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Advancing the therapeutic effectiveness of paclitaxel in chronic lymphocytic leukemia through the simultaneous inhibition of NOTCH1 and SF3B1

Cancer Cell International volume 25, Article number: 104 (2025) Cite this article

Abstract

Background

Chemoresistance is still a significant obstacle to cancer therapy. Overexpression of the splicing factor 3b subunit 1 (SF3B1) and neurogenic locus notch homolog protein 1 (NOTCH1) factors is typically found in chronic lymphocytic leukemia (CLL), leading to the development of chemotherapy resistance.

Objective

The current investigation aims to evaluate the chemosensitivity of CLL cells by blocking NOTCH1 and SF3B1 using chitosan lactate (CL) nanoparticles (NPs).

Methods

We used CL-NPs loaded with anti-NOTCH1 and -SF3B1 small interfering RNAs (siRNAs) in combination with paclitaxel (PTX) to suppress NOTCH1 and SF3B1 in peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) isolated from CLL cases to assess the impact of this therapeutic strategy on leukemic cell chemosensitivity. Further, the competing endogenous RNA (ceRNA) network that regulates NOTCH1 and -SF3B1 was constructed and enriched.

Results

Our findings showed that CL-NPs loaded with anti-NOTCH1/-SF3B1 siRNAs-PTX significantly suppressed NOTCH1 and SF3B1 expression in PBMCs and BMMCs isolated from CLL cases in comparison with the untreated samples, leading to increased leukemic cell sensitivity to PTX and decreased the proliferative capacity of leukemic cells. The enrichment analysis highlighted the fundamental pathways where the NOTCH1- and SF3B1-associated ceRNA network exerts its influence in the context of CLL.

Conclusions

This study implies the efficacy of combined therapy by CL-NPs loaded with anti-NOTCH1/-SF3B1 siRNAs and PTX as a novel therapeutic strategy for CLL, even though further studies are required to warrant the findings.

Graphical abstract

Background

Approach to chronic lymphocytic leukemia (CLL) has markedly transformed during the preceding years following the development of novel targeted drugs like ibrutinib and venetoclax, inhibitors of Bruton tyrosine kinase, and anti-apoptotic B-cell leukemia/lymphoma 2 (BCL2), respectively. Although drug resistance continues to be a major obstacle in the treatment of CLL patients [1], developing novel combination therapies capable of overcoming resistance and achieving long-term remissions is an inevitable necessity.

Translational and human investigations demonstrated that some key biological pathways serve a critical role in the pathogenesis of CLL, among which splicing factor 3b subunit 1 (SF3B1) and neurogenic locus notch homolog protein 1 (NOTCH1) are the well-known genes, overexpressing in different cancerous cells [2,3,4], in particular, CLL [5, 6]. SF3B1 is a crucial member of the RNA splicing process that recognizes branch point sequences during the early stages of the splicing process. It is determined that 10% of CLL cases have mutated SF3B1 gene at the diagnosis time [7], which is closely correlated with a poor prognosis and treatment outcome [8, 9]. Most of these alterations are heterozygous missense lesions organized in the heavily conserved C-terminal domain known as the “HEAT repeats” [10]. Studies have revealed that mutations of SF3B1 induce aberrant splicing, which affects various cellular pathways, involving DNA repair, telomere protection, B-cell receptor, and the NOTCH1 signaling pathway [11, 12]. Therefore, the SF3B1 gene is considered another novel and promising therapeutic target for CLL.

NOTCH1, the other most frequently mutated gene in CLL, is found in approximately 5–20% of patients at their diagnosis [7, 13,14,15]. NOTCH1 signaling is implicated in the growth, survival, and apoptosis countering of malignant cells [16]. The majority of CLL-related NOTCH1 mutations happen in the PEST domain, creating a condensed, more stable product that enhances NOTCH1 signaling activation [13]. Alterations in the non-coding 3’ untranslated regions of NOTCH1 were also reported in CLL, resulting in an anomalous splicing process that removes the PEST domain. These mutations are also intrinsically associated with poor prognosis, resistance to treatment, and the disease’s transformation into the Richter syndrome [15, 17]. Moreover, alternative mechanisms of abnormal NOTCH1 signaling activation in CLL cells have been detected, independent of mutations in this gene [16]. There is reliable evidence that inhibiting either the SF3B1 or NOTCH1 molecules can improve the efficacy of combination therapies [18, 19]. Hence, we hypothesize that simultaneous blockade of SF3B1 and NOTCH1 may increase the anti-tumor effects of chemotherapy in leukemic cells.

Paclitaxel (Taxol®, PTX) is known as a most beneficial and effective chemotherapy drug implemented to manage a vast variety of cancers, including leukemia [20,21,22,23,24]. PTX is a microtubule-stabilizing component that disrupts normal microtubule dynamics, leading to mitotic inhibition and, ultimately, inducing apoptosis in malignancies [25]. PTX has also direct effects on mitochondria, inflicting ROS generation, which finally results in the apoptosis of cancer cells [26]. Despite the high potential of PTX, its therapeutic beneficiary is constrained by severe catastrophic effects, inclusively, rapid removal from blood circulation and resistance of cancer cells to PTX [27, 28]. Overexpression of the multidrug resistance gene (MDR-1) is one of the leading resistance mechanisms. This gene is a member of the adenosine triphosphate binding cassette (ABC) gene superfamily and encodes P-glycoprotein (Pgp, p170). There is evidence that PTX is a Pgp substrate and overexpression of Pgp results in PTX resistance [27]. Hence, an essential requirement is to design appropriate drug delivery systems, like NPs, to mitigate the toxic effects of PTX and enhance its delivery to cancer cells.

