Anaplastic Lymphoma Kinase Kinase Domain Research Articles

Abstract 223: Precision medicine-based platform to guide the treatment of EML4-ALK fusion lung cancers and other NSCLC

Abstract Background: Lung cancer (LC) remains the top cause of cancer-associated mortality worldwide, with a 10-year overall survival rate of only 5%. While most LCs are smoking related, in the US, 25% of non-small cell LC (NSCLC) are diagnosed in patients with little or no smoking history. Fusions involving anaplastic lymphoma kinase (ALK) are the oncogenic driver in ~3-7% of NSCLC. While inhibitors targeting the kinase domain of ALK (TKIs) have proven extremely effective, inevitably, resistance develops with limited effective treatment options. Additionally, NSCLCs without identified molecular alterations have limited treatment options beyond radiation, immunotherapy, chemotherapy, and resection. Methods: We developed a precision medicine-based platform (PMP) to screen patient-derived material (PDM) directly from the operating room with curated panels of drugs. PDM collected during clinically indicated procedures is plated in 3D-culture to generate patient-derived organoids (PDOs) and screened with drugs curated to each tumor type. PDOs are screened at therapeutically relevant doses, drawing from pharmacokinetic data for each drug. We have optimized an assay to rapidly screen for EML4-ALK fusions and can perform next-generation sequencing in real time (~7 days) to integrate with drug screening results. Results: To date, we have screened 83 cases, including 8 EML4-ALK NSCLC. We have demonstrated an ability to produce high quality data from low input samples (biopsies). In one EML4-ALK NSCLC we were able to collect PDM from two distinct anatomic spaces (pleural effusion and peritoneal fluid) and screen with the same panel of drugs, with nearly identical results, highlighting the reproducibility and consistency of our assay. Screening of EML4-ALK tumors which have progressed to second or higher line TKIs, demonstrate sensitivity to earlier generation ALK TKIs, a known phenomenon. Characterization of tumors with unknown clinical drivers identifies ~1/3 tumors with no prioritized variants and particularly poor response to chemotherapies. Our results recapitulate known resistance/progression in samples previously exposed to therapy, demonstrating a strong negative predictive value. Longitudinal assessment will be required to robustly assess positive predictive value (PPV). Conclusions: Our PMP captures robust and reproducible results that are consistent with known clinical pathogenesis. Moving forward, we are collecting longitudinal data from enrolled patients in parallel with clinical trials to demonstrate the PPV of our PMP. We additionally strive to demonstrate reproducibility to obtain Clinical Laboratory Improvement Amendments approval and to deliver results to patients and physicians to help guide clinical care. Citation Format: Nathan M. Merrill, Aaron Udager, Angel Qin, Kiran Lagisetty, Liwei Bao, Xu Cheng, Hamadi Madhi, Ananya Banerjee, Marziyeh Salehi Jahromi, Laura Goo, Varun Kathawate, Bryce Vandenburg, Marisa Aikins, Mark Slayton, Peter Ulintz, Zhaoping Qin, Chia-jen Liu, Habib Serhan, John Jefferies, Muhammad Sajawal Ali, Vishal Navani, Michael Monument, Johannes Kratz, Amber Smith, Andrew Chang, Gregory Kalemkerian, Sunitha Nagrath, Peggy Hsu, Matthew B. Soellner, Sofia D. Merajver. Precision medicine-based platform to guide the treatment of EML4-ALK fusion lung cancers and other NSCLC [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 223.

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.

Environment-Sensitive Fluorescent Probe Enables Assessment of Anaplastic Lymphoma Kinase Activity in Nonsmall Cell Lung Cancer.

Anaplastic lymphoma kinase (ALK) rearrangements have been identified as key oncogenic drivers of a subset of nonsmall cell lung cancer (NSCLC). The final chimeric protein of the fusion gene can be constitutively activated, which accounts for the growth and proliferation of ALK-rearranged tumors and thus strongly associates with cancer invasion and metastasis. Diagnostic tools enabling the visualization of ALK activity in a structure-function-based approach are highly desirable to determine ALK status and guide ALK tyrosine kinase inhibitor (ALK-TKI) treatment making. Here, we describe the design, synthesis, and application of a new environment-sensitive fluorescent probe HX16 by introducing an environment-sensitive fluorophore 4-sulfonamidebenzoxadiazole to visualize ALK activity in living cancer cells and tumor tissue slices (mouse model and human biopsy sample). HX16 is a multifunctional chemical tool based on the pharmacophore of ALK-TKI (ceritinib) and can specifically target the kinase domain of ALK with a high sensitivity. Using flow cytometry and confocal microscopy, HX16 enables visualization of ALK activity in various cancer cells with distinct ALK fusion genes, as well as xenograft mouse models. Importantly, HX16 was also applied to visualize ALK activity in a tumor biopsy from a NSCLC patient with ALK-echinoderm microtubule-associated protein-like-4 fusion gene for prediction of ALK-TKI sensitivity. These results demonstrate that strategically designed ALK-TKI-based probe allows the assessment of ALK activity in tumor tissues and hold promise as a useful diagnostic tool in predicting ALK-TKI therapy response.

