Switch Pocket Research Articles

CodeBreaK 200: Sotorasib (AMG510) Has Broken the KRAS G12C+ NSCLC Enigma Code.

Per the US FDA sotorasib approval summary, KRAS G12C mutation is found in approximately 14% of adenocarcinoma of the lung, primarily in patients with a history of smoking. Until recently, targeted therapies against KRAS G12C have been largely unsuccessful due to the small protein size of KRAS and thus lack of binding pockets in KRAS and rapid hydrolysis of GTP to GDP by KRAS enzymes from abundance of GTP in the cytoplasm. Sotorasib, a first-in-class covalent KRAS G12C inhibitor that binds to the switch pocket II in the KRAS G12C-GDP "off" state, received US FDA accelerated approval on May 21, 2021 in the US, based on a Phase II dose expansion cohort of CodeBreaK 100 trial. Sotorasib at 960 mg once daily achieved an ORR of 36% (95% CI: 28%, 45%), with a median response duration of 10 months (range 1.3+, 11.1) in 124 KRAS G12C+ NSCLC. At the European Society of Medical Oncology (ESMO) 2022 annual meeting, sotorasib achieved a statistically significant improved PFS over docetaxel (HR = 0.66; 95% CI: 0. 51-0.86; P = 0.002). The modest magnitude of PFS improvement of 1.1 months (from 4.5 months to 5.6 months) and the ORR of 28% led to a vigorous debate on whether sotorasib was indeed a true breakthrough. In this pros and cons debate, we argue thatsotorasib has achieved a true breakthrough.

Switch Pocket Kinase: An Emerging Therapeutic Target for the Design of Anticancer Agents

Protein kinases are amongst the most focused enzymes in the current century to design, synthesize and formulate drugs that ought to be effective in the treatment of various disordered and diseased states involving either overexpression or deficiency situations. The ATP pocket on the kinases is the active binding site for most of the kinase inhibitors. However, the kinase mutations prevent the binding of kinase inhibitors to the ATP pocket. The enzyme becomes inactive even in the mutated state when the switch pocket site on the enzyme is occupied by switch pocket inhibitors. This review comprises detailed information regarding various classical protein kinases and switch pocket kinase inhibitors with their mechanism of action so that new molecules can be designed to encounter mutations in the kinase enzyme.

New drugs in gastrointestinal stromal tumors.

Tyrosine kinase inhibitors (TKIs) are the backbone for advanced gastrointestinal stromal tumor (GIST) treatment. The increasing knowledge concerning the structure and the changing conformational status because of some mutations in KIT and PDGFRα, allowed the development of new efficient compounds, with the main goal to overcome resistance in GIST. This review summarizes the latest developments in the treatment of GIST patients. Amongst the several TKIs currently being studied in GIST, ripretinib, avapritinib and crenolanib had shown promising potent activity in preclinical studies and clinical trials. Ripretinib is a type II inhibitor that exerts its main action in the switch pocket of the activation loop, by mimicking the inhibition exerted by the regulatory region in this domain. Ripretinib is considered the new standard in the fourth line in advanced GIST. Avapritinib is a type I inhibitor synthesized to exerts its activity in the active conformation of the activation loop of KIT and PDFGRα. The relevant activity reported with avapritinib in patients carrying the D842 v mutation represents, for first time, an active therapeutic option in this resistant mutant. Crenolanib is a type I selective inhibitor of PDGFRα-resistant mutants, mainly D842 V, which is currently under clinical trial. New potent TKIs are being approved, adding value to the already three registered drugs. Other agents, such as MEK inhibitors, immunotherapy and TRK-targeted therapy are potential new options in specific subsets of GIST patients.

Abstract C087: Phase 1 study of DCC-3014, an oral inhibitor of CSF1R, to assess the safety, tolerability, pharmacokinetics, and pharmacodynamics in patients with advanced solid tumors, including diffuse-type tenosynovial giant cell tumor

