RESUMO
Approximately 20% of high-grade serous ovarian cancers (HGSOC) have CCNE1 amplification. CCNE1-amplified tumors are homologous recombination (HR) proficient and resistant to standard therapies. Therapy resistance is associated with increased numbers of polyploid giant cancer cells (PGCC). We sought to identify new therapeutic approaches for patients with CCNE1-amplified tumors. Using TCGA data, we find that the mTOR, HR, and DNA checkpoint pathways are enriched in CCNE1-amplified ovarian cancers. Furthermore, Interactome Mapping Analysis linked the mTOR activity with upregulation of HR and DNA checkpoint pathways. Indeed, we find that mTOR inhibitors (mTORi) downregulate HR/checkpoint genes in CCNE1-amplified tumors. As CCNE1-amplified tumors are dependent on the HR pathway for viability, mTORi proved selectively effective in CCNE1-amplified tumors. Similarly, via downregulation of HR genes, mTORi increased CCNE1-amplifed HGSOC response to PARPi. In contrast, overexpression of HR/checkpoint proteins (RAD51 or ATR), induced resistance to mTORi. In vivo, mTORi alone potently reduced CCNE1-amplified tumor growth and the combination of mTORi and PARPi increased response and tumor eradication. Tumors treated with mTORi demonstrated a significant reduction in ALDH+ PGCCs. Finally, as a proof of principle, we identified three patients with CCNE1 amplified tumors who were treated with an mTORi. All three obtained clinical benefits from the therapy. Our studies and clinical experience indicate mTORi are a potential therapeutic approach for patients with CCNE1-amplified tumors.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Neoplasias Ovarianas , Carcinoma Epitelial do Ovário , Ciclina E/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Células Gigantes/metabolismo , Células Gigantes/patologia , Recombinação Homóloga , Humanos , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Serina-Treonina Quinases TOR/genéticaRESUMO
The Pax family of developmental control genes are frequently deregulated in human disease. In the kidney, Pax2 is expressed in developing nephrons but not in adult proximal and distal tubules, whereas polycystic kidney epithelia or renal cell carcinoma continues to express high levels. Pax2 reduction in mice or cell culture can slow proliferation of cystic epithelial cells or renal cancer cells. Thus, inhibition of Pax activity may be a viable, cell-type-specific therapy. We designed an unbiased, cell-based, high-throughput screen that identified triazolo pyrimidine derivatives that attenuate Pax transactivation ability. We show that BG-1 inhibits Pax2-positive cancer cell growth and target gene expression but has little effect on Pax2-negative cells. Chromatin immunoprecipitation suggests that these inhibitors prevent Pax protein interactions with the histone H3K4 methylation complex at Pax target genes in renal cells. Thus, these compounds may provide structural scaffolds for kidney-specific inhibitors with therapeutic potential.
Assuntos
Antineoplásicos , Neoplasias , Fator de Transcrição PAX2 , Animais , Antineoplásicos/farmacologia , Proliferação de Células , Expressão Gênica , Rim/metabolismo , Metilação , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Fator de Transcrição PAX2/antagonistas & inibidores , Fator de Transcrição PAX2/genéticaRESUMO
Rationale: Aldehyde dehydrogenase (ALDH) enzymes are often upregulated in cancer cells and associated with therapeutic resistance. ALDH enzymes protect cells by metabolizing toxic aldehydes which can induce DNA double stand breaks (DSB). We recently identified a novel ALDH1A family inhibitor (ALDHi), 673A. We hypothesized that 673A, via inhibition of ALDH1A family members, could induce intracellular accumulation of genotoxic aldehydes to cause DSB and that ALDHi could synergize with inhibitors of the ATM and ATR, proteins which direct DSB repair. Methods: We used immunofluorescence to directly assess levels of the aldehyde 4-hydroxynonenal and comet assays to evaluate DSB. Western blot was used to evaluate activation of the DNA damage response pathways. Cell counts were performed in the presence of 673A and additional aldehydes or aldehyde scavengers. ALDH inhibition results were confirmed using ALDH1A3 CRISPR knockout. Synergy between 673A and ATM or ATR inhibitors was evaluated using the Chou-Talalay method and confirmed in vivo using cell line xenograft tumor studies. Results: The ALDHi 673A cellular accumulation of toxic aldehydes which induce DNA double strand breaks. This is exacerbated by addition of exogenous aldehydes such as vitamin-A (retinaldehyde) and ameliorated by aldehyde scavengers such as metformin and hydralazine. Importantly, ALDH1A3 knockout cells demonstrated increased sensitivity to ATM/ATR inhibitors. And, ALDHi synergized with inhibitors of ATM and ATR, master regulators of the DSB DNA damage response, both in vitro and in vivo. This synergy was evident in homologous recombination (HR) proficient cell lines. Conclusions: ALDHi can be used to induce DNA DSB in cancer cells and synergize with inhibitors the ATM/ATR pathway. Our data suggest a novel therapeutic approach to target HR proficient ovarian cancer cells.
