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H3B-6527 response in 40 HCC cell lines and 625 cell lines.

Legends describing each of the supplementary figures and tables

RNA-seq analysis of 24 CCLE HCC cell lines.

Crystallographic Data collection and Refinement Statistics.

Body weight data of H3B-6527 as single agent and in combination

RNA-seq analysis of TCGA HCC samples.

H3B-6527 efficacy studies in 30 PDX models show that 10 models are responsive

H3B-6527 effects in a HCC cell line Hep3B orthotopic xenografts in female nude mice.

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.

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.

// Shih-Min A. Huang 1,2,* , Anlai Wang 1,* , Rita Greco 1 , Zhifang Li 1 , Fangxian Sun 1 , Claude Barberis 3 , Michel Tabart 4 , Vinod Patel 3 , Laurent Schio 4 , Raelene Hurley 1 , Bo Chen 5 , Hong Cheng 1 , Christoph Lengauer 1,6 , Jack Pollard 1 , James Watters 1 , Carlos Garcia-Echeverria 4 , Dmitri Wiederschain 1 , Francisco Adrian 1 and JingXin Zhang 1 1 Sanofi Oncology, Cambridge, MA, 02139, USA 2 Current affiliation: Genentech, 1 DNA way, South San Francisco, CA, 94080, USA 3 Sanofi, Lead Generation and Candidate Realization, Waltham, MA USA 4 Sanofi Oncology, Discovery and Early Development, Vitry-Sur-Seine, France 5 Sanofi- Genzyme, GDB, Cambridge, MA, 02139, USA 6 Current affiliation: Blueprint Medicines, Cambridge, MA 02140, USA * These authors contributed equally to this work Correspondence: Shih-Min A. Huang, email: // JingXin Zhang, email: // Keywords : Pooled shRNA screen; MYC; JAK2; PIM; drug combination; myeloproliferative neoplasms Received : March 27, 2014 Accepted : May 7, 2014 Published : May 8, 2014 Abstract Inhibitors of JAK2 kinase are emerging as an important treatment modality for myeloproliferative neoplasms (MPN). However, similar to other kinase inhibitors, resistance to JAK2 inhibitors may eventually emerge through a variety of mechanisms. Effective drug combination is one way to enhance therapeutic efficacy and combat resistance against JAK2 inhibitors. To identify potential combination partners for JAK2 compounds in MPN cell lines, we performed pooled shRNA screen targeting 5,000 genes in the presence or absence of JAK2 blockade. One of the top hits identified was MYC, an oncogenic transcription factor that is difficult to inhibit directly, but could be targeted by modulation of upstream regulatory elements such as kinases. We demonstrate herein that PIM kinase inhibitors efficiently suppress MYC protein levels in MPN cell lines. Overexpression of MYC restores the viability of PIM inhibitor-treated cells, revealing causal relationship between MYC down-regulation and cell growth inhibition by PIM compounds. Combination of various PIM inhibitors with a JAK2 inhibitor results in significant synergistic growth inhibition of multiple MPN cancer cell lines and induction of apoptosis. Mechanistic studies revealed strong downregulation of phosphorylated forms of S6 and 4EBP1 by JAK2/PIM inhibitor combination treatment. Finally, such combination was effective in eradicating in vitro JAK2 inhibitor-resistant MPN clones, where MYC is consistently up-regulated. These findings demonstrate that simultaneous suppression of JAK2 and PIM kinase activity by small molecule inhibitors is more effective than either agent alone in suppressing MPN cell growth. Our data suggest that JAK2 and PIM combination might warrant further investigation for the treatment of JAK2-driven hematologic malignancies.

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

Inhibitors of JAK2 kinase are emerging as an important treatment modality for myeloproliferative neoplasms (MPN). However, similar to other kinase inhibitors, resistance to JAK2 inhibitors may eventually emerge through a variety of mechanisms. Effective drug combination is one way to enhance therapeutic efficacy and combat resistance against JAK2 inhibitors. To identify potential combination partners for JAK2 compounds in MPN cell lines, we performed pooled shRNA screen targeting 5,000 genes in the presence or absence of JAK2 blockade. One of the top hits identified was MYC, an oncogenic transcription factor that is difficult to inhibit directly, but could be targeted by modulation of upstream regulatory elements such as kinases. We demonstrate herein that PIM kinase inhibitors efficiently suppress MYC protein levels in MPN cell lines. Importantly, overexpression of MYC restores the viability of PIM inhibitor-treated cells, revealing causal relationship between MYC down-regulation and cell growth inhibition by PIM compounds. Combination of various PIM inhibitors with a JAK2 inhibitor results in significant synergistic growth inhibition of multiple MPN cancer cell lines and induction of apoptosis. Mechanistic studies revealed strong downregulation of phosphorylated forms of S6 and 4EBP1 by JAK2/PIM inhibitor combination treatment. Finally, such combination was effective in eradicating in vitro JAK2 inhibitor-resistant MPN clones, where MYC is consistently up-regulated. These findings demonstrate that simultaneous suppression of JAK2 and PIM kinase activity by small molecule inhibitors is more effective than either agent alone in suppressing MPN cell growth. Our data suggest that JAK2 and PIM combination might warrant further investigation for the treatment of JAK2-driven hematologic malignancies.

