RESUMEN
Small-cell lung cancer (SCLC) is an aggressive malignancy with limited therapeutic options. The dismal prognosis in SCLC is in part associated with an upregulation of BCL-2 family anti-apoptotic proteins, including BCL-XL and MCL-1. Unfortunately, the currently available inhibitors of BCL-2 family anti-apoptotic proteins, except BCL-2 inhibitors, are not clinically relevant because of various on-target toxicities. We, therefore, aimed to develop an effective and safe strategy targeting these anti-apoptotic proteins with DT2216 (our platelet-sparing BCL-XL degrader) and AZD8055 (an mTOR inhibitor) to avoid associated on-target toxicities while synergistically optimizing tumor response. Through BH3 mimetic screening, we identified a subset of SCLC cell lines that is co-dependent on BCL-XL and MCL-1. After screening inhibitors of selected tumorigenic pathways, we found that AZD8055 selectively downregulates MCL-1 in SCLC cells and its combination with DT2216 synergistically killed BCL-XL/MCL-1 co-dependent SCLC cells, but not normal cells. Mechanistically, the combination caused BCL-XL degradation and suppression of MCL-1 expression, and thus disrupted MCL-1 interaction with BIM leading to an enhanced apoptotic induction. In vivo, the DT2216 + AZD8055 combination significantly inhibited the growth of cell line-derived and patient-derived xenografts and reduced tumor burden accompanied by increased survival in a genetically engineered mouse model of SCLC without causing appreciable thrombocytopenia or other normal tissue injuries. Thus, these preclinical findings lay a strong foundation for future clinical studies to test DT2216 + mTOR inhibitor combinations in a subset of SCLC patients whose tumors are co-driven by BCL-XL and MCL-1.
RESUMEN
KRAS mutations are the most common oncogenic drivers. Sotorasib (AMG510), a covalent inhibitor of KRASG12C, was recently approved for the treatment of KRASG12C-mutated non-small cell lung cancer (NSCLC). However, the efficacy of sotorasib and other KRASG12C inhibitors is limited by intrinsic resistance in colorectal cancer (CRC) and by the rapid emergence of acquired resistance in all treated tumors. Therefore, there is an urgent need to develop novel combination therapies to overcome sotorasib resistance and to maximize its efficacy. We assessed the effect of sotorasib alone or in combination with DT2216 (a clinical-stage BCL-XL proteolysis targeting chimera [PROTAC]) on KRASG12C-mutated NSCLC, CRC and pancreatic cancer (PC) cell lines using MTS cell viability, colony formation and Annexin-V/PI apoptosis assays. Furthermore, the therapeutic efficacy of sotorasib alone and in combination with DT2216 was evaluated in vivo using different tumor xenograft models. We observed heterogeneous responses to sotorasib alone, whereas its combination with DT2216 strongly inhibited viability of KRASG12C tumor cell lines that partially responded to sotorasib treatment. Mechanistically, sotorasib treatment led to stabilization of BIM and co-treatment with DT2216 inhibited sotorasib-induced BCL-XL/BIM interaction leading to enhanced apoptosis in KRASG12C tumor cell lines. Furthermore, DT2216 co-treatment significantly improved the antitumor efficacy of sotorasib in vivo. Collectively, our findings suggest that due to cytostatic activity, the efficacy of sotorasib is limited, and therefore, its combination with a pro-apoptotic agent, i.e., DT2216, shows synergistic responses and can potentially overcome resistance.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Mutación , Piperazinas , Proteolisis , Proteínas Proto-Oncogénicas p21(ras)/genética , Piridinas , PirimidinasRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is associated with extensive dysregulation of the epigenome and epigenetic regulators, such as bromodomain and extraterminal motif (BET) proteins, have been suggested as potential targets for therapy. However, single-agent BET inhibition has shown poor efficacy in clinical trials, and no epigenetic approaches are currently used in PDAC. To circumvent the limitations of the current generation of BET inhibitors, we developed the compound XP-524 as an inhibitor of the BET protein BRD4 and the histone acetyltransferase EP300/CBP, both of which are ubiquitously expressed in PDAC tissues and cooperate to enhance tumorigenesis. XP-524 showed increased potency and superior tumoricidal activity than the benchmark BET inhibitor JQ-1 in vitro, with comparable efficacy to higher-dose JQ-1 combined with the EP300/CBP inhibitor SGC-CBP30. We determined that this is in part due to the epigenetic silencing of KRAS in vitro, with similar results observed using ex vivo slice cultures of human PDAC tumors. Accordingly, XP-524 prevented KRAS-induced, neoplastic transformation in vivo and extended survival in two transgenic mouse models of aggressive PDAC. In addition to the inhibition of KRAS/MAPK signaling, XP-524 also enhanced the presentation of self-peptide and tumor recruitment of cytotoxic T lymphocytes, though these lymphocytes remained refractory from full activation. We, therefore, combined XP-524 with an anti-PD-1 antibody in vivo, which reactivated the cytotoxic immune program and extended survival well beyond XP-524 in monotherapy. Pending a comprehensive safety evaluation, these results suggest that XP-524 may benefit PDAC patients and warrant further exploration, particularly in combination with immune checkpoint inhibition.
