Search

Single-cell technologies have enabled the characterization of the tumour microenvironment at unprecedented depth and have revealed vast cellular diversity among tumour cells and their niche. Anti-tumour immunity relies on cell–cell relationships within the tumour microenvironment1,2, yet many single-cell studies lack spatial context and rely on dissociated tissues3. Here we applied imaging mass cytometry to characterize the immunological landscape of 139 high-grade glioma and 46 brain metastasis tumours from patients. Single-cell analysis of more than 1.1 million cells across 389 high-dimensional histopathology images enabled the spatial resolution of immune lineages and activation states, revealing differences in immune landscapes between primary tumours and brain metastases from diverse solid cancers. These analyses revealed cellular neighbourhoods associated with survival in patients with glioblastoma, which we leveraged to identify a unique population of myeloperoxidase (MPO)-positive macrophages associated with long-term survival. Our findings provide insight into the biology of primary and metastatic brain tumours, reinforcing the value of integrating spatial resolution to single-cell datasets to dissect the microenvironmental contexture of cancer.

Table S1: List of shRNA vectors. Table S2: List of RTqPCR primers.Table S3. List of targeted compounds.

Cell cycle analysis after BETi/cobimetinib exposure in OVCAR4 cells

Legends for Figures S1, S2, S3 and S4. Supplementary Materials and Methods.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare but often lethal cancer that is diagnosed at a median age of 24 years. Optimal management of patients is not well defined, and current treatment remains challenging, necessitating the discovery of novel therapeutic approaches. The identification of SMARCA4-inactivating mutations invariably characterizing this type of cancer provided insights facilitating diagnostic and therapeutic measures against this disease. We show here that the BET inhibitor OTX015 acts in synergy with the MEK inhibitor cobimetinib to repress the proliferation of SCCOHT in vivo. Notably, this synergy is also observed in some SMARCA4-expressing ovarian adenocarcinoma models intrinsically resistant to BETi. Mass spectrometry, coupled with knockdown of newly found targets such as thymidylate synthase, revealed that the repression of a panel of proteins involved in nucleotide synthesis underlies this synergy both in vitro and in vivo, resulting in reduced pools of nucleotide metabolites and subsequent cell-cycle arrest. Overall, our data indicate that dual treatment with BETi and MEKi represents a rational combination therapy against SCCOHT and potentially additional ovarian cancer subtypes.

Supp Table S1. List of IFNγ pathway genes investigated for TCGA analysis

Supp Figure S4. JAK1/2 inhibitor Ruxolitinib increases melanoma sensitivity to VSV-d51

Supplementary Fig. S1. Heatmap of mRNA expression for individual melanosomal differentiation antigens and a MITF transcriptional signature using a published dataset of melanoma samples (2). Supplementary Fig. S2. GPNMB expression is induced in multiple BRAF-mutant cells in response to MAPK pathway inhibition. Supplementary Fig. S3. Gene expression of MITF and GPNMB in melanoma cells following MAPK pathway inhibition. Supplementary Fig. S4. Dabrafenib and Trametinib stimulate MITF nuclear localization. Supplementary Fig. S5. Correlation of MITF and GPNMB mRNA expression in melanoma samples from patients that were on-treatment or that progressed on therapy with MAPK pathway 7 inhibitors. RT-qPCR data for MITF and GPNMB was expressed as the ratio relative to pretreatment levels. Supplementary Fig. S6. Correlation between the expression of individual melanosomal genes with MITF mRNA expression in melanoma samples from patients that were on-treatment or that progressed on therapy with MAPK pathway inhibitors. RT-qPCR data for each gene and MITF was normalized to GAPDH and expressed as the ratio relative to pre-treatment levels. Supplementary Fig. S7. MAPK pathway inhibition results in elevated levels of soluble GPNMB released from melanoma cells. Table S1: Clinical characteristics of patients biopsied for evaluation of MITF and GPNMB mRNA expression Table S2: Correlation coefficients between individual gene expression and MITF or a MITF Signature Table S3: Complete Responses Following Trametinib Treatment in Cohorts Injected with A375 or WM-2664 Melanoma Cells. Table S4: List of Primers for RT-qPCR Analyses

Supplementary legends and methods

Contains supplementary methods, references and figure legends.

Purpose: To determine if BRAF and/or MEK inhibitor–induced GPNMB expression renders melanomas sensitive to CDX-011, an antibody-drug conjugate targeting GPNMB.Experimental Design: The Cancer Genome Atlas melanoma dataset was interrogated for a panel of MITF-regulated melanosomal differentiation antigens, including GPNMB. BRAF-mutant melanoma cell lines treated with BRAF or MEK inhibitors were assessed for GPNMB expression by RT-qPCR, immunoblot, and FACS analyses. Transient siRNA-mediated knockdown approaches were used to determine if MITF is requirement for treatment-induced GPNMB upregulation. GPNMB expression was analyzed in serial biopsies and serum samples from patients with melanoma taken before, during, and after disease progression on MAPK inhibitor treatment. Subcutaneous injections were performed to test the efficacy of MAPK inhibitors alone, CDX-011 alone, or their combination in suppressing melanoma growth.Results: A MITF-dependent melanosomal differentiation signature is associated with poor prognosis in patients with this disease. MITF is increased following BRAF and MEK inhibitor treatment and induces the expression of melanosomal differentiation genes, including GPNMB. GPNMB is expressed at the cell surface in MAPK inhibitor–treated melanoma cells and is also elevated in on-treatment versus pretreatment biopsies from melanoma patients receiving MAPK pathway inhibitors. Combining BRAF and/or MEK inhibitors with CDX-011, an antibody-drug conjugate targeting GPNMB, is effective in causing melanoma regression in preclinical animal models and delays the recurrent melanoma growth observed with MEK or BRAF/MEK inhibitor treatment alone.Conclusions: The combination of MAPK pathway inhibitors with an antibody-drug conjugate targeting GPNMB is an effective therapeutic option for patients with melanoma. Clin Cancer Res; 22(24); 6088–98. ©2016 AACR.

