TSPYL5 is upregulated in γ-radiation-exposed or gefitinib-treated NSCLC cells that show CSC-like properties.
The relevance of TSPYL5 expression to radiation-resistance of CSC-like NSCLC cells has been examined with NSCLC cells (A549 and H460) treated with fractionated γ-radiation33 [2 (or 1) Gy × 3 with a 3-day interval, a total of 6 (or 3) Gy]. Exposure of NSCLC cells to γ-radiation increased the cellular levels of ALDH1 activity and CD44 expression (Fig. 1a, Supplementary Fig. 1a), which are representative biomarkers that impart chemo- or radio-resistant CSC properties to cancer cells34-36. These radiation-exposed cells exhibited a high sphere-formation ability, which represents the self-renewal potential (Fig. 1b). Radiation also activated the EMT program as shown by invasion and migration assay (Fig. 1c). CSC-like properties of these cells were further demonstrated by the increased expression of CSC biomarkers, including ALDH1 (ALDH1A1 and ALDH1A3 isoforms), CD44, SOX-2 and OCT-4 (Fig. 1d, Supplementary Fig. 1b). EMT markers, such as N-cadherin, Snail, and Vimentin, were also increased, thus providing a distinct advantage for metastatic dissemination (Fig. 1d). Cells that survived even after the higher dose of fractionated γ-irradiation (2 Gy × 10 with a 3-day interval, a total dose of 20 Gy) also showed the increase of CSC and EMT properties as demonstrated by specific markers (Supplementary Fig. 1c). Interestingly, in all of these radiation-exposed CSC-like cells with low or high dose, TSPYL5 expression increased in proportion to the radiation dose. Gefitinib-resistant cells, which were selected by a stepwise escalation of EGFR TKI gefitinib treatment, also showed CSC-like properties: ALDH1 activity and CD44 expression were increased (Fig. 1e), and the sphere-formation and metastatic capacities were enhanced (Fig. 1f, g). TSPYL5 also upregulated in these cells along with the ALDH1 and CD44 elevation (Fig. 1h).
To further analyze the relevance of TSPYL5 in lung CSCs, CSC-like subpopulations were sorted from A549 NSCLC cells via Aldefluor staining (Fig. 2a). The subpopulation of CSC-like cells with high ALDH1 expression (ALhigh) showed typical properties of self-renewal potential and invasion/migration capacity (Fig. 2b). Cancer stemness markers (ALDH1, CD44, OCT-4, and SOX-2) highly increased when comparing to the cell subpopulation with low ALDH1 expression (ALlow) (Fig. 2b, c). The ALDH1high subpopulation sorted from H460 cells also showed typical CSC-like characteristics and the expression of cellular markers (Fig. 2d–f). Similar to the radiation-exposed and gefitinib-resistant cells, both ALDH1high CSC-like subpopulations (A549 and H460) showed higher levels of TSPYL5 compared with corresponding ALDH1low cells (Fig. 2c, f). CD44high cells sorted from A549 NSCLC cells also showed stem-like and metastatic properties with high levels of ALDH1 and TSPYL5 compared with CD44low cells (Fig. 2g–i).
Collectively, radiation-exposed and gefitinib-resistant NSCLC cells showed cellular characteristics similar to CSC-like ALDH1high or CD44high cells, which of all expressed high levels of TSPYL5. These results suggest an association among ALDH1, CD44, and TSPYL5 expression in response to radiation, gefitinib, or cancer stemness.
TSPYL5 enhances CSC-like properties in NSCLC cells by promoting ALDH1 and CD44 and inhibiting PTEN expression.
To evaluate whether TSPYL5 substantially enhances CSC-like properties in radiation-exposed, gefitinib-treated, or in ALDH1high cells, we examined alterations in self-renewal and EMT potential in response to TSPYL5 overexpression or knockdown. In A549 cells, which have high endogenous TSPYL5 levels (Supplementary Fig. 1d), TSPYL5 knockdown by siRNA markedly reduced sphere formation and decreased cellular invasiveness and motility (Fig. 3a). The spindle-shaped cell morphology of A549 cells also had returned to a cobblestone-like shape, which is characteristic of differentiated cells. With these cellular changes in self-renewal and metastatic capacities, the levels of CSC-associated factors (OCT-4 and SOX-2) and EMT markers (N-cadherin and Vimentin) were decreased (Fig. 3b). In contrast, TSPYL5 overexpression in H460 cells, which have low endogenous TSPYL5 levels (Supplementary Fig. 1d), enhanced the sphere-formation and metastatic capacities and induced morphological changes from a cobblestone-like shape to a spindle shape, which is a typical CSC or EMT characteristic (Fig. 3c). The elevation of EMT and CSC- associated factor levels were also confirmed by Western blotting (Fig. 3d).
