Oncogenic rearrangements in RET are present in 1-2% of lung adenocarcinoma (LAD) patients. Ponatinib is a multi-kinase inhibitor with low-nanomolar potency against the RET kinase domain. Here, we demonstrate that ponatinib exhibits potent anti-proliferative activity in RET fusion positive LC-2/ad LAD cells and inhibits phosphorylation of the RET fusion protein and signaling through ERK1/2 and AKT. Using distinct dose-escalation strategies, two ponatinib-resistant LC-2/ad cell lines, PR1 and PR2, were derived. PR1 and PR2 cell lines retained expression, but not phosphorylation of the RET fusion and lacked evidence of a resistance mutation in the RET kinase domain. Both resistant lines retained activation of the MAPK pathway. Next-generation RNA sequencing revealed an oncogenic NRAS p.Q61K mutation in the PR1 cell. PR1 cell proliferation was preferentially sensitive to siRNA knockdown of NRAS compared to knockdown of RET, more sensitive to MEK inhibition than the parental line, and NRAS-dependence was maintained in the absence of chronic RET inhibition. Expression of NRAS p.Q61K in RET fusion expressing TPC1 cells conferred resistance to ponatinib. PR2 cells exhibited increased expression of EGFR and AXL. EGFR inhibition decreased cell proliferation and phosphorylation of ERK1/2 and AKT in PR2 cells but not LC-2/ad cells. Although AXL inhibition enhanced PR2 sensitivity to afatinib, it was unable to decrease cell proliferation by itself. Thus, EGFR and AXL cooperatively rescued signaling from RET inhibition in the PR2 cells. Collectively, these findings demonstrate that resistance to ponatinib in RET-rearranged LAD is mediated by bypass signaling mechanisms that result in restored RAS/MAPK activation.

Ewing sarcoma (ES) is the second most common bone cancer in children, accounting for 2% of pediatric cancer diagnoses. Patients who present with metastatic disease at the time of diagnosis have a dismal prognosis, compared to the >70% 5-year survival of those with localized disease. Here, we utilized single-cell RNA sequencing (scRNA-seq) to characterize the transcriptional landscape of primary ES tumors, and to identify circulating tumor cells (CTCs) in peripheral blood at the time of diagnosis in order to further understand ES transcriptional heterogeneity and factors that drive metastasis. Methods: Viably frozen primary tumor and peripheral blood samples were obtained from 7 ES patients at the time of diagnosis and prior to the initiation of treatment. Tumors were dissociated into a single cell suspension and sorted for viability using FACS (fluorescence activated cell sorting) whereas peripheral blood samples were subjected to a size-based selection with the CellSieve microfiltration system. ScRNA-seq was performed using the 10xChromium platform and count matrices were generated using 10x Genomics Cell Ranger software. Quality control, integration, and cluster analysis was performed with Seurat. Results: Cluster analysis of integrated, primary tumor samples demonstrated that candidate ES cell clusters express FLI1 and ES marker NKX2-2 and cluster separately from immune cell populations. Additionally, using inferCNV, ES clusters were demonstrated to harbor chromosomal copy number alterations known to be associated with ES and that were previously identified clinically on preliminary pathology reports for each patient; further GSEA analysis showed significant overlap between published ES gene sets and genes upregulated in ES clusters. Further analysis of ES cells demonstrated that cell-cycle phase determined a cluster enriched in pro-proliferation gene signatures, and overall heterogeneity of expression of previously known therapeutic targets. Candidate ES CTCs were identified among the peripheral blood samples among clusters which corresponded with distinct immune cell populations. The candidate ES CTCs expressed NKX2-2 and demonstrated enrichment in oncogenic gene signatures. Conclusion: ScRNA-seq of primary ES tumors is feasible and demonstrates that ES tumor cells are largely homogeneous in nature; and candidate ES CTCs can be identified in peripheral blood at the time of diagnosis in ES patient and warrant further investigation as to their utility as a biomarker of metastatic disease. Citation Format: Sarah K. Nelson-Taylor, Avery Bodlack, Andrew Goodspeed, Amy Treece, Nathan Donaldson, Carrye Cost, Tim Garrington, Brian Greffe, Sandra Luna-Fineman, Jenna Sopfe, Masanori Hayashi. Single cell RNA sequencing of primary Ewing sarcoma tumors and identification of circulating tumor cells in patient-matched peripheral blood samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1681.

