Reverse effect of Semaphorin-3F on rituximab resistance in diffuse large B-cell lymphoma via the Hippo pathway : Chinese Medical Journal

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Original Article

Reverse effect of Semaphorin-3F on rituximab resistance in diffuse large B-cell lymphoma via the Hippo pathway

Li, Qiong1,2,3,; Ma, Naya1,2,; Li, Xinlei1,2; Yang, Chao1,2; Zhang, Wei1,2; Xiong, Jingkang1,2; Zhu, Lidan1,2; Li, Jiali1,2; Wen, Qin1,2; Gao, Lei1,2; Yang, Cheng1,2; Rao, Lingyi1,2; Gao, Li1,2; Zhang, Xi1,2,3,; Rao, Jun1,2,3,

Editor(s): Hao, Xiuyuan

Author Information
Chinese Medical Journal ():10.1097/CM9.0000000000002686, April 28, 2023. | DOI: 10.1097/CM9.0000000000002686

Abstract

Introduction

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma and is a heterogeneous disease, and it can be classified into germinal center B-cell-like, activated B-cell-like, and unclassifiable DLBCL. [1] Incorporation of an anti-CD20 monoclonal antibody (rituximab, R) into standard CHOP regimens (cyclophosphamide, doxorubicin, vincristine, and prednisone) has been demonstrated to improve survival for DLBCL. [2–4] Nevertheless, approximately 30–40% of patients who receive RCHOP succumb to the disease due to relapse or treatment resistance, and patients who are resistant to rituximab-containing regimens have poor prognosis. [5,6] Thus, further exploration of the mechanism of resistance to rituximab is critical for the improvement of the prognosis in DLBCL patients.

Semaphorin-3F (SEMA3F) is an axon guidance factor that repels axons and collapses growth cones. [7,8] Recently, studies have demonstrated that SEMA3F is an essential regulator of leukocyte diapedesis contributing to the pathogenesis of systemic inflammation after surviving cardiac arrest. [9] Cumulative evidence also suggested that SEMA3F could inhibit tumor growth, angiogenesis, and metastasis in various tumors, [10–12] and our previous work also demonstrated that SEMA3F suppressed the stemness of cancer cells by inactivating Rac1 and inhibited tumor metastasis by downregulating the ASCL2 (achaete-scute family bHLH transcription factor 2)–CXCR4 C-X-C motif chemokine receptor 4 signaling pathway. [13,14] Recently, some researchers have demonstrated that DLBCL patients with higher CXCR4 expression had a poor prognosis, and upregulation of CXCR4 could be observed in rituximab-sensitive cell lines but not in resistant cell lines, suggesting that CXCR4 could reflect the rituximab response but was not the mechanism of rituximab resistance. [15–17] These studies showed that crosstalk between SEMA3F signaling and rituximab-induced anti-lymphoma effects might exist, and exploration of SEMA3F-mediated signaling might help unveil the biological mechanism of rituximab resistance.

In the current study, we retrospectively analyzed SEMA3F expression in three dependent GEO datasets and DLBCL patients from our own center who received RCHOP treatment. Furthermore, we examined the growth inhibition and treatment efficacy mediated by SEMA3F in in vitro and in vivo lymphoma models (WW domain-containing transcription regulator protein 1) to identify the effects of the axon guidance factor SEMA3F on rituximab resistance as well as its therapeutic value in DLBCL.

Methods

Human tissues and cell culture

Between 2015 and 2018, human formalin-fixed paraffin-embedded tissues were obtained from 112 newly diagnosed DLBCL patients who were treated with RCHOP regimens as first-line therapy. All consecutive patients who were deemed appropriate for this study during the period were included without selection. Histological diagnoses were established independently by at least two experienced senior pathologists according to the World Health Organization classification. The germinal center B cell (GCB) subtype and non-GCB subtype were diagnosed using immunohistochemistry (IHC) with CD10, BCL6, and MUM1 antibodies according to the Hans' algorithm. [18] All patients underwent baseline staging using laboratory, radiographic, and bone marrow examinations. Eastern Cooperative Oncology Group (ECOG) performance status was assessed at diagnosis. The stage was evaluated in accordance with the Ann Arbor staging system. The International Prognostic Index (IPI) was calculated based on serum lactate dehydrogenase, stage, and ECOG performance status. Normal lymph nodes were obtained from department of pathology, Xinqiao hospital. This study was approved by the Xinqiao Hospital Ethics committees (No.2022-17401), and all patients provided signed informed consent.

The human DLBCL cell lines OCI-Ly1, OCI-Ly3, OCI-Ly7, and TMD8 were obtained from the Beckman Research Center (City of Hope, USA). Cells were cultured in Iscove's modified Dulbecco's medium (IMDM) (Gibco, CA, USA) containing 10% fetal bovine serum (FBS) (Gibco, CA, USA) and were incubated at 37℃ in a humidified incubator with 5% CO2. All cell lines were genotyped by short tandem repeat analysis and were confirmed to be negative for mycoplasma with the detection kit.

