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. Author manuscript; available in PMC: 2024 May 1.
Published in final edited form as: Drug Resist Updat. 2023 Mar 24;68:100958. doi: 10.1016/j.drup.2023.100958

The heterogeneous transition state of resistance to RET kinase inhibitors converges on ERK1/2-driven Aurora A/B kinases

Xueqing Hu a,b, Xuan Liu a,b, Ujjwol Khatri a,b, Jie Wu a,b,*
PMCID: PMC10149623  NIHMSID: NIHMS1887626  PMID: 36990046

Abstract

Aim:

While most patients with RET-altered cancer responded to the RET protein tyrosine kinase inhibitors (TKIs) pralsetinib (BLU667) and selpercatinib (LOXO292), few achieved a complete response. Heterogeneity in residual tumors makes it difficult to target their diverse genetic alterations individually. The aim of this study is to characterize the cancer cells that persist under continuous RET TKI treatment and identify the shared vulnerability of these cells.

Methods:

We analyzed residual RET-altered cancer cells under prolonged RET TKI treatment by whole exome sequencing (WES), RNA-seq analysis, and drug-sensitivity screening. These were followed by tumor xenograft experiments of mono- and combinational drug treatments.

Results:

BLU667- and LOXO292-tolerated persisters were cellularly heterogeneous, contained slowly proliferating cells, regained low levels of active ERK1/2, and displayed plasticity in growth rate, which we designated as in the transition state of resistance (TSR). TSR cells were genetically heterogeneous. Aurora A/B kinases were among the most significantly upregulated genes and that the MAPK pathway activity had significantly higher transcript footprints. MEK1/2 and Aurora kinase inhibitors were the most effective drugs when combined with a RET kinase inhibitor. In a TSR tumor model, combination of BLU667 with an Aurora kinase or a MEK1/2 kinase inhibitor caused TSR tumor regression.

Conclusion:

Our experiments reveal that the heterogeneous TSR cancer cells under continuous RET TKI treatment converge on the targetable ERK1/2-driven Aurora A/B kinases. The discovery of the targetable convergent point in the genetically heterogeneous TSR points to an effective combination therapy approach to eliminate the residual tumors.

Keywords: Protein kinases, therapy resistance, residual tumors, RET, ERK1/2, Aurora, targeted therapy

1. Introduction

Pralsetinib (BLU667) and selpercatinib (LOXO292) gave high rates of durable response in oncogenic RET-positive non-small cell lung cancer (NSCLC) and thyroid cancer with the median duration of response (DOR) in 18.4–28.6 months (Drilon et al., 2023; Griesinger et al., 2022; Hu et al., 2023). However, <10% of patients who responded to BLU667 or LOXO292 achieved a complete response. While under the control of the TKIs, the tumor persisters will accumulate mutations (Subbiah et al., 2021a; Subbiah et al., 2021b), and eventually acquire on-target or target-bypass mechanisms to resist the TKIs for rapid tumor growth, resulting in disease progression (Lin et al., 2020; Rosen et al., 2021; Rosen et al., 2022; Shen et al., 2021; Solomon et al., 2020; Subbiah et al., 2021a; Subbiah et al., 2021b). New generations of TKIs can be developed to circumvent on-target mechanisms of resistance (Cross et al., 2014; Drilon et al., 2018; Drilon et al., 2019; Janne et al., 2015). While these new TKIs are effective in extending the duration of disease control, they are subject to the same problem of incomplete tumor response faced by the earlier generation of TKIs (Lin et al., 2018; Repetto et al., 2022; Thress et al., 2015).

A crucial question is how to eradicate residual tumors before they evolve to the fast-growing resistant tumors that eventually become non-manageable. Residual tumors and their mechanisms of adaptation to the targeting TKIs are heterogeneous (Bivona and Doebele, 2016; Dagogo-Jack and Shaw, 2018; Hata et al., 2016; Lin et al., 2020; Maynard et al., 2020; Rosen et al., 2022; Rotow and Bivona, 2017; Vasan et al., 2019). Therefore, it is a daunting task to target individual mechanisms identified from tumor biopsy (Maynard et al., 2020) or cell-free DNA (Subbiah et al., 2021a). An approach to solve this dilemma is to find a targetable convergent point of the diverse mechanisms that allow residual tumors to maintain a near-steady size while the original oncogenic kinase is suppressed by the TKI before they become fast-growing resistant tumors.

In the present study, we analyzed residual RET-altered human thyroid and NSCLC cells after prolonged treatment with the RET-selective TKIs BLU667 or LOXO292. These cells and their cell-derived xenografts (CDXs) grew very slowly in the presence of a RET TKI, but resumed robust growth upon release from RET kinase inhibition. These cells/tumors differ from the resistant cells/tumors because they grow very slowly, resembling the residual tumors in patients who are undergoing RET TKI therapy. By transcriptomic and drug screening analyses, we found that these heterogeneous TSR cells converged on the ERK1/2-driven Aurora A/B under a RET TKI treatment. In a BLU667-TSR tumor model, combination of BLU667 with an Aurora kinase or a MEK1/2 inhibitor resulted in tumor regression. These results reveal that the heterogeneous TSR cells under a RET TKI treatment converge on the targetable ERK1/2-driven Aurora A/B kinases.

