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. Author manuscript; available in PMC: 2021 Dec 1.
Published in final edited form as: Lung Cancer. 2020 Sep 29;150:26–35. doi: 10.1016/j.lungcan.2020.09.023

Inhibition of ACK1 delays and overcomes acquired resistance of EGFR mutant NSCLC cells to the third generation EGFR inhibitor, osimertinib

Jiajia Gu 1,2,*, Luxi Qian 1,2,*, Guojing Zhang 2, Nupam P Mahajan 3, Taofeek K Owonikoko 2, Suresh S Ramalingam 2, Shi-Yong Sun 2
PMCID: PMC7722235  NIHMSID: NIHMS1636978  PMID: 33049499

Abstract

Objectives:

The emergence of acquired resistance to the third generation EGFR inhibitor, osimertinib (AZD9291 or TAGRISSO™), is an unavoidable huge clinical challenge. The involvement of ACK1, a non-receptor tyrosine kinase with an oncogenic function, in regulating cell response to osimertinib has not been investigated and thus is the focus of this study.

Material and Methods:

Drug effects on cell growth were evaluated by measuring cell numbers and colony formation. Apoptosis was monitored with flow cytometry for annexin V-positive cells and Western blotting for protein cleavage. Intracellular protein and mRNA alterations were detected with Western blotting and qRT-PCR, respectively. Drug effects on delaying osimertinib acquired resistance were determined using colony formation in vitro and xenografts in nude mice in vivo, respectively. Cell senescence was assayed by β-galactosidase staining.

Results:

Inhibition of ACK1 with the novel ACK1 inhibitor, (R)-9b synergized with osimertinib in inhibiting the growth of EGFR mutant NSCLC cell lines. Similar results were also generated with ACK1 gene knockdown. The combination of osimertinib and (R)-9b enhanced induction of apoptosis. In both in vitro and in vivo long-term resistance delay assays, the combination of (R)-9b and osimertinib clearly delayed the emergence of osimertinib-resistance. Further, the (R)-9b and osimertinib combination was also effective in inhibiting the growth of EGFR mutant NSCLC cell lines with acquired resistance to osimertinib, which possess elevated levels of ACK1, and the growth of osimertinib-resistant tumors in vivo. In some resistant cell lines, the combinations induced senescence in addition to induction of apoptosis.

Conclusions:

These novel findings suggest that ACK1 inhibition might be a potential and innovative strategy for delaying and overcoming osimertinb acquired resistance.

Keywords: ACK1, osimertinib, acquired resistance, EGFR, apoptosis, lung cancer

1. Introduction

Lung cancer, which consists of approximately 80% non-small cell lung cancer (NSCLC) and 20% small cell lung cancer, has remained leading cause of all cancer deaths worldwide. The advances in targeted therapies against several driver mutations such as EGFR and ALK and immunotherapy have drastically improved 5-years survival of NSCLC patients (up to 19%) for the past decade in the USA [1]. Treatment of NSCLCs with activating EGFR mutations (e.g., 19del and L858R) using EGFR-tyrosine kinase inhibitors (EGFR-TKIs) represents the first successful targeted therapy against lung cancer and have substantially benefited patients. EGFR-TKIs have progressed to third generation, among which, osimertinib (AZD9291 or TAGRESSO) selectively and irreversibly inhibits EGFR activating and T790M mutants without affecting wild-type EGFR. It is now an approved drug for NSCLC patients with activating EGFR mutations (first-line) or those who have become resistant to the first generation EGFR-TKIs through the T790M mutation (second-line). However, all patients eventually relapse and become resistant to osimertinib, resulting in treatment failure with limited long-term benefit. Therefore, management of acquired resistance is a major clinical challenge.

Activated Cdc42-associated kinase 1 (ACK1), also called tyrosine kinase non-receptor 2 (TNK2), although initially identified as a non-receptor tyrosine kinase (NRTK) that specifically binds to the GTP-bound form of Cdc42, in recent years has emerged to be an oncogenic kinase involved in multiple malignancies [2, 3]. It is aberrantly activated, amplified, or mutated in many types of human cancers including lung cancer [2, 3]; this has led to specific efforts toward targeting ACK1 for cancer therapy, resulting in ACK1 inhibitor (R)-9b, used in this study [4, 5].