Chitosan-based nanoparticles (NPs) are effective gene and drug carriers by their distinctive qualities, including non-toxicity, low immunogenicity, good biocompatibility, and biodegradability. Chitosan is a linear polysaccharide obtained from chitin’s deacetylation and is structurally made up of N-acetyl-D-glucosamine and β-(1 → 4)-linked 2-amino-2-deoxy-β-D-glucose (deacetylated D-glucosamine) [29]. Chitosan is only soluble in dilute acids because of its amino groups, and this is why lactic acid is added to enhance chitosan solubility in neutral pH solutions. In previous studies, we have demonstrated that chitosan lactate (CL) NPs are suitable targeted nanocarriers of drugs and small interfering RNA (siRNA) in cancer patients [30, 31]. Thereby, in the current investigation, we evaluated the efficacy of CL-NPs loaded with anti-NOTCH1 and -SF3B1 siRNAs and PTX co-delivery to CLL cells as a novel therapeutic approach. The clinical translation of this therapeutic strategy can be an effective solution for the targeted management of CLL patients.

Materials and methods

Reagents

Paclitaxel was purchased from Selleckchem Company (Houston, TX, USA). Chitosan (MW: 100–150 kDa; deacetylation degree (DD): 95%), sodium tripolyphosphate (TPP), ethanol, and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (Mannheim, USA). RPMI-1640 medium, fetal bovine serum (FBS), and penicillin-streptomycin solution were provided by Gibco (Grand Island Biological Company, NY). Specific human siRNA against NOTCH1, SF3B1, and control siRNA were acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The MTT assay kits were produced by Merck (Germany).

Patient samples

Peripheral blood and bone marrow specimens were acquired from 11 CLL patients, involving 3 relapsed patients and 8 de novo subjects, following written informed consent based on the Declaration of Helsinki and approval from the ethical committee of Tabriz University of Medical Sciences (ethic code: IR.TBZMED.REC.1399.960). CLL patients were diagnosed according to the standards specified by the World Health Organization. The clinical and biological attributes of these patients are listed in Supplementary Table S1 [32].

Cell isolation and culture

Peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) were obtained using Ficoll gradient centrifugation and washed twice in phosphate-buffered saline (PBS). The isolated cells were cultured in RPMI-1640 medium containing 20% FBS and 100 U/mL penicillin-100 µg/mL streptomycin [33,34,35].

Synthesis of nanoparticles

Synthesis of chitosan lactate complex-based NPs was done following our earlier approaches with some alterations [36]. In brief, the copolymer was generated by mixing and stirring chitosan (200 mg) with lactic acid (7 ml) for 25 min. The generated solution was then mixed and stirred with distilled water (15 ml) for 12 h. The resultant complex was finally lyophilized and stored, ready for further implementation. Afterward, the generated mixture was dialyzed in distilled water and centrifugated. In the next step, CL solvent (1 mg/ml in PBS, pH = 7) was shaken/stirred with PTX ethanol solution (2 mg/mL) and TPP (0.5 ml, 0.6 mg/ml) at room temperature for 24 h. Loading of anti-NOTCH1 and -SF3B1 siRNA molecules was done by adding 30 µL of siRNA (equal to 5 µg) to PTX- under stirring (3000 rpm) for 2 h, which resulted in the final form of the NPs, NPs-SF3B1/NOTCH1 siRNA-PTX. The free siRNA was removed from the solution through dialysis by using a 1.0-K molecular weight cutoff membrane.

Evaluation of NPs

Scanning electron microscopy (SEM)

Scanning electron microscopy (SEM) (HITACHI; H9500 model, Japan) was exploited to analyze the morphology of the prepared NPs [37]. Briefly, a single drop of the siRNA-PTX-NPs mixture was put on a lam, allowed to dry in an incubator, and then coated with an argon atmosphere with a thin layer of carbon. Subsequently, samples were analyzed using Anix Emica software.

Drug encapsulation efficiency

The PTX encapsulation efficiency (EE) was examined by employing a UV/Vis spectrophotometer. PTX-loaded NP solutions were centrifuged at 10,000×g for 10 min. The amount of PTX in the supernatant (free or unencapsulated PTX) was analyzed by UV/Vis spectrophotometer at the wavelength of 227 nm against an appropriate blank. The drug loading (DL%) and encapsulation efficiency (EE%) were then computed according to the formulas below [38, 39]:

$$DL\% = \frac{{amount\:of\:drug\:in\:NPs}}{{amount\:of\:drug - loaded\:NPs}} \times 100$$
$$EE\% = \frac{{Wi - Wf}}{{Wi}} \times 100$$

where Wi and Wf are the initial weight of the drug and the drug weight in the supernatant (free or unencapsulated PTX), respectively.

siRNA/PTX release profile assay

As previously reported [40], the siRNA and PTX release profiles of NPs were investigated. siRNA-PTX-NPs were placed on a dialysis membrane in 5 mL PBS at different pHs (5.5 and 7.4). Following that, the dialysis bag was floated in 45 ml PBS as the release medium during shaking. Finally, small amounts of medium (1 ml) were collected at specified periods (0, 2, 4, 8, 16, 24, 36, 48, 72 h) and examined using a UV spectrophotometer at 227 and 490 nm for detecting PTX and siRNA, respectively.

