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CCR Translation for This Article from Phase I Pharmacokinetic and Pharmacodynamic Study of the First-in-Class Spliceosome Inhibitor E7107 in Patients with Advanced Solid Tumors

Supplementary Data - PDF file 104K, Supplementary Data: Eligibility criteria and Pharmacodynamic analysis: methodology sections

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, dose-limiting 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/m2, 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 half-life 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. Clin Cancer Res; 19(22); 6296–304. ©2013 AACR.

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., Small molecule modulators of RNA splicing have therapeutic potential in tumours bearing spliceosome mutations. Here, the authors identify BCL2 genes have differential sensitivities to SF3b-targeting splicing modulators and combination of SF3b-targeting splicing modulators and BCLxL inhibition induces synergistic cytotoxicity in cancer cells.

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.

Hypoxia-inducible factor-1 (HIF-1) is the chief transcription factor regulating hypoxia-driven gene expression. HIF-1 overexpression is associated with poor prognosis in several cancers and therefore represents an attractive target for novel antitumor agents. We explored small molecule inhibitors of the HIF-1 pathway. Using high-throughput-screening, we identified benzanilide compound 1 (IC50=560 nM) as a seed. Subsequent extensive derivatization led to the discovery of compounds 43a and 51d, with anti-HIF-1 activities in vitro (IC50=21 and 0.47 nM, respectively), and in vivo. Additionally, 43a (12.5-100mg/kg) also displayed in vivo anti-tumor efficacy, without influencing body weight.

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. Citation Format: Daniel Aird, Teng Teng, Chia-Ling Huang, Ermira Pazolli, Deepti Banka, Kahlin Cheung-Ong, Cheryl Eifert, Craig Furman, Jeremy Wu, Michael Seiler, Silvia Buonamici, Peter Fekkes, Craig Karr, James Palacino, Eunice Park, Peter Smith, Lihua Yu, Yoshiharu Mizui, Markus Warmuth, Agustin Chicas, Laura Corson, Ping Zhu. Sensitivity to splicing modulation of BCL2 family genes reveals cancer therapeutic strategies for splicing modulators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 281.

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(1–6). Cancer cells bearing these mutations are preferentially dependent on wild-type (WT) spliceosome function(7–11), 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.

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 Bact 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.

Hypoxia-inducible factor 1 (HIF-1) activates the transcription of genes that play crucial roles in the adaptation of cancer cells to hypoxia. HIF-1α overexpression has been associated with poor prognosis in patients with various types of cancer. Here, we describe ER-400583-00 as a novel HIF-1 inhibitor. ER-400583-00 suppressed the production of HIF-1α protein in response to hypoxia, with a half-maximal inhibitory concentration value of 3.7 nM in human U251 glioma cells. The oral administration of 100 mg/kg ER-400583-00 to mice bearing U251 tumor xenografts resulted in a rapid suppression of HIF-1α that persisted for 24 h. Immunohistochemical analysis revealed that ER-400583-00 suppressed the proliferation of cancer cells most prominently in areas distal to the region of blood perfusion, where HIF-1α-expressing hypoxic cancer cells were located. These hypoxic cancer cells were resistant to radiation therapy. ER-400583-00 showed a synergistic interaction with radiation therapy in terms of antitumor activity. These data suggest that HIF-1 blockade by small compounds may have therapeutic value in cancer, especially in combination with radiation therapy.

Pladienolide is a naturally occurring macrolide that binds to the SF3b complex to inhibit mRNA splicing. It has not been fully validated whether the splicing impairment is a relevant mechanism for the potent antitumor activity of pladienolide. We established pladienolide-resistant clones from WiDr and DLD1 colorectal cancer cells that were insensitive to the inhibitory action of pladienolide on cell proliferation and splicing. An mRNA-Seq differential analysis revealed that these two cell lines have an identical mutation at Arg1074 in the gene for SF3B1, which encodes a subunit of the SF3b complex. Reverse expression of the mutant protein transferred pladienolide resistance to WiDr cells. Furthermore, immunoprecipitation analysis using a radiolabeled probe showed that the mutation impaired the binding affinity of paldienolide to its target. These results clearly demonstrate that pladienolide exerts its potent activity by targeting SF3b and also suggest that inhibition of SF3b is a promising drug target for anticancer therapy.

