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1.
Cancer Cell ; 37(5): 720-734.e13, 2020 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-32359397

RESUMO

Renal medullary carcinoma (RMC) is a highly lethal malignancy that mainly afflicts young individuals of African descent and is resistant to all targeted agents used to treat other renal cell carcinomas. Comprehensive genomic and transcriptomic profiling of untreated primary RMC tissues was performed to elucidate the molecular landscape of these tumors. We found that RMC was characterized by high replication stress and an abundance of focal copy-number alterations associated with activation of the stimulator of the cyclic GMP-AMP synthase interferon genes (cGAS-STING) innate immune pathway. Replication stress conferred a therapeutic vulnerability to drugs targeting DNA-damage repair pathways. Elucidation of these previously unknown RMC hallmarks paves the way to new clinical trials for this rare but highly lethal malignancy.

2.
Int J Radiat Biol ; : 1-8, 2020 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-32073931

RESUMO

Purpose: Head and neck cancers (HNSCC) are routinely treated with radiotherapy; however, normal tissue toxicity remains a concern. Therefore, it is important to validate treatment modalities combining molecularly targeted agents with radiotherapy to improve the therapeutic ratio. The aim of this study was to assess the ability of the PARP inhibitor niraparib (MK-4827) alone, or in combination with cell cycle checkpoint abrogating drugs targeting Chk1 (MK-8776) or Wee1 (MK-1775), to radiosensitize HNSCCs in the context of HPV status.Materials and methods: PARP1, PARP2, Chk1 or Wee1 shRNA constructs were analyzed from an in vivo shRNA screen of HNSCC xenografts comparing radiosensitization differences between HPV(+) and HPV(-) tumors. Radiosensitization by niraparib alone or in combination with MK-8776 or MK-1775 was assessed by clonogenic survival in HPV(-) and HPV(+) cells; and the role of p16 in determining response was explored. Relative expressions of DNA repair genes were compared by PCR array in HPV(+) and HPV(-) cells, and following siRNA-mediated knockdown of TRIP12 in HPV(-) cells.Results: In vivo shRNA screening showed a modest preferential radiosensitization by Wee1 and PARP2 in HPV(-) and Chk1 in HPV(+) tumor models. Niraparib alone enhanced the radiosensitivity of all HNSCC cell lines tested. However, HPV(-) cells were sensitized to a greater degree, as suggested by the shRNA screen. When combined with MK-8776 or MK-1775, radiosensitization was further enhanced in an HPV dependent manner with HPV(+) cells enhanced by MK-8776 and HPV(-) cells enhanced by MK-1775. A PCR array for DNA repair genes showed PARP and HR proteins BRCA1 and RAD51 were much lower in HPV(+) cells than in HPV(-). Similarly, directly knocking down p16-dependent TRIP12 decreased expression of these same genes. Overexpressing p16 decreased TRIP12 expression and increased radiosensitivity in HPV(-) HN5. However, while PARP inhibition led to significant radiosensitization in the control, it led to no further significant radiosensitization in p16 overexpressing cells. Forced p16 expression in HPV(-) HN5 increased accumulation in G1 and subG1 and limited progression to S phase, thus reducing effectiveness of PARP inhibition.Conclusions: Niraparib effectively radiosensitizes HNSCCs with a greater benefit seen in HPV(-). HPV status also plays a role in response to MK-8776 or MK-1775 when combined with niraparib due to differences in DNA repair mechanisms. This study suggests that using cell cycle abrogators in combination with PARP inhibitors may be a beneficial treatment option in HNSCC, but also emphasizes the importance of HPV status when considering effective treatment strategies.

3.
JCI Insight ; 4(21)2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31557131

RESUMO

Transcriptomic profiling classifies pancreatic ductal adenocarcinoma (PDAC) into several molecular subtypes with distinctive histological and clinical characteristics. However, little is known about the molecular mechanisms that define each subtype and their correlation with clinical outcome. Mutant KRAS is the most prominent driver in PDAC, present in over 90% of tumors, but the dependence of tumors on oncogenic KRAS signaling varies between subtypes. In particular, the squamous subtype is relatively independent of oncogenic KRAS signaling and typically displays much more aggressive clinical behavior versus the progenitor subtype. Here, we identified that yes-associated protein 1 (YAP1) activation is enriched in the squamous subtype and associated with poor prognosis. Activation of YAP1 in progenitor subtype cancer cells profoundly enhanced malignant phenotypes and transformed progenitor subtype cells into squamous subtype. Conversely, depletion of YAP1 specifically suppressed tumorigenicity of squamous subtype PDAC cells. Mechanistically, we uncovered a significant positive correlation between WNT5A expression and YAP1 activity in human PDAC and demonstrated that WNT5A overexpression led to YAP1 activation and recapitulated a YAP1-dependent but Kras-independent phenotype of tumor progression and maintenance. Thus, our study identifies YAP1 oncogene as a major driver of squamous subtype PDAC and uncovers the role of WNT5A in driving PDAC malignancy through activation of the YAP pathway.

