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Cellular components of the tumor microenvironment, including myeloid cells, play important roles in the progression of lung adenocarcinoma (LUAD) and its response to therapy. Here, we characterize the function of the ubiquitin ligases Siah1a/2 in regulating the differentiation and activity of alveolar macrophages (AMs) and assess the implication of Siah1a/2 control of AMs for carcinogen-induced LUAD. Macrophage-specific genetic ablation of Siah1a/2 promoted accumulation of AMs with an immature phenotype and increased expression of pro-tumorigenic and pro-inflammatory Stat3 and β-catenin gene signatures. Administration of urethane to wild-type mice promoted enrichment of immature-like AMs and lung tumor development, which was enhanced by macrophage-specific Siah1a/2 ablation. The pro-fibrotic gene signature seen in Siah1a/2-ablated immature-like macrophages was associated with increased tumor infiltration of CD14+ myeloid cells and poorer survival of LUAD patients. Single-cell RNA-seq confirmed the presence of a cluster of immature-like AMs expressing a pro-fibrotic signature in lungs of LUAD patients, a signature enhanced in smokers. These findings identify Siah1a/2 in alveolar macrophages as gatekeepers of lung cancer development.

Supplementary Table S1: Clinical information related to the PDX used in Fig1A

Supplemental Methods, Figures, Figure legends, Table and Movie Legends

Supplementary Movie S1 - 3D representation around the Y axis of A375 cell nucleus

Supp fig 1-8: Figure S1. Inhibition of SLC1A5 Expression in A431 cells. Figure S2. Screen and Validation of Hits. Figure S3. IMD-0354 inhibited Glutamine Uptake. Figure S4. IMD-0354 Suppresses Glutamine Uptake Independent of IKK-NF- B Signaling Suppression. Figure S5. Profiles of IMD-0354-Treated A375 Cells Matched with Small-Molecule Inhibitors and Melanoma Subtypes. Figure S6. Expression of Glutamine Deprivation-Related Genes. Figure S7. Inhibition of Melanoma Growth with IMD-0354 in combination with LDHA or GLS1 Inhibitors. Figure S8. Immunohistochemistry Staining of Xenograft Tumor Samples.

Supplementary Table S3: List of the genes that were identified by RNAseq to be significantly deregulated upon SPANX KD.

Supplementary Table S2: List of SPANX-interacting proteins identified by LC-MS/MS

Supplementary Movie S2 - 3D representation around the X axis of A375 cell nucleus

Mechanisms regulating nuclear organization control fundamental cellular processes, including the cell and chromatin organization. Their disorganization, including aberrant nuclear architecture, has been often implicated in cellular transformation. Here, we identify Lamin A, among proteins essential for nuclear architecture, as SPANX (sperm protein associated with the nucleus on the X chromosome), a cancer testis antigen previously linked to invasive tumor phenotypes, interacting protein in melanoma. SPANX interaction with Lamin A was mapped to the immunoglobulin fold-like domain, a region critical for Lamin A function, which is often mutated in laminopathies. SPANX downregulation in melanoma cell lines perturbed nuclear organization, decreased cell viability, and promoted senescence-associated phenotypes. Moreover, SPANX knockdown (KD) in melanoma cells promoted proliferation arrest, a phenotype mediated in part by IRF3/IL1A signaling. SPANX KD in melanoma cells also prompted the secretion of IL1A, which attenuated the proliferation of naïve melanoma cells. Identification of SPANX as a nuclear architecture complex component provides an unexpected insight into the regulation of Lamin A and its importance in melanoma.Implications:SPANX, a testis protein, interacts with LMNA and controls nuclear architecture and melanoma growth.

Supp Tables 1-2: Table S1 List of hits selected from the primary screen. Table S2. Chemical structures of hits selected for validation.

Supplementary Table

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.

