GART Functions as a Novel Methyltransferase in the RUVBL1/β‐Catenin Signaling Pathway to Promote Tumor Stemness in Colorectal Cancer

Abstract Tumor stemness is associated with the recurrence and incurability of colorectal cancer (CRC), which lacks effective therapeutic targets and drugs. Glycinamide ribonucleotide transformylase (GART) fulfills an important role in numerous types of malignancies. The present study aims to identify the underlying mechanism through which GART may promote CRC stemness, as to developing novel therapeutic methods. An elevated level of GART is associated with poor outcomes in CRC patients and promotes the proliferation and migration of CRC cells. CD133+ cells with increased GART expression possess higher tumorigenic and proliferative capabilities both in vitro and in vivo. GART is identified to have a novel methyltransferase function, whose enzymatic activity center is located at the E948 site. GART also enhances the stability of RuvB‐like AAA ATPase 1 (RUVBL1) through methylating its K7 site, which consequently aberrantly activates the Wnt/β‐catenin signaling pathway to induce tumor stemness. Pemetrexed (PEM), a compound targeting GART, combined with other chemotherapy drugs greatly suppresses tumor growth both in a PDX model and in CRC patients. The present study demonstrates a novel methyltransferase function of GART and the role of the GART/RUVBL1/β‐catenin signaling axis in promoting CRC stemness. PEM may be a promising therapeutic agent for the treatment of CRC.


Supplementary Materials and Methods
Patients.Patients were considered eligible for this study if they were histologically diagnosed with metastatic colorectal cancer (CRC).CRC patients had received second-line standard chemotherapy, with progression and/or recurrence before enrollment.Pemetrexed (PEM) was evaluated as a third or fourth-line treatment.Other criteria for eligibility included measurable disease by the Response Evaluation Criteria in Solid Tumors, an Eastern Cooperative Oncology Group performance status of ≤2, adequate blood counts (neutrophils ≥1500/mm 3 and platelets ≥1x10 5 /mm 3 ), renal function within normal limits, total bilirubin ≤1.5 mg/dL, and transaminases ≤2.5 times the upper limit of normal.Tumor response included complete response (CR), partial response (PR), stable disease (SD) and progression disease (PD).All toxicities were graded according to the National Cancer Institute's Common Toxicity Criteria for adverse events, version 5.0 (CTCAE 5.0).Retreatment at the start of each cycle required adequate hematologic function (absolute neutrophil count ≥1500/mm 3 and platelets ≥1x10 5 /mm 3 ) and resolution of all toxicities to ≤CTC grade 2.

Treatment
The treatment was continued until CRC development, unacceptable toxicity, withdrawal of patient consent, or dead.

Statistical analysis.
The primary end point of this study was progression-free survival (PFS), and the secondary end points were disease control rate (DCR), objective response rate (ORR), overall survival (OS) and safety.PFS was defined as the time period from the initiation of PEM based chemotherapy regimens and the first evaluation of RECIST1.1 for solid tumors as disease progression or death from any cause, whichever occurred first.OS was defined as the time from initiation of PEM based chemotherapy regimens to death from any cause or the last follow-up.DCR was defined as the percentage of patients whose tumors had shrunk or remained stable for a certain period of time in the evaluable number of cases, including complete response (CR), partial response (PR), and stable response (SD).ORR referred to the percentage of evaluable cases in which tumor shrinkage had reached a certain level and remained constant, including complete response (CR) and partial response (PR).
PFS and OS times were estimated by the Kaplan-Meier method.
Univariate subgroup analyses of age, sex, primary site, degree of differentiation, presence of liver metastases, and baseline CEA level were performed by log-rank test.

.
PEM treatment study in CRC patients were conducted by Dr. Cui Xing in the Second Affiliated Hospital of Shandong University of Traditional Chinese Medicine.A total of 12 patients received PEM monotherapy.The dose of PEM was 500 mg/m 2 at Day 1, and the dose of other chemotherapy drugs was calculated based on the body surface area of patients according to the recommendation of NCCN guidelines.Each cycle lasted for 21 d.Evaluation.Baseline tumor measurements by computed tomography were obtained within 28 d before starting study treatment.Physical examinations, including medical history, laboratory studies, and assessment of performance status, were conducted at the beginning of each 3-week cycle.Tumor response was evaluated every 2 cycles by computed tomography imaging and tumor measurement done using the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria. 1

Reference1
Figure S1.The cell-derived xenograft model.(A) Establishment of CRC subcutaneous Figure S1.The cell-derived xenograft model.(A) Establishment of CRC subcutaneous

Figure S3 .
Figure S3.Schematic diagram of establishing the liver metastasis model.

Figure S4 .
Figure S4.Statistical analysis for Figure 3C-D in the manuscript.(A & B) Statistical

Figure S5 .
Figure S5.Schematic diagram of establishing the CRC patient-derived tumor xenograft

Figure S7 .
Figure S7.IHC results indicate that RUVBL1 and β-catenin are highly expressed in CRC

Figure
Figure S8.IF assays confirm that GART is co-located with RUVBL1 in both of cytoplasm

Figure S9 .
Figure S9.IF assays indicate RUVBL1 is co-located with β-catenin in both of cytoplasm

Figure S10 .
Figure S10.Schematic diagram of the APC min/+ mouse-induced CRC model.