RESUMO
Venetoclax is a promising agent in the treatment of acute myeloid leukemia, though its antileukemic activity is limited to combination therapies. Mcl-1 downregulation, Bim upregulation, and DNA damage have been identified as potential ways to enhance venetoclax activity. In this study, we combine venetoclax with the dual PI3K and histone deacetylase inhibitor CUDC-907, which can downregulate Mcl-1, upregulate Bim, and induce DNA damage, as well as downregulate c-Myc. We establish that CUDC-907 and venetoclax synergistically induce apoptosis in acute myeloid leukemia cell lines and primary acute myeloid leukemia patient samples ex vivo. CUDC-907 downregulates CHK1, Wee1, RRM1, and c-Myc, which were found to play a role in venetoclax-induced apoptosis. Interestingly, we found that venetoclax treatment enhances CUDC-907-induced DNA damage potentially through inhibition of DNA repair. In vivo results show that CUDC-907 enhances venetoclax efficacy in an acute myeloid leukemia cell line derived xenograft mouse model, supporting the development of CUDC-907 in combination with venetoclax for the treatment of acute myeloid leukemia.
Assuntos
Leucemia Mieloide Aguda , Fosfatidilinositol 3-Quinases , Animais , Apoptose , Compostos Bicíclicos Heterocíclicos com Pontes , Linhagem Celular Tumoral , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Camundongos , Morfolinas , Pirimidinas , Sulfonamidas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Induction therapy for patients with acute myeloid leukemia (AML) has remained largely unchanged for over 40 years, while overall survival rates remain unacceptably low, highlighting the need for new therapies. The PI3K/Akt pathway is constitutively active in the majority of patients with AML. Given that histone deacetylase inhibitors have been shown to synergize with PI3K inhibitors in preclinical AML models, we investigated the novel dual-acting PI3K and histone deacetylase inhibitor CUDC-907 in AML cells both in vitro and in vivo We demonstrated that CUDC-907 induces apoptosis in AML cell lines and primary AML samples and shows in vivo efficacy in an AML cell line-derived xenograft mouse model. CUDC-907-induced apoptosis was partially dependent on Mcl-1, Bim, and c-Myc. CUDC-907 induced DNA damage in AML cells while sparing normal hematopoietic cells. Downregulation of CHK1, Wee1, and RRM1, and induction of DNA damage also contributed to CUDC-907-induced apoptosis of AML cells. In addition, CUDC-907 treatment decreased leukemia progenitor cells in primary AML samples ex vivo, while also sparing normal hematopoietic progenitor cells. These findings support the clinical development of CUDC-907 for the treatment of AML.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Pirimidinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Biomarcadores , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Genes myc , Humanos , Imunofenotipagem , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pemetrexed (PMX) is a 5-substituted pyrrolo[2,3-d]pyrimidine antifolate used for therapy of nonsquamous nonsmall cell lung cancer (NS-NSCLC). PMX is transported by the reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT). Unlike RFC, PCFT is active at acidic pH levels characterizing the tumor microenvironment. By real-time reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, PCFT transcripts and proteins were detected in primary NS-NSCLC specimens. In six NS-NSCLC cell lines (A549, H1437, H460, H1299, H1650, and H2030), PCFT transcripts and proteins were detected by real-time RT-PCR and western blots, respectively. 6-Substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates related to PMX [compound 1 (C1) and compound 2 (C2), respectively] are selective substrates for PCFT over RFC. In the NS-NSCLC cell lines, both [(3)H]PMX and [(3)H]C2 were transported by PCFT. C1 and C2 inhibited proliferation of the NS-NSCLC cell lines; A549, H460, and H2030 cells were more sensitive to C1 than to PMX. C1 and C2 inhibited glycinamide ribonucleotide formyltransferase in de novo purine nucleotide biosynthesis. When treated at pH 6.8, which favors PCFT uptake, C1 and C2 inhibited clonogenicity of H460 cells greater than PMX; PMX inhibited clonogenicity more than C1 or C2 at pH 7.2, which favors RFC transport over PCFT. Knockdown of PCFT in H460 cells resulted in decreased [(3)H]PMX and [(3)H]C2 transport and decreased growth inhibition by C1 and C2, and to a lesser extent by PMX. In vivo efficacy of C1 was seen toward H460 tumor xenografts in severe-combined immunodeficient mice. Our results suggest that 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates offer significant promise for treating NS-NSCLC by selective uptake by PCFT.
