RESUMO
BACKGROUND: Qualitative and quantitative defects in natural killer (NK) cells have been noted in patients with acute myeloid leukemia (AML), providing rationale for infusion of donor-derived NK cells. We previously showed that decitabine enhances expression of NKG2D ligands in AML with additive cytotoxicity when NK cells and Fc (fragment crystallizable region)-engineered CD33 monoclonal antibody (CD33mAb) was used. We conducted a phase 1 study evaluating decitabine and haploidentical NK cells in relapsed AML. Using patient samples from this study, we evaluated whether ex vivo donor-derived expanded NK cells with or without CD33mAb was effective in decitabine-treated AML. METHODS: Bone marrow aspirates were collected from patients at pre- and post-NK cell infusion. NK cells from healthy donors were expanded for 14 days using irradiated K562 feeder cells displaying membrane-bound IL-21 (mbIL-21). Patient samples were used to test in vitro activity of mbIL-21 NK cells ± CD33m Ab-dependent cellular cytotoxicity (ADCC) and AML patient derived xenograft (PDX) mice were developed to test in vivo activity. RESULTS: Upon incubation with primary AML blasts, mbIL-21 NK cells showed variable donor-dependent intra-cellular interferon-γ production, which increased with CD33mAb-coated AML. ADCC assays revealed mbIL-21 NK cells effectively lysed primary AML blasts with higher activity on CD33mAb-coated AML. Importantly, CD33mAb-dependent enhanced cytotoxicity by mbIL-21 NK cells was maintained in AML cells from patients even 24 days post-decitabine treatment. In vivo infusion of mbIL-21 NK cells in AML PDX mice, treated with CD33mAb, reduced the tumor burden. DISCUSSION: These data show the therapeutic utility of mbIL-21 NK cells that can be further potentiated by addition of CD33mAb in AML.
Assuntos
Anticorpos Monoclonais/farmacologia , Membrana Celular/metabolismo , Citotoxicidade Imunológica/efeitos dos fármacos , Fragmentos Fc das Imunoglobulinas/metabolismo , Interleucinas/metabolismo , Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/imunologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Idoso , Animais , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Feminino , Humanos , Interleucina-2/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/transplante , Leucemia Mieloide Aguda/terapia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Pessoa de Meia-Idade , Ligação Proteica/efeitos dos fármacos , Engenharia de Proteínas , Vidarabina/análogos & derivados , Vidarabina/farmacologiaRESUMO
Diseases with clonal hematopoiesis such as myelodysplastic syndrome and acute myeloid leukemia have high rates of relapse. Only a small subset of acute myeloid leukemia patients are cured with chemotherapy alone. Relapse in these diseases occurs at least in part due to the failure to eradicate leukemic stem cells or hematopoietic stem cells in myelodysplastic syndrome. CD123, the alpha chain of the interleukin-3 receptor heterodimer, is expressed on the majority of leukemic stem cells and myelodysplastic syndrome hematopoietic stem cells and in 80% of acute myeloid leukemia. Here, we report indiscriminate killing of CD123+ normal and acute myeloid leukemia / myelodysplastic syndrome cells by SL-401, a diphtheria toxin interleukin-3 fusion protein. SL-401 induced cytotoxicity of CD123+ primary cells/blasts from acute myeloid leukemia and myelodysplastic syndrome patients but not CD123- lymphoid cells. Importantly, SL-401 was highly active even in cells expressing low levels of CD123, with minimal effect on modulation of the CD123 target in acute myeloid leukemia. SL-401 significantly prolonged survival of leukemic mice in acute myeloid leukemia patient-derived xenograft mouse models. In addition to primary samples, studies on normal cord blood and healthy marrow show that SL-401 has activity against normal hematopoietic progenitors. These findings indicate potential use of SL-401 as a "bridge-to-transplant" before allogeneic hematopoietic cell transplantation in acute myeloid leukemia / myelodysplastic syndrome patients.
Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Subunidade alfa de Receptor de Interleucina-3/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Síndromes Mielodisplásicas/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Animais , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Subunidade alfa de Receptor de Interleucina-3/análise , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Proteínas Recombinantes de Fusão/uso terapêutico , Células Tumorais CultivadasRESUMO
B-cell receptor (BCR) antagonists such as the BTK inhibitor ibrutinib have proven to effectively target chronic lymphocytic leukemia (CLL) tumor cells, leading to impressive response rates in these patients. However patients do still relapse on ibrutinib, and the progressive disease is often quite aggressive requiring immediate treatment. Several strategies are being pursued to treat patients who relapse on ibrutinib therapy. As the most common form of relapse is the development of a mutant form of BTK which limits ibrutinib binding, agents which lead to degradation of the BTK protein are a promising strategy. Our study explores the efficacy of the Hsp90 inhibitor, SNX-5422, in CLL. The SNX Hsp90 inhibitor was effective in primary CLL cells, as well as B-cell lines expressing either BTK wild type or C481 mutant BTK, which has been identified as the primary resistance mechanism to ibrutinib in CLL patients. Furthermore the combination of SNX-5422 and ibrutinib provided a remarkable in vivo survival benefit in the Eµ-TCL1 mouse model of CLL compared to the vehicle or single agent groups (51 day median survival in the vehicle and ibrutinib groups versus 100 day median survival in the combination). We report here preclinical data suggesting that the Hsp90 inhibitor SNX-5422, which has been pursued in clinical trials in both solid tumor and hematological malignancies, is a potential therapy for ibrutinib resistant CLL.
Assuntos
Adenina/análogos & derivados , Antineoplásicos/uso terapêutico , Benzamidas/uso terapêutico , Glicina/uso terapêutico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Indazóis/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Piperidinas/uso terapêutico , Adenina/farmacologia , Adenina/uso terapêutico , Animais , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Glicina/farmacologia , Humanos , Indazóis/farmacologia , Leucemia Linfocítica Crônica de Células B/patologia , Camundongos , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
Acute myeloid leukemia (AML) is a hematopoietic stem-cell-derived leukemia with often successive derived driver mutations. Late onset acquisition of internal tandem duplication in FLT3 (FLT3-ITD) at a high variant allele frequency often contributes to full transformation to a highly proliferative, rapidly progressive disease with poor outcome. The FLT3-ITD mutation is targetable with approved FLT3 small molecule inhibitors, including midostaurin and gilteritinib. However, outside of patients receiving allogeneic transplant, most patients fail to respond or relapse, suggesting alternative approaches of therapy will be required. We employed genome-wide pooled CRISPR knockout screening as a method for large-scale identification of targets whose knockout produces a phenotypic effect that enhances the antitumor properties of FLT3 inhibitors. Among the candidate targets we identified the effect of XPO1 knockout to be synergistic with midostaurin treatment. Next, we validated the genetic finding with pharmacologic combination of the slowly reversible XPO1 inhibitor selinexor with midostaurin and gilteritinib in FLT3-ITD AML cell lines and primary patient samples. Lastly, we demonstrated improved survival with either combination therapy compared to its monotherapy components in an aggressive AML murine model, supporting further evaluation and rapid clinical translation of this combination strategy.
RESUMO
Acute myeloid leukemia (AML) is a heterogeneous and complex disease, and treatments for this disease have not been curative for the majority of patients. In younger patients, internal tandem duplication of FLT3 (FLT3-ITD) is a common mutation for which two inhibitors (midostaurin and gilteritinib) with varied potency and specificity for FLT3 are clinically approved. However, the high rate of relapse or failed initial response of AML patients suggests that the addition of a second targeted therapy may be necessary to improve efficacy. Using an unbiased large-scale CRISPR screen, we genetically identified BCL2 knockout as having synergistic effects with an approved FLT3 inhibitor. Here, we provide supportive studies that validate the therapeutic potential of the combination of FLT3 inhibitors with venetoclax in vitro and in vivo against multiple models of FLT3-ITD-driven AML. Our unbiased approach provides genetic validation for co-targeting FLT3 and BCL2 and repurposes CRISPR screening data, utilizing the genome-wide scope toward mechanistic understanding.
