Wei-Ping Lu

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.

Targeting the KIT activating switch control pocket: a novel mechanism to inhibit neoplastic mast cell proliferation and mast cell activation

Activating mutations in the receptor tyrosine kinase KIT, most notably KIT D816V, are commonly observed in patients with systemic mastocytosis. Thus, inhibition of KIT has been a major focus for treatment of this disorder. Here we investigated a novel approach to such inhibition. Utilizing rational drug design, we targeted the switch pocket (SP) of KIT, which regulates its catalytic conformation. Two SP inhibitors thus identified, DP-2976 and DP-4851, were examined for effects on neoplastic mast cell proliferation and mast cell activation. Autophosphorylation of both wild-type and, where also examined, KIT D816V activation was blocked by these compounds in transfected 293T cells, HMC 1.1 and 1.2 human mast cell lines, and in CD34+-derived human mast cells activated by stem cell factor (SCF). Both inhibitors induced apoptosis in the neoplastic mast cell lines and reduced survival of primary bone marrow mast cells from patients with mastocytosis. Moreover, the SP inhibitors more selectively blocked SCF potentiation of FcɛRI-mediated degranulation. Overall, SP inhibitors represent an innovative mechanism of KIT inhibition whose dual suppression of KIT D816V neoplastic mast cell proliferation and SCF-enhanced mast cell activation may provide significant therapeutic benefits.

Switch control pocket inhibitors of p38-MAP kinase. Durable type II inhibitors that do not require binding into the canonical ATP hinge region

Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or the doubly phosphorylated form of p38-alpha kinase.

Abstract 2690: DCC-2618 is a potent inhibitor of wild-type and mutant KIT, including refractory Exon 17 D816 KIT mutations, and exhibits efficacy in refractory GIST and AML xenograft models

Introduction: KIT kinase mutations are causative of a number of human cancers, including gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM), mast cell leukemia (MCL), and subtypes of melanoma and acute myeloid leukemia (AML). DCC-2618 is a robust Type II switch pocket control inhibitor which potently inhibits exon 17 KIT mutations that are resistant to conventional TKIs. Experimental procedures: DCC-2618 was tested for inhibition of KIT isoforms using a standard PK/LDH coupled spectrophotometric assay. CHO cells were transiently transfected to express mutant KIT or PDGFRα constructs. Transfected cells were treated with a range of DCC-2618 and levels of phosphorylated KIT or PDGFRα in cell lysates were determined by ELISA or western blot. Cell proliferation of several cell lines was measured using the fluorescent dye resazurin. Experiments were performed in triplicate. In vivo xenograft models were performed at Molecular Imaging, Inc. (Ann Arbor, MI) or Molecular Response, LLC (San Diego, CA). Summary of results: DCC-2618 inhibited various forms of KIT with nanomolar potency: WT (IC50 4 nM), V654A (8 nM), T670I (18 nM), D816H (5 nM), D816V (14 nM). In CHO cells transiently transfected with both single and double (primary/secondary) KIT mutants, DCC-2618 robustly inhibited exon 17, exon 9/13, exon 9/14, and exon 9/17 KIT mutants, as well as exon 11/17 KIT mutants, including exon 17 D816V, D816G, D820A, D820E, D820Y, N822K, N822Y, N822H, and Y823D primary or secondary mutations. DCC-2618 inhibited wild type KIT phosphorylation in the MO7e cell line (IC50 36 nM). DCC-2618 potently inhibited KIT activation in human GIST cell lines, including GIST T1 (exon 11 deletion, IC50 2 nM), GIST 430 (exon 11 deletion/exon 13 V654A, IC50 7 nM), and GIST 48 (exon 11 V560D/exon 17 D820A, IC50 53 nM). In the murine mastocytosis P815 cell line expressing the exon 17 D816Y mutation, DCC-2618 potently inhibited cell proliferation (IC50 2 nM). In vivo, DCC-2618 administration at 50 mg/kg afforded an ED90 for inhibition of KIT phosphorylation in the GIST T1 xenograft model, corresponding to an EC90 concentration of ∼ 470 ng/mL. When give twice daily, this oral dose resulted in almost complete tumor stasis. This dose of DCC-2618 produced tumor regressions in a patient derived xenograft (PDX) GIST expressing KIT exon 11 delW557K558/exon 17 Y823D, and also in a KIT exon 17 N822K AML xenograft model. Conclusion: DCC-2618 is a potent inhibitor of singly and doubly mutated KIT characterized by primary exon 9 or exon 11 mutations paired with secondary mutations in exons 13, 14 or 17. DCC-2618 inhibits exon 17 mutations, including the D816V mutation refractory to currently marketed KIT inhibitors. DCC-2618 has the potential to treat KIT mutant-driven cancers including GIST, systemic mastocytosis, AML, or melanoma. DCC-2618 has been selected for formal IND-enabling clinical development. Citation Format: Bryan D. Smith, Molly M. Hood, Scott C. Wise, Michael D. Kaufman, Wei-Ping Lu, Thomas Rutkoski, Daniel L. Flynn, Michael C. Heinrich. DCC-2618 is a potent inhibitor of wild-type and mutant KIT, including refractory Exon 17 D816 KIT mutations, and exhibits efficacy in refractory GIST and AML xenograft models. [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 2690. doi:10.1158/1538-7445.AM2015-2690

