Tumor-B-cell interactions promote isotype switching to an immunosuppressive IgG4 antibody response through upregulation of IL-10 in triple negative breast cancers.

BACKGROUND: Triple negative breast cancer (TNBC) is an aggressive breast cancer for which there is currently no targeted therapy. Tumor-infiltrating B-cells (TIB) have been observed in tumor tissues of TNBC patients, but their functional role is unclear. IgG4 is one of four antibody subclasses of IgG expressed and secreted by B cells. Unlike other IgG isotypes, IgG4 has an immunosuppressive function and is induced by Th2-type cytokines. In cancers such as melanoma, IgG4 has been linked with advanced disease and poor patient survival. Therefore, we sought to determine if IgG4 + B cells are present and determine the mechanisms driving isotype switching in TNBC. METHODS: We performed co-culture assays to examine expression of Th2 cytokines by TNBC cells with and without the presence of B cells. We also performed in vitro class switching experiments with peripheral B cells with and without co-culture with TNBC cells in the presence or absence of an IL-10 blocking antibody. We examined expression of CD20+ TIB, IgG4 and Th2 cytokines by immunohistochemistry in 152 TNBC samples. Statistical analysis was done using Log-Rank and Cox-proportional hazards tests. RESULTS: Our findings indicate that B cells interact with TNBC to drive chronic inflammatory responses through increased expression of inflammatory cytokines including the TH2 cytokines IL-4 and IL-10. In vitro class switching studies show that interactions between TNBC cell lines and B cells drive isotype switching to the IgG4 isotype in an IL-10 dependent manner. In patient tissues, expression of IgG4 correlates with CD20 and tumor expression of IL-10. Both IgG4 and tumor IL-10 are associated to shorter recurrence free survival (RFS) and overall survival (OS) in TNBC. In a multi-variant analysis, IL-10 was associated with poor outcomes indicating that tumor IL-10 may drive immune escape. CONCLUSIONS: These findings indicate that interactions between TIB and TNBC results in activation of chronic inflammatory signals such as IL-10 and IL-4 that drive class switching to an IgG4 + subtype which may suppress antibody driven immune responses. The presence of IgG4 + B cells may serve as a biomarker for poor prognosis.

NUMB as a Therapeutic Target for Melanoma.

The upregulation of the adaptor protein NUMB triggers melanocytic differentiation from multipotent skin stem cells, which share many properties with aggressive melanoma cells. Although NUMB acts as a tumor suppressor in various human cancer types, little is known about its role in melanoma. In this study, we investigated the role of NUMB in melanoma progression and its regulatory mechanism. Analysis of The Cancer Genome Atlas melanoma datasets revealed that high NUMB expression in melanoma tissues correlates with improved patient survival. Moreover, NUMB expression is downregulated in metastatic melanoma cells. NUMB knockdown significantly increased the invasion potential of melanoma cells in a three-dimensional collagen matrix in vitro and in the lungs of a mouse model in vivo; it also significantly upregulated the expression of the NOTCH target gene CCNE. Previous studies suggested that Wnt signaling increases NUMB expression. By mimicking Wnt stimulation through glycogen synthase kinase-3 inhibition, we increased NUMB expression in melanoma cells. Furthermore, a glycogen synthase kinase-3 inhibitor reduced the invasion of melanoma cells in a NUMB-dependent manner. Together, our results suggest that NUMB suppresses invasion and metastasis in melanoma, potentially through its regulation of the NOTCH‒CCNE axis and that the inhibitors that upregulate NUMB can exert therapeutic effects in melanoma.

Costimulation of γδTCR and TLR7/8 promotes Vδ2 T-cell antitumor activity by modulating mTOR pathway and APC function.

