Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are standard of care in combination with endocrine therapy in advanced hormone receptor-positive (HR+), human epidermal growth factor receptor 2 negative (HER2-) breast cancer. Despite improved progression-free and overall survival, almost all patients develop CDK4/6 inhibitor resistance and experience disease progression on treatment. Aberrant activation of CDK2/cyclin E is a key resistance mechanism by which tumors can evade CDK4/6 blockade. Therefore, patients with HR+/HER2- breast cancer could benefit from treatment with a selective CDK2 inhibitor in combination with CDK4/6 inhibitors both in the resistant and first-line (1L) settings. BLU-222 is a novel, potent, and selective small-molecule inhibitor of CDK2 with favorable pharmacokinetic properties when administered orally, currently in early-stage clinical development. In pre-clinical studies using the MCF-7 xenograft model of HR+ CDK4/6-responsive breast cancer, treatment with BLU-222 combined with the CDK4/6 inhibitor ribociclib led to pronounced and durable tumor regression superior to ribociclib alone. In a derived palbociclib resistant MCF-7 xenograft model, ribociclib had no anti-tumor activity while BLU-222 led to a strong and durable anti-tumor response (83% tumor growth inhibition [TGI]) that was further improved when given in combination with ribociclib (110% TGI). To further explore the mechanism of aberrant CDK2 activation in CDK4/6 resistant, HR+ breast cancer, we engineered isogenic T47D cell lines to overexpress cyclin E1 (CCNE1) with or without p16, an endogenous inhibitor of CDK4/6 activity. In in vitro proliferation assays, co-expression of CCNE1 and p16 sensitized T47D cells to BLU-222 by approximately 10-fold compared to the parental control (110 nM vs 1078 nM, respectively). CDK4/6 inhibition with ribociclib had no anti-proliferative effect in the CCNE1 overexpressing cell lines regardless of p16 expression status. In vivo, treatment of the empty vector control T47D xenografts with ribociclib led to tumor stasis, while ribociclib in combination with BLU-222 led to tumor regression. T47D xenografts overexpressing both CCNE1 and p16 were resistant to ribociclib however CDK2 inhibition with BLU-222 single-agent treatment led to tumor regression. Finally, the activity of BLU-222 was evaluated in a patient-derived xenograft (PDX) model of CDK4/6 inhibitor-resistant HR+/HER2- breast cancer where the patient had progressed on 1L palbociclib/fulvestrant and 2L abemaciclib/fulvestrant therapy. In this PDX model, BLU-222 in combination with ribociclib led to tumor stasis, even in the absence of fulvestrant. In conclusion, these data support CDK2/cyclin E activation as a key vulnerability in CDK4/6 resistant, HR+/HER2- breast cancer and provide a rationale for the study of BLU-222 in patients with disease progression following CDK4/6 inhibitor treatment. Additionally, the improved durability of response when BLU-222 is combined with CDK4/6 inhibitors in the CDK4/6-naïve setting supports the combination of these agents as 1L treatment. BLU-222 is currently under investigation in VELA (NCT05252416), a phase 1/2, first-in-human trial for patients with cyclin E aberrant cancers and HR+/HER2- breast cancer. Citation Format: Victoria Brown, Nealia House, Phil Ramsden, Karen Ho, Hsin-Jung Wu, Erik Wilker, Jian Guo, Maxine Chen, Douglas Wilson, Neil Bifulco, Steve Wenglowsky, Yoon Jong Choi, Kerrie Faia. CDK2 inhibition with BLU-222 in combination with ribociclib demonstrates robust antitumor activity in pre-clinical models of CDK4/6 inhibitor-naïve and -resistant HR+/HER2- breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-07.

