RESUMO
We developed neratinib-resistant HER2-mutant cancer cells by gradual dose escalation. RNA sequencing identified TORC1 signaling as an actionable mechanism of drug resistance. Primary and acquired neratinib resistance in HER2-mutant breast cancer patient-derived xenografts (PDXs) was also associated with TORC1 hyperactivity. Genetic suppression of RAPTOR or RHEB ablated P-S6 and restored sensitivity to the tyrosine kinase inhibitor. The combination of the TORC1 inhibitor everolimus and neratinib potently arrested the growth of neratinib-resistant xenografts and organoids established from neratinib-resistant PDXs. RNA and whole-exome sequencing revealed RAS-mediated TORC1 activation in a subset of neratinib-resistant models. DNA sequencing of HER2-mutant tumors clinically refractory to neratinib, as well as circulating tumor DNA profiling of patients who progressed on neratinib, showed enrichment of genomic alterations that converge to activate the mTOR pathway.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Quinolinas/farmacologia , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Receptor ErbB-2/efeitos dos fármacos , Receptor ErbB-2/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
The 'Competing interests' statement of this Article has been updated; please see the accompanying Amendment. The original Article has not been corrected online.
RESUMO
Cancers expressing mutant RAS are associated with a weaker response to chemotherapy and a shorter overall patient survival. We have demonstrated that the irreversible inhibitor of ERBB1/2/4, neratinib, inhibits ERBB1/2/4 and causes their internalization and autolysosomal degradation. Fellow-traveler membrane proteins with RTKs, including mutant K-/N-RAS, were also degraded. We discovered that the CDK4/6 inhibitor palbociclib increased autophagosome and then autolysosome levels in a time dependent fashion, did not reduce mTOR activity, and interacted with temsirolimus to kill. Neratinib and palbociclib interacted in a greater than additive manner to increase autophagosome and then autolysosome levels in a time dependent fashion, and to cause tumor cell killing. Killing required the expression of ATM and AMPKα, Beclin1 and ATG5, BAX and BAK and of AIF, but not of caspase 9. In some cells over-expression of BCL-XL was protective whereas in others it was ineffective. The lethality of [neratinib + palbociclib] was modestly enhanced by the PDE5 inhibitor sildenafil and strongly enhanced by the HDAC inhibitor sodium valproate. This was associated with K-RAS degradation and a greater than additive increase in autophagosome and autolysosome levels. Killing by the three-drug combination required ATM and AMPKα, and, to a greater extent, Beclin1 and ATG5. In vivo, [valproate + palbociclib] and [neratinib + valproate + palbociclib] interacted to suppress the growth of a carboplatin/paclitaxel resistant PDX ovarian tumors that express a mutant N-RAS. Our data support performing a future three-drug trial with these agents.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Piperazinas/farmacologia , Piridinas/farmacologia , Quinolinas/farmacologia , Ácido Valproico/farmacologia , Animais , Sinergismo Farmacológico , Feminino , Inibidores de Histona Desacetilases/administração & dosagem , Inibidores de Histona Desacetilases/farmacologia , Humanos , Camundongos , Neoplasias Ovarianas/tratamento farmacológico , Piperazinas/administração & dosagem , Piridinas/administração & dosagem , Quinolinas/administração & dosagem , Ácido Valproico/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Regorafenib is approved for the treatment of colorectal cancer and hepatocellular carcinoma. In the trial NCT02466802, we have discovered that regorafenib can be safely combined with the phosphodiesterase 5 inhibitor sildenafil in advanced solid tumor patients. The present studies determined whether the approved ERBB1/2/4 and RAS downregulating drug neratinib, could enhance the lethality of [regorafenib + sildenafil]. Neratinib enhanced [regorafenib + sildenafil] lethality in a greater than additive fashion in colon cancer cells. The drug combination reduced the expression of mutant K-RAS and of multiple histone deacetylase (HDAC) proteins that required autophagosome formation. It caused green fluorescent protein or red fluorescent protein-tagged forms of K-RAS V12 to localize into large intracellular vesicles. Compared with [regorafenib + sildenafil], the three-drug combination caused greater and more prolonged activation of the ATM-AMPK-ULK-1 pathway and caused a greater suppression and prolonged inactivation of mammalian target of rapamycin, AKT, and p70 S6K. Approximately 70% of enhanced lethality caused by neratinib required ataxia-telangiectasia-mutated (ATM)-AMP-dependent protein kinase (AMPK) signaling whereas knockdown of Beclin1, ATG5, FADD, and CD95 completely prevented the elevated killing effect. Exposure of cells to [regorafenib + sildenafil] reduced the expression of the checkpoint immunotherapy biomarkers programmed death-ligand 1, ornithine decarboxylase, and indoleamine 2,3-dioxygenase-1 and increased the expression of major histocompatibility complex A (MHCA), which also required autophagosome formation. Knockdown of specific HDAC proteins recapitulated the effects observed using chemical agents. In vivo, using mouse cancer models, neratinib significantly enhanced the antitumor efficacy of [regorafenib + sildenafil]. Our data support performing a new three drug Phase I trial combining regorafenib, sildenafil, and neratinib.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Compostos de Fenilureia/farmacologia , Piridinas/farmacologia , Quinolinas/farmacologia , Citrato de Sildenafila/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Inibidores da Fosfodiesterase 5/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: The human epidermal growth factor receptor 2 (ERBB2) may harbour somatic mutations that drive breast tumorigenesis. Here, we study prevalence, tumour characteristics and disease outcome of ERBB2 mutations in a large unselected cohort of metastatic breast cancer (mBC) patients. METHODS: We retrospectively included all mBC patients with sufficient primary breast tumour, diagnosed between 2000 and 2015 (n = 775). Genomic DNA was subjected to a targeted-resequencing assay to identify hotspot mutations in exon 8, 17, 19, 20, and 21 of ERBB2. We studied demographics, tumour characteristics, median distant disease-free survival (DDFS), using a time-to-event analysis and time to progression (TTP) and overall survival (OS) upon metastasis, using Kaplan-Meier and log-rank statistics to assess differences between ERBB2-mutation statuses. RESULTS: ERBB2 mutations were observed in 1.8% of the samples (13/721). Patient and tumour characteristics were independent of ERBB2 mutations. Luminal ERBB2-mutated (ERBB2mut+) cases (n = 5) had a shorter DDFS than ERBB2mut- cases (median DDFS 0.8 vs. > 4.0 years, p = 0.02). ER-positive ERBB2mut+ patients who received an aromatase inhibitor (AI) as first-line treatment (stage IV disease) had a worse TTP vs. ERBB2mut- patients (n = 3 vs. 156; median TTP 103 vs. 311 days, p = 0.04). OS for all subtypes was lower for ERBB2mut+ vs. ERBB2mut- cases (n = 11 vs. 669; median OS 1.1 vs. 2.3 years, p = 0.46). CONCLUSION: ERBB2mut+ are rare in patients in whom mBC developed and no evidence was found for an association with specific types of BC or patient characteristics, although outcomes of ERBB2mut+ carriers might be worse. The latter, however, needs to be validated in larger populations.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Receptor ErbB-2/genética , Adulto , Idoso , Neoplasias da Mama/mortalidade , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Mutação , Estudos Retrospectivos , Resultado do TratamentoRESUMO
