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Altiratinib (DCC-2701) was designed based on the rationale of engineering a single therapeutic agent able to address multiple hallmarks of cancer (1). Specifically, altiratinib inhibits not only mechanisms of tumor initiation and progression, but also drug resistance mechanisms in the tumor and microenvironment through balanced inhibition of MET, TIE2 (TEK), and VEGFR2 (KDR) kinases. This profile was achieved by optimizing binding into the switch control pocket of all three kinases, inducing type II inactive conformations. Altiratinib durably inhibits MET, both wild-type and mutated forms, in vitro and in vivo. Through its balanced inhibitory potency versus MET, TIE2, and VEGFR2, altiratinib provides an agent that inhibits three major evasive (re)vascularization and resistance pathways (HGF, ANG, and VEGF) and blocks tumor invasion and metastasis. Altiratinib exhibits properties amenable to oral administration and exhibits substantial blood–brain barrier penetration, an attribute of significance for eventual treatment of brain cancers and brain metastases. Mol Cancer Ther; 14(9); 2023–34. ©2015 AACR.

Supplementary Materials and Methods. Supplementary table 1: XRay crystallography parameters for DP-4157/MET complex. Supplementary table 2: Source and description of kinases. Supplementary table 3: Selectivity of profile of altiratinib inhibition from Reaction Biology 295 kinase panel (fold selectivity versus IC50 value of 2.7 nM MET inhibition). Supplementary table 4: A. Inhibition of MET phosphorylation in the MKN-45 xenograft model after a single oral dose of altiratinib (30 mg/kg); B. Inhibition of MET phosphorylation in the MKN-45 xenograft model after a single oral dose of altiratinib (10 mg/kg). Supplementary figure 1: Co-crystal structures of altiratinib analog DP-4157. Supplementary figure 2: Comparison of altiratinib with other clinical stage MET inhibitors regarding potency versus activation loop mutant forms D1228H, D1228N, Y1230D, Y1230C, and Y1230H. Supplementary figure 3: Determination of off-rate, residency time, and tight-binding inhibitor constant Kd for altiratinib binding to the MET kinase domain. Supplementary figure 4: Michaelis-Menton analysis of altiratinib inhibition of MET with respect to ATP. Supplementary figure 5: Kinome tree interaction profile for altiratinib vs 295 human kinases. Altiratinib was evaluated using Kinase HotspotSM platform from Reaction Biology (Malvern, PA). Supplementary figure 6: Inhibitory potencies for inhibiting HUVEC cell signaling activated by HGF/MET (black), VEGF-A/VEGFR2 (melon), and ANG2/TIE2 (tan). Comparative potencies are shown for altiratinib, E-7050, cabozantinib, and MGCD-265. Supplementary figure 7: Upper panel: inhibition of capillary tube formation by various concentrations of altiratinib. Tube formation was initiated by addition of 200 ng/mL of angiopoietin 2 (ANG2). Lower table: titrated IC50 values for inhibition of capillary tube formation induced by ANG2, HGF, and VEGF-A. Supplementary figure 8: Inhibition of proliferation in the mutant B-RAF melanoma SK-MEL-28 cell line in the absence or presence of HGF or MRC-5 fibroblast conditioned medium. Supplementary figure 9: Ratio of brain: plasma concentrations of altiratinib through 24 hr after administration of a single dose of 5 mg/kg IV to C57/Black mice.

Based on the structure of an early lead identified in Deciphera's proprietary compound collection of switch control kinase inhibitors and using a combination of medicinal chemistry guided structure activity relationships and structure-based drug design, a novel series of potent acyl urea-based CSF1R inhibitors was identified displaying high selectivity for CSF1R versus the other members of the Type III receptor tyrosine kinase (RTK) family members (KIT, PDGFR-α, PDGFR-β, and FLT3), VEGFR2 and MET. Based on in vitro biology, in vitro ADME and in vivo PK/PD studies, compound 10 was selected as an advanced lead for Deciphera's CSF1R research program.

