Use of fgfr inhibitors in the manufacture of a medicament for treating craniopharyngioma

Abstract

The application discloses use of FGFR inhibitors in the preparation of a drug for treating craniopharyngioma, and belongs to the technical field of biological medicines. Single-cell transcriptome (scRNA-seq) and high-resolution spatial transcriptome (ST) analysis are performed on ACP and PCP, and tumor-related unique structures composed of different cell states are identified. It is found that the FGF signaling pathway related to SASP is a potential therapeutic target of ACP, and it is proved by FGFR inhibitors Pemigatinib and Infigratinib that inhibition of the FGF4-FGFR2 pathway can inhibit the progression of ACP tumors in an animal model.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of FGFR inhibitors in the preparation of drugs for treating craniopharyngioma. Background Technology

[0002] Craniopharyngioma (CP) is a common intracranial tumor located in the sellar and suprasellar regions, originating from embryonic remnants of the Rathke's bursa epithelium. Due to its sensitive sellar and suprasellar location, CP frequently compresses and damages important structures of the pituitary gland, hypothalamus, and visual organs. Based on its occurrence and age distribution, CP is histologically classified into two main subtypes: ACP and PCP. ACP accounts for 90% of all CPs and can occur at all ages, but exhibits a bimodal distribution. In contrast, PCP occurs almost exclusively in adults and is typically a solid tumor. Similar to CP, Rathke's cleft cyst (RCC) also forms from remnants of the embryonic Rathke's bursa and is often confused with PCP. Due to their underlying histopathological and embryological similarities, the two conditions are considered to be different stages of the same disease. However, due to the scarcity and difficulty in obtaining RCC specimens, only a very small number of RCC cases are thought to have spontaneously developed into PCP.

[0003] ACP and PCP exhibit distinct morphological and histological features, as well as different epidemiological and biological behaviors. ACP can be described using the three 90% rules: 90% of tumors are predominantly cystic, 90% show typical prominent calcifications, and 90% show contrast agent absorption within the cyst wall. In contrast to the cystic structure of ACP, PCP is mostly a solid tumor with calcifications being rare. PCP presents as differentiated squamous epithelium, forming a pseudoepithelial and fibrovascular core (FVC). ACP is composed of… CTNNB1 Somatic mutations in the gene (encoding the β-catenin protein) drive this process, increasing β-catenin stability and leading to activation of the WNT pathway. Two mouse models targeting pituitary stem cells have demonstrated this. CTNNB1 Mutations can drive tumorigenesis and the formation of tumors similar to human acute cerebral inflammatory syndrome (ACP). However, these tumors do not originate from SOX2+ stem cells expressing oncogenic β-catenin mutations, but rather develop from SOX2- cells, which are transformed by SOX2+-derived cluster cells via stromal-associated polysaccharide-associated (SASP) pathways. Recent pathological studies of ACP have made some progress. However, the SASP-related structures of ACP at single-cell and spatial resolution, as well as the key SASP-related signaling pathways leading to tumorigenesis, remain not fully elucidated. Summary of the Invention

[0004] To address the aforementioned shortcomings in the prior art, this invention provides a novel use of FGFR inhibitors in the preparation of drugs for treating craniopharyngioma. This invention has found that FGFR inhibitors Pemigatinib and Infigratinib, which inhibit the FGF4-FGFR2 pathway, can effectively inhibit the proliferation of craniopharyngioma.

[0005] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows:

[0006] The purpose of this invention is to provide the use of FGFR inhibitors in the preparation of medicaments for the treatment of craniopharyngioma.

[0007] Furthermore, FGFR inhibitors are inhibitors that suppress the FGF4-FGFR2 pathway.

[0008] Furthermore, FGFR inhibitors are Pemigatinib or Infigratinib.

[0009] Furthermore, the craniopharyngioma subtype is ACP.

