A High-Throughput Assay to Identify Small-Molecule Modulators of Alternative Pre-mRNA Splicing

Alternative splicing (AS) is an efficient mechanism that involves the generation of transcriptome and protein diversity from a single gene. Defects in pre–messenger RNA (mRNA) splicing are an important cause of numerous diseases, including cancer. AS of pre-mRNA as a target for cancer therapy has not been well studied. We have reported previously that a splicing factor, polypyrimidine tract-binding protein (PTB), is overexpressed in ovarian tumors compared with matched normal controls, and knockdown of PTB expression by short-hairpin RNA impairs ovarian tumor cell growth, colony formation, and invasiveness. Given the complexity of PTB’s molecular functions, a chemical method for controlling PTB activity might provide a therapeutic and experimental tool. However, no commercially available PTB inhibitors have yet been described. To expand our ability to find novel inhibitors, we developed a robust, fluorometric, cell-based high-throughput screening assay in 96-well plates that reports on the splicing activity of PTB. In an attempt to use the cells for large-scale chemical screens to identify PTB modulators, we established cell lines stably expressing the reporter gene. Our results suggest that this high-throughput assay could be used to identify small-molecule modulators of PTB activity. Based on these findings and the role that upregulated PTB has on cell proliferation and malignant properties of tumors, targeting PTB for inhibition with small molecules offers a promising strategy for cancer therapy

Regulatory Effects of Sestrin 3 (SESN3) in BCR-ABL Expressing Cells

<div><p>Chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL) are characterized by the presence of the BCR-ABL oncoprotein, which leads to activation of a plethora of pro-mitogenic and pro-survival pathways, including the mTOR signaling cascade. We provide evidence that in BCR-ABL expressing cells, treatment with tyrosine kinase inhibitors (TKIs) results in upregulation of mRNA levels and protein expression of sestrin3 (SESN3), a unique cellular inhibitor of mTOR complex 1 (mTORC1). Such upregulation appears to be mediated by regulatory effects on mTOR, as catalytic inhibition of the mTOR kinase also induces SESN3. Catalytic mTOR inhibition also results in upregulation of SESN3 expression in cells harboring the TKI-insensitive T315I-BCR-ABL mutant, which is resistant to imatinib mesylate. Overexpression of SESN3 results in inhibitory effects on different Ph+ leukemic cell lines including KT-1-derived leukemic precursors, indicating that SESN3 mediates anti-leukemic responses in Ph+ cells. Altogether, our findings suggest the existence of a novel mechanism for the generation of antileukemic responses in CML cells, involving upregulation of SESN3 expression.</p></div

Induction of SESN3 mRNA expression by ponatinib in T315I-BCR-ABL expressing cells.

<p><b>A</b>. Ba/F3 cells stably transfected with T315I-BCR-ABL were treated with ponatinib (10 nM) or imatinib (1 µM) for 12 hours as indicated. RNA was extracted and expression of SESN3 mRNA was determined by quantitative RT-PCR, using β-actin for normalization. Data are expressed as fold increase in the treated samples over untreated samples and represent means ± S.E. of 3 independent experiments. <b>B</b>. BV173R cells were treated with ponatinib (100 nM) for 12 hours. RNA was extracted and expression of SESN3 mRNA was determined by quantitative RT-PCR, using GAPDH for normalization. Data are expressed as fold increase in the treated samples over untreated samples and represent means ± S.E. of 4 independent experiments.</p

Differential effects of SESN3 and SESN2 overexpression on mTOR and MAPK signaling effectors.

<p><b>A</b>. KT-1 cells were transiently nucleofected with either empty vector (E.V.) or SESN3 expressing plasmid and were analyzed for the presence of ROS by flow cytometry, following 30 minutes of staining with DCFDA at the time-points indicated. Data are as percent control empty vector for each time-point and represent means ± SE of 3 independent experiments. <b>B</b>. KT1 cells were transiently nucleofected with either empty vector (E.V.) or SESN2 expressing plasmid. Cells were lysed 48 hours post-nucleofection Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against SESN2 or GAPDH as indicated. <b>C</b>. KT1 cells were transiently nucleofected with either empty vector (E.V.) or SESN3 expressing plasmid. Cells were lysed 24 hours post-nucleofection Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against SESN3 or GAPDH as indicated. <b>D</b>. KT-1 cells were transiently nucleofected with either empty vector (E.V.) or SESN3 expressing plasmid. Expression of mTOR was quantified at 24 hours post-nucleofection either by quantitative RT-PCR. Data are expressed as fold increase in the Sesn3-nucleofected samples over E.V.-nucleofected samples normalized to GAPDH and represent means ± S.E. of 3 independent experiments. <b>E</b>. KT1 cells were transiently nucleofected with either empty vector (E.V.), SESN2 or SESN3 expressing plasmids. Cells were lysed 48 hours post-nucleofection and equal amounts of protein from cell lysates from the same experiment for each panel were resolved separately by SDS-PAGE and immunoblotted with the indicated antibodies. <b>F–G</b>. KT1 cells were transiently nucleofected with either empty vector (E.V.), SESN2 or SESN3 expressing plasmids. Cells were lysed 48 hours post-nucleofection and equal amounts of protein were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Blots were subsequently stripped and reprobed with the respective antibodies against the total form of the protein. Densitometry analysis of the representative blots is shown.</p

Inhibitory effects of SESN3 but not SESN2 on primitive BCR-ABL expressing leukemic progenitors.

