RESUMEN
How the KRAS oncogene drives cancer growth remains poorly understood. Therefore, we established a systemwide portrait of KRAS- and extracellular signal-regulated kinase (ERK)-dependent gene transcription in KRAS-mutant cancer to delineate the molecular mechanisms of growth and of inhibitor resistance. Unexpectedly, our KRAS-dependent gene signature diverges substantially from the frequently cited Hallmark KRAS signaling gene signature, is driven predominantly through the ERK mitogen-activated protein kinase (MAPK) cascade, and accurately reflects KRAS- and ERK-regulated gene transcription in KRAS-mutant cancer patients. Integration with our ERK-regulated phospho- and total proteome highlights ERK deregulation of the anaphase promoting complex/cyclosome (APC/C) and other components of the cell cycle machinery as key processes that drive pancreatic ductal adenocarcinoma (PDAC) growth. Our findings elucidate mechanistically the critical role of ERK in driving KRAS-mutant tumor growth and in resistance to KRAS-ERK MAPK targeted therapies.
Asunto(s)
Carcinoma Ductal Pancreático , Quinasas MAP Reguladas por Señal Extracelular , Regulación Neoplásica de la Expresión Génica , Sistema de Señalización de MAP Quinasas , Mutación , Neoplasias Pancreáticas , Proteínas Proto-Oncogénicas p21(ras) , Transcriptoma , Animales , Humanos , Ratones , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Células HEK293RESUMEN
We and others have recently shown that proteins involved in the DNA damage response (DDR) are critical for KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) cell growth in vitro. However, the CRISPR-Cas9 library that enabled us to identify these key proteins had limited representation of DDR-related genes. To further investigate the DDR in this context, we performed a comprehensive, DDR-focused CRISPR-Cas9 loss-of-function screen. This screen identified valosin-containing protein (VCP) as an essential gene in KRAS-mutant PDAC cell lines. We observed that genetic and pharmacologic inhibition of VCP limited cell growth and induced apoptotic death. Addressing the basis for VCP-dependent growth, we first evaluated the contribution of VCP to the DDR and found that loss of VCP resulted in accumulation of DNA double-strand breaks. We next addressed its role in proteostasis and found that loss of VCP caused accumulation of polyubiquitinated proteins. We also found that loss of VCP increased autophagy. Therefore, we reasoned that inhibiting both VCP and autophagy could be an effective combination. Accordingly, we found that VCP inhibition synergized with the autophagy inhibitor chloroquine. We conclude that concurrent targeting of autophagy can enhance the efficacy of VCP inhibitors in KRAS-mutant PDAC.
RESUMEN
Mutational loss of CDKN2A (encoding p16INK4A) tumor-suppressor function is a key genetic step that complements activation of KRAS in promoting the development and malignant growth of pancreatic ductal adenocarcinoma (PDAC). However, pharmacologic restoration of p16INK4A function with inhibitors of CDK4 and CDK6 (CDK4/6) has shown limited clinical efficacy in PDAC. Here, we found that concurrent treatment with both a CDK4/6 inhibitor (CDK4/6i) and an ERK-MAPK inhibitor (ERKi) synergistically suppresses the growth of PDAC cell lines and organoids by cooperatively blocking CDK4/6i-induced compensatory upregulation of ERK, PI3K, antiapoptotic signaling, and MYC expression. On the basis of these findings, a Phase I clinical trial was initiated to evaluate the ERKi ulixertinib in combination with the CDK4/6i palbociclib in patients with advanced PDAC (NCT03454035). As inhibition of other proteins might also counter CDK4/6i-mediated signaling changes to increase cellular CDK4/6i sensitivity, a CRISPR-Cas9 loss-of-function screen was conducted that revealed a spectrum of functionally diverse genes whose loss enhanced CDK4/6i growth inhibitory activity. These genes were enriched around diverse signaling nodes, including cell-cycle regulatory proteins centered on CDK2 activation, PI3K-AKT-mTOR