ACK1 gene amplification and overexpression was first reported in lung tumors wherein 6–35 fold elevated ACK1 RNA levels were observed in 20 out of 47 aggressive lung tumors [6]. ACK1 copy number gains has also been reported in lung squamous cell carcinomas [7]. Analysis of TCGA database showed that 28.5% of these tumors exhibited ACK1 gene amplification [8]. Significant upregulation of ACK1 expression was also detected in NSCLC tissues compared to their adjacent normal tissues, which was reversely correlated with the survival of NSCLC patients [9]. EGF, like many other ligands, induces activation of the ACK1, suggesting that ACK1 functions downstream EGFR [10–12]. However, ACK1 interacts with and promotes EGFR internalization and lysosomal degradation [13–15]. Interestingly, in renal cancer cell lines resistant to gefetinib, silencing of ACK1 sensitized them to gefitinib [16] although the underlying mechanisms are not clear.

There was no study for demonstrating the impact of ACK1 inhibition on the therapeutic efficacies of EGFR-TKIs, including osimertinib, in EGFR mutant NSCLC cells while we started this project. With the aim of developing effective strategies for delaying and/or overcoming acquired resistance to osimertinib, we identified that (R)-9b and osimertinib combination synergistically decreased the survival of EGFR mutant NSCLC cells including those with acquired resistance to osimertinib. Coincidentally, a newly published report has identified ACK1 activation as a mechanism mediating acquired resistance to the novel 3rd generation EGFR-TKI, ASK120067 [17]. The current study focused on determining whether targeting ACK1 using the new class of ACK1 specific inhibitor, (R)-9b, impacts emergence of acquired resistance to osimertinib, including overcoming acquired resistance in EGFR mutant NSCLC cells.

2. Material and Methods

2.1. Reagents

Osimertinib and antibodies against PARP and caspase-3 were the same as described previously [18]. ACK1 antibody was purchased from Santa Cruz Biotechnology (sc-28336; Santa Cruz, CA). Other antibodies used in this studies were described in our previous studies [19]. (R)-9b synthesis was described previously [4, 5]. 98% pure GLP-grade compound was synthesized and used in this study. AIM-100 was purchased from MedChem Express LLC (Monmouth Junction, NJ).

2.2. Cell lines and cell culture

The EGFR mutant NSCLC cell lines, PC-9, HCC827, H1975, the osimertinib-resistant cell lines, PC-9/AR (AZD9291-resistant), PC-9/GR/AR (gefitinib-resistant/AZD9291-resistant) and HCC827/AR, and cell culture conditions for these cell lines were the same as described previously [18, 20].

2.3. Cell survival and apoptosis assays

Cells seeded in 96-well-plates for 24 h were treated with the tested agents. After 3 days, viable cells were determined using sulforhodamine B (SRB) assay as described previously [21]. Combinational index (CI) for drug synergy was calculated by CompuSyn software (ComboSyn, Inc.; Paramus, NJ). Cell apoptosis was detected with an annexin V/7-AAD apoptosis detection kit (BD Biosciences; San Jose, CA) following the manufacturer’s protocol. PARP and caspase cleavage were detected by Western blot analysis as additional indicators of apoptosis.

2.4. Western blot analysis

The procedure for detecting proteins of interest from whole-cell protein lysates were described previously [18, 20].

2.5. Quantitative real time reverse transcription PCR (qRT-PCR)

Cells receiving given treatments were collected in Trizol (Millipore Sigma; St. Louis, MO) for preparation of total cellular RNA. Reverse transcription was then performed to generate cDNA templates using the OneScript® cDNA Synthesis Kit from abm Inc (Richmond, BC). qRT-PCR reactions were performed using SYBR-Green (Bio-Rad) according to the manufacturer’s instructions. The primers used for ACK1 were 5’-ACTTTGGGCTGATGCGAGCACT-3’, (forward) and 5’-AAGGTGCGTGTCTTCAGGCTCT-3’ (reverse). GAPDH was amplified as an internal control using the primers, 5’-GACATCAAGAAGGTGGTGAA-3’ (forward) and 5’-TGTCATACCAGGAAATGAGC-3’ (reverse).

