Cancer Immunology, Immunotherapy

Mesothelin (MSLN) is a GPI-anchored cell surface glycoprotein that is overexpressed in various solid tumors, including mesothelioma, triple-negative breast cancer, colon, ovarian and pancreatic cancer, with restricted normal tissue expression. To explore the immunogenicity and immunotherapeutic potential of MSLN to T cells with native receptor specificity, 29 individuals of diverse HLA backgrounds were interrogated for T cell activity against MSLN. Twenty one (72%) subjects (21/29) mounted a specific T cell response when repetitively challenged with MSLN antigen. Reactive cells were Th1 polarized, polyfunctional, predominantly detected in the CD8+ T cell compartment and cytotoxic toward autologous and MSLN+/HLA-matched tumor cell lines in conventional 2D in vitro assays. Furthermore, these cells produced potent anti-tumor effects in a novel 3D tumor spheroid model system established to evaluate the safety and potency of reactive cells against tumors including pancreatic, cervical, and colorectal cancer and mesothelioma. Taken together, these findings establish the feasibility of targeting MSLN using adoptively transferred T cells with native antigen specificity.

The immune tumor microenvironment (TME) is increasingly recognized as a dynamic ecosystem where B cells play pivotal roles in modulating therapeutic responses, particularly in the context of immune checkpoint blockade (ICB) therapy. While B cells have traditionally been viewed as bystanders in tumor immunity, recent evidence suggests they may actively influence anti-tumor immunity, albeit with conflicting reports regarding their pro-tumor or anti-tumor roles. This study explores the crucial roles played by B cells and their secreted extracellular vesicles (EVs) in shaping melanoma responses to ICB therapy. We show a significant enrichment of B cells in ICB therapy responders compared to non-responders, pre-treatment, through retrospective analyses of melanoma patient tumors. Functional assays demonstrate that B cell depletion impairs T cell-mediated tumor cytotoxicity, underscoring the importance of B cells in anti-tumor responses. To investigate the clinical relevance, EVs were isolated from melanoma patient tumors, and fractioned into tumor and immune subpopulations. MiRNA profiling of CD19+ EVs identifies miR-99a-5p as a top candidate, among several others, upregulated in responders. Functional assays show that miR-99a-5p silencing in B cells diminishes T cell-mediated anti-tumor activity, suggesting its role in promoting B cell-mediated immune responses. Mechanistically, miR-99a-5p influences B cell maturation within the TME by mediating class-switch recombination. Our findings highlight the important role of B cells and their derived EVs in shaping the efficacy of melanoma immunotherapy, paving the way for novel therapeutic strategies targeting B cell-related pathways. Graphical abstract (created with Biorender) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/628150v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@144bbe6org.highwire.dtl.DTLVardef@18c1e9eorg.highwire.dtl.DTLVardef@2e37aaorg.highwire.dtl.DTLVardef@487e18_HPS_FORMAT_FIGEXP M_FIG C_FIG

The clinical development of Natural Killer (NK) cell-mediated immunotherapy marks a milestone in the development of new cancer therapies and has gained traction due to the intrinsic ability of the NK cell to target and kill tumour cells. To fully harness the tumour killing ability of NK cells, we need to improve NK cell persistence and overcome suppression of NK cell activation in the tumour microenvironment. The trans-membrane, protein tyrosine phosphatase CD45, regulates NK cell homeostasis, with genetic loss of CD45 in mice resulting in increased numbers of mature NK cells [1-3]. This suggests that CD45-deficient NK cells might display enhanced persistence following adoptive transfer. However, here we demonstrated that adoptive transfer of CD45-deficiency did not enhance NK cell persistence in mice, and instead, the homeostatic disturbance of NK cells in CD45-deficient mice stemmed from a developmental defect in the common lymphoid progenitor population. The enhanced maturation within the CD45-deficient NK cell compartment was intrinsic to the NK cell lineage, and independent of the developmental defect. CD45 is not a conventional immune checkpoint candidate, as systemic loss is detrimental to T and B cell development [4-6], compromising the adaptive immune system. Nonetheless, this study suggests that inhibition of CD45 in progenitor or stem cell populations may improve the yield of in vitro generated NK cells for adoptive therapy.

