Modulation of RNA splicing associated with Wnt signaling pathway using FD-895 and pladienolide B

Materials and Methods

Ethical statement

Informed consent was taken of the healthy donor prior to collection of blood samples from San Diego Blood Bank according to the regulation of the Institutional Review Board and Ethics committee at UC San Diego and maintained strict compliance with the Helsinki Declaration.

Compounds

FD-895 was prepared through total synthesis [32]. Pladienolide B (sc-391691, Santa Cruz Biotechnology, Santa Cruz, CA, USA), etoposide (E1383, Sigma-Aldrich, St Louis, MO, USA) and cisplatin (479306, Sigma-Aldrich, St Louis, MO, USA) were obtained commercially (Figure 1). Oligonucleotides were purchased via custom synthesis (Integrated DNA Technologies).

Figure 1. In vitro cytotoxicity induced by FD-895 and pladienolide B in different cancer cell lines, and normal human primary PBMCs. Cancer Cells were exposed to FD-895 (100 nM to 2 uM), pladienolide B (100 nM to 2 uM), etoposide (1 μM to 30 μM), and cisplatin (1 μM to 30 μM) for 48 h. Apoptosis were measured in MCF-7 (A), MDA-MB-468 (B), HCT-116 (C) and HeLa (D) cells using MTS assay. The absorbance of the control (cells without treatment) was subtracted from the treated cells of each cell line. (E) Normal PBMC cells were exposed to FD-895, and pladienolide B. Cells were stained with propidium iodide and DiOC₆ to differentiate dead and viable cells by using flow cytometer. Data presented in form of % specific induced apoptosis (% SIA). To assess the compound specific induced apoptosis vs. background spontaneous cell death from in vitro culture conditions, we calculated the percentage of SIA using the following formula: % SIA = [(compound induced apoptosis – media only spontaneous apoptosis)/(100- media only spontaneous apoptosis)] × 100. The data shows the results of samples analyzed in duplicate with the mean and its respective SD. (F) Structures of pladienolide-B and FD-895.

Cell culture methods

The MCF-7 (RRID:CVCL_0031), MDA-MB-468 (RRID:CVCL_0419), HS578T (breast cancer, RRID:CVCL_0332), A2780 (RRID:CVCL_0134), SKOV3 (ovarian cancer, RRID:CVCL_0532), 786-O (renal adenocarcinoma, RRID:CVCL_1051), HeLa (Cervical cancer, RRID:CVCL_0030), and HEK-293 cell lines were obtained from ATCC. MCF7 was cultured in DMEM (catalog # 12800017, GIBCO, Grand Island, USA) + 10% fetal bovine serum (FBS, catalog # FB-02, Omega Scientific, Tarzana, CA, USA) +2 mM L-glutamine and 1% Pen/Strep (catalog # 15140148, Invitrogen Corporation, CA, USA) supplemented with 0.01 mg/mL human recombinant insulin. Other cell lines were maintained in DMEM supplemented with 10% FBS, 2 mM L-glutamine, and 1% of Pen/Strep. Additionally, two ovarian cancer cell lines with differential cisplatin sensitivity, OV-2008 (sensitive, RRID:CVCL_0473) and it’s resistant variant C13 were obtained from Prof. Stephen Howell (UC San Diego). To complete the set, a final colon cancer cell line HCT116 (RRID:CVCL_0291) was obtained from the Johns Hopkins School of Medicine, Baltimore, MD. The suspension cell lines including Jeko-1, JVM2, and Mino cell lines were cultured in RPMI-1640 (catalog # R7509, Sigma) supplemented with 10% FBS along with 1% Pen-Strep. All cell lines were incubated at 37°C in an atmosphere of 5% CO₂ and routinely monitored for Mycoplasma infections by PCR analyses.

