Deficiency of Setd2 in mesenchymal stem cells facilitates the progression of myelodysplastic syndrome to leukemia

Deficiency of Setd2 in mesenchymal stem cells facilitates the progression of myelodysplastic syndrome to leukemia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Deficiency of Setd2 in mesenchymal stem cells facilitates the progression of myelodysplastic syndrome to leukemia Rou-Jia Wang, Zi-Juan Li, Bing-Yi Chen, Juan Guo, Ying Tao, Hong-Ping Li, and 17 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7140205/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Apr, 2026 Read the published version in Molecular Medicine → Version 1 posted 14 You are reading this latest preprint version Abstract While previous studies have indicated that H3K36me3, which is mediated by Setd2 , may regulate the cell fate of mesenchymal stem cells (MSCs) both in vitro and in vivo, the specific role of MSCs in the onset and progression of MDS remains unclear. Thus, the histone methyltransferase Setd2 is implicated in MDS-associated leukemia. This study utilized NUP98-HOXD13 (NHD13) mice with targeted deletion of Setd2 in MSCs. Here, we found that Setd2 -deficient mice undergo faster leukemia transformation than control mice do, as evidenced by the abnormal differentiation of hematopoietic stem progenitor cells in the bone marrow, abnormal hematopoiesis, and increased number of blast cells. Compared with that of control mice, the morphology of NHD13 mouse MSCs with Setd2 deficiency was irregular, and the support function of hematopoietic cells was compromised. This study demonstrated that targeted deletion of Setd2 in MSCs has a beneficial effect on the progression of MDS. Furthermore, we identified increased expression of coagulation factor XII as a key leukemic transformation mediator in Setd2 -deficient MSCs. Moreover, we found that Setd2 expression is significantly lower in high-risk MDS patients than in low-risk MDS patients, further suggesting that the targeted deletion of Setd2 in MSCs is associated with MDS progression. Collectively, our results suggest that Setd2 in MSCs suppresses MDS progression to leukemia through coagulation factor XII-mediated suppression of the stem cell support capacity of MSCs. Overall, this study sheds light on the pathogenesis of MDS and provides a therapeutic strategy for regulating the microenvironment in patients with MDS who cannot be cured by haematopoietic stem cell transplantation. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Myelodysplastic syndrome (MDS) comprises a heterogeneous group of malignant clonal hematologic disorders that originate from hematopoietic stem or progenitor cells. Characterized by ineffective hematopoiesis and a high risk of transformation to acute leukemia, these conditions present significant clinical challenges. The pathogenesis of MDS encompasses somatic cell mutations, chromosomal abnormalities, epigenetic modifications, and alterations in the bone marrow microenvironment [ 1 ]. Mesenchymal stem cells (MSCs) constitute a critical component of the hematopoietic microenvironment, playing an essential role in maintaining and promoting self-renewal and regulating the differentiation of hematopoietic stem cells via the production and secretion of hematopoietic factors and cytokines [ 2 ]. MSCs possess immunomodulatory properties, enabling them to exert regulatory and inhibitory effects through direct or indirect interactions with a variety of immune cells, including T cells, B cells, natural killer (NK) cells, and dendritic cells derived from monocytes and neutrophils [ 3 ]. MSCs constitute a critical component of the bone marrow hematopoietic microenvironment, regulating the activity of hematopoietic cells to facilitate the development of normal blood cells and support the survival and proliferation of progenitor cells [ 4 ]. MDS clones can induce the transformation of normal MSCs into MDS-like MSCs, highlighting the significant role of MDS clones in this process. Simultaneously, MSCs exert a counteracting effect on MDS cells, and stromal cell clones serve as the origin of MDS clones in certain patients. Consequently, further investigations into the mechanisms of MDS development and transformation from the perspective of reprogrammed MSC clones may provide novel insights for treating MDS within the microenvironment. The histone methyltransferase Setd2 is the sole enzyme capable of catalyzing H3K36me3 in mammals. Setd2 associates with elongating RNA polymerase II and generates H3K36me3-modified nucleosomes within actively transcribed genomic regions, thereby providing docking sites for various critical chromatin regulators. Our previous research demonstrated that deletion of Setd2 in hematopoietic cells of NHD13 transgenic mice accelerated the progression from MDS to leukemia, indicating that Setd2 functions as a tumor suppressor during the NHD13-driven transformation from MDS to leukemia [ 6 ]. Thus, in this study, we built upon our previous work by constructing a mouse model of MSC-targeted knockout of Setd2 . Therefore, the aim of this study was to evaluate the regulatory effect of MSC-targeted knockout of Setd2 on hematopoietic cells, explore the role of the microenvironment in hematopoietic cells during the incidence and development of MDS, and explore new therapeutic directions. Materials and methods Mice The background used for the mice in this study was C57BL/6J. The sources of each mouse were as follows: Setd2 fl/fl and NHD13 mice were donated by Lan Wang and Xiaojian Sun Laboratory, Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences; Prx1-Cre mice and Lep-Cre mice were donated by Bo Zhou Laboratory of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. All the mice were raised in an SPF-level animal room at the Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences. This research was approved by the Animal Care and Use Committee of Sixth People's Hospital affiliated with Shanghai Jiao Tong University School of Medicine (2022–0239). Patients The cDNA and protein samples of 10 MDS patients and 3 control subjects (Supplementary Table 1) were taken from the Sixth People's Hospital affiliated with Shanghai Jiao Tong University School of Medicine. The research was approved by the Ethics Committee of Sixth People's Hospital affiliated with Shanghai Jiao Tong University School of Medicine (2022-YS-051). Cell culture Murine MSCs were cultured in α-MEM (Dakewe Biotech Co., China) supplemented with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin. Mouse mononuclear cells were cultured in 1640 medium (Meilunbio, China) supplemented with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin. Establishment of the Lep-Cre NHD13 Setd2 △/△ transplantation model Donor: NHD13, regardless of sex, 3 mice. Receptors: Setd2 fl/fl , female, 5 mice; Lep-CreSetd2 △/△ , female, 6 mice. The receptors were divided into two groups: the experimental group ( Lep-Cre NHD13 Setd2 △/△ group) and the control group (NHD13 Setd2 fl/fl group). The recipient mice were placed in a breathable paper box and subjected to γ radiation exposure at a total dose of 9.5 Gy. (The radiation source was provided by the Animal Room of the Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences). After irradiation, donor cells were injected into the tail vein of the mice. In the control group (NHD13 Setd2 fl/fl group, n = 5), each mouse was injected with 3*10 6 (1.5*10 7 /mL bone marrow cells) bone marrow cells. In the experimental group ( Lep-Cre NHD13 Setd2 △/△ group, n = 6), each mouse was injected with 3*10 6 (1.5*10 7 /mL bone marrow cells) bone marrow cells. SA-β-gal assay The medium was removed from the Petri dish, and the cells were fixed and flushed. Staining solution (Beyotime, China) was added to the cells, which were subsequently incubated at 37°C overnight and observed under a microscope. Cell cycle analysis The cell samples were fixed overnight in ethanol cooled at -20°C. The cells were collected the next day and incubated at 37°C for 30 minutes after RNase treatment. The cells were washed with PBS, PI was added, the cells were rinsed, and then, flow cytometry analysis was performed. Apoptosis analysis Apoptosis was measured via a PI/Annexin V-APC kit (Sangon Biotech, China). The instructions of the reagent kit were followed to perform the experiment. The data were analysed via flow cytometry. Real-time PCR analysis The RNA samples were collected via the FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme, China). Follow the steps in the manual to operate it. HiScript II Q RT SuperMix (Vazyme, China) was used for reverse transcription of RNA, and ChamQ SYBR qPCR Master Mix (Vazyme, China) was used for real-time PCR analysis. The primers used are listed in Table 2 of the Supplementary Material. Western blotting analysis The samples were separated via SDS‒PAGE and subjected to constant pressure electrophoresis. The protein was transferred onto a PVDF membrane under constant current. The PVDF membrane was removed for sealing. The membranes were incubated with the primary antibody overnight, washed 3 times, and incubated with the secondary antibody. The membrane was washed 3 times and developed with an enhanced chemiluminescence (ECL) developer. Wright's staining A peripheral blood smear or bone marrow/spleen smear was taken and dried naturally, and the staining range was delineated with an immunohistochemical pen. An appropriate volume of Wright's staining solution was added for 1 minute, and 2 times the volume of the buffer solution was added for 5–10 minutes. The samples were rinsed under running water and observed with a regular optical microscope with a neutral resin seal. Hematoxylin‒eosin (HE) staining After the paraffin slices were baked in a 37°C oven for 2 hours, they were dehydrated in xylene and 100% ethanol, 95% ethanol, 85% ethanol, or 75% ethanol. The samples were dyed with hematoxylin dye for 3–5 minutes and rinsed with tap water for 1–2 minutes. The 1% hydrochloric acid alcohol mixture was differentiated for a few seconds, the 1% ammonia solution was returned to blue for a few seconds, and then the mixture was washed with water for 1–2 minutes. The samples were dyed with eosin dye solution for 1–3 minutes. Anhydrous ethanol (I) and (II) are rehydrated for 5 minutes each, and xylene (I) and (II) are transparent for 5 minutes. The samples were sealed with neutral gum and subjected to microscopic examination. Giemsa fibrosis staining The tissue was held in a 10% formaldehyde fixative and was routinely dehydrated and embedded, sliced, and dewaxed in water. A Reticulin Stain Kit (Solarbio, China) was used for staining, and the experiments were conducted according to the instructions. Cell proliferation assay The cell suspensions were prepared and added to a 96-well plate, which was precultured in an incubator. A Cell Counting Kit-8 (Dojindo Laboratories, Japan) was used to measure the absorbance of each sample, after which a proliferation curve was drawn according to the corresponding formula. Statistical analysis The data are presented via GraphPad Prism 8 software, and all measurement data are expressed as the means ± standard errors. All experimental data were obtained from at least three independent experiments, and significant differences between groups were analysed via ANOVA and t tests. Survival analysis was performed via the log-rank test. P < 0.05 was considered statistically significant. Results Setd2 expression is related to the MDS risk level We collected MSCs from three control patients (MALT lymphoma patients), four low-risk MDS patients, and six high-risk MDS patients to measure SETD2 expression via q‒PCR and western blot (WB) analysis. The results revealed that SETD2 expression was significantly lower in high-risk MDS patients than in low-risk MDS patients (Fig. 1 A and 1 B). Moreover, when the complete blood counts of MDS patients with low SETD2 expression or normal SETD2 expression were compared, we found that both the platelet and white blood cell counts of MDS patients with low SETD2 expression decreased but the CD34 + expression level increased (Fig. 1 C, 1 D and 1 E), which suggests that low SETD2 expression is associated with MDS progression. Through Wright's staining of bone marrow smears, we found that patients with low expression of SETD2 exhibited significant pathological hematopoiesis in erythroid and megakaryocyte lineages (Fig. 1 F and 1 G are simple examples). In addition, the corresponding hematoxylin‒eosin staining of bone marrow revealed clustered blast cells in patients with low expression of SETD2 (Fig. 1 H and 1 I are simple exemplars). MSC-targeted knockout of Setd2 shortens survival and accelerates the progression of MDS in mice We crossed Lep-Cre/Prx1-CreSetd2 △/△ mice with NHD13 mice to generate Lep-Cre/Prx1-Cre NHD13 Setd2 △/△ mice as the experimental group (NHD13 mice as the control group). Lep-Cre/Prx1-Cre mice were identified as MSC-targeted knockout mice. Lep-Cre/Prx1-Cre has a corresponding binding site only on MSCs, whereas it is absent on hematopoietic stem progenitor cells. Consequently, this approach has been widely adopted in MSC studies. First, we validated the knockout efficiency in primary murine MSCs. The mRNA expression of Setd2 in murine MSCs was assessed via qPCR analysis, which revealed a significant reduction in Setd2 expression in MSCs from Prx1-Cre NHD13 Setd2 Δ/Δ mice (Fig. 2 A). Through WB detection of Setd2 expression and H3K36Me3 levels in mice, we found that the expression of Setd2 and the level of H3K36Me3 in Prx1-Cre NHD13 Setd2 △/△ mice were also significantly reduced (Fig. 2 B). We