RESUMO
BACKGROUND: The PI 3-kinase (PI3K) pathway has been implicated as a target for melanoma therapy. METHODS: Given the high degree of genetic heterogeneity in melanoma, we sought to understand the breadth of variation in PI3K signalling in the large NZM panel of early passage cell lines developed from metastatic melanomas. RESULTS: We find the vast majority of lines show upregulation of this pathway, and this upregulation is achieved by a wide range of mechanisms. Expression of all class-IA PI3K isoforms was readily detected in these cell lines. A range of genetic changes in different components of the PI3K pathway was seen in different lines. Coding variants or amplification were identified in the PIK3CA gene, and amplification of the PK3CG gene was common. Deletions in the PIK3R1 and PIK3R2 regulatory subunits were also relatively common. Notably, no genetic variants were seen in the PIK3CD gene despite p110δ being expressed in many of the lines. Genetic variants were detected in a number of genes that encode phosphatases regulating the PI3K signalling, with reductions in copy number common in PTEN, INPP4B, INPP5J, PHLLP1 and PHLLP2 genes. While the pan-PI3K inhibitor ZSTK474 attenuated cell growth in all the lines tested, isoform-selective inhibition of p110α and p110δ inhibited cell growth in only a subset of the lines and the inhibition was only partial. This suggests that functional redundancy exists between PI3K isoforms. Furthermore, while ZSTK474 was initially effective in melanoma cells with induced resistance to vemurafenib, a subset of these cell lines concurrently developed partial resistance to PI3K inhibition. Importantly, mTOR-selective or mTOR/PI3K dual inhibitors effectively inhibited cell growth in all the lines, including those already resistant to BRAF inhibitors and ZSTK474. CONCLUSIONS: Overall, this indicates a high degree of diversity in the way the PI3K pathway is activated in different melanoma cell lines and that mTOR is the most effective point for targeting the growth via the PI3K pathway across all of these cell lines.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Melanoma/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/uso terapêutico , Neoplasias Cutâneas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Humanos , Isoenzimas , Melanoma/tratamento farmacológico , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Fosfatidilinositol 3-Quinase/genética , Fosfatidilinositol 3-Quinases/genética , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Triazinas/uso terapêutico , Regulação para Cima , Vemurafenib/uso terapêuticoRESUMO
As part of our investigation into pyrazolo[1,5-a]pyridines as novel p110α selective PI3 kinase inhibitors, we report a range of analogues with improved aqueous solubility by the addition of a basic amine. The compounds demonstrated comparable p110α potency and selectivity to earlier compounds but with up to 1000× greater aqueous solubility, as the hydrochloride salts. The compounds also displayed good activity in a cellular assay of PI3 kinase activity.
Assuntos
Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Hidrazonas/síntese química , Hidrazonas/farmacologia , Hidrazonas/toxicidade , Camundongos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/toxicidade , Pirazóis/síntese química , Pirazóis/toxicidade , Piridinas/síntese química , Piridinas/toxicidade , SolubilidadeRESUMO
A novel series of TGX-221 analogues was prepared and tested for their potency against the p110α, p110ß, and p110δ isoforms of the PI3K enzyme, and in two cellular assays. The biological results were interpreted in terms of a p110ß comparative model, in order to account for their selectivity towards this isoform. A CH2NH type linker is proposed to allow binding into the specificity pocket proposed to accommodate the high p110ß-selectivity of TGX-221, although there was limited steric tolerance for substituents on the pendant ring with the 2-position most favourable for substitution.
Assuntos
Antineoplásicos/farmacologia , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Subunidades Proteicas/antagonistas & inibidores , Pirimidinonas/farmacologia , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Classe Ia de Fosfatidilinositol 3-Quinase/química , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Morfolinas/síntese química , Inibidores de Proteínas Quinases/síntese química , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Pirimidinonas/síntese química , Relação Estrutura-AtividadeRESUMO
We have made a novel series of pyrazolo[1,5-a]pyridines as PI3 kinase inhibitors, and demonstrated their selectivity for the p110α isoform over the other Class Ia PI3 kinases. We investigated the SAR around the pyrazolo[1,5-a]pyridine ring system, and found compound 5x to be a particularly potent example (p110α IC(50) 0.9nM). This compound inhibits cell proliferation and phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity, and showed in vivo activity in an HCT-116 human xenograft model.
