Synergistic Efficacy of Chidamide and LB100 in Sézary Syndrome via TNC Downregulation and PI3K/AKT/mTOR Dephosphorylation

ABSTRACT

Primary cutaneous T‐cell lymphoma (CTCL) manifests as a distinct variant of T‐cell non‐Hodgkin's lymphoma, predominantly impacting skin tissues and constituting approximately 75%–80% of cutaneous lymphoma cases, exhibiting diverse clinical presentations. Sezary syndrome (SS) is a rare subtype of CTCL. The therapeutic approach of SS frequently incorporates multiple chemotherapeutic compounds, encompassing specific histone deacetylase inhibitor agents. Nevertheless, similar to numerous chemotherapeutic protocols, existing monotherapy approaches for SS encounter limitations regarding sustained therapeutic outcomes and optimal effectiveness. This investigation seeks to examine the therapeutic impact and molecular pathways of combining Chidamide with LB100 in the treatment of SS. The synergistic effects of Chidamide and LB100 were evaluated in SS cell lines (Hut78 and H9) using the Cell Counting Kit‐8, Chou‐Talalay combination index, colony formation assays, and mouse xenograft models. Apoptosis was examined by Annexin V‐APC/PI staining. The molecular mechanisms were explored through transcriptome sequencing, Western blotting, immunohistochemistry, and Lentivirus transfection. Chidamide and LB100 demonstrated synergistic antitumor activity in SS cell lines (Hut78 and H9) in vitro, with a combination index (CI) < 1. This synergy was also confirmed in Hut78 xenograft mice. Mechanistically, the two agents downregulated Tenascin C (TNC) and caused dephosphorylation of the PI3K/AKT/mTOR signaling cascade. This study also preliminarily validated the effect of the combination of the two drugs on other subtypes of CTCL. Overall, these findings provide potential new therapeutic strategies for CTCL (especially SS), although further clinical evaluation is required to validate these results.

Chidamide combined with LB100 can effectively promote the death of Sézary Syndrome by inhibiting the expression of TNC and the phosphorylation of PI3K/AKT/mTOR.

Abbreviations

Bcl‐2

B‐cell lymphoma/leukemia‐2

CCK‐8

cell counting kit‐8

CHI

chidamide

CI

combination index

CTCL

cutaneous T‐cell lymphoma

DMSO

dimethyl sulfoxide

ECM

extracellular matrix system

FBS

fetal bovine serum

FITC

fluorescein isothiocyanate

GAPDH

glyceraldehyde‐3‐phosphate dehydrogenase

IC₅₀

50% inhibitory concentration

IHC

immunohistochemistry

KEGG

Kyoto Encyclopedia of Genes and Genomes

LPD

CD30+ lymphoproliferative disorders

MF

mycosis fungoides

NSG

NOD/scid interleukin‐2 receptor γ‐chain‐deficient

p‐AKT

phospho‐AKT

PBS

phosphate buffer saline

PI

propidiumiodide

p‐mTOR

phospho‐mTOR

PP2A

protein phosphatase 2A

p‐PI3K

phospho‐PI3K

PTCL

peripheral T‐cell lymphoma

PVDF

polyvinylidene fluoride

RIPA

radio immunoprecipitation assay

SDS‐PAGE

sodium dodecyl sulfate‐polyacrylamide gel electrophoresis

SS

Sézary Syndrome

TNC

Tenascin C

WB

western blotting

1. Introduction

Cutaneous T‐cell lymphoma (CTCL) exists as a subtype of non‐Hodgkin's lymphoma, representing approximately 12% of all lymphomas globally. The condition manifests through malignant T cells penetrating the skin layers [1, 2]. CTCL includes various subtypes like mycosis fungoides (MF), Sézary Syndrome (SS), and CD30+ lymphoproliferative disorders (LPD) [3]. Among them, SS is rare (2%–5%), It is an aggressive form of CTCL that typically presents with erythroderma (erythema and scale affecting > 80% of body surface area), lymphadenopathy, and leukemic blood involvement [4, 5]. Patients with advanced‐stage SS have a poor prognosis with a median survival of less than 5 years. While monotherapies such as alemtuzumab, histone deacetylase inhibitors, pralatrexate, gemcitabine, and doxorubicin offer some degree of therapeutic benefit, most patients still exhibit poor responses or fail to achieve long‐term remission [6]. Recent studies have shown that the combination of mogamulizumab and other systemic therapies yields favorable outcomes, but little is known about its clinical effectiveness besides single case studies [5]. Consequently, there is a critical need for alternative combination therapies to improve clinical outcomes for patients with SS.

