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Supplementary Table 1 from A Small-Molecule Inhibitor of Bcl-XL Potentiates the Activity of Cytotoxic Drugs In vitro and In vivo

Inhibition of the prosurvival members of the Bcl-2 family of proteins represents an attractive strategy for the treatment of cancer. We have previously reported the activity of ABT-737, a potent inhibitor of Bcl-2, Bcl-XL, and Bcl-w, which exhibits monotherapy efficacy in xenograft models of small-cell lung cancer and lymphoma and potentiates the activity of numerous cytotoxic agents. Here we describe the biological activity of A-385358, a small molecule with relative selectivity for binding to Bcl-XL versus Bcl-2 (Ki's of 0.80 and 67 nmol/L for Bcl-XL and Bcl-2, respectively). This compound efficiently enters cells and co-localizes with the mitochondrial membrane. Although A-385358 shows relatively modest single-agent cytotoxic activity against most tumor cell lines, it has an EC50 of L for survival. In addition, A-385358 enhances the in vitro cytotoxic activity of numerous chemotherapeutic agents (paclitaxel, etoposide, cisplatin, and doxorubicin) in several tumor cell lines. In A549 non–small-cell lung cancer cells, A-385358 potentiates the activity of paclitaxel by as much as 25-fold. Importantly, A-385358 also potentiated the activity of paclitaxel in vivo. Significant inhibition of tumor growth was observed when A-385358 was added to maximally tolerated or half maximally tolerated doses of paclitaxel in the A549 xenograft model. In tumors, the combination therapy also resulted in a significant increase in mitotic arrest followed by apoptosis relative to paclitaxel monotherapy. (Cancer Res 2006; 66(17): 8731-9)

Introduction: Bruton’s tyrosine kinase (BTK), a member of the Tec family, plays a crucial role in signaling through B-cell receptor (BCR). BTK inhibition blocks BCR signals and prevents B-cell activation and growth. First-generation BTK inhibitors such as ibrutinib covalently binds to a cysteine residue (C481) of BTK. Their most frequent acquired resistance is the development of a serine mutation in the binding site (C481S). Next generation BTK inhibitors such as LOXO-305 and ARQ 531 are being developed to overcome this resistance to first-generation inhibitors. Methods: HMPL-760 was tested in biochemical assays using recombinant human wild type (WT) and C481S mutant BTKs. Its selectivity was carried out using Eurofins Cerep KinaseProfilerTM panel. Cellular activity of HMPL-760 was evaluated in HEK293 cells stably transfected with BTKWT or BTKC481S, and other tumor cell lines, which are either human diffuse large B cell lymphoma (DLBCL) or mantle cell lymphoma (MCL) cell lines. The in vivo antitumor activity and PKPD correlation of HMPL-760 was studied in HBL-1 xenograft mouse models bearing BTKWT or BTKC481S respectively. Results: In biochemical assays, HMPL-760 strongly inhibits BTK kinase activities towards wild-type BTK (BTKWT) and C481S mutant (BTKC481S), and binds to BTK in a reversible way. HMPL-760 demonstrates high selectivity in a panel containing 413 kinases. In cellular assays, HMPL-760 displays strong anti-proliferative activities in B-cell lymphoma cells (TMD-8, OCI-LY10, REC-1, HBL-1 and HBL-1-BTKC481S) harboring either BTKWT or BTKC481S (GI50: 0.0015-0.046 μM). In human whole blood assay, HMPL-760 inhibits activation of B-cells at nanomolar concentrations measured by inhibition of immunoglobulin-induced CD69 expression in CD19+cells. HMPL-760 shows ≥ 10-fold inhibitory potency than ARQ 531 in both BTKWT and BTKC481S cells, and ~3-fold higher inhibitory potency than that of LOXO-305 in BTKC481S cells. In cellular assay by detecting p-BTK after compound washout, HMPL-760 maintains a longer duration of target inhibition than LOXO-305 in both BTK wild type (HBL-1) and BTK mutant (HBL-1-BTKC481S) cell lines. HMPL-760 displays dose-dependent antitumor efficacy in multiple human B cell lymphoma xenograft models in mice when orally administered at 3~50 mg/kg once daily. Complete tumor regression occurs in most of the tested models at the high dose levels. HMPL-760 shows much stronger antitumor efficacy than LOXO-305 and ARQ 531 at similar dose level, which may be associated with HMPL-760’s higher drug exposures and more sustainable inhibition on BTK phosphorylation in the tumor tissues. Conclusion: HMPL-760 is a reversible, selective, highly potent, BTK inhibitor targeting both BTKWT and BTKC481S. The first-in-human Phase 1 clinical trials of HMPL-760 are under way in patients with r/r B-NHL (NCT05190068, NCT05176691). Citation Format: Linfang Wang, Junqing Liang, Zhihu Gao, Jia Hu, Weigang He, Xianwen Yang, Fangfang Mao, Wei Zhang, Ying Yu, Qihang Zhang, Na Yang, Chun Zhang, Jian Wang, Yu Cai, Xiong Li, Weiguo Qing, Guangxiu Dai, Yongxin Ren, Michael Shi, Weiguo Su. HMPL-760 is a highly potent and selective reversible BTK inhibitor, targeting BTK and BTKC481S in B-cell malignancies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4020.

Background: The delta isoform of PI3K (PI3Kδ) plays an important role in B-cell development and function by mediating the signaling of key receptors on B cells. Aberrant activation of PI3Kδ signaling pathway promotes the survival and proliferation of malignant B cells, making selective inhibition of this isoform a promising therapeutic approach for the treatment of B cell malignances. Amdizalisib (HMPL-689, Amdiz), discovered and being currently developed in a pivotal phase II clinical trial (NCT04849351) by HUTCHMED, is a highly potent and selective PI3Kδ inhibitor. Based on encouraging clinical data from the phase Ib study (2021 ESMO, abstract# 8330), Amdiz is a designated breakthrough therapy in China for treatment of relapsed and refractory follicular lymphoma. Herein, we report the pre-clinical anti-tumor activity of Amdiz. Methods: Kinase activity was measured by Transcreener™ Fluorescence Polarization assay. The kinome selectivity profile of Amdiz was evaluated in Eurofins KinaseProfiler™ panel containing 323 kinases. Cell based phosphorylation of AKT was determined by using Acumen Explorer system. CD63 expression in basophils was evaluated by FACS assay. Inhibitory effects of Amdiz alone or in combination with other agents on cell viability were investigated in a panel of B cell lymphoma cell lines by CellTiter-Glo luminescent or CCK-8 assay. The ability of Amdiz to inhibit the activation of B cells in animals was evaluated in naïve rats using an ex vivo assay, and B cell activation was determined by evaluating CD86 expression using FACS analysis. Human B cell lymphoma cell line derived xenograft models were used to determine the anti-tumor activity of Amdiz in combination with other agents such as rituximab, BTK inhibitor, and venetoclax. Results: Amdiz is a highly selective inhibitor of PI3Kδ, showing more than 250-fold selectivity over other PI3K isoforms and no significant inhibition over other 319 protein kinases at the concentration of 1 µM. Amdiz potently inhibited PI3Kδ in biochemical, cellular and human whole blood assays with IC50s ranging from 0.8-3 nM. Its inhibitory effects on cell viability were also evaluated in a panel of B -cell lymphoma cell lines. Results showed that Amdiz potently inhibited cell survival with IC50s from 0.005 to 5 μM. Amdiz showed a long-lasting and strong inhibition on B cell activation in a rat pharmacodynamics (PD) study at a dose as low as 0.1 mg/kg. Moreover, Amdiz significantly improved anti-tumor activity of standard-of-care agents as well as targeted agents in multiple B-cell lymphoma models both in vitro and in vivo. Conclusion: Amdiz is a highly potent and selective PI3Kδ inhibitor with strong activity against B-cell lymphoma in pre-clinical studies, supporting clinical evaluation as either a single agent or in combination with other therapeutic agents for the treatment of B-cell malignancies. Citation Format: Jia Hu, Jianhan Wang, Xiaoming Dai, Jianlin He, Junqing Liang, Ying Yu, Juntao Yu, Na Yang, Linfang Wang, Yu Cai, Xiong Li, Weiguo Qing, Yongxin Ren, Weiguo Su. Amdizalisib (HMPL-689), a highly selective PI3Kδ inhibitor, exhibits potent anti-tumor activity in pre-clinical B-cell lymphoma 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 5454.

