Anaplastic Lymphoma Kinase Phosphorylation Research Articles

Lysophosphatidic Acid Stimulates Mitogenic Activity and Signaling in Human Neuroblastoma Cells through a Crosstalk with Anaplastic Lymphoma Kinase.

Lysophosphatidic acid (LPA) is a well-documented pro-oncogenic factor in different cancers, but relatively little is known on its biological activity in neuroblastoma. The LPA effects and the participation of the tyrosine kinase receptor anaplastic lymphoma kinase (ALK) in LPA mitogenic signaling were studied in human neuroblastoma cell lines. We used light microscopy and [3H]-thymidine incorporation to determine cell proliferation, Western blot to study intracellular signaling, and pharmacological and molecular tools to examine the role of ALK. We found that LPA stimulated the growth of human neuroblastoma cells, as indicated by the enhanced cell number, clonogenic activity, and DNA synthesis. These effects were curtailed by the selective ALK inhibitors NPV-TAE684 and alectinib. In a panel of human neuroblastoma cell lines harboring different ALK genomic status, the ALK inhibitors suppressed LPA-induced phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), which are major regulators of cell proliferation. ALK depletion by siRNA treatment attenuated LPA-induced ERK1/2 activation. LPA enhanced ALK phosphorylation and potentiated ALK activation by the ALK ligand FAM150B. LPA enhanced the inhibitory phosphorylation of the tumor suppressor FoxO3a, and this response was impaired by the ALK inhibitors. These results indicate that LPA stimulates mitogenesis of human neuroblastoma cells through a crosstalk with ALK.

Abstract 987: Novel ALK mutations in EML4::ALK+ NSCLC resistant to TKIs identified by liquid biopsy

Abstract The identification of Anaplastic Lymphoma Kinase (ALK) chromosomal translocations led to the development of effective targeted therapies in a subset of Non-Small Cell Lung Cancer (NSCLC) patients. Several drugs are currently in clinical practice for advanced ALK+ NSCLC. However, resistance remains a challenge in these patients. The possibility to analyze circulating tumor DNA (ctDNA) with non-invasive methods can greatly improve prognostic capabilities, by allowing early detection of relapse and of drug-resistant mutations from peripheral blood. We report a series of 15 consecutive relapsed NSCLC patients (12 ALK+, 3 ROS1+) treated at the Fondazione IRCCS-San Gerardo dei Tintori (Monza, Italy) that were investigated by liquid biopsy at tyrosine kinase inhibitor (TKI) failure, through amplicon deep sequencing of the ALK/ROS1 kinase domains. Median DNA yield was 4.8 ng per ml of plasma and mean fragment size was 176 bp. Mutational hotspot coding regions of ALK and ROS1 genes were amplified by high-fidelity PCR and sequenced. Mean coverage of called variants was 24,687x. The threshold for mutation calling was set at 0.6%, after correcting for mapping quality, minimum coverage, and strand bias. Analysis of background signal on healthy control DNA identified 0.0024 mismatches per mapped base. Overall, plasma ctDNA analysis detected an ALK mutation in 7/18 relapse samples from 7/15 patients. No ROS1 mutations were found in ROS1 fusion-positive patients. Among 12 patients carrying an EML4::ALK fusion, 10 variants potentially driving resistance were found. Along with mutations that were previously identified (L1196M/G1202R, E1154K, F1174L), novel ALK variants were detected such as L1198R, T1211I, C1235R, C1237Y, L1196P. Mutations were characterized for sensitivity to ALK inhibitors in a BaF3-EML4::ALK cell model, using cell proliferation assays and western blotting to evaluate the effects on ALK phosphorylation. C1237Y and L1196P mutants appeared to resist all TKIs; hence, we further tested their sensitivity to novel investigational drugs, zotizalkib and repotrectinib. Both mutants demonstrated resistance, suggesting that these two mutations confer broad resistance to TKIs. Molecular modelling of C1237Y and L1196P mutants was run to elucidate the mechanisms of resistance. The ADP-C1237Y complex showed a much smaller root-mean-square deviation than ADP-WT, indicating a higher affinity/activity of the mutant. L1196 interacts hydrophobically with a methyl group of lorlatinib, P1196 is predicted to lose such interaction, while ATP binding is not affected. The presence of C1237Y and L1196P variants, is a relevant finding due to their resistance, even after the exposure to the fourth-generation inhibitor zotizalkib. The clonal selection of these mutations, made by second/third generation ALK TKIs may lead to a complete refractory status, which would then render TKIs useless in a sequential therapeutic strategy. Citation Format: Federica Malighetti, Matteo Villa, Elisa Sala, Geeta Sharma, Giulia Arosio, Maria Gemelli, Chiara Manfroni, Diletta Fontana, Nicoletta Cordani, Raffaella Meneveri, Alfonso Zambon, Rocco Piazza, Fabio Pagni, Diego Cortinovis, Luca Mologni. Novel ALK mutations in EML4::ALK+ NSCLC resistant to TKIs identified by liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 987.

