EML4-ALK Fusion Protein Research Articles

Abstract 1657: Microfluidics-based screening platform identifies a novel therapeutic approach to targeting EML4-ALK driven cancers

Abstract To achieve proliferative dysregulation, a hallmark of cancer, tumor cells utilize a variety of mechanisms to maintain constitutive oncogenic signaling in the absence of extracellular cues. One such mechanism occurs in cells expressing the EML4-ALK fusion protein. This fusion, found in about 4% of non-small cell lung cancer (NSCLC), is a result of an inversion within chromosome 2, placing the 3’ end of the ALK (anaplastic lymphoma kinase) gene downstream of the 5’ end of the EML4 (echinoderm microtubule associated protein-like 4) gene. The resulting fusion protein contains the oligomerization domain and additional disordered sequence elements of EML4 upstream of a functional ALK kinase domain. EML4-ALK is a critical driver for these tumors, promoting aberrant ALK-dependent oncogenic signaling. While tumors harboring the EML4-ALK fusion initially respond to ALK tyrosine kinase inhibitors (TKIs), resistance remains a significant clinical challenge requiring new therapeutic approaches. Recently, it has come to be understood that rather than responding through extracellular proliferative cues, the EML4-ALK fusion protein elicits constitutive ALK signaling through the formation of cytoplasmic biomolecular condensates. These condensates are the result of phase separation and have been shown to be causal for disease phenotypes in cell and animal models driven by EML4-ALK, establishing condensate modulation as a novel means by which to treat TKI-resistant tumors. Here, we have developed a novel proprietary microfluidics-based high throughput screening platform (termed PhaseScanTM) to identify small-molecule modulators of EML4-ALK condensates. Using this technology, we identified compounds that disrupt EML4-ALK condensates in vitro and in cells through a novel mechanism, distinct from that of known ALK TKIs, providing utility in both TKI-resistant as well as TKI-naïve tumors. Further profiling of the mechanism of action (MoA) revealed specific modification within the disordered region of EML4, disruption of EML4 oligomerization, and defective ALK signaling in cancer cells. Thus, our PhaseScanTM methodology enabled the identification of novel, functionally active chemical matter that would have otherwise been challenging to identify utilizing any conventional hit identification methodology. Citation Format: Richard C. Centore, Matthew Watson, Julia Doh, Jerome Cattin, Cinzia Sgambato, Amal Alex, Janhavi Sawant, Prathima Radhakrishnan, Jasmine Cornish, Alex Howarth, Nagakumar Bharatham, William E. Arter, Seema Qamar, Kadi L. Saar, Douglas Williamson, Andrew Seeber, Neils Groenewegen, Magdalena Czekalska, Tadas Kartanas, Niklas Ermann, Ahmed Taher, Tuomas Knowles, Shilpi Arora. Microfluidics-based screening platform identifies a novel therapeutic approach to targeting EML4-ALK driven cancers [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 1657.

EML4-ALK fusion protein in Lung cancer cells enhances venous thrombogenicity through the pERK1/2-AP-1-tissue factor axis

BackgroundAccumulating evidence links the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) rearrangement to venous thromboembolism (VTE) in non-small cell lung cancer (NSCLC) patients. However, the corresponding mechanisms remain unclear.MethodHigh-throughput sequencing analysis of H3122 human ALK-positive NSCLC cells treated with ALK inhibitor/ dimethyl sulfoxide (DMSO) was performed to identify coagulation-associated differential genes between EML4-ALK fusion protein inhibited cells and control cells. Sequentially, we confirmed its expression in NSCLC patients’ tissues and in the plasma of a subcutaneous xenograft mouse model. An inferior vena cava (IVC) ligation model was used to assess clot formation potential. Additionally, pathways involved in tissue factor (TF) regulation were explored in ALK-positive cell lines H3122 and H2228. Statistical significance was determined by Student t-test and one-way ANOVA using SPSS.ResultsSequencing analysis identified a significant downregulation of TF after inhibiting EML4-ALK fusion protein activity in H3122 cells. In clinical NSCLC cases, TF expression was increased especially in ALK-positive NSCLC tissues. Meanwhile, H3122 and H2228 with high TF expression exhibited shorter plasma clotting time and higher TF activity versus ALK-negative H1299 and A549 in cell culture supernatant. Mice bearing H2228 tumor showed a higher concentration of tumor-derived TF and TF activity in plasma and the highest adjusted IVC clot weights. Limiting EML4-ALK protein phosphorylation downregulated extracellular regulated protein kinases 1/2 (ERK1/2)-activating the protein-1(AP-1) signaling pathway and thus attenuated TF expression.ConclusionEML4-ALK fusion protein may enhance venous thrombogenicity by regulating coagulation factor TF expression. There was potential involvement of the pERK1/2-AP-1 pathway in this process.