Purpose:Next-generation sequencing studies and CRISPR-Cas9 screens have established mutations in the IFNγ-JAK-STAT pathway as an immune checkpoint inhibitor (ICI) resistance mechanism in a subset of patients with melanoma. We hypothesized ICI resistance mutations in the IFNγ pathway would simultaneously render melanomas susceptible to oncolytic virus (OV) therapy.Experimental Design:Cytotoxicity experiments were performed with a number of OVs on a matched melanoma cell line pair generated from a baseline biopsy and a progressing lesion with complete JAK2 loss from a patient that relapsed on anti-PD-1 therapy, in melanoma lines following JAK1/2 RNA interference (RNAi) and pharmacologic inhibition and in Jak2 knockout (KO) B16-F10 mouse melanomas. Furthermore, we estimated the frequency of genetic alterations in the IFNγ-JAK-STAT pathway in human melanomas.Results:The melanoma line from an anti-PD-1 progressing lesion was 7- and 22-fold more sensitive to the modified OVs, herpes simplex virus 1 (HSV1-dICP0) and vesicular stomatitis virus (VSV-Δ51), respectively, compared with the line from the baseline biopsy. RNAi, JAK1/2 inhibitor studies, and in vivo studies of Jak2 KOs B16-F10 melanomas revealed a significant increase in VSV-Δ51 sensitivity with JAK/STAT pathway inhibition. Our analysis of The Cancer Genome Atlas data estimated that approximately 11% of ICI-naïve cutaneous melanomas have alterations in IFNγ pathway genes that may confer OV susceptibility.Conclusions:We provide mechanistic support for the use of OVs as a precision medicine strategy for both salvage therapy in ICI-resistant and first-line treatment in melanomas with IFNγ-JAK-STAT pathway mutations. Our study also supports JAK inhibitor–OV combination therapy for treatment-naïve melanomas without IFN signaling defects.See related commentary by Kaufman, p. 3278

FIGURE S1: LY3009120 is an effective inhibitor of BRAF class II mutant melanoma cells. FIGURE S2: Bioinformatic characterization of PDX models. FIGURE S3: Inhibitory effects of BRAF and MEK inhibitors in melanoma cells. FIGURE S4: Representative images from cell growth clonogenic assays. FIGURE S5: Encorafenib and binimetinib are an effective combination for class II BRAF mutant melanoma. FIGURE S6: Dual MAPK inhibition slows progression of class II BRAF mutant melanoma brain metastases.

Purpose:Dual MAPK pathway inhibition (dMAPKi) with BRAF and MEK inhibitors improves survival in BRAF V600E/K mutant melanoma, but the efficacy of dMAPKi in non-V600 BRAF mutant tumors is poorly understood. We sought to characterize the responsiveness of class II (enhanced kinase activity, dimerization dependent) BRAF mutant melanoma to dMAPKi.Experimental Design:Tumors from patients with BRAF wild-type (WT), V600E (class I), and L597S (class II) metastatic melanoma were used to generate patient-derived xenografts (PDX). We assembled a panel of melanoma cell lines with class IIa (activation segment) or IIb (p-loop) mutations and compared these with WT or V600E/K BRAF mutant cells. Cell lines and PDXs were treated with BRAFi (vemurafenib, dabrafenib, encorafenib, and LY3009120), MEKi (cobimetinib, trametinib, and binimetinib), or the combination. We identified 2 patients with BRAF L597S metastatic melanoma who were treated with dMAPKi.Results:BRAFi impaired MAPK signaling and cell growth in class I and II BRAF mutant cells. dMAPKi was more effective than either single MAPKi at inhibiting cell growth in all class II BRAF mutant cells tested. dMAPKi caused tumor regression in two melanoma PDXs with class II BRAF mutations and prolonged survival of mice with class II BRAF mutant melanoma brain metastases. Two patients with BRAF L597S mutant melanoma clinically responded to dMAPKi.Conclusions:Class II BRAF mutant melanoma is growth inhibited by dMAPKi. Responses to dMAPKi have been observed in 2 patients with class II BRAF mutant melanoma. These data provide rationale for clinical investigation of dMAPKi in patients with class II BRAF mutant metastatic melanoma.See related commentary by Johnson and Dahlman, p. 6107.