BackgroundDendritic cells (DCs) are promising targets for cancer immunotherapies owing to their central role in the initiation and the control of immune responses. Their functions encompass a wide range of mechanisms mediated by different DC subsets. Several studies have identified human tumor- associated DC (TA-DC) populations through limited marker-based technologies, such as immunostaining or flow cytometry. However, tumor infiltration, spatial organization and specific functions in response to immunotherapy of each DC subset remain to be defined. MethodsHere, we implemented a multiplexed immunofluorescence analysis pipeline coupled with bio-informatic analyses to decipher the tumor DC landscape and its spatial organization within melanoma patients lesions, and its association with patients response to immune checkpoint inhibitors (ICI). For this aim, we analyze a cohort of 41 advanced melanoma patients treated with anti- PD1 alone or associated with anti-CTLA4. Distance and cell network analyses were performed to gain further insight into the spatial organization of tumor-associated DCs. A Digital Spatial Profiling analysis further characterized ecosystem of tumor-infiltrating DCs. ResultsPlasmacytoid DCs (pDCs) were the most abundant DC population, followed by conventional cDC1 and mature DCs, present in equal proportions. In contrast to CD8+ T cell frequency, and despite varying densities, all DC subsets were associated with a favorable response to ICI. Distance and cell network analyses demonstrated that tumor-infiltrating DCs were largely organized in dense areas with high homotypic connections, except for cDC1 that exhibited a more scattered distribution. We identified four patterns of ecosystems with distinct preferential interactions between DC subsets. Significantly, the proximity and interactions between CD8+ T cells and cDC1 were positively associated with patients response to ICI. ConclusionsOur study unravels the complex spatial organization of DC subsets and their interactions in melanoma patient lesions, shedding light on their pivotal role in shaping the response to ICI. Our discoveries regarding the spatial arrangement of cDC1, especially with CD8+ T cells, provide valuable clues for improving immunotherapeutic strategies in melanoma patients. What is already known on this topicDendritic cells (DCs) are promising targets for cancer immunotherapies owing to their central role in the initiation and the control of immune responses. Although conventional type 1 dendritic cells (cDC1) were proposed to contribute to immunotherapy response, their precise functions and interactions with other immune populations in human cancers are largely unknown. What this study addsThis study provides a precise characterization of the spatial distribution and organization of tumor- infiltrating DCs in a large cohort of advanced melanoma patients, and in correlation with response to immunotherapy. While DCs are organized in dense areas with high homotypic connections, cDC1 exhibit a more scattered distribution and form heterotypic aggregates with other DC subsets. More importantly, a close connection between cDC1 and CD8 T cell is uniquely correlated with the patients response to immunotherapy. How this study might affect research, practice or policyThis study improves our understanding of CD8-DC spatial organization within the tumor microenvironment and will have a broad spectrum of implications in the design of anti-tumor immune-activating compounds and the design of biomarkers of response to immunotherapy for melanoma patients.

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematological malignancies but has been clinically less effective in solid tumors. Engineering macrophages with CARs has emerged as a promising approach to overcome some of the challenges faced by CAR-T cells due to the macrophages ability to easily infiltrate tumors, phagocytose their targets, and reprogram the immune response. We engineered CAR-macrophages (CAR-Ms) to target chondroitin sulfate proteoglycan 4 (CSPG4), an antigen expressed in melanoma, and several other solid tumors. CSPG4-targeting CAR-Ms exhibited specific phagocytosis of CSPG4-expressing melanoma cells. Combining CSPG4-targeting CAR-Ms with CD47 blocking antibodies synergistically enhanced CAR-M-mediated phagocytosis and effectively inhibited melanoma spheroid growth in 3D. Furthermore, CSPG4-targeting CAR-Ms inhibited melanoma tumor growth in mouse models. These results suggest that CSPG4-targeting CAR-M immunotherapy is a promising solid tumor immunotherapy approach for treating melanoma. STATEMENT OF SIGNIFICANCEWe engineered macrophages with CARs as an alternative approach for solid tumor treatment. CAR-macrophages (CAR-Ms) targeting CSPG4, an antigen expressed in melanoma and other solid tumors, phagocytosed melanoma cells and inhibited melanoma growth in vivo. Thus, CSPG4-targeting CAR-Ms may be a promising strategy to treat patients with CSPG4-expressing tumors.

