The Experts below are selected from a list of 14293584 Experts worldwide ranked by ideXlab platform
Betty Chan - One of the best experts on this subject based on the ideXlab platform.
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h3b 8800 an orally available small molecule splicing modulator induces lethality in spliceosome mutant cancers
Nature Medicine, 2018Co-Authors: Michael Seiler, Akihide Yoshimi, Rachel Darman, Betty Chan, Gregg F Keaney, Michael Thomas, Anant A Agrawal, Benjamin Caleb, Alfredo Csibi, Eckley SeanAbstract:Genomic analyses of cancer have identified recurrent point mutations in the RNA splicing factor-encoding genes SF3B1, U2AF1, and SRSF2 that confer an alteration of function. Cancer cells bearing these mutations are preferentially dependent on wild-type (WT) spliceosome function, but clinically relevant means to therapeutically target the spliceosome do not currently exist. Here we describe an orally available modulator of the SF3b complex, H3B-8800, which potently and preferentially kills spliceosome-mutant epithelial and hematologic tumor cells. These killing effects of H3B-8800 are due to its direct interaction with the SF3b complex, as evidenced by loss of H3B-8800 activity in drug-resistant cells bearing mutations in genes encoding SF3b components. Although H3B-8800 modulates WT and mutant spliceosome activity, the preferential killing of spliceosome-mutant cells is due to retention of short, GC-rich introns, which are enriched for genes encoding spliceosome components. These data demonstrate the therapeutic potential of splicing modulation in spliceosome-mutant cancers.
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the cryo em structure of the sf3b spliceosome complex bound to a splicing modulator reveals a pre mrna substrate competitive mechanism of action
Genes & Development, 2018Co-Authors: Lorenzo I Finci, Betty Chan, Anant A Agrawal, Xiaofeng Zhang, Xiuliang Huang, Qiang Zhou, Jennifer J Tsai, Teng Teng, Sean Irwin, Craig KarrAbstract:Somatic mutations in spliceosome proteins lead to dysregulated RNA splicing and are observed in a variety of cancers. These genetic aberrations may offer a potential intervention point for targeted therapeutics. SF3B1, part of the U2 small nuclear RNP (snRNP), is targeted by splicing modulators, including E7107, the first to enter clinical trials, and, more recently, H3B-8800. Modulating splicing represents a first-in-class opportunity in drug discovery, and elucidating the structural basis for the mode of action opens up new possibilities for structure-based drug design. Here, we present the cryogenic electron microscopy (cryo-EM) structure of the SF3b subcomplex (SF3B1, SF3B3, PHF5A, and SF3B5) bound to E7107 at 3.95 A. This structure shows that E7107 binds in the branch point adenosine-binding pocket, forming close contacts with key residues that confer resistance upon mutation: SF3B1R1074H and PHF5AY36C The structure suggests a model in which splicing modulators interfere with branch point adenosine recognition and supports a substrate competitive mechanism of action (MOA). Using several related chemical probes, we validate the pose of the compound and support their substrate competitive MOA by comparing their activity against both strong and weak pre-mRNA substrates. Finally, we present functional data and structure-activity relationship (SAR) on the PHF5AR38C mutation that sensitizes cells to some chemical probes but not others. Developing small molecule splicing modulators represents a promising therapeutic approach for a variety of diseases, and this work provides a significant step in enabling structure-based drug design for these elaborate natural products. Importantly, this work also demonstrates that the utilization of cryo-EM in drug discovery is coming of age.
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Splicing modulators act at the branch point adenosine binding pocket defined by the PHF5A–SF3b complex
Nature Communications, 2017Co-Authors: Teng Teng, Michael Seiler, Benjamin Caleb, Daniel Aird, Silvia Buonamici, Jennifer Hc Tsai, Xiaoling Puyang, Shouyong Peng, Sudeep Prajapati, Betty ChanAbstract:Pladienolide, herboxidiene and spliceostatin have been identified as splicing modulators that target SF3B1 in the SF3b subcomplex. Here we report that PHF5A, another component of this subcomplex, is also targeted by these compounds. Mutations in PHF5A-Y36, SF3B1-K1071, SF3B1-R1074 and SF3B1-V1078 confer resistance to these modulators, suggesting a common interaction site. RNA-seq analysis reveals that PHF5A-Y36C has minimal effect on basal splicing but inhibits the global action of splicing modulators. Moreover, PHF5A-Y36C alters splicing modulator-induced intron-retention/exon-skipping profile, which correlates with the differential GC content between adjacent introns and exons. We determine the crystal structure of human PHF5A demonstrating that Y36 is located on a highly conserved surface. Analysis of the cryo-EM spliceosome B^act complex shows that the resistance mutations cluster in a pocket surrounding the branch point adenosine, suggesting a competitive mode of action. Collectively, we propose that PHF5A–SF3B1 forms a central node for binding to these splicing modulators. A number of natural occurring small-molecule splicing modulators are known. Here, the authors combine chemogenomic, structural and biochemical methods and show that these compounds also target the spliceosome-associated protein PHF5A and propose a potential modulator binding site in the PHF5A–SF3B1 complex.
