RESUMO
BACKGROUND: Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein present on many cancers. Zilovertamab vedotin (ZV) is an antibodydrug conjugate comprising a monoclonal antibody recognizing extracellular ROR1, a cleavable linker, and the anti-microtubule cytotoxin monomethyl auristatin E. METHODS: In this phase 1, first-in-human, dose-escalation study, we accrued patients with previously treated lymphoid cancers to receive ZV every 3 weeks until the occurrence of cancer progression or unacceptable toxicity had occurred. RESULTS: We enrolled 32 patients with tumor histologies of mantle cell lymphoma (MCL) (n=15), chronic lymphocytic leukemia (n=7), diffuse large B-cell lymphoma (DLBCL) (n=5), follicular lymphoma (n=3), Richter transformation lymphoma (n=1), or marginal zone lymphoma (n=1). Patients had received a median of four previous drug and/or cellular therapies. Starting dose levels were 0.5 (n=1), 1.0 (n=3), 1.5 (n=3), 2.25 (n=11), and 2.5 (n=14) mg per kg of body weight (mg/kg). Pharmacokinetic and pharmacodynamic data documented systemic ZV exposure and exposure-dependent ZV targeting of ROR1 on circulating tumor cells. As expected with an monomethyl auristatin E-containing antibodydrug conjugate, adverse events (AEs) included acute neutropenia and cumulative neuropathy resulting in a recommended ZV dosing regimen of 2.5 mg/kg every 3 weeks. No clinically concerning AEs occurred to suggest ROR1-mediated toxicities or nonspecific ZV binding to normal tissues. ZV induced objective tumor responses in 7 of 15 patients with MCL (47%; 4 partial and 3 complete) and in 3 of 5 patients with DLBCL (60%; 1 partial and 2 complete); objective tumor responses were not observed among patients with other tumor types. CONCLUSIONS: In heavily pretreated patients, ZV demonstrated no unexpected toxicities and showed evidence of antitumor activity, providing clinical proof of concept for selective targeting of ROR1 as a potential new approach to cancer therapy. (ClinicalTrials.gov number, NCT03833180.)
Assuntos
Linfoma de Célula do Manto , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase , Humanos , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Linfoma de Célula do Manto/tratamento farmacológico , Imunoconjugados/uso terapêutico , Imunoconjugados/farmacologia , Linfoma Difuso de Grandes Células B/tratamento farmacológicoRESUMO
Mantle cell lymphoma (MCL) is a rare, aggressive and incurable subtype of non-Hodgkin's B-cell lymphoma. The principal barrier is frequent clinical relapse to multiple lines of therapies, including new FDA-approved biologics and cell therapy. Brexucabtagene autoleucel, the first and only FDA approved chimeric antigen receptor (CAR) T product in MCL, demonstrated unprecedented efficacy in overcoming resistance to Bruton's tyrosine kinase inhibitors. However, relapses have inevitably occurred and once relapsed these patients display a very poor clinical outcome. Currently, there is no optional therapy specifically designed for these patients. The development of tailored and more efficacious therapies is therefore critical and represents a new medical need. We found that while the receptor tyrosine kinase-like orphan receptor 1 (ROR1) is expressed across most of the MCL cells, it is significantly elevated in CAR T-relapsed MCL tumors. To see whether this aberrant ROR1 expression contributed to CAR T resistance, we targeted ROR1 using VLS-101, a monomethyl auristatin E conjugated anti-ROR1 antibody. VLS-101 showed potent anti-MCL activity in vitro in ROR1-expressing MCL cell lines and ex vivo in primary patient samples. Importantly, VLS-101 safely induced tumor regression in PDX models resistant to CAR T-cell therapy, ibrutinib and/or venetoclax. These data advocate for targeting ROR1 as a viable approach in the treatment of ROR1-positive MCL tumors, especially those with failure to prior therapies. These data also provide strong evidence for future enrollment of post-CD19 CAR T-cell relapsed MCL patients in a first in-human phase 1b VLS-101 trial. The upcoming testing in a clinical setting will provide important insights on this new therapeutic development aiming to overcome the CAR T resistance via targeting ROR1, which is a rising unmet clinical need in MCL.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Imunoconjugados/uso terapêutico , Linfoma de Célula do Manto/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/antagonistas & inibidores , Animais , Antineoplásicos Imunológicos/imunologia , Humanos , Imunoconjugados/imunologia , Imunoterapia Adotiva , Linfoma de Célula do Manto/imunologia , Linfoma de Célula do Manto/terapia , Camundongos , Recidiva Local de Neoplasia/imunologia , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/imunologia , Células Tumorais CultivadasRESUMO
