Anaplastic Lymphoma Kinase Kinase Research Articles

Therapeutic strategies and mechanisms of drug resistance in anaplastic lymphoma kinase (ALK)-rearranged lung cancer.

Anaplastic lymphoma kinase (ALK) gene encoding the receptor tyrosine kinase ALK is expressed as a fusion gene in a variety of carcinomas. The expression of ALK is nearly undetectable in adults, and its activation is normally regulated by its ligands, FAM150A/B. However, ALK gene rearrangements result in constitutive ALK fusion proteins expression via the active promoter of fusion partner genes. ALK fusion proteins dimerize in a ligand-independent manner and lead to the dysregulation of cell proliferation via abnormal constitutive activation of ALK tyrosine kinase. Many ALK tyrosine kinase inhibitors (TKIs) have been developed to date, three of which are currently in clinical use for the treatment of ALK-rearranged non-small cell lung cancer (NSCLC). ALK TKIs often achieve marked tumor regression in NSCLC patients with ALK rearrangements; however, ALK TKI-resistant tumors inevitably emerge within a few years in most cases. In this review, we summarize diverse ALK TKI resistance mechanisms identified in NSCLC with ALK rearrangements, and review potential therapeutic strategies to overcome ALK TKI resistance in these patients.

P1.06-026 Adenosquamous Carcinoma of the Lung: A Single Institution Experience in the Era of Molecular Testing

Adenosquamous carcinoma (ADS) lung is rare subtype of non-small cell lung cancer (NSCLC) that compromises 0.4-4% of all lung cancers and is thought to carry a worse prognosis than adenocarcinoma (AD) or squamous cell carcinoma (SC). Epidermal growth factor receptor (EGFR) and Anaplastic Lymphoma Kinase (ALK) mutations have been observed in patients (pts) with this rare subtype. In the recent years EGFR tyrosine kinase inhibitors (Gefitinib, Afatinib and Erlotinib) and ALK inhibitors (Critzotinib, Ceritinib) have prolonged progression free survival in high-stage adenocarcinomas of the lung. The current NCCN guidelines recommend EGFR and ALK mutation testing in metastatic ADS lung cancer patients. However the frequency of these mutations as well as outcomes of patients with ADS lung is not known in this era of targeted therapies. We retrospectively identified all pts seen in our oncology clinic for ADS during the last 10 years (1/1/2005 - 1/1/2015). Overall survival (OS) was estimated by Kaplan-Meier methods. 16 pts were identified, median age at diagnosis was 71y (52-85y), 63% male, 81% had a smoking history, 87% had ECOG performance status 0-1. 37% had Stage I, 18% had stage II, 18% had stage III and 25% had stage IV disease at diagnosis, 13% developed metastatic disease after treatment for stage III disease. 75% of pts diagnosed with metastatic disease after 2012 were tested for EGFR and ALK, while none diagnosed prior to 2012 were tested. All pts were negative for EGFR and ALK mutations. All pts with Stage I and II received only surgery; pts with stage III got multimodality treatment with chemotherapy, radiation, and surgery. All the pts with metastatic disease received chemotherapy, with regimens similar to those for AD or SC of lung. The median OS for pts with localized disease was 48.3 months (48.0- NA). The median OS for pts with metastatic disease was 5.4 months (2.3-9.2). Our small analysis showed that pts with localized ADS of lung had similar outcomes to those of historical pts with localized AD or SC of the lung. However, pts with metastatic ADS of the lung had worse outcomes than historical pts with metastatic AD or SC of the lung even with similar chemotherapy regimens. Few pts had EGFR and ALK testing, but this is becoming more routine as we have better targeted therapies if they carry mutation.

The Truncated Isoform of the Receptor Tyrosine Kinase ALK Generated by Alternative Transcription Initiation (ALKATI) Induces Chromatin Structural Changes in the Nucleus in a Kinase Activity-Dependent Manner.

