Nrf2 overexpression increases risk of high tumor mutation 1 burden in acute myeloid leukemia through inhibiting MSH2 2

13 Background : Nuclear factor erythroid 2-related factor 2 (Nrf2, also called NFE2L2) 14 has been shown to play a pivotal role in preventing cancer cells from being affected by 15 chemotherapy. Gene mutation is a crucial reason of chemotherapy-resistance in acute 16 myeloid leukemia (AML). However, the relationship between Nrf2 and tumor mutation 17 burden and its mechanism in regulating chemotherapy-resistance remains unclear. 18 Methods : The whole-exome sequencing analysis were used to measure tumor mutation. 19 RNA sequencing, Oncomine, qRT-PCR, Western blotting and immunocytochemistry 20 were employed to detect differences in genes and proteins. The KEGG pathway 21 enrichment analysis and GeneMANIN were performed pathway analysis. Functional 22 assays, such as annexin V/PI, Hoechst33342 staining and DCFH were performed to 23 examine the apoptosis and reactive oxygen species (ROS) of AML cells in vitro. 24 Subcutaneous xenograft model was established to investigate in vivo growth. 25 Results : Nrf2 expression was associated with tumor mutation burden in AML. Patients 26 with Nrf2 overexpression had higher frequency of gene mutation and drug resistance. Nrf2 overexpression protected the AML cells from apoptosis induced by cytarabine in 28 vitro and increased the risk of gene mutant drug resistance in vivo. Furthermore, Nrf2 29 overexpression inhibited MSH2 protein expression, which caused DNA mismatch 30 repair (MMR) deficiency. Mechanistically, the inhibition of MSH2 by Nrf2 was in a 31 ROS-independent manner. Further studies showed that an increased activation of 32 JNK/c-Jun signaling in Nrf2 overexpression cells, which inhibited the expression of 33 MSH2 protein. 34 Conclusions : Our findings provided evidence that high Nrf2 expression inhibited 35 MSH2 expression, caused MMR deficiency and increased the tumor mutation burden, 36 which can induce gene instability-dependent drug resistance in AML. This study 37 demonstrates the reason why the high Nrf2 expression leads to the increase of gene 38 mutation frequency in AML, and provides a new strategy for clinical practice. 39 Immunochemistry; WB: Western blot .NAC: N-acetylcysteine; JNK: c-Jun N-terminal kinase.

However, the existing reports are mostly limited to the effect of high Nrf2 86 expression on gene instability-independent drug resistance, and there are few reports of 87 Nrf2 participating in the regulation of gene instability. In early study, we compared the 88 differentially expressed genes in the two groups of AML patients with high or low Nrf2 89 expression by transcriptome sequencing (RNAseq), and found that the high Nrf2 and so on [29,30]. Normally, cells are equipped with DNA damage response pathways 96 and damage to DNA is detected and repaired [31]. Defective mismatch repair cells 97 exhibit a higher frequency of mutation in both coding and noncoding microsatellite 98 sequences. MMR deficiency leading to microsatellite instability (MSI) has been 99 5 recognized as a distinct tumorigenesis pathway [32]. Additionally, DNA repair defects 100 are associated with the development of resistance to chemotherapeutics, in both solid 101 tumors and haematological malignancies [33,34]. 102 In this study, we sought to investigate the role of Nrf2 in AML gene instability-103 dependent drug resistance. We found that Nrf2 was significantly up-regulated in AML 104 with high tumor mutation burden and drug resistance. Further analysis revealed that 105 Nrf2 overexpression inhibited MSH2, thereby promoting chemotherapy resistance of 106 AML cells both in vitro and in vivo. Mechanistically, the role of Nrf2 in causing DNA 107 MMR deficiency was achieved by regulating JNK/c-Jun signaling.

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Patients' specimens and cell lines 110 We collected 33 bone marrow specimens of AML patients from September 2018 to

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Transfection of Nrf2 was performed using manufacturer's instructions. Cells (THP1 133 and Kasumi-1) respectively transfected with empty vector (EV) were used as controls.

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After expansion and maintenance in RPMI-1640 medium supplemented with 10% FBS 135 for 5 days, stable THP-1 and Kasumi-1 cell lines expressing L-Nrf2 or si-Nrf2 were 136 selected by puromycin (1.5μg/ml and 2μg/ml respectively).     cells of mutated group was significantly higher than that in non-mutated group (P < 258 0.05, Fig. 1g). Therefore, we preliminarily concluded that the high expression of Nrf2 259 in AML was related to the high tumor mutation burden. Nrf2-Low group (Fig. 2a) Ara-C for 24 h (Fig. 3d, e). Besides, we confirmed that Ara-C led to the accumulation 290 of MSH2 protein in a concentration-dependent manner ( Figure S1A, B). Moreover, we   (Fig. 4f). However, after treatment with Ara-C, MSH2 expression was still 310 weaken in the Nrf2 overexpression group, but increased in the EV group (Fig. 4g).

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Therefore, these data demonstrated that Nrf2 overexpression promoted tumor growth 312 and inhibited MSH2, which contributed to a higher risk of mutant drug resistance in 313 vivo. showed that ROS was higher than that before 2μM Ara-C treatment of cells in each 322 group. However, the increased ROS in the Nrf2 overexpressing group was significantly 323 lower than that of the EV group and the control group (P < 0.05, Fig. 5a). The apoptotic 324 rate was measured by Annexin V/PI assay after treating Nrf2-overexpressing cells with 325 2μM Ara-C for 24 h. The apoptosis of Nrf2 overexpressing group was significantly 326 decreased in comparison with EV and control group (P < 0.05, Fig. 5b).

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If Ara-C induced ROS production can account for the increase of MSH2 protein in showed that MSH2 expression in the Nrf2 overexpression group pretreated with H2O2 333 15 was still weakened (Fig. 5c, d). In addition, we pretreated the Nrf2 down-regulation 334 group with ROS scavenger NAC (5 mM) for 2 h and then exposure to Ara-C for 24 h.

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Western blot assays showed that THP-1 and Kasumi-1 cells treated with Ara-C in 336 combination with NAC weaken MSH2 expression upon Nrf2 knockdown, and there 337 was no significant difference compared with EV group (Fig. 5e, f)  Chemoresistance is one of the major difficulties during cancer chemotherapy.

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According to whether the occurrence of drug resistance is related to gene mutation, it 361 can be divided into gene instability-dependent and independent drug resistance.