2.6. Small hairpin RNA (shRNA), small interfering RNA (siRNA) and gene knockdown

ACK1 shRNA (shACK1) #1 (TRCN0000197093) and #2 (TRCN0000199936) in pLKO.1 lentiviral vector were purchased from Millipore Sigma and used as recommended by the manufacturer for gene knockdown. ACK1 siRNA (siACK1) was purchased from Santa Cruz Biotechnology (sc-29632). Non-silencing scramble control siRNA (siCtrl) was described previously [22]. Transfection of these siRNA duplexes into the tested cell lines was conducted in 6-well plates using the HiPerFect transfection reagent (QIAGEN; Germantown, MD) following the manufacturer’s instructions. After 48 h, the cells were collected and re-plated in 24-well plates for further treatments.

2.7. Colony formation assay

The effects of the given drug treatments on colony formation in 12-well plates were conducted as previously described [18, 23].

2.8. In vitro assays for delaying emergence of osimertinib acquired resistance

Cells seeded in triplicate 96-well plates, when becoming 80% confluence, were exposed to the tested drugs. Fresh medium containing the same treatment was changed every four days. Positive wells, which were defined as ≥ 50% confluence, were scored every week. Delaying assay was also conducted in 24-well plates. Cells treatments were the same as we did in 96-well plates. The remained cells were stained with crystal violet. We shared solvent control and osimertinib alone treatment with another study that examined the activity of trametinib and osimertinib combination for delaying emergence of osimertinib acquired resistance [24].

2.9. Senescence-associated β-galactosidase (SA-β-gal) staining

Cell senescence was assessed with SA-β-gal staining as described previously [25]. In brief, cells were washed twice with PBS and then covered with enough fixation solution for 5 min at room temperature. Fixed cells were incubated with the X-Gal staining solution overnight at 37°C. After incubation, the cells were washed twice with PBS and once with methanol. After air dry, cells with blue color in plates were counted under microscopy.

2.10. Animal xenograft and treatments

Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Emory University and conducted as described previously [24]. Treatments include vehicle control, osimertinib (15 mg/kg/day, daily, og), (R)-9b (50 mg/kg/day, daily, sc) and their combination. We shared vehicle and osimertinib treatments with another study that examined the activity of trametinib and osimertinib combination for delaying emergence of osimertinib acquired resistance [24] in order to minimize the usage of animals. Tumor volumes were measured using caliper measurements and calculated with the formula V= (length × width2) /2. Mice were sacrificed when tumor sizes on average were over 800 mm3.

2.11. Statistical analysis

The statistical significance of differences in tumor sizes or weights between two groups was analyzed with two-sided unpaired Student’s t tests when the variances were equal. Data were examined as suggested by the same software to verify that the assumptions for use of the t tests held. Differences among multiple treatment were analyzed with ono-way ANOVA. Results were considered to be statistically significant at P < 0.05.

3. Results

3.1. ACK1 inhibition enhances the inhibitory effects of osimertinib, which increases ACK1 expression, on the growth of EGFR mutant NSCLC cells

We first determined the effects of osimertinib on the growth of three EGFR mutant NSCLC cell lines in the absence and presence of (R)-9b. Under the testing conditions for a 3-day treatment, the combination of (R)-9b and osimertinib was more active than osimertinib alone in suppressing the growth of the tested cell lines (Fig. 1A). The CIs were < 1 for the most combinations of (R)-9b at 1 μM with the varied concentrations of osimertinib as indicated, suggesting synergistic effects. Similar results were also generated when another ACK1 inhibitor named AIM-100 [26] was used (Fig. 1B). In a different colony formation assay in 12-well plates with repeating treatments for 5 days and 13 days, respectively, we clearly saw that the combination of osimertinib and (R)-9b was much more active than osimertinib or (R)-9b alone in inhibiting the growth of cell colonies (Fig. 1D). These results together demonstrate that inhibition of ACK1 enhances the efficacies of osimertinib against the growth of EGFR mutant NSCLC cells.

Fig. 1. (R)-9b or AIM-100 combined with osimertinib exerts enhanced effects on inhibiting the growth of EGFR mutant NSCLC cells after both short-time (A and B) and long-term (C) treatments, whereas osimertinib increases ACK1 expression (D and E).

Fig. 1.

