RESUMEN
Avoidance of apoptosis is critical for the development and sustained growth of tumours. The pro-survival protein myeloid cell leukemia 1 (MCL1) is overexpressed in many cancers, but the development of small molecules targeting this protein that are amenable for clinical testing has been challenging. Here we describe S63845, a small molecule that specifically binds with high affinity to the BH3-binding groove of MCL1. Our mechanistic studies demonstrate that S63845 potently kills MCL1-dependent cancer cells, including multiple myeloma, leukaemia and lymphoma cells, by activating the BAX/BAK-dependent mitochondrial apoptotic pathway. In vivo, S63845 shows potent anti-tumour activity with an acceptable safety margin as a single agent in several cancers. Moreover, MCL1 inhibition, either alone or in combination with other anti-cancer drugs, proved effective against several solid cancer-derived cell lines. These results point towards MCL1 as a target for the treatment of a wide range of tumours.
Asunto(s)
Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Modelos Biológicos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Tiofenos/farmacología , Tiofenos/uso terapéutico , Animales , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Femenino , Humanos , Leucemia/tratamiento farmacológico , Leucemia/metabolismo , Leucemia/patología , Linfoma/tratamiento farmacológico , Linfoma/metabolismo , Linfoma/patología , Masculino , Ratones , Modelos Moleculares , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/química , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Neoplasias/metabolismo , Pirimidinas/administración & dosificación , Tiofenos/administración & dosificación , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Recent studies have demonstrated that the immunomodulatory drugs (IMiDs) lead to the degradation of the transcription factors Ikaros and Aiolos. However, why their loss subsequently leads to multiple myeloma (MM) cell death remains unclear. Using CRISPR-Cas9 genome editing, we have deleted IKZF1/Ikaros and IKZF3/Aiolos in human MM cell lines to gain further insight into their downstream gene regulatory networks. Inactivation of either factor alone recapitulates the cell intrinsic action of the IMiDs, resulting in cell cycle arrest and induction of apoptosis. Furthermore, evaluation of the transcriptional changes resulting from their loss demonstrates striking overlap with lenalidomide treatment. This was not dependent on reduction of the IRF4-MYC "axis," as neither protein was consistently downregulated, despite cell death occurring, and overexpression of either factor failed to rescue for Ikaros loss. Importantly, Ikaros and Aiolos repress the expression of interferon-stimulated genes (ISGs), including CD38, and their loss led to the activation of an interferon-like response, contributing to MM cell death. Ikaros/Aiolos repressed CD38 expression through interaction with the nucleosome remodeling and deacetylase complex in MM. IMiD-induced loss of Ikaros or treatment with interferon resulted in an upregulation of CD38 surface expression on MM cells, priming for daratumumab-induced NK cell-mediated antibody-dependent cellular cytotoxicity. These results give further insight into the mechanism of action of the IMiDs and provide mechanistic rationale for combination with anti-CD38 monoclonal antibodies.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Sistemas CRISPR-Cas , Factor de Transcripción Ikaros/genética , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , HumanosRESUMEN
New therapeutic targets are needed to address the poor prognosis of patients with high-risk multiple myeloma. Myeloma cells usually express a range of the prosurvival BCL2 proteins. To define the hierarchy of their relative importance for maintaining the survival of myeloma cells, we targeted each of them in a large panel of cell lines, using pharmacological inhibitors or gene editing or by peptide-based approaches, alone or in combination. The majority of well-established immortalized cell lines (17/25) or low-passage myeloma cell lines (5/7) are readily killed when MCL1 is targeted, even including those cell lines sensitive to BCL2 inhibition. Targeting MCL1 also constrained the growth of myeloma in vivo. We also identified a previously unrecognized subset of myeloma that is highly BCLXL-dependent, and has the potential for cotargeting MCL1 and BCLXL. As MCL1 is pivotal for maintaining survival of most myelomas, it should be prioritized for targeting in the clinic once high-quality, validated inhibitors become available.
