RESUMO
Targeting androgen receptor (AR) has shown great therapeutic potential in triple-negative breast cancer (TNBC), yet its efficacy remains unsatisfactory. Here, we aimed to identify promising targeted agents that synergize with enzalutamide, a second-generation AR inhibitor, in TNBC. By using a strategy for screening drug combinations based on the Sensitivity Index (SI), we found that MK-8776, a selective checkpoint kinase1 (CHK1) inhibitor, showed favorable synergism with enzalutamide in AR-positive TNBC. The combination of enzalutamide and MK-8776 was found to exert more significant anti-tumor effects in TNBC than the single application of enzalutamide or MK-8776, respectively. Furthermore, a nanoparticle-based on hyaluronic acid (HA)-modified hollow-manganese dioxide (HMnO2), named HMnE&M@H, was established to encapsulate and deliver enzalutamide and MK-8776. This HA-modified nanosystem managed targeted activation via pH/glutathione responsiveness. HMnE&M@H repressed tumor growth more obviously than the simple addition of enzalutamide and MK-8776 without a carrier. Collectively, our study elucidated the synergy of enzalutamide and MK-8776 in TNBC and developed a novel tumor-targeted nano drug delivery system HMnE&M@H, providing a potential therapeutic approach for the treatment of TNBC.
RESUMO
Acute promyelocytic leukemia (APL) is a hematological disease characterized by the expression of the oncogenic fusion protein PML-RARα. The current treatment approach for APL involves differentiation therapy using all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). However, the development of resistance to therapy, occurrence of differentiation syndrome, and relapses necessitate the exploration of new treatment options that induce differentiation of leukemic blasts with low toxicity. In this study, we investigated the cellular and molecular effects of MK-8776, a specific inhibitor of CHK1, in ATRA-resistant APL cells. Treatment of APL cells with MK-8776 resulted in a decrease in PML-RARα levels, increased expression of CD11b, and increased granulocytic activity consistent with differentiation. Interestingly, we showed that the MK-8776-induced differentiating effect resulted synergic with ATO. We found that the reduction of PML-RARα by MK-8776 was dependent on both proteasome and caspases. Specifically, both caspase-1 and caspase-3 were activated by CHK1 inhibition, with caspase-3 acting upstream of caspase-1. Activation of caspase-3 was necessary to activate caspase-1 and promote PML-RARα degradation. Transcriptomic analysis revealed significant modulation of pathways and upstream regulators involved in the inflammatory response and cell cycle control upon MK-8776 treatment. Overall, the ability of MK-8776 to induce PML-RARα degradation and stimulate differentiation of immature APL cancer cells into more mature forms recapitulates the concept of differentiation therapy. Considering the in vivo tolerability of MK-8776, it will be relevant to evaluate its potential clinical benefit in APL patients resistant to standard ATRA/ATO therapy, as well as in patients with other forms of acute leukemias.
Assuntos
Leucemia Promielocítica Aguda , Humanos , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Caspase 3 , Tretinoína/farmacologia , Trióxido de Arsênio/farmacologia , Trióxido de Arsênio/uso terapêutico , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Diferenciação Celular , CaspasesRESUMO
Although targeting DNA-damage repair by inhibition of PARP exhibits weak or modest single-agent activity due to the existence of functional BRCA1/2 alleles, PARP inhibitors have been gradually applicable in BRCA-proficient cancers. Checkpoint kinase 1 (Chk1) inhibition selectively disrupts homologous recombination (HR)-mediated DNA repair and confers synthetic lethality in p53-deficient tumors, we therefore aim at expounding the chemopotentiating effects of Chk1 inhibition on PARPi in BRCA-proficient and p53-deficient cancer cells. Initially, BRCA wild-type, p53-null cells including AsPC-1 and H1299 demonstrated innate resistance to PARP inhibitor olaparib compared to BRCA1-mutant, p53-null MDA-MB-436 cells. We quantified the interaction between olaparib and a selective Chk1 inhibitor MK-8776, which produced synergistic effects under sub-IC50 concentrations in p53-depleted AsPC-1 and H1299 cells. Olaparib in combination with MK-8776 showed enhanced antitumor effects through prohibiting proliferation and secondarily inducing apoptosis in two cell lines. Of note, we observed that MK-8776 significantly sensitized cells to olaparib by broad DNA and chromosomal breaks. Mechanistically, MK-8776 abrogated olaparib-induced BRCA1 intranuclear foci formation, MCM7-mediated replication machineries, and ultimately triggered an accumulation of γH2AX, a well-recognized marker of DNA double-strand breaks. Additionally, we established ectopic expression of hotspot mutant p53 in H1299 cells. Introduction of p53R175 H promoted olaparib resistance as single-agent treatment, but the synergy between olaparib and MK-8776 was still achievable and the region of synergy was produced by lower combination concentrations. These data provide insight into how Chk1 inhibition could be effectively targeted and confer sensitivity to olaparib toward p53-deficient and HR-proficient cancers.
