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.

Supplementary Figure S2 shows two cases of patients treated at the RP2D. IHC at Clarient for p-ERK, p27 and Ki-67 were assessed for PD effect using monoclonal rabbit phospho-p44/42 MAPK (ERK1/2), 20G11 Clone; monoclonal mouse anti-Ki67, MIB-1 clone; and monoclonal mouse anti-p27, SX53G8 clone. In (A), the patient is a 56-year old female patient with rectal neuroendocrine cancer who is KRAS and BRAF wild-type. No differences in staining for the 3 biomarkers were observed when baseline and Day 28 samples were compared. In (B), the patient is a 26-year old male patient with hepatocellular carcinoma with unknown mutational status. No differences in staining for Ki-67 or p27 were observed when baseline and Day 28 samples were compared. The p-ERK staining is poor quality and not interpretable. Abbreviations: IHC, immunohistochemistry; PD, pharmacodynamics; RP2D, recommended phase 2 dose

Supplementary Figure S3 shows a heatmap of gene expression in the scale of log2 fold ratio between pre- and on-treatment tumor samples. The gene expression was obtained using the formula for a gene g: 2^[20-CT_g+HK]/1000 where CT is the cycle threshold used to detect the gene g. HK is the average over the cycle thresholds used to detect the three housekeeping genes: GUSB, PPIB, and PGK1. The columns of the heatmap represent the 11 target genes. The rows are the five patients labeled with their mutation status. The label is missing if a gene test was not done or not available. Abbreviation: RT-PCR, reverse transcriptase polymerase chain reaction.

Supplementary Figure S1 shows the study design. The study consisted of Part A (dose escalation) and Part B (dose confirmation). In Part A, 8 dosing levels starting with 50 mg twice daily (BID) were originally planned. Estimation of the maximum tolerated dose was conducted using a Continual Reassessment design. In Part B, 3 confirmation cohorts were included: Cohort A: advanced unresectable and metastatic melanoma with BRAF V600X mutations after treatment with BRAFi, MEKi or the combination of BRAFi/ MEKi; Cohort B, NRAS-mutant melanoma; and Cohort C, BRAF- or KRAS-mutant non-small cell lung cancer (NSCLC).

Supplementary Material and Methods

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.

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 (DCC3014; 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.

Abstract Mutations in ERK signaling drive a significant percentage of malignancies. LY3009120, a pan-RAF and dimer inhibitor, has preclinical activity in RAS- and BRAF-mutated cell lines including BRAF-mutant melanoma resistant to BRAF inhibitors. This multicenter, open-label, phase I clinical trial (NCT02014116) consisted of part A (dose escalation) and part B (dose confirmation) in patients with advanced/metastatic cancer. In part A, oral LY3009120 was dose escalated from 50 to 700 mg twice a day on a 28-day cycle. In part B, 300 mg LY3009120 was given twice a day. The primary objective was to identify a recommended phase II dose (RP2D). Secondary objectives were to evaluate safety, pharmacokinetics, and preliminary efficacy. Identification of pharmacodynamic biomarkers was exploratory. In parts A and B, 35 and 16 patients were treated, respectively (N = 51). In part A, 6 patients experienced eight dose-limiting toxicities. The RP2D was 300 mg twice a day. Common (>10%) any-grade drug-related treatment-emergent adverse events were fatigue (n = 15), nausea (n = 12), dermatitis acneiform (n = 10), decreased appetite (n = 7), and maculopapular rash (n = 7). The median duration of treatment was 4 weeks; 84% of patients completed one or two cycles of treatment. Exposures observed at 300 mg twice a day were above the preclinical concentration associated with tumor regression. Eight patients had a best overall response of stable disease; there were no complete or partial clinical responses. Despite adequate plasma exposure levels, predicted pharmacodynamic effects were not observed.

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.

Abstract 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.