Richter syndrome (RS) represents the transformation of chronic lymphocytic leukemia (CLL), typically to an aggressive lymphoma. Treatment options for RS are limited and the disease is often fatal. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is expressed on CLL cells and other cancers but not on healthy adult tissues, making it an attractive, tumor-specific therapeutic target. VLS-101 is being developed as an antibody-drug conjugate (ADC) for therapy of ROR1-expressing (ROR1+) cancers. VLS-101 comprises UC-961 (a humanized immunoglobulin G1 monoclonal antibody that binds an extracellular epitope of human ROR1), a maleimidocaproyl-valine-citrulline-para-aminobenzoate linker, and the antimicrotubule cytotoxin monomethyl auristatin E (MMAE). VLS-101 binding to ROR1 results in rapid cellular internalization and delivery of MMAE to induce tumor cell death. We studied 4 RS patient-derived xenografts (RS-PDXs) with varying levels of ROR1 expression (11%, 32%, 85%, and 99% of cells). VLS-101 showed no efficacy in the lowest-expressing RS-PDX but induced complete remissions in those with higher levels of ROR1 expression. Responses were maintained during the posttherapy period, particularly after higher VLS-101 doses. In systemic ROR1+ RS-PDXs, VLS-101 dramatically decreased tumor burden in all RS-colonized tissues and significantly prolonged survival. Animals showed no adverse effects or weight loss. Our results confirm ROR1 as a target in RS and demonstrate the therapeutic potential of using an ADC directed toward ROR1 for the treatment of hematological cancers. A phase 1 clinical trial of VLS-101 (NCT03833180) is ongoing in patients with RS and other hematological malignancies.
Assuntos
Aminobenzoatos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Sistemas de Liberação de Medicamentos , Imunoconjugados/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Oligopeptídeos/farmacologia , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/antagonistas & inibidores , Aminobenzoatos/química , Animais , Antineoplásicos Imunológicos/química , Humanos , Imunoconjugados/química , Leucemia Linfocítica Crônica de Células B/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Proteínas de Neoplasias/metabolismo , Oligopeptídeos/química , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PI3K/AKT and NOTCH1 signaling pathways are frequently dysregulated in T-cell acute lymphoblastic leukemias (T-ALL). Although we have shown that the combined activities of the class I PI3K isoforms p110γ and p110δ play a major role in the development and progression of PTEN-null T-ALL, it has yet to be determined whether their contribution to leukemogenic programing is unique from that associated with NOTCH1 activation. Using an Lmo2-driven mouse model of T-ALL in which both the PI3K/AKT and NOTCH1 pathways are aberrantly upregulated, we now demonstrate that the combined activities of PI3Kγ/δ have both overlapping and distinct roles from NOTCH1 in generating T-ALL disease signature and in promoting tumor cell growth. Treatment of diseased animals with either a dual PI3Kγ/δ or a γ-secretase inhibitor reduced tumor burden, prolonged survival, and induced proapoptotic pathways. Consistent with their similar biological effects, both inhibitors downregulated genes involved in cMYC-dependent metabolism in gene set enrichment analyses. Furthermore, overexpression of cMYC in mice or T-ALL cell lines conferred resistance to both inhibitors, suggesting a point of pathway convergence. Of note, interrogation of transcriptional regulators and analysis of mitochondrial function showed that PI3Kγ/δ activity played a greater role in supporting the disease signature and critical bioenergetic pathways. Results provide insight into the interrelationship between T-ALL oncogenic networks and the therapeutic efficacy of dual PI3Kγ/δ inhibition in the context of NOTCH1 and cMYC signaling. Mol Cancer Ther; 16(10); 2069-82. ©2017 AACR.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/genética , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptor Notch1/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Regulação Leucêmica da Expressão Gênica/genética , Humanos , Camundongos , Mutação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de SinaisRESUMO
Purpose: Targeting the B-cell receptor (BCR) pathway with inhibitors of Bruton tyrosine kinase (BTK) and PI3Kδ is highly effective for the treatment of chronic lymphocytic leukemia (CLL). However, deep remissions are uncommon, and drug resistance with single-agent therapy can occur. In vitro studies support the effectiveness of combing PI3Kδ and BTK inhibitors.Experimental Design: As CLL proliferation and survival depends on the microenvironment, we used murine models to assess the efficacy of the BTK inhibitor acalabrutinib combined with the PI3Kδ inhibitor ACP-319 in vivo We compared single-agent with combination therapy in TCL1-192 cell-injected mice, a model of aggressive CLL.Results: We found significantly larger reductions in tumor burden in the peripheral blood and spleen of combination-treated mice. Although single-agent therapy improved survival compared with control mice by a few days, combination therapy extended survival by over 2 weeks compared with either single agent. The combination reduced tumor proliferation, NF-κB signaling, and expression of BCL-xL and MCL-1 more potently than single-agent therapy.Conclusions: The combination of acalabrutinib and ACP-319 was superior to single-agent treatment in a murine CLL model, warranting further investigation of this combination in clinical studies. Clin Cancer Res; 23(19); 5814-23. ©2017 AACR.
