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1.
Clin Cancer Res ; 2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947692

RESUMO

PURPOSE: Activating mutations in PIK3CA promote resistance to HER2-targeted therapy in breast cancer, however inhibition of PI3K alone leads to escape via feedback upregulation of HER3. Combined inhibition of HER2, HER3 and PI3K overcomes this mechanism preclinically. EXPERIMENTAL DESIGN: This phase I study investigated the maximum tolerated dose (MTD) of alpelisib given in combination with trastuzumab and LJM716 (a HER3-targeted antibody) in patients with PIK3CA-mutant HER2-positive metastatic breast cancer (MBC) using the continual reassessment method. Secondary analyses included efficacy and exploratory correlative studies. RESULTS: Ten patients were treated initially with daily alpelisib (Arm A). Grade {greater than or equal to}3 adverse events seen in {greater than or equal to}2 patients included diarrhea (n=6), hypokalemia (n=3), abnormal liver enzymes (n=3), hyperglycemia (n=2), mucositis (n=2), and elevated lipase (n=2). The MTD of alpelisib in Arm A was 250mg daily. This prompted the opening of Arm B in which 11 patients received intermittently dosed alpelisib. Grade {greater than or equal to}3 adverse events seen in {greater than or equal to}2 patients included diarrhea (n=5), hypokalemia (n=3), and hypomagnesemia (n=2). The MTD of alpelisib in Arm B was 350mg given 4 days on, 3 days off. Among 17 patients assessed, 1 had a partial response, 14 had stable disease, and 2 had disease progression at best response. Five patients had stable disease for >30 weeks. mRNA profiling of pre-and on-treatment tissue demonstrated PIK3CA target engagement by alpelisib via induction of downstream signaling and feedback pathways. CONCLUSIONS: Combination treatment with alpelisib, trastuzumab and LJM716 was limited by gastrointestinal toxicity. Further efforts are warranted to target the PI3K pathway in HER2+ MBC.

2.
Nat Commun ; 12(1): 920, 2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33568647

RESUMO

Over 300 BRAF missense mutations have been identified in patients, yet currently approved drugs target V600 mutants alone. Moreover, acquired resistance inevitably emerges, primarily due to RAF lesions that prevent inhibition of BRAF V600 with current treatments. Therefore, there is a need for new therapies that target other mechanisms of activated BRAF. In this study, we use the Proteolysis Targeting Chimera (PROTAC) technology, which promotes ubiquitination and degradation of neo-substrates, to address the limitations of BRAF inhibitor-based therapies. Using vemurafenib-based PROTACs, we achieve low  nanomolar degradation of all classes of BRAF mutants, but spare degradation of WT RAF family members. Our lead PROTAC outperforms vemurafenib in inhibiting cancer cell growth and shows in vivo efficacy in a Class 2 BRAF xenograft model. Mechanistic studies reveal that BRAFWT is spared due to weak ternary complex formation in cells owing to its quiescent inactivated conformation, and activation of BRAFWT sensitizes it to degradation. This study highlights the degree of selectivity achievable with degradation-based approaches by targeting mutant BRAF-driven cancers while sparing BRAFWT, providing an anti-tumor drug modality that expands the therapeutic window.


Assuntos
Antineoplásicos/administração & dosagem , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Vemurafenib/administração & dosagem , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Mutação , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/fisiopatologia , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Ubiquitinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Clin Cancer Res ; 27(9): 2604-2612, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33593884

