ALK Fusion Proteins Research Articles

Phosphorylated STAT5 regulates p53 expression via BRCA1/BARD1-NPM1 and MDM2

Signal transducer and activator of transcription 5 (STAT5) and nucleophosmin (NPM1) are critical regulators of multiple biological and pathological processes. Although a reciprocal regulatory relationship was established between STAT5A and a NPM–ALK fusion protein in T-cell lymphoma, no direct connection between STAT5 and wild-type NPM1 has been documented. Here we demonstrate a mutually regulatory relationship between STAT5 and NPM1. Induction of STAT5 phosphorylation at Y694 (P-STAT5) diminished NPM1 expression, whereas inhibition of STAT5 phosphorylation enhanced NPM1 expression. Conversely, NPM1 not only negatively regulated STAT5 phosphorylation but also preserved unphosphorylated STAT5 level. Mechanistically, we show that NPM1 downregulation by P-STAT5 is mediated by impairing the BRCA1-BARD1 ubiquitin ligase, which controls the stability of NPM1. In turn, decreased NPM1 levels led to suppression of p53 expression, resulting in enhanced cell survival. This study reveals a new STAT5 signaling pathway regulating p53 expression via NPM1 and uncovers new therapeutic targets for anticancer treatment in tumors driven by STAT5 signaling.

Science Signaling Podcast for 18 October 2016: ALK combination therapies for lung cancer

Network analysis reveals potential targets for treating lung cancers driven by ALK fusion proteins (Zhang et al ., in 18 October 2016 issue).

Expression of ALK protein in 7 371 pulmonary adenocarcinoma samples, with analysis of clinicopathologic features

To study the expression of ALK protein in pulmonary adenocarcinoma as detected by Ventana immunohistochemistry, with correlation of clinicopathologic features. Immunohistochemical study for ALK protein using Ventana ALK (D5F3) kit was carried in 7 371 pulmonary adenocarcinoma samples. The clinicopathologic features were subsequently analyzed. ALK fusion protein was detected in 446 of the 7 371 lung adenocarcinoma samples studied (6.05%). The ALK positivity rate in small biopsy samples was higher than that in surgical specimens [9.02% (153/1 696) versus 5.16% (293/5 675); P<0.01]. ALK fusion protein expression correlated with patient age, sample type and smoking history. ALK positivity rate in each age group increased with younger patient age. ALK positivity rate was 45.45% (10/22) in patients younger than 30 years old. The positivity rate of ALK fusion protein in adenocarcinoma in-situ, minimally invasive adenocarcinoma and invasive adenocarcinoma was 0, 0.48% (2/418) and 5.63% (291/5 165), respectively. The differences of ALK positivity rate amongst different subtypes had statistical significance (P<0.01). Invasive mucinous adenocarcinoma had highest ALK positivity rate, followed by invasive adenocarcinoma with predominantly solid pattern. ALK fusion protein is more often found in young patients with pulmonary adenocarcinoma, especially in those younger than 30 years old. ALK fusion protein is rarely expressed in early-stage pulmonary adenocarcinoma. Invasive mucinous adenocarcinoma and invasive adenocarcinoma with solid pattern have higher ALK positivity rate than other adenocarcinoma subtypes.

Abstract 311: Novel role for MIG-6 in mediating TKI resistance in ALK-rearranged lung cancer