Abstract 4024: Overcoming ALK resistance with covalent cysteine-reactive inhibitors in lung cancer

Abstract The EML4-ALK fusion gene is the main oncogenic driver in 5% of non-small cell lung cancer and has been effectively targeted using ALK-specific tyrosine kinase inhibitors. While survival rates have improved steadily with each new generation of ALK TKI, resistance generally develops, including second site ALK kinase mutations as well as by-pass track activation. Since almost all anti-ALK approaches have focused on kinase domain inhibition, we sought to develop novel inhibitors with alternative mechanisms of action against ALK to overcome current drug resistance. Cysteine-reactive small molecule drugs may enable new mechanisms of target inhibition, and clinically successful examples of recent FDA-approved drugs in NSCLC include afatinib, osimertinib, sotorasib, and adagrasib.We aimed to explore and uncover novel cysteine-reactive covalent inhibitors of EML4-ALK. Using cysteine druggability mapping (CDM), a proteome-wide mass spectrometry target engagement assay, we first determined the landscape of reactive cysteines in ALK-positive cell lines and identified various targetable cysteines within the MAPK pathway and within the EML4-ALK fusion protein itself. To identify small molecules that modulate ALK activation, degradation, and downstream signaling, we conducted a high-content phosphorylated protein screen by testing 4,400 reactive cysteine compounds and found 20 potential hits that impacted ALK phosphorylation or expression. Using proteomic analysis, we have determined the comparative specificity of these compounds, and have ranked them based on p-ALK reduction efficiency and on their off-target binding patterns. We have initiated hit-to-lead development on a compound that binds to the EML4 portion of the fusion and we are performing a detailed analysis of its mechanism of action. To further optimize the potency of the compound, we designed, synthesized, and are testing several hundred analog compounds. By applying the hit compounds from the screen in ALK TKI-resistant models, we will be able to evaluate the ability of these compounds to overcome ALK resistance. To our knowledge, we have identified the first EML4-ALK covalent binder targeting the EML4 domain. Using the unique mechanism of action of the current ALK kinase inhibitor, we have uncovered a potential compound to overcome ALK resistance. Citation Format: Diane Yang, Stefan Harry, Edwin Zhang, Zavontae Oppong-Holmes, Alexander Daniel Carlin, Wenxin Yang, Abigail Smith, Maristela Onozato, Liron Bar-Peled, Anthony J. Iafrate. Overcoming ALK resistance with covalent cysteine-reactive inhibitors in lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4024.

Abstract 4041: The EML4-ALK fusion protein mediates reduced sensitivity to the combination of neratinib and dasatinib

