Insight into drug resistance mechanisms and discovery of potential inhibitors against wild-type and L1196M mutant ALK from FDA-approved drugs

Anaplastic lymphoma kinase (ALK) plays a crucial role in multiple malignant cancers. It is known as a well-established target for the treatment of ALK-dependent cancers. Even though substantial efforts have been made to develop ALK inhibitors, only crizotinib, ceritinib, and alectinib had been approved by the U.S. Food and Drug Administration for patients with ALK-positive non-small cell lung cancer (NSCLC). The secondary mutations with drug-resistance bring up difficulties to develop effective drugs for ALK-positive cancers. To give a comprehensive understanding of molecular mechanism underlying inhibitor response to ALK tyrosine kinase mutations, we established an accurate assessment for the extensive profile of drug against ALK mutations by means of computational approaches. The molecular mechanics-generalized Born surface area (MM-GBSA) method based on molecular dynamics (MD) simulation was carried out to calculate relative binding free energies for receptor-drug systems. In addition, the structure-based virtual screening was utilized to screen effective inhibitors targeting wild-type ALK and the gatekeeper mutation L1196M from 3180 approved drugs. Finally, the mechanism of drug resistance was discussed, several novel potential wild-type and L1196M mutant ALK inhibitors were successfully identified.     

Exploring the crizotinib resistance mechanism of NSCLC with the L1196M mutation using molecular dynamics simulation

Crizotinib is an anticancer tyrosine kinase inhibitor that is approved for use as a first-line treatment for some non-small-cell lung cancers. L1196M is the most frequently observed mutation in NSCLC patients. This mutation, known as the gatekeeper mutation in the ALK kinase domain, confers resistance to crizotinib by sterically blocking the binding of the drug. However, the molecular mechanism of crizotinib resistance caused by the L1196M mutation is still unclear. Molecular dynamics simulation was therefore utilized in this study to investigate the mechanism by which the L1196M mutation may affect crizotinib resistance. Our results suggest that larger fluctuations in some important regions of the mutant complex compared to the wild-type complex may contribute to the resistance of the mutant complex to crizotinib. Also, mutation-induced alterations to the secondary structure of the complex as well as unstable hydrogen-bonding patterns in the A-loop and P-loop regions decrease the total binding energy of the complex. This study therefore provides a molecular explanation for the resistance to crizotinib caused by the L1196M mutation, which could aid the design of more efficient and selective drugs.     

Hydrogen bonding analysis of phosphoric acid–N,N-dimethylformamide mixtures

Crizotinib is the most effective and the only drug that has been approved for the treatment of anaplastic lymphoma kinase (ALK)-positive lung cancer. Reports suggest that there is a development of an acquired resistance against crizotinib action due to the emergence of several mutations in the ALK gene and F1174L is one such mutation. In this study, we used molecular docking and molecular dynamics (MD) approach to decipher the effect of F1174L mutation in drug–target binding. Docking results suggest that crizotinib was found to adopt the most promising conformations to the native-type ALK by identifying the M1199 residue as a prospective partner for making a hydrogen bond as compared to the mutant-type ALK. MD results showed that the average atom, especially atoms of the native-type ALK-crizotinib complex, movements were less, displayed less fluctuation, fast convergence of energy, and changes in geometry. This shows the stable binding of crizotinib with the native-type ALK in comparison to the mutant-type ALK. We believe that this study could be useful for the logical design of stronger, more selective, and more consistent ALK inhibitor against drug-resistant F1174L mutation.     

Molecular docking studies shows tivozanib and lapatinib as potential inhibitors of EML4-ALK translocation mediated fusion protein in non small cell lung cancer

Identification of activating mutations in non-small cell lung cancers (NSCLC) has been a focus in recent years. This led to successful evidence of using tyrosine kinase inhibitors (TKIs) over the standard platinum doublet based chemotherapy as the first line treatment in the metastatic setting.The rearrangements of fusion protein EML4-ALK in NSCLC lead to the use of crizotinib for this class of tumors. Preclinical and Phase 1 clinical studies show that ceritinib is more effective against both crizotinib sensitive and resistant tumors. Although robust responses to crizotinib are observed in NSCLC harboring ALK mutations, majority of tumors eventually become resistant, posing a major challenge in treatment course. Thus, there is a need for the identification and development of second-generation of ALK inhibitors. Computer aided molecular docking data show Tivozanib and Lapatinib bind EML4-ALK with high score. Tivozanib is in clinical trials for renal cell cancer and Lapatinib is a known dual tyrosine kinase inhibitor effective in breast cancer patients with HER2 over-expression. Additional data on these compounds for use in EML4-ALK positive NSCLC will provide evidence for use in patients treated with crizotinib. Data shows the importance of computer aided molecular docking in developing candidates with improved activity for further consideration in vitro and in vivo validation.     