BackgroundImmunotherapy represented by the programmed death-1 (PD-1)/ligand 1 (PD-L1) monoclonal antibodies has led tumor treatment into a new era. However, the low overall response rate and high incidence of drug resistance largely damage the clinical benefits of existing immune checkpoint therapies. Recent studies correlate the response to PD-1/PD-L1 blockade with PD-L1 expression levels in tumor cells. Hence, identifying molecular targets and pathways controlling PD-L1 protein expression and stability in tumor cells is a major priority. MethodsIn this study, we first performed a Stress and Proteostasis CRISPR interference library-based screening to identify PD-L1 positive modulators. We then used in vitro and in vivo assays to investigate the biological function and mechanism of TRAF6 and its downstream YAP1/TFCP2 signaling in malignant melanoma. ResultsHere, we identified TRAF6 as a critical regulator of PD-L1 in melanoma cells. Suppression of TRAF6 expression down-regulates PD-L1 expression on the membrane surface of melanoma cells. We also found that PD-L1 protein abundance is regulated by YAP1/TFCP2 transcriptional complex. TRAF6 stabilizes YAP1 by K63 poly-ubiquitination modification, subsequently promoting the formation of YAP1/TFCP2 and PD-L1 transcription. Furthermore, inhibition of TRAF6 by Bortezomib enhanced cytolytic activity of CD8+ T cells by reduction of endogenous PD-L1. Notably, Bortezomib enhances anti-tumor immunity to an extent that is comparable to anti-PD-1 mAb therapies with no obvious toxicity. ConclusionsThese findings uncover a novel molecular mechanism for regulating PD-L1 protein abundance by a E3 ligase in cancer cells and reveal the potential of using TRAF6 inhibitors to stimulate internal anti-tumor immunological effect for TRAF6-PD-L1 overexpressing cancers.

Immune checkpoint blockade (ICB) therapies, targeting PD-1 or PD-L1, have transformed cancer treatment, particularly for aggressive cancers. However, many patients fail to benefit from ICBs due to tumor characteristics, including a non-inflammatory tumor microenvironment (TME) that impedes immune cell infiltration. This study investigated the potential of targeting the Atypical Chemokine Receptor 2 (ACKR2), known for scavenging CXCR3-related chemokines crucial for lymphocyte recruitment to tumors. Genetic targeting of ACKR2 in melanoma cells increased the release of essential chemokines associated with the inflamed TME. In mouse models, ACKR2 inhibition suppressed tumor growth, improved survival, and enhanced activated immune cell infiltration into the TME. Moreover, ACKR2 targeting synergized with anti-PD-1 therapy, overcoming resistance to anti-PD-1 and improving its efficacy. Analysis of melanoma patient data from The Cancer Genome Atlas (TCGA) revealed that patients with high levels of chemokines scavenged by ACKR2 had significantly better survival rates, with increased expression of NK cell and CD8 T cell markers indicating their presence in the TME. Notably, even in patients with high CD8 expression, those expressing low ACKR2 survived better than those expressing high ACKR2. This study emphasizes the clinical importance of targeting ACKR2 as an attractive strategy for the development of combination immunotherapies to treat cold tumors, which are clinically stratified to not be eligible for ICB-based therapy.

Following the breakthrough of immune check point inhibitors (ICIs), a new era of immuno-oncology agents has emerged and established immunotherapy as a part of cancer treatment. Despite the improving outcomes of ICIs, many patients with initial response are known to develop acquired resistance later. There is increasing interest in utilizing other stimulatory means, such as anti-pathogen immune responses to induce anti-tumor immune responses. The immunostimulatory effects of anti-pathogen-treated tumors in combinations with ICI are known to potentially amplify anti-tumor immunity resulting in increased tumor responses and improved outcomes. Anti-pathogen-treated tumors can become immune-infiltrated "hot" tumors and demonstrate higher treatment response rates and improved survival. Our research group has previously demonstrated that tumors can be converted from "cold" to "hot" by intratumoral injection of a commercially available seasonal influenza vaccine. In continuation with our work, in deciphering the role of anti-viral immunity in the context of tumor immunology, we studied the role of inactivated SARS-CoV-2 virus as anti-tumor agent. Here we report that intratumoral injections of inactivated SARS-CoV-2 convert the immunologically cold tumors to hot by generating anti-tumor-mediated CD8+ T cells. Our findings suggest that inactivated SARS-CoV-2 can be used as an immune modulator in immunotherapy for melanoma and triple-negative breast cancer.