2. Materials and methods

2.1. Antibodies and inhibitors

These are listed in Tables S1 and S2.

2.2. Cell lines

TPC1 and LC-2/ad cells were from the European Collection of Authenticated Cells Culture (ECACC). TT Cells were from American Type Culture Collection (ATCC). MZCRC1 cells were kindly provided by Dr. Gilbert J. Cote. Cell lines were authenticated using Thermo Fisher’s CLA IdentiFiler system through a 16-marker short tandem repeat (SR) analysis. Cells were free of mycoplasma. TPC1-derived TSR cells used for the animal experiment were verified to be free of pathogens by IDEXX BioAnalytics.

2.3. Cell culture, viability, live/dead, senescence, DNA synthesis, Cell cycle synchronization, and clonogenic growth assays

TPC1 cells were cultured in RPMI-1640 plus 5% fetal bovine serum (FBS). LC-2/ad cells were cultured in RPMI-1640/F-12 medium (1:1 mixture) plus 15% FBS. TT cells were cultured in F-12K medium plus 10% FBS. MZCRC1 cells were cultured in DMEM plus 10% FBS. In cell cultures that last more than one week, medium and drug were replaced weekly.

Viable cells were measured in 96-well plates using CellTiterGlo reagent (#G7572, Promega) as described (Liu et al., 2018). Live/dead cells were visualized using a LIVE/DEAD cell imaging kit (R37601, Thermo Fisher). Cell senescence was determined using the senescence β-galactosidase staining kit (#9860, Cell Signaling Technology).

For the DNA synthesis assay, cells were cultured in 2-well chamber slides (#154852, Thermo Fisher) with media plus 4 μM BLU667 or LOXO292. On Day 14 following drug treatment, 14 μM 5-ethynyl-2”-deoxyuridine (EdU) was added to the culture. On Day 21, cells were processed for detection of DNA synthesis using Click-iT Plus EdU imaging reagents (Alexa Fluor-555 picolyl azide version, #C10638, Thermo Fisher) (Salic and Mitchison, 2008). DNA was stained with 4,6-diamidino-2-phenylindole (DAPI) using the ProLong Diamond Antifade Mount with DAPI (#P36966, Thermo Fisher). Images were captured with a Leica SP8 Confocal White Light Laser system (Magnification 40X). Cells were enriched in G0/Early G1 phase by serum starvation for 48 h, in later G1/S phase by double thymidine block, and in M phase by nocodazole block similar to that described (Fan et al., 2015).

Clonogenic growth assay was performed in 24-well plates. Cell colonies were fixed with 10% methanol/10% acetic acid in phosphate-buffered saline (PBS) for 10 min and stained with 0.4% crystal violet for 3 min. The stained plates were scanned with an Optronix GelCount colony counter and quantified.

2.4. Immunoblotting and immunohistochemical (IHC) assays

Cell lysate preparation and immunoblotting assays were performed as described (Liu et al., 2018). Tissue sections (4 μm) from formalin-fixed, paraffin-embedded (FFPE) tissue blocks were processed for IHC staining utilizing an automated Leica Bond III. Ki67 antibody was used at 1:400 dilution for 60 min. The phospho-Histone H3 Serine-10 (H3S10ph) antibody was used at 1:1000 dilution for 60 min.

2.5. Whole exome sequencing (WES)

Genomic DNA from cells was isolated using a DNeasy Blood & Tissue Kit (#69506, Qiagen). DNA library construction and WES were performed by Novogene (Davis, CA). The Burrows-Wheeler Aligner (BWA) software (Li and Durbin, 2009) was used to map the paired-end clean reads to the reference genome (GRCh38). SNPs/Indels were called using the Genome Analysis Toolkit (GATK) (DePristo et al., 2011) from BAM files and variants were annotated using ANNOVAR (Wang et al., 2010). CNV analysis was performed using Control-FREE Copy Number and Allelic Content Caller (Control-FREEC) (Boeva et al., 2012). CNVs of BR8, BR10, and BR16 cells were normalized to the parental TPC1 cells.

Exonic SNPs that were missense and stopgain/stoploss, and indels and CNVs that were exonic were selected for further analyses. Genetic alternations that were also present in the parental TPC1 cells were filtered out. New genetic alterations in BR8, 10, and 16 cells were first compared with OncoKR genes (https://www.oncokb.org/cancerGenes) (Chakravarty et al., 2017) to narrow down the list to cancer-related genes, and further compared with the Catalogue of Somatic Mutations in Cancer (COSMIC) (https://cancer.sanger.ac.uk/cosmic) to identify cancer-related SNPs. Then, sequencing reads were manually inspected with integrative genomics viewer (IGV) to confirm that the SNP or indel was not detected in the parental TPC1 cells.

