RESUMO
Despite the therapeutic efficacy of ibrutinib in chronic lymphocytic leukemia (CLL), complete responses are infrequent, and acquired resistance to Bruton agammaglobulinemia tyrosine kinase (BTK) inhibition is being observed in an increasing number of patients. Combination regimens that increase frequency of complete remissions, accelerate time to remission, and overcome single agent resistance are of considerable interest. We previously showed that the XPO1 inhibitor selinexor is proapoptotic in CLL cells and disrupts B-cell receptor signaling via BTK depletion. Herein we show the combination of selinexor and ibrutinib elicits a synergistic cytotoxic effect in primary CLL cells and increases overall survival compared with ibrutinib alone in a mouse model of CLL. Selinexor is effective in cells isolated from patients with prolonged lymphocytosis following ibrutinib therapy. Finally, selinexor is effective in ibrutinib-refractory mice and in a cell line harboring the BTK C481S mutation. This is the first report describing the combined activity of ibrutinib and selinexor in CLL, which represents a new treatment paradigm and warrants further evaluation in clinical trials of CLL patients including those with acquired ibrutinib resistance.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Hidrazinas/farmacologia , Leucemia Linfocítica Crônica de Células B/patologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Triazóis/farmacologia , Adenina/análogos & derivados , Animais , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Sinergismo Farmacológico , Humanos , Hidrazinas/administração & dosagem , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/mortalidade , Camundongos , Piperidinas , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , Triazóis/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Congenital thrombotic thrombocytopenic purpura (cTTP) is a rare, recessively inherited genetic disorder with varying clinical presentation that is caused by ADAMTS13 mutations. Several studies have found limited associations between ADAMTS13 mutations and cTTP phenotype. The use of in silico tools that examine multiple mutation characteristics may better predict phenotype. We analysed 118 ADAMTS13 mutations found in 144 cTTP patients reported in the literature and examined associations of several mutation characteristics, including N-terminal proximity, the evolutionary conservation of the affected amino acid position, as well as amino acid charge/phosphorylation and genetic codon usage to disease phenotype. Structure-altering mutations were examined for their impact on ADAMTS13 function based on existing ADAMTS13 crystallographic data (AA 77-685). Our in silico data indicate that: (i) The position of the mutation in the N- or C-terminus, (ii) evolutionary conservation and (iii) codon usage of the affected mutation position are associated with disease parameters, such as age of onset, organ damage and fresh frozen plasma prophylaxis. In conclusion, the usage of multiple in silico tools presents a promising strategy in refining predictions for the diverse presentation of cTTP. Enhancing our utilization of in silico tools to find genotype-phenotype associations will create better-tailored approaches for individual patient treatment.
Assuntos
Proteínas ADAM/genética , Mutação , Púrpura Trombocitopênica Trombótica/genética , Proteínas ADAM/química , Proteínas ADAM/metabolismo , Proteína ADAMTS13 , Adolescente , Sequência de Aminoácidos , Criança , Pré-Escolar , Códon , Estudos de Coortes , Análise Mutacional de DNA/métodos , Feminino , Estudos de Associação Genética , Humanos , Lactente , Recém-Nascido , Masculino , Fosforilação , Púrpura Trombocitopênica Trombótica/sangueRESUMO
Richter's Transformation (RT) is a poorly understood and fatal progression of chronic lymphocytic leukemia (CLL) manifesting histologically as diffuse large B-cell lymphoma. Protein arginine methyltransferase 5 (PRMT5) is implicated in lymphomagenesis, but its role in CLL or RT progression is unknown. We demonstrate herein that tumors uniformly overexpress PRMT5 in patients with progression to RT. Furthermore, mice with B-specific overexpression of hPRMT5 develop a B-lymphoid expansion with increased risk of death, and Eµ-PRMT5/TCL1 double transgenic mice develop a highly aggressive disease with transformation that histologically resembles RT; where large-scale transcriptional profiling identifies oncogenic pathways mediating PRMT5-driven disease progression. Lastly, we report the development of a SAM-competitive PRMT5 inhibitor, PRT382, with exclusive selectivity and optimal in vitro and in vivo activity compared to available PRMT5 inhibitors. Taken together, the discovery that PRMT5 drives oncogenic pathways promoting RT provides a compelling rationale for clinical investigation of PRMT5 inhibitors such as PRT382 in aggressive CLL/RT cases.