Abstract 4320: Interrogation of autophagy inhibition in anaplastic large cell lymphoma (ALCL)

Abstract The objectives of this study are to evaluate if anaplastic lymphoma kinase (ALK) inhibition activates autophagy in ALK+ ALCL cells and to determine if that activation can be mitigated using autophagy inhibition. ALCL is an aggressive T-cell non-Hodgkin lymphoma that often presents as advanced stage disease. With the addition of crizotinib or brentuximab vedotin to the ALCL99 chemotherapy backbone, the recently reported Children’s Oncology Group ANHL12P1 study demonstrated a 2-year event-free survival for ALCL patients of 79%. Thus, even with multi-agent chemotherapy combined with targeted therapy, improved therapeutic efficacy is needed. Autophagy is activated when mTORC1 is inhibited, and as ALK signaling is known to promote mTORC1 activity, ALK inhibition is hypothesized to activate autophagy. If present, ALK inhibitor-induced autophagy activation would represent a novel therapeutic opportunity targetable with emerging autophagy inhibitors. An ALK+ ALCL cell line, Karpas 299, was treated with drug combinations for 3 hours, lysates prepared, and proteins separated by molecular weight using standard procedures. ALK inhibitor target engagement was assessed by western blot using phospho-ALKY1604 intensity in 9-point dose response curves. Target engagement of autophagy inhibition (ULK-101 directed against ULK1/2 and hydroxychloroquine (HCQ) targeting the lysosome) was assessed by western blot using phospho-ATG14S29 and LC3B-II intensity in 6-point dose response curves. Cell viability was evaluated using a luminescent CellTiter-Glo assay. Karpas299 cells resistant to crizotinib at 50nM (KCrizR50) were generated by gradually increasing crizotinib concentration in the media until cells grew at a rate similar to parental cells. Crizotinib, ceritinib, and lorlatinib all effectively inhibit ALK phosphorylation in Karpas 299 cells with low nanomolar potency. ULK-101 effectively inhibited pATG14S29 at 5 µM and HCQ effectively inhibited autophagic flux, as evidenced by increased LC3B-II intensity, at 20 µM. When treated with the lowest ALK inhibitor dose needed to achieve complete pALK inhibition (IC99), pATG14 intensity increased representing increased autophagic activity. When combined with ALK inhibition, ULK-101 effectively decreased pATG14S29, blocking the ALK inhibitor-induced autophagy activation. Decreased viability in Karpas 299 cells was observed after crizotinib, ceritinib and lorlatinib treatments with nanomolar potency. KCrizR50 cells had a potency higher than parental Karpas 299 cells for all three ALK inhibitors. ALK inhibition activates autophagy in ALCL cells and autophagy activation is effectively mitigated through concurrent autophagy inhibition. ALK inhibitor resistance in Karpas 299 cells will be further evaluated to determine whether autophagy contributes to the resistant phenotype. Citation Format: Katie L. Buelow, Stephanie Celano, Gregory Poole, Katie Martin, Jeffrey MacKeigan, David J. Hoogstra. Interrogation of autophagy inhibition in anaplastic large cell lymphoma (ALCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4320.

Preclinical evidence for anaplastic lymphoma kinase inhibitors as novel therapeutic treatments for cholangiocarcinoma.

Bile duct cancer (cholangiocarcinoma, CCA) has a poor prognosis for patients, and despite recent advances in targeted therapies for other cancer types, it is still treated with standard chemotherapy. Anaplastic lymphoma kinase (ALK) has been shown to be a primary driver of disease progression in lung cancer, and ALK inhibitors are effective therapeutics in aberrant ALK-expressing tumors. Aberrant ALK expression has been documented in CCA, but the use of ALK inhibitors has not been investigated. Using CCA cell lines and close-to-patient primary cholangiocarcinoma cells, we investigated the potential for ALK inhibitors in CCA. ALK, cMET, and ROS1 expression was determined in CCA patient tissue by immunohistochemistry and digital droplet polymerase chain reaction, and that in cell lines was determined by immunoblot and immunofluorescence. The effect on cell viability and mechanism of action of ALK, cMet, and ROS1 inhibitors was determined in CCA cell lines. To determine whether ceritinib could affect primary CCA cells, tissue was taken from four patients with biliary tract cancer, without ALK rearrangement, mutation, or overexpression, and grown in three-dimensional tumor growth assays in the presence or absence of humanized mesenchymal cells. ALK and cMet but not ROS were both upregulated in CCA tissues and cell lines. Cell survival was inhibited by crizotinib, a c-met/ALK/ROS inhibitor. To determine the mechanism of this effect, we tested c-Met-specific and ALK/ROS-specific inhibitors, capmatinib and ceritinib, respectively. Whereas capmatinib did not affect cell survival, ceritinib dose-dependently inhibited survival in all cell lines, with IC50 ranging from 1 to 9 µM and co-treatments with gemcitabine and cisplatin further sensitized cells, with IC50 ranging from IC50 0.60 to 2.32 µM. Ceritinib did not inhibit cMet phosphorylation but did inhibit ALK phosphorylation. ALK was not mutated in any of these cell lines. Only ceritinib inhibited 3D growth of all four patient samples below mean peak serum concentration, in the presence and absence of mesenchymal cells, whereas crizotinib and capmatinib failed to do this. Ceritinib appeared to exert its effect more through autophagy than apoptosis. These results indicate that ceritinib or other ALK/ROS inhibitors could be therapeutically useful in cholangiocarcinoma even in the absence of aberrant ALK/ROS1 expression.