Cellular uptake

Fluorescent microscopy was utilized to study the cellular uptake of the synthesized NPs CLL cells (1 × 105) were grown in 6-well plates at 5% CO2 and 37 °C. Cultivated cells were treated fluorescein isothiocyanate (FITC)-conjugated siRNA-PTX-NPs and incubated overnight at 37 °C. After being washed with PBS, the transfected cells were visualized by a fluorescent microscope (Olympus, Tokyo, Japan).

Cytotoxicity assay

MTT tests were conducted to assess the cytotoxicity of various therapeutic groups. Briefly, 3 × 105 cells were cultured in 96-well microplates and treated with various therapeutic groups for 24 and 48 h. Afterward, 20 µl of MTT reagent (5 mg/ml) was introduced to each well and incubated for 4 h. After centrifuging the cell culture plates and discarding the supernatants, 150 µl of DMSO was added to dissolve the blue crystals formed by viable cells. The absorbance was assessed by an ELISA microplate Reader (BioTek, USA) at a wavelength between 570 and 630 nm.

Gene expression analysis

The mRNA levels related to the target genes of CLL cells were gauged employing quantitative real-time polymerase chain reaction (qRT-PCR). According to the manufacturer’s guidelines, total RNAs were isolated using the AccuZol reagent (Bioneer, South Korea). Next, RNAs were reverse transcribed to complementary DNA (cDNA) with the cDNA Synthesis Kit (Takara Bio Inc., Japan). Subsequently, qRT-PCR was done by exploiting the SYBR Green Master Mix (Takara Bio Inc., Japan) and a Light-Cycler 480 real-time qRT-PCR system (Roche). The 2 –ΔΔCt approach was employed to measure the mRNA expression levels of the targeted genes. β-actin was utilized as the reference gene to normalize the results. In Table 1, the primer sequences are stated [41,42,43].

Table 1 Primer sequences were utilized to evaluate the genes of interest expression
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Protein levels assay

The expression levels of proteins were measured using the QuantiSir™ General Gene Knockdown Quantification Kit. The cells were washed three times (5 min at 1000 rpm), and the cell pellet was suspended in an extraction buffer, vortexed, and incubated on ice for 10 min. Pellet cell debris was centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was transferred to a fresh microtube and kept at -80 °C. The protein extract was diluted with protein capture buffer (1:1). 10 µL of diluted protein extract was added to the central region of each strip well. Next, strip wells were incubated for 90 min at 37 °C without humidity until the solution evaporated and the wells dried. For the blank, 10 µL of protein capture buffer was added. 150 µL of blocking buffer was then added to the wells and incubated at 37 °C for 45 min. After washing, the GAPDH Control Antibody was diluted with antibody buffer at a 1:100 ratio to reach 1 µg/ml concentration. The antibodies specific to the target proteins were diluted with antibody buffer to 1 µg/ml concentration. 50 µl of diluted GAPDH control antibodies and specific antibodies for the target proteins were separately added to wells. Afterward, the mixture was incubated for 60 min at room temperature on an orbital shaker set at 100 rpm. The wells were aspirated and then washed with 150 µl of 1X Wash Buffer four times. The Detection Antibody was diluted with Antibody Buffer at a 1:1000 ratio. Subsequently, a secondary antibody was added to the wells and incubated at room temperature for 30 min. The wells were again aspirated and washed five times with 150 µl of diluted 1X Wash Buffer. Next, 100 µl of developing solution was added to the wells and incubated at room temperature for 10 min away from light. Eventually, 50 µl of stop solution was added to the wells, and the absorbance was determined on a microplate reader at 450 nm. The percentage of the target protein level was computed by the subsequent formula:

$$\displaylines{\Pr otein\,\% = \cr \frac{{{{O{D_T}\left( {treated\,sample - blank} \right)} \mathord{\left/{\vphantom {{O{D_T}\left( {treated\,sample - blank} \right)} {O{D_C}\left( {untrated\,control - blank} \right)}}} \right.\kern-\nulldelimiterspace} {O{D_C}\left( {untrated\,control - blank} \right)}}}}{{{{O{D_T}\left( {untreated\,control - blank} \right)} \mathord{\left/{\vphantom {{O{D_T}\left( {untreated\,control - blank} \right)} {O{D_C}\left( {treated\,sample - blank} \right)}}} \right.\kern-\nulldelimiterspace} {O{D_C}\left( {treated\,sample - blank} \right)}}}} \cr \times 100\,\% \cr} $$

BrdU assay

To examine the inhibitory effect of siRNA-PTX-NPs on CLL cells’ growth, a BrdU assay was done. Briefly, CLL cells (2 × 104) were grown in 96-well plates for 24 h. The cells were then treated with several therapeutic agents. After 24 h, 20 µl of BrdU solution was added to each well, and the cells were incubated at 37 °C for another 24 h. The cells were then centrifuged at 300 g for 10 min, and the supernatant was thrown away. Following that, 200 µl of FixDenat solution was applied to each well of the plate and incubated for 30 min at 20 °C. After that, the FixDenat solution was taken away, 100 µl of anti-BrdU antibody conjugated to peroxidase (anti-BrdU-POD) was added, and the cells were incubated for 90 min at 20 °C. After washing, the wells were incubated with tetramethylbenzidine for 1 h. Finally, the absorbance was established by exploiting an ELISA reader at 630 nm.

