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.
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 Å. This structure shows that E7107 binds in the branch point adenosine-binding pocket, forming close contacts with key residues that confer resistance upon mutation: SF3B1
and PHF5A
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 PHF5A
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.
Dysregulation of RNA splicing by spliceosome mutations or in cancer genes is increasingly recognized as a hallmark of cancer. Small molecule splicing modulators have been introduced into clinical ...trials to treat solid tumors or leukemia bearing recurrent spliceosome mutations. Nevertheless, further investigation of the molecular mechanisms that may enlighten therapeutic strategies for splicing modulators is highly desired. Here, using unbiased functional approaches, we report that the sensitivity to splicing modulation of the anti-apoptotic BCL2 family genes is a key mechanism underlying preferential cytotoxicity induced by the SF3b-targeting splicing modulator E7107. While BCL2A1, BCL2L2 and MCL1 are prone to splicing perturbation, BCL2L1 exhibits resistance to E7107-induced splicing modulation. Consequently, E7107 selectively induces apoptosis in BCL2A1-dependent melanoma cells and MCL1-dependent NSCLC cells. Furthermore, combination of BCLxL (BCL2L1-encoded) inhibitors and E7107 remarkably enhances cytotoxicity in cancer cells. These findings inform mechanism-based approaches to the future clinical development of splicing modulators in cancer treatment.
Activation of the fibroblast growth factor receptor FGFR4 by FGF19 drives hepatocellular carcinoma (HCC), a disease with few, if any, effective treatment options. While a number of pan-FGFR ...inhibitors are being clinically evaluated, their application to FGF19-driven HCC may be limited by dose-limiting toxicities mediated by FGFR1-3 receptors. To evade the potential limitations of pan-FGFR inhibitors, we generated H3B-6527, a highly selective covalent FGFR4 inhibitor, through structure-guided drug design. Studies in a panel of 40 HCC cell lines and 30 HCC PDX models showed that FGF19 expression is a predictive biomarker for H3B-6527 response. Moreover, coadministration of the CDK4/6 inhibitor palbociclib in combination with H3B-6527 could effectively trigger tumor regression in a xenograft model of HCC. Overall, our results offer preclinical proof of concept for H3B-6527 as a candidate therapeutic agent for HCC cases that exhibit increased expression of FGF19.
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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
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.
Uridine-5'-diphospho-N-acetylgalactosamine (UDP-GalNAc) is required in the formation of the outer filamentous wall of Giardia and is synthesized by inducible enzymes in the cytosol of encysting ...trophozoites. In this study, an inducible enzyme activity that is associated with a particle population isolated from encysting Giardia is reported, and this activity exclusively incorporates 1-(14)CGalNAc (from UDP-(14)CGalNAc) into an ethanol precipitate with the same properties as the filamentous cyst wall of GIARDIA: This ethanol precipitate exhibits characteristics of Giardia cyst wall filaments in that both contain GalNAc as the only sugar moieties and are SDS-insoluble, proteinase- and alkali-resistant and acid-hydrolysable. However, since the precise chemical nature of the ethanol precipitate remains unknown, this enzyme activity is referred to tentatively as cyst wall synthase (CWS). CWS activity peaks in cells between 24 and 36 h of encystment and exhibits a high affinity and marked specificity for UDP-GalNAc as its substrate. UDP-N-acetylglucosamine, UDP-glucose, UDP-galactose, D-glucosamine and D-galactosamine were not incorporated into the ethanol precipitate. Partially purified CWS activity exhibits an apparent K(m) of 0.048 mM for UDP-GalNAc, a V(max) of 0.70 nmol x min(-1) (mg protein)(-1) and a requirement for divalent cations in the following order of preference: Ca(2+), Mg(2+)>Co(2+)>>>Mn(2+), Zn(2+). EDTA inhibits CWS activity.
