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1.
Pediatr Blood Cancer ; 67(4): e28133, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-31876116

RESUMEN

BACKGROUND: The aim of this study was to improve the predictive power of patient-derived xenografts (PDXs, also known as mouse avatars) to more accurately reflect outcomes of clofarabine-based treatment in pediatric acute lymphoblastic leukemia (ALL) patients. PROCEDURE: Pharmacokinetic (PK) studies were conducted using clofarabine at 3.5 to 15 mg/kg in mice. PDXs were established from relapsed/refractory ALL patients who exhibited good or poor responses to clofarabine. PDX engraftment and response to clofarabine (either as a single agent or in combinations) were assessed based on stringent objective response measures modeled after the clinical setting. RESULTS: In naïve immune-deficient NSG mice, we determined that a clofarabine dose of 3.5 mg/kg resulted in systemic exposures equivalent to those achieved in pediatric ALL patients treated with clofarabine-based regimens. This dose was markedly lower than the doses of clofarabine used in previously reported preclinical studies (typically 30-60 mg/kg) and, when scheduled consistent with the clinical regimen (daily × 5), resulted in 34-fold lower clofarabine exposures. Using a well-tolerated clofarabine/etoposide/cyclophosphamide combination regimen, we then found that the responses of PDXs better reflected the clinical responses of the patients from whom the PDXs were derived. CONCLUSIONS: This study has identified an in vivo clofarabine treatment regimen that reflects the clinical responses of relapsed/refractory pediatric ALL patients. This regimen could be used prospectively to identify patients who might benefit from clofarabine-based treatment. Our findings are an important step toward individualizing prospective patient selection for the use of clofarabine in relapsed/refractory pediatric ALL patients and highlight the need for detailed PK evaluation in murine PDX models.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Medicina de Precisión/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Antimetabolitos Antineoplásicos/farmacología , Clofarabina/farmacología , Ciclofosfamida/farmacología , Etopósido/farmacología , Humanos , Ratones
2.
Int J Biol Macromol ; 190: 214-223, 2021 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-34481852

RESUMEN

Antibody fragments are promising building blocks for developing targeted therapeutics, thus improving treatment efficacy while minimising off-target toxicity. Despite recent advances in targeted therapeutics, patients with Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL), a high-risk malignancy, lack specific and effective targeted treatments. Cytokine receptor-like factor 2 (CRLF2) is overexpressed in 50% of Ph-like ALL cases, conferring the survival of leukemia blasts through activation of the JAK/STAT signalling pathway. Targeting such a vital cell-surface protein could result in potent anti-leukaemic efficacy and reduce the likelihood of relapse associated with antigen loss. Herein, we developed a novel single-chain variable fragment (scFv) against CRLF2 based on a monoclonal antibody raised against the recombinant extracellular domain of human TSLPRα chain. The scFv fragment demonstrated excellent binding affinity with CRLF2 protein in the nanomolar range. Cellular association studies in vitro using an inducible CRLF2 knockdown cell line and ex vivo using patient-derived xenografts revealed the selective association of the scFv with CRLF2. The fragment exhibited significant receptor antagonistic effects on STAT5 signalling, suggesting possible therapeutic implications in vivo. This study is the first to describe the potential use of a novel scFv for targeting Ph-like ALL.


Asunto(s)
Fragmentos de Inmunoglobulinas/metabolismo , Cromosoma Filadelfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Receptores de Citocinas/metabolismo , Proteínas Recombinantes/metabolismo , Animales , Línea Celular Tumoral , Niño , Endocitosis , Células HEK293 , Humanos , Ratones , Fosforilación , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Anticuerpos de Cadena Única/aislamiento & purificación
3.
Leukemia ; 35(11): 3101-3112, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-33895784

RESUMEN

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk ALL subtype with high rates of relapse and poor patient outcome. Activating mutations affecting components of the JAK-STAT signaling pathway occur in the majority of Ph-like ALL cases. The use of JAK inhibitors represents a potential treatment option for Ph-like ALL, although we and others have shown that CRLF2-rearranged Ph-like ALL responds poorly to single-agent JAK inhibitors in the preclinical setting. Therefore, the aim of this study was to identify effective combination treatments against CRLF2-rearranged Ph-like ALL, and to elucidate the underlying mechanisms of synergy. We carried out a series of high-throughput combination drug screenings and found that ruxolitinib exerted synergy with standard-of-care drugs used in the treatment of ALL. In addition, we investigated the molecular effects of ruxolitinib on Ph-like ALL by combining mass spectrometry phosphoproteomics with gene expression analysis. Based on these findings, we conducted preclinical in vivo drug testing and demonstrated that ruxolitinib enhanced the in vivo efficacy of an induction-type regimen consisting of vincristine, dexamethasone, and L-asparaginase in 2/3 CRLF2-rearranged Ph-like ALL xenografts. Overall, our findings support evaluating the addition of ruxolitinib to conventional induction regimens for the treatment of CRLF2-rearranged Ph-like ALL.