Preliminary Results of Phase 2 Open Label Study of Lorlatinib Monotherapy in Relapsed/Refractory ALK + Lymphomas Previously Treated with Other Tyrosine Kinase Inhibitors

Introduction: Both aggressive T and B cell lymphoma may harbour Anaplastic Lymphoma Kinase (ALK) translocations: ALK+ Anaplastic Large Cell lymphoma (ALCL) offer a good outcome with first line therapy with 5-year survival of 79%, but relapses occur in half of the patients (pts) and salvage therapy still represents a challenge for clinicians; ALK+ large B cell lymphoma (LBCL) are very rare cases but with dismal prognosis. In addition other hematologic neoplasms can be caused by ALK fusions. Crizotinib, an ALK/MET tyrosine kinase inhibitor (TKI), provides long-term response in up to 70% relapsed/refractory (R/R) ALK+ ALCL and can obtain a similar but less durable response rates in ALK+ LBCL. In both cases the loss of response to crizotinib is frequently a fatal event. Lorlatinib, a selective ALK and ROS1 TKI, demonstrated preclinical inhibition of all reported ALK kinase domain mutations responsible for resistance to Crizotinib. Methods: From 2019 to 2022 six relapsed/refractory ALK + neoplasm pts previously treated with at least one ALK-inhibitor were enrolled in a phase 2 open label monocentric study with lorlatinib monotherapy (EudraCT2016-003970-41): Three patients had diagnosis of ALK+ LBCL (# 1, 2, 3) and 2 (#5 and #6) had ALK+ ALCL. One patient (#4) with an ALK+ histiocytosis was also included. Median age was 22 years (range 19-57), Ratio M/F was 1, all pts were in stage IV. Median prior therapy lines was 3 (range 2-5); all pts previously received Crizotinib, two pts received also Alectinib and 1 Ceritinib as TKI treatment. Lorlatinib was administered daily at dose of 100 mg. Clinical, laboratory, radiological and molecular data were collected at screening and during scheduled follow-up. Response was evaluated with 18F-FDG PET/CT (and with contrast enhanced (ce) CT scan) according Lugano Classification, and with ALK RT-PCR on peripheral blood and bone marrow. Side effects were classified according to CTCAEv4.03. PFS and OS were determined from Lorlatinib start to relapse and/or death. The primary endpoint was the evaluation of ORR considering 18F-FDG PET/CT scan and ALK- RT-PCR. Secondary endpoints were: PFS, OS, toxicity, QoL. Results: ALK rearrangement fusion pattern was detected in 6/6 pts: 3 pts with LBCL harboured Clathrin-ALK (CTCL::ALK) rearrangement and 2 ALCL pts the ATIC::ALK fusion; the ALK+ histiocytosis patient harbored an EML4::ALK rearrangement. Median follow-up was 13.7 months (IQR 22-55). At month +1 (M1) 3/6 patients had complete response (CR) defined as complete metabolic and complete radiologic response (CMR/CR) according to PET/CT scans and ALK RT-PCR negativity. The remaining 3 pts had a partial response (PR), for an Overall Response Rate (ORR) of 100% (95%CI: 0.6-1.0). One pt is still on treatment and in CR at 45 months, receiving Lorlatinib 100 mg daily with neither toxicities nor need for Allogenic Stem Cell Transplantation (ASCT); 2 CR pts underwent ASCT after 1 month, resumed lorlatinib after ASCT and are still in CR. 2 pts with PR relapsed within month +3: one pt received salvage therapy without benefit, the other one underwent ASCT but progressed 3 months later. Both pts had ALK+ BCL with CTCL-ALK rearrangement. 1 pts in PR AT M1 (ALK+ istiocytosis) obtain CR at M3 after surgical resection of a residual lung mass and is still receiving Lorlatinib at month 36. Median PFS and OS were not reached: PFS at 6 and 12 months is 83% and 62% respectively; OS at 6 and 12 months is 83% and 55%. Safety: 2/6 pts had thrombocytopenia grade 1, whereas 3/6 pts showed grade I-II hypertriglyceridemia/hypercholesterolemia and muscle cramps. Conclusions: Lorlatinib represents a safe and effective salvage therapy for ALK+ Lymphoma patients failing first line TKI treatment. The obtainment of CR at M1 seems to represent a pivotal prognostic factor. Lorlatinib also offers a promising bridging regimen to salvage ASCT especially for ALK+ BCL patients, with high response rates and no additional toxicities, .

Comparative Atomistic Insights on Apo and ATP-I1171N/S/T in Nonsmall-Cell Lung Cancer.

Anaplastic lymphoma kinase (ALK) rearrangements occur in about 5% of nonsmall cell lung cancer (NSCLC) patients. Despite being first recognized as EML4-ALK, fusions with several additional genes have been identified, all of which cause constitutive activation of the ALK kinase and subsequently lead to tumor development. ALK inhibitors first-line crizotinib, second-line ceritinib, and alectinib are effective against NSCLC patients with these rearrangements. Patients progressing on crizotinib had various mutations in the ALK kinase domain. ALK fusion proteins are activated by oligomerization through the fusion partner, which leads to the autophosphorylation of the kinase's domain and consequent downstream activation. The proposed computational study focuses on understanding the activation mechanism of ALK and ATP binding of wild-type (WT) and I1171N/S/T mutations. We analyzed the conformational change of ALK I1171N/S/T mutations and ATP binding using molecular docking and molecular dynamics simulation approaches. According to principal component analysis and free energy landscape, it is clear that I1171N/S/T mutations in Apo and ATP showed different energy minima/unstable structures compared to WT-Apo. The results revealed that I1171N/S/T mutations and ATP binding significantly supported a change toward an active-state conformation, whereas WT-Apo remained inactive. We demonstrated that I1171N/S/T mutations are persistent in an active state and independent of ATP. The I1171S/T mutations showed greater intermolecular H-bonds with ATP than WT-ATP. The molecular mechanics Poisson-Boltzmann surface area analysis revealed that the I1171N/S/T mutation binding energy was similar to that of WT-ATP. This study shows that I1171N/S/T can form stable bonds with ATP and may contribute to a constitutively active kinase. Based on the Y1278-C1097 H-bond and E1167-K1150 salt bridge interaction, I1171N strongly promotes the constitutively active kinase independent of ATP. This structural mechanism study will aid in understanding the oncogenic activity of ALK and the basis for improving the ALK inhibitors.