Western blotting assay

Nuclear and cytoplasmic fractions were isolated using NE-PER ® Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Rockford, IL, USA). Western blotting was carried out by standard procedures as previously described. [13] The primary antibodies used for the assay were as follows: anti-human SEMA3F (1:1000) (Chemicon, Temecula, CA, USA), anti-human MST2, MST1, MOB1, p-MOB 1, LATS1/2, YAP/TAZ, cleaved caspase-3, BCL2, p-YAP Ser397, p-YAP Ser127, and CCND2 (1:500) (Cell Signaling Technology, Danvers, MA, USA), anti-human β-actin (1:1000) (Cell Signaling Technology), anti-human CD20 (1:1000) (Abcam Cambridge, MA, USA), anti-human TAZ (1:1000) (Abcam), and anti-human Lamin B1 (1:1000) (Novus Biologicals, Littleton, CO, USA).

Real-time reverse transcription-polymerase chain reaction (PCR)

Total RNA of tumor cells was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). Detailed procedures were performed as previously described. [13,14] The specific primers for each gene are listed in Supplementary Table 1 [https://links.lww.com/CM9/B546].

Table 1 - Demographic and clinical characteristics of DLBCL patients of our cohort ( N = 112) between 2015 and 2018.
Prognostic variables Expression of SEMA3F
n Low High χ 2 P-value
Gender
Female 38 17 21 0.279 0.598
Male 74 37 37
Age
>44 years 60 26 34 1.233 0.267
≤44 years 52 28 24
aaIPI
2 55 22 33 2.921 0.087
>2 57 32 25
Histological subtype
GCB 53 28 25 0.859 0.354
Non-GCB 59 26 33
Bone marrow involvement at diagnosis
Yes 22 14 8 2.608 0.106
No 90 40 50
aaIPI: Age adjusted International Prognostic Index; DLBCL: Diffuse large B-cell lymphoma; GCB: Germinal center B cell.

In vitro cell viability, complement-dependent cytotoxicity (CDC), colony formation, and apoptosis assays

Cells transfected with sh-SEMA3F, Lv-SEMA3F lentivirus, or control lentivirus were seeded in 96-well plates (2000 cells per well). Cell viability was determined on day 1–6 using a Cell Titer-Glo Luminescent Cell Viability Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. The half maximal inhibitory concentration (IC50) of rituximab (Roche, Kaiseraugst, Switzerland) for cells was determined by exposing cells to rituximab or saline at increasing concentrations from 1 µg/mL to 64 µg/mL, and IC50 was calculated using the least square fit of four parameter-sigmoidal curves executed using Prism ver. 9 (GraphPad Software, San Diego, CA, USA). Cell viability was measured 48 h after treatment. For CDC effects induced by rituximab, cells were seeded in 96-well plates (2000 cells per well) and incubated overnight before treatment with rituximab. After 30 min of incubation, 20% heat-inactivated human serum was applied, and 12 h later, cell viability was evaluated. For the cell colony formation assay, cells (500 cells per well) were seeded in 6-well plates in a 1 mL mixture with an equal volume of complete medium and 0.7% soft agar every 3 days. After the medium was added, the plates were further incubated for 14 days at 37℃ in a humidified incubator with 5% CO2. Cell apoptosis was evaluated using Annexin V and 7-aminoactinomycin D (7-ADD) on a BD LSR-II flow cytometer (BD Biosciences, San Jose, CA, USA) based on the manufacturer's guidance. Cell cycle was performed as followed, when cells were collected, then washed and fixed by 70% iced ethanol thereafter. After, resuspended cells were incubated with 1 μL of Triton-X100, 2 μL of RNase (0.5 mg/mL) and 10 μL of propidium iodide (1 mg/mL, Sigma Chemical, St. Louis, MO, USA) for 30 min of cell staining. The DNA contents were detected by FACScan laser flow cytometer (Beckman Coulter, Fullerton, CA, USA).

Immunofluorescence assays and IHC staining

Cells on glass coverslips were fixed for 20 min with 4% paraformaldehyde without permeabilization. After being washed three times with phosphate buffer saline (PBS) for 5 min each time, the fixed cells were then incubated in a protein-blocking solution for 20 min at room temperature. The primary antibody against SEMA3F (Chemicon) and CD20 (Abcam) was added, and the mixtures were incubated overnight at 4℃, followed by incubation with Alexa Fluor 647 or 488 (Invitrogen) at 37℃ for 30 min. The cells were counterstained with Hoechst 33342 to reveal the nuclei. All samples were then analyzed by Confocal laser scanning microscope (CLSM LSM 800, Zeiss, Oberkochen, Germany).

IHC staining was performed using the Dako REAL Envision Detection system(DAKO, CA, USA) according to the manufacturer's protocol. The primary antibodies used for IHC were anti-human TAZ (1:400, Abcam) and anti-human SEMA3F (1:500, Chemicon). Semiquantification of TAZ and SEMA3F expression was performed independently by two histopathologists according to the staining intensity and percentage of positive tumor cells in a blinded manner.

