Targeting CDA-Directed Metabolism With 5-Formyl-2'-Deoxycytidine For ALK Inhibitor–Resistant Lung Cancer Therapy

Background: Acquired resistance to inhibitors of anaplastic lymphoma kinase (ALK) is a major clinical challenge for ALK fusion–positive non-small-cell lung cancer (NSCLC). In the absence of secondary ALK mutations, epigenetic reprogramming is one of the main mechanisms of drug resistance as it leads to phenotype switching that occurs during the epithelial-to-mesenchymal transition (EMT). While drug-induced epigenetic reprogramming is believed to alter the sensitivity of cancer cells to anticancer treatments, there is still much to learn about overcoming drug resistance. Methods: We used an in vitro model of ceritinib-resistant NSCLC and employed genome-wide DNA methylation analysis in combination with single-cell (sc) RNA-seq to identify cytidine deaminase (CDA), a pyrimidine salvage pathway enzyme, as a candidate drug target. Molecular biology was used to characterize the role of CDA in drug resistance. Integrated analysis of scRNA-seq and scATAC-seq identified gene regulatory networks in resistant cells. Clinical relevance of CDA was evaluated using TCGA datasets, patient-derived cells, and tumor biopsies. Results: CDA was hypomethylated and upregulated in ceritinib-resistant cells. CDA-overexpressing cells were rarely but definitively detected in the na¨ıve cell population by scRNA-seq, and their abundance increased in the acquired-resistance population. Knockdown of CDA had antiproliferative e↵ects on resistant cells and reversed the EMT phenotype. Treatment with epigenome-related nucleosides such as 5-formyl-2’-deoxycytidine selectively ablated CDA-overexpressing resistant cells via accumulation of DNA damage. Conclusions: Targeting CDA metabolism using epigenome-related nucleosides represents a potential new therapeutic strategy for overcoming ALK-inhibitor resistance in NSCLC.

Elucidating the Characteristics of Two Tumor Cell Populations in Small Cell Variant of Anaplastic Lymphoma Kinase-Positive Anaplastic Large Cell Lymphoma

The small cell variant of anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (SC-ALCL) is a subtype defined as comprising two distinct tumor cell populations in immunohistochemistry: small cells staining negative or weakly positive for ALK and CD30 protein; and large cells staining strongly positive for those proteins. Although the constitution of these two different cell populations might contribute to the poor prognosis, detailed characterization of each population by molecular-based approaches has not been done due to the difficulty of cell separation from solid tumor sample. In this paper, we have analyzed the characterization of each tumor cell population using the patient sample from SC-ALCL leukemic phase obtained from peripheral blood by molecular-based approaches. Flow cytometric analysis revealed two distinct abnormal populations. The major population comprising 74% of total leukocytes was positive for CD3 and CD8, and negative for CD4, CD5, CD25 and CD30. The minor population comprising 5% of total leukocytes was positive for CD25, CD30, CD11b and CD13. Fluorescent in situ hybridization with ALK break-apart probes and RT-PCR analysis confirmed that most of the cells including both populations were rearranged with NPM-ALK gene. To elucidate the cause underlying the distinct levels of ALK protein expression in each population, we separated those tumor cells by CD30-PE antibody and a magnetic bead separation kit, extracted DNA and RNA from each population, and compared NPM-ALK mRNA expression levels by droplet digital polymerase chain reaction. The expression level of NPM-ALK mRNA in the CD30-negative population was ten-fold less than that in the CD30-positive population (Table). This result indicated that the two tumor cell populations expressed the distinct level of NPM-ALK mRNA, although both populations possessed NPM-ALK fusion gene. To assess the effectiveness of various chemotherapies in each tumor cell population, we monitored each population in peripheral blood by flow cytometric analysis (small cell; CD30-5-8+, large cell; CD30+) over time. Intriguingly, CD30-negative population behaved as chemo-resistant cells in clinical course, however alectinib, a second-generation ALK-inhibitor, eradicated both populations inducing first complete remission. This study revealed two definitive features regarding the small CD30-negative population of SC-ALCL, with a lower expression level of NPM-ALK mRNA transcripts and chemoresistance. The cause of distinct ALK staining levels in immunohistochemistry was revealed depending on the distinct expression level of NPM-ALK mRNA transcripts. Finally, since both populations were sensitive to ALK-inhibitor, early administration of ALK-inhibitor might be the reasonable option for SC-ALCL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Functional Consequence and Therapeutic Targeting of Cryptic ALK Fusions (ALK fus) in Monosomy7 AML

