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Notch signaling can have either an oncogenic or tumor suppressive function in cancer depending on the cancer type and cellular context. While Notch can be oncogenic in early prostate cancer, we identified significant downregulation of the Notch pathway during prostate cancer progression from adenocarcinoma to neuroendocrine prostate cancer where it functions as a tumor suppressor. Activation of Notch in neuroendocrine and Rb1/Trp53-deficient prostate cancer models led to phenotypic conversion towards a more indolent non-neuroendocrine state with glandular features and expression of luminal lineage markers. This was accompanied by up-regulation of MHC and type I interferon and immune cell infiltration. Overall, these data support Notch signaling as a suppressor of neuroendocrine differentiation in advanced prostate cancer and provides insights into how Notch signaling influences lineage plasticity and the tumor microenvironment.
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CTL recognition of non-mutated tumor-associated antigens (TAA), present on cancer cells but also in healthy tissues, is an important element of cancer immunity, but the mechanism of its selectivity for cancer cells and opportunities for its enhancement remain elusive. In this study, we found that CTL expression of the NK receptors (NKR) DNAM-1 and NKG2D was associated with the effector status of CD8+ tumor-infiltrating lymphocytes (TIL) and long-term survival of melanoma patients. Using MART-1 and NY-ESO-1 as model TAAs, we demonstrated that DNAM-1 and NKG2D regulate T-cell receptor (TCR) functional avidity and set the threshold for TCR activation of human TAA-specific CTLs. Superior costimulatory effects of DNAM-1 over CD28 involved enhanced TCR signaling, CTL killer function and polyfunctionality. Double transduction of human CTLs with TAA-specific TCR and NKRs resulted in strongly enhanced antigen sensitivity, without a reduction in the antigen specificity and selectivity of killer function. In addition, the elevation of NKR-Ligand expression on cancer cells by chemotherapy also increased CTL recognition of cancer cells expressing low levels of TAA. Our data help to explain the ability of self-antigens to mediate tumor rejection in the absence of autoimmunity and support the development of dual-targeting adoptive T cell therapies that use NKRs to enhance the potency and selectivity of recognition of TAA-expressing cancer cells.
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Recent progress in single-cell profiling technologies has revealed significant phenotypic and transcriptional heterogeneity in tumor-infiltrating CD8+ T cells. However, the transition between the different states of intratumoral antigen-specific CD8+ T cells remains elusive. Here, we sought to examine the generation, transcriptomic states, and the clinical relevance of melanoma-infiltrating CD8+ T cells expressing a chemokine receptor and T-cell differentiation marker, CX3C chemokine receptor 1 (CX3CR1). Analysis of single-cell datasets revealed distinct human melanoma-infiltrating CD8+ T-cell clusters expressing genes associated with effector T-cell function but with distinguishing expression of CX3CR1 or PDCD1. No obvious impact of CX3CR1 expression in melanoma on the response to immune checkpoint inhibitor therapy was observed while increased pretreatment and on-treatment frequency of a CD8+ T-cell cluster expressing high levels of exhaustion markers was associated with poor response to the treatment. Adoptively transferred antigen-specific CX3CR1- CD8+ T cells differentiated into the CX3CR1+ subset in mice treated with FTY720, which inhibits lymphocyte egress from secondary lymphoid tissues, suggesting the intratumoral generation of CX3CR1+ CD8+ T cells rather than their trafficking from secondary lymphoid organs. Furthermore, analysis of adoptively transferred antigen-specific CD8+ T cells, in which the Cx3cr1 gene was replaced with a marker gene confirmed that CX3CR1+ CD8+ T cells could directly differentiate from the intratumoral CX3CR1- subset. These findings highlight that tumor antigen-specific CX3CR1- CD8+ T cells can fully differentiate outside the secondary lymphoid organs and generate CX3CR1+ CD8+ T cells in the tumor microenvironment, which are distinct from CD8+ T cells that express markers of exhaustion. SIGNIFICANCE: Intratumoral T cells are composed of heterogeneous subpopulations with various phenotypic and transcriptional states. This study illustrates the intratumoral generation of antigen-specific CX3CR1+ CD8+ T cells that exhibit distinct transcriptomic signatures and clinical relevance from CD8+ T cells expressing markers of exhaustion.
