Application of CSF1r inhibitor in preparation of NK / t cell lymphoma treatment drug

Abstract

Disclosed is an application of a CSF1R inhibitor in the preparation of an NK / T cell lymphoma treatment drug. Further disclosed are a use as an NK / T cell lymphoma marker, a use of a CSF1 detection reagent in the preparation of an NK / T cell lymphoma prognosis kit, and a use of the CSF1 detection reagent in the preparation of an NK / T cell lymphoma primary screening kit. The present invention can be promoted and applied to primary screening, prognosis and treatment of clinical NK / T cell lymphoma patients.

Description

Application of CSF1R inhibitors in the preparation of drugs for treating NK / T cell lymphoma Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of a CSF1R inhibitor for preparing a drug for treating NK / T cell lymphoma. Background Art

[0002] NK / T-cell lymphoma is a highly heterogeneous and aggressive type of malignant non-Hodgkin's lymphoma. It is highly prevalent in my country, severely impacting the lives and health of its citizens. Clinically, NK / T-cell lymphoma heterogeneity manifests itself in varying tumor invasion sites, with the vast majority of patients presenting with lesions invading the nasal cavity or nasopharynx. NK / T-cell lymphoma also exhibits diverse pathological morphology. Even within the same stage, NK / T-cell lymphoma patients exhibit complex and heterogeneous responses to treatment, with nearly one-third experiencing relapse or refractory disease due to drug intolerance.

[0003] Tumor heterogeneity and treatment resistance are major challenges that urgently need to be addressed in the clinical setting of NK / T-cell lymphoma. Traditional treatment options for NK / T-cell lymphoma are limited, primarily determined by the site of tumor invasion (nasal or extranasal) and the extent of tumor invasion. For the past decade, treatment has primarily relied on multidrug combinations with non-anthracycline agents and concurrent / sequential radiotherapy. Currently, combination chemotherapy with various combinations of drugs such as levoaspartase (or pegaspargase), gemcitabine, oxaliplatin, etoposide, cisplatin, ifosfamide, methotrexate, dexamethasone, and chidamide, combined with concurrent radiotherapy, has achieved 5-year overall survival rates of 70% or higher for early-stage NK / T-cell lymphoma. However, for advanced, relapsed, and refractory NK / T-cell lymphoma, which accounts for approximately 30% of the total, treatment options are more challenging and limited, resulting in a poorer prognosis. In recent years, immunotherapies and other targeted small molecule drugs have been successfully applied to a variety of tumors, providing new insights into the clinical treatment of cancer patients. A multinational research team explored the efficacy of a mainstream immunotherapy drug, the immune checkpoint inhibitor PD-1 monoclonal antibody, in patients with NK / T cell lymphoma. However, the results showed that only a small number of patients responded to this immunotherapy regimen long-term.

[0004] Therefore, it is still urgent to discover new tumor markers for NK / T cell lymphoma, achieve more accurate and effective prognostic judgment for NK / T cell lymphoma patients, and use them as new targets for the treatment of NK / T cell lymphoma.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a technical solution that can accurately and effectively judge the prognosis of NK / T cell lymphoma in response to the above technical problems.

[0007] To achieve the above objectives, the present invention provides the use of a CSF1R (colony stimulating factor-1 receptor) inhibitor for preparing a drug for treating NK / T cell lymphoma.

[0008] Preferably, the CSF1R inhibitor includes but is not limited to any one or more of BLZ945, ARRY382, Axatilimab, Cabiralizumab, Edicotinib, Emactuzumab, Pexidartinib, Pimicotinib, Surufatinib or Vimseltinib. Most preferably, the CSF1R inhibitor is BLZ945.

[0009] On the other hand, the present invention also provides use of CSF1 as a NK / T cell lymphoma marker.

[0010] On the other hand, the present invention also provides use of a CSF1 detection reagent for preparing a NK / T cell lymphoma prognosis kit.

[0011] On the other hand, the present invention also provides the use of a CSF1 detection reagent for preparing a NK / T cell lymphoma screening kit.

[0012] Preferably, the CSF1 detection reagent comprises a PCR amplification primer pair, and the nucleotide sequences of the PCR amplification primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0013] The expression levels of CSF1 in various cell types in the peripheral blood and tumor tissues of patients with NK / T cell lymphoma were detected using single-cell transcriptome sequencing, among which CSF1 was significantly highly expressed in tumor cells.

[0014] The expression of CSF1 in NK / T cell lymphoma tumor cells can be detected by immunofluorescence co-staining of paraffin-embedded wax block sections of tumor tissue, in which CSF1 is widely and abundantly expressed in tumor cells.