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cancer associated SF3B1 hotspot mutations induce cryptic 3 splice site selection through use of a different branch point
Cell Reports, 2015Co-Authors: Rachel Darman, Michael Seiler, Betty Chan, Anant A Agrawal, Daniel Aird, Shouyong Peng, Kian-huat Lim, Suzanna L Bailey, Erica B Bhavsar, Simona CollaAbstract:Recurrent mutations in the spliceosome are observed in several human cancers, but their functional and therapeutic significance remains elusive. SF3B1, the most frequently mutated component of the spliceosome in cancer, is involved in the recognition of the branch point sequence (BPS) during selection of the 3' splice site (ss) in RNA splicing. Here, we report that common and tumor-specific splicing aberrations are induced by SF3B1 mutations and establish aberrant 3' ss selection as the most frequent splicing defect. Strikingly, mutant SF3B1 utilizes a BPS that differs from that used by wild-type SF3B1 and requires the canonical 3' ss to enable aberrant splicing during the second step. Approximately 50% of the aberrantly spliced mRNAs are subjected to nonsense-mediated decay resulting in downregulation of gene and protein expression. These findings ascribe functional significance to the consequences of SF3B1 mutations in cancer.
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abstract 5564 total synthesis of 6 deoxypladienolide d and assessment of splicing inhibitory activity in a mutant SF3B1 cancer cell line
Cancer Research, 2015Co-Authors: Kenzo Arai, Betty Chan, Craig Karr, Silvia Buonamici, Laura Corson, Atsushi Endo, Baudouin Gerard, Minghong Hao, Kazunobu Kira, Linda LeeAbstract:Hotspot mutations in several components of the spliceosome have been reported in various hematological (CLL, MDS, etc.) and solid tumor (melanoma, pancreatic, etc.) malignancies. SF3B1 is a component of the U2 snRNP complex of the spliceosome and is involved in the recognition of 3′-splice sites during early spliceosomal assembly. We and others have demonstrated that mutations in SF3B1 result in neomorphic activity and trigger the production of aberrantly spliced transcripts. Thus, the discovery of small molecule modulators of SF3B1 splicing activity may have therapeutic potential in cancers harboring SF3B1 mutations. Members of the pladienolide family of natural products have been shown to affect RNA splicing through interaction with SF3B1. We have found that one particular natural product in this family, 6-deoxypladienolide D, demonstrates potent growth inhibition and cellular lethality in Panc 05.04 cells (a hotspot mutant SF3B1 cancer cell line). Due to the limited natural supply of 6-deoxypladienolide D and our interest in identifying chemical matter able to modulate splicing in these newly-identified mutant SF3B1 cancers, a total synthesis of 6-deoxypladienolide D using versatile and modular fragments was initiated. We will describe the first total synthesis of the natural product 6-deoxypladienolide D. Two noteworthy synthetic attributes are: 1) a late-stage allylic oxidation which proceeds with full chemo-, regio-, and diastereoselectivity and 2) the use of cost-effective starting materials and reagents to enable access to 6-deoxypladienolide D and its analogs for biological evaluation. We will show that 6-deoxypladienolide D demonstrates: 1) high binding affinity to the SF3b complex, 2) ability to modulate canonical pre-mRNA splicing, and 3) modulation of aberrant splicing induced by mutant SF3B1. Citation Format: Kenzo Arai, Silvia Buonamici, Betty Chan, Laura Corson, Atsushi Endo, Baudouin Gerard, Ming-Hong Hao, Craig Karr, Kazunobu Kira, Linda Lee, Xiang Liu, Jason T. Lowe, Tuoping Luo, Lisa A. Marcaurelle, Yoshiharu Mizui, Marta Nevalainen, Morgan Welzel O9Shea, Eun Sun Park, Samantha A. Perino, Sudeep Prajapati, Mingde Shan, Peter G. Smith, Parcharee Tivitmahaisoon, John Yuan Wang, Markus Warmuth, Kuo-Ming Wu, Lihua Yu, Huiming Zhang, Guo Zhu Zheng, Gregg F. Keaney. Total synthesis of 6-deoxypladienolide D and assessment of splicing inhibitory activity in a mutant SF3B1 cancer cell line. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5564. doi:10.1158/1538-7445.AM2015-5564
Linda Lee - One of the best experts on this subject based on the ideXlab platform.