Cell death detection by ELISA

Cell apoptosis was measured using the Cell Death detection ELISA kit (Sigma-Aldrich, Mannheim, USA) based on the manufacturer’s protocol. Briefly, cells (1 × 104) were seeded in a 12-well plate and incubated overnight in a 5% CO2 moistened incubator at 37 °C. Following several therapeutic components implemented to stimulate and incubate the cells for 24 h, lysing buffer added to the cells for 30 min at 25 °C and centrifuged at 200 × g for 10 min. Afterward, in order to assess the quantity of cytoplasmic histone-associated-DNA-fragments, 20 µl of the supernatant was evaluated. Lastly, absorbance level was measured using an ELISA reader at 405 nm wavelength.

Reactive oxygen species detection by ELISA

The ROS generation was measured using a Human Reactive Oxygen Species ELISA kit (My BioSource, CA, USA). The cell suspension was diluted with PBS to a cell concentration of about a million/ml while exploring the cell composition. Several freeze-thaw cycles were utilized to repeatedly harm cells to release the internal components. The resulting suspension was then centrifuged for around 20 min at 2000 RPM and collected supernatant. Standard well-received 50 µl of standard. After adding 40 µl of sample-to-sample wells, 10 µl of anti-ROS antibody was also added to each well. After that, 50 µl of streptavidin-HRP was added to both the standard and sample wells (except the blank control wells). Subsequently, the wells were mixed, and the plates were sealed. They were then incubated for 60 min at 37 °C. Then, the plates were washed five times with a buffer after the sealer was eliminated. Additionally, during each cycle, wells were soaked with a minimum of 0.35 ml wash buffer for 30 s. All wells were aspirated and washed five times with wash buffer for automated washing while overfilling wells. The plates were then blotted dry using paper towels or any other absorbent agents. Lately, 50 µl of substrate solution A and 50 µl of substrate solution B were added to each well, in that order. Afterward, the plates were incubated at 37 °C in the dark for 10 min after being sealed with a fresh sealer. Subsequently, each well was allocated 50 µl of Stop Solution, which rapidly transformed the blue color into yellow. Within 10 min after adding the stop solution, each well’s OD value was evaluated with a microplate reader, which was set to 450 nm.

NOTCH1 and SF3B1 regulatory network

The miRNAs targeting NOTCH1 and SF3B1 were predicted based on the miRDB database (https://mirdb.org/) [44]. Also, lncRNAs sponging selected miRNAs in bone marrow were retrieved using miRNet (https://www.mirnet.ca/) [45]. Finally, a competitive endogenous RNA (ceRNA) network, including miRNAs targeting both NOTCH1 and SF3B1 genes and lncRNAs sponging selected miRNAs, was retrieved. Furthermore, the ceRNA network was enriched utilizing the NcPath database (http://ncpath.pianlab.cn/#/Home) [46].

Statistical analysis

All the tests were taken in triplicate and repeated two times, and mean ± standard error (SEM) was used to demonstrate the results on all graphs. Statistical analysis was performed using GraphPad Prism version 8.4.3). Normality of data distribution was assessed using the Shapiro-Wilk test. For comparisons among more than three groups, one-way ANOVA followed by Tukey’s post hoc test was applied for normally distributed data, while the Kruskal-Wallis test followed by Dunn’s post hoc test was used for non-normally distributed data. Pairwise comparisons between two groups were performed using Student’s t-test (for normally distributed data) or the Mann-Whitney U test (for non-normally distributed data). The Spearman’s rho method was used to evaluate the potential correlation between variables. A p-value of < 0.05 was considered statistically significant. The following symbols were applied to indicate statistically significant findings: * P < 0.05, ** P < 0.01, and *** P < 0.001.

Results

Characteristics of synthesized siRNA-PTX-CL-NPs

The siRNA-PTX -NPs had an average size of 161 nm, a zeta potential of about 17 mV, and a PDI of 0.37, implying the appropriate physicochemical properties of these NPs for effectively delivering siRNA and PTX to CLL targets. Moreover, the encapsulation efficiency of anti-NOTCH1 and -SF3B1 siRNA in CL-NPs was 86% using UV/vis spectrophotometry at 480 nm. Also, the encapsulating efficiency and loading capacity of PTX were 78.7% and 8.6%, respectively.

Moreover, SEM analysis confirmed that the siRNA-PTX-NPs had a spherical morphology and a uniform distribution (Fig. 1a). These NPs also showed no signs of instability and aggregation.

Fig. 1
figure 1

Characteristics of siRNA-PTX-NPs. The morphology of the synthesized NPs was analyzed via SEM (a). In vitro release profiles of PTX and siRNA from NPs in neutral (PH = 7.2) and acidic (PH = 5.5) conditions were shown using a UV spectrophotometer (b). Cellular uptake of FITC-conjugated siRNA-PTX-NPs was assessed using light microscope and fluorescent microscope (c). (CL: Chitosan lactate, NP: Nanoparticle, PTX: Paclitaxel, SEM: scanning electron microscope)

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The synthesized NPs were also assessed regarding how they released PTX/siRNA in PBS with two different pHs, including 7.2 and 5.5, which mimic normal and tumor conditions, respectively. According to Fig. 1b, acidic conditions enhanced the release of both siRNA and PTX.

siRNA-PTX-NPs effectively transfected leukemic targets and downregulated the expression of SF3B1 and NOTCH1

The in vitro intracellular uptake of NPs was studied using FITC-conjugated siRNA-PTX-NPs. As shown in Fig. 1c, CLL cells treated with FITC-conjugated siRNA-PTX-NPs displayed a strong green fluorescence around the cell nucleus, implying the prominent role of CL-NPs in transferring siRNA into the leukemia cells.