Assuntos
Linfócitos T CD8-Positivos , Receptor 1 de Quimiocina CX3C , Linfócitos do Interstício Tumoral , Melanoma , Transcriptoma , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Melanoma/imunologia , Melanoma/genética , Melanoma/patologia , Animais , Humanos , Camundongos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Cloridrato de Fingolimode/farmacologia , Cloridrato de Fingolimode/uso terapêutico , Camundongos Endogâmicos C57BL , Análise de Célula Única , Relevância ClínicaRESUMO
Tyrosine kinase inhibitors (TKIs) that block the vascular endothelial growth factor receptors (VEGFRs) not only disrupt tumor angiogenesis but also have many unexpected side effects that impact tumor cells directly. This includes the induction of molecular markers associated with senescence, a form of cellular aging that typically involves growth arrest. We have shown that VEGFR TKIs can hijack these aging programs by transiently inducting senescence markers (SMs) in tumor cells to activate senescence-associated secretory programs that fuel drug resistance. Here we show that these same senescence-mimicking ("senomimetic") VEGFR TKI effects drive an enhanced immunogenic signaling that, in turn, can alter tumor response to immunotherapy. By using a live cell sorting method to detect ß-galactosidase, a commonly used SM, we found that subpopulations of SM-expressing (SM+) tumor cells have heightened IFN signaling and increased expression of IFN-stimulated genes (ISGs). These ISGs increase under the control of the STimulator of the INterferon Gene (STING) signaling pathway, which we found could be directly activated by several VEGFR TKIs. TKI-induced SM+ cells could stimulate or suppress CD8 T-cell activation depending on host-tumor cell contact while tumors grown from SM+ cells were more sensitive to PDL1 inhibition in vivo, suggesting that offsetting immune-suppressive functions of SM+ cells can improve TKI efficacy overall. Our findings may explain why some (but not all) VEGFR TKIs improve outcomes when combined with immunotherapy and suggest that exploiting senomimetic drug side effects may help identify TKIs that uniquely "prime" tumors for enhanced sensitivity to PDL1-targeted agents.
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Tyrosine kinase inhibitors (TKIs) that block the vascular endothelial growth factor receptors (VEGFRs) disrupt tumor angiogenesis but also have many unexpected side-effects that impact tumor cells directly. This includes the induction of molecular markers associated with senescence, a form of cellular aging that typically involves growth arrest. We have shown that VEGFR TKIs can hijack these aging programs by transiently inducting senescence-markers (SMs) in tumor cells to activate senescence-associated secretory programs that fuel drug resistance. Here we show that these same senescence-mimicking ('senomimetic') VEGFR TKI effects drive an enhanced immunogenic signaling that, in turn, can alter tumor response to immunotherapy. Using a live-cell sorting method to detect beta-galactosidase, a commonly used SM, we found that subpopulations of SM-expressing (SM+) tumor cells have heightened interferon (IFN) signaling and increased expression of IFN-stimulated genes (ISGs). These ISG increases were under the control of the STimulator of INterferon Gene (STING) signaling pathway, which we found could be directly activated by several VEGFR TKIs. TKI-induced SM+ cells could stimulate or suppress CD8 T-cell activation depending on host:tumor cell contact while tumors grown from SM+ cells were more sensitive to PD-L1 inhibition in vivo, suggesting that offsetting immune-suppressive functions of SM+ cells can improve TKI efficacy overall. Our findings may explain why some (but not all) VEGFR TKIs improve outcomes when combined with immunotherapy and suggest that exploiting senomimetic drug side-effects may help identify TKIs that uniquely 'prime' tumors for enhanced sensitivity to PD-L1 targeted agents.
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The MUC1-C protein is aberrantly expressed in adenocarcinomas of epithelial barrier tissues and contributes to their progression. Less is known about involvement of MUC1-C in the pathogenesis of squamous cell carcinomas (SCC). Here, we report that the MUC1 gene is upregulated in advanced head and neck SCCs (HNSCC). Studies of HNSCC cell lines demonstrate that the MUC1-C subunit regulates expression of (i) RIG-I and MDA5 pattern recognition receptors, (ii) STAT1 and IFN regulatory factors, and (iii) downstream IFN-stimulated genes. MUC1-C integrates chronic activation of the STAT1 inflammatory pathway with induction of the ∆Np63 and SOX2 genes that are aberrantly expressed in HNSCCs. In extending those dependencies, we demonstrate that MUC1-C is necessary for NOTCH3 expression, self-renewal capacity, and tumorigenicity. The findings that MUC1 associates with ∆Np63, SOX2 and NOTCH3 expression by single-cell RNA sequencing analysis further indicate that MUC1-C drives the HNSCC stem cell state and is a target for suppressing HNSCC progression. SIGNIFICANCE: This work reports a previously unrecognized role for MUC1-C in driving STAT1-mediated chronic inflammation with the progression of HNSCC and identifies MUC1-C as a druggable target for advanced HNSCC treatment.