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abstract 5564 total synthesis of 6 deoxypladienolide d and assessment of splicing inhibitory activity in a mutant SF3B1 cancer cell line
Cancer Research, 2015Co-Authors: Kenzo Arai, Betty Chan, Craig Karr, Silvia Buonamici, Laura Corson, Atsushi Endo, Baudouin Gerard, Minghong Hao, Kazunobu Kira, Linda LeeAbstract:Hotspot mutations in several components of the spliceosome have been reported in various hematological (CLL, MDS, etc.) and solid tumor (melanoma, pancreatic, etc.) malignancies. SF3B1 is a component of the U2 snRNP complex of the spliceosome and is involved in the recognition of 3′-splice sites during early spliceosomal assembly. We and others have demonstrated that mutations in SF3B1 result in neomorphic activity and trigger the production of aberrantly spliced transcripts. Thus, the discovery of small molecule modulators of SF3B1 splicing activity may have therapeutic potential in cancers harboring SF3B1 mutations. Members of the pladienolide family of natural products have been shown to affect RNA splicing through interaction with SF3B1. We have found that one particular natural product in this family, 6-deoxypladienolide D, demonstrates potent growth inhibition and cellular lethality in Panc 05.04 cells (a hotspot mutant SF3B1 cancer cell line). Due to the limited natural supply of 6-deoxypladienolide D and our interest in identifying chemical matter able to modulate splicing in these newly-identified mutant SF3B1 cancers, a total synthesis of 6-deoxypladienolide D using versatile and modular fragments was initiated. We will describe the first total synthesis of the natural product 6-deoxypladienolide D. Two noteworthy synthetic attributes are: 1) a late-stage allylic oxidation which proceeds with full chemo-, regio-, and diastereoselectivity and 2) the use of cost-effective starting materials and reagents to enable access to 6-deoxypladienolide D and its analogs for biological evaluation. We will show that 6-deoxypladienolide D demonstrates: 1) high binding affinity to the SF3b complex, 2) ability to modulate canonical pre-mRNA splicing, and 3) modulation of aberrant splicing induced by mutant SF3B1. Citation Format: Kenzo Arai, Silvia Buonamici, Betty Chan, Laura Corson, Atsushi Endo, Baudouin Gerard, Ming-Hong Hao, Craig Karr, Kazunobu Kira, Linda Lee, Xiang Liu, Jason T. Lowe, Tuoping Luo, Lisa A. Marcaurelle, Yoshiharu Mizui, Marta Nevalainen, Morgan Welzel O9Shea, Eun Sun Park, Samantha A. Perino, Sudeep Prajapati, Mingde Shan, Peter G. Smith, Parcharee Tivitmahaisoon, John Yuan Wang, Markus Warmuth, Kuo-Ming Wu, Lihua Yu, Huiming Zhang, Guo Zhu Zheng, Gregg F. Keaney. Total synthesis of 6-deoxypladienolide D and assessment of splicing inhibitory activity in a mutant SF3B1 cancer cell line. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5564. doi:10.1158/1538-7445.AM2015-5564
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Abstract C8: Targeting MCL1-dependent cancers with SF3B splicing modulators
Apoptosis Necrosis and Autophagy, 2015Co-Authors: Daniel Aird, Betty Chan, Ermira Pazolli, Craig Furman, Linda Lee, Kaiko Kunii, Eun Sun Park, Craig Karr, Michelle Aicher, Silvia BuonamiciAbstract:Myeloid cell leukemia 1 (MCL1) is a member of the BCL2 family of proteins governing the apoptosis pathway and is one of the most frequently amplified genes in cancer. MCL1 overexpression often results in dependence on MCL1 for survival and is linked to resistance to anticancer therapies. However, the development of direct MCL1 inhibitors has proven challenging and new modalities for targeting MCL1 are required. Alternative splicing of MCL1 converts the anti-apoptotic MCL1 long (MCL1L) isoform to the BH3-only MCL1 short (MCL1S) isoform, which has been reported to be pro-apoptotic. Thus, changing MCL1 isoform levels through modulation of RNA splicing may represent an attractive approach to targeting MCL1-amplified cancers. To this end, we tested a collection of small molecule SF3B modulators that impact RNA splicing on MCL1-dependent and MCL1-independent NSCLC cell lines. SF3B modulators induced rapid downregulation of the long form and upregulation of the short- and intron-containing form of MCL1 across models; however, apoptosis was only observed in MCL1-dependent cells. Importantly, SF3B modulators preferentially killed MCL1-dependent cell lines and sensitivity correlated with MCL1 amplification. To dissect the mechanism of SF3B modulator-induced cytotoxicity, we overexpressed either the cDNA for the BH3-only short isoform or the full length isoform of MCL1. Surprisingly, overexpression of MCL1S cDNA had no significant effect on cells by itself and did not sensitize cells to SF3B modulator cytotoxicity. Conversely, MCL1L-specific shRNA knockdown was sufficient to kill MCL1-dependent cells and SF3B modulator cytotoxicity was rescued by expression of MCL1L cDNA. Together, these results argue that MCL1L modulation and not MCL1S upregulation is the effector of SF3B modulator cytotoxicity. In immunocompromised mice bearing MCL1-dependent xenograft models, SF3B1 modulator treatment resulted in significant downregulation of MCL1 levels accompanied by induction of apoptosis and robust efficacy at well-tolerated doses. Moreover, MCL1L cDNA expression in MCL1-dependent models rescued apoptosis induced by SF3B1 modulator treatment. These studies provide proof-of-concept that splicing modulation is an effective strategy for targeting cancers dependent on MCL1. Citation Format: Daniel Aird, Ermira Pazolli, Craig Furman, Linda Lee, Kaiko Kunii, Eun Sun Park, Craig Karr, Betty Chan, Michelle Aicher, Silvia Buonamici, John Yuan Wang, Jacob Feala, Lihua Yu, Markus Warmuth, Peter Smith, Peter Fekkes, Ping Zhu, Baudouin Gerard, Yoshiharu Mizui, Laura Corson. Targeting MCL1-dependent cancers with SF3B splicing modulators. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C8.