BACKGROUND: Gamma delta (γδ) T cells are attractive effector cells for cancer immunotherapy. Vδ2 T cells expanded by zoledronic acid (ZOL) are the most commonly used γδ T cells for adoptive cell therapy. However, adoptive transfer of the expanded Vδ2 T cells has limited clinical efficacy. METHODS: We developed a costimulation method for expansion of Vδ2 T cells in PBMCs by activating γδ T-cell receptor (γδTCR) and Toll-like receptor (TLR) 7/8 using isopentenyl pyrophosphate (IPP) and resiquimod, respectively, and tested the functional markers and antitumoral effects in vitro two-dimensional two-dimensional and three-dimensional spheroid models and in vivo models. Single-cell sequencing dataset analysis and reverse-phase protein array were employed for mechanistic studies. RESULTS: We find that Vδ2 T cells expanded by IPP plus resiquimod showed significantly increased cytotoxicity to tumor cells with lower programmed cell death protein 1 (PD-1) expression than Vδ2 T cells expanded by IPP or ZOL. Mechanistically, the costimulation enhanced the activation of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB/Akt)-the mammalian target of rapamycin (mTOR) pathway and the TLR7/8-MyD88 pathway. Resiquimod stimulated Vδ2 T-cell expansion in both antigen presenting cell dependent and independent manners. In addition, resiquimod decreased the number of adherent inhibitory antigen-presenting cells (APCs) and suppressed the inhibitory function of APCs by decreasing PD-L1 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression in these cells during in vitro Vδ2 T-cell expansion. Finally, we showed that human Vδ2 T cells can be expanded from PBMCs and spleen of humanized NSG mice using IPP plus resiquimod or ZOL, demonstrating that humanized mice are a promising preclinical model for studying human γδ T-cell development and function. CONCLUSIONS: Vδ2 T cells expanded by IPP and resiquimod demonstrate improved anti-tumor function and have the potential to increase the efficacy of γδ T cell-based therapies.

Neural Crest-Like Stem Cell Transcriptome Analysis Identifies LPAR1 in Melanoma Progression and Therapy Resistance.

Metastatic melanoma is challenging to clinically address. Although standard-of-care targeted therapy has high response rates in patients with BRAF-mutant melanoma, therapy relapse occurs in most cases. Intrinsically resistant melanoma cells drive therapy resistance and display molecular and biologic properties akin to neural crest-like stem cells (NCLSC) including high invasiveness, plasticity, and self-renewal capacity. The shared transcriptional programs and vulnerabilities between NCLSCs and cancer cells remains poorly understood. Here, we identify a developmental LPAR1-axis critical for NCLSC viability and melanoma cell survival. LPAR1 activity increased during progression and following acquisition of therapeutic resistance. Notably, genetic inhibition of LPAR1 potentiated BRAFi ± MEKi efficacy and ablated melanoma migration and invasion. Our data define LPAR1 as a new therapeutic target in melanoma and highlights the promise of dissecting stem cell-like pathways hijacked by tumor cells. SIGNIFICANCE: This study identifies an LPAR1-axis critical for melanoma invasion and intrinsic/acquired therapy resistance.

Tumor-infiltrating mast cells are associated with resistance to anti-PD-1 therapy.

Anti-PD-1 therapy is used as a front-line treatment for many cancers, but mechanistic insight into this therapy resistance is still lacking. Here we generate a humanized (Hu)-mouse melanoma model by injecting fetal liver-derived CD34+ cells and implanting autologous thymus in immune-deficient NOD-scid IL2Rγnull (NSG) mice. Reconstituted Hu-mice are challenged with HLA-matched melanomas and treated with anti-PD-1, which results in restricted tumor growth but not complete regression. Tumor RNA-seq, multiplexed imaging and immunohistology staining show high expression of chemokines, as well as recruitment of FOXP3+ Treg and mast cells, in selective tumor regions. Reduced HLA-class I expression and CD8+/Granz B+ T cells homeostasis are observed in tumor regions where FOXP3+ Treg and mast cells co-localize, with such features associated with resistance to anti-PD-1 treatment. Combining anti-PD-1 with sunitinib or imatinib results in the depletion of mast cells and complete regression of tumors. Our results thus implicate mast cell depletion for improving the efficacy of anti-PD-1 therapy.

Challenges in the humanized mouse model for cancer: A commentary.

The complexity of the tumor microenvironment has been a challenge for understanding the mechanisms of therapy resistance. The development of improved animal models that closely mimic human disease is key for understanding and treating diseases. Recently, a new humanized mouse model has been developed that enables the study of human immune cells in tumor host-cell interactions. In this commentary we highlight the critical aspects of mast cells in immune therapy resistance. These mast cells release cytokines that downmodulate HLA class I on the malignant cells making them inaccessible the cytotoxic activity of T cells.

