RESUMO
The mechanism by which the wild-type KRAS allele imparts a growth inhibitory effect to oncogenic KRAS in various cancers, including lung adenocarcinoma (LUAD), is poorly understood. Here, using a genetically inducible model of KRAS loss of heterozygosity (LOH), we show that KRAS dimerization mediates wild-type KRAS-dependent fitness of human and murine KRAS mutant LUAD tumor cells and underlies resistance to MEK inhibition. These effects are abrogated when wild-type KRAS is replaced by KRASD154Q, a mutant that disrupts dimerization at the α4-α5 KRAS dimer interface without changing other fundamental biochemical properties of KRAS, both in vitro and in vivo. Moreover, dimerization has a critical role in the oncogenic activity of mutant KRAS. Our studies provide mechanistic and biological insights into the role of KRAS dimerization and highlight a role for disruption of dimerization as a therapeutic strategy for KRAS mutant cancers.
Assuntos
Adenocarcinoma de Pulmão , Inibidores Enzimáticos/farmacologia , Neoplasias Pulmonares , MAP Quinase Quinase Quinases/antagonistas & inibidores , Mutação de Sentido Incorreto , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/enzimologia , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Substituição de Aminoácidos , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Perda de Heterozigosidade , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Camundongos Knockout , Multimerização Proteica/genética , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
RAS oncogenes (collectively NRAS, HRAS and especially KRAS) are among the most frequently mutated genes in cancer, with common driver mutations occurring at codons 12, 13 and 611. Small molecule inhibitors of the KRAS(G12C) oncoprotein have demonstrated clinical efficacy in patients with multiple cancer types and have led to regulatory approvals for the treatment of non-small cell lung cancer2,3. Nevertheless, KRASG12C mutations account for only around 15% of KRAS-mutated cancers4,5, and there are no approved KRAS inhibitors for the majority of patients with tumours containing other common KRAS mutations. Here we describe RMC-7977, a reversible, tri-complex RAS inhibitor with broad-spectrum activity for the active state of both mutant and wild-type KRAS, NRAS and HRAS variants (a RAS(ON) multi-selective inhibitor). Preclinically, RMC-7977 demonstrated potent activity against RAS-addicted tumours carrying various RAS genotypes, particularly against cancer models with KRAS codon 12 mutations (KRASG12X). Treatment with RMC-7977 led to tumour regression and was well tolerated in diverse RAS-addicted preclinical cancer models. Additionally, RMC-7977 inhibited the growth of KRASG12C cancer models that are resistant to KRAS(G12C) inhibitors owing to restoration of RAS pathway signalling. Thus, RAS(ON) multi-selective inhibitors can target multiple oncogenic and wild-type RAS isoforms and have the potential to treat a wide range of RAS-addicted cancers with high unmet clinical need. A related RAS(ON) multi-selective inhibitor, RMC-6236, is currently under clinical evaluation in patients with KRAS-mutant solid tumours (ClinicalTrials.gov identifier: NCT05379985).
Assuntos
Antineoplásicos , Mutação , Neoplasias , Proteína Oncogênica p21(ras) , Proteínas Proto-Oncogênicas p21(ras) , Animais , Humanos , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Guanosina Trifosfato/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Proteína Oncogênica p21(ras)/antagonistas & inibidores , Proteína Oncogênica p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Anaplastic large cell lymphomas (ALCLs) frequently carry oncogenic fusions involving the anaplastic lymphoma kinase (ALK) gene. Targeting ALK using tyrosine kinase inhibitors (TKIs) is a therapeutic option in cases relapsed after chemotherapy, but TKI resistance may develop. By applying genomic loss-of-function screens, we identified PTPN1 and PTPN2 phosphatases as consistent top hits driving resistance to ALK TKIs in ALK+ ALCL. Loss of either PTPN1 or PTPN2 induced resistance to ALK TKIs in vitro and in vivo. Mechanistically, we demonstrated that PTPN1 and PTPN2 are phosphatases that bind to and regulate ALK phosphorylation and activity. In turn, oncogenic ALK and STAT3 repress PTPN1 transcription. We found that PTPN1 is also a phosphatase for SHP2, a key mediator of oncogenic ALK signaling. Downstream signaling analysis showed that deletion of PTPN1 or PTPN2 induces resistance to crizotinib by hyperactivating SHP2, the MAPK, and JAK/STAT pathways. RNA sequencing of patient samples that developed resistance to ALK TKIs showed downregulation of PTPN1 and PTPN2 associated with upregulation of SHP2 expression. Combination of crizotinib with a SHP2 inhibitor synergistically inhibited the growth of wild-type or PTPN1/PTPN2 knock-out ALCL, where it reverted TKI resistance. Thus, we identified PTPN1 and PTPN2 as ALK phosphatases that control sensitivity to ALK TKIs in ALCL and demonstrated that a combined blockade of SHP2 potentiates the efficacy of ALK inhibition in TKI-sensitive and -resistant ALK+ ALCL.