Myeloid cells are prevalent in solid cancers, but they frequently exhibit a pro-tumor phenotype, hindering cancer immunotherapy. Their abundance makes engineered myeloid cell therapy an intriguing approach to tackle challenges posed by solid cancers, such as tumor trafficking and infiltration along with tumor cell heterogenicity and immunosuppressive tumor microenvironment (TME). Solid cancers often upregulate the checkpoint molecule PD-L1 to evade immune responses. Thus, we devised an adoptive cell therapy strategy based on myeloid cells expressing a Chimeric Antigen Receptor (CAR)-like immune receptor (CARIR). The extracellular domain of CARIR is derived from the natural inhibitory receptor PD-1, while the intracellular domain(s) are derived from CD40 and/or CD3{xi}. To assess the efficacy of CARIR-engineered myeloid cells, we conducted proof-of-principle experiments using co-culture and flow cytometry-based phagocytosis assays in vitro. Additionally, we employed a fully immune-competent syngeneic tumor mouse model to evaluate the strategys effectiveness in vivo. Co-culturing CARIR-expressing human monocytic THP-1 cells with PD-L1+ target cells lead to upregulation of the co-stimulatory molecule CD86 along with expression of proinflammatory cytokines TNF-1 and IL-1{beta}. Moreover, CARIR expression significantly enhanced phagocytosis of multiple PD-L1+ human solid tumor cell lines in vitro. Similar outcomes were observed with CARIR-expressing human primary macrophages. In experiments conducted on Balb/c mice bearing aggressive 4T1 mammary tumors, infusing murine myeloid cells expressing a murine version of CARIR significantly slowed tumor growth and prolonged survival. Taken together, our results demonstrate that adoptive transfer of PD-1 CARIR-engineered myeloid cells may be an effective strategy in treating PD-L1+ solid tumors. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/577873v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1657eborg.highwire.dtl.DTLVardef@e61ae3org.highwire.dtl.DTLVardef@ef3df4org.highwire.dtl.DTLVardef@cc90ea_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefWe described here an adoptive cell therapy approach employing PD-L1-specific CAR-like immune receptor (CARIR) modified myeloid cells as a potential immune cell therapy strategy for treating PD-L1+ solid cancer. O_LICARIR expression directed human THP-1 macrophages to recognize PD-L1+ target cells, which led to an upregulation of co-stimulatory molecule CD86 and production of proinflammatory cytokines TNF- and IL-1{beta}. C_LIO_LICARIR expression in human THP-1 macrophages had increased % phagocytosis and killing against PD-L1+ tumor cells in vitro. C_LIO_LIAdoptive transfer of CARIR transduced myeloid cells in immunocompetent syngeneic mice with established aggressive 4T1 tumor significantly slowed tumor growth and prolonged survival. C_LI

BackgroundThe emergence of immune checkpoint inhibitors (ICIs) has transformed the treatment landscape of metastatic melanoma. However, despite its success, reliable biomarkers for predicting primary resistance are not available in clinical practice. This study seeks to identify predictors of primary resistance based on novel gene expression signatures using pre-treatment multidimensional profiling in melanoma patients. MethodsThe transcriptomic profile of the tumor microenvironment was analyzed using tissue samples from 46 metastatic cutaneous melanoma patients collected prior to the initiation of ICIs therapy. A primary resistance predictive model was trained with the Discovery FFPE RNA-seq sub-cohort and validated using an independent external cohort of 54 samples. Additionally, liquid biopsy samples from peripheral blood mononuclear cells were analyzed in 8 patients using single-cell RNA sequencing (scRNA-seq) and in 46 patients using flow cytometry to characterize the distribution and abundance of the different immune cell populations. ResultsWe identified an 82-gene transcriptomic signature composed of tumor- and immune-related genes that stratifies metastatic cutaneous melanoma patients based on primary resistance to ICIs, with key markers including CXCL13, WDR63, MZB1, FDCSP, IGKC and GRIK3. This signature was enriched for pathways related to B cell activation and immune cell communication and achieved an AUC of 0.814 in predictive modeling. Immune deconvolution guided by scRNA-seq revealed four immune cell subsets (Plasma cells, Pre-B cells, Memory CD4 T cells, and Naive CD4 T cells) as prognostic indicators of resistance. Some of these subpopulations were validated by flow cytometry before and after treatment. ConclusionsWe propose a transcriptomic biomarker signature that accurately predicts primary resistance to ICIs in metastatic cutaneous melanoma. Through the integration of immune deconvolution with circulating immune cell profiles, we derived an ImmuneSignature linked to patient survival. By combining these approaches, we provide a framework for enhancing the prediction of immunotherapy outcomes and offer a novel strategy for identifying therapeutic targets to overcome resistance. Our findings lead to more effective and personalized immunotherapy guidance.