Epigenetic modifications, particularly histone alterations, serve a pivotal function in CTCL progression and emergence. Histone acetylation, a thoroughly investigated post‐translational alteration, remains under the control of two antagonistic enzymes: Histone acetyltransferases (HATs) and Histone deacetylases (HDACs) [7]. HDACs are key epigenetic regulators that deacetylate lysine residues, condense chromatin, and suppress the transcription of downstream genes. Targeting HDACs to modulate gene expression has emerged as a promising therapeutic strategy for hematological malignancies [8]. Chidamide (tucidinostat), an orally administered selective HDAC inhibitor (HDACi), demonstrates the capacity to trigger apoptosis and halt growth in leukemia cells, as evidenced through both in vitro and in vivo investigations [9]. Chidamide has also been found to enhance the efficacy of chemotherapy in various cancers, encompassing acute myeloid leukemia, multiple myeloma, and triple‐negative breast cancer, when combined with drugs like claribine, venetoclax, and enzalutamide [10, 11, 12]. Despite these promising results, no combination therapy involving chidamide has been established for SS, and the effectiveness of monotherapy remains limited [13]. Therefore, exploring novel and more effective chidamide‐based combination therapies is critical for improving the prognosis of patients with SS, particularly those with acute forms who urgently need remission.

Protein Phosphatase 2A (PP2A) is a serine/threonine phosphatase with the capacity to regulate multiple cancer‐related signaling pathways. Suppression of PP2A activity has emerged as a potential therapeutic approach across diverse cancer types [14, 15]. LB100 functions as a water‐soluble, small‐molecule competitive PP2A inhibitor that modulates cell cycle control and reduces tumor cell growth through PP2A suppression [16]. This compound exhibits substantial anti‐tumor properties according to multiple preclinical investigations [17, 18, 19]. Additionally, LB100 has been shown to enhance the functionality of cytotoxic T cells [20].

Given that both chidamide and LB100 have been successfully combined with other therapeutic agents to enhance efficacy, and both are associated with T‐cell toxicity, this investigation seeks to examine the prospective enhanced anti‐SS activity through their combined application. Results indicate that chidamide and LB100 synergistically exert anti‐SS effects both in vitro and in vivo. Mechanistically, the combination of these two agents may downregulate the TNC gene level in SS cells, leading to reduced phosphorylation of the PI3K/AKT/mTOR signaling cascade, thereby inducing apoptosis and cell cycle arrest. In addition, the combination of the two drugs also showed synergistic inhibitory effects on another CTCL subtype cell line: HH.

2. Methodologies and Materials

2.1. Cell Lines, Reagents and Antibodies

SS cell lines H9 and Hut78 were obtained from Nanjing Daona Biotechnology Co. Ltd. MF cell line HH was obtained from Maisen Cell Technology Co. LTD. These cells underwent cultivation in RPMI 1640 medium comprising 10% fetal bovine serum (FBS) and were kept at 37°C within a moisture‐controlled incubator containing 5% CO₂.

Chidamide (Cat. No. HY‐109015) and LB100 (Cat. No. HY‐18597) were procured from MedChemExpress. Chidamide underwent dissolution in dimethyl sulfoxide (DMSO, Sigma, USA) to establish a stock concentration of 20 mM, while LB100 was dissolved in ultrapure water. Both compounds were subsequently adjusted to the desired concentrations utilizing culture media for experimental use.

Antibody details are depicted in the Table S1.

2.2. Cell Proliferation Assay

Cell viability was evaluated via the Cell Counting Kit‐8 (CCK‐8) assay. CTCL cells (2 × 10⁴ cells/well) were placed into 96‐well plates and exposed to the designated drugs for the specified durations at 37°C in an atmosphere comprising 5% CO₂. Following drug exposure, cells underwent incubation with 10 μL of CCK‐8 solution (Shanghai Yisheng Biotechnology Co. Ltd.) for 4 additional hours at 37°C. Absorbance at 450 nm was recorded utilizing a microplate reader (Bio‐Rad, Model 680). The combination index (CI) of the two agents was computed employing CompuSyn software (ComboSyn Inc., Paramus, NJ, USA). Half‐maximal inhibitory concentrations (IC₅₀) were computed with GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA).

2.3. Colony Formation Assay

To evaluate colony development, methylcellulose medium (2 mL; Stem Cell Technologies, Canada) was introduced to log‐phase cells placed in 6‐well plates following exposure to the selected drugs for the indicated durations. The cells underwent incubation at 37°C with 5% CO₂ over a 2‐week period, succeeded by microscopic observation and enumeration of the resulting colonies.