Supplementary Figures

Supplementary Tables S1 and S2 - PDF file 100K, Supplementary Table S1. The top 20 upregulated genes in WM793 cells treated for 24 h with SBI-087702 as compared to DMSO-treated cells; Supplementary Table S2. The top 20 downregulated genes in WM793 cells treated for 24 h with SBI-0087702 as compared to DMSO-treated cells

Purpose: Sensitize melanomas to apoptosis and inhibit their growth and metastatic potential by compounds that mimic the activities of activating transcription factor 2 (ATF2)-driven peptides.Experimental Design: Small-molecule chemical library consisting of 3,280 compounds was screened to identify compounds that elicit properties identified for ATF2 peptide, including (a) sensitization of melanoma cells to apoptosis, (b) inhibition of ATF2 transcriptional activity, (c) activation of c-Jun NH2-terminal kinase (JNK) and c-Jun transcriptional activity, and (d) inhibition of melanoma growth and metastasis in mouse models.Results: Two compounds, celastrol (CSL) and acetyl isogambogic acid, could, within a low micromolar range, efficiently elicit cell death in melanoma cells. Both compounds efficiently inhibit ATF2 transcriptional activities, activate JNK, and increase c-Jun transcriptional activities. Similar to the ATF2 peptide, both compounds require JNK activity for their ability to inhibit melanoma cell viability. Derivatives of CSL were identified as potent inducers of cell death in mouse and human melanomas. CSL and a derivative (CA19) could also efficiently inhibit growth of human and mouse melanoma tumors and reduce the number of lung metastases in syngeneic and xenograft mouse models.Conclusions: These studies show for the first time the effect of CSL and acetyl isogambogic acid on melanoma. These compounds elicit activities that resemble the well-characterized ATF2 peptide and may therefore offer new approaches for the treatment of this tumor type.

Supplementary Methods Legends and References - PDF file 163K, Supplementary Methods, Legends for Figures, and References

Purpose: Effective therapy for malignant melanoma, the leading cause of death from skin cancer, remains an area of significant unmet need in oncology. The elevated expression of PKCϵ in advanced metastatic melanoma results in the increased phosphorylation of the transcription factor ATF2 on threonine 52, which causes its nuclear localization and confers its oncogenic activities. The nuclear-to-mitochondrial translocation of ATF2 following genotoxic stress promotes apoptosis, a function that is largely lost in melanoma cells, due to its confined nuclear localization. Therefore, promoting the nuclear export of ATF2, which sensitizes melanoma cells to apoptosis, represents a novel therapeutic modality.Experimental Design: We conducted a pilot high-throughput screen of 3,800 compounds to identify small molecules that promote melanoma cell death by inducing the cytoplasmic localization of ATF2. The imaging-based ATF2 translocation assay was conducted using UACC903 melanoma cells that stably express doxycycline-inducible GFP-ATF2.Results: We identified two compounds (SBI-0089410 and SBI-0087702) that promoted the cytoplasmic localization of ATF2, reduced cell viability, inhibited colony formation, cell motility, and anchorage-free growth, and increased mitochondrial membrane permeability. SBI-0089410 inhibited the 12-O-tetradecanoylphorbol-l3-acetate (TPA)–induced membrane translocation of protein kinase C (PKC) isoforms, whereas both compounds decreased ATF2 phosphorylation by PKCϵ and ATF2 transcriptional activity. Overexpression of either constitutively active PKCϵ or phosphomimic mutant ATF2T52E attenuated the cellular effects of the compounds.Conclusion: The imaging-based high-throughput screen provides a proof-of-concept for the identification of small molecules that block the oncogenic addiction to PKCϵ signaling by promoting ATF2 nuclear export, resulting in mitochondrial membrane leakage and melanoma cell death. Clin Cancer Res; 19(10); 2710–22. ©2013 AACR.

Supplementary Figures S1 - S6 - PDF file 945K, Supplementary Figure S1. Effect of select compounds on ATF2 nuclear-mitochondrial Translocation; Supplementary Figure S2. SBI-0089410 or SBI-0087702 do not affect the subcellular localization of Stat3 or beta-catenin. Supplementary Figure S3. The expression of ATF252E attenuates apoptosis induced by SBI-0089410 or SBI-0087702; Supplementary Figure S4. SBI-0089410 or SBI-0087702-induced inhibition of cell motility and cell death are reversed by the stable expression of ATF252E; Supplementary Figure S5. itochondrial potential changes induced by SBI-0089410 or SBI-0087702 in melanoma cells; Supplementary Figure S6. The effect of SBI-0089410 or SBI-0087702 on ATF2 phosphorylation, PKC translocation and activation, and the viability of PLX4032-resistant melanoma cell lines.