Assuntos
Antineoplásicos/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Antagonistas do Ácido Fólico/metabolismo , Neoplasias Pulmonares/metabolismo , Transportador de Folato Acoplado a Próton/metabolismo , Animais , Antineoplásicos/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Antagonistas do Ácido Fólico/administração & dosagem , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Pirimidinas/administração & dosagem , Pirimidinas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Venetoclax (VEN), in combination with low dose cytarabine (AraC) or a hypomethylating agent, is FDA approved to treat acute myeloid leukemia (AML) in patients who are over the age of 75 or cannot tolerate standard chemotherapy. Despite high response rates to these therapies, most patients succumb to the disease due to relapse and/or drug resistance, providing an unmet clinical need for novel therapies to improve AML patient survival. ME-344 is a potent isoflavone with demonstrated inhibitory activity toward oxidative phosphorylation (OXPHOS) and clinical activity in solid tumors. Given that OXPHOS inhibition enhances VEN antileukemic activity against AML, we hypothesized that ME-344 could enhance the anti-AML activity of VEN. Here we report that ME-344 enhanced VEN to target AML cell lines and primary patient samples while sparing normal hematopoietic cells. Cooperative suppression of OXPHOS was detected in a subset of AML cell lines and primary patient samples. Metabolomics analysis revealed a significant reduction of purine biosynthesis metabolites by ME-344. Further, lometrexol, a purine biosynthesis inhibitor, synergistically enhanced VEN-induced apoptosis in AML cell lines. Interestingly, AML cells with acquired AraC resistance showed significantly increased purine biosynthesis metabolites and sensitivities to ME-344. Furthermore, synergy between ME-344 and VEN was preserved in these AraC-resistant AML cells. In vivo studies revealed significantly prolonged survival upon combination therapy of ME-344 and VEN in NSGS mice bearing parental or AraC-resistant MV4-11 leukemia compared to the vehicle control. This study demonstrates that ME-344 enhances VEN antileukemic activity against preclinical models of AML by suppressing OXPHOS and/or purine biosynthesis.
Assuntos
Isoflavonas , Leucemia Mieloide Aguda , Sulfonamidas , Humanos , Animais , Camundongos , Fosforilação Oxidativa , Leucemia Mieloide Aguda/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes , Isoflavonas/farmacologia , Purinas/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêuticoRESUMO
Eradication of acute myeloid leukemia (AML) is therapeutically challenging; many patients succumb to AML despite initially responding to conventional treatments. Here, we showed that the imipridone ONC213 elicits potent antileukemia activity in a subset of AML cell lines and primary patient samples, particularly in leukemia stem cells, while producing negligible toxicity in normal hematopoietic cells. ONC213 suppressed mitochondrial respiration and elevated α-ketoglutarate by suppressing α-ketoglutarate dehydrogenase (αKGDH) activity. Deletion of OGDH, which encodes αKGDH, suppressed AML fitness and impaired oxidative phosphorylation, highlighting the key role for αKGDH inhibition in ONC213-induced death. ONC213 treatment induced a unique mitochondrial stress response and suppressed de novo protein synthesis in AML cells. Additionally, ONC213 reduced the translation of MCL1, which contributed to ONC213-induced apoptosis. Importantly, a patient-derived xenograft from a relapsed AML patient was sensitive to ONC213 in vivo. Collectively, these findings support further development of ONC213 for treating AML. SIGNIFICANCE: In AML cells, ONC213 suppresses αKGDH, which induces a unique mitochondrial stress response, and reduces MCL1 to decrease oxidative phosphorylation and elicit potent antileukemia activity. See related commentary by Boët and Sarry, p. 950.
