A folate receptor 3 SNP promotes mitochondria‐induced clonogenicity of CML leukemia cells: Implications for treatment free remission

Dear Editor, Treatment-free remission (TFR) is an emerging goal of chronic myeloid leukemia (CML) due to long-term costs and toxicity.1,2 Previous studies had shown that 55% of selected CML patients suffered molecular relapse after tyrosine kinase inhibitor (TKI) cessation, while the rest remained in a TFR.3,4 Here, we highlighted the biological role and indicator of folate receptor 3 (FOLR3) and its SNP in TFR. We collected bone marrow samples from CML patients at the time of TKI cessation. These patients were followed up for 24 months; from them 7 relapsed and 7 nonrelapsed were sequenced (Table S1). Among top differentially expressed genes (DEGs) (Figure 1A), the FOLR3, which was not expressed in any relapsed samples and highly expressed in 3/7 non-relapsed samples, was the most significantly gene (Fig S1B). Published expression profiles (Figure 1B) indicated FOLR3 highly expressed in bone marrow. We found that FOLR3 was highly expressed in TKI responders by analysing two public CML datasets, GSE146715 and GSE25356 (Figure 1C, D). Besides, we identified a TA insertion (SNP rs139130389, termed FOLR3 SNP+) in the third exon of FOLR3 gene in three nonrelapsed CML samples with FOLR3 overexpression (Figure 1E). The FOLR3 SNP+ encoded a functional protein; only a partial folate receptor domain was encoded by the FOLR3 SNP(Figure 1E; Figure S1). Average proportion of FOLR3 SNP+ genotype in human is 11.08%, with Africans having the highest frequency (Figure 1F). We retrospectively studied 87 CML patients who discontinued TKI outside of clinical trials. TFR at 48 months for the FOLR3 SNP+ andFOLR3SNPpatientswere 84.4% (95%CI: 74.2%94.6%) and 52.4% (95% CI: 44.6%-60.2%), respectively (Figure 1G, P= .0407). Fifteen of 87 patients carried the FOLR3 SNP, only two relapsed but they successfully achieved secondary withdrawal by resuming TKI treatment (Table S6). The distributions of immune cells were not significantly different between relapsed and non-relapsed samples in