Asunto(s)
Terapia Molecular Dirigida , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Edición Génica , Humanos , Ligandos , Péptidos/farmacología , Bibliotecas de Moléculas Pequeñas/farmacologíaAsunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Tasa de Mutación , Proteínas Proto-Oncogénicas c-bcl-2/genética , Sulfonamidas/uso terapéutico , Sustitución de Aminoácidos/genética , Antineoplásicos/uso terapéutico , Estudios de Cohortes , Análisis Mutacional de ADN , Progresión de la Enfermedad , Resistencia a Antineoplásicos/genética , Frecuencia de los Genes , Glicina/genética , Humanos , Leucemia Linfocítica Crónica de Células B/patología , Modelos Moleculares , Mutación , Proteínas Proto-Oncogénicas c-bcl-2/química , Células Tumorales Cultivadas , Valina/genéticaAsunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/administración & dosificación , Resistencia a Antineoplásicos/genética , Linfoma Folicular/genética , Mutación Missense , Proteínas Proto-Oncogénicas c-bcl-2/genética , Sulfonamidas/administración & dosificación , Sustitución de Aminoácidos , Humanos , Linfoma Folicular/tratamiento farmacológico , Masculino , Persona de Mediana EdadRESUMEN
Although microRNAs (miRNAs) are increasingly linked to various physiologic processes, including hematopoiesis, their function in the myeloid development is poorly understood. We detected up-regulation of miR-29a and miR-142-3p during myeloid differentiation in leukemia cell lines and CD34(+) hematopoietic stem/progenitor cells. By gain-of-function and loss-of-function experiments, we demonstrated that both miRNAs promote the phorbol 12-myristate 13-acetate-induced monocytic and all-trans-retinoic acid-induced granulocytic differentiation of HL-60, THP-1, or NB4 cells. Both the miRNAs directly inhibited cyclin T2 gene, preventing the release of hypophosphorylated retinoblastoma and resulting in induction of monocytic differentiation. In addition, a target of miR-29a, cyclin-dependent kinase 6 gene, and a target of miR-142-3p, TGF-ß-activated kinase 1/MAP3K7 binding protein 2 gene, are involved in the regulation of both monocytic and granulocytic differentiation. A significant decrease of miR-29a and 142-3p levels and an obvious increase in their target protein levels were also observed in blasts from acute myeloid leukemia. By lentivirus-mediated gene transfer, we demonstrated that enforced expression of either miR-29a or miR-142-3p in hematopoietic stem/progenitor cells from healthy controls and acute myeloid leukemia patients down-regulated expression of their targets and promoted myeloid differentiation. These findings confirm that miR-29a and miR-142-3p are key regulators of normal myeloid differentiation and their reduced expression is involved in acute myeloid leukemia development.
Asunto(s)
Diferenciación Celular/genética , Leucemia Mieloide Aguda/genética , MicroARNs/fisiología , Células Mieloides/fisiología , Antineoplásicos/farmacología , Carcinógenos/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/fisiología , Células HEK293 , Células HL-60 , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , MicroARNs/genética , MicroARNs/metabolismo , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Transfección , Tretinoina/farmacologíaRESUMEN
Introduction: BCL-2 family proteins are important for tumour cell survival and drug resistance in multiple myeloma (MM). Although proteasome inhibitors are effective anti-myeloma drugs, some patients are resistant and almost all eventually relapse. We examined the function of BCL-2 family proteins in stromal-mediated resistance to carfilzomib-induced cytotoxicity in MM cells. Methods: Co-cultures employing HS5 stromal cells were used to model the interaction with stroma. MM cells were exposed to CFZ in a 1-hour pulse method. The expression of BCL-2 family proteins was assessed by flow cytometry and WB. Pro-survival proteins: MCL-1, BCL-2 and BCL-XL were inhibited using S63845, ABT-199 and A-1331852 respectively. Changes in BIM binding partners were examined by immunoprecipitation and WB. Results: CFZ induced dose-dependent cell death of MM cells, primarily mediated by apoptosis. Culture of MM cells on HS-5 stromal cells resulted in reduced cytotoxicity to CFZ in a cell contact-dependent manner, upregulated expression of MCL-1 and increased dependency on BCL-XL. Inhibiting BCL-XL or MCL-1 with BH-3 mimetics abrogated stromal-mediated protection only at high doses, which may not be achievable in vivo. However, combining BH-3 mimetics at sub-therapeutic doses, which alone were without effect, significantly enhanced CFZ-mediated cytotoxicity even in the presence of stroma. Furthermore, MCL-1 inhibition led to enhanced binding between BCL-XL and BIM, while blocking BCL-XL increased MCL-1/BIM complex formation, indicating the cooperative role of these proteins. Conclusion: Stromal interactions alter the dependence on BCL-2 family members, providing a rationale for dual inhibition to abrogate the protective effect of stroma and restore sensitivity to CFZ.