signaling, SRC family kinases, HDAC proteins, autophagy-activating pathways, chromosome regulation and maintenance, and DNA damage and repair pathways. Novel therapeutic combinations were validated using siRNA and small-molecule inhibitor-based approaches. In addition, genes whose loss imparts a survival advantage were identified (e.g., RB1, PTEN, FBXW7), suggesting possible resistance mechanisms to CDK4/6 inhibition. In summary, this study has identified novel combinations with CDK4/6i that may have clinical benefit to patients with PDAC. SIGNIFICANCE: CRISPR-Cas9 screening and protein activity mapping reveal combinations that increase potency of CDK4/6 inhibitors and overcome drug-induced compensations in pancreatic cancer.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Línea Celular Tumoral , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Neoplasias PancreáticasRESUMEN
Cancer-associated mutations in the guanosine triphosphatase (GTPase) RHOA are found at different locations from the mutational hotspots in the structurally and biochemically related RAS. Tyr42-to-Cys (Y42C) and Leu57-to-Val (L57V) substitutions are the two most prevalent RHOA mutations in diffuse gastric cancer (DGC). RHOAY42C exhibits a gain-of-function phenotype and is an oncogenic driver in DGC. Here, we determined how RHOAL57V promotes DGC growth. In mouse gastric organoids with deletion of Cdh1, which encodes the cell adhesion protein E-cadherin, the expression of RHOAL57V, but not of wild-type RHOA, induced an abnormal morphology similar to that of patient-derived DGC organoids. RHOAL57V also exhibited a gain-of-function phenotype and promoted F-actin stress fiber formation and cell migration. RHOAL57V retained interaction with effectors but exhibited impaired RHOA-intrinsic and GAP-catalyzed GTP hydrolysis, which favored formation of the active GTP-bound state. Introduction of missense mutations at KRAS residues analogous to Tyr42 and Leu57 in RHOA did not activate KRAS oncogenic potential, indicating distinct functional effects in otherwise highly related GTPases. Both RHOA mutants stimulated the transcriptional co-activator YAP1 through actin dynamics to promote DGC progression; however, RHOAL57V additionally did so by activating the kinases IGF1R and PAK1, distinct from the FAK-mediated mechanism induced by RHOAY42C. Our results reveal that RHOAL57V and RHOAY42C drive the development of DGC through distinct biochemical and signaling mechanisms.
Asunto(s)
Neoplasias Gástricas , Animales , Humanos , Ratones , Actinas , Guanosina Trifosfato , Quinasas p21 Activadas , Proteínas Proto-Oncogénicas p21(ras) , Receptor IGF Tipo 1 , Proteína de Unión al GTP rhoA/genética , Transducción de Señal , Neoplasias Gástricas/genéticaRESUMEN
Mutational activation of the KRAS oncogene is found in ~95% of pancreatic ductal adenocarcinoma (PDAC), the major form of pancreatic cancer. With substantial experimental evidence that continued aberrant KRAS function is essential for the maintenance of PDAC tumorigenic growth, the National Cancer Institute has identified the development of effective anti-KRAS therapies as one of four major initiatives for pancreatic cancer research. The recent clinical success in the development of an anti-KRAS therapy targeting one specific KRAS mutant (G12C) supports the significant potential impact of anti-KRAS therapies. However, KRASG12C mutations comprise only 2% of KRAS mutations in PDAC. Thus, there remains a dire need for additional therapeutic approaches for targeting the majority of KRAS-mutant PDAC. Among the different directions currently being pursued for anti-KRAS drug development, one of the most promising involves inhibitors of the key KRAS effector pathway, the three-tiered RAF-MEK-ERK mitogen-activated protein kinase (MAPK) cascade. We address the promises and challenges of targeting ERK MAPK signaling as an anti-KRAS therapy for PDAC. In particular, we also summarize the key role of the MYC transcription factor and oncoprotein in supporting ERK-dependent growth of KRAS-mutant PDAC.