Molecular heterogeneity and co-altered genes in MET-amplified ALK-positive lung cancer: Implications for MET targeted therapy

ObjectivesMET amplification is a common mechanism of resistance to second- and third-generation anaplastic lymphoma kinase (ALK) inhibitors. In case series of MET-amplified ALK-rearranged (ALK + ) lung cancer, durability of responses to combinations targeting ALK and MET is variable, suggesting heterogeneity across tumors. However, little is known about the molecular composition of this subset of ALK-rearranged (ALK + ) NSCLC. Materials and methodsWe queried tissue and plasma databases to compile a group of > 50 specimens with ALK rearrangements and concurrent MET amplification. Fluorescence in-situ hybridization (FISH) and next-generation sequencing (NGS) were utilized to quantify the range of MET copies and describe the global molecular landscape of co-altered genes. ResultsBy FISH, high-level amplification (overall MET/centromere 7 probe ratio ≥ 5) was detected in 75 % of MET-amplified ALK + NSCLC tissue specimens. Intralesional heterogeneity of MET copies was observed, with high-level amplification identified even in cells from tumors with overall low-level MET amplification. Analysis of 48 MET-amplified ALK + NSCLC plasma specimens suggested that high-level amplification is rarely (17 %) detected in plasma. In both tissue and plasma, EML4-ALK variant 1 was the predominant variant (51 %) identified in MET-amplified specimens. ALK kinase domain mutations were only present in a minority of MET-amplified ALK + NSCLCs. MET-amplified ALK + NSCLC plasma specimens were enriched for TP53 mutations (81 % vs 45 %, p = 0.002), EGFR amplification (17 % vs 4 %, p < 0.001), and MYC amplification (21 % vs 3 %, p < 0.001) compared to ALK + NSCLC specimens lacking MET amplification. ConclusionsMET-amplified, ALK + NSCLC often presents with high-level and heterogeneous amplification in tissue, seldom overlaps with ALK mutations, and frequently co-occurs with alterations associated with aggressive tumor biology.

Precision medicine-based platform to guide the treatment of EML4-ALK fusion lung cancers and other non-small cell lung cancers.

146 Background: Lung cancer remains the number one cause of cancer-associated mortality worldwide, with a 10-year survival rate of only 5%. While most lung cancers are smoking related, 25% of non-small cell lung cancer (NSCLC) are diagnosed in patients with little or no smoking history. Fusions involving anaplastic lymphoma kinase (ALK) are the oncogenic driver in ~3–7% of NSCLC. While inhibitors targeting the kinase domain of ALK have proven extremely effective in the treatment of ALK-positive NSCLC, inevitably, resistance develops with limited effective treatment options. Methods: We developed a precision medicine-based platform to screen patient-derived material (PDM) directly from the operating room with curated panels of approved and IND drugs. PDM collected during clinically indicated procedures is dissociated into single cells, plated in culture to generate patient-derived organoids (PDOs), and screened with drugs that are curated to each tumor based on treatment history and clinically relevant sequencing. PDOs are screened at therapeutically relevant levels, drawing from pharmacokinetic data for each drug, when available. We have optimized an assay to rapidly screen for EML4-ALK fusions and can perform next-generation DNA and RNA sequencing in real time (~7 days) to integrate with drug screening results. Results: To date, we have screened 51NSCLC cases, including 7 ALK-positive NSCLC. Screening in highly progressed EML4-ALK patients demonstrates sensitivity to earlier generation ALK TKIs, a known phenomenon in the literature. Our screening results recapitulate known clinical resistance/progression in samples previously exposed to therapy, demonstrating a strong negative predictive value. Longitudinal assessment will be required to robustly assess the positive predictive value (PPV). In one EML4-ALK NSCLC (JTI026) we were able to collect PDM from two distinct regions (pleural effusion and thoracentesis) and screen them with the same panel of drugs, with identical results. These data highlight the reproducibility of our assay. Conclusions: Our precision medicine-based platform captures robust and reproducible results that are consistent with known clinical pathogenesis. Moving forward, we are collecting longitudinal data from enrolled patients in parallel with clinical trials to demonstrate PPV of our platform. We additionally strive to demonstrate reproducibility to obtain Clinical Laboratory Improvement Amendments (CLIA) approval and to deliver results to patients and physicians to help guide clinical care. [Table: see text]

Abstract 3884: Targeting the ARID1A mutations overcomes primary resistance to ALK inhibitors in EML4-ALK positive NSCLC