Abstract Background: DCC-3014 is an orally administered kinase inhibitor targeting the switch pocket of colony-stimulating factor 1 receptor (CSF1R). DCC-3014 exhibits approximately 100-fold selectivity from kinases homologous to CSF1R, such as KIT, and greater selectivity against 300 other human kinases. Tumor-associated macrophages (TAMs) are dependent on CSF1R kinase activity for proliferation and maintenance of the immunosuppressive phenotype. TAMs are known to enable tumor cells escape from immune surveillance. DCC-3014 is designed to exhibit an antitumor effect by inhibition of immunosuppressive TAMs or other CSF1R signaling-dependent pro-tumor activities. In addition, tenosynovial giant cell tumor (TGCT) is a rare, monoarticular disease known to be caused by the translocation in the CSF1 gene leading to overexpression of CSF1. Anti-CSF1R therapies have shown clinical activity in diffuse TGCT. Methods: This study is a multicenter, first-in-human study of DCC-3014 to determine a recommended Phase 2 dose (RP2D) or maximally tolerated dose (MTD) with a 3+3 dose escalation design and evaluate the safety, efficacy, pharmacokinetics (PK) and pharmacodynamics (PD) of DCC-3014 in advanced solid tumors, including diffuse-type TGCT [NCT03069469]. Results: As of June 2, 2019, the study is ongoing in the dose escalation phase and has enrolled 32 patients with advanced solid tumors in 7 cohorts. The first cohort was initiated at 10 mg daily. Based on preliminary PK analysis from the first cohort, subsequent cohorts utilized a loading dose followed by a maintenance dosing schedule to achieve steady-state exposures more rapidly. The median age of patients was 61 years old and 69% were female with the median of 4 lines of prior anti-cancer treatment. No dose limiting toxicities (DLTs) have been seen in the assessed cohorts. Most commonly seen treatment-emergent adverse events (TEAEs >20%) were constipation (38%), vomiting (34%), diarrhea (31%), nausea (31%), decreased appetite (25%), abdominal pain (22%), and dyspnea (22%) whereas diarrhea, nausea and fatigue were reported as treatment-related AEs seen in >10% of all patients (13%). TEAEs were mostly Grade 1 or 2. Serious AEs (SAEs) were seen in 16 patients, none of which were related to DCC-3014. Exposure was approximately dose proportional. PD effects were seen for dose levels at 10 mg and above, and induction of the CSF1R ligand, CSF-1, was seen up to 148-fold from baseline. The RP2D or MTD has not been selected or reached. Conclusion: DCC-3014 in this study was generally well tolerated in patients with advanced solid tumors and showed approximately dose-proportional exposure and PD changes as expected. Further evaluation is ongoing to determine the RP2D or MTD. Data from the TGCT cohort will be presented at a future meeting. Citation Format: Matthew H Taylor, Stephen Leong, Ying Su, Cynthia B Leary, Xiaoyan Li, Keisuke Kuida, Rodrigo Ruiz-Soto, Todd Bauer. Phase 1 study of DCC-3014, an oral inhibitor of CSF1R, to assess the safety, tolerability, pharmacokinetics, and pharmacodynamics in patients with advanced solid tumors, including diffuse-type tenosynovial giant cell tumor [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 C087. doi:10.1158/1535-7163.TARG-19-C087

Abstract B055: Phase 1b/2 study of rebastinib (DCC-2036) in combination with paclitaxel: Preliminary safety, efficacy, pharmacokinetics and pharmacodynamics in patients with advanced or metastatic solid tumors

Abstract Background: Rebastinib is an orally administered, kinase inhibitor targeting the switch pocket of tunica interna endothelial cell kinase (TIE2). TIE2 is primarily expressed in endothelial cells, playing a role in angiogenesis. In addition, TIE2 is expressed in a subset of macrophages, TIE2 expressing macrophages (TEMs), which have pro-angiogenic, pro-metastatic, and immunosuppressive properties. Accumulating evidence suggests that chemotherapies, such as paclitaxel, increase the recruitment and activity of TEMs, leading to chemotherapy resistance. This study aims to investigate the efficacy of anti-TIE2 therapy against chemo-resistant advanced solid tumors. Methods: This is an open-label, Phase 1b/2, multicenter study with two parts. Part 1 evaluated two doses of rebastinib at 50 and 100 mg twice daily (BID) with paclitaxel administered weekly at 80 mg/m2 to determine the recommended phase 2 dose (RP2D) by enrolling at least 15 pts in each arm. The objective of Part 1 is to identify a recommended Phase 2 dose. Pts with locally advanced or metastatic solid tumors for which paclitaxel was considered appropriate treatment were enrolled. Part 2 has four disease-specific cohorts: triple-negative breast cancer, inflammatory breast cancer, platinum-resistant ovarian cancer and metastatic endometrial cancer to further evaluate the safety, tolerability and efficacy of the RP2D. This abstract focuses on data from Part 1. Results: A total of 43 pts were enrolled in Part 1 (24 pts at 50 mg BID and 19 pts at 100 mg BID) as of June 21, 2019. The median age was 61 years; 79% were female. Most frequent diagnoses were ovarian (11 pts), breast (8 pts) and endometrial cancer (7 pts). The median number of prior anti-cancer therapies was four (4). Median duration of treatment is 56 days. Treatment-emergent adverse events (TEAEs) were reported for 41 pts. Most TEAEs were previously observed in the rebastinib first-in-human study or were known AEs of paclitaxel. TEAEs >10% include mostly Grade (Gr) 1 or 2 fatigue (30%), constipation (21%), dry mouth (21%), nausea and alopecia (19% each), anemia and UTI (16%), hypokalemia, hypomagnesemia, peripheral neuropathy, and dyspnea (14% each), diarrhea, stomatitis, vomiting, sepsis, dysgeusia, rash and ALT elevation (12% each). Twenty-four pts experienced at least one ≥Gr 3 TEAE. Six pts had ≥Gr 3 AEs related to rebastinib. No significant difference in TEAEs was observed between the two dose levels. The RP2D was determined for rebastinib at 100 mg BID in combination with weekly paclitaxel at 80 mg/m2. Exposure of rebastinib was dose-proportional. Plasma TIE2 and its ligand, ANG2, were induced in a dose-dependent manner. Evidence of observed preliminary clinical activity in heavily pre-treated patients will be presented. Conclusion: Preliminary results demonstrated that the combination of rebastinib and paclitaxel was generally well tolerated. This combination is currently under evaluation in four disease-specific cohorts in Part 2. Clinical trial information: NCT03601897. Citation Format: Filip Janku, Michael Birrer, Debra Richardson, Christina Chu, Sanjay Goel, Ying Su, Bahar Matin, Keisuke Kuida, Rodrigo Ruiz-Soto, Erika Paige Hamilton. Phase 1b/2 study of rebastinib (DCC-2036) in combination with paclitaxel: Preliminary safety, efficacy, pharmacokinetics and pharmacodynamics in patients with advanced or metastatic solid tumors [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 B055. doi:10.1158/1535-7163.TARG-19-B055