Chromatin immunoprecipitation assay (ChIP) analyses and luciferase reporter assay

ChIP analysis and luciferase reporter assays were performed as previously described. [14]

Data resources and preprocessing

The expression profiling of the Lymphoma/Leukemia Molecular Profiling Project (LLMPP) cohort was downloaded from the GEO dataset (GSE10846), and 414 DLBCL patient samples were collected before treatment initiation (CHOP regimen, n = 181; RCHOP regimen, n = 233). [19] Two other gene expression profiles were also downloaded from the GEO dataset (GSE4475, GSE31312). [20] Eighty-five patients who received the CHOP regimens from GSE4475 were included for further analysis, and 472 patients who received RCHOP regimens from GSE31312 were included for the survival analysis. All datasets were tested on the GPL570 platform http://www.affymetrix.com/support/technical/byproduct.affx? product=hg-u133-plus.

Xenograft mouse model assays

Six-week-old NOD/SCID mice (SPF Biotechnology Co., Ltd., Beijing, China) were injected with 5 × 10 6 OCI-Ly1 cells that had been transfected subcutaneously with mock or SEMA3F shRNA lentiviral, or DLBCL cells from a patient sample. When the tumor volume reached approximately 100 mm 3, the mice were randomly divided into three groups ( n = 5 per group): (1) control group: PBS; (2) rituximab group: 25 µg/g; (3) verteporfin group: verteporfin (Novartis, Visudyne, USA) (100 mg/kg); (4) rituximab + verteporfin group: rituximab (25 µg/g) plus verteporfin (100 mg/kg). All agents were dispersed in 0.1 mL of PBS and injected intraperitoneally every other day for 14 days. Tumor size was measured before injection every 3 days, and the values were calculated using the following formula: (0.5 × length × width 2). Then, tumor tissues were dissected and fixed in 10% formalin for hematoxylin-eosin (H&E) staining and IHC staining. The animal experiments were approved by the Institutional Animal Care and Use Committee of Xinqiao Hospital, Army Medical University (AMU) (AMUWEC2020547), and strictly complied with the recommendations in the Guide for the Care and Use of Laboratory Animals of AMU.

Statistical analyses

All data were analyzed using SPSS 17.0 software (IBM Inc, Armonk, NY, USA). The correlation of SEMA3F with the clinicopathologic features of patients was assessed by the Pearson χ2 test. Overall survival (OS) was defined from disease confirmation to the end of follow-up or death. The date of diagnosis to the date of disease progression, death, or last follow-up was used as progression-free survival (PFS). Survival estimates were obtained by using the Kaplan–Meier method, and comparisons were made by the log-rank test. We used the probe sets (206832_s_at and 37278_at) to estimate the association of SEMA3F and TAZ mRNA expression and rituximab efficacy. A Cox proportional regression model was used to calculate the survival hazard ratio (HR). Statistical differences were considered significant if the two-sided P-value was <0.05.

Results

High expression of SEMA3F predicted longer survival in patients with RCHOP treatment but not in patients treated with CHOP regimen

To evaluate the prognostic value of SEMA3F expression in DLBCL patients, we used three published datasets (GSE10846, GSE31312, GSE4475) for analysis. First, we found that SEMA3F expression was not significantly different between the patients with GCB and activated B cell (ABC) DLBCL [Figure 1A]. The cut-off value was determined by X-tile software, in the LLMPP cohort, no significant difference in 10-year overall survival was observed between the patients with high and low SEMA3F expression ( P = 0.618) [Figure 1B]. When the whole cohort was divided into CHOP-treated and RCHOP-treated groups, in patients treated with CHOP regimens, no significant difference in 10y-OS was observed between the patients with high and low SEMA3F expression ( P = 0.303) [Figure 1C]. In contrast, subgroup analysis of the RCHOP-treated patients showed that 10y-OS was significantly worse in the patients with low expression of SEMA3F ( P = 0.015) [Figure 1D], suggesting that SEMA3F expression might predict OS in the patients treated with RCHOP regimens but not the CHOP-treated patients. To validate the results, we used two additional datasets. In GSE4475, most of the patients only received CHOP regimens, and no significant difference was found in the patients with low and high expression [Figure 1E]. In GSE31312, we found that the patients with higher SEMA3F expression showed longer OS and PFS than those with the RCHOP regimen [Figure 1F,G]. Next, we examined whether the treatment strategy could influence the outcome of patients with different SEMA3F expression levels. In the patients with high SEMA3F expression, RCHOP treatment showed a superior survival benefit (HR = 0.423, P <0.001) [Figure 1H], and similar trends were observed in the patients with low SEMA3F expression (HR = 0.534, P = 0.016) [Figure 1I]. These results suggested that the RCHOP treatment strategy showed much better therapeutic effects for patients with high expression of SEMA3F.

