Cell culture

NSCLC cell lines, HBE4-E6E7 cells, HEK293T cells and NIH3T3 cells were purchased from the ATCC. HBE4-E6E7 cells were maintained in keratinocyte serum free medium (K-SFM; Cat.17005-042, GIBCO) with 10% fetal bovine serum, 0.05 mg/ml bovine pituitary extract (BPE), 5 ng/ml epidermal growth factor (EGF) and 10 ng/ml cholera toxin. Other cells were maintained in Dulbecco’s modified Eagle’s medium/F-12 medium with 10% fetal bovine serum and antibiotics. All cell lines were validated by short tandem repeat (STR) DNA fingerprinting at MD Anderson Cancer Center (Houston, TX) and negative for mycoplasma infection.

RNases purification and activity assay

Each RNase cDNA was cloned into a prokaryotic expression vector PSJ3 with N-terminal 6His tag. Plasmids expressing His-tagged RNase were used to transform E. coli BL21 cells. A single transformant was selected and cultured in 5-ml LB medium at 37 °C overnight. When OD reached >1.2, cells were transferred to a 250-ml LB medium and incubated at 37 °C with shaking for about 2 h. The cells were induced with 0.5 mM IPTG for 20–24 h at 16 °C with shaking to express RNase when OD increased to between 0.5 and 0.6. After IPTG induction, cells were collected, lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl), sonicated, supernatant loaded onto the HisTrap column (GE) slowly. The column was washed with wash buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM imidazole, and RNases eluted by elution buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 300 mM imidazole. The RNase activity was detected by an Ambion RNase Alert Lab Test Kit (Life Technologies AM1964) following the manufacturer’s protocol.

Stable cell lines

For ALK, RNase1, and EML4-ALK stable expression cell lines, the cDNA of each of the above genes was cloned into a modified pCDH-CMV-MCS-EF1-Puro vector (CD510B-1; System Biosciences) or pCDH-CMV-MCS-EF1-Neo (CD514B-1; System Biosciences) vector. Site-specific mutant of RNase1 was generated by a Site-Directed Mutagenesis Kit (NEB E0554S). The lentiviral shRNAs were used to knock down ALK and RNase1 (Sigma). HEK293T cells were transfected with the indicated plasmids and packaging plasmids (VSV-G and dvpr) to generate viruses. H1299, H322, H1355 and NIH3T3 cells were infected with above viruses for 6–8 h with polybrene (10 g/ml) and then subjected to puromycin (2 g/ml) or G418 (2000 g/ml) selection to generate stable transfectants. Lipofectamine 2000 (Life Technologies) was used for HEK293T transfection.

Antibodies and reagents

Antibodies against RNase1 (Sigma HPA001140), ALK (Cell Signaling 3633), phosphorylated ALK (Tyr1604; Cell Signaling 3341S), phosphorylated ERK1/2 (Thr202/Tyr204; Cell Signaling 9101), ERK1/2 (Cell Signaling 9102), STAT3 (Cell Signaling 9132), phosphorylated STAT 3 (Cell Signaling 9145S), PD-L1 (Cell Signaling 13684 s), and tubulin (Sigma B-5-1-2) were used for immunoblotting, immunohistochemistry, immunofluorescence, and Duolink assays. PE anti-mouse CD274 (B7-H1, PD-L1) Antibody (Biolegend 124307) was used for flow cytometry. For ALK inhibitor crizotinib (PF-02341066), ceritinib (S7083) and alectinib (CH5424802) were purchased from Selleckchem. The ELISA Kit for human RNase1 (SEA297Hu) and FAM150 (MBS9328956) were used to measure their plasma concentration. Plasma (5 µl) from patients or mice were added to 195 µl phosphate-buffered saline (PBS; 1:40 dilution) and then subjected to ELISA assay based on the manufacturer’s protocol.

