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New therapeutic strategies in neuroblastoma: combined targeting of a novel tyrosine kinase inhibitor and liposomal siRNAs against ALK.
ABSTRACT: Many different aberrations in the Anaplastic Lymphoma Kinase (ALK) were found to be oncogenic drivers in several cancers including neuroblastoma (NB), therefore ALK is now considered a critical player in NB oncogenesis and a promising therapeutic target. The ALK-inhibitor crizotinib has a limited activity against the various ALK mutations identified in NB patients. We tested: the activity of the novel ALK-inhibitor X-396 administered alone or in combination with Targeted Liposomes carrying ALK-siRNAs (TL[ALK-siRNA]) that are active irrespective of ALK gene mutational status; the pharmacokinetic profiles and the biodistribution of X-396; the efficacy of X-396 versus crizotinib treatment in NB xenografts; whether the combination of X-396 with the TL[ALK-siRNA] could promote long-term survival in NB mouse models. X-396 revealed good bioavailability, moderate half-life, high mean plasma and tumor concentrations. X-396 was more effective than crizotinib in inhibiting in vitro cell proliferation of NB cells and in reducing tumor volume in subcutaneous NB models in a dose-dependent manner. In orthotopic NB xenografts, X-396 significantly increased life span independently of the ALK mutation status. In combination studies, all effects were significantly improved in the mice treated with TL[ALK-siRNA] and X-396 compared to mice receiving the single agents. Our findings provide a rational basis to design innovative molecular-based treatment combinations for clinical application in ALK-driven NB tumors.
Project description:ALK has been identified as a novel therapeutic target in neuroblastoma (NB), but resistance to ALK inhibitors (such as crizotinib) is well recognized. We recently published that the crizotinib sensitivity in NB cells strongly correlates with the crizotinib-ALK binding, and ?-catenin effectively hinders this interaction and confers crizotinib resistance. Here, we asked if these observations hold true for the stem-like cells in NB cells, which were purified based on their responsiveness to a Sox2 reporter. Compared to bulk, reporter unresponsive (RU) cells, reporter responsive (RR) cells had significantly higher neurosphere formation ability, expression of CD133/nestin and chemo-resistance. Using the cellular thermal shift assay, we found that RR cells exhibited significantly weaker crizotinib-ALK binding and higher crizotinib resistance than RU cells. The suboptimal crizotinib-ALK binding in RR cells can be attributed to their high ?-catenin expression, since siRNA knockdown of ?-catenin restored the crizotinib-ALK binding and lowered the crizotinib resistance to the level of RU cells. Enforced expression of ?-catenin in RU cells resulted in the opposite effects. To conclude, high expression of ?-catenin in the stem-like NB cells contributes to their crizotinib resistance. Combining ?-catenin inhibitors and ALK inhibitors may be useful in treating NB patients.
Project description:RNA interference molecules have some advantages as cancer therapeutics, including a proved efficacy on both wild-type (WT) and mutated transcripts and an extremely high sequence-specificity. The most significant hurdle to be overcome if exogenous small interfering RNAs (siRNA) is to be used therapeutically is the specific, effective, nontoxic delivery of siRNA to its intracellular site of action. At present, human applications are confined almost exclusively to targets within the liver, where the delivery systems naturally accumulate, and extra-hepatic targets remain a challenge. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that has recently been shown to contribute to the cell growth and progression of human neuroblastoma (NB). We investigated its potential as a therapeutic target in NB by generating anti-GD?-targeted nanoparticles that carry ALK-directed siRNA, which are specifically and efficiently delivered to GD?-expressing NB cells. Relative to free ALK-siRNA, anti-GD?-targeted liposomal formulations of ALK-siRNA had low plasma clearance, increased siRNA stability, and improved binding, uptake, silencing and induction of cell death, and specificity for NB cells. In NB xenografts, intravenous (i.v.) injection of the targeted ALK-siRNA liposomes showed gene-specific antitumor activity with no side effects. ALK-selective siRNA entrapped in anti-GD?-targeted nanoparticles is a promising new modality for NB treatment.