Abstract Background: DCC-2618 is a potent switch control inhibitor of KIT and PDGFRα kinases maintains potent inhibition of mutant forms across all exon regions in preclinical models. Gastrointestinal stromal tumor (GIST) is an important disease to achieve a proof-of-concept due to the heterogeneity of resistance mutations in KIT which emerge on treatment with approved KIT inhibitors. In later lines of therapy resistance mechanisms independent of the KIT gene have also been described. Methods: The ongoing phase 1, PK-guided dose escalation study of DCC-2618 given orally BID [28-day cycle] tested doses from 20 mg to 200 mg in patients (pts) with advanced solid tumors including GIST (NCT02571036). We report preliminary longitudinal results of plasma cell free (cf) DNA sequencing by Guardant 360 collected throughout the study and levels of circulating tumor cells (CTCs) based on a viral telomerase promoter-driven GFP expression assay. Results: To date, 24 out of 31 enrolled pts had metastatic KITm GIST refractory to standard therapy. A high, total mean exposure of DCC-2618 and its active metabolite was achieved at 100 and 150 mg BID, affording steady state Cmax >5 µM in Cycle 1. Starting with 50 mg BID dose level, concentrations of total drug exceeding IC90 of the most resistant mutations to DCC-2618 were achieved. Next-generation sequencing of plasma cfDNA revealed a total of 40 KIT mutations in 16 of 18 GIST pts at baseline. DCC-2618 led to rapid decrease and/or clearance of the heterogeneous array of KIT mutations from plasma cfDNA including exons 9, 11, 13, 14, 17, and 18. Independent of suppressed KIT mutation burden, longitudinal monitoring of cfDNA revealed changes in non-KIT oncogenic mutations which may contribute to heterogenous mechanisms of resistance. KIT mutation burden will be correlated with metabolic response assessment by PET scans and exposure to DCC-2618. CTCs have been detected in blood from all GIST patients at baseline using a non-biased assay capable of identifying sarcoma cells. Preliminary result show that CTCs with immunofluorescent detection of KIT or PDGFRα, corresponding to their respective mutational status, show 1 of 3 patterns when compared to radiologic response: most pts show relatively stable low levels at stable disease (SD), a minority of pts with prolonged SD a decline in CTCs and 2 pts with progressive disease had significant increase in KIT positive CTCs. Conclusions: DCC-2618 and its active metabolite achieved high plasma concentrations sufficient to inhibit the most resistant KIT mutations at well-tolerated exposures. Monitoring of cfDNA KIT mutation frequency demonstrates rapid clearance of a broad spectrum of KIT mutations in this heavily pretreated GIST patient population and suggests candidate resistance genes that are independent of KIT. Our data provide a first signal that CTC monitoring might represent a potential marker for tumor control in KIT mutant GIST. Citation Format: Filip Janku, Albi Razak, Michael Gordon, David Brooks, Daniel Flynn, Anu Gupta, Michael Kaufman, Cynthia Leary, Bryan Smith, Deb Westwood, Neeta Somaiah, Elena Helman, Eric Gerstenberger, Oliver Rosen, Suzanne George. Translational research in a phase I proof-of-concept study supports that DCC-2618 is a pan-KIT inhibitor [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 LB-039. doi:10.1158/1538-7445.AM2017-LB-039

2507 Background: LY3009120, a pan-Raf and dimer inhibitor, demonstrates inhibition of phospho-Mek/Erk and tumor growth inhibition in several non-clinical cancer models with BRAF, NRAS, or KRAS mutations. This is the first-in-human phase 1 study of LY3009120 in patients (pts) with advanced cancer. Methods: The safety and tolerability of LY3009120 was evaluated in pts with cancer aged 18 years or older who had an ECOG performance status ≤1, at least 1 unidimensionally measurable lesion (RECIST 1.1), and adequate organ function (NCT02014116; I6X-MC-JBDA; Eli Lilly & Co.). The study sought to determine a recommended phase 2 dose using the toxicity band method and the safety, pharmacokinetic, and preliminary efficacy of LY3009120. Pharmacodynamic (PD) biomarkers, including pERK, p27 and Ki67, were evaluated in tumor tissue. The dose escalation phase evaluated dosages from 50 mg to 500 mg by mouth twice daily in pts with advanced cancers. Results: 34 pts (3 at 50 mg, 4 at 100 mg, 3 at 200 mg, 15 at 300 mg, 7 at 400 mg, and 2 at 500 mg) in dose escalation and 1 pt in dose expansion (1 at 300 mg) received at least one dose of LY3009120 by January 2, 2016 (median age = 47.4 yrs, range: 26-82 ). Most pts had a gene mutation (BRAF, n = 7; N/KRAS, n = 18); the most common cancer types included colon (n = 9), non–small cell lung cancer (n = 8), and pancreatic (n = 5). There were 6 dose-limiting toxicities in the dose escalation phase: 2 pts at 300 mg (G3 dermatitis acneiform [n = 1] and G2 blurred vision [n = 1]); 2 pts at 400 mg (G2 increased ALT with G3 hyperbilirubinemia [n = 1] and G3 increased ALT [n = 1]); 2 pts at 500 mg: (G3 arthralgia/myalgia [n = 1] and G3 stomatitis/pain [n = 1]). Based on these data, the maximum tolerated dose for LY3009120 was determined to be 300 mg twice daily. Treatment-emergent adverse events related to LY3009120 occurring in ≥10% of pts included fatigue (34%), nausea (31%), decreased appetite (20%), and dermatitis acneiform (20%) (Grade 1,2). A dose proportional increase in exposure was observed, but not at the 400 mg dose. The best response was stable disease in 5 pts. PD effect by rtPCR was not observed in tested paired tumor samples. Conclusions: LY3009120 is well tolerated at doses of 300 mg twice daily. Updated data from dose expansion will be presented in the meeting. Clinical trial information: NCT02014116.