Assuntos
Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/química , Pirazóis/química , Piridinas/química , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Sítios de Ligação , Linhagem Celular Tumoral , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piridinas/síntese química , Piridinas/farmacologia , Transplante HeterólogoRESUMO
Structure-activity relationship studies of the pyrazolo[1,5-a]pyridine class of PI3 kinase inhibitors show that substitution off the hydrazone nitrogen and replacement of the sulfonyl both gave a loss of p110α selectivity, with the exception of an N-hydroxyethyl analogue. Limited substitutions were tolerated around the phenyl ring; in particular the 2,5-substitution pattern was important for PI3 kinase activity. The N-hydroxyethyl compound also showed good inhibition of cell proliferation and inhibition of phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity. It had suitable pharmacokinetics for evaluation in vivo, and showed tumour growth inhibition in two human tumour cell lines in xenograft studies. This work has provided suggestions for the design of more soluble analogues.
Assuntos
Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/química , Pirazóis/química , Piridinas/química , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Sítios de Ligação , Linhagem Celular Tumoral , Simulação por Computador , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piridinas/síntese química , Piridinas/farmacocinética , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
Genetic alterations in PI3K (phosphoinositide 3-kinase) signalling are common in cancer and include deletions in PTEN (phosphatase and tensin homologue deleted on chromosome 10), amplifications of PIK3CA and mutations in two distinct regions of the PIK3CA gene. This suggests drugs targeting PI3K, and p110α in particular, might be useful in treating cancers. Broad-spectrum inhibition of PI3K is effective in preventing growth factor signalling and tumour growth, but suitable inhibitors of p110α have not been available to study the effects of inhibiting this isoform alone. In the present study we characterize a novel small molecule, A66, showing the S-enantiomer to be a highly specific and selective p110α inhibitor. Using molecular modelling and biochemical studies, we explain the basis of this selectivity. Using a panel of isoform-selective inhibitors, we show that insulin signalling to Akt/PKB (protein kinase B) is attenuated by the additive effects of inhibiting p110α/p110ß/p110δ in all cell lines tested. However, inhibition of p110α alone was sufficient to block insulin signalling to Akt/PKB in certain cell lines. The responsive cell lines all harboured H1047R mutations in PIK3CA and have high levels of p110α and class-Ia PI3K activity. This may explain the increased sensitivity of these cells to p110α inhibitors. We assessed the activation of Akt/PKB and tumour growth in xenograft models and found that tumours derived from two of the responsive cell lines were also responsive to A66 in vivo. These results show that inhibition of p110α alone has the potential to block growth factor signalling and reduce growth in a subset of tumours.