Supplementary Methods and Figure Legends

Abstract Aberrant signaling through the fibroblast growth factor 19 (FGF19)/fibroblast growth factor receptor 4 (FGFR 4) signaling complex has been shown to cause hepatocellular carcinoma (HCC) in mice and has been implicated to play a similar role in humans. We have developed BLU9931, a potent and irreversible small-molecule inhibitor of FGFR4, as a targeted therapy to treat patients with HCC whose tumors have an activated FGFR4 signaling pathway. BLU9931 is exquisitely selective for FGFR4 versus other FGFR family members and all other kinases. BLU9931 shows remarkable antitumor activity in mice bearing an HCC tumor xenograft that overexpresses FGF19 due to amplification as well as a liver tumor xenograft that overexpresses FGF19 mRNA but lacks FGF19 amplification. Approximately one third of patients with HCC whose tumors express FGF19 together with FGFR4 and its coreceptor klotho β (KLB) could potentially respond to treatment with an FGFR4 inhibitor. These findings are the first demonstration of a therapeutic strategy that targets a subset of patients with HCC. Significance: This article documents the discovery of BLU9931, a novel irreversible kinase inhibitor that specifically targets FGFR4 while sparing all other FGFR paralogs and demonstrates exquisite kinome selectivity. BLU9931 is efficacious in tumors with an intact FGFR4 signaling pathway that includes FGF19, FGFR4, and KLB. BLU9931 is the first FGFR4-selective molecule for the treatment of patients with HCC with aberrant FGFR4 signaling. Cancer Discov; 5(4); 424–37. ©2015 AACR. See related commentary by Packer and Pollock, p. 355 This article is highlighted in the In This Issue feature, p. 333

Supplementary Figure S1. Potency against FGFR family and kinome selectivity. Supplementary Figure S2. Kinases with a cysteine at the equivalent position to Cys552 in FGFR4. Supplementary Figure S3. MALDI Mass Spectrometry of FGFR4. Supplementary Figure S4. Induction of Caspase 3/7 activity in Hep 3B cells. Supplementary Figure S5. FGFR4 turnover rates in Hep 3B cells. Supplementary Figure S6. Analysis of TCGA HCC samples. Supplementary Table S1. Kinetic parameters of FGFR4 inhibition by BLU9931. Supplementary Table S2. Pharmacokinetic parameters for BLU9931.

Background & Aims Fibrolamellar carcinoma (FLC) is a rare, difficult-to-treat liver cancer primarily affecting pediatric and adolescent patients, and for which precision medicine approaches have historically not been possible. The DNAJB1-PRKACA gene fusion was identified as a driver of FLC pathogenesis. We aimed to assess whether FLC tumors maintain dependency on this gene fusion and determine if PRKACA is a viable therapeutic target. Methods FLC patient-derived xenograft (PDX) shRNA cell lines were implanted subcutaneously into female NOD-SCID mice and tumors were allowed to develop prior to randomization to doxycycline (to induce knockdown) or control groups. Tumor development was assessed every 2 days. To assess the effect of treatment with novel selective PRKACA small molecule kinase inhibitors, BLU0588 and BLU2864, FLC PDX tumor cells were implanted subcutaneously into NOD-SCID mice and tumors allowed to develop. Mice were randomized to treatment (BLU0588 and BLU2864, orally, once daily) or control groups and tumor size determined as above. Results Knockdown of DNAJB1-PRKACA reversed a FLC-specific gene signature and reduced PDX tumor growth in mice compared to the control group. Furthermore, FLC PDX tumor growth was significantly reduced with BLU0588 and BLU2864 treatment versus control ( P = 0.003 and P = 0.0005, respectively). Conclusions We demonstrated, using an inducible knockdown and small molecule approaches, that FLC PDX tumors were dependent upon DNAJB1 - PRKACA fusion activity. In addition, this study serves as a proof-of-concept that PRKACA is a viable therapeutic target for FLC and warrants further investigation.

Background and Aims:Fibrolamellar carcinoma (FLC) is a rare, difficult-to-treat liver cancer primarily affecting pediatric and adolescent patients, and for which precision medicine approaches have historically not been possible. The DNAJB1-PRKACA gene fusion was identified as a driver of FLC pathogenesis. We aimed to assess whether FLC tumors maintain dependency on this gene fusion and determine if PRKACA is a viable therapeutic target. Methods:FLC patient-derived xenograft (PDX) shRNA cell lines were implanted subcutaneously into female NOD-SCID mice and tumors were allowed to develop prior to randomization to doxycycline (to induce knockdown) or control groups. Tumor development was assessed every 2 days. To assess the effect of treatment with novel selective PRKACA small molecule kinase inhibitors, BLU0588 and BLU2864, FLC PDX tumor cells were implanted subcutaneously into NOD-SCID mice and tumors allowed to develop. Mice were randomized to treatment (BLU0588 and BLU2864, orally, once daily) or control groups and tumor size determined as previously. Results:Knockdown of DNAJB1-PRKACA reversed a FLC-specific gene signature and reduced PDX tumor growth in mice compared to the control group. Furthermore, FLC PDX tumor growth was significantly reduced with BLU0588 and BLU2864 treatment vs control (P = .003 and P = .0005, respectively). Conclusion:We demonstrated, using an inducible knockdown and small molecule approaches, that FLC PDX tumors were dependent upon DNAJB1-PRKACA fusion activity. In addition, this study serves as a proof-of-concept that PRKACA is a viable therapeutic target for FLC and warrants further investigation.