monitored the survival of primary Lep-Cre/Prx1-Cre NHD13 Setd2 Δ/Δ mice (n = 15) and NHD13 mice (n = 15). Our results demonstrated that the median survival of Lep-Cre/Prx1-Cre NHD13 Setd2 Δ/Δ mice (265/192 days) was significantly shorter than that of NHD13 mice (301 days). Notably, the survival of Prx1-Cre NHD13 Setd2 Δ/Δ mice (median survival 192 days) was markedly shorter than that of NHD13 mice. These findings indicate that MSC-targeted knockout of Setd2 promotes the initiation and progression of MDS to leukemia in NHD13 mice (Fig. 2 C and Supplementary Table 3). Therefore, we focused on Prx1-Cre NHD13 Setd2 △/△ mice as the research model and conducted corresponding research on Lep-Cre NHD13 Setd2 △/△ mice. Compared with the spleen size and weight of primary WT mice, NHD13 mice, and Prx1-Cre NHD13 Setd2 △/△ mice at the age of 5 months, the spleen size and weight of the mice in the knockout group were significantly greater than those of the NHD13 mice (Fig. 2 D and 2 E). Next, we compared the expression of c-Kit + in the mice from the three groups. We performed flow cytometry analysis to detect the expression of c-Kit + in primary WT mice, NHD13 mice, and Prx1-Cre NHD13 Setd2 △/△ mice. We found that the percentage of the hematopoietic stem and progenitor cell marker c-Kit + in the bone marrow and spleen of Prx1-Cre NHD13 Setd2 △/△ mice was significantly greater than that in NHD13 mice (Fig. 2 F and 2 G), indicating that the leukemia of the mice in the knockout group tended to be greater than that of the NHD13 mice. These results suggest that microenvironmental remodelling mediated by targeted knockout of Setd2 in MSCs controls the development of hematopoietic cells. MSC-targeted knockout of Setd2 hinders the differentiation of multiple hematopoietic cell types in mice The peripheral blood of the mice in the three groups was collected every four weeks from the sixth week after birth. The main follow-up procedures included peripheral blood routine, erythroid differentiation, myeloid differentiation, and lymphoid (B, T cells) differentiation. Statistical analysis of the results of routine blood analysis revealed that, starting at week 14, the white blood cell counts of the mice in the Setd2 -targeted knockout group increased significantly. However, the platelet counts in the NHD13 group and targeted knockout mouse group remained lower than those in the WT group did, and the platelet count in the knockout group decreased more significantly (Fig. 3 A). There was no significant difference in hemoglobin follow-up between the NHD13 mouse group and the knockout mouse group. A separate comparison of the blood routine of the mice followed up to week 22 (Fig. 3 B) revealed that the white blood cell counts of the Prx1-Cre NHD13 Setd2 △/△ mice were significantly greater than those of the NHD13 and WT mice. The hemoglobin levels of NHD13 mice and Prx1-Cre NHD13 Setd2 △/△ mice were lower than those of WT mice, but there was no significant difference between NHD13 mice and Prx1-Cre NHD13 Setd2 △/△ mice. Compared with WT mice, both NHD13 mice and Prx1-Cre NHD13 Setd2 △/△ mice presented a decrease in platelet count, and compared with NHD13 mice, Prx1-Cre NHD13 Setd2 △/△ mice presented a significant decrease in platelet count. The aforementioned follow-up results indicate that Prx1-Cre NHD13 Setd2 △/△ mice exhibit elevated leukocyte levels and a significant reduction in platelet count. Consequently, compared with NHD13 mice, Prx1-Cre NHD13 Setd2 △/△ mice demonstrate accelerated progression toward leukemia and impaired megakaryocyte development. All of these factors lead to shorter survival times in Prx1-Cre NHD13 Setd2 △/△ mice than in NHD13 mice. In terms of myeloid differentiation, as shown in Fig. 3 C, the percentage of Mac-1 + Gr-1 + cells in the peripheral blood, spleen, or bone marrow was greater in the Setd2 knockout group than in the NHD13 group, indicating the blocking effect of Setd2 fl/fl targeted knockout on myeloid differentiation. The number of early myeloid cells in the bone marrow increases, indicating a tendency toward leukemic transformation. With respect to lymphocyte differentiation, as illustrated in Fig. 3 C, the number of bone marrow T cells in the Prx1-Cre NHD13 Setd2 △/△ group was significantly lower than that in the NHD13 group. Although there was no significant difference in the expression of B cells between Prx1-Cre NHD13 Setd2 △/△ mice and NHD13 mice, the expression of Pro-B and Pre-B cells was significantly greater in the Prx1-Cre NHD13 Setd2 △/△ mouse group (Figure S1 A and S1B). As the disease progresses, lymphocyte differentiation is also inhibited, which has an inhibitory effect on the development and function of the immune system in mice. Compared with those in the NHD13 group, erythroid differentiation in the bone marrow and spleen of the Setd2 knockout group was inhibited, and the proportion of mature red blood cells in the RIV was significantly lower than that in the NHD13 group (Fig. 3 D and 3 E). For primary mice with another Cre system, Lep-Cre NHD13 Setd2 △/△ , owing to its slower onset than Prx1-Cre NHD13 Setd2 △/△ , we included primary mice at approximately 1 year of age, control mice, and normal mice for the study. We found that the proportion of mature red blood cells in the experimental group decreased compared with that in the control group, and the differentiation of the red blood system was hindered, with a slight increase in the proportion of c-Kit + cells (Figure S2 A and S2B). Afterwards, we compared myeloid and lymphoid differentiation between the Lep-Cre NHD13 Setd2 △/△ group and the control group and found that the number of early myeloid cells in the experimental group increased and that myeloid differentiation was hindered (Figure S2 C). Moreover, the number of B and T lymphocytes in the Lep-Cre NHD13 Setd2 △/△ group was lower than that in the control group, indicating that the differentiation of the lymphoid lineage was also hindered (Figure S2 C). MSC-targeted knockout of Setd2 tends toward leukemia transformation We subsequently conducted further research on bone marrow hematopoietic stem progenitor cells (HSPCs) from the WT, NHD13, and Prx1-Cre NHD13 Setd2 △/△ groups to investigate the effects of MSC genetic aberrations on their hematopoietic support function, as well as the effects of changes in the microenvironment on hematopoietic cell function. We isolated Lin − cells from the entire bone marrow cell population (Fig. 4 A). Figure 4 B clearly shows that the number of Lin- cells in the Prx1-CreNHD13Setd2 △/△ cohort was notably elevated. Additionally, there was an increase in the proportion and absolute count of LK cells. After targeted knockout of Setd2 in MSCs, the number of early immature cells increased, and hematopoietic stem progenitor cell differentiation shifted toward progenitor cells in these mice. We also observed that the number of hematopoietic stem cells (HSCs) decreased and that hematopoietic function was damaged. This may have caused Prx1-Cre NHD13 Setd2 △/△ mice to have an increase in early immature cells and impaired differentiation of the erythroid myeloid lineage compared with NHD13 mice, which accelerated the transformation to leukemia and reduced the number of platelets. These results suggest that changes in the microenvironment caused by MSCs counteract the function of hematopoietic cells, affecting disease incidence and development. Using flow cytometry analysis, LSK cells can be subdivided by the markers CD135, CD34, CD48, and CD150, and hematopoietic stem cells can be categorized into three categories: multipotent blood donors (MPPs), long-range hematopoietic stem cells (LT-HSCs), and short-term hematopoietic stem cells (ST-HSCs). In the hematopoietic system, long-term refilled hematopoietic stem cells, situated at the apex of the hematopoietic hierarchy, maintain a reservoir of primitive multipotentials throughout their lifetime through their self-renewal and asymmetric cell division potentials. The loss of normal LT-HSC and ST-HSC functions is a hallmark of natural stem cell aging and several hematopoietic disorders, most notably the development and progression of hematological malignancies. MPPs are pluripotent progenitor cells derived from hematopoietic stem cells that have lost their self-renewal potential but can still fully differentiate into all lineages. As shown in Fig. 4 B, compared with NHD13 mice, Prx1-Cre NHD13 Setd2 △/△ mice presented a significant increase in the absolute number of MPPs and ST-HSCs, whereas the number of LT-HSCs decreased. These results suggest that, after targeted knockout of Setd2 in MSCs, the long-term hematopoietic capacity of murine hematopoietic stem cells is impaired and that short-term hematopoietic function is enhanced compared with the reactivity of per-knockout cells. MPP further produces low-potential progenitor cells, commonly known as lymphoid and myeloid progenitor cells (CLPs and CMPs). We performed flow cytometry analysis to further classify haematopoietic progenitor cells (KKs) into three categories—GMP, MEP, and CMP—using markers such as CD34 and CD1632. An examination of the outcomes depicted in Fig. 3 B revealed that, compared with the NHD13 cohort, the selected knockout of Setd2 in MSCs resulted in an increase in partially differentiated MEP and GMP cells within the myeloid progenitor cell population. The expression of CLP was significantly lower in the Prx1-Cre NHD13 Setd2 △/△ group than in the NHD13 group, indicating that excessive proliferation of the myeloid system in the bone marrow inhibited the proliferation of the lymphoid system (Figure S1 C and S1D). These results suggest that, compared with the NHD13 group, the Setd2 knockout group has differentiated obstructions and impaired hematopoietic function. For the primary mice with another Cre system, Lep-Cre NHD13 Setd2 △/△ , the hematopoietic stem progenitor cell population also exhibited a decrease in the proportion of stem cells and progenitor cells and hindered stem progenitor cell differentiation (Figure S2 D). Further grouping of hematopoietic stem cells and hematopoietic progenitor cells was subsequently performed. As shown in Figure S2 E and S2F, the absolute numbers of each group of progenitor cells (GMP/MEP/CMP) in the experimental group decreased compared with those in the control group, whereas the absolute numbers of LT-HSCs and ST-HSCs decreased, indicating a trend toward bone marrow failure. Thus, when the Lep-Cre NHD13 Setd2 △/△ mice and Prx1-Cre NHD13 Setd2 △/△ mice were compared, the Prx1-Cre NHD13 Setd2 △/△ mice tended toward leukemic transformation, whereas the Lep-Cre mice tended toward the development of ineffective hematopoiesis and bone marrow failure. Next, we studied the morphology of the mice. We performed Wright's staining on peripheral blood smears, bone marrow cells and spleen cells, and the bone marrow, spleen, and liver biopsy tissues of the mice were sliced and subsequently subjected to hematoxylin and eosin (HE) staining. Wright staining (Fig. 4 C) revealed that the peripheral blood smears of the knockout group exhibited notable increases in naive neutrophils and erythrocytes compared with those of the NHD13 group. A bone marrow smear revealed changes in the number of megaloblastic cells in the knockout group, with an increase in early-stage myeloid cells. The same is true for spleen cells. H&E staining analysis (Fig. 4 D) revealed that the proportion of hematopoietic cells in the bone marrow of the Setd2 knockout group mice decreased, with venous sinus dilation and a significant increase in early myeloid cells. There was a significant increase in the number of spleen megakaryocytes in the Setd2 knockout group. Liver cell proliferation was suppressed in mice in the Setd2 knockout group, and extramedullary hematopoietic cells infiltrated these cells. Since the increase in megakaryocytes is closely related to fibrosis, Gomori fibrosis staining was performed on spleen sections from the three groups of mice to determine the degree of fibrosis. As shown in Fig. 4 E, compared with the NHD13 group, the Setd2 knockout group presented a significant increase in the number of splenic fibre strands and aggravated fibrosis. A comparison of the MSC morphology of the mice in the three groups (Fig. 4 F) revealed that the MSC morphology of the Setd2 knockout group was irregular, and aging staining analysis suggested that MSC aging was faster in the Setd2 knockout group than in the NHD13 group. These results confirm the flow cytometry results presented above and provide additional evidence of the inhibitory effect of MSC-targeted knockout of Setd2 on the differentiation of NHD13 hematopoietic stem cells and the accelerated incidence and development of this disease. To establish a control with primary mice, we established Lep-Cre NHD13 Setd2 △/△ and NHD13 Setd2 fl/fl transplantation mouse models (Figure S3A). We first compared the survival of the mice in the two groups and found no significant difference (Figure S3B). Peripheral blood follow-up was performed on the mice from both groups every four weeks beginning four weeks after transplantation. As shown in Figure S3D, the white blood cells of the mice in both groups significantly increased after transplantation, whereas the haemoglobin and platelet levels gradually decreased. There were no significant differences in the number of peripheral blood red blood cells or c-Kit + cells in the mice, as detected by flow cytometry analysis (Figure S3C and S3E). However, the differentiation of myeloid cells in the mice in the Lep-Cre NHD13 Setd2 △/△ group was hindered compared with that in the