Background: Mutations in the RAS/MAPK pathway are a frequent driver of cancer, with oncogenic RAS or RAF mutations occurring in >30% of all cancers. First generation BRAF inhibitors are approved for use for tumors with Class I BRAF mutations (V600X). However, these drugs are not efficacious in RAF dimer mutant and RAS mutant cancers due to paradoxical activation of RAF dimers. Herein, we describe DCC-3084, a potent and selective investigational Switch Control inhibitor of BRAF and CRAF kinase dimers that targets Class I, II and III BRAF mutations, BRAF fusions, and BRAF/CRAF heterodimers. DCC-3084 combines with inhibitors of additional nodes in the MAPK pathway to potentially target a large unmet medical need in RAS and RAF mutant cancers. Methods: Inhibition of RAF kinases, including off-rate analysis, was measured using recombinant enzymes. X-ray crystallography was used for structure-based drug design. Cellular proliferation was measured using resazurin to monitor cell viability. Synergy in cells was measured using BLISS scores and curve shift analysis. Inhibition of ERK or RSK phosphorylation was measured by AlphaLISA or ELISA. Pharmacokinetics (PK) in the plasma, brain and CSF compartments were measured following oral dosing in Wistar rats. RAF and RAS mutant mouse xenograft models were used to assess PK, pharmacodynamics (PD), and efficacy. Results: DCC-3084 is a potent and selective Switch Control inhibitor of RAF dimers that was designed to target Class I, II, III BRAF mutants, BRAF fusions, and BRAF/CRAF heterodimers. DCC-3084 inhibits BRAF and CRAF, exhibiting slow off-rates (t1/2 >20 hr). Potent single-agent inhibition of MAPK pathway signaling and cellular proliferation was observed in a wide range of Class I, II, III BRAF and BRAF fusion altered cell lines. Synergy was observed in combination with inhibitors of other nodes in the RAS/MAPK pathway in RAS mutant cell lines. DCC-3084 was demonstrated to be CNS penetrable and exhibited dose dependent oral exposure with robust inhibition of the RAS/MAPK pathway in PK/PD models. DCC-3084 accumulated in tumor tissue relative to plasma, further demonstrating a favorable pharmaceutical profile. Oral treatment of DCC-3084 as a single agent resulted in tumor regression in BRAF mutant and KRAS Q61K mutant mouse xenograft models and tumor growth inhibition in KRAS G12C/D mutant models. Additionally, DCC-3084 in combination with a MEKi resulted in tumor regression in KRAS mutant models. Conclusions: The Switch Control inhibitor DCC-3084 broadly inhibits Class I, II and III BRAF mutations, BRAF fusions, and BRAF/CRAF heterodimers leading to tumor regression in preclinical models. The overall preclinical profile of DCC-3084 supports IND-enabling activities towards clinical development in a key area of unmet medical need in RAS and RAF mutant cancers. Citation Format: Stacie L. Bulfer, Bertrand Le Bourdonnec, Jeffery D. Zwicker, Yu Mi Ahn, Gada Al-Ani, Hikmat Al-Hashimi, Chase Crawley, Kristin M. Elliott, Saqib Faisal, Andrew M. Harned, Cale L. Heiniger, Molly M. Hood, Salim Javed, Michael Kennedy, Joshua W. Large, Cynthia B. Leary, Wei-Ping Lu, Kylie Luther, Max D. Petty, Hunter R. Picard, Justin T. Proto, Yeni K. Romero, Forrest A. Stanley, Kristen L. Stoltz, Daniel C. Tanner, Hanumaiah Telikepalli, Mary J. Timson, Lakshminarayana Vogeti, Subha Vogeti, Sihyung Yang, Lexy H. Zhong, Bryan D. Smith, Daniel L. Flynn. DCC-3084, a RAF dimer inhibitor, broadly inhibits BRAF class I, II, III, BRAF fusions, and RAS-driven solid tumors leading to tumor regression in preclinical models. [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 4045.