Assuntos
Quinase do Linfoma Anaplásico/antagonistas & inibidores , Antineoplásicos/farmacologia , Linfoma Anaplásico de Células Grandes/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Quinase do Linfoma Anaplásico/metabolismo , Animais , Linhagem Celular Tumoral , Crizotinibe/farmacologia , Humanos , Linfoma Anaplásico de Células Grandes/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
BACKGROUND: Gastric cancer (GC) is the third leading cause of cancer-related death worldwide, with a poor prognosis for patients with advanced disease. Since the oncogenic role of KRAS mutants has been poorly investigated in GC, this study aims to biochemically and biologically characterize different KRAS-mutated models and unravel differences among KRAS mutants in response to therapy. METHODS: Taking advantage of a proprietary, molecularly annotated platform of more than 200 GC PDXs (patient-derived xenografts), we identified KRAS-mutated PDXs, from which primary cell lines were established. The different mutants were challenged with KRAS downstream inhibitors in in vitro and in vivo experiments. RESULTS: Cells expressing the rare KRAS A146T mutant showed lower RAS-GTP levels compared to those bearing the canonical G12/13D mutations. Nevertheless, all the KRAS-mutated cells displayed KRAS addiction. Surprisingly, even if the GEF SOS1 is considered critical for the activation of KRAS A146T mutants, its abrogation did not significantly affect cell viability. From the pharmacologic point of view, Trametinib monotherapy was more effective in A146T than in G12D-mutated models, suggesting a vulnerability to MEK inhibition. However, in the presence of mutations in the PI3K pathway, more frequently co-occurrent in A146T models, the association of Trametinib and the AKT inhibitor MK-2206 was required to optimize the response. CONCLUSION: A deeper genomic and biological characterization of KRAS mutants might sustain the development of more efficient and long-lasting therapeutic options for patients harbouring KRAS-driven GC.
Assuntos
Neoplasias Gástricas , Humanos , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Fosfatidilinositol 3-Quinases/metabolismo , Mutação , Inibidores de Proteínas Quinases/farmacologia , Linhagem Celular TumoralRESUMO
BACKGROUND: The discovery of functionally relevant KRAS effectors in lung and pancreatic ductal adenocarcinoma (LUAD and PDAC) may yield novel molecular targets or mechanisms amenable to inhibition strategies. Phospholipids availability has been appreciated as a mechanism to modulate KRAS oncogenic potential. Thus, phospholipid transporters may play a functional role in KRAS-driven oncogenesis. Here, we identified and systematically studied the phospholipid transporter PITPNC1 and its controlled network in LUAD and PDAC. METHODS: Genetic modulation of KRAS expression as well as pharmacological inhibition of canonical effectors was completed. PITPNC1 genetic depletion was performed in in vitro and in vivo LUAD and PDAC models. PITPNC1-deficient cells were RNA sequenced, and Gene Ontology and enrichment analyses were applied to the output data. Protein-based biochemical and subcellular localization assays were run to investigate PITPNC1-regulated pathways. A drug repurposing approach was used to predict surrogate PITPNC1 inhibitors that were tested in combination with KRASG12C inhibitors in 2D, 3D, and in vivo models. RESULTS: PITPNC1 was increased in human LUAD and PDAC, and associated with poor patients' survival. PITPNC1 was regulated by KRAS through MEK1/2 and JNK1/2. Functional experiments showed PITPNC1 requirement for cell proliferation, cell cycle progression and tumour growth. Furthermore, PITPNC1 overexpression enhanced lung colonization and liver metastasis. PITPNC1 regulated a transcriptional signature which highly overlapped with that of KRAS, and controlled mTOR localization via enhanced MYC protein stability to prevent autophagy. JAK2 inhibitors were predicted as putative PITPNC1 inhibitors with antiproliferative effect and their combination with KRASG12C inhibitors elicited a substantial anti-tumour effect in LUAD and PDAC. CONCLUSIONS: Our data highlight the functional and clinical relevance of PITPNC1 in LUAD and PDAC. Moreover, PITPNC1 constitutes a new mechanism linking KRAS to MYC, and controls a druggable transcriptional network for combinatorial treatments.