RESUMO

PURPOSE: KRAS G12C is the most common KRAS mutation in primary lung adenocarcinoma. Phase I clinical trials have demonstrated encouraging clinical activity of KRAS G12C inhibitors in the metastatic setting. We investigated disease-free survival (DFS) and tumor genomic features in patients with surgically resected KRAS G12C-mutant lung adenocarcinoma. EXPERIMENTAL DESIGN: Patients who underwent resection of stage I-III lung adenocarcinoma and next-generation sequencing (NGS) were evaluated. Exclusion criteria were receipt of induction therapy, incomplete resection, and low-quality NGS. Mutations were classified as KRAS wild-type (KRAS wt), G12C (KRAS G12C), or non-G12C (KRAS other). DFS was compared between groups using the log-rank test; factors associated with DFS were assessed using Cox regression. Mutual exclusivity and cooccurrence, tumor clonality, and mutational signatures were assessed. RESULTS: In total, 604 patients were included: 374 KRAS wt (62%), 95 KRAS G12C (16%), and 135 KRAS other (22%). Three-year DFS was not different between KRAS-mutant and KRAS wt tumors. However, 3-year DFS was worse in patients with KRAS G12C than KRAS other tumors (log-rank P = 0.029). KRAS G12C tumors had more lymphovascular invasion (51% vs. 37%; P = 0.032) and higher tumor mutation burden [median (interquartile range), 7.0 (5.3-10.8) vs. 6.1 (3.5-9.7); P = 0.021], compared with KRAS other tumors. KRAS G12C mutation was independently associated with worse DFS on multivariable analysis. Our DFS findings were externally validated in an independent The Cancer Genome Atlas cohort. CONCLUSIONS: KRAS G12C mutations are associated with worse DFS after complete resection of stage I-III lung adenocarcinoma. These tumors harbor more aggressive clinicopathologic and genomic features than other KRAS-mutant tumors. We identified a high-risk group for whom KRAS G12C inhibitors may be investigated to improve survival.

4.
Mol Cell ; 81(4): 708-723.e5, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33606974

RESUMO

The PI3K pathway regulates cell metabolism, proliferation, and migration, and its dysregulation is common in cancer. We now show that both physiologic and oncogenic activation of PI3K signaling increase the expression of its negative regulator PTEN. This limits the duration of the signal and output of the pathway. Physiologic and pharmacologic inhibition of the pathway reduces PTEN and contributes to the rebound in pathway activity in tumors treated with PI3K inhibitors and limits their efficacy. Regulation of PTEN is due to mTOR/4E-BP1-dependent control of its translation and is lost when 4E-BP1 is deleted. Translational regulation of PTEN is therefore a major homeostatic regulator of physiologic PI3K signaling and plays a role in reducing the pathway activation by oncogenic PIK3CA mutants and the antitumor activity of PI3K pathway inhibitors. However, pathway output is hyperactivated in tumor cells with coexistent PI3K mutation and loss of PTEN function.


Assuntos
Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Homeostase , Neoplasias/enzimologia , PTEN Fosfo-Hidrolase/biossíntese , Biossíntese de Proteínas , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células CHO , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/genética , Cricetulus , Humanos , Mutação , Neoplasias/genética , PTEN Fosfo-Hidrolase/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
5.
Clin Cancer Res ; 27(8): 2226-2235, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33509808

RESUMO

PURPOSE: Cutaneous and unknown primary melanomas frequently harbor alterations that activate the MAPK pathway. Whether MAPK driver detection beyond BRAF V600 is clinically relevant in the checkpoint inhibitor era is unknown. EXPERIMENTAL DESIGN: Patients with melanoma were prospectively offered tumor sequencing of 341-468 genes. Oncogenic alterations in 28 RTK-RAS-MAPK pathway genes were used to construct MAPK driver groups. Time to treatment failure (TTF) was determined for patients who received first-line programmed cell death protein 1 (PD-1) monotherapy, nivolumab plus ipilimumab, or subsequent genomically matched targeted therapies. A Cox proportional hazards model was constructed for TTF using driver group and clinical variables. RESULTS: A total of 670 of 696 sequenced melanomas (96%) harbored an oncogenic RTK-RAS-MAPK pathway alteration; 33% had ≥1 driver. Nine driver groups varied by clinical presentation and mutational burden. TTF of PD-1 monotherapy (N = 181) varied by driver, with worse outcomes for NRAS Q61 and BRAF V600 versus NF1 or other alterations (median 4.2, 7.5, 22, and not reached; P < 0.0001). Driver group remained significant, independent of tumor mutational burden and clinical features. TTF did not vary by driver for nivolumab plus ipilimumab (N = 141). Among 172 patients with BRAF V600 wild-type melanoma who progressed on checkpoint blockade, 27 were treated with genomically matched therapy, and eight (30%) derived clinical benefit lasting ≥6 months. CONCLUSIONS: Targeted capture multigene sequencing can detect oncogenic RTK-RAS-MAPK pathway alterations in almost all cutaneous and unknown primary melanomas. TTF of PD-1 monotherapy varies by mechanism of ERK activation. Oncogenic kinase fusions can be successfully targeted in immune checkpoint inhibitor-refractory melanoma.