Abstract Introduction: The anaplastic lymphoma kinase (ALK) gene encodes a receptor tyrosine kinase involved in cellular proliferation, differentiation, and cell death. ALK becomes oncogenic when it forms a fusion gene. The EML4-ALK fusion gene has been identified mainly in non-small cell lung cancer (NSCLC). It is unknown whether neratinib, an irreversible pan-HER tyrosine kinase inhibitor (TKI) targeting EGFR, HER2 and HER4, is effective in cancers harboring EML4-ALK gene fusion. The objective of this study was to assess in vitro efficacy of neratinib in combination with dasatinib (Src/Abl TKI) or crizotinib (ALK TKI) in EML4-ALK+ NSCLC. Methods: The antiproliferative effects of neratinib, dasatinib and crizotinib were assessed in the CRISPR/Cas9-modified EML4-ALK fusion-A549 (EML4-ALK+) and parental A459 (A549-Par) NSCLC cell lines by 5-day acid phosphatase assay. IC50 values were calculated using CalcuSyn software. To assess the synergy between neratinib and dasatinib, and neratinib and crizotinib, matrix assays were performed and analyzed using Combenefit software. To further assess the efficacy of the TKIs, changes in signaling pathways were assessed by Western blotting, and apoptosis induction and cell migration were measured with the Incucyte® S3 imaging system. Results: Neratinib, crizotinib, and dasatinib displayed nanomolar IC50 values in both cell lines. As expected, EML4-ALK+ cells were more sensitive to crizotinib than A549-Par cells (IC50 = 595 nM vs 1 µM, p ≤ 0.05). EML4-ALK expression led to numerical increases in neratinib IC50 value (326.37 ± 44.34 nM in EML4-ALK+ vs 247 ± 32.65 nM in A549-Par) and dasatinib IC50 value (39.95 ± 5.67 nM in EML4-ALK+ vs 27.75 ± 18.53 nM in A549-Par) but these changes were not statistically significant. Matrix assays showed that neratinib-crizotinib (NC) was more effective than crizotinib alone in both cell lines, with more potent sensitivity in EML4-ALK+ cells. The neratinib-dasatinib (ND) combination was synergistic in the A549-Par cell line but only additive in the EML4-ALK+ cell line, suggesting a reduced sensitivity to the ND combination. EML4-ALK+ cells had sustained pERK1/2 levels after treatment with all TKIs, except NC, compared to A549-Par cells. Neratinib alone was significantly less effective at inhibiting pAkt (99.2 ± 5.3 % in EML4-ALK+ vs 64.9 ± 19.0 %, p ≤ 0.05) and pERK1/2 (91.3 ± 4.6 % in EML4-ALK+ vs 54.9 ± 15.9 %, p ≤ 0.01) in EML4-ALK+ cells than in A549-Par cells at 24h. In A549-Par cells, ND caused the highest apoptotic induction, followed by NC. Apoptosis levels induced by ND in EML4-ALK+ cells were 4-fold lower at 72hr than in A549-Par cells (p ≤ 0.0001). Dasatinib and ND were more effective than crizotinib or NC at preventing cell migration in both cell lines. Conclusion: EML4-ALK potentially decreases sensitivity to the neratinib-dasatinib combination. This warrants further investigation in other EML4-ALK+ models. Citation Format: Myra E. Castel, Neil T. Conlon, Lisa D. Eli, Alvin Wong, John Crown, Denis M. Collins. The EML4-ALK fusion protein mediates reduced sensitivity to the combination of neratinib and dasatinib. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4041.

Molecular Anatomy of the EML4-ALK Fusion Protein for the Development of Novel Anticancer Drugs.

The EML4 (echinoderm microtubule-associated protein-like 4)-ALK (anaplastic lymphoma kinase) fusion gene in non-small-cell lung cancer (NSCLC) was first identified in 2007. As the EML4-ALK fusion protein promotes carcinogenesis in lung cells, much attention has been paid to it, leading to the development of therapies for patients with NSCLC. These therapies include ALK tyrosine kinase inhibitors and heat shock protein 90 inhibitors. However, detailed information on the entire structure and function of the EML4-ALK protein remains deficient, and there are many obstacles to overcome in the development of novel anticancer agents. In this review, we describe the respective partial structures of EML4 and ALK that are known to date. In addition to their structures, noteworthy structural features and launched inhibitors of the EML4-ALK protein are summarized. Furthermore, based on the structural features and inhibitor-binding modes, we discuss strategies for the development of novel inhibitors targeting the EML4-ALK protein.