Assuntos
Leucemia Mieloide Aguda , Fosforilação Oxidativa , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Linhagem Celular Tumoral , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , ApoptoseRESUMO
Venetoclax (VEN), in combination with low dose cytarabine (AraC) or a hypomethylating agent, is FDA approved to treat acute myeloid leukemia (AML) in patients who are over the age of 75 or cannot tolerate standard chemotherapy. Despite high response rates to these combination therapies, most patients succumb to the disease due to relapse and/or drug resistance, providing an unmet clinical need for novel therapies to improve AML patient survival. ME-344 is a potent isoflavone with demonstrated inhibitory activity toward oxidative phosphorylation (OXPHOS) and clinical activity in solid tumors. Given that OXPHOS inhibition enhances VEN antileukemic activity against AML, we hypothesized that ME-344 could enhance the anti-AML activity of VEN. Here we report that ME-344 synergized with VEN to target AML cell lines and primary patient samples while sparing normal hematopoietic cells. Cooperative suppression of OXPHOS was detected in a subset of AML cell lines and primary patient samples. Metabolomics analysis revealed a significant reduction of purine biosynthesis metabolites by ME-344. Further, lometrexol, an inhibitor of purine biosynthesis, synergistically enhanced VEN-induced apoptosis in AML cell lines. Interestingly, AML cells with acquired resistance to AraC showed significantly increased purine biosynthesis metabolites and sensitivities to ME-344. Furthermore, synergy between ME-344 and VEN was preserved in these AraC-resistant AML cells. These results translated into significantly prolonged survival upon combination of ME-344 and VEN in NSGS mice bearing parental or AraC-resistant MV4-11 leukemia. This study demonstrates that ME-344 enhances VEN antileukemic activity against preclinical models of AML by suppressing OXPHOS and/or purine biosynthesis.
RESUMO
Uptake of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates with four or three bridge carbons [compound 1 (C1) and compound 2 (C2), respectively] into solid tumors by the proton-coupled folate transporter (PCFT) represents a novel therapeutic strategy that harnesses the acidic tumor microenvironment. Although these compounds are not substrates for the reduced folate carrier (RFC), the major facilitative folate transporter, RFC expression may alter drug efficacies by affecting cellular tetrahydrofolate (THF) cofactor pools that can compete for polyglutamylation and/or binding to intracellular enzyme targets. Human tumor cells including wild-type (WT) and R5 (RFC-null) HeLa cells express high levels of PCFT protein. C1 and C2 inhibited proliferation of R5 cells 3 to 4 times more potently than WT cells or R5 cells transfected with RFC. Transport of C1 and C2 was virtually identical between WT and R5 cells, establishing that differences in drug sensitivities between sublines were independent of PCFT transport. Steady-state intracellular [³H]THF cofactors derived from [³H]5-formyl-THF were depleted in R5 cells compared with those in WT cells, an effect exacerbated by C1 and C2. Whereas C1 and C2 polyglutamates accumulated to similar levels in WT and R5 cells, there were differences in polyglutamyl distributions in favor of the longest chain length forms. In severe combined immunodeficient mice, the antitumor efficacies of C1 and C2 were greater toward subcutaneous R5 tumors than toward WT tumors, confirming the collateral drug sensitivities observed in vitro. Thus, solid tumor-targeted antifolates with PCFT-selective cellular uptake should have enhanced activities toward tumors lacking RFC function, reflecting contraction of THF cofactor pools.
Assuntos
Antineoplásicos/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Transportador de Folato Acoplado a Próton/metabolismo , Pirimidinas/farmacologia , Pirróis/farmacologia , Proteína Carregadora de Folato Reduzido/metabolismo , Tiofenos/farmacologia , Animais , Antineoplásicos/metabolismo , Transporte Biológico , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Espaço Extracelular/metabolismo , Feminino , Antagonistas do Ácido Fólico/metabolismo , Humanos , Camundongos , Camundongos SCID , Transplante de Neoplasias , Ácidos Pteroilpoliglutâmicos/metabolismo , Pirimidinas/metabolismo , Pirróis/metabolismo , Relação Estrutura-Atividade , Tetra-Hidrofolatos/metabolismo , Tiofenos/metabolismo , Microambiente TumoralRESUMO
The proton-coupled folate transporter (PCFT) is a proton-folate symporter with an acidic pH optimum. By real-time reverse transcription-polymerase chain reaction, PCFT was expressed in the majority of 53 human tumor cell lines, with the highest levels in Caco-2 (colorectal adenocarcinoma), SKOV3 (ovarian), and HepG2 (hepatoma) cells. A novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate (compound 1) was used to establish whether PCFT can deliver cytotoxic drug under pH conditions that mimic the tumor microenvironment. Both 1 and pemetrexed (Pmx) inhibited proliferation of R1-11-PCFT4 HeLa cells engineered to express PCFT without the reduced folate carrier (RFC) and of HepG2 cells expressing both PCFT and RFC. Unlike Pmx, 1 did not inhibit proliferation of R1-11-RFC6 HeLa cells, which express RFC without PCFT. Treatment of R1-11-PCFT4 cells at pH 6.8 with 1 or Pmx inhibited colony formation with dose and time dependence. Transport of [(3)H]compound 1 into R1-11-PCFT4 and HepG2 cells was optimal at pH 5.5 but appreciable at pH 6.8. At pH 6.8, [(3)H]compound 1 was metabolized to (3)H-labeled polyglutamates. Glycinamide ribonucleotide formyltransferase (GARFTase) in R1-11-PCFT4 cells was inhibited by 1 at pH 6.8, as measured by an in situ GARFTase assay, and was accompanied by substantially reduced ATP levels. Compound 1 caused S-phase accumulation and a modest level of apoptosis. An in vivo efficacy trial with severe combined immunodeficient mice implanted with subcutaneous HepG2 tumors showed that compound 1 was active. Our findings suggest exciting new therapeutic possibilities to selectively deliver novel antifolate drugs via transport by PCFT over RFC by exploiting the acidic tumor microenvironment.