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Multiple Myeloma (MM) is an incurable plasma cell malignancy, that despite an unprecedented increase in overall survival, lacks truly risk-adapted or targeted treatments. A proportion of patients with MM depend on BCL-2 for survival and recently the BCL-2 antagonist venetoclax has shown clinical efficacy and safety in t(11;14) and BCL-2 overexpressing MM. However, only a small proportion of MM patients rely on BCL-2 (~20%), there is a need to broaden the patient population outside of t(11;14) that can be treated with venetoclax. Therefore, we took an unbiased screening approach and screened epigenetic modifiers to enhance venetoclax sensitivity in two non-BCL-2 dependent MM cell lines. The demethylase inhibitor 5-azacytidine was one of the lead hits from the screen, and the enhanced cell killing of the combination was confirmed in additional MM cell lines. Using dynamic BH3 profiling and immunoprecipitations we identified the potential mechanism of synergy is due to increased NOXA expression, through the integrated stress response. Knockdown of PMAIP1 or PKR partially rescues cell death of the venetoclax and 5-azacytidine combination treatment. The addition of a steroid to the combination treatment did not enhance the cell death and interestingly we found enhanced death of the immune cells with steroid addition, suggesting that a steroid-sparing regimen may be more beneficial in MM. Lastly, we show for the first time in primary MM patient samples, that 5-azacytidine enhances the response to venetoclax ex-vivo, across diverse anti-apoptotic dependencies (BCL-2 or MCL-1) and diverse cytogenetic backgrounds. Overall, our data identifies 5-azacytidine and venetoclax as an effective treatment combination and this could be a tolerable steroid-sparing regimen, particularly for elderly MM patients.
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We report the development of the first metallo-PROTAC, specifically a Pt-PROTAC, that can effectively degrade select Pt(II)-binding proteins. The Pt-PROTAC prototype successfully degraded thioredoxin-1 and thioredoxin reductase-1 in multiple myeloma cancer cell lines. Metallo-PROTACs will have important applications in the identification of metal binding proteins and as chemotherapeutic agents.
Assuntos
Proteínas de Transporte , Neoplasias , Humanos , Proteólise , ProteínasRESUMO
INTRODUCTION: In KRAS-mutant NSCLC, co-occurring alterations in LKB1 confer a negative prognosis compared with other mutations such as TP53. LKB1 is a tumor suppressor that coordinates several signaling pathways in response to energetic stress. Our recent work on pharmacologic and genetic inhibition of histone deacetylase 6 (HDAC6) revealed the impaired activity of numerous enzymes involved in glycolysis. On the basis of these previous findings, we explored the therapeutic window for HDAC6 inhibition in metabolically-active KRAS-mutant lung tumors. METHODS: Using cell lines derived from mouse autochthonous tumors bearing the KRAS/LKB1 (KL) and KRAS/TP53 mutant genotypes to control for confounding germline and somatic mutations in human models, we characterize the metabolic phenotypes at baseline and in response to HDAC6 inhibition. The impact of HDAC6 inhibition was measured on cancer cell growth in vitro and on tumor growth in vivo. RESULTS: Surprisingly, KL-mutant cells revealed reduced levels of redox-sensitive cofactors at baseline. This is associated with increased sensitivity to pharmacologic HDAC6 inhibition with ACY-1215 and blunted ability to increase compensatory metabolism and buffer oxidative stress. Seeking synergistic metabolic combination treatments, we found enhanced cell killing and antitumor efficacy with glutaminase inhibition in KL lung cancer models in vitro and in vivo. CONCLUSIONS: Exploring the differential metabolism of KL and KRAS/TP53-mutant NSCLC, we identified decreased metabolic reserve in KL-mutant tumors. HDAC6 inhibition exploited a therapeutic window in KL NSCLC on the basis of a diminished ability to compensate for impaired glycolysis, nominating a novel strategy for the treatment of KRAS-mutant NSCLC with co-occurring LKB1 mutations.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Animais , Camundongos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/uso terapêutico , Desacetilase 6 de Histona/genética , Desacetilase 6 de Histona/metabolismo , Desacetilase 6 de Histona/uso terapêutico , Linhagem Celular Tumoral , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , MutaçãoRESUMO
