AML (acute myelogenous leukemia) post-chemotherapy recurrent leukemia stem cell marker with regulation function

Abstract

The invention relates to the field of medical biology, in particular to an AML (acute myelogenous leukemia) chemotherapy recurrent leukemia stem cell marker with a regulation function, and particularly relates to a PUF60 serving as the AML chemotherapy recurrent leukemia stem cell marker with the regulation function.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a stem cell marker for relapsed leukemia after AML chemotherapy with regulatory function, and particularly to PUF60 as a stem cell marker for relapsed leukemia after AML chemotherapy with regulatory function. Background Technology

[0002] Acute myeloid leukemia (AML) is a malignant proliferative disease of the hematopoietic system, characterized by a large number of undifferentiated or abnormally differentiated primitive cells that impair normal hematopoietic function and seriously endanger people's lives and health. The conventional treatment for AML is chemotherapy, which can effectively reduce the number of leukemia cells and bring many patients to temporary remission. However, relapse is common afterward, severely impacting patient survival. AML relapse remains a major cause of treatment failure and patient death.

[0003] The formation of AML primarily results from the transformation of mutated and epigenetically abnormal primitive hematopoietic cells into leukemia stem cells (LSCs). LSCs occupy the top of the abnormal hematopoietic hierarchy in AML, possessing self-renewal and differentiation capabilities, continuously generating downstream leukemia cells; they also exhibit quiescent state maintenance and drug resistance characteristics, and their presence is a root cause of AML relapse after chemotherapy. Therefore, it is urgent to screen and study key molecules regulating LSC function as potential targets for LSC markers and interventions in AML. This is of great significance for improving the efficacy of AML chemotherapy.

[0004] PUF60 (Poly(U) Binding Splicing Factor 60) is an RNA splicing factor that binds to the PolyU sequence, i.e., an RNA-binding protein (RBP). Characterized by its dual DNA and RNA binding functions, it primarily participates in RNA splicing, transcriptional regulation, and the maintenance of mRNA stability. It can form complexes with proteins such as FUBP1 to regulate the transcription of important genes like MYC; it can also synergistically promote the recognition of RNA 3' splice sites with the splicing factor U2AF2, regulating alternative splicing. PUF60 has been reported to be associated with the progression of various solid tumors, including lung cancer and breast cancer, but its function and regulation in AML LSC have not been reported. Existing literature suggests that PUF60 may be a potential oncogen, but its specific role in relapsed AML, especially its mechanism related to leukemia stem cells (LSC), remains unclear.

[0005] Current research lacks studies that use proteomics to identify PUF60 as a biomarker for relapsed AML leukemia stem cells (LSCs), as well as comprehensive validation demonstrating that its inhibition reduces cancer cell proliferation, drug resistance, and in vivo activity. Given the high relapse rate of AML (approximately 50-70%) and the fact that LSCs are a root cause of chemotherapy resistance, such targeted research could reveal new biomarkers and therapeutic targets, improving patient survival rates.

[0006] Existing research largely focuses on PUF60 fusion variants and its broad-spectrum inhibitory effects. For example, early studies identified the presence of PUF60-TYW1 fusions in AML, which may interfere with the function of tumor suppressor genes. However, these findings have not explored the expression patterns or functional impact of PUF60 in LSC subsets. Although AML proteomics studies have identified various LSC biomarkers, there are no reports of PUF60 being specifically highly expressed as a protein in relapsed AML LSCs using proteomics methods. AML relapse often stems from chemotherapy resistance in LSCs; developing novel biomarkers could improve early detection and targeted therapy.