ARID2 Deficiency Correlates with the Response to Immune Checkpoint Blockade in Melanoma.

The SWI/SNF chromatin remodeler family includes the BAF and PBAF complexes. ARID2, encoding a PBAF complex subunit, is frequently mutated in melanoma independently of BRAF/RAS mutations. Emerging evidence shows that SWI/SNF complexes regulate tumor immunity; for instance, the loss of PBRM1, another PBAF complex subunit, enhances susceptibility to immune checkpoint inhibitors in melanoma. Notably, ARID2 mutations are more frequent in melanoma than PBRM1 mutations. However, the role of ARID2 as a modulator of tumor immunity remains unclear. In this study, we show that ARID2 knockout sensitizes melanoma to immune checkpoint inhibitors. Anti‒PD-L1 treatment restricts tumor growth in mice bearing ARID2-knockout melanoma cells, correlating with an increase in the infiltration of cytotoxic CD8+ T cells. Furthermore, ARID2 deficiency leads to signal transducer and activator of transcription 1 upregulation, which subsequently causes increased expression of T-cell‒attracting chemokines such as CXCL9, CXCL10, and CCL5. These results demonstrate that ARID2 is an immunomodulator and a potential biomarker that indicates immune checkpoint inhibitor effectiveness in patients with melanoma.

IspH inhibitors kill Gram-negative bacteria and mobilize immune clearance.

Isoprenoids are vital for all organisms, in which they maintain membrane stability and support core functions such as respiration1. IspH, an enzyme in the methyl erythritol phosphate pathway of isoprenoid synthesis, is essential for Gram-negative bacteria, mycobacteria and apicomplexans2,3. Its substrate, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), is not produced in metazoans, and in humans and other primates it activates cytotoxic Vγ9Vδ2 T cells at extremely low concentrations4-6. Here we describe a class of IspH inhibitors and refine their potency to nanomolar levels through structure-guided analogue design. After modification of these compounds into prodrugs for delivery into bacteria, we show that they kill clinical isolates of several multidrug-resistant bacteria-including those from the genera Acinetobacter, Pseudomonas, Klebsiella, Enterobacter, Vibrio, Shigella, Salmonella, Yersinia, Mycobacterium and Bacillus-yet are relatively non-toxic to mammalian cells. Proteomic analysis reveals that bacteria treated with these prodrugs resemble those after conditional IspH knockdown. Notably, these prodrugs also induce the expansion and activation of human Vγ9Vδ2 T cells in a humanized mouse model of bacterial infection. The prodrugs we describe here synergize the direct killing of bacteria with a simultaneous rapid immune response by cytotoxic γδ T cells, which may limit the increase of antibiotic-resistant bacterial populations.

Pre-clinical modeling of cutaneous melanoma.

Metastatic melanoma is challenging to manage. Although targeted- and immune therapies have extended survival, most patients experience therapy resistance. The adaptability of melanoma cells in nutrient- and therapeutically-challenged environments distinguishes melanoma as an ideal model for investigating therapy resistance. In this review, we discuss the current available repertoire of melanoma models including two- and three-dimensional tissue cultures, organoids, genetically engineered mice and patient-derived xenograft. In particular, we highlight how each system recapitulates different features of melanoma adaptability and can be used to better understand melanoma development, progression and therapy resistance.

B cells sustain inflammation and predict response to immune checkpoint blockade in human melanoma.

Tumor associated inflammation predicts response to immune checkpoint blockade in human melanoma. Current theories on regulation of inflammation center on anti-tumor T cell responses. Here we show that tumor associated B cells are vital to melanoma associated inflammation. Human B cells express pro- and anti-inflammatory factors and differentiate into plasmablast-like cells when exposed to autologous melanoma secretomes in vitro. This plasmablast-like phenotype can be reconciled in human melanomas where plasmablast-like cells also express T cell-recruiting chemokines CCL3, CCL4, CCL5. Depletion of B cells in melanoma patients by anti-CD20 immunotherapy decreases tumor associated inflammation and CD8+ T cell numbers. Plasmablast-like cells also increase PD-1+ T cell activation through anti-PD-1 blockade in vitro and their frequency in pretherapy melanomas predicts response and survival to immune checkpoint blockade. Tumor associated B cells therefore orchestrate and sustain melanoma inflammation and may represent a predictor for survival and response to immune checkpoint blockade therapy.