Assuntos
Carcinoma Ductal Pancreático , Proteínas de Membrana Transportadoras , Neoplasias Pancreáticas , Humanos , Autofagia/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Pulmão/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Neoplasias PancreáticasRESUMO
BACKGROUND & AIMS: Acinar to ductal metaplasia is the prerequisite for the initiation of Kras-driven pancreatic ductal adenocarcinoma (PDAC), and candidate genes regulating this process are emerging from genome-wide association studies. The adaptor protein p130Cas emerged as a potential PDAC susceptibility gene and a Kras-synthetic lethal interactor in pancreatic cell lines; however, its role in PDAC development has remained largely unknown. METHODS: Human PDAC samples and murine KrasG12D-dependent pancreatic cancer models of increasing aggressiveness were used. p130Cas was conditionally ablated in pancreatic cancer models to investigate its role during Kras-induced tumorigenesis. RESULTS: We found that high expression of p130Cas is frequently detected in PDAC and correlates with higher histologic grade and poor prognosis. In a model of Kras-driven PDAC, loss of p130Cas inhibits tumor development and potently extends median survival. Deletion of p130Cas suppresses acinar-derived tumorigenesis and progression by means of repressing PI3K-AKT signaling, even in the presence of a worsening condition like pancreatitis. CONCLUSIONS: Our observations finally demonstrated that p130Cas acts downstream of Kras to boost the PI3K activity required for acinar to ductal metaplasia and subsequent tumor initiation. This demonstrates an unexpected driving role of p130Cas downstream of Kras through PI3K/AKT, thus indicating a rational therapeutic strategy of targeting the PI3K pathway in tumors with high expression of p130Cas.
Assuntos
Adenocarcinoma , Carcinoma Ductal Pancreático , Proteína Substrato Associada a Crk , Neoplasias Pancreáticas , Células Acinares/patologia , Adenocarcinoma/patologia , Animais , Carcinogênese , Carcinoma Ductal Pancreático/patologia , Transformação Celular Neoplásica/patologia , Proteína Substrato Associada a Crk/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Metaplasia/patologia , Camundongos , Neoplasias Pancreáticas/patologia , Pancreatite/induzido quimicamente , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Neoplasias PancreáticasRESUMO
PURPOSE OF REVIEW: Although the recent development of direct KRASG12C inhibitors (G12Ci) has improved outcomes in KRAS mutant cancers, responses occur only in a fraction of patients, and among responders acquired resistance invariably develops over time. Therefore, the characterization of the determinants of acquired resistance is crucial to inform treatment strategies and to identify novel therapeutic vulnerabilities that can be exploited for drug development. RECENT FINDINGS: Mechanisms of acquired resistance to G12Ci are heterogenous including both on-target and off-target resistance. On-target acquired resistance includes secondary codon 12 KRAS mutations, but also acquired codon 13 and codon 61 alterations, and mutations at drug binding sites. Off-target acquired resistance can derive from activating mutations in KRAS downstream pathway (e.g., MEK1), acquired oncogenic fusions (EML4-ALK, CCDC176-RET), gene level copy gain (e.g., MET amplification), or oncogenic alterations in other pro-proliferative and antiapoptotic pathways (e.g., FGFR3, PTEN, NRAS). In a fraction of patients, histologic transformation can also contribute to the development of acquire resistance. We provided a comprehensive overview of the mechanisms that limit the efficacy of this G12i and reviewed potential strategies to overcome and possibly delay the development of resistance in patients receiving KRAS directed targeted therapies.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Desenvolvimento de Medicamentos , MutaçãoRESUMO