Assuntos
Antineoplásicos , Neoplasias , Proteína Supressora de Tumor p53/genética , Quinase 1 do Ponto de Checagem , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Ftalazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologiaRESUMO
AbstractsChk1 inhibition can selectively improve gemcitabine sensitivity in p53-deficient cells by checkpoints abrogation throughout the cell cycle. However, the dependency of p53 status is still controversial for predicting the priority of such synergy. This study aimed at expounding the differential therapeutic properties of gemcitabine sensitization by Chk1 inhibition potentially affected by p53 status. We introduced wild-type and hotspot mutant p53 in p53-null H1299 cells, and quantified combination of gemcitabine with two Chk1 inhibitors using Chou-Talalay method. As a result, depletion of p53 preferentially produced synergistic effects. Wild-type and mutant p53 also conferred drug synergy but gradually showed compromised potency of growth inhibition. These data provide increased evidence that p53 status is a weak predictor for identifying an effective synergy, but genetic loss of p53 is relatively favorable for combination treatment. Further efforts on validation in more cell lines and clinical models could improve the predictive validity in this study.
Assuntos
Gencitabina , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Desoxicitidina/farmacologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Linhagem Celular Tumoral , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
BACKGROUND: Challenges exist in the clinical treatment of luminal estrogen receptor α (ERα)-positive breast cancers (BCs) both to prevent resistance to endocrine therapy (ET) and to treat ET-resistant metastatic BCs (MBC). Therefore, we evaluated if kinases could be new targets for the treatment of luminal primary and MBCs. METHODS: ~ 170 kinase inhibitors were applied to MCF-7 cells either with adaptative or genetic resistance to ET drugs and both ERα levels and cell proliferation were measured. Robust-Z-score calculation identified AZD7762 (CHK1/CHK2 inhibitor) as a positive hit. Subsequently, Kaplan-Meier analyses of CHK1 and CHK2 impact on ERα-positive BC patients relapse-free-survival (RFS), bioinformatic evaluations of CHK1 and CHK2 expression and activation status as a function of ERα activation status as well as drug sensitivity studies in ERα-positive BC cell lines, validation of the impact of the ATR:CHK1 and ATM:CHK2 pathways on the control of ERα stability and BC cell proliferation via inhibitor- and siRNA-based approaches, identification of the molecular mechanism required for inhibitor-dependent ERα degradation in BC and the impact of CHK1 and CHK2 inhibition on the 17ß-estradiol (E2):ERα signaling, synergy proliferation studies between ET-drugs and clinically relevant CHK1 inhibitors in different luminal BC cell lines, were performed. RESULTS: A reduced CHK1 expression correlates with a longer RFS in women with ERα-positive BCs. Interestingly, women carrying luminal A BC display an extended RFS when expressing low CHK1 levels. Accordingly, CHK1 and ERα activations are correlated in ERα-positive BC cell lines, and the ATR:CHK1 pathway controls ERα stability and cell proliferation in luminal A BC cells. Mechanistically, the generation of DNA replication stress rather than DNA damage induced by ATR:CHK1 pathway inhibition is a prerequisite for ERα degradation. Furthermore, CHK1 inhibition interferes with E2:ERα signaling to cell proliferation, and drugs approved for clinical treatment of primary and MBC (4OH-tamoxifen and the CDK4/CDK6 inhibitors abemaciclib and palbociclib) exert synergic effects with the CHK1 inhibitors in clinical trials for the treatment of solid tumors (AZD7762, MK8776, prexasertib) in preventing the proliferation of cells modeling primary and MBC. CONCLUSIONS: CHK1 could be considered as an appealing novel pharmacological target for the treatment of luminal primary and MBCs.
