H3B-6527 Is a Potent and Selective Inhibitor of FGFR4 in FGF19-Driven Hepatocellular Carcinoma.

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. Cancer Res; 77(24); 6999-7013. ©2017 AACR.

The Dual Estrogen Receptor α Inhibitory Effects of the Tissue-Selective Estrogen Complex for Endometrial and Breast Safety.

The conjugated estrogen /: bazedoxifene tissue-selective estrogen complex (TSEC) is designed to minimize the undesirable effects of estrogen in the uterus and breast tissues and to allow the beneficial effects of estrogen in other estrogen-target tissues, such as the bone and brain. However, the molecular mechanism underlying endometrial and breast safety during TSEC use is not fully understood. Estrogen receptor α (ERα)-estrogen response element (ERE)-DNA pull-down assays using HeLa nuclear extracts followed by mass spectrometry-immunoblotting analyses revealed that, upon TSEC treatment, ERα interacted with transcriptional repressors rather than coactivators. Therefore, the TSEC-mediated recruitment of transcriptional repressors suppresses ERα-mediated transcription in the breast and uterus. In addition, TSEC treatment also degraded ERα protein in uterine tissue and breast cancer cells, but not in bone cells. Interestingly, ERα-ERE-DNA pull-down assays also revealed that, upon TSEC treatment, ERα interacted with the F-box protein 45 (FBXO45) E3 ubiquitin ligase. The loss-of- and gain-of-FBXO45 function analyses indicated that FBXO45 is involved in TSEC-mediated degradation of the ERα protein in endometrial and breast cells. In preclinical studies, these synergistic effects of TSEC on ERα inhibition also suppressed the estrogen-dependent progression of endometriosis. Therefore, the endometrial and breast safety effects of TSEC are associated with synergy between the selective recruitment of transcriptional repressors to ERα and FBXO45-mediated degradation of the ERα protein.

Cancer-Associated SF3B1 Hotspot Mutations Induce Cryptic 3' Splice Site Selection through Use of a Different Branch Point.

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.

Proteomic analysis of coregulators bound to ERα on DNA and nucleosomes reveals coregulator dynamics.

Chromatin immunoprecipitation studies have mapped protein occupancies at many genomic loci. However, a detailed picture of the complexity of coregulators (CoRs) bound to a defined enhancer along with a transcription factor is missing. To address this, we used biotin-DNA pull-down assays coupled with mass spectrometry-immunoblotting to identify at least 17 CoRs from nuclear extracts bound to 17ß-estradiol (E2)-liganded estrogen receptor-α on estrogen response elements (EREs). Unexpectedly, these complexes initially are biochemically stable and contain certain atypical corepressors. Addition of ATP dynamically converts these complexes to an "activated" state by phosphorylation events, primarily mediated by DNA-dependent protein kinase. Importantly, a "natural" ERE-containing enhancer and nucleosomal EREs recruit similar complexes. We further discovered the mechanism whereby H3K4me3 stimulates ERα-mediated transcription as compared with unmodified nucleosomes. H3K4me3 templates promote specific CoR dynamics in the presence of ATP and AcCoA, as manifested by CBP/p300 and SRC-3 dismissal and SAGA and TFIID stabilization/recruitment.

Defects in DNA degradation revealed in crystal structures of TREX1 exonuclease mutations linked to autoimmune disease.

Mutations within the human TREX1 3' exonuclease are associated with Aicardi-Goutières Syndrome (AGS) and familial chilblain lupus (FCL). Both AGS and FCL are autoimmune diseases that result in increased levels of interferon alpha and circulating antibodies to DNA. TREX1 is a member of the endoplasmic reticulum (ER)-associated SET complex and participates in granzyme A-mediated cell death to degrade nicked genomic DNA. The loss of TREX1 activity may result in the accumulation of double-stranded DNA (dsDNA) degradation intermediates that trigger autoimmune activation. The X-ray crystal structures of the TREX1 wt apoprotein, the dominant D200H, D200N and D18N homodimer mutants derived from AGS and FCL patients, as well as the recessive V201D homodimer mutant have been determined. The structures of the D200H and D200N mutant proteins reveal the enzyme has lost coordination of one of the active site metals, and the catalytic histidine (H195) is trapped in a conformation pointing away from the active site. The TREX1 D18N and V201D mutants are able to bind both metals in the active site, but with inter-metal distances that are larger than optimal for catalysis. Additionally, all of the mutant structures reveal a reduced mobility in the catalytic histidine, providing further explanation for the loss of catalytic activity. The structures of the mutant TREX1 proteins provide insight into the dysfunction relating to human disease. Additionally, the TREX1 apoprotein structure together with the previously determined wild type substrate and product structures allow us to propose a distinct mechanism for the TREX1 exonuclease.

Mutations in the gene encoding the 3'-5' DNA exonuclease TREX1 are associated with systemic lupus erythematosus.