2.4. Flow‐Cytometric Analysis of Apoptosis

H9 and Hut78 cells underwent treatment with the specified drugs at designated time intervals, then stained with Annexin V‐APC and PI using the Apoptosis Kit (Multi Sciences, Lianke Bio, China) per the manufacturer's protocol. The assessment of apoptosis was executed via FACScan flow cytometer (BD Biosciences), while data interpretation was accomplished employing FlowJo software (v10, Tree Star, Ashland, USA).

2.5. Transcriptome Sequencing

The content of transcriptome sequencing mainly includes differential expression analysis, principal component analysis (PCA), GO and KEGG enrichment analyses. Detailed analysis methods are described in the Appendix S1.

2.6. Western Blotting (WB)

The samples underwent lysis using RIPA buffer (Beyotime Biotechnology, Cat. No. P0013B), followed by SDS‐PAGE (Life) separation of total cell extracts and subsequent transfer to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Specific antibodies were utilized to detect target proteins, with quantification performed through ImageJ software (Bio‐Rad, California, USA).

2.7. Xenograft Tumor Model

Female NOD/scid interleukin‐2 receptor γ‐chain‐deficient (NSG) mice aged 5–6 weeks were procured from the Animal Laboratory Center of Nantong University (Nantong, China). Hut78 cells underwent suspension in serum‐free medium at 1.5 × 10⁶ cells/200 μL and were introduced subcutaneously into the upper forelimb area of each mouse. After 7 days, when tumor diameters reached between 5 and 7 mm, the xenograft models were arbitrarily split into four cohorts (n = 5) and received daily treatments for 2 weeks with the vehicle, chidamide (20 mg/kg), LB100 (3 mg/kg), or a combination of both agents. Chidamide was given via oral administration, whereas LB100 was injected intraperitoneally. Tumor dimensions were recorded every 3 days utilizing calipers, and tumor volume (mm³) was determined employing the formula: (length × width²)/2. Following the 14‐day therapeutic course, mice underwent euthanasia, and both tumors and major internal organs were collected for subsequent analysis. The animal protocols were sanctioned by the Institutional Animal Care and Use Committee at Nantong University, in accordance with prevailing guidelines on animal care and welfare.

2.8. Immunohistochemistry (IHC) and HE Staining

The mouse tumor specimens, embedded in paraffin, underwent sectioning to create 3–5 μm thick slices for placement on charged slides for IHC and HE tests. The detailed operation process is shown in the Appendix S1.

2.9. Lentiviral Short Hairpin RNA (shRNA) Transfection

Lentivirus transduction particles containing small interfering RNA (siRNA) targeting TNC and negative control siRNA (Shanghai GeneChem Co. Ltd.) were transduced into H9 and Hut78 cells. Following exposure to lentiviral particles, the target cells were assessed for successful transduction after an appropriate incubation period. Table S2 provides detailed descriptions of the specific sequence used and information on virus titers and MOI values.

2.10. Statistical Analysis

Statistical analyses were executed employing GraphPad Prism 9 software, with two‐tailed t‐tests applied for comparisons between two cohorts, one‐way ANOVA facilitating examination of multiple cohorts involving a single variable, and two‐way ANOVA enabling analysis of multiple cohorts incorporating multiple variables. For transcriptome sequencing, correlations between samples were assessed by calculating the Pearson correlation coefficient using the R language. A log2 fold change ≥ 1 was interpreted as upregulation, whereas a log2 fold change ≤ −1 was considered indicative of downregulation. Statistical significance was established at p < 0.05.

3. Results

3.1. The Synergistic Effect of Chidamide Plus LB100 in SScells

This study investigated the interaction synergy effect between chidamide and LB100 in SS cells. H9 and Hut78 cells were selected as representative SS cell models based on previously published research [21]. Following 48 h of exposure, chidamide demonstrated concentration‐dependent decreases in cellular viability within both H9 and Hut78 cells, displaying IC₅₀ measurements of 686.0 and 231.4 nM, respectively. Incorporation of LB100 alongside chidamide markedly improved its anti‐SS effectiveness, lowering the IC₅₀ measurements of chidamide across both cellular lines (Figure 1A,B and Table 1). For evaluating the combined impact of chidamide and LB100, the Chou‐Talalay methodology was utilized to determine the CI based on cellular viability outcomes. CI values of < 1, =1, or > 1 signify synergistic, additive, or antagonistic interactions, respectively. As illustrated in Figure 1C, the majority of concentration combinations between chidamide and LB100, excluding the minimal dose combination (not depicted in the figure), yielded CI values < 1, indicating synergistic effects between the two drugs in eliminating SS cells. Similar results were obtained at 24 and 72 h of drug administration (Figure S1 and Table S3).