Savolitinib is a novel small-molecule selective cMet inhibitor. This work characterized its pharmacokinetics in preclinical phase, established the preclinical relationships between PK, cMet modulation and anti-tumor efficacy. In vitro and in vivo animal studies were performed for PK characterization. Savolitinib showed good absorption, moderate tissue distribution, low to intermediate clearance, and low accumulation. Hepatic oxidative metabolism followed by urinary and biliary excretions was the major elimination pathway. Based on preclinical PK data, human PK profiles were predicted using empirical methods. Pharmacodynamic studies for evaluating cMet inhibition and anti-tumor efficacy were conducted in nude mice bearing Hs746t xenograft. PK/PD models were built to link the PD measurements to nude mouse PK. The established integrated preclinical PK/PD model contained a two-compartment non-linear PK model, a biomarker link model and a tumor growth transit model. The IC50 of cMet inhibition and the concentration achieving half of the maximal Hs746t tumor reduction by savolitinib were equal to 12.5 and 3.7 nM (free drug), respectively. Based on the predicted human PK data, as well as the established PK/PD model in nude mouse, the human PD (cMet inhibition) profiles were also simulated. This research supported clinical development of savolitinib. Understanding the preclinical PK/PD relationship of savolitinib provides translational insights into the cMet-targeted drug development.

VEGF/VEGFR signal axis has been proven to be an important target for development of novel cancer therapies. One challenging aspect in small molecular VEGFR inhibitors is to achieve sustained target inhibition at tolerable doses previously seen only with the long-acting biologics. It would require high potency (low effective drug concentrations) and sufficient drug exposures at tolerated doses so that the drug concentration can be maintained above effective drug concentration for target inhibition within the dosing period. Fruquintinib (HMPL-013) is a small molecule inhibitor with strong potency and high selectivity against VEGFR family currently in Phase II clinical studies. Analysis of Phase I pharmacokinetic data revealed that at the maximum tolerated dose of once daily oral administration fruquintinib achieved complete VEGFR2 suppression (drug concentrations were maintained above that required to produce >85% inhibition of VEGFR2 phosphorylation in mouse) for 24 hours/day. In this article, the preclinical data for fruquintinib will be described, including kinase enzyme activity and selectivity, cellular VEGFR inhibition and VEGFR-driven functional activity, in vivo VEGFR phosphorylation inhibition in the lung tissue in mouse and tumor growth inhibition in a panel of tumor xenograft and patient derive xenograft models in mouse. Pharmacokinetic and target inhibition data are also presented to provide a correlation between target inhibition and tumor growth inhibition.

HGF/c-Met signaling has been implicated in human cancers. Herein we describe the invention of a series of novel triazolopyrazine c-Met inhibitors. The structure-activity relationship of these compounds was investigated, leading to the identification of compound 28, which demonstrated favorable pharmacokinetic properties in mice and good antitumor activities in the human glioma xenograft model in athymic nude mice.

// Yongxin Ren 1, * , Jianming Zheng 2, * , Shiming Fan 1, * , Linfang Wang 1 , Min Cheng 1 , Dongxia Shi 1 , Wei Zhang 1 , Renxiang Tang 1 , Ying Yu 1 , Longxian Jiao 1 , Jun Ni 1 , Haibin Yang 3 , Huaqing Cai 3 , Fang Yin 1 , Yunxin Chen 1 , Feng Zhou 1 , Weihan Zhang 3 , Weiguo Qing 1 and Weiguo Su 3 1 Department of Oncology Research, Hutchison MediPharma Limited, Shanghai, China 2 Department of Pathology, Changhai Hospital, the Second Military Medical University, Shanghai, China 3 Department of Chemistry, Hutchison MediPharma Limited, Shanghai, China * These authors are contributed equally to this work Correspondence to: Yongxin Ren, email: yongxinr@hmplglobal.com Weiguo Su, email: weiguos@hmplglobal.com Keywords: esophageal cancer, patient derived xenograft models, EGFR targeted therapy, theliatinib Received: December 01, 2016 Accepted: March 27, 2017 Published: April 19, 2017 ABSTRACT Targeted therapy is not yet approved for esophageal cancer (EC). In this study, we first evaluated EGFR gene and protein expression in 70 Chinese EC patient tumor samples collected during surgery. We then established 23 patient-derived EC xenograft (PDECX) models and assessed the efficacy of theliatinib, a potent and highly selective EGFR inhibitor currently in Phase I clinical study, in 9 PDECX models exhibiting various EGFR expression levels. Immunohistochemical analysis showed that 50 patient tumor samples (71.4%) had high EGFR expression. Quantitative PCR showed that eight tumors (11.6%) had EGFR gene copy number gain, and fluorescence in situ hybridization (FISH) revealed that four tumors had EGFR gene amplification. These results suggest that EGFR protein may be overexpressed in many EC tumors without gene amplification. Also detected were rare hot-spot mutations in EGFR and PIK3CA , whereas no mutations were found in K-Ras or B-Raf . Theliatinib exhibited strong antitumor activity in PDECX models with high EGFR expression, including remarkable tumor regression in two PDECX models with both EGFR gene amplification and protein overexpression. However, the efficacy of theliatinib was diminished in models with PI3KCA mutations or FGFR1 overexpression in addition to high EGFR expression. This study demonstrates that theliatinib could potentially benefit EC patients with high EGFR protein expression without mutations or aberrant activities of associated factors, such as PI3KCA or FGFR1.

