Questionable Plasmid Involvement in Streptomyces ATP Nucleotide 3’-Pyrophosphokinase Formation

A few actinomycetes produce ATP nucleotide 3’-pyrophosphokinase, a mechanistic stringent factor analog with broad acceptor spectrum. The plasmid-like DNA of an ATP nucleotide S’mpyrophosphokinase producer Streptomyces violascens was more persistent than the enzyme production after acriflavin treatment. A 7.lKbp long ATP nucleotide 3’- pyrophosphokinase gene-containing fragment, cloned from another producer Stwptomyces more-okaensis, was found to hybridize to both its Xba I- and Spe I-digested genomic DNA at about 400Kbp size, and also with the undigested DNA very close to or at the origin on pulsed field gel electrophoresis-Southern blotting analyses. The gene thus appears to reside not on a plasmid, but on the chromosome

Phase I pharmacokinetic and pharmacodynamic study of the first-in-class spliceosome inhibitor E7107 in patients with advanced solid tumors

Purpose: To assess the safety, tolerability, pharmacokinetics, pharmacodynamics, and clinical activity of E7107 administered as 5-minute bolus infusions on days 1, 8, and 15 in a 28-day schedule. Experimental Design: Patients with solid tumors refractory to standard therapies or with no standard treatment available were enrolled. Dose levels of 0.6 to 4.5 mg/m2 were explored. Results: Forty patients [24M/16F, median age 61 years (45-79)] were enrolled. At 4.5 mg/m2, doselimiting toxicity (DLT) consisted of grade 3 diarrhea, nausea, and vomiting and grade 4 diarrhea, respectively, in two patients. At 4.0 mg/m2, DLT (grade 3 nausea, vomiting, and abdominal cramps) was observed in one patient. Frequently occurring side effects were mainly gastrointestinal. After drug discontinuation at 4.0 mg/m 2, one patient experienced reversible grade 4 blurred vision. The maximum tolerated dose (MTD) is 4.0 mg/m2. No complete or partial responses during treatment were observed; one patient at 4.0 mg/m2 had a confirmed partial response after drug discontinuation. Pharmacokinetic analysis revealed a large volume of distribution, high systemic clearance, and a plasma elimination halflife of 5.3 to 15.1 hours. Overall drug exposure increased in a dose-dependent manner. At the MTD, mRNA levels of selected target genes monitored in peripheral blood mononuclear cells showed a reversible 15- to 25-fold decrease, whereas unspliced pre-mRNA levels of DNAJB1 and EIF4A1 showed a reversible 10- to 25-fold increase. Conclusion: The MTD for E7107 using this schedule is 4.0 mg/m2. Pharmacokinetics is dose-dependent and reproducible within patients. Pharmacodynamic analysis revealed dose-dependent reversible inhibition of pre-mRNA processing of target genes, confirming proof-of-principle activity of E7107

H3B-8800, an Orally Bioavailable Modulator of the SF3b Complex, Shows Efficacy in Spliceosome-Mutant Myeloid Malignancies