Extensive research has been conducted on the heterogenicity of immune cells within the tumor microenvironment, like cancer cell heterogenicity, particularly with the emergence of single cell analysis. While inducing factors have been used to artificially alter immune cell fate in vitro and dynamic cancer cell plasticity has been recently discovered, it remains unknown whether tumor infiltrating immune cells acquire plasticity and dynamics that contribute to heterogenicity. In this study, we explored mitochondrial DNA mutation combining with chromosome single nucleotide polymorphism to construct phylogenetic trees of immune cells within multiple solid tumors, together with precise cell type and subtype definition based on single cell RNA sequencing data. Based on these lineage tracing landscapes, we systematically identified cell state transitions and fate changes among different immune cell subtypes and types within multiple solid tumors. Interestingly, immune cells demonstrated a high level of plasticity for transitioning between different states, transdifferentiating from one type to another or dedifferentiating to a progenitor stage, in varying frequencies across different cancers. Moreover, most of these cell state transitions and cell fate changes discovered here were previously unknown. The cell changes may arise from extrinsic growth factors and cytokines secreted by tumor microenvironment cells, but intrinsic genetic mutations, particularly those related to ribosomes, may also be involved. Our data reveal that immune cell complexity extends beyond heterogenicity and also encompasses plasticity similar to that of cancer cells. Understanding the underlying mechanism of these cell changes will help elucidate the role of immune cells in cancer development and manipulating the cell change direction may ultimately enhance the efficiency of current immunotherapy.

PURPOSETumor Mutational Burden (TMB) is a crucial biomarker for predicting the effectiveness of cancer immunotherapy. However, ongoing debates about the optimal cutoff significantly impact the clinical application of immunotherapy. The purpose of our study is to comprehensively evaluate TMB cutoffs for predicting immunotherapy outcomes across multiple studies, considering both cancer type and outcome endpoint, using statistically principled approaches. METHODSWe analyzed data from PredictIO, curated through a PubMed search for studies involving immune checkpoint blockade treatment in the adjuvant setting, excluding those involving combinations with chemotherapy, targeted treatment, or radiation. The included studies, published between January 2015 and June 2022, required tumor sequencing data from whole exomes or targeted gene panels for TMB assessment. Outcome endpoints included clinical benefit rate (CBR), progression-free survival (PFS), and overall survival (OS). CBR was defined as the rate of complete response, partial response, or stable disease lasting at least six months, according to RECIST 1.1 criteria. OS and PFS were defined as the interval from treatment initiation to death and disease progression, respectively. TMB was uniformly derived from tumor sequencing data, representing the count of nonsynonymous mutations relative to the target sequencing size. TMB cutoffs were evaluated for outcome associations using study replicability analysis, alongside individual-study analysis and random-effects meta-analysis for comparison. RESULTSThe data provided sufficient evidence of replicable outcome associations for specific TMB cutoffs in melanoma, lung cancer, and bladder cancer. The FDA-recommended cutoff of 10 mutations per megabase showed replicable associations in melanoma for OS (p-value < 0.01) and CBR (p-value < 0.01), though more replicable cutoffs were identified for the latter. Lower cutoffs of 4 and 2 were found to be replicable in lung cancer for CBR (p-value = 0.04) and in bladder cancer for OS (p-value < 0.01), respectively. No cutoff was deemed replicable for the other cancer type and outcome combinations, due to no association, inadequate power, or insufficient data. CONCLUSIONA pan-cancer cutoff of 10 mutations per megabase may not be optimal for predicting immunotherapy outcomes. Further studies are needed to determine appropriate cutoffs specific to cancer types and outcomes through statistically principled replicability analyses.