RESUMEN
Although the implantation of intact tumor fragments is a common practice to generate orthotopic xenografts to study tumor invasion and metastasis, the direct implantation of tumor cell suspensions is necessary when prior manipulations of tumor cells are required. However, the establishment of orthotopic xenografts using tumor cell suspensions is not mature, and a comparative study directly comparing their engraftment and metastatic capabilities is lacking. It is unclear whether tumor fragments are superior to cell suspensions for successful engraftment and metastasis. In this study, we employed three GC cell lines with varying metastatic capacities to stably express firefly luciferase for monitoring tumor progression in real time. We successfully minimized the risk of cell leakage during the orthotopic injection of tumor cell suspensions without Corning Matrigel by systematically optimizing the surgical procedure, injection volume, and needle size options. Comparable high engraftment and metastatic rates between these two methods were demonstrated using MKN-45 cells with a strong metastatic ability. Importantly, our approach can adjust the rate of tumor progression flexibly and cuts the experimental timeline from 10-12 weeks (for tumor fragments) to 4-5 weeks. Collectively, we provided a highly reproducible procedure with a shortened experimental timeline and low cost for establishing orthotopic GC xenografts via the direct implantation of tumor cell suspensions.
RESUMEN
Intrinsic apoptosis is principally governed by the BCL-2 family of proteins, but some non-BCL-2 proteins are also critical to control this process. To identify novel apoptosis regulators, we performed a genome-wide CRISPR-Cas9 library screen, and it identified the mitochondrial E3 ubiquitin ligase MARCHF5/MITOL/RNF153 as an important regulator of BAK apoptotic function. Deleting MARCHF5 in diverse cell lines dependent on BAK conferred profound resistance to BH3-mimetic drugs. The loss of MARCHF5 or its E3 ubiquitin ligase activity surprisingly drove BAK to adopt an activated conformation, with resistance to BH3-mimetics afforded by the formation of inhibitory complexes with pro-survival proteins MCL-1 and BCL-XL. Importantly, these changes to BAK conformation and pro-survival association occurred independently of BH3-only proteins and influence on pro-survival proteins. This study identifies a new mechanism by which MARCHF5 regulates apoptotic cell death by restraining BAK activating conformation change and provides new insight into how cancer cells respond to BH3-mimetic drugs. These data also highlight the emerging role of ubiquitin signalling in apoptosis that may be exploited therapeutically.
