Publications

The remarkable capacity of immunotherapies to induce durable regression in some patients with metastatic cancer relies heavily on T cell recognition of tumor-presented antigens. As checkpoint-blockade therapy has limited efficacy, tumor antigens have the potential to be exploited for complementary treatments, many of which are already in clinical trials. The surge of interest in this topic has led to the expansion of the tumor antigen landscape with the emergence of new antigen categories. Nonetheless, how different antigens compare in their ability to elicit efficient and safe clinical responses remains largely unknown. Here, we review known cancer peptide antigens, their attributes and the relevant clinical data and discuss future directions.

Protein degradation by proteasomes is important for the immune response against tumors. Antigens generated by the proteasome promote immune cell infiltration into tumors and improve tumors’ responses to immunotherapy. For example, immunoproteasomes – a subset of proteasomes induced by inflammatory signals – may improve the response of melanomas to immune checkpoint inhibitors (ICI) by eliciting tumor inflammation. Yet, it is unclear whether and how protein degradation by proteasomes impacts cancer progression and contributes to immune evasion and resistance. Here, we profile the proteasome-cleaved peptides in lung cancers and find that PSME4 serves as a novel inhibitory regulator of the immunoproteasome, playing an anti-inflammatory role in cancer. Biochemical assays combined with scRNA-seq, immunopeptidomics and in vivo analyses demonstrate that PSME4 promotes an immunosuppressive environment around the tumor and abrogates anti-tumor immunity by inhibiting antigen presentation and attenuating tumor inflammation. Furthermore, we find that PSME4 expression is correlated with responsiveness to ICI across several cancer types. Our findings suggest that PSME4-mediated regulation of proteasome activity is a novel mechanism of immune evasion in non-small-cell lung carcinoma and may be targeted therapeutically for restoring anti-tumor immunity.

Over the last decade, it has become clear that the genomic landscapes of tumors profoundly impact their immunogenicity and how tumor cells interact with immune cells. Whereas past discoveries mainly focused on the interplay between tumor immunogenicity and tumor mutational burden (TMB), under the assumption that a higher mutation load would give rise to a better patient response to immune checkpoint blockade (ICB) therapies, we and others have underlined intratumor heterogeneity (ITH) as an important determinant of the magnitude of the anti-tumor response and the nature of the tumor microenvironment. In this Review, we define TMB vs. ITH and how the two factors are being inferred from data, examine key findings in the cancer immunogenomics literature deciphering the complex crosstalk between TMB, ITH and anti-tumor immunity in human cancers and in-vivo models, and discuss the mutual influence of ITH and immunity - how the anti-tumor response can give rise to tumors with higher ITH, and how higher ITH can put shackles on the anti-tumor response.

Neoantigens are now recognized drivers of the antitumor immune response. Recurrent neoantigens, shared among groups of patients, have thus become increasingly coveted therapeutic targets. Here, we report on the data-driven identification of a robustly presented, immunogenic neoantigen that is derived from the combination of HLA-A*01:01 and RAS.Q61K. Analysis of large patient cohorts indicated that this combination applies to 3% of patients with melanoma. Using HLA peptidomics, we were able to demonstrate robust endogenous presentation of the neoantigen in 10 tumor samples. We detected specific reactivity to the mutated peptide within tumor-infiltrating lymphocytes (TILs) from 2 unrelated patients, thus confirming its natural immunogenicity. We further investigated the neoantigen-specific clones and their T cell receptors (TCRs) via a combination of TCR sequencing, TCR overexpression, functional assays, and single-cell transcriptomics. Our analysis revealed a diverse repertoire of neoantigen-specific clones with both intra- and interpatient TCR similarities. Moreover, 1 dominant clone proved to cross-react with the highly prevalent RAS.Q61R variant. Transcriptome analysis revealed a high association of TCR clones with specific T cell phenotypes in response to cognate melanoma, with neoantigen-specific cells showing an activated and dysfunctional phenotype. Identification of recurrent neoantigens and their reactive TCRs can promote "off-the-shelf" precision immunotherapies, alleviating limitations of personalized treatments.