// Ryan E. Henry 1,* , Evan R. Barry 1,* , Lillian Castriotta 1 , Brendon Ladd 1 , Aleksandra Markovets 1 , Garry Beran 2 , Yongxin Ren 3 , Feng Zhou 3 , Ammar Adam 1 , Michael Zinda 1 , Corinne Reimer 1 , Weiguo Qing 3 , Weiguo Su 3 , Edwin Clark 1 , Celina M. D’Cruz 1 and Alwin G. Schuller 1 1 AstraZeneca Pharmaceuticals PLC, Oncology Bioscience, Waltham, MA, USA 2 AstraZeneca Pharmaceuticals PLC, Oncology Bioscience, Alderley Park, UK 3 Hutchison Medi Pharma Ltd, Zhangjiang Hi-Tech Park, Shanghai, China * These authors have contributed equally to this work Correspondence to: Alwin G. Schuller, email: // Celina M. D’Cruz, email: // Keywords : MET, MYC, NSCLC, acquired resistance, savolitinib Received : January 14, 2016 Accepted : July 13, 2016 Published : July 26, 2016 Abstract Lung cancer is the most common cause of cancer death globally with a significant, unmet need for more efficacious treatments. The receptor tyrosine kinase MET has been implicated as an oncogene in numerous cancer subtypes, including non-small cell lung cancer (NSCLC). Here we explore the therapeutic potential of savolitinib (volitinib, AZD6094, HMPL-504), a potent and selective MET inhibitor, in NSCLC. In vitro , savolitinib inhibits MET phosphorylation with nanomolar potency, which correlates with blockade of PI3K/AKT and MAPK signaling as well as MYC down-regulation. In vivo , savolitinib causes inhibition of these pathways and significantly decreases growth of MET-dependent xenografts. To understand resistance mechanisms, we generated savolitinib resistance in MET -amplified NSCLC cell lines and analyzed individual clones. We found that upregulation of MYC and constitutive mTOR pathway activation is a conserved feature of resistant clones that can be overcome by knockdown of MYC or dual mTORC1/2 inhibition. Lastly, we demonstrate that mechanisms of resistance are heterogeneous, arising via a switch to EGFR dependence or by a requirement for PIM signaling. This work demonstrates the efficacy of savolitinib in NSCLC and characterizes acquired resistance, identifying both known and novel mechanisms that may inform combination strategies in the clinic.

The p53 tumor suppressor retains its wild-type conformation and transcriptional activity in half of all human tumors, and its activation may offer a therapeutic benefit. However, p53 function could be compromised by defective signaling in the p53 pathway. Using a small-molecule MDM2 antagonist, nutlin-3, to probe downstream p53 signaling we find that the cell-cycle arrest function of the p53 pathway is preserved in multiple tumor-derived cell lines expressing wild-type p53, but many have a reduced ability to undergo p53-dependent apoptosis. Gene array analysis revealed attenuated expression of multiple apoptosis-related genes. Cancer cells with mdm2 gene amplification were most sensitive to nutlin-3 in vitro and in vivo, suggesting that MDM2 overexpression may be the only abnormality in the p53 pathway of these cells. Nutlin-3 also showed good efficacy against tumors with normal MDM2 expression, suggesting that many of the patients with wild-type p53 tumors may benefit from antagonists of the p53–MDM2 interaction.

Both vascular endothelial growth factor receptor (VEGFR) and fibroblast growth factor receptor (FGFR) singling pathways can mediate tumor angiogenesis. Colony stimulating factor 1 receptor (CSF1R) plays an important role on functions of macrophages. Recently the roles of the VEGFR, FGFR and CSF1R in regulation of T cells, tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells, thereby increasing tumor immune evasion, have been demonstrated[1-3]. Therefore, blockade of tumor angiogenesis and tumor immune evasion by simultaneously targeting VEGFR, FGFR and CSF1R kinases may represent a promising approach for anti-cancer therapy. We report here the preclinical studies for sulfatinib (HMPL-012), a potent and highly selective small molecule tyrosine kinase inhibitor against VEGFR, FGFR1 and CSF1R. Sulfatinib inhibited VEGFR1, 2, and 3, FGFR1 and CSF1R kinases with IC50s in a range of 1~24 nM, and it strongly blocked VEGF induced VEGFR2 phosphorylation in HEK293KDR cells and CSF1 stimulated CSF1R phosphorylation in RAW264.7 cells with IC50 of 2 and 79 nM, respectively. Sulfatinib also attenuated VEGF or FGF stimulated HUVEC cells proliferation with IC50 < 50 nM. In animal studies, a single oral dosing of sulfatinib inhibited VEGF stimulated VEGFR2 phosphorylation in lung tissues of nude mice in an exposure-dependent manner. Furthermore, elevation of FGF23 levels in plasma 24 hours post dosing suggested suppression of FGFR signaling. Sulfatinib demonstrated potent tumor growth inhibition in multiple human xenograft models and decreased CD31 expression remarkably, suggesting strong inhibition on angiogenesis through VEGFR and FGFR signaling. In a syngeneic murine colon cancer model CT-26, sulfatinib demonstrated moderate tumor growth inhibition after single agent treatment. Flow cytometry and immunohistochemistry analysis revealed an increase of CD8+ T cells and a significant reduction in TAMs, (CD163+ or F4/80+CD11b+CD45+) and CSF1R+ TAMs in tumor tissue indicating strong effect on CSF1R. Interestingly, combination of sulfatinib with a PD-L1 antibody resulted in enhanced anti-tumor effect. These results suggested that sulfatinib has a strong effect in modulating angiogenesis and cancer immunity. In summary, sulfatinib is a novel angio-immuno kinase inhibitor targeting VEGFR, FGFR1 and CSF1R kinases that could simultaneously block tumor angiogenesis and immune evasion. This unique feature seems to support sulfatinib as an attractive candidate for exploration of possible combinations with checkpoint inhibitors against various cancers. Sulfatinib is currently in multiple clinical trials including two Phase III trials against neuroendocrine tumors. Reference: 1. Voron T, et al. J Exp Med. 2015; 212(2):139-48. 2. Ries CH, et al. Cancer Cell. 2014; 25(6):846-59. 3. Katoh M, et al. Int. J. of Mol Med. 2016; 38: 3-15. Citation Format: jinghong Zhou, Jun Ni, Min Cheng, Na Yang, Junqing Liang, Liang Ge, Wei Zhang, Jianxing Tang, qiaoling Sun, Fu Li, Jia Hu, Dongxia Shi, Hongbo Chen, Jingwen Long, Junen Sun, Fang Yin, Xuelei Ge, Hong Jia, Feng Zhou, Yongxin ren, Weiguo Qing, Weiguo Su. Preclinical evaluation of sulfatinib, a novel angio-immuno kinase inhibitor targeting VEGFR, FGFR1 and CSF1R kinases [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 4187. doi:10.1158/1538-7445.AM2017-4187