TREX1 acts in concert with the SET complex in granzyme A-mediated apoptosis, and mutations in TREX1 cause Aicardi-Goutières syndrome and familial chilblain lupus. Here, we report monoallelic frameshift or missense mutations and one 3' UTR variant of TREX1 present in 9/417 individuals with systemic lupus erythematosus but absent in 1,712 controls (P = 4.1 x 10(-7)). We demonstrate that two mutant TREX1 alleles alter subcellular targeting. Our findings implicate TREX1 in the pathogenesis of SLE.

The crystal structure of TREX1 explains the 3' nucleotide specificity and reveals a polyproline II helix for protein partnering.

The TREX1 enzyme processes DNA ends as the major 3' --> 5' exonuclease activity in human cells. Mutations in the TREX1 gene are an underlying cause of the neurological brain disease Aicardi-Goutières syndrome implicating TREX1 dysfunction in an aberrant immune response. TREX1 action during apoptosis likely prevents autoimmune reaction to DNA that would otherwise persist. To understand the impact of TREX1 mutations identified in patients with Aicardi-Goutières syndrome on structure and activity we determined the x-ray crystal structure of the dimeric mouse TREX1 protein in substrate and product complexes containing single-stranded DNA and deoxyadenosine monophosphate, respectively. The structures show the specific interactions between the bound nucleotides and the residues lining the binding pocket of the 3' terminal nucleotide within the enzyme active site that account for specificity, and provide the molecular basis for understanding mutations that lead to disease. Three mutant forms of TREX1 protein identified in patients with Aicardi-Goutières syndrome were prepared and the measured activities show that these specific mutations reduce enzyme activity by 4-35,000-fold. The structure also reveals an 8-amino acid polyproline II helix within the TREX1 enzyme that suggests a mechanism for interactions of this exonuclease with other protein complexes.

Physical and structural basis for the strong interactions of the -ImPy- central pairing motif in the polyamide f-ImPyIm.

The polyamide f-ImPyIm has a higher affinity for its cognate DNA than either the parent analogue, distamycin A (10-fold), or the structural isomer, f-PyImIm (250-fold), has for its respective cognate DNA sequence. These findings have led to the formulation of a two-letter polyamide "language" in which the -ImPy- central pairings associate more strongly with Watson-Crick DNA than -PyPy-, -PyIm-, and -ImIm-. Herein, we further characterize f-ImPyIm and f-PyImIm, and we report thermodynamic and structural differences between -ImPy- (f-ImPyIm) and -PyIm- (f-PyImIm) central pairings. DNase I footprinting studies confirmed that f-ImPyIm is a stronger binder than distamycin A and f-PyImIm and that f-ImPyIm preferentially binds CGCG over multiple competing sequences. The difference in the binding of f-ImPyIm and f-PyImIm to their cognate sequences was supported by the Na(+)-dependent nature of DNA melting studies, in which significantly higher Na(+) concentrations were needed to match the ability of f-ImPyIm to stabilize CGCG with that of f-PyImIm stabilizing CCGG. The selectivity of f-ImPyIm beyond the four-base CGCG recognition site was tested by circular dichroism and isothermal titration microcalorimetry, which shows that f-ImPyIm has marginal selectivity for (A.T)CGCG(A.T) over (G.C)CGCG(G.C). In addition, changes adjacent to this 6 bp binding site do not affect f-ImPyIm affinity. Calorimetric studies revealed that binding of f-ImPyIm, f-PyImIm, and distamycin A to their respective hairpin cognate sequences is exothermic; however, changes in enthalpy, entropy, and heat capacity (DeltaC(p)) contribute differently to formation of the 2:1 complexes for each triamide. Experimental and theoretical determinations of DeltaC(p) for binding of f-ImPyIm to CGCG were in good agreement (-142 and -177 cal mol(-)(1) K(-)(1), respectively). (1)H NMR of f-ImPyIm and f-PyImIm complexed with their respective cognate DNAs confirmed positively cooperative formation of distinct 2:1 complexes. The NMR results also showed that these triamides bind in the DNA minor groove and that the oligonucleotide retains the B-form conformation. Using minimal distance restraints from the NMR experiments, molecular modeling and dynamics were used to illustrate the structural complementarity between f-ImPyIm and CGCG. Collectively, the NMR and ITC experiments show that formation of the 2:1 f-ImPyIm-CGCG complex achieves a structure more ordered and more thermodynamically favored than the structure of the 2:1 f-PyImIm-CCGG complex.

Design of a hairpin polyamide, ZT65B, for targeting the inverted CCAAT box (ICB) site in the multidrug resistant (MDR1) gene.