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Antagonistas do Ácido Fólico/metabolismo , Neoplasias/metabolismo , Transportador de Folato Acoplado a Próton/metabolismo , Pirimidinas/metabolismo , Animais , Relação Dose-Resposta a Droga , Feminino , Antagonistas do Ácido Fólico/administração & dosagem , Células HeLa , Células Hep G2 , Humanos , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Neoplasias/tratamento farmacológico , Pirimidinas/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
About 25% of patients with acute myeloid leukemia (AML) harbor FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations and their prognosis remains poor. Gilteritinib is a FLT3 inhibitor approved by the US FDA for use in adult FLT3-mutated relapsed or refractory AML patients. Monotherapy, while efficacious, shows short-lived responses, highlighting the need for combination therapies. Here we show that gilteritinib and CUDC-907, a dual inhibitor of PI3K and histone deacetylases, synergistically induce apoptosis in FLT3-ITD AML cell lines and primary patient samples and have striking in vivo efficacy. Upregulation of FLT3 and activation of ERK are mechanisms of resistance to gilteritinib, while activation of JAK2/STAT5 is a mechanism of resistance to CUDC-907. Gilteritinib and CUDC-907 reciprocally overcome these mechanisms of resistance. In addition, the combined treatment results in cooperative downregulation of cellular metabolites and persisting antileukemic effects. CUDC-907 plus gilteritinib shows synergistic antileukemic activity against FLT3-ITD AML in vitro and in vivo, demonstrating strong translational therapeutic potential.
Assuntos
Leucemia Mieloide Aguda , Tirosina Quinase 3 Semelhante a fms , Compostos de Anilina/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Feminino , Humanos , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Morfolinas/farmacologia , Pirazinas/farmacologia , Pirimidinas/farmacologia , Células THP-1 , Ensaios Antitumorais Modelo de Xenoenxerto , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Venetoclax, an FDA-approved Bcl-2 selective inhibitor for the treatment of chronic lymphocytic leukemia and acute myeloid leukemia (AML), is tolerated well in elderly patients with AML and has good overall response rates; however, resistance remains a concern. In this study, we show that targeting CDK9 with voruciclib in combination with venetoclax results in synergistic antileukemic activity against AML cell lines and primary patient samples. CDK9 inhibition enhances venetoclax activity through downregulation of Mcl-1 and c-Myc. However, downregulation of Mcl-1 is transient, which necessitates an intermittent treatment schedule to allow for repeated downregulation of Mcl-1. Accordingly, an every other day schedule of the CDK9 inhibitor is effective in vitro and in vivo in enhancing the efficacy of venetoclax. Our preclinical data provide a rationale for an intermittent drug administration schedule for the clinical evaluation of the combination treatment for AML.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Quinase 9 Dependente de Ciclina/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sulfonamidas/administração & dosagem , Adolescente , Adulto , Animais , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Benzopiranos/administração & dosagem , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Xenoenxertos , Humanos , Imino Furanoses/administração & dosagem , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-myc/genética , Adulto JovemRESUMO
Targeting oxidative phosphorylation (OXPHOS) is a promising strategy to improve treatment outcomes of acute myeloid leukemia (AML) patients. IACS-010759 is a mitochondrial complex I inhibitor that has demonstrated preclinical antileukemic activity and is being tested in Phase I clinical trials. However, complex I deficiency has been reported to inhibit apoptotic cell death through prevention of cytochrome c release. Thus, combining IACS-010759 with a BH3 mimetic may overcome this mechanism of resistance leading to synergistic antileukemic activity against AML. In this study, we show that IACS-010759 and venetoclax synergistically induce apoptosis in OXPHOS-reliant AML cell lines and primary patient samples and cooperatively target leukemia progenitor cells. In a relatively OXPHOS-reliant AML cell line derived xenograft mouse model, IACS-010759 treatment significantly prolonged survival, which was further enhanced by treatment with IACS-010759 in combination with venetoclax. Consistent with our hypothesis, IACS-010759 treatment indeed retained cytochrome c in mitochondria, which was completely abolished by venetoclax, resulting in Bak/Bax- and caspase-dependent apoptosis. Our preclinical data provide a rationale for further development of the combination of IACS-010759 and venetoclax for the treatment of patients with AML.
