RNase1-driven ALK-activation is an oncogenic driver and therapeutic target in non-small cell lung cancer

Abstract

Targeted therapy has achieved significant success in the treatment of non-small cell lung cancer (NSCLC), particularly in patients harboring common oncogenic driver mutations such as EGFR, KRAS, and ALK rearrangement. However, ~35-50% of NSCLC patients without tyrosine kinase mutation or rearrangement (non-mutated) cannot benefit from these targeted treatments, highlighting the urgent need for novel therapeutic strategies for this patient population. In this study, we report a non-canonical role of human secretory ribonuclease 1 (RNase1), which binds to and activates wild-type ALK in lung cancer cells, thereby triggering its downstream signaling pathway. RNase1-driven ALK-activation (RDAA) cells exhibit enhanced cell proliferation, migration, and colony formation. Additionally, RDAA facilitates tumor formation in fibroblast models, further underscoring its oncogenic potential in vivo. Importantly, RDAA lung cancer cells exhibit marked sensitivity to FDA-approved ALK inhibitors. Tumor growth suppression and survival were substantially improved in both RDAA-positive NSCLC cell line-derived and patient-derived xenograft tumor models treated with ALK inhibitors. Monoclonal antibodies against RNase1 and phosphorylated-ALK were used to analyze two different human NSCLC tissue cohorts by immunohistochemical staining identified 10.4% (5/48) and 8.5% (100/1173) patients who were RDAA positive, respectively. Notably, among the nine RDAA-positive NSCLC patients who accepted ALK inhibitor treatment, five achieved objective response including two who experienced complete response (CR). Together, the current study identifies RDAA as an oncogenic driver and proposes an effective targeted therapy strategy for non-mutated NSCLC patients.

Fig. 1

RNase1 binds to and activates ALK as its ligand in lung cancer cells. a Each of the 13 recombinantly purified 6´ N-terminal His-tagged RNases (10 µg) was incubated with H1299 lysate followed by Ni-His beads pull down and Western blot analysis with the indicated antibodies. b Each of the 13 RNases was added into the H1299 culture medium at a final concentration of 1 mg/ml. After 30 min incubation, cells were lysed and subjected to Western blotting with the indicated antibodies. A phospho-ALK antibody was used to detect ALK Y1604 phosphorylation. c Western blot analysis of ALK phosphorylation sites in HEK293 cells. Flag-tagged wild type or Tyr mutant ALK plasmids were transfected into HEK293 cells and then IP with Flag-beads. A pan-phosphorylated Tyr antibody (4G10) was used to detect ALK phosphorylation. d Plasmids expressing Myc-tagged ALK and C-terminal Flag-tagged RNase1 (R1) were transfected into HeLa cells. Cell lysates were harvested and subjected to co-immunoprecipitation (co-IP) assay. RNase1 was detected using the Flag antibody. RNase5 (R5) was used as a negative control. e In vitro binding affinity assay of ALK and RNase1. Kd, dissociation constant. BSA was used as a negative control. f Immunofluorescence microscopy of H1299 cells with or without RNase1 expression. Yellow dots and white arrows both indicate co-localization of ALK and RNase1. Scale bar, 20 mm. g H1299 cells with or without RNase1 expression were subjected to Duo Link assay. Red dots indicate binding between ALK and RNase1. Scale bar, 20 mm. h Time course analysis of ALK activation in H1299 cells by RNase1 (1 mg/ml). ALK phos-Y1604 was used as an indicator of ALK activation. i Co-IP of ALK and RNAse1 from HEK293T cells transfected with wild-type (WT) or catalytic-deficient (CD) RNase1-expressing plasmid followed by Western blotting with the indicated antibodies