Resistance to mitochondrial apoptosis predicts inferior treatment outcomes in patients with diverse tumor types, including T-cell acute lymphoblastic leukemia (T-ALL). However, the genetic basis for variability in this mitochondrial apoptotic phenotype is poorly understood, preventing its rational therapeutic targeting. Using BH3 profiling and exon sequencing analysis of childhood T-ALL clinical specimens, we found that mitochondrial apoptosis resistance was most strongly associated with activating mutations of JAK3. Mutant JAK3 directly repressed apoptosis in leukemia cells, because its inhibition with mechanistically distinct pharmacologic inhibitors resulted in reversal of mitochondrial apoptotic blockade. Inhibition of JAK3 led to loss of MEK, ERK and BCL2 phosphorylation, and BH3 profiling revealed that JAK3-mutant primary T-ALL patient samples were characterized by a dependence on BCL2. Treatment of JAK3-mutant T-ALL cells with the JAK3 inhibitor tofacitinib in combination with a spectrum of conventional chemotherapeutics revealed synergy with glucocorticoids, in vitro and in vivo. These findings thus provide key insights into the molecular genetics of mitochondrial apoptosis resistance in childhood T-ALL, and a compelling rationale for a clinical trial of JAK3 inhibitors in combination with glucocorticoids for patients with JAK3-mutant T-ALL.
Assuntos
Leucemia-Linfoma Linfoblástico de Células T Precursoras , Apoptose , Glucocorticoides , Humanos , Janus Quinase 3/genética , Mutação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Linfócitos T/patologiaRESUMO
Due to developments in modern chemistry, previously uundruggable substrates are now targetable thanks to selective degradation using the ubiquitin-proteasomal degradation system. PROteolysis TArgeting Chimeras (PROTACs) are heterobifunctional molecules designed specifically to degrade target proteins. They are of significant interest to industry and academia as they are highly specific and can target previously undruggable target proteins from transcription factors to enzymes. More than 15 degraders are expected to be evaluated in clinical trials by the end of 2021. Herein, we describe recent advances in the design and development of PROTAC-mediated degradation of histone deacetylases (HDACs). PROTAC-mediated degradation of HDACs can offer some significant advantages over direct inhibition, such as the use of substoichiometric doses and the potential to disrupt enzyme-independent HDAC function. We discuss the potential implication of the degradation of HDACs in comparison with HDAC knockout studies. Along with the selection of HDAC inhibitors and E3 ligase ligands for the design of PROTACs. The potential utility of HDAC PROTACs in various disease pathologies from cancer to inflammation to neurodegeneration is driving the interest in this field.
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Inibidores de Histona Desacetilases , Histona Desacetilases , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , ProteóliseRESUMO
Triple-negative breast cancer (TNBC) is a subtype of breast cancer without a targeted form of therapy. Unfortunately, up to 70% of patients with TNBC develop resistance to treatment. A known contributor to chemoresistance is dysfunctional mitochondrial apoptosis signaling. We set up a phenotypic small-molecule screen to reveal vulnerabilities in TNBC cells that were independent of mitochondrial apoptosis. Using a functional genetic approach, we identified that a "hit" compound, BAS-2, had a potentially similar mechanism of action to histone deacetylase inhibitors (HDAC). An in vitro HDAC inhibitor assay confirmed that the compound selectively inhibited HDAC6. Using state-of-the-art acetylome mass spectrometry, we identified glycolytic substrates of HDAC6 in TNBC cells. We confirmed that inhibition or knockout of HDAC6 reduced glycolytic metabolism both in vitro and in vivo. Through a series of unbiased screening approaches, we have identified a previously unidentified role for HDAC6 in regulating glycolytic metabolism.