PRIMARY MUCINOUS ADENOCARCINOMA OF THE LUNG WITH SIGNET-RING CELLS AND ALK REARRANGEMENT: A CASE REPORT

TYPE: Case Report TOPIC: Lung Pathology INTRODUCTION: The presence of malignant signet-ring cells is more commonly seen in the adenocarcinomas, particularly those of the gastrointestinal tract and breast. In a study with 24,171 patients with signet-ring cell carcinoma (SRCC), it was found that only 3.1% of the cases have a primary pulmonary origin, making the study of SRCC of the lung restricted to reports and case series. CASE PRESENTATION: We present the case of a 70-year-old patient with a history of retroperitoneal liposarcoma and papillary thyroid carcinoma in whom a primary pulmonary adenocarcinoma of mucinous pattern with signet-ring cells and ALK rearrangement was diagnosed. DISCUSSION: The World Heart Organization (WHO) had included the SRCC subtype. However, since 2015 WHO classification, it is recognized only as cytologic features rather than primary histologic subtype. The incidence of primary SRCC of the lung is between 0.14 to 1.9% of all lung tumors. The differences in incidence rates lie in the definition since clear diagnostic criteria have not been established. Its presentation is slightly more common in men (52.7%) and Caucasian patients (82.2%), with a mean overall survival duration of 8 months. Patients presenting with the EML4-ALK fusion protein have unique pathological and physiological characteristics. Several reports have shown that it predominates in a young female population with adenocarcinoma and without exposure to tobacco. However, these are not consistent data in all studies. CONCLUSIONS: Lung cancer has a very heterogeneous presentation. We emphasize the importance of approaching these patients with histopathological, immunohistochemical, and molecular biology studies. DISCLOSURE: Nothing to declare. KEYWORD: pulmonary signet ring cell carcinoma

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.

A fully automated assay to detect the expression of pan-cytokeratins and of EML4-ALK fusion protein in circulating tumour cells (CTCs) predicts outcome of non-small cell lung cancer (NSCLC) patients

BackgroundIn advanced non-small cell lung cancer (NSCLC) a recent meta-analysis confirms circulating tumour cells (CTCs) as an independent prognostic indicator of progression-free survival (PFS) and overall survival (OS). However, further investigations are necessary to predict and dynamically monitor the therapy in NSCLC patients using CTCs. To this aim, we combined the classical standard assay (SA) with an expanded cytokeratins profile (EA) and quantified the expression of EML4-ALK fusion protein in CTCs.MethodsThe CellSearch (CS) platform—first marked in vitro diagnostic use (IVD) from Food and Drug Administration (FDA), and “gold standard” for quantifying CTCs - detects EpCAM and cytokeratins (CKs) 8, 18, and 19. Since NSCLC shows different CKs profile, we customized the SA, to recognize CK 4, 5, 6, 7, 8, 10, 13, 14, 18, and 19 (EA). Using both assays we designed a prospective, multi-center study, primarily aimed to enumerate CTCs in advanced NSCLC. Secondarily, we developed an integration of the EA to quantify the expression of EML4-ALK fusion protein in CTCs, and correlated them with PFS and OS.ResultsEA identified a significantly much more number of CTC-positive patients (115 out of 180) than SA (103 out of 192; Chi-square =4.0179, with 1 degrees of freedom, P=0.04502). Similar to SA, EA levels were still associated with patient’ outcomes. Furthermore, the expression of EML4-ALK on CTCs allowed stratifying NSCLC patients according to a statistically significant difference in PFS.ConclusionsWe proposed here two novel automated tests, to characterize the expression of specific molecules on CTCs. We demonstrated that these integrated assays are robust and actionable in prospective clinical studies, and in the future could allow clinicians to improve both choice and length of treatment in individual NSCLC patient.