Abstract INTRODUCTION: Patients with ALK-rearranged non-small cell lung cancer (NSCLC) derive significant anti-tumor responses when treated with ALK tyrosine kinase inhibitors (TKIs), such as crizotinib, the first-in-class ALK TKI. However despite the high response rates to these agents, acquired resistance to ALK TKI therapy remains a significant barrier to overcome in order to maximize therapeutic responses in patients with ALK+ lung cancer. In crizotinib-resistant tumors, EGFR activation has been demonstrated to mediate acquired resistance in several independent studies. The tumor suppressor gene product MIG-6 (encoded by ERFFI1) acts as a natural inhibitor of signaling through EGFR (ErbB1) and other ErbB family members. However, a role for MIG-6 in ALK+ lung cancer has not yet been elucidated. Here, we investigated the regulation of human MIG-6 in ALK+ lung cancer cells. DESIGN: MIG-6 expression, protein interaction and phosphorylation status were evaluated in ALK+ lung cancer cell lines and human tumor samples. The impact of MIG-6 loss and gain of function on ErbB receptor activity and on cell proliferation/survival were determined in several models of TKI-sensitive and TKI-resistant ALK+ lung cancer. RESULTS: MIG-6 protein level is regulated by EML4-ALK fusion protein at both transcriptional and post-translational levels. Genetic or pharmacologic knock-down of ALK significantly reduced MIG-6 protein levels in ALK+/TKI sensitive cells. We established a novel interaction between MIG-6 and ALK fusion proteins (both EML4-ALK and other fusion partners), and this association was dependent on ALK kinase activity. MIG-6 can also be tyrosine-phosphorylated by EML4-ALK and be threonine-phosphorylated through EML4-ALK-dependent MAPK activation. The reduced MIG-6 protein level was accompanied by increased total and phosphorylated ErbB family members (EGFR, HER2, and HER3) in ALK+/TKI sensitive lung cancer cells. Consistent with this observation, MIG-6 protein level is also attenuated in ALK+/TKI resistant cells, following the decreased EML4-ALK activity, while EGFR signaling activity is remarkably up-regulated in these resistant cells. Crizotinib-resistant cells were re-sensitized upon exposure to EGFR/HER2 inhibitors in combination with crizotinib. In addition, MIG-6 reconstitution impeded the development of early adaptive resistance in crizotinib-sensitive cells and was also able to inhibit the proliferation of crizotinib-resistant cells. CONCLUSION: Our study presents an in-depth mechanistic understanding of how ErbB family members, such as EGFR, are up-regulated at the time of crizotinib resistance and provides insights into the early escape mechanisms tumor may have to evade ALK TKI therapy. Citation Format: Huan Qiao, Yingjun Yan, Matthew A. Smith, Eric B. Haura, Marc Ladanyi, Christine M. Lovly. Novel role for MIG-6 in mediating TKI resistance in ALK-rearranged lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 311.

Abstract 856: Number of ALK-amplified circulating tumor cells predicts progression-free survival in ALK-rearranged non-small cell lung cancer patients treated by crizotinib

Abstract The duration of clinical response is unpredictable in ALK-rearranged NSCLC patients treated by crizotinib but all patients invariably develop resistance. Using filter-adapted fluorescence in situ hybridization (FA-FISH) we previously reported ALK-rearrangement detection in circulating tumor cells (CTCs) from ALK-rearranged patients. Here we report the monitoring of ALK-rearranged (≥1×3’/5’, ≥1x(3’ and 5’)) and ALK-amplified (&amp;gt;2×3’/5’) CTCs at baseline and at an early time point under crizotinib therapy in an extended cohort of ALK-rearranged patients. The correlation between CTC subsets harboring distinct FISH patterns and clinical parameters including progression-free survival (PFS) and overall survival (OS) is presented. Forty ALK-rearranged patients were recruited. Blood samples were collected at baseline to crizotinib and at 1-3 months. Abnormal ALK-FISH patterns were examined in CTCs using immunofluorescence staining (DAPI/CD45) combined with FA-FISH after isolation by size of epithelial tumor cells (ISET) filtration. CTCs were defined into five distinct subsets according to the number of FISH ‘break-apart’ signals (3’ and 5’) or isolated red signals (3’) and/or native copies (3’/5’). All ALK-abnormal cells were validated by a cytogenetician. Clinical data was collected retrospectively. Confirming our previous data, ALK rearrangement was detected in CTCs: 32/39 patients (82%) had ≥4 ALK-rearranged CTCs per 1 ml of blood. Age &amp;gt;60 years and smoking status &amp;gt;15 pack years were associated with a higher mean number of ALK-amplified CTCs (p = 0.0423 and p = 0.0407 respectively). No statistically significant correlation was observed between the different CTC subsets with abnormal FISH patterns at baseline and PFS or OS. However we observed a statistically significant correlation (p = 0.0196) between the evolution under crizotinib of the numbers of ALK-amplified CTCs and PFS. An increase in the numbers of ALK-amplified CTCs during treatment is associated with a PFS of 6.5 months, while a decrease in this subset is associated with a PFS of 25 months. There was no corresponding correlation with OS. The evolution under crizotinib therapy of numbers of ALK-amplified CTCs is significantly correlated with PFS. Although not dominant, amplification of the ALK gene has been reported to be one mechanism of acquired resistance to crizotinib therapy in tumor biopsies. CTCs with an amplification of ALK do not express the oncogenic ALK fusion protein and are therefore not targeted by crizotinib. FISH patterns suggested these CTCs may have a high degree of ploidy and chromosomal instability which may contribute to promote the emergence of CTC subclones with a high metastatic potential. Our data suggest that number of ALK-amplified CTCs may be a predictive biomarker allowing prediction of ALK-rearranged NSCLC patients who are at risk of early resistance to crizotinib. Citation Format: Emma Pailler, Marianne Oulhen, Kirsty Ross, Fanny Billiot, Nathalie Auger, Isabelle Borger, Maud NgoCamus, Jordi Remon-Masip, David Planchard, Jean-Charles Soria, Benjamin Besse, Françoise Farace. Number of ALK-amplified circulating tumor cells predicts progression-free survival in ALK-rearranged non-small cell lung cancer patients treated by crizotinib. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 856.

