RESUMEN
ABSTRACT: Fanconi anemia (FA) is an inherited DNA repair disorder characterized by bone marrow (BM) failure, developmental abnormalities, myelodysplasia, leukemia, and solid tumor predisposition. Allogeneic hematopoietic stem cell transplantation (allo-HSCT), a mainstay treatment, is limited by conditioning regimen-related toxicity and graft-versus-host disease (GVHD). Antibody-drug conjugates (ADCs) targeting hematopoietic stem cells (HSCs) can open marrow niches permitting donor stem cell alloengraftment. Here, we report that single dose anti-mouse CD45-targeted ADC (CD45-ADC) facilitated stable, multilineage chimerism in 3 distinct FA mouse models representing 90% of FA complementation groups. CD45-ADC profoundly depleted host stem cell enriched Lineage-Sca1+cKit+ cells within 48 hours. Fanca-/- recipients of minor-mismatched BM and single dose CD45-ADC had peripheral blood (PB) mean donor chimerism >90%; donor HSCs alloengraftment was verified in secondary recipients. In Fancc-/- and Fancg-/- recipients of fully allogeneic grafts, PB mean donor chimerism was 60% to 80% and 70% to 80%, respectively. The mean percent donor chimerism in BM and spleen mirrored PB results. CD45-ADC-conditioned mice did not have clinical toxicity. A transient <2.5-fold increase in hepatocellular enzymes and mild-to-moderate histopathological changes were seen. Under GVHD allo-HSCT conditions, wild-type and Fanca-/- recipients of CD45-ADC had markedly reduced GVHD lethality compared with lethal irradiation. Moreover, single dose anti-human CD45-ADC given to rhesus macaque nonhuman primates on days -6 or -10 was at least as myeloablative as lethal irradiation. These data suggest that CD45-ADC can potently promote donor alloengraftment and hematopoiesis without significant toxicity or severe GVHD, as seen with lethal irradiation, providing strong support for clinical trial considerations in highly vulnerable patients with FA.
Asunto(s)
Anemia de Fanconi , Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Inmunoconjugados , Antígenos Comunes de Leucocito , Animales , Anemia de Fanconi/terapia , Ratones , Enfermedad Injerto contra Huésped/patología , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Acondicionamiento Pretrasplante/métodos , Trasplante Homólogo , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
BACKGROUND: Mutations in the recombinase-activating genes 1 and 2 (RAG1, RAG2) cause a spectrum of phenotypes, ranging from severe combined immune deficiency to combined immune deficiency with immune dysregulation (CID-ID). Hematopoietic cell transplantation is a curative option. Use of conditioning facilitates robust and durable stem cell engraftment and immune reconstitution but may cause toxicity. Transplantation from haploidentical donors is associated with poor outcome in patients with CID-ID. OBJECTIVES: We sought to evaluate multilineage engraftment and immune reconstitution after conditioning with CD45-antibody drug conjugate (CD45-ADC) as a single agent in hypomorphic mice with Rag1 mutation treated with congenic and haploidentical hematopoietic cell transplantation. METHODS: Rag1-F971L mice, a model of CID-ID, were conditioned with various doses of CD45-ADC, total body irradiation, or isotype-ADC, and then given transplants of total bone marrow cells from congenic or haploidentical donors. Flow cytometry was used to assess chimerism and immune reconstitution. Histology was used to document reconstitution of thymic architecture. RESULTS: Conditioning with CD45-ADC as a single agent allowed robust engraftment and immune reconstitution, with restoration of thymus, bone marrow, and peripheral compartments. The optimal doses of CD45-ADC were 1.5 mg/kg and 5 mg/kg for congenic and haploidentical transplantation, respectively. No graft-versus-host disease was observed. CONCLUSIONS: Conditioning with CD45-ADC alone allows full donor chimerism and immune reconstitution in Rag1 hypomorphic mice even following haploidentical transplantation, opening the way for the implementation of similar approaches in humans.