Dear Editor, Treatment-free remission (TFR) is an emerging goal of chronic myeloid leukemia (CML) due to long-term costs and toxicity. 1,2 Previous studies had shown that 55% of selected CML patients suffered molecular relapse after tyrosine kinase inhibitor (TKI) cessation, while the rest remained in a TFR. 3,4 Here, we highlighted the biological role and indicator of folate receptor 3 (FOLR3) and its SNP in TFR.
We collected bone marrow samples from CML patients at the time of TKI cessation. These patients were followed up for 24 months; from them 7 relapsed and 7 nonrelapsed were sequenced (Table S1). Among top differentially expressed genes (DEGs) ( Figure 1A), the FOLR3, which was not expressed in any relapsed samples and highly expressed in 3/7 non-relapsed samples, was the most significantly gene (Fig S1B). Published expression profiles ( Figure 1B) indicated FOLR3 highly expressed in bone marrow. We found that FOLR3 was highly expressed in TKI responders by analysing two public CML datasets, GSE14671 5 and GSE2535 6 ( Figure 1C, D). Besides, we identified a TA insertion (SNP rs139130389, termed FOLR3 SNP+) in the third exon of FOLR3 gene in three nonrelapsed CML samples with FOLR3 overexpression (Figure 1E). The FOLR3 SNP+ encoded a functional protein; only a partial folate receptor domain was encoded by the FOLR3 SNP-( Figure 1E; Figure S1). Average proportion of FOLR3 SNP+ genotype in human is 11.08%, with Africans having the highest frequency ( Figure 1F). We retrospectively studied 87 CML patients who discontinued TKI outside of clinical trials. TFR at 48 months for the FOLR3 SNP+ and FOLR3 SNP-patients were 84.4% (95% CI: 74.2%-94.6%) and 52.4% (95% CI: 44.6%-60.2%), respectively (Figure 1G, P = .0407). Fifteen of 87 patients carried the FOLR3 SNP, only two relapsed but they successfully achieved secondary withdrawal by resuming TKI treatment (Table S6).
The distributions of immune cells were not significantly different between relapsed and non-relapsed samples in  Figure S1). 7 Therefore, we established different FOLR3 SNP expression subtypes in CD34 + cells from newly diagnosed CML patients and K562 cells through lentiviral transfection (details in Supporting Information). The proliferation, cell cycle ( Figure 2A; Figure S2), colonyforming capacities ( Figure 2B) and capacity of folic acid uptake ( Figure 2C) of FOLR3 SNP+ CD34+ and K562 cells were higher than those of control, siFOLR3 and FOLR3 SNP-groups. Folate-free medium could cancel the difference of colony-forming units among different groups of K562 cells ( Figure 2D). Besides, we found FOLR3 SNP+ K562 cells exhibited the lowest BCR-ABL1 expression in the four groups ( Figure 2E) but higher sensitivity to TKI (Figure 2H). Metabolomics analysis of FOLR3 SNP+/-CD34 + indicated they were enriched in fatty acid metabolism, biosynthesis, and elongation pathways ( Figure 2F). The glycerophospholipid and fatty acid biosynthesis were more active in FOLR3 SNP+ K562 cells ( Figure 2F). Subcutaneous tumorigenesis by K562 cells and small animal PET scanning were performed on three representative mice of each group at 21 days after engraftment. An increase in the maximal standard uptake value of 18 F-FDG was found in FOLR3 SNP+ group ( Figure 2G).
To explore the outcome of continuously proliferated cells, we conducted colony forming assay on cells cultured for 21 days after lentivirus transfection. The colonyforming capacity of FOLR3 SNP+ CD34 + cells remarkably decreased compared to that of siFOLR3 and FOLR3 SNP-cells ( Figure 2I). The percentages of FOLR3 SNP-CD34 + cells at G2/M stage were higher than that of the FOLR3 SNP+ counterpart ( Figure 2J). Compared with the other arms, the proliferation ( Figure 2K), CyclinE2 and p21 ( Figure 2L, FigS3) of FOLR3 SNP+ CD34 + cells noticeably decreased, while ROS levels ( Figure 2M) and senescence-associated secretory phenotype-related genes IL-6 and MMP9 substantially increased ( Figure 2N, Figure  S3). Compared with CD34 + cells from cord blood, healthy mobilization and non-treated CML patients, we found that   , and Rot/AA (antimycin/rotenone) were added to the system. Data were obtained using the Seahorse XF24 analyzer. F, Quantified indexes, maximal respiration capacity and spare respiration capacity of CD34 + cells with different FOLR3 SNP were calculated by the Seahorse XF24 analyzer. G, Maximal respiration capacity and proton leakage in K562 cells with different FOLR3 SNP were quantified by the Seahorse XF24 analyzer. H, SHMT2 mRNA levels in CD34 + and K562 cells with different FOLR3 SNP were determined by qRT-PCR. The results are presented as 2 −ΔΔct . P values were tested by the paired t-test. * P < .05, ** P < .01, *** P < .001 CD34 + cells in CML patients treated with TKI presented more senescence phenotypes (P < .05) ( Figure 2O). The longer TKI treatment lasted, the higher of β-gal activity exhibited, and the fewer colonies produced ( Figure 2P).
To explore the mechanism, we performed RNA-seq for CD34+ cells from 3 newly diagnosed CML patients and K562 cells ( Figure 3A), which were transfected with lentivirus to differentially express FOLR3 SNP (blank, FOLR3 SNP+, FOLR3 SNP-and siFOLR3, Table S3). The 220 upregulated DEGs in FOLR3 SNP+ CD34+ vs blank comparison were significantly enriched in mitochondrionrelated processes, such as ATP hydrolysis, ATPase activity and mitochondrial electron transport ( Figure 3B; Figure S4C). Similarly, 229 upregulated DEGs identified in the FOLR3 SNP+ K562 versus blank comparison were also enriched in mitochondrion-related processes ( Figure 3B). Besides, the FOLR3 SNP+ groups had significantly higher mitochondrion-related gene expression, as well as oxidative phosphorylation, ATP synthesis and ROS production ( Figure 3C).
Transmission electron microscopy indicated that FOLR3 SNP+ K562 cells exhibited rich amounts of lipid droplets, which were not observed in SNP+ CD34+ cells ( Figure 3D). The FOLR3 SNP-groups had fewer mitochondria than SNP+ groups ( Figure 3D). FOLR3 SNP+ CD34 + and K562 cells had higher oxygen consumption rates ( Figure 3E), maximal respiration and spare respiratory capacity ( Figure 3F; Figure S5A). In K562 cells, FOLR3 SNP significantly increased maximal respiration and proton leakage; however, differences in basal respiration between each group were not notable ( Figure 3G; Figure S5A). Notably, mitochondrial membrane potential and ATP concentrations of FOLR3 SNP+ CD34 + cell was significantly higher ( Figure S5B, C). Serine hydroxymethyltransferase 2, a key enzyme in folate-dependent mitochondrial translation and oxidative phosphorylation, 8 highly expressed in SNP+ CD34 + and K562 cells ( Figure 3H; Figure S5D).
In conclusion, we detected FOLR3 SNP rs139130389 only in the TFR group. FOLR3 SNP+ CML cells proliferated actively and exhibited greater colony-forming ability via elevating mitochondrion activity. Proliferating cells were relatively lower BCR-ABL1 but more sensitive to TKI. Further, continuous proliferation of stem cells would induce replicative senescence. 9 We speculate patients achieve TFR because their aging CML-LSCs failed to produce malignant clones after discontinuation. As a result, CML-LSCs senescence might be a key point of discontinuation and the time needed to take medicine would be personalized for CML-LSCs to accumulate senescence. The idea of senescence will provide an outlook on future challenges of CML-LSCs elimination.

A C K N O W L E D G M E N T S
The authors thank all of the doctors and patients who have contributed to this work. We would like to thank Pei Zhang and An-Na Du from The Core Facility and Technical Support, Wuhan Institute of Virology, for their help with producing transmission electron microscope micrographs.

C O N F L I C T O F I N T E R E S T
The authors declare no conflict of interest.

F U N D I N G
This work was supported by the National Natural Science Foundation of China (31822030 and 31771458 to A.Y.G., 81500136 to X.Z., 81700142 to Q.L., 81873440 to Y.Y.), Na Shen, Teng Liu and Wen Liu contributed equally to this work.
Yong You, An-Yuan Guo and Xiaojian Zhu contributed equally for the last authorship.