<p><b>A</b>. KT-1 cells were transiently nucleofected with either empty vector (E.V.) or a SESN3 expressing plasmid. Levels of SESN3 were quantified at 24 hours post-nucleofection by immunoblotting. <b>B</b>. KT-1 cells transiently nucleofected with either empty vector (E.V.) or SESN3 were plated in methylcellulose 24 hours post-nucleofection. Leukemic CFU-L colonies were allowed to develop in clonogenic assays in methylcellulose and scored on day 6. Data are expressed as percentage of control untreated colonies and represent means ± S.E. of 5 independent experiments. <b>C</b>. KT-1 cells were transiently nucleofected with either empty vector (E.V.) or SESN2 expressing plasmid. Levels of SESN2 were quantified at 24 hours post-nucleofection by immunoblotting. <b>D</b>. KT-1 cells transiently nucleofected with either empty vector (E.V.) or SESN2 expressing plasmid were incubated in clonogenic assays in methylcellulose. Leukemic CFU-L colonies were scored on day 6 and data are expressed as percentage of control untreated colonies and represent means ± S.E. of 4 independent experiments. <b>E</b>. BV173R cells were transiently nucleofected with either empty vector (E.V.) or SESN2 or SESN3 expressing plasmid. 48 hours post-transfection, equal number of cells were plated and allowed to proliferate for 120 hours. Proliferation was measured by WST-1 assay at the indicated times. Data are expressed as the absorbance at 450 nm and represent means ± S.E. from 3 independent experiments, * p = 0.0018 comparing SESN3 nucleofected cells vs. E.V. nucleofected cells on day 4, ** p = 0.0068 comparing SESN3 nucleofected cells vs. E.V. nucleofected on day 5.</p

mTOR inhibition but not BCR-ABL inhibition upregulates SESN3 in T315I-BCR-ABL expressing cells.

<p><b>A</b>. BV173 or BV173R cells were treated with imatinib mesylate (5 µM) for 12 hours. RNA was extracted and expression of SESN3 mRNA was determined by quantitative RT-PCR, using GAPDH for normalization. Data are expressed as fold increase in the treated samples over untreated samples and represent means ± S.E. of 3 independent experiments. <b>B</b>. BV173 or BV173R cells were treated with either imatinib (5 µM) or nilotinib (100 nM) for 16 hours. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against SESN3 or GAPDH as indicated. <b>C</b>. Ba/F3 cells stably transfected with WT-BCR-ABL or T315I-BCR-ABL were treated with either imatinib (1 µM) or nilotinib (100 nM) for 16 hours. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against SESN3 or GAPDH as indicated. <b>D</b>. BV173 and BV173R cells were treated with OSI-027 (5 µM) for 12 hours. RNA was extracted and expression of SESN3 mRNA was determined by quantitative RT-PCR, using GAPDH for normalization. Data are expressed as fold increase in the treated samples over untreated samples and represent means ± S.E. of 3 independent experiments. <b>E</b>. BV173 or BV173R cells were treated with OSI-027 (5 µM) for 16 hours. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against SESN3 or GAPDH as indicated. <b>F</b>. Ba/F3 cells stably transfected with WT-BCR-ABL or T315I-BCR-ABL were treated with OSI-027 (5 µM) for 16 hours. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against SESN3 or GAPDH as indicated.</p

A High-Throughput Assay to Identify Small-Molecule Modulators of Alternative Pre-mRNA Splicing

Alternative splicing (AS) is an efficient mechanism that involves the generation of transcriptome and protein diversity from a single gene. Defects in pre-mRNA splicing are an important cause of numerous diseases, including cancer. AS of pre-mRNA as a target for cancer therapy has not been well studied. We have reported previously that a splicing factor, polypyrimidine tract-binding protein (PTB) is overexpressed in ovarian tumors, compared to matched normal controls, and knockdown (KD) of PTB expression by shRNA impairs ovarian tumor cell growth, colony formation and invasiveness. Given the complexity of PTB’s molecular functions, a chemical method for controlling PTB activity might provide a therapeutic and experimental tool. However, no commercially available PTB inhibitors have yet been described. To expand our ability to find novel inhibitors, we developed a robust, fluorometric, cell-based high throughput screening HTS assay in 96-well plates that reports on the splicing activity of PTB. In an attempt to use the cells for large-scale chemical screens to identify PTB modulators, we established cell lines stably expressing the reporter gene. Our results suggest that this high throughput assay could be used to identify small molecule modulators of PTB activity. Based on these findings and the role that upregulated PTB has on cell proliferation and malignant properties of tumors targeting PTB for inhibition with small molecules offers a promising strategy for cancer therapy