Supplementary Figures S1-S11 from Preclinical Studies of Celastrol and Acetyl Isogambogic Acid in Melanoma

Melanoma development involves members of the AGC kinase family, including AKT, PKC, and, most recently, PDK1, as elucidated recently in studies of Braf::Pten mutant melanomas. Here, we report that PDK1 contributes functionally to skin pigmentation and to the development of melanomas harboring a wild-type PTEN genotype, which occurs in about 70% of human melanomas. The PDK1 substrate SGK3 was determined to be an important mediator of PDK1 activities in melanoma cells. Genetic or pharmacologic inhibition of PDK1 and SGK3 attenuated melanoma growth by inducing G1 phase cell-cycle arrest. In a synthetic lethal screen, pan-PI3K inhibition synergized with PDK1 inhibition to suppress melanoma growth, suggesting that focused blockade of PDK1/PI3K signaling might offer a new therapeutic modality for wild-type PTEN tumors. We also noted that responsiveness to PDK1 inhibition associated with decreased expression of pigmentation genes and increased expression of cytokines and inflammatory genes, suggesting a method to stratify patients with melanoma for PDK1-based therapies. Overall, our work highlights the potential significance of PDK1 as a therapeutic target to improve melanoma treatment. Cancer Res; 75(7); 1399–412. ©2015 AACR.

SBI-756 inhibits AKT, mTOR signaling and alters cell cycle.

Supplementary Table and Figure Legends. Legend to Supplementary Tables S1-S2 and Supplementary Figures S1-S6.

Pharmacological properties of top four BI-69A11 analogs (S1); Blood chemistry following SBI-756 administration (S2); List of biotinylated-BI-69A11-bound proteins, identified by LC/MS/MS (S3); RPPA based analyses identify proteins that were significantly inhibited by SBI-756, compared to DMSO treatment at the 24 h time point (S4); Gene variants identified in SBI-756-resistant clones but not SBI-756-sensitive melanoma cell cultures (S5); The top 10 enriched canonical pathways identified using the Ingenuity Pathway Analysis of genes mutated in SBI-756 resistant melanoma (S6); Ingenuity Pathway Analysis of genes differentially expressed between SBI-756-sensitive and - resistant melanoma cells (S7).

Supplementary Table S2. Outline of genetic changes in the tumors and cell lines used.

Supplementary Figures S1-S6. BrafV600E::Cdkn2a-/- delay development of tumors in PtenWT::Pdk-/-genotype (S1); Inhibition of SGK1 or SGK3 resembles changes in signaling seen upon PDK1 inhibition (S2); SGK and PDK1 inhibition affect cell cycle progression (S3); Synthetic lethal screen for PDK1i combination drugs (S4); RPPA analysis of signaling components that are affected upon inhibition of PDK1 (S5); PDK1 KD reduces the expression of genes involved in pigmentation and metastasis (S6).

Tumor microenvironment components, including T and myeloid cells, play important roles in lung adenocarcinoma (LADC) progression and response to therapy. Here, we identify a role for Siah1a/2 ubiquitin ligases in controlling alveolar macrophage (AM) differentiation and urethane-induced LADC. Genetic ablation of Siah1a/2 in AMs enriched their immature state, coinciding with increased pro-tumorigenic and inflammatory gene signatures and abundance of Siah1a/2 substrates NRF2 and β-catenin. Urethane administration to mice enriched the population of monocytic and immature-like AMs, which were more prevalent in the lungs of mice carrying Siah1a/2-ablated macrophages. While resembling transitional profibrotic macrophages often seen in lung fibrosis, enrichment of immature AMs coincided with the development of more frequent and larger lung tumors in urethane-treated mice harboring Siah1a/2 ablated macrophages compared with controls. Gene expression signature of Siah1a/2 ablated immature-like AMs is associated with increased infiltration of CD14+ immune cells and worse survival of LADC patients. Our findings identify Siah1a/2 as gatekeepers of cancer development by controlling AM differentiation and profibrotic phenotypes contributing to carcinogen-induced lung cancer.

Tumour dependency on specific metabolic signals has been demonstrated and often guided numerous therapeutic approaches. We identify melanoma addiction to the mitochondrial protein glutaryl-CoA dehydrogenase (GCDH), which functions in lysine metabolism and controls protein glutarylation. GCDH knockdown induced cell death programmes in melanoma cells, an activity blocked by inhibition of the upstream lysine catabolism enzyme DHTKD1. The transcription factor NRF2 mediates GCDH-dependent melanoma cell death programmes. Mechanistically, GCDH knockdown induces NRF2 glutarylation, increasing its stability and DNA binding activity, with a concomitant transcriptional upregulation of ATF4, ATF3, DDIT3 and CHAC1, resulting in cell death. In vivo, inducible inactivation of GCDH effectively inhibited melanoma tumour growth. Correspondingly, reduced GCDH expression correlated with improved survival of patients with melanoma. These findings identify melanoma cell addiction to GCDH, limiting apoptotic signalling by controlling NRF2 glutarylation. Inhibiting the GCDH pathway could thus represent a therapeutic approach to treat melanoma.