FIGURE 1.

Chidamide and LB100 synergistically decrease cell viability in H9 and Hut78 cells. (A, B) CCK8 assay detecting cell viability after 48 h of combined drug treatment; (C) The Chou‐Talalay methodology calculating the combination index (CI). (D) Representative images of monoclonal formation. (E, F) Quantification plots of monoclonal cells. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

TABLE 1.

IC₅₀ values of H9 and Hut78 cells exposed to chidamide alone or in combination with LB100 for 48 h.

Drugs	IC50 (nM) of H9	Fold	IC50 (nM) of Hut78	Fold
	Single combination		Single combination
Chidamide	686.0		231.4
LB10 0 (0.1 μM) + Chidamide	605.0	1.13	211.3	1.09
LB100 (1 μM) + Chidamide	335.4	2.04	123.1	1.87
LB100 (2 μM) + Chidamide	222.7	3.08	91.27	2.53

To further validate the cooperative impact of chidamide and LB100 on SS cell proliferation, an additional colony formation assay was conducted. Based on insights from the preliminary study comparing single‐agent and dual‐therapy treatments, H9 and Hut78 cells were split into four experimental cohorts: a control cohort, a chidamide‐only treatment cohort (450 nM), an LB100‐only treatment cohort (4 μM), and a combination therapy cohort (chidamide 450 nM and LB100 4 μM) and maintained for 48 h (the same regimen was applied in subsequent vitro experiments). The assay clearly suggested that while both individual agents inhibited colony formation, the level of suppression was significantly enhanced when chidamide and LB100 were used together (Figure 1D–F). This finding highlights the enhanced effectiveness of the integrated therapeutic approach in reducing the proliferative potential of SS cells.

3.2. Co‐Treatment With Chidamide and LB100 Triggers SS Cell Apoptosis

To further investigate the cytotoxicity induced by chidamide and LB100, cell apoptosis was examined. The Annexin‐V‐APC/PI staining assay validated the synergistic SS‐killing effect of chidamide combined with LB100 in both H9 (Figure 2A) and Hut78 (Figure 2B) cells. Administration of LB100 or chidamide independently triggered slight to moderate apoptotic effects, whereas their concurrent application produced a more substantial apoptotic outcome. Moreover, WB analysis suggested that Bcl‐2, an anti‐apoptotic protein, exhibited markedly decreased expression in cells receiving both agents compared to control and individual treatment cohorts. Conversely, Cleaved‐caspase3, a pro‐apoptotic protein, displayed significantly elevated levels in the dual‐treatment cohort. Additionally, Cyclin D1, a cell cycle regulatory protein, showed reduced expression in the combination treatment cohort (Figure 2C–E). These observations indicate that the concurrent application of chidamide and LB100 promotes both apoptotic activity and cell cycle inhibition in SS cells.

FIGURE 2.

Therapeutic synergy of chidamide and LB100 in promoting apoptosis in H9 and Hut78 cells. (A, B) Flow cytometry analysis assessed apoptotic rates in SS cells, accompanied by corresponding statistical graphs of apoptotic populations. (C) Western blotting analysis detecting changes in Bcl‐2, Cleaved‐caspase‐3, and Cyclin D1 expression. (D, E) Grayscale quantification of the Western blotting bands.

3.3. Effects of Chidamide Combined With LB100 on TNC Level and Phosphorylation of PI3K/AKT/mTOR Pathway

To better understand how chidamide in combination with LB100 eliminates SS cells within 48 h, gene expression patterns were examined through eukaryotic reference transcriptome sequencing in untreated H9 and Hut78 cells, alongside those exposed to the combined treatment (Figure 3). Analysis of gene expression data through PCA demonstrated clear differentiation of drug interventions across the initial two principal components (Figure 4A).

FIGURE 3.

(A) The main steps of transcriptome sequencing (B) Bar chart showing the total gene expression levels of all samples based on transcriptome sequencing results. (C) Heatmap of co‐differentially expressed genes (co‐DEGs) in H9 and Hut78 cells treated with chidamide and LB100.

FIGURE 4.

