RESUMO
The common participation of oncogenic KRAS proteins in many of the most lethal human cancers, together with the ease of detecting somatic KRAS mutant alleles in patient samples, has spurred persistent and intensive efforts to develop drugs that inhibit KRAS activity. However, advances have been hindered by the pervasive inter- and intra-lineage diversity in the targetable mechanisms that underlie KRAS-driven cancers, limited pharmacological accessibility of many candidate synthetic-lethal interactions and the swift emergence of unanticipated resistance mechanisms to otherwise effective targeted therapies. Here we demonstrate the acute and specific cell-autonomous addiction of KRAS-mutant non-small-cell lung cancer cells to receptor-dependent nuclear export. A multi-genomic, data-driven approach, utilizing 106 human non-small-cell lung cancer cell lines, was used to interrogate 4,725 biological processes with 39,760 short interfering RNA pools for those selectively required for the survival of KRAS-mutant cells that harbour a broad spectrum of phenotypic variation. Nuclear transport machinery was the sole process-level discriminator of statistical significance. Chemical perturbation of the nuclear export receptor XPO1 (also known as CRM1), with a clinically available drug, revealed a robust synthetic-lethal interaction with native or engineered oncogenic KRAS both in vitro and in vivo. The primary mechanism underpinning XPO1 inhibitor sensitivity was intolerance to the accumulation of nuclear IκBα (also known as NFKBIA), with consequent inhibition of NFκB transcription factor activity. Intrinsic resistance associated with concurrent FSTL5 mutations was detected and determined to be a consequence of YAP1 activation via a previously unappreciated FSTL5-Hippo pathway regulatory axis. This occurs in approximately 17% of KRAS-mutant lung cancers, and can be overcome with the co-administration of a YAP1-TEAD inhibitor. These findings indicate that clinically available XPO1 inhibitors are a promising therapeutic strategy for a considerable cohort of patients with lung cancer when coupled to genomics-guided patient selection and observation.
Assuntos
Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Carioferinas/antagonistas & inibidores , Carioferinas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Feminino , Proteínas Relacionadas à Folistatina/genética , Genes Letais/genética , Via de Sinalização Hippo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Mutação , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/metabolismo , Porfirinas/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Verteporfina , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas de Sinalização YAP , Proteína Exportina 1RESUMO
Hybrid oncocytic/chromophobe tumor (HOCT) of the kidney represents a poorly understood clinicopathologic entity with pathologic features that overlap between benign renal oncocytoma (RO) and malignant chromophobe renal cell carcinoma (ChRCC). Consequently, characterization of HOCT and its separation from the foregoing entities are clinically important. The aim of this study was to describe the pathologic and molecular features of HOCT and to compare them with those of RO and ChRCC. We retrospectively identified a cohort of 73 cases with renal oncocytic tumors (19 RO, 27 HOCT, and 27 ChRCC) for whom clinical follow-up data were available by 2 tertiary care hospitals. All cases were sporadic except for 2 HOCTs that were associated with Birt-Hogg-Dubé syndrome. Lesional tissues were retrieved for molecular analysis. We performed targeted gene sequencing of all exons of 261 cancer related genes on a subset of HOCT samples (n = 16). Gene expression profiling of a customized codeset was conducted on 19 RO, 24 HOCT, and 27 ChRCC samples. Clinicopathologic characteristics as well as DNA copy number alterations, mutational and transcriptional features of HOCT derived from sequencing and expression profiling data are described and compared to those in RO and ChRCC. HOCTs were more frequently multifocal and did not exhibit mutations in genes that are recurrently mutated in RO or ChRCC but showed copy number alterations primarily involving losses in chromosomes 1 and X/Y. The mRNA transcript data show that HOCT can be separated from RO and ChRCC. Hence, HOCT appears to represent a distinct renal tumor entity with genomic features that are intermediate between those of RO and ChRCC.