Abstract 2400: Strong biological bias for ALK intron 19 breakpoints in NSCLC

Abstract Introduction: EML4-ALK translocations are detected in 4-8 % of non-small cell lung cancer (NSCLC). While different EML4-ALK variants are defined by different breakpoints in the EML4 gene, most frequently located in intron 6 or 13, ALK breakpoint is almost invariably in intron 19. Rare reports describe EML4-ALK translocations with breakpoints in intron 17 or intron 18 of the ALK gene. Despite all these ALK breakpoints potentially generate oncogenic fusions, the reasons of this strong imbalance toward intron 19 (exon 20) breakpoints in ALK positive NSCLC are currently unknown. The aim of this study is to investigate the mechanisms that underlie ALK translocation in NSCLC. Methods: Eight different EML4-ALK fusions were either overexpressed with a retroviral system or generated with CRISPR/Cas9 lentivirus from the endogenous loci. EML4 exons 1-6 (E6) or exons 1-13 (E13) with different ALK exons: E6;A18, E6;A19, E6;A20 or E6;A21 and E13;A18, E13;A19, E13;A20, E13;A21. The fusions E6;A21 and E13;A21 contained an early stop codon not producing functional proteins. These EML4-ALK fusions were expressed in NIH3T3 and PC9 by retroviral vectors or engineered in PC9 (an EGFR-dependent lung cancer cell line that is sensitive to inhibition with the EGFR inhibitor osimertinib) and BEAS-2B cells by CRISPR/Cas9. HTGTS was performed on engineered PC9 cells. NSG immunodeficient mice were used for in vivo experiments with NIH3T3 and BEAS-2B cells. Results: The retroviral overexpression system showed that all EML4-ALK fusion proteins were expressed leading to an actively phosphorylated ALK in NIH3T3 and PC9 cells, except E6;A21 and E13;A21 fusions where no protein was detected, as expected. Accordingly, all active variants were able to induce NIH3T3 cell transformation and tumor formation in vivo and conferred resistance to osimertinib in PC9 cells. In contrast, when EML4-ALK translocations were induced by CRISPR/Cas9 in PC9 cells, only E6;A20 and E13;A20 variants rapidly overcame osimertinib inhibition while other variants rescued osimertinib-inihibited cells slowly and less efficiently. Surprisingly, the E6;A20 and E13;A20 fusions were invariably found in all resistant clones independently of the translocation induced by CRISPR/Cas9. Similarly, all tumors formed in vivo by BEAS-2B cells contained the E6;A20 or E13;A20 variants irrespective of the translocation induced originally. By cloning translocation junctions at large scale with HTGTS, we found that clones initially forced to break into ALK intron 17 eventually acquired a second breakpoint in ALK intron 19. Conclusions: Our data show that all EML4-ALK fusion variants were equally oncogenic when overexpressed. In contrast, when EML4-ALK variants were generated from the endogenous loci there was a strong selection bias toward ALK fusions originating in intron 19 suggesting that intron 19 variants have the strongest oncogenic potential in lung epithelial cells. Citation Format: Giulia C. Leonardi, Taek-Chin Cheong, Chiara Ambrogio, Tao Chen, Wei-Tien Tai, Elif Karaca, Ines Mota, Massimo Libra, Mark M. Awad, Rafael Blasco-Patino, Roberto Chiarle. Strong biological bias for ALK intron 19 breakpoints in NSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2400.

Abstract 4900: An ALK fusion gene regulates different signaling pathways in mortal versus immortalized normal human cells for cellular senescence and transformation

Abstract Accumulating results of clinical trials lead targeted therapies to be the first choice for unresectable or recurrent lung cancer with driver mutations. Echinoderm Microtubule Associated Protein Like 4 (EML4) - Anaplastic lymphoma kinase (ALK) fusion is known as such a driver mutation. It presents in 3-6% of non-small cell lung carcinoma (NSCLC). EML4-ALK fusion protein generate the constitutive ALK kinase activity in NSCLC. The basic understanding of EML4-ALK remains insufficient due to the lack of functional studies using normal human cells. We investigated the role of EML4-ALK in mortal and immortalized normal human cells. The expression of EML4-ALK in normal, mortal human fibroblasts caused accumulated DNA damage, telomere shortening and the early induction of cellular senescence with senescence-associated beta-galactosidase activity and upregulation of p16INK4A and p21WAF1. In contrast, when EML4-ALK was expressed in telomerase reverse transcriptase (hTERT)-immortalized normal human fibroblasts and bronchial epithelial cells, the cells showed accelerated proliferation in vitro and anchorage-independent growth in soft agar, revealing its transformation activity. No chromosome aberrations, no mutations or loss of p53, nor impairment of the p16INK4A response was associated with this in vitro transformation, likely reflecting certain clinical features of EML4-ALK-positive NSCLC. In both mortal and immortalized cells, EML4-ALK induced the phosphorylation of STAT3, which is involved in both cellular senescence and transformation. Our data validate that EML4-ALK functions as an oncogene, although an additional oncogenic event(s) seems to be required for full tumorigenicity in vivo. This study also suggests that telomerase-mediated immortalization manifests the oncogenic activity of EML4-ALK, switching from its senescence-inducing activity. The RNA-seq analysis showed that the STAT3-induced cytokine/interferon signaling pathways were most significantly upregulated by EML4-ALK in mortal fibroblasts, consistent with accumulated DNA damage and senescence induction. The blood coagulation pathway activated by EML4-ALK in hTERT-immortalized cells may contribute to increased risk of disseminated intravenous coagulation in patients with EML4-ALK-positive cancer. These results suggest that EML4-ALK regulates the different signaling pathways in mortal versus immortalized normal human cells to induce the different cellular outcomes. Citation Format: Masaru Matsumoto, Akihiko Miyanaga, Jessica Beck, Izumi Horikawa, Mohammed Khan, Delphine Lissa, Masahiro Seike, Akihiko Gemma, Hiroyuki Mano, Curtis Harris. An ALK fusion gene regulates different signaling pathways in mortal versus immortalized normal human cells for cellular senescence and transformation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4900.