4.
Cancers (Basel) ; 11(9)2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31500168

RESUMO

Our poor understanding of the intricate biology of cancer and the limited availability of preclinical models that faithfully recapitulate the complexity of tumors are primary contributors to the high failure rate of novel therapeutics in oncology clinical studies. To address this need, patient-derived xenograft (PDX) platforms have been widely deployed and have reached a point of development where we can critically review their utility to model and interrogate relevant clinical scenarios, including tumor heterogeneity and clonal evolution, contributions of the tumor microenvironment, identification of novel drugs and biomarkers, and mechanisms of drug resistance. Colorectal cancer (CRC) constitutes a unique case to illustrate clinical perspectives revealed by PDX studies, as they overcome limitations intrinsic to conventional ex vivo models. Furthermore, the success of molecularly annotated "Avatar" models for co-clinical trials in other diseases suggests that this approach may provide an additional opportunity to improve clinical decisions, including opportunities for precision targeted therapeutics, for patients with CRC in real time. Although critical weaknesses have been identified with regard to the ability of PDX models to predict clinical outcomes, for now, they are certainly the model of choice for preclinical studies in CRC. Ongoing multi-institutional efforts to develop and share large-scale, well-annotated PDX resources aim to maximize their translational potential. This review comprehensively surveys the current status of PDX models in translational CRC research and discusses the opportunities and considerations for future PDX development.

5.
Sci Transl Med ; 11(488)2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996079

RESUMO

Eradicating triple-negative breast cancer (TNBC) resistant to neoadjuvant chemotherapy (NACT) is a critical unmet clinical need. In this study, patient-derived xenograft (PDX) models of treatment-naïve TNBC and serial biopsies from TNBC patients undergoing NACT were used to elucidate mechanisms of chemoresistance in the neoadjuvant setting. Barcode-mediated clonal tracking and genomic sequencing of PDX tumors revealed that residual tumors remaining after treatment with standard frontline chemotherapies, doxorubicin (Adriamycin) combined with cyclophosphamide (AC), maintained the subclonal architecture of untreated tumors, yet their transcriptomes, proteomes, and histologic features were distinct from those of untreated tumors. Once treatment was halted, residual tumors gave rise to AC-sensitive tumors with similar transcriptomes, proteomes, and histological features to those of untreated tumors. Together, these results demonstrated that tumors can adopt a reversible drug-tolerant state that does not involve clonal selection as an AC resistance mechanism. Serial biopsies obtained from patients with TNBC undergoing NACT revealed similar histologic changes and maintenance of stable subclonal architecture, demonstrating that AC-treated PDXs capture molecular features characteristic of human TNBC chemoresistance. Last, pharmacologic inhibition of oxidative phosphorylation using an inhibitor currently in phase 1 clinical development delayed residual tumor regrowth. Thus, AC resistance in treatment-naïve TNBC can be mediated by nonselective mechanisms that confer a reversible chemotherapy-tolerant state with targetable vulnerabilities.


Assuntos
Doxorrubicina/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Ciclofosfamida/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Camundongos SCID , Terapia Neoadjuvante , Transcriptoma/genética , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Nature ; 568(7752): 410-414, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30918400