The development of therapies targeting tumor angiogenesis, tumor driver gene alterations and tumor immune evasion has made tremendous advancement in improving overall survival. However, efficacy may be limited and resistance often develops rapidly when targeting a single axis of tumorigenesis. Therefore, it is worthwhile to explore rational combination of therapies based on tumor-specific features. Fruquintinib is a potent and selective oral VEGFR inhibitor currently in Phase III clinical trials for non-small-cell lung cancer (NSCLC) and colorectal cancer (CRC). We report here the evaluation of anti-tumor effect of fruquintinib in preclinical animal tumor models in combination with therapies targeting tumor driver gene alterations such as EGFR and c-MET or with immune checkpoints. In NSCLC xenograft models with EGFR activation such as activating mutations, gene amplification or protein overexpression, fruquintinib plus an EGFR tyrosine kinase inhibitor such as gefitinib or theliatinib (HMPL-309) was found to be more efficacious than either monotherapy. For instance, in PC-9 subcutaneous tumor model carrying EGFR exon 19 deletion, single agent treatment with fruquintinib at 2 mg/kg and gefitinib at 5 mg/kg produced the tumor growth inhibition (TGI) of 58% and 63%, respectively, while the combination treatment resulted in a TGI of 100% and tumor regression was observed in 11 of 16 mice treated with combinational therapy. In multiple xenograft models derived from lung cancer or renal cell cancer with c-MET activation (amplification or over-expression), addition of fruquintinib to a c-MET inhibitor savolitinib (AZD6094, HMPL-504) also improved the tumor growth inhibition substantially. At the end of the efficacy studies, CD31 and phosphorylation of EGFR, c-MET, AKT and ERK were analyzed with immunohistochemistry and western blotting method in tumor tissues. The results suggested that the enhanced anti-tumor effect in combination therapy could be attributed to the simultaneous blockade of cell signaling in tumor cells (EGFR or c-MET) and VEGFR suppression in the tumor microenvironment. Up-regulation of the immune inhibitory checkpoints induced by VEGF is one of the important mechanisms for tumor cells to escape immune surveillance. In a syngeneic murine tumor model, co-administration of fruquintinib and anti-PD-L1 antibody was found to provide improved anti-tumor effect compared to fruquintinib or anti-PD-L1 single agent alone. Studies to understand the mechanism responsible for the combination effect are under way. All combinations with fruquintinib described above were well tolerated. The efficacy observed in these models suggested that simultaneous blockade of tumor angiogenesis and tumor cell signaling or immune evasion may be a promising approach in improving treatment outcomes. Citation Format: Yongxin Ren, Qiaoling Sun, Jingwen Long, Shiming Fan, Renxiang Tang, Wei Zhang, Xuelei Ge, Jianxing Tang, Linfang Wang, Dongxia Shi, Hongbo Chen, Min Cheng, Weiguo Qing, Weiguo Su. Evaluation of fruquintinib, a potent and selective oral VEGFR inhibitor, in combination with targeted therapies or immune checkpoint inhibitors in preclinical tumor models [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 2089. doi:10.1158/1538-7445.AM2017-2089

HGF/c-Met signaling has been implicated in human cancers. Herein we describe the invention of a series of novel triazolopyrazine c-Met inhibitors. The structure-activity relationship of these compounds was investigated, leading to the identification of compound 28, which demonstrated favorable pharmacokinetic properties in mice and good antitumor activities in the human glioma xenograft model in athymic nude mice.

Purpose To investigate the incidence of cMET gene copy number changes and protein overexpression in Chinese gastric cancer (GC) and to preclinically test the hypothesis that the novel, potent and selective cMET small-molecule inhibitor volitinib, will deliver potent anti-tumor activity in cMET-dysregulated GC patient-derived tumor xenograft (PDX) models. Experimental design A range of assays were used and included; in vitro cell line panel screening and pharmacodynamic (PD) analysis, cMET fluorescence in-situ hybridization (FISH) and immunohistochemical (IHC) tissue microarray (TMA) analysis of Chinese GC (n = 170), and anti-tumor efficacy testing and PD analysis of gastric PDX models using volitinib. Results The incidence of cMET gene amplification and protein overexpression within Chinese patient GC tumors was 6% and 13%, respectively. Volitinib displayed a highly selective profile across a gastric cell line panel, potently inhibiting cell growth only in those lines with dysregulated cMET (EC50 values 0.6 nM/L–12.5 nM/L). Volitinib treatment led to pharmacodynamic modulation of cMET signaling and potent tumor stasis in 3/3 cMET-dysregulated GC PDX models, but had negligible activity in a GC control model. Conclusions This study provides an assessment of tumor cMET gene copy number changes and protein overexpression incidence in a cohort of Chinese GC patients. To our knowledge, this is the first study to demonstrate anti-tumor efficacy in a panel of cMET-dysregulated gastric cancer PDX models, using a novel selective cMET-inhibitor (volitinib). Thus, the translational science presented here provides strong rationale for the investigation of volitinib as a therapeutic option for patients with GC tumors harboring amplified cMET.

Introduction: Spleen Tyrosine Kinase (SYK), a non-receptor type of tyrosine kinase, is a member of Syk/ZAP70 tyrosine kinase family. It plays a pivotal role in the regulation of B-cell receptor (BCR) signal pathway, which regulates proliferation, differentiation and survival of B lymphocytes. The abnormal activation of BCR singling is closely related to transformation and development of B cell lymphoma. Targeting BCR downstream molecules, such as Bruton' tyrosine kinase (BTK) and phosphoinositide-3-kinase δ (PI3Kδ) has emerged as new therapeutic approaches and inhibitors of BTK and PI3Kδ were approved recently by FDA for treatment of some subtypes of B-cell malignancies. Currently, a couple small molecular inhibitors against SYK, another BCR downstream molecule, are under the early clinical development and showed initial efficacy in B cell lymphomas. HMPL-523, discovered and currently being developed in Phase I clinical trial by Hutchison MediPharma, is a novel, highly potent and selective SYK inhibitor (IC50: 0.025 μM). The anti-tumor activity of HMPL-523 was evaluated in this study. Methods: Inhibitory effects of HMPL-523 on cell viability were investigated in a panel of B cell lymphoma cell lines with SYK/BCR dysregulation by CellTiter-Glo luminescent or CCK-8 assay. The effect of HMPL-523 on SYK signaling pathway was detected by western blot. Annexin-V- positive and PI-negative population was recognized as apoptotic cells by FACS. Nude mice bearing B cell lymphoma xenograft tumors with SYK/BCR dysregulation were used to determine anti-tumor activity of HMPL-523 in vivo. Result: HMPL-523 blocked phosphorylation of BLNK, downstream protein of Syk, in human mantle cell line REC-1 and human plasma cell line ARH-7777 with IC50 of 0.105 µM and 0.173 μM, respectively. HMPL-523 also inhibited cell viability of Ba/F3 Tel-Syk with IC50 of 0.033 μM. Furthermore, inhibitory effects of HMPL-523 on cell viability were evaluated in a panel of B -cell lymphoma cell lines with SYK/BCR deregulation. Results showed that HMPL- 523 potently inhibited cell survival with IC50s from 0.4 to 2 μM. Consistent with the effect on cell viability, HMPL-523 increased the apoptotic rate of REC-1 cells. Moreover, HMPL-523 showed the synergistic activities on killing human diffused large B cell lymphoma (DLBCL) in combination with other drugs such as BTK inhibitor, PI3Kδ inhibitors and Bcl2 family inhibitor. The detailed mechanisms underlying the synergism are still under investigation. Anti-tumor activity of HMPL-523 was determined in Syk dependent xenograft models. Daily oral administration of 100 mg/kg HMPL-523 showed potent anti-tumor activity in B cell lymphoma REC-1 (TGI: 59%). Conclusion:HMPL-523 is a highly potent SYK inhibitor with good activity against B-cell lymphoma in pre-clinical in vitro and in vivo models, supporting further clinical research for HMPL-523 as either single agent or combination drug with other agents to treat B-cell malignancies e.g. DLBCL Disclosures Yang: Hutchison MediPharma Ltd: Employment, Research Funding. Deng:Hutchison MediPharma Ltd: Employment, Research Funding. Sun:Hutchison MediPharma Ltd: Employment, Research Funding. Liang:Hutchison MediPharma Ltd: Employment, Research Funding. Wang:Hutchison MediPharma Ltd: Employment, Research Funding. Fan:Hutchison MediPharma Ltd: Employment, Research Funding. Tang:Hutchison MediPharma Ltd: Employment, Research Funding. Yu:Hutchison MediPharma Ltd: Employment, Research Funding. Sun:Hutchison MediPharma Ltd: Employment, Equity Ownership. Zhou:Hutchison MediPharma Ltd: Employment, Research Funding. Dai:Hutchison MediPharma Ltd: Employment, Research Funding. Qing:Hutchison MediPharma Ltd: Employment, Research Funding. Su:Hutchison MediPharma Ltd: Employment, Research Funding. Ren:Hutchison MediPharma Ltd: Employment, Research Funding.

