EML4-ALK causes the early induction of cellular senescence in normal, mortal human fibroblasts
We transduced normal human fibroblasts CRL-2097, which are mortal, or have a limited replicative lifespan, with a lentiviral vector expressing the Tet repressor (generating CRL-2097/TR), and then with either a lentiviral vector encoding EML4-ALK or the control vector (Fig. 1a). While the control cells with no EML4-ALK expression reached approximately 11 or 12 population doubling levels (PDL) before proliferation arrest, the EML4-ALK-expressing cells ceased to proliferate earlier at PDL 6 after lentiviral transduction (Fig. 1b). In addition to the cells shown in (a) (observed through day 88 or 40), those retrovirally transduced with oncogenic Ras (H-RasV12) and its control vector (pBabe) were examined (Supplementary Fig. S1A-B; observed through day 20). Both the control and EML4-ALK-expressing cells, upon proliferation arrest, were similarly positive for senescence-associated b-galactosidase (SA-β-gal) (Fig. 1c). This early induction of cellular senescence by EML4-ALK was reminiscent of oncogenic Ras-induced senescence , although H-RasV12 induced senescence earlier after fewer PDL in this CRL-2097 strain (Fig. 1b, Supplementary Fig. S1A and S1B).
The EML4-ALK-induced senescence is associated with upregulated p16INK4A and p21WAF1
The expression levels of p16INK4A and p21WAF1, two markers of cellular senescence [25, 26], were examined in CRL-2097/TR with EML4-ALK or H-RasV12 expression, along with their control cells (Fig. 1d). The p16INK4A protein expression was increased during the replicative lifespan in both EML4-ALK-expressing and control cells, although its levels at senescence were lower than observed in H-RasV12-induced senescence. An increase in p21WAF1 protein expression was also observed similarly in the EML4-ALK-expressing and control cells, and to a lesser degree in H-RasV12-induced senescence. These findings suggest that the similar levels of upregulation of p16INK4A and p21WAF1 occur with the EML4-ALK-induced senescence and natural replicative senescence, although the former undergoes fewer PDL than the latter to achieve those expression levels.
The EML4-ALK-induced senescence is associated with accumulated DNA damage
A phosphorylated H2AX (g-H2AX)  was detected by immunofluorescence staining in CRL-2097/TR fibroblasts with EML4-ALK expression (EML4-ALK-Dox+ in Fig. 1b) and with the control vector (Vector in Fig. 1b) when approaching senescent proliferation arrest (at PDL 6 and PDL 12, respectively) (Fig. 1e-f). The early induction of cellular senescence in the EML4-ALK-expressing cells was associated with significantly more accumulation of g-H2AX foci (Fig. 1e-f), suggesting that EML4-ALK leads to an accelerated rate of persistent DNA damage. The level of p53 protein phosphorylated at serine 15, another indicator of DNA damage [27, 28], was increased in both control and EML4-ALK-expressing cells upon senescence (Supplementary Fig. S2A).
A quantitative PCR-based measurement of telomere length showed that the vector control cells underwent progressive telomere shortening through their replicative senescence at PDL 12 (Supplementary Fig. S2B). The EML4-ALK-expressing cells had the telomere length shorter than that of the original cells before lentiviral transduction, similar to that of the control cells at a comparable PDL (PDL 7) and longer than that of the replicatively senescent control cells at PDL 12 (Supplementary Fig. S2B). These data suggest that normal, mortal human fibroblasts with and without EML4-ALK expression undergo telomere shortening at similar rates per PDL, and that the accelerated accumulation of DNA damage in the EML4-ALK-expressing cells may be of non-telomeric origin .
The EML4-ALK-induced senescence depends on its ALK kinase activity
The EML4-ALK-expressing CRL-2097/TR with Dox addition were treated with an ALK tyrosine kinase inhibitor (TKI) Crizotinib at 25 nM (a concentration close to the reported IC50 value ) and were monitored for cell proliferation. The treatment with Crizotinib inhibited the autophosphorylation of EML4-ALK while not affecting the expression level of EML4-ALK, confirming its TKI activity against the ALK kinase activity (Fig. 2a). The expression of EML4-ALK without Crizotinib reproducibly induced the proliferation arrest at PDL 6 (Fig. 2b). In contrast, the Crizotinib-treated cells bypassed this early proliferation arrest and underwent approximately 4 more PDL, similarly to the control cells without EML4-ALK expression (Fig. 2b).