[0010] Another object of the present invention is to provide a drug for the prevention and treatment of craniopharyngioma, which includes the above-mentioned FGFR inhibitor.

[0011] Furthermore, FGFR inhibitors are Pemigatinib or Infigratinib.

[0012] Furthermore, the drug also includes pharmaceutically acceptable excipients of Pemigatinib or Infigratinib.

[0013] The beneficial effects of this invention are:

[0014] This invention performed single-cell transcriptome (scRNA-seq) and high-resolution spatial transcriptome (ST) analyses on ACP and PCP, identifying tumor-associated unique structures composed of different cellular states. The SASP-related FGF signaling pathway was identified as a potential therapeutic target for ACP, and the FGFR inhibitors pemigatinib and infigratinib confirmed that inhibiting the FGF4-FGFR2 pathway can suppress ACP tumor progression in animal models. Attached Figure Description

[0015] Figure 1Structural spatial analysis identification diagrams for ACP and PCP; where A is a workflow diagram of ST; B is a H&E staining and cell type diagram of ACP and PCP patient samples, with the boxed area shown at high magnification in diagrams C and D; C and D are annotated spatial diagrams (left) and matched H&E images (right) of ACP437 (C) and PCP473 (D), with the boxed area shown at high magnification, arrows indicating macrophages filled with microcysts, and arrowheads indicating empty microcysts, scale bar, 100 μm; E is a UMAP visualization of PCP epithelial cells based on scRNA-seq data; F is a UMAP diagram showing the pseudo-temporal developmental trajectory of PCP epithelial cell subsets, with orange arrows indicating the developmental order between cells; G is an annotated spatial diagram of PCP473 epithelial cell subsets (left and middle) and the corresponding H&E image (right), with the boxed area shown at high magnification in the middle panel, scale bar, 200 μm; H is the inferred developmental trajectory of PCP epithelial cell subpopulations at spatial resolution, with black arrows indicating the developmental order across spatial regions; I is a representative mIHC image of PCP pathological tissue stained with antibodies, with the boxed area displayed at high magnification in the right panel, scale bar 20 μm; J is a schematic diagram of multi-layered spatial tissue of PCP epithelial subtypes.

[0016] Figure 2 This section presents spatial hierarchy analysis diagrams of tumor cell developmental trajectories and SASP-related structures in ACP. A represents a UMAP visualization of ACP epithelial cells based on scRNA-seq data; B is a dynamic heatmap of gene expression patterns within the ACP epithelial cell state trajectory; C shows the UMAP diagram displaying the ACP epithelial cell developmental trajectory; D represents the differentiation status of ACP epithelial subtypes; E is a similarity analysis diagram of gene expression patterns of individual ACP epithelial subtypes between ST and scRNA-seq data; F is a spatial distribution map of each ACP epithelial subtype in a single specimen, scale bar 1 mm; G is an annotated spatial map of the epithelial subtypes of ACP499 and ACP511 (left and center) and their corresponding H&E images (right), with the boxed area displayed at high magnification in the middle panel, scale bar 200. μm; H is a representative mIHC image stained with antibodies selected from ACP pathological tissue, scale bar, 10μm; I is a diagram of the inferred developmental trajectory of ACP epithelial subtypes at spatial resolution, with black arrows indicating the developmental sequence across spatial regions; J is a schematic diagram of the multi-layered spatial organization of ACP epithelial subtypes;