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total synthesis of 6 deoxypladienolide d and assessment of splicing inhibitory activity in a mutant SF3B1 cancer cell line
Organic Letters, 2014Co-Authors: Kenzo Arai, Betty Chan, Craig Karr, Silvia Buonamici, Laura Corson, Atsushi Endo, Baudouin Gerard, Minghong Hao, Kazunobu Kira, Linda LeeAbstract:A total synthesis of the natural product 6-deoxypladienolide D (1) has been achieved. Two noteworthy attributes of the synthesis are (1) a late-stage allylic oxidation which proceeds with full chemo-, regio-, and diastereoselectivity and (2) the development of a scalable and cost-effective synthetic route to support drug discovery efforts. 6-Deoxypladienolide D (1) demonstrates potent growth inhibition in a mutant SF3B1 cancer cell line, high binding affinity to the SF3b complex, and inhibition of pre-mRNA splicing.
Teresa V. Bowman - One of the best experts on this subject based on the ideXlab platform.
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The splicing factor SF3B1 regulates erythroid maturation and proliferation via TGFβ signaling in zebrafish
Blood advances, 2019Co-Authors: Adriana De La Garza, Varun Gupta, Rosannah C. Cameron, Ellen Fraint, Sara Nik, Teresa V. BowmanAbstract:The spliceosomal component Splicing Factor 3B, subunit 1 (SF3B1) is one of the most prevalently mutated factors in the bone marrow failure disorder myelodysplastic syndrome. There is a strong clinical correlation between SF3B1 mutations and erythroid defects, such as refractory anemia with ringed sideroblasts, but the role of SF3B1 in normal erythroid development is largely unknown. Loss-of-function zebrafish mutants for SF3B1 develop a macrocytic anemia. Here, we explore the underlying mechanism for anemia associated with SF3B1 deficiency in vivo. We found that SF3B1 mutant erythroid progenitors display a G0/G1 cell-cycle arrest with mutant erythrocytes showing signs of immaturity. RNA-sequencing analysis of SF3B1 mutant erythroid progenitors revealed normal expression of red blood cell regulators such as gata1, globin genes, and heme biosynthetic factors, but upregulation of genes in the transforming growth factor β (TGFβ) pathway. As TGFβ signaling is a known inducer of quiescence, the data suggest that activation of the pathway could trigger SF3B1 deficiency-induced anemia via cell-cycle arrest. Indeed, we found that inhibition of TGFβ signaling released the G0/G1 block in erythroid progenitors. Surprisingly, removal of this checkpoint enhanced rather than suppressed the anemia, indicating that the TGFβ-mediated cell-cycle arrest is protective for SF3B1-mutant erythrocytes. Together, these data suggest that macrocytic anemia arising from SF3B1 deficiency is likely due to pleiotropic and distinct effects on cell-cycle progression and maturation.