Disrupted antiviral immune responses are associated with severe COVID-19, the disease caused by SAR-CoV-2. Here, we show that the 73-amino-acid protein encoded byORF9cof the viral genome contains a putative transmembrane domain, interacts with membrane proteins in multiple cellular compartments, and impairs antiviral processes in a lung epithelial cell line. Proteomic, interactome, and transcriptomic analyses, combined with bioinformatic analysis, revealed that expression of only this highly unstable small viral protein impaired interferon signaling, antigen presentation, and complement signaling, while inducing IL-6 signaling. Furthermore, we showed that interfering with ORF9c degradation by either proteasome inhibition or inhibition of the ATPase VCP blunted the effects of ORF9c. Our study indicated that ORF9c enables immune evasion and coordinates cellular changes essential for the SARS-CoV-2 life cycle.One-sentence summarySARS-CoV-2 ORF9c is the first human coronavirus protein localized to membrane, suppressing antiviral response, resembling full viral infection.

Functional T cell exhaustion develops with persistent antigen exposure during chronic viral infection and cancer. Greater insights into T cell exhaustion are needed to promote immunity to viruses and tumors. RNF5 (RING-finger protein 5) is an E3 ubiquitin ligase that functions in protein degradation. We previously found enhanced anti-tumor responses in RNF5−/− mice, which was coupled with greater recovery and function of CD8+ T cell in melanoma tumors. Tumor-infiltrating RNF5−/− CD8+ T cells expressed high levels of inhibitory receptors (IR), yet greater functionality compared to WT CD8+ T cells, suggesting greater activation. To further assess RNF5-dependent regulation of CD8+ T cell exhaustion, we used the chronic LCMV Cl13 infection that has defined states of progressive T cell exhaustion. Virus-specific CD8+ T cells were significantly increased in RNF5−/− mice leading to greater numbers of cytokine producers, which expressed high levels of IRs. They also expressed higher levels of TOX, a transcriptional regulator of T cell exhaustion and persistence. With low dose infection, RNF5−/− mice displayed decreased morbidity and CD8+ T cells acquired a highly functional memory phenotype rather than exhaustion. To identify T cell intrinsic role(s) of RNF5, we analyzed anti-viral responses in bone marrow chimeras lacking RNF5 only in T cells and used an in vitro T cell exhaustion assay. RNF5−/− CD8+ T cells also expressed high levels of IRs and TOX, as did the in vitro exhausted T cells yet importantly displayed enhanced cytokine production compared to WT cells. These data reveal a T cell-intrinsic role of RNF5 in promoting exhaustion in settings of chronic antigen stimulation.

Stringent transcriptional regulation is crucial for normal cellular biology and organismal development. Perturbations in the proper regulation of transcription factors can result in numerous pathologies, including cancer. Thus, understanding how transcription factors are regulated and how they are dysregulated in disease states is key to the therapeutic targeting of these factors and/or the pathways that they regulate. Activating transcription factor 2 (ATF2) has been studied in a number of developmental and pathological conditions. Recent findings have shed light on the transcriptional, post-transcriptional, and post-translational regulatory mechanisms that influence ATF2 function, and thus, the transcriptional programs coordinated by ATF2. Given our current knowledge of its multiple levels of regulation and function, ATF2 represents a paradigm for the mechanistic complexity that can regulate transcription factor function. Thus, increasing our understanding of the regulation and function of ATF2 will provide insights into fundamental regulatory mechanisms that influence how cells integrate extracellular and intracellular signals into a genomic response through transcription factors. Characterization of ATF2 dysfunction in the context of pathological conditions, particularly in cancer biology and response to therapy, will be important in understanding how pathways controlled by ATF2 or other transcription factors might be therapeutically exploited. In this review, we provide an overview of the currently known upstream regulators and downstream targets of ATF2.