The initiating oncogenic event in almost half of human lung adenocarcinomas is still unknown, a fact that complicates the development of selective targeted therapies. Yet these tumours harbour a number of alterations without obvious oncogenic function including BRAF-inactivating mutations. Inactivating BRAF mutants in lung predominate over the activating V600E mutant that is frequently observed in other tumour types. Here we demonstrate that the expression of an endogenous Braf(D631A) kinase-inactive isoform in mice (corresponding to the human BRAF(D594A) mutation) triggers lung adenocarcinoma in vivo, indicating that BRAF-inactivating mutations are initiating events in lung oncogenesis. Moreover, inactivating BRAF mutations have also been identified in a subset of KRAS-driven human lung tumours. Co-expression of Kras(G12V) and Braf(D631A) in mouse lung cells markedly enhances tumour initiation, a phenomenon mediated by Craf kinase activity, and effectively accelerates tumour progression when activated in advanced lung adenocarcinomas. We also report a key role for the wild-type Braf kinase in sustaining Kras(G12V)/Braf(D631A)-driven tumours. Ablation of the wild-type Braf allele prevents the development of lung adenocarcinoma by inducing a further increase in MAPK signalling that results in oncogenic toxicity; this effect can be abolished by pharmacological inhibition of Mek to restore tumour growth. However, the loss of wild-type Braf also induces transdifferentiation of club cells, which leads to the rapid development of lethal intrabronchiolar lesions. These observations indicate that the signal intensity of the MAPK pathway is a critical determinant not only in tumour development, but also in dictating the nature of the cancer-initiating cell and ultimately the resulting tumour phenotype.
Assuntos
Adenocarcinoma/genética , Mutação com Perda de Função , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas B-raf/genética , Adenocarcinoma/patologia , Alelos , Animais , Carcinogênese/genética , Progressão da Doença , Feminino , Genes da Neurofibromatose 1 , Humanos , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
Activation-induced cytidine deaminase (AID) is a B-cell-specific enzyme that targets immunoglobulin genes to initiate class switch recombination and somatic hypermutation. In addition, through off-target activity, AID has a much broader effect on genomic instability by initiating oncogenic chromosomal translocations and mutations involved in the development and progression of lymphoma. AID expression is tightly regulated in B cells and its overexpression leads to enhanced genomic instability and lymphoma formation. The phosphatidylinositol 3-kinase δ (PI3Kδ) pathway regulates AID by suppressing its expression in B cells. Drugs for leukaemia or lymphoma therapy such as idelalisib, duvelisib and ibrutinib block PI3Kδ activity directly or indirectly, potentially affecting AID expression and, consequently, genomic stability in B cells. Here we show that treatment of primary mouse B cells with idelalisib or duvelisib, and to a lesser extent ibrutinib, enhanced the expression of AID and increased somatic hypermutation and chromosomal translocation frequency to the Igh locus and to several AID off-target sites. Both of these effects were completely abrogated in AID-deficient B cells. PI3Kδ inhibitors or ibrutinib increased the formation of AID-dependent tumours in pristane-treated mice. Consistently, PI3Kδ inhibitors enhanced AID expression and translocation frequency to IGH and AID off-target sites in human chronic lymphocytic leukaemia and mantle cell lymphoma cell lines, and patients treated with idelalisib, but not ibrutinib, showed increased somatic hypermutation in AID off-targets. In summary, we show that PI3Kδ or Bruton's tyrosine kinase inhibitors increase genomic instability in normal and neoplastic B cells by an AID-dependent mechanism. This effect should be carefully considered, as such inhibitors can be administered to patients for years.