Anaplastic lymphoma kinase (ALK) is a receptor-type tyrosine kinase that promotes cell growth upon stimulation with ligands such as midkine and pleiotrophin. Recently, a truncated isoform of ALK was identified in a variety of tumors. This isoform is expressed from a novel ALK transcript initiated from a de novo alternative transcription initiation (ATI) site in ALK intron 19 (referred to as ALKATI). ALKATI, which consists of only the intracellular kinase domain, localizes to the nucleus as well as the cytoplasm. However, its nuclear role is unknown. In this study, we determined that ALKATI promoted chromatin structural changes in the nucleus in a kinase activity-dependent manner. We found that expression of ALKATI increased the level of the heterochromatin marker Lys9 tri-methylated histone H3. In addition, we demonstrated that ALKATI phosphorylated the nuclear protein A-kinase anchoring protein 8 (AKAP8) and altered its subcellular localization from the insoluble fraction to the soluble fraction. These results suggest that ALKATI induces chromatin structural changes and heterochromatinization through phosphorylation of AKAP8 in the nucleus.

QSAR‐based targeting anaplastic lymphoma kinase (ALK) variants with noncognate inhibitors in pediatric acute lymphoblastic leukemia

Human anaplastic lymphoma kinase (ALK) is a potential target for the treatment of pediatric acute lymphoblastic leukemia. However, a number of residue mutations in ALK kinase domain have been observed to cause drug resistance in pediatric acute lymphoblastic leukemia chemotherapy. Here, a chemometrics quantitative structure‐activity relationship predictor was developed using a structure‐based panel of kinase‐inhibitor activity data. The predictor was validated rigorously through internal cross‐validation and external blind test to ensure its statistical reliability, which was then used to computationally construct a systematic activity profile of 13 noncognate kinase inhibitors against both wild‐type ALKwt and cancer‐related variants ALKvt. It is revealed that most noncognate inhibitors exhibit weak potency on ALKwt, but some of them are able to selectively target ALKvt over ALKwt. The chemometrics findings were then evaluated by using a kinase inhibition protocol; results showed that few noncognate inhibitors are 2‐ to 5‐fold higher potent against ALK variant than wild‐type kinase.

Clinical data and role of ceritinib a second-generation ALK tyrosine kinase inhibitor for the treatment of ALK positive non-small cell lung cancer

Rearrangements in anaplastic lymphoma kinase ( ALK ) gene and echinoderm microtubule-associated protein-like 4 ( EML4 ) gene were first described in 2007. This genomic aberration is found in about 2–8% of non-small cell lung cancer (NSCLC) patients. In patients with adenocarcinoma lacking EGFR and KRAS mutations, the prevalence of EML4-ALK translocation could be as high as 42.8% (1). In these patients, ALK rearrangements serve as a key and strong oncogenic driver for NSCLC and represent a critical therapeutic target susceptible to targeted ALK kinase inhibition (2,3).

Resistance to Crizotinib in Advanced Non-Small Cell Lung Cancer (NSCLC) with ALK Rearrangement: Mechanisms, Treatment Strategies and New Targeted Therapies.

Non-small cell lung cancers (NSCLCs) harboring anaplastic lymphoma kinase (ALK) rearrangement are generally responsive to treatment with ALK tyrosine kinase inhibitors (TKIs). Crizotinib is the first-in-class TKI approved as front-line or salvage therapy in advanced ALK-rearranged NSCLC. Unfortunately, drug resistance develops after initial benefit, through a variety of mechanisms preserving or not the dominance of ALK signaling in the crizotinib-resistant state. The distinction between patients who preserve ALK dominance (secondary mutations alone or in combination with the number of copy ALK gain) compared to those that have decreased ALK dominance (separate or second oncogenic drivers, with or without concurrent persistence of the original ALK signal) is important in order to overcome resistance. Novel second-generation ALK inhibitors are currently in clinical development with promising results in ALK-rearranged NSCLC, as well as in crizotinib-resistant patients. Among these, ceritinib in the United States was granted by Food and Drug Administration accelerated approval for treatment of patients with ALK-rearranged, metastatic NSCLC with progression disease on or intolerance to crizotinib. Fully understanding of the different mechanisms of resistance to crizotinib will help us to continue to exploit personalized medicine approaches overcoming crizotinib resistance in these patients in the future. This review aims to discuss on strategy overcoming crizotinib-resistance starting from molecular mechanisms of resistance until novel ALK kinase inhibitors in ALK-rearranged NSCLC patients.