PURPOSE: We examined the role of ERBB2-activating mutations in endocrine therapy resistance in estrogen receptor positive (ER+) breast cancer. EXPERIMENTAL DESIGN: ERBB2 mutation frequency was determined from large genomic databases. Isogenic knock-in ERBB2 mutations in ER+ MCF7 cells and xenografts were used to investigate estrogen-independent growth. Structural analysis was used to determine the molecular interaction of HER L755S with HER3. Small molecules and siRNAs were used to inhibit PI3Kα, TORC1, and HER3. RESULTS: Genomic data revealed a higher rate of ERBB2 mutations in metastatic versus primary ER+ tumors. MCF7 cells with isogenically incorporated ERBB2 kinase domain mutations exhibited resistance to estrogen deprivation and to fulvestrant both in vitro and in vivo, despite maintaining inhibition of ERα transcriptional activity. Addition of the irreversible HER2 tyrosine kinase inhibitor neratinib restored sensitivity to fulvestrant. HER2-mutant MCF7 cells expressed higher levels of p-HER3, p-AKT, and p-S6 than cells with wild-type HER2. Structural analysis of the HER2 L755S variant implicated a more flexible active state, potentially allowing for enhanced dimerization with HER3. Treatment with a PI3Kα inhibitor, a TORC1 inhibitor or HER3 siRNA, but not a MEK inhibitor, restored sensitivity to fulvestrant and to estrogen deprivation. Inhibition of mutant HER2 or TORC1, when combined with fulvestrant, equipotently inhibited growth of MCF7/ERBB2 V777L xenografts, suggesting a role for TORC1 in antiestrogen resistance induced by ERBB2 mutations. CONCLUSIONS: ERBB2 mutations hyperactivate the HER3/PI3K/AKT/mTOR axis, leading to antiestrogen resistance in ER+ breast cancer. Dual blockade of the HER2 and ER pathways is required for the treatment of ER+/HER2 mutant breast cancers.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Receptor alfa de Estrogênio/genética , Receptor ErbB-2/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Estrogênios/metabolismo , Feminino , Fulvestranto/efeitos adversos , Fulvestranto/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Xenoenxertos , Humanos , Células MCF-7 , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Mutação/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Quinolinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-3/genética , Mutações Sintéticas Letais/efeitos dos fármacos , Mutações Sintéticas Letais/genéticaRESUMO
AIMS: Oprozomib is an oral, second-generation, irreversible proteasome inhibitor currently in clinical development for haematologic malignancies, including multiple myeloma and other malignancies. Oprozomib is a rare example of a small molecule drug that demonstrates cytochrome P450 (CYP) mRNA suppression. This unusual property elicits uncertainty regarding the optimal approach for predicting its drug-drug interaction (DDI) risk. The current study aims to understand DDI potential during early clinical development of oprozomib. METHODS: To support early development of oprozomib (e.g. inclusion/exclusion criteria, combination study design), we used human hepatocyte data and physiologically-based pharmacokinetic (PBPK) modelling to predict its CYP3A4-mediated DDI potential. Subsequently, a clinical DDI study using midazolam as the substrate was conducted in patients with advanced malignancies. RESULTS: The clinical DDI study enrolled a total of 21 patients, 18 with advanced solid tumours. No patient discontinued oprozomib due to a treatment-related adverse event. The PBPK model prospectively predicted oprozomib 300 mg would not cause a clinically relevant change in exposure to CYP3A4 substrates (≤30%), which was confirmed by the results of this clinical DDI study. CONCLUSIONS: These results indicate oprozomib has a low potential to inhibit the metabolism of CYP3A4 substrates in humans. The study shows that cultured human hepatocytes are a more reliable system for DDI prediction than human liver microsomes for studying this class of compounds. Developing a PBPK model prior to a clinical DDI study has been valuable in supporting clinical development of oprozomib.