Development of a Brigatinib degrader (SIAIS117) as a potential treatment for ALK positive cancer resistance

EML4-ALK and NPM-ALK fusion proteins possess constitutively activated ALK (anaplastic lymphoma kinase) activity, which in turn leads to the development of non-small cell lung cancer and anaplastic large-cell lymphomas (ALCLs). FDA-approved ALK inhibitor drugs cause significant cancer regression. However, drug resistance eventually occurs and it becomes a big obstacle in clinic. Novel proteolysis targeting chimera (PROTAC) technology platform provides a potential therapeutic strategy for drug resistance. Herein, we designed and synthesized a series of ALK PROTACs based on Brigatinib and VHL-1 conjunction, and screened SIAIS117 as the best degrader which not only blocked the growth of SR and H2228 cancer cell lines, but also degraded ALK protein. In addition, SIAIS117 also showed much better growth inhibition effect than Brigatinib on 293T cell line that exogenously expressed G1202R-resistant ALK proteins. Furthermore, it also degraded G1202R mutant ALK protein in vitro. At last, it has the potentially anti-proliferation ability of small cell lung cancer. Thus, we have successfully generated the degrader SIAIS117 that can potentially overcome resistance in cancer targeted therapy.

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.

Leptomeningeal recurrence after long-term alectinib therapy for non-small cell lung cancer harboring an EML4-ALK fusion protein

The recent approval of anaplastic lymphoma kinase (ALK) inhibitors for the treatment of ALK-rearranged non-small cell lung cancer (NSCLC) has dramatically transformed cancer therapy. However, leptomeningeal metastases (LM) are frequent and often devastating complications of ALK-rearranged NSCLC, and treatment against LM remains challenging. Herein we report a case of a 19-year-old male diagnosed with ALK-rearranged NSCLC with LM. He experienced heavy treatment before introduction of alectinib therapy, which continued for approximately 5.5years with marked efficacy. However, he experienced recurrence of a bulbar metastasis after discontinuation of alectinib. Reintroduction of standard-dose alectinib therapy resolved the lesion again. Our findings suggest that ALK-tyrosine kinase inhibitor therapy should be continued in patients showing a long-term complete response, unless intolerable toxicities are present, and that rechallenge treatment with alectinib may represent a therapeutic option for central nervous system metastases.

Abstract 2156: Lung niche factors for progenitor cell self-renewal sustain lung cancer stem cells and contribute to drug resistance to kinase inhibitors in non-small cell lung cancer

