James Robert Henry

Reactivation of Mitogen-activated Protein Kinase (MAPK) Pathway by FGF Receptor 3 (FGFR3)/Ras Mediates Resistance to Vemurafenib in Human B-RAF V600E Mutant Melanoma

Background: B-RAF V600E melanomas rapidly develop resistance to B-RAF inhibitors in the clinic. Results: FGFR3/Ras signaling is elevated and induces resistance to vemurafenib in vemurafenib-resistant cells. Conclusion: FGFR3/Ras confers resistance to B-RAF inhibition via MAPK pathway reactivation. Significance: A novel mechanism of resistance to B-RAF inhibitors is described and potential therapeutic strategies are suggested. Oncogenic B-RAF V600E mutation is found in 50% of melanomas and drives MEK/ERK pathway and cancer progression. Recently, a selective B-RAF inhibitor, vemurafenib (PLX4032), received clinical approval for treatment of melanoma with B-RAF V600E mutation. However, patients on vemurafenib eventually develop resistance to the drug and demonstrate tumor progression within an average of 7 months. Recent reports indicated that multiple complex and context-dependent mechanisms may confer resistance to B-RAF inhibition. In the study described herein, we generated B-RAF V600E melanoma cell lines of acquired-resistance to vemurafenib, and investigated the underlying mechanism(s) of resistance. Biochemical analysis revealed that MEK/ERK reactivation through Ras is the key resistance mechanism in these cells. Further analysis of total gene expression by microarray confirmed a significant increase of Ras and RTK gene signatures in the vemurafenib-resistant cells. Mechanistically, we found that the enhanced activation of fibroblast growth factor receptor 3 (FGFR3) is linked to Ras and MAPK activation, therefore conferring vemurafenib resistance. Pharmacological or genetic inhibition of the FGFR3/Ras axis restored the sensitivity of vemurafenib-resistant cells to vemurafenib. Additionally, activation of FGFR3 sufficiently reactivated Ras/MAPK signaling and conferred resistance to vemurafenib in the parental B-RAF V600E melanoma cells. Finally, we demonstrated that vemurafenib-resistant cells maintain their addiction to the MAPK pathway, and inhibition of MEK or pan-RAF activities is an effective therapeutic strategy to overcome acquired-resistance to vemurafenib. Together, we describe a novel FGFR3/Ras mediated mechanism for acquired-resistance to B-RAF inhibition. Our results have implications for the development of new therapeutic strategies to improve the outcome of patients with B-RAF V600E melanoma.

A Novel, Selective Inhibitor of Fibroblast Growth Factor Receptors That Shows a Potent Broad Spectrum of Antitumor Activity in Several Tumor Xenograft Models

The fibroblast growth factor receptors (FGFR) are tyrosine kinases that are present in many types of endothelial and tumor cells and play an important role in tumor cell growth, survival, and migration as well as in maintaining tumor angiogenesis. Overexpression of FGFRs or aberrant regulation of their activities has been implicated in many forms of human malignancies. Therefore, targeting FGFRs represents an attractive strategy for development of cancer treatment options by simultaneously inhibiting tumor cell growth, survival, and migration as well as tumor angiogenesis. Here, we describe a potent, selective, small-molecule FGFR inhibitor, (R)-(E)-2-(4-(2-(5-(1-(3,5-Dichloropyridin-4-yl)ethoxy)-1H-indazol-3yl)vinyl)-1H-pyrazol-1-yl)ethanol, designated as LY2874455. This molecule is active against all 4 FGFRs, with a similar potency in biochemical assays. It exhibits a potent activity against FGF/FGFR-mediated signaling in several cancer cell lines and shows an excellent broad spectrum of antitumor activity in several tumor xenograft models representing the major FGF/FGFR relevant tumor histologies including lung, gastric, and bladder cancers and multiple myeloma, and with a well-defined pharmacokinetic/pharmacodynamic relationship. LY2874455 also exhibits a 6- to 9-fold in vitro and in vivo selectivity on inhibition of FGF- over VEGF-mediated target signaling in mice. Furthermore, LY2874455 did not show VEGF receptor 2–mediated toxicities such as hypertension at efficacious doses. Currently, this molecule is being evaluated for its potential use in the clinic. Mol Cancer Ther; 10(11); 2200–10. ©2011 AACR.