Primary and Metastatic Cutaneous Melanomas Express ALK Through Alternative Transcriptional Initiation.

A number of common driver mutations have been identified in melanoma, but other genetic or epigenetic aberrations are also likely to play a role in the pathogenesis of melanoma and present potential therapeutic targets. Translocations of the anaplastic lymphoma kinase (ALK), for example, have been reported in spitzoid melanocytic neoplasms leading to kinase-fusion proteins that result in immunohistochemically detectable ALK expression. In this study, we sought to determine whether ALK was also expressed in nonspitzoid primary and metastatic cutaneous melanomas. ALK immunohistochemistry was performed on 603 melanomas (303 primary and 300 metastatic tumors) from 600 patients. ALK immunohistochemistry expression was identified in 7 primary and 9 metastatic tumors. In 5 of 7 primary tumors and in 6 of 9 metastatic lesions, the majority of tumor cells were immunoreactive for ALK. In the other 2 primary and 3 metastatic lesions, positive staining was identified in less than half of the tumor cells. ALK positivity was found in the presence or absence of BRAF or NRAS mutations. In contrast to prior observations with ALK-positive Spitz tumors, none of the ALK-positive melanomas harbored a translocation. Instead, the ALK-positive melanomas predominantly expressed the recently described ALK isoform, ALK, which lacks the extracellular and transmembrane domains of wild-type ALK, consists primarily of the intracellular tyrosine kinase domain, and originates from an alternative transcriptional initiation site within the ALK gene. The findings are clinically relevant as patients with metastatic melanoma who have ALK expression may potentially benefit from treatment with ALK kinase inhibitors.

Brigatinib, an anaplastic lymphoma kinase inhibitor, abrogates activity and growth in ALK-positive neuroblastoma cells, Drosophila and mice.

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor which has been implicated in numerous solid and hematologic cancers. ALK mutations are reported in about 5-7% of neuroblastoma cases but the ALK-positive percentage increases significantly in the relapsed patient population. Crizotinib, the first clinically approved ALK inhibitor for the treatment of ALK-positive lung cancer has had less dramatic responses in neuroblastoma. Here we investigate the efficacy of a second-generation ALK inhibitor, brigatinib, in a neuroblastoma setting. Employing neuroblastoma cell lines, mouse xenograft and Drosophila melanogaster model systems expressing different constitutively active ALK variants, we show clear and efficient inhibition of ALK activity by brigatinib. Similar abrogation of ALK activity was observed in vitro employing a set of different constitutively active ALK variants in biochemical assays. These results suggest that brigatinib is an effective inhibitor of ALK kinase activity in ALK addicted neuroblastoma that should be considered as a potential future therapeutic option for ALK-positive neuroblastoma patients alone or in combination with other treatments.