Asunto(s)
Ubiquitina-Proteína Ligasas , Proteína Destructora del Antagonista Homólogo bcl-2 , Proteína bcl-X/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Apoptosis/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
BACKGROUND: New therapeutic targets are needed to improve the outcomes for gastric cancer (GC) patients with advanced disease. Evasion of programmed cell death (apoptosis) is a hallmark of cancer cells and direct induction of apoptosis by targeting the pro-survival BCL2 family proteins represents a promising therapeutic strategy for cancer treatment. Therefore, understanding the molecular mechanisms underpinning cancer cell survival could provide a molecular basis for potential therapeutic interventions. METHOD: Here we explored the role of BCL2L1 and the encoded anti-apoptotic BCL-XL in GC. Using Droplet Digital PCR (ddPCR) technology to investigate the DNA amplification of BCL2L1 in GC samples and GC cell lines, the sensitivity of GC cell lines to selective BCL-XL inhibitors A1155463 and A1331852, pan-inhibitor ABT-263, and VHL-based PROTAC-BCL-XL was analyzed using (CellTiter-Glo) CTG assay in vitro. Western Blot (WB) was used to detect the protein expression of BCL2 family members in GC cell lines and the manner in which PROTAC-BCL-XL kills GC cells. Co-immunoprecipitation (Co-IP) was used to investigate the mechanism of A1331852 and ABT-263 kills GC cell lines. DDPCR, WB, and real-time PCR (RTPCR) were used to investigate the correlation between DNA, RNA, protein levels, and drug activity. RESULTS: The functional assay showed that a subset of GC cell lines relies on BCL-XL for survival. In gastric cancer cell lines, BCL-XL inhibitors A1155463 and A1331852 are more sensitive than the pan BCL2 family inhibitor ABT-263, indicating that ABT-263 is not an optimal inhibitor of BCL-XL. VHL-based PROTAC-BCL-XL DT2216 appears to be active in GC cells. DT2216 induces apoptosis of gastric cancer cells in a time- and dose-dependent manner through the proteasome pathway. Statistical analysis showed that the BCL-XL protein level predicts the response of GC cells to BCL-XL targeting therapy and BCL2L1 gene CNVs do not reliably predict BCL-XL expression. CONCLUSION: We identified BCL-XL as a promising therapeutic target in a subset of GC cases with high levels of BCL-XL protein expression. Functionally, we demonstrated that both selective BCL-XL inhibitors and VHL-based PROTAC BCL-XL can potently kill GC cells that are reliant on BCL-XL for survival. However, we found that BCL2L1 copy number variations (CNVs) cannot reliably predict BCL-XL expression, but the BCL-XL protein level serves as a useful biomarker for predicting the sensitivity of GC cells to BCL-XL-targeting compounds. Taken together, our study pinpointed BCL-XL as potential druggable target for specific subsets of GC.
Asunto(s)
Antineoplásicos , Neoplasias Gástricas , Humanos , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Variaciones en el Número de Copia de ADN , Quimera Dirigida a la Proteólisis , Línea Celular Tumoral , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéuticoRESUMEN
Expression profiling of microRNAs (miRNAs) in most diseases might be popular and provide the possibility for diagnostic implication, but few studies have accurately quantified the expression level of dysregulated miRNAs in acute myeloid leukemia (AML). In this study, we analyzed the peripheral blood mononuclear cells (PBMCs) from 10 AML patients (subtypes M1 to M5) and six normal controls by miRNA microarray and identified several differentially expressed miRNAs. Among them miR-29a and miR-142-3p were selectively encountered in Northern blot analysis and their significantly decreased expression in AML was further confirmed. Quantitative real-time PCR in 52 primarily diagnosed AML patients and 100 normal controls not only verified the expression properties of these 2 miRNAs, but also established that the expression level of miR-142-3p and miR-29a in PBMCs could be used as novel diagnostic markers. A better diagnostic outcome was achieved by combining miR-29a and miR-142-3p with about 90% sensitivity, 100% specificity, and an area under the ROC curve (AUC) of 0.97. Our results provide insights into the involvement of miRNAs in leukemogenesis, and offer candidates for AML diagnosis and therapeutic strategy.