Assuntos
Adenoma Oxífilo/genética , Adenoma Oxífilo/patologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Neoplasias Renais/genética , Neoplasias Renais/patologia , Idoso , Biomarcadores Tumorais/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , TranscriptomaRESUMO
BACKGROUND: Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL) yielded clinical benefit in patients with checkpoint blockade immunotherapy-refractory non-small cell lung cancer (NSCLC) prompting a renewed interest in TIL-ACT. This preclinical study explores the feasibility of producing a NSCLC TIL product with sufficient numbers and enhanced attributes using an improved culture method. METHODS: TIL from resected NSCLC tumors were initially cultured using (1) the traditional method using interleukin (IL)-2 alone in 24-well plates (TIL 1.0) or (2) IL-2 in combination with agonistic antibodies against CD3 and 4-1BB (Urelumab) in a G-Rex flask (TIL 3.0). TIL subsequently underwent a rapid expansion protocol (REP) with anti-CD3. Before and after the REP, expanded TIL were phenotyped and the complementarity-determining region 3 ß variable region of the T-cell receptor (TCR) was sequenced to assess the T-cell repertoire. RESULTS: TIL 3.0 robustly expanded NSCLC TIL while enriching for CD8+ TIL in a shorter manufacturing time when compared with the traditional TIL 1.0 method, achieving a higher success rate and producing 5.3-fold more TIL per successful expansion. The higher proliferative capacity and CD8 content of TIL 3.0 was also observed after the REP. Both steps of expansion did not terminally differentiate/exhaust the TIL but a lesser differentiated population was observed after the first step. TIL initially expanded with the 3.0 method exhibited higher breadth of clonotypes than TIL 1.0 corresponding to a higher repertoire homology with the original tumor, including a higher proportion of the top 10 most prevalent clones from the tumor. TIL 3.0 also retained a higher proportion of putative tumor-specific TCR when compared with TIL 1.0. Numerical expansion of TIL in a REP was found to perturb the clonal hierarchy and lessen the proportion of putative tumor-specific TIL from the TIL 3.0 process. CONCLUSIONS: We report the feasibility of robustly expanding a T-cell repertoire recapitulating the clonal hierarchy of the T cells in the NSCLC tumor, including a large number of putative tumor-specific TIL clones, using the TIL 3.0 methodology. If scaled up and employed as a sole expansion platform, the robustness and speed of TIL 3.0 may facilitate the testing of TIL-ACT approaches in NSCLC.
Assuntos
Complexo CD3/imunologia , Carcinoma Pulmonar de Células não Pequenas/genética , Interleucina-2/metabolismo , Neoplasias Pulmonares/genética , Linfócitos do Interstício Tumoral/imunologia , Pesquisa Translacional Biomédica/métodos , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Multiple synchronous lung tumors (MSLT), particularly within a single lobe, represent a diagnostic and treatment challenge. While histologic assessment was once the only method to possibly distinguish multiple primary lung cancers, there is a growing interest in identifying unique genomic features or mutations to best characterize these processes. METHODS: In order to differentiate multiple primary lung malignancies from intrapulmonary metastases in patients with MSLT, we performed whole exome sequencing (WES) on 10 tumor samples from 4 patients with MSLT. RESULTS: Shared mutations between tumors from the same patient varied from 0-91%. Patient 3 shared no common mutations; however, in Patients 2 and 4, identical mutations were identified among all tumors from each patient, suggesting that the three tumors identified in Patient 3 represent separate primary lung cancers, while those of Patients 1, 2 and 4 signify hematogenous and lymphatic spread. CONCLUSIONS: A high proportion of shared mutations between different lung tumors is likely indicative of intrapulmonary metastatic disease, while tumors with distinct genomic profiles likely represent multiple primary malignancies driven by distinct molecular events. Application of genomic profiling in the clinical setting may prove to be important to precise management of patients with MSLT.
RESUMO
BACKGROUND: Genome-wide association studies are widely used to map genomic regions contributing to lung cancer (LC) susceptibility, but they typically do not identify the precise disease-causing genes/variants. To unveil the inherited genetic variants that cause LC, we performed focused exome-sequencing analyses on genes located in 121 genome-wide association study-identified loci previously implicated in the risk of LC, chronic obstructive pulmonary disease, pulmonary function level, and smoking behavior. METHODS: Germline DNA from 260 case patients with LC and 318 controls were sequenced by utilizing VCRome 2.1 exome capture. Filtering was based on enrichment of rare and potential deleterious variants in cases (risk alleles) or controls (protective alleles). Allelic association analyses of single-variant and gene-based burden tests of multiple variants were performed. Promising candidates were tested in two independent validation studies with a total of 1773 case patients and 1123 controls. RESULTS: We identified 48 rare variants with deleterious effects in the discovery analysis and validated 12 of the 43 candidates that were covered in the validation platforms. The top validated candidates included one well-established truncating variant, namely, BRCA2, DNA repair associated gene (BRCA2) K3326X (OR = 2.36, 95% confidence interval [CI]: 1.38-3.99), and three newly identified variations, namely, lymphotoxin beta gene (LTB) p.Leu87Phe (OR = 7.52, 95% CI: 1.01-16.56), prolyl 3-hydroxylase 2 gene (P3H2) p.Gln185His (OR = 5.39, 95% CI: 0.75-15.43), and dishevelled associated activator of morphogenesis 2 gene (DAAM2) p.Asp762Gly (OR = 0.25, 95% CI: 0.10-0.79). Burden tests revealed strong associations between zinc finger protein 93 gene (ZNF93), DAAM2, bromodomain containing 9 gene (BRD9), and the gene LTB and LC susceptibility. CONCLUSION: Our results extend the catalogue of regions associated with LC and highlight the importance of germline rare coding variants in LC susceptibility.