LBA87 - INVICTUS: A phase III, interventional, double-blind, placebo-controlled study to assess the safety and efficacy of ripretinib as ≥ 4th-line therapy in patients with advanced gastrointestinal stromal tumors (GIST) who have received treatment with prior anticancer therapies (NCT03353753)

LBA87 - INVICTUS: A phase III, interventional, double-blind, placebo-controlled study to assess the safety and efficacy of ripretinib as ≥ 4th-line therapy in patients with advanced gastrointestinal stromal tumors (GIST) who have received treatment with prior anticancer therapies (NCT03353753)

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

Abstract 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 790: Altiratinib is a potent inhibitor of TRK kinases and is efficacious in TRK-fusion driven cancer models

Abstract Introduction: TRK kinases have been implicated in a variety of cancers, wherein TRK gene fusions, amplifications and mutations have been shown to drive tumor growth. TRK kinases have also been shown to play key roles in metastasis. Recently, large-scale sequencing efforts have identified TRK fusions at a low but significant frequency across all major cancers. Altiratinib is a potent single-digit nanomolar inhibitor of TRK, MET, TIE2, and VEGFR2 kinases. Altiratinib inhibits TRKA, TRKB, and TRKC phosphorylation in both WT TRK and TRK-fusion cell lines and inhibits cell proliferation in these cell lines. In vivo, altiratinib suppressed TRK phosphorylation for >18 hr after a single oral dose and showed significant inhibition of tumor growth in TRKA and TRKC-fusion xenograft efficacy studies. Altiratinib is currently in a Phase 1 clinical trial for patients with advanced solid tumors. Experimental procedures: Altiratinib was tested for inhibition of various recombinant kinases using a standard PK/LDH coupled spectrophotometric continuous assay. In cell assays, cells were treated with a dose response of compound. Levels of phosphorylated TRK in cell lysates were determined Western blot. Cell proliferation 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). Summary of results: Altiratinib potently inhibited TRK kinase activity in biochemical assays (IC50 values of 0.9 nM, 4.6 nM, and 0.8 nM for TRKA, B, and C, respectively). Altiratinib inhibited phosphorylation of TRKA in the TPM3-TRKA fusion cell line KM-12 (IC50 = 1.4 nM) and of TRKC in ETV6-TRKC transformed NIH-3T3 cells (IC50 = 0.5 nM). Altiratinib inhibited NGF-stimulated phosphorylation of wild-type TRKA in K562 (IC50 = 0.7 nM) and SK-N-SH cells (IC50 = 1.2 nM) and BDNF-stimulated phosphorylation of TRKB in ATRA-transformed SK-N-SH cells (IC50 = 0.24 nM). In compound washout experiments, altiratinib inhibited TRKA phosphorylation in KM-12 cells for > 24 hr after compound washout, due to its durable Type II switch pocket binding mode to the kinase. In cell lines driven by TRK fusions, altiratinib also inhibited cell proliferation (KM-12 IC50 = 3.8 nM; NIH-3T3 ETV6/TRKC IC50 = 0.5 nM). In vivo, altiratinib inhibited phosphorylation of TRK and downstream targets in the signaling pathway for >12 hr after a single oral dose. Altiratinib treatment led to tumor regression in TRK-fusion xenograft efficacy studies. Conclusions: Altiratinib exhibits potent inhibition of oncogenic TRK fusions in vitro and in vivo. Combined with its inhibition of the tumor and microenvironment targets MET, TIE2, and VEGFR2 kinases, altiratinib provides the potential to treat cancers driven by TRK fusions. Altiratinib is currently in a Phase 1 clinical trial in patients with solid tumors. Citation Format: Bryan D. Smith, Cynthia B. Leary, Benjamin A. Turner, Michael D. Kaufman, Scott C. Wise, Maria E. R. Garcia-Rendueles, James A. Fagin, Daniel L. Flynn. Altiratinib is a potent inhibitor of TRK kinases and is efficacious in TRK-fusion driven cancer 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 790. doi:10.1158/1538-7445.AM2015-790