6.
Cancer Res ; 80(19): 4233-4243, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32641410

RESUMO

Despite significant advances in cancer precision medicine, a significant hurdle to its broader adoption remains the multitude of variants of unknown significance identified by clinical tumor sequencing and the lack of biologically validated methods to distinguish between functional and benign variants. Here we used functional data on MAP2K1 and MAP2K2 mutations generated in real-time within a co-clinical trial framework to benchmark the predictive value of a three-part in silico methodology. Our computational approach to variant classification incorporated hotspot analysis, three-dimensional molecular dynamics simulation, and sequence paralogy. In silico prediction accurately distinguished functional from benign MAP2K1 and MAP2K2 mutants, yet drug sensitivity varied widely among activating mutant alleles. These results suggest that multifaceted in silico modeling can inform patient accrual to MEK/ERK inhibitor clinical trials, but computational methods need to be paired with laboratory- and clinic-based efforts designed to unravel variabilities in drug response. SIGNIFICANCE: Leveraging prospective functional characterization of MEK1/2 mutants, it was found that hotspot analysis, molecular dynamics simulation, and sequence paralogy are complementary tools that can robustly prioritize variants for biologic, therapeutic, and clinical validation.See related commentary by Whitehead and Sebolt-Leopold, p. 4042.

7.
Cancer Discov ; 10(8): 1129-1139, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32430388

RESUMO

Most patients with KRAS G12C-mutant non-small cell lung cancer (NSCLC) experience clinical benefit from selective KRASG12C inhibition, whereas patients with colorectal cancer bearing the same mutation rarely respond. To investigate the cause of the limited efficacy of KRASG12C inhibitors in colorectal cancer, we examined the effects of AMG510 in KRAS G12C colorectal cancer cell lines. Unlike NSCLC cell lines, KRAS G12C colorectal cancer models have high basal receptor tyrosine kinase (RTK) activation and are responsive to growth factor stimulation. In colorectal cancer lines, KRASG12C inhibition induces higher phospho-ERK rebound than in NSCLC cells. Although upstream activation of several RTKs interferes with KRASG12C blockade, we identify EGFR signaling as the dominant mechanism of colorectal cancer resistance to KRASG12C inhibitors. The combinatorial targeting of EGFR and KRASG12C is highly effective in colorectal cancer cells and patient-derived organoids and xenografts, suggesting a novel therapeutic strategy to treat patients with KRAS G12C colorectal cancer. SIGNIFICANCE: The efficacy of KRASG12C inhibitors in NSCLC and colorectal cancer is lineage-specific. RTK dependency and signaling rebound kinetics are responsible for sensitivity or resistance to KRASG12C inhibition in colorectal cancer. EGFR and KRASG12C should be concomitantly inhibited to overcome resistance to KRASG12C blockade in colorectal tumors.See related commentary by Koleilat and Kwong, p. 1094.This article is highlighted in the In This Issue feature, p. 1079.

8.
Mol Oncol ; 14(8): 1833-1849, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32336014

RESUMO

The mutation of K-RAS represents one of the most frequent genetic alterations in cancer. Targeting of downstream effectors of RAS, including of MEK and ERK, has limited clinical success in cancer patients with K-RAS mutations. The reduced sensitivity of K-RAS-mutated cells to certain MEK inhibitors (MEKi) is associated with the feedback phosphorylation of MEK by C-RAF and with the reactivation of mitogen-activated protein kinase (MAPK) signaling. Here, we report that the RAF dimer inhibitors lifirafenib (BGB-283) and compound C show a strong synergistic effect with MEKi, including mirdametinib (PD-0325901) and selumetinib, in suppressing the proliferation of K-RAS-mutated non-small-cell lung cancer and colorectal cancer (CRC) cell lines. This synergistic effect was not observed with the B-RAFV600E selective inhibitor vemurafenib. Our mechanistic analysis revealed that RAF dimer inhibition suppresses RAF-dependent MEK reactivation and leads to the sustained inhibition of MAPK signaling in K-RAS-mutated cells. This synergistic effect was also observed in several K-RAS mutant mouse xenograft models. A pharmacodynamic analysis supported a role for the synergistic phospho-ERK blockade in enhancing the antitumor activity observed in the K-RAS mutant models. These findings support a vertical inhibition strategy in which RAF dimer and MEKi are combined to target K-RAS-mutated cancers, and have led to a Phase 1b/2 combination therapy study of lifirafenib and mirdametinib in solid tumor patients with K-RAS mutations and other MAPK pathway aberrations.