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 4889: The highly specific CSF1R inhibitor DCC-3014 exhibits immunomodulatory and anti-invasive activities in cancer models

The role of tumor-associated macrophages (TAMs) in promoting an invasive and immunosuppressed tumor microenvironment is well established. TAMs mediate tumor growth, angiogenesis, invasiveness, and immunosuppression through the secretion and response to a variety of factors. There are few highly specific small molecule inhibitors of CSF1R kinase in clinical development. Such specific CSF1R inhibitors are sought for use in combination with other oncology immune checkpoint inhibitors and/or chemotherapeutic agents. DCC-3014, a highly specific CSF1R inhibitor, was developed based on Deciphera9s switch control inhibitor platform. Experimental procedures: DCC-3014 was evaluated in human kinase assays, including CSF1R, highly related kinases FLT3, KIT, and PDGFRa/b, and 300 additional kinases. Cellular studies included evaluation in the monocytic cell lines THP-1, MNFS-60, and a human whole blood assay. DCC-3014 was also evaluated in a human osteoclast TRAP assay. In vivo, DCC-3014 was evaluated in the murine cFOS PK/PD model. DCC-3014 was evaluated as a single agent and in combination with a murine anti-PD1 antibody in the murine syngeneic MC38 colorectal cancer model, a model characterized by high TAM infiltration. Results: DCC-3014 exhibited nanomolar (IC50 5 nM) potency for inhibition of CSF1R, sparing highly related kinases FLT3, KIT, PDGFRa, and PDGFRb, by > 100-fold, and sparing other kinases by > 1,000 fold. Cellular inhibition of CSF1R was resilient to high levels of the CSF1R ligand MCSF (10-1,000 ng/mL), due to its high residency time for binding to CSF1R and its binding mode which maintains CSF1R in a “switch off” state. DCC-3014 inhibited CSF1R in THP-1 monocytes (IC50 11 nM), MNFS-60 monocytes (IC50 4 nM), human osteoclasts (IC50 9 nM), and in a human whole blood monocyte/pERK assay (IC50 260 nM). In vivo, DCC-3014 exhibited sustained inhibition of CSF1R in the murine cFOS PK/PD model, affording 90+% inhibition at 15 mg/kg through 24 h post dose. At steady state (6 days of dosing), DCC-3014 robustly inhibited CSF1R at 3 mg/kg daily. In the MC38 colorectal cancer model, DCC-3014 (10 mg/kg daily) inhibited infiltrating TAMs, repolarized the adaptive immune cell population to an anti-tumoral profile, and depleted circulating CD16+ monocyte populations. Additionally, DCC-3014 was evaluated in combination with a murine anti-PD1 antibody and demonstrated additive effects compared to single agent cohorts. DCC-3014 exhibits optimized biopharmaceutical properties, including a favorable ADME and PK profile in preclinical studies. Conclusion: DCC-3014 is a highly specific CSF1R inhibitor and finds potential utility as a macrophage immunomodulatory agent for clinical evaluation in combination with other immune checkpoint inhibitors or chemotherapeutic agents. DCC-3014 is undergoing IND-enabling activities with a FIH study targeted for 2016. Citation Format: Bryan D. Smith, Cynthia B. Leary, Wei-Ping Lu, Michael D. Kaufman, Daniel L. Flynn. The highly specific CSF1R inhibitor DCC-3014 exhibits immunomodulatory and anti-invasive activities in cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4889.