Asunto(s)
Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Síndromes de Inmunodeficiencia , Humanos , Ratones , Animales , Acondicionamiento Pretrasplante , Trasplante de Médula Ósea , Síndromes de Inmunodeficiencia/terapia , Proteínas de Homeodominio/genéticaRESUMEN
Hematopoietic stem cell (HSC) gene therapy has curative potential; however, its use is limited by the morbidity and mortality associated with current chemotherapy-based conditioning. Targeted conditioning using antibody-drug conjugates (ADC) holds promise for reduced toxicity in HSC gene therapy. Here we test the ability of an antibody-drug conjugate targeting CD117 (CD117-ADC) to enable engraftment in a non-human primate lentiviral gene therapy model of hemoglobinopathies. Following single-dose CD117-ADC, a >99% depletion of bone marrow CD34 + CD90 + CD45RA- cells without lymphocyte reduction is observed, which results are not inferior to multi-day myeloablative busulfan conditioning. CD117-ADC, similarly to busulfan, allows efficient engraftment, gene marking, and vector-derived fetal hemoglobin induction. Importantly, ADC treatment is associated with minimal toxicity, and CD117-ADC-conditioned animals maintain fertility. In contrast, busulfan treatment commonly causes severe toxicities and infertility in humans. Thus, the myeloablative capacity of single-dose CD117-ADC is sufficient for efficient engraftment of gene-modified HSCs while preserving fertility and reducing adverse effects related to toxicity in non-human primates. This targeted conditioning approach thus provides the proof-of-principle to improve risk-benefit ratio in a variety of HSC-based gene therapy products in humans.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Inmunoconjugados , Animales , Busulfano/farmacología , Terapia Genética/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas , Inmunoconjugados/farmacología , Proteínas Proto-Oncogénicas c-kit/inmunología , Proteínas Proto-Oncogénicas c-kit/uso terapéutico , Macaca mulatta/inmunologíaRESUMEN
Tissue-resident myeloid (TRM) cells in adults have highly variable lifespans, and may be derived from early embryonic yolk sac, fetal liver, or bone marrow. Some of these TRM cells are known pathogenic participants in congenital and acquired diseases. Myeloablative conditioning and hematopoietic stem cell transplantation can replace long-lived brain TRM cells, resulting in clinical improvements in metabolic storage diseases. With the advent of antibody-drug conjugate (ADC)-targeted cell killing as a cell-selective means of transplant conditioning, we assessed the impact of anti-CD45-ADC on TRM cells in multiple tissues. Replacement of TRM cells ranged from 40% to 95% efficiencies in liver, lung, and skin tissues, after a single anti-CD45-ADC dose and bone marrow hematopoietic cell transfer. Of note, the population size of TRM cells in tissues returned to pretreatment levels, suggesting a regulated control of TRM cell abundance. As expected, brain microglia were not affected, but brain monocytes and macrophages were 50% replaced. Anti-CD45-ADC and adoptive cell transfer were then tested in the chronic acquired condition, atherosclerosis exacerbated by Tet2 mutant clonal hematopoiesis. Plaque-resident myeloid cells were efficiently replaced with anti-CD45-ADC and wild-type bone marrow cells. Notably, this reduced existent atherosclerotic plaque burden. Overall, these results indicate that the anti-CD45-ADC clears both hematopoietic stem and TRM cells from their niches, enabling cell replacement to achieve disease modification in a resident myeloid cell-driven disease.