CRISPR/Cas9 gene editing represents a powerful tool for investigating fusion oncogenes in cancer biology. Successful experiments require that sgRNAs correctly associate with their target sequence and initiate double stranded breaks which are subsequently repaired by endogenous DNA repair systems yielding fusion chromosomes. Simple tests to ensure sgRNAs are functional are not generally available and often require single cell cloning to identify successful CRISPR-editing events. Here, we describe a novel method relying on acquisition of IL3-independence in Ba/F3 cells to identify sgRNA pairs that generate oncogenic gene rearrangements of the Ret and Ntrk1 tyrosine kinases. The rearrangements were confirmed with PCR, RT-PCR and sequencing and Ba/F3 cells harboring Ret or Ntrk1 rearrangements acquired sensitivity to RET and TRK inhibitors, respectively. Adenoviruses encoding Cas9 and sgRNA pairs inducing the Kif5b-Ret and Trim24-Ret rearrangements were intratracheally instilled into mice and yielded lung adenocarcinomas. A cell line (TR.1) established from a Trim24-Ret positive tumor exhibited high in vitro sensitivity to the RET inhibitors LOXO-292 and BLU-667 and orthotopic TR.1 cell-derived tumors underwent marked shrinkage upon LOXO-292 treatment. Thus, the method offers an efficient means to validate sgRNAs that successfully target their intended loci for the generation of novel, syngeneic murine oncogene-driven tumor models.

<p>Supplementary Figures 1-8 Fig. S1: Cell viability of LC-2/ad, PR1, and PR2 cells treated with alectinib or cabozantinib. Fig. S2: RET break-apart FISH analysis of LC-2/ad, PR1, PR2, and H2228. Fig. S3: Phosphatase inhibitor treatment of PR1 and PR2 restores phospho-RET. Fig S4: NRAS Q61K increases NRAS GTP loading and induces RET inhibitor resistance in TPC1 cells. Fig S5: PR1 cells remain resistant to ponatinib after withdrawal from chronic ponatinib treatment. Fig S6: PR2 cells have lost RET signaling dependence. Fig S7: AXL signaling contributes to EGFR-mediated resistance to ROS1 inhibitors in HCC78-TAER cells. Fig S8: PR2 cells demonstrate plasticity when withdrawn from chronic ponatinib and display dual dependence on RET and EGFR. Supplementary Tables 1-3 Table S1: Normalized gene expression of RTKs in LC-2/ad, PR1 and PR2 cells. Table S2: Normalized gene expression of RTK ligands in LC-2/ad, PR1 and PR2 cells. Table S3: Normalized gene expression of protein tyrosine phosphatases in LC-2/ad, PR1, and PR2 cell lines.</p>

<p>Supplementary Figures 1-8 Fig. S1: Cell viability of LC-2/ad, PR1, and PR2 cells treated with alectinib or cabozantinib. Fig. S2: RET break-apart FISH analysis of LC-2/ad, PR1, PR2, and H2228. Fig. S3: Phosphatase inhibitor treatment of PR1 and PR2 restores phospho-RET. Fig S4: NRAS Q61K increases NRAS GTP loading and induces RET inhibitor resistance in TPC1 cells. Fig S5: PR1 cells remain resistant to ponatinib after withdrawal from chronic ponatinib treatment. Fig S6: PR2 cells have lost RET signaling dependence. Fig S7: AXL signaling contributes to EGFR-mediated resistance to ROS1 inhibitors in HCC78-TAER cells. Fig S8: PR2 cells demonstrate plasticity when withdrawn from chronic ponatinib and display dual dependence on RET and EGFR. Supplementary Tables 1-3 Table S1: Normalized gene expression of RTKs in LC-2/ad, PR1 and PR2 cells. Table S2: Normalized gene expression of RTK ligands in LC-2/ad, PR1 and PR2 cells. Table S3: Normalized gene expression of protein tyrosine phosphatases in LC-2/ad, PR1, and PR2 cell lines.</p>