There is a lack of appropriate melanoma models that can be used to evaluate the efficacy of novel therapeutic modalities. Here, we discuss the current state of the art of melanoma models including genetically engineered mouse, patient-derived xenograft, zebrafish, and ex vivo and in vitro models. We also identify five major challenges that can be addressed using such models, including metastasis and tumor dormancy, drug resistance, the melanoma immune response, and the impact of aging and environmental exposures on melanoma progression and drug resistance. Additionally, we discuss the opportunity for building models for rare subtypes of melanomas, which represent an unmet critical need. Finally, we identify key recommendations for melanoma models that may improve accuracy of preclinical testing and predict efficacy in clinical trials, to help usher in the next generation of melanoma therapies.

Hotspot mutations of the oncogenes BRAF and NRas are the most common genetic alterations in cutaneous melanoma. Still, the nanoscale organization and signal coupling of these proteins remain incompletely understood, particularly upon expression of oncogenic NRas mutants. Here we employed single-molecule localization microscopy to study the nanoscale organization of NRas and BRAF at the plasma-membrane (PM) of melanoma cells. NRas and BRAF resided in self-clusters that did not associate well in resting cells. In EGF-activated cells, NRas clusters became more diffused while overall protein levels at the PM increased; thus allowing enhanced association of NRas and BRAF and downstream signaling. In multiple melanoma cell lines, mutant NRas resided in more pronounced self-clusters relative to WT NRas yet associated more with the clustered and more abundant BRAF. In cells resistant to trametinib, a clinical MEK inhibitor (MEKi), a similar co-clustering of NRas and BRAF was observed upon EGF activation. Strikingly, treatment of cells expressing mutant NRas with trametinib reversed the effect of mutant NRas expression by restoring the non-overlapping self-clusters of NRas and BRAF and by reducing their PM levels and elevated pERK levels caused by mutant NRas. Our results indicate a new mechanism for signal regulation of NRas in melanoma through its nanoscale dynamic organization and a new mechanism for MEKi function in melanoma cells carrying NRas mutations but lacking MEK mutations.

NRAS mutations are the most common alterations among RAS isoforms in cutaneous melanoma, with patients harboring these aggressive tumors having a poor prognosis and low survival rate. The main line of treatment for these patients is MAPK pathway-targeted therapies, such as MEK inhibitors, but, unfortunately, the response to these inhibitors is variable due to tumor resistance. Identifying genetic modifiers involved in resistance toward MEK-targeted therapy may assist in the development of new therapeutic strategies, enhancing treatment response and patient survival. Our whole-genome CRISPR-Cas9 knockout screen identified the target Kelch domain-containing F-Box protein 42 (FBXO42) as a factor involved in NRAS-mutant melanoma-acquired resistance to the MEK1/2 inhibitor trametinib. We further show that FBXO42, an E3 ubiquitin ligase, is involved in the TAK1 signaling pathway, possibly prompting an increase in active P38. In addition, we demonstrate that combining trametinib with the TAK1 inhibitor, takinib, is a far more efficient treatment than trametinib alone in NRAS-mutant melanoma cells. Our findings thus show a new pathway involved in NRAS-mutant melanoma resistance and provide new opportunities for novel therapeutic options.

Although clonal neo-antigen burden is associated with improved response to immune therapy, the functional basis for this remains unclear. Here we study this question in a novel controlled mouse melanoma model that enables us to explore the effects of intra-tumor heterogeneity (ITH) on tumor aggressiveness and immunity independent of tumor mutational burden. Induction of UVB-derived mutations yields highly aggressive tumors with decreased anti-tumor activity. However, single-cell-derived tumors with reduced ITH are swiftly rejected. Their rejection is accompanied by increased T cell reactivity and a less suppressive microenvironment. Using phylogenetic analyses and mixing experiments of single-cell clones, we dissect two characteristics of ITH: the number of clones forming the tumor and their clonal diversity. Our analysis of melanoma patient tumor data recapitulates our results in terms of overall survival and response to immune checkpoint therapy. These findings highlight the importance of clonal mutations in robust immune surveillance and the need to quantify patient ITH to determine the response to checkpoint blockade.