Assuntos
Família Aldeído Desidrogenase 1/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Dano ao DNA , Inibidores Enzimáticos/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Aldeído Oxirredutases/deficiência , Aldeído Oxirredutases/genética , Aldeídos/metabolismo , Aldeídos/toxicidade , Animais , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Sinergismo Farmacológico , Inibidores Enzimáticos/administração & dosagem , Feminino , Técnicas de Inativação de Genes , Humanos , Camundongos , Medicina de Precisão , Inibidores de Proteínas Quinases/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
There is strong evidence that inhibition of one or more Aldehyde Dehydrogenase 1A (ALDH1A) isoforms may be beneficial in chemotherapy-resistant ovarian cancer and other tumor types. While many previous efforts have focused on development of ALDH1A1 selective inhibitors, the most deadly ovarian cancer subtype, high-grade serous (HGSOC), exhibits elevated expression of ALDH1A3. Herein, we report continued development of pan-ALDH1A inhibitors to assess whether broad spectrum ALDH1A inhibition is an effective adjunct to chemotherapy in this critical tumor subtype. Optimization of the CM39 scaffold, aided by metabolite ID and several new ALDH1A1 crystal structures, led to improved biochemical potencies, improved cellular ALDH inhibition in HGSOC cell lines, and substantial improvements in microsomal stability culminating in orally bioavailable compounds. We demonstrate that two compounds 68 and 69 are able to synergize with chemotherapy in a resistant cell line and patient-derived HGSOC tumor spheroids, indicating their suitability for future in vivo proof of concept experiments.
Assuntos
Aldeído Desidrogenase/antagonistas & inibidores , Aldeído Desidrogenase/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Aldeído Desidrogenase/farmacologia , Feminino , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Ovarian cancer is typified by the development of chemotherapy resistance. Chemotherapy resistance is associated with high aldehyde dehydrogenase (ALDH) enzymatic activity, increased cancer "stemness," and expression of the stem cell marker CD133. As such, ALDH activity has been proposed as a therapeutic target. Although it remains controversial which of the 19 ALDH family members drive chemotherapy resistance, ALDH1A family members have been primarily linked with chemotherapy resistant and stemness. We identified two ALDH1A family selective inhibitors (ALDH1Ai). ALDH1Ai preferentially kills CD133+ ovarian cancer stem-like cells (CSCs). ALDH1Ai induce necroptotic CSC death, mediated, in part, by the induction of mitochondrial uncoupling proteins and reduction in oxidative phosphorylation. ALDH1Ai is highly synergistic with chemotherapy, reducing tumor initiation capacity and increasing tumor eradication in vivo. These studies link ALDH1A with necroptosis and confirm the family as a critical therapeutic target to overcome chemotherapy resistance and improve patient outcomes.
Assuntos
Família Aldeído Desidrogenase 1/antagonistas & inibidores , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Necroptose , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/metabolismo , Retinal Desidrogenase/antagonistas & inibidores , Antígeno AC133/genética , Antígeno AC133/metabolismo , Família Aldeído Desidrogenase 1/metabolismo , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Fosforilação Oxidativa , Retinal Desidrogenase/metabolismoRESUMO
Bone morphogenetic protein (BMP) signaling is critical in renal development and disease. In animal models of chronic kidney disease (CKD), re-activation of BMP signaling is reported to be protective by promoting renal repair and regeneration. Clinical use of recombinant BMPs, however, requires harmful doses to achieve efficacy and is costly because of BMPs' complex synthesis. Therefore, alternative strategies are needed to harness the beneficial effects of BMP signaling in CKD. Key aspects of the BMP signaling pathway can be regulated by both extracellular and intracellular molecules. In particular, secreted proteins like noggin and chordin inhibit BMP activity, whereas kielin/chordin-like proteins (KCP) enhance it and attenuate kidney fibrosis or CKD. Clinical development of KCP, however, is precluded by its size and complexity. Therefore, we propose an alternative strategy to enhance BMP signaling by using small molecules, which are simpler to synthesize and more cost-effective. To address our objective, here we developed a small-molecule high-throughput screen (HTS) with human renal cells having an integrated luciferase construct highly responsive to BMPs. We demonstrate the activity of a potent benzoxazole compound, sb4, that rapidly stimulated BMP signaling in these cells. Activation of BMP signaling by sb4 increased the phosphorylation of key second messengers (SMAD-1/5/9) and also increased expression of direct target genes (inhibitors of DNA binding, Id1 and Id3) in canonical BMP signaling. Our results underscore the feasibility of utilizing HTS to identify compounds that mimic key downstream events of BMP signaling in renal cells and have yielded a lead BMP agonist.