Project description:The anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor that is involved in the pathogenesis of different types of human cancers, including neuroblastoma (NB). In NB, ALK overexpression, or point mutations, are associated with poor prognosis and advanced stage disease. Inhibition of ALK kinase activity by small-molecule inhibitors in lung cancers carrying ALK translocations has shown therapeutic potential. However, secondary mutations may occur that, generate tumor resistance to ALK inhibitors. To overcome resistance to ALK inhibitors in NB, we adopted an alternative RNA interference (RNAi)-based therapeutic strategy that is able to knockdown ALK, regardless of its genetic status [mutated, amplified, wild-type (WT)]. NB cell lines, transduced by lentiviral short hairpin RNA (shRNA), showed reduced proliferation and increased apoptosis when ALK was knocked down. In mice, a nanodelivery system for ALK-specific small interfering RNA (siRNA), based on the conjugation of antibodies directed against the NB-selective marker GD(2) to liposomes, showed strong ALK knockdown in vivo in NB cells, which resulted in cell growth arrest, apoptosis, and prolonged survival. ALK knockdown was associated with marked reductions in vascular endothelial growth factor (VEGF) secretion, blood vessel density, and matrix metalloproteinases (MMPs) expression in vivo, suggesting a role for ALK in NB-induced neoangiogenesis and tumor invasion, confirming this gene as a fundamental oncogene in NB.
Project description:ALK receptor tyrosine kinase has been shown to be a therapeutic target in neuroblastoma. Germline ALK activating mutations are responsible for the majority of hereditary neuroblastoma and somatic ALK activating mutations are also frequently observed in sporadic cases of advanced NB. Crizotinib, a first-line therapy in the treatment of advanced non-small cell lung cancer (NSCLC) harboring ALK rearrangements, demonstrates striking efficacy against ALK-rearranged NB. However, crizotinib fails to effectively inhibit the activity of ALK when activating mutations are present within its kinase domain, as with the F1174L mutation. Here we show that a new ALK inhibitor AZD3463 effectively suppressed the proliferation of NB cell lines with wild type ALK (WT) as well as ALK activating mutations (F1174L and D1091N) by blocking the ALK-mediated PI3K/AKT/mTOR pathway and ultimately induced apoptosis and autophagy. In addition, AZD3463 enhanced the cytotoxic effects of doxorubicin on NB cells. AZD3463 also exhibited significant therapeutic efficacy on the growth of the NB tumors with WT and F1174L activating mutation ALK in orthotopic xenograft mouse models. These results indicate that AZD3463 is a promising therapeutic agent in the treatment of NB.
Project description:The crizotinib-resistant ALK(F1174L) mutation arises de novo in neuroblastoma (NB) and is acquired in ALK translocation-driven cancers, lending impetus to the development of novel anaplastic lymphoma kinase (ALK) inhibitors with different modes of action. The diaminopyrimidine TAE684 and its derivative ceritinib (LDK378), which are structurally distinct from crizotinib, are active against NB cells expressing ALK(F1174L). Here we demonstrate acquired resistance to TAE684 and LDK378 in ALK(F1174L)-driven human NB cells that is linked to overexpression and activation of the AXL tyrosine kinase and epithelial-to-mesenchymal transition (EMT). AXL phosphorylation conferred TAE684 resistance to NB cells through upregulated extracellular signal-regulated kinase (ERK) signaling. Inhibition of AXL partly rescued TAE684 resistance, resensitizing these cells to this compound. AXL activation in resistant cells was mediated through increased expression of the active form of its ligand, GAS6, that also served to stabilize the AXL protein. Although ectopic expression of AXL and TWIST2 individually in TAE684-sensitive parental cells led to the elevated expression of mesenchymal markers and invasive capacity, only AXL overexpression induced resistance to TAE684 as well. TAE684-resistant cells showed greater sensitivity to HSP90 inhibition than did their parental counterparts, with downregulation of AXL and AXL-mediated ERK signaling. Our studies indicate that aberrant AXL signaling and development of an EMT phenotype underlie resistance of ALK(F1174L)-driven NB cells to TAE684 and its derivatives. We suggest that the combination of ALK and AXL or HSP90 inhibitors be considered to delay the emergence of such resistance.
Project description:The therapeutic targeting of anaplastic lymphoma kinase (ALK) has been a burgeoning area of research since 2007 when ALK fusions were initially identified in patients with non-small cell lung cancer. The field has rapidly progressed through development of the first-generation ALK inhibitor, crizotinib, to an understanding of mechanisms of acquired resistance to crizotinib and is currently witnessing an explosion in the development of next-generation ALK inhibitors such as ceritinib, alectinib, PF-06463922, AP26113, X-396, and TSR-011. As with most targeted therapies, acquired resistance appears to be an inevitable outcome. Current preclinical and clinical studies are focused on the development of rational therapeutic strategies, including novel ALK inhibitors, as well as rational combination therapies to maximize disease control by delaying or overcoming acquired therapeutic resistance. This review summarizes the existing clinical data and ongoing research pertaining to the clinical application of ALK inhibitors in patients with non-small cell lung cancer.