Flow cytometry analyses

Normal PBMCs were treated with FD-895 (100 nM to 2.0 μM), and pladienolide B (100 nM to 2.0 μM), for 48 h. Cell viability was determined by flow cytometry after staining with conventional live staining with 40 μM 3,3′dihexyloxacarbocyanine iodide (DiOC₆; Life Technologies, Carlsbad, CA, USA) and 15 μM (10 μg/mL) of propidium iodide (PI; Sigma-Aldrich, St Louis, MO, USA). Data were analyzed by using FlowJo software (version 6.4.7; Tree Star). Using this assay, viable cells excluded PI and stained brightly positive for DiOC₆ as it targets metabolically active mitochondria of alive cells [30–33].

Calculation of % specific induced apoptosis (SIA)

To discriminate the compound specific induced apoptosis vs. background spontaneous cell death from in vitro culture conditions, we calculated the percentage of specific induced apoptosis (% SIA) using the following formula: >% SIA = [(compound induced apoptosis – media only spontaneous apoptosis)/(100- media only spontaneous apoptosis)] × 100.

Cell proliferation assays

The cell proliferation assays were conducted in adherent cell lines (HCT116, MCF-7, MDA-MB-468, HS578T, OV-2008, A2780, SKOV3, 786-O, HEK-293, and HeLa) by using CellTiter 96 AQueous non-radioactive colorimetric method (G5421, Promega, Madison, WI, USA). Briefly, a total of 3000 cells/well were seeded in a 96-well flat-well plate followed by treatment with FD-895 (100 nM to 2.0 μM), pladienolide B (100 nM to 2.0 μM), cisplatin (1 μM to 30 μM) or etoposide (1 μM to 30 μM) in triplicate for 48 h at 37°C. Following the incubation, 20 μL of CellTiter 96 AQueous solution (Promega, Madison, WI, USA) was added directly to each well. Non-treated cells were considered as the control. After staining, the plates were incubated for an additional 2 h and then read on a 96-well plate reader (Molecular Devices, Sunnyvale, CA, USA). Absorbance readings were recorded absorbance at 490 nm using empty wells (air) for background collection.

Reverse transcriptase PCR (RT-PCR) analyses

HCT116, MCF-7, MDA-MB-468, HeLa, Jeko-1, JVM-2 and Mino cells (10⁶ cells/well) were treated with 100 nM FD-895, 100 nM pladienolide B, 30 μM cisplatin or 30 μM etoposide for 4 h. RNA isolation was done using mirVana miRNA Isolation Kit (Ambion, Austin, Texas). The 200 ng of RNA was subjected to DNase I (Life Technologies, Carlsbad, CA, USA). The cDNA was prepared by using SuperScript III Reverse Transcriptase Kit (Life Technologies, Carlsbad, CA, USA), and PCR reactions were performed in 20 μL of reaction volume. PCR conditions were 95°C for 3 min; 35 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 45 s; followed by 72°C for 5 min. PCR products were separated on a 2% agarose gel and stained with ethidium bromide. Details of the primers used for RT-PCR are described in Table 1.

Table 1. Sequences of primers used in the RT-PCR.

Primer	Sequence
DNAJB1-FW	5′-GAACCAAAATCACTTTCCCCAAGGAAGG-3′
DNAJB1-RV	5′-AATGAGGTCCCCACGTTTCTCGGGTGT-3′
RNU6A-FW	5′-CGCTTCGGCAGCACATATAC-3′
RNU6A-RV	5′-GAATTTGCGTGTCATCCTT-3′
LEF1-FP	5′-AGGAACATCCCCACACTGAC-3′;
LEF1-RP	5′-AGGTCTTTTTGGCTCCTGCT-3′
CCND1-FP	5′-AATGACCCCGCACGATTTC-3′
CCND1-RP	5′-TCAGGTTCAGGCCTTGCAC-3′
FN1-FP	5′-ACCTACGGATGACTCGTGCTTT-3′
FN1-RP	5′-TTCAGACATTCGTTCCCACTCA-3′
GSK3β-FP	5′-ATCAAGGCACATCCTTGGAC-3′
GSK3β-RP	5′-CAATTGCCTCTGGTGGAGTT-3′
LRP5-FP	5′-GCCTGCAACAAGTGGACA-3′
LRP5-RP	5′-CCTGCAGCACTATGTCTGTGA-3′
FP and RP denote forward and reverse primers respectively.