NHD13 Setd2 fl/fl group, with an increase in early myeloid cells (Figure S3E). As a continuation of previous studies, the expression of PD-L1 in the peripheral blood of mice in the two groups was detected by flow cytometry analysis[ 7 ]. The expression of PD-L1 in the Lep-Cre NHD13 Setd2 △/△ group was significantly greater than that in the NHD13 Setd2 fl/fl group (Figure S3F), suggesting that MDS-associated tumors escape via PD-1/PD-L1 overexpression[ 7 ]. Therefore, we believe that the impact of haematopoietic stem cell transplantation on the bone marrow microenvironment is limited compared with that of primary stem cell transplantation. Targeted knockout of Setd2 in MSCs may affect the disease progression of MDS through coagulation factor XII (F12) To investigate the possible mechanism by which MSCs with targeted knockout of Setd2 regulate hematopoietic cells, we analysed differential protein expression in the bone marrow supernatants of Prx1-Cre NHD13 Setd2 △/△ mice and NHD13 mice via mass spectrometry analysis (Fig. 5 A). We compared the expression profiles of bone marrow supernatant proteins between the two groups of mice and identified 38 upregulated proteins and 47 downregulated proteins with statistically significant differences. The qPCR results revealed that the expression of 38 proteins whose expression was upregulated in MSCs indicated that F12 was upregulated in Prx1-Cre NHD13 Setd2 △/△ MSCs (Fig. 5 B). After contact with the anion surface, F12 automatically becomes F12a. The heavy chain of F12a consists of six domains, one of which is the Fn1 domain [ 8 ]. The expression of the HSC niche factor CXCL12 and the transcription factors Foxc1 and Ebf3 in Prx1-Cre NHD13 Setd2 △/△ MSCs was significantly increased, but that of another HSC niche factor, SCF, was significantly decreased (Fig. 5 C). Compared with NHD13 Setd2 fl/fl MSCs, Prx1-Cre NHD13 Setd2 △/△ MSCs promoted the inflammatory response and chemotaxis and inhibited stem cell proliferation and mobilization. The gene expression of Fn1 was significantly upregulated according to RNA-seq analysis (Fig. 5 D). Therefore, F12 was selected by combining the upregulated proteins from bone marrow supernatants and upregulated genes identified via RNA-seq analysis (Fig. 5 E). Patients with acute leukemia presented markedly elevated levels of peak thrombin, extracellular trap markers, and F12a. The F12a level is significantly associated with the presence of acute leukemia [ 9 ]. In this study, the Setd2 knockout group presented significantly greater F12 expression in the bone marrow supernatant (Fig. 5 F) and greater Fn1 expression (Fig. 5 G) in the MSCs than did the NHD13 group did, indicating a trend toward leukemia transformation in the Setd2 knockout group. In patients with high-risk MDS, the expression of F12 in the bone marrow supernatant was significantly greater than that in patients with low-risk MDS (Fig. 5 H), and the expression of Fn1 in the MSCs of high-risk MDS patients was significantly greater than that in the MSCs of low-risk MDS patients (Fig. 5 I). These results suggest that targeted knockout of Setd2 in MSCs affects the expression of the F12 protein in the microenvironment through the secretion of Fn1 by MSCs. Targeted knockout of Setd2 impaired the hematopoietic stem progenitor cell support function of MSCs We cocultured murine bone marrow mononuclear cells with murine MSCs and performed cell proliferation, cell cycle, and colony formation assays on the cocultured Lin − c-Kit + mononuclear cells. D-Pro-Phe-Arg-chloromethylketone (PCK), an inhibitor of coagulation factor XII and plasma kallikrein, plays an important role in thrombosis and inflammation [ 10 ]. CCK8 (Cell Counting Kit-8) analysis was performed on mononuclear cells from three coculture groups of mice and on those treated with PCK. The proliferation rate of mononuclear cells in the Setd2 knockout group significantly decreased but increased after PCK treatment (Fig. 6 A). Apoptosis analysis of cocultured mononuclear cells revealed that the early and late apoptosis rates of mice in the Setd2 knockout group increased (Fig. 6 B). The proportion of these cells in the G2/M phase was lower in the NHD13 group than in the WT group, whereas the S phase was significantly prolonged and the G0/G1 phase was shortened. Thus, we speculate that the hematopoietic stem progenitor cells of the mice in the Setd2 knockout group are more likely to be blocked in the G0/G1 phase than those of the mice in the NHD13 group, which are blocked in the S phase (Fig. 6 C). Colony formation assays were performed on hematopoietic stem progenitor cells from three groups of mice after coculture and three groups of mice after PCK. The self-renewal ability of the Setd2 knockout group significantly decreased but increased after PCK treatment (Fig. 6 D). In summary, targeted knockout of Setd2 in MSCs impairs the support function of MSCs through F12, affecting the self-renewal and cell proliferation capabilities of HSPCs (Figure S4). PCK partially rescues the function of HSPCs by inhibiting the effect of F12. Discussion Previous studies have shown that mesenchymal stem cells derived from MDS patients (MDS-MSCs) exhibit reduced proliferation and cloning ability, cell morphological changes, increased cellular senescence, impaired immune regulation, and a reduced ability to support hematopoietic stem cell growth and differentiation [ 11 ]. Compared with normal human MSCs, MDS-MSCs can affect cytokines in the microenvironment and can be affected by an aberrant microenvironment and therapeutic drugs, resulting in certain functional changes and further affecting the progression of MDS [ 12 ]. In our study, we first found that mice with Setd2 knockout had significantly shorter survival times. This result aligns with other previous Setd2 results. We previously reported that deletion of Setd2 in hematopoietic cells of NHD13 transgenic mice accelerates the progression of MDS to leukemia, indicating that Setd2 is a tumor suppressor in the process of NHD13-driven MDS to leukemia transformation [ 6 ]. SETD2 mutations/variants are closely associated with overall survival (OS), and they have been identified as risk factors for progression-free survival (PFS), especially with low expression of SETD2 [ 13 ]. The Setd2 variant has been reported to be a risk factor for poor prognosis in patients with AML. The SETD2 levels appeared to be higher than the HR-MDS levels for the LR-MDS in both the transcript and protein measurements. During the transition from MDS to AML, there is often an increase in c-Kit + expression, leading to splenomegaly in mice. As expected, mice in the Setd2 knockout group presented a significant increase in the absolute value of white blood cells and c-Kit + expression, with an increase in early-stage myeloid cells in bone marrow smears. Our results revealed that targeted knockout of Setd2 in MSCs affects MDS progression and survival in mice with MDS. The impaired support function of MSCs with mutations in hematopoietic stem progenitor cells is manifested in several ways. We found that targeted knockout of Setd2 in MSCs resulted in an increase in peripheral blood white blood cells, a decrease in hemoglobin and platelet levels, and an obstruction of differentiation of the erythroid, myeloid, and lymphoid lineages in mice. Clustering analysis of hematopoietic stem progenitor cells revealed an increase in hematopoietic progenitor cells, a decrease in hematopoietic stem cells, and a decrease in long-term hematopoietic capacity. In particular, there are barriers to maturation and differentiation in myeloid cells, resulting in an increase in immature cells and a tendency for MDS to transform into leukemia. On the basis of the decrease in LT-HSCs and increase in ST-HSCs shown in Fig. 3 and the increase in apoptosis in MSCs cocultured with Setd2 knockout mononuclear cells shown in Fig. 4 , the cell cycle was blocked in the G0/G1 phase. We speculate that hematopoietic stem progenitor cells with differentiation disorders fail to differentiate appropriately and eventually undergo apoptosis, resulting in acute compensatory hematopoiesis in the bone marrow in the short term, long-term impaired hematopoietic function, and pathological hematopoiesis and hematopoiesis failure. Our study sheds light on the impact of the bone marrow microenvironment on the pathogenesis and progression of MDS from the MSC perspective. Research has shown that the TET2, KDM6A, BCOR, EZH2 and ASXL genes in MDS-MSCs are prone to mutation, and DNA methylation and chromosomal abnormalities frequently occur in MSCs, confirming that there is a correlation between genetic variation in DNA methylation genes and random chromosomal losses and the pathogenesis of MDS [ 14 ]. Moreover, loss of Setd2 promotes Kras-induced acinar-to-ductal metaplasia and epithelial‒mesenchymal transition during pancreatic carcinogenesis [ 15 ]. Notably, Setd2 deficiency sensitized KRAS-mutant lung cancer to histone chaperone inhibition [ 16 ]. Setd2 frequently cooccurs with IDH2, NRAS and CEBPA mutations [ 17 ]. The histone methyltransferase Setd2 is a tumor suppressor that functions by trimethylating lysine 36 in histone H3. A recent study confirmed that loss-of-function Setd2 mutations facilitate the initiation of leukemia and impair DNA damage recognition, thereby contributing to therapy resistance [ 18 ]. Another study demonstrated that Setd2 is required for the self-renewal of HSCs and that Setd2- deficient HSCs contribute to the development of MDS [ 19 ]. Our results are consistent with these findings. As shown by the colony-forming unit (CFU) assay in Fig. 4 , the self-renewal capacity of hematopoietic stem progenitors was significantly reduced after targeted knockout of Setd2 in MSCs. In addition, we observed that Setd2 expression was significantly lower in high-risk MDS patients than in low-risk MDS patients (Fig. 5 G and 5 H), corroborating our findings in murine models. Targeted knockout of Setd2 in MSCs affects hematopoietic stem progenitor cells through aberrant secretion of MSC-produced proteins, thereby impairing their hematopoietic function, but the mechanism is still unclear. Studies have demonstrated that the pathological cells of MDS contain procoagulant substances and that the tissue-clotting kinase carried by MSCs mediates the expression of procoagulant activity in the extracellular vesicles of MSCs, leading to coagulation dysfunction in MDS patients [ 20 ]. The level of F12 is significantly correlated with the presence of acute leukemia [ 9 ]. In our study, we screened for coagulation-related F12 and Fn1 via mass spectrometry and RNA-seq analysis and validated the results via ELISA and qPCR analysis. Thus, we demonstrated that MSCs with targeted knockout of Setd2 upregulate F12 levels in the microenvironment by secreting elevated amounts of Fn1, thereby creating a hypercoagulable environment and promoting MDS progression. PCK can partially rescue the hypercoagulable environment and seemingly reverse the effects of high-F12 conditioned media from Setd2 null cells on the cell cycle. In summary, our study confirms that MSCs interact with HSPCs through the microenvironment and ultimately influence the incidence and development of MDS in a mouse model of MDS with MSC-directed mutations. Through this study, we find a therapeutic approach involving microenvironment regulators for MDS patients harboring mutations that are refractory to allogeneic hematopoietic stem cell transplantation (such as NRAS and KRAS) and strive for greater benefits. Declarations Funding This work is funded by the National Natural Science Foundation of China (Grant no. 8207010691), the Clinical Research Plan of SHDC (NO. SHDC2020CR6004) and the Shanghai Municipal Science and Technology Commission, Science and Technology Innovation Action Plan (Grant 24YF2756500). Ethics approval and consent to participate All animal experiments in our study were carried out in accordance with the Helsinki Declaration, and approved by the Animal Care and Use Committee of Sixth People's Hospital affiliated with Shanghai Jiao Tong University School of Medicine (2022--0239). The research was approved by the Ethics Committee of Sixth People's Hospital affiliated with Shanghai Jiao Tong University School of Medicine (2022-YS-051). Consent for publication Not applicable. Competing interests The authors declare no competing interests. Conflict of interest The authors declare that they have no conflicts of interest. Author contributions Conceptualization: Chun-Kang Chang, Lan Wang and You-Shan Zhao Methodology: Rou-Jia Wang, Zi-Juan Li, Bing-Yi Chen, Juan Guo, Ying Tao, Hong-Ping Li and Ming-Yue Fei Investigation: Rou-Jia Wang, Zi-Juan Li, Bing-Yi Chen, Juan Guo, Ying Tao, Hong-Ping Li and Ming-Yue Fei, Mu-Ying Zhao, Lei Shi, Si-Da Zhao, Zheng Zhang, Ji-Ying Su, Lu-Xi Song, Qi He, Dong Wu, Ling-Yun Wu, Jia-Ying Zhang, Li-Juan Zong, Xiao-Jian Sun Visualization: Bin-He Chang Writing—original draft: Rou-Jia Wang and Zi-Juan Li Writing—review and editing: Chun-Kang Chang, Lan Wang and You-Shan Zhao References Awada H, Thapa B, Visconte V. The genomics of myelodysplastic syndromes: origins of disease evolution, biological pathways, and prognostic implications. Cells. 2020;9(11):2512. Lyu T, Zhang B, Li M, Jiao X, Song Y. Research progress on exosomes derived from mesenchymal stem cells in hematological malignancies. Hematol Oncol. 