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Progressão da Doença , Neoplasias de Cabeça e Pescoço , Mucina-1 , Carcinoma de Células Escamosas de Cabeça e Pescoço , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Mucina-1/genética , Mucina-1/metabolismo , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/metabolismo , Linhagem Celular Tumoral , Camundongos , Animais , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT1/genética , Receptor Notch3/genética , Receptor Notch3/metabolismoRESUMO
Extracts of the Chilean soapbark tree, Quillaja Saponaria (QS) are the source of potent immune-stimulatory saponin compounds. This study compared the adjuvanticity and toxicity of QS-18 and QS-21, assessing the potential to substitute QS-18 in place of QS-21 for vaccine development. QS-18, the most abundant QS saponin fraction, has been largely overlooked due to safety concerns. We found that QS-18 spontaneously inserted into liposomes, thereby neutralizing hemolytic activity, and following administration did not induce local reactogenicity in a footpad swelling test in mice. With high-dose intramuscular administration, transient weight loss was minor, and QS-18 did not induce significantly more weight loss compared to a liposome vaccine adjuvant system lacking it. Two days after administration, no elevation of inflammatory cytokines was detected in murine serum. In a formulation including cobalt-porphyrin-phospholipid (CoPoP) for short peptide sequestration, QS-18 did not impact the formation of peptide nanoparticles. With immunization, QS-18 peptide particles induced higher levels of cancer neoepitope-specific and tumor-associated antigen-specific CD8+ T cells compared to QS-21 particles, without indication of greater toxicity based on mouse body weight. T cell receptor sequencing of antigen-specific CD8+ T cells showed that QS-18 induced significantly more T cell transcripts. In two murine cancer models, vaccination with QS-18 peptide particles induced a similar therapeutic effect as QS-21 particles, without indication of increased toxicity. Antigen-specific CD8+ T cells in the tumor microenvironment were found to express the exhaustion marker PD-1, pointing to the rationale for exploring combination therapy. Taken together, these data demonstrate that QS-18, when formulated in liposomes, can be a safe and effective adjuvant to induce tumor-inhibiting cellular responses in murine models with potential to facilitate or diminish costs of production for vaccine adjuvant systems. Further studies are warranted to assess liposomal QS-18 immunogic, reactogenic and toxicological profiles in mice and other animal species.
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Adjuvantes Imunológicos , Vacinas Anticâncer , Lipossomos , Quillaja , Animais , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Quillaja/química , Adjuvantes Imunológicos/administração & dosagem , Feminino , Camundongos Endogâmicos C57BL , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Camundongos , Saponinas de Quilaia , Citocinas , Saponinas/administração & dosagem , Saponinas/farmacologia , Linhagem Celular Tumoral , Vacinas de Subunidades ProteicasRESUMO
Mimotopes of short CD8+ T-cell epitopes generally comprise one or more mutated residues, and can increase the immunogenicity and function of peptide cancer vaccines. We recently developed a two-step approach to generate enhanced mimotopes using positional peptide microlibraries and herein applied this strategy to the broadly used H-2Kb-restricted murine leukemia p15E tumor rejection epitope. The wild-type p15E epitope (sequence: KSPWFTTL) was poorly immunogenic in mice, even when combined with a potent peptide nanoparticle vaccine system and did not delay p15E-expressing MC38 tumor growth. Following positional microlibrary functional screening of over 150 mimotope candidates, two were identified, both with mutations at residue 3 (p15E-P3C; "3C," and p15E-P3M; "3M") that better induced p15E-specific CD8+ T cells and led to tumor rejection. Although 3M was more immunogenic, 3C effectively delayed tumor growth in a therapeutic setting relative to the wild-type p15E. As 3C had less H-2Kb affinity relative to both p15E and 3M, 15 additional mimotope candidates (all that incorporated the 3C mutation) were assessed that maintained or improved predicted MHC-I affinity. Valine substitution at position 2 (3C2V, sequence: KVCWFTTL) led to improved p15E-specific immunogenicity, tumor rejection, and subsequent long-term antitumor immunity. 3C, 3M, and 3C2V mimotopes were more effective than p15E in controlling MC38 and B16-F10 tumors. T-cell receptor (TCR) sequencing revealed unique TCR transcripts for mimotopes, but there were no major differences in clonality. These results provide new p15E mimotopes for further vaccine use and illustrate considerations for MHC-I affinity, immunogenicity, and functional efficacy in mimotope design. SIGNIFICANCE: The MHC-I-restricted p15E tumor rejection epitope is expressed in multiple murine cancer lines and is used as a marker of antitumor cellular immunity, but has seen limited success as a vaccine immunogen. An in vivo screening approach based on a positional peptide microlibraries is used to identify enhanced p15E mimotopes bearing amino acid mutations that induce significantly improved functional immunogenicity relative to vaccination with the wild-type epitope.
