Jennifer Landsberg

5′-triphosphate-siRNA: turning gene silencing and Rig-I activation against melanoma

Genetic and epigenetic plasticity allows tumors to evade single-targeted treatments. Here we direct Bcl2-specific short interfering RNA (siRNA) with 5′-triphosphate ends (3p-siRNA) against melanoma. Recognition of 5′-triphosphate by the cytosolic antiviral helicase retinoic acid–induced protein I (Rig-I, encoded by Ddx58) activated innate immune cells such as dendritic cells and directly induced expression of interferons (IFNs) and apoptosis in tumor cells. These Rig-I–mediated activities synergized with siRNA-mediated Bcl2 silencing to provoke massive apoptosis of tumor cells in lung metastases in vivo. The therapeutic activity required natural killer cells and IFN, as well as silencing of Bcl2, as evidenced by rescue with a mutated Bcl2 target, by site-specific cleavage of Bcl2 messenger RNA in lung metastases and downregulation of Bcl-2 protein in tumor cells in vivo. Together, 3p-siRNA represents a single molecule–based approach in which Rig-I activation on both the immune- and tumor cell level corrects immune ignorance and in which gene silencing corrects key molecular events that govern tumor cell survival.

Ultraviolet-radiation-induced inflammation promotes angiotropism and metastasis in melanoma

Intermittent intense ultraviolet (UV) exposure represents an important aetiological factor in the development of malignant melanoma. The ability of UV radiation to cause tumour-initiating DNA mutations in melanocytes is now firmly established, but how the microenvironmental effects of UV radiation influence melanoma pathogenesis is not fully understood. Here we report that repetitive UV exposure of primary cutaneous melanomas in a genetically engineered mouse model promotes metastatic progression, independent of its tumour-initiating effects. UV irradiation enhanced the expansion of tumour cells along abluminal blood vessel surfaces and increased the number of lung metastases. This effect depended on the recruitment and activation of neutrophils, initiated by the release of high mobility group box 1 (HMGB1) from UV-damaged epidermal keratinocytes and driven by Toll-like receptor 4 (TLR4). The UV-induced neutrophilic inflammatory response stimulated angiogenesis and promoted the ability of melanoma cells to migrate towards endothelial cells and use selective motility cues on their surfaces. Our results not only reveal how UV irradiation of epidermal keratinocytes is sensed by the innate immune system, but also show that the resulting inflammatory response catalyses reciprocal melanoma–endothelial cell interactions leading to perivascular invasion, a phenomenon originally described as angiotropism in human melanomas by histopathologists. Angiotropism represents a hitherto underappreciated mechanism of metastasis that also increases the likelihood of intravasation and haematogenous dissemination. Consistent with our findings, ulcerated primary human melanomas with abundant neutrophils and reactive angiogenesis frequently show angiotropism and a high risk for metastases. Our work indicates that targeting the inflammation-induced phenotypic plasticity of melanoma cells and their association with endothelial cells represent rational strategies to specifically interfere with metastatic progression.

Melanomas resist T-cell therapy through inflammation-induced reversible dedifferentiation

Adoptive cell transfer therapies (ACTs) with cytotoxic T cells that target melanocytic antigens can achieve remissions in patients with metastatic melanomas, but tumours frequently relapse. Hypotheses explaining the acquired resistance to ACTs include the selection of antigen-deficient tumour cell variants and the induction of T-cell tolerance. However, the lack of appropriate experimental melanoma models has so far impeded clear insights into the underlying mechanisms. Here we establish an effective ACT protocol in a genetically engineered mouse melanoma model that recapitulates tumour regression, remission and relapse as seen in patients. We report the unexpected observation that melanomas acquire ACT resistance through an inflammation-induced reversible loss of melanocytic antigens. In serial transplantation experiments, melanoma cells switch between a differentiated and a dedifferentiated phenotype in response to T-cell-driven inflammatory stimuli. We identified the proinflammatory cytokine tumour necrosis factor (TNF)-α as a crucial factor that directly caused reversible dedifferentiation of mouse and human melanoma cells. Tumour cells exposed to TNF-α were poorly recognized by T cells specific for melanocytic antigens, whereas recognition by T cells specific for non-melanocytic antigens was unaffected or even increased. Our results demonstrate that the phenotypic plasticity of melanoma cells in an inflammatory microenvironment contributes to tumour relapse after initially successful T-cell immunotherapy. On the basis of our work, we propose that future ACT protocols should simultaneously target melanocytic and non-melanocytic antigens to ensure broad recognition of both differentiated and dedifferentiated melanoma cells, and include strategies to sustain T-cell effector functions by blocking immune-inhibitory mechanisms in the tumour microenvironment.