AbstractTumor-infiltrating myeloid cells promote tumor progression by mediating angiogenesis, tumor cell intravasation, and metastasis, which can offset the effects of chemotherapy, radiation, and antiangiogenic therapy. Here, we show that the kinase switch control inhibitor rebastinib inhibits Tie2, a tyrosine kinase receptor expressed on endothelial cells and protumoral Tie2-expressing macrophages in mouse models of metastatic cancer. Rebastinib reduces tumor growth and metastasis in an orthotopic mouse model of metastatic mammary carcinoma through reduction of Tie2+ myeloid cell infiltration, antiangiogenic effects, and blockade of tumor cell intravasation mediated by perivascular Tie2Hi/Vegf-AHi macrophages in the tumor microenvironment of metastasis (TMEM). The antitumor effects of rebastinib enhance the efficacy of microtubule inhibiting chemotherapeutic agents, either eribulin or paclitaxel, by reducing tumor volume, metastasis, and improving overall survival. Rebastinib inhibition of angiopoietin/Tie2 signaling impairs multiple pathways in tumor progression mediated by protumoral Tie2+ macrophages, including TMEM-dependent dissemination and angiopoietin/Tie2-dependent angiogenesis. Rebastinib is a promising therapy for achieving Tie2 inhibition in cancer patients. Mol Cancer Ther; 16(11); 2486–501. ©2017 AACR.

2515 Background: DCC-2618 is a potent switch control inhibitor of KIT and PDGFR kinases active in a broad range of mutations. GIST is an important disease to achieve a proof-of-concept due to the heterogeneity of KIT resistance mutations, which emerge on treatment with approved KIT inhibitors. Methods: This was a PK-guided dose escalation study of oral DCC-2618 (QD or BID q28 days) in advanced solid tumors. FDG-PET scans were used to assess changes in FDG uptake in GIST pts after 3 wks of therapy. Next generation sequencing (NGS) of plasma cell-free (cf) DNA was performed throughout the study to assess and quantify KIT and other molecular alterations in drug targets and potential mechanisms of resistance. Results: 38 pts were enrolled (30 GIST; 4 glioma; 1 mastocytosis, 3 other carcinoma) to 8 dose levels: BID doses: 20 (4 pts), 30 (4), 50 (5), 100 (6), 150 (6) and 200 mg (3); QD doses: 100 (5) and 150 mg (4). Safety of evaluable pts is as follows: G3 or G4 adverse effects (regardless of attribution and occurring in > 1 pt) included anemia (5), lipase increase (4), hypertension (2). Two of the G3/4 lipase increase at 100 mg BID and 200 mg BID were DLTs. All G3/4 lipase increase were asymptomatic. G1/2 AEs (considered at least possibly related to DCC-2618) and occurring in ≥15% (n > 5) of pts include fatigue (12), alopecia & lipase increase (7), weight decrease (6). Starting with 50 mg BID dose level, trough concentrations of total drug exceeded the IC90 of the least sentivitive KIT mutations. Plasma concentrations > 5μM were achieved starting at 100 mg BID and the selection of the expansion phase dose is being finalized. Of 18 pts with KIT mutant GIST assessed by FDG PET, 14 (78%) had partial metabolic response per EORTC criteria. RANO/RECIST partial responses (PRs) were reported in 3 patients (1 GBM with PDGFRA/KIT amplifications and 2 GIST with Ex 11 & 17 / Ex 11 & 18 mutations, respectively). NGS of plasma cfDNA revealed 44 KIT mutations in baseline samples from 19 of 21 pts with GIST. Conclusions: DCC-2618 is well tolerated with encouraging preliminary activity in GIST pts with a broad spectrum of mutations and prior therapies. PR was also seen in a pt with GBM with PDGFRA/KIT amplifications. Clinical trial information: NCT02571036.

Non-clinical data suggest that PDGFRa plays an important role in the development and progression of human gliomas. To date, few PDGFRa inhibitors with CNS activity have been available. DCC-2618 was designed to potently inhibit the broadest range of mutations (mut) in KIT & PDGFRa kinases that emerge during tumor progession or on treatment. In a dose-escalation study (NCT# 02571036) of oral DCC-2618 (QD or BID q28 days), pts with advanced malignancies with a molecular rationale for activity were eligible. MRI scans were performed initially every 2 cycles then every 3 cycles. We enrolled 4 GBM pts and 1 anaplastic astrocytoma (AA) pt with PDGFRa muts/ amplifications who had progressed after standard temozolomide chemoradiation (GBM) or temozolomide only (AA) and had received 0 to 5 salvage therapies. Three pts (2 GBM and 1 AA) had a triple amplification of PDGFRa, KIT and KDR (4q12 amplicon). Two GBM pts had activating PDGFRa mutations. Pts were treated at 20 mg (1 pt), 50 mg BID (2 pts) or 100 mg QD (2 pts). The per-protocol population (N=48) received doses up to 200 mg BID and DCC-2618 was well tolerated. One GBM pt with mut PDGFRa progressed after 6 weeks and one stopped treatment due to a tumor-related hemaorrhage on C1D12. Two of the three pts with triple amplifications progressed after 2 cycles while the third pt (GBM, 20 mg BID) achieved a PR per RANO after 9 cycles. This pt is currently in cycle 20 with a remarkable 94% tumor reduction. The durable partial response of >18 months in a GBM patient (94% tumor reduction) warrants further evaluation of DCC-2618 in gliomas. An expansion cohort for pts with KIT- and PDGFRa driven tumors was initiated to be able to better select the patient population with a likely benefit.

Abstract 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 Deciphera's 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 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.

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.