Animal experiments

All animal experiments were performed under the guidelines and protocols approved by the MD Anderson Institutional Animal Care and Use Committee (IACUC). The nude male mice were purchased from Jackson Laboratory. In in vivo tumorigenesis assay, 1 × 106 indicated stable NIH-3T3 cells were subcutaneously injected into 6-week-old male nude mice (for NIH3T3 control, ALK, RNase1 and RDAA group, n = 8/group; for EML4-ALK group, n = 5/group). Tumor volumes were monitored using external calipers and calculated by (length × width2)/2. In the subcutaneous model, 6 groups of 48 nude mice were subcutaneously injected with 2 × 105 stable H1299-control, H1299-RDAA or H1299-EML4-ALK cells. Tumor-bearing mice in both groups were further placed in two groups (n = 8/group) for treatment with ceritinib (25 mg/kg/day), crizotinib (50 mg/kg/day), or vehicle control. Drugs were administrated via oral every day for 2 weeks. Tumor volume was measured every 3 days. In the orthotopic model, subcutaneous H1299-Control and H1299-RDAA tumors were cut into cubes (about 1 mm3) and then were transplanted into the lung tissue of nude mice under isoflurane anesthetized. Mice were given ceritinib (25 mg/kg/day) or matched vehicle control at 7 days after inoculation, and continuous for two weeks. The orthotopic tumors were evaluated by micro-CT scanner (Fig. 3f). At the endpoint of experiments, mice were killed by CO2 exposure and cervical dislocation according to the guidelines.

Clinical samples study

Paired plasma samples from the 48 patients with NSCLC were analyzed by ELISA to measure RNase1 plasma concentration (SEA297Hu). For immunohistochemistry, paraffin-embedded archived lung cancer tissue sections were submerged in xylene to remove the paraffin, and rehydrated with an ethanol gradient. H2O2 (3%) was used to block endogenous peroxidase activity, and antigen retrieval was carried out by incubating sections in sodium citrate with microwave heating. Slides were cooled to room temperature for 1 h. The sections were blocked with 5% goat serum (Beyotime Biotechnology) and then incubated overnight with ALK (1:50, CST) or p-ALK (1:100, CST) antibody at 4 °C. After washing with PBS, sections were incubated with HRP-conjugated secondary antibody (Abcam) at room temperature for 1 h. Sections were developed for visualization using diaminobenzene. Study of tissue staining and plasma samples from the patients was approved by the Ethics Committee of Cancer Hospital Affiliated to Harbin Medical University (IRB number: KY2019-18).

Clinical study for RDAA+ patients

The clinical therapy study for RDAA+ patients received approval from the Biomedical Ethics Review Committee of West China Hospital of Sichuan University (Ethics Approval No. 2021-1669) and was duly registered with the Chinese Clinical Trial Registry (Registration No: ChiCTR2100054794, accessible at https://www.chictr.org.cn/showproj.html?proj=144966, Statistical Analysis Plan (SAP) and Study Protocol of the clinical study was shown in Supplementary Materials). Prior to undergoing treatment, patients were provided comprehensive information and subsequently signed an informed consent form, adhering strictly to the principles outlined in the Declaration of Helsinki.

Flowchart of the participant selecting process is shown in Fig. 5, and the eligibility criteria for patient inclusion were meticulously defined as follows:

a) Age requirement of ≥18 years, encompassing both genders. (b) Confirmed histological or cytological diagnosis of locally advanced or metastatic non-small cell lung cancer (NSCLC). (c) Molecular testing (Next-Generation Sequencing, NGS) ensuring the absence of known EGFR, KRAS, BRAF sensitive mutations, NTRK, ALK, ROS1, RET rearrangements, or MET gene alterations (amplification and exon 14 jump mutations) in tumor samples. (d) Expression of PD-L1 below 20%, validated through the VENTANA PD-L1(SP263) assay. (e) RDAA-positive confirmation of biopsy tissue specimens through laboratory testing conducted at West China Hospital. (f) Documented disease progression post-standard treatment (according to RECIST 1.1 criteria) or demonstrated reluctance or intolerance to first-line therapy. (g) Eastern Cooperative Oncology Group (ECOG) performance status score of 0−2. (h) Anticipated survival of ≥3 months. (i) Satisfactory physiological parameters and organ function. (J) Asymptomatic central nervous system (CNS) involvement not necessitating steroid or anticonvulsant therapy for metastasis. (k) Presence of a measurable lesion as per RECIST 1.1 criteria. (l) Resolution of drug-related toxicity reactions, excluding alopecia, to grade 2 or lower (according to CTCAE 4.03 criteria). (m) Demonstrated willingness and capacity to adhere to trial protocols and follow-up procedures, coupled with a comprehensive understanding of the trial’s nature, as evidenced by voluntary signature on the written informed consent form.