Anaplastic Lymphoma Kinase (ALK) Kinase Domain Mutation Following ALK Inhibitor(s) Failure in Advanced ALK Positive Non–Small-Cell Lung Cancer: Analysis and Literature Review

BackgroundSecondary anaplastic lymphoma kinase (ALK) mutation may occur in patients with advanced ALK-positive non–small cell lung cancer treated with ALK inhibitors, but its nature is not well-known. Patients and MethodsWe analyzed tumor specimens after the failure of treatment with ALK inhibitor(s) (crizotinib, alectinib, and ceritinib) for secondary ALK kinase domain mutation, EGFR, K-ras, and PIK3CA mutations. The literature regarding acquired ALK-inhibitor(s) resistance was also reviewed. ResultsAmong 59 patients who received ALK inhibitor(s) during the period of December 2010 to April 2015, 7 had re-biopsied tumor specimens for analyses following ALK inhibitor(s) failure. One had G1202R after crizotinib and alectinib failure, and 6 were wild type. No EGFR, K-ras, or PIK3CA mutations were found. In our review of the literature and taken together with our patients, 25 of the 88 (28%) patients with crizotinib failure had secondary ALK mutation; L1196M mutation was most common (n = 11). Patients with secondary ALK mutation other than L1196M had a longer progression-free survival after crizotinib than patients with L1196M (median, 12.0 vs. 7.0 months; P = .04). Of the 9 patients with alectinib failure, 5 had I1171 mutation and 2 had G1202R. Of the 11 patients with ceritinib failure, 2 had G1202R, 1 had F1174C, and 1 had both G1202R and F1174V. I1171 mutation, G1202R, and F1174 mutations were also found in crizotinib-failed patients. ConclusionsSome acquired ALK mutations may cause co-resistance to other ALK inhibitors. Re-biopsy for ALK mutation analysis might be suggested prior to choosing a second-line ALK inhibitor treatment.

A New ALK Inhibitor for Crizotinib-Refractory Non–Small-Cell Lung Cancer

Patients with ALK -rearranged non–small-cell lung cancer (NSCLC) benefit from anaplastic lymphoma kinase (ALK) inhibitors such as crizotinib and ceritinib, although almost all experience progression, and many develop central nervous system (CNS) metastases as the first site of progression. Now, investigators have conducted an industry-supported, global, single-arm, phase II study to assess the efficacy and safety of the newly approved, oral tyrosine kinase ALK inhibitor alectinib (600 mg twice daily) in 138 patients with crizotinib-refractory ALK -positive disease. Of these, 61% had …

Overcoming resistance to first/second generation epidermal growth factor receptor tyrosine kinase inhibitors and ALK inhibitors in oncogene-addicted advanced non-small cell lung cancer.

Epidermal growth factor receptor (EGFR) activating mutations and anaplastic lymphoma kinase (ALK) gene rearrangement in advanced non-small cell lung cancer (NSCLC) represent the two oncogenic events with an impact on current clinical practice. EGFR tyrosine kinase inhibitors (TKIs) and crizotinib are the standard of care for the treatment of EGFR mutant and ALK gene rearranged advanced NSCLC patients. Unfortunately, despite initial clinical benefit, acquired resistance to EGFR-TKIs or crizotinib usually develops after an average of 10-12 months of treatment. The aim of this review is to describe the mechanisms of resistance to first/second generation EGFR-TKIs and crizotinib. In particular, we focus on strategies to overcome resistance due to secondary EGFR T790M mutation and mutations of the ALK domain.

Assessment of ALK gene fusions in lung cancer using the differential expression and exon integrity methods.

Anaplastic lymphoma kinase (ALK) gene fusion is a driving mutation underlying the development of non-small cell lung cancer (NSCLC). Accurate detection of ALK fusion is critical for the use of ALK inhibitors in the treatment of NSCLC. Commonly utilized methods for ALK detection include fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). However, these methods are time-consuming and costly. In the present study, a method for assessing ALK gene fusion based on the differential expression levels of the ALK kinase and non-kinase domains was developed and evaluated, with the aim of providing a convenient and reliable method for the detection of ALK fusion. In addition, another method was established to determine the integrity of exons 19-20 and 20-21 of ALK, two genomic loci that are typically broken in ALK fusions. These novel methods were applied to detect ALK fusion in 100 NSCLC patients, and were compared with IHC and FISH methods. The novel methods developed in the present study successfully detected ALK fusions in 10 samples. The concordances between the novel methods and IHC and FISH were 100%. Furthermore, the differential expression method was able to detect ALK fusions in cell-free urine samples, which was advantageous over FISH and IHC. The novel methods developed in the present study are cost-effective and easy to perform, and may provide simple and convenient techniques for the clinical assessment of ALK fusions, facilitating the use of targeted therapy for NSCLC.