Abstract Introduction: Anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitors (TKIs) have improved an initial clinical response of non-small-cell lung cancer (NSCLC) patients with ALK-rearrangements. However, a subset of patients shows a poor response to ALK-TKIs. Here, we aimed to identify novel mechanism of primary resistance in preclinical model from a patient who presented an impressive resistance to sequential treatment with ALK TKIs. Experimental design: The PDC YU-1076 cells were established from pleural effusion of the patient at the time he was receiving chemotherapy and radiotherapy after failure of ALK-TKI treatment. Sanger sequencing confirmed ML4-ALK variant 1 detected at the initial diagnosis. No resistance mutations in the ALK kinase domain were detected. To investigate co-occurring genetic alterations, we performed whole exome sequencing (WES) on YU-1076 cells and the matched blood sample. We further investigated the molecular profile of YU-1076 cells using RNA-sequencing analysis. Results: YU-1076 cells exhibited cross-resistance to clinically available ALK-TKIs including crizotinib, ceritinib, alectinib, and lorlatinib. However, immunoblot analysis of YU-1076 cells treated with incremental doses of TKIs unexpectedly revealed an effective reduction of ALK activity and downstream signals. We noticed that YU-1076 cells gradually changed from a small, round shape to a fibroblast-like shape following the treatment of ALK-TKIs. Transcriptome analysis confirmed enrichment for gene signatures related epithelial-to-mesenchymal transition (EMT) in ALK TKI-treated YU-1076 cells, suggesting that ALK-TKIs treatment promotes YU-1076 cells toward a more mesenchymal phenotype. WES analysis identified a major chromatin remodeling complex subunit, AT-rich interacting domain 1A (ARID1A), as well as MYC amplification, CDKN2A loss, and TP53 mutations. We focused on a synthetic lethal strategies using the SFK inhibitor dasatinib and the EZH2 inhibitor GSK126 in ARID1A mutant cancers. The combination of dasatinib with ALK-TKIs restored the sensitivity to the ALK TKIs in YU-1076 cells, accompanied by suppression of EMT marker genes VIM and CDH2 and increase of apoptotic markers BIM, PARP, and CAS3. Congruently, dasatinib significantly impaired tumor growth in YU-1076-xenografts. GSK126 also induced synergistic inhibition of cell growth with upregulation of apoptosis marker genes in YU-1076 cells, but did not affect the expression of EMT marker genes. Conclusion: Our data indicate that ARID1A could be potentially used as a predictive biomarker for unfavorable ALK-TKI response. In this context, a combination strategy of ALK TKI with dasatinib may be effective in overcoming primary resistance. Citation Format: Seung Yeon Oh, You Won Lee, Eun Ji Lee, Ju Young Kim, Sewon Park, Ju Yeon Park, Su-Jin Choi, Mi Ra Yu, Sowon Aum, Jiyun Lee, Chang Gon Kim, Jii Bum Lee, Sun Min Lim, Min Hee Hong, Mi Ran Yun, Byoung Chul Cho. Targeting the ARID1A mutations overcomes primary resistance to ALK inhibitors in EML4-ALK positive NSCLC. [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 3884.

Deciphering the Mechanism of Gilteritinib Overcoming Lorlatinib Resistance to the Double Mutant I1171N/F1174I in Anaplastic Lymphoma Kinase.

Anaplastic lymphoma kinase (ALK) is validated as a therapeutic molecular target in multiple malignancies, such as non-small cell lung cancer (NSCLC). However, the feasibility of targeted therapies exerted by ALK inhibitors is inevitably hindered owing to drug resistance. The emergence of clinically acquired drug mutations has become a major challenge to targeted therapies and personalized medicines. Thus, elucidating the mechanism of resistance to ALK inhibitors is helpful for providing new therapeutic strategies for the design of next-generation drug. Here, we used molecular docking and multiple molecular dynamics simulations combined with correlated and energetical analyses to explore the mechanism of how gilteritinib overcomes lorlatinib resistance to the double mutant ALK I1171N/F1174I. We found that the conformational dynamics of the ALK kinase domain was reduced by the double mutations I1171N/F1174I. Moreover, energetical and structural analyses implied that the double mutations largely disturbed the conserved hydrogen bonding interactions from the hinge residues Glu1197 and Met1199 in the lorlatinib-bound state, whereas they had no discernible adverse impact on the binding affinity and stability of gilteritinib-bound state. These discrepancies created the capacity of the double mutant ALK I1171N/F1174I to confer drug resistance to lorlatinib. Our result anticipates to provide a mechanistic insight into the mechanism of drug resistance induced by ALK I1171N/F1174I that are resistant to lorlatinib treatment in NSCLC.

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.

Development of a novel ALK rearrangement screening test for non-small cell lung cancers.