Introduction: Gastrointestinal stromal tumors (GISTs) are typically driven by primary mutations in KIT exons 9 or 11. Heterogeneous drug-resistant secondary mutations arise in patients treated with FDA approved KIT inhibitors, including imatinib and sunitinib. Drug resistant secondary mutations are found at multiple regions in the ATP pocket (encoded by exons 13 and 14) or activation switch (encoded by exons 17 and 18) of KIT kinase. In addition, multiple drug-resistant clones can arise within a tumor or in metastatic tumor sites. An inhibitor that can broadly and potently inhibit the spectrum of KIT mutations is highly sought. Ripretinib has been FDA approved as a 4th line treatment for GIST and has broad activity against KIT mutations, including clinical potency in patients with mutations in KIT exons 11, 17, or 18. DCC-3009 was designed as a next generation KIT inhibitor that broadly and potently inhibits primary KIT mutations in exons 9 and 11 and secondary drug-resistant mutations across exons 13, 14, 17, and 18. DCC-3009 is a potent and selective inhibitor in enzyme and cell-based assays, and has demonstrated efficacy in xenograft models driven by drug resistant KIT mutations. Methods: DCC-3009 was tested for inhibition of KIT mutants using standard enzyme and cell-based assays. Levels of phosphorylated KIT were determined by Western blot or ELISA. Proliferation was measured using the fluorescent dye resazurin. KIT mutant xenograft or patient-derived xenograft models were performed at Crown Biosciences or Labcorp, AAALAC accredited facilities, with the approval of Animal Care and Use Committees. Results: In BaF3 cells transfected with KIT mutants, DCC-3009 was shown to potently inhibit the spectrum of known primary and secondary drug-resistant mutations in GIST. The pan-mutant KIT profile of DCC-3009 was shown in vitro to be superior to 2nd and 3rd line standard of care therapies sunitinib and regorafenib. DCC-3009 was selective for KIT when screened against a large panel of kinases. DCC-3009 has optimized pharmaceutical properties for oral administration. In pharmacokinetic/pharmacodynamic studies DCC-3009 achieved sufficient free drug levels to significantly inhibit drug-resistant KIT mutants for 12 hr post dose. In xenograft studies, treatment with DCC-3009 twice daily led to tumor regression in drug-resistant models with KIT exon 9/13, 11/13 or 11/17 mutations. Conclusions: DCC-3009 is a pan-exon mutant KIT inhibitor exhibiting high potency in KIT mutants in pre-clinical models spanning exons 9, 11, 13, 14, 17 and 18. In vivo, DCC-3009 exhibited efficacy in drug-resistant models with KIT exon 9/13, 11/13 or 11/17 mutations. Based on this profile, DCC-3009 has entered formal preclinical development. Citation Format: Bryan D. Smith, Subha Vogeti, Timothy M. Caldwell, Hanumaiah Telikepalli, Yu Mi Ahn, Gada Al-Ani, Stacie L. Bulfer, Andrew Greenwood, Cale L. Heiniger, Joshua W. Large, Cynthia B. Leary, Wei-Ping Lu, Kylie Luther, William C. Patt, Max D. Petty, Yeni K. Romero, Forrest A. Stanley, Kristen L. Stoltz, Daniel C. Tanner, Sihyung Yang, Yu Zhan, Bertrand Le Bourdonnec, Daniel L. Flynn. Pan-exon mutant KIT inhibitor DCC-3009 demonstrates tumor regressions in preclinical gastrointestinal stromal tumor models. [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 4033.

Based on knowledge of kinase switch-control inhibition and using a combination of structure-based drug design and standard medicinal chemistry principles, we identified a novel series of dihydropyrimidone-based CSF1R kinase inhibitors displaying exquisite selectivity for CSF1R versus a large panel of kinases and non-kinase protein targets. Starting with lead compound 3, an SAR optimization campaign led to the discovery of vimseltinib (DCC-3014; compound 20) currently undergoing clinical evaluation for the treatment of Tenosynovial Giant Cell Tumor (TGCT), a locally aggressive benign tumor associated with substantial morbidity. 2021 Elsevier ltd. All rights reserved.