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Figure 1:
SEMA3F expression and significance in DLBCL patients. The expression level of SEMA3F in GCB-DLBCL and ABC-DLBCL groups (A). Kaplan–Meier survival curves estimated OS of patients with different expressions of SEMA3F based on dataset GSE10846 (B), patients treated with CHOP regimens with different expressions of SEMA3F based on dataset GSE10846 (C), patients treated with RCHOP regimens with different expressions of SEMA3F based on dataset GSE10846 (D), patients treated with CHOP regimens with different expressions of SEMA3F based on dataset GSE4475 (E), patients treated with RCHOP regimens with different expressions of SEMA3F based on dataset GSE31312 (F), patients treated with RCHOP regimens with different expressions of SEMA3F based on dataset GSE31312 (G). Comparison of CHOP with RCHOP treatment effects with high expression of SEMA3F (H) and in low expression of SEMA3F (I). Representative IHC images of SEMA3F staining in normal lymph node and human DLBCL patients' sample. Scale bar, 50 µm (J). IHC score of SEMA3F was lower in rituximab-resistant patients compared to rituximab-sensitive patients (K). Kaplan–Meier survival curves estimated the OS of patients treated with RCHOP regimens with different expressions of SEMA3F in our cohort (L). ABC: Activated B cell; CHOP: Cyclophosphamide, doxorubicin, vincristine, and prednisone;DLBCL: Diffuse large B-cell lymphoma; GCB: Germinal center B cell; ABC: activated B cell; GSE: Gene Expression Omnibus Series; IHC: Immunohistochemistry; OS: Overall survival; RCHOP: Rituximab plus cyclophosphamide, vincristine, doxorubicin, and prednisone. NS: P >0.05; * P <0.05.

In our own cohort, we defined resistant patients as those not achieving complete remission or developing rapid disease progression after 6–8 cycles of RCHOP treatment. [21] The patients' demographics and clinical characteristics are summarized in Table 1. IHC was conducted on tumor sections from 112 patients, as shown in Figure 1J. SEMA3F expression was lower in DLBCL patients than in normal lymph nodes. The resistant patients showed lower SEMA3F IHC scores than the sensitive patients [Figure 1K]; moreover, Kaplan–Meier survival curves showed that the OS of the patients with high SEMA3F expression was significantly superior to that of the patients with low SEMA3F expression ( P = 0.004) [Figure 1L]. The results of univariate and multivariate analyses for risk factors for OS of patients are summarized in Table 2. Multivariate analysis revealed that histological grade and SEMA3F expression were independent prognostic indicators of the OS of patients.

Table 2 - Univariate and multivariate analyses of the OS of patients with DLBCL of our cohort ( n = 112) between 2015 and 2018.
Prognostic variables Univariate analysis Multivariate analysis
HR (95% CI) P-value HR (95% CI) P-value
Gender 0.325 (0.096–1.105) 0.072
Age (years) 0.272 (0.104–0.713) 0.030 0.205 (0.091–0.592) 0.051
Bone marrow involvement 1.254 (0.484–3.245) 0.641
Histological grade 3.097 (1.195–8.024) 0.020 2.808 (1.075–7.331) 0.035
aaIPI 2.912 (1.128–7.517) 0.026 2.419 (0.813–7.194) 0.112
SEMA3F 0.277 (0.106–0.720) 0.008 0.205 (0.071–0.592) 0.003
aaIPI: Age adjusted International Prognostic Index; CI: Confidence interval; HR: Hazard ratio; OS: Overall survival; –: Not applicable.

SEMA3F inhibited the proliferation of DLBCL cells and regulated the cell response to rituximab

To determine the significance of SEMA3F expression in DLBCL, we used normal lymph nodes and four DLBCL cell lines for in vitro assays. We found that SEMA3F expression was lower than that in lymph nodes [Figure 2A], and different protein expression patterns were also investigated by Western blotting [Figure 2B]. Then, Ly3 and Ly7 were selected for further analysis. Transfection of Lv-SEMA3F led to higher protein and mRNA expression in both cell lines [Figure 2C,D]. We observed that the cell lines with high expression of SEMA3F (Ly3-SEMA3F, Ly7-SEMA3F) showed significantly decreased cell viability [Figure 2E, F], and overexpression of SEMA3F significantly induced apoptosis compared to that of the control cells [Figure 2G, Supplementary Figure 1A, https://links.lww.com/CM9/B546]. Moreover, we found that the cell cycle could be arrested at the G1 phase [Figure 2H]. To compare the sensitivity of rituximab on cells, we performed in vitro cell viability analyses and found that the mean IC50 of rituximab was much lower for cells with high expression of SEMA3F [Figure 2I, J]. Rituximab-mediated CDC activity was also evaluated by incubating cells with different concentrations of rituximab (2.5–20 µmol/L), as shown in Figure 2K. The survival ratio of the Ly3-SEMA3F cells was lower than that of the mock cells at the same rituximab concentration. Collectively, these results demonstrated that regulation of SEMA3F inhibited cell proliferation and enhanced the cell response to rituximab.