Structural basis for drug resistance mechanisms against anaplastic lymphoma kinase

Drug resistance to anaplastic lymphoma kinase (ALK) inhibitors (crizotinib and ceritinib) is caused by mutation in the region encoding kinase domain of ALK. Compounds with potential ability to inhibit all strains of ALK are a solution to tackle the problem of drug resistance. In this study, we delineated positions of residues possessing the ability to make ALK drug resistant upon mutation by assessing them using five parameters (conservation index, binding-site root-mean-square deviation, protein structure stability, change in ATP, and drug-binding affinity). Four residual positions (Leu 1122, Thr 1151, Phe 1245, and Gly 1269) were ascertained. This study will be beneficial for designing drugs with better proficiency against ALK and the issues of drug resistance. This study can be taken as a pipeline for investigating drug-resistant mutations in other diseases as well.     

Genomic Aberrations in Crizotinib Resistant Lung Adenocarcinoma Samples Identified by Transcriptome Sequencing

ALK-break positive non-small cell lung cancer (NSCLC) patients initially respond to crizotinib, but resistance occurs inevitably. In this study we aimed to identify fusion genes in crizotinib resistant tumor samples. Re-biopsies of three patients were subjected to paired-end RNA sequencing to identify fusion genes using deFuse and EricScript. The IGV browser was used to determine presence of known resistance-associated mutations. Sanger sequencing was used to validate fusion genes and digital droplet PCR to validate mutations. ALK fusion genes were detected in all three patients with EML4 being the fusion partner. One patient had no additional fusion genes. Another patient had one additional fusion gene, but without a predicted open reading frame (ORF). The third patient had three additional fusion genes, of which two were derived from the same chromosomal region as the EML4-ALK. A predicted ORF was identified only in the CLIP4-VSNL1 fusion product. The fusion genes validated in the post-treatment sample were also present in the biopsy before crizotinib. ALK mutations (p.C1156Y and p.G1269A) detected in the re-biopsies of two patients, were not detected in pre-treatment biopsies. In conclusion, fusion genes identified in our study are unlikely to be involved in crizotinib resistance based on presence in pre-treatment biopsies. The detection of ALK mutations in post-treatment tumor samples of two patients underlines their role in crizotinib resistance.     

The R1275Q Neuroblastoma Mutant and Certain ATP-competitive Inhibitors Stabilize Alternative Activation Loop Conformations of Anaplastic Lymphoma Kinase

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that, when genetically altered by mutation, amplification, chromosomal translocation or inversion, has been shown to play an oncogenic role in certain cancers. Small molecule inhibitors targeting the kinase activity of ALK have proven to be effective therapies in certain ALK-driven malignancies and one such inhibitor, crizotinib, is now approved for the treatment of EML4-ALK-driven, non-small cell lung cancer. In neuroblastoma, activating point mutations in the ALK kinase domain can drive disease progression, with the two most common mutations being F1174L and R1275Q. We report here crystal structures of the ALK kinase domain containing the F1174L and R1275Q mutations. Also included are crystal structures of ALK in complex with novel small molecule ALK inhibitors, including a classic type II inhibitor, that stabilize previously unobserved conformations of the ALK activation loop. Collectively, these structures illustrate a different series of activation loop conformations than has been observed in previous ALK crystal structures and provide insight into the activating nature of the R1275Q mutation. The novel active site topologies presented here may also aid the structure-based drug design of a new generation of ALK inhibitors.     