Macrophages can be co-opted to contribute to neoplastic, neurologic, and inflammatory diseases. Colony-stimulating factor 1 receptor (CSF1R)-dependent macrophages and other inflammatory cells can suppress the adaptive immune system in cancer and contribute to angiogenesis, tumor growth, and metastasis. CSF1R-expressing osteoclasts mediate bone degradation in osteolytic cancers and cancers that metastasize to bone. In the rare disease tenosynovial giant cell tumor (TGCT), aberrant CSF1 expression and production driven by a gene translocation leads to the recruitment and growth of tumors formed by CSF1R-dependent inflammatory cells. Small molecules and antibodies targeting the CSF1/CSF1R axis have shown promise in the treatment of TGCT and cancer, with pexidartinib recently receiving FDA approval for treatment of TGCT. Many small-molecule kinase inhibitors of CSF1R also inhibit the closely related kinases KIT, PDGFRA, PDGFRB, and FLT3, thus CSF1R suppression may be limited by off-target activity and associated adverse events. Vimseltinib (DCC-3014) is an oral, switch control tyrosine kinase inhibitor specifically designed to selectively and potently inhibit CSF1R by exploiting unique features of the switch control region that regulates kinase conformational activation. In preclinical studies, vimseltinib durably suppressed CSF1R activity in vitro and in vivo, depleted macrophages and other CSF1R-dependent cells, and resulted in inhibition of tumor growth and bone degradation in mouse cancer models. Translationally, in a phase I clinical study, vimseltinib treatment led to modulation of biomarkers of CSF1R inhibition and reduction in tumor burden in TGCT patients.

Ripretinib (DCC-2618) was designed to inhibit the full spectrum of mutant KIT and PDGFRA kinases found in cancers and myeloproliferative neoplasms, particularly in gastrointestinal stromal tumors (GISTs), in which the heterogeneity of drug-resistant KIT mutations is a major challenge. Ripretinib is a "switch-control" kinase inhibitor that forces the activation loop (or activation "switch") into an inactive conformation. Ripretinib inhibits all tested KIT and PDGFRA mutants, and notably is a type II kinase inhibitor demonstrated to broadly inhibit activation loop mutations in KIT and PDGFRA, previously thought only achievable with type I inhibitors. Ripretinib shows efficacy in preclinical cancer models, and preliminary clinical data provide proof-of-concept that ripretinib inhibits a wide range of KIT mutants in patients with drug-resistant GISTs.

Altiratinib (DCC-2701) was designed based on the rationale of engineering a single therapeutic agent able to address multiple hallmarks of cancer (1). Specifically, altiratinib inhibits not only mechanisms of tumor initiation and progression, but also drug resistance mechanisms in the tumor and microenvironment through balanced inhibition of MET, TIE2 (TEK), and VEGFR2 (KDR) kinases. This profile was achieved by optimizing binding into the switch control pocket of all three kinases, inducing type II inactive conformations. Altiratinib durably inhibits MET, both wild-type and mutated forms, in vitro and in vivo. Through its balanced inhibitory potency versus MET, TIE2, and VEGFR2, altiratinib provides an agent that inhibits three major evasive (re)vascularization and resistance pathways (HGF, ANG, and VEGF) and blocks tumor invasion and metastasis. Altiratinib exhibits properties amenable to oral administration and exhibits substantial blood–brain barrier penetration, an attribute of significance for eventual treatment of brain cancers and brain metastases. Mol Cancer Ther; 14(9); 2023–34. ©2015 AACR.

SummaryLY3009120 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.