SummaryUnderstanding the mechanism underlying the regulation of the androgen receptor (AR), a central player in the development of castration-resistant prostate cancer (CRPC), holds promise for overcoming the challenge of treating CRPC. We demonstrate that the ubiquitin ligase Siah2 targets a select pool of NCOR1-bound, transcriptionally-inactive AR for ubiquitin-dependent degradation, thereby promoting expression of select AR target genes implicated in lipid metabolism, cell motility, and proliferation. Siah2 is required for prostate cancer cell growth under androgen-deprivation conditions in vitro and in vivo, and Siah2 inhibition promotes prostate cancer regression upon castration. Notably, Siah2 expression is markedly increased in human CRPCs. Collectively, we find that selective regulation of AR transcriptional activity by the ubiquitin ligase Siah2 is important for CRPC development.

The tricarboxylic acid (TCA) cycle is the central hub of oxidative metabolism, running in the classic forward direction to provide carbon for biosynthesis and reducing agents for generation of ATP. Our metabolic tracer studies in melanoma cells showed that in hypoxic conditions the TCA cycle is largely disconnected from glycolysis. By studying the TCA branch point metabolites, acetyl CoA and citrate, as well as the metabolic endpoint glutamine and fatty acids, we developed a comprehensive picture of the rewiring of the TCA cycle that occurs in hypoxia. Hypoxic tumor cells maintain proliferation by running the TCA cycle in reverse. The source of carbon for acetyl CoA, citrate, and fatty acids switches from glucose in normoxia to glutamine in hypoxia. This hypoxic flux from glutamine into fatty acids is mediated by reductive carboxylation. This reductive carboxylation is catalyzed by two isocitrate dehydrogenases, IDH1 and IDH2. Their combined action is necessary and sufficient to effect the reverse TCA flux and maintain cellular viability.

Background: The E3 ubiquitin ligases SIah1 and 2 mediate a cellular response to hypoxia through its control of prolyl-hydroxylases and consequently HIF1α stability. Given the impact of hypoxia on heart function under stress, we have evaluated the effect of genetic deletion of Siah1 and 2 on cardiac function in response to myocardial infarction. Objective: To determine the physiological significance of the ubiquitin ligase Siah genes under in vivo ischemia condition. Materials and methods: To substantiate the role of Siah2 in ischemia-induced cell death, we tested difference in the response of myocardial tissue to ischemia using Siah1a +/− /Siah2 −/− mutant mice and their wildtype littermates. Analysis of heart tissue from Siah1a +/− /Siah2 −/− mice subjected to a model of 24 hours anterior descending coronary artery occlusion revealed their resistance to ischemic injury when compared with their littermates. Cardiac function, measured as EF %, was significantly less impaired in the mutant mice. Heart tissue obtained from Siah1a +/− /Siah2 −/− mice subjected to myocardial ischemia, exhibited a significantly lower degree of apoptosis (Tunel Staining), and myocardial infarct size (Evans blue, TTC). Conclusions: Collectively, these data establish that reduced expression of Siah1/2 exerts a cardioprotective function under ischemic conditions. The underlying molecular mechanisms linking cardioprotection to ischemic challenge will be discussed.

Metabolic rewiring is an established hallmark of cancer, but the details of this rewiring at a systems level are not well characterized. Here we acquire this insight in a melanoma cell line panel by tracking metabolic flux using isotopically labeled nutrients. Metabolic profiling and flux balance analysis were used to compare normal melanocytes to melanoma cell lines in both normoxic and hypoxic conditions. All melanoma cells exhibited the Warburg phenomenon; they used more glucose and produced more lactate than melanocytes. Other changes were observed in melanoma cells that are not described by the Warburg phenomenon. Hypoxic conditions increased fermentation of glucose to lactate in both melanocytes and melanoma cells (the Pasteur effect). However, metabolism was not strictly glycolytic, as the tricarboxylic acid (TCA) cycle was functional in all melanoma lines, even under hypoxia. Furthermore, glutamine was also a key nutrient providing a substantial anaplerotic contribution to the TCA cycle. In the WM35 melanoma line glutamine was metabolized in the “reverse” (reductive) direction in the TCA cycle, particularly under hypoxia. This reverse flux allowed the melanoma cells to synthesize fatty acids from glutamine while glucose was primarily converted to lactate. Altogether, this study, which is the first comprehensive comparative analysis of metabolism in melanoma cells, provides a foundation for targeting metabolism for therapeutic benefit in melanoma.