RESUMO
PURPOSE: To investigate the efficacy of the combination of the FLT3 inhibitors midostaurin or gilteritinib with the Bcl-2 inhibitor venetoclax in FLT3-internal tandem duplication (ITD) acute myeloid leukemia (AML) and the underlying molecular mechanism. EXPERIMENTAL DESIGN: Using both FLT3-ITD cell lines and primary patient samples, Annexin V-FITC/propidium iodide staining and flow cytometry analysis were used to quantify cell death induced by midostaurin or gilteritinib, alone or in combination with venetoclax. Western blot analysis was performed to assess changes in protein expression levels of members of the JAK/STAT, MAPK/ERK, and PI3K/AKT pathways, and members of the Bcl-2 family of proteins. The MV4-11-derived xenograft mouse model was used to assess in vivo efficacy of the combination of gilteritinib and venetoclax. Lentiviral overexpression of Mcl-1 was used to confirm its role in cell death induced by midostaurin or gilteritinib with venetoclax. Changes of Mcl-1 transcript levels were assessed by RT-PCR. RESULTS: The combination of midostaurin or gilteritinib with venetoclax potently and synergistically induces apoptosis in FLT3-ITD AML cell lines and primary patient samples. The FLT3 inhibitors induced downregulation of Mcl-1, enhancing venetoclax activity. Phosphorylated-ERK expression is induced by venetoclax but abolished by the combination of venetoclax with midostaurin or gilteritinib. Simultaneous downregulation of Mcl-1 by midostaurin or gilteritinib and inhibition of Bcl-2 by venetoclax results in "free" Bim, leading to synergistic induction of apoptosis. In vivo results show that gilteritinib in combination with venetoclax has therapeutic potential. CONCLUSIONS: Inhibition of Bcl-2 via venetoclax synergistically enhances the efficacy of midostaurin and gilteritinib in FLT3-mutated AML.See related commentary by Perl, p. 6567.
Assuntos
Compostos de Anilina/farmacologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Pirazinas/farmacologia , Estaurosporina/análogos & derivados , Tirosina Quinase 3 Semelhante a fms/genética , Animais , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Sinergismo Farmacológico , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Duplicação Gênica , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Estaurosporina/farmacologia , Sulfonamidas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Folate-dependent one-carbon (C1) metabolism is compartmentalized into the mitochondria and cytosol and supports cell growth through nucleotide and amino acid biosynthesis. Mitochondrial C1 metabolism, including serine hydroxymethyltransferase (SHMT) 2, provides glycine, NAD(P)H, ATP, and C1 units for cytosolic biosynthetic reactions, and is implicated in the oncogenic phenotype across a wide range of cancers. Whereas multitargeted inhibitors of cytosolic C1 metabolism, such as pemetrexed, are used clinically, there are currently no anticancer drugs that specifically target mitochondrial C1 metabolism. We used molecular modeling to design novel small-molecule pyrrolo[3,2-d]pyrimidine inhibitors targeting mitochondrial C1 metabolism at SHMT2. In vitro antitumor efficacy was established with the lead compounds (AGF291, AGF320, AGF347) toward lung, colon, and pancreatic cancer cells. Intracellular targets were identified by metabolic rescue with glycine and nucleosides, and by targeted metabolomics using a stable isotope tracer, with confirmation by in vitro assays with purified enzymes. In addition to targeting SHMT2, inhibition of the cytosolic purine biosynthetic enzymes, ß-glycinamide ribonucleotide formyltransferase and/or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase, and SHMT1 was also established. AGF347 generated significant in vivo antitumor efficacy with potential for complete responses against both early-stage and upstage MIA PaCa-2 pancreatic tumor xenografts, providing compelling proof-of-concept for therapeutic targeting of SHMT2 and cytosolic C1 enzymes by this series. Our results establish structure-activity relationships and identify exciting new drug prototypes for further development as multitargeted antitumor agents.