Upregulation of PD-L1 by EML4-ALK fusion protein mediates the immune escape in ALK positive NSCLC: Implication for optional anti-PD-1/PD-L1 immune therapy for ALK-TKIs sensitive and resistant NSCLC patients

ABSTRACTDriver mutations were reported to upregulate programmed death-ligand 1 (PD-L1) expression. However, how PD-L1 expression and immune function was affected by ALK-TKIs and anti-PD-1/PD-L1 treatment in ALK positive non-small-cell lung cancer (NSCLC) remains poorly understood. In the present study, western-blot, real-time PCR, flow cytometry and immunofluorescence were employed to explore how PD-L1 was regulated by ALK fusion protein. ALK-TKIs and relevant inhibitors were used to identify the downstream signaling pathways involved in PD-L1 regulation. Cell apoptosis, viability and Elisa test were used to study the immune suppression by ALK activation and immune reactivation by ALK-TKIs and/or PD-1 blocking in tumor cells and DC-CIK cells co-culture system. We found that PD-L1 expression was associated with EGFR mutations and ALK fusion genes in NSCLC cell lines. Over-expression of ALK fusion protein increased PD-L1 expression. PD-L1 mediated by ALK fusion protein increased the apoptosis of T cells in tumor cells and DC-CIK cells co-culture system. Inhibiting ALK by sensitive TKIs could enhance the production of IFNγ. Anti-PD-1 antibody was effective in both crizotinib sensitive and resistant NSCLC cells. Synergistic tumor killing effects were not observed with ALK-TKIs and anti-PD-1 antibody combination in co-culture system. ALK-TKIs not only directly inhibited tumor viability but also indirectly enhanced the antitumor immunity via the downregulation of PD-L1. Anti-PD-1/PD-L1 antibodies could be an optional therapy for crizotinib sensitive, especially crizotinib resistant NSCLC patients with ALK fusion gene. Combination of ALK-TKIs and anti-PD-1/PD-L1 antibodies treatment for ALK positive NSCLC warrants more data before moving into clinical practice.

Abstract 497: Understanding oncogenic fusions: Lessons learned from inflammatory myofibroblastic tumor

Abstract Oncogenic kinase fusions are validated targets for cancer therapy. With the advent of next generation sequencing (NGS) based clinical diagnostic tools, the detection of these kinase fusions is rapidly increasing across multiple adult and pediatric tumor types. However, there remains a need to better understand the functional impact of these fusions in order to help direct clinical therapies. To study fusion biology, we have made use of a large collection of inflammatory myofibroblastic tumor (IMT) samples. IMT is a rare mesenchymal malignancy, which typically occurs in children. We have recently demonstrated that IMTs harbor multiple therapeutically actionable kinase fusions, including ALK, ROS1, and PDGFRB fusions, using a targeted capture-based NGS assay in a CLIA laboratory (Foundation Medicine). Our initial results demonstrated both previously described ALK fusions, including TPM3-, TPM4-, TFG-, and RANBP2-ALK as well as novel ALK fusions, including PRKAR1A-ALK and LMNA-ALK. Although the presence of ALK fusions within a tumor has been correlated with response to ALK inhibitor therapy, the role that the 5′ prime partner gene may play in the functional biology of the fusion has not been systematically investigated. To address this, we stably transfected cDNAs encoding LMNA-ALK, RANBP2-ALK, FN1-ALK, TFG-ALK, and PRKAR1A-ALK into BA/F3 cells. All X-ALK (X = the fusion partner) variants were tyrosine phosphorylated and their subcellular distribution was in agreement with that observed in the primary tumors harboring the identical fusion. Subcellular localization was altered as a function of the fusion partner. For example, LMNA-ALK was predominantly cytoplasmic while RANBP2-ALK was predominantly perinuclear. Additionally, we compared proliferation rates, downstream signaling, and sensitivity to various structurally different ALK inhibitors amongst all of the X-ALK fusions. Overall, our results suggest that the specific fusion partner may affect the properties of the ALK fusion protein, including sensitivity to ALK inhibitors currently in clinical use. To date, most ALK fusions are detected by immunohistochemistry for ALK overexpression or by “break-apart” fluorescence in situ hybridization (FISH), techniques which may be falsely negative in some settings and in others cannot discern specific fusion present. As the role of NGS increases in clinical diagnostics, our findings may provide further biological and clinical insights into these kinase fusions. Citation Format: Merrida A. Childress, Abha Gupta, Doron Lipson, Geoff Otto, Tina Brennan, Catherine T. Chung, Scott C. Borinstein, Jeffrey S. Ross, Phillip J. Stephens, Vincent A. Miller, Cheryl M. Coffin, Jason L. Hornick, Christine M. Lovly. Understanding oncogenic fusions: Lessons learned from inflammatory myofibroblastic tumor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 497. doi:10.1158/1538-7445.AM2015-497