We explored further the association of TSPYL5 with the expression of ALDH1 and CD44. Knockdown of TSYPL5 expression in A549 cells resulted in downregulation of ALDH1 and CD44 (Fig. 3e), whereas TSPYL5 overexpression increased the expression of ALDH1 and CD44 in H460 cells (Fig. 3f). TSPYL5 altered ALDH1 and CD44 at the transcriptional levels, which subsequently influenced the corresponding protein expression levels (Fig. 3e, f). ALDH1 also can modulate the EMT-associated, CSC-like properties of the NSCLC cells: upregulation or knockdown of ALDH1A1 or ALDH1A3 influenced sphere-formation and invasion/ migration capacities as well as altered the expression of corresponding CSC and EMT markers (Supplementary Fig. 2a–d). However, differently from TSPYL5, the upregulation or knockdown of ALDH1A1 or ALDH1A3 did not alter the CD44 levels. TSPYL5 level was not changed also (Supplementary Fig. 2b, d), despite the changes in CSC-like properties (Supplementary Fig. 2a, c). These results implicate that all of them, ALDH1, CD44 as well as TSPYL5, can regulate cancer stemness; however, ALDH1 and CD44 are downstream regulatory targets of TSPYL5.
Also, we found that TSPYL5 negatively regulates PTEN expression (Fig. 3e, f), which antagonizes PI3K function by catalyzing PIP3 dephosphorylation, leading to AKT inactivation37,38. PTEN transcript and protein levels were downregulated by TSLYL5 overexpression (Fig. 3e, f), as partially shown in our previous study27.
Loss of PTEN stabilizes TSPYL5 via AKT-dependent phosphorylation, which reinforces CSC properties and therapeutic resistance in NSCLC cells.
PTEN/PI3K/AKT pathway is involved in the acquisition of resistance to targeted drugs28-30, and is crucial for CSC maintenance31,32. Decreased PTEN expression and consequent AKT activation was consistently observed in radiation-exposed, gefitinib-resistant, and CSC-like ALDH1high NSCLC cells (Fig. 4a). Therefore, we evaluated the CSC-like properties and related gene expression levels in response to PTEN overexpression or AKT knockdown to confirm whether AKT activation is essential for these CSC-like properties in NSCLC cells. Sphere-formation assay showed that AKT knockdown inhibited the self-renewal potential of the cells (Fig. 4b upper panels). Furthermore, AKT knockdown resulted in increased PTEN expression, whereas ALDH1 and CD44 expression was decreased (Fig. 4b lower panels). In response to PTEN overexpression, AKT was in an inactive dephosphorylated state. Also, ALDH1 and CD44 expression was suppressed, which was also confirmed by the decrease of self-renewal potential (Fig. 4c). In these AKT-inactivated cells, TSPYL5 protein levels also decreased (Fig. 4b, c). However, TSPYL5 transcript levels were not altered in response to AKT knockdown or PTEN overexpression. In contrast, AKT overexpression or PTEN knockdown enhanced the sphere-formation ability of H460 cells (Fig. 4d, e). In these cells, AKT was activated by phosphorylation and the expression levels of ALDH1 and CD44 were increased. Also, TSPYL5 protein levels were upregulated; however, its transcript levels were not altered again. Reduced protein level of TSPYL5, but not its transcript level, were also confirmed in ALDH1low A549 cells compared with the CSC-like ALDH1high cells (Fig. 2c, Supplementary Fig. 3). These results imply that in CSC-like NSCLC cells, AKT activation by the loss of PTEN enhances TSPYL5 expression at the protein level, and it depends on AKT-dependent phosphorylation, whereas TSPYL5 regulates PTEN expression at the transcription level.