Asunto(s)
Reordenamiento Génico , Nitrilos/farmacología , Preparaciones Farmacéuticas/administración & dosificación , Cromosoma Filadelfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Pirazoles/farmacología , Pirimidinas/farmacología , Receptores de Citocinas/genética , Animales , Apoptosis , Proliferación Celular , Quimioterapia Combinada , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
4.
Mol Cancer Res ; 18(12): 1767-1776, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32801162

RESUMEN

Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) is characterized by aberrant activation of signaling pathways and high risk of relapse. Approximately 50% of Ph-like ALL cases overexpress cytokine receptor-like factor 2 (CRLF2) associated with gene rearrangement. Activated by its ligand thymic stromal lymphopoietin (TSLP), CRLF2 signaling is critical for the development, proliferation, and survival of normal lymphocytes. To examine activation of tyrosine kinases regulated by TSLP/CRLF2, phosphotyrosine (P-Tyr) profiling coupled with stable isotope labeling of amino acids in cell culture (SILAC) was conducted using two CRLF2-rearranged (CRLF2r) Ph-like ALL cell lines stimulated with TSLP. As a result, increased P-Tyr was detected in previously reported TSLP-activated tyrosine kinases and substrates, including JAK1, JAK2, STAT5, and ERK1/2. Interestingly, TSLP also increased P-Tyr of insulin growth factor 1 receptor (IGF1R) and fibroblast growth factor receptor 1 (FGFR1), both of which can be targeted with small-molecule inhibitors. Fixed-ratio combination cytotoxicity assays using the tyrosine kinase inhibitors BMS-754807 and ponatinib that target IGF1R and FGFR1, respectively, revealed strong synergy against both cell line and patient-derived xenograft (PDX) models of CRLF2r Ph-like ALL. Further analyses also indicated off-target effects of ponatinib in the synergy, and novel association of the Ras-associated protein-1 (Rap1) signaling pathway with TSLP signaling in CRLF2r Ph-like ALL. When tested in vivo, the BMS-754807/ponatinib combination exerted minimal efficacy against 2 Ph-like ALL PDXs, associated with low achievable plasma drug concentrations. Although this study identified potential new targets in CRLF2r Ph-like ALL, it also highlights that in vivo validation of synergistic drug interactions is essential. IMPLICATION: Quantitative phosphotyrosine profiling identified potential therapeutic targets for high-risk CRLF2-rearranged Ph-like ALL.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Imidazoles/administración & dosificación , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Pirazoles/administración & dosificación , Piridazinas/administración & dosificación , Receptores de Citocinas/genética , Triazinas/administración & dosificación , Tirosina/metabolismo , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Línea Celular Tumoral , Citocinas/metabolismo , Sinergismo Farmacológico , Femenino , Reordenamiento Génico , Humanos , Imidazoles/farmacología , Marcaje Isotópico , Janus Quinasa 1/metabolismo , Janus Quinasa 2/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Fosforilación/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Pirazoles/farmacología , Piridazinas/farmacología , Factor de Transcripción STAT5/metabolismo , Triazinas/farmacología , Proteínas Supresoras de Tumor/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
5.
Cancer Cell ; 34(6): 906-921.e8, 2018 12 10.
Artículo en Inglés | MEDLINE | ID: mdl-30537513

RESUMEN

Glucocorticoids play a critical role in the treatment of lymphoid malignancies. While glucocorticoid efficacy can be largely attributed to lymphocyte-specific apoptosis, its molecular basis remains elusive. Here, we studied genome-wide lymphocyte-specific open chromatin domains (LSOs), and integrated LSOs with glucocorticoid-induced RNA transcription and chromatin modulation using an in vivo patient-derived xenograft model of acute lymphoblastic leukemia (ALL). This led to the identification of LSOs critical for glucocorticoid-induced apoptosis. Glucocorticoid receptor cooperated with CTCF at these LSOs to mediate DNA looping, which was inhibited by increased DNA methylation in glucocorticoid-resistant ALL and non-lymphoid cell types. Our study demonstrates that lymphocyte-specific epigenetic modifications pre-determine glucocorticoid resistance in ALL and may account for the lack of glucocorticoid sensitivity in other cell types.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Cromatina/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Glucocorticoides/farmacología , Linfocitos/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Apoptosis/efectos de los fármacos , Azacitidina/administración & dosificación , Azacitidina/farmacología , Cromatina/genética , Cromatina/metabolismo , Dexametasona/administración & dosificación , Dexametasona/farmacología , Resistencia a Antineoplásicos/genética , Glucocorticoides/administración & dosificación , Humanos , Linfocitos/metabolismo , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo
6.
Cancer Res ; 76(9): 2766-2777, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-26960974

