Optimization of Antitumor Modulators of Pre-mRNA Splicing

Alternative splicing generates a diversity of messenger RNA (mRNA) transcripts from a single mRNA precursor and contributes to the complexity of our proteome. Splicing is perturbed by a variety of mechanisms in cancer. Recurrent mutations in splicing factors have emerged as a hallmark of several hematologic malignancies. Splicing factor mutations tend to occur in the founding clone of myeloid cancers, and these mutations have recently been identified in blood cells from normal, healthy elderly individuals with clonal hematopoiesis who are at increased risk of subsequently developing a hematopoietic malignancy, suggesting that these mutations contribute to disease initiation. Splicing factor mutations change the pattern of splicing in primary patient and mouse hematopoietic cells and alter hematopoietic differentiation and maturation in animal models. Recent developments in this field are reviewed here, with an emphasis on the clinical consequences of splicing factor mutations, mechanistic insights from animal models, and implications for development of novel therapies targeting the precursor mRNA splicing pathway. (Blood. 2017;129(10):1260-1269 IntroductionThe control of messenger RNA (mRNA) processing is a crucial component of gene regulation. The precursor messenger RNA (premRNA) sequence contains exons that flank introns, which are removed during splicing.1 Human genes are remarkably complex, containing an average of 8 exons, with introns composing up to 90% of the pre-mRNA sequence.2,3 Up to 94% of human genes generate multiple mRNA isoforms. [4][5][6] The spliceosome is a multiprotein complex consisting of 5 small nuclear ribonucleoproteins (snRNPs) and more than 150 proteins that recognizes the elements near intron-exon boundaries and the branch site that catalyzes the excision of intronic regions. Additional trans-acting factors including SR proteins bind the cis-regulatory sequences (exonic and intronic silencers and enhancers) to promote or prevent spliceosome assembly. [7][8][9] The complexity of regulatory elements that control proper splicing makes this process not only critical to maintain tissue homeostasis, but also highly susceptible to hereditary and somatic mutations that are involved in disease. This review will discuss the implications of aberrant splicing on human disease, with a focus on the impact of somatic mutations in trans-acting splicing factors in hematologic malignancies. Splicing and diseaseA comprehensive analysis of The Cancer Genome Atlas (TCGA) project RNA sequencing (RNA-seq) data from 16 tumor types and matched normal tissue revealed evidence of global alternative splicing involving a variety of splicing junctions, including cassette exons, retrained introns, and competing 59 and 39 splice sites (SSs). 10 In particular, acute myeloid leukemia (AML) cells showed the largest number of alternative spliced events among tumor types.10 Recent evidence from analysis of paired DNA and RNA-seq data from TCGA has shown that somatic mutations affecting splicing regulatory sequences (sp...

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“…[85][86][87] Primary human CLL cells undergo apoptosis in a dose-dependent fashion after exposure to sudemycin D1 (SD1) in vitro.…”

Section: Splicing Modulatorsmentioning

confidence: 99%

Splicing factor gene mutations in hematologic malignancies

Sáez

Walter

Graubert

2017

Blood

112

108

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“…Structure-activity relationship (SAR) data for the three compounds and related molecules have been steadily emerging (Sakai et al 2002;Mizui et al 2004;Lagisetti et al 2008Lagisetti et al , 2013Lagisetti et al , 2014Albert et al 2009;Fan et al 2011;Gundluru et al 2011;Muller et al 2011;Villa et al 2012Villa et al , 2013Gao et al 2013;Ghosh and Chen 2013;Arai et al 2014;Effenberger et al 2014;Ghosh et al 2014a,b,c;He et al 2014). SSA (1), which is similar to FR901464 (Nakajima et al 1996b), meayamycin (Albert et al 2009), thailanstatins (Liu et al 2013), and sudemycins (Fan et al 2011), differs in structure relative to the other two compounds.…”

Section: Introductionmentioning

confidence: 99%

Interchangeable SF3B1 inhibitors interfere with pre-mRNA splicing at multiple stages