Abstract A53: The specific FMS kinase inhibitor, DCC-3014, durably inhibits FMS kinase in vivo and blocks cancer bone invasiveness

Within the tumor microenvironment, tumor-associated macrophages (TAMs) rely on signaling through FMS kinase to promote tumor growth, angiogenesis, vasculogenesis, and metastasis. In addition, FMS-expressing macrophages communicate with the adaptive immune system, which can lead to cancer immunotolerance. Osteoclasts also depend on FMS kinase for differentiation and growth, and play a direct role in the ability of a tumor to metastasize to the bone and progress. Taken together, FMS kinase is an excellent target for small molecule therapy of tumor growth and bone metastasis. Using Deciphera Pharmaceuticals9 approach to kinase inhibition, potent and specific FMS kinase inhibitors have been identified that achieve single digit nanomolar inhibition in biochemical and cellular assays of FMS activity. Due to their binding mode, these inhibitors retain potency in the presence of high ATP concentrations and also exhibit long off-rates from FMS kinase. Development candidate DCC-3014 is highly selective, inhibiting no other kinases within 100-fold of FMS potency, including related kinases KIT, PDGFRs, FLT3, and VEGFR2. DCC-3014 exhibits potent and durable inhibition of FMS in vivo in a pharmacokinetic/pharmacodynamic model. In a variety of cancer models, DCC-3014 or related analogs slow tumor growth, decrease tumor-promoting macrophages, and protect against osteolytic bone invasion. DCC-3014 is currently undergoing formal preclinical development. Citation Format: Bryan D. Smith, Michael D. Kaufman, Cynthia B. Leary, Molly M. Hood, Wei-Ping Lu, Benjamin A. Turner, Subha Vogeti, Scott C. Wise, Daniel L. Flynn. The specific FMS kinase inhibitor, DCC-3014, durably inhibits FMS kinase in vivo and blocks cancer bone invasiveness. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A53. doi:10.1158/1538-7445.CHTME14-A53

Abstract PR01: Rebastinib, a selective TIE2 kinase inhibitor, decreases TIE2-expressing macrophages, reduces metastasis, and increases survival in murine cancer models