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) remains recalcitrant to all forms of cancer treatment and carries a five-year survival rate of only 8%1. Inhibition of oncogenic KRAS (hereafter KRAS*), the earliest lesion in disease development that is present in more than 90% of PDACs, and its signalling surrogates has yielded encouraging preclinical results with experimental agents2-4. However, KRAS*-independent disease recurrence following genetic extinction of Kras* in mouse models anticipates the need for co-extinction strategies5,6. Multiple oncogenic processes are initiated at the cell surface, where KRAS* physically and functionally interacts to direct signalling that is essential for malignant transformation and tumour maintenance. Insights into the complexity of the functional cell-surface-protein repertoire (surfaceome) have been technologically limited until recently and-in the case of PDAC-the genetic control of the function and composition of the PDAC surfaceome in the context of KRAS* signalling remains largely unknown. Here we develop an unbiased, functional target-discovery platform to query KRAS*-dependent changes of the PDAC surfaceome, which reveals syndecan 1 (SDC1, also known as CD138) as a protein that is upregulated at the cell surface by KRAS*. Localization of SDC1 at the cell surface-where it regulates macropinocytosis, an essential metabolic pathway that fuels PDAC cell growth-is essential for disease maintenance and progression. Thus, our study forges a mechanistic link between KRAS* signalling and a targetable molecule driving nutrient salvage pathways in PDAC and validates oncogene-driven surfaceome annotation as a strategy to identify cancer-specific vulnerabilities.


Assuntos
Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/patologia , Pinocitose , Sindecana-1/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Proliferação de Células , Progressão da Doença , Feminino , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Masculino , Camundongos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais
7.
Sci Transl Med ; 11(483)2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30867319

RESUMO

Mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitors have failed to show clinical benefit in Kirsten rat sarcoma (KRAS) mutant lung cancer due to various resistance mechanisms. To identify differential therapeutic sensitivities between epithelial and mesenchymal lung tumors, we performed in vivo small hairpin RNA screens, proteomic profiling, and analysis of patient tumor datasets, which revealed an inverse correlation between mitogen-activated protein kinase (MAPK) signaling dependency and a zinc finger E-box binding homeobox 1 (ZEB1)-regulated epithelial-to-mesenchymal transition. Mechanistic studies determined that MAPK signaling dependency in epithelial lung cancer cells is due to the scaffold protein interleukin-17 receptor D (IL17RD), which is directly repressed by ZEB1. Lung tumors in multiple Kras mutant murine models with increased ZEB1 displayed low IL17RD expression, accompanied by MAPK-independent tumor growth and therapeutic resistance to MEK inhibition. Suppression of ZEB1 function with miR-200 expression or the histone deacetylase inhibitor mocetinostat sensitized resistant cancer cells to MEK inhibition and markedly reduced in vivo tumor growth, showing a promising combinatorial treatment strategy for KRAS mutant cancers. In human lung tumor samples, high ZEB1 and low IL17RD expression correlated with low MAPK signaling, presenting potential markers that predict patient response to MEK inhibitors.


Assuntos
Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Mutação/genética , Neoplasias/genética , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptores de Interleucina-17/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Animais , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Células Epiteliais/patologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases , Mesoderma/patologia , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias/tratamento farmacológico , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico
8.
Cancer Cell ; 35(2): 204-220.e9, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30753823

RESUMO

Alterations in chromatin remodeling genes have been increasingly implicated in human oncogenesis. Specifically, the biallelic inactivation of the SWI/SNF subunit SMARCB1 results in the emergence of extremely aggressive pediatric malignancies. Here, we developed embryonic mosaic mouse models of malignant rhabdoid tumors (MRTs) that faithfully recapitulate the clinical-pathological features of the human disease. We demonstrated that SMARCB1-deficient malignancies exhibit dramatic activation of the unfolded protein response (UPR) and ER stress response via a genetically intact MYC-p19ARF-p53 axis. As a consequence, these tumors display an exquisite sensitivity to agents inducing proteotoxic stress and inhibition of the autophagic machinery. In conclusion, our findings provide a rationale for drug repositioning trials investigating combinations of agents targeting the UPR and autophagy in SMARCB1-deficient MRTs.


Assuntos
Autofagia , Estresse do Retículo Endoplasmático , Proteostase , Tumor Rabdoide/metabolismo , Proteína SMARCB1/deficiência , Proteína Supressora de Tumor p53/metabolismo , Animais , Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibidores de Proteassoma/farmacologia , Proteostase/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Tumor Rabdoide/tratamento farmacológico , Tumor Rabdoide/genética , Tumor Rabdoide/patologia , Proteína SMARCB1/genética , Transdução de Sinais , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Resposta a Proteínas não Dobradas
9.
Cell Rep ; 26(2): 469-482.e5, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30625329