Assuntos
Inibidores do Citocromo P-450 CYP3A/farmacocinética , Citocromo P-450 CYP3A/metabolismo , Neoplasias/tratamento farmacológico , Oligopeptídeos/farmacocinética , Inibidores de Proteassoma/farmacocinética , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Inibidores do Citocromo P-450 CYP3A/administração & dosagem , Desenvolvimento de Medicamentos , Interações Medicamentosas , Feminino , Hepatócitos , Humanos , Masculino , Microssomos Hepáticos , Midazolam/administração & dosagem , Midazolam/farmacocinética , Pessoa de Meia-Idade , Modelos Biológicos , Neoplasias/sangue , Neoplasias/patologia , Oligopeptídeos/administração & dosagem , Cultura Primária de Células , Inibidores de Proteassoma/administração & dosagem , Adulto JovemRESUMO
PURPOSE: The phase III ExteNET trial showed improved invasive disease-free survival in patients with HER2+ breast cancer treated with neratinib versus placebo after trastuzumab-based adjuvant therapy. The benefit from neratinib appeared to be greater in patients with ER+/HER2+ tumors. We thus sought to discover mechanisms that may explain the benefit from extended adjuvant therapy with neratinib.Experimental Design: Mice with established ER+/HER2+ MDA-MB-361 tumors were treated with paclitaxel plus trastuzumab ± pertuzumab for 4 weeks, and then randomized to fulvestrant ± neratinib treatment. The benefit from neratinib was evaluated by performing gene expression analysis for 196 ER targets, ER transcriptional reporter assays, and cell-cycle analyses. RESULTS: Mice receiving "extended adjuvant" therapy with fulvestrant/neratinib maintained a complete response, whereas those treated with fulvestrant relapsed rapidly. In three ER+/HER2+ cell lines (MDA-MB-361, BT-474, UACC-893) but not in ER+/HER2- MCF7 cells, treatment with neratinib induced ER reporter transcriptional activity, whereas treatment with fulvestrant resulted in increased HER2 and EGFR phosphorylation, suggesting compensatory reciprocal crosstalk between the ER and ERBB RTK pathways. ER transcriptional reporter assays, gene expression, and immunoblot analyses showed that treatment with neratinib/fulvestrant, but not fulvestrant, potently inhibited growth and downregulated ER reporter activity, P-AKT, P-ERK, and cyclin D1 levels. Finally, similar to neratinib, genetic and pharmacologic inactivation of cyclin D1 enhanced fulvestrant action against ER+/HER2+ breast cancer cells. CONCLUSIONS: These data suggest that ER blockade leads to reactivation of ERBB RTKs and thus extended ERBB blockade is necessary to achieve durable clinical outcomes in patients with ER+/HER2+ breast cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Biomarcadores Tumorais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Quimioterapia Adjuvante , Modelos Animais de Doenças , Feminino , Fulvestranto/administração & dosagem , Humanos , Imuno-Histoquímica , Camundongos , Quinolinas/administração & dosagem , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mutations in ERBB2, the gene encoding epidermal growth factor receptor (EGFR) family member HER2, are common in and drive the growth of "HER2-negative" (not ERBB2 amplified) tumors but are rare in "HER2-positive" (ERBB2 amplified) breast cancer. We analyzed DNA-sequencing data from HER2-positive patients and used cell lines and a patient-derived xenograft model to test the consequence of HER2 mutations on the efficacy of anti-HER2 agents such as trastuzumab, lapatinib, and neratinib, an irreversible pan-EGFR inhibitor. HER2 mutations were present in ~7% of HER2-positive tumors, all of which were metastatic but not all were previously treated. Compared to HER2 amplification alone, in both patients and cultured cell lines, the co-occurrence of HER2 mutation and amplification was associated with poor response to trastuzumab and lapatinib, the standard-of-care anti-HER2 agents. In mice, xenografts established from a patient whose HER2-positive tumor acquired a D769Y mutation in HER2 after progression on trastuzumab-based therapy were resistant to trastuzumab or lapatinib but were sensitive to neratinib. Clinical data revealed that six heavily pretreated patients with tumors bearing coincident HER2 amplification and mutation subsequently exhibited a statistically significant response to neratinib monotherapy. Thus, these findings indicate that coincident HER2 mutation reduces the efficacy of therapies commonly used to treat HER2-positive breast cancer, particularly in metastatic and previously HER2 inhibitor-treated patients, as well as potentially in patients scheduled for first-line treatment. Therefore, we propose that clinical studies testing the efficacy of neratinib are warranted selectively in breast cancer patients whose tumors carry both amplification and mutation of ERBB2/HER2.