Tackling Crizotinib Resistance: The Pathway from Drug Discovery to the Pediatric Clinic.

Neuroblastoma is a childhood malignancy that has not yet benefitted from the rapid progress in the development of small-molecule therapeutics for cancer. An opportunity to take advantage of pharmaceutical innovation in this area arose when the identification of ALK fusion proteins in non-small cell lung cancer (NSCLC) occurred in parallel to the discovery of point mutations of ALK in neuroblastomas. ALK is now known to be a marker of poor outcome in neuroblastoma, and therefore, urgent development of specific ALK inhibitors to treat this devastating disease is a necessity. However, the translation of small molecules from adult directly into pediatric practice has thus far been challenging, due to mutation-specific structural variances in the ALK kinase domain. We discuss how the most recent structural and biological characterizations of ALK are directing preclinical and clinical studies of ALK inhibitors for both NSCLC and neuroblastoma.

Immunotherapy for Lung Cancer: Has it Finally Arrived?

The possible link between infection/inflammation/immune activation and a cancer patient’s outcome from both a causative and outcome point of view has long been postulated. Substantial progress in the understanding of tumor-associated antigens/epitopes, immune cellular subpopulations, cytokine pathways/expression, the tumor microenvironment, and the balance between tumor-immune suppression and stimulation have been made over the past decade. This knowledge has heralded a new era of tumor immunotherapy utilizing vaccines, immune checkpoint inhibition, and oncolytic viruses. Despite significant progress in the molecular era now with targeted therapeutics such as EGFR tyrosine kinase inhibitors and ALK fusion protein inhibitors that have significantly improved the outcome of these specific lung cancer subpopulations, the overall 5 year survival for all non-small cell lung cancer (NSCLC) is still <20%. Unlike malignancies such as malignant melanoma, renal cell carcinoma, and neuroblastoma given their documented spontaneous remission rates lung cancer historically has been felt to be resistant to immune approaches likely related to an immunosuppressive tumor microenvironment and/or lack of immune recognition. Defining responding populations, understanding the mechanism(s) underlying durable immune responses, and the role of chemotherapy, radiation, oncolytic viruses, and other tumor disrupting agents in augmenting immune responses have led to improved optimization of immune therapeutic strategies. The purpose of this review is to focus on the recent advances in lung immunotherapy with an emphasis on recent clinical trials in the last 5 years in NSCLC.

O3-9-6 - Molecular Targeting Therapy and Csf Shunt for Patients with Leptomeningeal Metastasis From Lung Adenocarcinoma