Assuntos
Adenosina/farmacologia , Benzamidas/administração & dosagem , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Proteínas Tirosina Quinases/genética , Pirazinas/administração & dosagem , Quinolinas/farmacologia , Tirosina Quinase da Agamaglobulinemia , Animais , Apoptose/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Camundongos , Proteínas de Neoplasias/genética , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Tirosina Quinases/antagonistas & inibidores , Microambiente Tumoral/efeitos dos fármacosRESUMO
Purpose: Acalabrutinib (ACP-196) is a novel, potent, and highly selective Bruton tyrosine kinase (BTK) inhibitor, which binds covalently to Cys481 in the ATP-binding pocket of BTK. We sought to evaluate the antitumor effects of acalabrutinib treatment in two established mouse models of chronic lymphocytic leukemia (CLL).Experimental Design: Two distinct mouse models were used, the TCL1 adoptive transfer model where leukemic cells from Eµ-TCL1 transgenic mice are transplanted into C57BL/6 mice, and the human NSG primary CLL xenograft model. Mice received either vehicle or acalabrutinib formulated into the drinking water.Results: Utilizing biochemical assays, we demonstrate that acalabrutinib is a highly selective BTK inhibitor as compared with ibrutinib. In the human CLL NSG xenograft model, treatment with acalabrutinib demonstrated on-target effects, including decreased phosphorylation of PLCγ2, ERK, and significant inhibition of CLL cell proliferation. Furthermore, tumor burden in the spleen of the mice treated with acalabrutinib was significantly decreased compared with vehicle-treated mice. Similarly, in the TCL1 adoptive transfer model, decreased phosphorylation of BTK, PLCγ2, and S6 was observed. Most notably, treatment with acalabrutinib resulted in a significant increase in survival compared with mice receiving vehicle.Conclusions: Treatment with acalabrutinib potently inhibits BTK in vivo, leading to on-target decreases in the activation of key signaling molecules (including BTK, PLCγ2, S6, and ERK). In two complementary mouse models of CLL, acalabrutinib significantly reduced tumor burden and increased survival compared with vehicle treatment. Overall, acalabrutinib showed increased BTK selectivity compared with ibrutinib while demonstrating significant antitumor efficacy in vivo on par with ibrutinib. Clin Cancer Res; 23(11); 2831-41. ©2016 AACR.
Assuntos
Benzamidas/administração & dosagem , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Pirazinas/administração & dosagem , Adenina/análogos & derivados , Transferência Adotiva/métodos , Tirosina Quinase da Agamaglobulinemia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Camundongos , Camundongos Transgênicos , Piperidinas , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Tirosina Quinases/genética , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Irreversible inhibition of Bruton's tyrosine kinase (BTK) by ibrutinib represents an important therapeutic advance for the treatment of chronic lymphocytic leukemia (CLL). However, ibrutinib also irreversibly inhibits alternative kinase targets, which potentially compromises its therapeutic index. Acalabrutinib (ACP-196) is a more selective, irreversible BTK inhibitor that is specifically designed to improve on the safety and efficacy of first-generation BTK inhibitors. METHODS: In this uncontrolled, phase 1-2, multicenter study, we administered oral acalabrutinib to 61 patients who had relapsed CLL to assess the safety, efficacy, pharmacokinetics, and pharmacodynamics of acalabrutinib. Patients were treated with acalabrutinib at a dose of 100 to 400 mg once daily in the dose-escalation (phase 1) portion of the study and 100 mg twice daily in the expansion (phase 2) portion. RESULTS: The median age of the patients was 62 years, and patients had received a median of three previous therapies for CLL; 31% had chromosome 17p13.1 deletion, and 75% had unmutated immunoglobulin heavy-chain variable genes. No dose-limiting toxic effects occurred during the dose-escalation portion of the study. The most common adverse events observed were headache (in 43% of the patients), diarrhea (in 39%), and increased weight (in 26%). Most adverse events were of grade 1 or 2. At a median follow-up of 14.3 months, the overall response rate was 95%, including 85% with a partial response and 10% with a partial response with lymphocytosis; the remaining 5% of patients had stable disease. Among patients with chromosome 17p13.1 deletion, the overall response rate was 100%. No cases of Richter's transformation (CLL that has evolved into large-cell lymphoma) and only one case of CLL progression have occurred. CONCLUSIONS: In this study, the selective BTK inhibitor acalabrutinib had promising safety and efficacy profiles in patients with relapsed CLL, including those with chromosome 17p13.1 deletion. (Funded by the Acerta Pharma and others; ClinicalTrials.gov number, NCT02029443.).