9.
Clin Cancer Res ; 26(12): 2932-2945, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32122926

RESUMO

PURPOSE: ROS1 tyrosine kinase inhibitors (TKI) provide significant benefit in lung adenocarcinoma patients with ROS1 fusions. However, as observed with all targeted therapies, resistance arises. Detecting mechanisms of acquired resistance (AR) is crucial to finding novel therapies and improve patient outcomes. EXPERIMENTAL DESIGN: ROS1 fusions were expressed in HBEC and NIH-3T3 cells either by cDNA overexpression (CD74/ROS1, SLC34A2/ROS1) or CRISPR-Cas9-mediated genomic engineering (EZR/ROS1). We reviewed targeted large-panel sequencing data (using the MSK-IMPACT assay) patients treated with ROS1 TKIs, and genetic alterations hypothesized to confer AR were modeled in these cell lines. RESULTS: Eight of the 75 patients with a ROS1 fusion had a concurrent MAPK pathway alteration and this correlated with shorter overall survival. In addition, the induction of ROS1 fusions stimulated activation of MEK/ERK signaling with minimal effects on AKT signaling, suggesting the importance of the MAPK pathway in driving ROS1 fusion-positive cancers. Of 8 patients, 2 patients harbored novel in-frame deletions in MEK1 (MEK1delE41_L54) and MEKK1 (MEKK1delH907_C916) that were acquired after ROS1 TKIs, and 2 patients harbored NF1 loss-of-function mutations. Expression of MEK1del or MEKK1del, and knockdown of NF1 in ROS1 fusion-positive cells activated MEK/ERK signaling and conferred resistance to ROS1 TKIs. Combined targeting of ROS1 and MEK inhibited growth of cells expressing both ROS1 fusion and MEK1del. CONCLUSIONS: We demonstrate that downstream activation of the MAPK pathway can mediate of innate acquired resistance to ROS1 TKIs and that patients harboring ROS1 fusion and concurrent downstream MAPK pathway alterations have worse survival. Our findings suggest a treatment strategy to target both aberrations.

10.
Cancer Discov ; 10(5): 674-687, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32213539

RESUMO

Amplification of and oncogenic mutations in ERBB2, the gene encoding the HER2 receptor tyrosine kinase, promote receptor hyperactivation and tumor growth. Here we demonstrate that HER2 ubiquitination and internalization, rather than its overexpression, are key mechanisms underlying endocytosis and consequent efficacy of the anti-HER2 antibody-drug conjugates (ADC) ado-trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd) in lung cancer cell lines and patient-derived xenograft models. These data translated into a 51% response rate in a clinical trial of T-DM1 in 49 patients with ERBB2-amplified or -mutant lung cancers. We show that cotreatment with irreversible pan-HER inhibitors enhances receptor ubiquitination and consequent ADC internalization and efficacy. We also demonstrate that ADC switching to T-DXd, which harbors a different cytotoxic payload, achieves durable responses in a patient with lung cancer and corresponding xenograft model developing resistance to T-DM1. Our findings may help guide future clinical trials and expand the field of ADC as cancer therapy. SIGNIFICANCE: T-DM1 is clinically effective in lung cancers with amplification of or mutations in ERBB2. This activity is enhanced by cotreatment with irreversible pan-HER inhibitors, or ADC switching to T-DXd. These results may help address unmet needs of patients with HER2-activated tumors and no approved targeted therapy.See related commentary by Rolfo and Russo, p. 643.This article is highlighted in the In This Issue feature, p. 627.