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A and B, The indicated cell lines seeded in 96-well plates were treated with different concentrations of osimertinib (Osim) as indicated and its combination with 1 μM (R)-9b (A) or with different concentrations of osimertinib or AIM-100 alone as indicated and the combination of osimertinib and AIM-100 (B). After 3 days, cell numbers were then estimated with the SRB assay. The data were means ± SDs of four replicate determinations. The CI for each combination was indicated inside the graphs. C, PC-9 cells plated in 12-well plates were treated with DMSO, 500 nM osimertinib, 3 μM (R)-9b and osimetinib plus (R)-9b. The same treatments were repeated every 3 days. The cells were stained with crystal violet. D and E, The given cell lines were exposed to the indicated concentrations of osimertinib (D) or 100 nM osimertinib for 8 h and then harvested for preparation of whole-cell protein lysates or total cellular RNA and subsequent detection of ACK1 with both Western blot analysis (D) and qRT-PCR (E). The data in E are means ± SDs of triplicate determinations.

We were also curious about whether osimertinib alters ACK1 expression in EGFR mutant NSCLC cell lines. In both PC-9 and HCC827 cells, we found that osimertinib at the tested concentration ranges between 100 and 500 nM increased the levels of ACK1 detected by Western blotting (Fig. 1D). In agreement, ACK1 mRNA expression was also significantly higher in osimertinib-treated cells than in DMSO-treated cells (Fig. 1E). This finding suggests a scientific rationale for co-targeting EGFR and ACK1.

To further demonstrate the impact of ACK1 inhibition on cell response to osimertinib, we used ACK1 siRNA to knock down ACK1 gene expression in HCC827 and H1975 cells and then determine cell responses to osimertinib. Under the condition that ACK1 levels were partially decreased in siACK1-transfected cells (Fig. 2A), the growth of both HCC827 and H1795 cells was significantly reduced upon treatment with 250 nM osimertinib for 5 days in comparison in comparison with the cells transfected with siCtrl, the growth of which was only minimally inhibited by osimertinib (Figs. 2B and 2C). Moreover, we further use ACK1 shRNAs to knock down ACK1, which in general generates more robust and sustained knockdown, and then checked its impact on cell response to osimertinib. Indeed, both tested ACK1 shRNAs generated robust reduction of ACK1 levels in both HCC827 and H1975 cells (Fig. 2D). While ACK1 knockdown per se under the tested condition inhibited the growth of HCC827 and H1975 cells, osimertinib (500 nM) treatment plus ACK1 knockdown showed the most effective effects on suppressing the growth of both HCC827 and H1975 cells in comparison with osimertinib or ACK1 knockdown alone, both, in a 4-days and 8-days exposures (Figs. 2E and 2F). Hence, similar to inhibition of ACK1 kinase activity by small molecule inhibitors, genetic ablation of ACK1 also sensitizes EGFR mutant cells to osimertinib.

Fig. 2. ACK1 knockdown with both ACK1 siRNA (A-C) and shRNAs (D-F) sensitizes EGFR mutant NSCLC cells to osimertinib.

Fig. 2.

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A-C, The given cell lines transfected with siCtrl or siACK1 for 48 h were collected either for Western blotting to validate ACK1 reduction (A) or re-plated in a 24-well cell culture plate. On the second day, the cells were treated with 250 nM osimertinib (Osim). After 5 days, the cells were fixed, stained, pictured (B) and quantified (C). D-F, Both HCC827 and H1975 cell were infected with lentiviruses carrying pLKO.1 and shACK1, respectively, for 48 h and then plated in 12-well plates. On the second day, the cells were treated with 500 nM osimertinib for the indicated days and then fixed, stained and pictured (E). The results on day 8 were also quantified (F). ACK1 knockdown after 48 h infection was validated with Western blotting (D).

3.2. ACK1 inhibition combined with osimertinib enhances suppression of EGFR/ERK signaling and induction of apoptosis in EGFR mutant NSCLC cells

We then determined whether ACK1 inhibition combined with osimertninib treatment enhances apoptosis. After 48 h treatment with osimertinib alone, (R)-9b alone and their combination, respectively, we found that the combination of (R)-9b and osimertinib were significantly more effective than either agent alone in inducing apoptosis, as assayed by annexin V staining/flow cytometry in both PC-9 and HCC827 cells (Fig. 3A). In agreement, the combination of (R)-9b and osimertinib was also more potent than either agent alone in inducing cleavage of caspase-8, caspase-3 and PARP in these two cell lines (Fig. 3B). Similarly, the combination of osimertinib and AIM-100 also enhanced induction of apoptosis in comparison with the effect caused by either agent alone (Fig. 3C). Together ACK1 inhibition combined with osimertinib enhances induction of apoptosis in EGFR mutant NSCLC cells.