Assuntos
Antineoplásicos/farmacologia , Carbono/metabolismo , Citosol/metabolismo , Mitocôndrias/metabolismo , Pirimidinas/farmacologia , Pirróis/farmacologia , Animais , Antineoplásicos/química , Vias Biossintéticas/efeitos dos fármacos , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Citosol/efeitos dos fármacos , Feminino , Concentração Inibidora 50 , Metabolômica , Camundongos SCID , Mitocôndrias/efeitos dos fármacos , Purinas/biossíntese , Pirimidinas/química , Pirróis/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Novel fluorinated 2-amino-4-oxo-6-substituted pyrrolo[2,3- d]pyrimidine analogues 7-12 were synthesized and tested for selective cellular uptake by folate receptors (FRs) α and ß or the proton-coupled folate transporter (PCFT) and for antitumor efficacy. Compounds 8, 9, 11, and 12 showed increased in vitro antiproliferative activities (â¼11-fold) over the nonfluorinated analogues 2, 3, 5, and 6 toward engineered Chinese hamster ovary and HeLa cells expressing FRs or PCFT. Compounds 8, 9, 11, and 12 also inhibited proliferation of IGROV1 and A2780 epithelial ovarian cancer cells; in IGROV1 cells with knockdown of FRα, 9, 11, and 12 showed sustained inhibition associated with uptake by PCFT. All compounds inhibited glycinamide ribonucleotide formyltransferase, a key enzyme in the de novo purine biosynthesis pathway. Molecular modeling studies validated in vitro cell-based results. NMR evidence supports the presence of an intramolecular fluorine-hydrogen bond. Potent in vivo efficacy of 11 was established with IGROV1 xenografts in severe compromised immunodeficient mice.
Assuntos
Flúor/química , Receptor 1 de Folato/metabolismo , Transportador de Folato Acoplado a Próton/metabolismo , Nucleotídeos de Purina/biossíntese , Pirimidinas/química , Pirimidinas/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Transporte Biológico , Células CHO , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cricetulus , Ácido Fólico/metabolismo , Humanos , Camundongos , Pirimidinas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tumor-targeted specificities of 6-substituted pyrrolo[2,3- d]pyrimidine analogues of 1, where the phenyl side-chain is replaced by 3',6' (5, 8), 2',5' (6, 9), and 2',6' (7, 10) pyridyls, were analyzed. Proliferation inhibition of isogenic Chinese hamster ovary (CHO) cells expressing folate receptors (FRs) α and ß were in rank order, 6 > 9 > 5 > 7 > 8, with 10 showing no activity, and 6 > 9 > 5 > 8, with 10 and 7 being inactive, respectively. Antiproliferative effects toward FRα- and FRß-expressing cells were reflected in competitive binding with [3H]folic acid. Only compound 6 was active against proton-coupled folate receptor (PCFT)-expressing CHO cells (â¼4-fold more potent than 1) and inhibited [3H]methotrexate uptake by PCFT. In KB and IGROV1 tumor cells, 6 showed <1 nM IC50, â¼2-3-fold more potent than 1. Compound 6 inhibited glycinamide ribonucleotide formyltransferase in de novo purine biosynthesis and showed potent in vivo efficacy toward subcutaneous IGROV1 tumor xenografts in SCID mice.