Assuntos
Antineoplásicos/administração & dosagem , Benzamidas/administração & dosagem , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazinas/administração & dosagem , Administração Oral , Tirosina Quinase da Agamaglobulinemia , Idoso , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Benzamidas/efeitos adversos , Benzamidas/farmacocinética , Deleção Cromossômica , Diarreia/induzido quimicamente , Intervalo Livre de Doença , Relação Dose-Resposta a Droga , Feminino , Cefaleia/induzido quimicamente , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Masculino , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/farmacocinética , Pirazinas/efeitos adversos , Pirazinas/farmacocinética , RecidivaRESUMO
Agents that target B-cell receptor (BCR) signaling in lymphoid malignancies including idelalisib (GS-1101) and fostamatinib which inhibit the delta isoform of PI3 kinase (PI3Kd) and spleen tyrosine kinase (Syk) respectively have shown significant clinical activity. By disrupting B-cell signaling pathways, idelalisib treatment has been associated with a dramatic lymph node response, but eradication of disease and relapse in high risk disease remain challenges. Targeting the BCR signaling pathway with simultaneous inhibition of PI3Kd and Syk has not yet been reported. We evaluated the pre-clinical activity of idelalisib combined with the novel and selective Syk inhibitor GS-9973 in primary peripheral blood and bone marrow Chronic Lymphocytic Leukemia (CLL) samples. Both PI3Kd and Syk inhibition reduced CLL survival and in combination induced synergistic growth inhibition and further disrupted chemokine signaling at nanomolar concentrations including in bone marrow derived and poor risk samples. Simultaneous targeting of these kinases may significantly increase clinical activity.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Indazóis/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Purinas/farmacologia , Pirazinas/farmacologia , Quinazolinonas/farmacologia , Apoptose/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Indazóis/administração & dosagem , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Linfocítica Crônica de Células B/enzimologia , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Purinas/administração & dosagem , Pirazinas/administração & dosagem , Quinazolinonas/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Quinase SykRESUMO
In a phase 1 trial, idelalisib (GS-1101, CAL-101), a selective inhibitor of the lipid kinase PI3Kδ, was evaluated in 54 patients with relapsed/refractory chronic lymphocytic leukemia (CLL) with adverse characteristics including bulky lymphadenopathy (80%), extensive prior therapy (median 5 [range 2-14] prior regimens), treatment-refractory disease (70%), unmutated IGHV (91%), and del17p and/or TP53 mutations (24%). Patients were treated at 6 dose levels of oral idelalisib (range 50-350 mg once or twice daily) and remained on continuous therapy while deriving clinical benefit. Idelalisib-mediated inhibition of PI3Kδ led to abrogation of Akt phosphorylation in patient CLL cells and significantly reduced serum levels of CLL-related chemokines. The most commonly observed grade ≥3 adverse events were pneumonia (20%), neutropenic fever (11%), and diarrhea (6%). Idelalisib treatment resulted in nodal responses in 81% of patients. The overall response rate was 72%, with 39% of patients meeting the criteria for partial response per IWCLL 2008 and 33% meeting the recently updated criteria of PR with treatment-induced lymphocytosis.(1,2) The median progression-free survival for all patients was 15.8 months. This study demonstrates the clinical utility of inhibiting the PI3Kδ pathway with idelalisib. Our findings support the further development of idelalisib in patients with CLL. These trials were registered at clinicaltrials.gov as #NCT00710528 and #NCT01090414.