Assuntos
Leucemia Linfocítica Crônica de Células B , Linfoma Difuso de Grandes Células B , Animais , Camundongos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma Difuso de Grandes Células B/patologiaRESUMO
Rare, recurrent balanced translocations occur in a variety of cancers but are often not functionally interrogated. Balanced translocations with the immunoglobulin heavy chain locus (IGH; 14q32) in chronic lymphocytic leukemia (CLL) are infrequent but have led to the discovery of pathogenic genes including CCND1, BCL2, and BCL3. Following identification of a t(X;14)(q28;q32) translocation that placed the mature T cell proliferation 1 gene (MTCP1) adjacent to the immunoglobulin locus in a CLL patient, we hypothesized that this gene may have previously unrecognized importance. Indeed, here we report overexpression of human MTCP1 restricted to the B cell compartment in mice produces a clonal CD5+/CD19+ leukemia recapitulating the major characteristics of human CLL and demonstrates favorable response to therapeutic intervention with ibrutinib. We reinforce the importance of genetic interrogation of rare, recurrent balanced translocations to identify cancer driving genes via the story of MTCP1 as a contributor to CLL pathogenesis.
Assuntos
Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Translocação Genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Proteína 3 do Linfoma de Células B , Ciclina D1 , Feminino , Regulação da Expressão Gênica , Genes de Cadeia Pesada de Imunoglobulina , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Oncogenes/genética , Proteínas Proto-Oncogênicas c-bcl-2RESUMO
BACKGROUND: Exportin 1 (XPO1/CRM1) is a key mediator of nuclear export with relevance to multiple cancers, including chronic lymphocytic leukemia (CLL). Whole exome sequencing has identified hot-spot somatic XPO1 point mutations which we found to disrupt highly conserved biophysical interactions in the NES-binding groove, conferring novel cargo-binding abilities and forcing cellular mis-localization of critical regulators. However, the pathogenic role played by change-in-function XPO1 mutations in CLL is not fully understood. METHODS: We performed a large, multi-center retrospective analysis of CLL cases (N = 1286) to correlate nonsynonymous mutations in XPO1 (predominantly E571K or E571G; n = 72) with genetic and epigenetic features contributing to the overall outcomes in these patients. We then established a mouse model with over-expression of wildtype (wt) or mutant (E571K or E571G) XPO1 restricted to the B cell compartment (Eµ-XPO1). Eµ-XPO1 mice were then crossed with the Eµ-TCL1 CLL mouse model. Lastly, we determined crystal structures of XPO1 (wt or E571K) bound to several selective inhibitors of nuclear export (SINE) molecules (KPT-185, KPT-330/Selinexor, and KPT-8602/Eltanexor). RESULTS: We report that nonsynonymous mutations in XPO1 associate with high risk genetic and epigenetic features and accelerated CLL progression. Using the newly-generated Eµ-XPO1 mouse model, we found that constitutive B-cell over-expression of wt or mutant XPO1 could affect development of a CLL-like disease in aged mice. Furthermore, concurrent B-cell expression of XPO1 with E571K or E571G mutations and TCL1 accelerated the rate of leukemogenesis relative to that of Eµ-TCL1 mice. Lastly, crystal structures of E571 or E571K-XPO1 bound to SINEs, including Selinexor, are highly similar, suggesting that the activity of this class of compounds will not be affected by XPO1 mutations at E571 in patients with CLL. CONCLUSIONS: These findings indicate that mutations in XPO1 at E571 can drive leukemogenesis by priming the pre-neoplastic lymphocytes for acquisition of additional genetic and epigenetic abnormalities that collectively result in neoplastic transformation.