2021;39(2):162–9. Zhao J, Chen J, Huang F, Wang J, Su W, Zhou J, et al. Human gingiva tissue-derived MSC ameliorates immune-mediated bone marrow failure of aplastic anemia via suppression of Th1 and Th17 cells and enhancement of CD4 + Foxp3 + regulatory T cells differentiation. Am J Transl Res. 2019;11(12):7627–43. Massaro F, Corrillon F, Stamatopoulos B, Nathalie Meuleman L, Lagneaux D, Bron. Aging of bone marrow mesenchymal stromal cells: hematopoiesis disturbances and potential role in the development of hematologic cancers. Cancers (Basel). 2020;13(1):68. Ming H, Sun X-J, Zhang Y-L, Kuang Y, Hu C-Q, Wu W-L, et al. Histone H3lysine 36 methyltransferase Hypb/Setd2 is required for embryonic vascular remodelling. Proc Natl Acad Sci USA. 2010;107(7):2956–61. Chen B-Y, Song J, Chen C-LHS-B, Zhang Q, Xu C-H, et al. SETD2 deficiency accelerates MDS-associated leukemogenesis via S100a9 in NHD13 mice and predicts poor prognosis in MDS. Blood. 2020;135(25):2271–85. Roujia Wang Y, Zhao Z, Li J, Guo S, Zhao L, Song, et al. S100a9 deficiency accelerates MDS-associated tumor escape via PD-1/PD-L1 overexpression. Acta Biochim Biophys Sin (Shanghai). 2023;55(2):194–201. Baglia FA, Jameson BA, Walsh PN. Identification and characterization of a binding site for factor XIIa in the Apple 4 domain of coagulation factor XI. J Biol Chem 1993Feb 25;268(6):3838–44. Kim TY, Gu J-Y, Jung HS, et al. Elevated extracellular trap formation and contact system activation in acute leukemia. J Thromb Thrombolysis. 2018;46(3):379–85. Nickel KF, Long AT, Fuchs TA, Youngil Koh I, Kim. Hyun Kyung Kim. Factor XII as a Therapeutic Target in Thromboembolic and Inflammatory Diseases. Arterioscler Thromb Vasc Biol. 2017;37(1):13–20. Sarhan D, Wang J, Sunil Arvindam U, Caroline Hallstrom MR, Verneris B, Grzywacz, et al. Mesenchymal stromal cells shape the MDS microenvironment by inducing suppressive monocytes that dampen NK cell function. JCI Insight. 2020;5(5):e130155. Bulycheva E, Rauner M, Medyouf H, Theurl I, Bornhäuser M, Hofbauer LC et al. Myelodysplasia is niche:novel concepts Emerg Ther Leuk 2015,29(2):259–68. Li J, Peng Z, Luo F, Chen Y. SET Domain Containing 2 Deficiency in Myelodysplastic Syndrome. Front Genet. 2020;11:794. Bandara WMMS, Rathnayake AJIS, Neththikumara NF, Hemali WW, Goonasekera, Vajira HW, Dissanayake. Comparative analysis of the genetic variants in haematopoietic stem/progenitor and mesenchymal stem cell compartments in de novo myelodysplastic syndromes. Blood Cells Mol Dis,2021(88):102535. Ningning Niu P, Lu Y, Yang R, He L, Zhang J, Shi, et al. Loss of Setd2 promotes Kras-induced acinar-to-ductal metaplasia and epithelia-mesenchymal transition during pancreatic carcinogenesis. Gut. 2020;69(4):715–26. Xie Y, Sahin M, Wakamatsu T, Inoue-Yamauchi A, Zhao W, Han S, et al. SETD2 regulates chromatin accessibility and transcription to suppress lung tumorigenesis. JCI Insight. 2023;8(4):e154120. Zhang X, Wang Z, Sun J, Liu L, Qin J, Huang A, et al. New insights into the clinical characteristics of SETD2-mutated acute myeloid leukaemia. Br J Haematol. 2023;202(1):111–5. Sheng Y, Ji Z, Zhao H, Wang J, Cheng C, Xu W, et al. Downregulation of the histone methyltransferase SETD2 promotes imatinib resistance in chronic myeloid leukaemia cells. Cell Prolif. 2019;52(4):e12611. Zhang Y-L, Sun J-W, Xie Y-Y, Zhou Y, Ping Liu J-C, Song, et al. Setd2 deficiency impairs hematopoietic stem cell self-renewal and causes malignant transformation. Cell Res. 2018;28(4):476–90. Zannoni J, Mauz N,Seyve L, Mathieu Meunier K, Pernet-Gallay J, Brault et al. Tumor microenvironment and clonal monocytes from chronic myelomonocytic leukemia induce a procoagulant climate. Blood Adv 2019,3(12):1868–80. Additional Declarations No competing interests reported. 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(C-E) Blood parameters and CD34\u003csup\u003e+\u003c/sup\u003e expression in individuals with low Setd2 expression and normal Setd2 expression. (F, G) Wright's staining of bone marrow smears from individuals with low Setd2 expression and normal expression. (H, I) Hematoxylin‒eosin staining of the bone marrow of \u003cem\u003eindividuals with\u003c/em\u003e low Setd2 expression and normal expression. *P≤0.05, **P≤0.01, ***P≤0.001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7140205/v1/ecd51f727a895ea891c21ff9.png"},{"id":88419345,"identity":"be0810da-ec9c-4cca-bfa5-c79f20de25e9","added_by":"auto","created_at":"2025-08-06 09:18:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6028813,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMSC-targeted knockout of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSetd2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e shortens survival and promotes the progression of MDS in mice. \u003c/strong\u003e(A, B) qPCR and western blot analysis of \u003cem\u003eSetd2\u003c/em\u003e expression in WT, NHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eand \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003emice (n≥3). (C) Survival time of NHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl \u003c/em\u003e\u003c/sup\u003eand \u003cem\u003eLep-Cre/Prx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△ \u003c/em\u003e\u003c/sup\u003emice. (D, E) Spleen enlargement in NHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice. (F, G) Flow cytometry analysis of c-Kit\u003csup\u003e+ \u003c/sup\u003eexpression\u003csup\u003e \u003c/sup\u003ein the bone marrow and spleen of WT, NHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice. *P≤0.05, **P≤0.01, ***P≤0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7140205/v1/ebf2ada0c7204ea066631c3d.png"},{"id":88419352,"identity":"938863dc-1ff0-4e91-abd0-5f5fd5d8724b","added_by":"auto","created_at":"2025-08-06 09:18:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6051322,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMSC-targeted knockout of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSetd2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e blocked the differentiation of multiple hematopoietic cells in mice. \u003c/strong\u003e(A, B) Complete blood counts of WT, NHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eand \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice. (C) Flow cytometry analysis of Mac-1\u003csup\u003e+\u003c/sup\u003eGr-1\u003csup\u003e+\u003c/sup\u003e, B220\u003csup\u003e+\u003c/sup\u003e and CD3e\u003csup\u003e+ \u003c/sup\u003eexpression in WT, NHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eand \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice. (D, E) Flow cytometry analysis of erythroid expression in WT, NHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl \u003c/em\u003e\u003c/sup\u003eand \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice. *P≤0.05, **P≤0.01.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7140205/v1/94154addadc1fef12e47ac74.png"},{"id":88419344,"identity":"e65d56fd-f034-40f4-a9de-61e3409752ff","added_by":"auto","created_at":"2025-08-06 09:18:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMSC-targeted knockout of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSetd2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e tended toward leukemia conversion. \u003c/strong\u003e(A, B) Flow cytometry analysis of LK and LSK expression in WT, NHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl \u003c/em\u003e\u003c/sup\u003eand \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice. (C) Wright's staining of peripheral blood smears, bone marrow smears and spleen smears. (D) Hematoxylin‒eosin staining of the bone marrow, spleen and liver. (E) Gomori fibrosis staining of spleens from WT, NHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl \u003c/em\u003e\u003c/sup\u003eand \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice. (F) MSC morphology of WT, NHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl \u003c/em\u003e\u003c/sup\u003eand \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice. *P≤0.05, **P≤0.01.\u003c/p\u003e","description":"","filename":"placeholderimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7140205/v1/bbbcbd3b2e6566ebbc2b9089.png"},{"id":88419357,"identity":"2e015337-5f87-4c1d-b051-105aa5368cfe","added_by":"auto","created_at":"2025-08-06 09:18:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3655881,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeted knockout of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSetd2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in MSCs affects the disease progression of MDS through coagulation factor XII (F12). \u003c/strong\u003e(A-C) Comparison of mass spectra and RNA-Seq results between NHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl \u003c/em\u003e\u003c/sup\u003eand \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice. (D, E) ELISA analysis of F12 expression in the bone marrow supernatant and qPCR analysis of Fn1 expression in the MSCs of NHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl \u003c/em\u003e\u003c/sup\u003eand \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice. (F-H) ELISA analysis of F12 expression in the bone marrow supernatant and western blot analysis of Fn1 expression in the MSCs of patients. *P≤0.05, **P≤0.01.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7140205/v1/e8dcaafb039bea6bc48881cb.png"},{"id":88419350,"identity":"3b314a1f-723c-4243-ba69-654778b569c7","added_by":"auto","created_at":"2025-08-06 09:18:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":12177528,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeted knockout of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSetd2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eimpairs the hematopoietic stem progenitor cell support function of MSCs.\u003c/strong\u003e (A-D) Proliferation rates, apoptosis rates, cell cycles and self-renewal ability of mononuclear cells in three groups of mice after coculture and three groups of mice with PCK. *P≤0.05, **P≤0.01.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7140205/v1/3e04edd0b8d558d809cf07f2.png"},{"id":108437716,"identity":"51f6c9bf-f9af-4d9c-b368-4bdadefed736","added_by":"auto","created_at":"2026-05-04 16:02:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":44402634,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7140205/v1/8be683af-92ed-4634-9bbf-476d45433db1.pdf"},{"id":88419342,"identity":"6763e4ae-5208-45d8-9d52-f0df506a58e0","added_by":"auto","created_at":"2025-08-06 09:18:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1558762,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7140205/v1/5af6148d7e6b1ec5dc3f9ec9.pdf"},{"id":88419346,"identity":"d59a9851-1955-4a43-a848-3b63d6ff103a","added_by":"auto","created_at":"2025-08-06 09:18:50","extension":"rar","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4622142,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile2.rar","url":"https://assets-eu.researchsquare.com/files/rs-7140205/v1/a1a2f5464365c141f9b36d9f.rar"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eDeficiency\u003cem\u003e \u003c/em\u003eof \u003cem\u003eSetd2\u003c/em\u003e in mesenchymal stem cells facilitates the progression of myelodysplastic syndrome to leukemia\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMyelodysplastic syndrome (MDS) comprises a heterogeneous group of malignant clonal hematologic disorders that originate from hematopoietic stem or progenitor cells. Characterized by ineffective hematopoiesis and a high risk of transformation to acute leukemia, these conditions present significant clinical challenges. The pathogenesis of MDS encompasses somatic cell mutations, chromosomal abnormalities, epigenetic modifications, and alterations in the bone marrow microenvironment [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Mesenchymal stem cells (MSCs) constitute a critical component of the hematopoietic microenvironment, playing an essential role in maintaining and promoting self-renewal and regulating the differentiation of hematopoietic stem cells via the production and secretion of hematopoietic factors and cytokines [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. MSCs possess immunomodulatory properties, enabling them to exert regulatory and inhibitory effects through direct or indirect interactions with a variety of immune cells, including T cells, B cells, natural killer (NK) cells, and dendritic cells derived from monocytes and neutrophils [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMSCs constitute a critical component of the bone marrow hematopoietic microenvironment, regulating the activity of hematopoietic cells to facilitate the development of normal blood cells and support the survival and proliferation of progenitor cells [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. MDS clones can induce the transformation of normal MSCs into MDS-like MSCs, highlighting the significant role of MDS clones in this process. Simultaneously, MSCs exert a counteracting effect on MDS cells, and stromal cell clones serve as the origin of MDS clones in certain patients. Consequently, further investigations into the mechanisms of MDS development and transformation from the perspective of reprogrammed MSC clones may provide novel insights for treating MDS within the microenvironment.