Background: Cyclin-dependent kinases (CDK) are a class of enzymes that, along with their regulatory cyclin binding partners, drive cell cycle progression. Cell cycle dysregulation is a hallmark of cancer and targeting its genetic drivers can confer therapeutic benefits. Cyclin E1 (CCNE1) gene alterations are common in patients with ovarian cancer and can be found in ~20% of high-grade serous ovarian cancer cases, which tend to be platinum therapy resistant and therefore represent a high medical need. Cyclin E1 is the canonical binding partner of CDK2, which becomes constitutively active when CCNE1 is amplified and overexpressed. Selectively inhibiting CDK2 is an attractive therapeutic option for CCNE1-amplified tumors and may limit off-target CDK-driven toxicities. Here we report preclinical validation studies leading to the development of an orally available CDK2 inhibitor, BLU-222, for the treatment of ovarian cancer harboring a CCNE1 amplification. Methods: BLU-222 selectivity was measured by enzyme assays, cellular target engagement assays (NanoBRET), and proliferation assays in a panel of ovarian cancer cell lines. In vitro cellular potency was assessed by phospho-Rb levels. In vivo antitumor activity of BLU-222 as a single agent or in combination with carboplatin was measured in an OVCAR-3 cell line-derived xenograft (CDX) tumor model harboring a CCNE1 amplification. Results: BLU-222 demonstrated selectivity, both biochemically and in cells, with low nanomolar potency for CDK2 vs other CDK family members (CDK1, -4, -6, -7, and -9). In a panel of ovarian cancer cell lines, those with CCNE1 amplifications were highly sensitive to BLU-222. Consistent with the in vitro profiling, BLU-222 exhibited significant antitumor activity in the OVCAR-3 CDX model. While administration of single-agent carboplatin led to stasis in vivo, the combination of BLU-222 + carboplatin induced durable tumor regression even after treatment cessation. Conclusions: These data provide a strong rationale for advancing BLU-222 towards clinical development in patients with CCNE1-amplified ovarian cancer. Citation Format: Victoria Brown, Phil Ramsden, Nealia House, Richard Vargas, Jian Guo, Ruduan Wang, Riadh Lobbardi, Maxine Chen, Douglas Wilson, Joseph Kim, Neil Bifulco, Michelle Maynard, Emanuele Perola, Dean Zhang, Steve Wenglowsky, Yoon Jong Choi. BLU-222, an investigational, potent, and selective CDK2 inhibitor, demonstrated robust antitumor activity in CCNE1-amplified ovarian cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2306.