The regulation of TSPYL5 expression by AKT-dependent phosphorylation was further examined using an AKT inhibitor assay, in which AKT phosphorylation was completely suppressed by treatment of MK2206. As expected, MK2206 treatment resulted in decreased TSPYL5 protein levels with unaltered TSPYL5 transcript levels (Fig. 4f). It showed that AKT-dependent phosphorylation stabilizes TSPYL5 protein, possibly by inhibiting its degradation. Interestingly, elevated PTEN expression was detected soon after the TSPYL5 protein levels were decreased in response to AKT inhibition. The self-renewal capacity of the A549 cells was also reduced by AKT inhibition (Supplementary Fig. 4), which may be associated with the decrease of TSPYL5.
Overall, aberrantly activated AKT in therapy-resistant or CSC-like cells has been shown to maintain the expression of TSPYL5 protein through AKT-dependent phosphorylation. From the aspect of TSPYL5, it maintains AKT activation and itself via an AKT/TSPYL5/PTEN regulatory loop in a positive-feedback manner. Without AKT-dependent stabilization, TSPYL5 quickly disappeared despite sufficient expression of its transcript. The decrease of TSPYL5 by inhibition of AKT consequently induced PTEN expression and again suppressed AKT activation. TSPYL5 acts as a CSC-associated factor by upregulating ALDH1 and CD44 expression, as well as by the AKT/TSPYL5/PTEN cyclic signaling loop.
AKT-mediated phosphorylation of TSPYL5 at threonine-120 is essential for its stabilization and nuclear translocation.
Aberrant AKT activation caused by oncogenic mutations or PTEN loss is associated with poor prognosis in cancer39,40. Also, the activated AKT contributes to various cellular processes either directly or indirectly via post-translational modifications of substrate proteins, including phosphorylation, ubiquitination, or SUMOylation41-45, which consequently determine the fate of the target proteins. The intracellular localization of TSPYL5 in response to AKT inhibition was examined by fluorescence microscopy in NSCLC cells to investigate the function of phosphorylated TSPYL5 as a CSC-associated factor. In A549 cells, TSPYL5 was highly expressed and localized in the nucleus as well as in the cytoplasm (Fig. 5a, upper panels). However, treatment with either MK2206 (AKT inhibitor) or LY294002 (PI3K inhibitor) significantly diminished the nuclear localization of TSPYL5 (Fig. 5a, low panels). Of course, AKT and PI3K inhibition decreased the total TSPYL5 levels, which was accompanied by CD44 and ALDH1 downregulation (Fig. 5b). Consistently, the nuclear accumulation of TSPYL5 was diminished in response to AKT or PI3K inhibition in H460 cells with TSPYL5 overexpression (Fig. 5c), thus resulting in its predominant cytoplasmic localization. TSPYL5 has been reported as a nucleosome protein25; however, the mechanism of its nuclear localization has not yet been elucidated. Our results suggest that AKT-mediated phosphorylation of TSPYL5 promotes not only its stabilization but also nuclear translocation. In addition, this leads us to speculate that nuclear TSPYL5 functions as a transcriptional regulator of specific genes that are important for characterizing EMT-associated, CSC-like properties, such as ALDH1 and CD44.
To further confirm the phosphorylation-dependent function of TSPYL5, the amino acid residue that is phosphorylated by AKT was examined. Until now, there have been no studies on the role of TSPYL5 post-translational modifications. However, proteomic studies have revealed several post-translational modification sites of TSPYL5, namely, four phosphorylation sites (T12046, S180, S201, S22746,47). When presuming phosphorylation sites using phosphorylation-site prediction software NetPhos 2.0 (http://www.cbs.dtu.dk/services/NetPhos), four threonine residues (T120, T177, T326, and T409) were expected to be potential phosphorylation sites of TSPYL5 with a 50–90% probability (Supplementary Table 1). Also, we compared the AKT substrate phosphorylation motif48RXRXXS/T with the whole TSPYL5 amino acid sequence, and found two potential protein sequence segments similar to the AKT substrate motif around S11 (RSRGRKSS11RAKNRGK) and T120 (SERLAADT120VFVGTAG). Of these backgrounds, we considered the T120 residue of TSPYL5 as a potential AKT phosphorylation site and performed the mutant studies.