Asunto(s)
Antineoplásicos/farmacología , Proteína p300 Asociada a E1A/antagonistas & inhibidores , Inhibidores de Puntos de Control Inmunológico/farmacología , Proteínas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Animales , Antineoplásicos/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Proteína p300 Asociada a E1A/química , Regulación de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Ratones , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/química , Relación Estructura-Actividad , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pancreatic cancer is the third most common cause of cancer-related deaths in the United States. Although gemcitabine is the standard of care for most patients with pancreatic cancer, its efficacy is limited by the development of resistance. This resistance may be attributable to the evasion of apoptosis caused by the overexpression of BCL-2 family antiapoptotic proteins. In this study, we investigated the role of BCL-XL in gemcitabine resistance to identify a combination therapy to more effectively treat pancreatic cancer. We used CRISPR-Cas9 screening to identify the key genes involved in gemcitabine resistance in pancreatic cancer. Pancreatic cancer cell dependencies on different BCL-2 family proteins and the efficacy of the combination of gemcitabine and DT2216 (a BCL-XL proteolysis targeting chimera or PROTAC) were determined by MTS, Annexin-V/PI, colony formation, and 3D tumor spheroid assays. The therapeutic efficacy of the combination was investigated in several patient-derived xenograft (PDX) mouse models of pancreatic cancer. We identified BCL-XL as a key mediator of gemcitabine resistance. The combination of gemcitabine and DT2216 synergistically induced cell death in multiple pancreatic cancer cell lines in vitro In vivo, the combination significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice compared with the individual agents in pancreatic cancer PDX models. Their synergistic antitumor activity is attributable to DT2216-induced degradation of BCL-XL and concomitant suppression of MCL-1 by gemcitabine. Our results suggest that DT2216-mediated BCL-XL degradation augments the antitumor activity of gemcitabine and their combination could be more effective for pancreatic cancer treatment.
Asunto(s)
Antineoplásicos/uso terapéutico , Desoxicitidina/análogos & derivados , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias Pancreáticas/tratamiento farmacológico , Piperazinas/uso terapéutico , Proteína bcl-X/metabolismo , Animales , Antineoplásicos/farmacología , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Humanos , Ratones , Ratones Endogámicos NOD , Neoplasias Pancreáticas/patología , Piperazinas/farmacología , GemcitabinaRESUMEN
PROteolysis-TArgeting Chimeras (PROTACs) have emerged as an innovative drug development platform. However, most PROTACs have been generated empirically because many determinants of PROTAC specificity and activity remain elusive. Through computational modelling of the entire NEDD8-VHL Cullin RING E3 ubiquitin ligase (CRLVHL)/PROTAC/BCL-xL/UbcH5B(E2)-Ub/RBX1 complex, we find that this complex can only ubiquitinate the lysines in a defined band region on BCL-xL. Using this approach to guide our development of a series of ABT263-derived and VHL-recruiting PROTACs, we generate a potent BCL-xL and BCL-2 (BCL-xL/2) dual degrader with significantly improved antitumor activity against BCL-xL/2-dependent leukemia cells. Our results provide experimental evidence that the accessibility of lysines on a target protein plays an important role in determining the selectivity and potency of a PROTAC in inducing protein degradation, which may serve as a conceptual framework to guide the future development of PROTACs.