Fig. 3. The combination of osimertinib with (R)-9b or AIM-100 enhances induction of apoptosis (A-C) and suppression of ERK phosphorylation (D) in EGFR mutant NSCLC cells.

Fig. 3.

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A-C, The given cell lines were exposed to DMSO, 5 nM osimertinib (Osim), 1 μM (R)-9b and osimertinib plus (R)-9b (A and B) or to DMSO, 10 nM osimertinib, 2 μM (PC-9) or 5 μM (H1975) AIM1000 and osimertinib plus AIM-100 (C). After 72 h (A and C) or 48 h (B), the cells were harvested for detection of annexin V-positive cells using flow cytometry (A and C) and caspase/PARP cleavage using Western blotting (B). The data in A and C are means ± SDs of duplicate determinations. ** P < 0.01 at least compared with all other groups. CF, cleaved form. D, The indicated cell lines were treated with DMSO, 5 nM osimertinib, 1 μM (R)-9b or the combination of osimertinib and (R)-9b. After 12 h, the cells were harvested for preparation of whole-cell protein lysates and subsequent Wester blotting.

We then determined the effects of osimertinib combined with (R)-9b on modulation of EGFR and its downstream signaling including Mcl-1 and Bim levels, both of which are involved in mediating osimertinib-induced apoptosis in EGFR mutant NSCLC cells [18]. The combination exhibited enhanced effect on reducing p-ERK levels in both PC-9 and HCC827 cells, where it augmented suppression of EGFR phosphorylation primarily in PC-9 cells (Fig. 3D). Moreover, the combination was also more effective than either agent alone in decreasing the levels of p-Akt and Mcl-1 and in increasing Bim levels in HCC827 cells (Fig. 3D). Thus, the combination of osimertinib and (R)-9b enhances suppression of EGFR/ERK signaling.

3.3. ACK1 inhibition delays development of acquired resistance to osimertinib in vitro

Given the above data indicating that ACK1 inhibition combined with osimertinib exerts enhanced effects on inhibiting the growth of EGFR mutant NSCLC cells, including enhanced induction of apoptosis, we then examined whether inhibition of ACK1 delays or prevents emergence of acquired resistance to osimertinib. To this end, we treated PC-9 cells (seeded in 24-well cell culture plates) with osimertinib at 500 nM, a concentration close to clinically achievable plasma levels of osimertinib in patients who take 80 mg osimertinib daily [27], in the absence and presence of (R)-9b. The same treatments were repeated every 4 days for up to 40 days. As presented in Fig. 4A, osimertinib at 500 nM was very effective to eliminate PC-9 cells in the beginning of the treatment (e.g., day 4). As the treatments were prolonged, we started to detect the growth of survived clones at day 30 and day 39 in wells exposed to osimertinib alone. However, the wells co-treated with osimertinib and (R)-9b combination showed no visible cell growth or clones even after 64 days. Significantly, (R)-9b alone at the tested concentration had limited effect on suppressing the growth of PC-9 cells. Similar results were also generated in HCC827 cells although the effects were not as drastic as observed in PC-9 cells (Fig. 4A). Thus, it is clear that osimertinib combined with (R)-9b delays or prevents development of acquired resistance to osimertinib in the in vitro assay.

Fig. 4. The presence of (R)-9b delays the onset of osimertinib resistance (A and B) and effectively inhibits the osimertinib-resistant cells (A) including enhancing senescence (C) in vitro.

Fig. 4.

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A and C, PC-9 cells seeded in 24-well plates were exposed to DMSO, 500 nM osimertinib (Osim), 1 μM (R)-9b and osimertinib plus (R)-9b, respectively. The same treatments were repeated every 4 days. The cells were stained and pictured on the indicated days. After 30 days, the cells treated with osimertinib alone were switched to the treatment with the combination of osimertinib and (R)-9b. On day 50 and 64, the cells were also stained for SA-β-gal (C). B, PC-9 cells seeded in 96-well plates were exposed to the treatments as described in A. Cell densities were scored weekly for 10 weeks. The data are the means ± SDs of triplicated 60 replicate determinations.