Quantitative reverse transcriptase-PCR (qRT-PCR) analyses

The HeLa cells were treated with 100 nM FD-895 or 100 nM pladienolide B for 6 h, 12 h, or 24 h, and the RNA isolation and cDNA preparation were done as described above. The amounts of mRNA of LEF1 (Lymphoid enhancer-binding factor-1), FN1 (fibronectin 1), and CCND1 genes were determined using Power SYBR Green PCR master mix (Applied Biosystems, Foster City, CA) real-time qRT-PCR using specific primers [34]. PCR was conducted using 5 picomoles of each primer and 20 ng of the obtained cDNA. PCR conditions were 50°C for 2 min; 95°C for 10 min; 40 cycles of 94°C for 15 s, and 60°C for 1 min. The mRNA levels were calculated using the 2^−ΔΔCT method [35]. GAPDH was used as a control for normalization.

Pathway reporter arrays

Cignal Finder Reporter Array (336821, Qiagen/SABiosciences, Frederick, MD, USA) was used to assess 45 different signaling pathways. HeLa cells were seeded into wells (50,000 cells/well) of the Cignal Finder 96-well plates (CCA-901L, Qiagen, SABiosciences, Frederick, MD, USA) for introducing pathway reporters into cells by reverse transfection according to the manufacturer’s protocol. Briefly, reporter DNA constructs in each plate well were re-suspended with 50 μL Opti-MEM and then mixed with 50 μL diluted Lipofectamine 2000 transfection (Life Technologies, Carlsbad, CA, USA) reagent. Cells were suspended in Opti-MEM (Life Technologies, Carlsbad, CA, USA) supplemented with 10% of FBS and 0.1 mM non-essential amino acids at a density of 6 × 10⁵ cells/mL, and then 50 μL of the cell suspension was added into each plate well and mixed with DNA resident in the plate and added transfection reagent. The cells were incubated for 3 h. Following transfection; the cells were treated with vehicle (Opti-MEM) or 100 nM FD-895 for 3 h in Opti-MEM media. Luciferase and renilla expression were determined (Qiagen/SABiosciences Corp., Frederick, MD, USA).

Western blot analyses

HeLa cells were treated with 100 nM FD-895 or 100 nM pladienolide B for 12 h, 24 h, and 48 h for β-catenin, LEF1, LRP6 (LDL Receptor Related Protein 6), and phospho-LRP6. The cells were then washed with PBS (2 × 5 mL) and lysed with modified RIPA buffer at 4°C. Untreated cells were used as a control. The whole-cell protein was quantified according to the Bradford method [36]. Lysates in sample buffer (2% Sodium dodecyl sulfate (SDS), 10% glycerol, 80 mM Tris•HCl (pH 6.8), 720 mM β-mercaptoethanol and 0.001% bromophenol blue) were denatured at 95°C for 5 min. Total cellular proteins (30 μg via Bradford analyses) were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) using a 4–20% Criterion Precast Gel (Bio-Rad, Hercules, CA), and the proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). After blocking with 5% bovine serum albumin (BSA) for 1 h in Tris-buffered saline, 0.1% Tween 20 (TBST, 20 mM Tris•HCl, 137 mM NaCl, 0.1% Tween-20 pH 7.6), the membrane was incubated with the following primary antibody overnight at 4°C. The primary antibodies include rabbit mAb anti-LEF1, rabbit anti-Phospho-LRP6 (Ser1490), rabbit anti-LRP6, rabbit mAb anti-β-catenin, and mouse Ab anti-β-actin were obtained from Cell Signaling Technology (Beverly, MA) and used at a dilution of 1:1000. After primary mAb staining and washing thrice with TBST, the membranes were incubated with HRP-labeled anti-rabbit (sc-2030, Santa Cruz Biotechnology) or HRP-labeled anti-mouse (sc-2031, Santa Cruz Biotechnology) secondary antibodies with a dilution of 1:5000 dilutions for 40 min at rt. After incubation, the membrane was washed thrice with TBST and developed using an enhanced chemiluminescence (ECL) kit (Pierce Thermo Scientific Inc., Rockford, IL).