Abstract Background: Cyclin dependent kinases (CDK) comprise a family of proteins which are activated upon cyclin binding to promote cell cycle progression. Historically, CDK family inhibitors have had limited utility in the clinic due to 1) toxicities associated with broad spectrum CDK inhibition; and 2) varied and unpredictable efficacy due to lack of a patient selection strategy. The recent approval of three selective CDK4/6 drugs (palbociclib, ribociclib and abemaciclib) has revived the pursuit of therapies against a related interphase kinase, CDK2. Cyclin E gene (CCNE) alterations are prevalent in cancers with high unmet medical need and are emerging as a potential mechanism of clinical resistance to targeted therapies (e.g. CDK4/6 therapies). Methods: Here we report preclinical validation leading to the development of a selective CDK2 inhibitor for the treatment of cancers harboring CCNE alterations. We highlight advanced, orally bioavailable compounds exhibiting single-digit low nanomolar biochemical and cellular potency (pRB), exquisite CDK family selectivity (CDK1, 4, 6, 7 and 9), and whole kinome selectivity. We have characterized the phenotypic and mechanistic consequences of targeting CDK2, employing both genetic and pharmacologic methods across cell lines and xenograft models (e.g. OVCAR-3, MKN-1, HCC1569). Results: In a panel of cell lines, CCNE1-amplified lines exhibited profound sensitivity to selective CDK2 inhibition by halting cells at the G1/S phase of the cell cycle. Upon further inspection, CDK2 inhibition induced multiple markers of senescence, e.g. SA-b-gal and IL-6, in CCNE1-amplified but not in CCNE non-amplified cell lines. Consistent with this, these compounds exhibit robust anti-tumor activity in multiple CCNE1-amplified xenograft models, which was sustained after removal of treatment with these compounds. Conclusions: These data provide a strong rationale for advancing this class of compounds toward clinical development in CCNE-altered cancers. Citation Format: Yoon J. Choi, Steve Wenglowsky, Victoria Brown, Neil Bifulco, Yeon S. Choi, Jian Guo, Megan Hatlen, Joseph Kim, Tim LaBranche, Riadh Lobbardi, Emanuele Perola, Emily Rozsahegyi, Michelle Maynard, Phil Ramsden, Grace Silva, Faith Stevison, Richard Vargas, Ruduan Wang, Doug Wilson, Rich Woessner, Dean Zhang, Rob Meissner, Klaus Hoeflich, Marion Dorsch. Development of a selective CDK2-E inhibitor in CCNE driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1279.

Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide with no clinically confirmed oncogenic driver. Although preclinical studies implicate the FGF19 receptor FGFR4 in hepatocarcinogenesis, the dependence of human cancer on FGFR4 has not been demonstrated. Fisogatinib (BLU-554) is a potent and selective inhibitor of FGFR4 and demonstrates clinical benefit and tumor regression in patients with HCC with aberrant FGF19 expression. Mutations were identified in the gatekeeper and hinge-1 residues in the kinase domain of FGFR4 upon disease progression in 2 patients treated with fisogatinib, which were confirmed to mediate resistance in vitro and in vivo. A gatekeeper-agnostic, pan-FGFR inhibitor decreased HCC xenograft growth in the presence of these mutations, demonstrating continued FGF19-FGFR4 pathway dependence. These results validate FGFR4 as an oncogenic driver and warrant further therapeutic targeting of this kinase in the clinic. SIGNIFICANCE: Our study is the first to demonstrate on-target FGFR4 kinase domain mutations as a mechanism of acquired clinical resistance to targeted therapy. This further establishes FGF19-FGFR4 pathway activation as an oncogenic driver. These findings support further investigation of fisogatinib in HCC and inform the profile of potential next-generation inhibitors.See related commentary by Subbiah and Pal, p. 1646.This article is highlighted in the In This Issue feature, p. 1631.

Abstract When on-target resistance mutations occur in response to potent and selective therapeutics, the target is often considered a validated driver of disease. Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide whose drivers remain unvalidated because approved therapies utilize multi-kinase inhibitors or immune modulation. In this study, we validate for the first time a driver of disease in the FGF19 expressing subset of HCC through the identification of Fisogatinib resistance mutations in FGFR4. Fisogatinib (formerly known as BLU-554), an investigational agent, was developed to specifically inhibit the tyrosine kinase activity of FGFR4. Approximately one third of HCC patients harbor aberrant expression of fibroblast growth factor 19 (FGF19), a ligand hypothesized to drive paracrine FGFR4 activation and enhance mitogenic signaling in HCC cells. The identification of FGF19 amplifications in a subset of HCC patients through genomic profiling is further suggestive of the involvement of the FGF19/FGFR4 signaling axis in oncogenesis. The first-in-human, phase I study of Fisogatinib in patients with advanced HCC (NCT02508467) suggests that expression of FGF19 as assessed by immunohistochemistry is responsible for oncogenesis and predictive of response to treatment. We identified FGFR4 gatekeeper (V550) and hinge-1 (C552) mutations upon disease progression in two patients treated with Fisogatinib. These resistance mutations had been predicted by our structure-based studies and were also identified through in vitro and in vivo Fisogatinib resistance screens. We confirm by liquid chromatography tandem mass-spectrometry that Fisogatinib binding to FGFR4 is prevented when either the gatekeeper or the hinge-1 mutation is present and demonstrate the capability of these mutations to mediate resistance to Fisogatinib through genetic engineering. Using a gatekeeper-agnostic pan-FGFR inhibitor we show, in preclinical models, continued FGF19/FGFR4 pathway dependence. These results validate FGF19/FGFR4 as an oncogenic driver and inform the profile of potential next-generation inhibitors designed to facilitate improved patient outcomes. Citation Format: Megan A Hatlen, Oleg Schmidt-Kittler, Cori-Ann Sherwin, Emily Rozsahegyi, Nooreen Rubin, Michael Sheets, Joseph L Kim, Chandra Miduturu, Neil Bifulco, Natasja Brooijmans, Hongliang Shi, Timothy Guzi, Andy Boral, Christoph Lengauer, Marion Dorsch, Richard D Kim, Yoon-Koo Kang, Beni B Wolf, Klaus P Hoeflich. Identification of resistance mechanisms to FGFR4 targeted therapy in hepatocellular carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A118. doi:10.1158/1535-7163.TARG-19-A118