Another normal human fibroblast strain MRC-5 (without a Tet repressor) was transduced with the lentiviral vector encoding wild-type EML4-ALK or a kinase-dead mutant of EML4-ALK (K589M) , along with the control vector. The K589M mutant of EML4-ALK was constitutively expressed at a higher level than the wild-type counterpart but was not autophosphorylated (Fig. 2c). Since the MRC-5 fibroblasts used in this experiment were closer to natural replicative senescence than the CRL-2097 fibroblasts used above, the control vector-transduced cells underwent at most 5 PDL before they ceased to proliferate (Fig. 2d). The expression of wild-type EML4-ALK, again in these fibroblasts approaching natural replicative senescence, caused earlier induction of proliferation arrest (Fig. 2d) with SA-b-gal (Fig. 2e), which was similar to oncogenic Ras-induced senescence (Supplementary Fig. S1C-D). Importantly, the K589M mutant-expressing cells did not show early proliferation arrest and behaved like the control vector-transduced cells (Fig. 2d-e), further supporting that the ALK kinase activity mediates the early induction of cellular senescence by EML4-ALK.
EML4-ALK accelerates cell proliferation and promotes anchorage-independent growth in hTERT-immortalized normal human fibroblasts
Although EML4-ALK-positive NSCLC rarely have other accompanying genetic alterations , they still have a mechanism to maintain telomere length and function, in most cases via hTERT activation. Consistent with the previous findings , ALK fusion-positive NSCLC tumor tissues, as well as negative ones, were confirmed to express hTERT (Supplementary Fig. S3A and B). We thus hypothesized that EML4-ALK might cooperate with the expression of hTERT, leading to telomerase activation and cell immortalization, to cause cellular transformation in normal human cells. To test this hypothesis, hTERT was retrovirally transduced into CRL-2097 to establish an hTERT-immortalized normal human cell line (hTERT-CRL-2097), which maintained elongated telomeres (Supplementary Fig. S4A). This cell line had normal karyotype (Supplementary Fig. S4B), maintained normal p16INK4A response to oncogenic Ras (Supplementary Fig. S4C) and retained wild-type TP53 (below in Fig. 3e), thus not coincident with the changes frequently associated with human cell immortalization. These hTERT-CRL-2097 cells were transduced with the wild-type EML4-ALK vector or the control vector, and the constitutive expression and autophosphorylation of the EML4-ALK protein was confirmed (Fig. 3a). Unlike in mortal CRL-2097, the expression of EML4-ALK in this immortalized cell line did not induce senescent proliferation arrest, but instead resulted in accelerated cell proliferation (Fig. 3b). The hTERT-CRL-2097 cells, with or without EML4-ALK expression, accumulated no or little DNA damage (g-H2AX foci in Fig. 3c and Supplementary Fig. S5), consistent with no induction of cellular senescence and in contrast to DNA damage accumulation in mortal CRL-2097 fibroblasts (the leftmost bar in Fig. 3c; and Fig. 1e above). Furthermore, the anchorage-independent formation of cell colonies in soft-agar medium was enhanced in the EML4-ALK-expressing hTERT-CRL-2097 cells, which was inhibited by treatment with the ALK TKI Crizotinib (Fig. 3d).
In another hTERT-immortalized normal human fibroblast cell line, hTERT-BJ (Supplementary Fig. S6A), the expression of wild-type EML4-ALK, but not of the K589M mutant, accelerated cell proliferation (Supplementary Fig. S6B) and promoted anchorage-independent growth in soft agar (Supplementary Fig. S6C). The ability of wild-type EML4-ALK to promote anchorage-independent growth was again abrogated by treatment with the ALK TKI Crizotinib (Supplementary Fig. S6C). From our reproducible results in two lines of hTERT-immortalized normal human fibroblasts, we conclude that EML4-ALK has in vitro transformation activity in these cells through its constitutive ALK kinase activity.