The patient exclusion criteria were rigorously outlined as follows:

(a) Confirmation of known oncogenic driver mutations such as EGFR, KRAS, BRAF, NTRK, ALK, ROS1, RET rearrangement, or MET gene alterations (amplification and exon 14 jump mutations), or PD-L1 expression ≥20%. (b) Pathological findings suggestive of mixed tumors. (c) Concurrent administration of other systemic anti-tumor therapies. (d) History of malignancy other than lung cancer within the past 3 years (excluding cured cutaneous basal cell tumors, endoscopically resected early gastrointestinal tumors, and cervical carcinoma in situ). (e) Participation in another investigational drug clinical trial within 4 weeks or undergoing major surgery (including stem cell transplantation or organ transplantation, immunotherapy, or radiation therapy) prior to the initial drug dose. (f) Presence of severe cardiovascular disease or cardiovascular abnormalities within 6 months preceding the first dose. (g) Significant medical conditions affecting drug absorption, distribution, metabolism, and excretion (e.g., swallowing dysfunction, active gastrointestinal disorders, etc.). (h) Positive serology for active hepatitis B, hepatitis C viruses, HIV, and syphilis. (i) History of prior interstitial lung disease, drug-induced interstitial lung disease, radiation pneumonitis requiring steroid therapy, or evidence of clinically active interstitial lung disease. (j) Women of childbearing potential or breastfeeding females with a positive serum pregnancy test within 7 days before treatment initiation, or both males and females not using effective contraception or planning to conceive during treatment and up to 3 months post-treatment completion. (k) Known history of anaphylactic reaction to ensartinib or any of its excipients. (l) Use of medications within 14 days prior to the first dose or requiring a combination of medications associated with a risk of QTc prolongation and/or torsades de pointes, or a strong CYP3A inhibitor or inducer during treatment. (m) Anticoagulant therapy with warfarin or any other coumarin derivative. (n) Presence of severe acute or chronic medical conditions, deemed by the investigator to increase the risk associated with study participation or interfere with the interpretation of study results.

Clinical trial procedure

Before undergoing screening assessment, patients must receive full information and sign an informed consent form. The screening phase must be completed within 28 days prior to dosing. Enrollment in the study was contingent upon the investigator confirming adherence to inclusion criteria and absence of exclusion criteria. Subsequently, patients were provided with the study drug and a dosing diary card. RDAA-positive screening was uniformly conducted by the central laboratory (Institute of Thoracic Oncology, West China Hospital).

Upon initiating therapy, ensartinib was administered orally at a dose of 225 mg once daily until disease progression, development of intolerable toxicity, decision by the investigator or patient to withdraw, loss to follow-up, initiation of other antineoplastic therapy, or death. Patients were assessed every 6 weeks for the first 24 weeks of the trial for safety assessments (including physical examination, vital signs, ECOG performance status score, routine blood tests, blood biochemistry, coagulation studies, routine urine analysis, routine stool examination with occult blood test, 12-lead electrocardiogram, and hormone panel), tumor evaluations (thoracic/abdominal/pelvic contrast-enhanced CT scans, cranial MRI), and quality of life assessments. After 24 weeks, visits occured every 9 weeks with the same assessments as before. Patients experiencing their first remission or disease control (complete response, partial response, or stable disease according to RECIST 1.1 criteria) at a visit will undergo a review 4 weeks later to confirm the evaluation results.

For patients completing the trial or withdrawing informed consent, all adverse events (AEs) and drug combinations must be documented up to 30 days after the last trial dose, and any new AEs occurring within 30 days of the last trial dose must be reported. All AEs must be monitored until resolved or stabilized unless deemed unrelated to the trial medication and attributable to the primary disease. Non-severe AEs, determined by the investigator to be unrelated to the trial medication, will not be recorded for patients who initiate other antineoplastic therapy.

End-of-Treatment (EOT) visit evaluations were conducted promptly after the patient discontinues the trial drug. Any patient who terminated treatment or withdrew from treatment for reasons other than disease progression should undergo a safety assessment promptly, while continuing to undergo tumor assessments at the same frequency as during the treatment period until disease progression occurs or other antineoplastic therapy was initiated. However, patients whose treatment termination was due to disease progression should only undergo safety assessment, and no further tumor assessments should be conducted. If a patient discontinues treatment at the final visit due to toxicity or other reasons and did not continue to take the test drug thereafter, the visit was considered an end-of-treatment/withdrawal visit.