The goal of this study is to identify a novel inhibitor with anti-inflammatory and antiproliferative properties for the treatment of psoriasis. Compound f152A1 \[(3 S ,5 Z ,8 S ,11 E )-8,9,16-trihydroxy-14-methoxy-3-methyl-3,4,9,10-tetrahydro-1 H -benzo[c\]\[1\]oxacyclotetradecine1,7(8 H )-dione] was identified as the main active metabolite with strong inhibitory activity against tumor necrosis factor-α (TNFα) transcription in a fraction originated from the fermentation broth of the fungus Curvularia verruculosa. Although active in cell-based assays, f152A1 was unstable in plasma and liver microsome preparations, thus limiting its pharmaceutical utilization. To improve the metabolic properties of f152A1, a medicinal chemistry program was undertaken, resulting in the generation of over 400 analogs of f152A1. Eventually, E6201 [(3 S ,4 R ,5 Z ,8 S ,9 S ,11 E )-14-(ethylamino)-8,9,16-trihydroxy-3,4-dimethyl-3,4,9,19-tetrahydro-1 H -2-benzoxacyclotetradecine-1,7(8 H )-dione] was identified as a promising analog in this series. In the present study, we characterized the in vitro activities of E6201 and discovered that the compound inhibits lipopolysaccharide-activated TNFα reporter activity in THP-1-33 cells with an IC50 value of 50 nM and selectively inhibits mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)-1 and MEK kinase-1 in cell-free biochemical assays. In addition, E6201 showed inhibitory activity in several other cell-based systems: 1) phosphorylation of c-jun N-terminal kinase and p38 MAPKs; 2) nuclear factor-κB and activated protein-1 activation in various cell types; 3) interleukin (IL)-2 production from human lymphocytes; 4) hyperproliferation of human keratinocytes; 5) IL-8 production from human keratinocytes; and 6) proinflammatory cytokine production from human peripheral blood mononuclear cells. Based on the data presented here, E6201 may be beneficial for treatment of inflammatory and hyperproliferative diseases such as psoriasis through its anti-inflammatory activities on immune cells and antihyperproliferative activities on keratinocytes.

Pladienolide is a naturally occurring antitumor macrolide that was discovered by using a cell-based reporter gene expression assay controlled by the human vascular endothelial growth factor promoter. Despite the unique mechanisms of action and prominent antitumor activities of pladienolides B and D in diverse in vitro and in vivo systems, their target protein has remained unclear. We used 3H-labeled, fluorescence-tagged and photoaffinity/biotin (PB)-tagged 'chemical probes' to identify a 140-kDa protein in splicing factor SF3b as the binding target of pladienolide. Immunoblotting of an enhanced green fluorescent protein fusion protein of SF3b subunit 3 (SAP130) revealed direct interaction between the PB probe and SAP130. The binding affinities of pladienolide derivatives to the SF3b complex were highly correlated with their inhibitory activities against reporter gene expression and cell proliferation. Furthermore, pladienolide B impaired in vivo splicing in a dose-dependent manner. Our results demonstrate that the SF3b complex is a pharmacologically relevant protein target of pladienolide and suggest that this splicing factor is a potential antitumor drug target.

We have discovered seven novel 12-membered macrolides, pladienolides A to G, from Streptomyces platensis Mer-11107, with pladienolide B the most potently inhibiting hypoxia induced-VEGF expression and proliferation of the U251 cancer cell line. A growth inhibitory study using a 39-cell line drug-screening panel demonstrated that pladienolide B has strong antitumor activities in vitro. A COMPARE analysis reveals that it has a unique antitumor spectrum that sets it apart from anticancer drugs currently in clinical use. This result suggests that pladienolide B has a novel mechanism of action. A series of xenograft studies were conducted to evaluate the in vivo potency of pladienolides. Pladienolide B extensively inhibited tumor growth in xenograft models. In the most sensitive model, using BSY-1 xenografts, tumors were completely regressed by administration of pladienolide B. For the reason of their novel mechanism of action and excellent in vivo efficacy, pladienolides appear to have major potential for use in cancer treatment.

Dysregulation of RNA splicing can cause various forms of cancer and neuromuscular disorders. Thus, developing compounds with splicing-modulating activity represents a promising therapeutic approach for these diseases. Natural products such as pladienolide, herboxidiene, and spliceostatin have been identified as potent splicing modulators that bind SF3B1, a member of the SF3b subcomplex that assembles into the U2 snRNP. Using integrated chemogenomic, structural and biochemical approaches, we show that PHF5A, another core component of the SF3b complex, is also targeted by these modulators. Whole exome sequencing of E7107 (pladienolide analogue) and herboxidiene resistant clones identified common mutations in either PHF5A-Y36, SF3B1-K1071, SF3B1-R1074, or SF3B1-V1078, which confers resistance to these modulators as assessed by splicing modulation and cell growth inhibition, suggesting a common site of interaction for these splicing modulators. We determine the crystal structure of human PHF5A and find that Y36 is located on the surface in a region of high sequence conservation. Analysis of the cryo-EM spliceosome Bact complex from yeast shows that these mutations cluster in a well-defined pocket surrounding the branch point adenosine suggesting a possible competitive mode of action for these modulators. Whole-transcriptome RNA-seq analysis reveals that PHF5A Y36C alters the profile of splicing modulators from inducing intron-retention events to exon-skipping events. Furthermore, the differential in GC content between adjacent introns and exons correlates with the relative intron strength, making some splicing events more susceptible to modulation. Collectively, we propose that PHF5A-SF3B1 is a central node for binding to these small-molecule splicing modulators offering novel approaches to modulate specific splicing events. Citation Format: Teng Teng, Jennifer Tsai, Xiaoling Puyang, Michael Seiler, Shouyong Peng, Daniel Aird, Silvia Buonamici, Benjamin Caleb, Betty Chan, Laura Corson, Jacob Feala, Peter Fekkes, Craig Karr, Manav Korpal, Yoshiharu Mizui, Eunice Park, James Palacino, Peter Smith, Vanitha Subramanian, Jeremy Wu, Lihua Yu, Agustin Chicas, Markus Warmuth, Nicholas Larsen, Ping Zhu. A chemogenomic approach reveals the action of splicing modulators at the branch point adenosine binding pocket defined by the PHF5A/SF3b complex [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 126. doi:10.1158/1538-7445.AM2017-126