Assuntos
Antineoplásicos/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Purinas/uso terapêutico , Quinazolinonas/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/mortalidade , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Purinas/administração & dosagem , Purinas/efeitos adversos , Purinas/farmacocinética , Quinazolinonas/administração & dosagem , Quinazolinonas/efeitos adversos , Quinazolinonas/farmacocinética , Recidiva , Resultado do TratamentoRESUMO
Although hyperactivation of the Ras-Erk signaling pathway is known to underlie the pathogenesis of juvenile myelomonocytic leukemia (JMML), a fatal childhood disease, the PI3K-Akt signaling pathway is also dysregulated in this disease. Using genetic models, we demonstrate that inactivation of phosphatidylinositol-3-kinase (PI3K) catalytic subunit p110δ, but not PI3K p110α, corrects gain-of-function (GOF) Shp2-induced granulocyte macrophage-colony-stimulating factor (GM-CSF) hypersensitivity, Akt and Erk hyperactivation, and skewed hematopoietic progenitor distribution. Likewise, potent p110δ-specific inhibitors curtail the proliferation of GOF Shp2-expressing hematopoietic cells and cooperate with mitogen-activated or extracellular signal-regulated protein kinase kinase (MEK) inhibition to reduce proliferation further and maximally block Erk and Akt activation. Furthermore, the PI3K p110δ-specific inhibitor, idelalisib, also demonstrates activity against primary leukemia cells from individuals with JMML. These findings suggest that selective inhibition of the PI3K catalytic subunit p110δ could provide an innovative approach for treatment of JMML, with the potential for limiting toxicity resulting from the hematopoietic-restricted expression of p110δ.
Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Leucemia Mielomonocítica Juvenil/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Proliferação de Células/efeitos dos fármacos , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Leucemia Mielomonocítica Juvenil/genética , Camundongos , Camundongos Knockout , Inibidores de Proteínas Quinases/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
CLL cell trafficking between blood and tissue compartments is an integral part of the disease process. Idelalisib, a phosphoinositide 3-kinase delta (PI3Kδ) inhibitor causes rapid lymph node shrinkage, along with an increase in lymphocytosis, prior to inducing objective responses in CLL patients. This characteristic activity presumably is due to CLL cell redistribution from tissues into the blood, but the underlying mechanisms are not fully understood. We therefore analyzed idelalisib effects on CLL cell adhesion to endothelial and bone marrow stromal cells (EC, BMSC). We found that idelalisib inhibited CLL cell adhesion to EC and BMSC under static and shear flow conditions. TNFα-induced VCAM-1 (CD106) expression in supporting layers increased CLL cell adhesion and accentuated the inhibitory effect of idelalisib. Co-culture with EC and BMSC also protected CLL from undergoing apoptosis, and this EC- and BMSC-mediated protection was antagonized by idelalisib. Furthermore, we demonstrate that CLL cell adhesion to EC and VLA-4 (CD49d) resulted in the phosphorylation of Akt, which was sensitive to inhibition by idelalisib. These findings demonstrate that idelalisib interferes with integrin-mediated CLL cell adhesion to EC and BMSC, providing a novel mechanism to explain idelalisib-induced redistribution of CLL cells from tissues into the blood.
Assuntos
Células da Medula Óssea/patologia , Células Endoteliais/patologia , Inibidores Enzimáticos/farmacologia , Integrinas/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Inibidores de Fosfoinositídeo-3 Quinase , Purinas/farmacologia , Quinazolinonas/farmacologia , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Integrina alfa4beta1/metabolismo , Camundongos , Transdução de Sinais/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Células Estromais/patologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Phosphatidylinositol 3-kinases (PI3K) participate in numerous signaling pathways, and control distinct biological functions. Studies using pan-PI3K inhibitors suggest roles for PI3K in osteoclasts, but little is known about specific PI3K isoforms in these cells. Our objective was to determine effects of isoform-selective PI3K inhibitors on osteoclasts. The following inhibitors were investigated (targets in parentheses): wortmannin and LY294002 (pan-p110), PIK75 (α), GDC0941 (α, δ), TGX221 (ß), AS252424 (γ), and IC87114 (δ). In addition, we characterized a new potent and selective PI3Kδ inhibitor, GS-9820, and explored roles of PI3K isoforms in regulating osteoclast function. Osteoclasts were isolated from long bones of neonatal rats and rabbits. Wortmannin, LY294002, GDC0941, IC87114, and GS-9820 induced a dramatic retraction of osteoclasts within 15-20 min to 65-75% of the initial area. In contrast, there was no significant retraction in response to vehicle, PIK75, TGX221, or AS252424. Moreover, wortmannin and GS-9820, but not PIK75 or TGX221, disrupted actin belts. We examined effects of PI3K inhibitors on osteoclast survival. Whereas PIK75, TGX221, and GS-9820 had no significant effect on basal survival, all blocked RANKL-stimulated survival. When studied on resorbable substrates, osteoclastic resorption was suppressed by wortmannin and inhibitors of PI3Kß and PI3Kδ, but not other isoforms. These data are consistent with a critical role for PI3Kδ in regulating osteoclast cytoskeleton and resorptive activity. In contrast, multiple PI3K isoforms contribute to the control of osteoclast survival. Thus, the PI3Kδ isoform, which is predominantly expressed in cells of hematopoietic origin, is an attractive target for anti-resorptive therapeutics.