Abstract A mechanism underlying anti-cancer drug resistance relies on the existence of cancer stem cells (CSCs). However, CSCs are either absent or are few in number in xenograft tumors and syngeneic mouse models. We developed new lung cancer mouse models with CSCs to elucidate the role of CSCs in resistance to targeted therapeutics in non-small cell lung cancer (NSCLC). Mouse lung organoids containing progenitor cells were cultured in complete medium containing lung niche factors, FGF-10 and HGF, and were infected with a retrovirus encoding a Kras mutation (G12D), an Egfr deletion, and the EML4-ALK fusion protein. These induced CSCs were transplanted into mice, and the established tumors were cultured in vitro to test for drug sensitivity to trametinib, erlotinib, and crizotinib in the presence or absence of niche factors. The tumors that were poorly differentiated contained EpCAM-, Cd49f-, and Sca-1-positive CSCs. These mouse models allowed the prediction of clinical responses to targeted therapeutics, i.e., the tumor cells with the Egfr deletion and those with EML4-ALK fusion proteins showed strong responses to erlotinib and crizotinib, respectively. In addition, we found that Kras mutant cancer cells were resistant to trametinib due to activation of the HGF/Met pathway since HGF is an essential niche factor for lung progenitor cell proliferation. Combination treatment with trametinib and a Met inhibitor showed a synergistic effect in Kras mutant tumors. Based on these results, we propose a treatment approach that focuses on the use of niche factors for lung progenitor cells in NSCLC. Citation Format: Akinori Mitsui, Sachiko Yokokawa, Osamu Nagano, Hideyuki Saya. Lung niche factors for progenitor cell self-renewal sustain lung cancer stem cells and contribute to drug resistance to kinase inhibitors in non-small cell lung cancer [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 2156.

Abstract 2896: A genome-wide CRISPR/Cas9 pooled screen identifies EML4-ALK variant-specific synthetic lethal partners of HSP90 inhibitors

Abstract The chromosomal rearrangement generating the EML4-ALK fusion protein occurs in 5–7% of patients with non-small-cell lung cancer (NSCLC). Distinct breakpoints give rise to variants with different EML4 fragments and an invariable ALK kinase domain. Patients treated with ALK tyrosine kinase inhibitors, such as crizotinib or alectinib, inevitably develop resistance to the drugs and need alternative therapies. EML4-ALK v1 and v3a/b, the most common variants of the fusion oncogene, display distinct cellular localization and different sensitivity to the inhibition of HSP90 chaperoning activity. In the clinic, HSP90 inhibitors have shown encouraging results in EML4-ALK+ NSCLC patients, but the responses have been heterogeneous. Currently, there is no EML4-ALK variant-based classification of patients and this might have contributed to confounding results in clinical trials. Moreover, the identification of additional biomarkers of sensitivity could further improve patient stratification and suggest potential combinatorial regimes. As chaperones are molecular hubs enriched for synthetic lethal partners, we performed a CRISPR/Cas9-based pooled screen in human NSCLC cell lines carrying different EML4-ALK variants in the presence of non-lethal concentrations of an HSP90 inhibitor. In the screen, we found already described as well as previously unreported EML4-ALK variant-specific synthetic lethal interactions. We are currently dissecting the mechanism behind these interactions, and evaluating their therapeutic potential for EML4-ALK+ NSCLC patients. Citation Format: Marco P. Licciardello, Paul A. Clarke, Paul Workman. A genome-wide CRISPR/Cas9 pooled screen identifies EML4-ALK variant-specific synthetic lethal partners of HSP90 inhibitors [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 2896.

Successful rechallenge with ceritinib after leukocytoclastic vasculitis during ceritinib treatment for non-small cell lung cancer harboring the EML4-ALK fusion protein

Anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitors (TKIs) dramatically improve progression-free survival compared to cytotoxic agents. It is therefore important to manage patients with ALK-TKIs until drug resistance occurs. Leukocytoclastic vasculitis (LCV) is a rare complication during cancer treatment and is associated with a variety of factors. Currently, it is unclear whether we should withdraw a treatment when drug-induced LCV develops.We report a 40-year-old man with advanced pulmonary adenocarcinoma harboring the EML4-ALK fusion protein who developed LCV during ceritinib treatment. Four weeks after withdrawing ceritinib, we could successfully perform rechallenge with ceritinib at the normal dose. Rapid and massive tumor apoptosis due to ceritinib treatment may lead to neoantigen release and immune complexes deposition.To the best of our knowledge, we report the first case of LCV in a patient during ALK-TKI treatment. Following this occurrence, we were able to successfully perform rechallenge with ceritinib. Therefore, key drugs used in a patient's treatment regimen should not be discontinued without careful evaluation, and we should also consider the possibility of rechallenge.

Identification of a potent kinase inhibitor targeting EML4-ALK fusion protein in non-small cell lung cancer.

