RESUMO
B-cell maturation antigen (BCMA) is a validated target for chimeric antigen receptor (CAR) T-cell therapy in multiple myeloma (MM). Despite promising objective response rates, most patients relapse, and low levels of BCMA on a subset of tumor cells has been suggested as a probable escape mechanism. BCMA is actively cleaved from the tumor cell surface by the ubiquitous multisubunit γ-secretase (GS) complex, which reduces ligand density on tumor cells for CAR T-cell recognition and releases a soluble BCMA (sBCMA) fragment capable of inhibiting CAR T-cell function. Sufficient sBCMA can accumulate in the bone marrow of MM patients to inhibit CAR T-cell recognition of tumor cells, and potentially limit efficacy of BCMA-directed adoptive T-cell therapy. We investigated whether blocking BCMA cleavage by small-molecule GS inhibitors (GSIs) could augment BCMA-targeted CAR T-cell therapy. We found that exposure of myeloma cell lines and patient tumor samples to GSIs markedly increased surface BCMA levels in a dose-dependent fashion, concurrently decreased sBCMA concentrations, and improved tumor recognition by CAR T cells in vitro. GSI treatment of MM tumor-bearing NOD/SCID/γc-/- mice increased BCMA expression on tumor cells, decreased sBCMA in peripheral blood, and improved antitumor efficacy of BCMA-targeted CAR T-cell therapy. Importantly, short-term GSI administration to MM patients markedly increases the percentage of BCMA+ tumor cells, and the levels of BCMA surface expression in vivo. Based on these data, a US Food and Drug Administration (FDA)-approved clinical trial has been initiated, combining GSI with concurrent BCMA CAR T-cell therapy. This trial was registered at www.clinicaltrials.gov as #NCT03502577.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Antígeno de Maturação de Linfócitos B/metabolismo , Imunoterapia Adotiva/métodos , Mieloma Múltiplo , Animais , Benzazepinas/farmacologia , Ensaios Clínicos como Assunto , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mieloma Múltiplo/terapia , Receptores de Antígenos Quiméricos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although Aurora A, B, and C kinases share high sequence similarity, especially within the kinase domain, they function distinctly in cell-cycle progression. Aurora A depletion primarily leads to mitotic spindle formation defects and consequently prometaphase arrest, whereas Aurora B/C inactivation primarily induces polyploidy from cytokinesis failure. Aurora B/C inactivation phenotypes are also epistatic to those of Aurora A, such that the concomitant inactivation of Aurora A and B, or all Aurora isoforms by nonisoform-selective Aurora inhibitors, demonstrates the Aurora B/C-dominant cytokinesis failure and polyploidy phenotypes. Several Aurora inhibitors are in clinical trials for T/B-cell lymphoma, multiple myeloma, leukemia, lung, and breast cancers. Here, we describe an Aurora A-selective inhibitor, LY3295668, which potently inhibits Aurora autophosphorylation and its kinase activity in vitro and in vivo, persistently arrests cancer cells in mitosis, and induces more profound apoptosis than Aurora B or Aurora A/B dual inhibitors without Aurora B inhibition-associated cytokinesis failure and aneuploidy. LY3295668 inhibits the growth of a broad panel of cancer cell lines, including small-cell lung and breast cancer cells. It demonstrates significant efficacy in small-cell lung cancer xenograft and patient-derived tumor preclinical models as a single agent and in combination with standard-of-care agents. LY3295668, as a highly Aurora A-selective inhibitor, may represent a preferred approach to the current pan-Aurora inhibitors as a cancer therapeutic agent.