\u003c/p\u003e\u003cp\u003eThe histone methyltransferase \u003cem\u003eSetd2\u003c/em\u003e is the sole enzyme capable of catalyzing H3K36me3 in mammals. \u003cem\u003eSetd2\u003c/em\u003e associates with elongating RNA polymerase II and generates H3K36me3-modified nucleosomes within actively transcribed genomic regions, thereby providing docking sites for various critical chromatin regulators. Our previous research demonstrated that deletion of \u003cem\u003eSetd2\u003c/em\u003e in hematopoietic cells of NHD13 transgenic mice accelerated the progression from MDS to leukemia, indicating that \u003cem\u003eSetd2\u003c/em\u003e functions as a tumor suppressor during the NHD13-driven transformation from MDS to leukemia [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThus, in this study, we built upon our previous work by constructing a mouse model of MSC-targeted knockout of \u003cem\u003eSetd2\u003c/em\u003e. Therefore, the aim of this study was to evaluate the regulatory effect of MSC-targeted knockout of \u003cem\u003eSetd2\u003c/em\u003e on hematopoietic cells, explore the role of the microenvironment in hematopoietic cells during the incidence and development of MDS, and explore new therapeutic directions.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eMice\u003c/p\u003e\u003cp\u003eThe background used for the mice in this study was C57BL/6J. The sources of each mouse were as follows: \u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e and NHD13 mice were donated by Lan Wang and Xiaojian Sun Laboratory, Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences; \u003cem\u003ePrx1-Cre\u003c/em\u003e mice and \u003cem\u003eLep-Cre\u003c/em\u003e mice were donated by Bo Zhou Laboratory of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. All the mice were raised in an SPF-level animal room at the Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences. This research was approved by the Animal Care and Use Committee of Sixth People's Hospital affiliated with Shanghai Jiao Tong University School of Medicine (2022\u0026ndash;0239).\u003c/p\u003e\u003cp\u003ePatients\u003c/p\u003e\u003cp\u003eThe cDNA and protein samples of 10 MDS patients and 3 control subjects (Supplementary Table\u0026nbsp;1) were taken from the Sixth People's Hospital affiliated with Shanghai Jiao Tong University School of Medicine. The research was approved by the Ethics Committee of Sixth People's Hospital affiliated with Shanghai Jiao Tong University School of Medicine (2022-YS-051).\u003c/p\u003e\u003cp\u003eCell culture\u003c/p\u003e\u003cp\u003eMurine MSCs were cultured in α-MEM (Dakewe Biotech Co., China) supplemented with 10% FBS, 100 U/mL penicillin and 100 \u0026micro;g/mL streptomycin. Mouse mononuclear cells were cultured in 1640 medium (Meilunbio, China) supplemented with 10% FBS, 100 U/mL penicillin and 100 \u0026micro;g/mL streptomycin.\u003c/p\u003e\u003cp\u003eEstablishment of the \u003cem\u003eLep-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e transplantation model\u003c/p\u003e\u003cp\u003eDonor: NHD13, regardless of sex, 3 mice. Receptors: \u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e, female, 5 mice; \u003cem\u003eLep-CreSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e, female, 6 mice. The receptors were divided into two groups: the experimental group (\u003cem\u003eLep-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e group) and the control group (NHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e group). The recipient mice were placed in a breathable paper box and subjected to γ radiation exposure at a total dose of 9.5 Gy. (The radiation source was provided by the Animal Room of the Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences). After irradiation, donor cells were injected into the tail vein of the mice. In the control group (NHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e group, n\u0026thinsp;=\u0026thinsp;5), each mouse was injected with 3*10\u003csup\u003e6\u003c/sup\u003e (1.5*10\u003csup\u003e7\u003c/sup\u003e/mL bone marrow cells) bone marrow cells. In the experimental group (\u003cem\u003eLep-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e group, n\u0026thinsp;=\u0026thinsp;6), each mouse was injected with 3*10\u003csup\u003e6\u003c/sup\u003e (1.5*10\u003csup\u003e7\u003c/sup\u003e/mL bone marrow cells) bone marrow cells.\u003c/p\u003e\u003cp\u003eSA-β-gal assay\u003c/p\u003e\u003cp\u003eThe medium was removed from the Petri dish, and the cells were fixed and flushed. Staining solution (Beyotime, China) was added to the cells, which were subsequently incubated at 37\u0026deg;C overnight and observed under a microscope.\u003c/p\u003e\u003cp\u003eCell cycle analysis\u003c/p\u003e\u003cp\u003eThe cell samples were fixed overnight in ethanol cooled at -20\u0026deg;C. The cells were collected the next day and incubated at 37\u0026deg;C for 30 minutes after RNase treatment. The cells were washed with PBS, PI was added, the cells were rinsed, and then, flow cytometry analysis was performed.\u003c/p\u003e\u003cp\u003eApoptosis analysis\u003c/p\u003e\u003cp\u003eApoptosis was measured via a PI/Annexin V-APC kit (Sangon Biotech, China). The instructions of the reagent kit were followed to perform the experiment. The data were analysed via flow cytometry.\u003c/p\u003e\u003cp\u003eReal-time PCR analysis\u003c/p\u003e\u003cp\u003eThe RNA samples were collected via the FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme, China). Follow the steps in the manual to operate it. HiScript II Q RT SuperMix (Vazyme, China) was used for reverse transcription of RNA, and ChamQ SYBR qPCR Master Mix (Vazyme, China) was used for real-time PCR analysis. The primers used are listed in Table\u0026nbsp;2 of the Supplementary Material.\u003c/p\u003e\u003cp\u003eWestern blotting analysis\u003c/p\u003e\u003cp\u003eThe samples were separated via SDS‒PAGE and subjected to constant pressure electrophoresis. The protein was transferred onto a PVDF membrane under constant current. The PVDF membrane was removed for sealing. The membranes were incubated with the primary antibody overnight, washed 3 times, and incubated with the secondary antibody. The membrane was washed 3 times and developed with an enhanced chemiluminescence (ECL) developer.\u003c/p\u003e\u003cp\u003eWright's staining\u003c/p\u003e\u003cp\u003eA peripheral blood smear or bone marrow/spleen smear was taken and dried naturally, and the staining range was delineated with an immunohistochemical pen. An appropriate volume of Wright's staining solution was added for 1 minute, and 2 times the volume of the buffer solution was added for 5\u0026ndash;10 minutes. The samples were rinsed under running water and observed with a regular optical microscope with a neutral resin seal.\u003c/p\u003e\u003cp\u003eHematoxylin‒eosin (HE) staining\u003c/p\u003e\u003cp\u003eAfter the paraffin slices were baked in a 37\u0026deg;C oven for 2 hours, they were dehydrated in xylene and 100% ethanol, 95% ethanol, 85% ethanol, or 75% ethanol. The samples were dyed with hematoxylin dye for 3\u0026ndash;5 minutes and rinsed with tap water for 1\u0026ndash;2 minutes. The 1% hydrochloric acid alcohol mixture was differentiated for a few seconds, the 1% ammonia solution was returned to blue for a few seconds, and then the mixture was washed with water for 1\u0026ndash;2 minutes. The samples were dyed with eosin dye solution for 1\u0026ndash;3 minutes. Anhydrous ethanol (I) and (II) are rehydrated for 5 minutes each, and xylene (I) and (II) are transparent for 5 minutes. The samples were sealed with neutral gum and subjected to microscopic examination.\u003c/p\u003e\u003cp\u003eGiemsa fibrosis staining\u003c/p\u003e\u003cp\u003eThe tissue was held in a 10% formaldehyde fixative and was routinely dehydrated and embedded, sliced, and dewaxed in water. A Reticulin Stain Kit (Solarbio, China) was used for staining, and the experiments were conducted according to the instructions.\u003c/p\u003e\u003cp\u003eCell proliferation assay\u003c/p\u003e\u003cp\u003eThe cell suspensions were prepared and added to a 96-well plate, which was precultured in an incubator. A Cell Counting Kit-8 (Dojindo Laboratories, Japan) was used to measure the absorbance of each sample, after which a proliferation curve was drawn according to the corresponding formula.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe data are presented via GraphPad Prism 8 software, and all measurement data are expressed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard errors. All experimental data were obtained from at least three independent experiments, and significant differences between groups were analysed via ANOVA and t tests. Survival analysis was performed via the log-rank test. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eSetd2\u003c/span\u003e expression is related to the MDS risk level\u003c/p\u003e\u003cp\u003eWe collected MSCs from three control patients (MALT lymphoma patients), four low-risk MDS patients, and six high-risk MDS patients to measure SETD2 expression via q‒PCR and western blot (WB) analysis. The results revealed that SETD2 expression was significantly lower in high-risk MDS patients than in low-risk MDS patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Moreover, when the complete blood counts of MDS patients with low SETD2 expression or normal SETD2 expression were compared, we found that both the platelet and white blood cell counts of MDS patients with low SETD2 expression decreased but the CD34\u003csup\u003e+\u003c/sup\u003e expression level increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), which suggests that low SETD2 expression is associated with MDS progression. Through Wright's staining of bone marrow smears, we found that patients with low expression of SETD2 exhibited significant pathological hematopoiesis in erythroid and megakaryocyte lineages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG are simple examples). In addition, the corresponding hematoxylin‒eosin staining of bone marrow revealed clustered blast cells in patients with low expression of SETD2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI are simple exemplars).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMSC-targeted knockout of \u003cem\u003eSetd2\u003c/em\u003e shortens survival and accelerates the progression of MDS in mice\u003c/p\u003e\u003cp\u003eWe crossed \u003cem\u003eLep-Cre/Prx1-CreSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice with NHD13 mice to generate \u003cem\u003eLep-Cre/Prx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice as the experimental group (NHD13 mice as the control group). \u003cem\u003eLep-Cre/Prx1-Cre\u003c/em\u003e mice were identified as MSC-targeted knockout mice. \u003cem\u003eLep-Cre/Prx1-Cre\u003c/em\u003e has a corresponding binding site only on MSCs, whereas it is absent on hematopoietic stem progenitor cells. Consequently, this approach has been widely adopted in MSC studies. First, we validated the knockout efficiency in primary murine MSCs. The mRNA expression of \u003cem\u003eSetd2\u003c/em\u003e in murine MSCs was assessed via qPCR analysis, which revealed a significant reduction in \u003cem\u003eSetd2\u003c/em\u003e expression in MSCs from \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/Δ\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Through WB detection of \u003cem\u003eSetd2\u003c/em\u003e expression and H3K36Me3 levels in mice, we found that the expression of \u003cem\u003eSetd2\u003c/em\u003e and the level of H3K36Me3 in \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice were also significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe monitored the survival of primary \u003cem\u003eLep-Cre/Prx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/Δ\u003c/em\u003e\u003c/sup\u003e mice (n\u0026thinsp;=\u0026thinsp;15) and NHD13 mice (n\u0026thinsp;=\u0026thinsp;15). Our results demonstrated that the median survival of \u003cem\u003eLep-Cre/Prx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/Δ\u003c/em\u003e\u003c/sup\u003e mice (265/192 days) was significantly shorter than that of NHD13 mice (301 days). Notably, the survival of \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/Δ\u003c/em\u003e\u003c/sup\u003e mice (median survival 192 days) was markedly shorter than that of NHD13 mice. These findings indicate that MSC-targeted knockout of \u003cem\u003eSetd2\u003c/em\u003e promotes the initiation and progression of MDS to leukemia in NHD13 mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and Supplementary Table\u0026nbsp;3). Therefore, we focused on \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice as the research model and conducted corresponding research on \u003cem\u003eLep-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice. Compared with the spleen size and weight of primary WT mice, NHD13 mice, and \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice at the age of 5 months, the spleen size and weight of the mice in the knockout group were significantly greater than those of the NHD13 mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eNext, we compared the expression of c-Kit\u003csup\u003e+\u003c/sup\u003e in the mice from the three groups. We performed flow cytometry analysis to detect the expression of c-Kit\u003csup\u003e+\u003c/sup\u003e in primary WT mice, NHD13 mice, and \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice. We found that the percentage of the hematopoietic stem and progenitor cell marker c-Kit\u003csup\u003e+\u003c/sup\u003e in the bone marrow and spleen of \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice was significantly greater than that in NHD13 mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG), indicating that the leukemia of the mice in the knockout group tended to be greater than that of the NHD13 mice. These results suggest that microenvironmental remodelling mediated by targeted knockout of \u003cem\u003eSetd2\u003c/em\u003e in MSCs controls the development of hematopoietic cells.