11.
Cancer Res ; 80(7): 1428-1437, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32015092

RESUMO

Genomic rearrangements leading to the aberrant expression of ERG are the most common early events in prostate cancer and are significantly enriched for the concomitant loss of PTEN. Genetically engineered mouse models reveal that ERG overexpression alone is not sufficient to induce tumorigenesis, but combined loss of PTEN results in an aggressive invasive phenotype. Here, we show that oncogenic ERG repressed PI3K signaling through direct transcriptional suppression of IRS2, leading to reduced RTK levels and activity. In accordance with this finding, ERG-positive human prostate cancers had a repressed AKT gene signature and transcriptional downregulation of IRS2. Although overexpression of IRS2 activated PI3K signaling, promoting cell migration in a PI3K-dependent manner, this did not fully recapitulate the phenotype seen with loss of PTEN as PI3K signaling is not as robust as observed in the setting of loss of PTEN. Importantly, deletions of the PTEN locus, which promotes active PI3K signaling, were among the most significant copy-number alterations that co-occurred with ERG genomic rearrangements. This work provides insight on how initiating oncogenic events may directly influence the selection of secondary concomitant alterations to promote oncogenic signaling during tumor evolution. SIGNIFICANCE: This work provides insight on how initiating oncogenic events may directly influence the selection of secondary concomitant alterations to promote tumorigenesis.


Assuntos
Proteínas Substratos do Receptor de Insulina/genética , Proteínas Oncogênicas/metabolismo , Neoplasias da Próstata/genética , Regulador Transcricional ERG/metabolismo , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Modelos Animais de Doenças , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Rearranjo Gênico , Humanos , Proteínas Substratos do Receptor de Insulina/metabolismo , Masculino , Camundongos , Proteínas Oncogênicas/genética , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Cultura Primária de Células , Regiões Promotoras Genéticas/genética , Próstata/patologia , Neoplasias da Próstata/patologia , RNA-Seq , Transdução de Sinais/genética , Regulador Transcricional ERG/genética , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Am J Med Genet A ; 182(3): 597-606, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31825160

RESUMO

The RASopathies are a group of genetic disorders that result from germline pathogenic variants affecting RAS-mitogen activated protein kinase (MAPK) pathway genes. RASopathies share RAS/MAPK pathway dysregulation and share phenotypic manifestations affecting numerous organ systems, causing lifelong and at times life-limiting medical complications. RASopathies may benefit from precision medicine approaches. For this reason, the Sixth International RASopathies Symposium focused on exploring precision medicine. This meeting brought together basic science researchers, clinicians, clinician scientists, patient advocates, and representatives from pharmaceutical companies and the National Institutes of Health. Novel RASopathy genes, variants, and animal models were discussed in the context of medication trials and drug development. Attempts to define and measure meaningful endpoints for treatment trials were discussed, as was drug availability to patients after trial completion.

13.
Clin Cancer Res ; 25(23): 7089-7097, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31515458

RESUMO

PURPOSE: While mutations in BRAF in metastatic colorectal cancer (mCRC) most commonly occur at the V600 amino acid, with the advent of next-generation sequencing, non-V600 BRAF mutations are increasingly identified in clinical practice. It is unclear whether these mutants, like BRAF V600E, confer resistance to anti-EGFR therapy. EXPERIMENTAL DESIGN: We conducted a multicenter pooled analysis of consecutive patients with non-V600 BRAF-mutated mCRCs identified between 2010 and 2017. Non-V600 BRAF mutations were divided into functional classes based on signaling mechanism and kinase activity: activating and RAS-independent (class 2) or kinase-impaired and RAS-dependent (class 3). RESULTS: Forty patients with oncogenic non-V600 BRAF-mutant mCRC received anti-EGFR antibody treatment [n = 12 (30%) class 2 and n = 28 (70%) class 3]. No significant differences in clinical characteristics were observed by mutation class. In contrast, while only 1 of 12 patients with class 2 BRAF mCRC responded, 14 of 28 patients with class 3 BRAF responded to anti-EGFR therapy (response rate, 8% and 50%, respectively, P = 0.02). Specifically, in first- or second-line, 1 of 6 (17%) patients with class 2 and 7 of 9 (78%) patients with class 3 BRAF mutants responded (P = 0.04). In third- or later-line, none of 6 patients with class 2 and 7 of 19 (37%) patients with class 3 BRAF mutants responded (P = 0.14). CONCLUSIONS: Response to EGFR antibody treatment in mCRCs with class 2 BRAF mutants is rare, while a large portion of CRCs with class 3 BRAF mutants respond. Patients with colorectal cancer with class 3 BRAF mutations should be considered for anti-EGFR antibody treatment.See related commentary by Fontana and Valeri, p. 6896.