Remodeling of the Collagen Matrix in Aging Skin Promotes Melanoma Metastasis and Affects Immune Cell Motility.

Physical changes in skin are among the most visible signs of aging. We found that young dermal fibroblasts secrete high levels of extracellular matrix (ECM) constituents, including proteoglycans, glycoproteins, and cartilage-linking proteins. The most abundantly secreted was HAPLN1, a hyaluronic and proteoglycan link protein. HAPLN1 was lost in aged fibroblasts, resulting in a more aligned ECM that promoted metastasis of melanoma cells. Reconstituting HAPLN1 inhibited metastasis in an aged microenvironment, in 3-D skin reconstruction models, and in vivo. Intriguingly, aged fibroblast-derived matrices had the opposite effect on the migration of T cells, inhibiting their motility. HAPLN1 treatment of aged fibroblasts restored motility of mononuclear immune cells, while impeding that of polymorphonuclear immune cells, which in turn affected regulatory T-cell recruitment. These data suggest that although age-related physical changes in the ECM can promote tumor cell motility, they may adversely affect the motility of some immune cells, resulting in an overall change in the immune microenvironment. Understanding the physical changes in aging skin may provide avenues for more effective therapy for older patients with melanoma. SIGNIFICANCE: These data shed light on the mechanochemical interactions that occur between aged skin, tumor, and immune cell populations, which may affect tumor metastasis and immune cell infiltration, with implications for the efficacy of current therapies for melanoma.See related commentary by Marie and Merlino, p. 19.This article is highlighted in the In This Issue feature, p. 1.

Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response.

Tumour cells evade immune surveillance by upregulating the surface expression of programmed death-ligand 1 (PD-L1), which interacts with programmed death-1 (PD-1) receptor on T cells to elicit the immune checkpoint response1,2. Anti-PD-1 antibodies have shown remarkable promise in treating tumours, including metastatic melanoma2-4. However, the patient response rate is low4,5. A better understanding of PD-L1-mediated immune evasion is needed to predict patient response and improve treatment efficacy. Here we report that metastatic melanomas release extracellular vesicles, mostly in the form of exosomes, that carry PD-L1 on their surface. Stimulation with interferon-γ (IFN-γ) increases the amount of PD-L1 on these vesicles, which suppresses the function of CD8 T cells and facilitates tumour growth. In patients with metastatic melanoma, the level of circulating exosomal PD-L1 positively correlates with that of IFN-γ, and varies during the course of anti-PD-1 therapy. The magnitudes of the increase in circulating exosomal PD-L1 during early stages of treatment, as an indicator of the adaptive response of the tumour cells to T cell reinvigoration, stratifies clinical responders from non-responders. Our study unveils a mechanism by which tumour cells systemically suppress the immune system, and provides a rationale for the application of exosomal PD-L1 as a predictor for anti-PD-1 therapy.

Age Correlates with Response to Anti-PD1, Reflecting Age-Related Differences in Intratumoral Effector and Regulatory T-Cell Populations.

Purpose: We have shown that the aged microenvironment increases melanoma metastasis, and decreases response to targeted therapy, and here we queried response to anti-PD1.Experimental Design: We analyzed the relationship between age, response to anti-PD1, and prior therapy in 538 patients. We used mouse models of melanoma, to analyze the intratumoral immune microenvironment in young versus aged mice and confirmed our findings in human melanoma biopsies.Results: Patients over the age of 60 responded more efficiently to anti-PD-1, and likelihood of response to anti-PD-1 increased with age, even when we controlled for prior MAPKi therapy. Placing genetically identical tumors in aged mice (52 weeks) significantly increased their response to anti-PD1 as compared with the same tumors in young mice (8 weeks). These data suggest that this increased response in aged patients occurs even in the absence of a more complex mutational landscape. Next, we found that young mice had a significantly higher population of regulatory T cells (Tregs), skewing the CD8+:Treg ratio. FOXP3 staining of human melanoma biopsies revealed similar increases in Tregs in young patients. Depletion of Tregs using anti-CD25 increased the response to anti-PD1 in young mice.Conclusions: While there are obvious limitations to our study, including our inability to conduct a meta-analysis due to a lack of available data, and our inability to control for mutational burden, there is a remarkable consistency in these data from over 500 patients across 8 different institutes worldwide. These results stress the importance of considering age as a factor for immunotherapy response. Clin Cancer Res; 24(21); 5347-56. ©2018 AACR See related commentary by Pawelec, p. 5193.