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.AM2017-1185

Aggrecanase-1, also known as ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4), cleaves at the Glu373-Ala374 site of aggrecan, thereby indicating aggrecan degradation. It is thought that ADAMTS4 plays a pivotal role in inflammatory joint diseases and cartilage degradation. To elucidate the mechanisms of regulation of ADAMTS4 gene expression, we cloned the 5′-flanking region of the human ADAMTS4 gene and characterized its promoter activity by means of reporter assay using porcine chondrocytes and NIH3T3 cells. Reporter gene analysis using deletion variants suggested that the region between −383 and +10 relative to the tentative transcription start site is necessary for full promoter activity; this region contains one Sp1 and three AP2 sites. In addition, the segment between −726 and −384 appears to contain silencer element(s). A complete deletion mutant of the nuclear factor I (NFI) binding site at −441 to −429 resulted in recovery of the promoter activity in chondrocytes, but not in NIH3T3 cells. Thus, the NFI site is involved in negative regulation of the human ADAMTS4 promoter activity in chondrocytes.

Tumor necrosis factor-alpha (TNFalpha) is a cytokine that induces pleiotropic inflammatory reactions. Soluble TNFalpha is released from its membrane-bound precursor by TNFalpha converting enzyme (TACE)/a disintegrin and metalloproteinase 17 (ADAM17). We have recently cloned the mouse TACE complete cDNA and the 5' flanking promoter region of the gene. Two versions of the mouse TACE cDNA having a coding region of 2541bp were obtained: one was about 4.1kb and the other was 4.4kb in length, in which only the length of the 3' UTR was different. Rapid amplification of 5' cDNA ends suggested that there were multiple transcriptional start sites. From the coding sequence, 827 amino acids were deduced which were 91.9 % identical to those of human TACE. Northern blot analysis indicated that the major transcript was approx. 4.4kb product in the mouse. The mouse TACE mRNA was ubiquitously expressed, and was particularly high in the lung. The proximal promoter contained multiple AP2 and Sp1 transcription factor binding sites and included a GC box and a CCAAT box, but lacked a consensus TATA box. Reporter gene analysis using RAW264.7 cells showed that the fragment at nt -290 to -1 from the translation start site has a strong promoter activity, and appeared to be essential for transcription of the mouse TACE mRNA. Finally, we found that the mouse TACE promoter at nt -2304 to -1 also worked in NIH3T3, 3T3L1 and C6 cell lines.

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, dose-limiting 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/m2, 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 half-life 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. Clin Cancer Res; 19(22); 6296–304. ©2013 AACR.

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.

Recent discoveries that splicing factors such SF3B1, U2AF1, SRSF2 are frequently mutated in multiple hematological malignancies including chronic lymphocytic leukaemia and myelodysplastic syndromes have generated interest in therapeutic approaches to target the splicesome dependency in cancer cells bearing mutations in splicing factors. Previously, several structurally unrelated natural compounds including pladienolide, herboxidiene, and FR901464 have been shown to exert potent anti-proliferative effects in cancer cells grown in vitro. Further optimization has led to the discovery of natural product analogs (e.g. E7107) with anti-tumor efficacy in vivo in xenograft models. Target identification has revealed the SF3B complex of the splicesome as the common action site for these compounds. Recent work has demonstrated biological and genetic evidence that single amino acid substitution (R1074H) in SF3B1 completely abolished the anti-proliferative effect of pladienolide derivative E7107, suggesting that SF3B1 is the direct binding partner for pladienolides. However, the same SF3B1 R1074H mutation does not provide equal level of protections for cells treated with herboxidiene derivatives, indicating differential mechanism of action for these two classes of splicing modulators. To identify targets for herboxidiene-like compounds, we have generated resistant HCT116 clones upon continuous administration of herboxidiene derivative H3B-37045 in vitro. Whole exome sequencing from 6 resistant clones revealed a common Y36C mutation in SF3B subunit component PHF5A (SF3B14b). Over-expression of PHF5A Y36C but not the wild-type form in parental HCT116 cells confirmed the protective effect of this mutation to H3B-37045. Surprisingly, PHF5A Y36C expression also conferred resistance to the pladienolide derivative E7107, which indicates that, unlike the SF3B1 R1074H mutation, PHF5A resides within a common node of action site among different splicing modulators. RNA-seq, biochemical and structure homology-modeling analysis suggested that PHF5A Y36C mutation disrupted the action of splicing modulators through interfering with the compounds’ interaction with the SF3B complex. Detailed analysis of the function of the Y36C mutant and wild-type PHF5A in the SF3B complex is currently ongoing. Understanding the function of PHF5A in splicing and the molecular mechanism of Y36C mutation shall provide new insights of the biological role of splicesome, and guide the development of next generation splicesome inhibitors. Citation Format: Teng Teng, Xiaoling Puyang, Shouyong Peng, Jacob Feala, Betty Chan, Jennifer Tsai, Benjamin Caleb, Craig Karr, Eunice Park, Laura Corson, Yoshiharu Mizui, Peter Smith, Nicholas Larsen, Lihua Yu, Markus Warmuth, Ping Zhu, Agustin Chicas. Identification of PHF5A as a common cellular target of splicing-modulating chemical probes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3013.