Discovery of a novel ALK/ROS1/FAK inhibitor, APG-2449, in preclinical non-small cell lung cancer and ovarian cancer models

BackgroundTyrosine kinase inhibitors (TKIs) are mainstays of cancer treatment. However, their clinical benefits are often constrained by acquired resistance. To overcome such outcomes, we have rationally engineered APG-2449 as a novel multikinase inhibitor that is highly potent against oncogenic alterations of anaplastic lymphoma kinase (ALK), ROS proto-oncogene 1 receptor tyrosine kinase (ROS1), and focal adhesion kinase (FAK). Here we present the preclinical evaluation of APG-2449, which exhibits antiproliferative activity in cells carrying ALK fusion or secondary mutations.MethodsKINOMEscan® and LANCE TR-FRET were used to characterize targets and selectivity of APG-2449. Water-soluble tetrazolium salt (WST-8) viability assay and xenograft tumorigenicity were employed to evaluate therapeutic efficacy of monotherapy or drug combination in preclinical models of solid tumors. Western blot, pharmacokinetic, and flow cytometry analyses, as well as RNA sequencing were used to explore pharmacokinetic–pharmacodynamic correlations and the mechanism of actions driving drug combination synergy.ResultsIn mice bearing wild-type or ALK/ROS1-mutant non-small-cell lung cancer (NSCLC), APG-2449 demonstrates potent antitumor activity, with correlations between pharmacokinetics and pharmacodynamics in vivo. Through FAK inhibition, APG-2449 sensitizes ovarian xenograft tumors to paclitaxel by reducing CD44+ and aldehyde dehydrogenase 1-positive (ALDH1+) cancer stem cell populations, including ovarian tumors insensitive to carboplatin. In epidermal growth factor receptor (EGFR)-mutated NSCLC xenograft models, APG-2449 enhances EGFR TKI-induced tumor growth inhibition, while the ternary combination of APG-2449 with EGFR (osimertinib) and mitogen-activated extracellular signal-regulated kinase (MEK; trametinib) inhibitors overcomes osimertinib resistance. Mechanistically, phosphorylation of ALK, ROS1, and FAK, as well as their downstream components, is effectively inhibited by APG-2449.ConclusionsTaken together, our studies demonstrate that APG-2449 exerts potent and durable antitumor activity in human NSCLC and ovarian tumor models when administered alone or in combination with other therapies. A phase 1 clinical trial has been initiated to evaluate the safety and preliminary efficacy of APG-2449 in patients with advanced solid tumors, including ALK+ NSCLC refractory to earlier-generation ALK inhibitors.Trial registrationClinicaltrial.gov registration: NCT03917043 (date of first registration, 16/04/2019) and Chinese clinical trial registration: CTR20190468 (date of first registration, 09/04/2019).

The neuronal tyrosine kinase receptor ligand ALKAL2 mediates persistent pain.

The anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase known for its oncogenic potential that is involved in the development of the peripheral and central nervous system. ALK receptor ligands ALKAL1 and ALKAL2 were recently found to promote neuronal differentiation and survival. Here, we show that inflammation or injury enhanced ALKAL2 expression in a subset of TRPV1+ sensory neurons. Notably, ALKAL2 was particularly enriched in both mouse and human peptidergic nociceptors, yet weakly expressed in nonpeptidergic, large-diameter myelinated neurons or in the brain. Using a coculture expression system, we found that nociceptors exposed to ALKAL2 exhibited heightened excitability and neurite outgrowth. Intraplantar CFA or intrathecal infusion of recombinant ALKAL2 led to ALK phosphorylation in the lumbar dorsal horn of the spinal cord. Finally, depletion of ALKAL2 in dorsal root ganglia or blocking ALK with clinically available compounds crizotinib or lorlatinib reversed thermal hyperalgesia and mechanical allodynia induced by inflammation or nerve injury, respectively. Overall, our work uncovers the ALKAL2/ALK signaling axis as a central regulator of nociceptor-induced sensitization. We propose that clinically approved ALK inhibitors used for non–small cell lung cancer and neuroblastomas could be repurposed to treat persistent pain conditions.

Tyrosine phosphatases regulate resistance to ALK inhibitors in ALK+ anaplastic large cell lymphoma

AbstractAnaplastic large cell lymphomas (ALCLs) frequently carry oncogenic fusions involving the anaplastic lymphoma kinase (ALK) gene. Targeting ALK using tyrosine kinase inhibitors (TKIs) is a therapeutic option in cases relapsed after chemotherapy, but TKI resistance may develop. By applying genomic loss-of-function screens, we identified PTPN1 and PTPN2 phosphatases as consistent top hits driving resistance to ALK TKIs in ALK+ ALCL. Loss of either PTPN1 or PTPN2 induced resistance to ALK TKIs in vitro and in vivo. Mechanistically, we demonstrated that PTPN1 and PTPN2 are phosphatases that bind to and regulate ALK phosphorylation and activity. In turn, oncogenic ALK and STAT3 repress PTPN1 transcription. We found that PTPN1 is also a phosphatase for SHP2, a key mediator of oncogenic ALK signaling. Downstream signaling analysis showed that deletion of PTPN1 or PTPN2 induces resistance to crizotinib by hyperactivating SHP2, the MAPK, and JAK/STAT pathways. RNA sequencing of patient samples that developed resistance to ALK TKIs showed downregulation of PTPN1 and PTPN2 associated with upregulation of SHP2 expression. Combination of crizotinib with a SHP2 inhibitor synergistically inhibited the growth of wild-type or PTPN1/PTPN2 knock-out ALCL, where it reverted TKI resistance. Thus, we identified PTPN1 and PTPN2 as ALK phosphatases that control sensitivity to ALK TKIs in ALCL and demonstrated that a combined blockade of SHP2 potentiates the efficacy of ALK inhibition in TKI-sensitive and -resistant ALK+ ALCL.