[0017] Figure 3The diagram shows the validation results of the SASP-related FGF signaling pathway as a potential therapeutic target for ACP. A represents a schematic diagram of the developmental hierarchy of ACP epithelial tumor cells; B represents a bubble diagram of the expression of the selected genes in ACP epithelial tumor cell subsets; C represents a diagram of ligand-receptor interactions among ACP epithelial tumor cell subsets involved in FGF signaling, inferred from the acpEpi_WNT cell subset; D represents a detection diagram targeting the acpEpi_Progenitor cell subset; and E represents the detection of FGF4 and FGFR2 in ACP and... A violin plot showing the expression patterns of PCP epithelial tumor cell subsets; F is a UMAP plot showing the expression patterns of FGF4 and FGFR2 in ACP and PCP epithelial tumor cell subsets; G is a plot showing the expression patterns of FGF4 and FGFR2 at spatial resolution in ACP499; H is a plot showing the expression patterns of FGF4 and FGFR2 at spatial resolution in ACP511. In G and H, the boxed areas are displayed at high magnification in the upper panel. The white dashed lines represent the acpEpi_WNT tumor cell subset. Scale bar: 50. μm; I is a representative immunostaining image of FGFR2 in ACP pathological tissue, scale bar, 20 μm; J is a flowchart of the ex vivo tumor pituitary culture process; K is an immunofluorescence staining image of β-catenin and Ki67 in tumor pituitary, scale bar, 20 μm; L is an immunofluorescence staining image of p-ERK1 / 2 in tumor pituitary, scale bar, 20 μm; M is a graph showing the percentage of Ki67+ cells in control and drug-treated tumor pituitary; N is a graph showing the percentage of p-ERK1 / 2+ cells in control and drug-treated tumor pituitary; O is a schematic diagram of delaying tumor growth by inhibiting the FGF signaling pathway. Detailed Implementation

[0018] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0019] The materials and experimental testing methods involved in this invention are as follows:

[0020] 1. Clinical specimens

[0021] With the written informed consent of the patients or their guardians, 14 specimens from 9 patients were collected at West China Hospital of Sichuan University. Among them, 9 fresh specimens were used for scRNA-seq and 5 frozen specimens were used for ST sequencing.

[0022] 2. Single-cell RNA sequencing and analysis

[0023] Fresh pathological tissue was rapidly minced and digested into a single-cell suspension for scRNA-seq. Libraries were constructed using the MGIEasyRNA library preparation set and then sequenced on the MGISEQ-2000 platform.

[0024] 3. Spatial transcriptomics analysis

[0025] Fresh postoperative tissue was embedded in OCT, rapidly frozen on dry ice, and stored at -80°C until use. Frozen tissue was optimized, fixed, H&E stained, imaged, and permeabilized according to the guidelines of the BMKMANU S1000 Spatial Tissue Optimization Kit and Gene Expression Kit.

[0026] 4. Mice

[0027] Ctnnb1lox （ex3） / + Allelic homozygous mice and mice carrying Sox2 CreERT2 / + Crossing mice produces mutant offspring (Sox2). CreERT2 / + ;Ctnnb1 lox（ex3） / + The experimental procedure was conducted in accordance with ethical guidelines.

[0028] 5. In vitro culture of mouse tumor pituitary glands

[0029] Tamoxifen was injected at E12.5, and the pituitary tumor was dissected at E17.5, and Millicell was used. ® Incubate in StandingCell Culture Inserts (Millipore, Cat#PICM01250) for 24 hours.

[0030] 6. Immunohistochemistry

[0031] For immunohistochemical (IHC) staining, FFPE sections were dewaxed, followed by antigen retrieval and blocking with 5% bovine serum albumin. For multiplex IHC (mIHC), slides were incubated sequentially with primary and secondary antibodies, and tyramine signal amplification was applied.

[0032] Example 1: Spatial Transcriptome Analysis to Identify Spatial Structural Features of ACP and PCP

[0033] Frozen sections of CP tissue from fresh tumor specimens from 5 patients (3 ACP and 2 PCP) were analyzed using the BMKMANU S1000 platform, which accurately identifies cellular structures to enable subsequent cell-based transcriptomics analysis at the single-cell level in a spatial dimension. Figure 1 (A)

[0034] First, quantitative analysis was performed based on Voronoi diagrams after cell segmentation, and cell types were defined by integrating ST data with scRNA-seq data using RCTD. Figure 1 The unique structure of CP, including the solid and cystic components of ACP, as well as glial scars and the FVC of PCP, can be clearly visualized. Figure 1 (B) proves the high quality of our ST data.