Pladienolide is a naturally occurring macrolide that binds to the SF3b complex to inhibit mRNA splicing. It has not been fully validated whether the splicing impairment is a relevant mechanism for the potent antitumor activity of pladienolide. We established pladienolide-resistant clones from WiDr and DLD1 colorectal cancer cells that were insensitive to the inhibitory action of pladienolide on cell proliferation and splicing. An mRNA-Seq differential analysis revealed that these two cell lines have an identical mutation at Arg1074 in the gene for SF3B1, which encodes a subunit of the SF3b complex. Reverse expression of the mutant protein transferred pladienolide resistance to WiDr cells. Furthermore, immunoprecipitation analysis using a radiolabeled probe showed that the mutation impaired the binding affinity of paldienolide to its target. These results clearly demonstrate that pladienolide exerts its potent activity by targeting SF3b and also suggest that inhibition of SF3b is a promising drug target for anticancer therapy.

The goal of this study is to identify a novel inhibitor with anti-inflammatory and antiproliferative properties for the treatment of psoriasis. Compound f152A1 [(3S,5Z,8S,11E)-8,9,16-trihydroxy-14-methoxy-3-methyl-3,4,9,10-tetrahydro-1H-benzo[c][1]oxacyclotetradecine1,7(8H)-dione] was identified as the main active metabolite with strong inhibitory activity against tumor necrosis factor-alpha (TNFalpha) transcription in a fraction originated from the fermentation broth of the fungus Curvularia verruculosa. Although active in cell-based assays, f152A1 was unstable in plasma and liver microsome preparations, thus limiting its pharmaceutical utilization. To improve the metabolic properties of f152A1, a medicinal chemistry program was undertaken, resulting in the generation of over 400 analogs of f152A1. Eventually, E6201 [(3S,4R,5Z,8S,9S,11E)-14-(ethylamino)-8,9,16-trihydroxy-3,4-dimethyl-3,4,9,19-tetrahydro-1H-2-benzoxacyclotetradecine-1,7(8H)-dione] was identified as a promising analog in this series. In the present study, we characterized the in vitro activities of E6201 and discovered that the compound inhibits lipopolysaccharide-activated TNFalpha reporter activity in THP-1-33 cells with an IC(50) value of 50 nM and selectively inhibits mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)-1 and MEK kinase-1 in cell-free biochemical assays. In addition, E6201 showed inhibitory activity in several other cell-based systems: 1) phosphorylation of c-jun N-terminal kinase and p38 MAPKs; 2) nuclear factor-kappaB and activated protein-1 activation in various cell types; 3) interleukin (IL)-2 production from human lymphocytes; 4) hyperproliferation of human keratinocytes; 5) IL-8 production from human keratinocytes; and 6) proinflammatory cytokine production from human peripheral blood mononuclear cells. Based on the data presented here, E6201 may be beneficial for treatment of inflammatory and hyperproliferative diseases such as psoriasis through its anti-inflammatory activities on immune cells and antihyperproliferative activities on keratinocytes.

With bioactivity-guided phenotype screenings, a potent anti-inflammatory compound f152A1 has been isolated, characterized and identified as the known natural product LL-Z1640-2. Metabolic instability precluded its use for the study on animal disease models. Via total synthesis, a potent, metabolically stabilized analog ER-803064 has been created; addition of the (S)-Me group at C4 onto f152A1 has resulted in a dramatic improvement on its metabolic stability, while preserving the anti-inflammatory activities.

