Abstract
Purpose
Activation of NFE2L2 has been linked to chemoresistance in cell line models. Recently, somatic mutations which activate NFE2L2, including mutations in NFE2L2, KEAP1, or CUL3, have been found to be associated with poor outcomes in patients with non-small cell lung cancer (NSCLC). However, the impact of these mutations on chemoresistance remains incompletely explored.
Experimental Design
We investigated the effect of Keap1 deletion on chemoresistance in cell lines from Trp53-based mouse models of lung squamous cell carcinoma (LSCC) and lung adenocarcinoma (LUAD). Separately, we identified 51 stage IV NSCLC patients with KEAP1, NFE2L2, or CUL3 mutations and a matched cohort of 52 wildtype patients. Time to treatment failure after front line platinum doublet chemotherapy and overall survival was compared between the two groups.
Results
Deletion of Keap1 in Trp53-null murine LUAD and LSCC resulted in increased clonogenic survival upon treatment with diverse cytotoxic chemotherapies. In NSCLC patients, median time to treatment failure (TTF) after first line chemotherapy for the KEAP1/NFE2L2/CUL3-mutant cohort was 2.8 months compared to 8.3 months in the control group (p < 0.0001). Median overall survival (OS) was 11.2 months in the KEAP1/NFE2L2/CUL3-mutant group and 36.8 months in the control group (p = 0.006).
Conclusions
Keap1 deletion confers chemoresistance in murine lung cancer cells. Patients with metastatic NSCLC with mutations in KEAP1, NFE2L2, or CUL3 have shorter time to treatment failure and overall survival after first line platinum doublet chemotherapy compared with matched controls. Novel approaches for improving outcomes in this subset of NSCLC patients are therefore needed.
Keywords: Keap1, NRF2, non-small cell lung cancer, chemotherapy, chemoresistance
Introduction
Despite significant advances in the treatment landscape for non-small cell lung cancer (NSCLC), the overall survival of advanced stage NSCLC remains poor (1,2). This is due in large part to the development of resistance to chemotherapy by cancer cells. Unfortunately, the molecular causes of intrinsic and acquired chemoresistance remain incompletely understood.
The KEAP1-NFE2L2 pathway regulates redox and metabolic homeostasis and has been implicated in chemoresistance in a variety of cancer types. Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2; also known as NRF2), is a transcription factor that regulates the transcription of antioxidant and drug detoxifying genes and thus enhances cellular survival. At homeostasis, an adaptor protein called Kelch-like ECH-associated protein 1 (KEAP1) recruits a CUL3-containing E3 ubiquitin ligase complex to NFE2L2 and leads to its proteasome-mediated degradation (3,4). Under oxidative and toxic stress, NFE2L2 is released from KEAP1, migrates into the nucleus and drives transcription of genes containing antioxidant response element (ARE) in their promoter regions (5). NFE2L2 target genes are involved in antioxidant metabolism and xenobiotic biotransformation reactions and thus protect cells from the effects of cytotoxic chemotherapy.
Recently, large-scale genomic analyses have revealed that genes in the KEAP1-NFE2L2 pathway are mutated in ~33% of lung squamous cell carcinoma (6) and ~22% of lung adenocarcinoma (7,8). Genetically engineered mouse model studies have indicated that KEAP1 deletion and NFE2L2 mutations confer a pro-survival phenotype and promote the development and aggressiveness of NSCLCs (9–11), suggesting a potential mechanism for selection of mutations in these genes during tumorigenesis. Previous studies have also suggested that NFE2L2 activation in cancer cells leads to treatment resistance to a variety of anti-cancer agents. For example, prior work from our group demonstrated that Keap1 deletion in a Trp53-based mouse model of lung squamous cell carcinoma confers radioresistance by interfering with reactive oxygen species (ROS) generation and that early stage NSCLC patients with KEAP1 or NFE2L2 mutations are at high risk for local recurrence after radiotherapy (11). A role of the KEAP1-NFE2L2 pathway in lung cancer chemoresistance is suggested by preclinical studies showing that KEAP1 loss or NFE2L2 overexpression is associated with resistance and NFE2L2 inhibition with sensitization to chemotherapeutics (12–17). In addition, recent studies have reported a prognostic association between activation of the KEAP1-NFE2L2 pathway and poor overall survival after chemotherapy. However, these studies were limited to NSCLC patients with KRAS mutations (18) or stratified NSCLC patients based on protein levels of NRF2 rather than KEAP1/NFE2L2 mutations (19–21).