In the tumor microenvironment, TIE2 expression on tissue macrophages, bone marrow derived TIE2-expressing monocytes (TEMs), osteoclasts, and vascular endothelial cells promotes tumor invasiveness, dissemination, and metastasis. Additionally, a subset of TIE2-expressing macrophages, located within specialized vascular structures known as tumor microenvironment for metastases (TMEMs), are linked to intravasation of cancer cells into circulation and dissemination to metastatic sites. Rebastinib is a picomolar inhibitor of TIE2 kinase, and exhibits an extraordinarily long off-rate from TIE2, measured to be over 24 hours in a cell-based assay. Herein, we examine the efficacy of rebastinib in the polyoma middle-T antigen (PyMT) syngeneic mouse breast cancer model. In this model, PyMT breast cancer cells are implanted in the mammary fat pad, and primary tumor growth leads to lung metastasis, which is known to be modulated by TEMs and TMEM vascular structures. We examined multiple dosing schedules of rebastinib in combination with anti-tubulin agents (ATAs). Rebastinib treatment in this model significantly ablated TEMs in the primary tumor stroma and caused a significant decrease in lung metastases. Furthermore, the combination of rebastinib with ATAs, even with once or twice weekly oral dosing of rebastinib, led to a significant further decrease in lung metastases compared to single-agent treatment with ATAs. Rebastinib also enhanced the activity of ATAs in reducing primary tumor growth and regrowth of tumor post-resection. TIE2 inhibition with targeted therapy represents a novel treatment approach for metastatic breast cancer and other cancers that rely on TEMs and TMEMs for growth and metastasis. As such, rebastinib has been selected for further clinical development in solid tumors with a Phase 1b trial being planned for 2014. This abstract is also presented as Poster A5. Citation Format: Daniel L. Flynn, Michael D. Kaufman, Cynthia B. Leary, Molly M. Hood, Wei-Ping Lu, Benjamin A. Turner, Scott C. Wise, Marc S. Rudoltz, Bryan D. Smith. Rebastinib, a selective TIE2 kinase inhibitor, decreases TIE2-expressing macrophages, reduces metastasis, and increases survival in murine cancer models. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR01. doi:10.1158/1538-7445.CHTME14-PR01

Abstract 3594: Conformational control of FMS kinase for treatment of human malignancies

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL FMS kinase is involved in the process of osteoclast maturation. Osteoclasts have been shown to play a role not only in arthritic diseases but also in a cancers ability to metastasize to the bone. This is especially true for breast, lung and prostate cancer. Taken together the ability to prevent osteoclast maturation and accumulation through FMS inhibition should be a good target for small molecule therapy of bone metastasis. Using Deciphera Pharmaceuticals approach to kinase inhibition, compounds have been designed that potently inhibit FMS kinase. These inhibitors can be highly selective with lead compounds inhibiting only a single kinase within 20 fold of FMS activity in a 300 kinase profile. A second class broadens the profile to include anti-angiogenic kinase targets in addition to potent FMS inhibition. This presentation will highlight the attributes and development status of these compounds for treatment of human malignancies. Decipheras FMS program has afforded potent inhibitors that achieve single digit nanomolar inhibition in biochemical assays of FMS activity. The inhibitors retain this same level of potency in the presence of high (5mM) ATP concentrations. These same compounds are also selective with some inhibiting as few as three kinases within 50 fold of FMS potency. In addition, proliferation and FMS phosphoprotein assays performed with M-NFS-60 and THP-1 cells have demonstrated excellent inhibitory profiles with achieved potencies in the low nanomolar range. In functional osteoclast differentiation models, key compounds have demonstrated single digit nanomolar inhibition as assessed by TRAP assays. In vivo evaluation of the inhibitors produced excellent tolerance in two week MTD studies. Potent and durable efficacy in pharmacokinetic/pharmacodynamic xenograft models was also observed demonstrating on target effects. In vivo models of bone invasion coupled with non-invasive translational image-based biomarkers potentially provide a powerful method for visualization and quantification of osteoclast activity and FMS inhibition. Early results in imaging of bone invasion will be presented using micro CT and fluorescent activateable probes. Decipheras FMS inhibitors show acceptable ADME properties in cell permeability, cytochrome p450 inhibition, microsomal clearance and are orally bioavailable in rat and dog. Key prototype compounds have been evaluated in a two week rat tolerability study. These data demonstrate that Decipheras technology has been used to identify potent and selective FMS inhibitors to be developed for treatment of cancers where metastasis to bone is an issue. 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 3594. doi:10.1158/1538-7445.AM2011-3594