Heterozygous mutations in several core members of the spliceosome complex have been reported in Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML). In particular high frequency SF3B1 hotspot mutations, a component of the U2 complex involved in the interaction with the branch point (BP) and recognition of the 3' splice sites (ss) during splicing, have been identified in Refractory Anemia with Ringed Sideroblasts (RARS) a subtype of MDS. Using computational analyses of RNAseq from several cancer types including RARS, we identified that SF3B1 hotspot mutations induce aberrant 3'ss selection by recognizing a cryptic AG located between 15 to 24 nucleotides upstream of the canonical AG. Experimental confirmation of these motif features was performed using minigenes in SF3B1 mutant cells. Furthermore, we discovered that SF3B1 mutant utilized a different BP from that used by SF3B1 wild-type providing novel mechanistic insights into changes in function induced by the hotspot mutations. The induction of aberrant splicing can introduce premature termination codons thus targeting mRNA for degradation by Nonsense Mediated Decay (NMD). We predicted that close to 50% of the aberrantly spliced genes would be subject to NMD and showed (using isogenic Nalm-6 cells engineered by AAV homology to express SF3B1K700E or SF3B1K700K) that several of these genes were downregulated at the transcript and protein levels. These downregulated genes/proteins might be involved in the pathogenesis of SF3B1 mutant cancers. Interestingly, pathway analysis of genes differentially expressed or aberrantly spliced in SF3B1 mutant compared to wild-type in RARS samples identified cell differentiation and epigenetics as the primary misregulated pathways. To study the impact of SF3B1 mutations on differentiation, we used the TF-1 differentiation cell model where erythroid differentiation is induced by treatment with erythropoietin (EPO). EPO treatment, as expected, induced erythroid differentiation in TF-1 cells transduced with SF3B1WT, but a block in erythroid differentiation was observed in TF-1 cells transduced with SF3B1K700E (the most common mutation in MDS and chronic lymphocytic leukemia (CLL)). Intriguingly, SF3B1G742D, which is found mutated in CLL but not MDS, did not block differentiation in this myeloid differentiation model, implying that specific SF3B1 mutations and splicing aberrations have important context dependent effects. Pathway analysis comparing SF3B1K700E vs. SF3B1WT or SF3B1G742D identified several genes involved in heme biosynthesis or downstream of GATA1 to be downregulated (such as, AHSP, ALAS2, CCL5, CD36, EPOR, GP1BB, HBB, HBE1, HBG1, PRG2) in SF3B1K700E cells only. This is consistent with the role of SF3B1K700E in RARS. In our analyses, we also identified that ABCB7 is aberrantly spliced and that the aberrant transcript is subject to NMD, causing downregulation of the canonical transcript and protein. ABCB7 is a mitochondrial transporter important in cellular iron metabolism and in heme production; moreover, partial loss of function mutation in ABCB7 has been identified in X-linked sideroblastic anemia and ataxia, demonstrating an iron overload phenotype in cells with defective ABCB7. Interestingly, when ABCB7 was knocked down in TF-1 cells we observed block in differentiation similar to that observed in SF3B1K700E cells suggesting a link between SF3B1 mutation and ABCB7 levels and impaired differentiation. Taken together, these data suggest that SF3B1 mutations induce aberrant splicing and as a consequence downregulation of several genes that contribute to the block in erythroid differentiation, one of the key biological defects observed in MDS. Disclosures Buonamici: H3 Biomedicine: Employment. Darman:H3 Biomedicine: Employment. Perino:H3 Biomedicine: Employment. Agrawal:H3 Biomedicine: Employment. Peng:H3 Biomedicine: Employment. Seiler:H3 Biomedicine: Employment. Feala:H3 Biomedicine: Employment. Bailey:H3 Biomedicine: Employment. Chan:H3 Biomedicine: Employment. Fekkes:H3 Biomedicine: Employment. Keaney:H3 Biomedicine: Employment. Kumar:H3 Biomedicine: Employment. Kunii:H3 Biomedicine: Employment. Lee:H3 Biomedicine: Employment. Mackenzie:Eisai: Employment. Matijevic:Eisai: Employment. Mizui:H3 Biomedicine: Employment. Myint:Eisai: Employment. Park:H3 Biomedicine: Employment. Pazolli:H3 Biomedicine: Employment. Thomas:H3 Biomedicine: Employment. Wang:H3 Biomedicine: Employment. Warmuth:H3 Biomedicine: Employment. Yu:H3 Biomedicine: Employment. Zhu:H3 Biomedicine: Employment. Furman:Acerta Pharma BV: Research Funding; Gilead: Consultancy; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Speakers Bureau. Ebert:Celgene: Consultancy; H3 Biomedicine: Consultancy; Genoptix: Consultancy, Patents & Royalties. Smith:H3 Biomedicine: Employment.

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.