2.6. Sanger DNA sequencing

mRNA was converted to cDNA by Tagman Reverse Transcription Reagents (#N8080234, ThermoFisher). The BRAF cDNA fragment containing the V600K601 region was isolated by PCR of cDNA using primers BRAF1655 (5’-AACTTATAGATATTGCACGACAGA) and BRAF1856r (5’-CACAAAATGGATCCAGACAACT). Sanger sequencing was performed using the PCR primers.

2.7. RNA-seq and bioinformatic analysis

Total RNA was isolated from cells using a modified protocol with the Direct-zol RNA MiniPrep kit (R2050, Zymol Research) and TRI reagent (T9424, Sigma). Briefly, cells were lysed in 1 ml TRI reagent. The cell lysate (1 ml) was mixed with 1-bromo-3-chloroproane (0.1 ml). After centrifugation, the upper aqueous phase was collected and mixed with an equal volume of ethanol. The subsequent purification steps using the Direct-zol RNA MiniPrep kit R2050 were performed according to the supplier’s protocol. cDNA library construction and RNA-seq were performed by Novogene (Davis, CA). The PE150 Illumina RNA-seq based on sequencing by synthesis was performed. STAR (v2.5), HTseq (v0.6.1), and DESeq2 (v2_1.6.3) software were used to map the clean reads to the reference genome (GRCh38), quantification of gene expression level, and for differential expression analysis. Transcripts with adjusted p value < 0.05 and |Log 2(Fold Change)| ≥ 1 were assigned as differentially expressed.

Gene Set Enrichment Analysis (GSEA) was performed using GSEA software (v4.0.3) based on Molecular Signatures Database (MSigDB) (v7.1, https://www.gsea-msigdb.org/gsea/index.jsp) (Subramanian et al., 2005). The hallmark gene sets and oncogenic signature gene sets were enriched and were visualized by ggplot2 R package (v3.3.5). The number of permutations was 1000 and the permutation type was gene-set. Signal 2 Noise was used as a metric for ranking genes. Other parameters were used as default. False discovery rate (FDR) q < 0.05 was considered statistically significant. PROGENy (v1.8.0) and dplyr (v0.8.5) R packages were used to infer the pathway activity and to calculate the statistical difference (Schubert et al., 2018).

2.8. RNA interference

Silencer Select Pre-Designed siRNAs targeting AURKA (siRNA s196, siRNA s197), AURKB (siRNA s19611; siRNA s19612), FoxM1 (siRNA s5248, siRNA s5249, siRNA s5250), and the negative control No. 2 siRNA (#4390846) were obtained from Thermo Fisher Scientific. RNA knockdown experiments were performed by reverse transfection using Lipofectamine RNAiMAX reagent (#13778075, Thermo Fisher) with 40 nM siRNA. Immunoblot analyses were performed using cell lysates 48 h after transfection. Viable cells were measured on Day 6.

2.9. Xenograft tumor assay

The animal experiment was approved by the IACUC of the University of Oklahoma Health Sciences Center. BR8, BR10, and BR16 cells in serum-free RPMI-1640 medium were mixed in a 2:1:2 ratio and inoculated s.c. (2×106 cells/0.1 ml/each) into the right flanks of ~6-week old female SHO mice (Charles River). After measurable tumors were established, mice were treated with BLU667 (30 mg/kg, qd) by oral gavage for seven days. Then, mice bearing similar-sized tumors were divided into six groups (n=7–8 mice/group), and treated with the indicated drugs by oral gavage as indicated in the figure legend. The tumor sizes and animal body weights were measured similar to that described (Shen et al., 2021).

2.10. Curve fitting and statistical analysis

Curve fitting was performed as described (Scott et al., 2011). Statistical analysis was performed using student’s unpaired t-test with Welch’s correction. p < 0.05 was considered statistically significant.

3. Results

3.1. Residual cells are proliferating while the RET kinase is suppressed

TPC1, LC-2/ad, TT, and MZCRC1 are RET oncogene-positive human papillary thyroid carcinoma (PTC), NSCLC, and medullary thyroid cancer (MTC) cell lines that are hypersensitive to RET TKIs (Subbiah et al., 2018a; Subbiah et al., 2018b). BLU667 or LOXO292 inhibited proliferation and induced death of these cells (Fig. 1A–C and Fig. S1). However, a small fraction of cells persisted even at high drug concentrations that were similar to the plasma Cmax in patients, at which RET kinase was completely inhibited (Fig. 1D). Three weeks after continuing BLU667 or LOXO292 treatment, some of the residual TPC1, TT, and MZCRC1 cells, but none of the residual LC-2/ad cells, were in senescence (Fig. 1C and Fig. S1C). All four cell lines had active proliferating cells (Fig. 1C, Fig. S1 and S2). These results revealed that the drug-tolerant persisters were not merely in a static state of quiescence. Rather, they comprised a heterogeneous population of cells, including proliferating, dying, and senescent cells.

Fig. 1.

Fig. 1.