Assuntos
Antineoplásicos/uso terapêutico , Aurora Quinase A/antagonistas & inibidores , Mitose/efeitos dos fármacos , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Células HeLa , Humanos , MasculinoRESUMO
Merestinib is an oral multi-kinase inhibitor targeting a limited number of oncokinases including MET, AXL, RON and MKNK1/2. Here, we report that merestinib inhibits neurotrophic receptor tyrosine kinases NTRK1/2/3 which are oncogenic drivers in tumors bearing NTRK fusion resulting from chromosomal rearrangements. Merestinib is shown to be a type II NTRK1 kinase inhibitor as determined by x-ray crystallography. In KM-12 cells harboring TPM3-NTRK1 fusion, merestinib exhibits potent p-NTRK1 inhibition in vitro by western blot and elicits an anti-proliferative response in two- and three-dimensional growth. Merestinib treatment demonstrated profound tumor growth inhibition in in vivo cancer models harboring either a TPM3-NTRK1 or an ETV6-NTRK3 gene fusion. To recapitulate resistance observed from type I NTRK kinase inhibitors entrectinib and larotrectinib, we generated NIH-3T3 cells exogenously expressing TPM3-NTRK1 wild-type, or acquired mutations G595R and G667C in vitro and in vivo. Merestinib blocks tumor growth of both wild-type and mutant G667C TPM3-NTRK1 expressing NIH-3T3 cell-derived tumors. These preclinical data support the clinical evaluation of merestinib, a type II NTRK kinase inhibitor (NCT02920996), both in treatment naïve patients and in patients progressed on type I NTRK kinase inhibitors with acquired secondary G667C mutation in NTRK fusion bearing tumors.
RESUMO
BACKGROUND: Notch signalling regulates stem cell development and survival and is deregulated in multiple malignancies. LY900009 is a small molecule inhibitor of Notch signalling via selective inhibition of the γ-secretase protein. We report the first-in-human phase I trial of LY900009. METHODS: Dose escalation (Part A) was performed in cohorts of three advanced cancer patients using a modified continual reassessment method and dose confirmation (Part B) was performed in ovarian cancer patients. LY900009 was taken orally thrice weekly (every Monday, Wednesday, and Friday) during a 28-d cycle. The primary objective determined the maximum tolerated dose (MTD); secondary end-points included toxicity, pharmacokinetics, pharmacodynamics, and antitumour activity. RESULTS: Thirty-five patients received LY900009 at dose levels ranging from 2-60 mg. Study drug-related adverse events were diarrhoea (46%), vomiting (34%), anorexia (31%), nausea (31%), and fatigue (23%). At 30 mg, a dose-limiting toxicity (grade III mucosal inflammation) was observed. LY900009 absorption was rapid, with median tmax at 1-4 h post-dose. LY900009 inhibited plasma levels of amyloid-ß peptide in a dose-dependent manner with 80-90% inhibition observed in the 30- to 60-mg cohorts. No responses were seen, but five patients had stable disease. Two patients (5.7%) with leiomyosarcoma and ovarian cancer received four cycles of therapy. One patient (15 mg) showed markedly increased glandular mucin consistent with pharmacologic inhibition of the Notch pathway. CONCLUSIONS: The recommended MTD schedule for future studies was 30 mg thrice weekly, which exceeds the target inhibition level observed in preclinical models to promote tumour regression in humans.