Refractory Anemia with Ringed Sideroblasts (RARS), a subtype of Myelodysplatic Syndrome (MDS), occurs with a high frequency of hotspot mutations in HEAT (Huntingtin, Elongation factor 3, protein phosphatase 2A, Targets of rapamycin 1 domains) domains of SF3B1. This protein component of the U2 snRNP complex of the spliceosome is essential in the proper selection and usage of 3' splice sites. RNAseq analysis of MDS and other tumor types in which SF3B1 hotspot mutations have been found show that alternative 3' splice site usage is the predominant cause of RNA transcript aberration. These modifications can result in mRNAs encoding novel peptides, or they can introduce premature termination codons into the pre-mRNA, most likely directing it to the Nonsense Mediated Decay (NMD) pathway for degradation. Using a predictive tool to determine the likelihood of a given aberrant transcript to be targeted for NMD, we determined that nearly 50% of the SF3B1-mutant-associated aberrant transcripts were candidates for degradation. We confirmed this experimentally by treating isogenic Nalm-6 cells (engineered by AAV homology to express SF3B1 K700E or K700K) with or without cycloheximide, an agent known to inhibit translation and RNA degradation by NMD. Investigation of the resulting RNAseq data showed significant rescue of gene expression only for the transcripts predicted to be NMD targets. Ingenuity Pathway Analysis indicated that many of the downregulated genes in SF3B1 mutant samples were involved in differentiation, which has been shown to be dysregulated in MDS. We tested the idea that such modifications in the transcriptome confer selective advantage or impair differentiation in SF3B1 mutant cells. We began by manipulating the expression of ABCB7, one of the genes identified in our RNAseq analysis to be downregulated by aberrant splicing and subsequent NMD. ABCB7 is a mitochondrial transporter important in cellular iron metabolism and, indirectly, in heme production. Additionally, loss of function of ABCB7 is causal in X-linked sideroblastic anemia and has been implicated in RARS MDS. We discovered in our SILAC proteomic analysis that ABCB7 protein was dramatically decreased in K700E SF3B1 Nalm-6 cells relative to K700K Nalm-6, in agreement with our RNAseq analysis. Using doxycycline-inducible shRNA expression, we knocked down ABCB7 mRNA and protein expression in TF-1 erythroblasts. These cells show significant decreases in erythropoeitin (EPO)-induced differentiation when expressing exogenous K700E SF3B1, but not K700R (a very conservative mutation) or WT SF3B1. With direct knock down of ABCB7, we observed a similar phenotype - impairment of EPO-induced differentiation in ABCB7 shRNA-induced cells by Day 7, with no overall decline in cell viability. Interestingly, knock down of SF3B1 expression with shRNA also reduces ABCB7 mRNA. However, it also promotes cell death. This is consistent with the heterozygous nature of SF3B1 hotspot mutations; severe loss of SF3B1 function is deleterious. We propose that hotspot SF3B1 mutants promote aberrant splicing of multiple genes, inducing a general “spliceosomal sickness” in addition to downregulating key genes (e.g. ABCB7) responsible for erythroid differentiation impairment, such as that observed in RARS. Citation Format: Rachel B. Darman, Samantha A. Perino, Michael Seiler, Shouyong Peng, Jacob Feala, Peter Fekkes, Gregg F. Keaney, Kaiko Kunii, Linda Lee, Kian Huat Lim, Yoshiya Oda, Khin Myint, Esther A. Obeng, Ermira Pazolli, Eun Sun Park, John Yuan Wang, Markus Warmuth, Lihua Yu, Ping Zhu, Yoshiharu Mizui, Benjamin L. Ebert, Peter G. Smith, Silvia Buonamici. Mutant SF3B1 downregulates proteins involved in differentiation, including ABCB7. [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 B125.

Heterozygous mutations in SF3B1, a component of the U2 complex involved in the recognition of 3′ splice sites (ss), have been reported with high frequency in refractory anemia with ring sideroblasts (RARS, a subtype of myelodysplastic syndrome, MDS) and have also been observed in chronic lymphocytic leukemia (CLL) and several solid tumors. To study the impact of SF3B1 mutations on splicing, RNAseq data obtained from breast cancer, melanoma, CLL and MDS samples with mutant (SF3B1MUT) or wild-type SF3B1 (SF3B1WT) were compared. The majority of aberrant junctions identified in the samples with mutant SF3B1 utilized an alternative 3′ss, suggesting its neomorphic function. Motif analysis of the sequences used by SF3B1MUT revealed the usage of a cryptic AG with a shorter and weaker polypyrimidine tract. Minigenes with modifications of these sites revealed the importance of both intronic and exonic sequence features for the recognition of the cryptic AG by SF3B1MUT. Of the aberrant junctions identified, several were common across all hotspot mutations and diseases; however, a unique aberrant splicing profile was found for each disease suggesting lineage and disease specific effects. The majority of splicing defects introduced a premature termination codon downstream of the cryptic AG leading to nonsense mediated decay (NMD) of aberrant transcripts and downregulation of gene expression, such as ABCB7. Gene-set enrichment analysis of aberrantly spliced and differentially expressed genes in SF3B1MUT MDS samples identified genes involved in cell differentiation and epigenetic pathways which are known to be deregulated in MDS. The impact on erythroid differentiation by SF3B1MUT was studied in transduced TF-1 cells following erythropoietin (EPO) stimulation. As expected, TF-1 SF3B1WT cells were able to differentiate normally after EPO treatment; however, expression of SF3B1K700E (the most common hotspot mutation found in RARS and CLL) induced a block in erythoid differentiation. This differentiation block was not observed with the expression of SF3B1G742D, a mutation found in CLL but not RARS, suggesting a context dependent role for SF3B1 mutations. Interestingly, the differentiation block observed in SF3B1K700E was associated with cytokine independent growth. Initial mining of RNAseq data from SF3B1MUT TF-1 cells highlighted several aberrantly spliced and NMD-downregulated genes previously implicated in MDS. Finally, a xenograft model was developed by subcutaneous implantation of transduced TF-1 cells. After several passages, an enrichment of TF-1 SF3B1K700E cells was observed, suggesting a growth advantage for SF3B1MUT cells over SF3B1WT cells. This data suggests that the K700E SF3B1 mutation can lead to a block in differentiation and competitive advantage as observed in human RARS. Citation Format: Silvia Buonamici, Samantha Perino, Kian Huat Lim, Jacob Feala, Rachel Darman, Esther Obeng, Richard R. Furman, Suzanna Bailey, Gregg Keaney, Pavan Kumar, Yoshiharu Mizui, Eunice Park, John Wang, Markus Warmuth, Lihua Yu, Ping Zhu, Benjamin L. Ebert, Peter Smith. Mutations in SF3B1 lead to aberrant splicing through cryptic 3′ splice site selection and impair hematopoietic cell differentiation. [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 2040. doi:10.1158/1538-7445.AM2015-2040