The invasive phenotype of metastatic cancer cells is accompanied by the formation of actin-rich invadopodia, which adhere to the extracellular matrix and degrade it. In this study, we explored the role of the tyrosine kinome in the formation of invadopodia in metastatic melanoma cells. Using a microscopy-based siRNA screen, we identified a series of regulators, the knockdown of which either suppresses (e.g., TYK2, IGFR1, ERBB3, TYRO3, FES, ALK, PTK7) or enhances (e.g., ABL2, AXL, CSK) invadopodia formation and function. Notably, the receptor tyrosine kinase AXL displayed a dual regulatory function, where both depletion or overexpression enhanced invadopodia formation and activity. This apparent contradiction was attributed to the capacity of AXL to directly stimulate invadopodia, yet its suppression upregulates the ERBB3 signaling pathway, which can also activate core invadopodia regulators and enhance invadopodia function. Bioinformatic analysis of multiple melanoma cell lines points to an inverse expression pattern of AXL and ERBB3. High expression of AXL in melanoma cells is associated with high expression of invadopodia components and an invasive phenotype. These results provide new insights into the complexity of metastasis-promoting mechanisms and suggest that targeting of multiple invadopodia signaling networks may serve as a potential anti-invasion therapy in melanoma. SIGNIFICANCE: These findings uncover a unique interplay between AXL and ERBB3 in invadopodia regulation that points to the need for combined therapy in order to prevent invadopodia-mediated metastasis in melanoma.

The Acknowledgements section in this Article is incomplete.“We thank Mr. N. K. Skamangas for technical support and Dr. A. Kovoor for the D2 receptor expression plasmid, Dr. H. Itoh for GαoA and Dr. N. Lambert for sharing the Gβγ-Venus constructs. This work was supported by NIH grants DA036596 and DA026405 (KAM). MYN is supported by Israel Science Foundation grant 432/12 and Chief Scientist Ministry of Health (the ERA-NET network) and grant no. 3-9543 from the Chief Scientist Office of the Ministry of Health, Israel via the ERA-net network. MK is supported by Israel Science Foundation grant numbers 1454/13, 1959/13, 2155/15. YS is supported by the Israel Science Foundation grant numbers 1604/13 and 877/13, the ERC (StG-335377), by the Henry Chanoch Krenter Institute for Biomedical Imaging and Genomics, the estate of Alice Schwarz-Gardos, the estate of John Hunter, the Knell Family, the Peter and Patricia Gruber Award and the Hamburger Family. Lady Davis Fellowship to ADP is gratefully acknowledged. NKH and RAS are supported by fellowships from the National Health and Medical Research Council of Australia.”

The quest for tumor-associated antigens (TAA) and neoantigens is a major focus of cancer immunotherapy. Here, we combine a neoantigen prediction pipeline and human leukocyte antigen (HLA) peptidomics to identify TAAs and neoantigens in 16 tumors derived from seven patients with melanoma and characterize their interactions with their tumor-infiltrating lymphocytes (TIL). Our investigation of the antigenic and T-cell landscapes encompassing the TAA and neoantigen signatures, their immune reactivity, and their corresponding T-cell identities provides the first comprehensive analysis of cancer cell T-cell cosignatures, allowing us to discover remarkable antigenic and TIL similarities between metastases from the same patient. Furthermore, we reveal that two neoantigen-specific clonotypes killed 90% of autologous melanoma cells, both in vitro and in vivo, showing that a limited set of neoantigen-specific T cells may play a central role in melanoma tumor rejection. Our findings indicate that combining HLA peptidomics with neoantigen predictions allows robust identification of targetable neoantigens, which could successfully guide personalized cancer immunotherapies.SIGNIFICANCE: As neoantigen targeting is becoming more established as a powerful therapeutic approach, investigating these molecules has taken center stage. Here, we show that a limited set of neoantigen-specific T cells mediates tumor rejection, suggesting that identifying just a few antigens and their corresponding T-cell clones could guide personalized immunotherapy. (c) 2018 AACR.

The NRAS oncoprotein is highly mutated in melanoma. However, to date, no comprehensive proteomic study has been reported for NRAS. Here, we utilized the endogenous epitope tagging (EET) approach for the identification of novel NRAS binding partners. Using EET, an epitope tag is added to the endogenously expressed protein, via modification of its genomic coding sequence. Existing EET systems are not robust, suffer from high background, and are labor-intensive. To this end, we present a polyadenylation signal-trap construct for N'-tagging that generates a polycistronic mRNA with the gene of interest. This system requires the integration of the tagging cassette in frame with the target gene to be expressed. Using this design, we demonstrate, for the first time, endogenous tagging of NRAS in melanoma cells allowing the identification of the E3 ubiquitin ligase c-CBL as a novel NRAS binding partner. Thus, our developed EET technology allows the characterization of new RAS effectors, which could be beneficial for the design of future drugs that inhibit constitutive signaling of RAS oncogenic mutants.