Asunto(s)
Carcinoma Ductal Pancreático , Sistema de Señalización de MAP Quinasas , Neoplasias Pancreáticas , Proteínas Proto-Oncogénicas p21(ras) , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismoRESUMEN
Oncogenic RAS signaling is an attractive target for fusion-negative rhabdomyosarcoma (FN-RMS). Our study validates the role of the ERK MAPK effector pathway in mediating RAS dependency in a panel of H/NRASQ61X mutant RMS cells and correlates in vivo efficacy of the MEK inhibitor trametinib with pharmacodynamics of ERK activity. A screen is used to identify trametinib-sensitizing targets, and combinations are evaluated in cells and tumor xenografts. We find that the ERK MAPK pathway is central to H/NRASQ61X dependency in RMS cells; however, there is poor in vivo response to clinically relevant exposures with trametinib, which correlates with inefficient suppression of ERK activity. CRISPR screening points to vertical inhibition of the RAF-MEK-ERK cascade by cosuppression of MEK and either CRAF or ERK. CRAF is central to rebound pathway activation following MEK or ERK inhibition. Concurrent CRAF suppression and MEK or ERK inhibition, or concurrent pan-RAF and MEK/ERK inhibition (pan-RAFi + MEKi/ERKi), or concurrent MEK and ERK inhibition (MEKi + ERKi) all synergistically block ERK activity and induce myogenic differentiation and apoptosis. In vivo assessment of pan-RAFi + ERKi or MEKi + ERKi potently suppress growth of H/NRASQ61X RMS tumor xenografts, with pan-RAFi + ERKi being more effective and better tolerated. We conclude that CRAF reactivation limits the activity of single-agent MEK/ERK inhibitors in FN-RMS. Vertical targeting of the RAF-MEK-ERK cascade and particularly cotargeting of CRAF and MEK or ERK, or the combination of pan-RAF inhibitors with MEK or ERK inhibitors, have synergistic activity and potently suppress H/NRASQ61X mutant RMS tumor growth.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Rabdomiosarcoma/genética , Animales , Apoptosis , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Femenino , Genes ras , Humanos , Ratones , Rabdomiosarcoma/patología , TransfecciónRESUMEN
The aggressive nature of pancreatic ductal adenocarcinoma (PDAC) mandates the development of improved therapies. As KRAS mutations are found in 95% of PDAC and are critical for tumor maintenance, one promising strategy involves exploiting KRAS-dependent metabolic perturbations. The macrometabolic process of autophagy is upregulated in KRAS-mutant PDAC, and PDAC growth is reliant on autophagy. However, inhibition of autophagy as monotherapy using the lysosomal inhibitor hydroxychloroquine (HCQ) has shown limited clinical efficacy. To identify strategies that can improve PDAC sensitivity to HCQ, we applied a CRISPR-Cas9 loss-of-function screen and found that a top sensitizer was the receptor tyrosine kinase (RTK) insulin-like growth factor 1 receptor (IGF1R). Additionally, reverse phase protein array pathway activation mapping profiled the signaling pathways altered by chloroquine (CQ) treatment. Activating phosphorylation of RTKs, including IGF1R, was a common compensatory increase in response to CQ. Inhibition of IGF1R increased autophagic flux and sensitivity to CQ-mediated growth suppression both in vitro and in vivo. Cotargeting both IGF1R and pathways that antagonize autophagy, such as ERK-MAPK axis, was strongly synergistic. IGF1R and ERK inhibition converged on suppression of glycolysis, leading to enhanced dependence on autophagy. Accordingly, concurrent inhibition of IGF1R, ERK, and autophagy induced cytotoxicity in PDAC cell lines and decreased viability in human PDAC organoids. In conclusion, targeting IGF1R together with ERK enhances the effectiveness of autophagy inhibitors in PDAC. SIGNIFICANCE: Compensatory upregulation of IGF1R and ERK-MAPK signaling limits the efficacy of autophagy inhibitors chloroquine and hydroxychloroquine, and their concurrent inhibition synergistically increases autophagy dependence and chloroquine sensitivity in pancreatic ductal adenocarcinoma.
