The absence of a novel intron 19-retaining ALK transcript (ALK-I19) and MYCN amplification correlates with an excellent clinical outcome in neuroblastoma patients.
ABSTRACT: ALK missense mutations are detected in 8% of neuroblastoma (NB) tumors at diagnosis and confer gain-of-function oncogenic effects. The mechanisms by which the expression of wild-type or mutant ALK, which is detectable in the majority of cases, is regulated are not well understood. We have identified a novel ALK transcript characterized by the retention of intron 19 (ALK-I19). ALK-I19 was detected in 4/4 NB cell lines, but not other non-NB cells with ALK aberrations. The functional significance of ALK-I19 was determined by specific siRNA knockdown of this transcript, which resulted in substantially decreased expression of the fully-spliced ALK transcripts (FS-ALK) and a significant reduction in cell growth. We also demonstrate that ALK-I19 is a precursor of FS-ALK. ALK-I19 was detected in 14/37 (38%) tumors from patients with newly diagnosed NB. ALK-I19 expression correlated with undifferentiated histology and strong ALK protein expression detectable by immunohistochemistry. Importantly, patients with tumors that did not express ALK-I19 and lacked MYCN amplification had an excellent clinical outcome, with 19/19 patients survived at 5-years. In conclusion, ALK-I19 is a novel ALK transcript that likely represents a marker of undifferentiated NB cells. The absence of ALK-I19 and MYCN amplification is a useful prognostic marker for NB patients.
Project description:The anaplastic lymphoma kinase (ALK) gene is overexpressed, mutated or amplified in most neuroblastoma (NB), a pediatric neural crest-derived embryonal tumor. The two most frequent mutations, ALK-F1174L and ALK-R1275Q, contribute to NB tumorigenesis in mouse models, and cooperate with MYCN in the oncogenic process. However, the precise role of activating ALK mutations or ALK-wt overexpression in NB tumor initiation needs further clarification. Human ALK-wt, ALK-F1174L, or ALK-R1275Q were stably expressed in murine neural crest progenitor cells (NCPC), MONC-1 or JoMa1, immortalized with v-Myc or Tamoxifen-inducible Myc-ERT, respectively. While orthotopic implantations of MONC- 1 parental cells in nude mice generated various tumor types, such as NB, osteo/ chondrosarcoma, and undifferentiated tumors, due to v-Myc oncogenic activity, MONC-1-ALK-F1174L cells only produced undifferentiated tumors. Furthermore, our data represent the first demonstration of ALK-wt transforming capacity, as ALK-wt expression in JoMa1 cells, likewise ALK-F1174L, or ALK-R1275Q, in absence of exogenous Myc-ERT activity, was sufficient to induce the formation of aggressive and undifferentiated neural crest cell-derived tumors, but not to drive NB development. Interestingly, JoMa1-ALK tumors and their derived cell lines upregulated Myc endogenous expression, resulting from ALK activation, and both ALK and Myc activities were necessary to confer tumorigenic properties on tumor-derived JoMa1 cells in vitro.
Project description:High-risk neuroblastoma (NB) is responsible for a disproportionate number of childhood deaths due to cancer. One indicator of high-risk NB is amplification of the neural MYC (MYCN) oncogene, which is currently therapeutically intractable. Identification of anaplastic lymphoma kinase (ALK) as an NB oncogene raised the possibility of using ALK tyrosine kinase inhibitors (TKIs) in treatment of patients with activating ALK mutations. 8-10% of primary NB patients are ALK-positive, a figure that increases in the relapsed population. ALK is activated by the ALKAL2 ligand located on chromosome 2p, along with ALK and MYCN, in the "2p-gain" region associated with NB. Dysregulation of ALK ligand in NB has not been addressed, although one of the first oncogenes described was v-sis that shares > 90% homology with PDGF. Therefore, we tested whether ALKAL2 ligand could potentiate NB progression in the absence of ALK mutation. We show that ALKAL2 overexpression in mice drives ALK TKI-sensitive NB in the absence of ALK mutation, suggesting that additional NB patients, such as those exhibiting 2p-gain, may benefit from ALK TKI-based therapeutic intervention.