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Inibidores de Proteínas Quinases/farmacologia , Quinolinas/farmacologia , Receptor ErbB-2/genética , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Lapatinib/farmacologia , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Nus , Mutação , Modelos de Riscos Proporcionais , Trastuzumab/farmacologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Endocrine therapies are the mainstay of treatment for oestrogen receptor (ER)-positive (ER+) breast cancer (BC). However, resistance remains problematic largely due to enhanced cross-talk between ER and growth factor pathways, circumventing the need for steroid hormones. Previously, we reported the anti-proliferative effect of everolimus (RAD001-mTORC1 inhibitor) with endocrine therapy in resistance models; however, potential routes of escape from treatment via ERBB2/3 signalling were observed. We hypothesised that combined targeting of three cellular nodes (ER, ERBB, and mTORC1) may provide enhanced long-term clinical utility. METHODS: A panel of ER+ BC cell lines adapted to long-term oestrogen deprivation (LTED) and expressing ESR1 wt or ESR1 Y537S , modelling acquired resistance to an aromatase-inhibitor (AI), were treated in vitro with a combination of RAD001 and neratinib (pan-ERBB inhibitor) in the presence or absence of oestradiol (E2), tamoxifen (4-OHT), or fulvestrant (ICI182780). End points included proliferation, cell signalling, cell cycle, and effect on ER-mediated transactivation. An in-vivo model of AI resistance was treated with monotherapies and combinations to assess the efficacy in delaying tumour progression. RNA-seq analysis was performed to identify changes in global gene expression as a result of the indicated therapies. RESULTS: Here, we show RAD001 and neratinib (pan-ERBB inhibitor) caused a concentration-dependent decrease in proliferation, irrespective of the ESR1 mutation status. The combination of either agent with endocrine therapy further reduced proliferation but the maximum effect was observed with a triple combination of RAD001, neratinib, and endocrine therapy. In the absence of oestrogen, RAD001 caused a reduction in ER-mediated transcription in the majority of the cell lines, which associated with a decrease in recruitment of ER to an oestrogen-response element on the TFF1 promoter. Contrastingly, neratinib increased both ER-mediated transactivation and ER recruitment, an effect reduced by the addition of RAD001. In-vivo analysis of an LTED model showed the triple combination of RAD001, neratinib, and fulvestrant was most effective at reducing tumour volume. Gene set enrichment analysis revealed that the addition of neratinib negated the epidermal growth factor (EGF)/EGF receptor feedback loops associated with RAD001. CONCLUSIONS: Our data support the combination of therapies targeting ERBB2/3 and mTORC1 signalling, together with fulvestrant, in patients who relapse on endocrine therapy and retain a functional ER.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Receptor alfa de Estrogênio/genética , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Receptor ErbB-2/genética , Receptor ErbB-3/genética , Inibidores da Aromatase/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Epidérmico/antagonistas & inibidores , Fator de Crescimento Epidérmico/genética , Estradiol/farmacologia , Estrogênios/metabolismo , Everolimo/farmacologia , Feminino , Fulvestranto/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/patologia , Quinolinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-3/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Tamoxifeno/farmacologiaRESUMO
The irreversible ERBB1/2/4 inhibitor neratinib has been shown in vitro to rapidly reduce the expression of ERBB1/2/4 and RAS proteins via autophagic/lysosomal degradation. We have recently demonstrated that neratinib and valproate interact to suppress the growth of 4T1 mammary tumors but had not defined whether the [neratinib + valproate] drug combination, in a mouse, had altered the biology of the 4T1 cells. Exposure of 4T1 mammary tumors to [neratinib + valproate] for three days resulted, two weeks later, in tumors that expressed less ERBB1, K-RAS, N-RAS, indoleamine-pyrrole 2,3-dioxygenase (IDO-1), ornithine decarboxylase (ODC) and had increased Class I MHCA expression. Tumors previously exposed to [neratinib + valproate] grew more slowly than those exposed to vehicle control and contained more CD8+ cells and activated NK cells. M1 but not M2 macrophage infiltration was significantly enhanced by the drug combination. In vitro exposure of 4T1 tumor cells to [neratinib + valproate] variably reduced the expression of histone deacetylases 1-11. In vivo, prior exposure of tumors to [neratinib + valproate] permanently reduced the expression of HDACs 1-3, 6 and 10. Combined knock down of HDACs 1/2/3 or of 3/10 rapidly reduced the expression IDO-1, and ODC and increased the expression of MHCA. H&E staining of normal tissues at animal nadir revealed no obvious cyto-architectural differences between control and drug-treated animals. We conclude that [neratinib + valproate] evolves 4T1 tumors to grow more slowly and to be more sensitive to checkpoint immunotherapy antibodies.