Asunto(s)
Inmunoconjugados , Adulto , Humanos , Inmunoconjugados/farmacología , Macrófagos , Monocitos , Médula Ósea , MicroglíaRESUMEN
Tissue resident myeloid cells (TRM) in adults have highly variable lifespans and may be derived from early embryonic yolk sac, fetal liver or bone marrow. Some of these TRM are known pathogenic participants in congenital and acquired diseases. Myeloablative conditioning and hematopoietic stem cell transplant can replace long-lived brain TRM resulting in clinical improvements in metabolic storage diseases. With the advent of antibody-drug-conjugate (ADC) targeted cell killing as a cell selective means of transplant conditioning, we assessed the impact of anti-CD45-ADC on TRM in multiple tissues. Replacement of TRM ranged from 40 to 95 percent efficiencies in liver, lung, and skin tissues, after a single anti-CD45-ADC dose and bone marrow hematopoietic cell transfer. Of note, the population size of TRM in tissues returned to pre-treatment levels suggesting a regulated control of TRM abundance. As expected, brain, microglia were not affected, but brain monocytes and macrophages were 50% replaced. Anti-CD45-ADC and adoptive cell transfer were then tested in the chronic acquired condition, atherosclerosis exacerbated by Tet2 mutant clonal hematopoiesis. Plaque resident myeloid cells were efficiently replaced with anti-CD45-ADC and wild-type bone marrow cells. Notably, this reduced existent atherosclerotic plaque burden. Overall, these results indicate that anti-CD45-ADC clears both HSC and TRM niches enabling cell replacement to achieve disease modification in a resident myeloid cell driven disease.
RESUMEN
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative treatment of patients with nonmalignant or malignant blood disorders. Its success has been limited by graft-versus-host disease (GVHD). Current systemic nontargeted conditioning regimens mediate tissue injury and potentially incite and amplify GVHD, limiting the use of this potentially curative treatment beyond malignant disorders. Minimizing systemic nontargeted conditioning while achieving alloengraftment without global immune suppression is highly desirable. Antibody-drug-conjugates (ADCs) targeting hematopoietic cells can specifically deplete host stem and immune cells and enable alloengraftment. We report an anti-mouse CD45-targeted-ADC (CD45-ADC) that facilitates stable murine multilineage donor cell engraftment. Conditioning with CD45-ADC (3 mg/kg) was effective as a single agent in both congenic and minor-mismatch transplant models resulting in full donor chimerism comparable to lethal total body irradiation (TBI). In an MHC-disparate allo-HSCT model, pretransplant CD45-ADC (3 mg/kg) combined with low-dose TBI (150 cGy) and a short course of costimulatory blockade with anti-CD40 ligand antibody enabled 89% of recipients to achieve stable alloengraftment (mean value: 72%). When CD45-ADC was combined with pretransplant TBI (50 cGy) and posttransplant rapamycin, cyclophosphamide (Cytoxan), or a JAK inhibitor, 90% to 100% of recipients achieved stable chimerism (mean: 77%, 59%, 78%, respectively). At a higher dose (5 mg/kg), CD45-ADC as a single agent was sufficient for rapid, high-level multilineage chimerism sustained through the 22 weeks observation period. Therefore, CD45-ADC has the potential utility to confer the benefit of fully myeloablative conditioning but with substantially reduced toxicity when given as a single agent or at lower doses in conjunction with reduced-intensity conditioning.
Asunto(s)
Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Inmunoconjugados , Animales , Quimerismo , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Inmunoconjugados/toxicidad , Ratones , Acondicionamiento Pretrasplante/métodosRESUMEN
BACKGROUND: Mutations in the recombinase-activating genes cause severe immunodeficiency, with a spectrum of phenotypes ranging from severe combined immunodeficiency to immune dysregulation. Hematopoietic stem cell transplantation is the only curative option, but a high risk of graft failure and poor immune reconstitution have been observed in the absence of myeloablation. OBJECTIVES: Our aim was to improve multilineage engraftment; we tested nongenotoxic conditioning with anti-CD45 mAbs conjugated with saporin CD45 (CD45-SAP). METHODS: Rag1-KO and Rag1-F971L mice, which represent models of severe combined immune deficiency and combined immune deficiency with immune dysregulation, respectively, were conditioned with CD45-SAP, CD45-SAP plus 2 Gy of total body irradiation (TBI), 2 Gy of TBI, 8 Gy of TBI, or no conditioning and