Identifying the angiogenic receptor expression in mantle cell lymphoma patients and murine models

Abstract

Mantle Cell Lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma, with frequent relapses and shorter responses with every subsequent treatment. MCL depends on growth factors and cytokines derived from microenvironmental cells for its growth and can alter the immune system to evade recognition and subsequent elimination. The soluble factors secreted by MCL can contribute to endothelial differentiation, lymphangiogenesis, and clonal selection under hypoxic conditions, thereby evading the DNA damage response. Targeting the tumor microenvironment and angiogenesis is an active area of research and development, as the angiogenic gene signatures in MCL remain poorly understood. To address this knowledge gap, we performed transcriptomic analyses of MCL patient cohorts and identified 10 key angiogenic genes upregulated in MCL. We focused on four receptors (FGFR1, VEGFR1, VEGFR2, and PDGFRB) that have receptor tyrosine kinase activity and are localized to the plasma membranes of MCL cells. These receptors were assessed for therapeutic targeting potential in four independent preclinical models, including patient-derived xenografts, cell-derived xenografts, bone marrow-derived xenografts, and a genetically engineered murine model of MCL. Our work establishes that simultaneous targeting of multiple kinases, such as FGFR1 and VEGFR2, is a promising therapeutic strategy for patients with MCL.

Supplementary Information

The online version contains supplementary material available at 10.1186/s40364-026-00975-3.

To the Editor,

Mantle Cell Lymphoma (MCL) is an aggressive B-cell lymphoma and relies on microenvironmental cues for growth and survival [1–3]. Soluble factors secreted by MCL tumors can promote endothelial differentiation and lymphangiogenesis [4], thereby evading cell death pathways. Targeting the tumor microenvironment and angiogenesis has shown responses in many cancers; however, the role of angiogenesis and its gene signatures in MCL remains poorly understood. To address this knowledge gap, we performed transcriptomic analyses of two MCL patient cohorts and a genetic murine model of MCL (tgSox11/Ccnd1), and overlapped the upregulated genes with known angiogenic genes (Fig. 1A). Our analysis identified 10 upregulated genes associated with angiogenic pathways (Supplementary Table 1). Among the upregulated genes, we focused on proteins with receptor tyrosine kinase activity and localized to cell membranes, thereby narrowing the candidate list to four receptors: FGFR1, FLT1 (VEGFR1), KDR (VEGFR2), and PDGFRB. We then validated the expression of these receptors in an independent cohort of 68 patients (Supplementary Table 2) compared with Healthy Donor (HD) CD19 + B cells (n = 15) (Fig. 1B-E). Our results did not show significant differences in the expression of these receptors between leukemic and conventional MCLs (Fig. S1A-D). Among the receptors, FGFR1 showed the highest expression in MCL (Fig. S1E). Furthermore, we performed an overall survival (OS) analysis of 68 MCL patients using the quartile method (Q1-Q4; n = 17 per quartile) based on ΔΔCt values for each gene, with a log-rank test cutoff of p < 0.01. The high expression of FGFR1 and VEGFR2 in MCL was associated with poor outcomes (Fig. 1F-G). However, higher expression of VEGFR1 and PDGFRB did not yield a significant difference. (Fig. 1H-I). Furthermore, the combined analysis of MCL patients with FGFR1^highand VEGFR2^high showed poorer outcomes than that of MCL patients with FGFR1^low and VEGFR2^low (Fig. 1J). Interestingly, FGFR1^highand VEGFR2^high MCL patients, also exhibit a significantly higher percentage of KI-67 expression but not PDGFRB^high and VEGFR1^high (Fig. 2A). Moreover, FGFR1^highVEGFR2^high patients also demonstrated a significant difference in the KI-67 expression (Fig. 2B). We further demonstrated that expression of FGFR1 and VEGFR2 is upregulated at the protein level in MCL cell lines (Fig. S1F), MCL patients, and in the murine MCL model tgSox11/Ccnd1 (Fig. 2C and S1G-H). Next, to determine whether targeting angiogenic receptors in MCL can improve outcomes, we compared the small molecules lucitanib, lenvatinib, and PD173074, which have been reported to inhibit both targets [5, 6]. The potency and efficacy of their engagement of both FGFR1 and VEGFR2 were confirmed using a fluorescent probe cellular binding assay [7] to determine cellular inhibitory constant (Ki) values (Fig. S2A-B). Because lucitanib effectively engages both targets in cells with higher potency compared to lenvatinib and PD173074 (Fig. S2C-D) and has established PK/PD parameters in-vivo, we chose lucitanib for further analysis. Next, we asked whether lucitanib treatment could impact both proliferation and cell death in MCL cells. As shown in Fig. 2D, lucitanib treatment induces significant cell death and reduced proliferation of MCL cells compared with DMSO-treated cells (Fig. 2E). As endothelial cells are critical to the lymphangiogenesis program [8], we performed a vascular permeability and branching assay on endothelial HUVEC cells using Jeko-1 conditioned media (CM) pretreated with DMSO or lucitanib for 24 h. We found that the addition of CM from the MCL cells significantly increased the vascular permeability of in-vitro vessels, thereby affecting their barrier function (Fig. 2F). In addition, HUVEC cells fail to develop branches, junctions, and nodes when cultured with CM from lucitanib-treated MCL (Fig. 2G and Fig. S3A). Our results suggest lucitanib exerts a direct anti-proliferative effect on MCL cells and confers an anti-angiogenic effect on the microenvironment, affecting endothelial HUVEC cells. We next asked if lucitanib can target tumor development in MCL mouse models. We used four independent MCL mouse models: (a) Cell-derived xenograft (CDX; Z-138 and Jeko-1), (b) Bone-Marrow (BM) derived MCL Xenograft (BMX; Jeko-1 and BM Stromal cells-HS5), (c) a murine genetically engineered mouse model (tgCyclinD1/Sox11), and (d) MCL patient-derived xenograft (PDX). As shown in Fig. 2H-I, treatment of NSG mice transplanted with MCL cells (CDX: Z138 and Jeko-1) with lucitanib significantly reduced tumor volumes and increased OS (Fig. S3B-C). Next, upon subcutaneous co-injection of HS-5 cells and Jeko-1 cells (BMX model), mice treated with lucitanib showed a significantly reduced tumor volume compared with control mice (Fig. 2J) and a significant increase in OS (Fig. S3D). In transgenic Sox11/Ccnd1 mice [9] that resemble MCL, we performed adaptive transplantation by injecting tgSox11/Ccnd1 splenocytes into C57BL/6 flanks and treated the recipients with lucitanib. We found that treatment with lucitanib reduced tumor growth compared with the vehicle control (Fig. 2K). Next, we confirmed the therapeutic effects of lucitanib in a human PDX model by monitoring B2M levels (Fig. 2L) and circulating CD45 + CD19+ cells to assess disease progression. (Fig. S3E) and found improved OS in the MCL PDX model compared to control (Fig. S3F).