[0015] CSF1 expressed by NK / T cell lymphoma tumor cells can effectively promote the formation of macrophages from monocytes, and its expression level is closely related to the proportion of macrophages in tumor tissue. In addition, the proportion of macrophages is also significantly correlated with the clinical prognosis of NK / T cell lymphoma patients.

[0016] The promoting effect of CSF1 on macrophages can be inhibited by CSF1R inhibitors. Therefore, CSF1 can be used as an important target for the treatment of NK / T cell lymphoma, and CSF1R inhibitors can be used to treat NK / T cell lymphoma patients.

[0017] The present invention provides a tumor marker, CSF1, for NK / T cell lymphoma, which can be widely used and applied in the initial screening of NK / T cell lymphoma patients in clinical practice. The expression level of this tumor marker, CSF1, is correlated with the formation of macrophages in NK / T cell lymphoma tissues and, in turn, with the clinical prognosis of NK / T cell lymphoma patients. This marker can be used for the precise classification and treatment of NK / T cell lymphoma patients in clinical practice. Using this tumor marker, CSF1, as an important target for the treatment of NK / T cell lymphoma patients, corresponding CSF1R inhibitors can be used to treat these patients, demonstrating clinical translational value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG1 shows the expression levels and expression ratios of CSF1 and CSF1R in various cell types in single-cell transcriptome sequencing data of NK / T cell lymphoma.

[0019] FIG2 shows the results of immunofluorescence co-staining experiments.

[0020] FIG3 shows the results of Western blotting.

[0021] FIG4 shows the interaction strength of the CSF1-CSF1R regulatory axis between tumor cell subsets and myeloid cell subsets obtained by cell communication analysis based on single-cell transcriptome sequencing data of NK / T cell lymphoma.

[0022] FIG5 is a schematic diagram of an in vitro co-culture experiment of cell supernatant derived from NK / T cell lymphoma tumor cells and monocytes (THP-1 cells).

[0023] FIG6 shows the mean fluorescence intensity of a macrophage marker (CD206) measured by flow cytometry of THP-1 cells in different treatment groups obtained from an in vitro co-culture experiment of monocytes with cell supernatants derived from NK / T cell lymphoma tumor cells.

[0024] FIG7 shows the results of survival analysis.

[0025] FIG8 is a schematic diagram of an in vivo mouse experiment to block the CSF1-CSF1R regulatory axis.

[0026] FIG9 shows the expression of macrophage markers (CD206) in mouse transplanted tumors detected by immunohistochemical staining (A) and a bar graph of the statistically obtained CD206-positive cell ratio (B).

[0027] FIG10 shows the tumor volumes measured for the CSF1R inhibitor group and the control group in the mouse experiment.

[0028] FIG11 shows the transplanted tumors (A) obtained from the CSF1R inhibitor group and the control group in the mouse experiment, as well as the final statistically obtained tumor mass (B) and mouse body weight (C). DETAILED DESCRIPTION

[0029] To more clearly understand the above-mentioned objects, features, and advantages of the present invention, the present invention is further described in detail below with reference to the accompanying drawings and specific examples. Those skilled in the art should understand that the examples are merely helpful for understanding the present invention and should not be construed as limiting the present invention. Unless otherwise specified, the sequencing and analysis methods used in the following examples are conventional methods.

[0030] Example 1. CSF1 is highly expressed in NK / T cell lymphoma patients

[0031] Single-cell transcriptome sequencing was performed on tumor tissue samples from 10 patients with NK / T-cell lymphoma from the Sun Yat-sen University Cancer Center. Briefly, the NK / T-cell lymphoma tissue samples were enzymatically digested to obtain a single-cell suspension. A commercial single-cell transcriptome library kit (10x Genomics) was then used to load the live cells and create a library. Finally, the resulting cDNA library was subjected to high-throughput sequencing to generate a sequence file.

[0032] The acquired single-cell transcriptome sequencing data were analyzed to identify all cell types present in tumor tissues from patients with NK / T-cell lymphoma, including normal NK cells, tumor NK cells, T cells, B cells, myeloid cells, and other cells. Figure 1 shows the expression levels and ratios of CSF1 and CSF1R in various cell types from single-cell transcriptome sequencing data from NK / T-cell lymphoma. CSF1 is primarily expressed in tumor NK cells, and its receptor, CSF1R, is specifically and highly expressed in myeloid cells.