Neurofibromin 1 (NF1), a tumor suppressor that negatively regulates RAS through its GTPase activity, is highly mutated in various types of sporadic human cancers, including melanoma. However, the binding partners of NF1 and the pathways in which it is involved in melanoma have not been characterized in an in depth manner. Utilizing a mass spectrometry analysis of NF1 binding partners, we revealed Calpain1 (CAPN1), a calcium-dependent neutral cysteine protease, as a novel NF1 binding partner that regulates NF1 degradation in melanoma cells. ShRNA-mediated knockdown of CAPN1 or treatment with a CAPN1 inhibitor stabilizes NF1 protein levels, downregulates AKT signaling and melanoma cell growth. Combination treatment of Calpain inhibitor I with MEKi Trametinib in different melanoma cells is more effective in reducing melanoma cell growth compared to treatment with Trametinib alone, suggesting that this combination may have a therapeutic potential in melanoma. This novel mechanism for regulating NF1 in melanoma provides a molecular basis for targeting CAPN1 in order to stabilize NF1 levels and, in doing so, suppressing Ras activation; this mechanism can be exploited therapeutically in melanoma and other cancers.

While synthetic lethality (SL) holds promise in developing effective cancer therapies, SL candidates found via experimental screens often have limited translational value. Here we present a data-driven approach, ISLE (identification of clinically relevant synthetic lethality), that mines TCGA cohort to identify the most likely clinically relevant SL interactions (cSLi) from a given candidate set of lab-screened SLi. We first validate ISLE via a benchmark of large-scale drug response screens and by predicting drug efficacy in mouse xenograft models. We then experimentally test a select set of predicted cSLi via new screening experiments, validating their predicted context-specific sensitivity in hypoxic vs normoxic conditions and demonstrating cSLi's utility in predicting synergistic drug combinations. We show that cSLi can successfully predict patients' drug treatment response and provide patient stratification signatures. ISLE thus complements existing actionable mutation-based methods for precision cancer therapy, offering an opportunity to expand its scope to the whole genome.

Protein zymography is the most commonly used technique to study the enzymatic activity of matrix metalloproteinases (MMPs) and their inhibitors. MMPs are proteolytic enzymes that promote extracellular matrix degradation. MMPs are frequently mutated in malignant melanomas as well as other cancers and are linked to increasing incidence of tumor metastasis. Substrate zymography characterizes MMP activity by their ability to degrade preferred substrates. Here we describe the collagen zymography technique to measure the active or latent form of MMPs using MMP-8 as an example, which is a frequently mutated MMP family member in malignant melanomas. The same technique can be used with the modification of substrate to detect metalloproteinase activity of other MMPs. Both wild-type and mutated forms of MMPs can be analyzed using a single gel using this method.

Ionotropic glutamate receptors (iGluRs) are large integral membrane multi-protein complexes that create ion channels in plasma membranes. Upon binding of receptor specific ligands (e.g., glutamate), increased efflux or influx of mono- or divalent cations (e.g., Ca2+) promotes synaptic transmission, cellular migration, and survival. Three classes of iGluRs were originally defined after their respective agonists: AMPA, kainate, and NMDA receptors (NMDARs). Recently, we examined iGluR families at the genetic level using Next-Generation Sequencing (NGS) (whole-exome sequencing (WES)) and discovered a high prevalence of somatic mutations within the gene for one of the NMDAR subunits, GRIN2A, specifically in malignant melanoma. Following confirmation of the somatic mutations, we focused on functional characterization of a subset of the GRIN2A mutants that demonstrated a loss of NMDAR functionality. We used gene expression and protein biochemistry to examine complex formation between GluN1 subunit (encoded by GRIN1) and GluN2A subunit (encoded by GRIN2A), anchorage-independent growth in soft agar and cellular migration. Furthermore, we used shRNA depletion of endogenous GRIN2A in melanoma cells expressing either wild-type GRIN2A or mutant GRIN2A and measured cellular proliferation compared to negative controls. Our data show that somatic mutation of certain residues in GluN2A results in increased survival and is the first such report to demonstrate the functional importance of GRIN2A mutations in melanoma and the significance ionotropic glutamate receptor signaling plays in malignant melanoma.