Chidamide combined with LB100 induced down‐regulation of TNC gene expression and enrichment of the PI3K/AKT/mTOR signaling cascade in SS cells. (A) PCA of gene expression data from transcriptome sequencing. (B) Venn diagram depicting the link between differentially modulated genes in the combination treatment cohort and the control cohort for both H9 and Hut78 cell lines. (C) RNA‐seq analysis showing the rate of change in TNC gene expression patterns. (D) Volcano plot from RNA sequencing showing TNC as one of the downregulated genes after combination treatment in Hut78 cells. (E) Bar plot and (F) bubble plot showing the enrichment analysis of co‐DEGs in Hut78 cells.

The Venn diagram depicted the intersection of differentially expressed genes in the combination treatment cohort of both cell lines relative to the control cohort and showed that among the 1109 differential genes identified, 122 genes were co‐upregulated or co‐downregulated in both cell lines after combined treatment (Figure 4B). Further analysis of gene expression changes in the combination treatment cohort revealed notable alterations in genes related to cytokine‐cytokine receptor interactions and the PI3K/AKT/mTOR pathway across both cell lines (Figure S2B,C and Figure 4E,F). Given the pivotal role of PI3K/AKT/mTOR signaling in driving cancer progression and targeting this pathway is a widely recognized strategy for overcoming therapeutic resistance, this pathway was prioritized for further investigation [22]. In addition, RNA sequencing volcano plot analysis identified TNC among the downregulated genes following combination treatment (Figure S2A and Figure 4D).

To validate TNC suppression and PI3K/AKT/mTOR pathway inactivation, WB (Figure 5A–C) and IHC (Figure S3C,D and Figure 5D) analysis was conducted. WB analysis validated that the TNC level followed a similar trend to that observed in the gene expression data, and dephosphorylation of PI3K/AKT/mTOR was evident. Immunohistochemical staining further corroborated these findings, showing slight differences in TNC level and phosphorylated mTOR and decreased expression of phosphorylated PI3K/AKT in the combined treatment cohort.

FIGURE 5.

(A) Western blotting analysis detecting changes in TNC expression and PI3K/AKT/mTOR pathway‐related proteins. (B, C) Grayscale quantification of Western blotting bands. (D) Immunohistochemical images showing p‐PI3K, p‐AKT, p‐mTOR, TNC, Bcl‐2, and Cleaved caspase‐3 levels in tumor tissues from treated mice.

3.4. TNC Acts as an Upstream Regulator of the PI3K/AKT/mTOR Pathway in SS

Earlier research demonstrated that TNC suppresses PI3K/AKT/mTOR signaling by binding to integrins, affecting the progression of glioblastoma, and notably inhibiting T cell activity [23]. This prompted an investigation into whether the decreased expression of TNC under combined treatment led to the dephosphorylation of the PI3K/AKT/mTOR pathway. We performed TNC knockdown in H9 and Hut78 cells and confirmed knockdown efficiency through WB (Figure 6A). We found that after knocking down TNC, the growth rate of cells slowed down slightly (Figure 6B). In addition, the protein levels of p‐PI3K, p‐AKT, and p‐mTOR in the shTNC groups also decreased (Figure 6C). Finally, we treated the shNC group and the shTNC group with the combined drugs for 48 h, and the CCK8 test showed that the proliferation inhibition effect of the shTNC groups was weaker than that of the shNC group (Figure 6D). These results suggest that TNC may play a carcinogenic role as a potential upstream regulator of the PI3K/AKT/mTOR pathway in SS cells and is one of the key factors responsible for the synergistic effect of chidamide combined with LB100 in SS cells.

FIGURE 6.

TNC plays an oncogenic role in SS cells as a potential upstream regulator of the PI3K/AKT/mTOR pathway. (A) Western blotting analysis detecting TNC that was knocked down in H9 and Hut78 cells. (B) Growth curves of SS cell lines under shNC and shTNC conditions. (C) Western blotting analysis detecting changes in PI3K/AKT/mTOR pathway‐related proteins after TNC knockdown. (D) CCK8 assay detecting the sensitivity of TNC knockdown SS cells to drug combination.

3.5. Chidamide and LB100 Are Active in a Xenograft Tumor Model

A xenograft mouse model was applied to validate the cooperative impact of chidamide and LB100 in inhibiting SS growth in vivo. Seven days after subcutaneously injecting Hut78 cells into 5–6 week‐old NSG mice, the animals were split into four cohorts: vehicle control, chidamide, LB100, and a combination of both treatments (Figure 7A). Notably, the combination treatment resulted in a more pronounced decrease in tumor burden, as evidenced by decreased tumor volume and weight versus the vehicle control or single‐agent treatments (Figure S3A and Figure 7B,D,E). No significant toxicity was observed in the combination‐treated mice, with no notable weight loss during the treatment period (Figure 7C). Furthermore, histological analysis revealed no significant changes, such as necrosis or inflammation, in the heart, liver, spleen, lung, or kidney sections between the combination and control cohorts (Figure S3B and Figure 7F).