[0035] By combining ST datasets and histopathological analysis, the unique cellular hierarchical structure of CP was resolved at the in situ level. First, ST analysis identified and accurately mapped the spatial in situ cell populations of three ACP samples (Figure 1, B). Cysts are one of the main diagnostic criteria for ACP and are filled with oily fluid, usually thought to be secreted by the tumor epithelium. ST analysis identified a series of microcysts in the ACP tumor region, which could be classified into two types (…). Figure 1 (C) Type I microcysts contain only a few cells or no cells, while another type of microcyst is filled with abundant macrophages and fibroblasts. Due to their close interaction with the surrounding tumor epithelial cells, microcyst-associated macrophages may play an important role in tumor progression.

[0036] In contrast to the specific distribution pattern of macrophages in ACP (within microcysts and surrounding calcified areas), myeloid cells in PCP exhibit a different distribution pattern, being both enriched in FVC and dispersed throughout the tumor slice. Figure 1 The median D (referring to the aforementioned inflammatory TME) reflects this. Furthermore, our ST analysis clearly observed three cell types within the FVC, including endothelial cells, fibroblasts, and macrophages (…). Figure 1 (D). Endothelial cells are distributed in the outermost layer of the FVC, in direct contact with the surrounding tumor epithelial cells, while fibroblasts and macrophages are arranged in a mixed manner within the FVC structure. We believe that the interaction between the cellular components of the FVC and the surrounding tumor epithelial cells may contribute to the progression of PCP.

[0037] To investigate the intrinsic tumor characteristics of PCP at single-cell and spatial resolution, we isolated tumor epithelial cells from scRNA-seq data of four PCP specimens and regrouped them into four tumor cell subpopulations. Figure 1(E). Furthermore, to analyze the developmental trajectory of epithelial tumor cells, a pseudo-temporal trajectory of epithelial tumor cell state evolution was analyzed using Slingshot. This indicates that the developmental trajectory of PCP epithelial tumor cells begins with the PCP_Progenitor cell subset, passes through the PCP_Mesenchymal cell subset, reaches the PCP_Differentiation cell subset, and finally differentiates into the PCP_Inflammation cell subset (…). Figure 1 F).

[0038] To further analyze the spatial organization of epithelial tumor cells, we integrated epithelial cells from ST data and regrouped them based on gene expression characteristics and cell cluster correlation scores with scRNA-seq data. PCP_Progenitor cells were mapped to epithelial columnar cells ( Figure 1 These cells (G) were mainly concentrated in the area surrounding the FVC, indicating the supporting role of the FVC in maintaining the progenitor cell state. PCP_Mesenchymal cells had the highest proportion, these cells were adjacent to progenitor cells and distributed throughout the tumor region. PCP_Differentiation cells were scattered around PCP_Mesenchymal cells, possibly originating from adjacent mesenchymal cells. PCP_Inflammation cells constituted a small fraction of the tumor cells, reflecting the inflammatory TME of PCP. stLearn analysis further confirmed the developmental trajectory of tumor cells at the spatial level from progenitor cells to mesenchymal cells, and finally to differentiated and inflammatory cells. Figure 1 (mIHC). Furthermore, mIHC staining revealed a stratified distribution of these cell subpopulations, with progenitor cells (KRT14+) surrounding FVCs, followed by adjacent mesenchymal-like cells (CD44+), and finally differentiated cells (SPRR3+) and inflammatory cells (100P+), indicating that the differentiation of these cell subpopulations was continuous. Figure 1 (I, J). In summary, different tumor cell states were defined through single-cell and spatial transcriptomic analyses, and tumor progenitor cells surrounding FCVs in PCP were identified.

[0039] Example 2: Single-cell and spatial transcriptomic analysis revealed the developmental trajectory of ACP and the cellular hierarchy associated with SASP.