Asunto(s)
Antineoplásicos/farmacología , Leucemia/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína bcl-X/metabolismo , Antineoplásicos/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Humanos , Leucemia/tratamiento farmacológico , Leucemia/genética , Lisina/química , Lisina/genética , Lisina/metabolismo , Modelos Moleculares , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Conformación Proteica , Proteolisis , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Proto-Oncogénicas c-bcl-2/genética , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/química , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Proteína bcl-X/química , Proteína bcl-X/genéticaRESUMEN
BCL-XL and BCL-2 are important targets for cancer treatment. BCL-XL specific proteolysis-targeting chimeras (PROTACs) have been developed to circumvent the on-target platelet toxicity associated with BCL-XL inhibition. However, they have minimal effects on cancer cells that are dependent on BCL-2 or both BCL-XL and BCL-2. Here we report a new series of BCL-PROTACs. The lead PZ703b exhibits high potency in inducing BCL-XL degradation and in inhibiting but not degrading BCL-2, showing a hybrid dual-targeting mechanism of action that is unprecedented in a PROTAC molecule. As a result, PZ703b is highly potent in killing BCL-XL dependent, BCL-2 dependent, and BCL-XL/BCL-2 dual-dependent cells in an E3 ligase (VHL)-dependent fashion. We further found that PZ703b forms stable {BCL-2:PROTAC:VCB} ternary complexes in live cells that likely contribute to the enhanced BCL-2 inhibition by PZ703b. With further optimization, analogues of PZ703b could potentially be developed as effective antitumor agents by co-targeting BCL-XL and BCL-2.
Asunto(s)
Descubrimiento de Drogas , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteína bcl-X/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Proteolisis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Relación Estructura-Actividad , Proteína bcl-X/metabolismoRESUMEN
BACKGROUND: Patients with advanced T cell lymphomas (TCLs) have limited therapeutic options and poor outcomes in part because their TCLs evade apoptosis through upregulation of anti-apoptotic Bcl-2 proteins. Subsets of TCL cell lines, patient-derived xenografts (PDXs), and primary patient samples depend on Bcl-xL for survival. However, small molecule Bcl-xL inhibitors such as ABT263 have failed during clinical development due to on-target and dose-limiting thrombocytopenia. METHODS: We have developed DT2216, a proteolysis targeting chimera (PROTAC) targeting Bcl-xL for degradation via Von Hippel-Lindau (VHL) E3 ligase, and shown that it has better anti-tumor activity but is less toxic to platelets compared to ABT263. Here, we examined the therapeutic potential of DT2216 for TCLs via testing its anti-TCL activity in vitro using MTS assay, immunoblotting, and flow cytometry and anti-TCL activity in vivo using TCL cell xenograft and PDX model in mice. RESULTS: The results showed that DT2216 selectively killed various Bcl-xL-dependent TCL cells including MyLa cells in vitro. In vivo, DT2216 alone was highly effective against MyLa TCL xenografts in mice without causing significant thrombocytopenia or other toxicity. Furthermore, DT2216 combined with ABT199 (a selective Bcl-2 inhibitor) synergistically reduced disease burden and improved survival in a TCL PDX mouse model dependent on both Bcl-2 and Bcl-xL. CONCLUSIONS: These findings support the clinical testing of DT2216 in patients with Bcl-xL-dependent TCLs, both as a single agent and in rational combinations.
Asunto(s)
Antineoplásicos/uso terapéutico , Linfoma de Células T/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Proteína bcl-X/antagonistas & inhibidores , Compuestos de Anilina/uso terapéutico , Compuestos de Anilina/toxicidad , Animales , Antineoplásicos/farmacología , Antineoplásicos/toxicidad , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Plaquetas/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Línea Celular Tumoral , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Femenino , Supervivencia de Injerto , Humanos , Hígado/patología , Linfoma de Células T/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Neoplasias/metabolismo , Trasplante de Neoplasias , Piperazinas , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Distribución Aleatoria , Bazo/patología , Sulfonamidas/uso terapéutico , Sulfonamidas/toxicidad , Ubiquitina-Proteína Ligasas/química , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína bcl-X/metabolismoRESUMEN