Assuntos
Inibidores Enzimáticos/uso terapêutico , Neoplasias Experimentais/patologia , Neoplasias Experimentais/prevenção & controle , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Animais , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Inibidores Enzimáticos/farmacocinética , Proteínas de Homeodomínio/fisiologia , Humanos , Camundongos , Camundongos Knockout , Neoplasias Experimentais/metabolismo , Isoformas de Proteínas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
The fact that 10% of colorectal cancer tumors harbor BRAF V600E mutations suggested targeting BRAF as a potential therapy. However, BRAF inhibitors have only limited single-agent efficacy in this context. The potential for combination therapy has been shown by the BEACON trial where targeting the EGF receptor with cetuximab greatly increased efficacy of BRAF inhibitors in BRAF-mutant colorectal cancer. Therefore, we explored whether efficacy of the mutant BRAF inhibitor vemurafenib could be enhanced by cotargeting of either oncogenic WNT/ß-catenin signaling or VEGFR signaling. We find the WNT/ß-catenin inhibitors pyrvinium, ICG-001 and PKF118-310 attenuate growth of colorectal cancer cell lines in vitro with BRAF-mutant lines being relatively more sensitive. Pyrvinium combined with vemurafenib additively or synergistically attenuated growth of colorectal cancer cell lines in vitro. The selective and potent VEGFR inhibitor axitinib was most effective against BRAF-mutant colorectal cancer cell lines in vitro, but the addition of vemurafenib did not significantly increase these effects. When tested in vivo in animal tumor models, both pyrvinium and axitinib were able to significantly increase the ability of vemurafenib to attenuate tumor growth in xenografts of BRAF-mutant colorectal cancer cells. The magnitude of these effects was comparable with that induced by a combination of vemurafenib and cetuximab. This was associated with additive effects on release from tumor cells and tumor microenvironment cell types of substances that would normally aid tumor progression. Taken together, these preclinical data indicate that the efficacy of BRAF inhibitor therapy in colorectal cancer could be increased by cotargeting either WNT/ß-catenin or VEGFRs with small-molecule inhibitors.
Assuntos
Neoplasias Colorretais , Proteínas Proto-Oncogênicas B-raf , Animais , Humanos , Vemurafenib/farmacologia , Cetuximab/farmacologia , Cetuximab/uso terapêutico , beta Catenina/genética , beta Catenina/metabolismo , Axitinibe/farmacologia , Axitinibe/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular Tumoral , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Via de Sinalização Wnt , Microambiente TumoralRESUMO
Melanoma is a disease associated with a very high mutation burden and thus the possibility of a diverse range of oncogenic mechanisms that allow it to evade therapeutic interventions and the immune system. Here, we describe the characterization of a panel of 102 cell lines from metastatic melanomas (the NZM lines), including using whole-exome and RNA sequencing to analyse genetic variants and gene expression changes in a subset of this panel. Lines possessing all major melanoma genotypes were identified, and hierarchical clustering of gene expression profiles revealed four broad subgroups of cell lines. Immunogenotyping identified a range of HLA haplotypes as well as expression of neoantigens and cancer-testis antigens in the lines. Together, these characteristics make the NZM panel a valuable resource for cell-based, immunological and xenograft studies to better understand the diversity of melanoma biology and the responses of melanoma to therapeutic interventions.
Assuntos
Biomarcadores Tumorais/genética , Exoma , Regulação Neoplásica da Expressão Gênica , Genômica/métodos , Melanoma/genética , Modelos Biológicos , Mutação , Humanos , Melanoma/secundário , Transdução de Sinais , Transcriptoma , Células Tumorais Cultivadas , Sequenciamento do ExomaRESUMO
Multiple therapeutic agents have been developed to target the phosphatidylinositol 3-kinase (PI3K) signaling pathway, which is frequently dysregulated in cancer promoting tumor growth and survival. These include pan PI3K inhibitors, which target class Ia PI3K isoforms and have largely shown limited single agent activity with narrow therapeutic windows in clinical trials. Here, we characterize SN32976, a novel pan PI3K inhibitor, for its biochemical potency against PI3K isoforms and mTOR, kinase selectivity, cellular activity, pharmacokinetics, pharmacodynamics and antitumor efficacy relative to five clinically-evaluated pan PI3K inhibitors: buparlisib, dactolisib, pictilisib, omipalisib and ZSTK474. SN32976 potently inhibited PI3K isoforms and mTOR, displaying preferential activity for PI3Kα and sparing of PI3Kδ relative to the other inhibitors, while showing less off-target activity than the clinical inhibitors in a panel of 