Assuntos
Cetuximab/uso terapêutico , Neoplasias Colorretais/patologia , Terapia de Alvo Molecular , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Receptores ErbB/antagonistas & inibidores , Feminino , Humanos , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Prognóstico , Estudos Prospectivos , Inibidores de Proteínas Quinases/uso terapêutico , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Sci Rep ; 9(1): 10865, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31350469

RESUMO

Small molecule inhibitors of BRAF and MEK have proven effective at inhibiting tumor growth in melanoma patients, however this efficacy is limited due to the almost universal development of drug resistance. To provide advanced insight into the signaling responses that occur following kinase inhibition we have performed quantitative (phospho)-proteomics of human melanoma cells treated with either dabrafenib, a BRAF inhibitor; trametinib, a MEK inhibitor or SCH772984, an ERK inhibitor. Over nine experiments we identified 7827 class I phosphorylation sites on 4960 proteins. This included 54 phosphorylation sites that were significantly down-modulated after exposure to all three inhibitors, 34 of which have not been previously reported. Functional analysis of these novel ERK targets identified roles for them in GTPase activity and regulation, apoptosis and cell-cell adhesion. Comparison of the results presented here with previously reported phosphorylation sites downstream of ERK showed a limited degree of overlap suggesting that ERK signaling responses may be highly cell line and cue specific. In addition we identified 26 phosphorylation sites that were only responsive to dabrafenib. We provide further orthogonal experimental evidence for 3 of these sites in human embryonic kidney cells over-expressing BRAF as well as further computational insights using KinomeXplorer. The validated phosphorylation sites were found to be involved in actin regulation, which has been proposed as a novel mechanism for inhibiting resistance development. These results would suggest that the linearity of the BRAF-MEK-ERK module is at least context dependent.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Imidazóis/farmacologia , Indazóis/farmacologia , Melanoma/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Oximas/farmacologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Piridonas/farmacologia , Pirimidinonas/farmacologia , Neoplasias Cutâneas/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Proteoma , Proteômica/métodos , Proteínas Proto-Oncogênicas B-raf/metabolismo , Neoplasias Cutâneas/patologia
15.
Cancer Cell ; 36(2): 139-155.e10, 2019 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-31327655

RESUMO

The mechanisms that enable immune evasion at metastatic sites are poorly understood. We show that the Polycomb Repressor Complex 1 (PRC1) drives colonization of the bones and visceral organs in double-negative prostate cancer (DNPC). In vivo genetic screening identifies CCL2 as the top prometastatic gene induced by PRC1. CCL2 governs self-renewal and induces the recruitment of M2-like tumor-associated macrophages and regulatory T cells, thus coordinating metastasis initiation with immune suppression and neoangiogenesis. A catalytic inhibitor of PRC1 cooperates with immune checkpoint therapy to reverse these processes and suppress metastasis in genetically engineered mouse transplantation models of DNPC. These results reveal that PRC1 coordinates stemness with immune evasion and neoangiogenesis and point to the potential clinical utility of targeting PRC1 in DNPC.


Assuntos
Adenocarcinoma/metabolismo , Movimento Celular , Autorrenovação Celular , Quimiocina CCL2/metabolismo , Células-Tronco Neoplásicas/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Neoplasias da Próstata/metabolismo , Evasão Tumoral , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/imunologia , Adenocarcinoma/secundário , Animais , Antineoplásicos Imunológicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Movimento Celular/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Quimiocina CCL2/genética , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos Nus , Camundongos SCID , Metástase Neoplásica , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/patologia , Células PC-3 , Complexo Repressor Polycomb 1/antagonistas & inibidores , Complexo Repressor Polycomb 1/genética , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Receptores Androgênicos/deficiência , Receptores Androgênicos/genética , Receptores CCR4/genética , Receptores CCR4/metabolismo , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Evasão Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Cancer Discov ; 9(9): 1182-1191, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31227518