treated by using transplantation with lineage-negative bone marrow cells from wild-type mice. Flow cytometry and immunohistochemistry were used to assess engraftment and immune reconstitution. Antibody responses to 2,4,6-trinitrophenyl-conjugated keyhole limpet hemocyanin were measured by ELISA, and presence of autoantibody was detected by microarray. RESULTS: Conditioning with CD45-SAP enabled high levels of multilineage engraftment in both Rag1 mutant models, allowed overcoming of B- and T-cell differentiation blocks and thymic epithelial cell defects, and induced robust cellular and humoral immunity in the periphery. CONCLUSIONS: Conditioning with CD45-SAP allows multilineage engraftment and robust immune reconstitution in mice with either null or hypomorphic Rag mutations while preserving thymic epithelial cell homeostasis.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Trasplante de Médula Ósea , Proteínas de Homeodominio/genética , Inmunoconjugados/farmacología , Antígenos Comunes de Leucocito/antagonistas & inhibidores , Saporinas/farmacología , Inmunodeficiencia Combinada Grave/terapia , Acondicionamiento Pretrasplante , Aloinjertos , Animales , Anticuerpos Monoclonales/efectos adversos , Proteínas de Homeodominio/inmunología , Inmunoconjugados/efectos adversos , Antígenos Comunes de Leucocito/genética , Antígenos Comunes de Leucocito/inmunología , Ratones , Ratones Noqueados , Saporinas/efectos adversos , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/inmunologíaRESUMEN
Conditioning chemotherapy is used to deplete hematopoietic stem cells in the recipient's marrow, facilitating donor cell engraftment. Although effective, a major issue with chemotherapy is the systemic genotoxicity that increases the risk for secondary malignancies. Antibody conjugates targeting hematopoietic cells are an emerging non-genotoxic method of opening the marrow niche and promoting engraftment of transplanted cells while maintaining intact marrow cellularity. Specifically, this platform would be useful in diseases associated with DNA damage or cancer predisposition, such as dyskeratosis congenita, Schwachman-Diamond syndrome, and Fanconi anemia (FA). Our approach utilizes antibody-drug conjugates (ADC) as an alternative conditioning regimen in an FA mouse model of autologous transplantation. Antibodies targeting either CD45 or CD117 were conjugated to saporin (SAP), a ribosomal toxin. FANCA knockout mice were conditioned with either CD45-SAP or CD117-SAP prior to receiving whole marrow from a heterozygous healthy donor. Bone marrow and peripheral blood analysis revealed equivalent levels of donor engraftment, with minimal toxicity in ADC-treated groups as compared with cyclophosphamide-treated controls. Our findings suggest ADCs may be an effective conditioning strategy in stem cell transplantation not only for diseases where traditional chemotherapy is not tolerated, but also more broadly for the field of blood and marrow transplantation.
RESUMEN
Hematopoietic stem cell transplantation (HSCT) is a curative therapy for blood and immune diseases with potential for many settings beyond current standard-of-care. Broad HSCT application is currently precluded largely due to morbidity and mortality associated with genotoxic irradiation or chemotherapy conditioning. Here we show that a single dose of a CD117-antibody-drug-conjugate (CD117-ADC) to saporin leads to > 99% depletion of host HSCs, enabling rapid and efficient donor hematopoietic cell engraftment. Importantly, CD117-ADC selectively targets hematopoietic stem cells yet does not cause clinically significant side-effects. Blood counts and immune cell function are preserved following CD117-ADC treatment, with effective responses by recipients to both viral and fungal challenges. These results suggest that CD117-ADC-mediated HSCT pre-treatment could serve as a non-myeloablative conditioning strategy for the treatment of a wide range of non-malignant and malignant diseases, and might be especially suited to gene therapy and gene editing settings in which preservation of immunity is desired.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/efectos de los fármacos , Inmunoconjugados/farmacología , Proteínas Proto-Oncogénicas c-kit/inmunología , Animales , Médula Ósea/efectos de los fármacos , Trasplante de Médula Ósea , Candida albicans/patogenicidad , Muerte Celular , Línea Celular , Femenino , Terapia Genética , Humanos , Inmunoconjugados/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Neoplasias , Donantes de TejidosRESUMEN