Resensitization to Crizotinib by the Lorlatinib ALK Resistance Mutation L1198F

In a patient who had metastatic anaplastic lymphoma kinase (ALK)-rearranged lung cancer, resistance to crizotinib developed because of a mutation in the ALK kinase domain. This mutation is predicted to result in a substitution of cysteine by tyrosine at amino acid residue 1156 (C1156Y). Her tumor did not respond to a second-generation ALK inhibitor, but it did respond to lorlatinib (PF-06463922), a third-generation inhibitor. When her tumor relapsed, sequencing of the resistant tumor revealed an ALK L1198F mutation in addition to the C1156Y mutation. The L1198F substitution confers resistance to lorlatinib through steric interference with drug binding. However, L1198F paradoxically enhances binding to crizotinib, negating the effect of C1156Y and resensitizing resistant cancers to crizotinib. The patient received crizotinib again, and her cancer-related symptoms and liver failure resolved. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT01970865.)

NPM-ALK Mediated Tyrosine Phosphorylation of ATP Citrate Lyase Regulates Lipid Metabolism and Promotes Oncogenesis of Anaplastic Large Cell Lymphoma

Introduction: Anaplastic large cell lymphoma (ALCL) is characterized by recurrent translocations involving anaplastic lymphoma kinase (ALK) and resulting in oncogenic chimeric fusion tyrosine kinases such as NPM-ALK. We integrated mass-spectrometry-driven phosphoproteomics and metabolomics of ALK+ ALCL and discovered that ATP citrate lyase (ACLY) is a candidate substrate of NPM-ALK at residue Y682. ACLY is a critical enzyme responsible for synthesis of acetyl-CoA and connects vital biosynthetic pathways such as carbohydrate and lipid metabolism. Functional regulation of ACLY by tyrosine phosphorylation is unknown and the significance of tyrosine phosphorylation of ACLY on cancer cell growth and metabolism have never been described.Methods: To identify novel substrates of NPM-ALK, we performed phospho-proteomic analyses of 3 ALK+ALCL derived cell lines. The MS-phosphoproteomic data were subjected to custom bioinformatic algorithms to identify highly statistically correlated phosphotyrosine proteins and phosphosites regulated by ALK kinase activity. We generated a phosphospecific antibody against a highly-ranked candidate (pACLY Y682) and utilized it in western blot analyses on ALK+ and ALK- ALCL lymphoma cell lines. Immunohistochemistry (IHC) on well-characterized primary human ALK+ (n=20) and ALK-ALCL biopsies (n=28) were performed. Co-immunoprecipitations (Co-IP) with HA-tagged ACLY-WT and ACLY-Y682F and ALK antibodies were performed and analyzed by western blot using the pACLY Y682 antibody. In vitro kinase assays were performed to investigate the direct role of NPM-ALK in phosphorylation of ACLY at Y682 using GST-tagged ACLY-WT or ACLY-Y682F peptides in the presence or absence of immunocomplexes containing active NPM-ALK or the kinase-defective K210R mutant. To study the impact of Y682F mutant on lipid metabolism, we generated ALCL-derived cell lines stably transduced to express GFP-tagged ACLY-WT or ACLY-Y682F and subjected them to metabolomic analysis for flux, β-oxidation or lipid synthesis using liquid chromatography/tandem mass spectrometry or gas chromatography platforms. The Seahorse XF24 Flux analyzer was used to measure oxygen consumption rates (OCR) in DEL stably expressing ACLY-WT and ACLY-Y682F. Cells stably expressing ACLY-WT and ACLY-Y682F were evaluated for cell proliferation, colony formation and tumor formation in SCID-BEIGE mouse xenograft models.Results: Phosphoproteomic analyses yielded a dataset containing a total of 626 tyrosine phosphoproteins. Bioinformatic analysis identified pACLY Y682 as a novel candidate substrate of NPM-ALK. Western blot analysis corroborated phosphoproteomic studies and demonstrated that phosphorylation of ALCY at Y682 is regulated by NPM-ALK activity. Expression of active NPM-ALK