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Residual cells are proliferating while the RET kinase is suppressed. (A, B) TPC1 and LC-2/ad cells were treated with BLU667 (BLU) or LOXO292 (LOX). Viable cells were measured on the given days (A), or on Day 5 (B). (C) Residual cells after 2–3 weeks of BLU667 treatment. In the Click-iT EdU assay, Edu (14 μM) was added during the last week of the culture. Residual cells were examined with a phase contrast microscope, stained with live/dead (calcein AM/BOBO-2 iodide) imaging reagent or β-galactosidase, or processed for the Click-iT-EdU assay of DNA synthesis. White arrows: live, non-senescent, or proliferating cells; red arrows: dead, senescent, or non-proliferating cells. (D) ERK1/2 was reactivated in TSR cells. Left panels: parental TPC1 cells were treated with solvent or BLU667 (5 μM) for 24 h, TSR cell colonies were isolated from cells cultured continuously with 5 μM BLU667. Middle and right panels: BLU667 was withdrawn from BR8, 10, and 16 cultures for four days before the treatment (5 h). (E) Comparison of cells growth +/− BLU667 (5 μM) for 5 days. (F) Comparisons of IC50s. Viable cells were measured on Day 5. (G) Clonogenic growth assay. Cells were treated with BLU667 in 24-well plates for nine days and stained with 0.4% crystal violet.

Initially, we attempted to isolate BLU667- or LOXO292-resistant CCDC6-RET kinase mutations in TPC1 and LC-2/ad cells, which would result in reactivation of the oncogenic RET fusion kinase. Cells were cultured in medium with 4–5 μM BLU667 or LOXO292 for ~50 days. Residual cells that were able to form slowly growing colonies were isolated and expanded, and screened for the presence of active RET by immunoblotting. None of the isolated cell colonies or pools of cell colonies had active RET (Fig. 1D and Fig. S3A–C), indicating that there was no RET mutation that disrupted the TKI-binding among these TPC1- or LC-2/ad-derived cells. These cells grew slowly in the presence of BLU667 or LOXO292, but resumed rapid cell growth when the TKI was removed (Fig. 1E and Fig. S3D). We consider these cells as being in a transition state of evolution to RET TKI resistance rather than as being RET TKI-resistant, because they lack a RET TKI-resistant RET mutant or a strong alternative oncogene to drive rapid cell growth in the presence of a RET TKI (Fig. S4).

Three independent TPC1-derived cell colonies isolated from 5 μM BLU667-cultures (BR8, BR10, BR16) were randomly selected for further analyses. cDNA sequencing confirmed that there was no mutation in the CCDC6-RET kinase domain (data not shown). Consistently, the subsequent WES of these cells (see below) did not detect RET kinase domain mutation. However, these cells had various levels of reactivated ERK1/2 (Fig. 1D and Fig. S3B). Withdrawal of BLU667 resulted in RET reactivation, a higher level of active ERK1/2 (Fig. 1D, middle and right panels), and significantly faster cell growth (Fig. 1E, G, and Fig. S3D). These cells had increased BLU667 IC50s (Fig. 1F) and were less sensitive to BLU667 in the colonenic growth assay (Fig. 1G). These BLU667-induced TSR cells cross-reacted to LOXO292 (Fig. S3D).

3.2. Heterogeneous genetic alternations in the transition state of BLU667-resistant cells

Using BR8, BR10, and BR16 cells as representative of TSR cells to BLU667, we took a snapshot of their genetic changes by WES. Nonsynonymous SNPs, indels, and CNVs in these cells were compared with those in the parental TPC1 cells to identify acquired genetic alterations in these TSR cells. We then used OncoKB (Chakravarty et al., 2017) and COSMIC databases to derive a list of acquired genetic alternations found in BR8/10/16 cells that are potentially cancer-relevant. Sequence reads of each SNP and indel on the list were further inspected manually using the IGV (Robinson et al., 2017) to confirm that the variant was not detected in the parental TPC1 cells. Nine different SNPs, three indels in two genes, and 98 CNVs of cancer-related genetic alterations were identified through this workflow in these TSR cell colonies (Table S3). Among these, one event (EGFR copy number gain) was common to all three cell colonies (Fig. 2A). BR8 cells had specifically acquired AXIN1(G565S), KEAP(D422A), TRIP11(S634C), and ACTB(P98L) mutations, and a ZNF750(T409M) mutation shared with BR16 cells. BR10 cells had acquired BRAF(K601E) (Fig. S5A), PCLO(S4313Y), and RABEP1(S449Y) mutations. BR16 cells had acquired a new KMT2C stopgain mutation. Several SNPs calls in these TSR cells were found to be enriched, but not newly acquired, from the parental cells. These included a missense mutation in the RB1 binding protein ARID4A and an indel in the lnc RNA MDS2 (Table S3).

Fig. 2.

Fig. 2.