Asunto(s)
Autofagia/fisiología , Carcinoma Ductal Pancreático/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Neoplasias Pancreáticas/metabolismo , Receptor IGF Tipo 1/metabolismo , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Glucólisis/efectos de los fármacos , Células HEK293 , Humanos , Hidroxicloroquina/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Fosforilación/efectos de los fármacos , Pirazoles/farmacología , Receptor IGF Tipo 1/antagonistas & inhibidores , Triazinas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Human papilloma virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is a common cancer worldwide with an unmet need for more effective, less toxic treatments. Currently, both the disease and the treatment of HNSCC cause significant mortality and morbidity. Targeted therapies hold new promise for patients with HPV-negative status whose tumors harbor oncogenic HRAS mutations. Recent promising clinical results have renewed interest in the development of farnesyltransferase inhibitors (FTIs) as a therapeutic strategy for HRAS-mutant cancers. With the advent of clinical evaluation of the FTI tipifarnib for the treatment of HRAS-mutant HNSCC, we investigated the activity of tipifarnib and inhibitors of HRAS effector signaling in HRAS-mutant HNSCC cell lines. First, we validated that HRAS is a cancer driver in HRAS-mutant HNSCC lines. Second, we showed that treatment with the FTI tipifarnib largely phenocopied HRAS silencing, supporting HRAS as a key target of FTI antitumor activity. Third, we performed reverse-phase protein array analyses to profile FTI treatment-induced changes in global signaling, and conducted CRISPR/Cas9 genetic loss-of-function screens to identify previously unreported genes and pathways that modulate sensitivity to tipifarnib. Fourth, we determined that concurrent inhibition of HRAS effector signaling (ERK, PI3K, mTORC1) increased sensitivity to tipifarnib treatment, in part by overcoming tipifarnib-induced compensatory signaling. We also determined that ERK inhibition could block tipifarnib-induced epithelial-to-mesenchymal transition, providing a potential basis for the effectiveness of this combination. Our results support future investigations of these and other combination treatments for HRAS mutant HNSCC.
Asunto(s)
Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Infecciones por Papillomavirus , Carcinoma de Células Escamosas/genética , Línea Celular Tumoral , Farnesiltransferasa/metabolismo , Farnesiltransferasa/uso terapéutico , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/genética , Humanos , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/genéticaRESUMEN
Missense mutations at the three hotspots in the guanosine triphosphatase (GTPase) RAS-Gly12, Gly13, and Gln61 (commonly known as G12, G13, and Q61, respectively)-occur differentially among the three RAS isoforms. Q61 mutations in KRAS are infrequent and differ markedly in occurrence. Q61H is the predominant mutant (at 57%), followed by Q61R/L/K (collectively 40%), and Q61P and Q61E are the rarest (2 and 1%, respectively). Probability analysis suggested that mutational susceptibility to different DNA base changes cannot account for this distribution. Therefore, we investigated whether these frequencies might be explained by differences in the biochemical, structural, and biological properties of KRASQ61 mutants. Expression of KRASQ61 mutants in NIH 3T3 fibroblasts and RIE-1 epithelial cells caused various alterations in morphology, growth transformation, effector signaling, and metabolism. The relatively rare KRASQ61E mutant stimulated actin stress fiber formation, a phenotype distinct from that of KRASQ61H/R/L/P, which disrupted actin cytoskeletal organization. The crystal structure of KRASQ61E was unexpectedly similar to that of wild-type KRAS, a potential basis for its weak oncogenicity. KRASQ61H/L/R-mutant pancreatic ductal adenocarcinoma (PDAC) cell lines exhibited KRAS-dependent growth and, as observed with KRASG12-mutant PDAC, were susceptible to concurrent inhibition of ERK-MAPK signaling and of autophagy. Our results uncover phenotypic heterogeneity among KRASQ61 mutants and support the potential utility of therapeutic strategies that target KRASQ61 mutant-specific signaling and cellular output.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Actinas , Carcinoma Ductal Pancreático/genética , GTP Fosfohidrolasas/genética , Humanos , Mutación , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Neoplasias PancreáticasRESUMEN
Cellular phenotype plasticity between the epithelial and mesenchymal states has been linked to metastasis and heterogeneous responses to cancer therapy, and remains a challenge for the treatment of triple-negative breast cancer (TNBC). Here, we used isogenic human breast epithelial cell lines, D492 and D492M, representing the epithelial and mesenchymal phenotypes, respectively. We employed a CRISPR-Cas9 loss-of-function screen targeting a 2240-gene 'druggable genome' to identify phenotype-specific vulnerabilities. Cells with the epithelial phenotype were more vulnerable to the loss of genes related to EGFR-RAS-MAPK signaling, while the mesenchymal-like cells had increased sensitivity to knockout of G2 -M cell cycle regulators. Furthermore, we discovered knockouts that sensitize to the mTOR inhibitor everolimus and the chemotherapeutic drug fluorouracil in a phenotype-specific manner. Specifically, loss of EGFR and fatty acid synthase (FASN) increased the effectiveness of the drugs in the epithelial and mesenchymal phenotypes, respectively. These phenotype-associated genetic vulnerabilities were confirmed using targeted inhibitors of EGFR (gefitinib), G2 -M transition (STLC), and FASN (Fasnall). In conclusion, a CRISPR-Cas9 loss-of-function screen enables the identification of phenotype-specific genetic vulnerabilities that can pinpoint actionable targets and promising therapeutic combinations.