Approximately 5–7% of non–small cell lung cancer (NSCLC) cases harbor an anaplastic lymphoma kinase (ALK) fusion gene and may benefit from ALK inhibitor therapy. To detect ALK fusion genes, we developed a novel test using reverse transcription polymerase chain reaction (RT-PCR) for the ALK kinase domain (KD). Since ALK expression is mostly silenced in the adult with the exception of neuronal tissue, the normal lung tissue, mesothelial lining, and inflammatory cells are devoid of ALK transcript, making ALK KD RT-PCR an ideal surrogate test for ALK fusion transcripts in lung or pleural effusion. The test was designed with a short PCR product (197 bp) to work for both malignant pleural effusion (MPE) and formalin-fixed, paraffin-embedded (FFPE) NSCLC samples. Using ALK IHC as a reference, the sensitivity of the test was 100% for both MPE and FFPE. The specificity was 97.6% for MPE and 97.4% for FFPE. Two false positive cases were found. One was a metastatic brain lesion which should be avoided in the future due to intrinsic ALK expression in the neuronal tissue. The other one resulted from ALK gene amplification. Due to potential false positivity, subsequent confirmation tests such as fluorescence in situ hybridization or multiplex PCR would be preferable. Nevertheless, the test is simple and inexpensive with no false negativity, making it a desirable screening test. It also offers an advantage over multiplex RT-PCR with the capability to detect novel ALK fusions. Indeed through the screening test, we found a novel ALK fusion partner (sperm antigen with calponin homology and coiled-coil domains 1 like gene, SPECC1L) with increased sensitivity to crizotinib in vitro. In summary, a novel RNA-based ALK KD analysis was developed for ALK rearrangement screening in MPE and FFPE specimens of NSCLC. This simple inexpensive test can be implemented as routine diagnostics.

Concomitance of a novel RMDN2-ALK fusion and an EML4-ALK fusion in a lung adenocarcinoma

The anaplastic lymphoma kinase (ALK) fusions/rearrangements in non-small cell lung cancer (NSCLC) act as oncogenic driver mutations. ALK tyrosine kinase inhibitors have anti-tumor activities in ALK-positive NSCLC. Although the EML4-ALK fusion is common in NSCLC, concomitance of an additional ALK fusion together with an EML4-ALK fusion is not common. Here, we present a lung adenocarcinoma with two ALK fusions, a novel RMDN2-ALK fusion accompanied by an EML4-ALK fusion, detected by a targeted next generation sequencing assay. The genomic translocation breakpoints of the RMDN2-ALK fusion were mapped to intron 2 for RMDN2 and exon 15 for ALK, and EML4-ALK breakpoints were mapped to intron 13 for EML4 and intron 19 for ALK. ALK break-apart FISH detected multiple ALK rearrangements, a gene fusion panel (NanoString) test confirmed the EML4-ALK fusion, and RNA-sequencing revealed two ALK fusions. The RMDN2 gene locates at the short arm of chromosome 2 between ALK and EML4 genes. The intact ALK kinase domain fused to RMDN2. Genome-wide copy number variants were found in multiple chromosome arms and the short arm of chromosome 2, suggestive of complex rearrangements. Further detailed analyses of breakpoints and copy number variants may shed light on mechanisms of their formation and pathogenesis in lung malignancies.

Pediatric Mesothelioma With ALK Fusions: A Molecular and Pathologic Study of 5 Cases.

Pediatric mesotheliomas are rare and their pathogenesis remains undefined. In this study, we report 5 cases of malignant mesothelioma in children, characterized by fusions involving the anaplastic lymphoma kinase (ALK) gene. Four cases occurred in females involving the abdominal cavity and were characterized by a pure epithelioid morphology. The fifth arose in the tunica vaginalis of a 15-year-old male and displayed a biphasic epithelioid-sarcomatoid phenotype. All cases demonstrated the classic morphologic and immunohistochemical features of malignant mesothelioma, including tubulopapillary architecture and cuboidal epithelioid cells with eosinophilic cytoplasm and uniform nuclei with vesicular chromatin. Immunohistochemically, all cases showed labeling for ALK, cytokeratins, WT1, and calretinin, while lacking expression of adenocarcinoma immunomarkers. Four cases demonstrated weak-moderate labeling for PAX8 protein, which resulted in diagnostic challenges with primary peritoneal serous carcinoma. The ALK genetic abnormalities were investigated by a combination of molecular methods. Archer FusionPlex was performed in 2 cases, showing fusions between ALK with either STRN or TPM1 genes, resulting in a transcript that retained the ALK kinase domain. One case was further studied by DNA targeted sequencing, but no additional genetic alterations were observed. In 1 case, cytogenetic analysis showed the presence of a t(2;15)(p23;q22) and fluorescence in situ hybridization confirmed the ALK gene break-apart. In the remaining 2 cases, ALK gene rearrangements were demonstrated by fluorescence in situ hybridization. Unlike adult mesotheliomas, which are tightly linked to asbestos exposure, often show loss of BAP1 expression and have complex karyotypes, ALK-rearranged mesothelioma appears to be similar to other fusion-positive mesotheliomas, such as those harboring EWSR1/FUS-ATF1 fusions, sharing significant morphologic overlap, occurring in young patients and displaying a simple, translocation-driven genetic profile.