The EML4-ALK-induced anchorage-independent growth occurs without chromosome aberrations, without loss or mutation of the TP53 gene, and with normal p16INK4A response
We performed karyotype analysis in hTERT-CRL-2097 cells transduced with the control vector, those expressing EML4-ALK and six of their derived clones isolated from soft-agar culture. All of these cells examined, as well as the original hTERT-CRL-2097 cells (as mentioned above and Supplementary Fig. S4B), maintained normal male karyotype 46,XY (Fig. 3e and Supplementary Fig. S7A-B). All cell lines listed in Fig. 3e had normal male karyotype 46, XY by G-banding (at least 10 well-spread metaphases per line were examined; an example is shown in Supplementary Fig. S7A). hTERT-CRL-2097, hTERT-CRL-2097+EML4-ALK and soft-agar clones #1 and #2 were also confirmed by SKY to have 46, XY (again, at least 10 metaphases per line were examined; an example is shown in Supplementary Fig. S7B). By direct sequencing of the RT-PCR products, the entire coding region of TP53 was shown to be wild-type without a clonal homozygous or heterozygous mutation. The polymorphic codon 72 of TP53 was heterozygous (Pro/Arg) in the original hTERT-CRL-2097 and remained heterozygous (Pro/Arg) in hTERT-CRL-2097 expressing EML4-ALK and their derived soft-agar clones (examples are shown in Supplementary Fig. S7C), indicating that no chromosome instability nor loss of heterozygosity (LOH) occurred at TP53 during EML4-ALK-induced acquisition of anchorage-independent growth.
Although loss or mutation of the TP53 gene is frequently associated with cell transformation and carcinogenesis , all of the EML4-ALK-expressing cells and their derived soft-agar clones had the wild-type TP53 sequence without any homozygous or heterozygous mutation (Fig. 3e). The original hTERT-CRL-2097 cells showed Pro/Arg heterozygosity at the polymorphic codon 72, which was maintained in the EML4-ALK-expressing cells and all the soft-agar clones (Fig. 3e and Supplementary Fig. S7C), indicating that there was no loss of a TP53 allele in these cells. The p16INK4A pathway, which is frequently impaired during cell transformation and carcinogenesis [26, 33], was suggested to remain intact during the EML4-ALK-mediated cell transformation by the finding that oncogenic Ras-induced p16INK4A upregulation  was observed in the EML4-ALK-expressing cells (Supplementary Fig. S7D).
STAT3 is activated by EML4-ALK in both mortal and immortalized normal human fibroblasts
We examined the activation status of three major factors downstream of EML4-ALK (i.e., STAT3, Akt and Erk1/2) by western blot (Fig. 4a and 4b). Mortal normal human fibroblasts CRL-2097/TR with and without EML4-ALK expression both showed increased levels of phosphorylated Akt when they became senescent, while the levels of phosphorylated Erk1/2 did not show a consistent change associated with EML4-ALK expression or increased PDL levels (Fig. 4a). Notably, a striking induction of STAT3 phosphorylation was observed in the EML4-ALK-expressing cells at PDL 4, which was decreased upon senescent proliferation arrest (PDL 6) but still at a higher level than that in the control cells at senescence (PDL 11) (Fig. 4a). Also in hTERT-immortalized CRL-2097 fibroblasts, the expression of EML4-ALK resulted in remarkable induction of phosphorylated STAT3, while no increase in phosphorylated Akt and a slight increase in phosphorylated Erk1/2 were associated with EML4-ALK expression (Fig. 4b). These findings suggest that STAT3 functions as a major downstream effector of EML4-ALK in both mortal and hTERT-immortalized normal human fibroblasts, consistent with its involvement in both cellular senescence and transformation [34-37]. MiR-21, a microRNA induced by STAT3 , was also upregulated by EML4-ALK in both mortal and hTERT-immortalized fibroblasts (Supplementary Figure S8). Although a decrease in phosphorylated Src was associated with cellular senescence in the presence or absence of EML4-ALK (Fig. 4a), the expression of EML4-ALK did not affect the phosphorylation level of Src (Fig. 4a and 4b), which is known to mediate acquired resistance to ALK TKIs .