[0007] More importantly, existing AML research emphasizes the central role of leukemia stem cells (LSCs) in disease relapse. LSCs are the root cause of chemotherapy resistance and are capable of self-renewing and remodeling tumors. Several proteomic studies have identified LSC-specific proteins, such as metabolism-related enzymes in the CD34+ subset, but no literature has yet identified PUF60 as a biomarker for relapsed AML LSCs using proteomic methods. For example, a 2022 proteomic analysis (Stratmann et al., "Proteogenomic analysis of acute myeloid leukemia associates relapsed disease with reprogrammed energy metabolism both in adults and children," Leukemia, December 26, 2022) revealed changes in the protein landscape of relapsed AML patients, including splicing factor abnormalities, but did not specifically mention PUF60. Similarly, the 2025 integrative omics study (Authors not fully listed in snippets, "Proteomic subtypes enrich current acute myeloid leukemia nomenclature and reflect intrinsic pathogenesis alongside aging," Blood, November 27, 2025.) assessed the stem cell characteristics of AML but did not include PUF60 in the list of core biomarkers.

[0008] Overall, high expression of PUF60 in acute myeloid leukemia (AML) is associated with poor patient prognosis, and preliminary studies have found that its inhibitors can induce cell cycle arrest and reduce cell viability in cell lines. However, existing studies are mostly based on in vitro cell line models, and there is a lack of in-depth exploration of the specific mechanism of action of PUF60 in leukemia stem cells (LSCs), the core cell population of AML relapse and treatment resistance. Although proteomics has identified several LSC-related biomarkers in AML, no studies have yet systematically identified whether PUF60 is a key protein specifically highly expressed in relapsed AML LSCs using proteomics methods, and there is also a lack of direct functional evidence regarding its role in regulating cell proliferation, chemotherapy resistance, and in vivo transplantation capacity in LSCs.

[0009] Given the high relapse rate of AML patients (50–70%) and the persistent presence of latent sclerosis (LSCs), which is a fundamental cause of chemotherapy failure and disease relapse, in-depth research into the expression patterns and functions of PUF60 in LSCs is expected not only to reveal novel biomarkers but also to provide important theoretical basis for developing targeted therapies for LSCs, potentially improving the clinical prognosis of AML patients. Therefore, elucidating the expression characteristics of PUF60 in AML LSCs and its regulatory role in LSC activity has significant scientific importance and clinical translational potential.

[0010] Therefore, in order to address the above shortcomings, this invention detects the expression level of PUF60 in AML-naïve to chemotherapy-relapsed LSCs and studies the regulatory effect of PUF60 on the activity of AML LSC cells, providing its application as a biomarker for AML LSCs and as a target for intervention in chemotherapy-induced relapse. Summary of the Invention

[0011] The high relapse rate and chemotherapy resistance in acute myeloid leukemia (AML) are primarily attributed to the persistent presence of leukemia stem cells (LSCs). Currently, there is a lack of specific molecular markers and effective intervention targets for relapsed AML LSCs. This invention aims to address this issue by identifying PUF60 as a key marker and functional regulator of relapsed AML LSCs using proteomics and other methods, and to verify its potential as a diagnostic marker and therapeutic target.

[0012] This invention utilizes protein mass spectrometry to perform differential protein analysis on LSC-enriched cell populations (CD34+) and non-LSC cell populations (CD34-) from treatment-naïve AML patients, screening for a group of RNA-binding proteins (RBPs) highly expressed in LSCs. Simultaneously, paired LSC-enriched cell populations from treatment-naïve and chemotherapy-relapsed AML patients were analyzed to screen for RBPs upregulated in relapsed LSCs. The intersection of the two sets of results yielded a group of "AML chemotherapy-relapsed LSC-specifically highly expressed RBPs," which includes PUF60.

[0013] Subsequent analysis using the TCGA database revealed that high PUF60 expression was most significantly associated with low survival rates in AML patients within this group of RBPs. Further analysis using the BloodSpot database confirmed that PUF60 expression was significantly higher in normal karyotype AML cells than in myelodysplastic syndrome (MDS) cells and healthy human bone marrow mononuclear cells; its expression was also significantly higher in different abnormal karyotype AML cells than in healthy human hematopoietic stem cells.