Supplementary Figure Legends 1-11 from Suppression of Hypoxia-Inducible Factor 2α Restores p53 Activity via Hdm2 and Reverses Chemoresistance of Renal Carcinoma Cells

p53 mutations are rarely detected in clear cell renal cell carcinoma (CCRCC), but, paradoxically, these tumors remain highly resistant to chemotherapy and death receptor–induced death. Here, we show that the accumulation of hypoxia-inducible factor 2α (HIF2α), a critical oncogenic event in CCRCC following the loss of von Hippel-Lindau (VHL) tumor suppressor protein, leads to Hdm2-mediated suppression of p53. Primary CCRCC specimens exhibiting strong hypoxic signatures show increased levels of activated nuclear phospho-Hdm2(Ser166), which is concomitant with low p53 expression. The abrogation of Hdm2-p53 interaction using the small-molecule Hdm2 inhibitor nutlin-3 or the downregulation of HIF2α via HIF2α-specific short hairpin RNA or wild-type VHL reconstitution restores p53 function and reverses the resistance of CCRCC cells to Fas-mediated and chemotherapy-induced cell death. These findings unveil a mechanistic link between HIF2α and p53 and provide a rationale for combining Hdm2 antagonists with chemotherapy for the treatment of CCRCC. [Cancer Res 2009;69(23):9056–64]

Supplementary Figures 1-11 from Suppression of Hypoxia-Inducible Factor 2α Restores p53 Activity via Hdm2 and Reverses Chemoresistance of Renal Carcinoma Cells

Supplementary Table 1. (Top) IC50 (nM), fold changes (FC) and (Bottom) % viability at 10uM drug treatment compared to DMSO control for BRAF mutant cell lines, A375, MALME-3M, 451Lu and IGR1 (expresses RAC1 P29S) are shown.

We updated our data set, please use thenew version(md5:ece00981be0f2616c1c0a57d10f3176e). All data supporting the findings of the publication "Single-cell spatial immune landscapeof primary and metastatic brain tumours", including masks of high-dimension tif images, single-cell segmentation, single-cell cell types, and patient data. The code used to produce the results of this study is available at https://github.com/walsh-quail-labs/IMC_Brain. Raw primary imaging data can be obtained from the authors directly upon reasonable request. Glioma channel index names: Channel Name Channel Index CD117 1 CD11c 2 CD14 3 CD163 4 CD16 5 CD20 6 CD31 7 CD3 8 CD4 9 CD68 10 CD8 11 CD94 12 DNA1 13 FoxP3 14 GFAP 15 HLA-DR 16 MPO 17 Olig2 18 P2PY12 19 Sox2 20 Sox9 21 BrM channel index names: Channel Name Channel Index CD117 1 CD11c 2 CD14 3 CD163 4 CD16 5 CD20 6 CD31 7 CD3 8 CD4 9 CD68 10 CD8a 11 CD94 12 DNA1 13 FoxP3 14 GFAP 15 HLA-DR 16 MPO 17 MeLanA 18 P2PY12 19 PMEL 20 PanCK 21

Data required to run the reproducible R notebooks and make the manuscript's figures.

Purpose: Next-generation sequencing studies and CRISPR-Cas9 screens have established mutations in the IFNγ-JAK-STAT pathway as an immune checkpoint inhibitor (ICI) resistance mechanism in a subset of patients with melanoma. We hypothesized ICI resistance mutations in the IFNγ pathway would simultaneously render melanomas susceptible to oncolytic virus (OV) therapy. Experimental Design: Cytotoxicity experiments were performed with a number of OVs on a matched melanoma cell line pair generated from a baseline biopsy and a progressing lesion with complete JAK2 loss from a patient that relapsed on anti-PD-1 therapy, in melanoma lines following JAK1/2 RNA interference (RNAi) and pharmacologic inhibition and in Jak2 knockout (KO) B16-F10 mouse melanomas. Furthermore, we estimated the frequency of genetic alterations in the IFNγ-JAK-STAT pathway in human melanomas. Results: The melanoma line from an anti-PD-1 progressing lesion was 7- and 22-fold more sensitive to the modified OVs, herpes simplex virus 1 (HSV1-dICP0) and vesicular stomatitis virus (VSV-Δ51), respectively, compared with the line from the baseline biopsy. RNAi, JAK1/2 inhibitor studies, and in vivo studies of Jak2 KOs B16-F10 melanomas revealed a significant increase in VSV-Δ51 sensitivity with JAK/STAT pathway inhibition. Our analysis of The Cancer Genome Atlas data estimated that approximately 11% of ICI-naïve cutaneous melanomas have alterations in IFNγ pathway genes that may confer OV susceptibility. Conclusions: We provide mechanistic support for the use of OVs as a precision medicine strategy for both salvage therapy in ICI-resistant and first-line treatment in melanomas with IFNγ-JAK-STAT pathway mutations. Our study also supports JAK inhibitor–OV combination therapy for treatment-naïve melanomas without IFN signaling defects. See related commentary by Kaufman, p. 3278

Melanoma is an immunogenic cancer with a high response rate to immune checkpoint inhibitors (ICIs). It harbors a high mutation burden compared with other cancers and, as a result, has abundant tumor-infiltrating lymphocytes (TILs) within its microenvironment. However, understanding the complex interplay between the stroma, tumor cells, and distinct TIL subsets remains a substantial challenge in immune oncology. To properly study this interplay, quantifying spatial relationships of multiple cell types within the tumor microenvironment is crucial. To address this, we used cytometry time-of-flight (CyTOF) imaging mass cytometry (IMC) to simultaneously quantify the expression of 35 protein markers, characterizing the microenvironment of 5 benign nevi and 67 melanomas. We profiled more than 220,000 individual cells to identify melanoma, lymphocyte subsets, macrophage/monocyte, and stromal cell populations, allowing for in-depth spatial quantification of the melanoma microenvironment. We found that within pretreatment melanomas, the abundance of proliferating antigen-experienced cytotoxic T cells (CD8 + CD45RO + Ki67 + ) and the proximity of antigen-experienced cytotoxic T cells to melanoma cells were associated with positive response to ICIs. Our study highlights the potential of multiplexed single-cell technology to quantify spatial cell-cell interactions within the tumor microenvironment to understand immune therapy responses.