Previous studies in a rat model of Sephadex induced lung inflammation showed that 4-Thiouridine (4SU), a thiol substituted nucleoside, was very effective in reducing edema, leukocyte influx and TNF levels in bronchoalvelolar lavage fluid. However, little is known about the factors and mechanisms underlying these effects. In the present study, we have used two separate mouse models of chronic inflammation, a model of dextran sulphate sodium (DSS) induced colitis and a model of antigen induced arthritis, to evaluate the anti-inflammatory effect of 4-thiouridine. We have analyzed a broad spectrum of inflammatory mediators in order to delineate the mechanisms behind a potential anti-inflammatory effect of 4SU. Colitis was induced in C57BL/6 mice by administration of 3.5% DSS in drinking water for 5 days and the potential anti-colitic effect of 4SU was assessed by monitoring the disease activity index (DAI), measurement of colon length and histopathological analysis of colon tissue. We analyzed tissue myeloperoxidase (MPO) activity, serum pro-inflammatory cytokines (IL-1ß, IL-6 and TNF), mRNA and protein expression of pro-inflammatory cytokines, COX-2, and NF-κB activity in colitis tissue. Intracolonic administration of 4SU (5 mg/kg & 10 mg/kg.) significantly inhibited MPO activity and reduced the levels of pro-inflammatory cytokines (IL-1ß, IL-6 and TNF) as well as COX-2. Further, NF-κB activation was also blocked by attenuating the phosphorylation of IkB kinase (IKK α/ß) in DSS-induced colitis tissues. Arthritis was induced by intra-articular injection of mBSA in the knee of NMRI mice pre-immunized with mBSA and 4SU was administered locally by direct injection into the knee joint. The antiarthritic potential of 4SU was calculated by histopathological scores and histochemical analysis of joint tissue. Further, immunohistochemistry was used to study inflammatory cell infiltration and expression of cytokines and adhesion molecules in the synovium. Local administration of 50-100 mg/kg 4SU at the time of arthritis onset clearly prevented development of joint inflammation and efficiently inhibited synovial expression of CD18, local cytokine production and recruitment of leukocytes to the synovium. Taken together, our data clearly demonstrates a potent anti-inflammatory effect of 4SU in two experimental models. In conclusion 4SU could be a new promising candidate for therapeutic modulation of chronic inflammatory diseases like ulcerative colitis and arthritis.
Asunto(s)
Antiinflamatorios/uso terapéutico , Artritis/tratamiento farmacológico , Colitis Ulcerosa/tratamiento farmacológico , Tiouridina/uso terapéutico , Animales , Antiinflamatorios/farmacología , Antígenos , Artritis/inmunología , Artritis/patología , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/inmunología , Colitis Ulcerosa/patología , Colon/efectos de los fármacos , Colon/inmunología , Colon/patología , Ciclooxigenasa 2/inmunología , Citocinas/sangre , Citocinas/genética , Citocinas/inmunología , Sulfato de Dextran , Modelos Animales de Enfermedad , Femenino , Articulación de la Rodilla/efectos de los fármacos , Articulación de la Rodilla/inmunología , Articulación de la Rodilla/patología , Masculino , Ratones Endogámicos C57BL , FN-kappa B/inmunología , Albúmina Sérica Bovina , Tiouridina/farmacologíaRESUMEN
Targeting BCL-XL via PROTACs is a promising strategy in reducing BCL-XL inhibition associated platelet toxicity. Recently, we reported potent BCL-XL PROTAC degraders that recruit VHL or CRBN E3 ligase. However, low protein expression or mutation of the responsible E3 ligase has been known to result in decreased protein degradation efficiency of the corresponding PROTACs. To overcome these mechanisms of resistance, PROTACs based on recruiting alternative E3 ligases could be generated. Thus, we designed and synthesized a series of PROTACs that recruit IAP E3 ligases for BCL-XL degradation. Among those PROTACs, compound 8a efficiently degrades BCL-XL in malignant T-cell lymphoma cell line MyLa 1929 while CRBN-based PROTACs that have high potency in other cancer cell lines show compromised potency, likely due to the low CRBN expression. Moreover, compared with the parent compound ABT-263, PROTAC 8a shows comparable cell killing effects in MyLa 1929 cells whereas the on-target platelet toxicity is significantly reduced. Our findings expand the anti-tumor spectra of BCL-XL degraders and further highlight the importance of selecting suitable E3 members to achieve effective cellular activity.
Asunto(s)
Antineoplásicos/farmacología , Descubrimiento de Drogas , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteolisis/efectos de los fármacos , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Proteína bcl-X/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Estructura Molecular , Relación Estructura-Actividad , Ubiquitina-Proteína Ligasas/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
Small molecules that selectively kill senescent cells (SCs), termed senolytics, have the potential to prevent and treat various age-related diseases and extend healthspan. The use of Bcl-xl inhibitors as senolytics is largely limited by their on-target and dose-limiting platelet toxicity. Here, we report the use of proteolysis-targeting chimera (PROTAC) technology to reduce the platelet toxicity of navitoclax (also known as ABT263), a Bcl-2 and Bcl-xl dual inhibitor, by converting it into PZ15227 (PZ), a Bcl-xl PROTAC, which targets Bcl-xl to the cereblon (CRBN) E3 ligase for degradation. Compared to ABT263, PZ is less toxic to platelets, but equally or slightly more potent against SCs because CRBN is poorly expressed in platelets. PZ effectively clears SCs and rejuvenates tissue stem and progenitor cells in naturally aged mice without causing severe thrombocytopenia. With further improvement, Bcl-xl PROTACs have the potential to become safer and more potent senolytic agents than Bcl-xl inhibitors.