Background: The incidence of leptomeningeal metastasis (LM) has increased in patients with lung adenocarcinoma (LAC) because of recent improvements in survival. New effective therapies for lung cancer have underscored the clinical significance of LM. LM associated with hydrocephalus (LM-H) has been particularly hard to be controlled of severe symptom by chemotherapy and/or radiotherapy. We reviewed the outcomes of patients with LAC-LM-H who underwent multidisciplinary treatment (EGFR-TKI or ALK-inhibitor, cerebrospinal fluid (CSF) shunt and/ or radiotherapy) in our institute.Methods: Data of patients with LAC-LM-H diagnosed by MR images and cytological examination were retrospectively analyzed. Seventeen LAC-LM-H patients were treated between June 2008 and November 2013. Patients underwent CSF shunt followed by EGFR-TKI or ALK-inhibitor therapy, if they had EGFR mutant or ALK fusion protein, systemic life expectancy longer than three months after control of their LM, and controlled extra CNS metastases. Overall survival was analysis with Kaplan-Meier survival curves.Results: The patients consisted of 4 men and 13 women with a median age at LM of 59 years (range 39-76). Sixteen patients were administrated EGFR-TKI, and one patient was ALK-inhibitor. Median overall survival (mOS) was 5.5 months from the onset of LM, and 3.5 months after CSF shunt. The longest survivors were alive for 20 months (ALK positive) and 15 months (EGFR mutant). Median OS in patients of ECOG performance status 1-2 significantly longer than those of PS3-4 (9 months, 4 months, p = 0.03). CSF shunt surgery yielded patients beneficial rapid improvement in 94% of patients of their performance status.Conclusion: Combination of molecular targeting therapy and CFS shunt is a safe and effective strategy in patients with LAC-LM-H. Further clinical investigations are needed to clarify appropriate assessments and treatment indication for patients with LAC-LM-H.

Abstract 2841: Characterization of ALK fusions in circulating tumor cells (CTCs) of NSCLC

Abstract Background: Many NSCLC patients have insufficient tumor sample or have high co-morbidities preventing access to tissue biopsies for FISH or IHC to determine ALK fusion and subsequent eligibility to Crizotinib therapy. We developed a gene/protein assay for the ALK fusion and examined CTCs and CTC subpopulation incidence and molecular characterization in newly diagnosed NSCLC patients. Methods: 10 samples from newly diagnosed NSCLC patients prior to therapy were recruited and blood specimens were collected and shipped to Epic Sciences. 2/10 had known ALK rearrangement through tissue analysis. All nucleated cells were plated onto glass slides and subjected to IF staining and CTC identification by fluorescent scanners and algorithmic analysis. CTCs, defined as classic (CK+ CD45- w/intact DAPI nuclei and morphologically distinct), apoptotic (CK+, CD45-, non-intact nuclei) and CK- (CK-, CD45-, intact and distinct) were identified. Samples were characterized with ALK IF to assess expression. Patients with known ALK rearrangements in tissue also had their CTCs assessed with ALK FISH. Results: Assays for the ALK protein and ALK gene rearrangement were developed and specificity confirmed utilizing H2228 (ALK positive) &amp; A549 (ALK negative) cells spiked into donor blood and run through the Epic Assay. Expression of ALK fusions was visualized in CTCs of 1/2 patients with known ALK rearrangements and 0/8 patients with no ALK rearrangements. ALK fusion protein expression was seen in classic, apoptotic, small and CK- CTCs. Conclusion: ALK fusion protein and ALK rearrangement assessment with FISH utilizing the Epic CTC platform demonstrates sensitivity and specificity to identification of patients eligible to Crizotinib. The identification of ALK in CTC subpopulations identifies unique tumor cell morphology and suggests evidence of epithelial plasticity. Further clinical studies are ensuing to determine the sensitivity and specificity of ALK in CTCs and the relevance of CTC subpopulations related to Crizotinib therapy and NSCLC progression. Citation Format: David Lu, Rachel Krupa, Natalee Bales, Jessica Louw, Dena Marrinucci, Ryan Dittamore. Characterization of ALK fusions in circulating tumor cells (CTCs) of NSCLC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2841. doi:10.1158/1538-7445.AM2014-2841

Intragenic anaplastic lymphoma kinase (ALK) rearrangements: translocations as a novel mechanism of ALK activation in neuroblastoma tumors.