[0014] In terms of functional validation, this invention confirms that: 1) In primary AML cells, the expression level of PUF60 in LSC-rich clusters is higher than that in non-LSC populations, and the expression level in LSCs of relapsed patients is higher than that in treatment-naïve patients; 2) In the AML cell line (THP-1), knockdown of PUF60 can significantly inhibit cell proliferation activity (CCK-8 experiment), reduce its resistance to doxorubicin (DOX), and weaken its colony-forming ability; while overexpression of PUF60 promotes cell proliferation; 3) In the mouse xenograft model (CLDX) derived from AML cell lines, compared with mice transplanted with control cells, mice transplanted with PUF60 knockdown cells have a lower engraftment rate of myeloid leukemia cells, a reduced infiltration of leukemia cells in peripheral blood and liver and spleen, and a significantly prolonged survival.

[0015] In a first aspect, the present invention provides the application of PUF60 as a molecular marker for relapsed acute myeloid leukemia (AML) leukemia stem cells (LSCs), which promotes the maintenance of LSC activity and the regulation of function.

[0016] Furthermore, the PUF60 is specifically highly expressed in CD34+ LSC-enriched cell populations of patients with relapsed AML after chemotherapy, and can be used for relapse rate assessment or disease risk assessment after chemotherapy.

[0017] In a first aspect, the present invention also provides a product for assessing disease progression, relapse risk, or prognosis in patients with acute myeloid leukemia, characterized in that the product comprises a reagent for detecting the expression level of PUF60 in a bone marrow sample of a subject, for stratified diagnosis or auxiliary detection of disease progression.

[0018] Furthermore, the product is a diagnostic reagent or kit used to assess prognosis or relapse risk by detecting the protein or mRNA expression level of PUF60 in the bone marrow CD34+ LSC of a subject and comparing it with reference values ​​for newly diagnosed patients or normal controls.

[0019] Furthermore, the expression level of PUF60 in the bone marrow LSCs of treatment-naïve AML patients was higher than that in the non-LSC population, and the expression level in the LSCs of patients who relapsed after chemotherapy was higher than that in treatment-naïve patients, supporting its use as a novel LSC biomarker.

[0020] In a second aspect, the present invention provides the use of a PUF60 inhibitor in the preparation of a medicament for the treatment or prevention of relapsed acute myeloid leukemia, wherein the inhibitor inhibits PUF60 to reduce the maintenance of LSC activity.

[0021] Furthermore, the drug contains an inhibitor that can suppress PUF60 gene expression, mRNA levels, or protein activity, for regulating AML LSC function and reducing chemotherapy resistance.

[0022] Preferably, the PUF60 inhibitor is an inhibitor that inhibits the activity or level of the PUF60 protein, or an inhibitor that inhibits the mRNA level of PUF60, and its inhibitory activity is reversible or irreversible.

[0023] More preferably, the inhibitors that inhibit PUF60 protein activity or protein level include antibodies against PUF60, proteins that inhibit PUF60 protein activity or protein level, peptides, enzymes, natural compounds, synthetic compounds, organic substances, and inorganic substances; the inhibitors that inhibit PUF60 protein activity or protein level refer to substances that can bind to PUF60 but do not produce a biological response upon binding, or the inhibitors can block, inhibit, or weaken agonist-mediated responses; inhibiting PUF60 protein activity or protein level refers to knocking out PUF60.

[0024] Preferably, the inhibitor that inhibits the mRNA level of PUF60 is its antisense nucleic acid sequence, siRNA, miRNA, shRNA, dsRNA, or other proteins, peptides, enzymes, or compounds that can inhibit the mRNA level of PUF60.

[0025] In a third aspect, the present invention provides a method for constructing an animal model for studying relapsed acute myeloid leukemia, characterized by including transplanting AML cell lines with PUF60 knockdown or overexpression or primary LSC-enriched cells into immunodeficient mice to evaluate the promoting effect of PUF60 on LSC activity.