Asunto(s)
Sistemas CRISPR-Cas , Mutación con Pérdida de Función , Fenotipo , Neoplasias de la Mama Triple Negativas/patología , Antineoplásicos/uso terapéutico , Proliferación Celular , Ensayos de Selección de Medicamentos Antitumorales , Transición Epitelial-Mesenquimal , Everolimus/uso terapéutico , Femenino , Fluorouracilo/uso terapéutico , Humanos , Transducción de Señal/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
We apply genetic screens to delineate modulators of KRAS mutant pancreatic ductal adenocarcinoma (PDAC) sensitivity to ERK inhibitor treatment, and we identify components of the ATR-CHK1 DNA damage repair (DDR) pathway. Pharmacologic inhibition of CHK1 alone causes apoptotic growth suppression of both PDAC cell lines and organoids, which correlates with loss of MYC expression. CHK1 inhibition also activates ERK and AMPK and increases autophagy, providing a mechanistic basis for increased efficacy of concurrent CHK1 and ERK inhibition and/or autophagy inhibition with chloroquine. To assess how CHK1 inhibition-induced ERK activation promotes PDAC survival, we perform a CRISPR-Cas9 loss-of-function screen targeting direct/indirect ERK substrates and identify RIF1. A key component of non-homologous end joining repair, RIF1 suppression sensitizes PDAC cells to CHK1 inhibition-mediated apoptotic growth suppression. Furthermore, ERK inhibition alone decreases RIF1 expression and phenocopies RIF1 depletion. We conclude that concurrent DDR suppression enhances the efficacy of ERK and/or autophagy inhibitors in KRAS mutant PDAC.
Asunto(s)
Carcinoma Ductal Pancreático/tratamiento farmacológico , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Daño del ADN , Mutación , Neoplasias Pancreáticas/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Apoptosis , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Proliferación Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Humanos , Ratones , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
To identify therapeutic targets for KRAS mutant pancreatic cancer, we conduct a druggable genome small interfering RNA (siRNA) screen and determine that suppression of BCAR1 sensitizes pancreatic cancer cells to ERK inhibition. Integrative analysis of genome-scale CRISPR-Cas9 screens also identify BCAR1 as a top synthetic lethal interactor with mutant KRAS. BCAR1 encodes the SRC substrate p130Cas. We determine that SRC-inhibitor-mediated suppression of p130Cas phosphorylation impairs MYC transcription through a DOCK1-RAC1-ß-catenin-dependent mechanism. Additionally, genetic suppression of TUBB3, encoding the ßIII-tubulin subunit of microtubules, or pharmacological inhibition of microtubule function decreases levels of MYC protein in a calpain-dependent manner and potently sensitizes pancreatic cancer cells to ERK inhibition. Accordingly, the combination of a dual SRC/tubulin inhibitor with an ERK inhibitor cooperates to reduce MYC protein and synergistically suppress the growth of KRAS mutant pancreatic cancer. Thus, we demonstrate that mechanistically diverse combinations with ERK inhibition suppress MYC to impair pancreatic cancer proliferation.