Asunto(s)
Envejecimiento/efectos de los fármacos , Compuestos de Anilina/farmacología , Plaquetas/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Sulfonamidas/farmacología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Compuestos de Anilina/química , Animales , Línea Celular , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Modelos Animales , Cultivo Primario de Células , Proteolisis/efectos de los fármacos , Sulfonamidas/química , Ubiquitina-Proteína Ligasas , Proteína bcl-X/antagonistas & inhibidores , Proteína bcl-X/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related death with a median survival time of 6-12 months. Most patients present with disseminated disease and the majority are offered palliative chemotherapy. With no approved treatment modalities for patients who progress on chemotherapy, we explored the effects of long-term gemcitabine administration on the tumor microenvironment to identify potential therapeutic options for chemorefractory PDAC. Using a combination of mouse models, primary cell line-derived xenografts, and established tumor cell lines, we first evaluated chemotherapy-induced alterations in the tumor secretome and immune surface proteins by high throughput proteomic arrays. In addition to enhancing antigen presentation and immune checkpoint expression, gemcitabine consistently increased the synthesis of CCL/CXCL chemokines and TGFß-associated signals. These secreted factors altered the composition of the tumor stroma, conferring gemcitabine resistance to cancer-associated fibroblasts in vitro and further enhancing TGFß1 biosynthesis. Combined gemcitabine and anti-PD-1 treatment in transgenic models of murine PDAC failed to alter disease course unless mice also underwent genetic or pharmacologic ablation of TGFß signaling. In the setting of TGFß signaling deficiency, gemcitabine and anti-PD-1 led to a robust CD8+ T-cell response and decrease in tumor burden, markedly enhancing overall survival. These results suggest that gemcitabine successfully primes PDAC tumors for immune checkpoint inhibition by enhancing antigen presentation only following disruption of the immunosuppressive cytokine barrier. Given the current lack of third-line treatment options, this approach warrants consideration in the clinical management of gemcitabine-refractory PDAC. SIGNIFICANCE: These data suggest that long-term treatment with gemcitabine leads to extensive reprogramming of the pancreatic tumor microenvironment and that patients who progress on gemcitabine-based regimens may benefit from multidrug immunotherapy.See related commentary by Carpenter et al., p. 3070 GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/15/3101/F1.large.jpg.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Humanos , Inmunoterapia , Ratones , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Proteómica , Microambiente Tumoral , GemcitabinaRESUMEN
Anti-apoptotic protein BCL-XL plays a key role in tumorigenesis and cancer chemotherapy resistance, rendering it an attractive target for cancer treatment. However, BCL-XL inhibitors such as ABT-263 cannot be safely used in the clinic because platelets solely depend on BCL-XL to maintain their viability. To reduce the on-target platelet toxicity associated with the inhibition of BCL-XL, we designed and synthesized PROTAC BCL-XL degraders that recruit CRBN or VHL E3 ligase because both of these enzymes are poorly expressed in human platelets compared to various cancer cell lines. We confirmed that platelet-toxic BCL-XL/2 dual inhibitor ABT-263 can be converted into platelet-sparing CRBN/VHL-based BCL-XL specific degraders. A number of BCL-XL degraders are more potent in killing cancer cells than their parent compound ABT-263. Specifically, XZ739, a CRBN-dependent BCL-XL degrader, is 20-fold more potent than ABT-263 against MOLT-4 T-ALL cells and has >100-fold selectivity for MOLT-4 cells over human platelets. Our findings further demonstrated the utility of PROTAC technology to achieve tissue selectivity through recruiting differentially expressed E3 ligases.