One of the challenges of using kinase inhibitors in the clinic is the potential emergence of resistance mechanisms that render them inefficacious in patients who were previously responsive. Imatinib is one of the best examples of this phenomenon. Despite initially impressive clinical responses, a significant proportion of CML patients become unresponsive to imatinib primarily due to the emergence of secondary missense mutations. Identifying these mutations and understanding resistance mechanisms are key to the design of second-generation inhibitors capable of overcoming resistance and for predicting potential clinical resistance to novel drugs. SAR302503 is a JAK2 inhibitor currently in Phase III clinical development as a treatment for myelofibrosis. In a Phase I/II trial, prolonged treatment (>4 years) with SAR302503 has resulted in durable responses (Pardanani et al. ASH 2011; Gotlib et al. EHA 2012). Because of the frequent emergence of resistance with other kinase inhibitors, we wanted to anticipate and understand potential resistance to JAK2 inhibitors in general and SAR302503 in particular. Resistance to ruxolitinib in vitro has recently been associated with reactivation of the JAK/STAT pathway due to the formation of JAK1/JAK2 and TYK2/JAK2 heterodimers (Koppikar et al. Nature 2012). To study potential clinical resistance to SAR302503 in vitro, two approaches were used: selective pressure and ENU induced mutagenesis using JAK2V617F expressing patient derived (SET-2 and HEL) and engineered (Ba/F3.JAK2V617F) cell lines. The results of these in vitro SAR302503 resistance screens indicate that: i) JAK2V617F cells are more prone to develop resistance to ruxolitinib than to SAR302503 after treatment with concentrations 4- or 10-fold over their respective IC50s; ii) resistance does not arise in cells treated with concentrations of SAR302503 above 3 μM even after a prolonged treatment (up to 12 weeks); iii) resistance to SAR302503 in the patient-derived cell lines SET-2 and HEL is accompanied by over-activation of the JAK/STAT pathway in the absence of JAK2 secondary mutations; and iv) resistance to SAR302503 in Ba/F3.JAK2V617F cells results from the mutation Y931C in all of the resistant clones analyzed. From these in vitro results and SAR302503 pharmacokinetic data in humans and preclinical species we can conclude: i) high-level resistance to SAR302503 in patients is unlikely because concentrations of free SAR302503 of approximately 6 μM are estimated to be reached and sustained in the tissues of myelofibrosis patients at a 500 mg daily dose level; ii) overactive JAK/STAT signaling in resistant clones can still be effectively inhibited by clinically achievable concentrations of SAR302503; and iii) clinical activity of SAR302503 may be less challenged than that of ruxolitinib in the event patients express the Y931C mutation. Citation Format: Raelene Hurley, Rita Greco, Fangxian Sun, Anlai Wang, Shih-Min A. Huang, Jenny Zhang, Claudia Lebedinsky, Pamela Cohen, Francisco Adrian. Anticipating clinical resistance to SAR302503, a JAK2 inhibitor in clinical development for the treatment of myelofibrosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2435. doi:10.1158/1538-7445.AM2013-2435

Numerous studies have recognized the critical role of STAT3 in malignant transformation and tumor progression. Constitutive STAT3 activation is frequently found in cancer cell lines and tumor samples and it is usually linked to the presence of IL-6. Autocrine or paracrine IL-6 loops have been described to provide tumor cells with the ability to proliferate, survive, migrate and metastasize. At the molecular level, IL-6 binding to its receptor results in activation of Jak/STAT3 signaling and other signaling cascades, namely PI3K/Akt, MEK/ERK1-2, with a well established role in cancer. We have evaluated the ability of SAR302503, a selective Jak2 inhibitor entering a PhIII clinical trial in myelofibrosis patients, to block these pathways in a panel of ∼20 tumor cell lines representing different cancer types (prostate, breast, lung, colorectal, pancreas, hepatocellular, etc). A 45 minutes treatment with different concentrations of SAR302503 (0.1-10 µM) was sufficient to block both basal or IL-6 induced STAT3 phosphorylation in a dose dependent manner. Compound concentrations equal or greater than 0.1 µM were able to reduce the phosphorylation levels of STAT3 by an extent greater than 50% in all the cell lines included in the study. The impact of SAR302503 in tumor cell proliferation and survival was evaluated using different assays including clonogenic assays. Complete inhibition of colony formation was achieved at concentrations of SAR302503 below 1 µM in most of the cell lines. The antitumor activity of SAR302503 was evaluated in mice xenotransplanted subcutaneously with DU145 human prostate cancer cells. Oral administration of SAR302503 for 10 days resulted in significant dose dependent tumor growth inhibition, near to stasis at the highest dose (T/C=19% at 100 mg/kg, bid). In summary, we demonstrate that SAR302503 negatively impacts the proliferation and survival of different solid tumor cells and our data supports a role for a selective Jak2 inhibition in treating solid tumors with activated Jak/STAT signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1796. doi:1538-7445.AM2012-1796