356 Discovery of novel pyrido[2,3-b]pyrazine as fibroblast growth factor receptor (FGFR-1, 2, 3 & 4) kinase inhibitors with nanomolar affinity

Background: Altiratinib is a Type II switch pocket inhibitor of MET kinase as well as TIE2 and VEGFR2 kinases. This profile provides an agent that exhibits anti-tumor activity in cancers driven by MET overexpression or genomic mutation, and also blocks angiogenic and metastatic processes mediated by the tumor microenvironment. Material and Methods: Altiratinib was evaluated in MET, TIE2, and VEGFR2 biochemical studies, including evaluation in a number of MET activation mutants. Cellular activity was evaluated in tumor cell lines exhibiting MET amplification (MKN-45 gastric, EBC-1 NSCLC) or overexpression (B16/F10 melanoma; U87 glioblastoma). In vivo pharmacokinetic/pharmacodynamic studies were performed in an MKN45 xenograft model. Efficacy was demonstrated in a battery of xenograft/ allograft models including: gastric (MKN-45), melanoma (B16/F10, A375), ovarian (SKOV-3), colorectal (COLO-205), lung (EBC-1), breast (PyMT), and glioblastoma (U87). Results: Altiratinib afforded balanced inhibition of MET, TIE2, and VEGFR2 kinases in the low nM range (IC50s 2−9 nM) and blocked HGF-, ANG-, or VEGFA-induced HUVEC activation and capillary tube formation. Altiratinib retained potency versus activation loop mutant forms of MET (D1228X, Y1230X, M1250X), inhibiting all forms with IC50 24 hr after a single 10mg/kg oral dose in an MKN-45 xenograft pharmacodynamic model. Altiratinib exhibited anti-tumor activity in melanoma (B16/F10, A375), gastric (MKN-45), lung (EBC-1), colorectal (COLO-205), breast (PyMT), ovarian (SKOV-3) and GBM (U87) xenograft or allograft models. In these in vivo studies, altiratinib was shown to inhibit tumor growth, angiogenesis, invasion and/or metastasis. Altiratinib also blocked recruitment of TIE2-expressing monocytes in the PyMT breast cancer model. In some models, altiratinib was shown to increase overall survival. In particular, in the i.c.v. orthotopically implanted U87 glioblastoma model, altiratinib extended survival by 1.7-fold vs vehicle (112 days vs 66 days), while the combination of altiratinib + bevacizumab extended survival by 2.5 fold vs vehicle and by 1.9-fold vs bevacizumab single agent (166 days vs. 88 days). Conclusions: Altiratinib is a balanced inhibitor of MET, TIE2, and VEGFR2 kinases. This profile provides robust inhibition of tumors driven by MET amplification or overexpression, and also provides the potential for altiratinib to block tumor microenvironment angiogenic resistance mechanisms and pro-tumoral effects of TIE2-expressing macrophages in the clinical setting. Altiratinib is currently in Phase 1 clinical trials in patients with solid tumors.

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 (WT) and, where also examined, KIT D816V 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 FcεRI-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.

Targeting the KIT Activating Switch Control Pocket: A Novel Mechanism to Inhibit Mast Cell Activation and KIT D816V Neoplastic Mast Cell Proliferation