Phase-separated foci of EML4-ALK facilitate signalling and depend upon an active kinase conformation.

Variants of the oncogenic EML4‐ALK fusion protein contain a similar region of ALK encompassing the kinase domain, but different portions of EML4. Here, we show that EML4‐ALK V1 and V3 proteins form cytoplasmic foci that contain components of the MAPK, PLCγ and PI3K signalling pathways. The ALK inhibitors ceritinib and lorlatinib dissolve these foci and EML4‐ALK V3 but not V1 protein re‐localises to microtubules, an effect recapitulated in a catalytically inactive EML4‐ALK mutant. Mutations that promote a constitutively active ALK stabilise the cytoplasmic foci even in the presence of these inhibitors. In contrast, the inhibitor alectinib increases foci formation of both wild‐type and catalytically inactive EML4‐ALK V3 proteins, but not a Lys‐Glu salt bridge mutant. We propose that EML4‐ALK foci formation occurs as a result of transient association of stable EML4‐ALK trimers mediated through an active conformation of the ALK kinase domain. Our results demonstrate the formation of EML4‐ALK cytoplasmic foci that orchestrate oncogenic signalling and reveal that their assembly depends upon the conformational state of the catalytic domain and can be differentially modulated by structurally divergent ALK inhibitors.

Mapping the Phospho-dependent ALK Interactome to Identify Novel Components in ALK Signaling

Protein-protein interactions (PPIs) play essential roles in Anaplastic Lymphoma Kinase (ALK) signaling. Systematic characterization of ALK interactors helps elucidate novel ALK signaling mechanisms and may aid in the identification of novel therapeutics targeting related diseases. In this study, we used the Mammalian Membrane Two-Hybrid (MaMTH) system to map the phospho-dependent ALK interactome. By screening a library of 86 SH2 domain-containing full length proteins, 30 novel ALK interactors were identified. Many of their interactions are correlated to ALK phosphorylation activity: oncogenic ALK mutations potentiate the interactions and ALK inhibitors attenuate the interactions. Among the novel interactors, NCK2 was further verified in neuroblastoma cells using co-immunoprecipitation. Modulation of ALK activity by addition of inhibitors lead to concomitant changes in the tyrosine phosphorylation status of NCK2 in neuroblastoma cells, strongly supporting the functionality of the ALK/NCK2 interaction. Our study provides a resource list of potential novel ALK signaling components for further study.

Anaplastic lymphoma kinase inhibitor NVP‑TAE684 suppresses the proliferation of human pancreatic adenocarcinoma cells.

Anaplastic lymphoma kinase (ALK) is known to be an important therapeutic target in various types of cancer. NVP‑TAE684, a well‑known inhibitor of ALK, was revealed to exert antitumor effects in several different malignancies. However, the molecular mechanisms responsible for these antitumor effects in cancer cells, including pancreatic adenocarcinoma cells, remain unknown. In the present study, NVP‑TAE684 was investigated for its antitumor effects towards pancreatic adenocarcinoma cells. MTT assay, western blot analysis, flow cytometry, caspase‑3/7 activity assay and Trypan blue exclusion assay were used and it was revealed that NVP‑TAE684 suppressed the proliferation of seven human pancreatic adenocarcinoma cell lines (AsPC‑1, Panc‑1, MIA PaCa‑2, Capan‑1, CFPAC‑1, Colo‑357 and BxPC‑3), and significantly increased G2/M arrest and apoptotic cell death. Furthermore, NVP‑TAE684 inhibited the phosphorylation of ALK at Y1604, as well as that of downstream mediators such as AKT(S473) and ERK1/2(Y202/T204). Notably, knocking down ALK with siRNAs also decreased proliferation and promoted G2/M arrest and apoptosis. Furthermore, inhibition of ALK with NVP‑TAE684 or siRNA synergistically enhanced gemcitabine‑induced cell death by inducing apoptosis. In conclusion, the findings of the present study indicated that NVP‑TAE684 exerted its antitumor effects by inducing G2/M arrest and apoptosis via the inhibition of the ALK signaling pathway, and suggests its potential use as an antitumor agent against pancreatic adenocarcinoma.

Monomerization of ALK Fusion Proteins as a Therapeutic Strategy in ALK-Rearranged Non-small Cell Lung Cancers.