Assuntos
Antineoplásicos/síntese química , Antagonistas do Ácido Fólico/síntese química , Pirimidinas/farmacologia , Pirróis/farmacologia , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Células CHO , Proliferação de Células/efeitos dos fármacos , Cricetulus , Receptor 1 de Folato/análise , Receptor 1 de Folato/metabolismo , Receptor 2 de Folato/análise , Antagonistas do Ácido Fólico/farmacologia , Xenoenxertos , Humanos , Camundongos , Camundongos SCID , Transportador de Folato Acoplado a Próton/metabolismo , Nucleotídeos de Purina/antagonistas & inibidores , Pirimidinas/química , Pirimidinas/farmacocinética , Pirróis/química , Pirróis/farmacocinéticaRESUMO
Folate uptake in epithelial ovarian cancer (EOC) involves the reduced folate carrier (RFC) and the proton-coupled folate transporter (PCFT), both facilitative transporters and folate receptor (FR) α. Although in primary EOC specimens, FRα is widely expressed and increases with tumor stage, PCFT was expressed independent of tumor stage (by real-time RT-PCR and IHC). EOC cell line models, including cisplatin sensitive (IGROV1 and A2780) and resistant (SKOV3 and TOV112D) cells, expressed a 17-fold range of FRα and similar amounts (within â¼2-fold) of PCFT. Novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates AGF94 and AGF154 exhibited potent antiproliferative activities toward all of the EOC cell lines, reflecting selective cellular uptake by FRα and/or PCFT over RFC. When IGROV1 cells were pretreated with AGF94 at pH 6.8, clonogenicity was potently inhibited, confirming cell killing. FRα was knocked down in IGROV1 cells with lentiviral shRNAs. Two FRα knockdown clones (KD-4 and KD-10) showed markedly reduced binding and uptake of [3H]folic acid and [3H]AGF154 by FRα, but maintained high levels of [3H]AGF154 uptake by PCFT compared to nontargeted control cells. In proliferation assays, KD-4 and KD-10 cells preserved in vitro inhibition by AGF94 and AGF154, compared to a nontargeted control, attributable to residual FRα- and substantial PCFT-mediated uptake. KD-10 tumor xenografts in severe-compromised immune-deficient mice were likewise sensitive to AGF94 Collectively, our results demonstrate the substantial therapeutic potential of novel 6-substituted pyrrolo[2,3-d]pyrimidine antifolates with dual targeting of PCFT and FRα toward EOCs that express a range of FRα, along with PCFT, as well as cisplatin resistance. Mol Cancer Ther; 16(5); 819-30. ©2017 AACR.
Assuntos
Receptor 1 de Folato/genética , Antagonistas do Ácido Fólico/administração & dosagem , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Transportador de Folato Acoplado a Próton/genética , Animais , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptor 1 de Folato/antagonistas & inibidores , Ácido Fólico/genética , Antagonistas do Ácido Fólico/síntese química , Humanos , Camundongos , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Targeted antifolates with heteroatom replacements of the carbon vicinal to the phenyl ring in 1 by N (4), O (8), or S (9), or with N-substituted formyl (5), acetyl (6), or trifluoroacetyl (7) moieties, were synthesized and tested for selective cellular uptake by folate receptor (FR) α and ß or the proton-coupled folate transporter. Results show increased in vitro antiproliferative activity toward engineered Chinese hamster ovary cells expressing FRs by 4-9 over the CH2 analogue 1. Compounds 4-9 inhibited de novo purine biosynthesis and glycinamide ribonucleotide formyltransferase (GARFTase). X-ray crystal structures for 4 with FRα and GARFTase showed that the bound conformations of 4 required flexibility for attachment to both FRα and GARFTase. In mice bearing IGROV1 ovarian tumor xenografts, 4 was highly efficacious. Our results establish that heteroatom substitutions in the 3-atom bridge region of 6-substituted pyrrolo[2,3-d]pyrimidines related to 1 provide targeted antifolates that warrant further evaluation as anticancer agents.