RESUMO
The irreversible ERBB1/2/4 inhibitor neratinib has been shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET, PDGFRα and mutant RAS proteins via autophagic degradation. Neratinib interacted in an additive to synergistic fashion with the approved PARP1 inhibitor niraparib to kill ovarian cancer cells. Neratinib and niraparib caused the ATM-dependent activation of AMPK which in turn was required to cause mTOR inactivation, ULK-1 activation and ATG13 phosphorylation. The drug combination initially increased autophagosome levels followed later by autolysosome levels. Preventing autophagosome formation by expressing activated mTOR or knocking down of Beclin1, or knock down of the autolysosome protein cathepsin B, reduced drug combination lethality. The drug combination caused an endoplasmic reticulum stress response as judged by enhanced eIF2α phosphorylation that was responsible for reducing MCL-1 and BCL-XL levels and increasing ATG5 and Beclin1 expression. Knock down of BIM, but not of BAX or BAK, reduced cell killing. Expression of activated MEK1 prevented the drug combination increasing BIM expression and reduced cell killing. Downstream of the mitochondrion, drug lethality was partially reduced by knock down of AIF, but expression of dominant negative caspase 9 was not protective. Our data demonstrate that neratinib and niraparib interact to kill ovarian cancer cells through convergent DNA damage and endoplasmic reticulum stress signaling. Cell killing required the induction of autophagy and was cathepsin B and AIF -dependent, and effector caspase independent.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Indazóis/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Piperidinas/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Quinolinas/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Feminino , Humanos , Indazóis/farmacologia , Neoplasias Ovarianas/patologia , Piperidinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinolinas/farmacologiaRESUMO
Somatic mutations of ERBB2 and ERBB3 (which encode HER2 and HER3, respectively) are found in a wide range of cancers. Preclinical modelling suggests that a subset of these mutations lead to constitutive HER2 activation, but most remain biologically uncharacterized. Here we define the biological and therapeutic importance of known oncogenic HER2 and HER3 mutations and variants of unknown biological importance by conducting a multi-histology, genomically selected, 'basket' trial using the pan-HER kinase inhibitor neratinib (SUMMIT; clinicaltrials.gov identifier NCT01953926). Efficacy in HER2-mutant cancers varied as a function of both tumour type and mutant allele to a degree not predicted by preclinical models, with the greatest activity seen in breast, cervical and biliary cancers and with tumours that contain kinase domain missense mutations. This study demonstrates how a molecularly driven clinical trial can be used to refine our biological understanding of both characterized and new genomic alterations with potential broad applicability for advancing the paradigm of genome-driven oncology.
Assuntos
Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Quinolinas/farmacologia , Quinolinas/uso terapêutico , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-3/antagonistas & inibidores , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Mutação de Sentido Incorreto , Neoplasias/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Quinolinas/efeitos adversos , Receptor ErbB-2/química , Receptor ErbB-2/genética , Receptor ErbB-3/química , Receptor ErbB-3/genética , Resultado do TratamentoRESUMO
The irreversible ERBB1/2/4 inhibitor, neratinib, down-regulates the expression of ERBB1/2/4 as well as the levels of MCL-1 and BCL-XL. Venetoclax (ABT199) is a BCL-2 inhibitor. At physiologic concentrations neratinib interacted in a synergistic fashion with venetoclax to kill HER2 + and TNBC mammary carcinoma cells. This was associated with the drug-combination: reducing the expression and phosphorylation of ERBB1/2/3; in an eIF2α-dependent fashion reducing the expression of MCL-1 and BCL-XL and increasing the expression of Beclin1 and ATG5; and increasing the activity of the ATM-AMPKα-ULK1 S317 pathway which was causal in the formation of toxic autophagosomes. Although knock down of BAX or BAK reduced drug combination lethality, knock down of BAX and BAK did not prevent the drug combination from increasing autophagosome and autolysosome formation. Knock down of ATM, AMPKα, Beclin1 or over-expression of activated mTOR prevented the induction of autophagy and in parallel suppressed tumor cell killing. Knock down of ATM, AMPKα, Beclin1 or cathepsin B prevented the drug-induced activation of BAX and BAK whereas knock down of BID was only partially inhibitory. A 3-day transient exposure of established estrogen-independent HER2 + BT474 mammary tumors to neratinib or venetoclax did not significantly alter tumor growth whereas exposure to [neratinib + venetoclax] caused a significant 7-day suppression of growth by day 19. The drug combination neither altered animal body mass nor behavior. We conclude that venetoclax enhances neratinib lethality by facilitating toxic BH3 domain protein activation via autophagy which enhances the efficacy of neratinib to promote greater levels of cell killing.