Abstract Mutations in RNA splicing factors confer an alteration of function and are common in patients with myelodysplastic syndrome (MDS, ~45%), chronic myelomonocytic leukemia (CMML, ~60%), and acute myeloid leukemia (AML) derived from these conditions. Recent data suggest that spliceosome-mutant cells are preferentially sensitive to genetic or pharmacologic splicing modulation compared with wildtype (WT) counterparts. Here, we describe the discovery of H3B-8800, a potent and orally bioavailable modulator of the SF3b complex, and demonstrate efficacy in models of spliceosome mutant myeloid malignancies including a novel xenograft system for CMML. H3B-8800 was identified through a medicinal chemistry approach aimed at identifying compounds with preferential lethality in spliceosome mutant cells. Using a scintillation proximity assay, we demonstrated that H3B-8800 potently binds to SF3b complexes containing either WT or mutant SF3B1 protein. Consistent with this, H3B-8800 showed dose-dependent modulation of splicing in in vitro biochemical splicing assays and cellular pharmacodynamic assays. Selectivity of H3B-8800 for the SF3b complex was confirmed through observing resistance in cells expressing SF3B1R1074H, an SF3B1 mutation previously shown to confer resistance to natural product splicing modulators. In the above biochemical and cellular assays, H3B-8800 affected splicing similarly regardless of spliceosome genotype. However, preferential inhibition of in vitro cell growth was observed in isogenic AML cells with endogenous knock-in of SF3B1K700E or SRSF2P95H mutations compared to WT counterparts. In animals xenografted with SF3B1K700E knock-in K562 cells, oral H3B-8800 treatment demonstrated dose-dependent splicing modulation and inhibited tumor growth, while no therapeutic impact was seen in WT controls. Similarly, anti-leukemic efficacy and improved survival were observed with H3B-8800 treatment in mice transplanted with Srsf2P95H/MLL-AF9 mouse AML cells, a result not seen in Srsf2 WT/MLL-AF9 counterpart leukemias. To understand the preferential effects on spliceosome mutant cells, RNA-seq analysis of isogenic K562 cells treated with H3B-8800 was performed. H3B-8800 induced intron retention and exon skipping, however these effects were not global and introns preferentially retained by H3B-8800 were shorter and more GC-rich compared to those unaffected by drug (Figure A). Interestingly, a substantial number of genes experiencing intron retention with H3B-8800 themselves encoded spliceosome components (Figure B). This suggests that the preferential effect of H3B-8800 on spliceosome mutant cells is due to the exquisite dependency of these cells on normal expression of spliceosome proteins. Next we aimed to understand the therapeutic potential of H3B-8800 in the context of CMML due to the high frequency of SRSF2 mutations and the need for improved outcome in this disorder. To this end, we developed a xenotransplantation model through direct intrafemoral injection of CD34+ cells from CMML patients into "NSGS" mice: a variant of NSG mice that express human IL3, SCF and GM-CSF. We specifically focused on CMML with 200,000 CD34+ cells achieved robust engraftment for all patients (n=7) with rapid lethality (median of 39 days). In vivo H3B-8800 administration substantially reduced leukemic burden in spliceosome-mutant but not spliceosome-WT CMML PDX (Figure C). Furthermore, 2.2-fold reductions in immunophenotypically-defined leukemia initiating cells were seen with H3B-8800 versus vehicle treatment in spliceosome-mutant CMML compared with no change in those mice engrafted with spliceosome-WT CMML. These data identify a novel therapeutic approach with selective lethality in myeloid cells bearing a spliceosome mutation. Despite the essential nature of splicing, CMML/AML cells without a spliceosome mutation were less sensitive to H3B-8800 compared with potent eradication of mutant counterparts. These data demonstrate the therapeutic potential of splicing modulation in spliceosome mutant cancers and H3B-8800 is currently undergoing clinical evaluation in patients with MDS, AML and CMML. Figure. Figure. Disclosures Buonamici: H3 Biomedicine: Employment. Thomas:H3 Biomedicine: Employment. Seiler:H3 Biomedicine: Employment. Chan:H3 Biomedicine: Employment. Caleb:H3 Biomedicine: Employment. Darman:H3 Biomedicine: Employment. Fekkes:H3 Biomedicine: Employment. Karr:H3 Biomedicine: Employment. Liu:H3 Biomedicine: Employment. Meeske:H3 Biomedicine: Employment. Mizui:Eisai: Employment. Pazolli:H3 Biomedicine: Employment. Prajapati:H3 Biomedicine: Employment. Wang:Eisai: Employment. Warmuth:H3 Biomedicine: Employment. Yu:H3 Biomedicine: Employment. Zhu:H3 Biomedicine: Employment. Smith:H3 Biomedicine: Employment

Abstract 1185: H3B-8800, a novel orally available SF3b modulator, shows preclinical efficacy across spliceosome mutant cancers