Background: Cancer cells activate autophagy, a catabolic process to resupply nutrients and recycle damaged organelles, in order to survive stresses such as limited nutrients and hypoxia, or chemotherapy treatments. RAS mutant cancers, in particular, have been found to require autophagy for tumor growth and survival.1,2 Treating RAS mutant tumors with inhibitors of the downstream MAPK pathway has been largely unsuccessful, as these drugs have been shown to further stimulate autophagy, allowing for tumor cell survival.3,4 Inhibiting autophagy in combination with MAPK pathway inhibition may represent a possible new treatment paradigm for RAS mutant cancers. Proof-of-concept for this strategy was obtained in cancer models and in a RAS mutant pancreatic cancer patient by blocking autophagy with derivatives of chloroquine, in combination with MAPK inhibitors.3,4 Chloroquines indirectly block autophagy via disruption of lysosomal function, which may also affect important normal cellular processes. Chloroquines accumulate in tissues, notably the brain, where autophagy may be vital for neuronal health. The potential exists to more selectively inhibit autophagy by targeting specific components of the autophagy pathway. ULK1/2 kinases initiate autophagy and provide the potential for a targeted approach for selectively inhibiting autophagy in RAS mutant cancers. Herein, we describe preclinical studies with the ULK inhibitor DCC-3116, designed as a potential inhibitor of autophagy in RAS mutant cancers. Methods: In vitro kinase assays were performed using cellular levels of ATP (1 mM) and a peptide substrate. In cell assays, ULK activity was assessed using an ELISA for phosphorylated ATG13. Autophagosome formation was measured using the dye, Cyto-ID. Autophagic flux was assessed using cells expressing the autophagy protein LC3 fused to luciferase. The synergy of DCC-3116 in combination with MAPK inhibitors was assessed in 2D or 3D cell growth assays. Xenograft models were used to assess pharmacokinetics (PK) and pharmacodynamics (PD), as well as efficacy in vivo. Results: DCC-3116 is a potent and selective inhibitor of ULK1/2, inhibiting no other kinases within 30-fold of ULK potency, and only 5 kinases within 100-fold. DCC-3116 inhibited phosphorylation of the ULK substrate ATG13 in cancer cell assays. DCC-3116 inhibited autophagosome formation, as well as degradation of the autophagy marker LC3. DCC-3116 exhibited synergy in vitro in combination with MAPK pathway inhibitors in inhibiting cancer cell growth. In PK/PD models, oral doses of DCC-3116 led to sustained inhibition of ATG13 phosphorylation. DCC-3116, in combination with MAPK inhibitors, exhibited additivity or synergy in inhibiting tumor growth in xenograft models. DCC-3116 exhibited low brain penetration in rats, minimizing inhibition of CNS autophagy. Conclusion: Selectively blocking autophagy via inhibition of ULK kinases, in combination with MAPK pathway inhibition, is a promising therapeutic approach for RAS mutant cancers. DCC-3116 warrants further study as an inhibitor of autophagy, and has been selected as a candidate for potential development in the treatment of RAS mutant cancers. Guo et al., Genes and Dev. 2011; 25: 460 Yang et al., Genes and Dev. 2011; 25: 717 Bryant et al., Nature Med. 2019; 25: 628 Kinsey et al., Nature Med. 2019; 25: 620 Citation Format: Bryan D Smith, Lakshminarayana Vogeti, Anu Gupta, Jarnail Singh, Gada Al-Ani, Stacie L Bulfer, Timothy M Caldwell, Mary J Timson, Subha Vogeti, Yu Mi Ahn, Hikmat Al-Hashimi, Chase K Crawley, Cale L Heiniger, Cynthia B Leary, Justin T Proto, Quanrong Shen, Hanumaiah Telikepalli, Karen Yates, Wei-Ping Lu, Daniel L Flynn. Preclinical studies with DCC-3116, an ULK kinase inhibitor designed to inhibit autophagy as a potential strategy to address mutant RAS cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B129. doi:10.1158/1535-7163.TARG-19-B129

In cancer models, TIE2 kinase plays an important role in angiogenesis, vasculogenesis, and tumor metastasis. TIE2 expression is largely restricted to vascular endothelial cells, tissue macrophages, and bone marrow derived TIE2-expressing monocytes (TEMs), which are proangiogenic, provasculogenic and enhance invasiveness. The hypoxic tumor environment engendered by damaging the vasculature with chemotherapy, radiation, or anti-angiogenic treatments leads to rebound tumor vascularization by an angiogenic switch from the VEGF pathway to the angiopoietin/TIE2 pathway. This leads to recruitment of provasculogenic TEMs from the bone marrow, leading to the growth of residual tumor cells and disease progression. Significantly, a subset of TIE2-expressing macrophages are located within specialized vascular structures known as tumor microenvironment for metastases (TMEMs). Recent observations have linked TIE2-expressing macrophages within TMEM structures to intravasation of cancer cells into circulation and subsequent dissemination to metastatic sites. We hypothesized that TIE2 inhibition should decrease migration and association of TEMs with blood vessels in the tumor stroma, therefore blocking their proangiogenic activity and leading to reduced tumor growth. TIE2 inhibition may also alter TMEM function, leading directly to a blockade of metastasis. 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 metastasis, which is known to be modulated by TEMs and TMEM vascular structures. We examined dosing rebastinib in combination with eribulin, an inhibitor of microtubule dynamics that recently was FDA-approved for treatment-refractory metastatic breast cancer. 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 and eribulin led to a significant further decrease in lung metastases compared to treatment with eribulin alone (Table 1). Rebastinib also enhanced the activity of eribulin in reducing primary tumor growth and regrowth of tumor post-resection. TIE2 inhibition 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 combination with eribulin for treatment-refractory metastatic breast cancer, with a Phase 1b trial being planned for late 2013. Rebastinib reduces lung metastases in the PyMT breast cancer modelTreatmentLung Metastases (% of Control)Vehicle100%Eribulin 1 mg/kg three times/week71%Rebastinib 10 mg/kg twice/week + Eribulin 1 mg/kg23%Eribulin 0.3 mg/kg three times/week71%Rebastinib 10 mg/kg twice/week + Eribulin 0.3 mg/kg51%Eribulin 0.1 mg/kg three times/week72%Rebastinib 10 mg/kg twice/week + Eribulin 0.1 mg/kg43% Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-15-12.