Objective: Oncogenic echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) (EML4-ALK) fusion proteins found in non-small cell lung cancers (NSCLC) are constitutively phosphorylated and activated by dimerization via the coiled-coil domain (cc) within EML4. Here, we investigated whether disruption of ALK fusion protein oligomerization via competitive cc mimetic compounds could be a therapeutic strategy for EML4-ALK NSCLC.Methods: A Ba/F3 cell model was created that expressed an ALK intracellular domain in which the dimer/monomer state is ligand-regulated. This novel cell model was used to investigate the effect of disrupting ALK fusion protein oligomerization on tumor cell growth in vitro and in vivo using nude mice. Subsequently, the antiproliferative effects of endogenous cc domain co-expression and mimetic cc peptides were assayed in EML4-ALK cancer cell lines.Results: Cells induced to express monomeric ALK in vitro did not survive. When transplanted into mice, induction of monomers abrogated tumor formation. Using a fluorescent protein system to quantify protein-protein interactions of EML4-ALK and EML4cc, we demonstrated that co-expression of EML4cc suppressed EML4-ALK assembly concomitant with decreasing the rate of tumor growth in vitro and in vivo. In EML4-ALK cancer cell lines, administration of synthetic EML4cc peptide elicited a decrease of phosphorylation of ALK leading to reduction in tumor cell growth.Conclusions: Our findings support the monomerization of ALK fusion proteins using EML4cc peptides for competitive inhibition of dimerization as a promising therapeutic strategy for EML4-ALK NSCLC. Further studies are warranted to explore the use of specific cc peptide as a therapeutic option for other lung cancers harboring driver fusion genes containing a cc or oligomerization domain within the fusion partner.

Anaplastic Lymphoma Kinase Regulates Internalization of the Dopamine D2 Receptor.

The dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) expressed in regions of the brain that control motor function, cognition, and motivation. As a result, D2R is involved in the pathophysiology of disorders such as schizophrenia and drug addiction. Understanding the signaling pathways activated by D2R is crucial to finding new therapeutic targets for these disorders. D2R stimulation by its agonist, dopamine, causes desensitization and internalization of the receptor. A previous study found that inhibitors of the receptor tyrosine kinase anaplastic lymphoma kinase (ALK) blocked D2R desensitization in neurons in the ventral tegmental area of the brain. In the present study, using a cell-based system, we investigated whether ALK regulates D2R internalization. The ALK inhibitor alectinib completely inhibited dopamine-induced D2R internalization. Since GPCRs can transactivate receptor tyrosine kinases, we also examined if D2R stimulation activated ALK signaling. ALK phosphorylation increased by almost 2-fold after dopamine treatment and ALK coimmunoprecipitated with D2R. To identify the signaling pathways downstream of ALK that might regulate D2R internalization, we used pharmacological inhibitors of proteins activated by ALK signaling. Protein kinase Cγ was activated by dopamine in an ALK-dependent manner, and a protein kinase C inhibitor completely blocked dopamine-induced D2R internalization. Taken together, these results identify ALK as a receptor tyrosine kinase transactivated by D2R that promotes its internalization, possibly through activation of protein kinase C. ALK inhibitors could be useful in enhancing D2R signaling. SIGNIFICANCE STATEMENT: Receptor internalization is a mechanism by which receptors are desensitized. In this study we found that agonist-induced internalization of the dopamine D2 receptor is regulated by the receptor tyrosine kinase ALK. ALK was also transactivated by and associated with dopamine D2 receptor. Dopamine activated protein kinase C in an ALK-dependent manner and a PKC inhibitor blocked dopamine D2 receptor internalization. These results indicate that ALK regulates dopamine D2 receptor trafficking, which has implications for psychiatric disorders involving dysregulated dopamine signaling.

Brefeldin Α Reduces STAT3 Dependent Survival of Acute Leukemia and Neuroblastoma Cells