Co-targeting BET and MEK as salvage therapy for MAPK and checkpoint inhibitor-resistant melanoma.

Despite novel therapies for melanoma, drug resistance remains a significant hurdle to achieving optimal responses. NRAS-mutant melanoma is an archetype of therapeutic challenges in the field, which we used to test drug combinations to avert drug resistance. We show that BET proteins are overexpressed in NRAS-mutant melanoma and that high levels of the BET family member BRD4 are associated with poor patient survival. Combining BET and MEK inhibitors synergistically curbed the growth of NRAS-mutant melanoma and prolonged the survival of mice bearing tumors refractory to MAPK inhibitors and immunotherapy. Transcriptomic and proteomic analysis revealed that combining BET and MEK inhibitors mitigates a MAPK and checkpoint inhibitor resistance transcriptional signature, downregulates the transcription factor TCF19, and induces apoptosis. Our studies demonstrate that co-targeting MEK and BET can offset therapy resistance, offering a salvage strategy for melanomas with no other therapeutic options, and possibly other treatment-resistant tumor types.

Induction of Telomere Dysfunction Prolongs Disease Control of Therapy-Resistant Melanoma.

Purpose: Telomerase promoter mutations are highly prevalent in human tumors including melanoma. A subset of patients with metastatic melanoma often fail multiple therapies, and there is an unmet and urgent need to prolong disease control for those patients.Experimental Design: Numerous preclinical therapy-resistant models of human and mouse melanoma were used to test the efficacy of a telomerase-directed nucleoside, 6-thio-2'-deoxyguanosine (6-thio-dG). Integrated transcriptomics and proteomics approaches were used to identify genes and proteins that were significantly downregulated by 6-thio-dG.Results: We demonstrated the superior efficacy of 6-thio-dG both in vitro and in vivo that results in telomere dysfunction, leading to apoptosis and cell death in various preclinical models of therapy-resistant melanoma cells. 6-thio-dG concomitantly induces telomere dysfunction and inhibits the expression level of AXL.Conclusions: In summary, this study shows that indirectly targeting aberrant telomerase in melanoma cells with 6-thio-dG is a viable therapeutic approach in prolonging disease control and overcoming therapy resistance. Clin Cancer Res; 24(19); 4771-84. ©2018 AACR See related commentary by Teh and Aplin, p. 4629.

Tumor-associated B-cells induce tumor heterogeneity and therapy resistance.

In melanoma, therapies with inhibitors to oncogenic BRAFV600E are highly effective but responses are often short-lived due to the emergence of drug-resistant tumor subpopulations. We describe here a mechanism of acquired drug resistance through the tumor microenvironment, which is mediated by human tumor-associated B cells. Human melanoma cells constitutively produce the growth factor FGF-2, which activates tumor-infiltrating B cells to produce the growth factor IGF-1. B-cell-derived IGF-1 is critical for resistance of melanomas to BRAF and MEK inhibitors due to emergence of heterogeneous subpopulations and activation of FGFR-3. Consistently, resistance of melanomas to BRAF and/or MEK inhibitors is associated with increased CD20 and IGF-1 transcript levels in tumors and IGF-1 expression in tumor-associated B cells. Furthermore, first clinical data from a pilot trial in therapy-resistant metastatic melanoma patients show anti-tumor activity through B-cell depletion by anti-CD20 antibody. Our findings establish a mechanism of acquired therapy resistance through tumor-associated B cells with important clinical implications.Resistance to BRAFV600E inhibitors often occurs in melanoma patients. Here, the authors describe a potential mechanism of acquired drug resistance mediated by tumor-associated B cells-derived IGF-1.

The Multifaceted Roles of B Cells in Solid Tumors: Emerging Treatment Opportunities.