Assuntos
Regulação Leucêmica da Expressão Gênica , Carioferinas/genética , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Receptores Citoplasmáticos e Nucleares/genética , Animais , Epigênese Genética , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estudos Retrospectivos , Transcriptoma , Proteína Exportina 1Assuntos
Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Haplótipos , Polimorfismo de Nucleotídeo Único , Púrpura Trombocitopênica Trombótica/genética , Púrpura Trombocitopênica Trombótica/metabolismo , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismo , Proteína ADAMTS13 , Criança , Pré-Escolar , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Púrpura Trombocitopênica Trombótica/diagnósticoRESUMO
PURPOSE: Aberrant Myc expression is a major factor in the pathogenesis of aggressive lymphoma, and these lymphomas, while clinically heterogeneous, often are resistant to currently available treatments and have poor survival. Myc expression can also be seen in aggressive lymphomas that are observed in the context of CLL, and we sought to develop a mouse model that could be used to study therapeutic strategies for aggressive lymphoma in the context of CLL. EXPERIMENTAL DESIGN: We crossed the Eµ-TCL1 mouse model with the Eµ-Myc mouse model to investigate the clinical phenotype associated with B-cell-restricted expression of these oncogenes. The resulting malignancy was then extensively characterized, from both a clinical and biologic perspective. RESULTS: Eµ-TCL1xMyc mice uniformly developed highly aggressive lymphoid disease with histologically, immunophenotypically, and molecularly distinct concurrent CLL and B-cell lymphoma, leading to a significantly reduced lifespan. Injection of cells from diseased Eµ-TCL1xMyc into WT mice established a disease similar to that in the double-transgenic mice. Both Eµ-TCL1xMyc mice and mice with disease after adoptive transfer failed to respond to ibrutinib. Effective and durable disease control was, however, observed by selective inhibition of nuclear export protein exportin-1 (XPO1) using a compound currently in clinical development for relapsed/refractory malignancies, including CLL and lymphoma. CONCLUSIONS: The Eµ-TCL1xMyc mouse is a new preclinical tool for testing experimental drugs for aggressive B-cell lymphoma, including in the context of CLL.
Assuntos
Leucemia Linfocítica Crônica de Células B/genética , Linfoma de Células B/genética , Neoplasias Primárias Múltiplas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas/genética , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Humanos , Carioferinas/antagonistas & inibidores , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/patologia , Masculino , Camundongos , Camundongos Transgênicos , Neoplasias Primárias Múltiplas/patologia , Estudo de Prova de Conceito , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Células Tumorais Cultivadas/transplante , Proteína Exportina 1RESUMO
Bromodomain and extra-terminal (BET) family proteins are key regulators of gene expression in cancer. Herein, we utilize BRD4 profiling to identify critical pathways involved in pathogenesis of chronic lymphocytic leukemia (CLL). BRD4 is overexpressed in CLL and is enriched proximal to genes upregulated or de novo expressed in CLL with known functions in disease pathogenesis and progression. These genes, including key members of the B-cell receptor (BCR) signaling pathway, provide a rationale for this therapeutic approach to identify new targets in alternative types of cancer. Additionally, we describe PLX51107, a structurally distinct BET inhibitor with novel in vitro and in vivo pharmacologic properties that emulates or exceeds the efficacy of BCR signaling agents in preclinical models of CLL. Herein, the discovery of the involvement of BRD4 in the core CLL transcriptional program provides a compelling rationale for clinical investigation of PLX51107 as epigenetic therapy in CLL and application of BRD4 profiling in other cancers.Significance: To date, functional studies of BRD4 in CLL are lacking. Through integrated genomic, functional, and pharmacologic analyses, we uncover the existence of BRD4-regulated core CLL transcriptional programs and present preclinical proof-of-concept studies validating BET inhibition as an epigenetic approach to target BCR signaling in CLL. Cancer Discov; 8(4); 458-77. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 371.
Assuntos
Regulação Leucêmica da Expressão Gênica , Isoxazóis/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Proteínas Nucleares/genética , Piridinas/uso terapêutico , Pirróis/uso terapêutico , Transdução de Sinais , Fatores de Transcrição/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Perfilação da Expressão Gênica , Humanos , Isoxazóis/farmacologia , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/fisiopatologia , Camundongos , Camundongos SCID , Proteínas Nucleares/metabolismo , Piridinas/farmacologia , Pirróis/farmacologia , Fatores de Transcrição/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Synonymous variations, which are defined as codon substitutions that do not change the encoded amino acid, were previously thought to have no effect on the properties of the synthesized protein(s). However, mounting evidence shows that these "silent" variations can have a significant impact on protein expression and function and should no longer be considered "silent". Here, the effects of six synonymous and six non-synonymous variations, previously found in the gene of ADAMTS13, the von Willebrand Factor (VWF) cleaving hemostatic protease, have been investigated using a variety of approaches. The ADAMTS13 mRNA and protein expression levels, as well as the conformation and activity of the variants have been compared to that of wild-type ADAMTS13. Interestingly, not only the non-synonymous variants but also the synonymous variants have been found to change the protein expression levels, conformation and function. Bioinformatic analysis of ADAMTS13 mRNA structure, amino acid conservation and codon usage allowed us to establish correlations between mRNA stability, RSCU, and intracellular protein expression. This study demonstrates that variants and more specifically, synonymous variants can have a substantial and definite effect on ADAMTS13 function and that bioinformatic analysis may allow development of predictive tools to identify variants that will have significant effects on the encoded protein.