RESUMO

The plasticity of a preexisting regulatory circuit compromises the effectiveness of targeted therapies, and leveraging genetic vulnerabilities in cancer cells may overcome such adaptations. Hereditary leiomyomatosis renal cell carcinoma (HLRCC) is characterized by oxidative phosphorylation (OXPHOS) deficiency caused by fumarate hydratase (FH) nullizyogosity. To identify metabolic genes that are synthetically lethal with OXPHOS deficiency, we conducted a genetic loss-of-function screen and found that phosphogluconate dehydrogenase (PGD) inhibition robustly blocks the proliferation of FH mutant cancer cells both in vitro and in vivo. Mechanistically, PGD inhibition blocks glycolysis, suppresses reductive carboxylation of glutamine, and increases the NADP+/NADPH ratio to disrupt redox homeostasis. Furthermore, in the OXPHOS-proficient context, blocking OXPHOS using the small-molecule inhibitor IACS-010759 enhances sensitivity to PGD inhibition in vitro and in vivo. Together, our study reveals a dependency on PGD in OXPHOS-deficient tumors that might inform therapeutic intervention in specific patient populations.


Assuntos
Fosforilação Oxidativa , Fosfogluconato Desidrogenase/genética , Mutações Sintéticas Letais , Animais , Linhagem Celular Tumoral , Feminino , Fumarato Hidratase/genética , Genômica/métodos , Glicólise , Humanos , Mutação com Perda de Função , Camundongos , Camundongos Nus
10.
Nat Commun ; 9(1): 5079, 2018 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-30498242

RESUMO

Most triple negative breast cancers (TNBCs) are aggressively metastatic with a high degree of intra-tumoral heterogeneity (ITH), but how ITH contributes to metastasis is unclear. Here, clonal dynamics during metastasis were studied in vivo using two patient-derived xenograft (PDX) models established from the treatment-naive primary breast tumors of TNBC patients diagnosed with synchronous metastasis. Genomic sequencing and high-complexity barcode-mediated clonal tracking reveal robust alterations in clonal architecture between primary tumors and corresponding metastases. Polyclonal seeding and maintenance of heterogeneous populations of low-abundance subclones is observed in each metastasis. However, lung, liver, and brain metastases are enriched for an identical population of high-abundance subclones, demonstrating that primary tumor clones harbor properties enabling them to seed and thrive in multiple organ sites. Further, clones that dominate multi-organ metastases share a genomic lineage. Thus, intrinsic properties of rare primary tumor subclones enable the seeding and colonization of metastases in secondary organs in these models.


Assuntos
Metástase Neoplásica/genética , Neoplasias de Mama Triplo Negativas/complicações , Neoplasias de Mama Triplo Negativas/genética , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/secundário , Modelos Animais de Doenças , Feminino , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Camundongos , Camundongos SCID , Metástase Neoplásica/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Cell Stem Cell ; 23(5): 700-713.e6, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30388424

RESUMO

Clonal hematopoiesis (CH), in which stem cell clones dominate blood production, becomes increasingly common with age and can presage malignancy development. The conditions that promote ascendancy of particular clones are unclear. We found that mutations in PPM1D (protein phosphatase Mn2+/Mg2+-dependent 1D), a DNA damage response regulator that is frequently mutated in CH, were present in one-fifth of patients with therapy-related acute myeloid leukemia or myelodysplastic syndrome and strongly correlated with cisplatin exposure. Cell lines with hyperactive PPM1D mutations expand to outcompete normal cells after exposure to cytotoxic DNA damaging agents including cisplatin, and this effect was predominantly mediated by increased resistance to apoptosis. Moreover, heterozygous mutant Ppm1d hematopoietic cells outcompeted their wild-type counterparts in vivo after exposure to cisplatin and doxorubicin, but not during recovery from bone marrow transplantation. These findings establish the clinical relevance of PPM1D mutations in CH and the importance of studying mutation-treatment interactions. VIDEO ABSTRACT.


Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Células Clonais/efeitos dos fármacos , Doxorrubicina/farmacologia , Hematopoese/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Mutação , Proteína Fosfatase 2C/genética , Idoso , Animais , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Cisplatino/química , Doxorrubicina/química , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Células HEK293 , Hematopoese/genética , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Pessoa de Meia-Idade , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteína Fosfatase 2C/metabolismo
12.
NPJ Breast Cancer ; 4: 9, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29736411

RESUMO

Tumor cells disseminate early in tumor development making metastasis-prevention strategies difficult. Identifying proteins that promote the outgrowth of disseminated tumor cells may provide opportunities for novel therapeutic strategies. Despite multiple studies demonstrating that the mesenchymal-to-epithelial transition (MET) is critical for metastatic colonization, key regulators that initiate this transition remain unknown. We serially passaged lung metastases from a primary triple negative breast cancer xenograft to the mammary fat pads of recipient mice to enrich for gene expression changes that drive metastasis. An unbiased transcriptomic signature of potential metastatic drivers was generated, and a high throughput gain-of-function screen was performed in vivo to validate candidates. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) was identified as a metastatic driver. CEACAM5 overproduction enriched for an epithelial gene expression pattern and facilitated tumor outgrowth at metastatic sites. Tissues from patients with metastatic breast cancer confirmed elevated levels of CEACAM5 in lung metastases relative to breast tumors, and an inverse correlation between CEACAM5 and the mesenchymal marker vimentin was demonstrated. Thus, CEACAM5 facilitates tumor outgrowth at metastatic sites by promoting MET, warranting its investigation as a therapeutic target and biomarker of aggressiveness in breast cancer.

13.
Cell Metab ; 27(5): 977-987.e4, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29628419

RESUMO

Adoptive T cell therapy (ACT) produces durable responses in some cancer patients; however, most tumors are refractory to ACT and the molecular mechanisms underlying resistance are unclear. Using two independent approaches, we identified tumor glycolysis as a pathway associated with immune resistance in melanoma. Glycolysis-related genes were upregulated in melanoma and lung cancer patient samples poorly infiltrated by T cells. Overexpression of glycolysis-related molecules impaired T cell killing of tumor cells, whereas inhibition of glycolysis enhanced T cell-mediated antitumor immunity in vitro and in vivo. Moreover, glycolysis-related gene expression was higher in melanoma tissues from ACT-refractory patients, and tumor cells derived from these patients exhibited higher glycolytic activity. We identified reduced levels of IRF1 and CXCL10 immunostimulatory molecules in highly glycolytic melanoma cells. Our findings demonstrate that tumor glycolysis is associated with the efficacy of ACT and identify the glycolysis pathway as a candidate target for combinatorial therapeutic intervention.


Assuntos
Glicólise , Imunoterapia Adotiva , Neoplasias Pulmonares/terapia , Melanoma/terapia , Linfócitos T/transplante , Animais , Linhagem Celular Tumoral , Quimiocina CXCL10/metabolismo , Feminino , Humanos , Fator Regulador 1 de Interferon/metabolismo , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Masculino , Melanoma/imunologia , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL
14.
Clin Cancer Res ; 23(23): 7263-7275, 2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-28947567

RESUMO

Purpose: Survival for pancreatic ductal adenocarcinoma (PDAC) patients is extremely poor and improved therapies are urgently needed. Tumor-infiltrating lymphocyte (TIL) adoptive cell therapy (ACT) has shown great promise in other tumor types, such as metastatic melanoma where overall response rates of 50% have been seen. Given this success and the evidence showing that T-cell presence positively correlates with overall survival in PDAC, we sought to enrich for CD8+ TILs capable of autologous tumor recognition. In addition, we explored the phenotype and T-cell receptor repertoire of the CD8+ TILs in the tumor microenvironment.Experimental Design: We used an agonistic 4-1BB mAb during the initial tumor fragment culture to provide 4-1BB costimulation and assessed changes in TIL growth, phenotype, repertoire, and antitumor function.Results: Increased CD8+ TIL growth from PDAC tumors was achieved with the aid of an agonistic 4-1BB mAb. Expanded TILs were characterized by an activated but not terminally differentiated phenotype. Moreover, 4-1BB stimulation expanded a more clonal and distinct CD8+ TIL repertoire than IL2 alone. TILs from both culture conditions displayed MHC class I-restricted recognition of autologous tumor targets.Conclusions: Costimulation with an anti-4-1BB mAb increases the feasibility of TIL therapy by producing greater numbers of these tumor-reactive T cells. These results suggest that TIL ACT for PDAC is a potential treatment avenue worth further investigation for a patient population in dire need of improved therapy. Clin Cancer Res; 23(23); 7263-75. ©2017 AACR.