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Neoplasias de Mama Triplo Negativas , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Detecção Precoce de Câncer , Desacetilase 6 de Histona/genética , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Humanos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
Metabolic reprogramming is a hallmark of cancer which contributes to essential processes required for cell survival, growth, and proliferation. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and its genomic classification has given rise to the design of therapies targeting tumors harboring specific gene alterations that cause aberrant signaling. Lung tumors are characterized with having high glucose and lactate use, and high heterogeneity in their metabolic pathways. Here we review how NSCLC cells with distinct mutations reprogram their metabolic pathways and highlight the potential metabolic vulnerabilities that might lead to the development of novel therapeutic strategies.
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Carcinoma Pulmonar de Células não Pequenas/genética , Reprogramação Celular/genética , Metabolismo/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Humanos , Mutação , Transdução de Sinais/genéticaRESUMO
The tendency of mitochondria to undergo or resist BCL2-controlled apoptosis (so-called mitochondrial priming) is a powerful predictor of response to cytotoxic chemotherapy. Fully exploiting this finding will require unraveling the molecular genetics underlying phenotypic variability in mitochondrial priming. Here, we report that mitochondrial apoptosis resistance in T cell acute lymphoblastic leukemia (T-ALL) is mediated by inactivation of polycomb repressive complex 2 (PRC2). In T-ALL clinical specimens, loss-of-function mutations of PRC2 core components (EZH2, EED, or SUZ12) were associated with mitochondrial apoptosis resistance. In T-ALL cells, PRC2 depletion induced resistance to apoptosis induction by multiple chemotherapeutics with distinct mechanisms of action. PRC2 loss induced apoptosis resistance via transcriptional up-regulation of the LIM domain transcription factor CRIP2 and downstream up-regulation of the mitochondrial chaperone TRAP1 These findings demonstrate the importance of mitochondrial apoptotic priming as a prognostic factor in T-ALL and implicate mitochondrial chaperone function as a molecular determinant of chemotherapy response.
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Apoptose , Resistencia a Medicamentos Antineoplásicos , Proteínas de Neoplasias/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Feminino , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas de Neoplasias/genética , Complexo Repressor Polycomb 2/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Targeted therapies designed to exploit specific molecular pathways in aggressive cancers are an exciting area of current research. Mixed Lineage Leukemia (MLL) mutations such as the t(4;11) translocation cause aggressive leukemias that are refractory to conventional treatment. The t(4;11) translocation produces an MLL/AF4 fusion protein that activates key target genes through both epigenetic and transcriptional elongation mechanisms. In this study, we show that t(4;11) patient cells express high levels of BCL-2 and are highly sensitive to treatment with the BCL-2-specific BH3 mimetic ABT-199. We demonstrate that MLL/AF4 specifically upregulates the BCL-2 gene but not other BCL-2 family members via DOT1L-mediated H3K79me2/3. We use this information to show that a t(4;11) cell line is sensitive to a combination of ABT-199 and DOT1L inhibitors. In addition, ABT-199 synergizes with standard induction-type therapy in a xenotransplant model, advocating for the introduction of ABT-199 into therapeutic regimens for MLL-rearranged leukemias.
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Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteína de Leucina Linfoide-Mieloide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/farmacologia , Animais , Linhagem Celular Tumoral , Genes bcl-2 , Histona-Lisina N-Metiltransferase/genética , Humanos , Metilação , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteína de Leucina Linfoide-Mieloide/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Accumulation of unfolded proteins within the endoplasmic reticulum (ER) triggers a highly conserved stress response mechanism termed the unfolded protein response (UPR). The UPR is a complex series of signaling pathways controlled by ER localized transmembrane receptors, PERK, ATF6 and IRE1α. Following activation IRE1α splices XBP-1 mRNA facilitating the formation of a potent transcription factor, spliced XBP-1. The BCL-2 family members, BAX and BAK, in addition to the mitochondrion also localize to the ER and have been demonstrated to directly interact with IRE1α promoting its activity. In this study we show that in addition to BAX and BAK, the anti-apoptotic BCL-2 protein can regulate IRE1α activity. Enhanced splicing of XBP-1 was observed in BCL-2 overexpressing cells implicating BCL-2 in the complex regulation of IRE1α activity.