Therapeutic options for non-small cell lung cancer (NSCLC) treatment have changed dramatically in recent years with the advent of novel immunotherapeutic approaches. Among these, immune checkpoint blockade (ICB), using monoclonal antibodies, has shown tremendous promise in a small proportion of patients. In order to better predict patients that will respond to ICB treatment, biomarkers such as tumor-associated CD8+ T cell frequency, tumor checkpoint protein status and mutational burden have been utilized, however, with mixed success. In this study, we hypothesized that significantly altering the suppressive tumor immune landscape in NSCLC could potentially improve ICB efficacy. Using sub-therapeutic doses of our Salmonella typhimurium-based therapy targeting the suppressive molecule indoleamine 2,3-dioxygenase (shIDO-ST) in tumor-bearing mice, we observed dramatic changes in immune subset phenotypes that included increases in antigen presentation markers, decreased regulatory T cell frequency and overall reduced checkpoint protein expression. Combination shIDO-ST treatment with anti-PD-1/CTLA-4 antibodies enhanced tumor growth control, compared to either treatment alone, which was associated with a significant intratumoral influx of CD8+ and CD4+ T lymphocytes. These results suggest that the success of ICB therapy may be more accurately predicted by taking into account multiple factors such as potential for antigen presentation and frequency of suppressive immune subsets in addition to markers already being considered. Alternatively, combination treatment with agents such as shIDO-ST could be used to create a more conducive tumor microenvironment for improving response rates to immunotherapy.

BackgroundI{kappa}B{zeta}, a rather unknown co-regulator of NF-{kappa}B, is mostly inducibly expressed and can either activate or repress a specific subset of NF-{kappa}B target genes. While its role as a transcriptional regulator of various cytokines and chemokines in immune cells has been revealed, I{kappa}B{zeta}s function in solid cancer remains unclear. MethodsWe investigated I{kappa}B{zeta} expression in melanoma, and assessed its impact on target gene expression, tumor growth, and response to immunotherapy in melanoma cell lines, mouse models, and patient samples. ResultsUnlike in other cell types, I{kappa}B{zeta} protein was found to be constitutively expressed in a subfraction of melanoma cell lines, and around 35% of melanoma cases. This atypical expression pattern of I{kappa}B{zeta} did not correlate with its mRNA levels or known driver mutations, but instead seemed to result from changes in its post-transcriptional or post-translational regulation. Deleting constitutively expressed I{kappa}B{zeta} abrogated the activity and chromatin association of STAT3 and p65, leading to reduced expression of the pro-proliferative cytokines IL-1{beta} and IL-6 in melanoma cells. Consequently, loss of tumor-derived I{kappa}B{zeta} suppressed self-sustained melanoma cell growth both in vitro and in vivo. Additionally, constitutive I{kappa}B{zeta} expression suppressed the induction of the chemokines CXCL9, CXCL10, and CCL5, which impaired the recruitment of NK and CD8+ T-cells to the tumor, causing resistance to -PD-1 immunotherapy in mice. Furthermore, the expression of tumor-derived I{kappa}B{zeta} also correlated with the absence of CD8+ T-cells in human melanoma samples and progressive disease during immunotherapy. ConclusionWe propose that tumor-derived I{kappa}B{zeta} could serve as a new therapeutic target and prognostic marker that characterizes melanoma with high tumor cell proliferation, cytotoxic T- and NK-cell exclusion, and unfavorable immunotherapy responses. Targeting I{kappa}B{zeta} expression might open up a new therapy option to re-establish the recruitment of cytotoxic cells, thereby resensitizing for immunotherapy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/613946v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@76389aorg.highwire.dtl.DTLVardef@17e3f1eorg.highwire.dtl.DTLVardef@161d491org.highwire.dtl.DTLVardef@1ca9f2d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Melanomas resistant to immune checkpoint blockade (ICB) therapies are frequently characterized by having a tumor-immune microenvironment (TiME) enriched with immunosuppressive tumor associated macrophages (TAMs). AXL tyrosine kinase is an emerging molecular target implicated in aggressive behavior by multiple cancers and both immunostimulatory and immunosuppressive functions of TAMs, however its role as a therapeutic target in melanoma has been poorly defined. Here, we define the relevance of AXL in melanoma with focus on efficacy of AXL-targeted therapy to restore sensitivity to anti-PD-1 therapy, shift TAM profiles, and augment anti-tumor immune responses in a TiME-specific context. We first demonstrated that the AXL+ tumor cell populations within melanoma are small, with TAMs representing the overwhelming source of AXL expression and activity. AXL inhibition effectively treated ICB-resistant melanoma and synergized with anti-PD-1 therapy in vivo; however, results were TAM-dependent. In vitro and in vivo, AXL inhibition in macrophages resulted in a powerful immunostimulatory reprogramming with net effect of disrupting checkpoint interactions in tumors. These results indicate that use of AXL as a selective biomarker or AXL inhibition as a therapeutic strategy to restore ICB responses may be cell-type or TiME-specific. Our data supports the modulation of TAM-enriched environments to enhance responses to immunotherapy that may facilitate future precise tailoring of therapies for refractory melanoma. Statement of SignificanceThis study investigates the macrophage-driven impact of AXL tyrosine kinase inhibition in ICB-resistant melanoma. AXL inhibition reduces TAMs in vivo and shifts immunosuppressive TAMs to immunostimulatory functions to improve response to ICB but may require immunostimulatory TAM-environments for maximal effect. These findings support the further exploration of AXL-targeting in TAMs for overcoming ICB resistance in precision melanoma treatment.

Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1) receptors, two clinically relevant targets for immunotherapy of cancer, are negative regulators of in immune cell activation and migration. However, optimizing therapeutic outcomes still requires fundamental research to reach a comprehensive insight into the coherent function of immune regulators. Here, we investigated the statistical dynamics of T cells migration as a measure of the functional response to these pathways in an experimental setup of immune checkpoint blockade. For this purpose, we used a previously developed 3-dimensional organotypic culture of patient-derived tumor spheroids. Experiment-based dynamical modeling remarked distinct characteristics of the receptors regulation followed through with the modification of their proportions in the immune modulation. We demonstrated that time-delayed kinetics of PD-1 activation just overrides its relatively more efficient cell-level function which potentially makes an operative contribution to the functional dominance of CTLA-4 in the tumor microenvironment. Simulation results showed good agreement with data for tumor cells reduction and active immune cells count observed in each experiment. These analyses propose a new mechanistic view on relative immunogenicity of PD-1 and CTLA-4 inhibitors manifested in literature and point the possible inherent obstacles in checkpoint inhibition-based immunotherapy of cancer to address in the future. SignificanceEx vivo monitoring of temporal response to PD-1 and CTLA-4 in the closure of T cell movement dynamics and elucidating their feasible commitment to the kinetic constraints at cell-level resolution. Delayed dynamics of migratory response to CTLA-4 inhibition revealed a mechanistic view on potential T cell reinvigoration following immune checkpoint blockade.

BackgroundThough the CXCR2 chemokine receptor is known to play a key role in cancer growth and response to therapy, a direct link between expression of CXCR2 in tumor progenitor cells during induction of tumorigenesis has not been established. MethodsTo characterize the role of CXCR2 during melanoma tumorigenesis, we generated tamoxifen-inducible tyrosinase-promoter driven BrafV600E/Pten-/-/Cxcr2-/- and NRasQ61R/INK4a-/-/Cxcr2-/- melanoma models. In addition, the effects of a CXCR1/CXCR2 antagonist, SX-682, on melanoma tumorigenesis were evaluated in BrafV600E/Pten-/- and NRasQ61R/INK4a-/- mice and in melanoma cell lines. Potential mechanisms by which Cxcr2 affects melanoma tumorigenesis in these murine models were explored using RNAseq, mMCP-counter, ChIPseq, and qRT-PCR; flow cytometry, and reverse phosphoprotein analysis (RPPA). ResultsGenetic loss of Cxcr2 or pharmacological inhibition of CXCR1/CXCR2 during melanoma tumor induction resulted in key changes in gene expression that reduced tumor incidence/growth and increased anti-tumor immunity. Interestingly, after Cxcr2 ablation, Tfcp2l1, a key tumor suppressive transcription factor, was the only gene significantly induced with a log2 fold-change greater than 2 in these three different melanoma models. ConclusionsHere, we provide novel mechanistic insight revealing how loss of Cxcr2 expression/activity in melanoma tumor progenitor cells results in reduced tumor burden and creation of an anti-tumor immune microenvironment. This mechanism entails an increase in expression of the tumor suppressive transcription factor, Tfcp2l1, along with alteration in the expression of genes involved in growth regulation, tumor suppression, stemness, differentiation, and immune modulation. These gene expression changes are coincident with reduction in the activation of key growth regulatory pathways, including AKT and mTOR.