Assuntos
Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Instabilidade Genômica/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Linfócitos B/enzimologia , Linfócitos B/patologia , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Citidina Desaminase/metabolismo , Inibidores Enzimáticos/efeitos adversos , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Switching de Imunoglobulina/efeitos dos fármacos , Cadeias Pesadas de Imunoglobulinas/genética , Isoquinolinas/efeitos adversos , Isoquinolinas/farmacologia , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/patologia , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Piperidinas , Proteínas Tirosina Quinases/antagonistas & inibidores , Purinas/efeitos adversos , Purinas/farmacologia , Pirazóis/efeitos adversos , Pirazóis/farmacologia , Pirimidinas/efeitos adversos , Pirimidinas/farmacologia , Quinazolinonas/efeitos adversos , Quinazolinonas/farmacologia , Recombinação Genética/efeitos dos fármacos , Hipermutação Somática de Imunoglobulina/efeitos dos fármacos , Translocação Genética/efeitos dos fármacosRESUMO
In Saccharomyces cerevisiae, absence of the checkpoint kinase Mec1 (ATR) is viable upon mutations that increase the activity of the ribonucleotide reductase (RNR) complex. Whether this pathway is conserved in mammals remains unknown. Here we show that cells from mice carrying extra alleles of the RNR regulatory subunit RRM2 (Rrm2(TG)) present supraphysiological RNR activity and reduced chromosomal breakage at fragile sites. Moreover, increased Rrm2 gene dosage significantly extends the life span of ATR mutant mice. Our study reveals the first genetic condition in mammals that reduces fragile site expression and alleviates the severity of a progeroid disease by increasing RNR activity.
Assuntos
Quebra Cromossômica , Sítios Frágeis do Cromossomo/genética , Dosagem de Genes/genética , Longevidade/genética , Proteínas Serina-Treonina Quinases/genética , Ribonucleosídeo Difosfato Redutase/genética , Animais , Linhagem Celular , Sobrevivência Celular , Células Cultivadas , Ativação Enzimática/genética , Fibroblastos/citologia , Humanos , Camundongos , Nucleosídeos/metabolismo , Análise de SobrevidaRESUMO
Increasing evidence suggests that Rho family GTPases could have a critical role in the biology of T-cell lymphoma. In ALK-rearranged anaplastic large cell lymphoma (ALCL), a specific subtype of T-cell lymphoma, the Rho family GTPases Cdc42 and Rac1 are activated by the ALK oncogenic activity. In vitro studies have shown that Cdc42 and Rac1 control rather similar phenotypes of ALCL biology such as the proliferation, survival, and migration of lymphoma cells. However, their role and possible redundancy in ALK-driven lymphoma development in vivo are still undetermined. We genetically deleted Cdc42 or Rac1 in a mouse model of ALK-rearranged ALCL to show that either Cdc42 or Rac1 deletion impaired lymphoma development, modified lymphoma morphology, actin filament distribution, and migration properties of lymphoma cells. Cdc42 or Rac1 deletion primarily affected survival rather than proliferation of lymphoma cells. Apoptosis of lymphoma cells was equally induced following Cdc42 or Rac1 deletion, was associated with upregulation of the proapoptotic molecule Bid, and was blocked by Bcl2 overexpression. Remarkably, Cdc42/Rac1 double deletion, but not Cdc42 or Rac1 single deletions, completely prevented NPM-ALK lymphoma dissemination in vivo. Thus, Cdc42 and Rac1 have nonredundant roles in controlling ALK-rearranged lymphoma survival and morphology but are redundant for lymphoma dissemination, suggesting that targeting both GTPases could represent a preferable therapeutic option for ALCL treatment.