Assuntos
Neoplasias da Mama , Receptor alfa de Estrogênio , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos/genética , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Células MCF-7 , Recidiva Local de Neoplasia/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
Pancreatic cancer is a fatal disease. The five-year survival for patients with all stages of this tumor type is less than 10%, with a majority of patients dying from drug resistant, metastatic disease. Gemcitabine has been a standard of care for the treatment of pancreatic cancer for over 20 years, but as a single agent gemcitabine is not curative. Since the only therapeutic option for the over 80 percent of pancreatic cancer patients ineligible for surgical resection is chemotherapy with or without radiation, the last few decades have seen a significant effort to develop effective therapy for this disease. This review addresses preclinical and clinical efforts to identify agents that target molecular characteristics common to pancreatic tumors and to develop mechanism-based combination approaches to therapy. Some of the most promising combinations include agents that inhibit transcription dependent on BET proteins (BET bromodomain inhibitors) or that inhibit DNA repair mediated by PARP (PARP inhibitors).
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Acetilação/efeitos dos fármacos , Animais , Antineoplásicos/uso terapêutico , Dano ao DNA , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Histonas/metabolismo , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas/antagonistas & inibidores , GencitabinaRESUMO
Mucosotropic human papillomaviruses (HPVs) cause prevalent anogenital infections, some of which can progress to cancers. It is imperative to identify efficacious drug candidates, as there are few therapeutic options. We have recapitulated a robust productive program of HPV-18 in organotypic raft cultures of primary human keratinocytes. The HPV E7 protein induces S phase reentry, along with DNA damage response (DDR) in differentiated cells to support viral DNA amplification. A number of small molecule inhibitors of DDR regulators are in clinical use or clinical trials to treat cancers. Here, we used our raft culture system to examine effects of inhibitors of ATR/Chk1 and ATM/Chk2 on HPV infection. The inhibitors impaired S-phase reentry and progression as well as HPV DNA amplification. The Chk1 inhibitor MK-8776 was most effective, reducing viral DNA amplification by 90-99% and caused DNA damage and apoptosis, preferentially in HPV infected cells. We found that this sensitivity was imparted by the E7 protein and report that MK-8776 also caused extensive cell death of cervical cancer cell lines. Furthermore, it sensitized the cells to cisplatin, commonly used to treat advanced cervical cancer. Based on these observations, the Chk1 inhibitors could be potential effective agents to be re-purposed to treat the spectrum of HPV infections in single or combination therapy.
Assuntos
DNA Viral/antagonistas & inibidores , Papillomavirus Humano 18/metabolismo , Queratinócitos/efeitos dos fármacos , Proteínas E7 de Papillomavirus/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Replicação Viral/efeitos dos fármacos , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Cisplatino/farmacologia , DNA Viral/genética , DNA Viral/metabolismo , Feminino , Células HeLa , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/fisiologia , Humanos , Queratinócitos/metabolismo , Queratinócitos/virologia , Masculino , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/prevenção & controle , Infecções por Papillomavirus/virologia , Inibidores de Proteínas Quinases/farmacologia , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia , Replicação Viral/genéticaRESUMO
P-glycoprotein (P-gp), which is encoded by the ATP-binding cassette (ABC) transporter subfamily B member 1 (ABCB1) gene, is one of the most pivotal ABC transporters that transport its substrates across the cell membrane. Its overexpression is one of the confirmed causes of multidrug resistance (MDR), which results in the failure of cancer treatment. Here, we report that checkpoint kinase (Chk) 1 inhibitor MK-8776, a drug candidate in clinical trial, can restore the sensitivity of chemotherapeutics that are substrates of P-gp in KB-C2, SW620/Ad300 cells and human embryonic kidney (HEK)293/ABCB1 cells that overexpress P-gp. MK-8776 remarkably enhanced the cellular [3H]-paclitaxel accumulation and suppressed the efflux function of P-gp without reducing its expression and affecting its cellular localization in cancer cells. Furthermore, MK-8776 (0-40 µM) stimulated the activity of ATPase in P-gp, which was 4.1-fold greater than the control. In addition, MK-8776 formed a cation-π bond and π-π interaction with key residues of the substrate-binding site in P-gp, as indicated by computer-aided molecular docking study. Our study indicated that MK-8776 may significantly enhance the sensitivity of chemotherapeutics that are substrates of P-gp, providing important information for its application in the reversal of MDR.