When non-mutagenic, wild-type TSPYL5 (WT-TSPYL5) was forcibly expressed in H460 cells, both nuclear and cytoplasmic TSPYL5 accumulation was observed, and the cellular levels of CD44 and ALDH1 were significantly upregulated (Fig. 5d, e). In contrast, the TSPYL5 alanine mutant (T120A-TSPYL5), of which potential phosphorylation site T120 is substituted with alanine, showed cytoplasmic accumulation only, and CD44 and ALDH1 levels were not increased. Of course, total T120A-TSPYL5 expression was lower than that of WT-TSPYL5 (Fig. 5e lower panel). The TSPYL5 aspartic acid mutant (T120D-TSPYL5), which is a mimic of phospho-T120-TSPYL5, acted similar to wild-type TSPYL5: T120D-TSPYL5 showed both nuclear and cytoplasmic localization, and CD44 and ALDH1 were elevated with its expression. The function and intracellular distributions of other threonine residue TSPYL5 mutants (T177A, T326A, and T409A) were not different from those of WT-TSPYL5 (Supplementary Fig. 5a, b; Fig. 5d, e). T120 mutation of TSPYL5 also showed suitable self-renewal and EMT potential in H460 cells (Supplementary Fig. 6). Altogether these results indicate that TSPYL5 phosphorylation at T120 is essential for TSPYL5 stabilization and nuclear translocation as well as the subsequent expression of CD44 and ALDH1 in CSC-like NSCLC cells.
To further confirm the phosphorylation of TSPYL5 at T120, TSPYL5 was detected using anti-pT120-TSPYL5 antibody that was generated with a phospho-peptide containing pT120 of TSPYL5 (see Methods). Anti-TSPYL5 antibody detected WT-TSPYL5, T120A-TSPYL5, or T120D-TSPYL in the cell lysates of transfected H460 cells (Fig. 5f). However, the anti-pT120-TSPYL5 antibody detected WT-TSPYL5 and T120D-TSPYL5, but not T120A-TSPYL5. Additionally, after immunoprecipitation with antibodies against phosphorylated threonine or serine (p-T/S), only WT- and T120D-TSPYL5, but not T120A-TSPYL5, were detected with anti-pT120-TSPYL5 antibody (Fig. 5f).
Immunoprecipitation assay with anti-AKT antibody showed that T120 of TSPYL5 is a target of AKT. Endogenous TSPYL5 and AKT were co-immunoprecipitated with specific antibodies of each other in A549 cells (Fig. 5g). Exogenously overexpressed TSPYL5 was also co-immunoprecipitated with AKT in H460 cells (Fig. 5h). However, mutant T120A-TSPYL5 did not precipitate AKT.
AKT-mediated phosphorylation of TSPYL5 at threonine-120 blocks its ubiquitination and induces its SUMOylation.
TSPYL5 stability was analyzed after treatment with MK2206 and proteasome inhibitor MG132 to examine whether T120 phosphorylation stabilizes TSPYL5 by interfering with its degradation process (Fig. 6a). Upon its overexpression in H460 cells, elevated WT-TSPYL5 levels were detected along with activated AKT (p-AKT). WT-TSPYL5 was reduced by MK2206 treatment; however, MG132 blocked its reduction. T120A-TSPYL5, an unstable TSPYL5 mutant, was not observed even when forcibly expressed, whereas detected after MG132 treatment. T120D-TSPYL5, a mimic of phospho-T120-TSPYL5, showed more elevated expression than WT-TSPYL5, and, even when MK2206 was treated, the remaining TSPYL5 was detected. These results suggest that AKT-dependent phosphorylation of TSPYL5 at T120 stabilizes it through inhibition of proteasomal degradation, which may be mediated by inhibition of ubiquitination. It was further confirmed by an in vivo ubiquitination assay (Fig. 6b). MK2206 treatment enhanced the ubiquitination of WT-TSPYL5 and its consequent degradation. T120D-TSPYL5 and T409A-TSPYL5 also showed similar ubiquitination and degradation patterns to those of WT-TSPYL5. However, T120A-TSPYL5 mutant, which cannot be a substrate for Akt-dependent phosphorylation, displayed elevated ubiquitination and degradation, even without AKT inhibition.