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 thus far has been unsuccessful. Alternative splicing of MCL1 converts the anti-apoptotic MCL1 long (MCL1-L) isoform to the BH3-only containing MCL1 short (MCL1-S) isoform. As a potential approach for targeting MCL1-dependent cancers, we explored the use of MCL1 splicing modulators. We screened a unique chemical library of compounds that span a range of splicing activities on various substrates in an in vitro assay. Interestingly, we found a subset of general splicing modulators, as well as a subset of SF3B1 inhibitors, that are capable of driving the distinctive alterations in MCL1 splicing that in turn can trigger preferential killing of MCL1-dependent cell lines. The best modulators induce a prominent down-regulation of MCL1-L, up-regulation of MCL1-S, and accumulation of intron-retained MCL1 transcript. Somewhat surprisingly, several additional avenues of investigation pointed to MCL1-L down-regulation rather than MCL1-S up-regulation as the driver of preferential killing of MCL1-dependent cells. This includes the fact that compound-induced cytotoxicity can be rescued by expression of a MCL1-L cDNA and MCL1-L specific shRNA knockdown is sufficient to kill MCL1-dependent cells. On the other hand, overexpression of MCL1-S cDNA had no significant effect on cells and splicing modulators that induced very high levels of MCL1-S mRNA in the absence potent MCL1-L down-regulation exhibit minimal cytotoxicity. Biochemical characterization and understanding of these MCL1 splicing modulators has enabled further optimization of compounds that can induce potent and preferential killing of MCL1-dependent cancer cell lines in vitro. Preliminary studies in mice bearing MCL1-dependent NSCLC xenografts confirmed current lead compounds can indeed induce rapid down-regulation of MCL1-L, induction of apoptosis, and antitumor activity. Collectively these data yield insight into mechanisms of MCL1 splicing modulation that can trigger acute apoptosis in MCL1-dependent cancers and provides support for the idea of using splicing modulators to target difficult-to-drug oncogenic drivers such as MCL1. Citation Format: Eun Sun Park, Michelle Aicher, Daniel Aird, Silvia Buonamici, Betty Chan, Cheryl Eifert, Peter Fekkes, Craig Furman, Baudouin Gerard, Craig Karr, Gregg Keaney, Kaiko Kunii, Linda Lee, Ermira Pazolli, Sudeep Prajapati, Takashi Satoh, Peter Smith, John Yuan Wang, Karen Wang, Markus Warmuth, Lihua Yu, Ping Zhu, Yoshiharu Mizui, Laura B. Corson. Targeting MCL1-dependent cancers through RNA splicing modulation. [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 2941. doi:10.1158/1538-7445.AM2015-2941

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 O'Shea, 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

Seven new macrolides having a 12-membered ring, which we termed pladienolides, were isolated from the fermentation broth of Streptomyces platensis Mer-11107. Six of the seven pladienolides inhibited hypoxia-induced reporter gene expression controlled by human VEGF promoter with IC50 values of 0.0018-2.89 microM. They also demonstrated growth-inhibitory activity against U251 human glioma cells in vitro. Pladienolides are highly potent inhibitors of both hypoxia signals and cancer cell proliferation, and thus may be useful as antitumor agents.

In the course of our screening using fermented broth from soil microorganisms, novel metabolites (pladienolides), possessing inhibitory activity against vascular endothelial growth factor (VEGF) expression and cancer cell proliferation, were isolated from Streptomyces platensis Mer-11107. Pladienolides A (1), B (2), C (3), D (4), E (5), F (6), and G (7) were found to be novel 12-membered macrolides by spectroscopic studies including 1H, 13C NMR, HMQC, HMBC, and NOE experiments. Pladienolides are unusual 12-membered macrolides having a long side chain at the carbon that bears a lactone oxygen.