Then, to investigate the efficacy of different therapeutic agents in suppressing target gene expression, the mRNA and protein levels of NOTCH1 and SF3B1 were assessed using qRT-PCR and colorimetric analysis, respectively. qRT-PCR analysis suggested that the expression capacity of NOTCH1 and SF3B1 genes in both PBMCs and BMMCs significantly declined after treatment with anti-NOTCH1 and -SF3B1 siRNAs-containing NPs (Fig. 2a, b). Similarly, the colorimetric analysis demonstrated that transfection of both PBMCs and BMMCs with anti-NOTCH1 and -SF3B1 siRNAs-containing NPs decreased the level of NOTCH1 and SF3B1 proteins (Fig. 2c-d).

Fig. 2
figure 2

siRNA-PTX-NPs suppressed NOTCH1 and SF3B1 at both the mRNA and protein levels. NOTCH1 and SF3B1 mRNA and protein expression levels were measured using qRT-PCR (a, b) and colorimetric analysis after treatment with various therapeutic agents (c-d). The data were normalized using β-actin mRNA expression levels as an internal control. The 2 –ΔΔCt technique was exploited to compute the relative mRNA expression level of the targeted genes. P-values < 0.05 (*), P-values < 0.01 (**), and P-values < 0.001 (***)

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Dual blocking of SF3B1 and NOTCH1 significantly enhanced the sensitivity of CLL cells to PTX and induced apoptosis

The IC50 of free PTX and CL-PTX was measured by treating CLL cells for 24 h with an increasing concentration of PTX. MTT results revealed that PTX delivery by CL-NPs significantly enhances the drug’s cytotoxicity and reduces its IC50 value in comparison with free PTX. As demonstrated in Fig. 3a, b, the IC50 of PTX was reduced from 7.5 to 2.60 µM after delivery with CL-NPs. Therefore, we used PTX in 2.60 µM in both free form and loaded in NPs in all tests.

Fig. 3
figure 3

Cell viability analysis using MTT. IC50 of free PTX (a). IC50 of siRNA-PTX-NPs (b). Cell viability of PBMCs and BMMCs after 24 and 48 h of treatment with different therapeutic agents (c, d). P-values < 0.05 (*), P-values < 0.01 (**), and P-values < 0.001 (***)

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Furthermore, we examined the cytotoxic potential of therapeutic agents on primary CLL cells using the MTT assay. According to Fig. 3c, d, siRNA-PTX-NPs were the most potent agents in reducing the viability of both PBMCs and BMMCs after 24–48 h of incubation. Nonetheless, both PTX-NPs and siRNA-NPs were also capable of suppressing cell viability after 24–48 h of incubation.

Notably, the ELISA-based apoptosis assay was implemented to measure the apoptotic effects of various therapeutic agents on CLL cells (Fig. 4a). Our findings disclosed that siRNA-PTX-NPs were the most potent agents in inducing apoptosis in both PBMCs and BMMCs, which confirmed the MTT assay findings of the current study. Strikingly, both anti-NOTCH1-siRNA-NPs and anti-SF3B1-siRNA-NPs were also capable of apoptosis induction in CLL cells. Furthermore, we observed that NP-PTX were able to induce a higher apoptosis level than free PTX. Conclusively, the exerted combination therapy revealed the highest levels of cell death compared to other therapeutic agents.

Fig. 4
figure 4

Silencing of SF3B1 and NOTCH1 significantly enhances the sensitivity of CLL cells to PTX-induced apoptosis. ELISA method was used to determine the cell death after treatment of CLL cells with different therapeutic agents (a). The qRT-PCR technique measured the mRNA expression levels of Bcl-2 and Bax after treatment of both PBMCs and BMMCs with different therapeutic agents (b, c). P-values < 0.05 (*), P-values < 0.01 (**), and P-values < 0.001 (***)

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Furthermore, to evaluate the pro-apoptotic and anti-apoptotic balance of CLL cells, the expression level of Bcl-2, as an anti-apoptotic factor, and Bax, as a pro-apoptotic factor, were measured. In accordance with Fig. 4b, c, the combination therapy of both PBMCs and BMMCs drastically increased Bax and decreased Bcl-2 expression, which is generally in favor of enhanced apoptosis.

Overall, all MTT, RT-qRT-PCR, and ELISA studies demonstrated that both PTX delivery by CL-NPs and simultaneous blocking of NOTCH1 and SF3B1 significantly improve anticancer effects of PTX and increase CLL cell sensitivity to PTX-induced apoptosis.

Combination therapy suppressed the CpG-mediated proliferation and enhanced ROS generation in leukemic cells

The BrdU was exploited to answer the question of how synthesized NPs inhibited the multiplication of leukemic cells. According to Fig. 5a, the studies revealed that treatment of CLL cells with anti-NOTCH1-siRNA-NPs and anti-SF3B1-siRNA-NPs alone had a slight effect on leukemic cell proliferation compared to therapy with free PTX. Furthermore, compared to free PTX treatment, cells treated with PTX-NPs demonstrated greater potential for suppressing proliferation. The highest suppressive effect on cell proliferation was discovered in cells treated with siRNA-PTX-NPs. It should be noted that B cells were pre-treated with CpG to proliferate.