We therefore set out to explore the impact of KEAP1/NFE2L2 mutations on chemoresistance and prognosis after chemotherapy in NSCLC regardless of KRAS mutational status. To this end, we employed NSCLC cells from Trp53-deletion based lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LSCC) mouse models without Kras mutations and found that Keap1 deletion confers resistance to diverse anticancer drugs including platinum reagents. We further demonstrate that KEAP1/NFE2L2 mutations are predictive of worse response to platinum doublet chemotherapy.
Methods
Animals
Keap1f/f mice (22,23) (C57BL/6J background) and Trp53f/f;R26tdTomato mice (24) (B6/129 background) were kindly gifted from T. Kensler (University of Pittsburgh, PA) and M. Winslow (Stanford University, CA), respectively. Trp53f/f;R26tdTomato and Keap1f/f;Trp53f/f;R26tdTomato mice between 4 weeks and 9 months of age were intranasally administered with Ad-Cre viruses for lung adenocarcinoma formation. Mice were housed in a designated pathogen-free area in a facility at the Stanford University School of Medicine accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. All care and treatment of experimental animals were in accordance with the guidelines of Stanford University School of Medicine institutional animal care and use committee guidelines.
Tumor Dissociation and Tumorsphere assay
As previously described (11), tumors generated from Trp53-null or Keap1;Trp53-null tracheal or lung cells were minced with a razor blade and suspended in 10 mL of L-15 Leibovitz medium (Thermo Fisher Scientific Inc.) supplemented with 0.5 mL of collagenase/hyaluronidase (Stem Cell Technologies). Tumors were digested for 1.5 to 2 hours at 37°C and 5% CO2 and manually dissociated by pipetting every 30 minutes. After digestion, 40 mL of blocking buffer was added and tumor cells were pelleted by centrifugation. Tumor cells were resuspended in 5 mL of trypsin/0.05% EDTA for 5 minutes and centrifuged with the addition of blocking buffer. The cell pellet was incubated with 100 Kunitz units of DNase I (Sigma) and Dispase (Stem Cell Technologies) for 5 minutes at 37°C and added with blocking buffer for centrifugation. After digestion, tumor cells were treated with ACK lysis buffer and filtered through a 40-μm cell strainer. Dissociated tumor cells were resuspended in MTEC/Plus (25) mixed at a 1:1 ratio with growth factor-reduced Matrigel (26). Cell/media/matrigel mixture (100 μL) was plated on top of a 24-well cell culture insert. 0.4 mL of media was provided to the lower chamber with the treatment of anticancer drugs. Sphere formation and growth were followed for 5–7 days. Tumorspheres (> 100 μm in diameter) were counted manually.
Non-Small Cell Lung Cancer Cohort
NSCLC patients who had tumor specimens analyzed using the Stanford Solid Tumor Actionable Mutation Panel (STAMP) (27) between January 2014 – August 2018 as part of routine clinical care were included. Two version of STAMP were used clinically during the era in which these patients were interrogated, one covering 198 genes (302 kb) and the other covering 130 genes (232 kb). We identified a total of 1021 NSCLC patients with STAMP results and found 178 patients with KEAP1, NFE2L2 or CUL3 mutations. From this cohort, patients were included if they had stage IV disease and were treated with a first-line platinum doublet. Patients must have had biopsy confirmed NSCLC and if diagnosed at an early stage, they could not have received chemotherapy in the adjuvant or neoadjuvant setting. Patients who were lost to follow up, who elected not to receive treatment or who were treated with first line immunotherapy or molecularly targeted agents were excluded (Fig. 2). For the control cohort, 843 patients with available STAMP testing who were wildtype (WT) for KEAP1/NFE2L2/CUL3 mutations were abstracted. Patients with stage IV disease treated with first line platinum doublet were selected. Patients were matched to the KEAP1/NFE2L2/CUL3 cohort on the basis of gender, age at diagnosis (+/− 10 years), platinum chemotherapy regimen, smoking history (former, current, never), and race/ethnicity.