Targeted genome editing in hematopoietic stem/progenitor cells (HSPCs) is an attractive strategy for treating immunohematological diseases. However, the limited efficiency of homology-directed editing in primitive HSPCs constrains the yield of corrected cells and might affect the feasibility and safety of clinical translation. These concerns need to be addressed in stringent preclinical models and overcome by developing more efficient editing methods. We generated a humanized X-linked severe combined immunodeficiency (SCID-X1) mouse model and evaluated the efficacy and safety of hematopoietic reconstitution from limited input of functional HSPCs, establishing thresholds for full correction upon different types of conditioning. Unexpectedly, conditioning before HSPC infusion was required to protect the mice from lymphoma developing when transplanting small numbers of progenitors. We then designed a one-size-fits-all IL2RG (interleukin-2 receptor common γ-chain) gene correction strategy and, using the same reagents suitable for correction of human HSPC, validated the edited human gene in the disease model in vivo, providing evidence of targeted gene editing in mouse HSPCs and demonstrating the functionality of the IL2RG-edited lymphoid progeny. Finally, we optimized editing reagents and protocol for human HSPCs and attained the threshold of IL2RG editing in long-term repopulating cells predicted to safely rescue the disease, using clinically relevant HSPC sources and highly specific zinc finger nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9). Overall, our work establishes the rationale and guiding principles for clinical translation of SCID-X1 gene editing and provides a framework for developing gene correction for other diseases.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Edición Génica/métodos , Marcación de Gen/métodos , Subunidad gamma Común de Receptores de Interleucina/genética , Subunidad gamma Común de Receptores de Interleucina/metabolismo , Ratones , Ratones SCIDRESUMEN
Hematopoietic stem cell transplantation (HSCT) offers curative therapy for patients with hemoglobinopathies, congenital immunodeficiencies, and other conditions, possibly including AIDS. Autologous HSCT using genetically corrected cells would avoid the risk of graft-versus-host disease (GVHD), but the genotoxicity of conditioning remains a substantial barrier to the development of this approach. Here we report an internalizing immunotoxin targeting the hematopoietic-cell-restricted CD45 receptor that effectively conditions immunocompetent mice. A single dose of the immunotoxin, CD45-saporin (SAP), enabled efficient (>90%) engraftment of donor cells and full correction of a sickle-cell anemia model. In contrast to irradiation, CD45-SAP completely avoided neutropenia and anemia, spared bone marrow and thymic niches, enabling rapid recovery of T and B cells, preserved anti-fungal immunity, and had minimal overall toxicity. This non-genotoxic conditioning method may provide an attractive alternative to current conditioning regimens for HSCT in the treatment of non-malignant blood diseases.
Asunto(s)
Daño del ADN/inmunología , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/inmunología , Antígenos Comunes de Leucocito/inmunología , Proteínas Inactivadoras de Ribosomas Tipo 1/genética , Proteínas Inactivadoras de Ribosomas Tipo 1/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Células Cultivadas , Daño del ADN/genética , Femenino , Mejoramiento Genético/métodos , Fenómenos Inmunogenéticos/genética , Inmunotoxinas , Ratones , Ratones Endogámicos C57BL , SaporinasRESUMEN
Apoptosis is generally believed to be a process that requires several hours, in contrast to non-programmed forms of cell death that can occur in minutes. Our findings challenge the time-consuming nature of apoptosis as we describe the discovery and characterization of a small molecule, named Raptinal, which initiates intrinsic pathway caspase-dependent apoptosis within minutes in multiple cell lines. Comparison to a mechanistically diverse panel of apoptotic stimuli reveals that Raptinal-induced apoptosis proceeds with unparalleled speed. The rapid phenotype enabled identification of the critical roles of mitochondrial voltage-dependent anion channel function, mitochondrial membrane potential/coupled respiration, and mitochondrial complex I, III, and IV function for apoptosis induction. Use of Raptinal in whole organisms demonstrates its utility for studying apoptosis in vivo for a variety of applications. Overall, rapid inducers of apoptosis are powerful tools that will be used in a variety of settings to generate further insight into the apoptotic machinery.