Immune Cell–Poor Melanomas Benefit from PD-1 Blockade after Targeted Type I IFN Activation

UNLABELLED Infiltration of human melanomas with cytotoxic immune cells correlates with spontaneous type I IFN activation and a favorable prognosis. Therapeutic blockade of immune-inhibitory receptors in patients with preexisting lymphocytic infiltrates prolongs survival, but new complementary strategies are needed to activate cellular antitumor immunity in immune cell-poor melanomas. Here, we show that primary melanomas in Hgf-Cdk4(R24C) mice, which imitate human immune cell-poor melanomas with a poor outcome, escape IFN-induced immune surveillance and editing. Peritumoral injections of immunostimulatory RNA initiated a cytotoxic inflammatory response in the tumor microenvironment and significantly impaired tumor growth. This critically required the coordinated induction of type I IFN responses by dendritic, myeloid, natural killer, and T cells. Importantly, antibody-mediated blockade of the IFN-induced immune-inhibitory interaction between PD-L1 and PD-1 receptors further prolonged the survival. These results highlight important interconnections between type I IFNs and immune-inhibitory receptors in melanoma pathogenesis, which serve as targets for combination immunotherapies. SIGNIFICANCE Using a genetically engineered mouse melanoma model, we demonstrate that targeted activation of the type I IFN system with immunostimulatory RNA in combination with blockade of immune-inhibitory receptors is a rational strategy to expose immune cell-poor tumors to cellular immune surveillance.

Complete regression of advanced primary and metastatic mouse melanomas following combination chemoimmunotherapy.

The development of therapeutic strategies which induce effective cellular antitumor immunity represents an important goal in cancer immunology. Here, we used the unique features of the genetically engineered Hgf-Cdk4(R24C) mouse model to identify a combination chemoimmunotherapy for melanoma. These mice develop primary cutaneous melanomas which grow progressively and metastasize in the absence of immunogenic foreign proteins such as oncogenes or antigens. Primary and metastatic tumors evade innate and adaptive immune defenses, although they naturally express melanocytic antigens which can be recognized by antigen-specific T cells. We found that primary melanomas continued to grow despite infiltration with adoptively transferred, in vivo-activated, tumor-specific CD8(+) T cells. To promote tumor immune defense, we developed a treatment protocol consisting of four complementary components: (a) chemotherapeutic preconditioning prior to (b) adoptive lymphocyte transfer and (c) viral vaccination followed by (d) adjuvant peritumoral injections of immunostimulatory nucleic acids. Lymphocyte ablation and innate antiviral immune stimulation cooperatively enhanced the expansion and the effector cell differentiation of adoptively transferred lymphocytes. The efficacy of the different treatment approaches converged in the tumor microenvironment and induced a strong cytotoxic inflammatory response enabling preferential recognition and destruction of melanoma cells. This combination chemoimmunotherapy caused complete regression of advanced primary melanomas in the skin and metastases in the lung with minimal autoimmune side effects. Our results in a clinically highly relevant experimental model provide a scientific rationale to evaluate similar strategies which unleash the power of innate and adaptive immune defense in future clinical trials.

MITF and c-Jun antagonism interconnects melanoma dedifferentiation with pro-inflammatory cytokine responsiveness and myeloid cell recruitment.

Inflammation promotes phenotypic plasticity in melanoma, a source of non-genetic heterogeneity, but the molecular framework is poorly understood. Here we use functional genomic approaches and identify a reciprocal antagonism between the melanocyte lineage transcription factor MITF and c-Jun, which interconnects inflammation-induced dedifferentiation with pro-inflammatory cytokine responsiveness of melanoma cells favouring myeloid cell recruitment. We show that pro-inflammatory cytokines such as TNF-α instigate gradual suppression of MITF expression through c-Jun. MITF itself binds to the c-Jun regulatory genomic region and its reduction increases c-Jun expression that in turn amplifies TNF-stimulated cytokine expression with further MITF suppression. This feed-forward mechanism turns poor peak-like transcriptional responses to TNF-α into progressive and persistent cytokine and chemokine induction. Consistently, inflammatory MITFlow/c-Junhigh syngeneic mouse melanomas recruit myeloid immune cells into the tumour microenvironment as recapitulated by their human counterparts. Our study suggests myeloid cell-directed therapies may be useful for MITFlow/c-Junhigh melanomas to counteract their growth-promoting and immunosuppressive functions.

Immunogenic cell death of human ovarian cancer cells induced by cytosolic poly(I:C) leads to myeloid cell maturation and activates NK cells

Owing to high rates of tumor relapse, ovarian cancer remains a fatal disease for which new therapeutic approaches are urgently needed. Accumulating evidence indicates that immune stimulation may delay or even prevent disease recurrence in ovarian cancer. In order to elicit proinflammatory signals that induce or amplify antitumor immune reactivity, we mimicked viral infection in ascites‐derived ovarian cancer cells. By transfection or electroporation we targeted the synthetic double‐stranded RNA poly(I:C) intracellularly in order to activate melanoma differentiation‐associated gene‐5 (MDA‐5), a sensor of viral RNA in the cytosol of somatic cells. Cancer cells reacted with enhanced expression of HLA‐class I, release of CXCL10, IL‐6, and type I IFN as well as tumor cell apoptosis. Monocytes and monocyte‐derived DCs (MoDCs) engulfed MDA‐5‐activated cancer cells, and subsequently upregulated HLA‐class I/II and costimulatory molecules, and secreted CXCL10 and IFN‐α. Further, this proinflammatory milieu promoted cytolytic activity and IFN‐γ secretion of NK cells. Thus, our data suggest that the engagement of MDA‐5 in a whole tumor cell vaccine is a promising approach for the immunotherapy of ovarian cancer.