Assuntos
Osteoclastos/efeitos dos fármacos , Osteoclastos/enzimologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Androstadienos/farmacologia , Animais , Reabsorção Óssea/prevenção & controle , Sobrevivência Celular/efeitos dos fármacos , Cromonas/farmacologia , Citoesqueleto/efeitos dos fármacos , Indazóis/farmacologia , Isoenzimas/antagonistas & inibidores , Morfolinas/farmacologia , Osteoclastos/citologia , Quinazolinas/farmacologia , Ligante RANK/metabolismo , Coelhos , Ratos , Sulfonamidas/farmacologia , WortmaninaRESUMO
Previously, we showed that inhibition of the protein kinase C ß (PKCß)/AKT pathway augments engagement of the histone deacetylase inhibitor (HDI)-induced apoptosis in lymphoma cells. In the present study, we investigated the cytotoxicity and mechanisms of cell death induced by the delta isoform-specific phosphatidylinositide 3-kinase (PI3K) inhibitor, GS-1101, in combination with the HDI, panobinostat (LBH589) and suberoylanilide hydroxamic acid (SAHA). Lymphoma cell lines, primary non-Hodgkin Lymphoma (NHL) and chronic lymphocytic leukaemia (CLL) cells were simultaneously treated with the HDI, LBH589 and GS-1101. An interaction of the LBH589/GS-1101 combination was formally examined by using various concentrations of LBH589 and GS-1101. Combined treatment resulted in a synergistic inhibition of proliferation and showed synergistic effect on apoptotic induction in all tested cell lines and primary NHL and CLL cells. This study indicates that interference with PI3K signalling dramatically increases HDI-mediated apoptosis in malignant haematopoietic cells, possibly through both AKT-dependent or AKT- independent mechanisms. Moreover, the increase in HDI-related apoptosis observed in PI3K inhibitor-treated cells appears to be related to the disruption of the extracellular signal-regulated kinase (ERK) signalling pathway. This study provides a strong rational for testing the combination of PI3K inhibitors and HDI in the clinic.
Assuntos
Apoptose/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Purinas/farmacologia , Quinazolinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Inibidores de Histona Desacetilases/toxicidade , Humanos , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/toxicidade , Indóis/farmacologia , Indóis/toxicidade , Concentração Inibidora 50 , Linfoma de Células B/metabolismo , Modelos Biológicos , Panobinostat , Fosfatidilinositol 3-Quinases/metabolismo , Purinas/toxicidade , Quinazolinonas/toxicidadeRESUMO
Colony stimulating factor-1 (CSF-1) stimulates mononuclear phagocytic cell survival, growth and differentiation into macrophages through activation and autophosphorylation of the CSF-1 receptor (CSF-1R). We have previously demonstrated that CSF-1-induced phosphorylation of Y721 (pY721) in the receptor kinase insert triggers its association with the p85 regulatory subunit of phosphoinositide 3'-kinase (PI3K). Binding of p85 PI3K to the CSF-1R pY721 motif activates the associated p110 PI3K catalytic subunit and stimulates spreading and motility in macrophages and enhancement of tumor cell invasion. Here we show that pY721-based signaling is necessary for CSF-1-stimulated PtdIns(3,4,5)P production. While primary bone marrow-derived macrophages and the immortalized bone marrow-derived macrophage cell line M-/-.WT express all three class IA PI3K isoforms, p110δ predominates in the cell line. Treatment with p110δ-specific inhibitors demonstrates that the hematopoietically enriched isoform, p110δ, mediates CSF-1-regulated spreading and invasion in macrophages. Thus GS-1101, a potent and selective p110δ inhibitor, may have therapeutic potential by targeting the infiltrative capacity of tumor-associated macrophages that is critical for their enhancement of tumor invasion and metastasis.