Anaplastic lymphoma kinase (ALK) has been demonstrated to be deregulated in sporadic as well as in familiar cases of neuroblastoma (NB). Whereas ALK-fusion proteins are common in lymphoma and lung cancer, there are few reports of ALK rearrangements in NB indicating that ALK mainly exerts its oncogenic capacity via activating mutations and/or overexpression in this tumor type. In this study, 332 NB tumors and 13 cell lines were screened by high resolution single nucleotide polymorphism microarray. Gain of 2p was detected in 23% (60/332) of primary tumors and 46% (6/13) of cell lines, while breakpoints at the ALK locus were detected in four primary tumors and two cell lines. These were further analyzed by next generation sequencing and a targeted enrichment approach. Samples with both ALK and MYCN amplification displayed complex genomic rearrangements with multiple breakpoints within the amplicon. None of the translocations characterized in primary NB tumors are likely to result in a chimeric protein. However, immunohistochemical analysis reveals high levels of phosphorylated ALK in these samples despite lack of initial exons, possibly due to alternative transcription initiation sites. Both ALK proteins predicted to arise from such alterations and from the abnormal ALK exon 4-11 deletion observed in the CLB-BAR cell line show strong activation of downstream targets STAT3 and extracellular signal-regulated kinase (ERK) when expressed in PC12 cells. Taken together, our data indicate a novel, although rare, mechanism of ALK activation with implications for NB tumorigenesis.

Rationale for co-targeting IGF-1R and ALK in ALK fusion-positive lung cancer.

The ALK tyrosine kinase inhibitor (TKI), crizotinib, shows significant activity in patients whose lung cancers harbor ALK fusions but its efficacy is limited by variable primary responses and acquired resistance. In work arising from the intriguing clinical observation of a patient with ALK fusion+ lung cancer who had an ‘exceptional response’ to an IGF-1R antibody, we define a therapeutic synergism between ALK and IGF-1R inhibitors. Similar to IGF-1R, ALK fusion proteins bind to the adaptor, IRS-1, and IRS-1 knockdown enhances the anti-tumor effects of ALK inhibitors. In models of ALK TKI resistance, the IGF-1R pathway is activated, and combined ALK/IGF-1R inhibition improves therapeutic efficacy. Consistent with this finding, IGF-1R/IRS-1 levels are increased in biopsy samples from patients progressing on crizotinib therapy. Collectively, these data support a role for the IGF-1R/IRS-1 pathway in both ALK TKI-sensitive and TKI-resistant states and provide biological rationale for further clinical development of dual ALK/IGF-1R inhibitors.

Future of ALK inhibition in non-small-cell lung cancer

Progress in defining molecular targets of oncogenesis and drugs to inhibit cancer growth in specific populations has led to augmented outcomes for patients and new expectations in the development of treatments. The EML4–ALK fusion protein was identified in patients with non-small-cell lung cancer (NSCLC) in 2007.1 Rearrangements in the ALK gene lead to constitutive signalling, triggering transforming properties. Up to now, two drugs have been approved by the US Food and Drug Administration for ALK-rearranged NSCLC—crizotinib and ceritinib.

EML4–ALK Fusions: Propelling Cancer but Creating Exploitable Chaperone Dependence

The crystal structure of a conserved tubulin-binding region of the EML1 protein reveals a highly atypical fold in one of its β-propeller domains. Disruption of the EML1 core region domain in many of the oncogenic EML4-ALK fusion protein variants that drive non-small cell lung cancer explains their dependence on the HSP90 molecular chaperone, provides a basis to allow more precise patient stratification for therapy, and suggests a more general model for other oncogenic fusion proteins.

ALK gene aberrations and the JUN/JUNB/PDGFR axis in metastatic NSCLC.

Anaplastic lymphoma kinase (ALK) gene aberrations are found in several tumor types including anaplastic large cell lymphoma (ALCL) and non-small cell lung cancer (NSCLC). Crizotinib, an inhibitor of ALK-fusion proteins, has shown clinical activity, but resistance mechanisms limit long-lasting disease control. It has been shown that ALK-NPM fusion upregulates platelet-derived growth factor receptor beta (PDGFRB) via JUN and transcription factor Jun B (JUNB) in ALCL. PDGFRB inhibition has been identified as therapy option for ALK-positive ALCL. Here, we investigated the ALK/JUN/JUNB/transcription factor Jun C (JUNC)/PDGFR axis in metastatic NSCLC with regard to ALK aberrations. We performed immunohistochemical analysis of platelet-derived growth factor receptor alpha (PDGFRA), PDGFRB, JUNB and JUNC expression in formalin-fixed, paraffin-embedded specimens of 15 NSCLC brain metastases (5 ALK translocations, 3 of them involving EML4, 5 ALK amplifications, 5 without ALK aberrations). We did not find a statistically significant difference in expression of PDGFRA, PDGFRB, JUNB or JUNC in tumor samples with normal ALK status, ALK amplification or ALK translocations (Kruskal-Wallis test p>0.05). Interestingly, PDGFRB was not expressed in tumor cells (but in vascular and stromal cells) in any of our cases. Our data argue against PDGFRB activation in association with ALK gene aberrations in metastatic NSCLC and thus seem to imply differential pathobiological roles of ALK alterations in lung cancer and lymphoma, possibly depending on different fusion partner genes. These results may be relevant for targeted therapies, as they indicate that inhibition of PDGFRB in ALK translocated NSCLC seems to be no therapeutic opportunity.