We further validated this finding by conducting a similar experiment in 96-well plates with 60 replicate wells for each treatment. Here we defined positive wells (i.e., with growth of resistant cells) as over 50% of cell confluence. We did not detect positive wells until week 5 post treatment with osimertinib alone. After 10 weeks, we detected 80% of positive wells exposed to osimertinib alone, but no positive wells were observed when exposed to the combination of (R)-9b and osimertinib (Fig. 4B). Again, (R)-9b alone at the tested condition did not affect the growth of PC-9 cells. The data from this experiment further demonstrate the effect of (R)-9b on delaying or preventing emergence of acquired resistance to osimertinib in vitro.

3.4. ACK1 inhibition delays development of acquired resistance to osimertinib in vivo

Following the above promising in vitro results, we next conducted in vivo study using mouse xenograft model to explore the effect of ACK1 inhibition on delaying or preventing development of acquired resistance to osimertinib. At the dosage of 15 mg/kg body weight/daily, which is equivalent to 80 mg daily in patients, osimertinib effectively inhibited the growth of PC-9 xenografts for the first 30 days. After that, tumors started to grow back even with daily continuous treatment with osimertinib (Fig. 5A). (R)-9b alone at the tested dosage did not affect the growth of PC-9 xenografts, however, osimertinib combined with (R)-9b effectively suppressed PC-9 tumor growth as osimertinib did for the first 30 days and remained effectively against the growth of PC-9 xenografts for up to 82 days (Fig. 5A). Among the 6 mice receiving the combinational treatment, two mice were tumor-free. Once the combination treatment halted (at day 82), we notices that these tumors started to grow (Fig. 5A), clearly suggesting that drug combination is critical to keep the tumor growth in check. The body weights of mice receiving the combinational treatment was same as those of mice treated with osimertinib alone (Fig. 5B), indicating that the combination of osimertinib with (R)-9b did not increase toxicity to mice while enhancing therapeutic efficacy.

Fig. 5. The presence of (R)-9b delays the onset of osimertinib resistance in vivo (A) without enhancing toxicity (B).

Fig. 5.

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The PC-9 xenografts in nude mice (6 mice/group) were treated with vehicle, osimertinib (Osim), (R)-9b or the combination of osimertinib and (R)-9b as described in the “Materials and Methods”. Tumor sizes and mouse body weights were measured every three days and presented as means ± SEMs.

3.5. ACK1 inhibition combined with osimertinib exerts enhanced effects on suppressing the growth of osimertinib-resistance EGFR mutant cells and tumors

Since osimertinib increases ACK1 expression as demonstrate above, we speculated that osimertinib-resistant cell lines may possess elevated ACK1 levels. Indeed, we detected elevated ACK1 levels in several osimertinb-resistant cell lines in comparison with their corresponding parental cell lines using both Western blotting (Fig. 6A) and qRT-PCR (Fig. 6B). Therefore, beyond the effect on delaying resistance as demonstrated above, we further determined whether the combination of (R)-9b and osimertinib is also effective against EGFR mutant NSCLC cell lines with acquired resistance to osimertinib or in overcoming acquired resistance to osimertinib. As presented in Fig. 6C, the combination of (R)-9b and osimertinib effectively decreased the survival of different osimertinib-resistant cell lines. The CIs for the most combinations were < 1, indicating synergistic effects against the growth of these resistant cell lines. In the long-term colony formation assay, we found that the combination of (R)-9b and osimertinib was also much more active than either agent alone in suppressing colony formation and growth of the resistant cell lines (Fig. 6D). These data further validated that suppression of ACK1 kinase activity by (R)-9b is critical for overcoming acquired resistance to osimerinib. In agreement, in the aforementioned in vitro osimertinib resistance delay study, exposure of the PC-9 resistant cells after 39 days to the combination of osimnertinib and (R)-9b effectively eliminated the majority of the cells (Fig. 4A).

Fig. 6. The combination of (R)-9b and osimertinib exerts enhanced effects on decreasing cell survival (C), inhibiting colony formation and growth (D) and increasing senescence cells (E) in different osimertinib-resistant NSCLC cell lines, which possess elevated levels of ACK1 (A and B).

Fig. 6.