Abstract A36: Targeting anaplastic lymphoma kinase in neuroblastoma

Abstract Over the last decade Anaplastic Lymphoma Kinase (ALK), a receptor tyrosine kinase (RTK), has been identified as a fusion partner in a diverse variety of translocation events resulting in oncogenic signaling in many different cancer types. In tumors where full-length ALK RTK itself is mutated, such as neuroblastoma, the picture regarding the role of ALK as an oncogenic driver is less clear. Neuroblastoma is a complex and heterogeneous tumor that arises from the neural crest-derived peripheral nervous system. Although high-risk neuroblastoma is rare, it often relapses and becomes refractory to treatment. Thus, neuroblastoma accounts for 10-15% of all childhood cancer deaths. Since most cases are in children under the age of two, understanding the role and regulation of ALK during neural crest development is an important goal in addressing neuroblastoma tumorigenesis. An impressive array of tyrosine kinase inhibitors (TKIs) that act to inhibit ALK have been FDA approved for using in ALK-driven cancers. ALK TKIs bind differently within the ATP-binding pocket of the ALK kinase domain and have been associated with different resistance mutations within ALK itself that arise in response to therapeutic use, particularly in ALK fusion-positive NSCLC. This patient population has highlighted the importance of considering the relevant ALK TKI to be used for a given ALK mutant variant. In this poster we discuss ALK in neuroblastoma, as well as the use of ALK TKIs and other strategies to inhibit tumor growth. Current efforts combining novel approaches and increasing our understanding of the oncogenic role of ALK in neuroblastoma are aimed at improving efficacy of ALK TKIs as precision medicine options in the clinic. Citation Format: Jikui Guan, Diana Cervantes-Madrid, Joachim Siaw, Wasi Alam, Ganesh Umapathy, Marcus Borenäs, Dan Lind, Joanna Szydzik, Kathrin Pfeifer, Patricia Mendoza-Garcia, Ruth Palmer, Bengt Hallberg. Targeting anaplastic lymphoma kinase in neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A36.

4558 Investigating the functional consequences of anaplastic lymphoma kinase (ALK) mutations arising upon Lorlatinib treatment

OBJECTIVES/GOALS: Neuroblastoma (NB) is an embryonal cancer of the sympathetic nervous system that affects mostly infants and young children. The complex genetic background present across NB patients results in diverse clinical response and difficulty in individualizing therapy. Currently, NB patients undergo a regimen of genotoxic chemotherapeutics, radiation therapy, and new immunotherapy that, while effective, has significant side effects, including excruciating pain. One promising avenue for targeted therapy in neuroblastoma focuses on anaplastic lymphoma kinase (ALK), a cell surface neural receptor tyrosine kinase. We previously identified activating point mutations within the tyrosine kinase domain of ALK as the primary cause of hereditary NB, and we and others subsequently showed that these same alterations are the most common somatic single-nucleotide mutations in the sporadic forms of the disease. Crizotinib, a first-generation small molecule ATP-competitive inhibitor of the ALK tyrosine kinase, showed limited anti-tumor activity in patients with relapsed NB harboring ALK F1174 and F1245 mutations. We have demonstrated that lorlatinib, a novel ATP-competitive ALK inhibitor, overcomes this de novo resistance in preclinical models of ALK-driven NB. Recent clinical trials with lorlatinib in patients with non-small cell lung cancer harboring an ALK fusion, and in patients with NB harboring ALK mutations show the emergence of multiple or compound ALK mutations as a mechanism of resistance. We postulate that these compound mutations disrupt the interaction between and ALK and cause resistance. In this study, we employ a computational approach to model mutated ALK in complex with lorlatinib as well as ATP to understand whether the new mutations alter the affinity or mode of lorlatinib/ATP binding to ALK, and thus cause suboptimal ALK inhibition. METHODS/STUDY POPULATION: We employ methods in computational structural biology and drug design, primarily based on molecular modeling, molecular dynamics (MD), and molecular docking. Based on existing crystal structures of wildtype ALK, we model the mutations and perform MD simulations in order to characterize the activation state of the protein as well as perform ensemble docking calculations to assess the binding affinities and modes in ALK-lorlatinib and ALK-ATP complexes. RESULTS/ANTICIPATED RESULTS: We expect that the compound mutations cause resistance to lorlatinib either by lowering protein affinity for the drug or increasing the affinity for ATP. Alternatively, the compound mutations may disrupt the protein activation state, in which case ALK may no longer be active, and another protein/pathway could be driving the resistance. DISCUSSION/SIGNIFICANCE OF IMPACT: The results of this study will enable the understanding of the mechanism of resistance to lorlatinib and facilitate the design of new ALK inhibitors, or help develop more optimal and mechanism-guided therapies aimed to overcome the resistance.

Anaplastic lymphoma kinase fusions as a target for TCR-directed cellular therapies

Abstract Chromosomal rearrangement derived gene fusions with the catalytic domain of anaplastic lymphoma kinase (ALK) drive oncogenesis in several cancers, including in ~5% of non-small-cell lung cancers (NSCLCs). While ALK tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of these patients the majority will develop resistance; novel treatments are required. We hypothesize that the ALK kinase domain may be a suitable target for directed immunotherapy. Full length ALK is normally expressed during fetal development but becomes highly restricted in adults, and anti-ALK immune responses are readily detected in other ALK-fusion+ cancers, correlating with good prognosis. Using peptide pool expansions and T cell activation assays, we have detected numerous anti-ALK T cell responses from normal donors and ALK+ NSCLC patients. These donors express a variety of HLA alleles, and some donors display reactivity to multiple pools spanning the kinase domain, suggestive of common T cell responses to multiple ALK epitopes. Leveraging a high-throughput microfluidic technique to clone and functionally express natively-paired T cell receptor (TCR) genes from these ALK-reactive T cell populations, we are screening for both the exact antigenic peptides and the HLA restrictions of these epitopes. TCRs thus isolated will then be tested against a panel of ALK+/− patient derived and cancer cell lines using in vitro and in vivo models, to assess potential utility in cytotoxic TCR-directed immunotherapies. These patient derived models include those with acquired TKI resistance: by comparing these mutational hotspots with putative epitope sites we expect to find TCRs capable of killing ALK+ cancer cells irrespective of prior treatment.