Asunto(s)
Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión Génica/genética , Leucemia Mieloide Aguda/genética , MicroARNs/metabolismo , Área Bajo la Curva , Northern Blotting , Perfilación de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/metabolismo , Leucocitos Mononucleares/metabolismo , Análisis por Micromatrices , Curva ROC , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
Dysregulated expression of BCL-2 family proteins allows cancer cells to escape apoptosis. To counter this, BH3-mimetic drugs that target and inhibit select BCL-2 prosurvival proteins to induce apoptosis have been developed for cancer therapy. Venetoclax, which targets BCL-2, has been effective as therapy for patients with chronic lymphocytic leukemia, and MCL-1-targeting BH3-mimetic drugs have been extensively evaluated in preclinical studies for a range of blood cancers. Recently, BCL-W, a relatively understudied prosurvival member of the BCL-2 protein family, has been reported to be abnormally upregulated in Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), and Hodgkin lymphoma patient samples. Therefore, to determine if BCL-W would be a promising therapeutic target for B-cell lymphomas, we have examined the role of BCL-W in the sustained growth of human BL- and DLBCL-derived cell lines. We found that CRISPR/CAS9-mediated loss or short hairpin RNA-mediated knockdown of BCL-W expression in selected BL and DLBCL cell lines did not lead to spontaneous apoptosis and had no effect on their sensitivity to a range of BH3-mimetic drugs targeting other BCL-2 prosurvival proteins. Our results suggest that BCL-W is not universally required for the sustained growth and survival of human BL and DLBCL cell lines. Thus, targeting BCL-W in this subset of B-cell lymphomas may not be of broad therapeutic benefit.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/farmacología , Linfoma de Burkitt/tratamiento farmacológico , Proteínas Reguladoras de la Apoptosis/metabolismo , Linfoma de Burkitt/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/farmacologíaRESUMEN
MCL1, a BCL2 relative, is critical for the survival of many cells. Its turnover is often tightly controlled through both ubiquitin-dependent and -independent mechanisms of proteasomal degradation. Several cell stress signals, including DNA damage and cell cycle arrest, are known to elicit distinct E3 ligases to ubiquitinate and degrade MCL1. Another trigger that drives MCL1 degradation is engagement by NOXA, one of its BH3-only protein ligands, but the mechanism responsible has remained unclear. From an unbiased genome-wide CRISPR-Cas9 screen, we discovered that the ubiquitin E3 ligase MARCH5, the ubiquitin E2 conjugating enzyme UBE2K, and the mitochondrial outer membrane protein MTCH2 co-operate to mark MCL1 for degradation by the proteasome-specifically when MCL1 is engaged by NOXA. This mechanism of degradation also required the MCL1 transmembrane domain and distinct MCL1 lysine residues to proceed, suggesting that the components likely act on the MCL1:NOXA complex by associating with it in a specific orientation within the mitochondrial outer membrane. MTCH2 has not previously been reported to regulate protein stability, but is known to influence the mitochondrial localization of certain key apoptosis regulators and to impact metabolism. We have now pinpointed an essential but previously unappreciated role for MTCH2 in turnover of the MCL1:NOXA complex by MARCH5, further strengthening its links to BCL2-regulated apoptosis.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Supervivencia Celular , Lisina/metabolismo , Ratones , Proteínas Mitocondriales/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/química , Factores de Elongación de Péptidos/metabolismo , Dominios Proteicos , Proteolisis , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Novel targets are required to improve the outcomes for patients with colorectal cancers. In this regard, the selective inhibitor of the pro-survival protein BCL2, venetoclax, has proven highly effective in several hematological malignancies. In addition to BCL2, potent and highly selective small molecule inhibitors of its relatives, BCLxL and MCL1, are now available, prompting us to investigate the susceptibility of colorectal cancers to the inhibition of one or more of these pro-survival proteins. While targeting BCLxL, but not BCL2 or MCL1, on its own had some impact, most (15/17) of the immortalized colorectal cancer cell lines studied were efficiently killed by the combined targeting of BCLxL and MCL1. Importantly, these in vitro findings were confirmed in a xenograft model and, interestingly, in all (5/5) patient derived tumor organoids evaluated. Our results lend strong support to the notion that BCLxL and MCL1 are highly promising targets for further evaluation in efforts to improve the treatment of colorectal cancers.