FIGURE 7.

Chidamide in combination treatment with LB100 demonstrates synergistic anticancer effectiveness in vivo. (A) Diagrammatic illustration of tumor implantation in mice and following drug administration protocol. (B) Tumor dimensions were assessed at three‐day intervals post‐tumor establishment. (C) The weight of mice was recorded at three‐day intervals following tumor establishment. (D) Tumor specimens were collected and imaged after animal euthanization. (E) The mass of tumors was determined following their removal from the mice. (F) H&E stained sections of heart, liver, spleen, lung, and kidney tissues from tumor‐bearing mice in the control and combination cohorts after 14 days of administration, examined under 10× magnification.

To elucidate the molecular mechanisms, immunohistochemical analysis was conducted to examine Bcl‐2, Cleaved caspase‐3, TNC, p‐PI3K, p‐AKT, and p‐mTOR levels within tumor specimens. As expected, concurrent administration enhanced cleaved caspase‐3 expression while diminishing p‐PI3K and p‐AKT levels. Moreover, decreased expression patterns of Bcl‐2, TNC, and p‐mTOR were detected (Figure S3C,D and Figure 5D). These findings indicate that chidamide plus LB100 downregulates TNC expression and suppresses PI3K/AKT/mTOR pathway activation, thereby synergistically inhibiting SS growth in vivo.

3.6. The Synergistic Effect of Chidamide Plus LB100 in Other CTCL Cells

SS is an extremely rare subtype of cutaneous T‐cell lymphoma. Given the promising synergistic effect of chidamide and LB100 in SS cells, we further explored the effect of the combination in other cell lines of CTCL. We selected HH cells from Mycosis Fungoides (MF) [21]. The 24/48/72 h CCK8 and CI index results of HH cells treated with the combination of the two drugs were similar to those of SS cells. Moreover, the WB results validated that the downregulation of TNC level and dephosphorylation of PI3K/AKT/mTOR followed a similar trend to that observed in SS cells basically (Figure S4 and Table S4). These results suggest that in addition to its effects on SS cells, chidamide combined with LB100 may act more broadly on CTCL cells by down‐regulating the expression of TNC. However, more basic experiments (such as animal experiments, sequencing analysis) and clinical trials are needed to verify this idea, and more cell line studies are needed, such as the LPD cells [21].

4. Discussion

Chidamide is an oral selective HDAC inhibitor that was approved for the treatment of peripheral T‐cell lymphoma (PTCL) in Japan and China [24]. It can also act on lymphoblastic leukemia (ALL), AML, and myelodysplastic syndrome [25, 26]. LB100 is a synthetic, water‐soluble, small‐molecule PP2A competitive inhibitor. It has also seen applications in cancer treatments [16]. To date, many clinical trials have been conducted and completed for chidamide, whereas relatively few results have been obtained for LB100. Specific clinical trial results are summarized in Table S5 concisely.

Given that neither drug has been tested in rare SS patients, this investigation examined the dual impact of chidamide and LB100 on SS by basic experiments, demonstrating that this combination exhibited synergistic effects in suppressing cell proliferation and enhancing apoptosis in SS cell lines. Furthermore, the combination demonstrated enhanced anti‐tumor activity in the Hut78 xenograft model in NSG mice. Importantly, no weight loss or significant toxic side effects were detected in vivo. These results provide experimental evidence and potential molecular mechanisms to support future clinical trials evaluating chidamide combined with LB100 for SS treatment, particularly in patients who do not respond well to monotherapy. The research indicates three principal molecular mechanisms potentially responsible for the effective apoptosis induced by this combination: (1) diminished Bcl‐2 expression, elevated Cleaved Caspase 3, and G1 phase cell‐cycle arrest; (2) TNC reduction; and (3) subsequent suppression of PI3K/AKT/mTOR pathway phosphorylation. The following sections elaborate on these mechanisms, while Figure 8 and Figure S5 illustrate the potential combined anti‐cancer mechanisms of chidamide and LB100.

FIGURE 8.

Mechanistic schematic of chidamide combined with LB100 in eliminating SS cells.