Asunto(s)
Compuestos de Anilina/farmacología , Antineoplásicos/farmacología , Plaquetas/efectos de los fármacos , Descubrimiento de Drogas , Proteolisis/efectos de los fármacos , Sulfonamidas/farmacología , Proteína bcl-X/antagonistas & inhibidores , Compuestos de Anilina/síntesis química , Compuestos de Anilina/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/química , Células Tumorales CultivadasRESUMEN
Core-shell nanostructures are promising platforms for combination drug delivery. However, their complicated synthesis process, poor stability, surface engineering, and low biocompatibility are major hurdles. Herein, a carboxymethyl chitosan-coated poly(lactide-co-glycolide) (cmcPLGA) core-shell nanostructure is prepared via a simple one-step nanoprecipitation self-assembly process. Engineered core-shell nanostructures are tested for combination delivery of loaded docetaxel and doxorubicin in a cancer-mimicked environment. The drugs are compartmentalized in a shell (doxorubicin, Dox) and a core (docetaxel, Dtxl) with loading contents of â¼1.2 and â¼2.06%, respectively. Carboxymethyl chitosan with both amine and carboxyl groups act as a polyampholyte in diminishing ζ-potential of nanoparticles from fairly negative (-13 mV) to near neutral (-2 mV) while moving from a physiological pH (7.4) to an acidic tumor pH (6) that can help the nanoparticles to accumulate and release the drug on-site. The dual-drug formulation was found to carry a clinically comparable 1.7:1 weight ratio of Dtxl/Dox, nanoengineered for the sequential release of Dox followed by Dtxl. Single and engineered combinatorial nanoformulations show better growth inhibition toward three different cancer cells compared to free drug treatment. Importantly, Dox-Dtxl cmcPLGA nanoparticles scored synergism with combination index values between 0.2 and 0.3 in BT549 (breast ductal carcinoma), PC3 (prostate cancer), and A549 (lung adenocarcinoma) cell lines, demonstrating significant cell growth inhibition at lower drug concentrations as compared to single-drug control groups. The observed promising performance of dual-drug formulation is due to the G2/M phase arrest and apoptosis.
RESUMEN
B-cell lymphoma extra large (BCL-XL) is a well-validated cancer target. However, the on-target and dose-limiting thrombocytopenia limits the use of BCL-XL inhibitors, such as ABT263, as safe and effective anticancer agents. To reduce the toxicity of ABT263, we converted it into DT2216, a BCL-XL proteolysis-targeting chimera (PROTAC), that targets BCL-XL to the Von Hippel-Lindau (VHL) E3 ligase for degradation. We found that DT2216 was more potent against various BCL-XL-dependent leukemia and cancer cells but considerably less toxic to platelets than ABT263 in vitro because VHL is poorly expressed in platelets. In vivo, DT2216 effectively inhibits the growth of several xenograft tumors as a single agent or in combination with other chemotherapeutic agents, without causing appreciable thrombocytopenia. These findings demonstrate the potential to use PROTAC technology to reduce on-target drug toxicities and rescue the therapeutic potential of previously undruggable targets. Furthermore, DT2216 may be developed as a safe first-in-class anticancer agent targeting BCL-XL.
Asunto(s)
Compuestos de Anilina/farmacología , Sulfonamidas/farmacología , Trombocitopenia/tratamiento farmacológico , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína bcl-X/genética , Compuestos de Anilina/química , Animales , Antineoplásicos/farmacología , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Xenoinjertos , Humanos , Ratones , Proteolisis , Sulfonamidas/química , Trombocitopenia/genética , Trombocitopenia/patología , Proteína bcl-X/antagonistas & inhibidoresRESUMEN
BCL-XL, an anti-apoptotic BCL-2 family protein, plays a key role in cancer cell survival. However, the potential of BCL-XL as an anti-cancer target has been hampered by the on-target platelet toxicity because platelets depend on BCL-XL to maintain their viability. Here we report the development of a PROTAC BCL-XL degrader, XZ424, which has increased selectivity for BCL-XL-dependent MOLT-4 cells over human platelets compared with conventional BCL-XL inhibitors. This proof-of-concept study demonstrates the potential of utilizing a PROTAC approach to achieve tissue selectivity.
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Isoquinolinas/química , Talidomida/análogos & derivados , Proteína bcl-X/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis/efectos de los fármacos , Plaquetas/citología , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Línea Celular Tumoral , Humanos , Isoquinolinas/metabolismo , Isoquinolinas/farmacología , Unión Proteica , Talidomida/química , Talidomida/metabolismo , Talidomida/farmacología , Ubiquitina-Proteína Ligasas , Proteína bcl-X/antagonistas & inhibidoresRESUMEN
Radiation-induced pulmonary fibrosis (RIPF) is a serious treatment complication that affects about 9%-30% cancer patients receiving radiotherapy for thoracic tumors. RIPF is characterized by progressive and irreversible destruction of lung tissues and deterioration of lung function, which can compromise quality of life and eventually lead to respiratory failure and death. Unfortunately, the mechanisms by which radiation causes RIPF have not been well established nor has an effective treatment for RIPF been developed. Recently, an increasing body of evidence suggests that induction of senescence by radiation may play an important role in RIPF and clearance of senescent cells (SnCs) with a senolytic agent, small molecule that can selectively kill SnCs, has the potential to be developed as a novel therapeutic strategy for RIPF. This review discusses some of these new findings to promote further study on the role of cellular senescence in RIPF and the development of senolytic therapeutics for RIPF.