Abstract Genomic characterization of hematologic and solid cancers has revealed recurrent somatic mutations affecting genes encoding the RNA splicing factors SF3B1, U2AF1, SRSF2 and ZRSR2. Recent data reveal that these mutations confer an alteration of function inducing aberrant splicing and rendering spliceosome mutant cells preferentially sensitive to splicing modulation compared with wildtype (WT) cells. Here we describe a novel orally bioavailable small molecule SF3B1 modulator identified through a medicinal chemistry effort aimed at optimizing compounds for preferential lethality in spliceosome mutant cells. H3B-8800 potently binds to WT or mutant SF3b complexes and modulates splicing in in vitro biochemical splicing assays and cellular pharmacodynamic assays. The selectivity of H3B-8800 was confirmed by observing lack of activity in cells expressing SF3B1R1074H, the SF3B1 mutation previously shown to confer resistance to other splicing modulators. Although H3B-8800 binds both WT and mutant SF3B1, it results in preferential lethality of cancer cells expressing SF3B1K700E, SRSF2P95H, or U2AF1S34F mutations compared to WT cells. In animals xenografted with SF3B1K700E knock-in leukemia K562 cells or mice transplanted with Srsf2P95H/MLL-AF9 mouse AML cells, oral H3B-8800 treatment demonstrated splicing modulation and inhibited tumor growth, while no therapeutic impact was seen in WT controls. These data were also evident in patient-derived xenografts (PDX) from patients with CMML where H3B-8800 resulted in a substantial reduction of leukemic burden only in SRSF2-mutant but not in WT CMML PDX models. Additionally, due to the high frequency of U2AF1 mutations in non-small cell lung cancer, H3B-8800 was tested in U2AF1S34F-mutant H441 lung cancer cells. Similar to the results from leukemia models, H3B-8800 demonstrated preferential lethality of U2AF1-mutant cells in vitro and in in vivo orthotopic xenografts at well tolerated doses. RNA-seq of isogenic K562 cells treated with H3B-8800 revealed dose-dependent inhibition of splicing. Although global inhibition of RNA splicing was not observed; H3B-8800 treatment led to preferential intron retention of transcripts with shorter and more GC-rich regions compared to those unaffected by drug. Interestingly, H3B-8800-retained introns commonly disrupted the expression of spliceosomal genes, suggesting that the preferential effect of H3B-8800 on spliceosome mutant cells is due to the dependency of these cells on expression of WT spliceosomal genes. These data identify a novel therapeutic approach with selective lethality in leukemias and lung cancers bearing a spliceosome mutation. Despite the essential nature of splicing, cancer cells without a spliceosome mutation were less sensitive to H3B-8800 compared with potent eradication of mutant counterparts. H3B-8800 is currently undergoing clinical evaluation in patients with MDS, AML, and CMML. Citation Format: Silvia Buonamici, Akihide Yoshimi, Michael Thomas, Michael Seiler, Betty Chan, Benjamin Caleb, Fred Csibi, Rachel Darman, Peter Fekkes, Craig Karr, Gregg Keaney, Amy Kim, Virginia Klimek, Pavan Kumar, Kaiko Kunii, Stanley Chun-Wei Lee, Xiang Liu, Crystal MacKenzie, Carol Meeske, Yoshiharu Mizui, Eric Padron, Eunice Park, Ermira Pazolli, Sudeep Prajapati, Nathalie Rioux, Justin Taylor, John Wang, Markus Warmuth, Huilan Yao, Lihua Yu, Ping Zhu, Omar Abdel-Wahab, Peter Smith. H3B-8800, a novel orally available SF3b modulator, shows preclinical efficacy across spliceosome mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1185. doi:10.1158/1538-7445.AM2017-118

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

Total Synthesis of 6‑Deoxypladienolide D and Assessment of Splicing Inhibitory Activity in a Mutant SF3B1 Cancer Cell Line

A total synthesis of the natural product 6-deoxypladienolide D (<b>1</b>) 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 (<b>1</b>) demonstrates potent growth inhibition in a mutant SF3B1 cancer cell line, high binding affinity to the SF3b complex, and inhibition of pre-mRNA splicing

Total Synthesis of 6‑Deoxypladienolide D and Assessment of Splicing Inhibitory Activity in a Mutant SF3B1 Cancer Cell Line

A total synthesis of the natural product 6-deoxypladienolide D (<b>1</b>) 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 (<b>1</b>) demonstrates potent growth inhibition in a mutant SF3B1 cancer cell line, high binding affinity to the SF3b complex, and inhibition of pre-mRNA splicing