Neutrophils represent the immune system’s first line of defense and are rapidly recruited into inflamed tissue. In cancer associated inflammation, phenotypic heterogeneity has been ascribed to this cell type, whereby neutrophils can manifest anti- or pro-metastatic functions depending on the cellular/micro-environmental context. Here, we demonstrate that pro-metastatic immature low-density neutrophils (iLDNs) more efficiently accumulate in the livers of mice bearing metastatic lesions compared with anti-metastatic mature high-density neutrophils (HDNs). Transcriptomic analyses reveal enrichment of a migration signature in iLDNs relative to HDNs. We find that conditioned media derived from liver-metastatic breast cancer cells, but not lung-metastatic variants, specifically induces chemotaxis of iLDNs and not HDNs. Chemotactic responses are due to increased surface expression of C3aR in iLDNs relative to HDNs. In addition, we detect elevated secretion of cancer-cell derived C3a from liver-metastatic versus lung-metastatic breast cancer cells. Perturbation of C3a/C3aR signaling axis with either a small molecule inhibitor, SB290157, or reducing the levels of secreted C3a from liver-metastatic breast cancer cells by short hairpin RNAs, can abrogate the chemotactic response of iLDNs both in vitro and in vivo, respectively. Together, these data reveal novel mechanisms through which iLDNs prefentially accumulate in liver tissue harboring metastases in response to tumor-derived C3a secreted from the liver-aggressive 4T1 breast cancer cells.

SummarycAMP signaling pathways are critical for both oncogenesis and tumor suppression. cAMP signaling is localized to multiple spatially distinct microdomains, but the role of cAMP microdomains in cancer cell biology is poorly understood. We developed a tunable genetic system that allows us to activate cAMP signaling in specific microdomains. We uncovered a previously unappreciated nuclear cAMP microdomain that functionally activates a tumor suppressive pathway in a broad range of cancers by inhibiting YAP, a key effector protein of the Hippo pathway, inside the nucleus. We show that nuclear cAMP induces a LATS-dependent pathway leading to phosphorylation of nuclear YAP solely at serine 397, export of YAP from the nucleus, without YAP protein degradation. Thus, nuclear cAMP inhibition of nuclear YAP is distinct from other known mechanisms of Hippo regulation. Pharmacologic targeting of specific cAMP microdomains remains an untapped therapeutic approach for cancer, and since Hippo pathway deregulation can lead to oncogenesis and chemotherapeutic resistance, drugs directed at the nuclear cAMP microdomain may provide new avenues for the treatment of cancer.

Although combination BRAF/MEK inhibition has produced significant survival benefits for BRAF p.V600 mutant melanomas, targeted therapies approved for BRAF non-p.V600 mutant melanomas remain limited. Through the analysis of 772 cutaneous melanoma exomes, we reveal that BRAF non-p.V600 mutations co-occurs more frequently with NF1 loss, but not with oncogenic NRAS mutations, than expected by chance. We present cell signaling data, which demonstrate that BRAF non-p.V600 mutants can signal as monomers and dimers within an NF1 loss context. Concordantly, BRAF inhibitors that inhibit both monomeric and dimeric BRAF synergize with MEK inhibition to significantly reduce cell viability in vitro and tumor growth in vivo in BRAF non-p.V600 mutant melanomas with co-occurring NF1 loss-of-function mutations. Our data suggest that patients harboring BRAF non-p.V600 mutant melanomas may benefit from current FDA-approved BRAF/MEK inhibitor combination therapy currently reserved for BRAF p.V600 mutant patients.

Depleting creatine levels in thermogenic adipocytes by inhibiting creatine biosynthesis reduces thermogenesis and causes obesity. However, whether creatine import from the circulation affects adipocyte thermogenesis is unknown. Here we show that deletion of the cell-surface creatine transporter (CrT) selectively in fat (AdCrTKO) substantially reduces adipocyte creatine and phosphocreatine levels, and reduces whole-body energy expenditure in mice. AdCrTKO mice are cold intolerant and become more obese than wild-type animals when fed a high-fat diet. Loss of adipocyte creatine transport blunts diet- and β3-adrenergic-induced thermogenesis, whereas creatine supplementation during high-fat feeding increases whole-body energy expenditure in response to β3-adrenergic agonism. In humans, CRT expression in purified subcutaneous adipocytes correlates with lower body mass index and increased insulin sensitivity. Our data indicate that adipocyte creatine abundance depends on creatine sequestration from the circulation. Given that it affects whole-body energy expenditure, enhancing creatine uptake into adipocytes may offer an opportunity to combat obesity and obesity-associated metabolic dysfunction. Creatine can be used for thermogenesis in adipocytes. Here Kazak et al. show that creatine uptake is required to sustain this thermogenic pathway. Knockdown of the creatine transporter, CrT, in adipocytes decreases thermogenesis and energy expenditure, whereas creatine supplementation increases energy expenditure in mice fed a high-fat diet.