The influence of tumor infiltrating lymphocytes on tumor growth and response to therapy is becoming increasingly apparent. While much work has focused on the role of T cell responses in anti-tumor immunity, the role of B cells in solid tumors is much less understood. Tumor infiltrating B cells have been found in a variety of solid tumors, including breast, ovarian, prostate, melanoma, and colorectal cancer. The function of B cells in solid tumors is controversial, with many studies reporting a pro-tumor effect, while other studies demonstrate a role for B cells in the anti-tumor immune response. In this review, we discuss the prognostic ability of B cells in solid tumors as well as the mechanisms by which B cells can either promote or suppress anti-tumor immunity. Additionally, we review current therapeutic strategies that may target both pro- and anti-tumor B cells.

The role of tumor microenvironment in melanoma therapy resistance.

Melanoma patients develop resistance to both chemotherapy and targeted-therapy drugs. Promising preclinical and clinical results with immune checkpoint inhibitors using antibodies directed against cytotoxic T-lymphocyte-associated protein 4 and programmed cell death protein 1 have re-energized the field of immune-based therapies in melanoma. However, similar to chemotherapy or targeted therapies, immune checkpoint blockade responds in only subsets of melanoma patients. A number of factors, including gene mutations, altered cell-signaling pathways and tumor heterogeneity can contribute to therapy resistance. Recent studies have highlighted the role of inflammatory tumor microenvironment on therapy resistance of cancer cells. Cancer cells either alone or in conjunction with the tumor stroma can contribute to an inflammatory microenvironment. Multimodal approaches of targeting the tumor microenvironment, in addition to malignant cells, may be necessary for better therapy responses.

Type I-polarized BRAF-pulsed dendritic cells induce antigen-specific CD8+ T cells that impact BRAF-mutant murine melanoma.

Existing therapies targeting the mutated BRAF oncodriver (BRAF(V600E)) successfully treat melanoma but are susceptible to resistance. This study assessed the potential of a dendritic cell-based BRAF(V600E) vaccine for the treatment of BRAF(V600E)-mutant melanoma. Type 1-polarized dendritic cells (DC1) pulsed with affinity-modified BRAF(V600E) peptide were administered to C57Bl/6 mice both before (prevention) and twice weekly after (treatment) the development of established tumor with B16 melanoma transfected to express BRAF(V600E) (B16(V600E)). The efficacy of the BRAF(V600E)-pulsed DC1 vaccine was corroborated in a novel transplantable BRAF(V600E)-mutant murine melanoma model (BRAF(V600E-/+); PTEN(-/-); CDK2NA(-/-)). Three-dimensional tumor measurements and survival were determined. Induction of BRAF(V600E)-specific CD8(+) T-cell responses after brief in-vitro sensitization was assessed by interferon-γ enzyme-linked immunosorbent assay and/or enzyme-linked immunospot. Mice receiving BRAF(V600E)-pulsed DC1 vaccines before B16(V600E) tumor challenge demonstrated increased tumor-doubling times (P<0.001) and improved survival (P=0.0186) compared with those that received ovalbumin (control)-pulsed DC1 vaccines. In mice bearing established B16(V600E) tumors (mean 32 mm(3)), BRAF-pulsed DC1 vaccines delayed tumor growth (P<0.001) and improved survival (P=0.0008), compared with untreated mice. Likewise, in mice bearing BRAF(V600E-/+); PTEN(-/-); CDK2NA(-/-) tumors, compared with controls, BRAF-DC1 vaccination recapitulated these effects by delaying tumor growth (P<0.001) and improving survival (P=0.002). Vaccination elicited specific CD8(+) T-cell recognition of BRAF(V600E)-pulsed antigen-presenting cells (P<0.05), as well as BRAF(V600E)-expressing cancer cells (P<0.001), measured by interferon-γ release in vitro. BRAF-(V600E)pulsed DC1 vaccines induce oncogene-specific CD8(+) T-cell immune responses that impact tumor growth and survival in preclinical models of BRAF(V600E)-mutant melanoma. Exploration of BRAF(V600E)-targeted vaccines, in combination with BRAF-targeted therapies and checkpoint inhibitors, is warranted.