RESUMO

We report the emergence of the novel MEK1 V211D gatekeeper mutation in a patient with BRAF K601E colon cancer treated with the allosteric MEK inhibitor binimetinib and the anti-EGFR antibody panitumumab. The MEK1 V211D mutation concurrently occurs in the same cell with BRAF K601E and leads to RAF-independent activity but remains regulated by RAF. The V211D mutation causes resistance to binimetinib by both increasing the catalytic activity of MEK1 and reducing its affinity for the drug. Moreover, the mutant exhibits reduced sensitivity to all the allosteric MEK inhibitors tested. Thus, this mutation serves as a general resistance mutation for current MEK inhibitors; however, it is sensitive to a newly reported ATP-competitive MEK inhibitor, which therefore could be used to overcome drug resistance. SIGNIFICANCE: We report a resistance mechanism to allosteric MEK inhibitors in the clinic. A MEK1 V211D mutation developed in a patient with BRAF K601E colon cancer on MEK and EGFR inhibitors. This mutant increases the catalytic activity of MEK1 and reduces its affinity for binimetinib, but remains sensitive to ATP-competitive MEK inhibitors.This article is highlighted in the In This Issue feature, p. 1143.


Assuntos
Substituição de Aminoácidos , Benzimidazóis/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , MAP Quinase Quinase 1/genética , Inibidores de Proteínas Quinases/uso terapêutico , Adulto , Regulação Alostérica , Animais , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Feminino , Humanos , MAP Quinase Quinase 1/química , Camundongos , Células NIH 3T3 , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas B-raf/genética
17.
J Thorac Oncol ; 14(5): 802-815, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30831205

RESUMO

INTRODUCTION: Multiple genetic mechanisms have been identified in EGFR-mutant lung cancers as mediators of acquired resistance (AR) to EGFR tyrosine kinase inhibitors (TKIs), but many cases still lack a known mechanism. METHODS: To identify novel mechanisms of AR, we performed targeted large panel sequencing of samples from 374 consecutive patients with metastatic EGFR-mutant lung cancer, including 174 post-TKI samples, of which 38 also had a matched pre-TKI sample. Alterations hypothesized to confer AR were introduced into drug-sensitive EGFR-mutant lung cancer cell lines (H1975, HCC827, and PC9) by using clustered regularly interspaced short palindromic repeats/Cas9 genome editing. MSK-LX138cl, a cell line with EGFR exon 19 deletion (ex19del) and praja ring finger ubiquitin ligase 2 gene (PJA2)/BRAF fusion, was generated from an EGFR TKI-resistant patient sample. RESULTS: We identified four patients (2.3%) with a BRAF fusion (three with acylglycerol kinase gene (AGK)/BRAF and one with PJA2/BRAF) in samples obtained at AR to EGFR TKI therapy (two posterlotinib samples and two posterlotinib and postosimertinib samples). Pre-TKI samples were available for two of four patients and both were negative for BRAF fusion. Induction of AGK/BRAF fusion in H1975 (L858R + T790M), PC9 (ex19del) and HCC827 (ex19del) cells increased phosphorylation of BRAF, MEK1/2, ERK1/2, and signal transducer and activator of transcription 3 and conferred resistance to growth inhibition by osimertinib. MEK inhibition with trametinib synergized with osimertinib to block growth. Alternately, a pan-RAF inhibitor as a single agent blocked growth of all cell lines with mutant EGFR and BRAF fusion. CONCLUSION: BRAF fusion is a mechanism of AR to EGFR TKI therapy in approximately 2% of patients. Combined inhibition of EGFR and MEK (with osimertinib and trametinib) or BRAF (with a pan-RAF inhibitor) are potential therapeutic strategies that should be explored.


