RESUMO
The current targeted therapy for BRAFV600E-mutant lung cancer consists of a dual blockade of RAF/MEK kinases often combining dabrafenib/trametinib (D/T). This regimen extends survival when compared to single-agent treatments, but disease progression is unavoidable. By using whole-genome CRISPR screening and RNA sequencing, we characterize the vulnerabilities of both persister and D/T-resistant cellular models. Oxidative stress together with concomitant induction of antioxidant responses is boosted by D/T treatment. However, the nature of the oxidative damage, the choice of redox detoxification systems, and the resulting therapeutic vulnerabilities display stage-specific differences. Persister cells suffer from lipid peroxidation and are sensitive to ferroptosis upon GPX4 inhibition in vivo. Biomarkers of lipid peroxidation are detected in clinical samples following D/T treatment. Acquired alterations leading to mitogen-activated protein kinase (MAPK) reactivation enhance cystine transport to boost GPX4-independent antioxidant responses. Similarly to BRAFV600E-mutant melanoma, histone deacetylase (HDAC) inhibitors decrease D/T-resistant cell viability and extend therapeutic response in vivo.
Assuntos
Adenocarcinoma de Pulmão , Resistencia a Medicamentos Antineoplásicos , Inibidores de Histona Desacetilases , Neoplasias Pulmonares , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Proteínas Proto-Oncogênicas B-raf , Humanos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Linhagem Celular Tumoral , Animais , Inibidores de Histona Desacetilases/farmacologia , Ferroptose/efeitos dos fármacos , Ferroptose/genética , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Oximas/farmacologia , Imidazóis/farmacologia , Piridonas/farmacologia , Pirimidinonas/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Mutação/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
KRAS is one of the most commonly mutated proteins in cancer, and efforts to directly inhibit its function have been continuing for decades. The most successful of these has been the development of covalent allele-specific inhibitors that trap KRAS G12C in its inactive conformation and suppress tumour growth in patients1-7. Whether inactive-state selective inhibition can be used to therapeutically target non-G12C KRAS mutants remains under investigation. Here we report the discovery and characterization of a non-covalent inhibitor that binds preferentially and with high affinity to the inactive state of KRAS while sparing NRAS and HRAS. Although limited to only a few amino acids, the evolutionary divergence in the GTPase domain of RAS isoforms was sufficient to impart orthosteric and allosteric constraints for KRAS selectivity. The inhibitor blocked nucleotide exchange to prevent the activation of wild-type KRAS and a broad range of KRAS mutants, including G12A/C/D/F/V/S, G13C/D, V14I, L19F, Q22K, D33E, Q61H, K117N and A146V/T. Inhibition of downstream signalling and proliferation was restricted to cancer cells harbouring mutant KRAS, and drug treatment suppressed KRAS mutant tumour growth in mice, without having a detrimental effect on animal weight. Our study suggests that most KRAS oncoproteins cycle between an active state and an inactive state in cancer cells and are dependent on nucleotide exchange for activation. Pan-KRAS inhibitors, such as the one described here, have broad therapeutic implications and merit clinical investigation in patients with KRAS-driven cancers.
Assuntos
Neoplasias , Proteínas Proto-Oncogênicas p21(ras) , Transdução de Sinais , Animais , Camundongos , Peso Corporal , Ativação Enzimática , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Nucleotídeos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Especificidade por SubstratoRESUMO
Inactive state-selective KRAS(G12C) inhibitors1-8 demonstrate a 30-40% response rate and result in approximately 6-month median progression-free survival in patients with lung cancer9. The genetic basis for resistance to these first-in-class mutant GTPase inhibitors remains under investigation. Here we evaluated matched pre-treatment and post-treatment specimens from 43 patients treated with the KRAS(G12C) inhibitor sotorasib. Multiple treatment-emergent alterations were observed across 27 patients, including alterations in KRAS, NRAS, BRAF, EGFR, FGFR2, MYC and other genes. In preclinical patient-derived xenograft and cell line models, resistance to KRAS(G12C) inhibition was associated with low allele frequency hotspot mutations in KRAS(G12V or G13D), NRAS(Q61K or G13R), MRAS(Q71R) and/or BRAF(G596R), mirroring observations in patients. Single-cell sequencing in an isogenic lineage identified secondary RAS and/or BRAF mutations in the same cells as KRAS(G12C), where they bypassed inhibition without affecting target inactivation. Genetic or pharmacological targeting of ERK signalling intermediates enhanced the antiproliferative effect of G12C inhibitor treatment in models with acquired RAS or BRAF mutations. Our study thus suggests a heterogenous pattern of resistance with multiple subclonal events emerging during G12C inhibitor treatment. A subset of patients in our cohort acquired oncogenic KRAS, NRAS or BRAF mutations, and resistance in this setting may be delayed by co-targeting of ERK signalling intermediates. These findings merit broader evaluation in prospective clinical trials.
Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Acetonitrilas/farmacologia , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular , Estudos de Coortes , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Piridinas/farmacologia , Piridinas/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recently reported to be effective in patients with lung cancer, KRASG12C inhibitors bind to the inactive, or guanosine diphosphate (GDP)bound, state of the oncoprotein and require guanosine triphosphate (GTP) hydrolysis for inhibition. However, KRAS mutations prevent the catalytic arginine of GTPase-activating proteins (GAPs) from enhancing an otherwise slow hydrolysis rate. If KRAS mutants are indeed insensitive to GAPs, it is unclear how KRASG12C hydrolyzes sufficient GTP to allow inactive stateselective inhibition. Here, we show that RGS3, a GAP previously known for regulating G proteincoupled receptors, can also enhance the GTPase activity of mutant and wild-type KRAS proteins. Our study reveals an unexpected mechanism that inactivates KRAS and explains the vulnerability to emerging clinically effective therapeutics.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Neoplasias Pulmonares/enzimologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas RGS/metabolismo , Animais , Extratos Celulares , Linhagem Celular Tumoral , Ativação Enzimática , Humanos , Hidrólise , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Camundongos Nus , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas RGS/genética , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Guanine nucleotide exchange factors (GEF) control the rate-limiting step of physiologic RAS activation. In this issue of Cancer Discovery, Hofmann and colleagues describe the discovery of a selective inhibitor targeting the GEF, SOS1, along with its preclinical effects in suppressing KRAS-mutant tumor growth.See related article by Hofmann et al., p. 142.
Assuntos
Neoplasias , Proteínas Proto-Oncogênicas p21(ras) , Fatores de Troca do Nucleotídeo Guanina , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno , Neoplasias/tratamento farmacológico , Neoplasias/genética , Nucleotídeos , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
KRAS GTPases are activated in one-third of cancers, and KRAS(G12C) is one of the most common activating alterations in lung adenocarcinoma1,2. KRAS(G12C) inhibitors3,4 are in phase-I clinical trials and early data show partial responses in nearly half of patients with lung cancer. How cancer cells bypass inhibition to prevent maximal response to therapy is not understood. Because KRAS(G12C) cycles between an active and inactive conformation4-6, and the inhibitors bind only to the latter, we tested whether isogenic cell populations respond in a non-uniform manner by studying the effect of treatment at a single-cell resolution. Here we report that, shortly after treatment, some cancer cells are sequestered in a quiescent state with low KRAS activity, whereas others bypass this effect to resume proliferation. This rapid divergent response occurs because some quiescent cells produce new KRAS(G12C) in response to suppressed mitogen-activated protein kinase output. New KRAS(G12C) is maintained in its active, drug-insensitive state by epidermal growth factor receptor and aurora kinase signalling. Cells without these adaptive changes-or cells in which these changes are pharmacologically inhibited-remain sensitive to drug treatment, because new KRAS(G12C) is either not available or exists in its inactive, drug-sensitive state. The direct targeting of KRAS oncoproteins has been a longstanding objective in precision oncology. Our study uncovers a flexible non-uniform fitness mechanism that enables groups of cells within a population to rapidly bypass the effect of treatment. This adaptive process must be overcome if we are to achieve complete and durable responses in the clinic.
Assuntos
Mutação , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Adaptação Biológica , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
PURPOSE: Combined MAPK pathway inhibition using dual BRAF and MEK inhibitors has prolonged the duration of clinical response in patients with BRAFV600E-driven tumors compared with either agent alone. However, resistance frequently arises. EXPERIMENTAL DESIGN: We generated cell lines resistant to dual BRAF/MEK inhibition and utilized a pharmacologic synthetic lethal approach to identify a novel, adaptive resistance mechanism mediated through the fibroblast growth factor receptor (FGFR) pathway. RESULTS: In response to drug treatment, transcriptional upregulation of FGF1 results in autocrine activation of FGFR, which potentiates extracellular signal-regulated kinases (ERK) activation. FGFR inhibition overcomes resistance to dual BRAF/MEK inhibitors in both cell lines and patient-derived xenograft (PDX) models. Abrogation of this bypass mechanism in the first-line setting enhances tumor killing and prevents the emergence of drug-resistant cells. Moreover, clinical data implicate serum FGF1 levels in disease prognosis. CONCLUSIONS: Taken together, these results describe a new, adaptive resistance mechanism that is more commonly observed in the context of dual BRAF/MEK blockade as opposed to single-agent treatment and reveal the potential clinical utility of FGFR-targeting agents in combination with BRAF and MEK inhibitors as a promising strategy to forestall resistance in a subset of BRAF-driven cancers.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Resistencia a Medicamentos Antineoplásicos , Fator 1 de Crescimento de Fibroblastos/metabolismo , MAP Quinase Quinase 1/antagonistas & inibidores , Melanoma/patologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Apoptose , Comunicação Autócrina , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Camundongos , Camundongos Nus , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Intrinsic and acquired drug resistance and induction of secondary malignancies limit successful chemotherapy. Because mutagenic translesion synthesis (TLS) contributes to chemoresistance as well as treatment-induced mutations, targeting TLS is an attractive avenue for improving chemotherapeutics. However, development of small molecules with high specificity and in vivo efficacy for mutagenic TLS has been challenging. Here, we report the discovery of a small-molecule inhibitor, JH-RE-06, that disrupts mutagenic TLS by preventing recruitment of mutagenic POL ζ. Remarkably, JH-RE-06 targets a nearly featureless surface of REV1 that interacts with the REV7 subunit of POL ζ. Binding of JH-RE-06 induces REV1 dimerization, which blocks the REV1-REV7 interaction and POL ζ recruitment. JH-RE-06 inhibits mutagenic TLS and enhances cisplatin-induced toxicity in cultured human and mouse cell lines. Co-administration of JH-RE-06 with cisplatin suppresses the growth of xenograft human melanomas in mice, establishing a framework for developing TLS inhibitors as a novel class of chemotherapy adjuvants.