Figure 2.
Consort Diagram detailing identification of KEAP1/NFE2L2/CUL3 mutant patients included in this study. Wildtype patients were identified from the 843 patients without KEAP1/NFE2L2/CUL3 mutations and were matched on gender, age at diagnosis (+/− 10 years), platinum chemotherapy regimen, smoking history (former, current, never), and race/ethnicity.
Demographic and clinical information including age at cancer diagnosis, gender, smoking history (former, current, never smoker), ethnicity, performance status and presence of brain metastases at diagnosis were abstracted from each patient’s medical record. Date of initiation of therapy was defined as the first day of infusional chemotherapy. Date of progression was defined as the date of progression as defined by the treating clinician, or death, whichever came first. Time to treatment failure (TTF) on first line treatment was calculated by subtracting the date of front-line therapy initiation from the date of treatment discontinuation due to clinical progression, toxicity, patient preference or death and reported in months. Overall survival (OS) was calculated by subtracting the date of start of chemotherapy from the date of death, also reported in months. Patients who died before radiographic reassessment were deemed to have progressive disease. The study was conducted in accordance with the ethical principles set forward in the Declaration of Helsinki. All patients provided their written consent to participate in a molecular analysis study approved by the Stanford University Institutional Review Board.
Statistical analysis
Statistical analysis was performed using Excel Version 14.7.3, RStudio version 1.1 and Prism 8. The Kaplan-Meier method was used to estimate PFS and OS. For analysis of PFS, patients who were alive with no evidence of disease progression at the time of the data abstraction (11/30/2018) or who were lost to follow up were censored. For OS, patients who were alive or lost to follow up at the time of data abstraction (11/30/2018) were censored. Comparison of survival curves was done using the Log-rank (Mantel-Cox) test. Significance was defined as P<0.05 and hazard ratio (HR) with 95% CI were reported. Univariate and multivariate analysis were performed with RStudio. A forward selection method was used in which variables with a p≤0.1 on univariate analysis were selected for input into multivariate analysis.
Results
Keap1 deletion confers NSCLC chemoresistance
In a previous study, we demonstrated that combined deletion of Trp53 and Keap1 in airway basal stem cells or peripheral lung cells leads to LSCC and LUAD, respectively (11). Additionally, we found that deletion of Keap1 confers radioresistance in mouse NSCLC and that KEAP1/NFE2L2 mutations are predictive of local failure and recurrence of NSCLC after radiotherapy in human patients. In this study, we set out to investigate whether Keap1 deletion in NSCLC also confers chemoresistance, since, like irradiation, many anti-cancer drugs kill cancer cells via generation of ROS and subsequent DNA damage. Additionally, transcriptional targets of NFE2L2 include genes involved in electrophile detoxification and drug efflux (28). We therefore hypothesized that Keap1 deletion in NSCLC will lead to chemoresistance. To test this hypothesis, we treated cancer cells from Trp53-deletion based Keap1-WT (Keap1WT;Trp53−/−, “P-“) and Keap1-deleted (Keap1−/−;Trp53−/−, “K/P-“) LUADs with several anti-cancer drugs including cisplatin, carboplatin, paclitaxel, and etoposide. In in vitro tumorsphere assays, K/P-LUAD cells were significantly more resistant than P-LUAD cells to all drugs tested, suggesting that Keap1 deletion confers resistance to anticancer drugs (Fig. 1A–B). Similarly, K/P-LSCC tumorspheres also displayed resistance to all drugs tested as compared with the P-LSCC cells (Fig. 1C–D). These data demonstrate that Keap1 deletion, which mimics KEAP1/NFE2L2 mutations found in human tumors, leads to in vitro chemoresistance in NSCLC models.
Figure 1.
KEAP1/NFE2L2/CUL3 mutations confer NSCLC chemoresistance.
(A-B) Relative number of P-LUAD and K/P-LUAD tumorspheres treated with vehicle or cisplatin (Cis, 20 μM), carboplatin (Carbo, 10 μM), paclitaxel (Pac, 0.5 μM), and etoposide (Eto, 0.3 μM) (N=3 biological replicates).