Inhibition of RAF Isoforms and Active Dimers by LY3009120 Leads to Anti-tumor Activities in RAS or BRAF Mutant Cancers

LY3009120 is a pan-RAF and RAF dimer inhibitor that inhibits all RAF isoforms and occupies both protomers in RAF dimers. Biochemical and cellular analyses revealed that LY3009120 inhibits ARAF, BRAF, and CRAF isoforms with similar affinity, while vemurafenib or dabrafenib have little or modest CRAF activity compared to their BRAF activities. LY3009120 induces BRAF-CRAF dimerization but inhibits the phosphorylation of downstream MEK and ERK, suggesting that it effectively inhibits the kinase activity of BRAF-CRAF heterodimers. Further analyses demonstrated that LY3009120 also inhibits various forms of RAF dimers including BRAF or CRAF homodimers. Due to these unique properties, LY3009120 demonstrates minimal paradoxical activation, inhibits MEK1/2 phosphorylation, and exhibits anti-tumor activities across multiple models carrying KRAS, NRAS, or BRAF mutation.

Discovery of 1-(3,3-Dimethylbutyl)-3-(2-fluoro-4-methyl-5-(7-methyl-2-(methylamino)pyrido[2,3-d]pyrimidin-6-yl)phenyl)urea (LY3009120) as a Pan-RAF Inhibitor with Minimal Paradoxical Activation and Activity against BRAF or RAS Mutant Tumor Cells

The RAS-RAF-MEK-MAPK cascade is an essential signaling pathway, with activation typically mediated through cell surface receptors. The kinase inhibitors vemurafenib and dabrafenib, which target oncogenic BRAF V600E, have shown significant clinical efficacy in melanoma patients harboring this mutation. Because of paradoxical pathway activation, both agents were demonstrated to promote growth and metastasis of tumor cells with RAS mutations in preclinical models and are contraindicated for treatment of cancer patients with BRAF WT background, including patients with KRAS or NRAS mutations. In order to eliminate the issues associated with paradoxical MAPK pathway activation and to provide therapeutic benefit to patients with RAS mutant cancers, we sought to identify a compound not only active against BRAF V600E but also wild type BRAF and CRAF. On the basis of its superior in vitro and in vivo profile, compound 13 was selected for further development and is currently being evaluated in phase I clinical studies.

Discovery of LY2457546: a multi-targeted anti-angiogenic kinase inhibitor with a novel spectrum of activity and exquisite potency in the acute myelogenous leukemia-Flt-3-internal tandem duplication mutant human tumor xenograft model

SummaryLY2457546 is a potent and orally bioavailable inhibitor of multiple receptor tyrosine kinases involved in angiogenic and tumorigenic signalling. In biochemical and cellular assays, LY2457546 demonstrates potent activity against targets that include VEGFR2 (KDR), PDGFRβ, FLT-3, Tie-2 and members of the Eph family of receptors. With activities against both Tie2 and Eph receptors, LY2457546 possesses an activity profile that distinguishes it from multikinase inhibitors. When compared head to head with sunitinib, LY2457546 was more potent for inhibition of endothelial tube formation in an in vitro angiogenesis co-culture model with an intermittent treatment design. In vivo, LY2457546 inhibited VEGF-driven autophosphorylation of lung KDR in the mouse and rat in a dose and concentration dependent manner. LY2457546 was well tolerated and exhibited efficacy in a 13762 syngeneic rat mammary tumor model in both once and twice daily continuous dosing schedules and in mouse human tumor xenograft models of lung, colon, and prostate origin. Additionally, LY2457546 caused complete regression of well-established tumors in an acute myelogenous leukemia (AML) FLT3-ITD mutant xenograft tumor model. The observed efficacy that was displayed by LY2457546 in the AML FLT3-ITD mutant tumor model was superior to sunitinib when both were evaluated using equivalent doses normalized to in vivo inhibition of pKDR in mouse lung. LY2457546 was well tolerated in non-clinical toxicology studies conducted in rats and dogs. The majority of the toxicities observed were similar to those observed with other multi-targeted anti-angiogenic kinase inhibitors (MAKs) and included bone marrow hypocellularity, hair and skin depigmentation, cartilage dysplasia and lymphoid organ degeneration and necrosis. Thus, the unique spectrum of target activity, potent in vivo anti-tumor efficacy in a variety of rodent and human solid tumor models, exquisite potency against a clinically relevant model of AML, and non-clinical safety profile justify the advancement of LY2457546 into clinical testing.