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Senescencia Celular , Fibrosis Pulmonar Idiopática/etiología , Fibrosis Pulmonar Idiopática/patología , Traumatismos por Radiación/complicaciones , Animales , Humanos , Modelos BiológicosRESUMEN
Autophagy is a homeostatic process that recycles damaged organelles and long-lived proteins by delivering them in double-membrane vesicles to lysosomes for degradation. Autophagy has a prominent role in survival, proliferation, and resistance of tumors in metabolic and chemotherapeutic stress conditions. Clinical trials with chloroquine-a known autophagy inhibitor-were unable to achieve complete autophagy inhibition in vivo, warranting the search for more potent autophagy inhibitors. In a process of exploring the mechanism of action of previously identified cytotoxic s-triazine analogs, we discovered that both IITZ-01 and IITZ-02 act as potent autophagy inhibitors. Treatment with these compounds resulted in the vacuolated appearance of cells due to their specific accumulation in lysosomes. In addition, these basic compounds also deacidify lysosomes as evidenced by the decrease in lysotracker red staining and inhibit maturation of lysosomal enzymes leading to lysosomal dysfunction. IITZ-01 and IITZ-02 enhance autophagosome accumulation but inhibit autophagosomal degradation by impairing lysosomal function, finally resulting in the inhibition of autophagy. Interestingly, compound IITZ-01 exhibited more than 10-fold potent autophagy inhibition along with 12- to 20-fold better cytotoxic action than CQ. IITZ-01 and IITZ-02 also abolished mitochondrial membrane potential and triggered apoptosis through the mitochondria-mediated pathway. Furthermore, IITZ-01 and IITZ-02 displayed potent antitumor action in vivo through autophagy inhibition and apoptosis induction in MDA-MB-231 breast cancer xenograft model with IITZ-01 exhibiting superior anticancer efficacy. Overall, these data demonstrate that IITZ-01 is potent autophagy inhibitor with single-agent anticancer activity and awaits further preclinical development as potential anticancer therapeutic.
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Antineoplásicos/uso terapéutico , Autofagia/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Autofagosomas/efectos de los fármacos , Autofagosomas/ultraestructura , Línea Celular Tumoral , Femenino , Humanos , Concentración de Iones de Hidrógeno , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ratones Endogámicos BALB C , Ratones Desnudos , Mitocondrias/efectos de los fármacos , Estructura Molecular , Distribución Aleatoria , Método Simple Ciego , Neoplasias de la Mama Triple Negativas/patología , Ensayo de Tumor de Célula Madre , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Osteoporosis is a major debilitating cause of fractures and decreases the quality of life in elderly patients. Bone homeostasis is maintained by bone forming osteoblasts and bone resorpting osteoclasts. Substantial evidences have shown that targeting osteoclasts using natural products is a promising strategy for the treatment of osteoporosis. In the current study, we investigated the osteoprotective effect of Abietic acid (AA) in in vitro and in vivo models of osteolysis. In vitro experiments demonstrated that, AA suppressed receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and F-actin ring formation in a concentration dependent manner. Mechanistically, AA abrogated RANKL-induced phosphorylation of IKKα/ß (ser 176/180), IkBα (ser 32), and inhibited the nuclear translocation of NF-κB. We also found that, AA attenuated the RANKL-induced phosphorylation of MAPKs and decreased the expression of osteoclast specific genes such as TRAP, DC-STAMP, c-Fos, and NFATc1. Consistent with in vitro results, in vivo Lipoploysaccharide (LPS)-induced osteolysis model showed that AA inhibited the LPS-induced serum surge in cytokines TNF-α and IL-6. µ-CT analysis showed that AA prevented the LPS-induced osteolysis. Furthermore, histopathology and TRAP staining results suggested that AA decreased the number of osteoclasts in LPS-injected mice. Taken together, we demonstrated that the osteoprotective action of AA is coupled with the inhibition of NF-κB and MAPK signaling and subsequent inhibition of NFATc1 and c-Fos activities. Hence, AA may be considered as a promising drug candidate for the treatment of osteoporosis.