Asunto(s)
Neoplasias Colorrectales/genética , Susceptibilidad a Enfermedades/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Humanos , RatonesRESUMEN
Multiple myeloma is an incurable and fatal cancer of immunoglobulin-secreting plasma cells. Most conventional therapies aim to induce apoptosis in myeloma cells but resistance to these drugs often arises and drives relapse. In this study, we sought to identify the best adjunct targets to kill myeloma cells resistant to conventional therapies using deep profiling by mass cytometry (CyTOF). We validated probes to simultaneously detect 26 regulators of cell death, mitosis, cell signaling, and cancer-related pathways at the single-cell level following treatment of myeloma cells with dexamethasone or bortezomib. Time-resolved visualization algorithms and machine learning random forest models (RFMs) delineated putative cell death trajectories and a hierarchy of parameters that specified myeloma cell survival versus apoptosis following treatment. Among these parameters, increased amounts of phosphorylated cAMP response element-binding protein (CREB) and the pro-survival protein, MCL-1, were defining features of cells surviving drug treatment. Importantly, the RFM prediction that the combination of an MCL-1 inhibitor with dexamethasone would elicit potent, synergistic killing of myeloma cells was validated in other cell lines, in vivo preclinical models and primary myeloma samples from patients. Furthermore, CyTOF analysis of patient bone marrow cells clearly identified myeloma cells and their key cell survival features. This study demonstrates the utility of CyTOF profiling at the single-cell level to identify clinically relevant drug combinations and tracking of patient responses for future clinical trials.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Transducción de Señal , Algoritmos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Bortezomib/farmacología , Bortezomib/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular , Dexametasona/farmacología , Dexametasona/uso terapéutico , Sinergismo Farmacológico , Citometría de Flujo , Humanos , Aprendizaje Automático , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal/efectos de los fármacos , Análisis de la Célula Individual , Factores de TiempoRESUMEN
Prosurvival BCL-2 family proteins are potent inhibitors of apoptosis and often overexpressed in lymphoid malignancies. In multiple myeloma (MM), MCL-1 expression contributes to survival of malignant plasma cells, and overexpression correlates with poor prognosis. In this study, we investigated whether sensitivity to the novel MCL-1 inhibitor S63845 could be predicted using cytogenetics, focusing on amplification of 1q21, the chromosomal region that contains the MCL1 locus. In addition, we studied the relation of MCL-1 inhibitor sensitivity with other diagnostic characteristics and BCL-2 family protein expression. In 31 human myeloma cell lines and in bone marrow aspirates from 47 newly diagnosed MM patients, we measured the effect of S63845 alone, or combined with BCL-2 inhibitor ABT-199 (venetoclax), and BCL-XL inhibitor A-1155463 or A-1331852 on cell viability. We demonstrated for the first time that MM cells from patients with 1q21 amplification are significantly more sensitive to inhibition of MCL-1. We suggest that this increased sensitivity results from high relative MCL1 expression resulting from amplification of 1q21. Additionally, and partially independent from 1q21 status, high serum ß2 microglobulin level and presence of renal insufficiency correlated with increased sensitivity to MCL-1 inhibitor treatment. Combining S63845 with other BH3 mimetics synergistically enhanced apoptosis compared with single inhibitors, and sensitivity to inhibitor combinations was found in a large proportion of MM insensitive to MCL-1 inhibition alone. Collectively, our data indicate that amplification of 1q21 identifies an MM subset highly sensitive to MCL-1 inhibitor treatment and can be used as a predictive marker to guide selection of therapy.
Asunto(s)
Antineoplásicos/farmacología , Cromosomas Humanos Par 1/genética , Resistencia a Antineoplásicos/genética , Amplificación de Genes , Mieloma Múltiple/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Ratones , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Interferencia de ARN , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Venetoclax is a first-in-class cancer therapy that interacts with the cellular apoptotic machinery promoting apoptosis. Treatment of patients suffering chronic lymphocytic leukaemia with this BCL-2 antagonist has revealed emergence of a drug-selected BCL-2 mutation (G101V) in some patients failing therapy. To understand the molecular basis of this acquired resistance we describe the crystal structures of venetoclax bound to both BCL-2 and the G101V mutant. The pose of venetoclax in its binding site on BCL-2 reveals small but unexpected differences as compared to published structures of complexes with venetoclax analogues. The G101V mutant complex structure and mutant binding assays reveal that resistance is acquired by a knock-on effect of V101 on an adjacent residue, E152, with venetoclax binding restored by a E152A mutation. This provides a framework for considering analogues of venetoclax that might be effective in combating this mutation.