[0026] The present invention provides a method for screening candidate drugs for the treatment of relapsed acute myeloid leukemia in a fourth aspect, characterized by comprising: a) providing an animal model constructed according to the aforementioned method; b) administering a candidate drug to the animal model; c) assessing the improvement of indicators such as leukemia cell engraftment rate, survival time, or tissue invasion in the animal model; d) screening the candidate drug based on the improvement, wherein the screening targets PUF60 inhibition to regulate LSC function.

[0027] This invention first utilizes protein mass spectrometry to perform differential protein analysis on the LSC-enriched cell population (CD34+) and non-LSC cell population (CD34-) of newly diagnosed AML patients, screening for a group of RNA-binding proteins (RBPs) highly expressed in LSCs. Simultaneously, paired LSC-enriched cell populations from newly diagnosed and chemotherapy-relapsed AML patients were analyzed to screen for RBPs upregulated in relapsed LSCs. The intersection of the two sets of results yielded a group of "AML chemotherapy-relapsed LSC-specifically highly expressed RBPs," which included PUF60. Subsequent analysis using the TCGA database revealed that high PUF60 expression was most significantly correlated with low survival rates in AML patients within this RBP group. Further analysis using the BloodSpot database confirmed that PUF60 expression was significantly higher in normal karyotype AML cells than in myelodysplastic syndrome (MDS) cells and healthy human bone marrow mononuclear cells; its expression was also significantly higher in different abnormal karyotype AML cells than in healthy human hematopoietic stem cells. In terms of functional validation, this invention demonstrates that: 1) in primary AML cells, PUF60 expression levels in LSC-rich clusters are higher than in non-LSC populations, and expression levels in relapsed patient LSCs are higher than in treatment-naïve patients; 2) in the AML cell line (THP-1), PUF60 knockdown significantly inhibits cell proliferation activity (CCK-8 assay), reduces doxorubicin (DOX) resistance, and weakens colony-forming ability; while PUF60 overexpression promotes cell proliferation; 3) in a mouse xenograft model (CLDX) derived from AML cell lines, compared with mice transplanted with control cells, mice transplanted with PUF60 knockdown cells showed reduced myeloid leukemia cell engraftment, decreased leukemia cell infiltration in peripheral blood and liver / spleen, and significantly prolonged survival. These findings, based on clinical samples and in vitro / in vivo models, indicate the crucial promoting role of PUF60 in maintaining AML LSC activity, self-renewal, chemotherapy resistance, and in vivo remodeling capacity.

[0028] The beneficial effects of this invention are as follows: 1. Provides a new biomarker: For the first time, PUF60 was identified as a highly expressed protein specifically for relapsed AMLSC through proteomics analysis, providing a potential molecular biomarker for early relapse monitoring, risk stratification, and prognostic assessment of AML.

[0029] 2. Revealed a new therapeutic target: The system elucidated the key promoting role of PUF60 in maintaining AML LSC activity, self-renewal, chemotherapy resistance and in vivo reconstitution capacity, and demonstrated that targeting PUF60 can effectively inhibit LSC, providing a theoretical basis and a clear target for developing new therapies targeting the root cause of AML (LSC).

[0030] 3. Possesses clinical translational potential: PUF60-based diagnostic kits can be used to assist in clinical decision-making; inhibitors of PUF60 (such as small molecule compounds and nucleic acid interference drugs) have the potential to be developed into novel AML treatments, especially for overcoming chemotherapy resistance and preventing relapse. Attached Figure Description

[0031] Figure 1 The heatmaps show the comparison of RNA-binding protein levels between newly diagnosed AML LSC-rich clusters and non-LSC populations (n=3) (A) and the comparison of RNA-binding protein levels between newly diagnosed AML and relapsed AML LSC-rich clusters after chemotherapy (n=2) (B), as well as the Venn diagram of the intersection of the two groups (C). Twenty-two RNA-binding proteins specifically highly expressed in the relapsed AML LSC-rich clusters after chemotherapy were screened out, including PUF60.