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STAT3 INHIBITION IS A SYNTHETIC LETHAL VULNERABILITY FOR SF3B1-MUTATED HEMATOPOIETIC STEM CELLS
Experimental Hematology, 2019Co-Authors: Kathryn S. Potts, Varun Gupta, Esther A Obeng, Rosannah C. Cameron, Aditi Shastri, Gaurav Choudhary, Mia Mckinstry, Xiaoying Bai, Amit Verma, Teresa V. BowmanAbstract:Hematopoietic stem and progenitor cells (HSPCs) maintain hematopoiesis throughout the lifetime of an organism. HSPC dysfunction can lead to disorders such as myelodysplastic syndrome (MDS). Spliceosome mutations are prevalent in MDS, but how splicing regulates HSPCs remains poorly understood. Using a zebrafish loss-of-function mutant for the top mutated splicing factor in MDS, splicing factor 3b, subunit 1 (SF3B1), we found that impaired splicing hindered HSPC specification. To identify SF3B1-regulated transcripts, we performed RNA-sequencing on kdrl:gfp+ endothelial cells from SF3B1 mutant and unaffected siblings at 24hpf. ∼900 genes were mis-spliced with significant enrichment for Signaling Transducer and Activator of Transcription 3 (Stat3) pathway components. Mis-spliced Stat3 signaling components observed in SF3B1 mutants were predicted to dampen protein levels or inhibit functionality, and Stat3 target genes were significantly downregulated in SF3B1 mutants. Constitutively-active Stat3 overexpression in SF3B1 mutants partially suppressed the HSPC defect. Induced mis-splicing of stat3 using morpholinos caused a significant HSPC decrease in SF3B1 heterozygotes at 28hpf, but elicited no effect in wildtype embryos, demonstrating a synthetic lethal interaction. Inhibiting Stat3 with small molecule STATTIC similarly reduced HSPCs in heterozygous but not wildtype embryos. We next tested if STAT3 inhibition selectively affected SF3B1-mutated human cells. K562 cells expressing the MDS-associated SF3B1-K666N mutation were more sensitive to STAT3 inhibition with STATTIC than wildtype cells. Similarly, primary SF3B1-mutant MDS patient cells produced fewer hematopoietic colonies when treated with STATTIC than controls. Together, STAT3 inhibition is a conserved synthetic lethal vulnerability for SF3B1-mutated cells with potential to treat splicing factor-mutated MDS.
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Spliceosomal component SF3B1 is essential for hematopoietic differentiation in zebrafish
Experimental hematology, 2016Co-Authors: Adriana De La Garza, Rosannah C. Cameron, Sara Nik, Sara G. Payne, Teresa V. BowmanAbstract:SF3B1 ( Splicing factor 3b , subunit 1 ) is one of the most commonly mutated factors in myelodysplastic syndrome (MDS). Although the genetic correlation between SF3B1 mutations and MDS etiology are quite strong, no in vivo model currently exists to explore how SF3B1 loss alters blood cell development. Using zebrafish mutants, we show here that proper function of SF3B1 is required for all hematopoietic lineages. As in MDS patients, zebrafish SF3B1 mutants develop a macrocytic-anemia–like phenotype due to a block in maturation at a late progenitor stage. The mutant embryos also develop neutropenia, because their primitive myeloid cells fail to mature and turn on differentiation markers such as l-plastin and myeloperoxidase. In contrast, production of definitive hematopoietic stem and progenitor cells (HSPCs) from hemogenic endothelial cells within the dorsal aorta is greatly diminished, whereas arterial endothelial cells are correctly fated. Notch signaling, imperative for the endothelial-to-hematopoietic transition, is also normal, indicating that HSPC induction is blocked in SF3B1 mutants downstream or independent of Notch signaling. The data demonstrate that SF3B1 function is necessary during key differentiation fate decisions in multiple blood cell types. Zebrafish SF3B1 mutants offer a novel animal model with which to explore the role of splicing in hematopoietic development and provide an excellent in vivo system with which to delve into the question of why and how SF3B1 dysfunction is detrimental to hematopoietic differentiation, which could improve MDS diagnosis and treatment.
Zhaoqi Liu - One of the best experts on this subject based on the ideXlab platform.
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Mutant SF3B1 promotes AKT and NF-kB driven mammary tumorigenesis.
The Journal of clinical investigation, 2021Co-Authors: Bo Liu, Zhaoqi Liu, Jorge S. Reis-filho, Sisi Chen, Caroline Erickson, Benjamin H. Durham, Qing Chang, Elisa De Stanchina, Yiwei SunAbstract:Mutations in the core RNA splicing factor SF3B1 are prevalent in leukemias and uveal melanoma but hotspot SF3B1 mutations are also seen in epithelial malignancies such as breast cancer. Although hotspot mutations in SF3B1 alter hematopoietic differentiation, whether SF3B1 mutations contribute to epithelial cancer development and progression is unknown. Here, we identify that SF3B1 mutations in mammary epithelial and breast cancer cells induce a recurrent pattern of aberrant splicing leading to activation of AKT and NF-kB, enhanced cell migration, and accelerated tumorigenesis. Transcriptomic analysis of human cancer specimens, MMTV-cre SF3B1K700E/WT mice, and isogenic mutant cell lines identified hundreds of aberrant 3' splice sites (3'ss) induced by mutant SF3B1. Consistently between mouse and human tumors, mutant SF3B1 promoted aberrant splicing (dependent on aberrant branchpoints as well as pyrimidines downstream of the cryptic 3'ss) and consequent suppression of PPP2R5A and MAP3K7, critical negative regulators of AKT and NF-kB. Coordinate activation of NF-kB and AKT signaling was observed in the knock-in models, leading to accelerated cell migration and tumor development in combination with mutant PIK3CA but also hypersensitizing cells to AKT kinase inhibitors. These data identify hotspot mutations in SF3B1 as an important contributor to breast tumorigenesis and reveal unique vulnerabilities in cancers harboring them.