OBJECTIVE: To assess whether Parkinson disease (PD) genes are somatically mutated in cutaneous melanoma (CM) tissue, because CM occurs in patients with PD at higher rates than in the general population and PD is more common than expected in CM cohorts.METHODS: We cross-referenced somatic mutations in metastatic CM detected by whole-exome sequencing with the 15 known PD (PARK) genes. We computed the empirical distribution of the sum of mutations in each gene (Smut) and of the number of tissue samples in which a given gene was mutated at least once (SSampl) for each of the analyzable genes, determined the 90th and 95th percentiles of the empirical distributions of these sums, and verified the location of PARK genes in these distributions. Identical analyses were applied to adenocarcinoma of lung (ADENOCA-LUNG) and squamous cell carcinoma of lung (SQUAMCA-LUNG). We also analyzed the distribution of the number of mutated PARK genes in CM samples vs the 2 lung cancers.RESULTS: Somatic CM mutation analysis (n = 246) detected 315,914 mutations in 18,758 genes. Somatic CM mutations were found in 14 of 15 PARK genes. Forty-eight percent of CM samples carried ?1 PARK mutation and 25% carried multiple PARK mutations. PARK8 mutations occurred above the 95th percentile of the empirical distribution for SMut and SSampl. Significantly more CM samples harbored multiple PARK gene mutations compared with SQUAMCA-LUNG (p = 0.0026) and with ADENOCA-LUNG (p

In this article, we discuss the complex involvement of a Rho-family GTPase, Rac1, in cell migration and in invadopodia-mediated matrix degradation. We discuss the involvement of invadopodia in invasive cell migration, and their capacity to promote cancer metastasis. Considering the regulation of invadopodia formation, we describe studies that demonstrate the role of Rac1 in the metastatic process, and the suggestion that this effect is attributable to the capacity of Rac1 to promote invadopodia formation. This notion is demonstrated here by showing that knockdown of Rac1 in melanoma cells expressing a wild type form of this GTPase, reduces invadopodia-dependent matrix degradation. Interestingly, we also show that excessive activity of Rac1, displayed by the P29S, hyperactive, "fast cycling" mutant of Rac1, which is present in 5-10% of melanoma tumors, inhibits invadopodia function. Moreover, knockdown of this hyperactive mutant enhanced matrix degradation, indicating that excessive Rac1 activity by this mutant can negatively regulate invadopodia formation and function. (C) 2016 Elsevier Inc. All rights reserved.

Recent technological advances in sequencing have flooded the field of cancer research with knowledge about somatic mutations for many different cancer types. Most cancer genomics studies focus on mutations that alter the amino acid sequence, ignoring the potential impact of synonymous mutations. However, accumulating experimental evidence has demonstrated clear consequences for gene function, leading to a widespread recognition of the functional role of synonymous mutations and their causal connection to various diseases. Here, we review the evidence supporting the direct impact of synonymous mutations on gene function via gene splicing; mRNA stability, folding, and translation; protein folding; and miRNA-based regulation of expression. These results highlight the functional contribution of synonymous mutations to oncogenesis and the need to further investigate their detection and prioritization for experimental assessment.

Despite the remarkable clinical response of melanoma harboring BRAF mutations to BRAF inhibitors (BRAFi), most tumors become resistant. Here, we identified the downregulation of the ubiquitin ligase RNF125 in BRAFi-resistant melanomas and demonstrated its role in intrinsic and adaptive resistance to BRAFi in cultures as well as its association with resistance in tumor specimens. Sox10/MITF expression correlated with and contributed to RNF125 transcription. Reduced RNF125 was associated with elevated expression of receptor tyrosine kinases (RTKs), including EGFR. Notably, RNF125 altered RTK expression through JAK1, which we identified as an RNF125 substrate. RNF125 bound to and ubiquitinated JAK1, prompting its degradation and suppressing RTK expression. Inhibition of JAK1 and EGFR signaling overcame BRAFi resistance in melanoma with reduced RNF125 expression, as shown in culture and in in vivo xenografts. Our findings suggest that combination therapies targeting both JAK1 and EGFR could be effective against BRAFi-resistant tumors with de novo low RNF125 expression.

