YES1 and MYC Amplifications as Synergistic Resistance Mechanisms to Different Generation ALK Tyrosine Kinase Inhibitors in Advanced NSCLC: Brief Report of Clinical and Preclinical Proofs

Introduction ALK tyrosine kinase inhibitors (TKIs) are the standard treatment for advanced ALK-positive NSCLC. Nevertheless, drug resistance inevitably occurs. Here, we report a case of a patient with metastatic ALK-positive lung adenocarcinoma with an impressive resistance to sequential treatment with ALK TKIs mediated by YES1 and MYC amplification in a contest of epithelial-to-mesenchymal transition and high progressive chromosomal instability. Methods The patient received, after chemotherapy and 7 months of crizotinib, brigatinib and lorlatinib with no clinical benefit to both treatments. A study of resistance mechanisms was performed with whole exome sequencing on different biological samples; primary cell lines were established from pleural effusion after lorlatinib progression. Results At whole exome sequencing analysis, YES1 and MYC amplifications were observed both in the pericardial biopsy and the pleural effusion samples collected at brigatinib and lorlatinib progression, respectively. Increasing chromosomal instability from diagnostic biopsy to pleural effusion was also observed. The addition of dasatinib to brigatinib or lorlatinib restored the sensitivity in primary cell lines; data were confirmed also in H3122_ALK-positive model overexpressing both YES1 and MYC. Conclusions In conclusion, YES1 and MYC amplifications are candidates to justify a rapid acquired resistance to crizotinib entailing primary brigatinib and lorlatinib resistance. In this context, a combination strategy of ALK TKI with dasatinib could be effective to overcome a rapid resistance.


Introduction
Patients with advanced NSCLC harboring ALK rearrangement experience extended benefit from sequential treatment with ALK tyrosine kinase inhibitors (TKIs), reaching survivals up to 5 years. 1 Acquired resistance to ALK TKIs inevitably occurs and is often mediated by acquisition of secondary ALK mutations. 2 Other resistance mechanisms mediated by activation of different bypass signaling pathways were described. 2 Moreover, epithelial-to-mesenchymal transition (EMT) 2 and small cell transformation have been reported. 2 Intrinsic resistance implies the absence of ALK TKI activity, therefore leading to poor outcomes: its mechanisms are poorly understood and this represents an important gap in the field of ALK TKI resistance. Here, we present a case report of a young never-smoker woman affected by an ALK-positive NSCLC tumor who presented an impressive resistance to sequential treatment with ALK TKIs mediated by YES1 and MYC amplifications in a complex context of EMT and high progressive chromosomal instability. Preclinical proofs of their role were provided both in primary cell lines and in vitro model.

Materials and Methods
For the extensive protocol, see the Supplementary Materials.

Case Report
A 33-year-old never-smoker woman was hospitalized in December 2015 for dyspnea with findings of pleural and pericardial effusion owing to a right lung adenocarcinoma ( Fig. 1A and B). Immunohistochemistry results revealed ALK positivity (Fig. 1C), confirmed with targeted RNA sequencing as EML4 (exon 13)-ALK (exon 20) variant 1. The patient received carboplatin-pemetrexed with tumor response after two courses (Fig. 1D); after the third cycle, dyspnea worsened and crizotinib was rapidly started in February 2016, with tumor response (Fig. 1E). After 7 months of crizotinib, subcarinal progression was treated with radiotherapy and crizotinib was continued. In December 2016, a pleuropericardial progression occurred with the need of pericardiocentesis which revealed ALK-positive adenocarcinoma cells. The second-generation ALK TKI brigatinib was administered from December 2016, without effect, as no clinical benefit was achieved and disease progression was documented after 2 months (Fig. 1F); a new pericardiocentesis was performed, followed by pleuro-pericardial window with a pleurocath positioning. The histologic results of pericardial localization confirmed ALK-positive adenocarcinoma ( Fig. 1G and H). In March 2017, lorlatinib was started, but the the third-generation ALK TKI was ineffective with a rapid clinical worsening with a massive pleural effusion. Informed consent to perform molecular analysis in her pleural effusion sample and in other tissue biopsy samples was collected, but the patient passed away in May 2017.

Molecular Study of Resistance
Tumor/normal whole exome sequencing (WES) was performed on all available tissue rebiopsy samples collected at progression of brigatinib and lorlatinib (pericardial biopsy and pleural effusion, respectively), on one liquid biopsy sample (collected during brigatinib treatment, 1 month before the pericardial biopsy), and on a matched control sample (Fig. 1). Single nucleotide variant analysis revealed low tumor mutational burden (<1 mutation per megabase) across all samples (Fig. 1I) and absence of secondary ALK mutations (Fig. 1J).
Somatic copy number alteration (SCNA) analysis across our samples revealed a temporally progressive chromosomal instability from the diagnostic biopsy to the pleural effusion samples (Fig. 1I). Gains for YES1, encoding a SFK, and MYC genes were observed both in the pericardial biopsy and the pleural effusion samples (Fig. 1J). An in-depth allele-specific copy number analysis of the pleural effusion sample revealed a complex genomic landscape (Fig. 1K) and highlights a strong copy-aberrant loss of heterozygosity of YES1 gene and a MYC allele-specific gain.
Similarity analysis of single nucleotide variants and SCNA profiles between the liquid biopsy and the other samples (Fig. 1L), together with phylogenetic tree reconstructions ( Fig. 1M), suggests a branching evolutionary tumor trajectory characterized by early dominant driver, emerging complex genomic landscape compatible with aneuploidy, and low background mutation rate.
The pericardial biopsy sample was further characterized by immunohistochemical analysis to evaluate the expression of cytokeratin, E-cadherin, and vimentin (Vim), suggesting the gain of mesenchymal phenotype and the acquisition of migratory properties ( Fig. 2A-F).
For the extensive protocol of the molecular study, see the Supplementary Materials.