Asunto(s)
Proteína Sustrato Asociada a CrK/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Microtúbulos/metabolismo , Neoplasias Pancreáticas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Acetamidas/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Calpaína/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Sinergismo Farmacológico , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Semivida , Humanos , Microtúbulos/efectos de los fármacos , Morfolinas/farmacología , Mutación/genética , Organoides/efectos de los fármacos , Organoides/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Piridinas/farmacología , Transcripción Genética/efectos de los fármacos , Tubulina (Proteína)/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/metabolismoRESUMEN
We address whether combinations with a pan-RAF inhibitor (RAFi) would be effective in KRAS mutant pancreatic ductal adenocarcinoma (PDAC). Chemical library and CRISPR genetic screens identify combinations causing apoptotic anti-tumor activity. The most potent combination, concurrent inhibition of RAF (RAFi) and ERK (ERKi), is highly synergistic at low doses in cell line, organoid, and rat models of PDAC, whereas each inhibitor alone is only cytostatic. Comprehensive mechanistic signaling studies using reverse phase protein array (RPPA) pathway mapping and RNA sequencing (RNA-seq) show that RAFi/ERKi induced insensitivity to loss of negative feedback and system failures including loss of ERK signaling, FOSL1, and MYC; shutdown of the MYC transcriptome; and induction of mesenchymal-to-epithelial transition. We conclude that low-dose vertical inhibition of the RAF-MEK-ERK cascade is an effective therapeutic strategy for KRAS mutant PDAC.
Asunto(s)
Apoptosis/genética , Carcinoma Ductal Pancreático/genética , Sistema de Señalización de MAP Quinasas/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Apoptosis/efectos de los fármacos , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Mutación/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Neoplasias PancreáticasRESUMEN
Allele-specific signaling by different KRAS alleles remains poorly understood. The KRAS G12R mutation displays uneven prevalence among cancers that harbor the highest occurrence of KRAS mutations: It is rare (â¼1%) in lung and colorectal cancers, yet relatively common (â¼20%) in pancreatic ductal adenocarcinoma (PDAC), suggesting context-specific properties. We evaluated whether KRASG12R is functionally distinct from the more common KRASG12D- or KRASG12V-mutant proteins (KRASG12D/V). We found that KRASG12D/V but not KRASG12R drives macropinocytosis and that MYC is essential for macropinocytosis in KRASG12D/V- but not KRASG12R-mutant PDAC. Surprisingly, we found that KRASG12R is defective for interaction with a key effector, p110α PI3K (PI3Kα), due to structural perturbations in switch II. Instead, upregulated KRAS-independent PI3Kγ activity was able to support macropinocytosis in KRASG12R-mutant PDAC. Finally, we determined that KRASG12R-mutant PDAC displayed a distinct drug sensitivity profile compared with KRASG12D-mutant PDAC but is still responsive to the combined inhibition of ERK and autophagy. SIGNIFICANCE: We determined that KRASG12R is impaired in activating a key effector, p110α PI3K. As such, KRASG12R is impaired in driving macropinocytosis. However, overexpression of PI3Kγ in PDAC compensates for this deficiency, providing one basis for the prevalence of this otherwise rare KRAS mutant in pancreatic cancer but not other cancers.See related commentary by Falcomatà et al., p. 23.This article is highlighted in the In This Issue feature, p. 1.
Asunto(s)
Carcinoma Ductal Pancreático/patología , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Mutación , Neoplasias Pancreáticas/patología , Pinocitosis , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Proliferación Celular , Fosfatidilinositol 3-Quinasa Clase I/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Myeloid cell leukemia 1 (Mcl-1) is an antiapoptotic member of the Bcl-2 family of proteins that when overexpressed is associated with high tumor grade, poor survival, and resistance to chemotherapy. Mcl-1 is amplified in many human cancers, and knockdown of Mcl-1 using RNAi can lead to apoptosis. Thus, Mcl-1 is a promising cancer target. Here, we describe the discovery of picomolar Mcl-1 inhibitors that cause caspase activation, mitochondrial depolarization, and selective growth inhibition. These compounds represent valuable tools to study the role of Mcl-1 in cancer and serve as useful starting points for the discovery of clinically useful Mcl-1 inhibitors. PDB ID CODES: Comp. 2: 5IEZ; Comp. 5: 5IF4.