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Genetic and transcriptomic features of TSR cells. (A) Venn diagram of genetic alterations in the exonic regions of three TSR cells. (B) Volcano plot of DEGs between TSR cells (R group) and parental cells transiently treated with BLU667 (B group). (C-D) Enriched hallmarks and oncogenic signatures in R vs. B, and examples of enrichment plots by GSEA. NES, normalized enrichment score. Only gene sets with a false discovery rate (FDR) q value < 0.05 are shown. (E) Comparison of PROGENy pathway activities between R and B groups. (F) Cell lysates of the parental TPC1 cells with (P+B) or without (P) transient BLU667 treatment (5 μM, 24 h), and three TSR cell colonies (BR8, 10, 16) cultured in BLU667, and were analyzed by immunoblotting. (G) Cell lysates of the parental LC-2/ad cells +/− BLU667 treatment as in (F), or from a pool of LOXO292-TSR cells, were analyzed by immunoblots.

Sanger sequencing of cDNA showed an approximately equal level of transcripts encoding K601 and E601 in BR10 cells, but not in other cells (Fig. S5B–C). BRAF K601 mutations were reported at low frequencies in COSMIC (Fig. S5D). In addition to other specific indels and CNVs, BR10 shared with BR8 the copy number loss of the CDKN2A tumor suppressor gene (Fig. 2A). Thus, this WES analysis of OncoKR-annotated variants in BR8/10/16 cells illustrated that TSR cells are genetically heterogeneous.

3.3. Transcriptomes of TSR cells are enriched with proliferating cell gene sets and MAPK activity footprints

To gain insight into the transcriptional reprogramming that mediates the TSR, we compared transcriptomes of mock-treated TPC1 cells (group P), TPC1 cells transiently treated with 5 μM BLU667 for 24h (group B), and BR8/BR10/BR16 cells cultured continuously with BLU667 (group R). Compared with group B, among the most significantly elevated transcripts in group R were G2/M genes, such as MYBL2, AURKA, AURKB, TPX2, FoxM1, INCENP, and genes that control cell proliferation such as CCND1 (cyclin D1), which is regulated by ERK1/2 (Lavoie et al., 1996) (Fig. 2B). Immunoblotting analysis of these proteins during cell cycle showed that phospho-Aurora, Aurora A/B, TPX2, and FoxM1 were expressed at the highest level in M phase (Fig. S6A, B). Conversely, these genes were among the most significantly down-regulated genes when the parental TPC1 cells were transiently treated with BLU667 (Fig. S7A). Consistently, mapping of differentially expressed genes (DEGs) between groups R and B by GSEA (Subramanian et al., 2005) showed that G2M and cell cycle-related genes dominated the enriched gene sets for group R in the Hallmark gene set collection (Fig. 2C). In the Oncogenic Signature gene set collection, cell cycle and growth factor response gene sets were significantly enriched in group R (Fig. 2D). To assess whether FoxM1 is a critical regulator of Aurora A/B, we knocked down FoxM1 with siRNAs. While all three FoxM1 siRNAs effectively knocked down FoxM1, they did not reduce Aurora A/B protein levels (Fig. S6D).

In transcript footprint analysis (Schubert et al., 2018), the activities of estrogen, MAPK, EGFR, VEGF, and PI3K pathways had significantly lower footprints in group B than in group P (Fig. S7B). Activities of EGFR, MAPK, VEGF, PI3K, and estrogen pathways (in the ascending order of p values) had significantly higher footprints in group R than in group B (Fig. 2E). Changes in the footprints of the MAPK pathway activities were greater than those of the PI3K pathway activities in both comparisons (Tables S4, S5).

Immunoblotting analysis showed that ERK1/2, but not AKT, was reactivated in BR8/10/16 cells (Fig. 2F). EGFR phosphorylation was increased when TPC1 cells were treated with BLU667 for 24 h, and further elevated in the BR8/10/16 cells cultured continuously with BLU667 (Fig. 2F), suggesting that the RET kinase inhibitor induced a rapid rewiring of the tyrosine kinase signaling pathway that was reinforced with subsequent EGFR copy number gain. In contrast, BLU667 and LOXO292 inhibited MET phosphorylation in TPC1 and LC-2/ad cells (Fig. 2F,G). MET phosphorylation in BR10 cells became resistant to BLU667 through an unknown mechanism (Fig. 2F), as no MET mutation or copy number gain was detected by WES (Table S3) or by Sanger sequencing of the MET kinase coding region (not shown). Nevertheless, inhibition of the MET kinase by capmatinib or crizotinib did not affect ERK1/2, cell growth, or clonogenic growth in BR10 cells or in a pool of TSR cells (Fig. S8). Consistent with the transcriptomic analysis, increased levels of Aurora A/B, TPX2, and MYBL2 proteins were detected in BR8, BR10, and BR16 cells by immunoblotting (Fig. 2F). Similarly, elevated phospho-ERK1/2, Aurora A/B, and EGFR, but no phospho-AKT, were observed in a pool of LC-2/ad TSR cells (Fig. 2G). Similar to LC-2/ad cells (Fig. 2G), transient treatment of TT and MZCRC1 cells did not increase EGFR tyrosine phosphorylation (not shown).