Bioinformatics analysis

Gene-gene interaction networks were predicted and generated with GeneMANIA (Gene Multiple Association Network Integration Algorithm) available at http://genemania.org [37].

Statistical analysis

The data presented as mean ± standard deviation (SD). The data was analyzed using GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA). Multiple groups were compared using Bonferroni correction and p < 0.05 was considered statistically significant.

Ethics approval and consent to participate

The study involved human, animal or cell lines as a material for experimental purpose and the ethical clearance was conducted before starting the study.

Consent for publication

All authors consent to the publication of the manuscript in "Aging". Further, figures or tables are original, so there was no requirement of taking permission or consent from anyone.

Availability of data and material

All data generated and analyzed during our study are included in the published article.

Results

In vitro cytotoxicity evaluation of FD-895 and pladienolide B in colon, breast, cervical cancer cell lines

We previously reported the apoptotic activity of FD-895 and pladienolide B in CLL-B, mantle cell lymphoma (MCL), and other B and T lymphoma cell lines [31] Further, we were interested in exploring the apoptotic activity of FD-895 and pladienolide B in different solid tumor cell lines (Figure 1A–1D). The chemical structure of both SPLMs has been shown in Figure 1F. Using an expanded panel of cell lines, we found that FD-895 and pladienolide B IC₅₀ ranged from 30.7 ± 2.2 to 415.0 ± 5.3 nM (Table 2) across the breast, colon, and cervix tumor cell lines (Figure 1A–1D). Upon treatment of normal PBMCs with FD-895 and pladienolide B, the lack of activity (IC₅₀ values <450 nM) was not achieved in normal PBMCs, an observation that suggests that both the FD-895 and pladienolide B spare the normal PBMCs, but not the leukemic B cells (Figure 1E) [30]. We also tested the normal cell line HEK-293 in response to FD-895 (100 nM to 2 μM) and found that there was non-significant cell death induced with varying concentrations of FD-895 when compared with control (p > 0.05), but cisplatin (30 μM) induced significant cell death in HEK-293 cells (p < 0.05, Supplementary Figure 1).

Table 2. IC₅₀ values for FD-895 and pladienolide B in selected tumor cell lines.

Cell lines	Type of Cancer	FD-895 (nM)	Pladienolide B (nM)
HCT116 (TP53, +/+)	Colon	34.1 ± 2.4	42.3 ± 3.1
HCT116 (TP53, +/−)	Colon	75.2 ± 2.5	65.6 ± 2.9
HCT116 (TP53, −/−)	Colon	100.7 ± 1.2	88.6 ± 2.0
MCF-7 (TP53, +/+)	Breast	51.7 ± 1.9	38.7 ± 3.8
MDA-MB-468 (TP53, +/−)	Breast	30.7 ± 2.1	38.5 ± 3.8
HS578T (TP53, −/−)	Breast	139.7 ± 1.1	112.0 ± 3.1
OV-2008	Ovarian	311.6 ± 2.2	344.5 ± 1.3
A2780	Ovarian	415.0 ± 5.2	337.0 ± 7.0
SKOV3	Ovarian	143.4 ± 2.1	128.7 ± 3.2
786-O	Renal	412.5 ± 2.4	293.6 ± 3.1
HeLa	Cervical	131.0 ± 3.3	118.4 ± 4.4

Cytotoxic evaluation of FD-895 and pladienolide B in ovarian cancer cells regardless of differential cisplatin sensitivity