Assuntos
Movimento Celular , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/citologia , Inibidores de Fosfoinositídeo-3 Quinase , Purinas/farmacologia , Quinazolinonas/farmacologia , Animais , Western Blotting , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Adesão Celular , Proliferação de Células , Classe I de Fosfatidilinositol 3-Quinases , Ensaio de Imunoadsorção Enzimática , Humanos , Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismoRESUMO
Phosphatidylinositol-3-kinase (PI3K) signaling is constitutive in most human cancers. Selective inhibition of PI3Kδ (p110δ) by GS-1101 has emerged as a promising therapy in chronic lymphocytic leukemia and indolent lymphomas. In aggressive non-Hodgkin lymphomas such as mantle cell lymphoma (MCL), however, efficacy has been observed, but the extent and duration of tumor control is modest. To determine if tumor killing by GS-1101 is cell cycle-dependent, we show in primary MCL cells by whole-transcriptome sequencing that, despite aberrant expression and recurrent mutations in Cyclin D1, mutations are rare in coding regions of CDK4, RB1 and other genes that control G1-S cell cycle progression or PI3K/AKT signaling. PI3Kδ is the predominant PI3K catalytic subunit expressed, and inhibition by GS-1101 transiently inhibits AKT phosphorylation but not proliferation in MCL cells. Induction of prolonged early G1-arrest (pG1) by selective inhibition of CDK4/CDK6 with PD 0332991 amplifies and sustains PI3Kδ inhibition, which leads to robust apoptosis. Accordingly, inhibition of PI3Kδ induces apoptosis of primary MCL tumor cells once they have ceased to cycle ex vivo, and this killing is enhanced by PD 0332991 inhibition of CDK4/CDK6. PIK3IP1, a negative PI3K regulator, appears to mediate pG1 sensitization to PI3K inhibition; it is markedly reduced in MCL tumor cells compared with normal peripheral B cells, profoundly induced in pG1 and required for pG1 sensitization to GS-1101. Thus, the magnitude and duration of PI3K inhibition and tumor killing by GS-1101 is pG1-dependent, suggesting induction of pG1 by CDK4/CDK6 inhibition as a strategy to sensitize proliferating lymphoma cells to PI3K inhibition.
Assuntos
Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Linfoma/metabolismo , Proteínas de Membrana/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Ciclina D1/genética , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/genética , Mutação/genética , Fosfatidilinositol 3-Quinases/genética , Piperazinas/farmacologia , Purinas/farmacologia , Piridinas/farmacologia , Quinazolinonas/farmacologiaRESUMO
Stroma induces treatment resistance in chronic lymphocytic leukemia (CLL), possibly because of alterations in the BCL-2 family of proteins, which are key regulators of apoptosis. We previously developed BH3 profiling, a functional assay that assesses mitochondrial depolarization in response to BH3-only peptides, to measure "apoptotic priming," the proximity of a cell to the apoptotic threshold. In the present study, we use BH3 profiling to show that CLL cells from the PB are highly primed. Increased priming is associated with improved clinical response and, unexpectedly, with unmutated IGHV status. Coculturing CLL cells in vitro with stroma decreases priming. Using matched PB, BM, and lymph node compartment samples, we found in vivo that BM-derived CLL cells are the least primed. CLL cells cocultured with stroma were treated with the PI3K δ-isoform inhibitor CAL-101 (GS1101). CAL-101 caused CLL cell de-adhesion, leading to increased CLL cell priming. Stimulation of CLL cells with anti-IgM or CXCL12 caused decreased priming that could be reversed by CAL-101. Our results show that inhibition of stromal interactions leading to displacement of CLL cells into the blood by CAL-101 in vivo may increase CLL cell priming, suggesting a mechanism by which agents inducing lymphocyte redistribution might facilitate improved clinical response when used in combination with other therapies.