The recent elucidation of the genomic landscape of head and neck squamous cell carcinoma (HNSCC) has provided a unique opportunity to develop selective cancer treatment options. These efforts will require the establishment of relevant HNSCC models for preclinical testing. Here, we performed full exome and transcriptome sequencing of a large panel of HNSCC-derived cells from different anatomical locations and human papillomavirus (HPV) infection status. These cells exhibit typical mutations in TP53, FAT1, CDK2NA, CASP8, and NOTCH1, and copy number variations (CNVs) and mutations in PIK3CA, HRAS, and PTEN that reflect the widespread activation of the PI3K-mTOR pathway. SMAD4 alterations were observed that may explain the decreased tumor suppressive effect of TGF-beta in HNSCC. Surprisingly, we identified HPV+ HNSCC cells harboring TP53 mutations, and documented aberrant TP53 expression in a subset of HPV+ HNSCC cases. This analysis also revealed that most HNSCC cells harbor multiple mutations and CNVs in epigenetic modifiers (e.g., EP300, CREBP, MLL1, MLL2, MLL3, KDM6A, and KDM6B) that may contribute to HNSCC initiation and progression. These genetically-defined experimental HNSCC cellular systems, together with the identification of novel actionable molecular targets, may now facilitate the pre-clinical evaluation of emerging therapeutic agents in tumors exhibiting each precise genomic alteration.

Purpose: Cancer immunotherapy with adoptive transfer of tumor-infiltrating lymphocytes (TIL) represents an effective treatment for patients with metastatic melanoma, with the objective regressions in up to 72% of patients in three clinical trials. However, the antigen targets recognized by these effective TILs remain largely unclear. Experimental Design: Melanoma patients 2359 and 2591 both experienced durable complete regressions of metastases ongoing beyond five years following adoptive TIL transfer. Two conventional screening approaches were carried out to identify the antigens recognized by these clinically effective TILs. In addition, a novel approach was developed in this study to identify mutated T-cell antigens by screening a tandem minigene library, which comprised nonsynonymous mutation sequences identified by whole-exome sequencing of autologous tumors. Results: Screening of an autologous melanoma cDNA library using a conventional approach led to the identification of previously undescribed nonmutated targets recognized by TIL 2359 or TIL 2591. In contrast, screening of tandem minigene libraries encoding tumor-specific mutations resulted in the identification of mutated kinesin family member 2C (KIF2C) antigen as a target of TIL 2359, and mutated DNA polymerase alpha subunit B (POLA2) antigen as a target of TIL 2591. Both KIF2C and POLA2 have been found to play important roles in cell proliferation. Conclusions: These findings suggest that the minigene screening approach can facilitate the antigen repertoire analysis of tumor reactive T cells, and lead to the development of new adoptive cell therapies with purified T cells that recognize candidate-mutated antigens derived from genes essential for the carcinogenesis. (C)2014 AACR.

Synonymous mutations, which do not alter the protein sequence, have been shown to affect protein function [Sauna ZE, Kimchi-Sarfaty C (2011) Nat Rev Genet 12(10):683-691]. However, synonymous mutations are rarely investigated in the cancer genomics field. We used whole-genome and -exome sequencing to identify somatic mutations in 29 melanoma samples. Validation of one synonymous somatic mutation in BCL2L12 in 285 samples identified 12 cases that harbored the recurrent F17F mutation. This mutation led to increased BCL2L12 mRNA and protein levels because of differential targeting of WT and mutant BCL2L12 by hsa-miR-671-5p. Protein made from mutant BCL2L12 transcript bound p53, inhibited UV-induced apoptosis more efficiently than WT BCL2L12, and reduced endogenous p53 target gene transcription. This report shows selection of a recurrent somatic synonymous mutation in cancer. Our data indicate that silent alterations have a role to play in human cancer, emphasizing the importance of their investigation in future cancer genome studies.