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A and B, The basal levels of ACK1 in the indicated cell lines were detected with Western blot analysis (A) and qRT-PCR (B), respectively. C, The indicated cell lines seeded in 96-well plates were treated with different concentrations of osimertinib (Osim) alone, (R)-9b alone or their respective combinations. After 3 days, cell numbers were then estimated with the SRB assay. The data were means ± SDs of four replicate determinations. CIs were labeled inside graphs. D, The indicated cell lines plated in 12-well plates with different densities were treated with DMSO, 500 nM osimertinib, 1 μM (R)-9b and AZD9291 plus (R)-9b. The same treatments were repeated every 3 days. The data from 200 cells/well experiment were presented as means ± SDs of triplicate determinations. E, PC-9/GR/AR cells were treated with DMSO, 500 nM osimertinib, 1 μM (R)-9b or the combination of osimertinib and (R)-9b and repeated the treatments every 3 days for 12 days. The cells were stained for SA-β-gal.

Moreover, in the above in vivo resistance-delaying experiment where osimertinib-treated tumors became resistant, we treated these tumors at day 92 with the combination of osimertinib and (R)-9b for additional two weeks and found that these tumors responded to the combination treatment, as evident by tumor size reduction or tumor shrinkage (Fig. 5A). Taken together, these data clearly indicates that the combination of (R)-9b and osimertinib is also active against the growth of osimertinib-resistant tumors in vivo.

3.6. ACK1 inhibition combined with osimertinib enhances cell senescence in osimertinib-resistant cells

Although the combination of (R)-9b with osimertinib effectively eliminated most of the resistant cells to osimertinib as demonstrated in Fig. 4A, there were some cells remained even after 25 days treatment. These cells became larger and started rounding up. To determine whether these cells are senescent cell populations, we conducted SA-β-gal staining and detected SA-β-gal-positive cells in resistant-cells post the combination treatment for 11 and 24 days, respectively (Fig. 4C). We detected rounding up and SA-β-gal-positive cells in another osimertinib-resistant cells line, PC-9/GR/AR, exposed to repeated treatment with (R)-9b and osimertinib combination for 12 days (Fig. 6E). These results suggest that sustained treatment of osimertinib-resistant cells with (R)-9b and osimertinib combination enhances cell senescence.

4. Discussion

In this study, inhibition of ACK1 with both ACK1 small molecule inhibitors such as (R)-9b and gene knockdown effectively enhanced the effects of osimertinib on suppressing the growth of several EGFR mutant NSCLC cell lines, likely through enhancing induction of apoptosis. Importantly, the combination of (R)-9b and osimertinib effectively delayed the emergence of acquired resistance to osimertinib both in vitro and in vivo. Further, the combination of (R)-9b and osimertinib also very effectively inhibited the growth of osimertinib-resistant cells and tumors. Thus, this study has provided strong evidence in support of targeting ACK1 in delaying and overcoming acquired resistance to osimertinib. Considering that there are hardly any therapeutic modalities available for NSCLC patients who have developed resistance against osimertinib and an excellent ACK1 inhibitor (R)-9b is now available, which is currently undergoing exhaustive pre-clinical studies, our findings makes this study timely and highly relevant for future clinical trials.

The scientific rationale for targeting the ACK1 to delaying or overcome acquired resistance comes from our findings that osimertinib increased ACK1 levels in EGFR mutant NSCLC cells, which was also elevated in several osimertinib-resistant NSCLC cell lines. While summarizing this work, a newly published study coincidently reported that ACK1 activity or phosphorylation was elevated in EGFR mutant NSCLC cells with acquired resistance to the novel 3rd generation EGFR-TKI, ASK120067 and inhibition of ACK1 with agents possessing ACK1 inhibitory activity enhanced ASK120067 activity against the growth of the resistant cells or tumors [17]. Together, these findings support ACK1 as a potential target for delaying and overcoming acquired resistance to osimertinib or other 3rd generation EGFR-TKIs. Given that EGFR-TKIs in general function through a similar action mechanism by targeting mutant EGFR, this strategy is likely valid to other EGFR-TKIs as well.

The major clinical obstacle for long-term benefit to EGFR mutant NSCLC patients treated with osimertinib is the emergence of acquired resistance. Therefore, effective management of acquired resistance is critical in the clinic to offer long-term benefit to EGFR mutant NSCLC patients receiving osimertinib treatment. In this study, we notified that two of 6 mice receiving the combination of (R)-9b and osimertinib were tumor-free at the end of the experiment, indicating cure. Moreover, this combination did not decrease mouse body weights while effectively delaying the development of acquired resistance to osimertinib, suggesting that (R)-9b is not toxic in animals and the safety of this combination is a realistic therapeutic strategy. Therefore, our results warrant the future testing of this strategy by targeting ACK1 in NSCLC patients for delaying and overcoming acquired resistance to osimertinib in the clinic.