Revealing Acquired Resistance Mechanisms of Kinase-Targeted Drugs Using an on-the-Fly, Function-Site Interaction Fingerprint Approach.

Although kinase-targeted drugs have achieved significant clinical success, they are frequently subject to the limitations of drug resistance, which has become a primary vulnerability to targeted drug therapy. Therefore, deciphering resistance mechanisms is an important step in designing more efficacious, antiresistant drugs. Here we studied two FDA-approved kinase drugs: Crizotinib and Ceritinib, which are first- and second-generation anaplastic lymphoma kinase (ALK) targeted inhibitors, to unravel drug-resistance mechanisms. We used an on-the-fly, function-site interaction fingerprint (on-the-fly Fs-IFP) approach, combining binding free-energy surface calculations with the Fs-IFPs. Establishing the potentials of mean force and monitoring the atomic-scale protein-ligand interactions, before and after L1196M-induced drug resistance, revealed insights into drug-resistance/antiresistant mechanisms. Crizotinib prefers to bind the wild-type ALK kinase domain, whereas Ceritinib binds more favorably to the mutated ALK kinase domain, in agreement with experimental results. We determined that ALK kinase-drug interactions in the region of the front pocket are associated with drug resistance. Additionally, we find that the L1196M mutation does not simply alter the binding modes of inhibitors but also affects the flexibility of the entire ALK kinase domain. Our work provides an understanding of the mechanisms of ALK drug resistance, confirms the usefulness of the on-the-fly Fs-IFP approach, and provides a practical paradigm to study drug-resistance mechanisms in prospective drug discovery.

Diverse Resistance Mechanisms to the Third-Generation ALK Inhibitor Lorlatinib in ALK-Rearranged Lung Cancer.

Lorlatinib is a third-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor with proven efficacy in patients with ALK-rearranged lung cancer previously treated with first- and second-generation ALK inhibitors. Beside compound mutations in the ALK kinase domain, other resistance mechanisms driving lorlatinib resistance remain unknown. We aimed to characterize the mechanisms of resistance to lorlatinib occurring in patients with ALK-rearranged lung cancer and design new therapeutic strategies in this setting. Resistance mechanisms were investigated in 5 patients resistant to lorlatinib. Longitudinal tumor biopsies were studied using high-throughput next-generation sequencing. Patient-derived models were developed to characterize the acquired resistance mechanisms, and Ba/F3 cell mutants were generated to study the effect of novel ALK compound mutations. Drug combinatory strategies were evaluated in vitro and in vivo to overcome lorlatinib resistance. Diverse biological mechanisms leading to lorlatinib resistance were identified. Epithelial-mesenchymal transition (EMT) mediated resistance in two patient-derived cell lines and was susceptible to dual SRC and ALK inhibition. We characterized three ALK kinase domain compound mutations occurring in patients, L1196M/D1203N, F1174L/G1202R, and C1156Y/G1269A, with differential susceptibility to ALK inhibition by lorlatinib. We identified a novel bypass mechanism of resistance caused by NF2 loss-of-function mutations, conferring sensitivity to treatment with mTOR inhibitors. This study shows that mechanisms of resistance to lorlatinib are diverse and complex, requiring new therapeutic strategies to tailor treatment upon disease progression.

GSK1838705a, an IGF-1R/ALK Inhibitor, Overcomes Resistance to Crizotinib in ALK-Positive ALCL