Introduction: Activating mutations and other genetic alterations in KIT and PDGFRA receptor tyrosine kinases have been identified in certain cancers and proliferative diseases, including most cases of gastrointestinal stromal tumors (GIST) and systemic mastocytosis, and small percentages of gliomas, lung cancer, and leukemias. The treatment of metastatic GIST has been transformed with KIT inhibitors, but heterogeneous drug-resistant mutations arise during therapy, with individual patients often having multiple KIT mutations in different tumor sites. PDGFRA variants in GIST and other cancers also have a significant unmet medical need. DCC-2618 is a kinase switch control inhibitor that potently inhibits the spectrum of exon 9, 11, 13, 14, 17 and 18 mutations in KIT and exons 12, 14 and 18 mutations in PDGFRA. DCC-2618 has been designed to bind as a type II kinase inhibitor that forces the mutant kinases, including strongly activated mutants such as D816V KIT and D842V PDGFRA, into inactive conformations. DCC-2618 has been observed to be potent in enzyme and cell-based assays, and has demonstrated consistent efficacy in xenograft models driven by PDGFRA and KIT alterations. Methods: DCC-2618, and an active human metabolite, DP-5439, were tested for inhibition of PDGFRA and KIT mutants using standard enzyme and binding assays, and a variety of cell-based assays. Levels of phosphorylated PDGFRA and KIT were determined by Western blot or ELISA. Proliferation was measured using the fluorescent dye resazurin. An x-ray crystal structure of an analog of DCC-2618 was determined at Emerald Biostructures. The H1703 PDGFRA-amplified lung cancer and GIST T1 mutant KIT xenograft models were performed at MI Bioresearch. A GIST PDX exon 17 mutant KIT xenograft model was run at Molecular Response. Results: DCC-2618 and the metabolite DP-5439 inhibited KIT and PDGFRA variants with nanomolar potency. In CHO cells transfected with KIT or PDGFRA variants, DCC-2618 was shown to inhibit the full spectrum of the clinically relevant primary and refractory drug-resistant mutations tested. DCC-2618 also inhibited phosphorylation of KIT or PDGFRA in cell lines with various drug-resistant KIT mutations or PDGFRA alterations. DCC-2618 was compared to the FDA-approved KIT inhibitors imatinib, sunitinib, regorafenib, and midostaurin, as well as other KIT and PDGFRA inhibitors. In vivo, treatment with DCC-2618 led to tumor regressions in KIT- and PDGFRA-driven xenograft models. Conclusions: DCC-2618 has been observed to be a potent inhibitor of KIT and PDGFRA alterations, including mutants, fusions, and amplifications. Based on this profile, DCC-2618 may have utility in the treatment of KIT and PDGFRA-driven cancers including GIST, systemic mastocytosis, and a subset of lung cancers, gliomas, and leukemias. DCC-2618 is currently in a Phase 1 clinical trial in KIT and PDGFRA driven cancers (ClinicalTrials.gov Identifier: NCT02571036). Citation Format: Bryan D. Smith, Michael D. Kaufman, Anu Gupta, Cynthia B. Leary, Wei-ping Lu, Stacie L. Bulfer, Gada Al-Ani, Jarnail Singh, Subha Vogeti, Michael C. Heinrich, Daniel L. Flynn. Inhibition of oncogenic and drug-resistant PDGFRA and KIT alterations by DCC-2618 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3925.