[0040] To further analyze the cellular hierarchical structure in ACP, tumor epithelial cells in scRNA-seq data were reclassified into four tumor cell subpopulations based on gene expression characteristics. Figure 2(A). The ACP_WNT cell subset is characterized by high expression levels of WNT pathway-related genes. The ACP_Progenitor cell subset highly expresses COL17A1 and SULF1 genes. Mesenchymal characteristic genes and pathways regulating mesenchymal stem cell differentiation, fatty acid response, and wound healing are specifically enriched in the ACP_Mesenchymal cell subset. Stress-related genes define the ACP_Stress cell subset. Since the ACP_WNT cell subset consists of ACP tumor maintainer cells rather than tumor initiation cells, we excluded it in subsequent pseudo-chronological analyses. Cell developmental trajectory analysis revealed that the tumorigenesis trajectory begins in the ACP_Progenitor cell subset, passes through the ACP_Mesenchymal cell subset, and ends in the ACP_Stress cell subset. Epithelial progenitor-related genes (such as KRT14, STMN2, and SULF1) are downregulated along the pseudo-chronological developmental trajectory, while stress-related genes (such as SLP1, KRT6A, and ANXA1) are upregulated along the pseudo-chronological trajectory. Figure 2 (B) CytoTRACE analysis further confirmed that the most differentiated cell subpopulation was the ACP_Stress cell subpopulation, while the cell subpopulation most similar to stem cells was the ACP_Progenitor cell subpopulation (B). Figure 2 (C, D)

[0041] ACP tumors are thought to be induced by a SASP-associated paracrine pathway, with SASP-associated signals inducing the transformation of surrounding cells into tumor-initiating cells. However, the in situ spatial distribution of the SASP-associated cellular hierarchy has not been fully elucidated. To gain a deeper understanding of the in situ cellular state of tumor cells in ACP, we regrouped tumor cells from ST data and directly mapped them to tumor tissue sections from three ACP patients. Accordingly, based on characteristic marker genes and correlation scores with cell populations defined in scRNA-seq data, tumor cells in the ST data were regrouped into four cell subpopulations ( Figure 2 (E). Interestingly, each cell subpopulation is located within a unique histopathological structure within the tumor section (E). Figure 2 (F, G). The ACP_Progenitor cell subset is mainly distributed in palisade-like basal cells and arranged on the microcapsule walls, while the ACP_WNT cell subset is located in the ring-shaped structures of finger-like epithelial processes. The distribution patterns of these two cell types indicate the SASP-related cellular hierarchy of ACP. ACP_Mesenchymal cells are distributed in the stellate network region, while ACP_Stress cells are distributed in both the palisade and stellate network regions (F, G). Figure 2(G, H). Consistent with the pseudo-temporal developmental trajectory of scRNA-seq data, stLearn analysis further confirmed spatially that epithelial cells differentiated from ACP_Progenitor cells into ACP_Mesenchymal cells and ACP_Stress cells at the spatial level. Figure 2 In addition, we observed mIHC signaling in progenitor cells (SULF1+), WNT cluster cells (LEF1+), mesenchymal-like cells (KRT7+), and stress-related cells (KRT6A+) in additional ACP specimens, distributed in the corresponding structures. Figure 2 Middle I).

[0042] In summary, scRNA-seq combined with ST analysis revealed the developmental trajectory of tumor epithelium and SASP-related cellular hierarchies in ACP. Figure 2 (J).

