Targeting WEE1 enhances the antitumor effect of KRAS-mutated non-small cell lung cancer harboring TP53 mutations

Abstract

The clinical development of Kirsten rat sarcoma virus (KRAS)-G12C inhibitors for the treatment of KRAS-mutant lung cancer is limited by the presence of co-mutations, intrinsic resistance, and the emergence of acquired resistance. Therefore, innovative strategies for enhancing apoptosis in KRAS-mutated non-small cell lung cancer (NSCLC) are urgently needed. Through CRISPR-Cas9 knockout screening using a library of 746 crRNAs and drug screening with a custom library of 432 compounds, we discover that WEE1 kinase inhibitors are potent enhancers of apoptosis, particularly in KRAS-mutant NSCLC cells harboring TP53 mutations. Mechanistically, WEE1 inhibition promotes G2/M transition and reduces checkpoint kinase 2 (CHK2) and Rad51 expression in the DNA damage response (DDR) pathway, which is associated with apoptosis and the repair of DNA double-strand breaks, leading to mitotic catastrophe. Notably, the combined inhibition of KRAS-G12C and WEE1 consistently suppresses tumor growth. Our results suggest targeting WEE1 as a promising therapeutic strategy for KRAS-mutated NSCLC with TP53 mutations.

Keywords: CHK2; DNA damage response pathway; G12C; KRAS; Kirsten rat sarcoma; TP53; WEE1; azenosertib; non-small cell lung cancer; sotorasib.

Graphical abstract

Figure 1

Target screening using CRISPR-Cas9 library in KRAS-mutated lung cancer (A) Schematic of functional genomic CRISPR-KO screening. (B) H23 and H358 cells were expressed using Cas9 and treated with a crRNA library for 7 days. Cell viability was assessed using an MTT assay at 72 h. (C) The Venn diagram shows the top 50 genes that suppress growth inhibition of H23 and H358 cells. (D) The top 10 genes that suppress growth inhibition of H23. (E) Cell viability of H23, H1355, H358, H1792, A549, H460, and SW1573 cells transfected with siRNAs targeting WEE1 for 72 h. Cell viability was quantified using an MTT assay. Bars represent mean ± SD of triplicate. (F) Cell viability of TP53-mutant and TP53 wild-type (WT) KRAS-mutated lung cancer cells transfected with siRNAs targeting WEE1 for 72 h were compared. Bars represent mean ± SD of triplicate. Statistical significance was determined using Student’s t test. ∗∗∗p < 0.001. (G) Cell lysates were analyzed by western blotting with the indicated antibodies. (H) Apoptosis of TP53-mutant and TP53-WT KRAS-mutated lung cancer cells transfected with siRNAs targeting WEE1 for 72 h were compared. Apoptosis was quantified using the Caspase-Glo 3/7 assay, and cell viability was quantified using an MTT assay. Bars represent mean ± SD of triplicate. Statistical significance was determined using Student’s t test. ∗p < 0.05 and ∗∗p < 0.01.

Figure 2

Drug screening identifies WEE1 inhibitors as potent enhancers of apoptosis (A) H23 and H358 cells were treated with each compound (1 μM) in the library. Cell viability was assessed using an MTT assay at 72 h. An overview of the growth inhibition of H23 cells by various pathway inhibitors is provided. Bars represent mean ± SD. Each inhibitor’s efficacy was compared to the control group using Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. (B) The top 25 agents that enhanced the growth inhibition of H23. Red clusters represent WEE1 inhibitors, green clusters CHK1 inhibitors, blue clusters PLK inhibitors, and gray clusters CDK inhibitors. (C) Venn diagram showing the top 50 agents that enhance growth inhibition of H23 and H358 cells. (D) Growth inhibition by various inhibitors was compared between TP53-mutant H23 and H358 cells and TP53-WT A549 and H460 cells. Bars represent mean ± SD. Statistical significance was determined using Student’s t test. ∗∗p < 0.01 and ∗∗∗∗p < 0.0001. (E and F) H23, H1355, H358, H1792, LU65, A549, H460, SW1573, and MRC-5 cells were treated with indicated concentrations of adavosertib or ZN-c3 (E). IC₅₀ was assessed by an MTT assay at 72 h and compared between the TP53-mutant and TP53-WT KRAS-mutated cell groups, as shown in (F). Bars represent mean ± SD of triplicate. Statistical significance was determined using Student’s t test. ∗∗p < 0.01. (G and H) H23, H1355, H358, H1792, LU65, A549, H460, SW1573, MRC-5, and IMR-90 cells were treated with adavosertib at the indicated concentrations for 48 h (G). Apoptosis was quantified using the Caspase-Glo 3/7 assay and compared for TP53-mutant and TP53-WT KRAS-mutated cell groups, as shown in (H). Bars represent mean ± SD of triplicate. Statistical significance was determined using Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Figure 3