Fig. 5
figure 5

Combination therapy suppressed the proliferation and enhanced ROS generation in leukemic cells. BrdU technique was used to evaluate the suppressive impact of different therapeutic agents on the proliferation of CLL cells (a). ELISA assay was employed to levels of ROS generation in both PBMCs and BMMCs after treatment with various therapeutic groups (b). The correlation between ROS and caspase 9 was evaluated in CLL cells (c)

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Since ROS production is one of the underlying mechanisms of apoptosis induction by PTX, we also assessed the effect of various therapies on ROS generation in both PBMCs and BMMCs. According to the ELISA analysis, inhibition of NOTCH1 and SF3B1 increased the generation of intracellular ROS. Additionally, the treatment with PTX-NPs induced higher levels of ROS synthesis compared to the treatment with free PTX. The greatest intensity of ROS production was seen in cells treated with siRNA-PTX-NPs (Fig. 5b).

Moreover, we investigated the correlation between ROS production and Apoptosis in PBMCs. As shown in Fig. 5c, there was a strong correlation (r = 0.78) between apoptosis and ROS production. However, the overall findings support the hypothesis that ROS is a major inducer of programmed cell death in CLL cells.

The possible CeRNA network that significantly modulates NOTCH1 and SF3B1 expression

Certain ceRNAs that modulate NOTCH1 and SF3B1 expression significantly could be targeted alongside NOTCH1 and SF3B1 to enhance the chemosensitivity of CLL cells. Hence, we constructed the ceRNA network that modulates the genes of interest. A total of 101 and 184 miRNAs were identified targeting NOTCH1 and SF3B1, respectively. Of them, 9 miRNAs were identified targeting both NOTCH1 and SF3B1 (Supplementary File 1). Also, 46 lncRNAs were identified, which sponge 5 miRNAs. No lncRNA was found in sponge hsa-miR-548n, hsa-miR-548c-3p, hsa-miR-12,136, and hsa-miR-340-5p (Supplementary File 1). Finally, a ceRNA network was constructed containing NOTCH1 and SF3B1, 9 miRNAs targeting both of them, and 46 lncRNAs sponging selected miRNAs (Fig. 6a). Among the 83 significantly enriched pathways identified, the MAPK, AMPK, FoxO, Rap1, mTOR, Ras, and PI3K-Akt signaling axis, as well as pathways correlated with Chronic myeloid leukemia, apoptosis, and Acute myeloid leukemia, were highlighted as the most relevant to CLL. Notably, cellular senescence emerged as the most significant pathway (q-value = 8.9 e-15, Fig. 6b).

Fig. 6
figure 6

The competing endogenous RNA (ceRNA) network that modulates NOTCH1 and SF3B1 genes. The ceRNA network, including NOTCH1 and SF3B1, 9 miRNAs targeting both genes and 46 lncRNAs sponging selected miRNAs were constructed (a). The enrichment analysis underscored the pivotal pathways where the ceRNA network, associated with NOTCH1 and SF3B1, exerts its influence in the context of chronic lymphocytic leukemia (b)

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Discussion

Genetic studies have proved genes that affect critical biological pathways, such as NOTCH1 and SF3B1. Alterations to these genes and their signaling pathways may result in the initiation and progression of CLL [47], thus making them attractive targets for this disease. It has been shown that NOTCH1 and SF3B1 are mostly dysregulated in CLL cells resulting in the mechanisms responsible for chemoresistance [8, 15]. Accordingly, in vitro, investigations have revealed that NOTCH1 signaling enhances CLL cell survival, which leads to treatment resistance [48]. In addition, Lopez-Guerra et al. reported that prohibiting NOTCH1 with the G-secretase inhibitor (GSI) revokes resistance to drug-induced apoptosis [18]. These results demonstrate that targeting NOTCH1 alone or commitment with other drugs for the therapeutic aims of CLL is fruitful, and can contribute to synergistic effects and attenuated chemoresistance. Additionally, SF3B1, a prominent component of the RNA splicing pathway, is mostly mutated or overexpressed in different forms of cancers, such as CLL [12, 49, 50]. Upregulation in SF3B1 is correlated with chemoresistance and prolonged overall survival in CLL [8, 9]. Studies have shown that targeting SF3B1 has anti-tumor impacts on CLL cells irrespective of mutation condition [19, 51, 52].

Several pharmacological agents have been developed to block SF3B1 and NOTCH1, separately. According to DGIdb (https://www.dgidb.org/) [53], 11 drugs were found having interaction with NOTCH1 and SF3B1 (Supplementary Fig. 1). While BRONTICTUZUMAB and H3B-8800 had the most interaction scores with NOTCH1 and SF3B1, blocking both NOTCH1 and SF3B1 could markedly improve the chemosensitivity of CLL cells. Therefore, CL-NPs were used to dual blocking of SF3B1 and NOTCH1.

Chitosan-based NPs are widely used to carry genes and drugs both inside and outside of living organisms [54]. Here, we used our previously developed CL nanocarriers to co-deliver anti-NOTCH1 and -SF3B1 siRNAs along with PTX to CLL cells. We chose CL instead of unmodified chitosan due to its water-solubility property, which allows drugs to be incorporated into particles [55].