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Quinolinas/farmacologia , Sulfonamidas/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Autofagia/efeitos dos fármacos , Neoplasias da Mama/patologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Linhagem Celular Tumoral , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inibidores , Feminino , Humanos , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Quinolinas/uso terapêutico , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Receptor ErbB-4/antagonistas & inibidores , Receptores de Estrogênio/metabolismo , Sulfonamidas/uso terapêutico , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The FDA approved irreversible inhibitor of ERBB1/2/4, neratinib, was recently shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET and mutant K-RAS via autophagic degradation. In the present studies, in a dose-dependent fashion, neratinib reduced the expression levels of mutant K-RAS or of mutant N-RAS, which was augmented in an additive to greater than additive fashion by the HDAC inhibitors sodium valproate and AR42. Neratinib could reduce PDGFRα levels in GBM cells, that was enhanced by sodium valproate. Knock down of Beclin1 or of ATG5 prevented neratinib and neratinib combined with sodium valproate / AR42 from reducing the expression of mutant N-RAS in established PDX and fresh PDX models of ovarian cancer and melanoma, respectively. Neratinib and the drug combinations caused the co-localization of mutant RAS proteins and ERBB2 with Beclin1 and cathepsin B. The drug combination activated the AMP-dependent protein kinase that was causal in enhancing HMG Co A reductase phosphorylation. Collectively, our data reinforce the concept that the irreversible ERBB1/2/4 inhibitor neratinib has the potential for use in the treatment of tumors expressing mutant RAS proteins.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Quinolinas/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inibidores , GTP Fosfo-Hidrolases/genética , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Humanos , Proteínas de Membrana/genética , Neoplasias/genética , Neoplasias/patologia , Fenilbutiratos/farmacologia , Fenilbutiratos/uso terapêutico , Proteínas Proto-Oncogênicas p21(ras)/genética , Quinolinas/uso terapêutico , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-4/antagonistas & inibidores , Ácido Valproico/farmacologia , Ácido Valproico/uso terapêuticoRESUMO
Patients whose NSCLC tumors become afatinib resistant presently have few effective therapeutic options to extend their survival. Afatinib resistant NSCLC cells were sensitive to clinically relevant concentrations of the irreversible pan-HER inhibitor neratinib, but not by the first generation ERBB1/2/4 inhibitor lapatinib. In multiple afatinib resistant NSCLC clones, HDAC inhibitors reduced the expression of ERBB1/3/4, but activated c-SRC, which resulted in higher total levels of ERBB1/3 phosphorylation. Neratinib also rapidly reduced the expression of ERBB1/2/3/4, c-MET and of mutant K-/N-RAS; K-RAS co-localized with phosphorylated ATG13 and with cathepsin B in vesicles. Combined exposure of cells to [neratinib + HDAC inhibitors] caused inactivation of mTORC1 and mTORC2, enhanced autophagosome and subsequently autolysosome formation, and caused an additive to greater than additive induction of cell death. Knock down of Beclin1 or ATG5 prevented HDAC inhibitors or neratinib from reducing ERBB1/3/4 and K-/N-RAS expression and reduced [neratinib + HDAC inhibitor] lethality. Neratinib and HDAC inhibitors reduced the expression of multiple HDAC proteins via autophagy that was causal in the reduced expression of PD-L1, PD-L2 and ornithine decarboxylase, and increased expression of Class I MHCA. In vivo, neratinib and HDAC inhibitors interacted to suppress the growth of 4T1 mammary tumors, an effect that was enhanced by an anti-PD-1 antibody. Our data support the premises that neratinib lethality can be enhanced by HDAC inhibitors, that neratinib may be a useful therapeutic tool in afatinib resistant NSCLC, and that [neratinib + HDAC inhibitor] exposure facilitates anti-tumor immune responses.