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Proteínas de Ligação a DNA/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Splicing de RNA , Fatores de Transcrição/genética , Resposta a Proteínas não Dobradas , Animais , Linhagem Celular , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/metabolismo , Endorribonucleases/análise , Endorribonucleases/metabolismo , Camundongos , Complexos Multienzimáticos/análise , Complexos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/análise , Ratos , Fatores de Transcrição de Fator Regulador X , Transdução de Sinais , Fatores de Transcrição/análise , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação a X-BoxRESUMO
UNLABELLED: Acute lymphoblastic leukemia (ALL) is a hematopoietic malignancy derived from immature B-lymphoid and T-lymphoid cells (T-ALL). In T-ALL, there is an early T-cell progenitor (ETP) subgroup that has a very high risk for relapse. In this study, we used mitochondrial BH3 profiling to determine antiapoptotic protein dependencies in T-ALL. We found that T-ALL cell lines and primary patient samples are dependent upon BCL-XL, except when the cancer bears an ETP phenotype, in which case it is BCL-2 dependent. These distinctions directly relate to differential sensitivity to the BH3 mimetics ABT-263 and ABT-199, both in vitro and in vivo. We thus describe for the first time a change of antiapoptotic protein dependence that is related to the differentiation stage of the leukemic clone. Our findings demonstrate that BCL-2 is a clinically relevant target for therapeutic intervention with ABT-199 in ETP-ALL. SIGNIFICANCE: ETP T-ALL is a treatment-resistant subtype of T-ALL for which novel targeted therapies are urgently needed. We have discovered, through BH3 profiling, that ETP-ALL is BCL-2 dependent and is very sensitive to in vitro and in vivo treatment with ABT-199, a drug well tolerated in clinical trials.
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Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Resistencia a Medicamentos Antineoplásicos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/farmacologia , Proteína bcl-X/metabolismo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Expressão Gênica , Humanos , Modelos Biológicos , Gradação de Tumores , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Fragmentos de Peptídeos/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/genéticaRESUMO
PURPOSE: Predictive biomarkers are required to identify patients who may benefit from the use of BH3 mimetics such as ABT-263. This study investigated the efficacy of ABT-263 against a panel of patient-derived pediatric acute lymphoblastic leukemia (ALL) xenografts and utilized cell and molecular approaches to identify biomarkers that predict in vivo ABT-263 sensitivity. EXPERIMENTAL DESIGN: The in vivo efficacy of ABT-263 was tested against a panel of 31 patient-derived ALL xenografts composed of MLL-, BCP-, and T-ALL subtypes. Basal gene expression profiles of ALL xenografts were analyzed and confirmed by quantitative RT-PCR, protein expression and BH3 profiling. An in vitro coculture assay with immortalized human mesenchymal cells was utilized to build a predictive model of in vivo ABT-263 sensitivity. RESULTS: ABT-263 demonstrated impressive activity against pediatric ALL xenografts, with 19 of 31 achieving objective responses. Among BCL2 family members, in vivo ABT-263 sensitivity correlated best with low MCL1 mRNA expression levels. BH3 profiling revealed that resistance to ABT-263 correlated with mitochondrial priming by NOXA peptide, suggesting a functional role for MCL1 protein. Using an in vitro coculture assay, a predictive model of in vivo ABT-263 sensitivity was built. Testing this model against 11 xenografts predicted in vivo ABT-263 responses with high sensitivity (50%) and specificity (100%). CONCLUSION: These results highlight the in vivo efficacy of ABT-263 against a broad range of pediatric ALL subtypes and shows that a combination of in vitro functional assays can be used to predict its in vivo efficacy.