increased the phosphorylation of ACLY at Y682 in HEK293T cells compared to the K210R mutant. In vitro kinase assays demonstrated that NPM-ALK phosphorylates ACLY at Y682. Immunoprecipitation (IP) of ALK and co-IP (HA) demonstrated interaction of NPM-ALK and ACLY in co-transfected HEK293T cell lysates. Stable expression of ACLY-Y682F or pharmacologic antagonism of ALK with crizotinib resulted in decreased ACLY activity, decreased lipid synthesis and increased fatty acid β-oxidation when compared to ACLY-WT. β-oxidation of 13 C-oleic acid-labeled fatty acid demonstrated increased labeling of +2-citrate (p<0.01) and +18-Oleyol carnitine (p<0.001) in ACLY-Y682F cells when compared to ACLY-WT. Similarly, the OCR was significantly increased in cells expressing ACLY-Y682F (p<0.001). IHC studies of ALCL biopsies (ALK+ and ALK-) demonstrated significant correlation between NPM-ALK and ACLY pY682- expression (p<0.01). Expression of ACLY-Y682F in the DEL cell line resulted in dramatically decreased cell proliferation, impaired clonogenic potential as observed by small colony size in colony formation assay and abrogated tumor growth in in vivo xenograft model when compared to ACLY-WT.Conclusion: We identify for the first time a direct tyrosine kinase-mediated mechanism for ACLY regulation. In this regard, our studies demonstrate a novel oncogenic mechanism whereby NPM-ALK controls lipid synthesis and b-oxidation by phosphorylation of ACLY at Y682 and this mechanism contributes to ALCL pathogenesis. Our findings have significant implications for novel therapies targeting tumor metabolism in ALCLs. DisclosuresNo relevant conflicts of interest to declare.

Abstract C82: Induction of resistances to crizotinib in lung patient derived xenograft

Abstract Background. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase, and its mutation, over-expression and gene fusion have been associated with various cancers. EML4-ALK fusion has been confirmed to be an oncogenic driver for a small subset of the lung cancers (∼4% of NSCLC)1, and becomes an excellent drug target that led to the development of the first effective therapy, crizotinib, a tyrosine kinase inhibitor for anaplastic lymphoma kinase, for this disease 2,3. However, like any other cancer therapy so far, the treatment always led to the development of resistance, rendering them ineffective in the end4. Understanding the mechanisms causing these resistances can potentially facilitate overcoming the resistance and extend patients' life5,6. However, lack of experimental model hinders this understanding. Method. We have recently established a large collection of patient derived xenografts (∼350 PDXs)7, and we screened some of them for alk gene fusions. We then tested the positive model for sensitivity to crizothinib, and keep it under several rounds of crizotinib treatments to select resistant model. The induced resistant model was subjected to genomic analysis to identify the changes that might potentially be responsible for the resistance. Results. We identified one model, NSCLC LU1656, containing EML4-ALK fusion with elevated ALK expression. It has also been shown to respond well to crizotinib in vivo. The continued treatment eventually led to the development of resistance to crizotinib (LU2445), a situation that might occur in patients in the clinic under the same treatment. We have performed transcriptome sequencing of the parental sensitive tumor (LU1656) and the selected resistant tumor (LU2445). So far, we found both models expressed similar high levels of expression of ALK, but no additional mutations in ALK gene. There are other alterations either genetically or epigenetically, some seemingly related to the ALK signaling pathways. However, their role in resistance still need to be confirmed. Conclusions. Induced crizotinib resistance in ALK-fusion lung PDX can be useful to investigate crizotinib resistant mechanism and future drugs overcoming the resistance.