Assuntos
Benzoxazóis/farmacologia , Proteínas Morfogenéticas Ósseas/agonistas , Proteínas Morfogenéticas Ósseas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Fosfoproteínas/metabolismo , Proteínas Smad/metabolismoRESUMO
Aldehyde dehydrogenase (ALDH) activity is commonly used as a marker to identify cancer stem-like cells. The three ALDH1A isoforms have all been individually implicated in cancer stem-like cells and in chemoresistance; however, which isoform is preferentially expressed varies between cell lines. We sought to explore the structural determinants of ALDH1A isoform selectivity in a series of small-molecule inhibitors in support of research into the role of ALDH1A in cancer stem cells. An SAR campaign guided by a cocrystal structure of the HTS hit CM39 (7) with ALDH1A1 afforded first-in-class inhibitors of the ALDH1A subfamily with excellent selectivity over the homologous ALDH2 isoform. We also discovered the first reported modestly selective single isoform 1A2 and 1A3 inhibitors. Two compounds, 13g and 13h, depleted the CD133+ putative cancer stem cell pool, synergized with cisplatin, and achieved efficacious concentrations in vivo following IP administration. Compound 13h additionally synergized with cisplatin in a patient-derived ovarian cancer spheroid model.
Assuntos
Aldeído Desidrogenase/antagonistas & inibidores , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Família Aldeído Desidrogenase 1 , Animais , Proliferação de Células , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/classificação , Feminino , Humanos , Camundongos , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , Retinal Desidrogenase , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pax genes encode developmental regulators that are expressed in a variety of tissues and control critical events in morphogenesis. In the kidney, Pax2 and Pax8 are expressed in embryonic development and in specific renal diseases associated with aberrant epithelial cell proliferation. Prior genetic and cell biological studies suggest that reducing the activity of Pax proteins in renal cancer or in polycystic kidney disease can slow the progression of these conditions. The Pax proteins may be critical for providing tissue and locus specificity to recruit epigenetic modifiers that control gene expression and chromatin structure. Although they are nuclear, targeting Pax proteins to inhibit function may be feasible with small molecules. Such inhibition of Pax protein function may provide novel therapies for subsets of renal disorders that are tissue- and cell type-specific and avoid systemic effects on non-Pax-expressing cells and tissues. Given the paucity of effective treatments for renal cancer and cystic disease, the Pax family of proteins represents new pharmaceutical targets that merit exploration and further development.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Renais/tratamento farmacológico , Fator de Transcrição PAX2/antagonistas & inibidores , Fator de Transcrição PAX8/antagonistas & inibidores , Doenças Renais Policísticas/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Progressão da Doença , Epigênese Genética/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Rim/citologia , Rim/crescimento & desenvolvimento , Rim/metabolismo , Neoplasias Renais/genética , Neoplasias Renais/patologia , Terapia de Alvo Molecular/métodos , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX2/metabolismo , Fator de Transcrição PAX8/genética , Fator de Transcrição PAX8/metabolismo , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/patologia , Domínios Proteicos/efeitos dos fármacos , Urotélio/citologia , Urotélio/efeitos dos fármacos , Urotélio/metabolismo , Urotélio/patologiaRESUMO
Obesity and its associated complications such as insulin resistance and non-alcoholic fatty liver disease are reaching epidemic proportions. In mice, the TGF-ß superfamily is implicated in the regulation of white and brown adipose tissue differentiation. The kielin/chordin-like protein (KCP) is a secreted regulator of the TGF-ß superfamily pathways that can inhibit both TGF-ß and activin signals while enhancing bone morphogenetic protein (BMP) signaling. However, KCP's effects on metabolism and obesity have not been studied in animal models. Therefore, we examined the effects of KCP loss or gain of function in mice that were maintained on either a regular or a high-fat diet. KCP loss sensitized the mice to obesity and associated complications such as glucose intolerance and adipose tissue inflammation and fibrosis. In contrast, transgenic mice that expressed KCP in the kidney, liver, and adipose tissues were resistant to developing high-fat diet-induced obesity and had significantly reduced white adipose tissue. Moreover, KCP overexpression shifted the pattern of SMAD signaling in vivo, increasing the levels of phospho (P)-SMAD1 and decreasing P-SMAD3. Adipocytes in culture showed a cell-autonomous effect in response to added TGF-ß1 or BMP7. Metabolic profiling indicated increased energy expenditure in KCP-overexpressing mice and reduced expenditure in the KCP mutants with no effect on food intake or activity. These findings demonstrate that shifting the TGF-ß superfamily signaling with a secreted protein can alter the physiology and thermogenic properties of adipose tissue to reduce obesity even when mice are fed a high-fat diet.