Recurrent heterozygous mutations of the spliceosome protein SF3B1 have been identified in myelodysplastic syndromes, chronic lymphocytic leukemia (CLL), breast, pancreatic and skin cancers. SF3B1 is a component of the U2 snRNP complex which binds to the pre-mRNA branch point site and is involved in recognition and stabilization of the spliceosome at the 3′ splice site. To understand the impact of SF3B1 mutations, we compared RNAseq profiles from tumor samples with SF3B1 hotspot mutations (SF3B1-MUT) or wild-type SF3B1 (SF3B1-WT) in breast cancer, melanoma and CLL. This analysis revealed significant increases in the usage of novel alternative splice junctions in SF3B1-MUT samples including selection of alternative 3′ splice sites and less frequently exon skipping. These events induce expression of alternative mRNAs that are translated into novel proteins or aberrant mRNAs that are decayed by cells. A common alternative splicing profile was shared across different hotspot mutations and lineages (e.g. ZDHHC16 and COASY); however, unique alternative splicing profiles were also observed suggesting lineage specific effects. RNAseq analysis of several cell lines with endogenous SF3B1 hotspot mutations confirmed the presence of the same spliced isoforms as observed in tumor samples. To prove that SF3B1-MUT were inducing alternative splicing, transient transfection of several SF3B1 hotspot mutations in 293FT cells induced the expression of the common alternatively spliced genes suggesting functional similarity. Selective shRNA depletion of mutant SF3B1 allele in SF3B1-MUT cells resulted in downregulation of the same splice isoforms. Furthermore, isogenic B-cell lines (NALM-6) expressing the most frequent SF3B1 mutation (K700E) were generated and profiled by RNAseq. As expected, similar alternatively spliced genes were observed in NALM-6 SF3B1-K700E cells exclusively. To investigate the role of nonsense-mediated mRNA decay (NMD) in eliminating aberrant mRNAs induced by SF3B1-MUT, we treated NALM-6 SF3B1-K700E cells with cycloheximide, a translation inhibitor known to inhibit NMD. In the treated samples, expression of several aberrant mRNAs was revealed and some of these transcripts were shown to be downregulated in patient samples. Taken together, these results confirm the association between different SF3B1 hotspot mutations and the presence of novel splice isoforms. We demonstrated that E7107, a potent and selective inhibitor of wild-type SF3B1, also binds and inhibits SF3B1-MUT protein. In addition, E7107 represses the expression of several common aberrant splice mRNA products in SF3B1-MUT cells in vitro and in vivo. When tested in a NALM-6 mouse model, E7107 induced tumor regression and increased the overall survival of animals implanted with NALM-6 SF3B1-K700E cells. These data suggest splicing inhibitors as a promising therapeutic approach for cancer patients carrying SF3B1 mutations. Citation Format: Silvia Buonamici, Kian Huat Lim, Jacob Feala, Eunice Park, Laura Corson, Michelle Aicher, Daniel Aird, Betty Chan, Erik Corcoran, Rachel Darman, Peter Fekkes, Gregg Keaney, Pavan Kumar, Kaiko Kunii, Linda Lee, Xiaoling Puyang, Jose Rodrigues, Anand Selvaraj, Michael Thomas, John Wang, Markus Warmuth, Lihua Yu, Ping Zhu, Peter Smith, Yoshiharu Mizui. SF3B1 mutations induce aberrant mRNA splicing in cancer and confer sensitivity to spliceosome inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2932. doi:10.1158/1538-7445.AM2014-2932

Glutamine:fructose-6-phosphate amidotransferase (GFAT) is the first and rate-limiting enzyme of the hexosamine biosynthesis pathway, which plays an important role in glucose toxicity and cellular insulin resistance. Thus, the mechanisms by which GFAT expression is regulated under physiological and pathological conditions are of interest in connection with diabetes. In this study, we cloned the 5′-flanking region of the mouse GFAT2 gene and characterized its promoter activity. Sequence analysis revealed several putative regulatory elements Sp1, a CCAAT box, AP-1 and AP-2, but no TATA box. Transfection experiments showed that the 5′-flanking region between −2462 to +38 relative to the transcription start site of the GFAT2 gene drives transcription in NIH3T3 cells and that the fragment from −141 to −9 has the highest transcription activity. Reporter assays using deletion and mutant variants suggested that the Sp1 sites at positions −83 to −78 and −22 to −17 both play an important role in the basal promoter activity of the mouse GFAT2 gene. Electrophoretic mobility shift assay showed DNA-protein binding at both Sp1 sites. We also compared the promoter activities of mouse GFAT1 and GFAT2 in several cell lines.

A cellular disintegrin, also called MDC and ADAM is a recently discovered gene family that encodes protein with disintegrin-like and metalloprotease-like domains. We have reported the identification of human cDNAs encoding novel ADAM family proteins that we named MDC2 and MDC3 because of their structural similarity to the MDC (Sagane, K. et al., 1998. Biochem. J. 334, 93-98). The Human Gene Nomenclature Committee assigned the gene symbols ADAM11 for the MDC, ADAM22 for the MDC2 and ADAM23 for the MDC3. Here we report the isolation of three novel murine cDNAs encoding the proteins closely related to the human ADAM11, ADAM22 and ADAM23. Their chromosomal locations in the mouse were identified by interspecies backcross mapping. The loci of these murine ADAM genes were in good accordance with the location of each human ortholog, ADAM11, ADAM22 and ADAM23. These findings suggest that three murine cDNAs that we have isolated are the murine ADAM11, ADAM22 and ADAM23 cDNAs. Northern blot analysis shows that all of these three murine ADAMs were highly expressed in the mouse brain. The structures of these ADAM proteins strongly suggest that they could function as integrin receptors. The implications of the cellular disintegrins in neural development are discussed.

Streptomyces ATP nucleotide 3'-pyrophosphokinase is an extracellular enzyme that transfers 5'-beta, gamma-pyrophosphoryl groups of ATP to a variety of nucleotides at the 3'-OH site. The enzyme gene was cloned from partially Sau3AI-digested chromosomal DNA of S. morookaensis in S. lividans TK24/pIJ699 and then in E. coli JM83/pUC12. Some transformants produced the active enzyme. The gene was sequenced by the dideoxynucleotide termination procedure. Its GC content was 72%. Its putative promoter regions, showing little homology to that of the Streptomyces consensus type, were pointed out. No sequence homology was found between the pyrophosphokinase and any other known genes including those of the most mechanistically similar bacterial stringent factor and related proteins. Northern hybridization analysis showed that the gene is constitutionally polycistronic and expressed under transcriptional control. Nuclease S1 mapping indicated that the gene transcription starts from its translation initiation site.