Assembly of a protective cyst wall by Giardia is essential for the survival of the parasite outside the host intestine and for transmission among susceptible hosts. The structure of the G. ...intestinalis filamentous cyst wall was studied by chemical methods, mass spectrometry, and 1H nuclear magnetic resonance spectroscopy. Isolated cyst wall material contains carbohydrate and protein in a ratio of 3:2 (w/w), and the carbohydrate moiety is composed of a β(1-3)-N-acetyl-d-galactopyranosamine homopolymer. Conformational analysis by molecular dynamics and persistence length calculations of GalNAc oligomers in solution demonstrated a flexible structure consisting of left- and right-handed helical elements. It is most likely that in the solid state, the polysaccharide forms ordered helices or possibly multiple helical structures having strong interchain interactions. The highly insoluble nature of the Giardia cyst wall must be due to these strong interchain interactions and, probably, a strong association between the carbohydrate and the protein moiety.
Recent studies have reported that the peroxisome proliferator–activated receptor gamma (PPARγ) pathway is activated in approximately 40% of patients with muscle-invasive bladder cancer. This led us ...to investigate pharmacological repression of PPARγ as a possible intervention strategy. Here, we characterize PPARγ antagonists and inverse agonists and find that the former behave as silent ligands, whereas inverse agonists (T0070907 and SR10221) repress downstream PPARγ target genes leading to growth inhibition in bladder cancer cell lines. To understand the mechanism, we determined the ternary crystal structure of PPARγ bound to T0070907 and the corepressor (co-R) peptide NCOR1. The structure shows that the AF-2 helix 12 (H12) rearranges to bind inside the ligand-binding domain, where it forms stabilizing interactions with the compound. This dramatic movement in H12 unveils a large interface for co-R binding. In contrast, the crystal structure of PPARγ bound to a SR10221 analog shows more subtle structural differences, where the compound binds and pushes H12 away from the ligand-binding domain to allow co-R binding. Interestingly, we found that both classes of compound promote recruitment of co-R proteins in biochemical assays but with distinct conformational changes in H12. We validate our structural models using both site-directed mutagenesis and chemical probes. Our findings offer new mechanistic insights into pharmacological modulation of PPARγ signaling.
Fibroblast growth factor receptors (FGFR) 2 and 3 have been established as drivers of numerous types of cancer with multiple drugs approved or entering late stage clinical trials. A limitation of ...current inhibitors is vulnerability to gatekeeper resistance mutations. Using a combination of targeted high-throughput screening and structure-based drug design, we have developed a series of aminopyrazole based FGFR inhibitors that covalently target a cysteine residue on the P-loop of the kinase. The inhibitors show excellent activity against the wild-type and gatekeeper mutant versions of the enzymes. Further optimization using SAR analysis and structure-based drug design led to analogues with improved potency and drug metabolism and pharmacokinetics properties.
Hepatocellular carcinoma (HCC) is an aggressive liver malignancy that is difficult to treat with no approved biomarker based targeted therapies. FGF19-FGFR4 signaling blockade has been recently ...identified as a promising avenue for treatment of a subset of HCC patients. Using HCC relevant xenograft and PDX models, we show that Lenvatinib, an approved multi-kinase inhibitor, strongly enhanced the efficacy of FGFR4 inhibitor H3B-6527. This enhanced combination effect is not due to enhanced FGFR4 inhibition and it is likely due to cell non-autonomous VEGFR activity of Lenvatinib. This cell non-autonomous mode of action was further supported by strong
in vivo
combination efficacy with the mouse specific VEGFR2 antibody, DC101, which cannot cell-autonomously inhibit pathways in human xenografts. Mechanistic studies showed that the combination resulted in enhanced efficacy through increased anti-angiogenic and anti-tumorigenic activities. Overall, our results indicate that this combination can be a highly effective treatment option for FGF19 driven HCC patients, and provide preclinical validation of a combination that can be readily tested in the clinical setting.
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