RESUMEN
Solute Carrier Family 3, Member 2 (SLC3A2 or 4F2hc) is a multifunctional glycoprotein that mediates integrin-dependent signaling, acts as a trafficking chaperone for amino acid transporters, and is involved in polyamine transportation. We identified SLC3A2 as a potential Anaplastic Lymphoma Kinase (ALK) interacting partner in a BioID-proximity labeling screen in neuroblastoma (NB) cells. In this work we show that endogenous SLC3A2 and ALK interact in NB cells and that this SLC3A2:ALK interaction was abrogated upon treatment with the ALK inhibitor lorlatinib. We show here that loss of ALK activity leads to decreased SLC3A2 expression and reduced SLC3A2 protein stability in a panel of NB cell lines, while stimulation of ALK with ALKAL2 ligand resulted in increased SLC3A2 protein levels. We further identified MARCH11, an E3 ligase, as a regulator of SLC3A2 ubiquitination downstream of ALK. Further, knockdown of SLC3A2 resulted in inhibition of NB cell growth. To investigate the therapeutic potential of SLC3A2 targeting, we performed monotreatment of NB cells with AMXT-1501 (a polyamine transport inhibitor), which showed only moderate effects in NB cells. In contrast, a combination lorlatinib/AMXT-1501 treatment resulted in synergistic inhibition of cell growth in ALK-driven NB cell lines. Taken together, our results identify a novel role for the ALK receptor tyrosine kinase (RTK), working in concert with the MARCH11 E3 ligase, in regulating SLC3A2 protein stability and function in NB cells. The synergistic effect of combined ALK and polyamine transport inhibition shows that ALK/MARCH11/SLC3A2 regulation of amino acid transport is important for oncogenic growth and survival in NB cells.
Asunto(s)
Quinasa de Linfoma Anaplásico , Proliferación Celular , Cadena Pesada de la Proteína-1 Reguladora de Fusión , Neuroblastoma , Transducción de Señal , Humanos , Quinasa de Linfoma Anaplásico/metabolismo , Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Quinasa de Linfoma Anaplásico/genética , Neuroblastoma/patología , Neuroblastoma/metabolismo , Cadena Pesada de la Proteína-1 Reguladora de Fusión/metabolismo , Cadena Pesada de la Proteína-1 Reguladora de Fusión/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Lactamas/farmacología , Aminopiridinas/farmacología , Ubiquitinación/efectos de los fármacos , Pirazoles/farmacologíaRESUMEN
High-risk neuroblastoma (NB) is a significant clinical challenge. MYCN and Anaplastic Lymphoma Kinase (ALK), which are often involved in high-risk NB, lead to increased replication stress in cancer cells, suggesting therapeutic strategies. We previously identified an ATR (ataxia telangiectasia and Rad3-related)/ALK inhibitor (ATRi/ALKi) combination as such a strategy in two independent genetically modified mouse NB models. Here, we identify an underlying molecular mechanism, in which ALK signaling leads to phosphorylation of ATR and CHK1, supporting an effective DNA damage response. The importance of ALK inhibition is supported by mouse data, in which ATRi monotreatment resulted in a robust initial response, but subsequent relapse, in contrast to a 14-d ALKi/ATRi combination treatment that resulted in a robust and sustained response. Finally, we show that the remarkable response to the 14-d combined ATR/ALK inhibition protocol reflects a robust differentiation response, reprogramming tumor cells to a neuronal/Schwann cell lineage identity. Our results identify an ability of ATR inhibition to promote NB differentiation and underscore the importance of further exploring combined ALK/ATR inhibition in NB, particularly in high-risk patient groups with oncogene-induced replication stress.
Asunto(s)
Neuroblastoma , Proteínas Tirosina Quinasas Receptoras , Humanos , Ratones , Animales , Quinasa de Linfoma Anaplásico/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proliferación Celular , Línea Celular Tumoral , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética , Neuroblastoma/patología , Reparación del ADN , Daño del ADN , Proteínas de la Ataxia Telangiectasia Mutada/genéticaRESUMEN
Anaplastic lymphoma kinase (ALK) fusion variants in Non-Small Cell Lung Cancer (NSCLC) consist of numerous dimerizing fusion partners. Retrospective investigations suggest that treatment benefit in response to ALK tyrosine kinase inhibitors (TKIs) differs dependent on the fusion variant present in the patient tumor. Therefore, understanding the oncogenic signaling networks driven by different ALK fusion variants is important. To do this, we developed controlled inducible cell models expressing either Echinoderm Microtubule Associated Protein Like 4 (EML4)-ALK-V1, EML4-ALK-V3, Kinesin Family Member 5B (KIF5B)-ALK, or TRK-fused gene (TFG)-ALK and investigated their transcriptomic and proteomic responses to ALK activity modulation together with patient-derived ALK-positive NSCLC cell lines. This allowed identification of both common and isoform-specific responses downstream of these four ALK fusions. An inflammatory signature that included upregulation of the Serpin B4 serine protease inhibitor was observed in both ALK fusion inducible and patient-derived cells. We show that Signal transducer and activator of transcription 3 (STAT3), Nuclear Factor Kappa B (NF-κB) and Activator protein 1 (AP1) are major transcriptional regulators of SERPINB4 downstream of ALK fusions. Upregulation of SERPINB4 promotes survival and inhibits natural killer cell-mediated cytotoxicity, which has potential for therapeutic impact targeting the immune response together with ALK TKIs in NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Serpinas , Humanos , Quinasa de Linfoma Anaplásico/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Oncogenes , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/genética , Proteómica , Estudios Retrospectivos , Serpinas/genéticaRESUMEN