Adoptive cell therapy with tumor-infiltrating lymphocytes (TILs) represents an effective treatment for patients with metastatic melanoma. However, most of the Ag targets recognized by effective melanoma-reactive TILs remain elusive. In this study, patient 2369 experienced a complete response, including regressions of bulky liver tumor masses, ongoing beyond 7 y following adoptive TIL transfer. The screening of a cDNA library generated from the autologous melanoma cell line resulted in the isolation of a mutated protein phosphatase 1, regulatory (inhibitor) subunit 3B (PPP1R3B) gene product. The mutated PPP1R3B peptide represents the immunodominant epitope recognized by tumor-reactive T cells in TIL 2369. Five years following adoptive transfer, peripheral blood T lymphocytes obtained from patient 2369 recognized the mutated PPP1R3B epitope. These results demonstrate that adoptive T cell therapy targeting a tumor-specific Ag can mediate long-term survival for a patient with metastatic melanoma. This study also provides an impetus to develop personalized immunotherapy targeting tumor-specific, mutated Ags.

Keywords: Oncology

The neurotransmitter glutamate interacts with glutamate receptor proteins, leading to the activation of multiple signaling pathways. Dysfunction in the glutamatergic signaling pathway is well established as a frequent player in diseases such as schizophrenia, Alzheimer disease, and brain tumors (gliomas). Recently, aberrant functioning of this pathway has also been shown in melanoma. In both glioma and melanoma, glutamate secretion stimulates tumor growth, proliferation, and survival through activation of the mitogen-activated protein kinase and phosphoinositide 3-kinase/Akt pathways. In the future, extracellular glutamate levels and glutamatergic signaling may serve as biological markers for tumorigenicity and facilitate targeted therapy for melanoma. Clin Cancer Res; 18(16); 4240-6. (C) 2012 AACR.

Phosphatidylinositol 3-kinases (PI3Ks) are known to be important regulators of signaling pathways. To determine whether PI3Ks are genetically altered in cancers, we analyzed the sequences of the P13K gene family and discovered that one family member, PIK3CA, is frequently mutated in cancers of the colon and other organs. The majority of mutations clustered near two positions within the P13K helical or kinase domains. PIK3CA represents one of the most highly mutated oncogenes yet identified in human cancers and is useful as a diagnostic and therapeutic target.

We performed a mutational analysis of the 19 disintegrin-metalloproteinases (ADAMs) genes in human cutaneous metastatic melanoma and identified eight to be somatically mutated in 79 samples, affecting 34% of the melanoma tumors analyzed. Functional analysis of the two frequently mutated ADAM genes, ADAM29 and ADAM7 demonstrated that the mutations affect adhesion of melanoma cells to specific extracellular matrix proteins and in some cases increase their migration ability. This suggests that mutated ADAM genes could play a role in melanoma progression. (C) 2011 Wiley-Liss, Inc.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) mediate signals between G-protein coupled receptors and their downstream pathways, and have been shown to be mutated in cancer. In particular, GNAQ was found to be frequently mutated in blue nevi of the skin and uveal melanoma, acting as an oncogene in its mutated form. To further examine the role of heterotrimeric G proteins in melanoma, we performed a comprehensive mutational analysis of the 35 genes in the heterotrimeric G protein gene family in a panel of 80 melanoma samples. Somatic alterations in a G protein subunit were detected in 17% of samples spanning seven genes. The highest rates of somatic, non-synonymous mutations were found in GNG10 and GNAZ, neither of which has been previously reported to be mutated in melanoma. Our study is the first systematic analysis of the heterotrimeric G proteins in melanoma and indicates that multiple mutated heterotrimeric G proteins may be involved in melanoma progression.

As the upward spiral of novel cancer gene discoveries and novel molecular compounds continues to accelerate, a repetitive theme in molecular drug development remains the lack of activity of initially promising agents when given to patients in clinical trials. It is however invigorating that a few targeted agents directed against a select group of a few 'cancer gene superfamilies' have escaped this all to common fate, and have evolved into novel, clinically meaningful molecular therapy strategies. Targeting dysregulated signaling of the epidermal growth factor family of transmembrane receptors (Erbb family) has encompassed over the last decade an ever increasing role in personalized treatment approaches in an increasing number of human malignancies. Erbbs are receptor tyrosine kinases that are important regulators of several signaling pathways. Two of its family members (Erbb1/EGFR and Erbb2/HER2) have previously been shown to be somatically mutated of human cancers. To determine if this family is somatically mutated in melanoma, its sequences were recently analyzed and one of its members, Erbb4, was found to be somatically mutated in 19% of melanoma cases. Functional analysis of seven of its mutations was shown to increase its catalytic and transformation abilities as well as providing essential survival signals. Similar to other Erbb family members, mutant Erbb4 seems to confer "oncogene addiction" on melanoma cells, making it an attractive therapeutic target. Gaining further understanding into the oncogenic mechanism of Erbb4 may not only help in the development of targeted therapy in melanoma patients but might accelerate the acceptance of a novel taxonomy of cancer which is based on the genomic perturbations in cancer genes and cancer gene families and their response to targeted agents.