[0043] Example 3: Exploring the SASP-related FGF signaling pathway as a therapeutic target for ACP

[0044] 1. FGF signaling pathway as a therapeutic target for ACP

[0045] Previous studies have reported that activation of SASP signaling induces the transformation of surrounding cells, which then convert to tumor initiating cells via paracrine signals. Our single-cell and spatial transcriptomic analyses revealed SASP-associated cellular structures in which ACP_WNT cells act as signaling centers, and ACP_Progenitor cells are defined as tumor initiating cells (…). Figure 3 (A). Therefore, we hypothesize that targeting key SASP signaling may delay tumor growth. We first examined the expression patterns of major SASP signaling, including members of FGF, WNT, TGF, SHH, and NOTCH ligands, and their receptors in each tumor cell subpopulation ( ). Figure 3 (B)

[0046] Although multiple ligands can be detected in ACP_WNT cells, FGFR2 is the most predominant receptor expressed in ACP_Progenitor cells, suggesting that the FGF signaling pathway may play a crucial role in tumorigenesis and progression. Other receptors, such as WNT receptors FZD1 / 6 / 7, are mainly expressed in ACP_Mesenchymal cells, while the TGF receptor TGFR2 is highly expressed in ACP_Stress cells. Furthermore, CellChat analysis further confirmed that FGF4-FGFR2 is the most predominant ligand-receptor pair in the ACP FGF pathway. Figure 3(C, D). Further examination of the FGF4-FGFR2 signaling expression pattern revealed that FGFR2 expression was higher in ACP_Progenitor than in other cell populations. Figure 3 (E, F). FGF4 is expressed only by ACP_WNT cells and not by other cell types ( Figure 3 E, F). ST analysis further confirmed that FGF4 was expressed only in ACP_WNT cells, while FGFR2 was mainly expressed in peripheral ACP_Progenitor cells (E, F). Figure 3 (G, H). IHC analysis also confirmed that FGFR2 was highly expressed in progenitor cells located in the basal palisade region (G, H). Figure 3 I).

[0047] 2. The effect of inhibitors targeting the FGF4-FGFR2 pathway on ACP progression

[0048] To further verify the impact of the FGF4-FGFR2 pathway on ACP progression, two drugs, pemigatinib and infigratinib, were used to target this pathway, as detailed below:

[0049] Since ACP is a paracrine-driven tumor associated with SASP, patient-derived mature cell lines and tumor xenograft (PDX) models of ACP are currently lacking. Therefore, we used a mouse tumor model to validate the impact of the FGF4-FGFR2 pathway on ACP progression. Previous studies have shown that activation and expression of β-catenin in Sox2+ pituitary stem cells leads to the formation of tumors similar to human ACP. To activate β-catenin protein during embryonic development, a tamoxifen-induced Sox2-CreERT2-driven mouse model (Sox2... CreERT2 / + ;Ctnnb1 lox（ex3） / + Tamoxifen can induce knockout. Ctnnb1 The third exon of the gene activates the encoded β-catenin protein, which then enters the cell nucleus to perform its function.

[0050] Tamoxifen was administered intraperitoneally on day 12.5 of the embryonic period (E12.5), and the tumor-bearing pituitary gland was isolated and cultured for 24 hours at stage E17.5. Figure 3 (J). Proliferation assessment showed that, compared with the control group, treatment with FGFR inhibitors significantly reduced Ki67 proliferation in the tumor pituitary gland (J). Figure 3 The presence of p-ERK1 / 2 (M) in the pituitary gland indicates that treatment with FGFR inhibitors significantly inhibits tumor cell activity. Immunofluorescence showed a significant reduction in p-ERK1 / 2 signal in the tumor pituitary gland after inhibitor treatment. Figure 3 The presence of L and N in the middle indicates that the FGF4-FGFR2 pathway was successfully inhibited.

[0051] The above results indicate that FGFR inhibitors have anti-tumor efficacy against ACP, and the FGF4-FGFR2 pathway may be a potential therapeutic target. Figure 3 (O).

[0052] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The use of Pemigatinib or Infigratinib in the preparation of medicaments for the treatment of craniopharyngioma, characterized in that, The Pemigatinib or Infigratinib mentioned are inhibitors of the FGF4-FGFR2 pathway, and the craniopharyngioma subtype is ACP.

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