Abstract 1740KIT is a receptor protein tyrosine kinase which undergoes dimerization, autophysophyralation, and activation upon binding of its ligand, stem cell factor (SCF). Such activation is critical for the growth, differentiation and survival of mast cells. By enhancing Fcƒ’RI-mediated degranulation and inducing chemotaxis, KIT also contributes to mast cell functional responses. Targeting KIT is thus an attractive approach for the management of proliferative mast cell disorders. Activating mutations in the KIT tyrosine kinase domain, most notably KIT D816V, are commonly observed in the myeloproliferative disorder, systemic mastocytosis. The D816 mutation is located within the activating-switch component of KIT. These D816 mutations render the activating-switch of KIT constitutively active and capable of binding into the cognate switch pocket of KIT. Such aberrant switch function aggressively fluxes KIT to a catalytically active conformation. Switch pocket inhibitors block access to this switch pocket, thus inhibiting the ability of the mutationally activated D816 KIT mutants to adopt active conformations. Most tyrosine kinase inhibitors in clinical development target the KIT ATP binding pocket whose structure is often conserved among kinases resulting in off-target inhibition. In contrast, switch pockets are more unique among kinases. Utilizing rational drug design, the diversity in switch pockets that KIT uses to switch between the inactive and active conformations may be targeted to fine-tune selectivity of an inhibitor against the rest of the kinome. In this study, two such KIT “switch pocket” (SP) inhibitors, DP-2976 and DCC-2618, were examined for their effects on KIT activation, mast cell activation and mast cell proliferation; in comparison to the known ATP binding pocket inhibitors, imatinib and PKC412. To explore the effects of the SP inhibitors on KIT activity, wild type KIT and KIT D816V were expressed in 293T cells and their KIT autophosphorylation status assessed. DP-2976 and DCC-2618 blocked the inherent autophosphorylation of both wild type and KIT D816V in the transfected cells in the nanomolar range. Similar results were observed on constiutively active KIT in HMC 1.1 and 1.2 cells; and in CD34+ derived human mast cells in which KIT phosphorylation was induced by exposure to SCF (10 ng/ml). SP inhibitors had minimal effects on FcεRI mediated mast cell degranulation. They did completely block the SCF potentiation of FceRI-mediated degranulation. In contrast, PKC 412 inhibited degrangulation in an SCF independent manner suggesting a more generalized kinase targeting. To assess the anti-neoplastic activity of the SP inhibitors, they were next examined in HMC1.1 and HMC1.2 cell proliferation assays. Both DP-2976 and DCC-2618 significantly reduced cell numbers through induction of apoptosis with low nanomolar IC50 values. To examine whether such observations translate into clinical efficacy, ex vivo studies were performed. The survival of primary bone marrow mast cells from mastocytosis patients was significantly reduced when exposed to the SP inhibitors; a reduction comparable to PKC412. Overall, DP-2976 and DCC-2618 “switch pocket” inhibitors offer a novel and potent KIT inhibition profile whose selectivity and dual suppression of SCF enhanced mast cell activation and KIT D816V neoplastic proliferation may provide significant therapeutic benefits. Disclosures:Wise:Deciphera Pharmaceuticals LLC: Employment. Flynn:Deciphera Pharmaceuticals LLC: Employment, Equity Ownership.

A Phase 1 Study of DCC-2036, a Novel Oral Inhibitor of BCR-ABL Kinase, in Patients with Philadelphia Chromosome Positive (Ph+) Leukemias Including Patients with T315I Mutation

Abstract 601 Background.DCC-2036 is a novel and potent tyrosine kinase inhibitor (TKI) which binds to a novel region called the switch pocket, thereby preventing BCR-ABL from adopting a conformationally active state. Efficacy against multiple imatinib-resistant BCR-ABL mutants has been demonstrated both in vitro and in vivo (Chan et al., Cancer Cell 2011;19:556). Importantly, DCC-2036 retains full potency against the T315I mutant in preclinical efficacy studies. Methods.This study was designed to find the maximal tolerated dose (MTD) of DCC-2036 when administered daily as a single-agent on a 28-day cycle. Eligible patients included adults with Ph+ CML/ALL who were refractory/intolerant to ≥2 TKI’s or were T315I positive. Initially DCC-2036 capsules were administered orally once daily (QD) at increasing dose levels. Only 1 patient was enrolled in each of the lowest dose cohorts of 57mg QD and 114 mg QD. For higher doses, 3– 6 patients were enrolled into each ascending dose cohort with standard dose limiting toxicity (DLT) rules evaluating safety in cycle 1 to determine dose escalation. A transition from unformulated capsules (C) to formulated tablets (T) occurred after the 1200 mg QD dose level. Paired blood samples were obtained for PK and PD assessments. Results.30 patients (16 males, 14 females; median age 59, range 31 – 80) with CML including 19 in Chronic (CP); 8 in Accelerated (AP) and 3 in Blast (BP) Phase were enrolled. Enrolled patients had received 1–6 prior CML treatments, and 11 patients had the T315I mutation. To date, a total of 212.5 (median 5.6; range 0.2 – 23.4) 28-day cycles were administered over 10 dose levels either as C (7 dose levels) or T (3 dose levels). The 7 C dose levels were studied first and included 57 mg QD through 1200 mg QD. Following transition to T, evaluation continued with 100 mg QD, 100 mg twice daily (BID), and 200 mg BID. Two reversible DLTs (Grade 3 peripheral neuropathy and Grade 4 lower extremity weakness) occurred during the initial treatment cycle at the 200 mg T BID dose level. Evaluation of 6 patients at the 150 mg T BID dose level determined that dose to be the MTD.Preliminary safety data show that other Grade (Gr) 3/4 adverse events (AEs) were Gr 3 slurred speech and Gr 3 eruptive nevi. Gr 1/2 AEs included dry mouth, constipation, diarrhea, paresthesias, and retinal vein occlusion. There was 1 case of Gr 2 pancreatitis that recurred on rechallenge in a patient with previous pancreatitis with nilotinib.Preliminary responses include one major molecular response in a CP patient with T315I mutation who started on capsules and transitioned to 100 mg T QD. There was one complete cytogenetic response in a CP patient at 100 mg T BID, and one partial cytogenetic response in a CP patient who started on capsules and transitioned to 100 mg T BID. One patient with AP CML and T315I mutation had a complete hematologic response at 450 mg C QD. Another patient with AP CML had a partial hematologic response after receiving 200 mg BID for 1 cycle and then downdosing to 100 mg T BID. Four out of 8 patients receiving 100 mg tablets and evaluable for efficacy (completed 3 cycles of treatment) had responses.PK results indicate dose-related, nonlinear increases in both peak plasma concentration (Cmax) and exposure (AUC). PD results reveal both acute and steady state post-treatment reductions in phospho-protein levels on Days 1 and 8. Marked reductions in pSTAT5 and pCRKL have been observed in subjects with both CP and AP and appear to be required for clinical response. Conclusion.The MTD of DCC-2036 tablets is 150 mg BID. Preliminary results suggest that DCC-2036 is well tolerated and has anti-leukemia activity in subjects with refractory CML and T315I positive disease. PD results are consistent with inhibition of BCR-ABL signaling in this first-in-man study of a switch pocket tyrosine kinase inhibitor. Disclosures:Cortes:Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Chemgenex: Consultancy, Research Funding; Deciphera Pharmaceuticals: Research Funding. Bixby:Novartis: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau; GlaxoSmithKline: Speakers Bureau. Rafferty:Deciphera Pharmaceuticals: Employment. Berger:Deciphera Pharmaceuticals: Employment. Wise:Deciphera Pharmaceuticals LLC: Employment. Rutkoski:Deciphera Pharmaceuticals: Employment. Smith:Deciphera Pharmaceuticals: Employment. Van Etten:Deciphera Pharmaceuticals: Consultancy, Research Funding.