As part of this study, we evaluated the effects of PTX delivery by NPs on drug concentrations and the cytotoxicity against CLL. Our results illustrated that the PTX transfer by CL-NPs can lower the drug’s effective dose and increase its cytotoxicity compared to free PTX. Interestingly, the results also revealed that co-suppression of NOTCH1 and SF3B1 may increase the vulnerability of CLL targets to PTX-induced apoptosis. Accordingly, Soni and colleagues revealed that PTX and Thymoquinone-loaded polymer PLGA NPs synergically improve the oncolytic activity of PTX in MCF-7 breast cancer and decrease the adverse effects of this agent by reducing the effective dosage [56]. Another study also indicated that PEGylated-PTX-Dihydroartemisinin (DHA) nanocarriers minimized systemic side effects of high PTX doses by reducing effective doses while improving their anticancer activity [57].

We also evaluated the impact of simultaneous suppression of SF3B1 and NOTCH1 in combination with PTX using specific NPs on the apoptosis of CLL cells. Our results showed that combination therapy can elevate pro-apoptotic Bax mRNA expression and decrease anti-apoptotic Bcl-2 mRNA expression, inhibiting the survival and growth of malignant cells. Moreover, before/after the combination therapy, the levels of NOTCH1 and SF3B1 mRNAs and proteins were measured using qRT-PCR and colorimetric analysis, respectively. We confirmed that NOTCH1 and SF3B1 are expressed in CLL cells. However, treatment with siRNA-PTX-NPs reduced NOTCH1 and SF3B1 mRNA and protein levels in CLL cells.

Multiple studies have tried to target NOTCH1 or SF3B1 in various malignancies. Kashyap et al. demonstrated that targeting SF3B1 by FD895 and Pladienolide B induces apoptosis in primary leukemic cells found in CLL cases and lymphoma cell lineage irrespective of the SF3B1 mutation status [51]. According to the report by Larrayoz et al., spliceostatin A stimulates apoptosis in primary CLL cells by downregulating anti-apoptotic myeloid cell leukemia-1 (Mcl-1), independent of SF3B1 mutation [19]. On the other hand, PF 03084014, an oral GSI, preferentially promoted apoptosis in T-ALL and CLL cells, especially in cells with NOTCH1 mutations [18, 58]. Moreover, the combined therapy of PF-03084014 and fludarabine had synergistic oncolytic implications on CLL targets with NOTCH1 mutations. Besides, It upregulated the expression of the apoptosis-related gene Harakiri (HRK) while downregulating the expression of the invasion and chemotaxis genes including matrix metalloproteinase-9 (MMP 9) and Interleukin 32 (IL32) [18]. Moreover, OMP-52M51, a mAb targeting NOTCH1, exhibited desirable oncolytic activity in T-ALL animal models, CLL, and mantle cell lymphoma [59,60,61]. Notably, OMP-52M51 and dexamethasone combined therapy markedly improved the anti-NOTCH1 treatment effects [60].

The research into the endogenous RNA network has uncovered significant correlations between multiple microRNAs and key genes. Specifically, there is a strong link between NOTCH1 and SF3B1 expressions and a group of microRNAs, including miR-340-5p, hsa-mir-30a-5p, hsa-mir-30b-5p, hsa-mir-30c-5p, hsa-mir-30d-5p, and hsa-mir-30e-5p. Additionally, the microRNA-30 family, particularly noted for its potential as a suppressive agent, has been observed to have decreased expression among individuals with T-ALL. This finding suggests a possible role for miRNA-30 in the disease’s development and progression [62]. There is a complex, bidirectional relationship among the MYC proto-oncogene, miR-30, and the NOTCH1 and NOTCH2 genes, key players in cell division and growth regulation. Significantly, MYC, known for its role in promoting cell division, suppresses the expression of miR-30. In turn, miR-30 specifically targets and inhibits the activities of the NOTCH1 and NOTCH2 genes. Moreover, there is a notable positive feedback loop involving NOTCH1, which actively supports the transcription of MYC [63]. Furthermore, studies have shown a crucial link in pediatric T-ALL patients: low levels of miR-30a-5p in mononuclear cells from their bone marrow samples correlate with increased levels of serine/threonine kinase 39 (STK39). STK39 was proposed as a critical player in the development of leukemia [64]. Further research has established miR-340-5p as a tumor suppressor in several cancers, such as myeloma. In the process of myelomagenesis, an early and critical event is the silencing of miR-340-5p through methylation. This silencing adversely affects patient survival. miR-340-5p exerts its antitumor effects by targeting multiple oncogenes within the MAPK signaling pathway and influencing apoptosis. These findings underscore the significance of miR-340-5p as a suppressive factor in myeloma [65]. Therefore, our study demonstrates that by silencing long non-coding RNAs (lncRNAs), we can induce the activity of miR-340-5p and the miRNA-30 family. This induction subsequently leads to a decrease in the expressions of NOTCH1 and SF3B1, highlighting a potential therapeutic mechanism in cancer treatment.