A novel hairpin polyamide, ZT65B, containing a 3-methylpicolinate moiety was designed to target the inverted CCAAT box (ICB) of the human multidrug resistance 1 gene (MDR1) promoter. Binding of nuclear factor-Y (NF-Y) to the ICB site upregulates MDR1 gene expression and is, therefore, a good target for anticancer therapeutic agents. However, it is important to distinguish amongst different promoter ICB sites so that only specific genes will be affected. All ICB sites have the same sequence but they differ in the sequence of the flanking base pairs, which can be exploited in the design of sequence-specific polyamides. To test this hypothesis, ten ICB-containing DNA hairpins were designed with different flanking base pairs; the sequences ICBa and ICBb were similar to the 3'-ICB site of MDR1 (TGGCT). Thermal-denaturation studies showed that ZT65B effectively targeted ICBa and ICBb (DeltaTM=6.5 and 7.0 degrees C) in preference to the other DNA hairpins (<3.5 degrees C), with the exception of ICBc (5.0 degrees C). DNase I-footprinting assays were carried out with the topoisomerase IIalpha-promoter sequence, which contains five ICB sites; of these, ICB1 and ICB5 are similar to the ICB site of MDR1. ZT65B was found to selectively bind ICB1 and ICB5; footprints were not observed with ICB2, ICB3, or ICB4. A strong, positive induced ligand band at 325 nm in CD studies confirmed that ZT65B binds in the DNA minor groove. The selectivity of ZT65B binding to hairpins that contained the MDR1 ICB site compared to one that did not (ICBd) was confirmed by surface-plasmon studies, and equilibrium constants of 5x10(6)-1x10(7) and 4.6x10(5) M-1 were obtained with ICB1, ICB5,and ICB2 respectively. ZT65B and the previously published JH37 (J. A. Henry, et al. Biochemistry 2004, 43, 12 249-12 257) serve as prototypes for the design of novel polyamides. These can be used to specifically target the subset of ubiquitous gene elements known as ICBs, and thereby affect the expression of one or a few proteins.

Molecular recognition of DNA base pairs by the formamido/pyrrole and formamido/imidazole pairings in stacked polyamides.

Polyamides containing an N-terminal formamido (f) group bind to the minor groove of DNA as staggered, antiparallel dimers in a sequence-specific manner. The formamido group increases the affinity and binding site size, and it promotes the molecules to stack in a staggered fashion thereby pairing itself with either a pyrrole (Py) or an imidazole (Im). There has not been a systematic study on the DNA recognition properties of the f/Py and f/Im terminal pairings. These pairings were analyzed here in the context of f-ImPyPy, f-ImPyIm, f-PyPyPy and f-PyPyIm, which contain the central pairing modes, -ImPy- and -PyPy-. The specificity of these triamides towards symmetrical recognition sites allowed for the f/Py and f/Im terminal pairings to be directly compared by SPR, CD and DeltaT (M) experiments. The f/Py pairing, when placed next to the -ImPy- or -PyPy- central pairings, prefers A/T and T/A base pairs to G/C base pairs, suggesting that f/Py has similar DNA recognition specificity to Py/Py. With -ImPy- central pairings, f/Im prefers C/G base pairs (>10 times) to the other Watson-Crick base pairs; therefore, f/Im behaves like the Py/Im pair. However, the f/Im pairing is not selective for the C/G base pair when placed next to the -PyPy- central pairings.

Targeting the inverted CCAAT box 2 in the topoisomerase IIalpha promoter by JH-37, an imidazole-pyrrole polyamide hairpin: design, synthesis, molecular biology, and biophysical studies.

The topoisomerase IIalpha promoter is regulated through transcription factor interactions with five inverted CCAAT boxes (ICBs). In confluent cancer cells, binding of nuclear factor Y to ICB2 represses the expression of this gene, contributing to resistance to topoisomerase II poisons. The ICB sites within the topoisomerase IIalpha promoter are, therefore, potential targets for the design of anticancer drugs and gene control agents. The synthesis and DNA binding properties of a hairpin polyamide molecule (JH-37) that targets 5'-TTGGT-3' found in ICB2 and ICB3 sites are described. Gel shift and DNase I footprinting studies on the topoisomerase IIalpha promoter showed JH-37 to preferentially bind to ICB2,3 and ICB1 sites. The larger DeltaT(M) values for ICB2,3 (8-9 degrees C) over ICB1,4,5 (4-5 degrees C) indicated a preference of JH-37 for ICB2,3. CD titration studies confirmed the binding of JH-37 to the minor groove, with a 1:1 binding stoichiometry. Results from SPR studies showed JH-37 to bind most strongly to ICB2 (K = 3 x 10(7) M(-1)), followed by ICB1, the non-ICB sequence (TGCA), and finally the ICB mutant (ICB2m). The improved binding to ICB2 is largely due to a lower dissociation rate of the compound at the preferred site. To our knowledge, this is the first example on the use of SPR for studying the interactions of hairpin polyamides with DNA. Binding of JH-37 to ICB2 was corroborated by ITC studies, in which the DeltaG degrees of binding is driven by both enthalpy and entropy. With knowledge of the fundamental thermodynamic and kinetic properties that govern the molecular recognition of polyamides with DNA, we are poised to systematically edit the structure of JH-37 in order to further enhance its binding affinity and selectivity for ICB2,3. Our strategy for designing molecules that control gene expression is to target shorter, but multiple, binding sites that are in close array within the promoter. Binding of JH-37 to multiple ICB sites in the topoisomerase IIalpha promoter is an ideal test for this strategy. This approach is in contrast to the traditional strategy of targeting 15-16 base pairs, which has not been successful in actual biological systems due to poor cell uptake and distribution.