Fig. 1.

FGFR1 and VEGFR2 expression profiles and clinical outcomes in primary MCL patients. (A). Venn diagram depicting upregulated genes overlapping across MCL patient Cohort 1 (GSE159808), Cohort 2 (GSE46846), the genetic murine model of MCL, tgSox11/Ccnd1, and the angiogenesis gene set. (B-E). RT-qPCR analysis for the angiogenic receptors FGFR1 (B), VEGFR2 (C), VEGFR1 (D), PDGFRB (E), from MCL patients (n = 68) compared with CD19 + B cells derived from a healthy donor (n = 15). Statistical analysis was performed using a two-tailed unpaired Student’s t-test. (F-I). Kaplan-Meier plot for overall survival of MCL patients (n = 68) generated and analyzed with Mantel-Cox log-rank test. The MCL cohort was divided into four quartiles (n = 17 per quartile), with the top and bottom 25% of tumors, based on normalized ΔΔCt values, considered high- and low-expressing, respectively. The comparison of expression levels for Q1 versus Q4 is shown for FGFR1 (F), VEGFR2 (G), VEGFR1 (H), and PDGFRB (I). (J). Kaplan-Meier plot of overall survival in MCL patients (Q1 versus Q4) was generated and analyzed using the Mantel-Cox Log-rank test based on the expression levels of both FGFR1 and VEGFR2, with FGFR1^high and VEGFR2^high (n = 14) identified as poor outcomes compared with MCL patients with FGFR1^low and VEGFR2^low (n = 7)

Fig. 2.