While patient-derived xenograft (PDX) models are a valuable tool for studying human cancers, they associate with several limitations that make them less suitable for our study. CLL is a highly microenvironment-dependent disease, and PDX models do not fully replicate the complex interactions between leukemic cells and the human immune system or stromal components. Additionally, CLL cells often exhibit poor engraftment and slow growth in PDX models, which could lead to inconsistent results. Our ex vivo approach using patient-derived PBMCs and BMMCs allowed us to directly investigate the effects of this therapeutic strategy on human CLL cells in a controlled and reproducible manner; however, future studies could study the use of PDX models or other in vivo systems to further validate our findings.

While this study demonstrates plausible evidence for the efficacy of CL-NPs encapsulating anti-NOTCH1/-SF3B1 siRNAs in enhancing chemosensitivity, it is critical to admit its limitations. CLL is a uniquely human disease, and the lack of appropriate animal models creates a critical problem for translational research. Existing models do not comprehensively replicate the complexity of CLL, particularly the interactions between leukemic cells and the bone marrow microenvironment. Therefore, our ex vivo findings, while informative, need further validation in more physiologically relevant systems. Additionally, the inclusion of both peripheral blood and bone marrow samples in this study is a significant strength, as it allows for a more comprehensive study of the therapeutic modality across different disease compartments. The bone marrow microenvironment is known to play a critical role in CLL progression and chemoresistance, and these findings show that targeting NOTCH1 and SF3B1 in both PBMCs and BMMCs can markedly enhance chemosensitivity. Future studies should investigate the potential of this therapeutic strategy in more complex models and larger patient cohorts to further validate its clinical applicability.

In conclusion, our study confirmed that CL-based NPs are good carriers for the delivery of a combination therapy consisting of anti-NOTCH1 and -SF3B1 siRNAs and PTX. The synthesized chitosan lactate-based NPs enhanced their cell entry efficacy. Furthermore, these carriers had higher encapsulation efficiency and release profile, which makes them a promising therapeutic strategy. Moreover, this combination of multiple agents improved CLL cell cytotoxicity potential via enhancing ROS generation and cell sensitivity to PTX. Future studies are required to further analyze the potential of anti-NOTCH1 and -SF3B1 siRNAs and PTX for combination therapy.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

Not Applicable.

Funding

The research team is deeply grateful to Tabriz University of Medical Sciences for financial support of this study (grant numbers: 66209, 68175, and 75378).

Author information

Authors and Affiliations

  1. Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

    Shiva Abolhasani, Armin Mahmoud salehi Khesht, Bentolhoda Rashidi, Fatemeh Karimian Noukabadi, Vahid Karpisheh, Khatereh Torabi Poudeh, Seyyed Sina Hejazian & Farhad Jadidi

  2. Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran

    Atefeh Khodakarami & Rafieh Bagherifar

  3. Institute of Experimental Hematology, School of Medicine, Technical University of Munich, 81675, Munich, Germany

    Ali Masjedi

  4. Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, 81675, Munich, Germany

    Ali Masjedi

  5. Research Center for Integrative Medicine in Aging, Aging Research Institute, Tabriz University of Medical Sciences, Tabriz, Iran

    Sepideh Izadi

  6. Basic and Molecular Epidemiology of Gastrointestinal Disorders Research Centre, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Pooya Jalali

  7. Hematology, Oncology and Stem Cell Transplantation Research Center, Research Institute for Oncology, Hematology and Cell Therapy, Tehran University of Medical Sciences, Tehran, Iran

    Zahra Salehi

  8. Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

    AliAkbar Movassaghpour

  9. Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran

    Abbas Ali Hosseinpour Feizi & Farhad Jadidi

Authors
  1. Shiva Abolhasani
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  2. Armin Mahmoud salehi Khesht
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  3. Atefeh Khodakarami
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  4. Ali Masjedi
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  5. Bentolhoda Rashidi
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  6. Sepideh Izadi
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  7. Fatemeh Karimian Noukabadi
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  8. Vahid Karpisheh
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  9. Khatereh Torabi Poudeh
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  10. Pooya Jalali
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  11. Zahra Salehi
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  12. Rafieh Bagherifar
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  13. Seyyed Sina Hejazian
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  14. AliAkbar Movassaghpour
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  15. Abbas Ali Hosseinpour Feizi
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  16. Farhad Jadidi
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Contributions

SA the acquisition, analysis and has drafted the work or substantively revised it; AMK substantively revised; AK the acquisition; AM analysis; BR substantively revised; SI substantively revised; FKN substantively revised; VK interpretation of data; KTB substantively revised; PJ substantively revised; ZS has drafted the work; RB substantively revised; SSH analysis; AAM has made substantial contributions to the conception; AAHF has made substantial contributions to the conception; FJN has made substantial contributions to the conception, design of the work and substantively revised it, and has approved the submitted version. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Farhad Jadidi.

Ethics declarations

Ethics approval and consent to participate

Peripheral blood and bone marrow specimens were acquired from 11 untreated CLL cases following written informed consent based on the Declaration of Helsinki and approval from the ethical committee of Tabriz University of Medical Sciences (ethic code: IR.TBZMED.REC.1399.960).

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Not Applicable.

Competing interests

The authors declare no competing interests.

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Abolhasani, S., salehi Khesht, A.M., Khodakarami, A. et al. Advancing the therapeutic effectiveness of paclitaxel in chronic lymphocytic leukemia through the simultaneous inhibition of NOTCH1 and SF3B1. Cancer Cell Int 25, 104 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12935-025-03702-4

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12935-025-03702-4

Keywords

Cancer Cell International

ISSN: 1475-2867

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