[0032] Figure 2 This is a graph analyzing the impact of highly expressed RNA-binding proteins in 22 LSC-rich clusters of relapsed AML patients after chemotherapy on the survival of AML patients, using data from the TCGA database. A. Summary graph of the significance of the correlation between high expression of the corresponding RNA-binding protein and low survival rate of AML patients (significance is represented by p-value); B. Survival analysis graph of RNA-binding proteins that are significantly associated with high expression and low survival rate of AML patients.

[0033] Figure 3 The images show the gene expression of PUF60 in tumor cells of patients with normal karyotype AML, tumor cells of patients with MDS, and bone marrow mononuclear cells of healthy individuals in the BloodSpot database (A), and the gene expression of PUF60 in tumor cells of patients with different abnormal karyotype AML and hematopoietic stem cells of healthy individuals (B).

[0034] Figure 4 The figure shows the gene expression of PUF60 in the newly diagnosed AML LSC rich cluster and non-LSC population as detected by RT-q-PCR (A), and the gene expression of PUF60 in the newly diagnosed AML to the relapsed AML LSC rich cluster after chemotherapy (B).

[0035] Figure 5 This is a statistical graph showing the effects of PUF60 knockdown (A) and overexpression (B) on cell proliferation activity in the AML cell line THP-1, as detected by the CCK8 assay.

[0036] Figure 6 This is a statistical chart showing the drug resistance of PUF60 knocked-down AML cell line THP-1 and control THP-1 cells in a DOX concentration gradient resistance experiment.

[0037] Figure 7The images show the colony morphology (A) and statistical diagram (B) of the AML cell line THP-1 after the PUF60 gene was knocked down.

[0038] Figure 8 The images are from a mouse transplantation model of immunodeficient mice that were infused with the AML cell line THP-1 (either PUF60 knockdown or control group): A. Percentage of AML cells in bone marrow (top) and percentage of AML cells in peripheral blood (bottom); B. IHC staining images of bone marrow, liver, and spleen tissue sections; C. Statistical graph of IHC staining intensity of bone marrow, liver, and spleen tissue sections; D. Mouse survival chart. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] PUF60 promotes the maintenance of leukemia stem cell activity in acute myeloid leukemia (AML). This invention elucidates the functional regulation of PUF60 on leukemia stem cells in AML. It provides a novel leukemia stem cell biomarker that can be used for: assessing relapse rates after chemotherapy, disease risk assessment, prognostic evaluation, stratified diagnosis, and detecting or assisting in the detection of disease progression; and a kit for detecting the PUF60 gene or protein. Specifically, it verifies: 1. Screening was conducted to identify a group of RNA-binding proteins, including PUF60, with higher protein levels in an AML LSC-enriched cell population (CD34+) than in the non-LSC population. Another group of RNA-binding proteins, including PUF60, were screened to identify a group of RNA-binding proteins, including PUF60, with higher protein levels in a chemotherapy-relapsed AML LSC-enriched cell population than in a treatment-naïve AML LSC-enriched cell population. The intersection of the two groups yielded a population-specific high expression of RBP (containing PUF60) in the AML chemotherapy-relapsed LSC-enriched cell population.

[0041] 2. In the TCGA database analysis, among the LSC-rich clusters of RBP that showed high expression of RBP after AML chemotherapy relapse, high expression of PUF60 was most significantly associated with reduced survival rate in AML patients.

[0042] 3. In the BloodSpot database analysis, the gene expression level of PUF60 in tumor cells of patients with normal karyotype AML was significantly higher than that in tumor cells of patients with MDS, and also significantly higher than that in bone marrow mononuclear cells of healthy individuals; the gene expression level of PUF60 in tumor cells of patients with different abnormal karyotype AML was significantly higher than that in hematopoietic stem cells of healthy individuals.