A New BCR-ABL1 Mutation (L248R) Is Highly Resistant to Imatinib, Bosutinib, Nilotinib and Dasatinib, but Can Be Inhibited by AP-24534 and DCC-2036.

AbstractAbstract 3398Chronic Myeloid Leukemia (CML) treatment was radically modified by the discovery of imatinib (IM), a selective inhibitor of the fusion kinase Bcr-Abl. Second generation ATP-competitive tyrosine kinase inhibitors (TKIs) bosutinib (BOS), nilotinib (NIL) and dasatinib (DAS) further improved CML therapy. However, resistance to TKIs occurs in a variable proportion of patients; it can arise from different mechanisms but in the majority of cases it is due to Bcr-Abl point mutations that alter directly or indirectly the drug-protein binding. Over 70 mutations have been described in patients, with T315I showing resistance to IM, NIL, BOS and DAS. Here we report the discovery of a new P-loop mutant (L248R) that is highly resistant to all currently available inhibitors. L248R was identified in a lymphoid Blast Crisis CML patient. The patient initially presented with an IM-resistant F359I mutation. A cytogenetic and molecular response was obtained with BOS, but after 1 year haematological relapse developed; at this point a F359I/L248R clone was identified. L248R was previously reported only in an in vitro during a mutagenesis screen involving IM (Bradeen et al. 2006) and in a study with the T315I inhibitor SGX393 (O'Hare et al. 2008). L248R was never isolated from a clinical sample. Activity profile of BOS, IM, DAS and NIL against L248R, F359I and F359I/L248R was performed (Table). Stable transfectant Ba/F3 cells were generated and the TKIs anti-proliferative activity was determined. The relative IC50 increase over wild type Bcr-Abl (Relative Resistance, RR) was calculated. We classified RR values in four categories: sensitive (RR≤ 2), moderately resistant (RR between 2.1 and 4), resistant (RR between 4.1 and 10) or highly resistant (RR>10). In all cases a RR >10 was obtained, for L248R. Recently new compounds were developed to overcome resistance generated by the T315I mutant. Among them AP-24534 (AP), a panBcr-Abl inhibitor (O'Hare et al. 2009) and the switch pocket inhibitor DCC-2036 (DCC) were reported as potently active against the T315I mutation. Both compounds showed activity against L248R (RR 6.2 and 0.4), although the activity against the double mutant F359I/L248R was reduced especially for AP (RR 17.7 and 1.0). The activity profile of AP and DCC against L248R and a panel of 26 mutated forms of Bcr/Abl covering the most common mutations is also presented (Table). Activity of BOS, IM, DAS and NIL is also reported for comparison. According to our data, only mutation E255V is classified as highly resistant to AP, in addition to F359I/L248R: 6/26 mutations are considered “resistant” to AP (L248R, G250E, G252H, E255K, F359V, H396R) and 3/26 to DCC (D276G, E279K, F317V). The huge difference in the IC50 values for AP between Bcr/Abl wild type (2.1 nM) and parental Ba/F3 cells (>1000 nM) could render some of the mutants with high RR values to AP still sensitive to this inhibitor. It is also important to note that, according to our data, every mutation analysed shows sensitivity to at least one of the tested TKIs. In conclusion we describe a novel mutation that is highly resistant to the commonly used Bcr-Abl TKIs but which is inhibited by AP and DCC. Modelling data on the L248R mutant in complex with different TKIs will also be presented [Display omitted] IC 50 values are based on tritiated thymidine incorporation assay. Results are an average of at least 3 independent experiments Disclosures:Wise:Deciphera Pharmaceuticals: Employment. Flynn:Deciphera Pharmaceuticals: Employment. Gambacorti-Passerini:Pfizer pharmaceuticals: Research Funding.