[0033] On the other hand, the paraffin-embedded wax block sections of NK / T cell lymphoma tumor tissue were co-stained with immunofluorescence, and the NK / T cell lymphoma cell lines were detected by real-time fluorescence quantitative PCR and Western blotting. In immunofluorescence co-staining experiments, a CD56-specific antibody (Zsbio; Cat# ZM-0057) was used to identify tumor cells in tumor tissue paraffin sections, and a CSF1-specific antibody (Abcam; Cat# ab52864) was used to identify CSF1 protein in tumor tissue. The corresponding fluorescent signals were detected to determine the expression of CSF1 in NK / T cell lymphoma tumor cells. The primer sequences used in the real-time fluorescence quantitative PCR experiment on the NK / T cell lymphoma cell line are shown in Table 1 below (SEQ ID NO: 1 and SEQ ID NO: 2) to detect the expression level of CSF1 transcripts. Western blotting was also performed using the aforementioned CSF1-specific antibody to detect cell lysate specimens and the supernatant obtained from the separation of the NK / T cell lymphoma cell line to determine the expression and secretion of CSF1 in tumor cells.

[0034] Table 1: Real-time fluorescence quantitative PCR primer sequences

[0035] The oligonucleotide chain sequences of the above primers were synthesized by Guangzhou Paizhen Biotechnology Co., Ltd.

[0036] The above method further confirmed the expression of CSF1 in NK / T cell lymphoma tumor cells, and the presence of cell-secreted CSF1 molecules in the tumor cell supernatant (as shown in Figures 2 and 3). Figure 2 shows the expression of CSF1 in tumor cells in paraffin-embedded wax block sections of NK / T cell lymphoma tumor tissue detected by immunofluorescence co-staining experiments (CD56 and CSF1 double-positive cells). Figure 3 shows the expression and secretion of CSF1 protein in cell lysates and cell supernatants of NK / T cell lymphoma tumor cell lines (YT cells and KHYG-1 cells) confirmed by Western blotting.

[0037] Example 2. Highly expressed CSF1 in NK / T cell lymphoma induces the formation of tumor-associated macrophages

[0038] CellPhoneDB software was used to analyze cell communication using single-cell transcriptome sequencing data from the aforementioned NK / T cell lymphoma patients, revealing significant cell-cell interactions between tumor cell subsets and immune cell subsets. Figure 4 shows the strength of the CSF1-CSF1R regulatory axis interactions between tumor cell subsets and myeloid cell subsets. As shown in Figure 4, all tumor NK cell subsets (NK_C5_ISG15, NK_C6_ITM2A, NK_C7_CCL4, NK_C8_MT1E, NK_C9_CXCL13, and NK_C10_STMN1) were found to have strong cellular interactions with tumor-associated macrophages (TAMs) that promote cancer through the CSF1-CSF1R regulatory axis.

[0039] Furthermore, by an in vitro co-culture experiment of cell supernatants derived from NK / T cell lymphoma tumor cells and monocytes (THP-1 cells), it was observed whether the cell supernatants potentially containing CSF1 secreted by tumor cells have a promoting effect on the differentiation of monocytes into macrophages, and whether this effect can be further blocked by CSF1R inhibitors. Figure 5 is a schematic diagram of an in vitro co-culture experiment of cell supernatants derived from NK / T cell lymphoma tumor cells and monocytes (THP-1 cells). Cell supernatants from NK / T cell lymphoma tumor cell lines pretreated with CSF1R inhibitors or DSMO controls were used to culture THP-1 cells, thereby confirming the presence of secreted CSF1 in the supernatants of NK / T cell lymphoma tumor cells, and that it can promote the formation of macrophages from monocytes through the CSF1-CSF1R regulatory axis. The results of the co-culture experiment confirmed that cell supernatants derived from NK / T cell lymphoma tumor cells can promote the differentiation of monocytes into macrophages, and this promoting effect is inhibited by CSF1R specific inhibitors. Figure 6 shows the mean fluorescence intensity of the macrophage marker (CD206) of THP-1 cells in different treatment groups obtained from an in vitro co-culture experiment of cell supernatants derived from NK / T cell lymphoma tumor cells and monocytes, as measured by flow cytometry. A higher mean fluorescence intensity of CD206 indicates a greater number of THP-1 cells transformed into macrophages.

[0040] This suggests that NK / T cell lymphoma tumor cells can induce macrophage formation by expressing and secreting the CSF1 molecule. Furthermore, survival analysis was performed using the survival and survminer software in the R language on paired transcriptome sequencing data and patient prognostic information from 51 NK / T cell lymphoma patients. This survival analysis divided the 51 lymphoma patients into two groups based on the level of tumor-associated macrophage infiltration in tumor tissue: high tumor-associated macrophage infiltration (n=13) and low tumor-associated macrophage infiltration (n=38). The results of the survival analysis revealed that high tumor-associated macrophage infiltration in tumor tissue was associated with poor overall survival. Figure 7 shows the results of the survival analysis. As can be seen, the higher the tumor-associated macrophage infiltration in the patient's tumor tissue, the worse the prognosis. This suggests that the high enrichment of tumor-associated macrophages in NK / T cell lymphoma, caused by tumor cell expression of CSF1, affects patient survival.