Mouse PDX Trial Suggests Synergy of concurrent Inhibition of RAF and EGFR in Colorectal Cancer with BRAF or KRAS mutations

Purpose: To evaluate the antitumor efficacy of cetuximab in combination with LSN3074753, an analog of LY3009120 and pan-RAF inhibitor in 79 colorectal cancer patient-derived xenograft (PDX) models. Experimental Design: Seventy-nine well-characterized colorectal cancer PDX models were employed to conduct a single mouse per treatment group (n = 1) trial. Results: Consistent with clinical results, cetuximab was efficacious in wild-type KRAS and BRAF PDX models, with an overall response rate of 6.3% and disease control rate (DCR) of 20.3%. LSN3074753 was active in a small subset of PDX models that harbored KRAS or BRAF mutations. However, the combination treatment displayed the enhanced antitumor activity with DCR of 35.4%. Statistical analysis revealed that BRAF and KRAS mutations were the best predictors of the combinatorial activity and were significantly associated with synergistic effect with a P value of 0.01 compared with cetuximab alone. In 12 models with BRAF mutations, the combination therapy resulted in a DCR of 41.7%, whereas either monotherapy had a DCR of 8.3%. Among 44 KRAS mutation models, cetuximab or LSN3074753 monotherapy resulted in a DCR of 13.6% or 11.4%, respectively, and the combination therapy increased DCR to 34.1%. Molecular analysis suggests that EGFR activation is a potential feedback and resistant mechanism of pan-RAF inhibition. Conclusions: MAPK and EGFR pathway activations are two major molecular hallmarks of colorectal cancer. This mouse PDX trial recapitulated clinical results of cetuximab. Concurrent EGFR and RAF inhibition demonstrated synergistic antitumor activity for colorectal cancer PDX models with a KRAS or BRAF mutation. Clin Cancer Res; 23(18); 5547–60. ©2017 AACR.

Abstract DDT02-02: Identification of LY3009120 as a pan inhibitor of Raf isoforms and dimers with minimal paradoxical activation and activities against BRaf or Ras mutant tumor cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Mutations in KRas, NRas, BRaf and NF-1 that activate the Ras and mitogen-activated protein kinase (MAPK) pathway are among the most common oncogenic drivers in many cancers, including melanoma, lung, colorectal, and pancreatic cancer. Two BRaf selective inhibitors, vemurafenib and dabrafenib, have been approved for the treatment of melanoma patients harboring the BRaf V600E/K mutation. However, both compounds have been reported to promote paradoxical MAPK pathway activation in BRaf wild-type cells through induction of active Raf dimers. Therefore, they are believed to be contraindicated for treatment of cancers with BRaf wild type background. In this study, we have identified and characterized a pyrido-pyrimidine derivative inhibitor of all three Raf isoforms. A whole-cell mass spectrum-based analysis revealed that LY3009120 binds to ARaf, BRaf and CRaf isoforms with similar affinity in cells with activating mutations of BRaf or KRas, while vemurafenib or dabrafenib have little or modest CRaf activity. Additionally, LY3009120 induces BRaf-CRaf heterodimerization, but inhibits the phosphorylation of downstream MEK and ERK, indicating that it effectively inhibits the kinase activity of BRaf-CRaf heterodimer. Due to its activity against the three Raf isoforms and dimer, LY3009120 induces minimal paradoxical pathway activation in NRas or KRas mutant cells. These unique pharmacological properties of LY3009120 further distinguish it from selective BRaf inhibitors by its physiologically-relevant activities against tumor cells with NRas or KRas mutations. LY3009120 inhibits MEK phosphorylation and cell proliferation in vitro, and exhibits anti-tumor activity in multiple xenograft models carrying mutations in BRaf, NRas or KRas. LY3009120 is also active against melanoma cells with acquired resistance to vemurafenib or dabrafenib in the setting of MAPK reactivation and cyclin D1 upregulation caused by RTK/Ras activation, BRaf splice variants, or NRas Q61K mutation. Collectively, our findings identify LY3009120 as a potentially best-in-class inhibitor of three Raf isoforms and Raf dimer, with activity against tumor cells with BRaf, NRas or KRas mutations, as well as melanoma cells with acquired resistance to current BRaf therapies. These unique features support investigation of LY3009120 in clinical studies. Citation Format: Sheng-Bin Peng, James Henry, Michael Kaufman, Wei-Ping Lu, Bryan D. Smith, Subha Vogeti, Scott Wise, Youyan Zhang, Robert Van Horn, Xiaoyi Zhang, Tinggui Yin, Vipin Yadav, Lysiane Huber, Lisa Kays, Jennie Walgren, Denis McCann, Phenil Patel, Sean Buchanan, Ilaria Conti, James J. Starling, Daniel L. Flynn. Identification of LY3009120 as a pan inhibitor of Raf isoforms and dimers with minimal paradoxical activation and activities against BRaf or Ras mutant tumor cells. [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 DDT02-02. doi:10.1158/1538-7445.AM2014-DDT02-02