Preclinical Results
Primary cell lines were established from pleural effusion and double immunofluorescence staining samples for the epithelial markers EpCAM or cytokeratin, and the mesenchymal marker Vim was performed. The following two cell populations were distinguished: one adherent to the dish surface and the other spontaneously growing as spheroids We then evaluated the effect of ALK TKIs in inhibiting cell proliferation of primary cell lines. In respect to human EML4/ALK-rearranged H3122 cell line, sensitive to all ALK inhibitors (range concentration that inhibits 50%: 5-10 nM), 3 our primary cell lines, either adherent (Supplementary Fig. 2A) or growing as spheroids ( Fig. 3A), had resistance to these inhibitors.
To better define the role of YES1 and MYC amplification in conferring resistance to ALK inhibitors, H3122 cells and MYC-overexpressing H3122 cells (H3122MYC) 4 were infected with the lentiviral transfer vector carrying full-length human YES1 cDNA (H3122MYC/YES) as previously described. 4 Selected overexpressing clones were then analyzed for sensitivity to the alectinib, brigatinib, and lorlatinib. The sole overexpression of YES1 did not modify the responsiveness to these inhibitors. By contrast, H3122MYC was more resistant to all the inhibitors, as already reported, 4 and H3122MYC/YES had the highest resistance to the drugs ( Fig. 3B and Supplementary  Fig. 2B and C). With the aim to overcome ALK TKI resistance mediated by MYC/YES1 amplification, we combined ALK inhibitors with dasatinib, tested either in the patient's primary cell lines and in the H3122MYC/ YES.
The combination of dasatinib with lorlatinib or with brigatinib restored the sensitivity to the ALK TKIs in primary cells, inducing an additive or synergistic effect in inhibiting cell proliferation ( Fig. 3C and Supplementary Fig. 3A). Similarly, a strong synergistic effect was exerted when dasatinib was combined with ALK TKIs in H3122MYC/YES (Fig. 3D and Supplementary Fig. 3B and C), confirming the addiction of a SFK-targeting agent can overcome the resistance to ALK TKIs in the presence of the MYC/YES1 amplification.

Discussion
Here, we describe a case of a young never-smoker woman affected by advanced ALK-positive NSCLC who presented a dramatic clinical course to a sequential treatment with ALK TKIs, with an acquired resistance to crizotinib entailing primary resistance to brigatinib and lorlatinib. Results of WES analysis revealed the context of the disease with high genomic instability. The increasing level of SCNAs observed between the pericardial tissue and pleural effusion samples revealed a high aneuploidy profile that could lead to an aggressive tumor phenotype.
Analyzing more specifically the WES results, YES1 and MYC amplifications may be implicated in the resistance to the ALK TKIs. MYC amplification was already described as a potential primary resistance mechanism to crizotinib in an ALK-positive patient with NSCLC. 4 Regarding YES1, recently, Garmendia et al. 5 presented a novel evidence for its amplification as a mediator of carcinogenesis, revealing its overexpression induced metastatic spread in in vivo models and that it is a predictive marker of dasatinib response in NSCLC cells. YES1 is the only member of the SFK regulated mainly by gene amplification; it was described as a resistance mechanism to EGFR TKIs 6 and recently reported in two of 17 ALK-positive patients with NSCLC as putative resistance mechanism to ALK TKIs. 6 In our H3122MYC/YES model, we reported that YES1 amplification seems not able alone to guide ALK TKI resistance; it needs a trigger, as MYC amplification, to generate high TKI-resistant cells. In contrast with our findings, Sato et al. 7 reported that overexpression of either YES1 or YAP1 in ALK-positive cell lines conferred resistance to ALK TKIs. Nevertheless, according with that study, we observed that the addition of dasatinib to brigatinib or lorlatinib is able to restore sensitivity to ALK TKIs both in H3122MYC/YES and in primary cell lines.
Considering the absence of specific MYC inhibitor and the evidence that H3122MYC is highly sensitive to dasatinib, 8 our results of restoration of ALK TKI sensitivity in the primary cell lines and in H3122MYC/YES could be explained by this broad activity of dasatinib both on YES1 and MYC.
YES1 amplification is recently reported to be a noncanonical mechanism of YAP activation, 9 which itself had a potential role in ALK TKI resistance. 10,11 Moreover, this pathway was revealed to promote EMT in different cancer cell types. 12,13 In our case, immunocytochemical characterization of pleural effusion revealed EMT features, confirmed also in primary cell line, highlighting the potential putative role of YES1 in inducing EMT.
Furthermore, in our patient, mesenchymal microenvironment could have elicited genomic instability contributing to tumor evolution. Comaills et al. 14 revealed that increased mesenchymal marker expression is correlated with genomic instability in circulating tumor cells of patients with metastatic breast cancer. On the basis of these evidences, YES1 amplification could play a potential key role in inducing EMT and chromosomal instability and with the trigger played by MYC amplification in guiding resistance to ALK TKIs.
In conclusion, we provided an in-depth analysis of longitudinal samples derived from a patient with rapid resistance to three generations of ALK TKIs. YES1 and MYC amplification, in a context of high genomic instability associated with EMT, are candidates to justify the rapid dramatic evolution. Prompt administration of ALK TKI in combination with dasatinib could be an effective strategy in the case of rapid ALK TKI resistance.

Informed Consent
Informed consent was obtained from the patient for publication of these case report and any accompanying images.

Supplementary Data
Note: To access the supplementary material accompanying this article, visit the online version of the JTO Clinical and Research Reports at www.jtocrr.org and at https://doi.org/10.1016/j.jtocrr.2022.100278.