Anaplastic large cell lymphoma (ALCL) is a type of CD30-expressing non-Hodgkin's lymphoma (NHL), which accounts for 2% to 3% of adult non-Hodgkin's lymphoma,accounting for 15% to 30% of children with large cell lymphoma. Anaplastic lymphoma kinase (ALK) positive ALCL is highly invasive, and currently it is generally based on CHOP combined with chemotherapy. The proportion of patients with complete relief of symptoms is as high as 90%, but the proportion of recurrence is also as high as 40%. Crizotinib is the first generation of ALK inhibitors that have been approved for the treatment of ALK+ ALCL. Unfortunately, most patients treated with crizotinib relapse after a significant initial response. The median progression-free survival of clinical trials was 10.5 months. Various mutations in the ALK kinase domain and amplification of the ALK gene copy number, activation of the alternative pathway, and tumor heterogeneity are major causes of crizotinib resistance. Studies have shown that IGF-1R interacts with NPM-ALK to promote ALK+ALCL transformation, proliferation and migration. GSK is a small molecule kinase inhibitor that inhibits both IGF-IR and ALK. Therefore, GSK with simultaneous inhibition of the bidirectional potential of IGF-IR and ALK has a promising prospect in the targeted therapy of NPM-ALK+ALCL. This study explored the inhibitory effects of GSK on NPM-ALK+ALCL and crizotinib-resistant NPM-ALK+ALCL by in vivo and in vitro experiments. In vitro experiments: The sensitivity of ALCL cell line to GSK1838705a was detected by CCK8 and flow cytometry. The expression of phosphorylation of IGF-1R and NPM-ALK signaling pathway in Karpas299 and SR786 cell lines stimulated by GSK was detected by WB method. In order to study the crizotinib resistance mutation, we established ALK+ALCL crizotinib-resistant cell lines Karpas299-R and SR786-R, and identified the resistance of Karpas299-R and SR786-R cell lines by CCK8 and flow cytometry. The drug-resistant and non-resistant strains were stimulated with gradient concentrations of crizotinib and gradient GSK, and the IC50 of the two were compared by CCK8. The WB method was used to compare the phosphorylation levels of downstream signaling pathways in drug-resistant and non-resistant strains. In vivo experiment: The ALK+ALCL and resistant-ALK+ALCL mouse model was established, and three groups of mice treated with control, GSK single drug 30 mg/kg, GSK single drug 60 mg/kg, were established. The tumor volume and body weight of the four groups were compared. Immunohistochemistry was used to compare the expression levels of key signaling molecules and apoptotic proteins in each group. SPSS statistical software draws survival curves. As the concentration of GSK gradually increases, the survival rate of ALCL cells gradually decreases. The expression of pIGF-1R, pNPM-ALK, pSTAT3, pAKT, casepase3 and other molecules decreased in the downstream signaling pathway, and the expression level of cleaved-casepase3 increased.In the crizotinib-resistant cell line, with the increase of the concentration of GSK, the apoptosis rate of the cells increased and the phosphorylation level of the downstream molecules gradually decreased. Tumor volume of three groups of mouse models: control&gt;GSK single drug 30 mg/kg&gt;GSK single drug 60 mg/kg. Immunohistochemistry results showed that the expression level of key signaling molecules in GSK-treated CHOP-treated mice decreased, and the expression level of apoptotic proteins increased. In this research, we explored the effects of GSK1838705A on proliferation, apoptosis, and clonogenesis of ALCL cell lines. Subsequently, we established a crizotinib-resistant cell line and noticed that GSK1838705A can effectively reduce the viability of resistant ALCL cells and significantly restrain the transmission of downstream survival signaling pathways induced by IGF1R/IR phosphorylation. Besides, we discovered that GSK1838705A inhibited the development of both crizotinib-sensitive and crizotinib-resistant ALCL tumors in the ALCL mouse model established by subcutaneous tumorigenesis. Based on the results of previous clinical trials, we put forward to use GSK1838705A as an alternative treatment strategy to overcome crizotinib-resistant ALCL. Disclosures No relevant conflicts of interest to declare.

Identifying Novel Mechanisms of Resistance to Tyrosine Kinase Inhibitors in Anaplastic Large Cell Lymphoma

INTRODUCTION: Anaplastic large cell lymphomas (ALCL) frequently carry oncogenic fusion proteins as a consequence of chromosomal translocations of the anaplastic lymphoma kinase (ALK) gene. The fusion protein resulting from the translocation between dimerization domain of nucleophosmin (NPM) and intracellular tyrosine kinase domain of ALK activates several signaling pathways, promoting cell growth, transformation, migration, and survival of the cells. Chemotherapy has been used as a standard treatment approach for ALCL patients, yet about 30% of patients relapse. A more specific treatment method is based on targeting ALK tyrosine kinase using tyrosine kinase inhibitors (TKIs). Crizotinib is an ALK TKI that is approved for the treatment of ALK-rearranged lung cancer and has received Breakthrough Therapy designation for lymphoma because of its high activity in chemo refractory ALCL. However, as for lung cancer, also ALCL patient develop crizotinib resistance due to ALK mutations or unknown mechanisms. In this study, we aimed at elucidating unknown by-pass mechanisms of crizotinib resistance in ALCL. METHODS: We used Genome-wide CRISPR-Cas9 Knockout Screening (GeCKO) to identify candidate genes that contribute to resistance to crizotinib. Four different ALCL cell lines were infected with Lenti-GeCKO libraries. After treatment with crizotinib for 14 days, DNA isolation and next generation sequencing was performed on crizotinib resistant cells to identify candidate genes depleted by the GeCKO screening. Top candidates were selected for validation assays and further analyses. RESULTS: We identified two phosphatases, PTPN1 and PTPN2, in different ALCL cell lines as consistent top hits. Functional validation of these candidate genes showed that single loss of either PTPN1 or PTPN2 generate immediate resistance to crizotinib in ALCL cell lines. Analysis of downstream pathways showed that while loss of PTPN1 activates primarily the MAPK pathway, loss of PTPN2 promotes persistent STAT3 and MAPK activation in ALK inhibited cells. Remarkably, in PTPN1 knockout cells we observed hyperactivation of SHP2, an oncogenic phosphatase that positively regulates the RAS-MAPK pathway. On the other hand, over-expression of PTPN1 and PTPN2 partially inhibited SHP2 phosphorylation. A treatment that combined crizotinib and the recently developed SHP2 inhibitor completely blocked the sustained ERK phosphorylation and reverted the crizotinib resistance observed in PTPN1 and PTPN2 deficient lymphoma cells. CONCLUSIONS: GeCKO library screen identified PTPN1 and PTPN2 as specific genes that mediate crizotinib resistance in ALCL cell lines. Loss of PTPN1 and PTPN2 drives resistance by activating MAPK and/or JAK-STAT pathway. Combined inhibition of SHP2 is a potent therapeutic approach to overcome resistance to crizotinib in ALCL cells. Disclosures Gambacorti-Passerini: Bristol-Meyers Squibb: Consultancy; Pfizer: Honoraria, Research Funding.