Cyclic AMP (cAMP) signaling is localized to multiple spatially distinct microdomains, but the role of cAMP microdomains in cancer cell biology is poorly understood. Here, we present a tunable genetic system that allows us to activate cAMP signaling in specific microdomains. We uncover a nuclear cAMP microdomain that activates a tumor-suppressive pathway in a broad range of cancers by inhibiting YAP, a key effector protein of the Hippo pathway, inside the nucleus. We show that nuclear cAMP induces a LATS-dependent pathway leading to phosphorylation of nuclear YAP solely at serine 397 and export of YAP from the nucleus with no change in YAP protein stability. Thus, nuclear cAMP inhibition of nuclear YAP is distinct from other known mechanisms of Hippo regulation. Pharmacologic targeting of specific cAMP microdomains remains an untapped therapeutic approach for cancer; thus, drugs directed at the nuclear cAMP microdomain may provide avenues for the treatment of cancer.

Background The p66ShcA redox protein is the longest isoform of the Shc1 gene and is variably expressed in breast cancers. In response to a variety of stress stimuli, p66ShcA becomes phosphorylated on serine 36, which allows it to translocate from the cytoplasm to the mitochondria where it stimulates the formation of reactive oxygen species (ROS). Conflicting studies suggest both pro- and anti-tumorigenic functions for p66ShcA, which prompted us to examine the contribution of tumor cell-intrinsic functions of p66ShcA during breast cancer metastasis. Methods We tested whether p66ShcA impacts the lung-metastatic ability of breast cancer cells. Breast cancer cells characteristic of the ErbB2+/luminal (NIC) or basal (4T1) subtypes were engineered to overexpress p66ShcA. In addition, lung-metastatic 4T1 variants (4T1-537) were engineered to lack endogenous p66ShcA via Crispr/Cas9 genomic editing. p66ShcA null cells were then reconstituted with wild-type p66ShcA or a mutant (S36A) that cannot translocate to the mitochondria, thereby lacking the ability to stimulate mitochondrial-dependent ROS production. These cells were tested for their ability to form spontaneous metastases from the primary site or seed and colonize the lung in experimental (tail vein) metastasis assays. These cells were further characterized with respect to their migration rates, focal adhesion dynamics, and resistance to anoikis in vitro. Finally, their ability to survive in circulation and seed the lungs of mice was assessed in vivo. Results We show that p66ShcA increases the lung-metastatic potential of breast cancer cells by augmenting their ability to navigate each stage of the metastatic cascade. A non-phosphorylatable p66ShcA-S36A mutant, which cannot translocate to the mitochondria, still potentiated breast cancer cell migration, lung colonization, and growth of secondary lung metastases. However, breast cancer cell survival in the circulation uniquely required an intact p66ShcA S36 phosphorylation site. Conclusion This study provides the first evidence that both mitochondrial and non-mitochondrial p66ShcA pools collaborate in breast cancer cells to promote their maximal metastatic fitness.

Purpose: Dual MAPK pathway inhibition (dMAPKi) with BRAF and MEK inhibitors improves survival in BRAF V600E/K mutant melanoma, but the efficacy of dMAPKi in non-V600 BRAF mutant tumors is poorly understood. We sought to characterize the responsiveness of class II (enhanced kinase activity, dimerization dependent) BRAF mutant melanoma to dMAPKi. Experimental Design: Tumors from patients with BRAF wild-type (WT), V600E (class I), and L597S (class II) metastatic melanoma were used to generate patient-derived xenografts (PDX). We assembled a panel of melanoma cell lines with class IIa (activation segment) or IIb (p-loop) mutations and compared these with WT or V600E/K BRAF mutant cells. Cell lines and PDXs were treated with BRAFi (vemurafenib, dabrafenib, encorafenib, and LY3009120), MEKi (cobimetinib, trametinib, and binimetinib), or the combination. We identified 2 patients with BRAF L597S metastatic melanoma who were treated with dMAPKi. Results: BRAFi impaired MAPK signaling and cell growth in class I and II BRAF mutant cells. dMAPKi was more effective than either single MAPKi at inhibiting cell growth in all class II BRAF mutant cells tested. dMAPKi caused tumor regression in two melanoma PDXs with class II BRAF mutations and prolonged survival of mice with class II BRAF mutant melanoma brain metastases. Two patients with BRAF L597S mutant melanoma clinically responded to dMAPKi. Conclusions: Class II BRAF mutant melanoma is growth inhibited by dMAPKi. Responses to dMAPKi have been observed in 2 patients with class II BRAF mutant melanoma. These data provide rationale for clinical investigation of dMAPKi in patients with class II BRAF mutant metastatic melanoma. See related commentary by Johnson and Dahlman, p. 6107.