Augmentor α and β (FAM150) are ligands of the receptor tyrosine kinases ALK and LTK: Hierarchy and specificity of ligand–receptor interactions

Receptor tyrosine kinases (RTKs) are a class of cell surface receptors that, upon ligand binding, stimulate a variety of critical cellular functions. The orphan receptor anaplastic lymphoma kinase (ALK) is one of very few RTKs that remain without a firmly established protein ligand. Here we present a novel cytokine, FAM150B, which we propose naming augmentor-α (AUG-α), as a ligand for ALK. AUG-α binds ALK with high affinity and activates ALK in cells with subnanomolar potency. Detailed binding experiments using cells expressing ALK or the related receptor leukocyte tyrosine kinase (LTK) demonstrate that AUG-α binds and robustly activates both ALK and LTK. We show that the previously established LTK ligand FAM150A (AUG-β) is specific for LTK and only weakly binds to ALK. Furthermore, expression of AUG-α stimulates transformation of NIH/3T3 cells expressing ALK, induces IL-3 independent growth of Ba/F3 cells expressing ALK, and is expressed in neuroblastoma, a cancer partly driven by ALK. These experiments reveal the hierarchy and specificity of two cytokines as ligands for ALK and LTK and set the stage for elucidating their roles in development and disease states.

Clinical Implications of Variant ALK FISH Rearrangement Patterns

Break-apart fluorescence in situ hybridization (FISH) is the FDA-approved assay for detecting anaplastic lymphoma kinase (ALK) rearrangements in non-small-cell lung cancer (NSCLC), identifying patients who can gain dramatic benefit from ALK kinase inhibitors. Assay interpretation can be technically challenging, and either splitting of the 5' and 3' probes or loss of the 5' probe constitute rearrangement. We hypothesized that there may be clinical differences depending on rearrangement pattern on FISH. An IRB-approved database of NSCLC patients at Dana-Farber Cancer Institute was queried for ALK rearrangement. Clinical characteristics and response to crizotinib were reviewed. Immunohistochemistry (IHC) and targeted next-generation sequencing (NGS) were obtained when available. Of 1614 NSCLC patients with ALK testing, 82 patients (5.1%) had ALK rearrangement by FISH: 30 patients with split signals, 25 patients with 5' deletion, and 27 patients with details unavailable. Patients with 5' deletion were older (p = 0.01) and tended to have more extensive smoking histories (p = 0.08). IHC was positive for ALK rearrangement in all 27 patients with FISH split signals, whereas three of 21 patients with FISH 5' deletion had negative IHC (p = 0.05). Targeted NGS on two of three cases with discordant FISH and IHC results did not identify ALK rearrangement, instead finding driver mutations in EGFR and KRAS. Patients with 5' deletion treated with crizotinib had a smaller magnitude of tumor response (p = 0.03). Patients with 5' deletion on ALK FISH harbor features less typical of ALK-rearranged tumors, potentially indicating that some cases with this variant are false positives. Corroborative testing with IHC or NGS may be beneficial.

Current Strategies to Overcome Resistance to ALK-Inhibitor Agents.