(C-D) Relative number of P-LSCC and K/P-LSCC tumorspheres treated with vehicle or cisplatin (Cis, 20 μM), carboplatin (Carbo, 10 μM), paclitaxel (Pac, 0.5 μM), and etoposide (Eto, 0.3 μM) (N=3 biological replicates).
KEAP1/NFE2L2/CUL3 mutation status is a predictor of outcomes after first-line platinum doublet chemotherapy
Based on these preclinical data, we hypothesized that patients with KEAP1, NFE2L2, and CUL3 mutant metastatic NSCLC will have worse prognosis after first-line platinum doublet chemotherapy than their wild type counterparts. To test this hypothesis, we identified a cohort of 178 NSCLC patients with KEAP1/NFE2L2/CUL3 mutations on the Stanford Solid Tumor Actionable Mutation Panel. Of these, 51 patients met our inclusion criteria (Fig. 2) and were matched to 52 KEAP1/NFE2L2/CUL3 WT patients as described above (Table 1). Of the 51 patients with KEAP1/NFE2L2/CUL3 mutations, 35 had somatic mutations in KEAP1, 12 patients in NFE2L2, and 4 patients in CUL3. The average age was 69 in the KEAP1/NFE2L2/CUL3-mutant group and 65 in the control group. Across both groups, the majority of patients were male, white, former smokers, diagnosed with de novo metastatic disease and had adenocarcinoma histology. The most common first line chemotherapy regimen was carboplatin or cisplatin plus pemetrexed. There were a similar number of patients with brain metastases at diagnosis in both cohorts and Eastern Cooperative Oncology Group performance status was well matched. Median follow up was 39 months.
Table 1.
Patient and Tumor Characteristics.
| Characteristics | KEAP1/NFE2L2/CUL3 mutant (n= 51) | Control (n= 52) | p value |
|---|---|---|---|
| Age, mean (years) | 69 | 65 | 0.06 |
| Gender | |||
| Male | 31 | 28 | 0.82 |
| Female | 20 | 24 | |
| Race | |||
| White | 31 | 34 | 0.96 |
| Asian | 14 | 14 | |
| Black or African American | 5 | 2 | |
| Other | 1 | 2 | |
| Smoking History | |||
| Current | 3 | 2 | 0.97 |
| Previous | 44 | 41 | |
| Never | 4 | 9 | |
| Stage at Diagnosis | |||
| Recurrent stage I | 4 | 0 | 0.97 |
| Recurrent stage II | 2 | 1 | |
| Recurrent stage III | 0 | 0 | |
| De novo stage IV | 45 | 51 | |
| Brain Metastases at diagnosis | |||
| Yes | 20 | 17 | 0.76 |
| No | 31 | 35 | |
| ECOG at diagnosis | |||
| 0 | 15 | 7 | 0.94 |
| 1 | 18 | 26 | |
| 2 | 9 | 8 | |
| 3 | 1 | 4 | |
| Not documented | 8 | 7 | |
| Histology | |||
| Adenocarcinoma | 46 | 46 | 0.98 |
| Squamous | 4 | 4 | |
| Mixed | 1 | 1 | |
| Significant Co-mutations | |||
| TP53 | 34 | 29 | 0.89 |
| KRAS | 13 | 14 | |
| STK11 | 7 | 3 | |
| EGFR | 5 | 8 | |
| Chemotherapy | |||
| Platinum*/pemetrexed | 40 | 40 | 0.97 |
| Platinum*/etoposide | 4 | 2 | |
| Platinum*/taxol | 3 | 4 | |
| Platinum*/gemcitabine | 4 | 6 | |
Carboplatin or cisplatin. ECOG; Eastern Cooperative Oncology Group
We next explored the distribution of mutations in other genes within our cohort (Fig. 3). The most frequently mutated genes in the KEAP1/NFE2L2/CUL3 group were classic NSCLC driver genes including TP53 (N=34 (67%)), KRAS (N=13 (25%)), STK11 (N=7 (14%)) and EGFR (N=5 (10%)). Similarly, the most common mutations in the control group were found in TP53 (N=29 (55%)), KRAS (N=14 (26%)) and EGFR (N=8 (15%)). These frequencies are in line with those observed in large tumor genotyping studies (6–8), suggesting our cohort is representative of the molecular diversity within NSCLC. We observed similar rates of EGFR and KRAS mutations across both cohorts. MET (8% vs 0%, unadjusted p = 0.01) and NRAS (6% vs 0%, unadjusted p = 0.03) mutations were slightly more common in the control group and APC mutations were more common in the KEAP1/NFE2L2/CUL3 cohort (8% vs 0%, unadjusted p = 0.01), although these differences were not significant upon multi-hypothesis testing correction. Other targetable mutations such as ALK, ROS1 or BRAF were uncommon across both cohorts (range 2% - 4%; data not shown).