Lung cancer is the most common cause of cancer death globally with a significant, unmet need for more efficacious treatments. Aberrant receptor tyrosine kinase (RTK) signaling is a well-documented driver of disease onset and progression in multiple cancer types, including non-small cell lung cancer (NSCLC), where the cMET RTK contributes to tumor progression, maintenance and resistance to targeted therapies. Here, we explore the therapeutic potential of the potent and selective cMET inhibitor savolitinib (volitinib, AZD6094, HMPL-504) in NSCLC and begin to elucidate mechanisms of acquired savolitinib resistance in preclinical models. Using in vitro proliferation assays and immunoblot analysis, we determine that savolitinib rapidly inhibits cMET auto-phosphorylation/activation and reduces the viability of NSCLC cell lines NCI-H1993 and EBC-1 with a GI50 of 4.20 nM and 2.14 nM, respectively. In vivo, once daily treatment of NCI-H1993 xenografts with 3.0 mg/kg savolitinib significantly slows tumor growth, whereas treatment of EBC-1 xenografts with 30.0 mg/kg results in tumor stasis. Importantly, we observe tumor regressions in a patient-derived xenograft model of a NSCLC lymph node metastasis, HLXF-036LN, dosed with savolitinib 50.0 mg/kg once daily. Pharmacodynamic analysis of in vitro and in vivo models shows that savolitinib sensitivity correlates with blockade of PI3K/AKT and MAPK signaling, and interestingly, with cMYC (MYC) protein down-regulation. To elucidate mechanisms of acquired resistance in NSCLC, we generated savolitinib resistance in vitro using the NCI-H1993 and EBC-1 cell lines and further sub-cloned resistant NCI-H1993 cells to study the heterogeneity of resistance mechanisms. Using small-molecule screening, phospho-protein arrays and interrogation of signaling pathway activity by immunoblot, we identify 1) deregulated mTORC1/2 signaling and 2) the uncoupling of MYC expression from cMET activation as commonly contributing to resistance in all clones tested. RNA interference (siRNA) and MYC over-expression experiments confirm the novel finding that sustained MYC expression can partially drive resistance to a tyrosine kinase inhibitor such as savolitinib. Additionally, we identify clone-specific resistance mechanisms arising via a previously-described switch to EGFR dependence or by our novel finding of a de novo requirement for PIM signaling. Taken together, this work demonstrates the preclinical efficacy of savolitinib in NSCLC and provides an initial characterization of potential resistance mechanisms, identifying core resistance targets and clone-specific vulnerabilities that could be exploited to counter acquired savolitinib resistance that may emerge in the clinic. Citation Format: Ryan E. Henry, Evan R. Barry, Brendon Ladd, Aleksandra Markovets, Garry J. Beran, Yongxin Ren, Feng Zhou, Lillian Castriotta, Ammar Adam, Weiguo Qing, Weiguo Su, Edwin Clark, Celina M. D'Cruz, Alwin Schuller. Acquired resistance to the cMET inhibitor savolitinib in lung cancer models through EGFR/mTOR/MYC deregulation and adoption of PIM signaling. [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 LB-C22.

Renal cell carcinoma (RCC) is the most common type of kidney tumor in human. Approximately 80∼85% of RCC is clear cell renal cell carcinoma (ccRCC).[1] Although VEGF/VEGFR targeted therapies bring significant advances in the treatment of RCC, ultimate resistance occurs in most cases following a transient period of clinical benefit. [2] The hepatocyte growth factor (HGF) receptor c-Met activation emerges as one of the mechanisms for resistance to anti-VEGF/VEGFR therapies in ccRCC,[3] implying that a combinational inhibition of c-Met and VEGFR pathways may induce a synergistic anti-tumor effect and could produce additional clinical benefit. The aim of this study was to assess the effect of a combination strategy targeting the VEGFR and c-Met pathways in ccRCC xenograft models. Savolitinib (AZD6094, HMPL-504) is a highly selective inhibitor against c-Met. Fruquintinib (HMPL-013) strongly inhibits VEGFR1, 2 and 3. Both of them were discovered by Hutchison MediPharma and are currently being evaluated in clinical trials for the treatment of various cancers. Several subcutaneous xenograft models were established in nude mice with human ccRCC cell lines or patient derived tumors (PDX) to investigate the anti-tumor effect of combination of savolitinib with fruquntinib. Treatment with savolitinib or fruquintinib at clinically relevant dose only exhibited mild to moderate tumor growth inhibition as a single agent in all of tested models, but significantly increased anti-tumor effect was observed in all of tested models for the combination group. It seemed that the enhanced anti-tumor effect was associated with c-Met inhibition. In a ccRCC PDX model KIN1T1342, the increased anti-tumor effect was correlated with dose increment of savolitinib. Immunohistochemistry (IHC) analysis revealed that combination treatment produced stronger inhibition on tumor proliferation marker Ki67 and angiogenesis marker CD31, compared to either savolitinib or fruquntinib alone, indicating that the observed synergistic effect might be attributed to the dual inhibition on tumor signaling and tumor microenvironment. C-Met expression was observed in all tested models, and treatment with savolitinib effectively suppressed phospho-Met. To evaluate c-Met expression in Chinese ccRCC patients, Formalin-Fixed and Parrffin-Embedded (FFPE) tumor sections were collected from sixty-two treatment-naive patients during surgical resection. Positive c-Met expression was found in 69% (43/62) of ccRCC samples under IHC staining.Overall our data demonstrated that c-Met was widely expressed in Chinese ccRCC patients and provided a rationale to test the combined HGF/c-Met and VEGF/VEGFR pathway blockade in the treatment of ccRCC in the clinical trials. References: 1. Harshman LC et al. Cancer J. 2013;19: 316-323 2. Swanton C et al. Genome Med. 2010; 2(8): 53 3.Ciamporcero E et al. Mol Cancer Ther. 2015: 14 (1); 101-10. Citation Format: Yongxin Ren, Shiming Fan, Yunxin Chen, Renxiang Tang, Wei Zhang, Jianxing Tang, Linfang Wang, Dongxia Shi, Hongbo Chen, Min Cheng, Weiguo Qing, Weiguo Su. Synergistic effect of c-Met inhibitor savolitinib in combination with a VEGFR inhibitor fruquintinib in clear cell renal cell carcinoma xenograft models. [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 B189.