Assuntos
Adipócitos/metabolismo , Proteínas de Transporte/metabolismo , Gorduras na Dieta/efeitos adversos , Síndrome Metabólica/metabolismo , Obesidade/metabolismo , Transdução de Sinais , Adipócitos/patologia , Animais , Proteína Morfogenética Óssea 7/genética , Proteína Morfogenética Óssea 7/metabolismo , Proteínas de Transporte/genética , Gorduras na Dieta/farmacologia , Síndrome Metabólica/induzido quimicamente , Síndrome Metabólica/genética , Síndrome Metabólica/patologia , Camundongos , Camundongos Knockout , Obesidade/induzido quimicamente , Obesidade/genética , Obesidade/patologia , Especificidade de Órgãos/genética , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
The Pax gene family encodes DNA binding transcription factors that control critical steps in embryonic development and differentiation of specific cell lineages. Often, Pax proteins are re-expressed or ectopically expressed in cancer and other diseases of abnormal proliferation, making them attractive targets for tissue specific inhibition by small molecules. In this report, we used a homology model of the Pax2 paired domain and a virtual screen to identify small molecules that can inhibit binding of the paired domain to DNA and Pax2 mediated transcription activation. Candidates from the virtual screen were then confirmed in a cell based Pax2 transactivation assay. Subsequently, we tested analogs of these hits to identify a single compound that effectively blocked Pax2 activity and DNA binding with a Kd of 1.35-1.5 µM. The compound, termed EG1, was used to inhibit embryonic kidney development, a process directly dependent on Pax2 activity. Furthermore, we show that EG1 can inhibit proliferation of Pax2 positive renal and ovarian cancer cell lines but has little effect on Pax2 negative cancer cells. These data confirm that small molecules targeting the DNA binding paired domain can be identified and may be good lead compounds for developing tissue and cell-type specific anticancer therapies.
Assuntos
DNA/metabolismo , Fator de Transcrição PAX2/metabolismo , Bibliotecas de Moléculas Pequenas , Ativação Transcricional , Animais , Linhagem Celular Tumoral , Simulação por Computador , Células HEK293 , Humanos , Rim/crescimento & desenvolvimento , Rim/metabolismo , Fator de Transcrição PAX2/antagonistas & inibidoresRESUMO
BACKGROUND: Dihydroorotase (DHO) is a zinc metalloenzyme, although the number of active site zinc ions has been controversial. E. coli DHO was initially thought to have a mononuclear metal center, but the subsequent X-ray structure clearly showed two zinc ions, α and ß, at the catalytic site. Aquifex aeolicus DHO, is a dodecamer comprised of six DHO and six aspartate transcarbamoylase (ATC) subunits. The isolated DHO monomer, which lacks catalytic activity, has an intact α-site and conserved ß-site ligands, but the geometry of the second metal binding site is completely disrupted. However, the putative ß-site is restored when the complex with ATC is formed and DHO activity is regained. Nevertheless, the X-ray structure of the complex revealed a single zinc ion at the active site. The structure of DHO from the pathogenic organism, S. aureus showed that it also has a single active site metal ion. RESULTS: Zinc analysis showed that the enzyme has one zinc/DHO subunit and the addition of excess metal ion did not stimulate catalytic activity, nor alter the kinetic parameters. The metal free apoenzyme was inactive, but the full activity was restored upon the addition of one equivalent of Zn2+ or Co2+. Moreover, deletion of the ß-site by replacing the His180 and His232 with alanine had no effect on catalysis in the presence or absence of excess zinc. The 2.2 Å structure of the double mutant confirmed that the ß-site was eliminated but that the active site remained otherwise intact. CONCLUSIONS: Thus, kinetically competent A. aeolicus DHO has a mononuclear metal center. In contrast, elimination of the putative second metal binding site in amidohydrolyases with a binuclear metal center, resulted in the abolition of catalytic activity. The number of active site metal ions may be a consideration in the design of inhibitors that selectively target either the mononuclear or binuclear enzymes.