Abstract A94: Understanding of differing sensitivity in EML4-ALK NSCLC patients to crizotinib and geldanamycin.

Abstract Non-small cell lung cancer (NSCLC) is the most common form of lung cancer, afflicting nearly 200,000 people in the United States each year. Abnormal ALK is found in about 5% of NSCLC cases, meaning more than 5,000 new patients could benefit from the tyrosine kinase inhibitor (TKI) crizotinib. However, not all patients benefit from such treatment, with the clinical response to crizotinib differing among patients who harbor the same molecular abnormality. Similarly, patients with ALK fusion proteins have shown varying sensitivity to the HSP90 inhibitor geldanamycin in preclinical studies. We have chosen two NSCLC cell lines, H3122 and H2228, as a model to address these questions. Both cell lines harbor EML4-ALK fusions with differing sensitivity to the inhibitors crizotinib and geldanamycin. We used the established method of TMT peptide labeling coupled with serial peptide immunoprecipitation. To observe the effects of crizotinib and geldanamycin on ALK sensitive H3122 and non-sensitive H2228 NSCLC cell lines quantitative analysis was performed. Phosphotyrosine, acetylation, methylation, ATM/ATR substrate (s/tQ), Akt/AMPK subs AGC/CAMK/STE, and MAPK family kinase motif antibodies were used for immunoprecipitation allowing us to characterize and quantify post translational modifications before and after treatment. In this study, we identify extensive signaling networks downstream of ALK across multiple spaces, including phosphorylation, acetylation, and methylation. These differences may be clinically significant and highlight the possibility that ALK inhibitors alone may only be effective in a subset of NSCLC ALK-positive patients with EML4-ALK inversion. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A94. Citation Format: Klarisa Rikova, Benjamin Hall, Anthony Possemato, Keri Mroszczyk, Kimberly A. Lee, Joan MacNeill, Jian Min Ren, Ailan Guo, Daniel Mulhern, Yi Wang, Sean A. Beausoleil, Michael J. Comb. Understanding of differing sensitivity in EML4-ALK NSCLC patients to crizotinib and geldanamycin. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A94.

Prognostic significance and therapeutic potential of the activation of anaplastic lymphoma kinase/protein kinase B/mammalian target of rapamycin signaling pathway in anaplastic large cell lymphoma

BackgroudActivation of the protein kinase B/mammalian target of rapamycin (AKT/mTOR) pathway has been demonstrated to be involved in nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-mediated tumorigenesis in anaplastic large cell lymphoma (ALCL) and correlated with unfavorable outcome in certain types of other cancers. However, the prognostic value of AKT/mTOR activation in ALCL remains to be fully elucidated. In the present study, we aim to address this question from a clinical perspective by comparing the expressions of the AKT/mTOR signaling molecules in ALCL patients and exploring the therapeutic significance of targeting the AKT/mTOR pathway in ALCL.MethodsA cohort of 103 patients with ALCL was enrolled in the study. Expression of ALK fusion proteins and the AKT/mTOR signaling phosphoproteins was studied by immunohistochemical (IHC) staining. The pathogenic role of ALK fusion proteins and the therapeutic significance of targeting the ATK/mTOR signaling pathway were further investigated in vitro study with an ALK + ALCL cell line and the NPM-ALK transformed BaF3 cells.ResultsALK expression was detected in 60% of ALCLs, of which 79% exhibited the presence of NPM-ALK, whereas the remaining 21% expressed variant-ALK fusions. Phosphorylation of AKT, mTOR, 4E-binding protein-1 (4E-BP1), and 70 kDa ribosomal protein S6 kinase polypeptide 1 (p70S6K1) was detected in 76%, 80%, 91%, and 93% of ALCL patients, respectively. Both phospho-AKT (p-AKT) and p-mTOR were correlated to ALK expression, and p-mTOR was closely correlated to p-AKT. Both p-4E-BP1 and p-p70S6K1 were correlated to p-mTOR, but were not correlated to the expression of ALK and p-AKT. Clinically, ALK + ALCL occurred more commonly in younger patients, and ALK + ALCL patients had a much better prognosis than ALK-ALCL cases. However, expression of p-AKT, p-mTOR, p-4E-BP1, or p-p70S6K1 did not have an impact on the clinical outcome. Overexpression of NPM-ALK in a nonmalignant murine pro-B lymphoid cell line, BaF3, induced the cells to become cytokine-independent and resistant to glucocorticoids (GCs). Targeting AKT/mTOR inhibited growth and triggered the apoptotic cell death of ALK + ALCL cells and NPM-ALK transformed BaF3 cells, and also reversed GC resistance induced by overexpression of NPM-ALK.ConclusionsOverexpression of ALK due to chromosomal translocations is seen in the majority of ALCL patients and endows them with a much better prognosis. The AKT/mTOR signaling pathway is highly activated in ALK + ALCL patients and targeting the AKT/mTOR signaling pathway might confer a great therapeutic potential in ALCL.