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Figure 2:
SEMA3F overexpression inhibited DLBCL proliferation and enhanced sensitivity to rituximab. (A) Relative mRNA levels of SEMA3F in the normal lymph node, GCB-DLBCL, and non-GCB DLBCL cell lines. (B) Western blot of SEMA3F in DLBCL cell lines. (C,D) Relative protein and mRNA levels of SEMA3F in Ly3 and Ly7 cells transfected with SEMA3F lentivirus. (E) Cell viability of Ly3-Mock and Ly3-SEMA3F cell lines. (F) Cell viability of Ly7-Mock and Ly7-SEMA3F cell lines. (G) Flow cytometry analysis of apoptosis of DLBCL cells infected with SEMA3F overexpression lentivirus. (H) The cell phase distribution of Ly3-Mock and Ly3-SEMA3F cell lines. (I) IC50 of rituximab in Ly3-Mock and Ly3-SEMA3F cell lines was measured using linear regression analysis of the dose–response curves. (J) IC50 of rituximab for Ly7-Mock and Ly7-SEMA3F cell lines was measured using linear regression analysis of the dose–response curves. (K) Rituximab-induced CDC activity at different concentrations via CCK-8 assay. CDC: Complement-dependent cytotoxicity; DLBCL: Diffuse large B-cell lymphoma; GCB: Germinal center B cell; IC50: Half maximal inhibitory concentration; Ly3- or Ly7-Mock: Ly3 or Ly7 cells infected with control lentivirus; Ly3- or Ly7-SEMA3F: Ly3 or Ly7 cells infected with DsRED-SEMA3F lentivirus. * P <0.05, P <0.001.

We then generated stably SEMA3F-deficient cell lines using shRNA in Ly1 and TMD8 cell lines, which resulted in a significant loss of SEMA3F mRNA and protein expression in Ly1 cells, and shRNA#2 was used in followed experiments [Figure 3A,B]. The cells with sh-SEMA3F showed significantly enhanced cell viability [Figures 3C,D]; accordingly, the number and average size of colonies formed by the sh-SEMA3F cells were higher than those formed by the control cells [Figure 3E, Supplementary Figure 1B, https://links.lww.com/CM9/B546]. Knockdown of SEMA3F also increased the mean IC50 of rituximab [Figure 3F]. Moreover, we found that rituximab-induced apoptosis could be decreased by SEMA3F knockdown [Figure 3G], and rituximab-mediated CDC activity of sh-SEMA3F was depressed at the same rituximab concentration [Figure 3H]. Taken together, these results indicated that deletion of SEMA3F could reduce the efficacy of rituximab on DLBCL cells.

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Figure 3:
The effects of SEMA3F downregulation on tumor inhibition and sensitivity to rituximab. (A) SEMA3F knockdown efficiency in Ly1 cells line. (B) Quantitative expression of SEMA3F in Ly1 and TMD8 cells transfected with sh-SEMA3F lentivirus. (C) Cell viability of Ly1-Mock and Ly1-sh-SEMA3F cell lines. (D) Cell viability of TMD8-Mock and TMD8-sh-SEMA3F cell lines. (E) Quantitative analysis of colony formation by Ly1 and TMD8 cells infected with sh-SEMA3F lentivirus. (F) IC50 of rituximab for Ly1-Mock and Ly1-sh-SEMA3F cell lines was measured using linear regression analysis of the dose–response curves. (G) Flow cytometry analysis of apoptosis of DLBCL cells infected with SEMA3F knockdown lentivirus. (H) Rituximab-induced CDC activity in different concentrations via CCK-8 assay. CDC: Complement-dependent cytotoxicity; DLBCL: Diffuse large B-cell lymphoma; Ly1- or TMD8-Mock: Ly1 or TMD8 cells infected with lentivirus containing scrambled shRNA sequence; Ly1- or TMD8-sh-SEMA3F: Ly1 or TMD8-cells infected with eGFP-sh-SEMA3F lentivirus; R: rituximab. * P <0.01, P <0.05.

SEMA3F deficiency repressed the expression of CD20 via the Hippo signaling pathway