Leukemic stem cells have several dysregulated pathways and the Janus kinases (JAKs) and signal transducer and activator of transcription (STAT) pathway are prominent among them. High STAT3 expression in leukemia has been associated with an increased risk for relapse and decreased overall survival. In addition, phosphorylated STAT3 can be found following prolonged activation of the anaplastic lymphoma kinase (ALK) ligand binding site, but the level of phospho-STAT3 engendered by ligand-independent activation of ALK was exponentially higher. In ALK positive tumors, ALK-STAT3 activation promotes tumor cell outgrowth and directly prevents apoptosis. The role of ALK in several types of ALK-expressing solid tumors as well as the therapeutic use of ALK inhibitors in treating these cancers also have been reviewed in a number of recent papers.However, clinical resistance to ALK inhibitors invariably occurs. Amplified ALK or mutated ALK was identified in ~14% of neuroblastomas (NB), the most common and aggressive childhood malignancy, and phase I trial of ALK inhibitor such as crizotinib showed a lack of response in patients harboring certain ALK mutations. In this study, we implemented a high throughput chemical screen in 4 NB-derived cell lines to identify compounds with the potential of inhibiting oncogenic activity of ALK in NB, using a curated library of ~450 compounds. Brefeldin A (BFA),an inhibitor of the transport of proteins from the endoplasmic reticulum (ER) to the Golgi, was identified as a suitable candidate. It was previously reported that BFA inhibited phosphorylation of ALK and its downstream molecule, STAT3. Since an increasing number of studies have found that STAT3 abnormal expression and activation are accompanied by leukemia development, which indicates the potential role of STAT3 in the pathogenesis of leukemia, we analyzed the cytotoxicity of BFA against 12 T-ALL and 6 AML cell lines. Assessment of BFA antitumor effect using an MTT assay revealed BFA to possess potent cytotoxic activity across a broad spectrum of hematopoietic malignancies, with 10 T-ALL and 5 AML being especially responsive. For example, T-ALL cell lines, as well as AML lines, were potently inhibited by BFA ( IC50 values: ICH-TALL-UK, 168.1 nM; MOLT-4, 23.2nM; Jurkat, 66.6 nM; MOLT14, 97.3 nM; ICH-TALL-SM, 54.9 nM). In an expanded panel of 20 NB cell lines, those with or without MYCN-amplification or 11q loss of heterozygousity which have been identified as two major oncogenic events in NB pathogenesis, especially in the high-risk group were the most sensitive to low nanomolar concentrations of BFA. In NB cell lines harboring F1174L or R1275Q-mutated ALK,BFAshowed cytotoxicity.BFA reduced the phosphorylation of ALK in NIH3T3 cells stably expressed F1174L ALK or TGW, cell line harboring R1275Q-mutated ALK. Of the molecules that are phosphorylated downstream of ALK that have been reported to be important for ALCL cells, the phosphorylation of STAT3, AKT and ERK was inhibited by BFA. These results suggested that BFA can be used to target STAT3 signaling pathways for leukemia treatment. Furthermore these results indicated that the effect of BFA on ALK mutated NB cell lines is accompanied by inhibition of the phosphorylation of ALK and its downstream key effector molecule STAT3.These findings provide the significance of ALK and STAT3 as they relate to NB or leukemia, explores the significance of STAT3, AKT and ERK downstream of ALK, and touches on the potential for new chemotherapeutics targeting ALK and STAT3 to improve high risk NB and leukemia patient prognosis. Disclosures No relevant conflicts of interest to declare.

ELP3 Acetyltransferase is phosphorylated and regulated by the oncogenic anaplastic lymphoma kinase (ALK).

Protein lysine acetylation is one of the major posttranslational modifications (PTMs) with several thousands of proteins identified to be acetylated in mammalian tissues. Mechanistic studies have revealed important functions of acetylation in the regulation of protein function. Much less is known on how the acetyltransferases themselves are regulated. In the current study, we discover that the Elongator protein 3 (ELP3) acetyltransferase is modified by tyrosine phosphorylation. We demonstrate that the anaplastic lymphoma kinase (ALK) is the major tyrosine kinase responsible for ELP3 tyrosine phosphorylation. ELP3 is phosphorylated in tumor cells expressing oncogenic NPM-ALK fusion protein. We further identify Tyr202 as the major ALK phosphorylation site in ELP3. Importantly, the introduction of Y202 phosphorylation mutant ELP3 into ALK-positive tumor cells reduced cell growth and impaired gene expression. Collectively, our study reveals a novel regulatory mechanism for ELP3, provides an example that acetyltransferase itself can be regulated by PTM, and suggests a potential target for ALK-positive cancer therapies.

Abstract 2982: Monomerization of ALK fusion proteins as a therapeutic strategy in ALK rearranged cancers

Abstract Introduction: Since the discovery of the anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein like-4 (EML4) fusion protein, ALK tyrosine kinase inhibitors have been shown to have great anti-tumor activities against ALK rearranged non-small cell lung cancers (NSCLC). As previously reported, the EML4-ALK fusion proteins induce constitutive phosphorylation of ALK via the trimeric coiled-coil domain within EML4. ALK F1174L mutations known in neuroblastoma, can induce autophosphorylation without dimerization. We hypothesize that monomerization of ALK fusion proteins decreased tumorigenesis via competitive dissociation of the trimeric coiled-coil domain of EML4. Methods: We established Ba/F3 cells stably expressing wild-type (wt) ALK intracellular domain or F1174L mutant (mu) with using iDimerize system (Takara Bio). Using this system, monomer or dimer of ALK fusion proteins were controlled by adding AP20187 (Takara Bio). These Ba/F3 cells were subcutaneously injected into BALB nu/nu mice followed by intraperitoneal administration of AP20187 or Mock intermittently. A chemically synthesized peptide composed of amino acid sequences of coiled-coil domain within EML4, was administered for H3122 (EML4-ALK) or A549 (KRAS) cells and the viabilities were analyzed after 24 hours. In order to demonstrate the specificity of the peptide, a synthesized peptide in which two amino acids were substituted was also used as a control. Each peptide was solubilized in DMSO and the peptide concentration was optimized. Results: We successfully established the Ba/F3 stably expressing fusion-ALKwt, fusion-ALKmu under continuous administration of AP20187. Subsequently, ALK proteins were depolymerized by withdrawal of AP20187. The number of cells expressing ALKwt were decreased while the same of ALKmu were increased. The protein expression of phosphorylation of ALK or downstream survival signaling proteins were weakened after 24hours from AP20187 withdrawal. In fusion-ALKwt cells, tumor xenograft was developed in every mice with AP20187 (n=6) administration, while tumor was not developed in Mock controls (n=6). In fusion-ALKmu cells, tumor xenograft was developed in all mice with or without AP20187 administration (n=12). In vitro assay, cell growth of H3122 was 40% decreased by adding coiled-coil peptides, while increased cell growth was observed in control peptide groups and A549 cells. Conclusion: Monomerization of ALK intracellular domain induced cell death and suppressed tumorigenesis. The coiled-coil peptide specifically suppressed the activity of EML4-ALK cell line. Monomerization of ALK fusion proteins via coiled-coil peptide is a promising therapeutic strategy for ALK rearranged NSCLC. Citation Format: Noriko Hirai, Takaaki Sasaki, Yoshinobu Ohsaki. Monomerization of ALK fusion proteins as a therapeutic strategy in ALK rearranged cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2982.