The rearrangements of the anaplastic lymphoma kinase (ALK) gene are key drivers in the carcinogenesis of a portion of anaplastic large cell lymphomas (ALCL) and non-small cell lung cancers (NSCLC). Crizotinib, an orally available small molecule, has been the first ALK inhibitor to demonstrate a significant clinical activity in patients with ALK-positive tumors and, thus, to achieve the US food and drug administration approval for the treatment of advanced NSCLC harboring ALK-rearrangements. However, despite initially dramatic and quite durable responses in most cases, acquired resistance to crizotinib arises unavoidably often within the first year of treatment. Three main mechanisms of resistance to crizotinib have been identified to date: mutations in the ALK kinase domain, amplifications of ALK gene, and activation of escape signaling pathways. As ALK signaling dependence is retained in most cases become refractory to crizotinib, newer and more potent ALK-inhibitors have been developed and tested in clinical trials with encouraging activity results. Ceritinib has been recently approved by FDA for the treatment of locally advanced and metastatic NSCLC, and several more agents, including alectinib, ASP3026, and X396, are in active clinical development, demonstrating to be safe, selective and potent. Dual inhibition approaches targeting both ALK and the escape pathways bypassing ALK are currently under investigation. Moreover, being ALK a partner of the heat shock protein Hsp90, inhibitors of this chaperone have been proposed as potential alternative therapeutic strategies for ALKdriven tumors.

Structural insight into selectivity and resistance profiles of ROS1 tyrosine kinase inhibitors

Oncogenic ROS1 fusion proteins are molecular drivers in multiple malignancies, including a subset of non-small cell lung cancer (NSCLC). The phylogenetic proximity of the ROS1 and anaplastic lymphoma kinase (ALK) catalytic domains led to the clinical repurposing of the Food and Drug Administration (FDA)-approved ALK inhibitor crizotinib as a ROS1 inhibitor. Despite the antitumor activity of crizotinib observed in both ROS1- and ALK-rearranged NSCLC patients, resistance due to acquisition of ROS1 or ALK kinase domain mutations has been observed clinically, spurring the development of second-generation inhibitors. Here, we profile the sensitivity and selectivity of seven ROS1 and/or ALK inhibitors at various levels of clinical development. In contrast to crizotinib's dual ROS1/ALK activity, cabozantinib (XL-184) and its structural analog foretinib (XL-880) demonstrate a striking selectivity for ROS1 over ALK. Molecular dynamics simulation studies reveal structural features that distinguish the ROS1 and ALK kinase domains and contribute to differences in binding site and kinase selectivity of the inhibitors tested. Cell-based resistance profiling studies demonstrate that the ROS1-selective inhibitors retain efficacy against the recently reported CD74-ROS1(G2032R) mutant whereas the dual ROS1/ALK inhibitors are ineffective. Taken together, inhibitor profiling and stringent characterization of the structure-function differences between the ROS1 and ALK kinase domains will facilitate future rational drug design for ROS1- and ALK-driven NSCLC and other malignancies.

Ethanol activates midkine and anaplastic lymphoma kinase signaling in neuroblastoma cells and in the brain.

Alcohol engages signaling pathways in the brain. Midkine (MDK) is a neurotrophic factor that is over-expressed in the prefrontal cortex of alcoholics. MDK and one of its receptors, anaplastic lymphoma kinase (ALK), also regulate behavioral responses to ethanol in mice. The goal of this study was to determine whether MDK and ALK expression and signaling are activated by ethanol. We found that ethanol treatment of neuroblastoma cells increased MDK and ALK expression. We also assessed activation of ALK by ethanol in cells and found that ALK and ALK-dependent extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription 3 (STAT3) phosphorylation increased rapidly with ethanol exposure. Similarly, treatment of cells with recombinant MDK protein increased ALK, ERK and STAT3 phosphorylation, suggesting that ethanol may utilize MDK to activate ALK signaling. In support of this, transfection of cells with MDK siRNAs attenuated ALK signaling in response to ethanol. Ethanol also activates ERK signaling in the brain. We found that inhibition of ALK or knockout of MDK attenuated ethanol-induced ERK phosphorylation in mouse amygdala. These results demonstrate that ethanol engages MDK and ALK signaling, which has important consequences for alcohol-induced neurotoxicity and the regulation of behaviors related to alcohol abuse.