Figure 3.
Co-occurring genetic events in KEAP1/NFE2L2/CUL3 mutant and wildtype cancers.
Next, we compared treatment outcomes in patients with or without KEAP1, NFE2L2, or CUL3 mutations. The average time to treatment failure (TTF) for first line chemotherapy was 5.0 months in the KEAP1/NFE2L2/CUL3-mutant cohort versus 11.4 months in the WT group (p<0.001). Median TTF on first line chemotherapy for the KEAP1/NFE2L2/CUL3-mutant cohort was 2.8 months compared to 8.3 months in the control group (p <0.0001) (Fig. 4A). Median overall survival (OS) was 11.2 months in the KEAP1/NFE2L2/CUL3-mutant group and 36.8 months in the control group (p = 0.006) (Fig. 4B). Given prior reports of inferior outcomes in KRAS and KEAP1 co-mutated patients, we further examined the effect of KEAP1/NFE2L2/CUL3 mutations in KRAS wildtype patients. In KRAS wild type patients, KEAP1/NFE2L2/CUL3 mutations were associated with shorter time on treatment (2.6 vs 8.5 months, p = 0.0001) and shorter OS (11.2 vs 38.1 months, p = 0.0039) compared with KEAP1/NFE2L2/CUL3 wildtype patients, suggesting that the association between KEAP1/NFE2L2/CUL3 mutations and worse prognosis is not limited to patients with KRAS mutations (Fig. 5A–B).
Figure 4.
Association of prognosis and KEAP1/NFE2L2/CUL3 mutations. Kaplan Meier Survival Analyses for A) Time to Treatment Failure and B) Overall Survival for patients with metastatic non-small cell lung cancer after first line platinum doublet chemotherapy
Figure 5.
A) Time to treatment failure and B) overall survival for KRAS wildtype/KEAP1/NFE2L2/CUL3 mutant and KRAS wildtype/KEAP1/NFE2L2/CUL3 wildtype patients
In addition, as patients with Epidermal Growth Factor Receptor (EGFR) mutations on average have superior outcomes compared to EGFR wildtype patients, we additionally performed a subset analysis in which EGFR-mutant patients were removed from both cohorts (5 from the KEAP1/NFE2L2/CUL3 mutant cohort and 8 from the KEAP1/NFE2L2/CUL3 wildtype cohort). TTF remained shorter in KEAP1/NFE2L2/CUL3 mutant patients compared with KEAP1/NFE2L2/CUL3 wildtype patients (2.7 vs 8.2 months, p=0.0003). Similarly, KEAP1/NFE2L2/CUL3 mutant patients continued to display shorter OS than KEAP1/NFE2L2/CUL3 wildtype patients (10.1 months vs 34.1 months; p=0.02; Supplemental Fig. 1).
Finally, we also analyzed OS and PFS associations of other genes that were frequently mutated in our cohort. Univariate cox regression analysis was performed on STK11, TP53 and KRAS mutations. Genes with p≤0.1 in univariate analysis (UVA) were forward selected for multivariate analysis (MVA). STK11 mutations were negatively associated with TTF on UVA (HR 2.36 95% CI 1.09 – 5.08, p = 0.03) but not significant in MVA with KEAP1/NFE2L2/CUL3 (HR 2.07 95% CI 0.48–0.89 p=0.09). KEAP1/NFE2L2/CUL3 mutations were significant predictors of TTF (HR 2.19, 95% CI 1.42–3.38, p=0.00036) and overall survival (HR 2.17, 95% CI 1.34–3.52, p=0.0016). Thus, KEAP1-NFE2L2 pathway mutations are strongly associated with poor outcomes to first-line chemotherapy in advanced NSCLC.