After administration of chidamide combined with LB100, Bcl‐2 levels diminished while Cleaved Caspase 3 levels elevated. These observations align with the investigation by Cyrenne et al., which revealed that the combination of a Bcl‐2 inhibitor and HDACi exhibited synergistic lethality against CTCL cells [27]. Similarly, Cao et al. reported comparable changes in Bcl‐2 and Caspase 3 expression in multiple myeloma cells exposed to chidamide and venetoclax [11]. Additionally, research has shown that chidamide used in conjunction with the epidermal growth factor receptor tyrosine kinase inhibitor icotinib produced synergistic tumor‐suppressing effects in non‐small cell lung cancer (NSCLC), resulting in enhanced Cleaved Caspase 3 expression [28]. Thus, the downregulation of Bcl‐2 and upregulation of Cleaved Caspase 3 are critical factors driving efficient apoptosis in SS cells treated with chidamide and LB100. Furthermore, chidamide induces the cyclin‐dependent kinase inhibitor p21, leading to cell‐cycle arrest at the G1 phase [13, 29]. In the present study, LB100 enhanced chidamide's ability to arrest CTCL cells in the G1 phase.

We then proceeded to search for potential complementary causes of significant apoptosis and cycle arrest based on the pharmacological effects of both drugs. The PI3K/AKT/mTOR signaling cascade represents a fundamental intracellular mechanism that controls vital cellular processes [30, 31]. This signaling network exhibits excessive activation across numerous human malignancies [32, 33, 34]. PI3K possessing a regulatory p85 subunit along with a catalytic p110 subunit [35]. After extracellular growth factors and cytokines recognition, PI3K shifts to the plasma membrane where receptor tyrosine kinases or G‐protein coupled receptors initiate its activation. This mechanism enables the conversion of phosphatidylinositol‐4, 5‐bisphosphate (PIP2) into phosphatidylinositol‐3, 4, 5‐trisphosphate (PtdIns(3, 4, 5)P3; also known as PIP3) [36]. PIP3 acts as a lipid second messenger supporting phosphorylation of AKT at Thr308 and phosphorylation of AKT at Ser473 [37, 38]. Completely activated AKT then suppresses apoptosis and enhances cell survival and metabolism through activating downstream mTOR [39, 40, 41]. The potent inhibitory effect of LB100 on cancer cell viability is due in part to the fact that inhibition of PP2A can reduce AKT phosphorylation and inactivate AKT signaling, and LB100 also reduces the expression of PI3K p110α. However, the mechanism by which PP2A inhibition decreases PI3K p110α expression is not fully understood. Alternatively, PP2A inhibition may also lead to enhanced AKT phosphorylation in certain cancer cell lines, possibly because upstream targets of AKT are preferentially phosphorylated [42, 43, 44].

The above studies suggest that neither the regulation mechanism nor the success rate of PP2A inhibitors on the PI3K/AKT pathway is fully understood, raising the risk that LB100 as a single agent will have limited efficacy against cancer cells. However, Chidamide, as an HDACI, can inhibit histone deacetylase, leading to increased histone acetylation levels. This acetylation modification can alter chromatin structure, making it easier for genes that would otherwise be repressed to be transcribed. Some genes related to Extracellular Matrix System (ECM) synthesis, such as collagen and fibronectin, may therefore be activated or inhibited, thereby regulating ECM expression [45]. Moreover, the ECM can maintain cell viability by activating the PI3K/AKT/mTOR pathway through integrin interactions [46]. Coupled with earlier research, it has been established that the HDACi Chidamide suppresses PI3K/AKT/mTOR signaling, triggering cell cycle interruption and programmed cell death in colorectal cancer cells [47]. Therefore, we speculate that by regulating the ECM, Chidamide has the opportunity to stabilize the regulation of LB100 on the PI3K/AKT pathway, so that the phosphorylation of PI3K/AKT/mTOR is down‐regulated, and then it is better than single‐drug therapy and enhances the efficacy of LB100. Therefore, we focused on whether the combination of the two agents has a complementary effect in inhibiting PI3K/AKT/mTOR pathway activation via the ECM.

Transcriptome sequencing analysis and validation of protein and histology revealed that the combined treatment of chidamide and LB100 led to the downregulation of Tenascin‐C (TNC). TNC is a protein with spatiotemporal expression patterns in embryos [48], serving as a large ECM glycoprotein with a six‐armed quaternary and multi‐module structure [49]. Due to its role in regulating cell adhesion through binding with fibronectin and its involvement in tissue regeneration, TNC has gained increasing attention from both basic and clinical scientists in the field of oncology [50]. In cancer therapy, the downregulation of TNC is often viewed as a positive indicator of successful treatment, signifying a shift in cell status from proliferative to non‐proliferative [51, 52, 53]. Sequencing analysis also revealed notable activation of the PI3K/AKT/mTOR pathway after combination therapy. The phosphorylation levels of PI3K, AKT, and mTOR were evaluated and found to be downregulated at both the protein and tissue levels following combined treatment. Then, knockdown of TNC in H9 and Hut78 cells also reduced the phosphorylation level of PI3K/AKT/mTOR. This suggests that TNC may act as an upstream regulator of the PI3K/AKT/mTOR pathway in SS cells and be affected by this combination treatment. In addition, knockdown of TNC reduced the ability of cell proliferation and the effect of combination therapy.