Assuntos
Linfoma Difuso de Grandes Células B/metabolismo , Neuropeptídeos/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Sobrevivência Celular/genética , Deleção de Genes , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Neuropeptídeos/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Tirosina Quinases/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Mutations in the RAS-RAF-MEK-ERK pathway are frequent alterations in cancer and RASopathies, and while RAS oncogene activation alone affects 19% of all patients and accounts for approximately 3.4 million new cases every year, less frequent alterations in the cascade's downstream effectors are also involved in cancer etiology. RAS proteins initiate the signaling cascade by promoting the dimerization of RAF kinases, which can act as oncoproteins as well: BRAFV600E is the most common oncogenic driver, mutated in the 8% of all malignancies. Research in this field led to the development of drugs that target the BRAFV600-like mutations (Class I), which are now utilized in clinics, but cause paradoxical activation of the pathway and resistance development. Furthermore, they are ineffective against non-BRAFV600E malignancies that dimerize and could be either RTK/RAS independent or dependent (Class II and III, respectively), which are still lacking an effective treatment. This review discusses the recent advances in anti-RAF therapies, including paradox breakers, dimer-inhibitors, immunotherapies, and other novel approaches, critically evaluating their efficacy in overcoming the therapeutic limitations, and their putative role in blocking the RAS pathway.
Assuntos
Neoplasias , Quinases raf , Proteínas ras , Humanos , Proteínas ras/metabolismo , Proteínas ras/genética , Quinases raf/metabolismo , Quinases raf/antagonistas & inibidores , Quinases raf/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Animais , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Mutação , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Terapia de Alvo MolecularRESUMO
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.
RESUMO
The development of tailored therapies designed to specifically target driver oncogenes has initiated a revolutionary era in cancer biology. The availability of a growing number of selective inhibitors has generated novel experimental and clinical paradigms. These represent an opportunity and a challenge for researchers and clinicians to delve deeper into the intricate dynamics of cancer development and response to treatment. By directly inhibiting key driver oncogenes involved in tumor initiation and progression, scientists have an unprecedented opportunity to conduct longitudinal and clonal evolutionary studies of how cancer cells adapt, rewire, and exploit conflictive or overlapping signaling dependencies in response to treatment in vitro and in vivo. This challenge has to be progressively resolved to discover more effective and personalized cancer therapies.
Assuntos
Neoplasias , Oncogenes , Humanos , Neoplasias/genética , Transformação Celular Neoplásica/genética , Transdução de Sinais , MutaçãoRESUMO
Selection of the best tumor antigen is critical for the therapeutic success of chimeric antigen receptor (CAR) T cells in hematologic malignancies and solid tumors. The anaplastic lymphoma kinase (ALK) receptor is expressed by most neuroblastomas while virtually absent in most normal tissues. ALK is an oncogenic driver in neuroblastoma and ALK inhibitors show promising clinical activity. Here, we describe the development of ALK.CAR-T cells that show potent efficacy in monotherapy against neuroblastoma with high ALK expression without toxicity. For neuroblastoma with low ALK expression, combination with ALK inhibitors specifically potentiates ALK.CAR-T cells but not GD2.CAR-T cells. Mechanistically, ALK inhibitors impair tumor growth and upregulate the expression of ALK, thereby facilitating the activity of ALK.CAR-T cells against neuroblastoma. Thus, while neither ALK inhibitors nor ALK.CAR-T cells will likely be sufficient as monotherapy in neuroblastoma with low ALK density, their combination specifically enhances therapeutic efficacy.