Effenberger

Urabe

Prichard

et al. 2016

RNA

102

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The protein SF3B1 is a core component of the spliceosome, the large ribonucleoprotein complex responsible for pre-mRNA splicing. Interest in SF3B1 intensified when tumor exome sequencing revealed frequent specific SF3B1 mutations in a variety of neoplasia and when SF3B1 was identified as the target of three different cancer cell growth inhibitors. A better mechanistic understanding of SF3B1's role in splicing is required to capitalize on these discoveries. Using the inhibitor compounds, we probed SF3B1 function in the spliceosome in an in vitro splicing system. Formerly, the inhibitors were shown to block early steps of spliceosome assembly, consistent with a previously determined role of SF3B1 in intron recognition. We now report that SF3B1 inhibitors also interfere with later events in the spliceosome cycle, including exon ligation. These observations are consistent with a requirement for SF3B1 throughout the splicing process. Additional experiments aimed at understanding how three structurally distinct molecules produce nearly identical effects on splicing revealed that inactive analogs of each compound interchangeably compete with the active inhibitors to restore splicing. The competition indicates that all three types of compounds interact with the same site on SF3B1 and likely interfere with its function by the same mechanism, supporting a shared pharmacophore model. It also suggests that SF3B1 inhibition does not result from binding alone, but is consistent with a model in which the compounds affect a conformational change in the protein. Together, our studies reveal new mechanistic insight into SF3B1 as a principal player in the spliceosome and as a target of inhibitor compounds.

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“…2011; Lagisetti et al. 2013), and focused our work on the lead-optimization of the sudemycins, based on FR structure-activity relationships and a hypothetical consensus pharmacophore model (Lagisetti et al. 2008).…”

Section: Introductionmentioning

confidence: 99%

“…This work ultimately led to SD6, which is currently in preclinical development (Lagisetti et al. 2013). In the following we describe two types of novel pharmacodynamic assays that facilitate drug discovery and development of this intriguing class of innovative therapeutics; the first assay is useful for preclinical optimization of small-molecule agents that target the SF3B1 spliceosomal protein in animals, the second assay is an ex vivo validated, gel-based assay for the measurement of drug exposure in human leukocytes.…”

Section: Introductionmentioning

confidence: 99%

Pharmacodynamic assays to facilitate preclinical and clinical development of pre‐mRNA splicing modulatory drug candidates

Shi

Joyner

Shadrick

et al. 2015

Pharmacology Res & Perspec

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The spliceosome has recently emerged as a new target for cancer chemotherapy and novel antitumor spliceosome targeted agents are under development. Here, we describe two types of novel pharmacodynamic assays that facilitate drug discovery and development of this intriguing class of innovative therapeutics; the first assay is useful for preclinical optimization of small-molecule agents that target the SF3B1 spliceosomal protein in animals, the second assay is an ex vivo validated, gel-based assay for the measurement of drug exposure in human leukocytes. The first assay utilizes a highly specific bioluminescent splicing reporter, based on the skipping of exons 4–11 of a Luc-MDM2 construct, which specifically yields active luciferase when treated with small-molecule spliceosome modulators. We demonstrate that this reporter can be used to monitor alternative splicing in whole cells in vitro. We describe here that cell lines carrying the reporter can be used in vivo for the efficient pharmacodynamic analysis of agents during drug optimization and development. We also demonstrate dose- and time-dependent on-target activity of sudemycin D6 (SD6), which leads to dramatic tumor regression. The second assay relies on the treatment of freshly drawn human blood with SD6 ex vivo treatment. Changes in alternative splicing are determined by RT-PCR using genes previously identified in in vitro experiments. The Luc-MDM2 alternative splicing bioluminescent reporter and the splicing changes observed in human leukocytes should allow for the more facile translation of novel splicing modulators into clinical application.

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Optimization of Antitumor Modulators of Pre-mRNA Splicing

Cited by 61 publications

References 51 publications

Splicing factor gene mutations in hematologic malignancies

Splicing factor gene mutations in hematologic malignancies

Interchangeable SF3B1 inhibitors interfere with pre-mRNA splicing at multiple stages

Pharmacodynamic assays to facilitate preclinical and clinical development of pre‐mRNA splicing modulatory drug candidates

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