\u003c/p\u003e\u003cp\u003eMSC-targeted knockout of \u003cem\u003eSetd2\u003c/em\u003e hinders the differentiation of multiple hematopoietic cell types in mice\u003c/p\u003e\u003cp\u003eThe peripheral blood of the mice in the three groups was collected every four weeks from the sixth week after birth. The main follow-up procedures included peripheral blood routine, erythroid differentiation, myeloid differentiation, and lymphoid (B, T cells) differentiation. Statistical analysis of the results of routine blood analysis revealed that, starting at week 14, the white blood cell counts of the mice in the \u003cem\u003eSetd2\u003c/em\u003e-targeted knockout group increased significantly. However, the platelet counts in the NHD13 group and targeted knockout mouse group remained lower than those in the WT group did, and the platelet count in the knockout group decreased more significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). There was no significant difference in hemoglobin follow-up between the NHD13 mouse group and the knockout mouse group. A separate comparison of the blood routine of the mice followed up to week 22 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) revealed that the white blood cell counts of the \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice were significantly greater than those of the NHD13 and WT mice. The hemoglobin levels of NHD13 mice and \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice were lower than those of WT mice, but there was no significant difference between NHD13 mice and \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice. Compared with WT mice, both NHD13 mice and \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice presented a decrease in platelet count, and compared with NHD13 mice, \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice presented a significant decrease in platelet count. The aforementioned follow-up results indicate that \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice exhibit elevated leukocyte levels and a significant reduction in platelet count. Consequently, compared with NHD13 mice, \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice demonstrate accelerated progression toward leukemia and impaired megakaryocyte development. All of these factors lead to shorter survival times in \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice than in NHD13 mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn terms of myeloid differentiation, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, the percentage of Mac-1\u003csup\u003e+\u003c/sup\u003eGr-1\u003csup\u003e+\u003c/sup\u003e cells in the peripheral blood, spleen, or bone marrow was greater in the \u003cem\u003eSetd2\u003c/em\u003e knockout group than in the NHD13 group, indicating the blocking effect of \u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e targeted knockout on myeloid differentiation. The number of early myeloid cells in the bone marrow increases, indicating a tendency toward leukemic transformation. With respect to lymphocyte differentiation, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, the number of bone marrow T cells in the \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e group was significantly lower than that in the NHD13 group. Although there was no significant difference in the expression of B cells between \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice and NHD13 mice, the expression of Pro-B and Pre-B cells was significantly greater in the \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mouse group (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA and S1B). As the disease progresses, lymphocyte differentiation is also inhibited, which has an inhibitory effect on the development and function of the immune system in mice. Compared with those in the NHD13 group, erythroid differentiation in the bone marrow and spleen of the \u003cem\u003eSetd2\u003c/em\u003e knockout group was inhibited, and the proportion of mature red blood cells in the RIV was significantly lower than that in the NHD13 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eFor primary mice with another Cre system, \u003cem\u003eLep-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e, owing to its slower onset than \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e, we included primary mice at approximately 1 year of age, control mice, and normal mice for the study. We found that the proportion of mature red blood cells in the experimental group decreased compared with that in the control group, and the differentiation of the red blood system was hindered, with a slight increase in the proportion of c-Kit\u003csup\u003e+\u003c/sup\u003e cells (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA and S2B). Afterwards, we compared myeloid and lymphoid differentiation between the \u003cem\u003eLep-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e group and the control group and found that the number of early myeloid cells in the experimental group increased and that myeloid differentiation was hindered (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC). Moreover, the number of B and T lymphocytes in the \u003cem\u003eLep-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e group was lower than that in the control group, indicating that the differentiation of the lymphoid lineage was also hindered (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eMSC-targeted knockout of \u003cem\u003eSetd2\u003c/em\u003e tends toward leukemia transformation\u003c/p\u003e\u003cp\u003eWe subsequently conducted further research on bone marrow hematopoietic stem progenitor cells (HSPCs) from the WT, NHD13, and \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e groups to investigate the effects of MSC genetic aberrations on their hematopoietic support function, as well as the effects of changes in the microenvironment on hematopoietic cell function. We isolated Lin\u003csup\u003e\u0026minus;\u003c/sup\u003e cells from the entire bone marrow cell population (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB clearly shows that the number of Lin- cells in the Prx1-CreNHD13Setd2\u003csup\u003e△/△\u003c/sup\u003e cohort was notably elevated. Additionally, there was an increase in the proportion and absolute count of LK cells. After targeted knockout of \u003cem\u003eSetd2\u003c/em\u003e in MSCs, the number of early immature cells increased, and hematopoietic stem progenitor cell differentiation shifted toward progenitor cells in these mice. We also observed that the number of hematopoietic stem cells (HSCs) decreased and that hematopoietic function was damaged. This may have caused \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice to have an increase in early immature cells and impaired differentiation of the erythroid myeloid lineage compared with NHD13 mice, which accelerated the transformation to leukemia and reduced the number of platelets. These results suggest that changes in the microenvironment caused by MSCs counteract the function of hematopoietic cells, affecting disease incidence and development.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eUsing flow cytometry analysis, LSK cells can be subdivided by the markers CD135, CD34, CD48, and CD150, and hematopoietic stem cells can be categorized into three categories: multipotent blood donors (MPPs), long-range hematopoietic stem cells (LT-HSCs), and short-term hematopoietic stem cells (ST-HSCs). In the hematopoietic system, long-term refilled hematopoietic stem cells, situated at the apex of the hematopoietic hierarchy, maintain a reservoir of primitive multipotentials throughout their lifetime through their self-renewal and asymmetric cell division potentials. The loss of normal LT-HSC and ST-HSC functions is a hallmark of natural stem cell aging and several hematopoietic disorders, most notably the development and progression of hematological malignancies. MPPs are pluripotent progenitor cells derived from hematopoietic stem cells that have lost their self-renewal potential but can still fully differentiate into all lineages. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, compared with NHD13 mice, \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice presented a significant increase in the absolute number of MPPs and ST-HSCs, whereas the number of LT-HSCs decreased. These results suggest that, after targeted knockout of \u003cem\u003eSetd2\u003c/em\u003e in MSCs, the long-term hematopoietic capacity of murine hematopoietic stem cells is impaired and that short-term hematopoietic function is enhanced compared with the reactivity of per-knockout cells.\u003c/p\u003e\u003cp\u003eMPP further produces low-potential progenitor cells, commonly known as lymphoid and myeloid progenitor cells (CLPs and CMPs). We performed flow cytometry analysis to further classify haematopoietic progenitor cells (KKs) into three categories\u0026mdash;GMP, MEP, and CMP\u0026mdash;using markers such as CD34 and CD1632. An examination of the outcomes depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB revealed that, compared with the NHD13 cohort, the selected knockout of \u003cem\u003eSetd2\u003c/em\u003e in MSCs resulted in an increase in partially differentiated MEP and GMP cells within the myeloid progenitor cell population. The expression of CLP was significantly lower in the \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e group than in the NHD13 group, indicating that excessive proliferation of the myeloid system in the bone marrow inhibited the proliferation of the lymphoid system (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC and S1D). These results suggest that, compared with the NHD13 group, the \u003cem\u003eSetd2\u003c/em\u003e knockout group has differentiated obstructions and impaired hematopoietic function.\u003c/p\u003e\u003cp\u003eFor the primary mice with another Cre system, \u003cem\u003eLep-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e, the hematopoietic stem progenitor cell population also exhibited a decrease in the proportion of stem cells and progenitor cells and hindered stem progenitor cell differentiation (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eD). Further grouping of hematopoietic stem cells and hematopoietic progenitor cells was subsequently performed. As shown in Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eE and S2F, the absolute numbers of each group of progenitor cells (GMP/MEP/CMP) in the experimental group decreased compared with those in the control group, whereas the absolute numbers of LT-HSCs and ST-HSCs decreased, indicating a trend toward bone marrow failure. Thus, when the \u003cem\u003eLep-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice and \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice were compared, the \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice tended toward leukemic transformation, whereas the \u003cem\u003eLep-Cre\u003c/em\u003e mice tended toward the development of ineffective hematopoiesis and bone marrow failure.\u003c/p\u003e\u003cp\u003eNext, we studied the morphology of the mice. We performed Wright's staining on peripheral blood smears, bone marrow cells and spleen cells, and the bone marrow, spleen, and liver biopsy tissues of the mice were sliced and subsequently subjected to hematoxylin and eosin (HE) staining. Wright staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) revealed that the peripheral blood smears of the knockout group exhibited notable increases in naive neutrophils and erythrocytes compared with those of the NHD13 group. A bone marrow smear revealed changes in the number of megaloblastic cells in the knockout group, with an increase in early-stage myeloid cells. The same is true for spleen cells. H\u0026amp;E staining analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) revealed that the proportion of hematopoietic cells in the bone marrow of the \u003cem\u003eSetd2\u003c/em\u003e knockout group mice decreased, with venous sinus dilation and a significant increase in early myeloid cells. There was a significant increase in the number of spleen megakaryocytes in the \u003cem\u003eSetd2\u003c/em\u003e knockout group. Liver cell proliferation was suppressed in mice in the \u003cem\u003eSetd2\u003c/em\u003e knockout group, and extramedullary hematopoietic cells infiltrated these cells. Since the increase in megakaryocytes is closely related to fibrosis, Gomori fibrosis staining was performed on spleen sections from the three groups of mice to determine the degree of fibrosis. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, compared with the NHD13 group, the \u003cem\u003eSetd2\u003c/em\u003e knockout group presented a significant increase in the number of splenic fibre strands and aggravated fibrosis. A comparison of the MSC morphology of the mice in the three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF) revealed that the MSC morphology of the \u003cem\u003eSetd2\u003c/em\u003e knockout group was irregular, and aging staining analysis suggested that MSC aging was faster in the \u003cem\u003eSetd2\u003c/em\u003e knockout group than in the NHD13 group. These results confirm the flow cytometry results presented above and provide additional evidence of the inhibitory effect of MSC-targeted knockout of \u003cem\u003eSetd2\u003c/em\u003e on the differentiation of NHD13 hematopoietic stem cells and the accelerated incidence and development of this disease.