Next, we turned our attention to explore the activity of splice modulation on cell lines displaying sensitivity or resistance to cisplatin, as the latter is an issue in the treatment of solid tumors including ovarian [38], cervical cancer [39], gastric adenocarcinoma [40], prostate cancer [41], colorectal [42], and head and neck squamous cell carcinoma [43]. Here, we used two human ovarian cancer cell lines, one consisting of a cisplatin-sensitive parental line, OV2008, and the other stably cisplatin-resistant subline, OV2008/C13 derived by in vitro selection with cisplatin. We began by screening these cell lines for their induction of apoptosis when treated with FD-895, pladienolide B, cisplatin, or etoposide. FD-895 and pladienolide B induced significant apoptosis in both parental and cisplatin-resistant OV2008 cells (Figure 2A, 2B). We also observed that FD-895 and pladienolide B demonstrated significant apoptosis in A2780 and SKOV3 ovarian cancer cell lines as compared to cisplatin and etoposide (Figure 2C, 2D). These findings suggest that nanomolar concentrations of SPLMs have the potential to overcome cisplatin resistance. We also evaluated the apoptotic activity of FD-895 and pladienolide B in 786-O (renal) cancer cells. We found that both splice modulators were ten-fold more efficient at induced apoptosis than cisplatin and etoposide (Figure 2E).

Figure 2. FD-895 or by pladienolide B induced apoptosis in ovarian and renal cancer cells. Ovarian cancer cells (A) OV-2008, cisplatin sensitive (B) OV-2008 C13 mut, cisplatin resistant, (C) A2780, (D) SKOV3 or (E) renal cancer cells (786-O) were incubated with FD-895 (100 nM to 2 μM), pladienolide B (100 nM to 2 μM), etoposide (1 μM to 30 μM), or of cisplatin (1 μM to 30 μM) for 48 h. Cells viability were measured as using MTS assay. This experiment was repeated in triplicate independently. Data presented in form of % SIA. The data shows the results of samples analyzed in duplicate with the mean and its respective SD.

FD-895 and pladienolide B induced spliceosome modulation marked by intron retention in cancer cells

In previous studies, we found that FD-895 and pladienolide B induced IR in CLL and MCL cells [30–32]. Here, we expand our understanding of their ability to induce IR across an expanded panel of cancer cell lines, including Jeko-1, Mino, JVM2, HeLa, HCT116, MCF-7, and MDA-MB-468 cell lines. In brief, we incubated 10⁶ cells/well from each cell line with a 100 nM FD-895, 100 nM pladienolide B, 30 μM cisplatin or 30 μM etoposide for 4 h. After treatment, the levels of spliced and unspliced gene expression were evaluated by RT-PCR. We observed that cells treated with FD-895 or pladienolide B demonstrated IR, which was not observed with non-splice modulatory controls, cisplatin, or etoposide (Figure 3) using DNAJB1 (DnaJ Heat Shock Protein Family (Hsp40) Member B1) as a surrogate marker for spliceosome modulation [30]. IR was observed in cells treated with FD-895 or pladienolide B for DNAJB1 when compared to the intronless gene RNU6A used as a loading control RNA (Figure 3A–3G).

Figure 3. Intron retention of DNAJB1 gene in different cancer cell lines. Tumor cell lines were treated with 100 nM of FD-895, 100 nM pladienolide B, 30 μM cisplatin or 30 μM etoposide for 4 h. Analysis of IR of DNAJB1 mRNAs was evaluated by RT-PCR in (A–C) Mantle cell lymphoma cells, (Jeko-1, Mino and JMV-2), (D) HeLa, (E) MDA-MB-468 (F) HCT116 and (G) MCF-7 cells. RNU6A, an intronless gene was used as RNA quality and loading control.

FD-895 downregulates and modulates splicing of proteins involved in Wnt signaling

To investigate the intracellular signaling pathways affected by FD-895, we applied the Cignal 45-Pathway Reporter Array to simultaneously analyze FD-895 effect on 45 different signaling pathways [44]. HeLa cells, selected due to their SPLM sensitivity, were treated with FD-895 over 12 h and baseline-signaling profile was compared to vehicle control (Figure 4A). Interestingly, treatment with 100 nM FD-895 modulated a number of pathways including notch signaling, octamer-binding transcription factor 4 (OCT4), activating transcription factor 6 (ATF6), NANOG, and Wnt signaling pathway as early as within 1 h but decreased as the time progressed (Figure 4A).