The RAS-RAF-MEK-ERK signaling cascade is among the most frequently mutated pathways in human cancer. Approximately 50% of melanoma patients possess a druggable hotspot V600E/K mutation in the BRAF protein kinase. FDA-approved combination therapies of BRAF and MEK inhibitors are available that provide survival benefits to patients with a BRAF V600 mutation. Non-V600 BRAF mutants are found in many cancers, and are more prevalent than V600 mutations in certain tumor types. For example, between 50-80% of BRAF mutations in non-small cell lung cancer and 22-30% in colorectal cancer encode for non-V600 mutants. As next generation sequencing becomes increasingly used in clinical practice, oncologists are frequently identifying non-V600 BRAF mutations in their patient's tumors, but are uncertain of viable therapeutic options that could be employed for optimal treatment. From recent studies, a new classification system is emerging for BRAF mutations based on biochemical and signaling mechanisms associated with these mutants. Class I BRAF mutations affect amino acid V600 and signal as RAS-independent active monomers, class II mutations function as RAS-independent activated dimers, and class III mutations are kinase impaired but increase signaling through the MAPK pathway due to enhanced RAS binding and subsequent CRAF activation. These distinct classes of BRAF mutations predict response to targeted therapies and have important implications for future drug development. Herein, we discuss pre-clinical and clinical findings that may lead to improved treatments for all classes of BRAF mutant cancers.

Metastasis is the leading cause of cancer-related deaths, and obesity is associated with increased breast cancer (BC) metastasis. Preclinical studies have shown that obese adipose tissue induces lung neutrophilia associated with enhanced BC metastasis to this site. Here we show that obesity leads to neutrophil-dependent impairment of vascular integrity through loss of endothelial adhesions, enabling cancer cell extravasation into the lung. Mechanistically, neutrophil-produced reactive oxygen species in obese mice increase neutrophil extracellular DNA traps (NETs) and weaken endothelial junctions, facilitating the influx of tumor cells from the peripheral circulation. In vivo treatment with catalase, NET inhibitors or genetic deletion of Nos2 reversed this effect in preclinical models of obesity. Imaging mass cytometry of lung metastasis samples from patients with cancer revealed an enrichment in neutrophils with low catalase levels correlating with elevated body mass index. Our data provide insights into potentially targetable mechanisms that underlie the progression of BC in the obese population.

The high background tumor mutation burden in cutaneous melanoma limits the ability to identify significantly mutated genes (SMGs) that drive this cancer. To address this, we performed a mutation significance study of over 1,000 melanoma exomes, combined with a multi-omic analysis of 470 cases from The Cancer Genome Atlas. We discovered several SMGs with co-occurring loss-of-heterozygosity and loss-of-function mutations, including PBRM1, PLXNC1 and PRKAR1A, which encodes a protein kinase A holoenzyme subunit. Deconvolution of bulk tumor transcriptomes into cancer, immune and stromal components revealed a melanoma-intrinsic oxidative phosphorylation signature associated with protein kinase A pathway alterations. We also identified SMGs on the X chromosome, including the RNA helicase DDX3X, whose loss-of-function mutations were exclusively observed in males. Finally, we found that tumor mutation burden and immune infiltration contain complementary information on survival of patients with melanoma. In summary, our multi-omic analysis provides insights into melanoma etiology and supports contribution of specific mutations to the sex bias observed in this cancer. Alkallas et al. uncover new significantly mutated genes in a large cohort of cutaneous melanoma, including the RNA helicase DDX3X, through integrated analysis of genomic, transcriptomic and DNA methylation data.

Neutrophils represent the immune system's first line of defense and are rapidly recruited into inflamed tissue. In cancer associated inflammation, phenotypic heterogeneity has been ascribed to this cell type, whereby neutrophils can manifest anti- or pro-metastatic functions depending on the cellular/micro-environmental context. Here, we demonstrate that pro-metastatic immature low-density neutrophils (iLDNs) more efficiently accumulate in the livers of mice bearing metastatic lesions compared with anti-metastatic mature high-density neutrophils (HDNs). Transcriptomic analyses reveal enrichment of a migration signature in iLDNs relative to HDNs. We find that conditioned media derived from liver-metastatic breast cancer cells, but not lung-metastatic variants, specifically induces chemotaxis of iLDNs and not HDNs. Chemotactic responses are due to increased surface expression of C3aR in iLDNs relative to HDNs. In addition, we detect elevated secretion of cancer-cell derived C3a from liver-metastatic versus lung-metastatic breast cancer cells. Perturbation of C3a/C3aR signaling axis with either a small molecule inhibitor, SB290157, or reducing the levels of secreted C3a from liver-metastatic breast cancer cells by short hairpin RNAs, can abrogate the chemotactic response of iLDNs both in vitro and in vivo, respectively. Together, these data reveal novel mechanisms through which iLDNs prefentially accumulate in liver tissue harboring metastases in response to tumor-derived C3a secreted from the liver-aggressive 4T1 breast cancer cells.