442 kinases. The major metabolites of SN32976 were also potent PI3K inhibitors with similar selectivity for PI3Kα as the parent compound. SN32976 compared favorably with the clinically-evaluated PI3K inhibitors in cellular assays, inhibiting pAKT expression and cell proliferation at nM concentrations, and in animal models, inducing a greater extent and duration of pAKT inhibition in tumors than pictilisib, dactolisib and omipalisib at similarly tolerated dose levels and inhibiting tumor growth to a greater extent than dactolisib and ZSTK474 and with similar efficacy to pictilisib and omipalisib. These results suggest that SN32976 is a promising clinical candidate for cancer therapy with enhanced kinase selectivity and preferential inhibition of PI3Kα compared to first generation pan PI3K inhibitors, while retaining comparable anticancer activity.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Glucose/metabolismo , Humanos , Masculino , Camundongos , Fosforilação , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Ratos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BRAF and MEK inhibitors have improved outcomes for patients with BRAF-mutant melanoma, but their efficacy is limited by both intrinsic and acquired resistances. Activation of the PI3K pathway can mediate resistance to these agents, providing a strong rationale for combination therapy in melanoma. Here, a panel of nine low-passage human metastatic melanoma cell lines with BRAF mutations was tested in cell proliferation and protein expression assays for sensitivity to inhibitors of MEK (selumetinib) and BRAF (vemurafenib) as single agents and in combination with inhibitors of pan-PI3K (ZSTK474), pan-PI3K/mTOR (BEZ235), individual PI3K isoforms (p110α, A66; p110ß, TGX-221; p110γ, AS-252424; p110δ, idelalisib), or mTORC1/2 (KU-0063794). Selumetinib and vemurafenib potently inhibited cell proliferation in all cell lines, especially in those that expressed low levels of phosphorylated AKT (pAKT). ZSTK474 and BEZ235 also inhibited cell proliferation in all cell lines and enhanced the antitumor activity of selumetinib and vemurafenib in the majority of lines by either interacting synergistically or additively to increase potency or by inducing cytotoxicity by significantly increasing the magnitude of cell growth inhibition. Furthermore, ZSTK474 or BEZ235 combined with selumetinib to produce robust inhibition of pERK, pAKT, and pS6 expression and synergistic inhibition of NZM20 tumor growth. The inhibitors of individual PI3K isoforms or mTORC1/2 were less effective at inhibiting cell proliferation either as single agents or in combination with selumetinib or vemurafenib, although KU-0063794 synergistically interacted with vemurafenib and increased the magnitude of cell growth inhibition with selumetinib or vemurafenib in certain cell lines. Overall, these results suggest that the sensitivity of BRAF-mutant melanoma cells to BRAF or MEK inhibitors is at least partly mediated by activation of the PI3K pathway and can be enhanced by combined inhibition of the BRAF/MEK and PI3K/mTOR signaling pathways.
RESUMO
A range of 4-substituted derivatives of the pan class I PI 3-kinase inhibitor 2-(difluoromethyl)-1-[4,6-di-(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474) were prepared in a search for more soluble analogs. 4-Aminoalkoxy substituents provided the most potent derivatives, with the 4-O(CH2)3NMe2 analog (compound 14) being identified as displaying the best overall activity in combination with good aqueous solubility (25 mg/mL for the hydrochloride salt). This compound was tested in a U87MG xenograft model, but displayed less potency than ZSTK474 as a result of an unfavorable pharmacokinetic profile.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Triazinas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Células HCT116 , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Transdução de Sinais/efeitos dos fármacos , Solubilidade , Relação Estrutura-Atividade , Triazinas/síntese química , Triazinas/químicaRESUMO
A structure-activity relationship (SAR) study of the pan class I PI 3-kinase inhibitor 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474) identified substitution at the 4 and 6 positions of the benzimidazole ring as having significant effects on the potency of substituted derivatives. The 6-amino-4-methoxy analogue displayed a greater than 1000-fold potency enhancement over the corresponding 6-aza-4-methoxy analogue against all three class Ia PI 3-kinase enzymes (p110α, p110ß, and p110δ) and also displayed significant potency against two mutant forms of the p110α isoform (H1047R and E545K). This compound was also evaluated in vivo against a U87MG human glioblastoma tumor xenograft model in Rag1(-/-) mice, and at a dose of 50 mg/kg given by ip injection at a qd × 10 dosing schedule it dramatically reduced cancer growth by 81% compared to untreated controls.