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Neoplasias , Vacinas , Animais , Camundongos , Neoplasias/terapia , Peptídeos , Epitopos de Linfócito T/genética , Receptores de Antígenos de Linfócitos TRESUMO
Sarcomatoid renal cell carcinoma (sRCC) is histologically heterogeneous, with variable sarcomatoid amounts intermixed within epithelial carcinoma. However, the current classification for this aggressive disease is homogeneous and agnostic to the sarcomatoid proportion. We investigated whether sRCC subclassification has prognostic value and can reveal the biology underlying dedifferentiation and its clinical aggressiveness. On the basis of the intratumoral abundance of sarcomatoid features, cases were classified as sarcomatoid-high (≥10% sarcomatoid features) or sarcomatoid-low (<10% sarcomatoid features) in a cohort of 104 consecutive patients with sRCC undergoing nephrectomy at a single center. In comparison to sarcomatoid-low patients (n = 52), sarcomatoid-high patients (n = 52) had significantly shorter overall survival (median 14.5 vs 62.9 mo; p < 0.001), which was confirmed on multivariable analysis, and significantly shorter median metastasis-free survival among patients with clinically localized disease (10.7 vs 39.0 mo; p = 0.043). Transcriptomic analyses of 45 sRCC tumors revealed significant upregulation of nine hallmark pathways related to cell cycle/proliferation, epithelial-to-mesenchymal transition, reactive oxidative species, and interferon-α signaling among sarcomatoid-high (n = 24) versus sarcomatoid-low (n = 21) tumors. Categorization into transcriptomic clusters revealed predominance of proliferative, inflammatory, and immune effector phenotypes among sarcomatoid-high tumors, versus a hypoxia/angiogenesis phenotype among sarcomatoid-low tumors. Overall, these findings indicate prognostic value for sRCC subclassification into high versus low sarcomatoid groups and highlight key biology underlying the differences in clinical outcomes. PATIENT SUMMARY: Sarcomatoid renal cell carcinoma (sRCC) is a highly aggressive form of kidney cancer. The percentage of sarcomatoid features varies among tumors, but sRCC is still defined as a single kidney cancer type. Our results show that grouping patients according to their percentage of sarcomatoid features improves prediction of whether their tumors will become metastatic or lethal, and reveal molecular differences that may be important for this disease. Future assignment of sRCC to high and low sarcomatoid groups may help in guiding research and patient management.