Abstract LB-004: Mouse PDX Trial Suggests Combination Efficacy of Raf and EGFR Inhibition in Colorectal Cancer withBRaforKRasmutation

MAPK activation through KRas, NRas or BRaf mutation occurs in approximately 70% of colorectal cancer patients. Due to their epithelial origin, colorectal tumors generally have high levels of EGFR expression and activation. EGFR therapies such as cetuximab are effective for treatment of a subset of colorectal cancer, particularly patients with wild type (WT) KRas . EGFR signaling is also recently identified as a key resistance mechanism in BRaf mutant colorectal cancer to BRaf inhibitors. In this study, we have genetically characterized 78 patient-derived xenograft (PDX) models of colorectal tumors, and conducted an “n = 1” (single mouse per treatment group) trial in these PDX models with cetuximab, LSN3074753, a pan-Raf and Raf dimer inhibitor, and their combination in collaboration with Oncotest GmbH and Champions Oncology. Among these 78 PDX models, 42 (53.8%) have a KRas mutation, 12 (15.4%) have BRaf V600E or an atypical BRaf mutation, and 26 (33.3%) are WT KRas and BRaf . Consistent with clinical results, cetuximab is primarily active in WT KRas and BRaf PDX models, with disease control rate (DCR) of 53.8% (14/26) in this subgroup. These results suggest that the mouse n = 1 PDX trial paradigm could reliably predict clinical results. For pan-Raf and Raf dimer inhibitor LSN3074753, it is active in a subset of PDX models, particularly those with BRaf or KRas mutation(s), with DCR of 21.2% among models with a KRas or BRaf mutation. Importantly, a synergistic effect is observed when cetuximab and LSN3074753 are combined for treatment of these 78 PDX models. The overall DCR in the combination arm is 50% (39/78), while cetuximab or LSN3074753 alone has an overall DCR of 24 or 18%, respectively. Further statistical analyses reveal that BRaf mutations including V600E or other atypical mutations ( G469E, G76E, G596V, G203V , etc) are the best predictor of combination synergy, and are significantly associated with synergistic effect with a p value of 0.004. In models with BRaf mutations, the combination arm has a DCR of 50% (6/12), whereas cetuximab or LSN3074753 alone has a DCR of 8.3 or 17%, respectively. BRaf or KRas mutations are also significantly associated with combination synergy with p value of 0.01. Among 42 KRas mutation models, LSN3074753 or cetuximab alone has a DCR of 21.4 or 16.7%, and the combination arm has a DCR of 43%. Overall, these results indicate that combination of EGFR and Raf inhibition by cetuximab and a pan-Raf inhibitor has the potential for treatment of colorectal cancer patients with BRaf or KRas mutation. Citation Format: Yung-mae M. Yao, Gregory P. Donoho, Philip W. Iversen, Yue Wang Webster, Yong Gang Yue, James R. Henry, Gregory D. Plowman, Sheng-Bin Peng. Mouse PDX Trial Suggests Combination Efficacy of Raf and EGFR Inhibition in Colorectal Cancer with BRaf or KRas mutation. [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 LB-004. doi:10.1158/1538-7445.AM2015-LB-004

Abstract B21: Resistance to B-RAF inhibitor is mediated by reactivation of MAPK pathway by FGFR3/Ras signaling in B-RAF V600E mutant melanoma