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.
Project description:The invasive nature of surgical biopsies deters sequential application, and single biopsies often fail to reflect tumor dynamics, intratumor heterogeneity and drug sensitivities likely to change during tumor evolution and treatment. Implementing molecular characterization of cell-free neuroblastoma-derived DNA isolated from blood plasma could improve disease assessment for treatment selection and monitoring of patients with high-risk neuroblastoma. We established droplet digital PCR (ddPCR) protocols for MYCN and ALK copy number status in plasma from neuroblastoma patients. Our ddPCR protocol accurately discriminated between MYCN and ALK amplification, gain and normal diploid status in a large panel of neuroblastoma cell lines, and discrepancies with reported MYCN and ALK status were detected, including a high-level MYCN amplification in NB-1, a MYCN gain in SH-SY5Y, a high-level ALK amplification in IMR-32 and ALK gains in BE(2)-C, Kelly, SH-SY5Y and LAN-6. MYCN and ALK status were also reliably determined from cell-free DNA derived from medium conditioned by the cell lines. MYCN and ALK copy numbers of subcutaneous neuroblastoma xenograft tumors were accurately determined from cell-free DNA in the mouse blood plasma. In a final validation step, we accurately distinguished MYCN and ALK copy numbers of the corresponding primary tumors using retrospectively collected blood plasma samples from 10 neuroblastoma patients. Our data justify the further development of molecular disease characterization using cell-free DNA in blood plasma from patients with neuroblastoma. This expanded molecular diagnostic palette may improve monitoring of disease progression including relapse and metastatic events as well as therapy success or failure in high-risk neuroblastoma patients.
Project description:Activating mutations of the ALK receptor occur in a subset of neuroblastoma tumors. We previously demonstrated that Alk mutations cooperate with MYCN overexpression to induce neuroblastoma in mice and identified Ret as being strongly upregulated in MYCN/Alkmut tumors. By a genetic approach in vivo, we now document an oncogenic cooperation between activated Ret and MYCN overexpression in neuroblastoma formation. We show that MYCN/RetM919T tumors exhibit histological features and expression profiles close to MYCN/Alkmut tumors. We show that RET transcript levels decrease precedes RET protein levels decrease upon ALK inhibition in neuroblastoma cell lines. Etv5 was identified as a candidate transcription factor regulating Ret expression from murine MYCN/Alkmut tumor transcriptomic data. We demonstrate that ETV5 is regulated both at the protein and mRNA levels upon ALK activation or inhibition in neuroblastoma cell lines and that this regulation precedes RET modulation. We document that ALK activation induces ETV5 protein upregulation through stabilization in a MEK/ERK-dependent manner. We show that RNAi-mediated inhibition of ETV5 decreases RET expression. Reporter assays indicate that ETV5 is able to drive RET gene transcription. ChIP-seq analysis confirmed ETV5 binding on the RET promoter and identified an enhancer upstream of the promoter. Finally, we demonstrate that combining RET and ALK inhibitors reduces tumor growth more efficiently than each single agent in MYCN and AlkF1178L-driven murine neuroblastoma. Altogether, these results define the ERK-ETV5-RET pathway as a critical axis driving neuroblastoma oncogenesis downstream of activated ALK.