3.4. TSR cells are vulnerable to ERK1/2 and Aurora kinase inhibition

To identify targetable vulnerability of BLU667- and LOXO292-TSR cells, we screened a collection of enzyme inhibitors by clonogenic growth assay. TPC1 cells were cultured with medium containing 4 μM BLU667 or LOXO292 for eight weeks to form TSR cell colonies, followed by incubation with BLU667 or LOXO292 plus 0.5 μM test compound for two weeks. Compounds that showed TSR colony inhibition were tested again in the second experiment (Fig. 3). The most consistent TSR inhibition was obtained with MEK1/2 kinase inhibitors (trametinib, PD325901, selumetinib, binimetinib), as was the SHP2 inhibitor RMC4550 that inhibited the MEK1/2 pathway (Nichols et al., 2018). The EGFR TKIs (erlotinib, gefitinib, and afatinib) showed good activity in BLU667-TSR but had variable effects in LOXO292-TSR cells. EGFR TKIs at the screen concentration had little effect on the parental TPC1, LC-2/ad, TT and MZCRC1 cells, as would be expected from RET-altered cancer cells (Fig. S9A, B). The histone deacetylase inhibitor panobinostat also showed consistent activity. Consistent inhibition of both BLU667 and LOXO292 TSR cells was also obtained with lestaurtinib. Lestaurtinib is a multiple kinase inhibitor that was tested as a JAK2 and FLT3 inhibitor in clinical settings (Santos et al., 2010; Smith et al., 2004). However, because BLU667 cross-inhibits JAK2 (in vitro IC50: 2.4 nM) (Subbiah et al., 2018a), JAK2 was completely inhibited by BLU667 in TPC1 cells and BR8/10/16 cells, and the JAK2 inhibitor ruxolitinib had no effect on proliferation of BLU667 or LOXO292 TSR cells (Fig. S10A, B). The FLT3 inhibitor midostaurine did not give consistent results (Fig. 3C). Interestingly, it was suggested that lestaurtinib might inhibit Aurora kinase and RET (Gabler et al., 2013; Strock et al., 2003). To test these possibilities, we examined the effects of lestaurtinib on phospho-Aurora A/B/C and pRET in TPC1 cells. The results showed that lestaurtinib had a similar potency to that of the Aurora kinase inhibitors alisertinib and tozasertinib on inhibiting Aurora A/B/C phosphorylation in TPC1 cells, and that CCDC6-RET phosphorylation was inhibited by lestaurtinib (Fig. S10C).

Fig. 3.

Fig. 3.

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Vulnerability screening of BLU667- and LOXO292-TSR cells. (A, B) TPC1 cells were cultured in 24-well plates for eight weeks in medium containing 4 μM BLU667, followed by BLU667 plus the test compounds (0.5 μM) for two weeks. Cell colonies were visualized by crystal violet staining (B), and quantified (A). Selected compounds were tested again in a second experiment. (C, D) Experiments were performed as in (A) and (B), except LOXO292 was used.

Because our RNA-seq data implicated Aurora A/B kinases as the key players in these TSR cells, our subsequent experiments in TSR cells focused on Aurora kinases and ERK1/2. Among the Aurora kinase inhibitors that we evaluated, AMG900 (Bush et al., 2013; Geuns-Meyer et al., 2015) was the most potent (Fig. S10C, D). As shown in Fig. 4A, BR8/10/16 cells had phospho-Aurora A/B/C kinases that were not seen in the parental TPC1 cells treated with BLU667 (2 μM, 48 h). Combination of 50 nM AMG900 or 50 nM trametinib with 2 μM BLU667 blocked phospho-Aurora A/B/C kinases and induced apoptosis. The combined trametinib and BLU667 treatment blocked the increase in Aurora A/B protein and consequently their active phosphorylated form, indicating that the MEK1/2-ERK1/2 pathway drove Aurora A/B expression and activation. In the clonogenic growth assay, combination of BLU667 (2 μM) with AMG900 (>5 nM), alisertinib (>50 nM), tozasertib (100 nM), or trametinib (>25 nM) significantly reduced colony density of all three TSR cell colonies (Fig. 4B). In cell proliferation (Fig. S9C, D) and clonogenic growth assay (Fig. S9E), the parental cells were also inhibited by MEK1/2 and Aurora kinase inhibitors. Inhibition of Aurora kinases by AMG900 prevented Histone H3 Ser10 phosphorylation (H3S10ph) (Fig. S6C). H3S10ph is a known substrate of Aurora B (Hirota et al., 2005). H3S10ph not only is a mitotic marker, but this epigenetic modification also plays important roles in chromosome dynamics (Hirota et al., 2005; Komar and Juszczynski, 2020).

Fig. 4.

Fig. 4.