Management of dermatofibrosarcoma protuberans with fibrosarcomatous transformation: an evidence-based review of the literature

Fibrosarcomatous transformation represents a rare event in dermatofibrosarcoma protuberans (DFSP) with unpredictable biological behaviour. No guidelines for the adequate treatment of patients with this rare neoplasm have been published. Herein, we present a comprehensive review of the literature comprising 157 patients with transformed DFSP focussing on surgical and adjuvant treatment modalities for this tumour. In the cohort examined, local recurrence occurred in 36% of cases and was significantly lower in patients treated by wide excision with margins ≥2 cm when compared with those treated with local excision without defined margins (P = 0.01). Consistently, negative margin status was associated with a lower recurrence rate when compared with positive or unknown margin status (P = 0.01). Distant metastases were detected in 13% of patients, which is significantly higher when compared with ordinary dermatofibrosarcoma protuberans. Systemic dissemination was preceded by local recurrence in 81% of cases, and is therefore strongly associated with tumour recurrence (P ≤ 0.001). The present data confirm that wide excision with margins ≥2 cm represent the gold standard in the treatment of transformed dermatofibrosarcoma protuberans, and prevents recurrence as well as metastasis. When R0‐resection is not feasible, adjuvant radiation should be considered for cases with incomplete resection or unknown surgical margins. Irresectable or metastatic transformed DFSP harbouring the COL1A1‐PDGFB fusion gene should be treated with imatinib in the palliative setting or as an adjunctive treatment before surgery, although responses may be short‐lasting.

Autochthonous primary and metastatic melanomas in Hgf‐Cdk4R24C mice evade T‐cell‐mediated immune surveillance

Genetically engineered mouse models offer new opportunities to investigate the role of cell‐mediated immunity in the natural progression of melanoma in an immunocompetent host. Here we report that Hgf‐Cdk4R24C mice spontaneously develop a spectrum of primary melanomas with high penetrance during their first year of life. Malignant transformation proceeds in a stepwise manner from multiple melanocytic nevi to single nodular melanomas and disseminated metastases in most mice. Migrating melanoma cells invade the draining lymph nodes without activating the immune system. Autochthonous primary tumors are destroyed following experimental introduction of immune surveillance using an adoptive lymphocyte transfer approach. However, some tumor cells are able to survive, evade immune cell control, and recur both locally and systemically. Immune tolerance in recurring tumors may be supported by immunosuppressive Gr1+ myeloid cells. Taken together, our results demonstrate that primary and metastatic melanomas developing spontaneously in Hgf‐Cdk4R24C mice effectively evade cellular immune surveillance.

Reactive Neutrophil Responses Dependent on the Receptor Tyrosine Kinase c-MET Limit Cancer Immunotherapy

&NA; Inhibitors of the receptor tyrosine kinase c‐MET are currently used in the clinic to target oncogenic signaling in tumor cells. We found that concomitant c‐MET inhibition promoted adoptive T cell transfer and checkpoint immunotherapies in murine cancer models by increasing effector T cell infiltration in tumors. This therapeutic effect was independent of tumor cell‐intrinsic c‐MET dependence. Mechanistically, c‐MET inhibition impaired the reactive mobilization and recruitment of neutrophils into tumors and draining lymph nodes in response to cytotoxic immunotherapies. In the absence of c‐MET inhibition, neutrophils recruited to T cell‐inflamed microenvironments rapidly acquired immunosuppressive properties, restraining T cell expansion and effector functions. In cancer patients, high serum levels of the c‐MET ligand HGF correlated with increasing neutrophil counts and poor responses to checkpoint blockade therapies. Our findings reveal a role for the HGF/c‐MET pathway in neutrophil recruitment and function and suggest that c‐MET inhibitor co‐treatment may improve responses to cancer immunotherapy in settings beyond c‐MET‐dependent tumors. Graphical Abstract Figure. No caption available. HighlightsHGF/c‐MET signaling mobilizes neutrophils in response to cancer immunotherapiesNeutrophils acquire immunosuppressive properties in T cell‐inflamed tissuesc‐MET+ neutrophils suppress therapy‐induced T cell expansion and effector functionsConcomitant c‐MET inhibition enhances the efficacy of cancer immunotherapies &NA; Inhibitors of the receptor tyrosine kinase c‐MET are currently used in the clinic to target oncogenic signaling in tumor cells. Glodde et al. now show that c‐MET inhibition impairs reactive neutrophil recruitment to tumors and lymph nodes, potentiating T cell anti‐tumor immunity. Thus, c‐MET inhibitor co‐treatment may improve responses to cancer immunotherapy in settings beyond c‐MET‐dependent tumors.