Asunto(s)
Apoptosis , Ciclopentanos/química , Fluorenos/química , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/química , Caspasa 3/genética , Caspasa 3/metabolismo , Línea Celular Tumoral , Complemento C8/deficiencia , Complemento C8/genética , Ciclopentanos/farmacocinética , Ciclopentanos/toxicidad , Embrión no Mamífero/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/deficiencia , Proteína de Dominio de Muerte Asociada a Fas/genética , Fluorenos/farmacocinética , Fluorenos/toxicidad , Semivida , Humanos , Células Jurkat , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Pez CebraRESUMEN
The glycosyltransferase gene, Ext1, is essential for heparan sulfate production. Induced deletion of Ext1 selectively in Mx1-expressing bone marrow (BM) stromal cells, a known population of skeletal stem/progenitor cells, in adult mice resulted in marked changes in hematopoietic stem and progenitor cell (HSPC) localization. HSPC egressed from BM to spleen after Ext1 deletion. This was associated with altered signaling in the stromal cells and with reduced vascular cell adhesion molecule 1 production by them. Further, pharmacologic inhibition of heparan sulfate mobilized qualitatively more potent and quantitatively more HSPC from the BM than granulocyte colony-stimulating factor alone, including in a setting of granulocyte colony-stimulating factor resistance. The reduced presence of endogenous HSPC after Ext1 deletion was associated with engraftment of transfused HSPC without any toxic conditioning of the host. Therefore, inhibiting heparan sulfate production may provide a means for avoiding the toxicities of radiation or chemotherapy in HSPC transplantation for nonmalignant conditions.
Asunto(s)
Movilización de Célula Madre Hematopoyética/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Heparitina Sulfato/biosíntesis , N-Acetilglucosaminiltransferasas/metabolismo , Células del Estroma/metabolismo , Acondicionamiento Pretrasplante , Animales , Anticoagulantes/farmacología , Unión Competitiva/inmunología , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Experimental/metabolismo , Factor Estimulante de Colonias de Granulocitos/farmacología , Proteínas Fluorescentes Verdes/genética , Heparina/farmacología , Heparitina Sulfato/inmunología , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , N-Acetilglucosaminiltransferasas/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Células del Estroma/inmunología , Molécula 1 de Adhesión Celular Vascular/inmunología , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Effective glucose diet: We report the development and activity of glucose-conjugated LDH-A inhibitors designed for dual targeting of the Warburg effect (elevated glucose uptake and glycolysis) in cancer cells. Glycoconjugation could be applied to inhibitors of many enzymes involved in glycolysis or tumor metabolism.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Glucosa/química , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Glucosa/metabolismo , Glucólisis/efectos de los fármacos , Humanos , L-Lactato Deshidrogenasa/metabolismo , Modelos Moleculares , Estructura Molecular , Neoplasias/diagnóstico , Neoplasias/enzimología , Relación Estructura-ActividadRESUMEN
The evasion of apoptosis is a key characteristic of cancer, and thus strategies to selectively induce apoptosis in cancer cells hold considerable promise in personalized anticancer therapy. Structurally similar procaspase activating compounds PAC-1 and S-PAC-1 restore procaspase-3 activity through the chelation of inhibitory zinc ions in vitro, induce apoptotic death of cancer cells in culture, and reduce tumor burden in vivo. Ip or iv administrations of high doses of PAC-1 are transiently neurotoxic in vivo, while S-PAC-1 is safe even at very high doses and has been evaluated in a phase I clinical trial of pet dogs with spontaneously occurring lymphoma. Here we show that PAC-1 and S-PAC-1 have similar mechanisms of cell death induction at low concentrations (less than 50 µM), but at high concentrations PAC-1 displays unique cell death induction features. Cells treated with a high concentration of PAC-1 have a distinctive gene expression profile, unusual cellular and mitochondrial morphology, and an altered intracellular Ca(2+) concentration, indicative of endoplasmic reticulum (ER) stress-induced apoptosis. These studies suggest strategies for anticancer clinical development, specifically bolus dosing for PAC-1 and continuous rate infusion for S-PAC-1.