SUMOylation often functions as a regulatory mechanism for the transport of cellular proteins between the cytoplasm and nucleus49. We examined whether the SUMOylation of TSPYL5 is associated with its nuclear translocation and it depends on T120 phosphorylation. When A549 cells were treated with ginkgolic acid, a SUMOylation inhibitor that blocks the formation of the E1-SUMO intermediate50, nuclear TSPYL5 significantly disappeared (Fig. 6c). Moreover, an in vivo SUMOylation assay revealed that WT-TSPYL5 was significantly conjugated with SUMO1, whereas the T120A-TSPYL5 mutant was not (Fig. 6d). These results suggest that SUMOylation of TSPYL5 is essential for its nuclear entry and that phosphorylation of TSPYL5 at T120 is required for its SUMOylation. TSPYL5 mutants with an alanine substitution at T177, T326, or T409, which were all translocated into the nucleus the same as WT-TSPYL5 (Supplementary Fig. 5a, Fig. 5d), showed similar SUMO1 conjugation to that of WT-TSPYL5 (Supplementary Fig. 7). Collectively, we concluded that stabilized TSPYL5 via T120 phosphorylation receives a SUMOylation modification for its nuclear entry, thus promoting its function as a CSC-associated factor.
Phosphorylated TSPYL5 at threonine-120 functions as a transcriptional regulator for ALDH1, CD44, and PTEN, which enhances cancer stem-like properties.
CSC-like properties of NSCLC cells depending on the expression and phosphorylation of TSPYL5 were confirmed again by TSPYL5 overexpression in ALDH1low or CD44low A549 cells. When ALDH1low A549 cells were transfected with WT-TSPYL5 or T120D-TSPYL5, the cells exhibited a restored sphere-formation capacity of up to 70–80% of that of the ALDH1high cells. However, overexpression of the T120A-TSPYL5 mutant did not enhance the sphere-formation capacity of the ALDH1low cells (Fig. 7a). Also, when WT-TSPYL5 was overexpressed in CD44low cells, the EMT-associated, CSC-like properties were enhanced (Fig. 7b). These results indicate that TSPYL5 plays an important role in maintaining CSC-like properties by regulating ALDH1 and CD44 in NSCLC cells.
ALDH1 and CD44 were also upregulated in H460 cells with overexpression of either WT-TSPYL5 or T120D-TSPYL5, as shown by FACS analysis using Aldefluor or CD44 staining (Fig. 7c, d upper panels). The elevated expression levels of ALDH1 and CD44 in these cells were further confirmed by RT-PCR and Western blot analysis (Fig. 7c, d lower panels). Overexpression of WT-TSPYL5 or the T120D-TSPYL5 mutant also inhibited the cellular expression of PTEN at both the transcriptional and protein levels (Fig. 7d lower panels). In contrast, the T120A-TSPYL5 mutant did not affect the expression of ALDH1, CD44, or PTEN (Fig. 7c, d lower panels).
As shown above (Fig 5, 6), TSPYL5 is stabilized via phosphorylation by activated AKT, which blocks its ubiquitination and promotes its nuclear translocation. Moreover, phosphorylated TSPYL5 affects the expression of ALDH1, CD44, and PTEN at the transcriptional level. Therefore, we investigated whether TSPYL5 phosphorylation at threonine-120 is required for TSPYL5 to function as a transcriptional regulator of ALDH1, CD44, and PTEN by performing chromatin immunoprecipitation (ChIP) analyses (Fig. 7e, Supplementary Fig. 8). In A549 cells, the specific promoter regions of ALDH1, CD44, and PTEN were amplified from the immunoprecipitants that were collected using the anti-TSPYL5 antibody. Luciferase reporter assays also confirmed that WT-TSPYL5 and T120D-TSPYL5 function as positive transcriptional activators of CD44 and ALDH1 and as negative transcriptional regulators of PTEN (Fig. 7f). However, the T120A-TSPYL5 mutant did not affect the transcription of these genes, implicating that TSPYL5 phosphorylation at threonine 120 is necessary for its function as a transcriptional regulator.
TSPYL5-antagonistic peptides decrease TPSYL5 expression and consequently inhibit EMT-associated tumorigenicity in CSC-like NSCLC cells.
