Sudeep Prajapati

H3B-8800, an orally available small-molecule splicing modulator, induces lethality in spliceosome-mutant cancers

Genomic analyses of cancer have identified recurrent point mutations in the RNA splicing factor–encoding genes SF3B1, U2AF1, and SRSF2 that confer an alteration of function. Cancer cells bearing these mutations are preferentially dependent on wild-type (WT) spliceosome function, but clinically relevant means to therapeutically target the spliceosome do not currently exist. Here we describe an orally available modulator of the SF3b complex, H3B-8800, which potently and preferentially kills spliceosome-mutant epithelial and hematologic tumor cells. These killing effects of H3B-8800 are due to its direct interaction with the SF3b complex, as evidenced by loss of H3B-8800 activity in drug-resistant cells bearing mutations in genes encoding SF3b components. Although H3B-8800 modulates WT and mutant spliceosome activity, the preferential killing of spliceosome-mutant cells is due to retention of short, GC-rich introns, which are enriched for genes encoding spliceosome components. These data demonstrate the therapeutic potential of splicing modulation in spliceosome-mutant cancers.

Splicing modulators act at the branch point adenosine binding pocket defined by the PHF5A–SF3b complex

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.

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 (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.

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.

Abstract 3126: H3B6527, a selective and potent FGFR4 inhibitor for FGF19-driven hepatocellular carcinoma

Hepatocellular carcinoma (HCC) has limited treatment options and generally poor prognosis. Recent genomic studies have identified FGF19 as a driver oncogene in HCC. FGF19 is a gut secreted hormone that acts in the liver through FGFR4 to regulate bile acid synthesis. Consistent with the notion that FGF19 is a driver oncogene in HCC, transgenic mice overexpressing FGF19 form liver tumors and genetic ablation of FGFR4 prevented tumor formation. These data suggest targeting FGFR4 would have therapeutic benefit in HCC with altered FGF19 signaling. While a number of Pan-FGFR inhibitors are being clinically evaluated, their application to FGF19-driven HCC may be limited by their FGFR1-3 related dose limiting toxicities. Using structure guided drug design, we have generated a highly selective covalent FGFR4 inhibitor, H3B-6527. Biochemical and cellular selectivity assays showed that H3B-6527 is >300 fold selective towards FGFR4 compared to other FGFR isoforms. Addition of H3B-6527 to FGF19 amplified HCC cell lines led to dose dependent inhibition of FGF19/FGFR4 signaling and concomitant reduction in cell viability. In a panel of 40 HCC cell lines, H3B-6527 selectively reduced the viability of cells that harbor FGF19 amplification and showed no effect in FGF19 non-amplified HCC cell line models. Oral dosing of H3B-6527 to mice led to dose-dependent pharmacodynamic modulation of FGFR4 signaling and tumor regression in FGF19 altered HCC cell line derived xenograft models. H3B-6527 demonstrated inhibition of tumor growth in an orthotopic liver xenograft model of FGF19 altered HCC grown in nude mice. Importantly, the inhibition of tumor growth occurred at doses that were well tolerated in mice and no evidence of FGFR1-3 related toxicities were observed at efficacious doses. In a panel of 30 HCC patient-derived xenograft (PDX) models, H3B-6527 demonstrated tumor regressions in the context of FGF19-amplified tumors. In addition, H3B-6527 showed antitumor activity and tumor regressions in PDX models with high FGF19 expression but no FGF19 amplification. The mechanism for FGF19 overexpression in the absence of gene amplification is under investigation. In conclusion, our preclinical studies demonstrate that FGF19 expression is a predictive biomarker for response to FGFR4 inhibitor therapy. Genomic analysis of public and proprietary data sets indicates that at least approximately 30% of HCC patients exhibit altered FGF19 expression and could potentially benefit from H3B-6527 monotherapy treatment. Citation Format: Anand Selvaraj, Erik Corcoran, Heather Coffey, Sudeep Prajapati, Ming-Hong Hao, Nicholas Larsen, Jennifer Tsai, Takashi Satoh, Kana Ichikawa, Julie Jaya Joshi, Raelene Hurley, Jeremy Wu, Chia-Ling Huang, Suzanna Bailey, Craig Karr, Pavan Kumar, Victoria Rimkunas, Crystal Mackenzie, Nathalie Rioux, Amy Kim, Sandeep Akare, George Lai, Lihua Yu, Peter Fekkes, John Wang, Markus Warmuth, Peter Smith, Dominic Reynolds. H3B6527, a selective and potent FGFR4 inhibitor for FGF19-driven hepatocellular carcinoma [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 3126. doi:10.1158/1538-7445.AM2017-3126

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

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

Abstract 2941: Targeting MCL1-dependent cancers through RNA splicing modulation

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA 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

Abstract 5564: Total synthesis of 6-deoxypladienolide D and assessment of splicing inhibitory activity in a mutant SF3B1 cancer cell line

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 O9Shea, 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