\u003c/p\u003e\u003cp\u003eTo establish a control with primary mice, we established \u003cem\u003eLep-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e and NHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e transplantation mouse models (Figure S3A). We first compared the survival of the mice in the two groups and found no significant difference (Figure S3B). Peripheral blood follow-up was performed on the mice from both groups every four weeks beginning four weeks after transplantation. As shown in Figure S3D, the white blood cells of the mice in both groups significantly increased after transplantation, whereas the haemoglobin and platelet levels gradually decreased. There were no significant differences in the number of peripheral blood red blood cells or c-Kit\u003csup\u003e+\u003c/sup\u003e cells in the mice, as detected by flow cytometry analysis (Figure S3C and S3E). However, the differentiation of myeloid cells in the mice in the \u003cem\u003eLep-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e group was hindered compared with that in the NHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e group, with an increase in early myeloid cells (Figure S3E). As a continuation of previous studies, the expression of PD-L1 in the peripheral blood of mice in the two groups was detected by flow cytometry analysis[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The expression of PD-L1 in the \u003cem\u003eLep-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e group was significantly greater than that in the NHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e group (Figure S3F), suggesting that MDS-associated tumors escape via PD-1/PD-L1 overexpression[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, we believe that the impact of haematopoietic stem cell transplantation on the bone marrow microenvironment is limited compared with that of primary stem cell transplantation.\u003c/p\u003e\u003cp\u003eTargeted knockout of \u003cem\u003eSetd2\u003c/em\u003e in MSCs may affect the disease progression of MDS through coagulation factor XII (F12)\u003c/p\u003e\u003cp\u003eTo investigate the possible mechanism by which MSCs with targeted knockout of \u003cem\u003eSetd2\u003c/em\u003e regulate hematopoietic cells, we analysed differential protein expression in the bone marrow supernatants of \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e mice and NHD13 mice via mass spectrometry analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We compared the expression profiles of bone marrow supernatant proteins between the two groups of mice and identified 38 upregulated proteins and 47 downregulated proteins with statistically significant differences. The qPCR results revealed that the expression of 38 proteins whose expression was upregulated in MSCs indicated that F12 was upregulated in \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e MSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). After contact with the anion surface, F12 automatically becomes F12a. The heavy chain of F12a consists of six domains, one of which is the Fn1 domain [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The expression of the HSC niche factor CXCL12 and the transcription factors Foxc1 and Ebf3 in \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003e MSCs was significantly increased, but that of another HSC niche factor, SCF, was significantly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Compared with NHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e MSCs, \u003cem\u003ePrx1-Cre\u003c/em\u003eNHD13\u003cem\u003eSetd2\u003c/em\u003e\u003csup\u003e\u003cem\u003e△/△\u003c/em\u003e\u003c/sup\u003eMSCs promoted the inflammatory response and chemotaxis and inhibited stem cell proliferation and mobilization. The gene expression of Fn1 was significantly upregulated according to RNA-seq analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Therefore, F12 was selected by combining the upregulated proteins from bone marrow supernatants and upregulated genes identified via RNA-seq analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePatients with acute leukemia presented markedly elevated levels of peak thrombin, extracellular trap markers, and F12a. The F12a level is significantly associated with the presence of acute leukemia [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In this study, the \u003cem\u003eSetd2\u003c/em\u003e knockout group presented significantly greater F12 expression in the bone marrow supernatant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF) and greater Fn1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG) in the MSCs than did the NHD13 group did, indicating a trend toward leukemia transformation in the \u003cem\u003eSetd2\u003c/em\u003e knockout group. In patients with high-risk MDS, the expression of F12 in the bone marrow supernatant was significantly greater than that in patients with low-risk MDS (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH), and the expression of Fn1 in the MSCs of high-risk MDS patients was significantly greater than that in the MSCs of low-risk MDS patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). These results suggest that targeted knockout of \u003cem\u003eSetd2\u003c/em\u003e in MSCs affects the expression of the F12 protein in the microenvironment through the secretion of Fn1 by MSCs.\u003c/p\u003e\u003cp\u003eTargeted knockout of \u003cem\u003eSetd2\u003c/em\u003e impaired the hematopoietic stem progenitor cell support function of MSCs\u003c/p\u003e\u003cp\u003eWe cocultured murine bone marrow mononuclear cells with murine MSCs and performed cell proliferation, cell cycle, and colony formation assays on the cocultured Lin\u003csup\u003e\u0026minus;\u003c/sup\u003ec-Kit\u003csup\u003e+\u003c/sup\u003e mononuclear cells. D-Pro-Phe-Arg-chloromethylketone (PCK), an inhibitor of coagulation factor XII and plasma kallikrein, plays an important role in thrombosis and inflammation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. CCK8 (Cell Counting Kit-8) analysis was performed on mononuclear cells from three coculture groups of mice and on those treated with PCK. The proliferation rate of mononuclear cells in the Setd2 knockout group significantly decreased but increased after PCK treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Apoptosis analysis of cocultured mononuclear cells revealed that the early and late apoptosis rates of mice in the \u003cem\u003eSetd2\u003c/em\u003e knockout group increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The proportion of these cells in the G2/M phase was lower in the NHD13 group than in the WT group, whereas the S phase was significantly prolonged and the G0/G1 phase was shortened. Thus, we speculate that the hematopoietic stem progenitor cells of the mice in the \u003cem\u003eSetd2\u003c/em\u003e knockout group are more likely to be blocked in the G0/G1 phase than those of the mice in the NHD13 group, which are blocked in the S phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Colony formation assays were performed on hematopoietic stem progenitor cells from three groups of mice after coculture and three groups of mice after PCK. The self-renewal ability of the \u003cem\u003eSetd2\u003c/em\u003e knockout group significantly decreased but increased after PCK treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). In summary, targeted knockout of \u003cem\u003eSetd2\u003c/em\u003e in MSCs impairs the support function of MSCs through F12, affecting the self-renewal and cell proliferation capabilities of HSPCs (Figure S4). PCK partially rescues the function of HSPCs by inhibiting the effect of F12.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrevious studies have shown that mesenchymal stem cells derived from MDS patients (MDS-MSCs) exhibit reduced proliferation and cloning ability, cell morphological changes, increased cellular senescence, impaired immune regulation, and a reduced ability to support hematopoietic stem cell growth and differentiation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Compared with normal human MSCs, MDS-MSCs can affect cytokines in the microenvironment and can be affected by an aberrant microenvironment and therapeutic drugs, resulting in certain functional changes and further affecting the progression of MDS [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn our study, we first found that mice with \u003cem\u003eSetd2\u003c/em\u003e knockout had significantly shorter survival times. This result aligns with other previous \u003cem\u003eSetd2\u003c/em\u003e results. We previously reported that deletion of \u003cem\u003eSetd2\u003c/em\u003e in hematopoietic cells of NHD13 transgenic mice accelerates the progression of MDS to leukemia, indicating that \u003cem\u003eSetd2\u003c/em\u003e is a tumor suppressor in the process of NHD13-driven MDS to leukemia transformation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. \u003cem\u003eSETD2\u003c/em\u003e mutations/variants are closely associated with overall survival (OS), and they have been identified as risk factors for progression-free survival (PFS), especially with low expression of \u003cem\u003eSETD2\u003c/em\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The \u003cem\u003eSetd2\u003c/em\u003e variant has been reported to be a risk factor for poor prognosis in patients with AML. The \u003cem\u003eSETD2\u003c/em\u003e levels appeared to be higher than the HR-MDS levels for the LR-MDS in both the transcript and protein measurements. During the transition from MDS to AML, there is often an increase in c-Kit\u003csup\u003e+\u003c/sup\u003e expression, leading to splenomegaly in mice. As expected, mice in the \u003cem\u003eSetd2\u003c/em\u003e knockout group presented a significant increase in the absolute value of white blood cells and c-Kit\u003csup\u003e+\u003c/sup\u003e expression, with an increase in early-stage myeloid cells in bone marrow smears. Our results revealed that targeted knockout of \u003cem\u003eSetd2\u003c/em\u003e in MSCs affects MDS progression and survival in mice with MDS.\u003c/p\u003e\u003cp\u003eThe impaired support function of MSCs with mutations in hematopoietic stem progenitor cells is manifested in several ways. We found that targeted knockout of \u003cem\u003eSetd2\u003c/em\u003e in MSCs resulted in an increase in peripheral blood white blood cells, a decrease in hemoglobin and platelet levels, and an obstruction of differentiation of the erythroid, myeloid, and lymphoid lineages in mice. Clustering analysis of hematopoietic stem progenitor cells revealed an increase in hematopoietic progenitor cells, a decrease in hematopoietic stem cells, and a decrease in long-term hematopoietic capacity. In particular, there are barriers to maturation and differentiation in myeloid cells, resulting in an increase in immature cells and a tendency for MDS to transform into leukemia. On the basis of the decrease in LT-HSCs and increase in ST-HSCs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and the increase in apoptosis in MSCs cocultured with \u003cem\u003eSetd2\u003c/em\u003e knockout mononuclear cells shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the cell cycle was blocked in the G0/G1 phase. We speculate that hematopoietic stem progenitor cells with differentiation disorders fail to differentiate appropriately and eventually undergo apoptosis, resulting in acute compensatory hematopoiesis in the bone marrow in the short term, long-term impaired hematopoietic function, and pathological hematopoiesis and hematopoiesis failure.\u003c/p\u003e\u003cp\u003eOur study sheds light on the impact of the bone marrow microenvironment on the pathogenesis and progression of MDS from the MSC perspective. Research has shown that the TET2, KDM6A, BCOR, EZH2 and ASXL genes in MDS-MSCs are prone to mutation, and DNA methylation and chromosomal abnormalities frequently occur in MSCs, confirming that there is a correlation between genetic variation in DNA methylation genes and random chromosomal losses and the pathogenesis of MDS [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Moreover, loss of \u003cem\u003eSetd2\u003c/em\u003e promotes Kras-induced acinar-to-ductal metaplasia and epithelial‒mesenchymal transition during pancreatic carcinogenesis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Notably, \u003cem\u003eSetd2\u003c/em\u003e deficiency sensitized KRAS-mutant lung cancer to histone chaperone inhibition [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. \u003cem\u003eSetd2\u003c/em\u003e frequently cooccurs with IDH2, NRAS and CEBPA mutations [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe histone methyltransferase \u003cem\u003eSetd2\u003c/em\u003e is a tumor suppressor that functions by trimethylating lysine 36 in histone H3. A recent study confirmed that loss-of-function \u003cem\u003eSetd2\u003c/em\u003e mutations facilitate the initiation of leukemia and impair DNA damage recognition, thereby contributing to therapy resistance [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Another study demonstrated that \u003cem\u003eSetd2\u003c/em\u003e is required for the self-renewal of HSCs and that \u003cem\u003eSetd2-\u003c/em\u003edeficient HSCs contribute to the development of MDS [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Our results are consistent with these findings. As shown by the colony-forming unit (CFU) assay in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the self-renewal capacity of hematopoietic stem progenitors was significantly reduced after targeted knockout of \u003cem\u003eSetd2\u003c/em\u003e in MSCs. In addition, we observed that \u003cem\u003eSetd2\u003c/em\u003e expression was significantly lower in high-risk MDS patients than in low-risk MDS patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH), corroborating our findings in murine models.