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Dendritic cells (DC) are mediators between innate and adaptive immune responses to pathogens and tumors. DC development is determined by signaling through the receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3) in bone marrow myeloid progenitors. Recently the naming conventions for DC phenotypes have been updated to distinguish between "Conventional" DCs (cDCs) and plasmacytoid DCs (pDCs). Activating mutations of FLT3, including Internal Tandem Duplication (FLT3-ITD), are associated with poor prognosis for acute myeloid leukemia (AML) patients. Having a shared myeloid lineage it can be difficult to distinguish bone fide DCs from AML tumor cells. To date, there is little information on the effects of FLT3-ITD in DC biology. To further elucidate this relationship we utilized CITE-seq technology in combination with flow cytometry and multiplex immunoassays to measure changes to DCs in human and mouse tissues. We examined the cDC phenotype and frequency in bone marrow aspirates from patients with AML to understand the changes to cDCs associated with FLT3-ITD. When compared to healthy donor (HD) we found that a subset of FLT3-ITD+ AML patient samples have overrepresented populations of cDCs and disrupted phenotypes. Using a mouse model of FLT3-ITD+ AML, we found that cDCs were increased in percentage and number compared to control wild-type (WT) mice. Single cell RNA-seq identified FLT3-ITD+ cDCs as skewed towards a cDC2 T-bet- phenotype, previously shown to promote Th17 T cells. We assessed the phenotypes of CD4+ T cells in the AML mice and found significant enrichment of both Treg and Th17 CD4+ T cells in the bone marrow and spleen compartments. Ex vivo stimulation of CD4+ T cells also showed increased Th17 phenotype in AML mice. Moreover, co-culture of AML mouse-derived DCs and naïve OT-II cells preferentially skewed T cells into a Th17 phenotype. Together, our data suggests that FLT3-ITD+ leukemia-associated cDCs polarize CD4+ T cells into Th17 subsets, a population that has been shown to be negatively associated with survival in solid tumor contexts. This illustrates the complex tumor microenvironment of AML and highlights the need for further investigation into the effects of FLT3-ITD mutations on DC phenotypes and their downstream effects on Th polarization.
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Leucemia Mieloide Aguda , Tirosina Quinase 3 Semelhante a fms , Animais , Humanos , Camundongos , Células Dendríticas/patologia , Tirosina Quinase 3 Semelhante a fms/genética , Homeostase , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Mutação , Microambiente Tumoral/genéticaRESUMO
Compelling evidence shows that the frequency of T cells in the tumor microenvironment correlates with prognosis as well as response to immunotherapy. However, considerable heterogeneity exists within tumor-infiltrating T cells, and significance of their genomic and transcriptomic landscape on clinical outcomes remains to be elucidated. Signaling lymphocyte activation molecule 6 (SLAMF6) is expressed on intra-tumoral progenitor-exhausted T cells, which exhibit the capacity to proliferate, self-renew and produce terminally-exhausted T cells in pre-clinical models and patients. Here, we investigated the impact of SLAMF6 expression on prognosis in two immunologically different tumor types using publicly available databases. Our findings demonstrate that high SLAMF6 expression is associated with better prognosis, expression of TCF7 (encoding T-cell factor 1), and increased gene signatures associated with conventional type 1 dendritic cells and effector function of T cells in melanoma and breast cancer. Single-cell profiling of breast cancer tumor microenvironment reveals SLAMF6 expression overlaps CD8 T cells with a T-effector signature, which includes subsets expressing TCF7, memory and effector-related genes, analogous to progenitor-exhausted T cells. These findings illustrate the significance of SLAMF6 in the tumor as a marker for better effector responses, and provide insights into the predictive and prognostic determinants for cancer patients.
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Neoplasias da Mama , Melanoma , Humanos , Feminino , Melanoma/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Microambiente Tumoral/genética , Linfócitos T CD8-Positivos , Imunoterapia , Prognóstico , Família de Moléculas de Sinalização da Ativação Linfocitária/genética , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismoRESUMO
The current study in prostate cancer (PCa) focused on the genomic mechanisms at the cross-roads of pro-differentiation signals and the emergence of lineage plasticity. We explored an understudied cistromic mechanism involving RARγ's ability to govern AR cistrome-transcriptome relationships, including those associated with more aggressive PCa features. The RARγ complex in PCa cell models was enriched for canonical cofactors, as well as proteins involved in RNA processing and bookmarking. Identifying the repertoire of miR-96 bound and regulated gene targets, including those recognition elements marked by m6A, revealed their significant enrichment in the RARγ complex. RARγ significantly enhanced the AR cistrome, particularly in active enhancers and super-enhancers, and overlapped with the binding of bookmarking factors. Furthermore, RARγ expression led to nucleosome-free chromatin enriched with H3K27ac, and significantly enhanced the AR cistrome in G2/M cells. RARγ functions also antagonized the transcriptional actions of the lineage master regulator ONECUT2. Similarly, gene programs regulated by either miR-96 or antagonized by RARγ were enriched in alternative lineages and more aggressive PCa phenotypes. Together these findings reveal an under-investigated role for RARγ, modulated by miR-96, to bookmark enhancer sites during mitosis. These sites are required by the AR to promote transcriptional competence, and emphasize luminal differentiation, while antagonizing ONECUT2.