Asunto(s)
Antineoplásicos/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/metabolismo , Resistencia a Antineoplásicos/genética , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/genética , Sulfonamidas/metabolismo , Antineoplásicos/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Cristalización , Cristalografía por Rayos X , Humanos , Mutación , Unión Proteica , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Sulfonamidas/uso terapéutico , Resonancia por Plasmón de SuperficieRESUMEN
The BCL2 inhibitor venetoclax induces high rates of durable remission in patients with previously treated chronic lymphocytic leukemia (CLL). However, despite continuous daily treatment, leukemia recurs in most patients. To investigate the mechanisms of secondary resistance, we analyzed paired pre-venetoclax and progression samples from 15 patients with CLL progression enrolled on venetoclax clinical trials. The novel Gly101Val mutation in BCL2 was identified at progression in 7 patients, but not at study entry. It was first detectable after 19 to 42 months of therapy, and its emergence anticipated clinical disease progression by many months. Gly101Val reduces the affinity of BCL2 for venetoclax by â¼180-fold in surface plasmon resonance assays, thereby preventing the drug from displacing proapoptotic mediators from BCL2 in cells and conferring acquired resistance in cell lines and primary patient cells. This mutation provides new insights into the pathobiology of venetoclax resistance and provides a potential biomarker of impending clinical relapse. SIGNIFICANCE: Why CLL recurs in patients who achieve remission with the BCL2 inhibitor venetoclax has been unknown. We provide the first description of an acquired point mutation in BCL2 arising recurrently and exclusively in venetoclax-treated patients. The mutation reduces venetoclax binding and is sufficient to confer resistance.See related commentary by Thangavadivel and Byrd, p. 320.This article is highlighted in the In This Issue feature, p. 305.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Mutación , Proteínas Proto-Oncogénicas c-bcl-2/genética , Sulfonamidas/farmacología , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Leucemia Linfocítica Crónica de Células B/patología , Modelos Moleculares , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Conformación Proteica , Células Tumorales CultivadasRESUMEN
Myelopoiesis is under the control of a complex network containing various regulation factors. Deregulation of any important regulation factors may result in serious consequences including acute myeloid leukemia (AML). In order to find out the genes that may take a part in AML development, we analyzed data from AML cDNA microarray (GSE2191) in the NCBI data pool and noticed that heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is abnormally over-expressed in AML patients. Then we investigated the function and mechanisms of hnRNP A1 in myeloid development. A gradually decreased hnRNP A1 expression was detected during granulocytic differentiation in ATRA-induced-NB4 and HL-60 cells and cytokines-induced hematopoietic stem and progenitor cells. By function-loss and winning experiments we demonstrated hnRNP A1's inhibition role via inhibiting expression of C/EBPα, a key regulator of granulocytic differentiation, in the granulocytic differentiation. During granulocytic differentiation the decrease of hnRNP A1 reduces inhibition on C/EBPα expression, and the increased C/EBPα promotes the differentiation. We also demonstrated that miR-451 promotes granulocytic differentiation via targeting to and down-regulating hnRNP A1, and hnRNP A1 positively regulates c-Myc expression. Summarily, our results revealed new function and mechanisms of hnRNP A1 in normal granulocytiesis and the involvement of a feed-back loop comprising c-Myc, miR-451 and hnRNP A1 in AML development.
RESUMEN
The development of BH3 mimetics, which antagonize prosurvival proteins of the BCL-2 family, represents a potential breakthrough in cancer therapy. Targeting the prosurvival member MCL-1 has been an area of intense interest because it is frequently deregulated in cancer. In breast cancer, MCL-1 is often amplified, and high expression predicts poor patient outcome. We tested the MCL-1 inhibitor S63845 in breast cancer cell lines and patient-derived xenografts with high expression of MCL-1. S63845 displayed synergistic activity with docetaxel in triple-negative breast cancer and with trastuzumab or lapatinib in HER2-amplified breast cancer. Using S63845-resistant cells combined with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9) technology, we identified deletion of BAK and up-regulation of prosurvival proteins as potential mechanisms that confer resistance to S63845 in breast cancer. Collectively, our findings provide a strong rationale for the clinical evaluation of MCL-1 inhibitors in breast cancer.