\u003c/p\u003e\u003cp\u003eTargeted knockout of \u003cem\u003eSetd2\u003c/em\u003e in MSCs affects hematopoietic stem progenitor cells through aberrant secretion of MSC-produced proteins, thereby impairing their hematopoietic function, but the mechanism is still unclear. Studies have demonstrated that the pathological cells of MDS contain procoagulant substances and that the tissue-clotting kinase carried by MSCs mediates the expression of procoagulant activity in the extracellular vesicles of MSCs, leading to coagulation dysfunction in MDS patients [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The level of F12 is significantly correlated with the presence of acute leukemia [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In our study, we screened for coagulation-related F12 and Fn1 via mass spectrometry and RNA-seq analysis and validated the results via ELISA and qPCR analysis. Thus, we demonstrated that MSCs with targeted knockout of \u003cem\u003eSetd2\u003c/em\u003e upregulate F12 levels in the microenvironment by secreting elevated amounts of Fn1, thereby creating a hypercoagulable environment and promoting MDS progression. PCK can partially rescue the hypercoagulable environment and seemingly reverse the effects of high-F12 conditioned media from \u003cem\u003eSetd2\u003c/em\u003e null cells on the cell cycle.\u003c/p\u003e\u003cp\u003eIn summary, our study confirms that MSCs interact with HSPCs through the microenvironment and ultimately influence the incidence and development of MDS in a mouse model of MDS with MSC-directed mutations. Through this study, we find a therapeutic approach involving microenvironment regulators for MDS patients harboring mutations that are refractory to allogeneic hematopoietic stem cell transplantation (such as NRAS and KRAS) and strive for greater benefits.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is funded by the National Natural Science Foundation of China (Grant no. 8207010691), the Clinical Research Plan of SHDC (NO. SHDC2020CR6004) and the Shanghai Municipal Science and Technology Commission, Science and Technology Innovation Action Plan (Grant 24YF2756500).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments in our study were carried out in accordance with the Helsinki Declaration, and approved by the Animal Care and Use Committee of Sixth People\u0026apos;s Hospital affiliated with Shanghai Jiao Tong University School of Medicine (2022--0239). The research was approved by the Ethics Committee of Sixth People\u0026apos;s Hospital affiliated with Shanghai Jiao Tong University School of Medicine (2022-YS-051).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: Chun-Kang Chang, Lan Wang and You-Shan Zhao\u003c/p\u003e\n\u003cp\u003eMethodology: Rou-Jia Wang, Zi-Juan Li, Bing-Yi Chen, Juan Guo, Ying Tao, Hong-Ping Li and Ming-Yue Fei\u003c/p\u003e\n\u003cp\u003eInvestigation: Rou-Jia Wang, Zi-Juan Li, Bing-Yi Chen, Juan Guo, Ying Tao, Hong-Ping Li and Ming-Yue Fei, Mu-Ying Zhao, Lei Shi, Si-Da Zhao, Zheng Zhang, Ji-Ying Su, Lu-Xi Song, Qi He, Dong Wu, Ling-Yun Wu, Jia-Ying Zhang, Li-Juan Zong, Xiao-Jian Sun\u003c/p\u003e\n\u003cp\u003eVisualization: Bin-He Chang\u003c/p\u003e\n\u003cp\u003eWriting\u0026mdash;original draft: Rou-Jia Wang and Zi-Juan Li\u003c/p\u003e\n\u003cp\u003eWriting\u0026mdash;review and editing: Chun-Kang Chang, Lan Wang and You-Shan Zhao\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAwada H, Thapa B, Visconte V. The genomics of myelodysplastic syndromes: origins of disease evolution, biological pathways, and prognostic implications. Cells. 2020;9(11):2512.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLyu T, Zhang B, Li M, Jiao X, Song Y. Research progress on exosomes derived from mesenchymal stem cells in hematological malignancies. Hematol Oncol. 2021;39(2):162\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao J, Chen J, Huang F, Wang J, Su W, Zhou J, et al. Human gingiva tissue-derived MSC ameliorates immune-mediated bone marrow failure of aplastic anemia via suppression of Th1 and Th17 cells and enhancement of CD4\u0026thinsp;+\u0026thinsp;Foxp3\u0026thinsp;+\u0026thinsp;regulatory T cells differentiation. Am J Transl Res. 2019;11(12):7627\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMassaro F, Corrillon F, Stamatopoulos B, Nathalie Meuleman L, Lagneaux D, Bron. Aging of bone marrow mesenchymal stromal cells: hematopoiesis disturbances and potential role in the development of hematologic cancers. Cancers (Basel). 2020;13(1):68.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMing H, Sun X-J, Zhang Y-L, Kuang Y, Hu C-Q, Wu W-L, et al. Histone H3lysine 36 methyltransferase Hypb/Setd2 is required for embryonic vascular remodelling. Proc Natl Acad Sci USA. 2010;107(7):2956\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen B-Y, Song J, Chen C-LHS-B, Zhang Q, Xu C-H, et al. SETD2 deficiency accelerates MDS-associated leukemogenesis via S100a9 in NHD13 mice and predicts poor prognosis in MDS. Blood. 2020;135(25):2271\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRoujia Wang Y, Zhao Z, Li J, Guo S, Zhao L, Song, et al. S100a9 deficiency accelerates MDS-associated tumor escape via PD-1/PD-L1 overexpression. Acta Biochim Biophys Sin (Shanghai). 2023;55(2):194\u0026ndash;201.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBaglia FA, Jameson BA, Walsh PN. Identification and characterization of a binding site for factor XIIa in the Apple 4 domain of coagulation factor XI. J Biol Chem 1993Feb 25;268(6):3838\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim TY, Gu J-Y, Jung HS, et al. Elevated extracellular trap formation and contact system activation in acute leukemia. J Thromb Thrombolysis. 2018;46(3):379\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNickel KF, Long AT, Fuchs TA, Youngil Koh I, Kim. Hyun Kyung Kim. Factor XII as a Therapeutic Target in Thromboembolic and Inflammatory Diseases. Arterioscler Thromb Vasc Biol. 2017;37(1):13\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSarhan D, Wang J, Sunil Arvindam U, Caroline Hallstrom MR, Verneris B, Grzywacz, et al. Mesenchymal stromal cells shape the MDS microenvironment by inducing suppressive monocytes that dampen NK cell function. JCI Insight. 2020;5(5):e130155.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBulycheva E, Rauner M, Medyouf H, Theurl I, Bornh\u0026auml;user M, Hofbauer LC et al. Myelodysplasia is niche:novel concepts Emerg Ther Leuk 2015,29(2):259\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi J, Peng Z, Luo F, Chen Y. SET Domain Containing 2 Deficiency in Myelodysplastic Syndrome. Front Genet. 2020;11:794.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBandara WMMS, Rathnayake AJIS, Neththikumara NF, Hemali WW, Goonasekera, Vajira HW, Dissanayake. Comparative analysis of the genetic variants in haematopoietic stem/progenitor and mesenchymal stem cell compartments in de novo myelodysplastic syndromes. Blood Cells Mol Dis,2021(88):102535.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNingning Niu P, Lu Y, Yang R, He L, Zhang J, Shi, et al. Loss of Setd2 promotes Kras-induced acinar-to-ductal metaplasia and epithelia-mesenchymal transition during pancreatic carcinogenesis. Gut. 2020;69(4):715\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXie Y, Sahin M, Wakamatsu T, Inoue-Yamauchi A, Zhao W, Han S, et al. SETD2 regulates chromatin accessibility and transcription to suppress lung tumorigenesis. JCI Insight. 2023;8(4):e154120.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang X, Wang Z, Sun J, Liu L, Qin J, Huang A, et al. New insights into the clinical characteristics of SETD2-mutated acute myeloid leukaemia. Br J Haematol. 2023;202(1):111\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSheng Y, Ji Z, Zhao H, Wang J, Cheng C, Xu W, et al. Downregulation of the histone methyltransferase SETD2 promotes imatinib resistance in chronic myeloid leukaemia cells. Cell Prolif. 2019;52(4):e12611.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang Y-L, Sun J-W, Xie Y-Y, Zhou Y, Ping Liu J-C, Song, et al. Setd2 deficiency impairs hematopoietic stem cell self-renewal and causes malignant transformation. Cell Res. 2018;28(4):476\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZannoni J, Mauz N,Seyve L, Mathieu Meunier K, Pernet-Gallay J, Brault et al. Tumor microenvironment and clonal monocytes from chronic myelomonocytic leukemia induce a procoagulant climate. Blood Adv 2019,3(12):1868\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mome","sideBox":"Learn more about [Molecular Medicine](https://molmed.biomedcentral.com)","snPcode":"10020","submissionUrl":"https://submission.springernature.com/new-submission/10020/3","title":"Molecular Medicine","twitterHandle":"@MolecularMedic1","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7140205/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7140205/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWhile previous studies have indicated that H3K36me3, which is mediated by \u003cem\u003eSetd2\u003c/em\u003e, may regulate the cell fate of mesenchymal stem cells (MSCs) both in vitro and in vivo, the specific role of MSCs in the onset and progression of MDS remains unclear. Thus, the histone methyltransferase \u003cem\u003eSetd2\u003c/em\u003e is implicated in MDS-associated leukemia. This study utilized NUP98-HOXD13 (NHD13) mice with targeted deletion of \u003cem\u003eSetd2\u003c/em\u003e in MSCs. Here, we found that \u003cem\u003eSetd2\u003c/em\u003e-deficient mice undergo faster leukemia transformation than control mice do, as evidenced by the abnormal differentiation of hematopoietic stem progenitor cells in the bone marrow, abnormal hematopoiesis, and increased number of blast cells. Compared with that of control mice, the morphology of NHD13 mouse MSCs with \u003cem\u003eSetd2\u003c/em\u003e deficiency was irregular, and the support function of hematopoietic cells was compromised. This study demonstrated that targeted deletion of \u003cem\u003eSetd2\u003c/em\u003e in MSCs has a beneficial effect on the progression of MDS. Furthermore, we identified increased expression of coagulation factor XII as a key leukemic transformation mediator in \u003cem\u003eSetd2\u003c/em\u003e-deficient MSCs. Moreover, we found that \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eSetd2\u003c/span\u003e expression is significantly lower in high-risk MDS patients than in low-risk MDS patients, further suggesting that the targeted deletion of \u003cem\u003eSetd2\u003c/em\u003e in MSCs is associated with MDS progression. Collectively, our results suggest that \u003cem\u003eSetd2\u003c/em\u003e in MSCs suppresses MDS progression to leukemia through coagulation factor XII-mediated suppression of the stem cell support capacity of MSCs. Overall, this study sheds light on the pathogenesis of MDS and provides a therapeutic strategy for regulating the microenvironment in patients with MDS who cannot be cured by haematopoietic stem cell transplantation.\u003c/p\u003e","manuscriptTitle":"Deficiency of Setd2 in mesenchymal stem cells facilitates the progression of myelodysplastic syndrome to leukemia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-06 09:18:45","doi":"10.21203/rs.3.rs-7140205/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-25T11:56:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-22T02:37:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-21T14:54:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-15T17:33:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"80446274137930957913191798314891263391","date":"2025-09-12T16:55:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"223329354989511962293844943032711049480","date":"2025-09-12T14:34:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"320025719664241761808853491249787065746","date":"2025-09-12T13:54:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-19T13:38:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"89791017222251949714278702082743715652","date":"2025-07-29T09:47:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"14739889440858675996625304893242910281","date":"2025-07-29T07:07:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-28T11:32:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-23T05:29:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-23T05:28:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Medicine","date":"2025-07-16T12:45:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mome","sideBox":"Learn more about [Molecular Medicine](https://molmed.biomedcentral.com)","snPcode":"10020","submissionUrl":"https://submission.springernature.com/new-submission/10020/3","title":"Molecular Medicine","twitterHandle":"@MolecularMedic1","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"49364023-e493-4fbe-a9e9-1763b2b29db9","owner":[],"postedDate":"August 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T16:01:14+00:00","versionOfRecord":{"articleIdentity":"rs-7140205","link":"https://doi.org/10.1186/s10020-026-01492-7","journal":{"identity":"molecular-medicine","isVorOnly":false,"title":"Molecular Medicine"},"publishedOn":"2026-04-30 15:57:57","publishedOnDateReadable":"April 30th, 2026"},"versionCreatedAt":"2025-08-06 09:18:45","video":"","vorDoi":"10.1186/s10020-026-01492-7","vorDoiUrl":"https://doi.org/10.1186/s10020-026-01492-7","workflowStages":[]},"version":"v1","identity":"rs-7140205","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7140205","identity":"rs-7140205","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}