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Activation of the MUC1-C protein promotes lineage plasticity, epigenetic reprogramming, and the cancer stem cell (CSC) state. The present studies performed on enriched populations of triple-negative breast cancer (TNBC) CSCs demonstrate that MUC1-C is essential for integrating activation of glycolytic pathway genes with self-renewal and tumorigenicity. MUC1-C further integrates the glycolytic pathway with suppression of mitochondrial DNA (mtDNA) genes encoding components of mitochondrial Complexes I-V. The repression of mtDNA genes is explained by MUC1-C-mediated (i) downregulation of the mitochondrial transcription factor A (TFAM) required for mtDNA transcription and (ii) induction of the mitochondrial transcription termination factor 3 (mTERF3). In support of pathogenesis that suppresses mitochondrial ROS production, targeting MUC1-C increases (i) mtDNA gene transcription, (ii) superoxide levels, and (iii) loss of self-renewal capacity. These findings and scRNA-seq analysis of CSC subpopulations indicate that MUC1-C regulates self-renewal and redox balance by integrating activation of glycolysis with suppression of oxidative phosphorylation.
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We have dissected the role of Estrogen receptor beta (ERß) in prostate cancer (PCa) with a novel ERß ligand, OSU-ERb-12. Drug screens revealed additive interactions between OSU-ERB-12 and either epigenetic inhibitors or the androgen receptor antagonist, Enzalutamide (Enza). Clonogenic and cell biolody studies supported the potent additive effects of OSU-ERB-12 (100nM) and Enza (1µM). The cooperative behavior was in PCa cell lines treated with either OSU-ERB-12 plus Enza or combinations involving 17ß-estradiol (E2). OSU-ERb-12 plus Enza uniquely impacted the transcriptiome, accessible chromatin, and the AR, MYC and H3K27ac cistromes. This included skewed transcriptional responses including suppression of the androgen and MYC transcriptomes, and repressed MYC protein. OSU-ERb-12 plus Enza uniquely impacted chromatin accessibility at approximately 3000 nucleosome-free sites, enriched at enhancers, enriched for basic Helix-Loop-Helix motifs. CUT&RUN experiments revealed combination treatment targeting of MYC, AR, and H3K27ac again shaping enhancer accessibility. Specifically, it repressed MYC interactions at enhancer regions enriched for bHLH motifs, and overlapped with publicly-available bHLH cistromes. Finally, cistrome-transcriptome analyses identified ~200 genes that distinguished advanced PCa tumors in the SU2C cohort with high androgen and low neuroendocrine scores.
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Neuroblastoma is the most common extracranial solid tumor in children. MYCN amplification is detected in almost half of high-risk cases and is associated with poorly differentiated tumors, poor patient prognosis and poor response to therapy, including retinoids. We identify the aryl hydrocarbon receptor (AhR) as a transcription factor promoting the growth and suppressing the differentiation of MYCN-amplified neuroblastoma. A neuroblastoma specific AhR transcriptional signature reveals an inverse correlation of AhR activity with patients' outcome, suggesting AhR activity is critical for disease progression. AhR modulates chromatin structures, reducing accessibility to regions responsive to retinoic acid. Genetic and pharmacological inhibition of AhR results in induction of differentiation. Importantly, AhR antagonism with clofazimine synergizes with retinoic acid in inducing differentiation both in vitro and in vivo. Thus, we propose AhR as a target for MYCN-amplified neuroblastoma and that its antagonism, combined with current standard-of-care, may result in a more durable response in patients.
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Chronic inflammation promotes epigenetic reprogramming in cancer progression by pathways that remain unclear. The oncogenic MUC1-C protein is activated by the inflammatory NF-κB pathway in cancer cells. There is no known involvement of MUC1-C in regulation of the COMPASS family of H3K4 methyltransferases. We find that MUC1-C regulates (i) bulk H3K4 methylation levels, and (ii) the COMPASS SET1A/SETD1A and WDR5 genes by an NF-κB-mediated mechanism. The importance of MUC1-C in regulating the SET1A COMPASS complex is supported by the demonstration that MUC1-C and WDR5 drive expression of FOS, ATF3 and other AP-1 family members. In a feedforward loop, MUC1-C, WDR5 and AP-1 contribute to activation of genes encoding TRAF1, RELB and other effectors in the chronic NF-κB inflammatory response. We also show that MUC1-C, NF-κB, WDR5 and AP-1 are necessary for expression of the (i) KLF4 master regulator of the pluripotency network and (ii) NOTCH1 effector of stemness. In this way, MUC1-C/NF-κB complexes recruit SET1A/WDR5 and AP-1 to enhancer-like signatures in the KLF4 and NOTCH1 genes with increases in H3K4me3 levels, chromatin accessibility and transcription. These findings indicate that MUC1-C regulates the SET1A COMPASS complex and the induction of genes that integrate NF-κB-mediated chronic inflammation with cancer progression.