Assuntos
Comunicação Celular/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Linfócitos/imunologia , Mitocôndrias/imunologia , Células Estromais/imunologia , Anticorpos Anti-Idiotípicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/patologia , Comunicação Celular/efeitos dos fármacos , Quimiocina CXCL12/farmacologia , Técnicas de Cocultura , Humanos , Imunoglobulina M/imunologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Linfonodos/efeitos dos fármacos , Linfonodos/patologia , Linfócitos/efeitos dos fármacos , Linfócitos/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/imunologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fragmentos de Peptídeos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas/farmacologia , Purinas/farmacologia , Quinazolinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Células Estromais/efeitos dos fármacos , Células Estromais/patologia , Células Tumorais CultivadasRESUMO
Constitutive phosphoinositide 3-kinase (PI3K)/Akt activation is common in T cell acute lymphoblastic leukemia (T-ALL). Although four distinct class I PI3K isoforms (α, ß, γ, δ) could participate in T-ALL pathogenesis, none has been implicated in this process. We report that in the absence of PTEN phosphatase tumor suppressor function, PI3Kγ or PI3Kδ alone can support leukemogenesis, whereas inactivation of both isoforms suppressed tumor formation. The reliance of PTEN null T-ALL on the combined activities of PI3Kγ/δ was further demonstrated by the ability of a dual inhibitor to reduce disease burden and prolong survival in mice as well as prevent proliferation and promote activation of proapoptotic pathways in human tumors. These results support combined inhibition of PI3Kγ/δ as therapy for T-ALL.
Assuntos
Antineoplásicos/uso terapêutico , Inibidores de Fosfoinositídeo-3 Quinase , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Isoformas de Proteínas , Purinas/uso terapêutico , Quinazolinonas/uso terapêutico , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Classe I de Fosfatidilinositol 3-Quinases , Classe Ib de Fosfatidilinositol 3-Quinase/química , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Desenho de Fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Camundongos , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Purinas/química , Purinas/farmacologia , Quinazolinonas/química , Quinazolinonas/farmacologiaRESUMO
GS-1101 (CAL-101) is an oral PI3Kδ-specific inhibitor that has shown preclinical and clinical activity in non-Hodgkin lymphoma and chronic lymphocytic leukemia. To investigate the potential role of PI3Kδ in Hodgkin lymphoma (HL), we screened 5 HL cell lines and primary samples from patients with HL for PI3Kδ isoform expression and constitutive PI3K pathway activation. Inhibition of PI3Kδ by GS-1101 resulted in the inhibition of Akt phosphorylation. Cocultures with stroma cells induced Akt activation in HL cells, and this effect was blocked by GS-1101. Conversely, production of the stroma-stimulating chemokine, CCL5, by HL cells was reduced by GS-1101. GS-1101 also induced dose-dependent apoptosis of HL cells at 48 hours. Reductions in cell viability and apoptosis were enhanced when combining GS-1101 with the mTOR inhibitor everolimus. Our findings suggest that excessive PI3Kδ activity is characteristic in HL and support clinical evaluation of GS-1101, alone and in combination, as targeted therapy for HL.
Assuntos
Apoptose/efeitos dos fármacos , Microambiente Celular/efeitos dos fármacos , Purinas/farmacologia , Quinazolinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CCL5/metabolismo , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Relação Dose-Resposta a Droga , Doença de Hodgkin/enzimologia , Doença de Hodgkin/patologia , Humanos , Immunoblotting , Imuno-Histoquímica , Inibidores de Fosfoinositídeo-3 Quinase , Análise Serial de TecidosRESUMO
In lymphocytes, the phosphoinositide 3'-kinase (PI3K) isoform p110δ (PI3Kδ) transmits signals from surface receptors, including the B-cell receptor (BCR). CAL-101, a selective inhibitor of PI3Kδ, displays clinical activity in CLL, causing rapid lymph node shrinkage and a transient lymphocytosis. Inhibition of pro-survival pathways, the presumed mechanism of CAL-101, does not explain this characteristic pattern of activity. Therefore, we tested CAL-101 in assays that model CLL-microenvironment interactions in vitro. We found that CAL-101 inhibits CLL cell chemotaxis toward CXCL12 and CXCL13 and migration beneath stromal cells (pseudoemperipolesis). CAL-101 also down-regulates secretion of chemokines in stromal cocultures and after BCR triggering. CAL-101 reduces survival signals derived from the BCR or from nurse-like cells, and inhibits BCR- and chemokine-receptor-induced AKT and MAP kinase (ERK) activation. In stromal cocultures, CAL-101 sensitizes CLL cells toward bendamustine, fludarabine, and dexamethasone. These results are corroborated by clinical data showing marked reductions in circulating CCL3, CCL4, and CXCL13 levels, and a surge in lymphocytosis during CAL-101 treatment. Thus, CAL-101 displays a dual mechanism of action, directly decreasing cell survival while reducing interactions that retain CLL cells in protective tissue microenvironments. These data provide an explanation for the clinical activity of CAL-101, and a roadmap for future therapeutic development.