FGFR1 and VEGFR2 can be targeted by the dual inhibitor lucitanib in models of MCL in-vivo. (A). Expression of the proliferation marker KI-67 in primary MCL was analyzed in relation to high or low expression of the genes FGFR1, VEGFR2, VEGFR1, and PDGFRB (A) or in FGFR1^high/VEGFR2^high versus FGFR1^low/VEGFR2^low identified MCL patients (B). Statistical significance was determined using a two-tailed, unpaired Student’s t-test. (C). Western blot-based density graphs showing protein expression of human VEGFR2 and human FGFR1, normalized to β-actin (loading control), in MCL patients (n = 5) compared with healthy donors (n = 2). Protein levels of mouse Fgfr1 and mouse Vegfr2 in WT and tgSox11/Ccnd1 (DT) mice are also shown. The y-axis represents relative densitometry units (RDU). Statistical significance was determined using an unpaired t-test. (D). Annexin V/Sytox Blue assay was performed on MCL cells (Jeko-1 and Z-138) treated with DMSO or varying concentrations of lucitanib. Bar graphs represent the percentage of live cells (AnnexinV- and Sytox Blue- negative cells). A two-way ANOVA was used to assess statistical significance. (E). Cell proliferation was analyzed by MTT assay in MCL cells (Jeko-1 and Z-138) treated with DMSO or varying concentrations of lucitanib. Bar graphs showing fold change, normalized to the DMSO control, are plotted for different concentrations of lucitanib at 72 h post-treatment. A two-way ANOVA was used to assess statistical significance. (F). Quantification of HUVEC vessel permeability in normal EGM-2 media (Basal) versus Jeko-1-derived conditioned media (CM). The diffusion-based flux of a 20-kDa FITC-dextran tracer was measured to estimate vessel permeability. Statistical significance was determined using a t-test. (G). Capillary tube formation assay of HUVECs cultured on CM derived from Jeko-1 cells in EGM-2 media for 18 h in the presence or absence of 10 µM lucitanib for 24 h. ImageJ was used to quantify the number of nodes, junctions, and branches formed by HUVECs between groups. An unpaired t-test was used to assess statistical significance. (H-J). Tumor volume at the endpoint removal criterion (ERC) of the first mouse was measured after subcutaneous injection of Z138 (H), Jeko-1 (I), or Jeko-1 cells in combination with HS-5 cells (J), followed by treatment with vehicle control or lucitanib in NSG mice. An unpaired t-test was used to assess statistical significance. (K). Tumor volume at the ERC of the first mouse was measured after subcutaneous injection of Eµ-Sox11/Ccnd1 double-transgenic (DT) MCL mouse model cells, followed by treatment with vehicle control (n = 5) or lucitanib (n = 6) in C57BL/6 mice. Statistical significance was determined using an unpaired t-test. (L). Levels of β₂ microglobulin (B2M) analyzed by ELISA in normal C57BL/6 mice (Negative Control) or at the ERC of the first C57BL/6 mouse injected intravenously with a patient-derived xenograft, followed by subsequent treatment with vehicle control or lucitanib. An unpaired t-test was used to assess statistical significance

In summary, our study identified 10 key angiogenic upregulated markers in MCL patients. We focused on four receptors (FGFR1, VEGFR2, VEGFR1, and PDGFRB) to assess therapeutic potential in four independent preclinical MCL mouse models. VEGF/VEGFR2 signaling is known to drive endothelial cell proliferation [8]. Furthermore, its high expression has been associated with poorer clinical outcomes [10]. Interestingly, the SOX11:SMARCA4 complex in MCL can drive many oncogenic transcriptional programs, including VEGFA/VEGFR2 signaling [11]. FGFR1 expression and its signaling are deregulated in MCL [12, 13], and FGFR1 expression is highly elevated in trans-differentiation MCLs [14]. Since FGFR1 can compensate when VEGF pathways are targeted, it could therefore contribute to the limited efficacy of anti-VEGF-based therapies [15]. The crosstalk between VEGFR2 and FGFR1 promotes angiogenesis; thus, dual targeting of these receptors may be a rational therapy to block angiogenic signaling in MCL. We show that lucitanib, targeting multiple kinases, significantly reduces MCL proliferation and induces apoptosis in-vitro. It also decreases angiogenesis in endothelial cells in-vitro and tumorigenicity in-vivo. The in-vivo anti-tumor effect was confirmed across several models, highlighting lucitanib’s therapeutic potential in MCL. Although this report does not examine how MCL receptor expression is regulated or how these receptors mediate autocrine (cancer cells) or paracrine (endothelial cells) regulation of angiogenesis, our work establishes that targeting multiple kinases, such as FGFR1 and VEGFR2, is a promising therapeutic approach for treating MCL patients.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