These results and analyses suggest that chidamide combined with LB100 may inhibit the phosphorylation of the PI3K/AKT/mTOR pathway in SS cells by downregulating TNC expression specifically. The findings suggest an innovative therapeutic strategy and mechanistic insight for SS treatment. However, the precise role of TNC in mediating changes in the PI3K/AKT/mTOR pathway in the presence of these two drugs remains unclear. Additional studies are essential to elucidate whether TNC facilitates focal adhesion kinase (FAK) formation by interacting with integrins (heterodimers composed of ITGA and ITGB) in SS cells, thereby activating the downstream PI3K/AKT signaling cascade. This interaction may be effectively disrupted by chidamide and LB100. This hypothesis warrants further exploration. In addition to being in SS cells, we found that chidamide combined with LB100 was also able to act in the same mechanism on another CTCL isoform, the MF cell line HH. This suggests that these two drugs are promising to be widely used as treatment options for CTCL, but more experiments are still needed.

In conclusion, these findings highlight the strong preclinical efficacy and synergistic potential of the combined treatment with chidamide and LB100 in SS cells, likely attributable in part to the downregulation of TNC and the consequent dephosphorylation of the PI3K/AKT/mTOR pathway, with no notable side effects detected. The chidamide‐LB100 regimen could represent a promising alternative therapeutic option for SS, even in CTCL, in clinical settings.

Author Contributions

Nan Chen: data curation, validation, writing – original draft. Yuan Wang: resources. Lan Luo: data curation, software. Xin Hua: formal analysis, resources. Qingfeng Xue: data curation, formal analysis. Beiqi Gong: conceptualization, formal analysis. Yaping Zhang: investigation, visualization, writing – review and editing. Wenyu Shi: funding acquisition, methodology, writing – review and editing.

Ethics Statement

The animal protocols were sanctioned by the Institutional Animal Care and Use Committee at Nantong University, in accordance with prevailing guidelines on animal care and welfare.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Chidamide and LB100 synergistically decrease the cell viability of H9 and Hut78 cells. (A, B) CCK8 assay detecting cell viability after 24/48/72 h of LB100 alone treatment; (C, D, F, G) CCK8 assay detecting cell viability after 24 or 72 h of combined drug treatment; (E, H) The Chou‐Talalay methodology calculating the combination index (CI).

Figure S2: (A) Volcano plot from RNA sequencing depicting TNC as one of the downregulated genes by combination treatment in the H9 cell line. (B) Bar plot and (C) bubble plot showing the enrichment analysis of co‐DEGs in H9 cells.

Figure S3: (A) Images of the cadaver after the mice were sacrificed. (B) H&E stained images of heart, liver, spleen, lung, and kidney sections from tumor‐bearing mice, observed under a magnification of 20×. (C) Immunohistochemical images showing the expression of p‐PI3K, p‐AKT, p‐mTOR, TNC, Bcl‐2, and Cleaved caspase‐3 in tumor tissues, observed under a magnification of 10×. (D) Semi‐quantitative analysis of the percentage of positive brown‐stained pixel intensity.

Figure S4: Chidamide and LB100 synergistically decrease cell viability in MF cell(HH). (A) CCK8 assay detecting cell viability after 24/48/72 h of combined or single drug treatment; (B) The Chou‐Talay methodology calculating the ccombination index (CI). (C) Western blotting analysis detecting changes in TNC expression and PI3K/AKT/mTOR pathway‐related proteins.

Figure S5: (A) Schematic of the KEGG pathway showing TNC involvement in the PI3K/AKT/mTOR pathway.

Appendix S1: Methods of transcriptome sequencing, IHC and HE staining.

Table S1: Antibody details.

Table S2: RNAi sequences.

Table S3: IC50 values of H9 and Hut78 cells exposed to chidamide alone or in combination with LB100 for 24 and 72 h.

Table S4: IC50 values of HH cells exposed to chidamide alone or in combination with LB100 for 24/48/72 h.

Table S5: Clinical trials conducted with chidamide or LB100.