A series of pyrimidodiazepines was identified as potent Polo-like kinase 1 (PLK1) inhibitors. The synthesis and SAR are discussed. The lead compound 7 (RO3280) has potent inhibitory activity against PLK1, good selectivity against other kinases, and excellent in vitro cellular potency. It showed strong antitumor activity in xenograft mouse models.

Inhibition of the prosurvival members of the Bcl-2 family of proteins represents an attractive strategy for the treatment of cancer. We have previously reported the activity of ABT-737, a potent inhibitor of Bcl-2, Bcl-XL, and Bcl-w, which exhibits monotherapy efficacy in xenograft models of small-cell lung cancer and lymphoma and potentiates the activity of numerous cytotoxic agents. Here we describe the biological activity of A-385358, a small molecule with relative selectivity for binding to Bcl-XL versus Bcl-2 (Ki's of 0.80 and 67 nmol/L for Bcl-XL and Bcl-2, respectively). This compound efficiently enters cells and co-localizes with the mitochondrial membrane. Although A-385358 shows relatively modest single-agent cytotoxic activity against most tumor cell lines, it has an EC50 of

MET is a transmembrane tyrosine kinase receptor that is deregulated (gene amplification, mutation and over-expression) across multiple cancer types. Signaling through MET is normally activated through interactions with its specific ligand, hepatocyte growth factor (HGF). Aberrant MET/HGF activation can stimulate tumor growth, promote angiogenesis, induce metastasis and may contribute to resistance mechanisms in several tumor types. Several non-selective MET inhibitors have entered clinical development; results have been mixed based on potency, selectivity, and/or patient selection. Volitinib is a potent (IC50 4 nM) and selective (>650 fold selectivity over 265 kinases), small molecule inhibitor of MET. Recent evaluation of Volitinib across a panel of cancer cell lines demonstrated selectivity for MET-driven disease, with MET amplified cell lines being most sensitive (IC50s of 1nM) and also suggesting limited off target activity. Volitinib resistant cell lines with MET amplification were identified and used to better understand the relationship of concurrent mutations with response. In addition to cancer cell line selectivity, we are analyzing preclinical models of NSCLC (non small cell lung cancer) that are representative of key patient segments, namely MET amplification and over expression. In newly diagnosed NSCLC adenocarcinomas, focal MET amplification events represent ∼3% of the population while over expression of MET (without gene amplification) is observed in the majority of patients. In preclinical models, focal Met amplification in EBC-1 and NCI-H1993 caused significant tumor growth inhibition, confirming sensitivity to Volitinib. Given the prevalence of over expression in NSCLC, however, we sought to build a platform of evidence for the therapeutic use of Volitinib in preclinical models lacking amplification of the MET gene. Using a patient-derived xenograft model (PDX) of EGFR WT, KRAS WT and metastatic NSCLC disease (HLXF-036LN), we demonstrate that Volitinib induces tumor regression as monotherapy and has added therapeutic benefit when used in combination with taxotere. In addition, we show robust efficacy effects for additional preclinical models, LG0567, LG0645 and Calu-3, for either Volitinib alone or in combination with taxotere. In parallel pharmacodynamic studies, we demonstrate that Volitinib inhibits p-MET and downstream signaling in each model. Together, using an integrated platform of molecular characterization and MET FISH and IHC scores, corresponding antitumor responses to Volitinib are being evaluated for several patient segments. We are using these studies to inform the design of patient selection criteria for upcoming clinical trials in NSCLC. Citation Format: Celina D'Cruz, Melanie Frigault, Ammar Adam, Minhui Shen, Garry Beran, Evan Barry, Paul Gavine, Yongxin Ren, Shiming Fan, Feng Zhou, Weiguo Qing, Mike Zinda, Weiguo Su, Edwin Clark. Targeting MET in preclinical models to support the clinical development of Volitinib in NSCLC. [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 3114. doi:10.1158/1538-7445.AM2014-3114

Esophagus cancer is the fifth most common malignancy and the fourth leading cause of cancer mortality in China. According to Chinese cancer registry annual report in 2012, esophagus cancer accounts for nearly 1 in 10 of all cancer deaths. Despite the fact that much progress has been made in diagnosis and systemic chemotherapy regimens, the overall prognosis of esophagus cancer is disappointing. The 5-year survival rate, all stages included, is around 15∼25%. There remains a significant unmet medical need for esophagus cancer treatment. EGFR expression was reported in 30∼90% esophagus cancers and overexpression of EGFR was found to be associated with poorer survival. Unlike colon cancer, K-ras mutation was less frequently found in esophagus cancer (5∼10%), suggesting EGFR pathway blockade might bring therapeutic benefit to those patients with EGFR activation or overexpression. In this study, as of November 2013, 35 surgical esophagus tumor samples were collected from a local hospital in Shanghai. 31 of the 35 samples were identified as squamous cell carcinoma. EGFR gene amplification, protein expression and K-ras mutation were studied. In addition, 9 patient derived esophagus cancer xenograft models (PDX) were developed and anti-tumor effect of a novel and highly potent EGFR inhibitor was evaluated in 6 PDX models. Positive EGFR protein expression was found in 66% (23/35) of esophageal cancer samples. EGFR gene amplifications were observed in 9% patients (3/35). In 9 established PDX models, 7 of them showed EGFR expression and 2 with EGFR gene amplification. No K-ras mutation was observed in the 9 models. A novel and highly potent EGFR inhibitor (EGFRi), developed by Hutchison and currently being evaluated in phase I clinical trials in China, demonstrated potent anti-tumor activity in several PDX models with tumor growth inhibition in a range of 70% to >100%. One PDX model with EGFR gene amplification and overexpression exhibited the highest sensitivity to EGFRi with remarkable tumor regression. EGFR signaling transduction was evaluated and the EGFRi inhibited phosphorylation of EGFR and downstream signaling molecules AKT and ERK. In conclusion, EGFR expression and/or gene amplification was frequently found in Chinese esophagus cancer. EGFR inhibition resulted in potent anti-tumor effect in multiple patient derived esophagus cancer models carrying EGFR amplifications or high expression, suggesting the potential benefit that anti-EGFR agents might bring to esophagus cancer patients with abnormal EGFR activation. Citation Format: Yongxin Ren, Jianming Zheng, Linfang Wang, Wei Zhang, Fang Yin, Jinghong Zhou, Xuelei Ge, Shiming Fan, Renxiang Tang, Junen Sun, Weiguo Qing, Weiguo Su. A study on EGFR gene amplification and protein expression in Chinese esophagus cancer patients and antitumor effect of an EGFR inhibitor in patient-derived esophagus cancer models. [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 1730. doi:10.1158/1538-7445.AM2014-1730