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NF-kappa B , Neoplasias , Humanos , NF-kappa B/genética , NF-kappa B/metabolismo , Fator de Transcrição AP-1/metabolismo , Neoplasias/genética , Processos Neoplásicos , Inflamação/genética , Epigênese Genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mucina-1/genética , Mucina-1/metabolismoRESUMO
Dendritic cells (DC) are mediators of adaptive immune responses to pathogens and tumors. DC development is determined by signaling through the receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3) in bone marrow myeloid progenitors. Recently the naming conventions for DC phenotypes have been updated to distinguish between "Conventional" DCs (cDCs) and plasmacytoid DCs (pDCs). Activating mutations of FLT3, including Internal Tandem Duplication (FLT3-ITD), are associated with poor prognosis for leukemia patients. To date, there is little information on the effects of FLT3-ITD in DC biology. We examined the cDC phenotype and frequency in bone marrow aspirates from patients with acute myeloid leukemia (AML) to understand the changes to cDCs associated with FLT3-ITD. When compared to healthy donor (HD) we found that a subset of FLT3-ITD+ AML patient samples have overrepresented populations of cDCs and disrupted phenotypes. Using a mouse model of FLT3-ITD+ AML, we found that cDCs were increased in percentage and number compared to control wild-type (WT) mice. Single cell RNA-seq identified FLT3-ITD+ cDCs as skewed towards a cDC2 T-bet - phenotype, previously shown to promote Th17 T cells. We assessed the phenotypes of CD4+ T cells in the AML mice and found significant enrichment of both Treg and Th17 CD4+ T cells. Furthermore, co-culture of AML mouse- derived DCs and naïve OT-II cells preferentially skewed T cells into a Th17 phenotype. Together, our data suggests that FLT3-ITD+ leukemia-associated cDCs polarize CD4+ T cells into Th17 subsets, a population that has been shown to be negatively associated with survival in solid tumor contexts. This illustrates the complex tumor microenvironment of AML and highlights the need for further investigation into the effects of FLT3-ITD mutations on DC phenotypes.
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Multiple myeloma (MM) remains an incurable plasma cell (PC) malignancy. Although it is known that MM tumor cells display extensive intratumoral genetic heterogeneity, an integrated map of the tumor proteomic landscape has not been comprehensively evaluated. We evaluated 49 primary tumor samples from newly diagnosed or relapsed/refractory MM patients by mass cytometry (CyTOF) using 34 antibody targets to characterize the integrated landscape of single-cell cell surface and intracellular signaling proteins. We identified 13 phenotypic meta-clusters across all samples. The abundance of each phenotypic meta-cluster was compared to patient age, sex, treatment response, tumor genetic abnormalities and overall survival. Relative abundance of several of these phenotypic meta-clusters were associated with disease subtypes and clinical behavior. Increased abundance of phenotypic meta-cluster 1, characterized by elevated CD45 and reduced BCL-2 expression, was significantly associated with a favorable treatment response and improved overall survival independent of tumor genetic abnormalities or patient demographic variables. We validated this association using an unrelated gene expression dataset. This study represents the first, large-scale, single-cell protein atlas of primary MM tumors and demonstrates that subclonal protein profiling may be an important determinant of clinical behavior and outcome.