Abstract Introduction: Fibrolamellar Carcinoma (FLC) is a rare primary liver malignancy, affecting children and young adults without chronic liver disease. FLC tumors are largely resistant to chemotherapy, making the identification of effective treatment options urgently needed. Recent genomic data strongly suggest that DNAJB1-PRKACA kinase fusions are the drivers of the vast majority of FLC cases. However, it has not been assessed whether FLC tumors remain dependent on DNAJB1-PRKACA expression and whether PRKACA inhibition could be a therapeutic approach for FLC. Here we summarize the preclinical evaluation of PRKACA as a potential therapeutic target for FLC. Methods: We established a xenograft model from a FLC- patient and then developed inducible PRKACA shRNA cell lines from this model. We also designed potent tool compounds that selectively inhibit the PRKACA protein to assess PRKACA as a potential therapeutic target for FLC. Results: We characterized a patient-derived xenograft (PDX) model of FLC (LI5132) and confirmed DNAJB1-PRKACA fusion expression and constitutive PRKACA pathway activation measured by phospho-VASP. The model also shows fibrolamellar type histology by H&E staining and expression of typical FLC markers like cytokeratin 7 and CD68 by IHC. Using inducible PRKACA-specific shRNA cell lines from this PDX model we demonstrated that the FLC transcriptional gene signature correlates strongly with expression of the DNAJB1-PRKACAfusion protein. Importantly, we demonstrated for three inducible PRKACA shRNA cell-line-derived xenograft models that the in vivo tumor growth remained dependent on DNAJB1-PRKACA fusion expression (TGI-72%-78%, day 22). PRKACA knockdown tumors displayed reduced Ki67 index (6.4 %) when compared to non-induced controls (37.1 %) further confirming that proliferation of the tumors depends on the fusion expression. To investigate the PRKACA catalytic dependency of the FLC model, we designed potent and selective PRKACA inhibitors based on starting points from our proprietary kinase inhibitor library. These investigational compounds are the first selective and potent PRKACA inhibitors and provide excellent tools to assess in vitro and in vivo PRKACA dependency. These compounds achieved potent PRKACA pathway inhibition and dose-dependent inhibition of FLC-specific gene expression, including genes such as carbamoyl phosphate synthetase (CPS1) and forkhead box C1 (FoxC1). We established a pharmacokinetic/ pharmacodynamic relationship and demonstrated in vivo PRKACA pathway inhibition in PDX tumors, as measured by phospho-VASP. Importantly, oral delivery of a potent and selective PRKACA inhibitor achieved up to 80% PRKACA kinase inhibition and led to statistically significant FLC tumor growth inhibition (54%, day 34) on a tolerated schedule. These data demonstrate that FLC depends on PRKACA kinase activity. Conclusion: This study is the first evaluation of PRKACA kinase inhibition as a therapeutic approach for FLC. The results from these preclinical experiments provide strong evidence that FLC depends on PRKACA catalytic activity and that novel PRKACA inhibitors may significantly decrease tumor growth in vivo. Citation Format: Stefanie S Schalm, Erin O’Hearn, Kevin Wilson, Timothy LaBranche, Grace Silva, Lucian DiPietro, Neil Bifulco, Richard Woessner, Nicolas Stransky, Darshan Sappal, Adam Shutes, Robert Campbell, Riadh Lobbardi, Michael Palmer, Joseph Kim, Stephen Miller, Marion Dorsch, Christoph Lengauer, Timothy Guzi, Vivek Kadambi, Andrew Garner, Klaus P Hoeflich. Evaluating PRKACA as a therapeutic target for Fibrolamellar Carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B127. doi:10.1158/1535-7163.TARG-19-B127