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Abietanos/administración & dosificación , Inflamación/tratamiento farmacológico , Osteogénesis/genética , Osteólisis/tratamiento farmacológico , Ligando RANK/genética , Actinas/genética , Animales , Diferenciación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/patología , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , FN-kappa B/genética , Factores de Transcripción NFATC/genética , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacos , Osteólisis/inducido químicamente , Osteólisis/genética , Osteólisis/patología , Osteoporosis/inducido químicamente , Osteoporosis/tratamiento farmacológico , Osteoporosis/genética , Osteoporosis/patología , Fosfatidiletanolaminas/toxicidad , Fosforilación/efectos de los fármacos , Polilisina/análogos & derivados , Polilisina/toxicidad , Transducción de Señal/efectos de los fármacosRESUMEN
A total of twenty-two novel coumarin triazole hybrids (4a-4k and 6a-6k) were synthesized from orcinol in good to excellent yields of 70-94%. The structures of all the synthesized compounds were elucidated by spectroscopic techniques such as 1H NMR, 13C NMR, and HRMS. The anti-inflammatory potential of synthesized compounds was investigated against the proinflammatory cytokine, TNF-α on U937 cell line and compounds 4d, 4j, and 6j were found to exhibit promising anti-inflammatory activity. These three compounds were further screened against TNF-α on LPS-stimulated RAW 264.7 cells, which confirm their anti-inflammatory potential. Furthermore, the above said active compounds were tested for their inhibitory effect on RANKL-induced osteoclastogenesis in RAW 264.7 cells by using tartrate resistant acid phosphatase (TRAP) staining assay at 10⯵M. Molecular mechanism studies demonstrated that compound 4d exhibited dose dependent inhibition of RANKL-induced osteoclastogenesis by suppression of the NF-kB pathway. Thus, compound 4d is a promising candidate for further optimization to develop as a potent anti-osteoporotic agent.
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Cumarinas/farmacología , FN-kappa B/antagonistas & inhibidores , Osteogénesis/efectos de los fármacos , Receptor Activador del Factor Nuclear kappa-B/antagonistas & inhibidores , Resorcinoles/farmacología , Transducción de Señal/efectos de los fármacos , Triazoles/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Cumarinas/síntesis química , Cumarinas/química , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Estructura Molecular , FN-kappa B/metabolismo , Células RAW 264.7 , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Resorcinoles/química , Relación Estructura-Actividad , Triazoles/síntesis química , Triazoles/química , Células U937RESUMEN
Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor, which plays an important role in the cellular defense against oxidative stress by induction of anti-oxidant and cytoprotective enzymes. In the current study, we sought to investigate the osteoprotective effect of carnosic acid (CA), a phenolic (catecholic) diterpene. It is widely identified for its electrophilic nature under oxidative stress conditions and thus anticipated to counter osteoporosis by facilitation of Nrf2 signalling. Osteoclast differentiation was induced by incubation of RAW 264.7 (mouse macrophage) cells and mouse bone marrow macrophages (BMMs) in the presence of receptor activator of NF-κB ligand (RANKL) (100 ng/ml). After treatment, osteoclastogenesis was assessed using tartrate-resistant acid phosphatase (TRAP) assay. We observed that 6 h pretreatment with CA (1.25, 2.5, 5 µM) decreased RANKL-induced osteoclast formation and abolished RANKL-induced ROS generation by activating Nrf2 and its transcriptional targets. Further, CA also inhibited RANKL-induced activation of NF-κB and MAPK signalling. RANKL-induced mRNA expression of osteoclast related genes and transcription factors was also diminished by CA. In vivo osteolysis was developed in C57BL/6 male mice using lipopolysaccharide (LPS). Consistent with in vitro results, in vivo µ-CT analysis of femurs showed that bone mineral density (BMD), bone mineral content (BMC), and bone architecture parameters such as trabecular thickness (Tb.Th) and trabecular space (Tb.Sp) were positively modulated by CA in LPS-injected mice. The results obtained in this study support that CA inhibits RANKL-induced osteoclastogenesis by maintaining redox homeostasis through modulation of Nrf2 and NF-κB pathways. KEY MESSAGES: Carnosic acid (CA) inhibits RANKL-induced osteoclastogenesis. CA inhibits RANKL-induced oxidative stress by upregulating Nrf2 transcriptional targets. CA attenuates RANKL-induced NF-κB and MAPK signalling activation. CA decreases NFATc1 and c-Fos expression. In vivo µ-CT analysis reveals that CA prevents bone loss in LPS-injected mice.