Gastric cancer (GC) incidence rates are amongst the highest in Asian countries including; China, Japan and Korea(1). 5-year survival rates in early stage disease have improved through aggressive combinations of surgery and chemo/radiotherapy. In late stage disease however, despite Her2 molecular segmentation and trastuzumab therapy, prognosis remains dismal and novel therapeutic options are urgently required(2). The MET oncogene encodes the receptor tyrosine kinase for hepatocyte growth factor and controls genetic programs leading to cell growth, invasion and survival. Dysregulation of MET signaling is a common feature of a diverse range of human tumor types and thus represents a highly competitive and attractive therapeutic target. Volitinib (HMPL-504) is an orally bioavailable, highly selective and potent small molecule ATP-competitive inhibitor, which inhibits cMET autophosphorylation and downstream signaling (3). Volitinib is currently in Phase I clinical development. To evaluate the utility of volitinib in treating patients with GC, we established the incidence of cMET amplification and overexpression and attempted to correlate this with response to volitinib in primary GC tumor models. Elevated cMET gene copy number (amplification 5%, high polysomy 13%), and protein overexpression (12%, cases > IHC 2+, 16% > IHC1+) were detected in a cohort of 217 Chinese GC samples using fluorescent in situ hybridization (FISH) and immunohistochemical (IHC) approaches. In vitro proliferation assays were performed on a panel of 26 GC cell lines and profound sensitivity to volitinib was demonstrated in lines harboring elevated cMET gene copy number (GI50 range 6 to 49nM). In the cMET amplified GC xenograft model, Hs746t, once-daily oral dosing of volitinib (2.5mg/kg) elicited potent anti-tumour activity at well-tolerated doses (97% tumour growth inhibition after 16 days dosing, P Citation Format: Paul R. Gavine, Shiming Fan, Haihua Fu, Lu Han, Yuan Jie Liu, Jing Lv, Weiguo Qing, Yongxin Ren, Weiguo Su, Xinying Su, Huiying Wang, Liang Xie, Shirlian Xu, Wen Xu, XiaoLu Yin, Yongjuan Yu, Tianwei Zhang, Q.May Wang. Volitinib (HMPL504), a novel, selective and potent cMET inhibitor, is efficacious in primary tumor models of cMET-driven gastric cancer. [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 928. doi:10.1158/1538-7445.AM2013-928

It is well recognized that non-small cell lung carcinoma (NSCLC) patients with activating EGFR mutations will develop drug resistance after receiving EGFR-tyrosine kinase inhibitors (EGFR-TKI). About 20% of those patients with TKI resistance were identified to harbor Met gene amplification. The aim of this study includes two aspects: a) generating an acquired c-Met amplified EGFR-TKI resistant tumor line in preclinical setting, and b) exploring combination effect of c-met inhibitor volitinib and EGFR inhibitor gefitinib on the new tumor line. Volitinib is a novel, highly potent and selective c-Met inhibitor, currently being evaluated in the phase I clinical trial. HCC827(exon 19 del E746-A750), a human NSCLC cell line, was treated with increasing concentrations of EGFR inhibitor for about 6 months and gradually produced the resistance to EGFR-TKIs, such as gefitinib and erlotinib. One of the subclones, HCC827C4R was isolated and was confirmed to carry Met gene amplification in comparison to parent cell line HCC827. It was not sensitive to volitinib treatment in cell survival assay due to dual activation of EGFR and c-Met pathways. The results from signal pathway study demonstrated that in HCC827C4R cells, volitinib or gefitinib alone only inhibited the phosphorylation of c-Met or EGFR, respectively, but had no effect on the activation of downstream molecules such as Akt and ERK which drive tumor cell proliferation and other cell functions. In contrast, combination of volitinib and gefitinib significantly inhibited phosphorylation of EGFR, c-Met, Akt and ERK in HCC827C4R, and consequently led to a synergistic effect on inhibiting tumor cell growth in vitro. These results were further confirmed in HCC827C4R xenograft model in vivo at clinically relevant doses. Combination of volitinib and gefitinib induced significant tumor regression and displayed synergistic effect compared to treatment by either gefitinib or volitinib alone. Consistent with in vitro results, combination group strongly inhibited the downstream Akt and ERK phosphorylation. In addition, combination treatment was well tolerant and no significant drug-drug exposure interaction was observed. These data indicated that both c-Met and EGFR could contribute to activating downstream signaling pathway and control HCC827C4R cell growth. Blocking either pathway may not be strong enough to stop tumor growth. In conclusion, a cell line with activating EGFR mutation and c-Met gene amplification was generated with resistance to gefitinib and insensitivity to volitinib. The combination treatment with volitinib and an EGFR inhibitor gefitinib was highly effective in vitro and in vivo, suggesting that such combination could provide a safe and effective treatment in clinics for this particular patient population. Citation Format: Feng Zhou, Yongxin Ren, Yumin Cui, Hanyang Chen, Longxian Jiao, Guangxiu Dai, Shiming Fan, Junen Sun, Yongjuan Yu, Yang Sai, Yi Gu, Weiguo Qing, Weiguo Su. Synergistic effect of c-Met inhibitor volitinib in combination with EGFR inhibitor Gefitnib on EGFR-TKI resistant NSCLC model HCC827C4R harboring acquired Met gene amplification. [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 971. doi:10.1158/1538-7445.AM2013-971