Although promising effect for delaying the emergence of acquired resistance, we noted that residual tumors in the rest four mice with residual tumors treated with osimertinib and (R)-9b had a trend to grow back once we stopped the treatment. This suggests the necessity for continuous treatment, implying that although the cancer may not be cured in some patients, but we can significantly delay the emergence of acquired resistance. Further, it is highly likely that this efficacy can be further improved by optimizing the combination regimen. Our future studies are directed towards this goal.

Cellular senescence represents a permanent state of cell cycle arrest. It can function as a tumor suppressive mechanism, but also have a potential to enhance tumor progression though its reactivation and inflammation-promoting activity [28, 29]. Senescent cells activate several pro-survival factors such as Bcl-2 and become resistant to apoptosis [28]. The combination of (R)-9b and osimertinib apparently decreased cell numbers of osimertinib-resistant cells and induced shrinkage of osimertinib-resistant tumors in vivo, likely through induction of apoptosis. Whether the remained senescent cells represent resistant populations to the combinational treatment with (R)-9b plus osimertinib is unknown and needs further investigation. On the other hand, senescent cells can be cleared by the immune system owing to the senescence-associated secretory phenotype (SASP) [30]. Whether enhanced cellular senescence induced by the (R)-9b and osimertinib combination promotes immune clearance of osimertinib-resistant cells in vivo is also interesting topic for further investigation.

In this study, we have not provided mechanistic insights into how the combination of (R)-9b and osimertinib enhances induction of apoptosis and cell senescence in EGFR mutant cells. The combination clearly augments suppression of ERK phosphorylation, a key event downstream of EGFR, in the tested cell lines. Whether this event plays a critical role in enhancing induction of apoptosis and cell senescence needs further evaluation. Nonetheless, our findings presented in this study warrant further mechanistic investigation accounting for these promising biological activities.

5. Conclusions

The findings suggest that targeting ACK1 may be an innovative and effective strategy for delaying and overcoming acquired resistance to osimertinib as well other 3rd generation EGFR-TKIs in NSCLC patients. The availability of the ACK1 small molecule inhibitor, (R)-9b, warrants future testing of our findings in the clinic.

Highlights.

  • ACK1 inhibition synergizes with osimertinib against EGFR mutant NSCLC cells.

  • ACK1 inhibition delays emergence of osimertinib resistance in vitro and in vivo.

  • Targeting ACK1 restores the response of osimertinib-resistant cells to osimertinib.

  • Targeting ACK1 as a novel strategy to delay and overcome osimertinib resistance.

Acknowledgement

We are grateful to Dr. A. Hammond in our department for editing the manuscript. TKO, SSR and SYS are Georgia Research Alliance Distinguished Cancer Scientists.

Sources of funding

This study was supported in part by NIH/NCI R01 CA223220 (to SYS), UG1 CA233259 (to SSR), R01 CA208258 (to NPM) and R01 CA227025 (to NPM), Prostate Cancer Foundation (PCF) Challenge Grant (17CHAL06; to NPM), Emory Winship Cancer Institute lung cancer research pilot funds (to SYS) and Lee Foundation Award to the Winship Lung Cancer Program for supporting the pilot project.

Footnotes

Declaration of competing interest

NPM is named as inventor on patent “Inhibitors of ACK1/TNK2 Tyrosine Kinase” (patent no. 9,850,216 and 10,017,478), which have been licensed by TechnoGenesys, Inc.. NPM is a cofounder of TechnoGenesys, Inc., owns stock, and serves as consultant. SSR is on consulting/advisory board for AstraZeneca, BMS, Merck, Roche, Tesaro and Amgen. TKO is on consulting/advisory board for Novartis, Celgene, Lilly, Sandoz, Abbvie, Eisai, Takeda, Bristol-Myers Squibb, MedImmune, Amgen, AstraZeneca and Boehringer Ingelheim. Other people declare that they have no conflict of interest related to this work.

Availability of data and materials

All data are included in this manuscript.

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