Assuntos
Alanina/análogos & derivados , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Antineoplásicos/administração & dosagem , Dibenzazepinas/administração & dosagem , Neoplasias/tratamento farmacológico , Inibidores de Proteases/administração & dosagem , Receptores Notch/antagonistas & inibidores , Administração Oral , Adulto , Idoso , Alanina/administração & dosagem , Alanina/efeitos adversos , Alanina/farmacocinética , Secretases da Proteína Precursora do Amiloide/metabolismo , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Dibenzazepinas/efeitos adversos , Dibenzazepinas/farmacocinética , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Humanos , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Neoplasias/enzimologia , Neoplasias/patologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , Inibidores de Proteases/efeitos adversos , Inibidores de Proteases/farmacocinética , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento , Estados Unidos , Adulto JovemRESUMO
Hepatocyte growth factor (HGF) regulates various physiological and developmental processes in concert with other growth factors, cytokines and hormones. We examined interactions between cell signaling events elicited by HGF and the cytokine interleukin (IL)-4, in the IL-3-dependent murine myeloid cell line 32D transfected with the human HGF receptor, c-Met. HGF was a potent mitogen in these cells, and prevented apoptosis in response to IL-3 withdrawal. IL-4 showed modest anti-apoptotic activity, but no significant mitogenic activity. IL-4 synergistically enhanced HGF-stimulated DNA synthesis, whereas only additive prevention of apoptosis was observed. IL-4 did not enhance HGF-dependent tyrosine phosphorylation of c-Met or Shc. In contrast, HGF-stimulated activation of MAP kinases was enhanced by IL-4, suggesting that the IL-4 and HGF signaling pathways converge upstream of these events. Although phosphatidylinositol 3-kinase (PI3K) inhibitors diminished HGF-induced mitogenesis, anti-apoptosis, and MAP kinase activation, IL-4 enhanced HGF signaling persisted even in the presence of these inhibitors. IL-4 enhancement of HGF signaling was partially blocked in 32D/c-Met cells treated with inhibitors of MEK1 or c-Src kinases, completely blocked by expression of a catalytically inactive mutant of Janus kinase 3 (Jak3), and increased in 32D/c-Met cells overexpressing STAT6. Our results suggest that the IL-4 and HGF pathways converge at multiple levels, and that IL-4-dependent Jak3 and STAT6 activities modulate signaling events independent of PI3K to enhance HGF-dependent mitogenesis in myeloid cells, and possibly other common cellular targets.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Fator de Crescimento de Hepatócito/farmacologia , Interleucina-4/farmacologia , Mitógenos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Interleucina-3/farmacologia , Interleucina-4/antagonistas & inibidores , Janus Quinase 3 , MAP Quinase Quinase 1 , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Fator de Transcrição STAT6 , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Fatores de Tempo , Transativadores/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
The protein kinase family represents an enormous opportunity for drug development. However, the current limitation in structural diversity of kinase inhibitors has complicated efforts to identify effective treatments of diseases that involve protein kinase signaling pathways. We have identified a new structural class of protein serine/threonine kinase inhibitors comprising an aminoimidazo[1,2-a]pyridine nucleus. In this report, we describe the first successful use of this class of aza-heterocycles to generate potent inhibitors of cyclin-dependent kinases that compete with ATP for binding to a catalytic subunit of the protein. Co-crystal structures of CDK2 in complex with lead compounds reveal a unique mode of binding. Using this knowledge, a structure-based design approach directed this chemical scaffold toward generating potent and selective CDK2 inhibitors, which selectively inhibited the CDK2-dependent phosphorylation of Rb and induced caspase-3-dependent apoptosis in HCT 116 tumor cells. The discovery of this new class of ATP-site-directed protein kinase inhibitors, aminoimidazo[1,2-a]pyridines, provides the basis for a new medicinal chemistry tool to be used in the search for effective treatments of cancer and other diseases that involve protein kinase signaling pathways.
Assuntos
Quinases relacionadas a CDC2 e CDC28/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Piridinas/farmacologia , Trifosfato de Adenosina/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Quinases relacionadas a CDC2 e CDC28/química , Quinases relacionadas a CDC2 e CDC28/metabolismo , Caspase 3 , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cristalografia por Raios X , Quinase 2 Dependente de Ciclina , Desenho de Fármacos , Inibidores Enzimáticos/química , Células HCT116 , Humanos , Imidazóis/química , Concentração Inibidora 50 , Fosforilação/efeitos dos fármacos , Piridinas/química , Proteína do Retinoblastoma/metabolismo , Relação Estrutura-AtividadeRESUMO
The Hedgehog (Hh) pathway is involved in oncogenic transformation and tumor maintenance. The primary objective of this study was to select surrogate tissue to measure messenger ribonucleic acid (mRNA) levels of Hh pathway genes for measurement of pharmacodynamic effect. Expression of Hh pathway specific genes was measured by quantitative real time polymerase chain reaction (qRT-PCR) and global gene expression using Affymetrix U133 microarrays. Correlations were made between the expression of specific genes determined by qRT-PCR and normalized microarray data. Gene ontology analysis using microarray data for a broader set of Hh pathway genes was performed to identify additional Hh pathway-related markers in the surrogate tissue. RNA extracted from blood, hair follicle, and skin obtained from healthy subjects was analyzed by qRT-PCR for 31 genes, whereas 8 samples were analyzed for a 7-gene subset. Twelve sample sets, each with ≤500 ng total RNA derived from hair, skin, and blood, were analyzed using Affymetrix U133 microarrays. Transcripts for several Hh pathway genes were undetectable in blood using qRT-PCR. Skin was the most desirable matrix, followed by hair follicle. Whether processed by robust multiarray average or microarray suite 5 (MAS5), expression patterns of individual samples showed co-clustered signals; both normalization methods were equally effective for unsupervised analysis. The MAS5- normalized probe sets appeared better suited for supervised analysis. This work provides the basis for selection of a surrogate tissue and an expression analysis-based approach to evaluate pathway-related genes as markers of pharmacodynamic effect with novel inhibitors of the Hh pathway.