RESUMEN

Activating mutations in tyrosine kinases (TKs) drive pediatric high-risk acute lymphoblastic leukemia (ALL) and confer resistance to standard chemotherapy. Therefore, there is urgent need to characterize dysregulated TK signaling axes in patients with ALL and identify actionable kinase targets for the development of therapeutic strategies. Here, we present the first study to quantitatively profile TK activity in xenografted patient biopsies of high-risk pediatric ALL. We integrated a quantitative phosphotyrosine profiling method with 'spike-in' stable isotope labeling with amino acids in cell culture (SILAC) and quantified 1394 class I phosphorylation sites in 16 ALL xenografts. Moreover, hierarchical clustering of phosphotyrosine sites could accurately classify these leukemias into either B or T-cell lineages with the high-risk early T-cell precursor (ETP) and Ph-like ALL clustering as a distinct group. Furthermore, we validated this approach by using specific kinase pathway inhibitors to perturb ABL1, FLT3, and JAK TK signaling in four xenografted patient samples. By quantitatively assessing the tyrosine phosphorylation status of activated kinases in xenograft models of ALL, we were able to identify and validate clinically relevant targets. Therefore, this study highlights the application and potential of phosphotyrosine profiling for identifying clinically relevant kinase targets in leukemia.

7.
Mol Cancer Ther ; 14(2): 364-74, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25504635

RESUMEN

Genome-wide studies have identified a high-risk subgroup of pediatric acute lymphoblastic leukemia (ALL) harboring mutations in the Janus kinases (JAK). The purpose of this study was to assess the preclinical efficacy of the JAK1/2 inhibitor AZD1480, both as a single agent and in combination with the MEK inhibitor selumetinib, against JAK-mutated patient-derived xenografts. Patient-derived xenografts were established in immunodeficient mice from bone marrow or peripheral blood biopsy specimens, and their gene expression profiles compared with the original patient biopsies by microarray analysis. JAK/STAT and MAPK signaling pathways, and the inhibitory effects of targeted drugs, were interrogated by immunoblotting of phosphoproteins. The antileukemic effects of AZD1480 and selumetinib, alone and in combination, were tested against JAK-mutated ALL xenografts both in vitro and in vivo. Xenografts accurately represented the primary disease as determined by gene expression profiling. Cellular phosphoprotein analysis demonstrated that JAK-mutated xenografts exhibited heightened activation status of JAK/STAT and MAPK signaling pathways compared with typical B-cell precursor ALL xenografts, which were inhibited by AZD1480 exposure. However, AZD1480 exhibited modest single-agent in vivo efficacy against JAK-mutated xenografts. Combining AZD1480 with selumetinib resulted in profound synergistic in vitro cell killing, although these results were not translated in vivo despite evidence of target inhibition. Despite validation of target inhibition and the demonstration of profound in vitro synergy between AZD1480 and selumetinib, it is likely that prolonged target inhibition is required to achieve in vivo therapeutic enhancement between JAK and MEK inhibitors in the treatment of JAK-mutated ALL.


Asunto(s)
Quinasas Janus/antagonistas & inhibidores , Mutación/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirazoles/farmacología , Pirazoles/uso terapéutico , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Animales , Bencimidazoles/farmacología , Biopsia , Supervivencia Celular/efectos de los fármacos , Niño , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Estudios de Asociación Genética , Humanos , Quinasas Janus/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Terapia Molecular Dirigida , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Inhibidores de Proteínas Quinasas/farmacología , Resultado del Tratamiento , Ensayos Antitumor por Modelo de Xenoinjerto
8.
Cell Rep ; 5(4): 1047-59, 2013 Nov 27.
Artículo en Inglés | MEDLINE | ID: mdl-24268771

RESUMEN

To design rational therapies for JAK2-driven hematological malignancies, we functionally dissected the key survival pathways downstream of hyperactive JAK2. In tumors driven by mutant JAK2, Stat1, Stat3, Stat5, and the Pi3k and Mek/Erk pathways were constitutively active, and gene expression profiling of TEL-JAK2 T-ALL cells revealed the upregulation of prosurvival Bcl-2 family genes. Combining the Bcl-2/Bcl-xL inhibitor ABT-737 with JAK2 inhibitors mediated prolonged disease regressions and cures in mice bearing primary human and mouse JAK2 mutant tumors. Moreover, combined targeting of JAK2 and Bcl-2/Bcl-xL was able to circumvent and overcome acquired resistance to single-agent JAK2 inhibitor treatment. Thus, inhibiting the oncogenic JAK2 signaling network at two nodal points, at the initiating stage (JAK2) and the effector stage (Bcl-2/Bcl-xL), is highly effective and provides a clearly superior therapeutic benefit than targeting just one node. Therefore, we have defined a potentially curative treatment for hematological malignancies expressing constitutively active JAK2.


Asunto(s)
Resistencia a Antineoplásicos , Janus Quinasa 2/antagonistas & inhibidores , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Proteína bcl-X/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Compuestos de Bifenilo/farmacología , Línea Celular Tumoral , Supervivencia Celular , Perfilación de la Expresión Génica , Humanos , Janus Quinasa 2/genética , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , Nitrilos , Nitrofenoles/farmacología , Piperazinas/farmacología , Proteínas Proto-Oncogénicas/genética , Pirazoles/farmacología , Pirimidinas/farmacología , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Trasplante Heterólogo , Proteína bcl-X/genética
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