Assuntos
Imunoterapia Adotiva/métodos , Linfócitos do Interstício Tumoral/transplante , Neoplasias Pancreáticas/terapia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Modelos Lineares , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/agonistas
15.
Nature ; 542(7641): 362-366, 2017 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-28178232

RESUMO

Malignant neoplasms evolve in response to changes in oncogenic signalling. Cancer cell plasticity in response to evolutionary pressures is fundamental to tumour progression and the development of therapeutic resistance. Here we determine the molecular and cellular mechanisms of cancer cell plasticity in a conditional oncogenic Kras mouse model of pancreatic ductal adenocarcinoma (PDAC), a malignancy that displays considerable phenotypic diversity and morphological heterogeneity. In this model, stochastic extinction of oncogenic Kras signalling and emergence of Kras-independent escaper populations (cells that acquire oncogenic properties) are associated with de-differentiation and aggressive biological behaviour. Transcriptomic and functional analyses of Kras-independent escapers reveal the presence of Smarcb1-Myc-network-driven mesenchymal reprogramming and independence from MAPK signalling. A somatic mosaic model of PDAC, which allows time-restricted perturbation of cell fate, shows that depletion of Smarcb1 activates the Myc network, driving an anabolic switch that increases protein metabolism and adaptive activation of endoplasmic-reticulum-stress-induced survival pathways. Increased protein turnover renders mesenchymal sub-populations highly susceptible to pharmacological and genetic perturbation of the cellular proteostatic machinery and the IRE1-α-MKK4 arm of the endoplasmic-reticulum-stress-response pathway. Specifically, combination regimens that impair the unfolded protein responses block the emergence of aggressive mesenchymal subpopulations in mouse and patient-derived PDAC models. These molecular and biological insights inform a potential therapeutic strategy for targeting aggressive mesenchymal features of PDAC.


Assuntos
Mesoderma/patologia , Neoplasias Pancreáticas/patologia , Animais , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Estresse do Retículo Endoplasmático/genética , Feminino , Genes myc , Genes ras , Humanos , MAP Quinase Quinase 4/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Mesoderma/metabolismo , Camundongos , Mosaicismo , Proteína Oncogênica p55(v-myc)/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Proteólise , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína SMARCB1/deficiência , Proteína SMARCB1/metabolismo , Transcriptoma/genética
16.
Cancer Discov ; 6(6): 650-63, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27179036

RESUMO

UNLABELLED: The identification of genes maintaining cancer growth is critical to our understanding of tumorigenesis. We report the first in vivo genetic screen of patient-derived tumors, using metastatic melanomas and targeting 236 chromatin genes by expression of specific shRNA libraries. Our screens revealed unprecedented numerosity of genes indispensable for tumor growth (∼50% of tested genes) and unexpected functional heterogeneity among patients (<15% in common). Notably, these genes were not activated by somatic mutations in the same patients and are therefore distinguished from mutated cancer driver genes. We analyzed underlying molecular mechanisms of one of the identified genes, the Histone-lysine N-methyltransferase KMT2D, and showed that it promotes tumorigenesis by dysregulating a subset of transcriptional enhancers and target genes involved in cell migration. The assembly of enhancer genomic patterns by activated KMT2D was highly patient-specific, regardless of the identity of transcriptional targets, suggesting that KMT2D might be activated by distinct upstream signaling pathways. SIGNIFICANCE: Drug targeting of biologically relevant cancer-associated mutations is considered a critical strategy to control cancer growth. Our functional in vivo genetic screens of patient-derived tumors showed unprecedented numerosity and interpatient heterogeneity of genes that are essential for tumor growth, but not mutated, suggesting that multiple, patient-specific signaling pathways are activated in tumors. Cancer Discov; 6(6); 650-63. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 561.


Assuntos
Transformação Celular Neoplásica/genética , Estudos de Associação Genética , Testes Genéticos , Neoplasias/diagnóstico , Neoplasias/genética , Fenótipo , Animais , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Biologia Computacional/métodos , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Elementos Facilitadores Genéticos , Epigênese Genética , Epigenômica/métodos , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Xenoenxertos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Metástase Neoplásica , Proteínas de Neoplasias/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genética , Reprodutibilidade dos Testes
17.
Oncotarget ; 7(26): 39595-39608, 2016 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-27167191