Since CD20 expression on tumor B cells is responsible for the response of patients treated with rituximab, we further explored the role of SEMA3F in regulating the expression of CD20. Knockdown of SEMA3F inhibited CD20 mRNA and protein expression, and overexpression of SEMA3F upregulated CD20 expression [Figure 4A–C]. In addition, knockdown of SEMA3F resulted in downregulation of surface CD20 levels [Figure 4D], which was also validated by immunofluorescence microscopy [Figure 4E]. Recently, evidence has demonstrated that Hippo signaling plays a critical role in hematopoietic stem cell homeostasis and differentiation, and YAP/TAZ, as a key transcriptional coactivator of the Hippo pathway, has been shown to be an oncoprotein in DLBCL. [22] Other studies have suggested that the Hippo pathway might be involved in RCHOP resistance. [23] Thus, we tried to determine the role of the Hippo pathway in our model. Knockdown of SEMA3F resulted in reduced expression of LATS1/2 and upregulated expression of TAZ [Figure 4F]. Further examination of TAZ in the nuclear and cytoplasmic fractions showed that nuclear TAZ was elevated after SEMA3F knockdown and slightly upregulated in the cytoplasm [Figure 4G]. The alterations in BCL2, CCND2, CD20, and cleaved caspase-3 mediated by SEMA3F knockdown could be reversed by TAZ inhibitor (verteporfin) treatment [Figure 4H]. Moreover, the decreased rituximab-induced apoptosis of the sh-SEMA3F cells could be reversed by treatment with the inhibitor [Figure 4I], and the CDC activity of the sh-SEMA3F cells at each rituximab concentration could also be abolished by the inhibitor administration [Figure 4J]. These results demonstrated that the Hippo pathway played a critical role in the effects mediated by SEMA3F. Mechanistically, to uncover how YAP/TAZ regulated the expression of CD20, we investigated several key transcriptional regulators of B-cell differentiation. The results showed that SEMA3F knockdown had a slight effect on EBF1, IRF4, TCF3, and IKZF, with no effects on the expression of PAX5, and the administration of the inhibitor partially abrogated the expression of these factors [Supplementary Figure 2, https://links.lww.com/CM9/B546]. Upon nuclear accumulation of YAP/TAZ, they often form complexes with TEA domain (TEAD) transcription factors and then exert multiple functions. Next, we tried to determine whether TEAD could directly regulate the expression of CD20. Upstream of the CD20 promoter, we found an MCAT core motif (CATTCC) at -2095 bp, suggesting that TEAD might bind the promoter region of CD20. Using a ChIP assay followed by quantitative PCR, we observed enrichment for the binding of TEAD2 to the CD20 3΄ untranslated region relative to the binding of isotype-matched control IgG [Figure 4K]. A luciferase reporter assay showed that the signal of the TEAD2 vector was weaker than that of the control [Figure 4L], suggesting that the direct interaction of TEAD2 and the CD20 promoter was responsible for the transcriptional repression of CD20.

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Figure 4:
SEMA3F deficiency repressed expression of CD20 via Hippo signaling pathway. (A). Western blot analysis of CD20 in DLBCL cell lines with overexpression or knockdown expression of SEMA3F. (B) Quantitative analysis of CD20 in DLBCL cell lines with knockdown of SEMA3F. (C) Quantitative analysis of CD20 in DLBCL cell lines with overexpression of SEMA3F. (D) The levels of surface CD20 were analyzed with flow cytometry upon staining with FITC-conjugated anti-CD20 antibody. (E) Immunofluorescent staining of CD20 and SEMA3F in Ly1-Mock and Ly1-sh-SEMA3F cell lines. Scale bar, 20 μm. (F) Western blot of cascade proteins in Hippo pathway in Ly1 and TMD8 cells transfected with sh-SEMA3F lentivirus. (G) Western blot of TAZ in lysates of cytoplasm and nuclear fractions of Ly1-Mock and Ly1-sh-SEMA3F cell lines. (H) Western blot of BCL2, CCND2, CD20, and cleaved caspase-3 in Ly1-Mock, Ly1-sh-SEMA3F, and Ly1-sh-SEMA3F treated with YAP/TAZ inhibitor. (I) Apoptosis analysis of Ly1-Mock, Ly1-sh-SEMA3F treated with or without YAP/TAZ inhibitor or rituximab. (J) CDC activity mediated by combination with different rituximab concentrations and YAP/TAZ inhibitor via CCK-8 assay in Ly1-Mock, Ly1-sh-SEMA3F cells. (K) ChIP assay reveals binding of TEAD2 to the promoter of CD20. (L) Luciferase reporter assays of Ly1 cell co-transfected with TEAD2 vector or the corresponding control vector and reporter constructs containing CD20 promoter sequences. CDC: Complement-dependent cytotoxicity; ChIP: Chromatin immunoprecipitation assay; DLBCL: Diffuse large B-cell lymphoma; Ly1-Mock: Ly1 cells infected with lentivirus containing scrambled shRNA sequence; Ly1-sh-SEMA3F: Ly1 cells infected with eGFP-sh-SEMA3F lentivirus; MFI: Mean Fluorescence Intensity. * P <0.01, P <0.05.

SEMA3F deficiencies led to a weaker response to rituximab treatment in vivo

To validate the above findings in vivo, we established xenograft mouse models using Ly1-sh-SEMA3F cell lines when the tumor volume reached 100 mm 3. PBS, rituximab, and rituximab plus inhibitor were injected intraperitoneally. As expected, the xenografts with mock cells showed larger tumor shrinkage in tumor volume and weight with rituximab treatment, and when the xenograft mice were treated with rituximab plus inhibitor, the extent of the tumor volume and weight inhibition was nearly the same as that of those transduced with mock cells [Figure 5A,B]. Moreover, we tested the treatment effect of combined rituximab and inhibitor in patient-derived xenografts (PDX) mice; accordingly, treatment with the inhibitor alone showed a limited therapeutic efficiency in tumor volume and weight, administration of rituximab led to a larger reduction in tumor volume and weight, and a combination of rituximab and inhibitor showed a promising therapeutic efficiency [Figure 5C,D].