Figure 4. Effect of FD-895 on different pathways and effect on Wnt signaling in HeLa cells. (A) HeLa cells were use for introducing pathway reporters into cells via reverse transfection. Post-transfection, the cells were treated with vehicle or 100 nM FD-895 for 3 h. Luciferase and renilla expression was evaluated. HeLa cells were exposed to 100 nM of FD-895 or 100 nM pladienolide B for 6 h, 12 h or 24 h and expression of (B) LEF1, (C) FN1, and (D) CCND1 were determined by qRT-PCR. (E) HeLa cells were treated with 100 nM of FD-895 or 30 μM cisplatin for 4 h. Analysis of IR for GSK3β and LRP5 mRNAs was evaluated by using RT-PCR. (F) HeLa cells were treated with 100 nM of FD-895 or 100 nM pladienolide B for 6 h, 12 h or 24 h. Protein extracts were immunoblotted for β-catenin, phohspho-LRP6, LRP6, and β-actin.

Among these pathways, we found that the Wnt signaling pathway was activated at an early time point but downregulated as the duration of FD-895 prolonged, as monitored by the TCF/LEF reporter in cervical cancer cell line HeLa (Figure 4A).

Alternative RNA splicing events have been reported within the Wnt signaling pathway for various molecules such as in LRP6, ROR, Axin, APC, CK1, and Catenin, beta-1 (CTNNB1) [45]. We selected Wnt signaling pathway not only because of this but also because Wnt5A overexpression has been reported in multiple cancers and it is associated with disease progression, metastasis, and resistance to treatment [46]. Additionally, there were several reasons to select LRP5 and GSK-3β for further validation. We know that both lipoprotein receptor-related protein (LRP) 5 and 6 are crucial Wnt co-receptors and interact with other components of the Wnt signaling pathway. Additionally, both LRP5 and LRP6 are oncogenic proteins as well. Similarly, GSK-3β, a serine-threonine kinase and a negative regulator of the oncogenic Wnt/β-catenin signaling pathway [47]. Moreover, the role of Wnt signaling has been well established in CLL [34], and in our previous study, in the RNAseq data analysis, we found that both LRP5 and GSK-3β were found to have 3.44, and 5.1-fold IR, respectively in CLL-B cells treated with splice modulator compared with control CLL [30].

From this data, we selected Wnt signaling pathway to study further because as Wnt5A overexpressed and has been correlated with cervical carcinoma [46]. We began by exploring its effect on the TCF/LEF reporter system. As shown in Figure 4A, we observed that TCF/LEF reporter showed downregulation in 100 nM FD-895 treated HeLa cells after 12 h. We also investigated the effect of FD-895 and pladienolide B on mRNA expression of selected genes LEF1, FN1, and CCND1 [48–50] which involved in the Wnt/β-catenin signaling pathway. The HeLa cells were exposed to 100 nM of FD-895 or pladienolide B for different times and LEF1, CCND1, and FN1 mRNA levels measured by qRT-PCR. The expression of LEF1, CCND1, and FN1 was significantly decreased by 8.33, 6.25, and 5.26 fold respectively (Figure 4B–4D) as the incubation period increased with the maximum level appearing at 20% at 24 h. Further, we performed the RT-PCR to detect splicing in genes involved in Wnt signaling pathway like GSK3β and LRP5. HeLa cells treated with 100 nM FD-895 showed weak IR in GSK3β and LRP5, an observation that was not observed in non-splicing controls (30 μM cisplatin). In comparisons to chemotherapy, the macrolides significantly downregulated the expression of GSK3β in HeLa cell line (Figure 4E). We then conducted Western blot analyses to study the correlation between mRNA and protein. Treatment with 100 nM FD-895 or 100 nM pladienolide B treatment resulted in the downregulation of β-catenin, LEF1, total LRP6, and phospho-LRP6 protein levels (Figure 4F). Altogether, the results from qRT-PCR and Western blot data demonstrate downregulation of key Wnt signaling pathway molecules in the HeLa cell line.