TP53 mutation increases vulnerability to WEE1 inhibition in KRAS-mutated NSCLC (A) A549 and H460 cells were transfected with siRNAs targeting TP53 for 48 h, followed by transfection with siRNAs targeting WEE1 for 72 h. Cell lysates were analyzed by western blotting with the indicated antibodies. (B) The cell viability was assessed using an MTT assay. Bars represent mean ± SD of triplicate. Statistical significance was determined using Student’s t test. ∗∗∗p < 0.001. (C) Apoptosis was quantified using the Caspase-Glo 3/7 assay. Bars represent mean ± SD of triplicate. Statistical significance was determined using Student’s t test. ∗∗∗∗p < 0.0001. (D) Cell growth was analyzed after 7 days of si-WEE1 treatment using crystal violet staining. (E) H358 cells were transfected with pLV-hTP53, and cell lysates were analyzed with western blotting using the indicated antibodies. (F) H358/pLV-TP53 cells were treated with ZN-c3 at the indicated concentrations for 72 h. Cell viability was assessed using an MTT assay. Bars represent mean ± SD of triplicate. Statistical significance was determined using Student’s t test. ∗p < 0.05 and ∗∗∗p < 0.001.

Figure 4

WEE1 inhibition induces mitotic catastrophe in TP53-mutant KRAS-G12C cells (A and B) H358, H1792, A549, and H460 cells were treated with ZN-c3 (0.5 μM) and subjected to cell cycle analysis using Deep Blue staining at the indicated times. (C) H358 and H1792 cells were treated with 1 μM ZN-c3 for 48 h, subsequently fixed, and stained for α-tubulin (green) via immunofluorescence and for DNA with DAPI (blue). Mitotic catastrophe development was evaluated by analyzing nuclear morphology with a confocal microscope. Representative images of the formation of multinucleated cells are shown (white arrows). Scale bar: 10 μm. (D) Live-cell imaging of H358 and H1792 cells treated with 1 μM ZN-c3 was conducted and continuously monitored through microscopy. Scale bar: 10 μm. (E) H23, H1792, H2122, A549, and H460 cells were treated with ZN-c3 at the indicated concentrations for 48 h. Cell lysates were analyzed using western blotting with the indicated antibodies. (F) H23, H1792, H2122, A549, and H460 cells were transfected with siRNAs targeting WEE1 for 48 h. Cell lysates were analyzed using western blotting with the indicated antibodies.

Figure 5

WEE1 inhibitors enhance apoptosis in combination with a KRAS-G12C inhibitor (A–C) H358, LU65, H23, H1792, H2122, and SW1573 cells were treated with sotorasib for 72 h at the indicated concentration. The cell viability was assessed using an MTT assay (A). Cells treated with adavosertib or ZN-c3 are shown in (B) and (C). Bars represent mean ± SD of triplicate. (D) H358, LU65, H23, H1792, and H2122 cells were treated with 1 μM sotorasib and/or 1 μM ZN-c3. The cell viability was assessed using an MTT assay at 72 h. Bars represent mean ± SD of triplicate. Statistical significance was determined using Student’s t test. ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. (E) Apoptosis was quantified using the Caspase-Glo 3/7 assay at 48 h. Bars represent mean ± SD of triplicate. Statistical significance was determined using Student’s t test. ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. (F) Cell lysates were extracted at 48 h and analyzed by western blotting with the indicated antibodies. (G and H) H358 and H2122 cells were treated with ZN-c3 and sotorasib for 72 h at the indicated concentration. Cell viability was assessed using an MTT assay. 2D surface response for cell inhibition and 3D surface Bliss synergy response are shown. Data are presented as mean of triplicates.