Assuntos
Adenocarcinoma de Pulmão/genética , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética , Idoso , Receptores ErbB/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação
18.
Nature ; 567(7749): 521-524, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30867592

RESUMO

Histiocytic neoplasms are a heterogeneous group of clonal haematopoietic disorders that are marked by diverse mutations in the mitogen-activated protein kinase (MAPK) pathway1,2. For the 50% of patients with histiocytosis who have BRAFV600 mutations3-5, RAF inhibition is highly efficacious and has markedly altered the natural history of the disease6,7. However, no standard therapy exists for the remaining 50% of patients who lack BRAFV600 mutations. Although ERK dependence has been hypothesized to be a consistent feature across histiocytic neoplasms, this remains clinically unproven and many of the kinase mutations that are found in patients who lack BRAFV600 mutations have not previously been biologically characterized. Here we show ERK dependency in histiocytoses through a proof-of-concept clinical trial of cobimetinib, an oral inhibitor of MEK1 and MEK2, in patients with histiocytoses. Patients were enrolled regardless of their tumour genotype. In parallel, MAPK alterations that were identified in treated patients were characterized for their ability to activate ERK. In the 18 patients that we treated, the overall response rate was 89% (90% confidence interval of 73-100). Responses were durable, with no acquired resistance to date. At one year, 100% of responses were ongoing and 94% of patients remained progression-free. Cobimetinib treatment was efficacious regardless of genotype, and responses were observed in patients with ARAF, BRAF, RAF1, NRAS, KRAS, MEK1 (also known as MAP2K1) and MEK2 (also known as MAP2K2) mutations. Consistent with the observed responses, the characterization of the mutations that we identified in these patients confirmed that the MAPK-pathway mutations were activating. Collectively, these data demonstrate that histiocytic neoplasms are characterized by a notable dependence on MAPK signalling-and that they are consequently responsive to MEK inhibition. These results extend the benefits of molecularly targeted therapy to the entire spectrum of patients with histiocytosis.


Assuntos
Azetidinas/uso terapêutico , Transtornos Histiocíticos Malignos/tratamento farmacológico , Transtornos Histiocíticos Malignos/enzimologia , Histiocitose/tratamento farmacológico , Histiocitose/enzimologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Piperidinas/uso terapêutico , Azetidinas/farmacologia , Transtornos Histiocíticos Malignos/genética , Transtornos Histiocíticos Malignos/patologia , Histiocitose/genética , Histiocitose/patologia , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mutação , Piperidinas/farmacologia , Intervalo Livre de Progressão , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-raf/genética
19.
Nat Cell Biol ; 21(4): 534, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30842593

RESUMO

In the version of this Article originally published the same blot was inadvertently presented as both p-Rb and Cyclin A in Fig. 2a. This blot corresponds to the p-Rb panel, as can be seen in the unprocessed version of these blots in Supplementary Fig. 9. The corrected version of the panel is shown below, together with a completely uncropped image of both blots. In addition, in the 'Viral transduction' section of the Methods, the pLKO.1 plasmids encoding short hairpin RNAs targeting human Rnd1 were incorrectly listed as clones TRCN0000018338 and TRCN0000039977. The correct clone numbers are TRCN0000047434 and TRCN0000047435.

20.
Nat Med ; 25(2): 284-291, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30559419

RESUMO

Activating BRAF mutants and fusions signal as RAS-independent constitutively active dimers with the exception of BRAF V600 mutant alleles which can function as active monomers1. Current RAF inhibitors are monomer selective, they potently inhibit BRAF V600 monomers but their inhibition of RAF dimers is limited by induction of negative cooperativity when bound to one site in the dimer1-3. Moreover, acquired resistance to these drugs is usually due to molecular lesions that cause V600 mutants to dimerize4-8. We show here that PLX8394, a new RAF inhibitor9, inhibits ERK signaling by specifically disrupting BRAF-containing dimers, including BRAF homodimers and BRAF-CRAF heterodimers, but not CRAF homodimers or ARAF-containing dimers. Differences in the amino acid residues in the amino (N)-terminal portion of the kinase domain of RAF isoforms are responsible for this differential vulnerability. As a BRAF-specific dimer breaker, PLX8394 selectively inhibits ERK signaling in tumors driven by dimeric BRAF mutants, including BRAF fusions and splice variants as well as BRAF V600 monomers, but spares RAF function in normal cells in which CRAF homodimers can drive signaling. Our work suggests that drugs with these properties will be safe and useful for treating tumors driven by activating BRAF mutants or fusions.


Assuntos
Compostos Heterocíclicos com 2 Anéis/farmacologia , Mutação/genética , Multimerização Proteica , Proteínas Proto-Oncogênicas B-raf/genética , Transdução de Sinais , Sulfonamidas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas B-raf/metabolismo
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