In vitro activity of novel KIT/PDGFRA switch pocket kinase inhibitors against mutations associated with drug-resistant GI stromal tumors.

10007 Background: Most gastrointestinal stromal tumors (GISTs) harbor mutant KIT or PDGFRA kinases, which are targets of imatinib (IM) or sunitinib (SU). Resistance to IM or SU is commonly due to the acquisition of secondary mutations that markedly reduce the potency of kinase inhibitors. Both IM and SU bind to the KIT/PDGFRA ATP binding pocket. KIT and PDGFRA kinases possess negative and positive regulatory domains that modulate kinase activity by competitive binding into the switch pocket domain. Switch pocket kinase inhibitors (SPKIs) occupy this pocket and block enzymatic activity. We developed SPKIs with low nanomolar potency against wild-type KIT/PDGFRA kinases. Methods: We tested the activity of three SPKIs (DP-2976, DP-3636, DP-4444) in enzymatic assays using various wild-type and recombinant KIT kinases. In addition, a panel of kinase mutants was biochemically profiled for sensitivity to these SPKIs using several cell line models, including GIST cell lines derived from imatinib-resistant tumor clones. Results: All three SPKIs were very potent (5-10 nM IC50) against wild-type KIT and PDGFRA. In addition, these compounds maintained their potency against representative GIST-associated drug resistance mutations (V654A, T670I, D816H, D816V). DP-2976 and DP-3636 were also potent against the pan drug-resistant PDGFRA D842V mutation with IC50s of 50 and 20 nM, respectively. To confirm these observations in a GIST cellular context, we tested the relative potency of these compounds against two previously described IM-resistant GIST cell lines (exon 11 + V654A, exon 11 + D820A). All three compounds were significantly more potent than IM or SU for both cell lines. These results also correlated with enhanced inhibition of downstream signaling pathways, cellular proliferation and survival. Conclusions: DP-2976, DP-3636, and DP-4444 have markedly superior in vitro potency compared with IM or SU against a panel of GIST-relevant mutant kinases. Based on these results, we hypothesize that these compounds might have useful clinical activity against drug- resistant GIST. Further preclinical studies are indicated to develop these compounds for human clinical studies. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Deciphera Deciphera, MolecularMD Novartis Novartis

Biochemical and biophysical characterization of unique switch pocket inhibitors of p38α

Herein we describe the identification and characterization of a class of molecules that are believed to extend into a region of p38 known as the ‘switch pocket’. Although these molecules lack a canonical hinge binding motif, they show K i values as low as 100 nM against p38. We show that molecules that interact with this region of the protein demonstrate different binding kinetics than a canonical ATP mimetic, as well as a wide range of kinome profiles. Thus, the switch pocket presents new opportunities for kinome selectivity which could result in unique biochemical responses and offer new opportunities in the field of kinase drug discovery.

Switch Pocket Inhibitors of the ABL Tyrosine Kinase: Distinct Kinome Inhibition Profiles and in Vivo Efficacy in Mouse Models of CML and B-Lymphoblastic Leukemia Induced by BCR-ABL T315I