[0041] Example 3. CSF1-targeted therapeutic regimen inhibits tumor progression of NK / T cell lymphoma in mice

[0042] Figure 8 is a schematic diagram of an in vivo mouse experiment to block the CSF1-CSF1R regulatory axis. Peripheral immune cells were first isolated from human peripheral blood samples using human peripheral blood lymphocyte separation medium (Rayward; Cat# LTS1077). Four-week-old severely immunodeficient NCG mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were selected and injected with 1x10 cells into the tail vein of the NCG mice. 7 Normal human peripheral immune cells were used to construct humanized mice, and 5x10 cells were injected subcutaneously. 6 The tumor cells (YT cells) of NK / T cell lymphoma were used to construct a humanized mouse xenograft model of NK / T cell lymphoma. Then, mice in different groups were orally gavaged with 200 mg / kg of BLZ945 (CAS No.: 953769-46-5; Selleck; Cat#S7725), a small molecule drug that specifically inhibits CSF1R, and the control 20% Captisol (Aladdin; Cat#C125030) for ten days for comparison. This was to explore whether treatment targeting the CSF1-CSF1R regulatory axis can effectively alleviate tumor progression of NK / T cell lymphoma at the in vivo level.

[0043] The CSF1R inhibitor BLZ945 can specifically target CSF1R (i.e., the binding receptor of the tumor marker CSF1). BLZ945 is used as an example to illustrate the specific embodiment of the present invention. However, the CSF1R inhibitor of the present invention includes but is not limited to BLZ945. Other CSF1R inhibitors and even CSF1 inhibitors can also be applied to the present invention and fall within the scope of protection of the present invention, such as ARRY-382 (PF-07265804, CAS No.: 1313407-95-2), Axatilimab (SNDX-6352, CAS No.: 2155851-88-8), Cabiralizumab (FPA-008, CAS No.: 1613144-80-1), Edicotinib (JNJ -40346527, CAS No.: 1142363-52-7), Emactuzumab (RG-7155, CAS No.: 1448221-67-7), Pexidartinib (PLX3397, CAS No.: 1029044-16-3), Pimicotinib (ABSK021, CAS No.: 2253123-16-7), Surufatinib (HMPL-012, CAS No.: 1308672-74-3), Vimseltinib (DCC-3014, CAS No.: 1628606-05-2).

[0044] The experimental results are shown in Figure 9. Figure 9 shows the expression of a macrophage marker (CD206) in mouse xenografts as detected by immunohistochemical staining (A) and a bar graph of the percentage of CD206-positive cells (B), demonstrating the effect of blocking the CSF1-CSF1R regulatory axis on macrophage infiltration. As shown in Figure 9, macrophage infiltration in xenografts of mice treated with a CSF1R inhibitor was significantly reduced.

[0045] In addition, the tumor volumes of the CSF1R inhibitor group and the control group in the mouse experiment were measured, and the tumor mass and mouse weight of the transplanted tumors obtained from the CSF1R inhibitor group and the control group in the mouse experiment were calculated. Figure 10 shows the changes in tumor volume measured for the CSF1R inhibitor group and the control group in the mouse experiment. Figure 11 shows the transplanted tumors (A) obtained from the CSF1R inhibitor group and the control group in the mouse experiment, as well as the final statistical tumor mass (B) and mouse weight (C). As can be seen from Figures 10 and 11, the tumor volume and mass of the mice in the CSF1R inhibitor group were significantly reduced compared to the control group, indicating that the treatment regimen targeting CSF1 can significantly reduce the infiltration of macrophages that play a tumor-promoting function in NK / T cell lymphoma, thereby inhibiting the tumor progression of NK / T cell lymphoma.

[0046] It is worth noting that the above description is not a limitation of the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by technicians in this technical field within the essential scope of the present invention should also fall within the scope of protection of the present invention.

Claims

1. Application of CSF1R inhibitors in the preparation of drugs for the treatment of NK / T cell lymphoma.

2. The use according to claim 1, characterized in that: The CSF1R inhibitor is any one or more of BLZ945, ARRY382, Axatilimab, Cabiralizumab, Edicotinib, Emactuzumab, Pexidartinib, Pimicotinib, Surufatinib or Vimseltinib.

3. Use of CSF1 as a marker for NK / T cell lymphoma.

4. Use of CSF1 detection reagent for preparing NK / T cell lymphoma prognosis kit.

5. The use according to claim 4, wherein the CSF1 detection reagent comprises a PCR amplification primer pair, and the nucleotide sequences of the PCR amplification primer pair are shown in SEQ ID NO: 1 and SEQ ID NO:

2.

6. Use of CSF1 detection reagent for preparing NK / T cell lymphoma screening kit.

7. The use according to claim 6, wherein the CSF1 detection reagent comprises a PCR amplification primer pair, and the nucleotide sequences of the PCR amplification primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

Images