Monosomy 7 (Mono7) AML is one of the most established adverse prognostic markers in AML with over four decades of data on response and outcome in varied clinical trials. However, despite intensive and myeloablative therapies, outcome for this subtype of AML remains dismal with no meaningful advances in therapy in decades. As part of our recently completed discovery efforts in childhood AML (TARGET Pediatric AML; TpAML), we interrogated the transcriptome of 1068 children and young adults treated on COG AAML1031, including 28 cases of Mono7. We discovered novel cryptic fusions involving the Anaplastic Tyrosine Kinase (ALK) gene that were exclusively seen in 4 of 28 Mono7 patients (14.3%) and not detected in the remaining 1064 patients (p<0.001). These ALK fusions (ALK fus) included SPTBN1-ALK (n=3) or RANBP2-ALK (n=1) (figure 1A) and all 4 cases included the entire kinase domain of the ALK gene. Expression of ALK transcript was evaluated in Mono7 patients with and without ALK fus to determine whether these fusions lead to induction/upregulation of ALK. As demonstrated in figure 1B, the only patients with any ALK expression were those with ALK fus positive Mono7. Expression of the resultant onco-proteins of the ALK fus was confirmed by transfection of parental HEK293T cells with the oncofusion and western blot analysis of the protein lysates. As shown in the western blot in figure 1C, immunoblotting of the lysates from ALK fus cells with ALK antibody confirmed expression of the appropriate ALK+ fusion oncoprotein. Under experimental conditions, fusion involving SPTBN1-ALK appeared to generate higher levels of the fusion onco-protein compared to that of RANBP2-ALK fusion. Confirmation of the fusion onco-protein expression further validates the possibility of the functional significance and potential targetability of these fusions. The ALK gene codes for a receptor tyrosine kinase belonging to a family of protein kinases linked to unregulated cell growth which play a key role in CNS development. Alterations of the ALK gene are commonly seen in Non-Small Cell Lung Cancer (NSCLC), Anaplastic Large-Cell Lymphomas, and Neuroblastomas. We further inquired about the functionality of the ALK fus and whether they may confer cytokine-independent proliferation. The ALK fus were cloned from patient samples into pCSII, a direct GFP-tagged lentiviral backbone under the Ef1α promoter; followed by Sanger sequencing to verify the insert. IL-3 dependent Ba/F3 cells were transduced with the GFP tagged lentiviral vectors containing the SPTBN1-ALK or RANBP2-ALK fusion transcripts. Transduced cells were sorted to GFP homogeneity and growth parameters evaluated post-IL-3 withdrawal. In contrast to the parental line, which rapidly died in the absence of cytokines, cells expressing either SPTBN1-ALK or RANBP2-ALK sustained growth and rapidly proliferated in cytokine-free media, suggesting a conferred transformation event by the fusions, figure 1D. SPTBN1-ALK fusion appears to show more transforming potential with more rapid cytokine-free proliferation compared to that of RANBP2-ALK fusion. The observed differential growth pattern with the two ALK fus correlates with the differential expression of the two oncoproteins as presented above. We then sought to evaluate the efficacy of Crizotinib as a potential therapy for aberrant ALK fus positive Mono7 AML. Crizotinib has been FDA-approved and demonstrated efficacy in treating ALK positive NSCLC. ALK fus -positive cells were treated with varying doses of Crizotinib in cytokine-free media and cell viability and cell death were determined with the Promega Cell Titer Glow assay after 48 hours in culture. Fusion positive cells show sensitivity to Crizotinib with IC50s of 144nM and 95nM for the SPTBN1-ALK or RANBP2-ALK fusions, respectively. Here we demonstrate experimental data that when newly discovered cryptic ALK fus in Mono7 AML are functional (translation to fusion onco-protein, conferring cytokine independence) and show susceptibility to the ALK inhibitor crizotinib. This data may support effective therapeutic targeting of a subset of this highly refractory AML. Figure 1 Figure 1. Disclosures Hylkema: Quest Diagnostics Inc: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company. OffLabel Disclosure: Crizotinib is a first-generation ALK inhibitor that is FDA-approved for the treatment of Non-Small Cell Lung Cancer, Anaplastic Lymphoma, and Neuroblastoma.