RESUMO

PURPOSE: Though the efficacy of MEK inhibitors is being investigated in KRAS-mutant colorectal cancers (CRC), early clinical trials of MEK inhibitor monotherapy did not reveal significant antitumor activity. Resistance to MEK inhibitor monotherapy developed through a variety of mechanisms converging in ERK reactivation. Since ERK increases cyclin D expression and increases entry into the cell cycle, we hypothesized that the combination of MEK inhibitors and CDK4/6 inhibitors would have synergistic antitumor activity and cause tumor regression in vivo. RESULTS: The combination of MEK and CDK4/6 inhibitors synergistically inhibited cancer cell growth in vitro and caused tumor regression in vivo in cell line and patient-derived xenograft models. Combination therapy markedly decreased levels of phosphorylated ribosomal protein S6 both in vitro and in vivo and decreased Ki67 staining in vivo. EXPERIMENTAL DESIGN: We performed in vitro proliferation, colony formation, apoptosis, and senescence assays, and Western blots, on a panel of 11 KRAS mutant CRC cell lines treated with the MEK inhibitor MEK162, the CDK4/6 inhibitor palbociclib, or the combination. We also treated 4 KRAS mutant CRC cell line and patient-derived xenografts with the MEK inhibitor trametinib, the CDK4/6 inhibitor palbociclib, or the combination, and performed immunohistochemical and reverse phase protein array analysis. CONCLUSIONS: Combined inhibition of both MEK and CDK4/6 is effective in preclinical models of KRAS mutant CRC and justifies a planned phase II clinical trial in patients with refractory KRAS-mutant CRC.Efficacy of the combination of MEK and CDK4/6 inhibitors in vitro and in vivo in KRAS mutant colorectal cancer models.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , MAP Quinase Quinase Quinase 1/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células , Ensaios Clínicos como Assunto , Neoplasias Colorretais/genética , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Genes ras , Humanos , Camundongos , Camundongos Nus , Mutação , Transplante de Neoplasias , Fosforilação , Piperazinas/farmacologia , Piridinas/farmacologia , Resultado do Tratamento
18.
Cancer Discov ; 6(2): 202-16, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26645196

RESUMO

UNLABELLED: T cell-mediated immunotherapies are promising cancer treatments. However, most patients still fail to respond to these therapies. The molecular determinants of immune resistance are poorly understood. We show that loss of PTEN in tumor cells in preclinical models of melanoma inhibits T cell-mediated tumor killing and decreases T-cell trafficking into tumors. In patients, PTEN loss correlates with decreased T-cell infiltration at tumor sites, reduced likelihood of successful T-cell expansion from resected tumors, and inferior outcomes with PD-1 inhibitor therapy. PTEN loss in tumor cells increased the expression of immunosuppressive cytokines, resulting in decreased T-cell infiltration in tumors, and inhibited autophagy, which decreased T cell-mediated cell death. Treatment with a selective PI3Kß inhibitor improved the efficacy of both anti-PD-1 and anti-CTLA-4 antibodies in murine models. Together, these findings demonstrate that PTEN loss promotes immune resistance and support the rationale to explore combinations of immunotherapies and PI3K-AKT pathway inhibitors. SIGNIFICANCE: This study adds to the growing evidence that oncogenic pathways in tumors can promote resistance to the antitumor immune response. As PTEN loss and PI3K-AKT pathway activation occur in multiple tumor types, the results support the rationale to further evaluate combinatorial strategies targeting the PI3K-AKT pathway to increase the efficacy of immunotherapy.


Assuntos
Anticorpos/administração & dosagem , Melanoma/tratamento farmacológico , Melanoma/genética , PTEN Fosfo-Hidrolase/deficiência , Linfócitos T/imunologia , Aminopiridinas/administração & dosagem , Aminopiridinas/uso terapêutico , Animais , Anticorpos/uso terapêutico , Antígeno CTLA-4/imunologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Humanos , Imunoterapia/métodos , Melanoma/imunologia , Camundongos , Morfolinas/administração & dosagem , Morfolinas/uso terapêutico , Receptor de Morte Celular Programada 1/imunologia
19.
Cancer Discov ; 5(12): 1314-27, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26450788