Assuntos
Antineoplásicos/química , Receptor 1 de Folato/metabolismo , Antagonistas do Ácido Fólico/química , Transportador de Folato Acoplado a Próton/metabolismo , Nucleotídeos de Purina/antagonistas & inibidores , Pirimidinas/química , Pirróis/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/farmacologia , Xenoenxertos , Humanos , Camundongos SCID , Simulação de Acoplamento Molecular , Transplante de Neoplasias , Fosforribosilglicinamido Formiltransferase/antagonistas & inibidores , Nucleotídeos de Purina/biossíntese , Pirimidinas/síntese química , Pirimidinas/farmacologia , Pirróis/síntese química , Pirróis/farmacologia , Relação Estrutura-AtividadeRESUMO
2-Amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidine antifolate thiophene regioisomers of AGF94 (4) with a thienoyl side chain and three-carbon bridge lengths [AGF150 (5) and AGF154 (7)] were synthesized as potential antitumor agents. These analogues inhibited proliferation of Chinese hamster ovary (CHO) sublines expressing folate receptors (FRs) α or ß (IC50s < 1 nM) or the proton-coupled folate transporter (PCFT) (IC50 < 7 nM). Compounds 5 and 7 inhibited KB, IGROV1, and SKOV3 human tumor cells at subnanomolar concentrations, reflecting both FRα and PCFT uptake. AGF152 (6) and AGF163 (8), 2,4-diamino-5-substituted-furo[2,3-d]pyrimidine thiophene regioisomers, also inhibited growth of FR-expressing CHO and KB cells. All four analogues inhibited glycinamide ribonucleotide formyltransferase (GARFTase). Crystal structures of human GARFTase complexed with 5 and 7 were reported. In severe combined immunodeficient mice bearing SKOV3 tumors, 7 was efficacious. The selectivity of these compounds for PCFT and for FRα and ß over the ubiquitously expressed reduced folate carrier is a paradigm for selective tumor targeting.
Assuntos
Antineoplásicos/química , Receptor 1 de Folato/antagonistas & inibidores , Antagonistas do Ácido Fólico/química , Transportador de Folato Acoplado a Próton/antagonistas & inibidores , Pirimidinas/química , Pirróis/química , Tiofenos/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Transporte Biológico , Células CHO , Linhagem Celular Tumoral , Cricetulus , Cristalografia por Raios X , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Receptor 1 de Folato/química , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/farmacologia , Xenoenxertos , Humanos , Camundongos SCID , Modelos Moleculares , Transplante de Neoplasias , Pemetrexede/farmacologia , Fosforribosilglicinamido Formiltransferase/antagonistas & inibidores , Fosforribosilglicinamido Formiltransferase/química , Transportador de Folato Acoplado a Próton/química , Pirimidinas/síntese química , Pirimidinas/farmacologia , Pirróis/síntese química , Pirróis/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/farmacologiaRESUMO
XK469 (1) is among the most highly and broadly active antitumor agents to have been evaluated in our laboratories. Subsequent developmental studies led to the entry of (R)-(+) 1 (NSC 698215) into phase 1 clinical trials (NIH UO1-CA62487). The antitumor mechanism of action of 1 remains to be elucidated, which has prompted a sustained effort to elaborate a pharmacophoric pattern of 1. The present study focused on a strategy of synthesis and biological evaluation of topologically based, bioisosteric replacements of the quinoxaline moiety in the lead compound (1) by quinazoline (4a-d), 1,2,4-benzotriazine (12a-18b), and quinoline (21a-g) ring systems. The synthetic approach to each of the bioisosteres of 1 utilized the methodology developed in previous work (see Hazeldine, S. T.; Polin, L.; Kushner, J.; Paluch, J.; White, K.; Edelstein, M.; Palomino, E.; Corbett, T. H.; Horwitz, J. P. Design, Synthesis, and Biological Evaluation of Analogues of the Antitumor Agent 2-(4-[(7-Chloro-2-quinoxalinyl)oxy]phenoxy)propionic acid (XK469). J. Med. Chem. 2001, 44, 1758-1776.), which is extended to the procurement of the benzoxazole (23a,b), benzthiazole (23c,d), pyridine (25a,b), and pyrazine (27) congeners of 1. Only quinoline analogues, bearing a 7-halo (21a,b,d,e) or a 7-methoxy substituent (21g), showed antitumor activities (Br > Cl > CH(3)O > F approximately I), at levels comparable to or greater than the range of activities manifested by 1 and corresponding analogues. At high individual dosages, the (S)-(-) enantiomers of 1 and 21b,d all produce a reversible slowing of nerve-conduction velocity in the mice, the onset of which is characterized by a distinctive dysfunction of the hind legs, causing uncoordinated movements. The condition resolves within 5-10 min. However, at higher dosages, which approach a lethal level, the behavior extended to the front legs, lasting from 20 min to 1 h. By contrast, the (R)-(+) forms of these same agents did not induce the phenomenon of slowing of nerve-conduction velocity.