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Pan-cancer analysis identifies mutations in SUGP1 that recapitulate mutant SF3B1 splicing dysregulation
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Zhaoqi Liu, Yiwei Sun, Jian Zhang, James L. Manley, Tomin E. Perea-chamblee, Raul RabadanAbstract:The gene encoding the core spliceosomal protein SF3B1 is the most frequently mutated gene encoding a splicing factor in a variety of hematologic malignancies and solid tumors. SF3B1 mutations induce use of cryptic 3' splice sites (3'ss), and these splicing errors contribute to tumorigenesis. However, it is unclear how widespread this type of cryptic 3'ss usage is in cancers and what is the full spectrum of genetic mutations that cause such missplicing. To address this issue, we performed an unbiased pan-cancer analysis to identify genetic alterations that lead to the same aberrant splicing as observed with SF3B1 mutations. This analysis identified multiple mutations in another spliceosomal gene, SUGP1, that correlated with significant usage of cryptic 3'ss known to be utilized in mutant SF3B1 expressing cells. Remarkably, this is consistent with recent biochemical studies that identified a defective interaction between mutant SF3B1 and SUGP1 as the molecular defect responsible for cryptic 3'ss usage. Experimental validation revealed that five different SUGP1 mutations completely or partially recapitulated the 3'ss defects. Our analysis suggests that SUGP1 mutations in cancers can induce missplicing identical or similar to that observed in mutant SF3B1 cancers.
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Mutations in the RNA Splicing Factor SF3B1 Promote Tumorigenesis through MYC Stabilization.
Cancer discovery, 2020Co-Authors: Zhaoqi Liu, Akihide Yoshimi, Jiguang Wang, Hana Cho, Stanley Chun-wei Lee, Lillian Bitner, Timothy Chu, Harshal Shah, Bo LiuAbstract:Although mutations in the gene encoding the RNA splicing factor SF3B1 are frequent in multiple cancers, their functional effects and therapeutic dependencies are poorly understood. Here, we characterize 98 tumors and 12 isogenic cell lines harboring SF3B1 hotspot mutations, identifying hundreds of cryptic 3' splice sites common and specific to different cancer types. Regulatory network analysis revealed that the most common SF3B1 mutation activates MYC via effects conserved across human and mouse cells. SF3B1 mutations promote decay of transcripts encoding the protein phosphatase 2A (PP2A) subunit PPP2R5A, increasing MYC S62 and BCL2 S70 phosphorylation which, in turn, promotes MYC protein stability and impair apoptosis, respectively. Genetic PPP2R5A restoration or pharmacologic PP2A activation impaired SF3B1-mutant tumorigenesis, elucidating a therapeutic approach to aberrant splicing by mutant SF3B1. SIGNIFICANCE: Here, we identify that mutations in SF3B1, the most commonly mutated splicing factor gene across cancers, alter splicing of a specific subunit of the PP2A serine/threonine phosphatase complex to confer post-translational MYC and BCL2 activation, which is therapeutically intervenable using an FDA-approved drug.See related commentary by O'Connor and Narla, p. 765.This article is highlighted in the In This Issue feature, p. 747.
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Disease-Causing Mutations in SF3B1 Alter Splicing by Disrupting Interaction with SUGP1.
Molecular cell, 2019Co-Authors: Jian Zhang, Zhaoqi Liu, Abdullah Mahmood Ali, Yen K. Lieu, Jianchao Gao, Raul Rabadan, Azra Raza, Siddhartha Mukherjee, James L. ManleyAbstract:SF3B1, which encodes an essential spliceosomal protein, is frequently mutated in myelodysplastic syndromes (MDS) and many cancers. However, the defect of mutant SF3B1 is unknown. Here, we analyzed RNA sequencing data from MDS patients and confirmed that SF3B1 mutants use aberrant 3' splice sites. To elucidate the underlying mechanism, we purified complexes containing either wild-type or the hotspot K700E mutant SF3B1 and found that levels of a poorly studied spliceosomal protein, SUGP1, were reduced in mutant spliceosomes. Strikingly, SUGP1 knockdown completely recapitulated the splicing errors, whereas SUGP1 overexpression drove the protein, which our data suggest plays an important role in branchsite recognition, into the mutant spliceosome and partially rescued splicing. Other hotspot SF3B1 mutants showed similar altered splicing and diminished interaction with SUGP1. Our study demonstrates that SUGP1 loss is a common defect of spliceosomes with disease-causing SF3B1 mutations and, because this defect can be rescued, suggests possibilities for therapeutic intervention.