B2M

Human beta-2 Microglobulin

BMX

Bone marrow-derived MCL Xenograft

CCND1

Cyclin D1

CD19

Cluster of Differentiation 19

CD45

Cluster of Differentiation 45

CDX

Cell Derived Xenograft

CM

Conditioned Media

DMSO

Dimethyl Sulfoxide

DT

Double Transgenic

EGM-2

Endothelial Growth Media

ERC

Early Removal Criterion

FGFR1

Fibroblast Growth Factor Receptor 1

FLT1

fms-related receptor tyrosine kinase 1

HD

Healthy Donor

HUVECs

Human Umbilical Vein Endothelial Cells

KDR

Kinase Insert Domain Receptor

Ki

Inhibitory Constant

MCL

Mantle Cell Lymphoma

MTT

3-(4,5-di methyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide

Neg

Negative Control

NSG mice

NOD scid gamma Mice

OS

Overall Survival

PD

Pharmacodynamics

PDGFRB

Platelet-Derived Growth Factor Receptor Beta

PDX

Patient-derived xenograft

PK

Pharmacokinetics

RDU

Relative Densitometry Unit

SMARCA4

SWI/SNF-related, Matrix-associated, Actin-dependent Regulator of Chromatin, subfamily A, member 4

SOX11

SRY-box transcription factor 11

tgSOX11/CCND1

Transgenic SRY-box transcription factor 11 and Cyclin D1 expressing mouse

Veh

Vehicle Control

VEGF

Vascular Endothelial Growth Factor

VEGFA

Vascular Endothelial Growth Factor A

VEGFR1

Vascular Endothelial Growth Factor Receptor-1

VEGFR2

Vascular Endothelial Growth Factor Receptor-2

WT

Wild type

Author contributions

UJ, SS, AS, YW, SS, GPC, SLZ, HJ, JCH, JWT, JMB, RK, ASI, DK, NC, and MB performed the experiments and analyzed the data. UKN and KR provided clinical samples and FISH data for CCND1 on MCL samples. SP, JS, JP, RN, LA, and BP provided research resources and performed the analysis. AL and RD performed bioinformatics analysis of the data. PR, NM, RN, VA, PKL, RAB, and SP provided critical suggestions. LS, NJ, and NE contributed to the conception, organization, and writing of the manuscript. All authors have read and approved the final version of the manuscript.

Funding

Research reported in this publication was supported by Young investigator award from Pelotonia to LS, DSRP to LS and LA, Pelotonia Graduate fellowship to YW, AS and JCH, Pelotonia Postdoctoral fellowship to SS, Pelotonia Undergraduate fellowship to JWT and JMB, American Society of Hematology Global Research Award 2020, Washington, DC, USA (NJ), Ramalingaswami grant (BT/RLF/Re-entry/18/2017) from the Department of Biotechnology, Ministry of Science and Technology, Government of India (NJ), and CORE Research Grant (CRG/2020/003377), from Anusandhan National Research Foundation (ANRF), Government of India (NJ).

Data availability

The datasets used from public sources are mentioned in the supplemental files, and other data resources generated in this report are available on reasonable request from the corresponding authors.

Declarations

Ethics approval and consent

Samples were collected with informed consent from patients and in accordance with the Declaration of Helsinki and approval by the institutional human ethical review board of CSIR-Central Drug Research Institute (CDRI), Lucknow (CDRI/IEC/2023/PA11, CDRI/IEC/2023/PA12, CDRI/IEC/2023/PA13) (25th October 2022) and the Ohio State University.

Consent for publication

All authors have read and approved the final version of the manuscript.

Footnote

The institutional (CSIR-CDRI) communication number for this article is 11185.

Competing interests

R.A.B. received research support from Prelude Therapeutics and serves on the SAB for Sobi, Pierre Fabre, and Atara Therapeutics. J.W.S. is a co-founder and shareholder of EMBioSys, Inc. S.P. has research support from Amgen, Celgene/Bristol Myers Squibb Corporation, Regeneron, Genentech, imCORE, and Caribou, and serves as a consultant for Grail (advisory board). L.S. serves as a consultant for the Institute for Follicular Lymphoma Innovation. N.E. Research funding (to the institution) from Lilly, Incyte, ADC Therapeutics, Ipsen, Beigene, Advisory Board for CRISPR Therapeutics, Ipsen, Genetech. A.S. is employed and a shareholder at Astrazeneca. The remaining authors declare that they have no competing financial interests.