Multidrug-resistant (MDR) bacterial infections are a serious threat to public health. Among the most alarming resistance trends is the rapid rise in the number and diversity of β-lactamases, enzymes that inactivate β-lactams, a class of antibiotics that has been a therapeutic mainstay for decades. Although several new β-lactamase inhibitors have been approved or are in clinical trials, their spectra of activity do not address MDR pathogens such as Acinetobacter baumannii. This report describes the rational design and characterization of expanded-spectrum serine β-lactamase inhibitors that potently inhibit clinically relevant class A, C and D β-lactamases and penicillin-binding proteins, resulting in intrinsic antibacterial activity against Enterobacteriaceae and restoration of β-lactam activity in a broad range of MDR Gram-negative pathogens. One of the most promising combinations is sulbactam–ETX2514, whose potent antibacterial activity, in vivo efficacy against MDR A. baumannii infections and promising preclinical safety demonstrate its potential to address this significant unmet medical need. Development of a broad-spectrum β-lactamase inhibitor capable of combatting multidrug-resistant pathogens, including Acinetobacter baumannii.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide. Chemotherapy has proven ineffective, and Sorafenib remains the only approved targeted drug with no second or third line treatment options. Sorafenib slows the growth of advanced liver cancers and helps some patients live longer - by an average of about three months. There is a pressing need for more effective therapies. FGF19 is a highly controlled hormone normally expressed in the intestine, that acts in the liver to regulate bile acid synthesis and hepatocyte proliferation via activation of FGFR4. In 7% of patients with HCC, FGF19 is contained within a focal amplification on chromosome 11q13.3. Overexpression of FGF19 in transgenic mice produces liver tumors which are sensitive to treatment with a FGFR4 tool antibody. Additionally, the growth of tumor cells in xenograft models with FGF19 amplification is dependent on FGFR4 signaling. Thus, selective inhibition of FGFR4 might represent a viable strategy for treating this genetically defined subgroup of HCC patients. Herein, we describe our efforts to identify an ultraselective small molecule inhibitor of FGFR4 which spares the other FGFR isoforms with the intent to avoid FGFR1-3 driven, dose limiting toxicities like soft tissue mineralization. Utilizing structure based drug design, we prepared a series of inhibitor templates designed to covalently modify a target cysteine residue present in FGFR4, but not the other FGFR isoforms. Optimization of one of these templates led to the identification of BLU9931, a highly potent and exquisitely selective, covalent inhibitor of FGFR4. BLU9931 persistently inhibits FGFR4 mediated signaling in cancer cells as evidenced by decreased phosphorylation of FRS2 and ERK. Importantly, BLU9931 does not block signaling driven by FGFR1. Upon oral dosing in mice, BLU9931 is well tolerated and demonstrates robust and dose dependent induction of the FGFR4 target gene CYP7a1 in Hep3B cells, a FGF19 amplified HCC xenograft model. Upon extended dosing, BLU9931 causes sustained regression of tumors, including complete responses. We then explored if HCCs with alterations other than FGF19 amplification are also dependent on FGFR4 signaling. Dosing of molecularly annotated patient derived HCC xenografts with BLU9931 suggests that selective targeting of FGFR4 represents a viable option for the treatment of an expanded population segment of genomically defined HCC patients, much larger than originally anticipated. Citation Format: Margit Hagel, Chandra Miduturu, Mike Sheets, Weifan Weng, Nooreen Rubin, Neil Bifulco, Lucian DiPietro, Joseph Kim, Natasja Brooijmans, Nicolas Stransky, Christopher Winter, Christoph Lengauer, Timothy Guzi. First isoform selective inhibitor of FGFR4 for the treatment of genomically defined patients with hepatocellular carcinoma. [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 LB-324. doi:10.1158/1538-7445.AM2014-LB-324