Asunto(s)
Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Calcio/metabolismo , Línea Celular Tumoral , Perros , Femenino , Células HL-60 , Células HeLa , Humanos , Hidrazonas/farmacología , Ratones , Microscopía Confocal , Microscopía Electrónica de Transmisión , Piperazinas/farmacología , Zinc/metabolismoRESUMEN
The combined use of several drugs targeting different cellular functions is one approach to achieve tumor control in cancer. We studied the effects of Pseudomonas exotoxin A (PE)-based immunotoxins (ITs), the 9.2.27PE and the 425.3PE, together with 2 different triphenylmethyl derivatives, triphenylmethyl phosphonates and phosphonochloridates (TPMP)-I-2 and 4BI. Combining the triphenylmethyl derivatives with ITs enhanced the cytotoxic effect of the ITs, with TPMP-I-2 in combination with the 425.3PE (targeting the epidermal growth factor receptor) being the most promising combination. The cytotoxicity involving signs of apoptosis was observed in cancer cells from different origins in vitro. It is interesting to note that treatment with IT, TPMP-I-2, or 4BI alone or in combination resulted in strongly decreased protein levels of stearoyl-CoA desaturase. Stearoyl-CoA desaturase is the rate-limiting enzyme for converting saturated fatty acids into monounsaturated fatty acids needed for membrane genesis. Furthermore, the combination of 425.3PE and TPMP-I-2 prolonged the survival time of nude rats in a simulated micrometastatic cervical cancer model. In addition, we demonstrate that a combination of the 425.3PE and 4BI was more effective in reducing tumor growth in a breast cancer model in nude mice compared with either agent alone.
Asunto(s)
ADP Ribosa Transferasas/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Toxinas Bacterianas/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Exotoxinas/farmacología , Inmunotoxinas/farmacología , Neoplasias Cutáneas/tratamiento farmacológico , Estearoil-CoA Desaturasa/biosíntesis , Neoplasias del Cuello Uterino/tratamiento farmacológico , Factores de Virulencia/farmacología , ADP Ribosa Transferasas/inmunología , Animales , Apoptosis/efectos de los fármacos , Toxinas Bacterianas/inmunología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Caspasa 3/biosíntesis , Caspasa 3/genética , Línea Celular Tumoral , Membrana Celular/genética , Membrana Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Receptores ErbB/metabolismo , Exotoxinas/inmunología , Femenino , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Organofosfonatos/farmacología , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/biosíntesis , Poli(ADP-Ribosa) Polimerasas/genética , Ratas , Ratas Desnudas , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/mortalidad , Estearoil-CoA Desaturasa/genética , Compuestos de Tritilo/farmacología , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/mortalidad , Factores de Virulencia/inmunología , Exotoxina A de Pseudomonas aeruginosaRESUMEN
Highly invasive tumor cells are characterized by a metabolic switch, known as the Warburg effect, from "normal" oxidative phosphorylation to increased glycolysis even under sufficiently oxygenated conditions. This dependence on glycolysis also confers a growth advantage to cells present in hypoxic regions of the tumor. One of the key enzymes involved in glycolysis, the muscle isoform of lactate dehydrogenase (LDH-A), is overexpressed by metastatic cancer cells and is linked to the vitality of tumors in hypoxia. This enzyme may be considered as a potential target for new anticancer agents, since its inhibition cuts cancer energetic and anabolic supply, thus reducing the metastatic and invasive potential of cancer cells. We have discovered new and efficient N-hydroxyindole-based inhibitors of LDH-A, which are isoform-selective (over LDH-B) and competitive with both the substrate (pyruvate) and the cofactor (NADH). The antiproliferative activity of these compounds was confirmed on a series of cancer cell lines, and they proved to be particularly effective under hypoxic conditions. Moreover, NMR experiments showed that these compounds are able to reduce the glucose-to-lactate conversion inside the cell.