Assuntos
Leucemia Mieloide Aguda/terapia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/genética , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Compostos de Anilina/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Feminino , Técnicas de Inativação de Genes , Terapia Genética , Humanos , Leucemia Mieloide Aguda/genética , Camundongos SCID , Pirazinas/uso terapêutico , Estaurosporina/análogos & derivados , Estaurosporina/uso terapêutico , Sulfonamidas/uso terapêuticoRESUMO
BACKGROUND: Acute myeloid leukemia (AML) is the most common type of adult leukemia. Several studies have demonstrated that oncogenesis in AML is enhanced by kinase signaling pathways such as Src family kinases (SFK) including Src and Lyn, spleen tyrosine kinase (SYK), and bruton's tyrosine kinase (BTK). Recently, the multi-kinase inhibitor ArQule 531 (ARQ 531) has demonstrated potent inhibition of SFK and BTK that translated to improved pre-clinical in vivo activity as compared with the irreversible BTK inhibitor ibrutinib in chronic lymphocytic leukemia (CLL) models. Given the superior activity of ARQ 531 in CLL, and recognition that this molecule has a broad kinase inhibition profile, we pursued its application in pre-clinical models of AML. METHODS: The potency of ARQ 531 was examined in vitro using FLT3 wild type and mutated (ITD) AML cell lines and primary samples. The modulation of pro-survival kinases following ARQ 531 treatment was determined using AML cell lines. The effect of SYK expression on ARQ 531 potency was evaluated using a SYK overexpressing cell line (Ba/F3 murine cells) constitutively expressing FLT3-ITD. Finally, the in vivo activity of ARQ 531 was evaluated using MOLM-13 disseminated xenograft model. RESULTS: Our data demonstrate that ARQ 531 treatment has anti-proliferative activity in vitro and impairs colony formation in AML cell lines and primary AML cells independent of the presence of a FLT3 ITD mutation. We demonstrate decreased phosphorylation of oncogenic kinases targeted by ARQ 531, including SFK (Tyr416), BTK, and fms-related tyrosine kinase 3 (FLT3), ultimately leading to changes in down-stream targets including SYK, STAT5a, and ERK1/2. Based upon in vitro drug synergy data, we examined ARQ 531 in the MOLM-13 AML xenograft model alone and in combination with venetoclax. Despite ARQ 531 having a less favorable pharmacokinetics profile in rodents, we demonstrate modest single agent in vivo activity and synergy with venetoclax. CONCLUSIONS: Our data support consideration of the application of ARQ 531 in combination trials for AML targeting higher drug concentrations in vivo.
Assuntos
Antineoplásicos/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/metabolismoRESUMO
Prostate apoptosis response 4 (Par-4) is a tumor suppressor that prevents proliferation and induces cell death in several solid tumors. However, its role in B-cell malignancies has not been elucidated. To describe the role of Par-4 in chronic lymphocytic leukemia (CLL) pathogenesis, we developed a B-cell-specific human Par-4-overexpressing mouse model of CLL using the TCL1 leukemia model. While Par-4 transgenic mice did not display any obvious defects in B-cell development or function, disease burden as evidenced by abundance of CD19+CD5+ B cells in the peripheral blood was significantly reduced in Par-4 × TCL1 mice compared with TCL1 littermates. This conferred a survival advantage on the Par-4-overexpressing mice. In addition, a B-cell-specific knockout model displayed the opposite effect, where lack of Par-4 expression resulted in accelerated disease progression and abbreviated survival in the TCL1 model. Histological and flow cytometry-based analysis of spleen and bone marrow upon euthanasia revealed comparable levels of malignant B-cell infiltration in Par-4 × TCL1 and TCL1 individuals, indicating delayed but pathologically normal disease progression in Par-4 × TCL1 mice. In vivo analysis of splenic B-cell proliferation by 5-ethynyl-2-deoxyuridine incorporation indicated >50% decreased expansion of CD19+CD5+ cells in Par-4 × TCL1 mice compared with TCL1 littermates. Moreover, reduced nuclear p65 levels were observed in Par-4 × TCL1 splenic B cells compared with TCL1, suggesting suppressed NF-κB signaling. These findings have identified an in vivo antileukemic role for Par-4 through an NF-κB-dependent mechanism in TCL1-mediated CLL-like disease progression.