Synthesis and anti-tumor efficacy of novel 2, 4-diarylaminopyrimidine derivatives bearing N-(3-pyridinylmethyl) urea moiety as anaplastic lymphoma kinase inhibitors

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase responsible for development of various tumor types. In this study, we synthesized a series of novel 2,4-diarylaminopyrimidine derivatives possessing a unique N-(3-pyridinylmethyl)urea moiety as ALK inhibitors. The most promising analog 5m bearing a 3-methoxy-4-morpholinophenyl substituent significantly inhibited proliferation of ALK positive H3122 and Karpas-299 cells with IC50 values about 10 nM, which were comparable with positive control LDK378. Compound 5m suppressed phosphorylation of ALK and its downstream proteins, and showed low cytotoxicity on normal human primary fibroblast cells (BJ cells). The binding mode of 5m was proposed by docking simulation, which explains the important role of N-(3-pyridinylmethyl)urea moiety. Furthermore, compound 5m exhibited favorable liver microsomal stability and significant efficacy in H3122 xenograft mice model. Interestingly, compound 5m also showed broader anti-proliferative activity on other human tumor cell lines, which was different from other ALK inhibitors.

Abstract 902: In vitro and in vivo characterization of resistance to lorlatinib treatment in ALK mutated cancers

Abstract The development of targeted therapy has significantly improved the treatment of Anaplastic Lymphoma Kinase (ALK) dependent neoplasias such as Anaplastic Large Cell Lymphoma (ALCL) and Non-Small Cell Lung Cancer (NSCLC). Despite the efficacy of ALK inhibitor crizotinib, acquired resistance is a major challenge. Lorlatinib is a second generation inhibitor with improved activity and selectivity against ALK. To investigate resistance mechanisms that may arise on lorlatinib therapy, we selected in vitro and in vivo lorlatinib resistant tumor cells. In vivo, 10 mice carrying subcutaneous Karpas299 xenografts received vehicle or lorlatinib at increasing doses, starting at 0.1 mg/kg BID. As expected, tumor size was significantly reduced in lorlatinib group compared to controls. After initial partial responses, tumors relapsed and mice were shifted to an increased dose (0.25 mg/kg). Tumors relapsed again and mice were randomized into three groups, to achieve a maximum dose of 0.5, 1 or 2 mg/kg. In all cases tumors regrew after an initial response, indicating acquired resistance to lorlatinib. Upon mice sacrifice, tumors were collected and characterized. All ex vivo cell lines were resistant to lorlatinib as assessed by proliferation assay and 2/10 showed moderate drug addiction. However, in several cases, ALK phosphorylation did not fully correlate with the observed high drug resistance, suggesting the involvement of ALK-independent mechanisms. ALK kinase domain sequencing revealed N1178H mutation in 5/10 mice, alone or in combination with other mutations and L1196M substitution in 2/10 cases. Since both mutants have been described as sensitive or moderately resistant to lorlatinib in murine BaF3 cells, further characterization of these cell lines was performed. ALK N1178H mutant was found to be predominantly localized in the cytoplasm, possibly mimicking overexpression, in human cells. Whole-exome and mRNA sequencing of cells carrying the L1196M substitution showed significant alterations in the PI3K/AKT and RAS/MAPK pathways, as well as perturbations of RAC1-related small GTPases. Functional validation by small molecule inhibitors confirmed the involvement of these pathways in resistance to lorlatinib. In vitro selected lorlatinib-resistant cell lines, representing ALCL, NSCLC and neuroblastoma, showed both ALK mutations, including G1202R and C1156F/L1198F, and ALK-independent mechanisms of resistance. In one case, NSCLC cells acquired hyper-activation of EGFR; its blockage by erlotinib restored sensitivity to lorlatinib. Whole-exome sequencing and proteomic profiling of sensitive and resistant neuroblastoma cell lines revealed NF1 and EP300 mutations, as well as hyper-activation of ErbB4, in lorlatinib-resistant cells. Altogether, our data provide an extensive characterization of resistance mechanisms that may arise in different ALK positive cancers upon lorlatinib exposure. Citation Format: Geeta G. Sharma, Sara Redaelli, Monica Ceccon, Marina Zappa, Carlo Gambacorti-Passerini, Luca Mologni. In vitro and in vivo characterization of resistance to lorlatinib treatment in ALK mutated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 902.