Project description:The treatment of patients with advanced non-small cell lung cancer (NSCLC) harboring chromosomal rearrangements of anaplastic lymphoma kinase (ALK) has been revolutionized by the development of crizotinib, a small molecule inhibitor of the tyrosine kinases ALK, ROS1, and MET. Resistance to crizotinib invariably develops, however, through a variety of mechanisms. In the last few years, a flurry of new and more potent ALK inhibitors has emerged for the treatment of ALK-positive NSCLC, including ceritinib (LDK378), alectinib (RO5424802/CH5424802), AP26113, ASP3026, TSR-011, PF-06463922, RXDX-101, X-396, and CEP-37440. Cancers harboring ALK rearrangements may also be susceptible to treatment with heat shock protein 90 inhibitors. This review focuses on the pharmacologic and clinical properties of these compounds, either as monotherapies or in combination with other drugs. With so many ALK inhibitors in development, the challenges of how these agents should be studied and ultimately prescribed are also discussed.
Project description:BACKGROUND:Epithelioid inflammatory myofibroblastic sarcoma (eIMS) is characterised by perinuclear ALK localisation, CD30 expression and early relapse despite crizotinib treatment. We aimed to identify therapies to prevent and/or treat ALK inhibitor resistance. METHODS:Malignant ascites, from an eIMS patient at diagnosis and following multiple relapses, were used to generate matched diagnosis and relapse xenografts. RESULTS:Xenografts were validated by confirmation of RANBP2-ALK rearrangement, perinuclear ALK localisation and CD30 expression. Although brentuximab-vedotin (BV) demonstrated single-agent activity, tumours regrew during BV therapy. BV resistance was associated with reduced CD30 expression and induction of ABCB1. BV resistance was reversed in vitro by tariquidar, but combination BV and tariquidar treatment only briefly slowed xenograft growth compared with BV alone. Combining BV with either crizotinib or ceritinib resulted in marked tumour shrinkage in both xenograft models, and resulted in prolonged tumour-free survival in the diagnosis compared with the relapse xenograft. CONCLUSIONS:CD30 is a therapeutic target in eIMS. BV efficacy is limited by the rapid emergence of resistance. Prolonged survival with combination ALK and CD30-targeted-therapy in the diagnosis model provides the rationale to trial this combination in eIMS patients at diagnosis. This combination could also be considered for other CD30-positive, ALK-rearranged malignancies.
Project description:The presence of an ALK aberration correlates with inferior survival for patients with high-risk neuroblastoma. The emergence of ALK inhibitors such as crizotinib has provided novel treatment opportunities. However, certain ALK mutations result in de novo crizotinib resistance, and a phase I trial of crizotinib showed a lack of response in patients harboring those ALK mutations. Thus, understanding mechanisms of resistance and defining circumvention strategies for the clinic is critical.The sensitivity of human neuroblastoma-derived cell lines, cell line-derived, and patient-derived xenograft (PDX) models with varying ALK statuses to crizotinib combined with topotecan and cyclophosphamide (topo/cyclo) was examined. Cultured cells and xenografts were evaluated for effects of these drugs on proliferation, signaling, and cell death, and assessment of synergy.In neuroblastoma murine xenografts harboring the most common ALK mutations, including those mutations associated with resistance to crizotinib (but not in those with wild-type ALK), crizotinib combined with topo/cyclo enhanced tumor responses and mouse event-free survival. Crizotinib + topo/cyclo showed synergistic cytotoxicity and higher caspase-dependent apoptosis than crizotinib or topo/cyclo alone in neuroblastoma cell lines with ALK aberrations (mutation or amplification).Combining crizotinib with chemotherapeutic agents commonly used in treating newly diagnosed patients with high-risk neuroblastoma restores sensitivity in preclinical models harboring both sensitive ALK aberrations and de novo-resistant ALK mutations. These data support clinical testing of crizotinib and conventional chemotherapy with the goal of integrating ALK inhibition into multiagent therapy for ALK-aberrant neuroblastoma patients.
Project description:Resistance to anaplastic lymphoma kinase (ALK)-targeted therapy in ALK-positive non-small cell lung cancer has been reported, with the majority of acquired resistance mechanisms relying on bypass signaling. To proactively identify resistance mechanisms in ALK-positive neuroblastoma (NB), we herein employ genome-wide CRISPR activation screens of NB cell lines treated with brigatinib or ceritinib, identifying PIM1 as a putative resistance gene, whose high expression is associated with high-risk disease and poor survival. Knockdown of PIM1 sensitizes cells of differing MYCN status to ALK inhibitors, and in patient-derived xenografts of high-risk NB harboring ALK mutations, the combination of the ALK inhibitor ceritinib and PIM1 inhibitor AZD1208 shows significantly enhanced anti-tumor efficacy relative to single agents. These data confirm that PIM1 overexpression decreases sensitivity to ALK inhibitors in NB, and suggests that combined front-line inhibition of ALK and PIM1 is a viable strategy for the treatment of ALK-positive NB independent of MYCN status.