Survival Follow-up and Efficacy Assessment:

Patients who experienced disease progression or initiated other anti-tumor therapies were no longer subject to safety and tumor assessments but continued to be followed up via telephone every 12 weeks. During these follow-up calls, information regarding overall survival and subsequent treatment was collected until death or loss to follow-up. The outcome of this study was the reduction of tumor size after combination therapy, and the efficacy of the treatment regimen was determined according to the RECIST 1.1 criteria, which were as follows: complete remission (CR): disappearance of all target foci, and the short diameter of all pathological lymph nodes (both target and non-target) must be reduced to <10 mm; partial remission (PR): reduction of at least 30% in the sum diameter of the target foci compared to baseline; progression of disease (PD): relative increase in the sum of diameters of target lesions of at least 20%, referenced to the smallest of the sums of the diameters of all target lesions measured throughout the experimental study (or to baseline if baseline measurements are smallest), and in addition, an increase in the sum of the diameters of the diameters in absolute terms of at least 5 mm must be met (the presence of one or more new lesions is considered to be disease progression); stable disease (SD): a decrease in target lesion size that did not reach PR and an increase in target lesion size that did not reach PD, and in between, with reference to the smallest of the sums of the diameters of the target lesions studied.

Patients #05-#08 in this study received ensartinib treatment(225 mg /day). Simultaneously, patient #09 also met the inclusion criteria and voluntarily signed the consent form to receive ensartinib treatment. However, patient #09 resided in Zhejiang Province, which was 1853.3 km away from our study center located in Chengdu. Due to the unwillingness to travel such a long distance for treatment and follow-up, the patient was not enrolled in the clinical study. Instead, the patient was treated as an observational case and received the same ensartinib treatment and follow-up regimen at the local hospital and voluntarily signed informed consent to share the clinical data collected during the treatment period.

Additionally, during the period shortly before the start of this clinical study, we collaborated with the Department of Thoracic Surgery at Harbin Medical University Cancer Hospital to screen and identify 5 cases of RDAA-positive NSCLC patients. Four of them (Patient #01-#04) voluntarily signed the consent forms and began receiving crizotinib treatment (250–500 mg/day). The follow-up procedures for these 4 patients after treatment were consistent with those of patients #05-#08 and maintained for 3 years.

RDAA detection assay

The antibodies of RDAA, the protocol for immunohistochemical staining and positive determination of RDAA were supported by Betta Pharmaceuticals Co., Ltd and Hangzhou Repugene Technology Co., Ltd.

Immunohistochemical staining procedures by using RDAA antibodies:

The primary antibodies used in this study during the immunohistochemical staining were all murine monoclonal antibodies. After deparaffinizing and rehydrating, the paraffin sections were repaired with high-temperature microwave (EDTA antigen repair solution: pH 8.0 (Beijing Zhong Shan -Golden Bridge Biological Technology Co., Ltd., cat: ZLI-9067, China), heated for 8 min each, two times), blocked with 3%H2O2 (20–30 min) and 10% goat serum (30 min), respectively. Then 100ul diluted p-ALK Y1604 (dilution ratio 1:500, antibody concentration 0.5 mg/ml), p-ALK Y1282/1283 (1:250, Antibody stock concentration 0.5 mg/ml),RNase1 (dilution ratio 1:500, antibody stock concentration 0.5 mg/ml) were incubated to cover the surface of the tissue sections overnight at 4 °C(The production steps of RDAA-related antibodies have been described in the previous article and will not be repeated in this article). Subsequent overnight sections were incubated with mouse secondary antibody diluted in PBS (dilution ratio: 1:250, cat: 115-035-003, Jacson, United States) at room temperature for 40 min and then stained with SignalStain® DAB Substrate Kit (Cat. 8059S, CST, USA) (color reaction time: p-ALK Y1604: 40 s, p-ALK Y1282/1283:20 s, RNase1:40 s). After DAB color development, the slices were nucleated with hematoxylin, separated with hydrochloric acid alcohol, returned to blue with ammonia water, dehydrated with absolute ethanol, and then sealed.

Immunohistochemical staining scoring rules of RDAA antibodies:

The positive expression intensity of RDAA was evaluated in tissue sections with tumor area greater than 70% in the field of high-power microscope (10x eyepiece + 40x objective). Whether a lung cancer tumor section was judged to be RDAA positive or not was comprehensively determined according to IHC intensity scores of the three antibodies: p-ALK Y1604, p-ALK Y1282/1283 and RNase1.The score formula was as follows: score of the proportion of positive staining cells of tumor cells * Staining intensity score.

a.