[0043] 4. The gene expression level of PUF60 in the newly diagnosed AML LSC enriched cell population (CD34+) was significantly higher than that in the non-LSC population; the gene expression level of PUF60 in the relapsed AML LSC enriched cell population after chemotherapy was significantly higher than that in the newly diagnosed AML LSC enriched cell population.

[0044] 5. Compared with the control group AML cells THP-1, PUF60 knockdown THP-1 cells showed significantly reduced cell viability in the CCK8 assay; PUF60 overexpression THP-1 cells showed significantly increased proliferation activity.

[0045] 6. Compared with the control group AML cells THP-1, PUF60 knockdown THP-1 cells showed significantly reduced drug resistance in DOX concentration gradient resistance experiments.

[0046] 7. Compared with the control group AML cells THP-1, the number of CFUs formed by PUF60 knockdown THP-1 cells was significantly reduced.

[0047] Compared with the control group AML cells THP-1, immunodeficient mice infused with PUF60 knockdown THP-1 cells showed a significantly decreased bone marrow engraftment rate, reduced leukocyte infiltration in peripheral blood and liver and spleen, and a significantly increased survival rate.

[0048] Example 1: Differential proteomics analysis of AML LSC and relapsed LSC This embodiment aims to screen for proteins that are specifically highly expressed in AML LSC and relapsed LSC.

[0049] (1) Sample collection and processing: Bone marrow blood samples were collected from 3 newly diagnosed AML patients and sequential bone marrow blood samples from 2 matched (newly diagnosed-relapsed) AML patients. Bone marrow mononuclear cells (BMMNCs) were extracted using density gradient centrifugation (human leukocyte separation fluid, centrifugation at 1500 rpm for 5 min to remove plasma, followed by centrifugation at 1800 rpm for 18 min to separate mononuclear cells).

[0050] (2) Cell sorting: BMMNCs were stained with CD34-PE antibody and CD34+ cells (LSC-rich population) and CD34- cells (non-LSC population) were sorted by flow cytometry. For paired samples, CD34+ cells at two time points were sorted.

[0051] (3) Protein mass spectrometry detection and analysis: The sorted cell pellet was sent to Novogene for mass spectrometry detection. Data analysis: ① Comparison of CD34+ vs CD34- cells from newly treated patients yielded the "LSC high expression protein set" (p<0.05); ② Comparison of CD34+ cells from relapsed vs newly treated patients yielded the "relapsed LSC upregulated protein set" (p<0.05). The intersection of the two was used to obtain the "AML chemotherapy relapse LSC specific high expression RBP" list, which included PUF60.

[0052] Example 2: Correlation analysis between PUF60 expression and prognosis of AML patients To validate the clinical significance of the proteomics findings, the GEPIA2 online tool (based on the TCGA database) was used to analyze the relationship between PUF60 expression and overall survival (OS) in AML patients. In the "Survival Analysis" module, the gene name "PUF60" was entered, and the tumor type "LAML" (AML) was selected.

[0053] Survival curve analysis showed that the overall survival of AML patients in the high PUF60 expression group was significantly shorter than that in the low expression group (p<0.05), indicating that high PUF60 expression is a potential indicator of poor prognosis in AML.

[0054] Example 3: Expression profiling of PUF60 in different blood cell populations Cellular expression specificity of PUF60 was analyzed using the BloodSpot database.

[0055] (1) The “Leukemia MILE study” dataset was selected from the database to compare the mRNA expression levels of PUF60 in normal karyotype AML, MDS, and healthy donor bone marrow mononuclear cells. The results showed that the expression of PUF60 in normal karyotype AML was significantly higher than that in MDS and healthy controls.