"Pro-senescence therapy", which triggers both permanent cell cycle arrest and an immune response, has been proposed as a strategy for cancer treatment, but is still controversial. To assess this strategy in melanoma, we performed a high throughput microscopy-based senescence screen utilizing a panel of melanoma cell lines with different driver mutations and a collection of clinical and experimental drugs. We found that vemurafenib and trametinib, which inhibit BRAFV600E and MEK1/2, respectively, induced senescence in some but not all BRAF-mutant cell lines. In contrast, palbociclib, BKM-120 and crizotinib, which inhibit CDK4/6, PI3K, and MET/ALK/ROS1, respectively, triggered senescence in most cell lines, irrespective of BRAF/NRAS mutation status, and overcame intrinsic and acquired vemurafenib resistance. The combination of palbociclib and crizotinib synergized to further enhance the senescence response in all cell lines irrespective of BRAF/NRAS mutation status, increased the expression of SASP factors, such as IL-1 and {beta}, and HLA class I and other markers for recognition by NK and T cells. Further, this combination caused a significant increase in CD8+ T cells and pro-inflammatory macrophages in the tumor microenvironment and a marked reduction of mouse melanoma tumor growth that was dependent on CD8+ T cells, suggesting increased immune surveillance. Our findings suggest that pro-senescence therapy based on concomitant inhibition of both CDK4/6 and MET/ALK/ROS1 could be developed further as an alternative treatment strategy for melanoma. Significance: Pro-senescence therapy based on combined targeting of CDK4/6 with palbociclib and MET/ALK/ROS1 with crizotinib inhibits melanoma tumor growth through anti-tumor immune response activation, providing an alternative treatment strategy for melanoma.

Natural killer (NK) cells have emerged as an important clinical tool cellular immunotherapy. Whereas immune checkpoint blockade (ICB) or chimeric antigen receptor (CAR) T-cell therapy (CAR-T) therapy have been adopted as a first line treatments in different malignancies, such as melanoma, these approaches do not work for all patients. T cells require proper antigen presentation on tumor cells for recognition and to carry out their corresponding cytotoxic functions. Deficiency of tumor antigens, or high variability in those present, make T cell-based CAR-T and ICB ineffective. By contrast, NK cells are not limited by antigen presentation deficiencies, offering a potential alternative approach, yet their efficacy can suffer from immunosuppressive signals. Herein, we sought to develop in vitro and on-chip platforms to identify strategies for enhance, rather than suppress, NK cell homing to tumor cells. We explored the use of inhibition of kinases such as CK4/6 and AURKA to induce tumor cell production of chemokines that NK cells migrate towards in aggressive melanoma models. We evaluated chemokine-aided NK cell migration-homing capabilities and their therapeutic efficacy and found that treatment of both melanoma cell line and patient-tumor constructs (PTCs) with CDK4/6 and AURKA generally resulted in improved NK cell homing to tumor cells and accompanying tumor cell killing. Interestingly, this chemokine-guided NK cell migration did not generate as effective outcomes in models using a mildly aggressive melanoma cell line. For our studies, we used 3D tumor constructs in both static Transwell models and then in a bioengineered NK cell-functionalized tumor-on-a-chip (NK-TOC) platform. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/662476v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@17bf445org.highwire.dtl.DTLVardef@e1e1d9org.highwire.dtl.DTLVardef@1b29c2corg.highwire.dtl.DTLVardef@12b1f03_HPS_FORMAT_FIGEXP M_FIG C_FIG

Targeting tumour antigens is a major challenge in cancer-immunotherapy. We use active vaccination to induce antibodies targeting self-antigen Robo4, which is selectively expressed on tumour vascular endothelium, supporting vascular development. Our previous work showed that a conjugate of Robo4 with a foreign carrier protein can induce autoantibodies specific to Robo4, which inhibited angiogenesis and tumour growth. The current project aims to translate the vaccine protocol to exploit a carrier protein used in routine human vaccination schedules. The well-characterised, non-toxic fragment C of tetanus toxin (TTc) was selected as the carrier protein. Here we show that priming with the carrier TTc followed by boost with Robo4-TTc (R4-TTc) efficiently induces strong antibody responses to Robo4 and inhibits tumour growth in LLC1 and 4T1 tumour models. The growth inhibition was correlated with anti-Robo4 IgG1 titres. Furthermore, we observed decreased vessel formation and increased immune cell infiltration in tumours from R4-TTc vaccinated mice in the absence of detectable adverse effects on health. The data indicate that this vaccination strategy remodels tumour vessels and probably promotes immunogenic pathway activation, therefore repressing tumour growth. One Sentence SummaryA conjugate vaccine inducing antibody responses to tumour endothelial markers Robo4 can inhibit tumour growth.