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Mieloma Múltiplo , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Proteômica , Plasmócitos/metabolismoRESUMO
Lack of reliable predictive biomarkers is a major limitation of combination therapy with chemotherapy and anti-programmed cell death protein 1/programmed death-ligand 1 (anti-PD-1/PD-L1) therapy (chemo-immunotherapy). We previously observed that the increase of peripheral blood CD8+ T cells expressing CX3CR1, a marker of differentiation, correlates with response to anti-PD-1 therapy; however, the predictive and prognostic value of T-cell CX3CR1 expression during chemo-immunotherapy is unknown. Here, we evaluated the utility of circulating CX3CR1+CD8+ T cells as a predictive correlate of response to chemo-immunotherapy in patients with non-small cell lung cancer (NSCLC). At least 10% increase of the CX3CR1+ subset in circulating CD8+ T cells from baseline (CX3CR1 score) was associated with response to chemo-immunotherapy as early as 4 weeks with 85.7% overall accuracy of predicting response at 6 weeks. Furthermore, at least 10% increase of the CX3CR1 score correlated with substantially better progression-free (P = 0.0051) and overall survival (P = 0.0138) on Kaplan-Meier analysis. Combined single-cell RNA/T-cell receptor (TCR) sequencing of circulating T cells from longitudinally obtained blood samples and TCR sequencing of tumor tissue from the same patient who received a long-term benefit from the treatment demonstrated remarkable changes in genomic and transcriptomic signatures of T cells as well as evolution of TCR clonotypes in peripheral blood containing highly frequent tumor-infiltrating lymphocyte repertoires overexpressing CX3CR1 early after initiation of the treatment despite stable findings of the imaging study. Collectively, these findings highlight the potential utility of T-cell CX3CR1 expression as a dynamic blood-based biomarker during the early course of chemo-immunotherapy and a marker to identify frequent circulating tumor-infiltrating lymphocyte repertoires. Significance: Current approaches to combined chemotherapy and anti-PD-1/PD-L1 therapy (chemo-immunotherapy) for patients with NSCLC are limited by the lack of reliable predictive biomarkers. This study shows the utility of T-cell differentiation marker, CX3CR1, as an early on-treatment predictor of response and changes in genomic/transcriptomic signatures of circulating tumor-infiltrating lymphocyte repertoires in patients with NSCLC undergoing chemo-immunotherapy.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Prognóstico , Neoplasias Pulmonares/tratamento farmacológico , Antígeno B7-H1/análise , Linfócitos T CD8-Positivos/química , Imunoterapia/métodos , Receptores de Antígenos de Linfócitos T/genética , Receptor 1 de Quimiocina CX3C/genéticaRESUMO
African American (AA) prostate cancer associates with vitamin D3 deficiency, but vitamin D receptor (VDR) genomic actions have not been investigated in this context. We undertook VDR proteogenomic analyses in European American (EA) and AA prostate cell lines and four clinical cohorts. Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) analyses revealed that nonmalignant AA RC43N prostate cells displayed the greatest dynamic protein content in the VDR complex. Likewise, in AA cells, Assay for Transposase-Accessible Chromatin using sequencing established greater 1α,25(OH)2D3-regulated chromatin accessibility, chromatin immunoprecipitation sequencing revealed significant enhancer-enriched VDR cistrome, and RNA sequencing identified the largest 1α,25(OH)2D3-dependent transcriptome. These VDR functions were significantly corrupted in the isogenic AA RC43T prostate cancer cells, and significantly distinct from EA cell models. We identified reduced expression of the chromatin remodeler, BAZ1A, in three AA prostate cancer cohorts as well as RC43T compared with RC43N. Restored BAZ1A expression significantly increased 1α,25(OH)2D3-regulated VDR-dependent gene expression in RC43T, but not HPr1AR or LNCaP cells. The clinical impact of VDR cistrome-transcriptome relationships were tested in three different clinical prostate cancer cohorts. Strikingly, only in AA patients with prostate cancer, the genes bound by VDR and/or associated with 1α,25(OH)2D3-dependent open chromatin (i) predicted progression from high-grade prostatic intraepithelial neoplasia to prostate cancer; (ii) responded to vitamin D3 supplementation in prostate cancer tumors; (iii) differentially responded to 25(OH)D3 serum levels. Finally, partial correlation analyses established that BAZ1A and components of the VDR complex identified by RIME significantly strengthened the correlation between VDR and target genes in AA prostate cancer only. Therefore, VDR transcriptional control is most potent in AA prostate cells and distorted through a BAZ1A-dependent control of VDR function. Significance: Our study identified that genomic ancestry drives the VDR complex composition, genomic distribution, and transcriptional function, and is disrupted by BAZ1A and illustrates a novel driver for AA prostate cancer.