RESUMO
Survivin, a family member of the inhibitor of apoptosis proteins that is expressed during mitosis in a cell cycle-dependent manner and localized to different components of the mitotic apparatus, plays an important role in both cell division and inhibition of apoptosis. Survivin is expressed in a vast majority of human cancers, but not in normal adult tissues. Survivin expression is often correlated with poor prognosis in a wide variety of cancer patients. These features make survivin an attractive target against which cancer therapeutics could be developed. We have identified a survivin antisense oligonucleotide (ASO) that potently downregulated survivin expression in human cancer cells derived from lung, colon, pancreas, liver, breast, prostate, ovary, cervix, skin, and brain as measured by quantitative RT-PCR and immunoblotting analysis. Specific inhibition of survivin expression in multiple cancer cell lines by this ASO (LY2181308) induced caspase-3-dependent apoptosis, cell cycle arrest in the G(2)-M phase, and multinucleated cells. We also showed that inhibition of survivin expression by LY2181308 sensitized tumor cells to chemotherapeutic-induced apoptosis. Most importantly, in an in vivo human xenograft tumor model, LY2181308 produced significant antitumor activity as compared with saline or its sequence-specific control oligonucleotide and sensitized to gemcitabine, paclitaxel, and docetaxel. Furthermore, we showed that this antitumor activity was associated with significant inhibition of survivin expression in these xenograft tumors. On the basis of these, LY2181308 is being evaluated in a clinical setting (Phase II) in combination with docetaxel for the treatment of prostate cancer.
Assuntos
Antineoplásicos/farmacologia , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Neoplasias/fisiopatologia , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Células HeLa , Humanos , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Survivina , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A series of indolo[2,3-a]pyrrolo[3,4-c]carbazoles and their bis-indolylmaleimides precursors have been prepared in order to compare their activity as D1-CDK4 inhibitors. Both enzymatic and antiproliferative assays have shown that the structurally more constrained indolo[2,3-a]pyrrolo[3,4-c]carbazoles are consistently more active (8-42-fold) in head-to-head comparison with their bis-indolylmaleimides counterparts. Cell-cycle analysis using flow cytometry have also shown that the indolocarbazoles are selective G1 blockers while the bis-indolylmaleimides arrest cells in the G2/M phase.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Carbazóis/síntese química , Carbazóis/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Pirróis/síntese química , Pirróis/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Citometria de Fluxo , Humanos , Conformação Molecular , Relação Estrutura-AtividadeRESUMO
The synthesis of new analogues of Arcyriaflavin A in which one indole ring is replaced by an aryl or heteroaryl ring is described. These new series of aryl[a]pyrrolo[3,4-c]carbazoles were evaluated as inhibitors of Cyclin D1-CDK4. A potent and selective D1-CDK4 inhibitor, 7a (D1-CDK4 IC(50)=45 nM), has been identified. The potency, selectivity profile against other kinases, and structure-activity relationship (SAR) trends of this class of compounds are discussed.