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Figure 5:
SEMA3F deficiency led to a weaker response to rituximab treatment in vivo and SEMA3F lowTAZ high predicted a worse prognosis. (A) Mean tumor volume of xenograft mice model injected with Ly1-Mock and Ly1-sh-SEMA3F cell lines treated with or without rituximab, combination rituximab or YAP/TAZ inhibitor. (B) Mean tumor weights of xenograft mice model injected with Ly1-Mock and Ly1-sh-SEMA3F cell lines treated with or without rituximab, combination rituximab or YAP/TAZ inhibitor. (C) Mean tumor volumes of PDX xenograft mice model treated with or without rituximab, YAP/TAZ inhibitor, or combination rituximab and YAP/TAZ inhibitor. (D) Mean tumor weights of PDX xenograft mice model treated with rituximab, YAP/TAZ inhibitor, combination rituximab and YAP/TAZ inhibitor. (E) Correlation of SEMA3F and TAZ expression in database GSE31312. (F) Comparison of TAZ expression in SEMA3F high and low expression DLBCL samples based on database GSE4475. (G) Comparison of TAZ expression in SEMA3F high and low expression DLBCL samples based on our cohort. (H) Representative IHC images of SEMA3F and TAZ staining in human DLBCL patients' sample. Scale bar, 50 µm. (I) Kaplan–Meier survival curves estimated OS of patients with combined expression of SEMA3F and TAZ based on database GSE10846. (J) Kaplan–Meier survival curves estimated OS of patients with combined expression of SEMA3F and TAZ based on database GSE31312. (K) Kaplan–Meier survival curves estimated PFS of patients with combined expression of SEMA3F and TAZ based on database GSE31312. (L) Kaplan–Meier survival curves estimated OS of patients with the combined expression of SEMA3F and TAZ based on the database and our cohort. DLBCL: Diffuse large B-cell lymphoma; IHC: Immunohistochemistry; Ly1-Mock: Ly1 cells infected with lentivirus containing scrambled shRNA sequence; Ly1-sh-SEMA3F: Ly1 cells infected with eGFP-sh-SEMA3F lentivirus; OS: Overall survival; PDX: patient-derived xenografts; PFS: Progression-free survival; R: rituximab. * P <0.05, P <0.01.

SEMA3F expression was negatively correlated with TAZ expression in DLBCL tissue, and SEMA3F lowTAZ high predicted a worse prognosis

We further investigated the relevance of SEMA3F and TAZ expression in human DLBCL specimens. SEMA3F was negatively correlated with TAZ in the GSE31312 dataset [Figure 5E] but not in the GSE10846 and GSE4475 datasets; however, we found that the expression of TAZ was much higher in the patients with low SEMA3F expression than in those with high SEMA3F expression in GSE4475 and our cohort [Figure 5F,G]. IHC staining showed that the patients with higher levels of SEMA3F had lower TAZ expression [Figure 5H]. Moreover, patients were divided into four subgroups based on the expression of SEMA3F and TAZ, and we found that the patients with SEMA3F lowTAZ high showed the worst survival in the two dependent datasets and our cohort [Figure 5I–L].

Discussion

Although the addition of rituximab to the CHOP regimen improves the clinical outcome of DLBCL patients with a higher response rate and longer relapse-free survival and OS, remission failure and relapse still occur in approximately 30–40% of all patients. The data presented here demonstrated that SEMA3F expression could predict the prognosis of DLBCL patients treated with RCHOP regimens but not in patients treated with the CHOP strategy, and patients with high expression of SEMA3F benefited much more than those with low expression of SEMA3F. Rituximab-resistant patients showed lower expression of SEMA3F. Our study further revealed that SEMA3F regulated the cell response to rituximab via upregulation of CD20. The Hippo signaling pathway is responsible for the effects mediated by SEMA3F, and the combination of rituximab and a YAP/TAZ inhibitor showed a promising therapeutic effect in xenograft mouse models. Our results suggest that SEMA3F might serve as a prognostic marker or novel therapeutic target in the future.

Rituximab resistance is a major obstacle to improving DLBCL clinical outcomes; however, the exact mechanisms are not clearly defined. Indeed, a plausible mechanism of rituximab resistance would be the loss of CD20 expression on tumor cells due to clonal selective pressure; some studies have shown that the transcription factors PU.1, OCT2, and PAX5 can regulate the expression of CD20. [24–26] Slabicki et al[27] found that cAMP-responsive element modulator (CREM) and methyl-CpG binding domain protein 2 (MBD2) could affect CD20 expression by using the RNAi screening method. Recently, the evidence demonstrated that rituximab-induced downregulation of CD20 was mainly mediated by the deacetylation of histones by histone deacetylases. [28–30] Chidamide (an inhibitor of HDAC1, HDAC2, and HDAC3) could upregulate the expression of CD20 and many other plasma membrane genes, and the combination of chidamide and rituximab showed promising therapeutic efficiency in relapsed/refractory DLBCL patients. [31,32] Other HDAC inhibitors, such as pan-HDAC inhibitors and entinostat (targeting HDAC1 and HDAC3), have also been reported to enhance the antitumor activity of rituximab. [29,33] Moreover, some therapeutic agents, such as gemcitabine, [34] farnesyltransferase inhibitors, [35] bryostatin-1, and some cytokines, [36,37] could enhance CD20 expression by increasing the cytotoxic activity of rituximab. In addition, overexpression of membrane complement-regulatory proteins, [38] consumption of immune effectors in the tumor microenvironment, [39] and expression of antiapoptotic proteins contribute to rituximab resistance. In this study, we found that SEMA3F played a critical role in the upregulation of CD20 by inactivating the Hippo pathway, and our clinical data suggested that patients with high SEMA3F expression survived longer than those with low SEMA3F expression. Rituximab-resistant patients also showed lower expression of SEMA3F. Our results proved that SEMA3F could be identified as a novel therapeutic target for rituximab resistance in DLBCL patients.