ALK-fusion proteins play a fundamental role in the development of about 5% of non-small cell lung cancers. Herein, we identified the compound 5067-0952 as a potent ALK inhibitor, which inhibited cell growth, induced apoptosis, and suppressed the phosphorylation of ALK, subsequently blocking its downstream signaling pathway.

Inconsistent results in the analysis of ALK rearrangements in non-small cell lung cancer.

BackgroundIdentification of targetable EML4-ALK fusion proteins has revolutionized the treatment of a minor subgroup of non-small cell lung cancer (NSCLC) patients. Although fluorescence in situ hybridization (FISH) is regarded as the gold standard for detection of ALK rearrangements, ALK immunohistochemistry (IHC) is often used as screening tool in clinical practice. In order to unbiasedly analyze the diagnostic impact of such a screening strategy, we compared ALK IHC with ALK FISH in three large representative Swedish NSCLC cohorts incorporating clinical parameters and gene expression data.MethodsALK rearrangements were detected using FISH on tissue microarrays (TMAs), including tissue from 851 NSCLC patients. In parallel, ALK protein expression was detected using IHC, applying the antibody clone D5F3 with two different protocols (the FDA approved Ventana CDx assay and our in house Dako IHC protocol). Gene expression microarray data (Affymetrix) was available for 194 patients.ResultsALK rearrangements were detected in 1.7 % in the complete cohort and 2.0 % in the non-squamous cell carcinoma subgroup. ALK protein expression was observed in 1.8 and 1.4 % when applying the Ventana assay or the in house Dako protocol, respectively. The specificity and accuracy of IHC was high (> 98 %), while the sensitivity was between 69 % (Ventana) and 62 % (in house Dako protocol). Furthermore, only 67 % of the ALK IHC positive cases were positive with both IHC assays. Gene expression analysis revealed that 6/194 (3 %) tumors showed high ALK gene expression (≥ 6 AU) and of them only three were positive by either FISH or IHC.ConclusionThe overall frequency of ALK rearrangements based on FISH was lower than previously reported. The sensitivity of both IHC assays was low, and the concordance between the FISH and the IHC assays poor, questioning current strategies to screen with IHC prior to FISH or completely replace FISH by IHC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2646-x) contains supplementary material, which is available to authorized users.

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.

Genomic Alterations Observed in Colitis-Associated Cancers Are Distinct From Those Found in Sporadic Colorectal Cancers and Vary by Type of Inflammatory Bowel Disease

Patients with inflammatory bowel diseases, such as Crohn's disease (CD) and ulcerative colitis (UC), are at increased risk for small bowel or colorectal cancers (colitis-associated cancers [CACs]). We compared the spectrum of genomic alterations in CACs with those of sporadic colorectal cancers (CRCs) and investigated differences between CACs from patients with CD vs UC. We studied tumor tissues from patients with CACs treated at Memorial Sloan Kettering Cancer Center or Weill Cornell Medical College from 2003 through 2015. We performed hybrid capture-based next-generation sequencing analysis of >300 cancer-related genes to comprehensively characterize genomic alterations. We performed genomic analyses of 47 CACs (from 29 patients with UC and 18 with CD; 43 primary tumors and 4 metastases). Primary tumors developed in the ileum (n= 2), right colon (n= 18), left colon (n= 6), and rectosigmoid or rectum (n= 21). We found genomic alterations in TP53, IDH1, and MYC to be significantly more frequent, and mutations in APC to be significantly less frequent, than those reported in sporadic CRCs by The Cancer Genome Atlas or Foundation Medicine. We identified genomic alterations that might be targeted by a therapeutic agent in 17 of 47 (36%) CACs. These included the mutation encoding IDH1 R132; amplification of FGFR1, FGFR2, and ERBB2; and mutations encoding BRAF V600E and an EML4-ALK fusion protein. Alterations in IDH1 and APC were significantly more common in CACs from patients with CD than UC. In an analysis of CACs from 47 patients, we found significant differences in the spectrum of genomic alterations in CACs compared with sporadic CRCs. We observed a high frequency of IDH1 R132 mutations in patients with CD but not UC, as well as a high frequency of MYC amplification in CACs. Many genetic alterations observed in CACs could serve as therapeutic targets.