Oral Metronomic Topotecan Sensitizes Crizotinib Antitumor Activity in ALKF1174L Drug-Resistant Neuroblastoma Preclinical Models

BACKGROUND: Anaplastic lymphoma kinase (ALK) inhibitor crizotinib has proven to be effective in the treatment of ALK-mutated neuroblastoma, but crizotinib resistance was commonly observed in patients. We aimed to overcome crizotinib resistance by combining with the MEK inhibitor trametinib or low-dose metronomic (LDM) topotecan in preclinical neuroblastoma models. METHODS: We selected a panel of neuroblastoma cell lines carrying various ALK genetic aberrations to assess the therapeutic efficacy on cell proliferation in vitro. Downstream signals of ALK activation, including phosphorylation of ERK1/2, Akt as well as HIF-1α expression were evaluated under normoxic and hypoxic conditions. Tumor growth inhibition was further assessed in NOD/SCID xenograft mouse models. RESULTS: All NBL cell lines responded to crizotinib treatment but at variable ED50 levels, ranging from 0.25 to 5.58 μM. ALK-mutated cell lines SH-SY5Y, KELLY, LAN-5, and CHLA-20 are more sensitive than ALK wild-type cell lines. In addition, we demonstrated that under hypoxic conditions, all NBL cell lines showed marked decrease of ED50s when compared to normoxia except for KELLY cells. Taking into consideration the hypoxia sensitivity to crizotinib, combined treatment with crizotinib and LDM topotecan demonstrated a synergistic effect in ALK^F1174L-mutated SH-SY5Y cells. In vivo, single-agent crizotinib showed limited antitumor activity in ALK^F1174L-mutated SH-SY5Y and KELLY xenograft models; however, when combined with topotecan, significantly delayed tumor development was achieved in both SH-SY5Y and KELLY tumor models. CONCLUSIONS: Oral metronomic topotecan reversed crizotinib drug resistance in the ALK^F1174L-mutated neuroblastoma preclinical model.     

Virtual screening and further development of novel ALK inhibitors

Anaplastic lymphoma kinase (ALK) has been in the spotlight in recent years as a promising new target for therapy of non-small-cell lung cancer (NSCLC). Since the identification of the echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion gene in some NSCLC patients was reported in 2007, various research groups have been seeking ALK inhibitors. Above all, crizotinib (PF-02341066) has been under clinical trial, and its therapeutic efficacy of inhibiting ALK in NSCLC has been reported. Among anticancer drugs, drug resistance appears frequently necessitating various kinds of inhibitors. We identified novel ALK inhibitors by virtual screening from the public chemical library collected by the Chemical Biology Research Initiative (CBRI) at the University of Tokyo, and inhibitors that are more potent were developed.     

A major role for a set of non-active site mutations in the development of HIV-1 protease drug resistance

A major problem in the chemotherapy of HIV-1 infection is the appearance of drug resistance. In the case of HIV-1 protease inhibitors, resistance originates from mutations in the protease molecule that lower the affinity of inhibitors while still maintaining a viable enzymatic profile. Drug resistance mutations can be classified as active site or non-active site mutations depending on their location within the protease molecule. Active site mutations directly affect drug/target interactions, and their action can be readily understood in structural terms. Non-active site mutations influence binding from distal locations, and their mechanism of action is not immediately apparent. In this paper, we have characterized a mutant form of the HIV-1 protease, ANAM-11, identified in clinical isolates from HIV-1 infected patients treated with protease inhibitors. This mutant protease contains 11 mutations, 10 of which are located outside the active site (L10I/M36I/S37D/M46I/R57K/L63P/A71V/G73S/L90M/I93L) and 1 within the active site (I84V). ANAM-11 lowers the binding affinity of indinavir, nelfinavir, saquinavir, and ritonavir by factors of 4000, 3300, 5800, and 80000, respectively. Surprisingly, most of the loss in inhibitor affinity is due to the non-active site mutations as demonstrated by additional experiments performed with a protease containing only the 10 non-active site mutations (NAM-10) and another containing only the active site mutation (A-1). Kinetic analysis with two different substrates yielded comparable catalytic efficiencies for A-1, ANAM-11, NAM-10, and the wild-type protease. These studies demonstrate that non-active site mutations can be the primary source of resistance and that their role is not necessarily limited to compensate deleterious effects of active site mutations. Analysis of the structural stability of the proteases by differential scanning calorimetry reveals that ANAM-11 and NAM-10 are structurally more stable than the wild-type protease while A-1 is less stable. Together, the binding and structural thermodynamic results suggest that the non-active site mutants affect inhibitor binding by altering the geometry of the binding site cavity through the accumulation of mutations within the core of the protease molecule.     