[0056] (2) The “Normal hematopoiesis with AMLs” dataset was selected from the database to compare the expression of PUF60 in AML cells with different abnormal karyotypes and healthy human hematopoietic stem cells (HSCs). The results showed that PUF60 expression was significantly higher in all types of AML cells than in normal HSCs. These results suggest that the increased expression of PUF60 is AML-specific and stem cell-related.

[0057] Example 4: Validation of PUF60 expression in primary AML cells The expression of PUF60 in LSC and relapsed samples was verified at the mRNA level by qPCR.

[0058] (1) Samples and sorting: Bone marrow blood samples were collected from 10 newly treated and 8 relapsed AML patients, and BMMNCs were separated and CD34+ and CD34- cell populations were sorted by flow cytometry (the method is the same as in Example 1).

[0059] (2) RNA extraction and qPCR: Total RNA was extracted from cells using the Qiagen kit (#74104), and real-time quantitative PCR was performed after reverse transcription. The relative expression level of PUF60 was calculated using the 2^(-ΔΔCt) method with GAPDH as an internal control.

[0060] (3) Results: In treatment-naïve patients, the expression level of PUF60 in CD34+ cells was significantly higher than that in paired CD34- cells. In the paired (treatment-naïve-relapse) analysis, the expression level of PUF60 in CD34+ cells of relapsed samples was significantly higher than that in their treatment-naïve samples. This result confirms the findings of proteomics at the mRNA level.

[0061] Example 5: Effect of PUF60 on the proliferation activity of AML cell lines (1) Cell model construction: Stable knockdown (KD) and overexpression (OE) PUF60 cell lines were constructed in the AML cell line THP-1 using a lentiviral system, and empty vector virus transfected cells were used as controls (NC). The knockdown and overexpression efficiency were verified by qPCR.

[0062] (2) CCK-8 proliferation assay: PUF60-KD, PUF60-OE, and NC cells were seeded in 96-well plates at a density of 5000 cells per well. On days 0, 1, 2, and 3 of culture, 10 μL of CCK-8 reagent was added to each well, and after incubation for 1-2 hours, the absorbance at 450 nm (OD450) was measured using a microplate reader. Cell growth curves were plotted. The results showed that compared with the NC group, cell proliferation in the PUF60-KD group was significantly inhibited, while proliferation in the PUF60-OE group was accelerated.

[0063] Example 6: The effect of PUF60 on chemotherapy resistance in AML cells (1) Experimental treatment: PUF60-KD and NC THP-1 cells were seeded in 96-well plates (~10^4 cells / well) and different concentrations of doxorubicin (DOX: 0, 200, 400, 800 nM) were set up, with 6 replicates in each group.

[0064] (2) Drug resistance test: After DOX treatment for about 24 hours, CCK-8 assay was performed (method as in Example 5). The relative survival rate of cells in each group at different drug concentrations was calculated (with the untreated group as 100%). The results showed that at the same DOX concentration, the cell survival rate of the PUF60-KD group was significantly lower than that of the NC group, indicating that knocking down PUF60 enhanced the sensitivity of cells to DOX, i.e., reduced drug resistance.

[0065] Example 7: Effect of PUF60 on the self-renewal capacity of AML cells The effect of PUF60 on cell colony formation ability was evaluated using a colony formation assay. PUF60-KD and NC THP-1 cells were resuspended in methylcellulose semi-solid medium (StemCell, #H04435) and seeded in 6-well plates at a density of 1000-2000 cells per well, with 3 replicates per group. Cells were incubated at 37°C in a 5% CO2 incubator for 7-14 days. The number of colonies with >50 cells was counted under an inverted microscope. The results showed that the number of colonies formed in the PUF60-KD group was significantly less than that in the NC group, indicating that PUF60 knockdown weakened the self-renewal ability of AML cells.

[0066] Example 8: The role of PUF60 in an in vivo AML model (1) Mouse model construction: 6-8 week old female NSG immunodeficient mice were irradiated with 1 Gy before transplantation. PUF60-KD or NC THP-1 cells (e.g., 5×10^6 cells / mouse) were injected via the tail vein.