Discussion
In this study, we demonstrated that deletion of KEAP1 confers chemoresistance in preclinical models of LUAD and LSCC and that patients with metastatic NSCLC with KEAP1/NFE2L2/CUL3 mutations have significantly shorter time to treatment failure and overall survival when treated with front line platinum doublet chemotherapy. Our prior work demonstrated that KEAP1 deletion also confers NSCLC resistance to ionizing radiation and that KEAP1 and NFE2L2 mutations are associated with worse prognosis after radiotherapy. Together our findings indicate that KEAP1/NFE2L2/CUL3 mutations induce resistance to conventional cancer therapies and could serve as a biomarker to predict therapeutic responses for both localized and metastatic NSCLC patients.
Our findings build upon prior work showing that activation of the KEAP1-NFE2L2 pathway is associated with worse outcomes after chemotherapy. Prior studies have mostly focused on immunohistochemical NFE2L2 and/or KEAP1, and some have shown association of expression with outcomes in early or advanced stage NSCLC patients (18–20). Given the rapid adoption of next generation sequencing-based tumor genotyping, our demonstration that mutations in KEAP1/NFE2L2/CUL3 are associated with worse outcomes after first line chemotherapy has immediate clinical relevance.
In line with previous studies reporting the frequency of KEAP1/NFE2L2/CUL3 mutations in 15–30% of NSCLCs (6–8,29), we identified mutations in these genes in 178 out of 1021 patients (17%). Notably, there was no significant difference in frequency of driver mutations such as KRAS, EGFR, ALK, ROS1 or BRAF in between the two cohorts. Our results are also consistent with a recent study reporting that KEAP1/NFE2L2 mutations are associated with worse outcomes after platinum doublet chemotherapy in NSCLC patients with KRAS mutations (18). This study found that at a median follow up of 1.5 years, advanced NSCLC patients with mutations in KRAS and KEAP1/NFE2L2 had shorter overall survival and duration of platinum-based therapy than patients with only mutations in KRAS. Our findings expand upon these observations by demonstrating that patients with KEAP1/NFE2L2/CUL3 mutations have shorter time on treatment and overall survival regardless of KRAS mutation status and extending the median follow up duration to over three years. Due to the small number of NSCLC patients with KRAS mutations in our cohort, we were not powered to detect differences in outcomes within the KRAS mutant cohort.
The TTF and OS we observed in the KEAP1/NFE2L2/CUL3 wildtype cohort significantly exceeded the expected outcomes after first line platinum-based chemotherapy in advanced NSCLC (30). We believe this likely reflects referral bias in the types of patients seen at tertiary institutions, with patients generally being younger and healthier. In addition, our center also treats a higher percentage of Asian patients (~30% of our cohort) and prior studies have shown that Asians have improved progression free and overall survival after chemotherapy compared with Caucasian patients (31).
Limitations of our study include its retrospective nature and the single-institution origin of the patients. Furthermore, there were not enough patients in our cohort who received immunotherapy to explore the potential effect of KEAP1, NFE2L2 and CUL3 mutations on treatment with such agents, which is a significant limitation given that chemo-immunotherapy combinations are now standard of care for front line treatment of advanced NSCLC. However, prior work on KRAS and KEAP1/NFE2L2 mutated adenocarcinoma showed shorter progression free survival after frontline immunotherapy (18). Therefore, it is likely that the poor prognosis to chemotherapy conferred by KEAP1/NFE2L2 mutations would extend to chemo-immunotherapy combinations. However, further studies in this space are needed. Additionally, due to their low frequency of occurrence, we had insufficient power to test if NFE2L2 or CUL3 mutations are individually associated with outcome.
In conclusion, we show that Keap1 deletion results in chemoresistance in isogenic mouse models of LUAD and LSCC and that patients with mutations in the KEAP1-NEF2L2 pathway have significantly worse outcomes after front line platinum-based chemotherapy. Our findings add to the growing body of evidence that these mutations identify a particularly resistant subtype of NSCLC and have potential clinical implications. Future work should endeavor to understand if patients with KEAP1/NFE2L2/CUL3 mutations could benefit from more aggressive up-front therapy. Additionally, our work supports the importance of developing novel strategies to overcome treatment resistance conferred by KEAP1/NFE2L2/CUL3 mutations (32,33).
Supplementary Material
Table 2.