ZX-29, a novel ALK inhibitor,induces apoptosis via ER stress in ALK rearrangement NSCLC cells and overcomes cell resistance caused by an ALK mutation

Although anaplastic lymphoma kinase (ALK) inhibitors have good clinical efficacy, the inevitable development of drug resistance is the most common obstacle to their clinical application. There is an urgent need to develop more effective and selective ALK inhibitors to overcome the problem of drug resistance. Here, we screened a series of ALK inhibitors and found that ZX-29 displayed potent cytotoxic activity against ALK rearrangement non-small cell lung cancer (NSCLC) NCI-H2228 cells. Then, we investigated the antitumor effects of ZX-29. We demonstrated that ZX-29 time- and dose-dependently inhibited the viability of NCI-H2228 cells, induced cell cycle arrest in the G1 phase, and then they subsequently progressed into cell death. The type of cell death induced by ZX-29 was apoptosis through endoplasmic reticulum (ER) stress. Interestingly, ZX-29 induced protective autophagy, and inhibiting autophagy could enhance the antitumor effect of ZX-29. Furthermore, ZX-29 suppressed tumor growth in a mouse xenograft model. More importantly, ZX-29 could overcome the drug resistance caused by the ALK G1202R mutation. In conclusion, we demonstrated that ZX-29 showed excellent anti-ALK rearrangement NSCLC activity in vitro and in vivo and overcame the drug resistance caused by an ALK mutation. Therefore, ZX-29 is a promising antitumor drug targeting ALK rearrangement or ALK G1202R mutation NSCLC.     

N-[18F]-Fluoroacetylcrizotinib: A potentially potent and selective PET tracer for molecular imaging of non-small cell lung cancer

N-[¹⁸F]fluoroacetylcrizotinib, a fluorine-18 labeled derivative of the first FDA approved tyrosine kinase inhibitor (TKI) for the treatment of Anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC), crizotinib, was successfully synthesized for use in positron emission tomography (PET). Sequential in vitro biological evaluation of fluoracetylcrizotinib and in vivo biodistribution studies of [¹⁸F]fluoroacetylcrizotinib demonstrated that the biological activity of the parent compound remained unchanged, with potent ALK kinase inhibition and effective tumor growth inhibition. These results show that [¹⁸F]fluoroacetylcrizotinib has the potential to be a promising PET ligand for use in NSCLC imaging. The utility of PET in this context provides a non-invasive, quantifiable method to inform on the pharmacokinetics of an ALK-inhibitor such as crizotinib prior to a clinical trial, as well as during a trial in the event of acquired drug resistance.     

Exploring the drug resistance mechanism of active site,non-active site mutations and their cooperative effects in CRF01_AE HIV-1 protease: molecular dynamics simulations and free energy calculations

Human immunodeficiency virus type 1 protease is essential for virus replication and maturation and has been considered as one of the important drug target for the antiretroviral treatment of HIV infection. The majority of HIV infections are caused due to non-B subtypes in developing countries. Subtype AE is spreading rapidly and infecting huge population worldwide. Understanding the interdependence of active and non-active site mutations in conferring drug resistance is crucial for the development effective inhibitors in subtype AE protease. In this work, we have investigated the mechanism of resistance against indinavir (IDV) due to therapy selected active site mutation V82F, non-active site mutations PF82V and their cooperative effects PV82F in subtype AE-protease using molecular dynamics simulations and binding free energy calculations. The simulations suggested all the three complexes lead to decrease in binding affinity of IDV, whereas the PF82V complex resulted in an enhanced binding affinity compared to V82F and PV82F complexes. Large positional deviation of IDV was observed in V82F complex. The preservation of hydrogen bonds of IDV with active site Asp25/Asp25′ and flap residue Ile50/50′ via a water molecule is crucial for effective binding. Owing to the close contact of 80s loop with Ile50′ and Asp25, the alteration between residues Thr80 and Val82, further induces conformational change thereby resulting in loss of interactions between IDV and the residues in the active site cavity, leading to drug resistance. Our present study shed light on the effect of active, non-active site mutations and their cooperative effects in AE protease.

Communicated by Ramaswamy H. Sarma     

Will the clinical development of 4th-generation “double mutant active” ALK TKIs (TPX-0131 and NVL-655) change the future treatment paradigm of ALK+ NSCLC?