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BLU667-TSR cells are vulnerable to combined inhibition of BLU667 and ERK1/2 or Aurora A/B kinases. (A) Parental TPC1 cells (P) or BR8/10/16 cells were treated with BLU667 (2 μM), a combination of BLU667 with 50 nM AMG900 (BA) or 50 nM trametinib (BT) for 48 h. Cell lysates were analyzed by immunoblotting. (B) Clonogenic growth of BR8/10/16 cells treated with BLU667 in combination with AMG900, alisertinib, or tozasertinib, or trametinib for 11 days. Data were from two triplicate experiments. ns, not significantly different; all others were significantly different. (C) Knockdown of Aurora A/B kinases with siRNAs. TPC1 or a mixture of BR8/10/16 cells were transfected with siRNA for 48h and cell lysates were analyzed by immunoblots. (D) Relative numbers of viable cells after siRNA-transfected cells were cultured for five days. The parental cells were cultured without 2 μM BLU667 except the BLU677 treatment reference. The BR cells were cultured with 2 μM BLU667. (E) Clonogenic growth of BLU667 TSR LC-2/ad cells treated with a combination of BLU667 and the indicated Aurora kinase or MEK1/2 inhibitors for nine days. (F) Immunoblot confirmation of Aurora A/B knockdown with siRNAs in LC-2/ad BLU667 TSR cells. (G) Relative numbers of viable LC-2/ad or a mixture of LC-2/ad TSR cells five days after siRNA-transfected cells were cultured with 2 μM BLU667 as in panel D.

We next knocked down Aurora A/B using siRNAs (Fig. 4C). Interestingly, Aurora B knockdown also prevented phosphorylation of Aurora C. This is reminiscent of Aurora inhibitors in which the change of Aurora C phosphorylation correlated with Aurora B kinase phosphorylation (Fig. S10C). Thus, Aurora B appears to control Aurora C phosphorylation. Knockdown of Aurora A or B significantly reduced the viable cell numbers in both TPC1 and BR8/10/16 cells cultured with BLU667 (Fig. 4D). Similar to that observed in TPC1 and BR8/10/16 cells, combination of trametinib with BLU667 inhibited Aurora A/B phosphorylation and reduced Aurora A/B protein levels in LC-2/ad and LC-2/ad derived TSR cells (Fig. S10D, right panels). In combination with BLU667 (2 μM), AMG900 (50 nM), alisertinib (>50 nM), tozasertinib (100 nM), and trametinib (>25 nM) significantly inhibited the colony density of LC-2/ad TSR cells (Fig. 4E). Furthermore, in combination with BLU667, knockdown of Aurora A or Aurora B (Fig. 4F) significantly reduced viable cell numbers of LC-2/ad derived BLU667 TSR cells (Fig. 4G).

3.5. TSR tumors regress in response to combination treatment of BLU667 with Aurora or MEK1/2 kinase inhibitors

To test whether combination of a RET inhibitor with an Aurora or a MEK1/2 inhibitor suppresses TSR tumors in vivo, we first established a BLU667-TSR tumor model. A mixture of BR8/10/16 cells were injected s.c. into SHO mice to form tumors. After BLU667 treatment, the sizes of these tumors were maintained at a near-steady state (Fig. 5A), resembling residual tumors under the control of BLU667 in patients. We then either stopped the BLU667 treatment to simulate target mutation in RET TKI-resistant tumors that had reactivated RET kinase (Fig. 5D) and treated these tumors with AMG900, trametinib, or vehicle (mock), or continued the BLU667 treatment with or without combination with AMG900 or trametinib. As shown in Fig. 5A, upon BLU667 withdrawal (vehicle group), the tumors grew vigorously. AMG900, trametinib, or BLU667 as single agent significantly inhibited tumor growth, but did not reduce tumor sizes from the baseline (Fig. 5A, B). Importantly, combining BLU667 with AMG900 or trametinib significantly reduced the tumors by 78% (AMG900) or 82% (trametinib) from the baseline (Fig. 5A, B). The tumor weights from the BLU667 and AMG900 or trametinib combination treatment groups were significantly reduced from the BLU667 single agent treatment group (Fig. 5C, Fig. S11A). Animal body weights showed little change in all groups, suggesting that these treatments were tolerable (Fig. S11B).

Fig. 5.

Fig. 5.

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Combination of BLU667 with AMG900 or trametinib caused regression of BLU667-TSR tumors. Mice were injected s.c. with a mixture of BR8/10/16 cells. When the tumors were formed, mice were treated with BLU667 (30 mg/kg, qd) by oral gavage for 1 week to establish the near steady-sized TSR tumors. After that, mice were treated with the specified drugs or vehicle by oral gavage. (A) Tumor sizes were measured on the indicated days. The data shown are the mean ± SD. (B) Waterfall plot of tumor size change at the endpoint from the baseline. Each bar corresponds to a tumor in each mouse. A photograph of a representative tumor from each group is shown. (C) Tumors were dissected and weighed. (D) Two tumor tissue samples from each group were analyzed by immunoblots. V, vehicle; A, AMG900; T, trametinib; B, BLU667; BA, BLU667+AMG900; BT, BLU667+trametinib. (E) IHC staining of Ki67 and H3S10ph from FFPE tissue sections.