Figure 6

Dual treatment suppresses DNA damage response via CHK2 inhibition (A) Immunofluorescence staining with γH2AX-Alexa 488 and DAPI of H358 cells treated with 1 μM sotorasib, 1 μM sotorasib combined with 1 μM adavosertib, and 1 μM sotorasib combined with 1 μM ZN-c3 for 48 h. Scale bars: 100 μm. (B) H358 cells were treated with 1 μM sotorasib combined with ZN-c3 (1 μM) or adavosertib (1 μM). Cell lysates were extracted at 48 h and analyzed by western blotting with the indicated antibodies. (C) H358 cells were treated with 1 μM sotorasib and transfected with siRNAs targeting CHK2. Cell lysates at 48 h were analyzed by western blotting with the indicated antibodies. (D) Cell viability was assessed using an MTT assay at 72 h. Bars represent mean ± SD of triplicate. Statistical significance was determined using Student’s t test. ∗∗∗p < 0.001. (E) H358 cells were transfected with pLV-CHK2, and the cell lysates were analyzed by western blotting with the indicated antibodies. H358/pLV-CHK2 cells were treated with 1 μM sotorasib and 1 μM ZN-c3 for 72 h. (F) Cell viability was assessed using an MTT assay. Bars represent mean ± SD of triplicate. Statistical significance was determined using Student’s t test. ∗∗∗p < 0.001. (G) Schematic of the hypothetical roles of WEE1 and CHK2 in KRAS-mutated NSCLC cells.

Figure 7

WEE1 inhibition improves the therapeutic efficacy of sotorasib in xenograft models (A) Tumor volumes in mice bearing H358 xenografts treated with vehicle (control: n = 8), sotorasib (30 mg/kg: n = 8), ZN-c3 (60 mg/kg: n = 8), or a combination of ZN-c3 (60 mg/kg) and sotorasib (30 mg/kg) (n = 10). (B) Percentage changes in tumor volume after 29 days of treatment in the individual H358 xenografts treated with sotorasib and/or ZN-c3. (C) Tumor volumes in mice bearing H2122 xenografts treated with vehicle (control: n = 8), sotorasib (30 mg/kg: n = 8), ZN-c3 (60 mg/kg: n = 8), or a combination of ZN-c3 (60 mg/kg) and sotorasib (30 mg/kg) (n = 10). (D) Percentage changes in tumor volume after 28 days of treatment in the individual H2122 xenografts treated with sotorasib and/or ZN-c3. (E) Tumor volumes in mice bearing TM00233 PDXs treated with vehicle (control: n = 5), sotorasib (30 mg/kg: n = 5), ZN-c3 (60 mg/kg: n = 6), or the combination of ZN-c3 (60 mg/kg) and sotorasib (30 mg/kg) (n = 6). (F) Percentage changes in tumor volume after 20 days of treatment in the individual TM00233 xenografts treated with sotorasib and/or ZN-c3. (G) Tumor volumes in KU-001 PDXs treated with vehicle (control: n = 3), sotorasib (30 mg/kg: n = 4), ZN-c3 (60 mg/kg: n = 4), or a combination of ZN-c3 (60 mg/kg) and sotorasib (30 mg/kg) (n = 5). (H) Percentage changes in tumor volume after 17 days of treatment with sotorasib and/or ZN-c3. All bars are presented as mean ± SEM of experimental replicates. Significant differences were determined using Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.