Univariable (UVA) and multivariable (MVA) competing risk regression adjusted for the competing risk of death. Abbreviations: HR=hazard ratio; CI=confidence interval
| Time to Treatment Failure | Overall Survival | ||||
|---|---|---|---|---|---|
| Mutation | UVA | MVA KEAP1/NFE2L2/CUL3 | UVA | MVA KEAP1/NFE2L2/CUL3 | |
| KEAP1/NFE2L2/CUL3 | HR | 2.19 | 2.11 | 2.17 | |
| 95% CI | 1.42–3.38 | 0.473–1.37 | 1.34–3.52 | ||
| p | 3.6e−04 | 7.2e−04 | 1.6e−03 | ||
| STK11 | HR | 2.36 | 2.07 | 1.57 | |
| 95% CI | 1.09–5.08 | 0.48–0.89 | 0.65–3.8 | ||
| p | 0.03 | 0.09 | 0.32 | ||
| TP53 | HR | 1.17 | 1.08 | ||
| 95% CI | 0.80–1.72 | 0.67–1.73 | |||
| p | 0.41 | 0.75 | |||
| KRAS | HR | 1.02 | 1.09 | ||
| 95% CI | 0.67–1.56 | 0.63–1.89 | |||
| p | 0.91 | 0.75 | |||
Statement of Translational Relevance.
For patients with metastatic, driver-mutation negative, non-small cell lung cancer, chemotherapy remains an integral treatment modality, either in combination with immunotherapy or alone. While frequently efficacious in the short term, resistance to chemotherapy almost uniformly develops. The mechanisms of intrinsic resistance and therefore strategies to overcome it are poorly understood. The KEAP1-NFE2L2 pathway plays an important role in management of reactive oxygen species and mutations in this pathway lead to tumor aggressiveness and enhanced tumor survival. In this study, we investigated the role of KEAP1-NFE2L2 pathway in intrinsic chemoresistance. To do this we first exposed lung cancer cells from murine lung cancer models with Keap1 deletion to cytotoxic chemotherapies and showed that this led to increased clonogenic survival. We then retrospectively evaluated patients with mutations in KEAP1-NFE2L2 pathway that received upfront chemotherapy and compared their outcomes to patients without mutations. We found that patients with mutations in the KEAP1-NFE2L2 pathway have inferior time on therapy and overall survival suggesting that these mutations confer chemoresistance.
Acknowledgments
Grant support: This work was supported by grants from CIRM (TG2-01159, Y.J.; TB1-01194; N.H.), the NIH (P30CA147933, P01CA139490, and R01CA188298, M.D.), CRK Faculty Scholar Fund (M.D.), and the Virginia and D.K. Ludwig Foundation (M.D.). Y. Jeong was also supported by the DGIST Start-Up Fund Program (2018080012) and National Research Foundation (NRF) of the Ministry of Science and ICT in Korea (2019R1F1A1058990). M. Diehn was also supported by a Doris Duke Clinical Scientist Development Award and an NIH New Innovator Award (1-DP2-CA186569).
Disclosure of Potential Conflict of Interest
SKP reports grant support from EpicentRx, Forty Seven Inc, Bayer and serves as a consultant for AstraZeneca, AbbVie, G1 Therapeutics, and Janssen Pharmaceuticals. M. Das reports grant support from Verily, United Therapeutics, Celgene, and Abbvie and serves as a consultant for Bristol Myers Squibb. JWN reports grant support from Genentech/Roche, Merck, Novartis, Boehringer Ingelheim, Exelixis, ARIAD/Takeda, and Nektar and serves as a consultant for ARIAD/Takeda, AstraZeneca, Genentech/Roche, Lilly, Exelixis, Loxo Oncology and Jounce Therapeutics. HW reports honoraria from Novartis and AstraZeneca, research funding from Genentech, Roche, Pfizer, Eli Lilly, Celgene, AstraZeneca, MedImmune, Exelixis, Novartis, Clovis Oncology, Xcovery, Bristol-Myers Squibb, Gilead Sciences, Pharmacyclics, and ACEA Biosciences. M. Diehn reports ownership interest in CiberMed, is a consultant/advisory board member for Roche, AstraZeneca, and BioNTech, and has received grant support from Varian Medical Systems. All other authors declare no competing interests.
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