Assuntos
Proteínas Reguladoras de Apoptose/biossíntese , Carcinogênese/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Neoplasias Experimentais/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Animais , Carcinogênese/genética , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Targeted therapy with small molecules directed at essential survival pathways in leukemia represents a major advance, including the phosphatidylinositol-3'-kinase (PI3K) p110δ inhibitor idelalisib. Here, we found that genetic inactivation of p110δ (p110δD910A/D910A) in the Eµ-TCL1 murine chronic lymphocytic leukemia (CLL) model impaired B cell receptor signaling and B cell migration, and significantly delayed leukemia pathogenesis. Regardless of TCL1 expression, p110δ inactivation led to rectal prolapse in mice resembling autoimmune colitis in patients receiving idelalisib. Moreover, we showed that p110δ inactivation in the microenvironment protected against CLL and acute myeloid leukemia. After receiving higher numbers of TCL1 leukemia cells, half of p110δD910A/D910A mice spontaneously recovered from high disease burden and resisted leukemia rechallenge. Despite disease resistance, p110δD910A/D910A mice exhibited compromised CD4+ and CD8+ T cell response, and depletion of CD4+ or CD8+ T cells restored leukemia. Interestingly, p110δD910A/D910A mice showed significantly impaired Treg expansion that associated with disease clearance. Reconstitution of p110δD910A/D910A mice with p110δWT/WT Tregs reversed leukemia resistance. Our findings suggest that p110δ inhibitors may have direct antileukemic and indirect immune-activating effects, further supporting that p110δ blockade may have a broader immune-modulatory role in types of leukemia that are not sensitive to p110δ inhibition.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Classe I de Fosfatidilinositol 3-Quinases/imunologia , Tolerância Imunológica , Leucemia Linfoide/imunologia , Mutação de Sentido Incorreto , Neoplasias Experimentais/imunologia , Linfócitos T Reguladores/imunologia , Substituição de Aminoácidos , Animais , Linfócitos T CD8-Positivos/patologia , Classe I de Fosfatidilinositol 3-Quinases/genética , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Leucemia Linfoide/genética , Leucemia Linfoide/patologia , Leucemia Linfoide/terapia , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Linfócitos T Reguladores/patologiaRESUMO
The Bruton tyrosine kinase inhibitor (BTKi) ibrutinib has transformed chronic lymphocytic leukemia (CLL) therapy but requires continuous administration. These factors have spurred interest in combination treatments. Unlike with chemotherapy, CD20-directed antibody therapy has not improved the outcome of BTKi treatment. Whereas CD20 antigen density on CLL cells decreases during ibrutinib treatment, the B-cell activating factor (BAFF) and its receptor (BAFF-R) remain elevated. Furthermore, BAFF signaling via noncanonical NF-κB remains elevated with BTKi treatment. Blocking BAFF interaction with BAFF-R by using VAY-736, a humanized defucosylated engineered antibody directed against BAFF-R, antagonized BAFF-mediated apoptosis protection and signaling at the population and single-cell levels in CLL cells. Furthermore, VAY-736 showed superior antibody-dependent cellular cytotoxicity compared with CD20- and CD52-directed antibodies used in CLL. VAY-736 exhibited in vivo activity as a monotherapy and, when combined with ibrutinib, produced prolonged survival compared with either therapy alone. The in vivo activity of VAY-736 is dependent upon immunoreceptor tyrosine-based activation motif (ITAM)-mediated activation of effector cells as shown by using an ITAM-deficient mouse model. Collectively, our findings support targeting the BAFF signaling pathway with VAY-736 to more effectively treat CLL as a single agent and in combination with ibrutinib.
Assuntos
Anticorpos Monoclonais Humanizados/metabolismo , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Adenina/análogos & derivados , Animais , Humanos , Camundongos , Piperidinas , Pirazóis/farmacologia , Pirimidinas/farmacologiaRESUMO
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.