Assuntos
Antineoplásicos/farmacologia , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Pirazóis/farmacologia , Pirimidinas/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Antineoplásicos/química , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Expressão Gênica , Humanos , Concentração Inibidora 50 , Transporte Proteico , Pirazóis/química , Pirimidinas/química , Relação Estrutura-AtividadeRESUMO
Metronomic chemotherapy, which is defined as a low-dose and frequent administration of cytotoxic drugs without drug-free breaks, has been recently emerged as an alternative to traditional MTD therapy and has shown therapeutic benefit in breast cancer patients in numbers of clinical studies. Unlike MTD, metronomic chemotherapy acts by multiple mechanisms including antiangiogenic effect and immunomodulation, but the direct cytotoxic effect only playing a minor role due to the lowered dose. In this light, within the limits of p53-deficient breast cancer, we demonstrate the enhanced anticancer effect of metronomic chemotherapy using doxorubicin when combined with Chk1 inhibitor MK-8776 by specifically augmenting the direct cytotoxic effect on cancer cells. Since the oral drug is greatly favored in metronomic chemotherapy due to the frequent and potential long-term administration, we prepared an oral doxorubicin by producing an ionic complex with deoxycholic acid, which showed sufficient bioavailability and anticancer effect when administered orally. MK-8776 selectively enhanced the cytotoxic effect of low-concentration doxorubicin in p53-deficient breast cancer cells by abrogating the Chk1-dependent cell cycle arrest in vitro. Consistently, combining MK-8776 significantly improved the anticancer effect of the daily administered oral doxorubicin in p53-deficient breast cancer xenografts especially in a lower dose of doxorubicin without evident systemic toxicities. Combination therapy of MK-8776 and metronomic oral doxorubicin would be thus promising in the treatment of p53-deficient breast cancer benefited from the augmented direct cytotoxic effect and low risk of toxicities.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Doxorrubicina/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , Proteína Supressora de Tumor p53/genética , Administração Metronômica , Administração Oral , Animais , Antibióticos Antineoplásicos/uso terapêutico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Doxorrubicina/uso terapêutico , Feminino , Deleção de Genes , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/uso terapêutico , Pirimidinas/uso terapêuticoAssuntos
Antígenos CD34 , Sistemas de Liberação de Medicamentos , Leucemia Mieloide Aguda , Proteínas de Neoplasias , Células-Tronco Neoplásicas , Pirazóis/farmacologia , Pirimidinas/farmacologia , Antígenos CD34/genética , Antígenos CD34/metabolismo , Feminino , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologiaRESUMO
Small molecule inhibitors of the checkpoint proteins CHK1 and WEE1 are currently in clinical development in combination with the antimetabolite gemcitabine. It is unclear, however, if there is a therapeutic advantage to CHK1 vs. WEE1 inhibition for chemosensitization. The goals of this study were to directly compare the relative efficacies of the CHK1 inhibitor MK8776 and the WEE1 inhibitor AZD1775 to sensitize pancreatic cancer cell lines to gemcitabine and to identify pharmacodynamic biomarkers predictive of chemosensitization. Cells treated with gemcitabine and either MK8776 or AZD1775 were first assessed for clonogenic survival. With the exception of the homologous recombination-defective Capan1 cells, which were relatively insensitive to MK8776, we found that these cell lines were similarly sensitized to gemcitabine by CHK1 or WEE1 inhibition. The abilities of either the CDK1/2 inhibitor roscovitine or exogenous nucleosides to prevent MK8776 or AZD1775-mediated chemosensitization, however, were both inhibitor-dependent and variable among cell lines. Given the importance of DNA replication stress to gemcitabine chemosensitization, we next assessed high-intensity, pan-nuclear γH2AX staining as a pharmacodynamic marker for sensitization. In contrast to total γH2AX, aberrant mitotic entry or sub-G1 DNA content, high-intensity γH2AX staining correlated with chemosensitization by either MK8776 or AZD1775 (R2 0.83 - 0.53). In summary, we found that MK8776 and AZD1775 sensitize to gemcitabine with similar efficacy. Furthermore, our results suggest that the effects of CHK1 and WEE1 inhibition on gemcitabine-mediated replication stress best predict chemosensitization and support the use of high-intensity or pan-nuclear γH2AX staining as a marker for therapeutic response.