Asunto(s)
Antineoplásicos/síntesis química , Indoles/síntesis química , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Antineoplásicos/química , Antineoplásicos/farmacología , Ciclo Celular/efectos de los fármacos , Hipoxia de la Célula , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Glucosa/metabolismo , Humanos , Indoles/química , Indoles/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Lactato Deshidrogenasa 5 , Ácido Láctico/metabolismo , Modelos Moleculares , NAD/metabolismo , Ácido Pirúvico/metabolismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Cribrostatin 6 is a quinone-containing natural product that induces the death of cancer cell lines in culture, and its mechanism of action and scope of activity are unknown. Here we show that cribrostatin 6 has broad anticancer activity, potently inducing apoptotic cell death that is not preceded by any defined cell cycle arrest. Consistent with this data, we find that cribrostatin 6 treated cells have large amounts of reactive oxygen species (ROS) and, based on transcript profiling experiments and other data, this ROS generation is likely the primary mechanism by which cribrostatin 6 induces apoptosis. Given the success of certain ROS producers as anticancer agents, cribrostatin 6 has potential as a novel chemotherapeutic agent.
Asunto(s)
Isoquinolinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Células 3T3 , Acetilcisteína/farmacología , Animales , Camptotecina/farmacología , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citoprotección/efectos de los fármacos , ADN/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Etidio/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Humanos , Isoquinolinas/síntesis química , Isoquinolinas/química , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The identification of the mechanism of action and therapeutic potential of bioactive small molecules remains a considerable challenge in the field of drug discovery and chemical biology. Apart from traditional target identification techniques, new tools have emerged that can significantly aid mechanism elucidation efforts. The development of pattern matching algorithms that compare transcription profile data to analogous data on compounds with known cellular targets allows for mechanistic insights without the need to synthesize chemically modified probes. In addition, such methods can be used to connect small molecules to particular disease states, thus aiding the rational identification of candidate therapeutics. Another method with considerable potential is whole-genome RNAi screening, a technique that can identify critical upstream proteins involved in a small molecule's mechanism of action. Several proof-of-concept studies using compounds with known cellular targets suggest this tool will enable mechanistic characterization of bioactive small molecules with unknown mechanisms. This Review highlights recent successes in using these pattern matching and chemical genetic tools, with the goal of uncovering small molecule mechanisms and identifying therapeutic candidates for disease treatment.
Asunto(s)
Diseño de Fármacos , Análisis por Matrices de Proteínas/métodos , Interferencia de ARN/efectos de los fármacos , Precursores del ARN/efectos de los fármacos , ARN Interferente Pequeño/efectos de los fármacos , Preparaciones Farmacéuticas , Precursores del ARN/genética , ARN Interferente Pequeño/genéticaRESUMEN
A series of N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamides was synthesized by copper-catalyzed azide-alkyne cycloaddition (CuAAC) and afforded inhibitors of cancer cell growth. For example, compound 13e had an IC(50) of 46 nM against MCF-7 human breast tumor cells. Structure-activity relationship (SAR) studies demonstrated that (i) meta-phenoxy substitution of the N-1-benzyl group is important for antiproliferative activity and (ii) a variety of heterocyclic substitutions for the aryl group of the arylamide are tolerated. In silico COMPARE analysis of antiproliferative activity against the NCI-60 human tumor cell line panel revealed a correlation to clinically useful antimicrotubule agents such as paclitaxel and vincristine. This in silico correlation was supported by (i) in vitro inhibition of tubulin polymerization, (ii) G(2)/M-phase arrest in HeLa cells as assessed by flow cytometry, and (iii) perturbation of normal microtubule activity in HeLa cells as observed by confocal microscopy. The results demonstrate that N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamide is a readily accessible small molecule scaffold for compounds that inhibit tubulin polymerization and tumor cell growth.