RESUMO
We report a HER2T798I gatekeeper mutation in a patient with HER2L869R-mutant breast cancer with acquired resistance to neratinib. Laboratory studies suggested that HER2L869R is a neratinib-sensitive, gain-of-function mutation that upon dimerization with mutant HER3E928G, also present in the breast cancer, amplifies HER2 signaling. The patient was treated with neratinib and exhibited a sustained partial response. Upon clinical progression, HER2T798I was detected in plasma tumor cell-free DNA. Structural modeling of this acquired mutation suggested that the increased bulk of isoleucine in HER2T798I reduces neratinib binding. Neratinib blocked HER2-mediated signaling and growth in cells expressing HER2L869R but not HER2L869R/T798I In contrast, afatinib and the osimertinib metabolite AZ5104 strongly suppressed HER2L869R/T798I-induced signaling and cell growth. Acquisition of HER2T798I upon development of resistance to neratinib in a breast cancer with an initial activating HER2 mutation suggests HER2L869R is a driver mutation. HER2T798I-mediated neratinib resistance may be overcome by other irreversible HER2 inhibitors like afatinib.Significance: We found an acquired HER2 gatekeeper mutation in a patient with HER2-mutant breast cancer upon clinical progression on neratinib. We speculate that HER2T798I may arise as a secondary mutation following response to effective HER2 tyrosine kinase inhibitors (TKI) in other cancers with HER2-activating mutations. This resistance may be overcome by other irreversible HER2 TKIs, such as afatinib. Cancer Discov; 7(6); 575-85. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 539.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores de Proteínas Quinases/uso terapêutico , Quinolinas/uso terapêutico , Receptor ErbB-2/genética , Afatinib , Linhagem Celular Tumoral , Feminino , Humanos , Pessoa de Meia-Idade , Mutação , Fenótipo , Quinazolinas/farmacologia , Receptor ErbB-2/antagonistas & inibidoresRESUMO
PURPOSE: Tolerability, pharmacokinetics (PK), pharmacodynamics, and antitumor activity of carfilzomib, a selective proteasome inhibitor, administered twice weekly by 2-10-min intravenous (IV) infusion on days 1, 2, 8, 9, 15, and 16 in 28-day cycles, were assessed in patients with advanced solid tumors in this phase I/II study. METHODS: Adult patients with solid tumors progressing after ≥1 prior therapies were enrolled. The dose was 20 mg/m(2) in week 1 of cycle 1 and 20, 27, or 36 mg/m(2) thereafter. The maximum tolerated dose or protocol-defined maximum planned dose (MPD) identified during dose escalation was administered to an expansion cohort and to patients with small cell lung, non-small cell lung, ovarian, and renal cancer in phase II tumor-specific cohorts. RESULTS: Fourteen patients received carfilzomib during dose escalation. The single dose-limiting toxicity at 20/36 mg/m(2) was grade 3 fatigue, establishing the MPD as the expansion and phase II dose. Sixty-five additional patients received carfilzomib at the MPD. Adverse events included fatigue, nausea, anorexia, and dyspnea. Carfilzomib PK was dose proportional with a half-life <1 h. All doses resulted in at least 80 % proteasome inhibition in blood. Partial responses occurred in two patients in phase I, with 21.5 % stable disease after four cycles in evaluable patients in the expansion and phase II cohorts. CONCLUSION: Carfilzomib 20/36 mg/m(2) was well tolerated when administered twice weekly by 2-10-min IV infusion. At this dose and infusion rate, carfilzomib inhibited the proteasome in blood but demonstrated limited antitumor activity in patients with advanced solid tumors.