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Replicação do DNA/fisiologia , Desoxicitidina/análogos & derivados , Histonas/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Pirimidinonas/farmacologia , Análise de Variância , Biomarcadores Tumorais/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desoxicitidina/farmacologia , Humanos , Nucleosídeos/farmacologia , Neoplasias Pancreáticas , Inibidores de Proteínas Quinases/farmacologia , Roscovitina/farmacologia , Coloração e Rotulagem , GencitabinaRESUMO
PURPOSE: Cytosine arabinoside (AraC) remains the backbone of most treatment regimens for acute myeloid leukemia (AML). Incorporation of AraC into DNA activates checkpoint kinase 1 (Chk1), leading to cell-cycle arrest and diminished AraC cytotoxicity, which can be reversed by the selective Chk1 inhibitor MK-8776. Building on a Phase I trial, we conducted a phase II trial comparing timed sequential AraC with or without MK-8776. METHODS: Patients with relapsed or primary refractory AML were randomized 1:1 to receive either AraC with MK-8776 (Arm A); or AraC alone (Arm B). RESULTS: 32 patients were treated: 14 assigned to Arm A and 18 to Arm B. There were 5 (36%) complete responses (CR/CRi) and 1 (7%) partial response (PR) in Arm A, and 8 (44%) CR/CRis and 1 (6%) PR in Arm B. Median survival did not differ significantly between the two groups (5.9months in Arm A vs. 4.5 months in Arm B). MK-8776 led to a robust increase in DNA damage in circulating leukemic blasts as measured by increased γ-H2AX (16.9%±6.1% prior and 36.4%±6.8% at one hour after MK-8776 infusion, p=0.016). CONCLUSION: Response rates and survival were similar between the two groups in spite of evidence that MK-8776 augmented DNA damage in circulating leukemic blasts. Better than expected results in the control arm using timed sequential AraC and truncated patient enrollment may have limited the ability to detect clinical benefit from the combination.
Assuntos
Antineoplásicos/administração & dosagem , Citarabina/administração & dosagem , Leucemia Mieloide Aguda/tratamento farmacológico , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , Adulto , Idoso , Antineoplásicos/efeitos adversos , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Citarabina/efeitos adversos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/tratamento farmacológico , Pirazóis/efeitos adversos , Pirimidinas/efeitos adversosRESUMO
Radiotherapy is commonly used to treat a variety of solid tumors but improvements in the therapeutic ratio are sorely needed. The aim of this study was to assess the Chk1 kinase inhibitor, MK-8776, for its ability to radiosensitize human tumor cells. Cells derived from NSCLC and HNSCC cancers were tested for radiosensitization by MK-8776. The ability of MK-8776 to abrogate the radiation-induced G2 block was determined using flow cytometry. Effects on repair of radiation-induced DNA double strand breaks (DSBs) were determined on the basis of rad51, γ-H2AX and 53BP1 foci. Clonogenic survival analyses indicated that MK-8776 radiosensitized p53-defective tumor cells but not lines with wild-type p53. Abrogation of the G2 block was evident in both p53-defective cells and p53 wild-type lines indicating no correlation with radiosensitization. However, only p53-defective cells entered mitosis harboring unrepaired DSBs. MK-8776 appeared to inhibit repair of radiation-induced DSBs at early times after irradiation. A comparison of MK-8776 to the wee1 inhibitor, MK-1775, suggested both similarities and differences in their activities. In conclusion, MK-8776 radiosensitizes tumor cells by mechanisms that include abrogation of the G2 block and inhibition of DSB repair. Our findings support the clinical evaluation of MK-8776 in combination with radiation.