Asunto(s)
Antineoplásicos/farmacología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Animales , Antineoplásicos/química , Proteína 11 Similar a Bcl2/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Diseño de Fármacos , Descubrimiento de Drogas , Humanos , Inmunoprecipitación , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteína bcl-X/metabolismoRESUMEN
Myeloid cell leukemia-1 (Mcl-1) is a member of the Bcl-2 family of proteins responsible for the regulation of programmed cell death. Amplification of Mcl-1 is a common genetic aberration in human cancer whose overexpression contributes to the evasion of apoptosis and is one of the major resistance mechanisms for many chemotherapies. Mcl-1 mediates its effects primarily through interactions with pro-apoptotic BH3 containing proteins that achieve high affinity for the target by utilizing four hydrophobic pockets in its binding groove. Here we describe the discovery of Mcl-1 inhibitors using fragment-based methods and structure-based design. These novel inhibitors exhibit low nanomolar binding affinities to Mcl-1 and >500-fold selectivity over Bcl-xL. X-ray structures of lead Mcl-1 inhibitors when complexed to Mcl-1 provided detailed information on how these small-molecules bind to the target and were used extensively to guide compound optimization.
Asunto(s)
Descubrimiento de Drogas , Indoles/farmacología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Sulfonamidas/farmacología , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Indoles/síntesis química , Indoles/química , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/químicaRESUMEN
Myeloid cell leukemia-1 (Mcl-1) is an antiapoptotic member of the Bcl-2 family of proteins that is overexpressed and amplified in many cancers. Overexpression of Mcl-1 allows cancer cells to evade apoptosis and contributes to the resistance of cancer cells to be effectively treated with various chemotherapies. From an NMR-based screen of a large fragment library, several distinct chemical scaffolds that bind to Mcl-1 were discovered. Here, we describe the discovery of potent tricyclic 2-indole carboxylic acid inhibitors that exhibit single digit nanomolar binding affinity to Mcl-1 and greater than 1700-fold selectivity over Bcl-xL and greater than 100-fold selectivity over Bcl-2. X-ray structures of these compounds when complexed to Mcl-1 provide detailed information on how these small-molecules bind to the target, which was used to guide compound optimization.
Asunto(s)
Compuestos Heterocíclicos con 3 Anillos/química , Indoles/química , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Cristalografía por Rayos X , Compuestos Heterocíclicos con 3 Anillos/síntesis química , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Indoles/síntesis química , Indoles/farmacología , Células K562 , Modelos Moleculares , Conformación Molecular , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Unión Proteica , Relación Estructura-Actividad , Proteína bcl-X/química , Proteína bcl-X/metabolismoRESUMEN
Gas-solid chromatography was used to determine B(2s) (gas-solid virial coefficient) values for 12 alkanes (10 branched and 2 cyclic) interacting with a carbon powder (Carbopack B, Supelco). B(2s) values were determined by multiple size variant injections within the temperature range of 393 to 623 K with each alkane measured at 5 or 6 different temperatures. The temperature variations of the gas-solid virial coefficients were used to find the experimental adsorption energy or binding energy values (E( *)) for each alkane. A molecular mechanics based, rough-surface model was used to calculate the molecule-surface binding energy (E(cal)( *)) using augmented MM2 parameters. The surface model consisted of three parallel graphene layers with each layer containing 127 interconnected benzene rings and two separated nanostructures each containing 17 benzene rings arranged in a linear strip. As the parallel nanostructures are moved closer together, the surface roughness increases and molecule-surface interactions are enhanced. A comparison of the experimental and calculated binding energies showed excellent agreement with an average difference of 3.8%. Linear regressions of E( *) versus E(cal)( *) for the current data set and a combined current and prior alkane data set both gave excellent correlations. For the combined data set with 18 linear, branched and cyclic alkanes; a linear regression of E( *)=0.9848E(cal)( *) and r(2)=0.976 was obtained. The results indicate that alkane-surface binding energies may be calculated from MM2 parameters for some gas-solid systems.