A Clinical Research of Ceritinib Treating Patients With ALK-Positive Advanced Non-Small Cell Lung Cancer With Brain Metastasis

Objective As a second-generation oral anaplastic lymphoma kinase (ALK) inhibitor, ceritinib was recommended as posterior line therapy in patients with crizotinib resistance or intolerance. While in the real world, the clinical efficacy and safety of ceritinib, 450mg/d, taken with a low-fat meal in crizotinib resistant patients, especially with brain metastases, are still to be further investigated. Methods This retrospective analysis was conducted on patients with ALK- positive advanced non-small cell lung cancer (NSCLC) between May 2017 and December 2020 in our hospital, who were crizotinib resistant or intolerant. The aim was to analyze the clinical efficacy and safety of ceritinib as posterior line therapy in patients, especially in those with brain metastases, and to further explore the efficacy against rare fusion of ALK and the option after ALK inhibitor resistance. RECIST (1.1) criteria were adopted, and for relevant statistical analysis, RStudio was used. Results Of the 13 cases treated with ceritinib, 8 were treated as second-line therapy, 5 as third-line or fourth-line treatment. Of the total, 12 had brain metastases, 3 had received brain radiotherapy previously, and 1 had received surgical treatment for brain metastases previously. There were 8 cases evaluated as partial remission (PR), 2 evaluated as stable disease (SD), and 3 unevaluable for the response. There were 1 case of PKNOX2-ALK fusion and 1 case of IGR-ALK fusion observed as rare ALK fusion, and intracranial lesions were evaluated as PR for both. There were 6 cases received subsequent targeted therapy after progression of ceritinib, including 5 with alectinib and 1 with ensartinib, all of which showed response. In 13 cases, by the terminal time of follow-up, median progression-free survival (PFS) was 7.4 m, disease control rate (DCR) was 76.92%, overall response rate (ORR) was 61.54%, and median overall survival (OS) was 25.4m. In 12 cases with brain metastasis, 9 were suitable for intracranial response evaluation, of which, 1 case achieved CR for intracranial lesions, 7 achieved PR and 1 SD was observed, ORR was 88.89%, and DCR was 100%. The adverse events rate of any grade was 91.67%, the common adverse events were diarrhea (46.15%), AST level increased (41.67%), ALT level increased (33.33%), increased GGT (33.33%), increased ALP (25.00%), nausea (16.67%), increased creatinine (16.67%). Other reported AEs were cough, vomiting, dizziness, arthralgia and fatigue, 1 case (8.33%) each. Conclusion Ceritinib 450mg taken with taken with low-fat meal showed good efficacy and safety in crizotinib resistant patients with central nervous system metastasis, and also, it is effective in some cases with rare fusion of ALK.

Targeting anaplastic lymphoma kinase (ALK) gene alterations in neuroblastoma by using alkylating pyrrole-imidazole polyamides

Anaplastic lymphoma kinase (ALK) aberration is related to high-risk neuroblastomas and is an important therapeutic target. As acquired resistance to ALK tyrosine kinase inhibitors is inevitable, novel anti-ALK drug development is necessary in order to overcome potential drug resistance against ATP-competitive kinase inhibitors. In this study, to overcome ALK inhibitor resistance, we examined the growth inhibition effects of newly developed ALK-targeting pyrrole-imidazole polyamide CCC-003, which was designed to directly bind and alkylate DNA within the F1174L-mutated ALK gene. CCC-003 suppressed cell proliferation in ALK-mutated neuroblastoma cells. The expression of total and phosphorylated ALK was downregulated by CCC-003 treatment but not by treatment with a mismatch polyamide without any binding motif within the ALK gene region. CCC-003 preferentially bound to the DNA sequence with the F1174L mutation and significantly suppressed tumor progression in a human neuroblastoma xenograft mouse model. Our data suggest that the specific binding of CCC-003 to mutated DNA within the ALK gene exerts its anti-tumor activity through a mode of action that is distinct from those of other ALK inhibitors. In summary, our current study provides evidence for the potential of pyrrole-imidazole polyamide ALK inhibitor CCC-003 for the treatment of neuroblastoma thus offering a possible solution to the problem of tyrosine kinase inhibitor resistance.