RESUMO

UNLABELLED: Epigenetic regulators have emerged as critical factors governing the biology of cancer. Here, in the context of melanoma, we show that RNF2 is prognostic, exhibiting progression-correlated expression in human melanocytic neoplasms. Through a series of complementary gain-of-function and loss-of-function studies in mouse and human systems, we establish that RNF2 is oncogenic and prometastatic. Mechanistically, RNF2-mediated invasive behavior is dependent on its ability to monoubiquitinate H2AK119 at the promoter of LTBP2, resulting in silencing of this negative regulator of TGFß signaling. In contrast, RNF2's oncogenic activity does not require its catalytic activity nor does it derive from its canonical gene repression function. Instead, RNF2 drives proliferation through direct transcriptional upregulation of the cell-cycle regulator CCND2. We further show that MEK1-mediated phosphorylation of RNF2 promotes recruitment of activating histone modifiers UTX and p300 to a subset of poised promoters, which activates gene expression. In summary, RNF2 regulates distinct biologic processes in the genesis and progression of melanoma via different molecular mechanisms. SIGNIFICANCE: The role of epigenetic regulators in cancer progression is being increasingly appreciated. We show novel roles for RNF2 in melanoma tumorigenesis and metastasis, albeit via different mechanisms. Our findings support the notion that epigenetic regulators, such as RNF2, directly and functionally control powerful gene networks that are vital in multiple cancer processes.


Assuntos
Melanoma/genética , Melanoma/patologia , Complexo Repressor Polycomb 1/genética , Animais , Catálise , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Ciclina D2/genética , Ciclina D2/metabolismo , Progressão da Doença , Proteína p300 Associada a E1A/metabolismo , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Histona Desmetilases/metabolismo , Humanos , Proteínas de Ligação a TGF-beta Latente/genética , Proteínas de Ligação a TGF-beta Latente/metabolismo , Sistema de Sinalização das MAP Quinases , Melanoma/metabolismo , Camundongos , Metástase Neoplásica , Proteínas Nucleares/metabolismo , Oncogenes , Fosforilação , Complexo Repressor Polycomb 1/metabolismo , Prognóstico , Regiões Promotoras Genéticas , Fator de Crescimento Transformador beta/metabolismo
20.
Artigo em Inglês | MEDLINE | ID: mdl-26396593

RESUMO

BACKGROUND: Proteins that 'read' the histone code are central elements in epigenetic control and bromodomains, which bind acetyl-lysine motifs, are increasingly recognized as potential mediators of disease states. Notably, the first BET bromodomain-based therapies have entered clinical trials and there is a broad interest in dissecting the therapeutic relevance of other bromodomain-containing proteins in human disease. Typically, drug development is facilitated and expedited by high-throughput screening, where assays need to be sensitive, robust, cost-effective and scalable. However, for bromodomains, which lack catalytic activity that otherwise can be monitored (using classical enzymology), the development of cell-based, drug-target engagement assays has been challenging. Consequently, cell biochemical assays have lagged behind compared to other protein families (e.g., histone deacetylases and methyltransferases). RESULTS: Here, we present a suite of novel chromatin and histone-binding assays using AlphaLISA, in situ cell extraction and fluorescence-based, high-content imaging. First, using TRIM24 as an example, the homogenous, bead-based AlphaScreen technology was modified from a biochemical peptide-competition assay to measure binding of the TRIM24 bromodomain to endogenous histone H3 in cells (AlphaLISA). Second, a target agnostic, high-throughput imaging platform was developed to quantify the ability of chemical probes to dissociate endogenous proteins from chromatin/nuclear structures. While overall nuclear morphology is maintained, the procedure extracts soluble, non-chromatin-bound proteins from cells with drug-target displacement visualized by immunofluorescence (IF) or microscopy of fluorescent proteins. Pharmacological evaluation of these assays cross-validated their utility, sensitivity and robustness. Finally, using genetic and pharmacological approaches, we dissect domain contribution of TRIM24, BRD4, ATAD2 and SMARCA2 to chromatin binding illustrating the versatility/utility of the in situ cell extraction platform. CONCLUSIONS: In summary, we have developed two novel complementary and cell-based drug-target engagement assays, expanding the repertoire of pharmacodynamic assays for bromodomain tool compound development. These assays have been validated through a successful TRIM24 bromodomain inhibitor program, where a micromolar lead molecule (IACS-6558) was optimized using cell-based assays to yield the first single-digit nanomolar TRIM24 inhibitor (IACS-9571). Altogether, the assay platforms described herein are poised to accelerate the discovery and development of novel chemical probes to deliver on the promise of epigenetic-based therapies.

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