To further validate that TSPYL5 phosphorylation at threonine 120 plays a key role in the EMT-associated, CSC-like properties of NSCLC cells, we designed peptide inhibitors based on the TSPYL5 amino acid sequence to prevent threonine 120 phosphorylation by competitively binding to AKT (TS120 peptides: Fig. 8a). These peptides were PEGylated at their amino-termini to confer cell permeability and stability51, and their intracellular delivery was confirmed by fluorescence imaging (Fig. 8b). Treatment of NSCLC A549 cells with the PEGylated TSPYL5-derived peptides, particularly TS120-T and TS120-D, resulted in significantly reduced self-renewal and metastatic capacities, as shown by sphere-forming assay and invasion/migration assays (Fig. 8c). In contrast, TS120-A did not affect these cellular properties. Consistent with these results, TS120-T and TS120-D reduced the expression levels of ALDH1 and CD44, whereas TS120-A did not (Fig. 8d). Also, as expected, TS120-T and TS120-D peptides reduced TSPYL5 levels, which may be due to the competitive binding to AKT, thus resulting in decreased phosphorylation and stabilization of TSPYL5. Contrary to the effects observed in A549 cells, TS120-T peptides did not affect the self-renewal and metastatic capacities of H460 cells with a low level of endogenous TSPYL5 (Supplementary Fig. 9, Supplementary Fig. 1). These results suggest that TSPYL5 phosphorylation at threonine 120 is essential for EMT-associated CSC-like cell properties, and that PEGylated TS120-T or -D peptides can be used as inhibitory molecules of CSC-like cells with high TSPYL5 expression. The antagonistic effects of the TSPYL5 sequence-derived peptides were also investigated in vivo. Mice that were inoculated with ALDH1high A549 cells showed a gradual increase in tumor burden, which reached a volume of approximately 1,000 mm3 at day 52 after inoculation (Fig. 8e). However, when the TS120-T peptides were intratumorally injected every 3 days after the tumor volume reached approximately 13.4 mm3, the tumor growth dramatically regressed. On the contrary, TS120-A peptides did not significantly affect tumor growth. Additionally, in a metastatic lung cancer model that was established by intravenous injection of ALDH1high A549 cells into nude mice, TS120-T peptides markedly inhibited metastatic dissemination of the cancer cells to the lungs (Fig. 8f); however, TS120-A peptides did not. Interestingly, despite the inhibitory effects of the TS120-D peptides in cellular assays (Fig. 8c, d), significant inhibitory effects were not observed for in vivo models (Fig. 8e, f), which may be due to certain effects on peptides that only occur in vivo, such as proteolytic attack. Immunohistochemical analysis of tumor tissues from the in vivo models confirmed that TS120-T peptide treatment reduced the cellular levels of TSPYL5, thereby resulting in decreased CD44 and increased PTEN levels (Supplementary Fig. 10). As expected, treatment with TS120-A or TS120-D peptides did not alter the levels of TSPYL5, CD44, or PTEN in tumor cells from the in vivo models.
TSPYL5-antagonistic peptides sensitize NSCLCs to γ-radiation and EGFR tyrosine kinase inhibitors.
Finally, we investigated whether blocking the phosphorylation of TSPYL5 at threonine-120 could overcome the acquired resistance of NSCLC cells to γ-radiation or EGFR TKIs. Treatment of TSPYL5-expressing A549 cells with TS120-T or TS120-D peptides significantly increased the sensitivity of the cells to γ-radiation as shown by colony-forming assay, whereas TS120-A peptides did not (Fig. 9a). TS120-T peptide also inhibited the self-renewal and metastatic properties of the radiation-resistant A549 cells (Fig. 9b). We also examined whether targeting TSPYL5 phosphorylation could reverse the drug resistance of the NSCLC cells. Targeting TSPYL5 with TS120-T peptide improved the sensitivity of TSPYL5-expressing A549 cells to EGFR TKI gefitinib (Fig. 9c). Similarly, TS120-T peptide treatment inhibited the CSC-like properties and tumorigenicity of the gefitinib-resistant H460 cells (Fig. 9d). Treatment with the TS120-T peptides in other lung cancer cells with high levels of TSPYL5, such as H358 and H2009 (Supplementary Fig. 1), also inhibited cell growth and enhanced the sensitivity of the cells to γ-radiation and gefitinib (Supplementary Fig. 11d).
Collectively, these results suggest that phosphorylation of TSPYL5 at threonine-120 is essential for eliciting cancer stemness and inherent or acquired resistance of NSCLC cells to radiation or targeted drugs, and that TS120-T peptides may be used to sensitize CSC-like NSCLC cells to these therapies.