Tyrosine phosphorylation is important in signaling pathways underlying tumorigenesis. We performed a mutational analysis of the protein tyrosine kinase (PTK) gene family in cutaneous metastatic melanoma. We identified 30 somatic mutations affecting the kinase domains of 19 PTKs and subsequently evaluated the entire coding regions of the genes encoding these 19 PTKs for somatic mutations in 79 melanoma samples. We found ERBB4 mutations in 19% of individuals with melanoma and found mutations in two other kinases (FLT1 and PTK2B) in 10% of individuals with melanomas. We examined seven missense mutations in the most commonly altered PTK gene, ERBB4, and found that they resulted in increased kinase activity and transformation ability. Melanoma cells expressing mutant ERBB4 had reduced cell growth after shRNA-mediated knockdown of ERBB4 or treatment with the ERBB inhibitor lapatinib. These studies could lead to personalized therapeutics specifically targeting the kinases that are mutationally altered in individual melanomas.

Sequence mutations and gene amplifications lead to activation of the PIK3CA-AKT2 signaling pathway and have been reported in several types of neoplasms including ovarian cancer. Analysis of such genetic alterations, however, is usually complicated by contamination of normal cell DNA, artifacts associated with formalin-fixed tissues and the sensitivity of the techniques employed. In this study, we analyzed the sequence mutations in PIK3CA and AKT2 genes using purified tumor cells that were isolated from high-grade ovarian serous carcinomas and serous borderline tumors (SBTs) and assessed gene amplification using a dual-color FISH on tissue microarrays. Somatic sequence mutations in the kinase domain of AKT2 were not detected in any of the 65 ovarian tumors analyzed. Mutations of PIK3CA were rare, occurring only in one (2.3%) of 44 high-grade serous carcinomas and in only one (4.8%) of 21 SBTs. Dual-color FISH demonstrated that PIK3CA and AKT2 were not amplified in SBTs but amplified in 13.3% and 18.2% high-grade carcinomas, respectively. High-level amplification (>3 fold) was more frequently observed in AKT2 than in PIKCA. Unlike mutations in ERBB2, KRAS and BRAF which are mutually exclusive in SBTs, coamplification of PIK3CA and AKT2 was present in five high-grade carcinomas including the OVCAR3 cells. Amplification in either of the genes occurred in 27% high-grade serous carcinomas. In conclusion, the methods we employed provide unambiguous evidence that somatic sequence mutations of PIK3CA and ATK2 are rare in ovarian serous tumors but amplification of both genes may play an important role in the development of high-grade ovarian serous carcinoma.

PIK3CA is mutated in diverse human cancers, but the functional effects of these mutations have not been defined. To evaluate the consequences of PIK3CA alterations, the two most common mutations were inactivated by gene targeting in colorectal cancer (CRC) cells. Biochemical analyses of these cells showed that mutant PIK3CA selectively regulated the phosphorylation of AKT and the forkhead transcription factors FKHR and FKHRL1. PIK3CA mutations had little effect on growth under standard conditions, but reduced cellular dependence on growth factors. PIK3CA mutations resulted in attenuation of apoptosis and facilitated tumor invasion. Treatment with the PI3K inhibitor LY294002 abrogated PIK3CA signaling and preferentially inhibited growth of PIK3CA mutant cells. These data have important implications for therapy of cancers harboring PIK3CA alterations.

The phosphatidylinositol 3-kinases (PI3Ks) are known regulators of cellular growth and proliferation. It has recently been reported that somatic mutations within the PI3K subunit p110alpha (PIK3CA) are present in human colorectal and other cancers. Here we show that thirteen of fifty-three breast cancers (25%) contain somatic mutations in PIK3CA, with the majority of mutations located in the kinase domain. These results demonstrate that PIK3CA is the most mutated oncogene in breast cancer and support a role for PIK3CA in epithelial carcinogenesis.