Pharmacological inhibition of Receptor Protein Tyrosine Phosphatase β/ζ (PTPRZ1) modulates behavioral responses to ethanol

Pleiotrophin (PTN) and Midkine (MK) are neurotrophic factors that are upregulated in the prefrontal cortex after alcohol administration and have been shown to reduce ethanol drinking and reward. PTN and MK are the endogenous inhibitors of Receptor Protein Tyrosine Phosphatase (RPTP) β/ζ (a.k.a. PTPRZ1, RPTPβ, PTPζ), suggesting a potential role for this phosphatase in the regulation of alcohol effects. To determine if RPTPβ/ζ regulates ethanol consumption, we treated mice with recently developed small-molecule inhibitors of RPTPβ/ζ (MY10, MY33-3) before testing them for binge-like drinking using the drinking in the dark protocol. Mice treated with RPTPβ/ζ inhibitors, particularly with MY10, drank less ethanol than controls. MY10 treatment blocked ethanol conditioned place preference, showed limited effects on ethanol-induced ataxia, and potentiated the sedative effects of ethanol. We also tested whether RPTPβ/ζ is involved in ethanol signaling pathways. We found that ethanol treatment of neuroblastoma cells increased phosphorylation of anaplastic lymphoma kinase (ALK) and TrkA, known substrates of RPTPβ/ζ. Treatment of neuroblastoma cells with MY10 or MY33-3 also increased levels of phosphorylated ALK and TrkA. However, concomitant treatment of neuroblastoma cells with ethanol and MY10 or MY33-3 prevented the increase in pTrkA and pALK. These results demonstrate for the first time that ethanol engages TrkA signaling and that RPTPβ/ζ modulates signaling pathways activated by alcohol and behavioral responses to this drug. The data support the hypothesis that RPTPβ/ζ might be a novel target of pharmacotherapy for reducing excessive alcohol consumption.

Rhabdomyosarcoma cells are susceptible to cell death by LDK378 alone or in combination with sorafenib independently of anaplastic lymphoma kinase status.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that is often overexpressed in rhabdomyosarcoma (RMS). However, its oncogenic and functional role in RMS remains unclear. Therefore, we investigated the antitumor activity of LDK378 (ceritinib), a new second-generation ALK inhibitor approved for patients with ALK-positive non-small-cell lung cancers. Here, we report that LDK378 reduces cell viability and induces cell death in RMS cell lines at low micromolar IC50 concentrations irrespective of ALK expression levels or phosphorylation status. Compared with Karpas 299 non-Hodgkin's lymphoma cells carrying the NPM-ALK fusion gene, RMS cell lines proved to be far less sensitive to LDK378. The broad-range caspase inhibitor zVAD.fmk significantly protects RMS cells from LDK378-mediated cell death, indicating that LDK378 induces caspase-dependent apoptotic cell death. Before the onset of apoptosis, LDK378 reduces phosphorylation of AKT, S6 ribosomal protein, STAT3 and - to a lesser extent - phosphorylation of ERK, showing that it suppresses key survival pathways. Importantly, we identify a synergistic induction of cell death by combining subtoxic concentrations of LDK378 with the multitargeting kinase inhibitor sorafenib. Calculation of the combination index confirmed that this interaction is synergistic. Also, LDK378 cooperates with sorafenib to significantly reduce colony formation of RMS cells, showing that this combination affects long-term clonogenic growth. In conclusion, LDK378 induces caspase-dependent apoptotic cell death in RMS cells independent of their ALK status and synergizes at subtoxic concentrations with sorafenib to induce cell death. These findings have important implications for the use of LDK378 in RMS.

Targeting Anaplastic Lymphoma Kinase (ALK) in Rhabdomyosarcoma (RMS) with the Second-Generation ALK Inhibitor Ceritinib.

BackgroundThe receptor tyrosine kinase (RTK) anaplastic lymphoma kinase (ALK) has been implicated in the tumorigenesis of rhabdomyosarcoma (RMS). However, the exact role of ALK in RMS is debatable and remains to be elucidated.ObjectiveTo determine the in vitro and in vivo effects and mechanism of action of the second-generation ALK inhibitor ceritinib on RMS cell growth.MethodsEffects of ceritinib on cell proliferation, wound healing, cell cycle, and RTK signaling were determined in alveolar and embryonal rhabdomyosarcoma (ARMS, ERMS). In addition, possible synergistic effects of combined treatment with ceritinib and the Abl/Src family kinase inhibitor dasatinib were determined.ResultsCeritinib treatment led to decreased cell proliferation, cell cycle arrest, apoptosis, and decreased in vivo tumor growth for the ARMS subtype. ERMS cell lines were less affected and showed no cell cycle arrest or apoptosis. Both subtypes lacked intrinsic ALK phosphorylation, and ceritinib was shown to affect the IGF1R signaling pathway. High levels of phosphorylated Src (Tyr416) were present following ceritinib treatment, making combined treatment with a Src inhibitor a potential treatment option. Combined treatment of ceritinib and dasatinib showed synergistic effects in both ERMS and ARMS cell lines.ConclusionThis study shows that monotherapy with an ALK inhibitor, such as ceritinib, in RMS, has no effect on ALK signaling. However, the synergistic effects of ceritinib and dasatinib are promising, most probably due to targeting of IGF1R and Src. Electronic supplementary materialThe online version of this article (10.1007/s11523-017-0528-z) contains supplementary material, which is available to authorized users.