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Mutations in the RNA Splicing Factor SF3B1 Promote Transformation through MYC Stabilization
Blood, 2018Co-Authors: Akihide Yoshimi, Zhaoqi Liu, Hana Cho, Stanley Chun-wei Lee, Lillian Bitner, Timothy Chu, Wang Jiguang, Anthony R. Mato, Peter P. RuvoloAbstract:Mutations in the RNA splicing factor SF3B1 are recurrent in CLL and myeloid neoplasms but their functional role in promoting tumorigenesis remain poorly understood. While SF3B1 mutations have been identified as promoting use of aberrant 39 splice sites (39ss), consistent identification of mis-spliced transcripts and pathways that functionally link mutant SF3B1 to transformation remains elusive. Moreover, large-scale analyses of the impact of mutant SF3B1 on gene expression and gene regulatory networks, which may be distinct from aberrant splicing changes, remain to be performed. We therefore sought to elucidate the effects of SF3B1 mutations across hematopoietic malignancies and cancer lineages at the level of both mRNA splicing and expression. To this end, we collected RNA-seq data from 79 tumors and 12 isogenic cell lines harboring SF3B1 hotspot mutations. The most frequent hotspot, K700E, was the most common mutation in CLL and breast cancers while mutations at position R625 were restricted to melanomas ( Figure A, B ). Regulatory network analysis of differentially expressed genes in SF3B1 mutated CLL identified MYC as the top master regulator ( Figure C ). MYC activation in SF3B1 mutated CLL was also verified by differential expression analyses ( Figure D ) and was common to SF3B1 K700E mutant cancers while absent in cancers with mutations affecting R625. Taken together, these observations suggested that tumors harboring SF3B1 K700E mutations activate the MYC transcriptional program. We next sought to verify the effects of c-Myc activation by mutant SF3B1 in the B-cell lineage in vivo . We crossed Cd19 -cre SF3B1 K700E/+ mice with E μ- Myc transgenic mice to generate Cd19 -cre + control, SF3B1 K700E/+ , E μ- Myc Tg/+ , and SF3B1 K700E/+ E μ- Myc Tg/+ double-mutant mice. While control or single mutant primary mice did not develop disease over one year, double-mutant mice developed a lethal B-cell malignancy. This effect was consistent in serial transplantation, where mice transplanted with double-mutant cells had shorter survival compared to single-mutant controls ( Figure E ). These data provide the first evidence that SF3B1 mutations contribute to tumorigenesis in vivo . To understand the molecular mechanism for MYC activation across SF3B1 mutant human and mouse cells, we analyzed RNA-seq data from CLL patients, isogenic Nalm-6 cells, and splenic B-cells from the mouse models. This revealed a significant overlap in aberrant (39ss) events across SF3B1 mutant samples. Interestingly, mis-spliced events across mouse and human SF3B1 K700E mutant samples identified aberrant 39ss usage and decay of PPP2R5A ( Figure F ) , a gene whose product has previously been shown to regulate c-MYC protein stability and the only gene whose aberrant splicing was most prominent in K700E compared with R625 mutant SF3B1. PPP2R5A is a subunit of the PP2A phosphatase complex that dephosphorylates Serine 62 (S62) of c-MYC, resulting in an unstable form of c-MYC that is a substrate for proteasomal degradation. Consistent with this, SF3B1 K700E mutant cells exhibited dramatic increase in S62-phosphorylated c-MYC and increased stability of c-MYC protein. MYC expression, stability, and S62 phosphorylation could be abrogated in SF3B1 mutant cells by restoring PPP25RA expression. In addition to c-MYC S62 phosphorylation, PPP2R5A-containing PP2A reduced S70 phosphorylation of BCL2 (a modification important for apoptosis induction) in SF3B1 mutant cells. To functionally evaluate the importance of impaired PP2A enzymatic activity in SF3B1 mutant cells further, we assessed the therapeutic potential of the FDA-approved oral PP2A activator, FTY-720. SF3B1 mutant cells were more sensitive to FTY-720 treatment than SF3B1 WT counterparts, experiencing growth arrest at lower concentration ( Figure G ). Moreover, both S62-phosphorylated c-MYC and S70-phosphorylated BCL2 decreased in a dose-dependent manner upon treatment with FTY-720 ( Figure H ). Here through combined evaluation of the effects of the SF3B1 mutation on splicing, gene expression, and transcriptional networks across cancer types, we identify a novel mechanism by which mutant SF3B1-mediated alterations in RNA splicing contribute to activation of oncogenic MYC through effects on MYC proteolysis. Moreover, these data highlight a novel therapeutic approach targeting the impact of mutant SF3B1 on post-translational modification of MYC. Disclosures Mato: Janssen: Consultancy, Honoraria; Celgene: Consultancy; Prime Oncology: Speakers Bureau; TG Therapeutics: Research Funding; Regeneron: Research Funding; Abbvie: Consultancy; Sunesis: Honoraria, Research Funding; Acerta: Research Funding; AstraZeneca: Consultancy; Pharmacyclics: Consultancy, Honoraria, Research Funding.