ALK status of NSCLC reflected in CTCs

Lung cancer is the leading cause of cancer death worldwide, with non-small-cell lung cancer (NSCLC) accounting for the majority of cases. The discovery that the EML4–ALK fusion protein kinase is a potent oncogenic driver in 3–7% of patients with NSCLC led to the development of the ALK inhibitor crizotinib. This agent and an accompanying companion diagnostic test were granted FDA approval for the detection of ALK-rearrangements. The test is performed on tumour biopsies or fine-needle aspirates, but is hampered by the lack of available tumour tissue.

Abstract 2637: Examining the function of the serine protease, Granzyme B, in ALK-positive, anaplastic large cell lymphoma.

Abstract Anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma (ALK+ ALCL) is an aggressive non-Hodgkin lymphoma (NHL) that accounts for 10-20% of childhood NHL. The normal cellular counterpart of this malignancy is thought to be an activated T lymphocyte, based on the observations that tumour cells often exhibit clonal T cell receptor gene rearrangements and possess cytotoxic granules containing proteins such as Perforin and serine protease, Granzyme B (GzB). Chromosomal translocations involving the ALK tyrosine kinase gene are the defining characteristic of this lymphoma. These translocations result in the expression of oncogenic ALK fusion proteins (e.g. NPM-ALK), which are able initiate signalling events crucial for the pathogenesis of ALK+ ALCL. We have previously found that signalling initiated by NPM-ALK and the JunB transcription factor contributes to the phenotypic characteristics of ALK+ ALCL by promoting the expression of GzB. GzB is best characterized for its role as an effector molecule used by cytotoxic T lymphocytes and natural killer cells to kill virally infected and transformed cells; however, GzB can also be secreted from different cell types and has the ability to cleave extracellular matrix (ECM) proteins. Furthermore, GzB is expressed in some urothelial and bladder cancers and was shown to promote invasion of bladder cancer cell lines in vitro and GzB expression is associated with a greater level of tumour invasiveness in patients with urothelial cancer. Given these findings, and that NPM-ALK signalling promotes GzB expression in ALK+ ALCL, we hypothesized that GzB plays a role in the pathogenesis of this malignancy by influencing the interaction of ALK+ ALCL cells with ECM proteins. In this regard we hypothesize that GzB released by ALK+ ALCL cells can cleave ECM proteins which may affect the ability of ALK+ ALCL cells to adhere to these ECM proteins and may also promote tumour cell invasion. We have found that ALK+ ALCL cell lines express enzymatically active GzB that has the ability to cleave the ECM proteins, fibronectin and vitronectin. Furthermore, we have found that these cells also release active GzB and we are now examining if GzB expression influences adhesion of ALK+ ALCL cell lines to ECM proteins and whether GzB promotes the invasion of ALK+ ALCL cell lines in vitro. This work will determine if the expression of GzB is more than just a phenotypic characteristic of ALK+ ALCL, and whether this protein may contribute to the invasive potential of this lymphoma. If so, these findings may help to explain why tumour cells are found to be widely disseminated in ALK+ ALCL patients and murine xenograft models. Citation Format: Joel D. Pearson, Katelynn Rowe, Kayla Thew, Jing xi Zhang, Spencer Freeman, Robert J. Ingham. Examining the function of the serine protease, Granzyme B, in ALK-positive, anaplastic large cell lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2637. doi:10.1158/1538-7445.AM2013-2637