Fig. 2

RDAA is an oncogenic driver in vitro and in vivo. a Western blot analysis of H1299 stable cells expressing wild-type RNase1 and/or short hairpin RNA (shRNA) to knockdown ALK with the indicated antibodies. b The indicated H1299 cells were subjected to MTT assay and cell viability was quantified. **p < 0.01, Student’s t-test. c Cell counting of the indicated cells was performed in triplicate and normalized to control. Error bars, mean ± SD. **p < 0.01. d Colony formation assay of the indicated cells. The relative number of colonies formed was measured in triplicate. **p < 0.01. e Wound healing assay of the indicated cells. Representative images shown. Scale bar, 500 mm. Quantification shown in Supplementary Fig. 2d. f Nude mice were injected with NIH3T3 cells or those expressing ALK and/or RNase 1. Tumor size was measured every 3 days. Red arrows pointing to tumors developed. g Quantification of tumors in (f). N = 8, control, R1, ALK and R1-ALK co-expression group. N = 5, ALK mutant group. **p < 0.01, Student’s t-test. NS, not significant. h Western blot analysis of ALK-RNase1 tumors (N = 7). Actin was used as control. i Correlation between serum RNase1 concentration and tumor size. R = 0.89, Pearson’s Chi-Square test. j Western blot analysis of NIH3T3 cells with ALK and RNase1^high or RNase1^low expression. k Quantification of tumor size from mice injected with RNase1^high or RNase1^low NIH3T3 cells. N = 5. *p < 0.05, Student’s t test

Fig. 3

RDAA tumors are sensitive to ALK inhibitor in both subcutaneous and orthotopic mouse models. a Western blot analysis of ALK downstream signaling of RNase1-driven ALK-positive (RDAA) H1299 cells with the indicated antibody. Phospho-ERK1/2 T202/Y204 and phospho-STAT3 (Y705) specific antibodies were used to detect ERK1/2 and STAT3 phosphorylation, respectively. b RNA-seq analysis of control, RDAA and ALK-rearranged (EML4-ALK) H2228 lung cancer cells. c Cell counting of the indicated cells treated with or without (DMSO; control) the indicated ALK inhibitors. Experiments were performed in triplicate. d Mice received the indicate H1299 cells by subcutaneous injection, and when tumors reached 500 mm³, ALK inhibitor Ceritinib (25 mg/kg/d) was administered beginning day 14 for 2 weeks. Tumor size was measured every 3 days. N = 10. e Overall survival curve of mice in (d) starting the day of cell injection. f Western blot analysis of ALK phosphorylation with or without Ceritinib treatment. The tumor samples were collected from the mice in (e). g Representative Micro-CT scan images of mice who received an orthotopic injection of the indicated cells treated with or without Ceritinib (25 mg/kg/d). N = 7. Red arrows pointing to tumors. h Overall survival curve of mice in (e) starting the day of tumor transplantation. **p < 0.01, Log-rank (Mantel-Cox) test. i Representative 3D images of mice with orthotopic RDAA lung tumor before or after Ceritinib treatment. Area in pink indicate tumor size and location

Fig. 4

Identification of patients with RDAA NSCLC. a Quantification of plasma RNase1 in NSCLC patients (N = 48) and normal individuals (N = 15). RNase1 concentration was measured by ELISA as described in Methods. p < 0.01, Student’s t-test. b IHC staining of RNase1 in human lung tumors (N = 48) and normal lung (N = 10) tissues. RNase1 expression level was calculated based on the intensity and percentage of stained cells as described in Methods. Representative images shown. c Correlation analysis between plasma RNase1 concentration and RNase1 expression level in paired tumor tissues (N = 47). R = 0.84, Pearson’s Chi-Square test. d IHC staining of RNase1 expression and ALK phosphorylation levels in human NSCLC tissues. ALK p-Y1604, ALK p-Y1282/1283 and RNase1 specific antibodies were used for IHC staining. Representative images shown. e Diagnosis of RDAA NSCLC patients in 1173 NSCLC tissues. ALK p-Y1604, ALK p-Y1282/1283 and RNase1 were used as biomarkers to identify RDAA positive samples. f Correlation analysis between RNase1 expression and ALK phosphorylation in NSCLC tissues which used from (e). ALK p-Y1604 and 1282/1283 double positive means ALK phosphorylation positive (pALK + ), otherwise means ALK phosphorylation negative (pALK-)

Fig. 5

Patients with RDAA NSCLC benefit from ALK inhibitor in clinical study. a The flowchart of participant selecting process. b Clinical CT scan images of RDAA NSCLC patients before and after ALK inhibition treatment. Red arrows pointing to tumors. Patient #01 and Patient #09 demonstrated complete response (CR) based on Response Evaluation Criteria in Solid Tumors (RECIST). Representative images shown. c Best tumor percent change from baseline after ALK inhibition treatment of 9 RDAA NSCLC patients. SD, stable disease, black column represented; PR, partial response, yellow column represented; CR complete response, red column represented; PD progression disease, bottle green column represented. Total response rate is 88.9% (RECIST)