EML4-ALK regulates different signaling pathways in mortal and immortalized normal human fibroblasts
We performed RNA sequencing (RNA-seq) in duplicated samples of mortal CRL-2097/TR with and without EML4-ALK expression (Supplementary Fig. S9A; Supplementary Table S1) and hTERT-immortalized CRL-2097 with and without EML4-ALK expression (Supplementary Fig. S9B; Supplementary Table S2). The analysis of the differentially expressed genes to KEGG (Kyoto Encyclopedia of Genes and Genomes) Pathways (https://www.genome.jp/kegg/) identified the cytokine-cytokine receptor interaction pathway as significantly upregulated by EML4-ALK expression in mortal CRL-2097/TR fibroblasts, along with some related and overlapping pathways involving tumor necrosis factor (TNF) or viral infection (Table 1 and Supplementary Table S3). The interferon (IFN)-a/b and IFN-g signaling pathways, which have some genes in common with the above KEGG pathways, were also identified from Reactome Pathway Database (https://reactome.org/) as EML4-ALK-upregulated pathways in mortal CRL-2097/TR fibroblasts (Table 1 and Supplementary Table S3). Consistent with the EML4-ALK-induced phosphorylation of STAT3 (as above in Fig. 4), these cytokines and IFN signaling pathways include several STAT3-upregulated genes such as IL1B, CXCL8, SOCS3, IRF1 and IRF7 (Table 1 and Supplementary Table S3). These data suggest that STAT3 mediates the effect of EML4-ALK on activating the proinflammatory cytokine and IFN signaling cascades, which coordinately induce and maintain sustained DNA damage and senescent proliferative arrest in mortal normal human cells [37, 40].
In hTERT-immortalized CRL-2097 fibroblasts, instead of the above-mentioned cytokine and IFN signaling pathways, the complement and blood coagulation cascades signaling was identified as significantly upregulated by EML4-ALK expression (Table 1 and Supplementary Table S3). This pathway included A2M, PLAT and PLAU as STAT3-upregulated genes [41, 42], suggesting that STAT3 also mediates the modulation by EML4-ALK of blood coagulation, which may have clinical implications in increased risk of disseminated intravenous coagulation in patients with EML4-ALK-positive cancer . Consistently, a Japanese cohort also showed an upregulation of the blood coagulation pathway in EML4-ALK-positive lung cancer  (Supplementary Fig. S10). We also found that some integrin and non-integrin components of focal adhesion and extracellular matrix (ECM) interactions were downregulated by EML4-ALK in hTERT-immortalized CRL-2097 fibroblasts (Table 1 and Supplementary Table S3), which likely contributed to EML4-ALK-induced anchorage-independent growth via overcoming anoikis [44, 45].
EML4-ALK also has in vitro transforming activity in hTERT-immortalized normal human bronchial epithelial cells but does not cause in vivo tumorigenicity
The transforming activity of EML4-ALK was also tested in hTERT-immortalized, normal human bronchial epithelial cells, which represent a cell type more relevant to NSCLC pathogenesis. For this purpose, we used a previously established cell line, HBET1, which has a tetraploid karyotype with no or few structurally abnormal chromosomes (Supplementary Fig. S11), maintains elongated telomeres and does not show anchorage-independent growth or in vivo tumorigenicity . The HBET1 cells constitutively expressing EML4-ALK with its autophosphorylation (Fig. 5a) showed accelerated cell proliferation (Fig. 5b) and acquired anchorage-independent growth in soft agar (Fig. 5c), as observed above in hTERT-immortalized fibroblasts. The increased levels of phosphorylation of STAT3 and Akt, but not of Erk1/2 or Src, were associated with EML4-ALK expression in HBET1 cells (Fig. 5a).
To examine in vivo tumorigenicity, we injected hTERT-immortalized HBET1 and CRL-2097 cells with and without EML4-ALK, along with the soft-agar clones derived from the EML4-ALK-expressing cells, subcutaneously into immunodeficient NOD.SCID/Ncr mice (Fig. 5d). None of these cells were able to form a growing tumor, while EML4-ALK-expressing mouse NIH/3T3 cells as a positive control  consistently formed progressively growing tumors (Fig. 5d). These results suggest that the expression of EML4-ALK alone is not sufficient for hTERT-immortalized normal human cells to acquire in vivo tumorigenicity.