PIK3CA gene amplification and mutations have been identified in many types of tumors, suggesting the deregulation of PI3K/AKT/mTOR axis plays pivotal roles in tumorigenesis and development. K-Ras/Raf mutation is recognized as one of the key drug resistant factors for tumors against TKIs. HM-032 was reported here as a potent, selective and reversible ATP-competitive PI3K/mTOR dual inhibitor with special apoptotic activity in tumor cells harboring K-Ras mutations. HM-032 showed potent kinase inhibition with IC50 of 0.0008, 0.002, 0.001, 0.002 and 0.001 µM on PI3Kα, β, δ, ≤ and mTOR, respectively. In human prostate PC3 cells, it inhibited p-AKTS473, p-S6 and p-4EPB1 with IC50s of 0.002, 0.015 and 0.056 μM. Of the 89 tumor cell lines from lung, colon, breast, ovarian, and other tumor types, it was found that tumor cells with HER2 gene amplification, or PIK3CA mutation were relatively more sensitive to HM-032. Attractively, HM-032 showed apoptotic induction activity in the tumor cells harboring Ras/Raf mutations through inhibiting p-Erk and increasing gene expression of pro-apoptotic protein Bim. This result suggested that HM-032 might bring benefits to patients carrying K-Ras/Raf mutations in the clinic. Consistent with in vitro studies, HM-032 exhibited significant tumor growth inhibition in a dose dependent manner on multiple human xenograft models, including breast tumor MDA-MB-361(PIK3CAE545K, Her2 amplification), lung tumors H460 (PIK3CAE545K, K-RasQ61H) and A549 (LKB1 del, K-RasG12S), and glioblastoma U87MG (PTEN del) after 3 weeks oral dosing. Correspondingly, decrease of p-AKT and p-S6 was observed in the tumor xenograft tissues upon treatment with HM-032. In addition, HM-032 exhibited unique in vivo PK properties characterized by low clearance and long t1/2, which supported the intermittent dosing schedule in xenograft models. Based on the pre-clinical study results, HM-032 is anticipated to be a promising agent against solid tumors including carrying Ras/Raf mutations. 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 1808. doi:1538-7445.AM2012-1808

JAK2 kinase mutation V617F is prevalent in myeloproliferative diseases (MPD), including polycythemia vera (PV, 81-99%), essential thrombocytosis (ET, 41-72%) and primary myelofibrosis (PMF, 39-57%). This point mutation constitutively activates the JAK kinase and leads to oncogenic potential of host cells, and thus making JAK2 a promising molecular target for MPD therapy. HM-018 is a small molecule inhibitor against JAK kinase and the compound's preclinical anti-MPD effects from signal transduction to biological consequences were investigated. HM-018 was found to inhibit JAK kinase 1, 2, 3 and TYK with IC50 of 0.010, 0.006, 0.040 and 0.047 μM, respectively. The compound demonstrated >100 folds selectivity against a panel of 63 kinases. In accordance with enzymatic activity, HM-018 suppressed ligand dependent or constitutive JAK activation in multiple cell lines as evidenced by the decrease of STAT3/5 phosphorylation. As a result, JAK dependent cell proliferation was significantly inhibited by HM-018. EPO-mediated mouse PV model was utilized to evaluate the in vivo efficacy of the compound. HM-018 could shrink enlarged mouse spleen, a typical symptom of PV, in a dose dependent manner accompanied with decreased STAT5 phosphorylation both in animal spleen and in bone marrow after oral dosing for 7 days. To better mimic MPD development in a more clinically-relevant manner, JAK2-V617F-tranfected 32D cells were injected into mice intravenously, and it was observed that oral treatment of HM-018 not only prolonged the animal's life span, but also reduced MPD-related symptoms, such as spleen weight increase and organ invasion by malignant cells. Meanwhile, HM-018 exhibited a favorable pharmacokinetic profile and acceptable safety window in rats. Based on the preclinical data, HM-018 demonstrated anti-MPD potency both in vitro and in vivo, and the studies have provided rationale to further develop this compound as possible MPD therapeutics. 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 1797. doi:1538-7445.AM2012-1797

PI3KCA gene amplification and mutations have been identified in many types of tumors, suggesting the dysregulation of PI3K/AKT/mTOR axis plays pivotal roles in tumorigenesis and development. Reported here is the discovery of HM5016699 as a potent, reversible ATP-competitive PI3K/mTOR dual inhibitor for treatment of cancer. HM5016699 showed potent kinase inhibition with IC50 of 0.01, 0.206, 0.052, 0.051 and 0.059 µM on PI3Kα, β, γ, Δ and mTOR, respectively. It demonstrated high selectivity on PI3K family and mTOR over a panel of 274 kinases. Of the 67 tumor cell lines screened from lung, colon, breast, ovarian, and other tumor types, we found that the tumor cells with HER2 gene amplification or PTEN loss tended to be sensitive to HM5016699, while the tumor cells harboring PI3KCA mutations and wild type Ras/Raf were mostly sensitive to the compound. HM5016699 could also show p-Erk inhibition and apoptotic induction at higher concentrations in the tumor cells harboring Ras/Raf mutations, suggesting it might bring benefits to patients carrying Ras/Raf mutations in the clinic. Consistent with in vitro studies, HM5016699 exhibited dose dependent tumor growth inhibition on multiple human xenografts models. It was found that the tumors with PIK3CA mutation or PTEN deletion were highly sensitive to HM5016699 with ED50s ranging from 0.6 to 1.8 mg/kg in U87MG (pTEN null), SKOV3 (H1047) and H1975 (G118D), whereas in tumors with Ras/Raf mutations, such as DLD-1(G13D), A549 (G12S) and HT-29(V600E), its ED50s were from 3.0 to 15.0 mg/kg. The decreased phosphorylation of AKT and S6 was observed in the tumor xenograft tissues treated with HM5016699, which was well-correlated with compound exposure. Therefore, we concluded that HM5016699 demonstrated both in vitro and in vivo activity through acting on PI3K/mTOR pathway. HM5016699 exhibited a favorable pharmacokinetic profile and acceptable safety window in rats. The pre-clinical study results suggest that HM5016699 could be a promising anti-cancer agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4499. doi:10.1158/1538-7445.AM2011-4499

HM5016504 is a novel, highly potent and selective c-Met inhibitor under development by HMPL. In preclinical studies, it demonstrated strong in vitro activity against c-Met and its downstream signaling targets, thus blocking the related cellular functions, including proliferation, migration, invasion, scattering and the secretion of VEGF that plays a pivotal role in tumor angiogenesis. The cross-talk between c-Met and EGFR was investigated, and their contribution to downstream signaling cascade was identified in certain tumor cell lines with dysregulation of both c-Met and EGFR. HM5016504 could effectively inhibit the in vivo growth of a number of human tumor xenografts. Particularly, the tumor cells or xengorafts with high fold of c-Met gene amplification showed high sensitivity to HM5016504, such as gastric cancer SNU-5, Hs746T and lung cancer H1993 and EBC-1 cells or their respective xenografts. The compound not only showed activity in subcutaneous xenograft models, but also demonstrated efficacy in an orthotopic brain tumor model induced by U87MG, indicating it could pass blood brain barrier. In vitro and in vivo activities of HM5016504 were found to be closely correlated with the c-Met expression level and activation status. In addition, HM5016504 exhibited a reasonable pharmacokinetic profile and safety window based on the toxicology study results in rats, dogs, and monkeys. The pre-clinical study results suggest that HM5016504 could be a promising c-Met targeting anti-cancer agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3612. doi:10.1158/1538-7445.AM2011-3612