Melanoma is the most aggressive form of skin cancer. The RAF/MEK/ERK MAP kinase signaling plays a key role in melanoma progression and etiology, and is considered an important target for anti-melanoma therapies. Approximately 50% of melanomas carry an activating B-RAF V600E mutation that results in constitutive activation of B-RAF and downstream MAPK signaling. Recently, vemurafenib (PLX4032), a selective B-RAF inhibitor, was recently approved by U.S. FDA for treatment of metastatic and unresectable melanomas that carry an activating B-RAF V600E mutation. However, patients eventually developed resistance to vemurafenib and relapsed within an average of 7 months. To improve the clinical benefit of B-RAF inhibitors, it is critical to identify the molecular mechanisms that confer resistance to B-RAF inhibition. Recent reports indicate that multiple mechanisms may render mutant B-RAF expressing melanoma cells resistant to B-RAF inhibition. In this study, we generated vemurafenib-resistant cell lines by chronic treatment of the human B-RAF V600E melanoma cell lines, and investigated the underlying mechanism(s) of resistance. Our analysis revealed that MAPK pathway reactivation through Ras is the key resistance mechanism in these cells. Furthermore, microarray-based gene expression profiling confirmed a significant elevation of RTK and Ras gene signatures in the vemurafenib-resistant cells. Mechanistically, we found that the enhanced activation of FGFR3 is linked to elevated Ras and MAPK activation in the resistant cells, therefore conferring resistance to vemurafenib. Constitutive or inducible activation of FGFR3 sufficiently reactivated Ras/MAPK signaling and conferred resistance to vemurafenib in the parental B-RAF V600E melanoma cells. Additionally, genetic or pharmacological inhibition of the FGFR3/Ras axis restored the sensitivity of the resistant cells to vemurafenib. Finally, we demonstrated that vemurafenib-resistant cells are sensitive to MEK or pan-RAF inhibition and maintain their addiction to the MAPK pathway. Therefore, inhibition of MEK or pan-RAF activities is an effective therapeutic strategy to overcome acquired-resistance to vemurafenib. Together, we describe a novel FGFR3/Ras mediated mechanism for acquired-resistance to B-RAF inhibition. Our results shed new light on the complexity of the resistance mechanisms and have implications for the development of new therapeutic strategies to improve the outcome of patients with B-RAF V600E melanoma.

Abstract 3624: A novel, selective FGFR inhibitor that causes blockade of FGFR autophosphorylation shows a broad-spectrum of anti-tumor activity in preclinical xenograft tumor models

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Anti-angiogenic agents targeting VEGF/VEGFR including Bevacizumab, Sorafenib, and Sunitinib that target blood vessel formation in rapidly growing cancer cells are very effective cancer therapies in the clinic. However, these anti-angiogenic agents also have debilitating side effects, such as hypertension and bleeding, which thereby limit their use in many patients in which the anti-VEGF/VEGFR based therapies are contraindicated. Furthermore, the emergence of resistance to the anti-VEGF/VEGFR based therapies in the clinic due to over-expression of FGF2 or other growth factors could pose a serious challenge to their current use in the clinic. The FGFRs are receptor tyrosine kinases that are present in many types of tumor cells as well as endothelial cells, and play an important role in tumor cell growth, survival, and migration, and also in maintaining tumor angiogenesis. Over-expression of FGFRs or aberrant regulation of their activities has been implicated in many forms of human malignancies. Therefore, targeting FGFRs for cancer treatment represents an attractive strategy that has the potential to treat a variety of cancers by simultaneously inhibiting tumor cell growth, survival, and migration, and also tumor angiogenesis without the side effects associated with the VEGF/VEGFR based therapies. Here, we describe a selective small molecule inhibitor of FGFRs. The FGFR inhibitor is active against all four FGF receptors (IC50 = 2.6 to 7.7 nM) as revealed by biochemical autophosphorylation assays. It also exhibits a potent activity against FGFR or FGFR-mediated Erk phosphorylation in several cancer cell lines (IC50 = 0.6 to 1.5 nM). In addition, in vitro cell based assays have shown that this inhibitor is much more active at inhibiting the proliferation of FGFR-dependent than FGFR-independent cancer cell lines (IC50s = 3-2,600 nM). Furthermore, in vitro selectivity assays have established that this inhibitor exhibits a robust preferential activity in inhibiting FGF- over VEGF-mediated cord formation (IC50s = 0.6 nM for FGF and 3.6 nM for VEGF). Consistent with these in vitro selectivity data, in vivo target inhibition assays have also demonstrated that the FGFR inhibitor has a potent, yet much higher activity against FGF- than VEGF-mediated target phosphorylation in mice (TEC50s and TEC90s of 6 and 29 nM for FGFR, and 35 and 252 nM for VEGFR, respectively). Finally, the FGFR inhibitor shows an excellent broad-spectrum anti-tumor activity in xenograft tumor models representing several major cancer histologies including bladder cancer when dosed at TED50 (1.3 mg/kg) and especially at TED90 (3.2 mg/kg). Thus, the FGFR inhibitor exhibits a very well behaved PK/PD relationship. Currently, the FGFR inhibitor is being further evaluated for its potential utility in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3624.