Our current treatment paradigm of advanced anaplastic lymphoma kinase fusion (ALK+) non-small cell lung cancer (NSCLC) classifies the six currently approved ALK tyrosine kinase inhibitors (TKIs) into three generations. The 2nd-generation (2G) and 3rd-generation (3G) ALK TKIs are all “single mutant active” with varying potencies across a wide spectrum of acquired single ALK resistance mutations. There is a vigorous debate among clinicians which is the best upfront ALK TKI is for the first-line (1L) treatment of ALK+ NSCLC and the subsequent sequencing strategies whether it should be based on the presence of specific on-target ALK resistance mutations or not. Regardless, sequential use of “single mutant active” ALK TKIs will eventually lead to double ALK resistance mutations in cis. This has led to the creation of fourth generation (4G) “double mutant active” ALK TKIs such as TPX-0131 and NVL-655. We discuss the critical properties 4G ALK TKIs must possess to be clinically successful. We proposed conceptual first-line, second-line, and molecularly-based third-line registrational randomized clinical trials designed for these 4G ALK TKIs. How these 4G ALK TKIs would be used in the future will depend on which line of treatment the clinical trial design(s) is adopted provided the trial is positive. If approved, 4G ALK TKIs may usher in a new treatment paradigm for advanced ALK+ NSCLC that is based on classifying ALK TKIs based on the intrinsic functional capabilities (“singe mutant active” versus “double mutant active”) rather than the loosely-defined “generational” (first-, second-,third-,fourth-) classification and avoid the current clinical approaches of seemingly random sequential use of 2G and 3G ALK TKIs.     

ALK Kinase Domain Mutations in Primary Anaplastic Large Cell Lymphoma: Consequences on NPM-ALK Activity and Sensitivity to Tyrosine Kinase Inhibitors

ALK inhibitor crizotinib has shown potent antitumor activity in children with refractory Anaplastic Large Cell Lymphoma (ALCL) and the opportunity to include ALK inhibitors in first-line therapies is oncoming. However, recent studies suggest that crizotinib-resistance mutations may emerge in ALCL patients. In the present study, we analyzed ALK kinase domain mutational status of 36 paediatric ALCL patients at diagnosis to identify point mutations and gene aberrations that could impact on NPM-ALK gene expression, activity and sensitivity to small-molecule inhibitors. Amplicon ultra-deep sequencing of ALK kinase domain detected 2 single point mutations, R335Q and R291Q, in 2 cases, 2 common deletions of exon 23 and 25 in all the patients, and 7 splicing-related INDELs in a variable number of them. The functional impact of missense mutations and INDELs was evaluated. Point mutations were shown to affect protein kinase activity, signalling output and drug sensitivity. INDELs, instead, generated kinase-dead variants with dominant negative effect on NPM-ALK kinase, in virtue of their capacity of forming non-functional heterocomplexes. Consistently, when co-expressed, INDELs increased crizotinib inhibitory activity on NPM-ALK signal processing, as demonstrated by the significant reduction of STAT3 phosphorylation. Functional changes in ALK kinase activity induced by both point mutations and structural rearrangements were resolved by molecular modelling and dynamic simulation analysis, providing novel insights into ALK kinase domain folding and regulation. Therefore, these data suggest that NPM-ALK pre-therapeutic mutations may be found at low frequency in ALCL patients. These mutations occur randomly within the ALK kinase domain and affect protein activity, while preserving responsiveness to crizotinib.     

Will the clinical development of 4th-generation “double mutant active” ALK TKIs (TPX-0131 and NVL-655) change the future treatment paradigm of ALK+ NSCLC?

Our current treatment paradigm of advanced anaplastic lymphoma kinase fusion (ALK+) non-small cell lung cancer (NSCLC) classifies the six currently approved ALK tyrosine kinase inhibitors (TKIs) into three generations. The 2nd-generation (2G) and 3rd-generation (3G) ALK TKIs are all “single mutant active” with varying potencies across a wide spectrum of acquired single ALK resistance mutations. There is a vigorous debate among clinicians which is the best upfront ALK TKI is for the first-line (1L) treatment of ALK+ NSCLC and the subsequent sequencing strategies whether it should be based on the presence of specific on-target ALK resistance mutations or not. Regardless, sequential use of “single mutant active” ALK TKIs will eventually lead to double ALK resistance mutations in cis. This has led to the creation of fourth generation (4G) “double mutant active” ALK TKIs such as TPX-0131 and NVL-655. We discuss the critical properties 4G ALK TKIs must possess to be clinically successful. We proposed conceptual first-line, second-line, and molecularly-based third-line registrational randomized clinical trials designed for these 4G ALK TKIs. How these 4G ALK TKIs would be used in the future will depend on which line of treatment the clinical trial design(s) is adopted provided the trial is positive. If approved, 4G ALK TKIs may usher in a new treatment paradigm for advanced ALK+ NSCLC that is based on classifying ALK TKIs based on the intrinsic functional capabilities (“singe mutant active” versus “double mutant active”) rather than the loosely-defined “generational” (first-, second-,third-,fourth-) classification and avoid the current clinical approaches of seemingly random sequential use of 2G and 3G ALK TKIs.     