Analysis of tissue samples by immunoblotting showed that tumors from vehicle-, AMG900-, or trametinib-treated mice had active CCDC6-RET kinase, while the CCDC6-RET kinase activity was suppressed in tumors from mice treated with BLU667 and its combination with AMG900 or trametinib (Fig. 5D). Phospho-ERK1/2 was completely suppressed in tumors from mice treated with BLU667 plus trametinib, whereas phospho-Aurora kinases were suppressed in tumors from mice treated with BLU667 plus AMG900 (Fig. 5D). Tumors from mice treated with the combination of BLU667 with AMG900 or trametinib had the lowest levels of Aurora A and B kinases and increased cleaved caspase-3. IHC analysis of tumor tissue samples showed that the positive stain of the cell proliferation marker Ki67 and mitotic marker H3S10ph were reduced in AMG900-, trametinib-, and BLU667-treated groups compared with the vehicle-treated group (Fig. 5E). The tumor samples from the combined BLU667 with AMG900 or trametinib treatment group had the lowest Ki67- and H3S10ph-positive cells.

4. Discussion

While the RET-selective TKIs have high response rates in RET-altered cancers, they face the unmet challenge of incomplete response in most patients. It is imperative to eradicate the residual tumors or maximally reduce their sizes in order to achieve a cure or maximize the duration of response, turning an incurable metastatic disease into a manageable chronic condition. However, the diverse genomic or non-genomic mechanisms of RET TKI adaptation make targeting individual mechanisms cumbersome

In the present study, we found that the cellularly heterogeneous persisters after prolonged BLU667 or LOXO292 treatment contain a subset of slowly proliferating cells. Because they lack a secondary RET mutation that interferes with TKI binding, and are without a strong alternative oncogene to drive rapid growth when the RET kinase activity is suppressed, they are not considered RET TKI-resistant cells. Rather, they are more similar to residual tumors under the control of RET TKIs, but in the dynamic process of transition to resistant tumors. We designated these cells as TSR cells (Fig. S4). While different TSR cell colonies are molecularly heterogeneous, they share the feature of containing low levels of reactivated ERK1/2 and elevated Aurora A/B kinases while the RET kinase is suppressed. The partial ERK1/2 reactivation may be attributed to multiple mechanisms, such as rewiring of RET and EGFR tyrosine kinase signaling as previously observed in NSCLC cells (Chang et al., 2017; Vaishnavi et al., 2017) and implicated by our WES, immunoblotting, and drug screen data, or by acquisition of a possibly weak oncogene [BRAF(K601E)] detected in BR10 cells. Inhibition of MEK1/2 in combination with BLU667 in these TSR cells blocks the expression of Aurora A/B kinases (Fig. 4A), indicating that the expression of Aurora A/B is driven by ERK1/2 in TSR cells. Suppression of Aurora kinase activity with kinase inhibitors or knockdown of either Aurora A or B kinase inhibits the TSR cells cultured with BLU667, indicating that the elevated Aurora A/B kinases presented in the TSR cells are necessary to maintain the TSR cells under the control of the RET TKI. This notion is reinforced by our in vivo study in a TSR tumor model, in which combination of BLU667 with AMG900 or trametinib causes TSR tumor regression.

Specific knockdown of either Aurora A or B inhibited TSR cell growth (Fig. 4C, D, F, G), suggesting that both Aurora A and B are involved in mediating these TSR cells. In our tumor xenograft experiment, we first established measurable TSR tumors before starting the combination treatment. Conceivably, in the real world of patient care, the combination may start at the initiation of RET TKI treatment, rather than waiting for the depth of response to the RET TKI monotherapy to reach plateaus. Most of our data were generated from experiments using TPC1, LC-2/ad, and cells derived from them that contain the CCDC6-RET fusion oncogene. While it remains to be tested, we speculate that the conclusions derived from these CCDC6-RET+ cells are applicable to at least some of other molecular types of RET-altered cancers. This notion is supported in part by the observation that secondary EGFR mutation-independent mechanisms of EGFR TKI-resistance is also vulnerable to Aurora and other mitotic spindle assembly checkpoint inhibitors (Nilsson et al., 2020).

Together, our experiments identify the ERK1/2-driven Aurora A/B kinase as the targetable convergent point of human CCDC6-RET+ TSR tumors. The identification of the targetable convergent point of mechanistically heterogeneous TSR provides a key for solving the complex problem of residual disease.

Supplementary Material

Supplementary Materials

Acknowledgments

This work was supported by NIH grants R01CA242845, R01CA273168, P30CA225520, and P20GM103639; a Presbyterian Health Foundation SEED grant; Oklahoma Center for the Advancement of Science and Technology (OCAST) grant HR19–026; the Oklahoma Tobacco Settlement Endowment Trust, and the Peggy and Charles Stephenson Endowment to either the Wu laboratory or to the institutional core facilities. We thank the core facility staff at our institution for their assistance.

Footnotes

Competing Interests

The authors declare no competing interests.

Availability of data and materials

WES and RNS-seq data have been deposited in the NCBI Sequence Read Archive (SRA). The BioProject accession number for the WES data is PRJNA870066. The GEO accession number for the RNA-seq data is GSE211371.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Materials
NIHMS1887626-supplement-Supplementary_Materials.pdf (4.3MB, pdf)

Data Availability Statement

WES and RNS-seq data have been deposited in the NCBI Sequence Read Archive (SRA). The BioProject accession number for the WES data is PRJNA870066. The GEO accession number for the RNA-seq data is GSE211371.

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