Project description:ALK is a tyrosine kinase receptor and oncogene in neuroblastoma (NB). The receptor is activated by the ALKAL2 ligand, but it is unknown whether missregulation of this ligand may play a role in NB carcinogenesis. Here, a TH-MYCN driven neuroblastoma mice was created +/- ALK F1178S mutation and +/- ALKAL2 overexpression
Project description:The ALK(F1174L) mutation is associated with intrinsic and acquired resistance to crizotinib and cosegregates with MYCN in neuroblastoma. In this study, we generated a mouse model overexpressing ALK(F1174L) in the neural crest. Compared to ALK(F1174L) and MYCN alone, co-expression of these two oncogenes led to the development of neuroblastomas with earlier onset, higher penetrance, and enhanced lethality. ALK(F1174L)/MYCN tumors exhibited increased MYCN dosage due to ALK(F1174L)-induced activation of the PI3K/AKT/mTOR and MAPK pathways, coupled with suppression of MYCN pro-apoptotic effects. Combined treatment with the ATP-competitive mTOR inhibitor Torin2 overcame the resistance of ALK(F1174L)/MYCN tumors to crizotinib. Our findings demonstrate a pathogenic role for ALK(F1174L) in neuroblastomas overexpressing MYCN and suggest a strategy for improving targeted therapy for ALK-positive neuroblastoma.
Project description:The ALK^F1174L mutation is associated with intrinsic and acquired resistance to crizotinib and cosegregates with MYCN in neuroblastoma. In this study, we generated a mouse model overexpressing ALK^F1174L in the neural crest. Comapred to mice expressing ALK^F1174L or MYCN alone, combined expression of the two aberrations led to development of neuroblastoma with a shorter latency and higher penetrance. Here, we evaluated the transcriptional profiles of MYCN-driven neuroblastomas with or without the expression of ALK^F1174L to determine the pathogenic consequences of the ALK^F1174L/MYCN interaction in neuroblastoma. 10 mice were analysed in this study. Five ALK^F1174L/MYCN tumors were compared with five MYCN tumors. Total RNA was extracted, samples were labeled and processed using the Agilent Low Input Quick Amp two color Cy3(sample) and Cy5 (mouse reference) labeling kit and hybridized to Agilent SurePrint G3 Mouse Gene Expression arrays.
Project description:Activating germline mutations of anaplastic lymphoma kinase (ALK) occur in most cases of hereditary neuroblastoma (NB) and the constitutively active kinase activity of ALK promotes cell proliferation and survival in NB. Therefore, ALK kinase is a potential therapeutic target for NB. In this study, we show that the novel ALK inhibitor alectinib effectively suppressed cell proliferation and induces apoptosis in NB cell lines with either wild-type ALK or mutated ALK (F1174L and D1091N) by blocking ALK-mediated PI3K/Akt/mTOR signaling. In addition, alectinib enhanced doxorubicin-induced cytotoxicity and apoptosis in NB cells. Furthermore, alectinib induced apoptosis in an orthotopic xenograft NB mouse model. Also, in the TH-MYCN transgenic mouse model, alectinib resulted in decreased tumor growth and prolonged survival time. These results indicate that alectinib may be a promising therapeutic agent for the treatment of NB.
Project description:High-risk neuroblastoma (NB) often involves amplification of the neural MYC (MYCN) oncogene as well as mutations in ALK. Currently, high-risk NB presents significant clinical challenges, and additional therapeutic options are needed. Oncogenes such as MYCN and ALK result in increased replication stress in cancer cells, offering one such therapeutically exploitable option. Here, we followed up on earlier phosphoproteomic analyses that identified ATR activity in ALK-driven NB cell lines. We tested several ATR inhibitors, identifying BAY 1895344 as the most potent inhibitor of NB cell growth and proliferation. Using RNA-Seq, proteomics and phosphoproteomics we characterized the response of NB cells and tumours to ATR inhibition, identifying key components of the DNA damage response (DDR) as well as ATRX, MYCN, E2F and DCK among other ATR targets in NB cells. ATR inhibition with BAY 1895344 also produced robust responses in mouse NB models. Remarkably, a 2 week protocol combining ATR and ALK inhibition led to complete regression of NB tumours in two independent NB genetically modified mouse tumour models. These results suggest that NB patients, particularly in high-risk groups with oncogene induced replication stress, may benefit from inhibition of ATR as therapeutic intervention.