Purpose: To determine if BRAF and/or MEK inhibitor–induced GPNMB expression renders melanomas sensitive to CDX-011, an antibody-drug conjugate targeting GPNMB. Experimental Design: The Cancer Genome Atlas melanoma dataset was interrogated for a panel of MITF-regulated melanosomal differentiation antigens, including GPNMB. BRAF-mutant melanoma cell lines treated with BRAF or MEK inhibitors were assessed for GPNMB expression by RT-qPCR, immunoblot, and FACS analyses. Transient siRNA-mediated knockdown approaches were used to determine if MITF is requirement for treatment-induced GPNMB upregulation. GPNMB expression was analyzed in serial biopsies and serum samples from patients with melanoma taken before, during, and after disease progression on MAPK inhibitor treatment. Subcutaneous injections were performed to test the efficacy of MAPK inhibitors alone, CDX-011 alone, or their combination in suppressing melanoma growth. Results: A MITF-dependent melanosomal differentiation signature is associated with poor prognosis in patients with this disease. MITF is increased following BRAF and MEK inhibitor treatment and induces the expression of melanosomal differentiation genes, including GPNMB. GPNMB is expressed at the cell surface in MAPK inhibitor–treated melanoma cells and is also elevated in on-treatment versus pretreatment biopsies from melanoma patients receiving MAPK pathway inhibitors. Combining BRAF and/or MEK inhibitors with CDX-011, an antibody-drug conjugate targeting GPNMB, is effective in causing melanoma regression in preclinical animal models and delays the recurrent melanoma growth observed with MEK or BRAF/MEK inhibitor treatment alone. Conclusions: The combination of MAPK pathway inhibitors with an antibody-drug conjugate targeting GPNMB is an effective therapeutic option for patients with melanoma. Clin Cancer Res; 22(24); 6088–98. ©2016 AACR.

Large-scale genomic analyses of cutaneous melanoma have revealed insights into the aetiology and heterogeneity of this disease, as well as opportunities to further personalise treatment for patients with targeted and immune therapies. Herein, we review the proposed genomic classification of cutaneous melanoma from large-scale next-generation sequencing studies, including the largest integrative analysis of melanoma from The Cancer Genome Atlas (TCGA) Network. We examine studies that have identified molecular features of melanomas linked to immune checkpoint inhibitor response. In addition, we draw attention to low-frequency actionable mutations and highlight frequent non-coding mutations in melanoma where little is known about their biological function that may provide novel avenues for the development of treatment strategies for melanoma patients.

Anti-programmed cell death-1 (anti-PD-1) blockade in tumor therapy results in an expansion of CD8+ T cells and can restore the function of exhausted CD8+ T cells. We identified GSK-3 as a key upstream kinase of regulating PD-1 expression. Inhibition of GSK-3 in CD8+ T cells increased Tbx21 (Tbet), which suppressed Pdcd1 (PD-1) transcription (Taylor et al., Immunity 2016). In controlling spontaneous pulmonary metastasis of B16 melanoma and EL-4 lymphoma cells, GSK-3 blockade is as effective as anti-PD-1 blocking antibody (Taylor et al., Cancer Research 2018). Given the clinical importance in devising strategies to overcome tumor resistance to immune checkpoint blockade, we assessed whether the combined use of GSK-3 small-molecule inhibitor (SMI) and anti-PD-1 can overcome resistance to anti-PD-1 monotherapy. Indeed, GSK-3 SMI and anti-PD-1 synergized in therapy against solid B16 tumors that are resistant to monotherapy, leading to delayed tumor growth. Further, the synergy of GSK-3 SMI and anti-PD-1 therapy has been successfully confirmed by using adoptive transfer of OT1 cells into B16-OVA tumor-bearing mice. High-dimensional profiling of tumor-infiltrated lymphocytes (TILs) using mass cytometry (CyTOF) showed that the combination therapy gave an increase in cellularity of CD8+ effector memory T cells, which correlated to better tumor control. Transcriptome analysis of these CD8+ TILs highlighted the increased cytotoxicity as shown by significant changes of granzymes. Our findings outline a next-generation approach of potentiating PD-1 antibody therapy with SMIs of GSK-3 that synergizes as therapy by boosting the cellularity and cytotoxicity of tumor infiltrated CD8+ T cells. Citation Format: Janna Krueger, Alison Taylor, Alexandra Kazanova, Saloua Jeidane, Vinicius Motta, Ian Watson, Christopher E. Rudd. Inhibition of Glycogen Synthase Kinase 3 (GSK-3) synergizes with anti-PD-1 by potentiating tumor-infiltrated CD8+ T-cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A59.

Summary: Neutrophils are phenotypically heterogeneous and exert either anti- or pro-metastatic functions. We show that cancer-cell-derived G-CSF is necessary, but not sufficient, to mobilize immature low-density neutrophils (iLDNs) that promote liver metastasis. In contrast, mature high-density neutrophils inhibit the formation of liver metastases. Transcriptomic and metabolomic analyses of high- and low-density neutrophils reveal engagement of numerous metabolic pathways specifically in low-density neutrophils. iLDNs exhibit enhanced global bioenergetic capacity, through their ability to engage mitochondrial-dependent ATP production, and remain capable of executing pro-metastatic neutrophil functions, including NETosis, under nutrient-deprived conditions. We demonstrate that NETosis is an important neutrophil function that promotes breast cancer liver metastasis. iLDNs rely on the catabolism of glutamate and proline to support mitochondrial-dependent metabolism in the absence of glucose, which enables sustained NETosis. These data reveal that distinct pro-metastatic neutrophil populations exhibit a high degree of metabolic flexibility, which facilitates the formation of liver metastases. : Hsu et al. demonstrate that tumor-derived G-CSF, in concert with additional factors, mobilizes immature low-density neutrophils (iLDNs) that promote breast cancer liver metastasis. iLDNs are able to perform pro-metastatic functions under metabolically challenging conditions, such as low glucose, due to their enhanced global bioenergetic capacity. Keywords: neutrophil plasticity, metastasis, metabolic flexibility, NETosis