Clinical atovaquone-proguanil resistance of Plasmodium falciparum associated with cytochrome b codon 268 mutations

Plasmodium falciparum resistance to atovaquone-proguanil has so far been associated with Y268S or Y268N mutations in cytochrome b, although these changes were identified in only seven of the 11 treatment failures. Here, we describe 10 new cases of atovaquone-proguanil treatment failures among which the parasite resistance was confirmed in six cases, either by identifying correct plasma drug concentrations or by observing in vitro atovaquone resistance. Resistance was consistently associated with codon 268 mutations (Y268S or a previously unidentified mutation, Y268C). Notably, mutations were not detected before the treatment but only after the drug exposure.     

Gaining insight into crizotinib resistance mechanisms caused by L2026M and G2032R mutations in ROS1 via molecular dynamics simulations and free-energy calculations

ROS1 fusion kinase—highly expressed in a variety of human cancers—has emerged as an important and attractive target for anticancer drug design. Crizotinib, a well-known drug approved by the FDA as an ALK inhibitor to treat advanced NSCLC, also shows potent inhibitoy activity against ROS1. However, the development of serious resistance due to secondary mutations has been observed in clinical studies. To provide insight into the mechanisms of this drug resistance, molecular dynamics simulations and free-energy calculations were carried out for complexes of crizotinib with wild-type (WT) ROS1 as well as the mutated L2026M and G2032R forms. MD simulations indicated that the L2026M and G2032R systems are slightly less flexible than the WT system. Binding free energy calculations showed that the L2026M and G2032R mutations significantly reduce the binding affinity of crizotinib for ROS1, and that the resistance to crizotinib caused by the L2026M and G2032R mutations arises mostly from increases in entropic terms. Furthermore, calculations of per-residue binding free energies highlighted increased and decreased contributions of some residues in the L2026M and G2032R systems relative to those in the WT system. The present study therefore yielded detailed insight into the mechanisms of resistance to crizotinib caused by the L2026M and G2032R mutations, which should provide the basis for rational drug design to combat crizotinib resistance.

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Graphical Abstract Superposition of the average structures obtained from the last 10 ns of the molecular dynamics simulation trajectoriy for WT (green) and mutated ROS1 (cyan)

     

Internalization and down-regulation of the ALK receptor in neuroblastoma cell lines upon monoclonal antibodies treatment

Recently, activating mutations of the full length ALK receptor, with two hot spots at positions F1174 and R1275, have been characterized in sporadic cases of neuroblastoma. Here, we report similar basal patterns of ALK phosphorylation between the neuroblastoma IMR-32 cell line, which expresses only the wild-type receptor (ALK(WT)), and the SH-SY5Y cell line, which exhibits a heterozygous ALK F1174L mutation and expresses both ALK(WT) and ALK(F1174L) receptors. We demonstrate that this lack of detectable increased phosphorylation in SH-SY5Y cells is a result of intracellular retention and proteasomal degradation of the mutated receptor. As a consequence, in SH-SY5Y cells, plasma membrane appears strongly enriched for ALK(WT) whereas both ALK(WT) and ALK(F1174L) were present in intracellular compartments. We further explored ALK receptor trafficking by investigating the effect of agonist and antagonist mAb (monoclonal antibodies) on ALK internalization and down-regulation, either in SH-SY5Y cells or in cells expressing only ALK(WT). We observe that treatment with agonist mAbs resulted in ALK internalization and lysosomal targeting for receptor degradation. In contrast, antagonist mAb induced ALK internalization and recycling to the plasma membrane. Importantly, we correlate this differential trafficking of ALK in response to mAb with the recruitment of the ubiquitin ligase Cbl and ALK ubiquitylation only after agonist stimulation. This study provides novel insights into the mechanisms regulating ALK trafficking and degradation, showing that various ALK receptor pools are regulated by proteasome or lysosome pathways according to their intracellular localization.