Esther A Obeng - One of the best experts on this subject based on the ideXlab platform.
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STAT3 INHIBITION IS A SYNTHETIC LETHAL VULNERABILITY FOR SF3B1-MUTATED HEMATOPOIETIC STEM CELLS
Experimental Hematology, 2019Co-Authors: Kathryn S. Potts, Varun Gupta, Esther A Obeng, Rosannah C. Cameron, Aditi Shastri, Gaurav Choudhary, Mia Mckinstry, Xiaoying Bai, Amit Verma, Teresa V. BowmanAbstract:Hematopoietic stem and progenitor cells (HSPCs) maintain hematopoiesis throughout the lifetime of an organism. HSPC dysfunction can lead to disorders such as myelodysplastic syndrome (MDS). Spliceosome mutations are prevalent in MDS, but how splicing regulates HSPCs remains poorly understood. Using a zebrafish loss-of-function mutant for the top mutated splicing factor in MDS, splicing factor 3b, subunit 1 (SF3B1), we found that impaired splicing hindered HSPC specification. To identify SF3B1-regulated transcripts, we performed RNA-sequencing on kdrl:gfp+ endothelial cells from SF3B1 mutant and unaffected siblings at 24hpf. ∼900 genes were mis-spliced with significant enrichment for Signaling Transducer and Activator of Transcription 3 (Stat3) pathway components. Mis-spliced Stat3 signaling components observed in SF3B1 mutants were predicted to dampen protein levels or inhibit functionality, and Stat3 target genes were significantly downregulated in SF3B1 mutants. Constitutively-active Stat3 overexpression in SF3B1 mutants partially suppressed the HSPC defect. Induced mis-splicing of stat3 using morpholinos caused a significant HSPC decrease in SF3B1 heterozygotes at 28hpf, but elicited no effect in wildtype embryos, demonstrating a synthetic lethal interaction. Inhibiting Stat3 with small molecule STATTIC similarly reduced HSPCs in heterozygous but not wildtype embryos. We next tested if STAT3 inhibition selectively affected SF3B1-mutated human cells. K562 cells expressing the MDS-associated SF3B1-K666N mutation were more sensitive to STAT3 inhibition with STATTIC than wildtype cells. Similarly, primary SF3B1-mutant MDS patient cells produced fewer hematopoietic colonies when treated with STATTIC than controls. Together, STAT3 inhibition is a conserved synthetic lethal vulnerability for SF3B1-mutated cells with potential to treat splicing factor-mutated MDS.
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physiologic expression of SF3B1 k700e causes impaired erythropoiesis aberrant splicing and sensitivity to therapeutic spliceosome modulation
Cancer Cell, 2016Co-Authors: Michael Seiler, Esther A Obeng, Ryan J Chappell, Michelle C Chen, Dean R Campagna, Paul J Schmidt, Rebekka K Schneider, Allegra M LordAbstract:Summary More than 80% of patients with the refractory anemia with ring sideroblasts subtype of myelodysplastic syndrome (MDS) have mutations in Splicing Factor 3B, Subunit 1 ( SF3B1 ). We generated a conditional knockin mouse model of the most common SF3B1 mutation, SF3B1 K700E . SF3B1 K700E mice develop macrocytic anemia due to a terminal erythroid maturation defect, erythroid dysplasia, and long-term hematopoietic stem cell (LT-HSC) expansion. SF3B1 K700E myeloid progenitors and SF3B1 -mutant MDS patient samples demonstrate aberrant 3′ splice-site selection associated with increased nonsense-mediated decay. Tet2 loss cooperates with SF3B1 K700E to cause a more severe erythroid and LT-HSC phenotype. Furthermore, the spliceosome modulator, E7017, selectively kills SF3B1 K700E -expressing cells. Thus, SF3B1 K700E expression reflects the phenotype of the mutation in MDS and may be a therapeutic target in MDS.