Assuntos
Neuroblastoma , Humanos , Quinase do Linfoma Anaplásico/genética , Quinase do Linfoma Anaplásico/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Antígenos de Neoplasias , Linfócitos T , Linhagem Celular TumoralRESUMO
Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) show potent efficacy in several ALK-driven tumors, but the development of resistance limits their long-term clinical impact. Although resistance mechanisms have been studied extensively in ALK-driven non-small cell lung cancer, they are poorly understood in ALK-driven anaplastic large cell lymphoma (ALCL). Here, we identify a survival pathway supported by the tumor microenvironment that activates phosphatidylinositol 3-kinase γ (PI3K-γ) signaling through the C-C motif chemokine receptor 7 (CCR7). We found increased PI3K signaling in patients and ALCL cell lines resistant to ALK TKIs. PI3Kγ expression was predictive of a lack of response to ALK TKI in patients with ALCL. Expression of CCR7, PI3Kγ, and PI3Kδ were up-regulated during ALK or STAT3 inhibition or degradation and a constitutively active PI3Kγ isoform cooperated with oncogenic ALK to accelerate lymphomagenesis in mice. In a three-dimensional microfluidic chip, endothelial cells that produce the CCR7 ligands CCL19/CCL21 protected ALCL cells from apoptosis induced by crizotinib. The PI3Kγ/δ inhibitor duvelisib potentiated crizotinib activity against ALCL lines and patient-derived xenografts. Furthermore, genetic deletion of CCR7 blocked the central nervous system dissemination and perivascular growth of ALCL in mice treated with crizotinib. Thus, blockade of PI3Kγ or CCR7 signaling together with ALK TKI treatment reduces primary resistance and the survival of persister lymphoma cells in ALCL.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Linfoma Anaplásico de Células Grandes , Humanos , Animais , Camundongos , Crizotinibe/farmacologia , Crizotinibe/uso terapêutico , Receptores Proteína Tirosina Quinases/metabolismo , Quinase do Linfoma Anaplásico , Receptores CCR7/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Células Endoteliais/metabolismo , Fosfatidilinositol 3-Quinases , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Tirosina Quinases , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Linfoma Anaplásico de Células Grandes/tratamento farmacológico , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/patologia , Linhagem Celular Tumoral , Microambiente TumoralRESUMO
Anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC) is treated with ALK tyrosine kinase inhibitors (TKIs), but the lack of activity of immune checkpoint inhibitors (ICIs) is poorly understood. Here, we identified immunogenic ALK peptides to show that ICIs induced rejection of ALK+ tumors in the flank but not in the lung. A single-peptide vaccination restored priming of ALK-specific CD8+ T cells, eradicated lung tumors in combination with ALK TKIs and prevented metastatic dissemination of tumors to the brain. The poor response of ALK+ NSCLC to ICIs was due to ineffective CD8+ T cell priming against ALK antigens and is circumvented through specific vaccination. Finally, we identified human ALK peptides displayed by HLA-A*02:01 and HLA-B*07:02 molecules. These peptides were immunogenic in HLA-transgenic mice and were recognized by CD8+ T cells from individuals with NSCLC, paving the way for the development of a clinical vaccine to treat ALK+ NSCLC.
Assuntos
Vacinas Anticâncer , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Camundongos , Animais , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Quinase do Linfoma Anaplásico/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Vacinas Anticâncer/uso terapêutico , Receptores Proteína Tirosina Quinases/uso terapêutico , Linfócitos T CD8-Positivos/patologia , Vacinas de Subunidades Antigênicas/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/uso terapêutico , Camundongos Transgênicos , VacinaçãoRESUMO
The anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor that is involved in the pathogenesis of different types of human cancers, including neuroblastoma (NB). In NB, ALK overexpression, or point mutations, are associated with poor prognosis and advanced stage disease. Inhibition of ALK kinase activity by small-molecule inhibitors in lung cancers carrying ALK translocations has shown therapeutic potential. However, secondary mutations may occur that, generate tumor resistance to ALK inhibitors. To overcome resistance to ALK inhibitors in NB, we adopted an alternative RNA interference (RNAi)-based therapeutic strategy that is able to knockdown ALK, regardless of its genetic status [mutated, amplified, wild-type (WT)]. NB cell lines, transduced by lentiviral short hairpin RNA (shRNA), showed reduced proliferation and increased apoptosis when ALK was knocked down. In mice, a nanodelivery system for ALK-specific small interfering RNA (siRNA), based on the conjugation of antibodies directed against the NB-selective marker GD(2) to liposomes, showed strong ALK knockdown in vivo in NB cells, which resulted in cell growth arrest, apoptosis, and prolonged survival. ALK knockdown was associated with marked reductions in vascular endothelial growth factor (VEGF) secretion, blood vessel density, and matrix metalloproteinases (MMPs) expression in vivo, suggesting a role for ALK in NB-induced neoangiogenesis and tumor invasion, confirming this gene as a fundamental oncogene in NB.