High-risk neuroblastoma (NB) often involves MYCN amplification as well as mutations in ALK. Currently, high-risk NB presents significant clinical challenges, and additional therapeutic options are needed. Oncogenes like MYCN and ALK result in increased replication stress in cancer cells, offering therapeutically exploitable options. We have pursued phosphoproteomic analyses highlighting ATR activity in ALK-driven NB cells, identifying the BAY1895344 ATR inhibitor as a potent inhibitor of NB cell growth and proliferation. Using RNA-Seq, proteomics and phosphoproteomics we characterize NB cell and tumour responses to ATR inhibition, identifying key components of the DNA damage response as ATR targets in NB cells. ATR inhibition also produces robust responses in mouse models. Remarkably, a 2-week combined ATR/ALK inhibition protocol leads to complete tumor regression in two independent genetically modified mouse NB models. These results suggest that NB patients, particularly in high-risk groups with oncogene-induced replication stress, may benefit from ATR inhibition as therapeutic intervention.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Neuroblastoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Quinasa de Linfoma Anaplásico/genética , Quinasa de Linfoma Anaplásico/metabolismo , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Línea Celular Tumoral , Daño del ADN , Reparación del ADN , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Morfolinas/farmacología , Morfolinas/uso terapéutico , Neuroblastoma/genética , Neuroblastoma/patología , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirazoles/farmacología , Pirazoles/uso terapéutico , RNA-Seq , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Neuroblastoma (NB) is one of the most frequently diagnosed tumors in infants. NB is a neuroendocrine tumor type with various characteristics and features, and with diverse outcome. The most malignant NBs have a 5-year survival rate of only 40-50%, indicating the need for novel and improved treatment options. 177Lu-octreotate is routinely administered for treatment of neuroendocrine tumors overexpressing somatostatin receptors (SSTR). The aim of this study was to examine the biodistribution of 177Lu-octreotate in mice bearing aggressive human NB cell lines, in order to evaluate the potential usefulness of 177Lu-octreotate for treatment of NB. METHODS: BALB/c nude mice bearing CLB-BAR, CLB-GE or IMR-32 tumor xenografts (n = 5-7/group) were i.v. injected with 0.15 MBq, 1.5 MBq or 15 MBq 177Lu-octreotate and sacrificed 1 h, 24 h, 48 h and 168 h after administration. The radioactivity concentration was determined for collected tissue samples, tumor-to-normal-tissue activity concentration ratios (T/N) and mean absorbed dose for each tissue were calculated. Immunohistochemical (IHC) staining for SSTR1-5, and Ki67 were carried out for tumor xenografts from the three cell lines. RESULTS: High 177Lu concentration levels and T/N values were observed in all NB tumors, with the highest for CLB-GE tumor xenografts (72%IA/g 24 h p.i.; 1.5 MBq 177Lu-octreotate). The mean absorbed dose to the tumor was 6.8 Gy, 54 Gy and 29 Gy for CLB-BAR, CLB-GE and IMR-32, respectively, p.i. of 15 MBq 177Lu-octreotate. Receptor saturation was clearly observed in CLB-BAR, resulting in higher concentration levels in the tumor when lower activity levels where administered. IHC staining demonstrated highest expression of SSTR2 in CLB-GE, followed by CLB-BAR and IMR-32. CONCLUSION: T/N values for all three human NB tumor xenograft types investigated were high relative to previously investigated neuroendocrine tumor types. The results indicate a clear potential of 177Lu-octreotate as a therapeutic alternative for metastatic NB.
Asunto(s)
Lutecio/uso terapéutico , Neuroblastoma/radioterapia , Octreótido/análogos & derivados , Radioisótopos/uso terapéutico , Animales , Apoptosis , Proliferación Celular , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neuroblastoma/metabolismo , Neuroblastoma/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Citocinas/genética , Citocinas/metabolismo , Proteína Proto-Oncogénica N-Myc/metabolismo , Neuroblastoma/patología , Inhibidores de Proteínas Quinasas/farmacología , Regulación hacia Arriba , Quinasa de Linfoma Anaplásico/genética , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Mutación con Ganancia de Función , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo , Análisis de Secuencia de ARN , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Oncogenic receptor tyrosine kinases including anaplastic lymphoma kinase (ALK) are implicated in numerous solid and hematologic cancers. ALK mutations are reported in an estimated 9% of neuroblastoma and recent reports indicate that the percentage of ALK-positive cases increases in the relapsed patient population. Initial clinical trial results have shown that it is difficult to inhibit growth of ALK positive neuroblastoma with crizotinib, motivating investigation of next generation ALK inhibitors with higher affinity for ALK. Here, alectinib, a potent next generation ALK inhibitor with antitumor activity was investigated in ALK-driven neuroblastoma models. Employing neuroblastoma cell lines and mouse xenografts we show a clear and efficient inhibition of ALK activity by alectinib. Inhibition of ALK activity was observed in vitro employing a set of different constitutively active ALK variants in biochemical assays. The results suggest that alectinib is an effective inhibitor of ALK kinase activity in ALK addicted neuroblastoma and should be considered as a potential future therapeutic option for ALK-positive neuroblastoma patients alone or in combination with other treatments.