Bioinformatics based gene-gene interaction

Our next studies explored the effects of FD-895 and pladienolide B on select gene-gene interaction networks using GeneMANIA. We used DNAJB1, LEF1, CTNNB1, LRP6, and SF3B1 as “Input” genes for gene-gene interaction analysis and included SF3B1 in this analysis because SF3B1 is a component of the spliceosome-binding pocket of pladienolide B [51, 52]. As shown in Figure 5, we found the selected genes were part of the network directly or indirectly associated with SF3B1.

Figure 5. Gene-gene interaction networks among selected genes constructed by GeneMANIA. A gene-gene network was constructed with the search tool for the retrieval of interacting genes available in GeneMANIA annotation information for selected 5 genes, including physical interaction, genetic interaction, co-expression and shared pathways and protein structure domain. The central black nodes denote 5 selected genes used as an “INPUT”, and peripheral nodes denote gene interactions with the black nodes. The network also contains 20 normal human genes. The size of the circles indicates the degree of interaction.

We obtained total twenty genes from GeneMANIA analysis and among those top ten interactor molecule emerge in the network were based on the size of the circle including NELFE (negative elongation factor complex member E, rank-1), SF3B6 (splicing factor 3b subunit 6, rank-2), Wnt3A (Wnt family member 3A, rank-3), CTNNBIP1 (catenin beta interacting protein 1, rank-4), CDX1 (caudal type homeobox 1, rank-5), DKK1 (dickkopf Wnt signaling pathway inhibitor 1, rank-6), CTNNA1 (catenin alpha 1, rank-7), PIAS4 (protein inhibitor of activated STAT 4, rank-8), DKK4 (dickkopf Wnt signaling pathway inhibitor 4, rank-9), and TCF7L2 (transcription factor 7 like 2, rank-10). Among these genes few genes are involved in Wnt signaling such as Wnt3A, CTNNA1, DKK4, and TCF7L2 suggest that there is an important role of RNA splicing machinery in regulation of Wnt signaling pathway.

Abbreviations

CCND1: Cyclin D1; CTNNB1: Catenin, beta-1; CLL: Chronic lymphocytic leukemia; DLT: Dose limiting toxicity; FBS: Fetal bovine serum; FN1: Fibronectin 1; GeneMANIA: Gene Multiple Association Network Integration Algorithm; GSK3: Glycogen synthase kinase-3; IR: Intron retention; LEF1: Lymphoid enhancer-binding factor-1; LRP6: LDL Receptor Related Protein 6; MCL: Mantle cell lymphoma; MTD: Maximum tolerated dose; PBMCs: Peripheral blood mononuclear cells; RT-PCR: Reverse transcriptase PCR; SF3B1: Splicing Factor 3b Subunit 1; SPLMs: Splicing modulators; SRSF2: Serine And Arginine Rich Splicing Factor 2; U2AF1: U2 Small Nuclear RNA Auxiliary Factor 1.

Author Contributions

DK performed cell culture, cell proliferation, RT-PCR, qRT-PCR with assistance of ZH, and IS. DK performed Western blot and Flow cytometer with assistance of MKK. MKK carried out the bioinformatics analysis. DK, and MKK analyzed and interpreted the data, and wrote the manuscript. DK wrote the first draft of the manuscript and prepared draft of the figures. DK, MKK, JJL, MDB, and JEC edited the manuscript. JJL, and RV, and MDB prepared the samples of FD-895 used within this program. TJK and JEC provided regents, chemotherapeutic agents and cell lines data. JEC, and DK conceived and guided the research. All authors read and approved the final manuscript.

Acknowledgments

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest related to this study.

Funding

This work was supported by grant 285871 from the Lymphoma Research Foundation (LRF) to JEC, and MDB, the American Cancer Society (RSG-06-011-01-CDD) to MDB, the National Institutes of Health (PO1-CA081534)-CLL research consortium grant to JEC. MKK is the recipient of the TARE fellowship (TAR/2018/001054) from the Science and Engineering Research Board (SERB), Department of Science and Technology, Government of India and the study partly supported by the research grants to MKK (sanction #: 5/13/55/2020/NCD-III) from ICMR, New Delhi, India.

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