The score definition and interval of the proportion of positive staining of tumor cells were follows: 0 point: negative expression; 1 point: the proportion of positive cells was <15%; 2 points: 15–50% positive cells; 3 points: positive cells ≥50%.

b.

The definition and interval of cell staining intensity were as follows: 0: no positive; 1point: light yellow; 2 points: pale brown; 3 points: brown.

The definition of RDAA positive in tumor tissue samples from patients was as followed: two experienced pathologists independently selected three different fields of view for each section to score RDAA immunohistochemical intensity and took the average value. The tissue samples from lung cancer patient was determined to be RDAA positive when the three IHC intensity scores of p-ALK Y1604, p-ALK Y1282/1283 and RNase1 of the tumor were all greater than 3 points by two doctors. If the scores of two pathologists were inconsistent, the discussion and evaluation were conducted by a third authoritative pathology.

Duolink and immunofluorescence assay

Duolink assay was performed using the Duolink In Situ Red Starter Kit (Sigma DUO92101). Cells were fixed with 4% paraformaldehyde for 20 min, treated with 0.5% Triton X-100 for 20 min, and then blocked with 5% BSA for 1 h. After blocking, cells were incubated with ALK (1:100) or anti-RNase1 (1:200) antibody at 4 °C overnight and then subjected to Duolink assay according to the manufacturer’s protocol. The red spots were detected by fluorescence inverted microscope, and each red spot represents a cluster of protein-protein interaction. Nuclei were stained with DAPI (Invitrogen). For the immunofluorescence experiments, cells were fixed, blocked, and incubated with the primary antibodies under the same conditions of the Duolink assay. Cells were further incubated with a secondary antibody tagged with red or green fluorescein (Life Technologies) for 1 h at room temperature. All the fluorescence signals were observed under the Zeiss LSM 710 laser microscope.

Cell viability assay

H1299, H1355, H322 and PC-9 Cells were cultured at 5 × 105 cells per well in 6-well plates in 2 ml of medium and then treated with ALK inhibitors. At every experimental time point, 2 ml of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/ml) was added into each well, and then incubated at 37 °C for 2 h. After the removal of MTT solution, DMSO was added to each well to dissolve the water-insoluble purple precipitate. Absorbance at 595 nm was measured for each well.

Colony formation assay

Indicated cells were cultured in soft agar medium and seeded at 5 × 102 cells per well into 6-well plates. The cells were incubated for 2 weeks, and then treated with 4% paraformaldehyde for 20 min. After staining by 0.05% crystal violet for 15 min, the size and number of colonies were measured.

Binding affinity assay

H1299 cells with wild-type ALK expression were used for binding affinity assay. ALK antibody (3 μg/ml) or normal mouse IgG (negative control) were diluted into 0.2 M sodium phosphate buffer (pH 6.5) and captured in ELISA 96-well (100 μl/well) plates overnight at 4 °C. Each well was washed with PBST buffer (PBS with 0.05% Tween 20), blocked with 200 μl 1% BSA solution (containing 0.05% Tween 20) for 3 h, and then rinsed with PBST buffer 3 times. H1299 cell lysate was added to each well (100 μl/well), incubated overnight at 4 °C, and then rinsed 3 times with PBST buffer. Recombinant RNase1 protein was diluted to different concentrations in lysis buffer and added to each well. After incubation overnight at 4 °C, each well was washed with 400 μl of 1 M sucrose three times, and then incubated with 100 μl of streptavidin-conjugated HRP (1:2000 in blocking buffer) at room temperature for 2 h. Each well was washed again with 400 μl 1 M sucrose three times, and then incubated with 100 μl TMB as a peroxidase substrate for 30 min at room temperature. A stop solution (50 μl) was added to each well to end the reactions. The OD was measured at 450 nm using a BioTek Synergy™ Neo multi-mode reader. The dissociation constant (Kd) was calculated by the above binding data using the GraphPad Prism software (Prism Software Inc.).

Statistical analysis

All error bars are presented as mean standard deviation (SD). Statistical P values were calculated using a two-tailed, independent Student’s t-test, and P values less than 0.05 were considered significant. Significance of mean comparison is represented on the graphs as follow: *p < 0.05; **p < 0.01; NS, not significant. Kaplan-Meier survival analysis was used to compare patient survival using a log-rank test. The Pearson’s correlation coefficient (R) was used to evaluate a linear association between two variables of experimental data. An R value > 0.8 was considered as strong correlation.