SEMA3F is a secreted axon guidance molecule of the semaphorin family, which plays an important role in brain development. [40] However, SEMA3F was also reported to be downregulated in multiple tumors, such as colorectal cancer, lung cancer, hepatocellular cancer, and breast cancer, which could inhibit tumor formation and development. [41] Moreover, SEMA3F has a regulatory role in the formation of vascular and lymphatic vessels by interacting with the receptors plexin A1 and NRP2. [42,43] Our previous work showed that SEMA3F could inhibit colorectal cancer cell stemness via inactivation of Rac1 and downregulate CXCR4 by downregulating the transcription factor ASCL2 [14]. Rac1 GTPase has long been recognized as a critical regulatory protein in different cellular and molecular processes involved in cancer progression, including acute myeloid leukemia. The activation of Rac1 promotes leukemia development by enhancing leukemia cell homing and retention in the niche, and Rac1 inhibition selectively induces apoptosis in patient-derived leukemia cells but not in normal mononuclear cells, suggesting that Rac1 signaling also plays an important role in hematological malignancies. [44] Our study demonstrated that SEMA3F also exerted an inhibitory role in lymphoma cell proliferation and promoted cell apoptosis, and the expression of SEMA3F in normal lymph nodes was much higher than that in DLBCL patients. Notably, we found that SEMA3F had no obvious effects on the classic PI3K/AKT signaling pathway, suggesting that signaling mediated by SEMA3F might have tissue specificity. To date, the cause of SEMA3F downregulation remains unclear, and evidence has shown that histone deacetylase, p53, and RORα reduce SEMA3F expression. Therefore, understanding the reduction in SEMA3F in DLBCL might help to elucidate the development of DLBCL.

The Hippo signaling pathway is a conserved signaling pathway that regulates cell proliferation and organ size in Drosophila and mammals. [45] However, YAP and TAZ, as the key transcriptional coactivators of the Hippo pathway, have been demonstrated to be oncoproteins in multiple cancers, such as NK/T cell lymphoma and breast cancer. In the nucleus, YAP and TAZ primarily increase pro-proliferative gene expression through interaction with the TEAD family of transcription factors. [46,47] Cell–cell communication has been identified as the pivotal signal of YAP/TAZ regulation, and YAP/TAZ can act as sensors of cell density to limit proliferation in response to excessive cell contacts. [48] SEMA3F is a chemorepulsive factor that regulates axon and endothelial cell growth, and loss of SEMA3F might lead to the dysregulation of cell–cell communication. In this study, we found that the expression of SEMA3F in DLBCL patients was lower and that knockdown of SEMA3F expression could lead to overexpression and nuclear localization of YAP and TAZ. We also showed that SEMA3F lowTAZ high was associated with poor survival, confirming the role of SEMA3F in tumor progression. Moreover, we demonstrated that the transcription factor TEAD2 could bind to the promoter of CD20 and inhibit the expression of CD20, suggesting that the Hippo pathway played an important role in rituximab resistance.

In summary, our results demonstrated that SEMA3F regulated the rituximab response through the Hippo pathway by upregulating the expression of CD20, and the combination of a YAP/TAZ inhibitor and rituximab showed promising therapeutic effects. These results help elucidate the mechanism of refractory disease and suggest prognostic markers or novel therapeutic targets in the future.

Acknowledgments

We thank our colleagues for their helpful comments and Yanqi Zhang for the kindly help in statistics.

Funding

This study was supportedFoundation: by grants from the National Natural Science Fund (No. 82070208), National Natural Science Foundation of Chongqing (cstc2020jcyjmsxmX0433), the Major program of Chongqing Health Commission and Science and Technology Bureau Joint project (2022DBXM003), Chongqing Science and Health Joint medical research project (2023QNXM047), Military clinical medical innovation project of Xinqiao hospital (2021JSLC0003), the Science and technology innovation promotion project of AMU (2019XLC3020), and the Translational Research Grant of NCRCH (2020ZKZC02 and 2021WWB05).

Conflict of Interest

None.

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Keywords:

Lymphoma, Large B-Cell, Diffuse; Rituximab resistance; Apoptosis; WW domain-containing transcription regulator protein 1

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