Background SMARCB1 (INI1/BAF47/SNF5) encodes a part of a multiprotein complex that regulates gene expression through chromatin remodeling. SMARCB1 expression is lost or downregulated in multiple human tumors, including epithelioid sarcoma, meningioma and rhabdoid tumors of the brain, soft tissue and kidney. Methods A 46-gene or 50-gene next-generation sequencing AmpliSeq Cancer Panel (Life Technologies; San Francisco, CA, USA) was applied to ∼1400 primary or metastatic melanoma tissues. Results We identified eight cases of melanoma harboring mutations in SMARCB1. Immunohistochemistry demonstrated preservation of SMARCB1 protein expression in all cases. SMARCB1 mutations occurred together with TP53 mutations in five of the eight cases, suggesting a functional relationship between these tumor suppressors in melanoma. Conclusions Because single-base substitutions in SMARCB1 occur in a small subset of melanomas and do not affect SMARCB1 protein expression, such mutations would only be discovered by sequencing approaches. Our findings highlight the potential for next-generation sequencing platforms to identify mutations unexpected for melanoma that may contribute to its oncogenic potential. Though rare, the identification of SMARCB1 mutations adds to the growing literature regarding the role of epigenetic control mechanisms in melanoma progression and therapeutic resistance and provide a rationale for strategies targeting such alterations (via chromatin remodeling agents) in clinical trials.

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.

Following mutations in BRAF and NRAS, the RAC1 c.85C>T single-nucleotide variant (SNV) encoding P29S amino acid change represents the next most frequently observed protein-coding hotspot mutation in melanoma. However, the biologic and clinical significance of the RAC1 P29S somatic mutation in approximately 4% to 9% of patients remains unclear. Here, we demonstrate that melanoma cell lines possessing the RAC1 hotspot variant are resistant to RAF inhibitors (vemurafenib and dabrafenib). Enforced expression of RAC1 P29S in sensitive BRAF-mutant melanoma cell lines confers resistance manifested by increased viability, decreased apoptosis, and enhanced tumor growth in vivo upon treatment with RAF inhibitors. Conversely, RNAi-mediated silencing of endogenous RAC1 P29S in a melanoma cell line with a co-occurring BRAF V600 mutation increased sensitivity to vemurafenib and dabrafenib. Our results suggest RAC1 P29S status may offer a predictive biomarker for RAF inhibitor resistance in melanoma patients, where it should be evaluated clinically. Cancer Res; 74(17); 4845–52. ©2014 AACR.

Malignant melanoma is one of the most aggressive cancers and can disseminate from a relatively small primary tumor and metastasize to multiple sites, including the lung, liver, brain, bone, and lymph nodes. Elucidating the molecular and genetic changes that take place during the metastatic process has led to a better understanding of why melanoma is so metastatic. Herein, we describe the unique features that distinguish melanoma from other solid tumors and contribute to the malignant phenotype of melanoma cells. For example, although melanoma cells are highly antigenic, they are extremely efficient at evading host immune response. Melanoma cells share numerous cell surface molecules with vascular cells, are highly angiogenic, are mesenchymal in nature, and possess a higher degree of 'stemness' than do other solid tumors. Finally, analysis of melanoma mutations has revealed that the gene expression profile of malignant melanoma is different from that of other cancers. Elucidating these molecular and genetic processes in highly metastatic melanoma can lead to the development of improved treatment and individualized therapy options.

Recent advances in technological tools for massively parallel, high-throughput sequencing of DNA have enabled the comprehensive characterization of somatic mutations in a large number of tumour samples. In this Review, we describe recent cancer genomic studies that have assembled emerging views of the landscapes of somatic mutations through deep-sequencing analyses of the coding exomes and whole genomes in various cancer types. We discuss the comparative genomics of different cancers, including mutation rates and spectra, as well as the roles of environmental insults that influence these processes. We highlight the developing statistical approaches that are used to identify significantly mutated genes, and discuss the emerging biological and clinical insights from such analyses, as well as the future challenges of translating these genomic data into clinical impacts.

Summary Despite recent insights into melanoma genetics, systematic surveys for driver mutations are challenged by an abundance of passenger mutations caused by carcinogenic UV light exposure. We developed a permutation-based framework to address this challenge, employing mutation data from intronic sequences to control for passenger mutational load on a per gene basis. Analysis of large-scale melanoma exome data by this approach discovered six novel melanoma genes ( PPP6C , RAC1 , SNX31 , TACC1 , STK19 , and ARID2 ), three of which— RAC1 , PPP6C , and STK19 —harbored recurrent and potentially targetable mutations. Integration with chromosomal copy number data contextualized the landscape of driver mutations, providing oncogenic insights in BRAF- and NRAS-driven melanoma as well as those without known NRAS / BRAF mutations. The landscape also clarified a mutational basis for RB and p53 pathway deregulation in this malignancy. Finally, the spectrum of driver mutations provided unequivocal genomic evidence for a direct mutagenic role of UV light in melanoma pathogenesis.