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.

Kinases are an important class of targets for a number of therapeutic indications, and currently, kinase inhibitors constitute a significant fraction of recently approved drugs, particularly for cancer. Using a probe-based chemoproteomics platform, we have demonstrated the utility of the platform to monitor the interactions between inhibitors and native kinases. Herein, we profile kinase inhibitors in cell lines either sensitive or resistant to the compound for various periods of time. We observe that at early time points, inhibitor profiles for a compound are qualitatively similar in both the sensitive and resistant cells, suggesting the compound is capable of engaging its direct targets in both cell types. In contrast, at later time points, additional kinases are observed affected by compound treatment. We infer that these kinases do not directly bind the compound but rather reflect secondary effects that result from the engagement of the primary targets (pathway effects). Significantly, the pathway effects are only observed in the sensitive cells and not the resistant cells. Kinases affected through these pathway effects include kinases known to be critical for cell cycle progression including CDK1, Aurora A and B, PLK1, and MASTL. Finally, we demonstrate that during the treatment of tumor-bearing mice with an inhibitor, in addition to inhibition of the direct targets, pathway effects are observed recapitulating the results from the cell-based studies. Citation Format: Tyzoon K. Nomanbhoy, Heidi E. Brown, Jiangyue Wu, Subha Vogeti, Arwin Aban, Wendy Grant, Alemayehu Senait, Shuzhen Wu, Christa Dias, Geeta Sharma. Chemoproteomic profiling of native kinases during the treatment of cells with kinase inhibitors. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B23.

Palbociclib (PD0332991) is a cyclin dependent kinase 4 and 6 inhibitor being developed for treatment of breast cancer. In order to evaluate the kinome-wide impacts of Palbociclib in an in-vivo setting, nude mice inoculated with COLO-205 (human colon carcinoma) cells were administered Palbociclib at different doses (12.5, 75 and 150mg/kg once daily for 3 days. Tumors were collected at various time points after the final dose (1, 3, 6, 12, 24 & 48 hours), lysed and subjected to in-situ kinase profiling using the KinativTM technology. Tumor lysates were probed with an ATP acylphosphate desthiobiotin probe which covalently modifies lysine residues in the kinase active site. Samples are then denatured, trypsinized, and the probe-labeled peptides captured with streptavidin, eluted, and quantitated by targeted mass spectrometry. The presence of an inhibitor bound to the active site of a kinase prevents the probe binding, resulting in the loss of MS signal for peptides from that kinase. In addition, the upregulation or downregulation of kinase proteins can also result in changes in the MS signals in palbociclib versus vehicle-treated samples. A total of 180 kinases were quantified. As expected, direct targets of palbociclib were observed to be inhibited in this study (i.e., kinases inhibited by Palbociclib in Colo205 lysate), including CDK4 &6 as well as ERK5, JNK, CDK16 &17, PIK3C3 and PIP4K2C. The observed inhibition of these targets was both dose and time dependent. Significantly, the MS signals for additional kinases not observed to directly bind Palbociclib in Colo205 lysate were also strongly downregulated in this study. These include CDC2, AurA, AurB, PLK1 and MASTL; kinases critical for cell cycle progression. We hypothesize that the downregulation of these kinases was due to cell cycle arrest in G1 phase. Consistent with this hypothesis, the downregulation of these kinases corresponded well with the inhibition of phosphorylated (Ser780; pRb) as demonstrated by Western Blots of a part of tumor tissue used for the Kinome profiling. Taken together, these results highlight the utility of kinome wide profiling in tumor samples collected from xenograft mice to better understand both pharmacodynamics of target engagement as well as pathway effects elicited by compound treatment. Citation Format: Geeta Sharma, Subha Vogeti, Wendy Grant, Shuzhen Wu, Christa Dias, Tyzoon Nomanbhoy, Jiangyue Wu, Arwin Aban. In-situ Kinome wide profiling of Palbociclib treated COLO-205 human tumor xenograft samples. [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 3092. doi:10.1158/1538-7445.AM2015-3092

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 Pharmaceuticals' 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

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