Acquired resistance to the ABL tyrosine kinase inhibitor (TKI) imatinib is a significant clinical problem for patients with chronic myeloid leukemia (CML), where a major resistance mechanism is mutations in the ABL kinase domain that render the BCR-ABL enzyme insensitive to the drug. The gatekeeper mutation Thr 315 Ile (T315I) arises at high frequency in patients relapsing after sequential therapy with second-line TKIs such as dasatinib or nilotinib (Shah et al., J Clin Invest 2007; 117:2562), and confers pan-resistance to all ATP-competitive TKIs. Using structure-based rational drug design, we have developed a novel chemical class of compounds that bind to five distinct structural pockets that the ABL kinase uses to switch between the inactive and active conformations. Diversity in these “switch pockets” between kinases can be exploited to fine-tune the selectivity of an inhibitor against the rest of the kinome. Two development candidate ABL inhibitors have emerged from this effort: DCC-2036 and DCC-2157. Both compounds potently (IC50 = 0.8–4.0 nM) inhibit purified ABL in the switch-off (unphosphorylated) and switch-on (phosphorylated) states via a non-ATP-competitive mechanism, and avoid steric clash with Ile 315. Both drugs also impair proliferation and induce apoptosis of Ba/F3 cells expressing a wide variety of BCR-ABL TKI-resistant mutants, including T315I, many P-loop mutants, and the dasatinib-resistant mutant F317L. DCC-2036 is highly selective for ABL, FLT3, TIE2, and Src family kinases, and when dosed at 100 mg/kg/day by oral gavage, significantly prolonged the survival of mice with CML-like myeloproliferative disease induced by retroviral expression of BCR-ABL WT and T315I in bone marrow. Genetic studies in mice (Hu et al., Nat Genet 2004; 36:453) suggest that the Src inhibitory activity of DCC-2036 would be beneficial against Ph+ B-cell acute lymphoblastic leukemia (B-ALL), and indeed DCC-2036 was superior to dasatinib (Hu et al., PNAS 2006; 103:16870) in prolonging survival of mice with B-ALL induced by BCR-ABL T315I. By contrast, DCC-2157 is selective for ABL, KIT, and PDGFRA/B kinases, and pharmacodynamic studies demonstrated prolonged (>18 hour) inhibition of phospho-Stat5 in BCR-ABL+ leukemic cells following a single oral 100 mg/kg dose. Treatment of mice with CML-like disease induced by BCR-ABL T315I with DCC-2157 at 50 mg/kg every other day induced virtually complete suppression of leukemia and extensive prolongation of survival. These results demonstrate that switch pocket drug design technology yields ABL TKIs with distinct pharmacologic profiles that are promising new weapons for the treatment of de novo and drug-resistant Ph+ leukemias.

DCC-2036: A Novel Switch Pocket Inhibitor of ABL Tyrosine Kinase with Therapeutic Efficacy Against BCR-ABL T315I In Vitro and in a CML Mouse Model.

Therapy with ATP-competitive ABL kinase inhibitors, including imatinib (Gleevec), dasatinib (Sprycel), and nilotinib (Tasigna), has revolutionized the treatment of chronic myeloid leukemia (CML), but a substantial proportion of patients develop resistance to these agents. A major mechanism of acquired resistance is mutations in the ABL kinase domain that render BCR-ABL insensitive to the drug; in particular, mutation of the ABL kinase “gatekeeper” Thr315 residue to Ile (T315I) confers pan-resistance to all ATP-competitive ABL inhibitors. To address this need, we developed and characterized a novel chemical class of compounds that bind to five structural pockets involved in the endogenous “switch” mechanism used by the ABL kinase to conformationally control its activity state. Using a structure-based drug design approach, diversity in these “switch pockets” between kinases can be exploited to develop inhibitors with high potency and specificity. The resulting ABL inhibitors have several attractive features, as they:1.act through a non-ATP-competitive mechanism;2.avoid steric clash with Ile315;3.inhibit purified ABL that is either unphosphorylated (switch-off) or phosphorylated (switch-on) at Tyr393 with IC50 of 0.8–4.0 nM;4.co-crystallize with phospho-Y393-ABL WT and T315I; and5.have very prolonged residency time on the kinase (off-rate ∼400 min vs. 3 min for imatinib).A lead compound, DCC-2036, is highly selective for ABL, FLT3, and SRC family kinases, and has favorable pharmacokinetic and toxicity profiles in animals. In vitro, DCC-2036 inhibited proliferation and induced apoptosis of Ba/F3 cells expressing BCR-ABL WT or several common imatinib-resistant mutants (Y253F, T315I, M351T) with IC50 from 5 to 25 nM, without appreciable inhibition of parental Ba/F3 cells in IL-3 (IC50 >1000 nM). In Balb/c mice injected with Ba/F3-BCR-ABL T315I cells, pharmacodynamic studies indicated >8 hour inhibition of phospho-Stat5 and phospho-ABL in the leukemic cells following a single oral 100 mg/kg dose of DCC-2036, whereas daily dosing of DCC-2036 significantly prolonged survival. In the mouse retroviral bone marrow transduction/transplantation model of CML, DCC-2036 at 100 mg/kg/d also reduced peripheral blood leukocyte counts and significantly prolonged survival vs. vehicle-treated control mice. These results demonstrate that ABL switch pocket inhibitors are a promising new pharmacologic weapon for the treatment of de novo and drug-resistant CML, including BCR-ABL T315I.