[0067] (2) In vivo monitoring: Blood was collected weekly via the tail vein after transplantation, and the proportion of human CD45+ (hCD45+) cells in the peripheral blood of mice was detected by flow cytometry to assess the engraftment of leukemia cells. The survival time of the mice was recorded.

[0068] (3) Endpoint analysis: When mice died or reached the experimental endpoint, bone marrow, spleen and liver were taken. One part was made into a single-cell suspension and the hCD45+ cell infiltration ratio was detected by flow cytometry; the other part of the tissue was fixed with 4% paraformaldehyde, embedded in paraffin and then subjected to H&E staining and PUF60 immunohistochemical staining to observe pathological morphology and PUF60 protein localization.

[0069] (4) Results: Compared with mice transplanted with NC cells, mice transplanted with PUF60-KD cells showed significantly lower hCD45+ cell engraftment rates in peripheral blood and bone marrow, reduced hepatosplenomegaly and leukemia infiltration, and significantly prolonged median survival. In vivo experiments confirmed the key role of PUF60 in the in vivo growth and survival of AML cells.

[0070] This invention, a comprehensive study of the proteomic identification and functional inhibition of PUF60 in relapsed AML LSC, will significantly advance the field of AML and provide new diagnostic and treatment strategies.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of PUF60 as a molecular marker for relapsed acute myeloid leukemia (AML) leukemia stem cells (LSCs) promotes the maintenance of LSC activity and functional regulation.

2. The application according to claim 1, characterized in that, The PUF60 is specifically highly expressed in CD34+ LSC-enriched cell populations of patients with relapsed AML after chemotherapy, and can be used to assess relapse rate or disease risk after chemotherapy.

3. A product for assessing disease progression, relapse risk, or prognosis in patients with acute myeloid leukemia, characterized in that, The product contains reagents for detecting the expression level of PUF60 in bone marrow samples of subjects, for stratified diagnosis or to assist in the detection of disease progression.

4. The product according to claim 3, characterized in that, The product is a diagnostic reagent or kit used to assess prognosis or relapse risk by detecting the protein or mRNA expression level of PUF60 in the CD34+ LSC of a subject's bone marrow and comparing it with reference values ​​for newly diagnosed patients or normal controls.

5. The product according to claim 4, characterized in that, The expression level of PUF60 in the bone marrow LSCs of newly diagnosed AML patients was higher than that in the non-LSC population, and the expression level in the LSCs of patients who relapsed after chemotherapy was higher than that in newly diagnosed patients, supporting its status as a novel LSC biomarker.

6. The use of PUF60 inhibitors in the preparation of drugs for the treatment or prevention of relapsed acute myeloid leukemia, which inhibit PUF60 to reduce the maintenance of LSC activity.

7. The application according to claim 6, characterized in that, The drug contains inhibitors that can suppress PUF60 gene expression, mRNA levels, or protein activity, and is used to regulate AML LSC function and reduce chemotherapy resistance.

8. The application according to claim 7, characterized in that, The inhibitor is selected from antisense nucleic acids, siRNA, shRNA, miRNA, dsRNA, or small molecule compounds.

9. A method for constructing an animal model for studying relapsed acute myeloid leukemia, characterized in that, This includes transplanting PUF60 knockdown or overexpression AML cell lines or primary LSC-enriched cells into immunodeficient mice to evaluate the promoting effect of PUF60 on LSC activity.

10. A method for screening candidate drugs for the treatment of relapsed acute myeloid leukemia, characterized in that, include: a) Provide an animal model constructed according to the method of claim 9; b) Administer the candidate drug to the animal model; c) Evaluate the improvement in indicators such as leukemia cell engraftment rate, survival time, or tissue invasion in the animal model; d) Based on the improvement, screen the candidate drug for PUF60 inhibition to regulate LSC function.

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