Merlino, Chi-Ping Day. Developing a preclinical immunotherapy platform using syngeneic mouse models of human 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 5678.

PurposeComplete deficiency of microphthalmia transcription factor (MITF) in Mitfmi-vga9/mi-vga9 mice is associated with microphthalmia, retinal dysplasia, and albinism. We investigated the ability of dopachrome tautomerase (DCT) promoter-mediated inducible ectopic expression of Mitf-M to rescue these phenotypic abnormalities.MethodsA new mouse line was created with doxycycline-inducible ectopic Mitf-M expression on an Mitf-deficient Mitfmi-vga9 background (DMV mouse). Adult DMV mice were phenotypically characterized and tissues were collected for histology, immunohistochemistry, and evaluation of Mitf, pigmentary genes, and retinal pigment epithelium (RPE) gene expression.ResultsEctopic Mitf-M expression was specifically induced in the eyes, but was not detected in the skin of DMV mice. Inducible expression of Mitf-M partially rescued the microphthalmia, RPE structure, and pigmentation as well as a subset of the choroidal and iris melanocytes but not cutaneous melanocytes. RPE function and vision were not restored in the DMV mice.ConclusionsEctopic expression of Mitf-M during development of Mitf-deficient mice is capable of partially rescuing ocular and retinal structures and uveal melanocytes. These findings provide novel information about the roles of Mitf isoforms in the development of mouse eyes.

Preclinical therapeutic assessment currently relies on the growth response of established human cell lines xenografted into immunocompromised mice, a strategy that is generally not predictive of clinical outcomes. Immunocompetent genetically engineered mouse (GEM)-derived tumor allograft models offer highly tractable preclinical alternatives and facilitate analysis of clinically promising immunomodulatory agents. Imageable reporters are essential for accurately tracking tumor growth and response, particularly for metastases. Unfortunately, reporters such as luciferase and GFP are foreign antigens in immunocompetent mice, potentially hindering tumor growth and confounding therapeutic responses. Here we assessed the value of reporter-tolerized GEMs as allograft recipients by targeting minimal expression of a luciferase-GFP fusion reporter to the anterior pituitary gland (dubbed the "Glowing Head" or GH mouse). The luciferase-GFP reporter expressed in tumor cells induced adverse immune responses in wildtype mouse, but not in GH mouse, as transplantation hosts. The antigenicity of optical reporters resulted in a decrease in both the growth and metastatic potential of the labeled tumor in wildtype mice as compared to the GH mice. Moreover, reporter expression can also alter the tumor response to chemotherapy or targeted therapy in a context-dependent manner. Thus the GH mice and experimental approaches vetted herein provide concept validation and a strategy for effective, reproducible preclinical evaluation of growth and response kinetics for traceable tumors.

Exposure to ultraviolet (UV) radiation has been implicated in melanoma formation. Glenn Merlino and colleagues now show, in a mouse model, that UVB irradiation induces the recruitment of macrophages that produce interferon-γ to promote melanoma formation. The study suggests that agents that target interferon-γ may have therapeutic potential in patients with melanoma. Ultraviolet (UV) exposure has been implicated in melanoma formation. Here it is shown, in a mouse model, that UVB induces the recruitment of macrophages which produce interferon-γ to promote melanomagenesis. The study suggests that in melanomas, interferon-γ could be targeted therapeutically. Cutaneous malignant melanoma is a highly aggressive and frequently chemoresistant cancer, the incidence of which continues to rise. Epidemiological studies show that the major aetiological melanoma risk factor is ultraviolet (UV) solar radiation, with the highest risk associated with intermittent burning doses, especially during childhood1,2. We have experimentally validated these epidemiological findings using the hepatocyte growth factor/scatter factor transgenic mouse model, which develops lesions in stages highly reminiscent of human melanoma with respect to biological, genetic and aetiological criteria, but only when irradiated as neonatal pups with UVB, not UVA3,4. However, the mechanisms underlying UVB-initiated, neonatal-specific melanomagenesis remain largely unknown. Here we introduce a mouse model permitting fluorescence-aided melanocyte imaging and isolation following in vivo UV irradiation. We use expression profiling to show that activated neonatal skin melanocytes isolated following a melanomagenic UVB dose bear a distinct, persistent interferon response signature, including genes associated with immunoevasion. UVB-induced melanocyte activation, characterized by aberrant growth and migration, was abolished by antibody-mediated systemic blockade of interferon-γ (IFN-γ), but not type-I interferons. IFN-γ was produced by macrophages recruited to neonatal skin by UVB-induced ligands to the chemokine receptor Ccr2. Admixed recruited skin macrophages enhanced transplanted melanoma growth by inhibiting apoptosis; notably, IFN-γ blockade abolished macrophage-enhanced melanoma growth and survival. IFN-γ-producing macrophages were also identified in 70% of human melanomas examined. Our data reveal an unanticipated role for IFN-γ in promoting melanocytic cell survival/immunoevasion, identifying a novel candidate therapeutic target for a subset of melanoma patients.

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Abstract Nm23-H1 transcriptionally down-regulates expression of the lysophosphatidic acid receptor EDG2 and this down-regulation is critical for Nm23-H1–mediated motility suppression in vitro. We investigated the effect of altered EDG2 expression on Nm23-H1–mediated metastasis suppression in vivo. Clonal MDA-MB-435–derived tumor cell lines transfected with Nm23-H1 together with either a vector control or EDG2 had similar anchorage-dependent and anchorage-independent growth rates in vitro. However, a 45- and 300-fold inhibition of motility and invasion (P < 0.0001), respectively, was observed in Nm23-H1/vector lines, whereas coexpression of EDG2 restored activity to levels observed in the parental line. Using fluorescently labeled cells and ex vivo microscopy, the capacity of these cells to adhere, arrest, extravasate, and survive in the murine lung over a 24-h time course was measured. Only 5% of Nm23-H1/vector–transfected cells were retained in the murine lung 6 h following tail vein injection; coexpression of EDG2 enhanced retention 8- to 13-fold (P < 0.01). In a spontaneous metastasis assay, the primary tumor size of Nm23-H1/vector and Nm23-H1/EDG2 clones was not significantly different. However, restoration of EDG2 expression augmented the incidence of pulmonary metastasis from 51.9% to 90.4% (P = 2.4 × 10−5), comparable with parental MDA-MB-435 cells. To determine the relevance of this model system to human breast cancer, a cohort of breast carcinomas was stained for Nm23-H1 and EDG2 and a statistically significant inverse correlation between these two proteins was revealed (r = −0.73; P = 0.004). The data indicate that Nm23-H1 down-regulation of EDG2 is functionally important to suppression of tumor metastasis. [Cancer Res 2007;67(24):11751–9]

Supplementary Table 1, Figure Legend from Nm23-H1 Suppresses Metastasis by Inhibiting Expression of the Lysophosphatidic Acid Receptor EDG2