Differentially expressed genes with AZD5153 treatment

Supplementary Information and Figures 1-3 from Identification of Candidate Molecular Markers Predicting Sensitivity in Solid Tumors to Dasatinib: Rationale for Patient Selection

Abstract Purpose: Aberrant activation of EGFR is a hallmark of glioblastoma. However, EGFR inhibitors exhibit at best modest efficacy in glioblastoma. This is in sharp contrast with the observations in EGFR-mutant lung cancer. We examined whether activation of functionally redundant receptor tyrosine kinases (RTKs) conferred resistance to EGFR inhibitors in glioblastoma. Experimental Design: We collected a panel of patient-derived glioblastoma xenograft (PDX) lines that maintained expression of wild-type or mutant EGFR in serial xenotransplantation and tissue cultures. Using this physiologically relevant platform, we tested the abilities of several RTK ligands to protect glioblastoma cells against an EGFR inhibitor, gefitinib. Based on the screening results, we further developed a combination therapy cotargeting EGFR and insulin receptor (InsR)/insulin-like growth factor 1 receptor (IGF1R). Results: Insulin and IGF1 induced significant protection against gefitinib in the majority of EGFR-dependent PDX lines with one exception that did not express InsR or IGF1R. Blockade of the InsR/IGF1R pathway synergistically improved sensitivity to gefitinib or dacomitinib. Gefitinib alone effectively attenuated EGFR activities and the downstream MEK/ERK pathway. However, repression of AKT and induction of apoptosis required concurrent inhibition of both EGFR and InsR/IGF1R. A combination of gefitinib and OSI-906, a dual InsR/IGF1R inhibitor, was more effective than either agent alone to treat subcutaneous glioblastoma xenograft tumors. Conclusions: Our results suggest that activation of the InsR/IGF1R pathway confers resistance to EGFR inhibitors in EGFR-dependent glioblastoma through AKT regulation. Concurrent blockade of these two pathways holds promise to treat EGFR-dependent glioblastoma. Clin Cancer Res; 22(7); 1767–76. ©2015 AACR.

Cell line authentication information

Protein modulation by AZD5153 in hematological cell lines

Supplementary Figures 1-8 from The Mechanisms of Differential Sensitivity to an Insulin-like Growth Factor-1 Receptor Inhibitor (BMS-536924) and Rationale for Combining with EGFR/HER2 Inhibitors

Supplemental Table 1: Treatment-related AEs experienced by {greater than or equal to}10% of patients on any schedule, and treatmentrelated grade {greater than or equal to}3 AEs reported in at least 1 patient; Supplemental Table 2: Summary of hepatotoxicities reported with pevonedistat on each dosing schedule and overa ll

Supplementary Figure 1. AZD5153 shows reduced BRD4 binding activity when BD2 function is abolished; Supplementary Figure 2. AZD5153 modulates MYC protein levels across hematologic cancer cell lines; Supplementary Figure 3. Gene Ontology (GO) analysis of the down-regulated transcripts by AZD5153 across all cell line types; Supplementary Figure 4. IC50 and percent maximal cell kill by AZD5153 and I-BET762 in five hematologic cell lines; Supplementary Figure 5. Tumor growth inhibition across six hematological xenograft Models

Supplementary Table 1. EGFR and PTEN mutational status in glioblastoma samples; Supplementary Figure 1. Expression of EGFR in glioblastoma cells cultured in serum-free medium; Supplemental Figure 2. Insulin, IGF1, and PDGF-AB confers resistance to gefitinib; Supplemental Figure 3. Inhibition of InsR/IGF1R improves response to EGFR inhibitors in Glioblastoma; Supplemental Figure 4. (A) GBM46 cells were starved overnight and stimulated with100 ng/ml insulin, IGF1 or EGF for 30 minutes in the presence of absence of 1 ô€ˆM gefitinib. (B) GBM12 and (C) GBM46 were treated with gefitinib {plus minus} OSI-906 for 24 hours and collected for immunoblotting; Supplemental Figure 5. (A) GBM12 GBM39 were infected with control lentivirus or lentivirus directing expression of Myr-AKT1 and selected with puromycin for 2 days; Supplemental Figure 6. Inhibition of the PI3K/AKT pathway sensitizes glioblastoma cells to EGFR Inhibitors; Supplemental Figure 7. (A) PC9 cells were treated with gefitinib {plus minus} OSI-906 and cellular response was determined as described in Fig. 2A; Supplemental Figure 8. (A) Animals bearing GBM76 and (B) GBM39 subcutaneous tumors were treated as described in Fig. 4 and methods. Median animal weights throughout the course of treatment were presented.

Abstract We discuss the integer sequence transform a 1→ b, where bn is the number of real roots of the polynomial a 0 + a 1 x + a 2 x 2 + · · · + anxn . It is shown that several sequences a give the trivial sequence b = (0, 1, 0, 1, 0, 1, . . .), i.e., bn = n mod 2, among them the Catalan numbers, central binomial coefficients, n! and for a fixed k. We also look at some sequences a for which b is more interesting such as an = (n + 1) k for k ≥ 3. Further, general procedures are given for constructing real sequences an for which bn is either always maximal or minimal.

3085 Background: BRD4 is a bromodomain and extraterminal (BET) protein that regulates oncogenic programs by modifying gene transcription and additional mechanisms. AZD5153 is a novel, reversible BRD4 inhibitor with bivalent mechanism of action and enhanced antitumor activity in preclinical models. This phase 1, multicenter, dose escalation study (NCT03205176) assesses AZD5153’s safety, pharmacokinetics (PK), and pharmacodynamics (PD). We report here preliminary, unvalidated data from AZD5153 monotherapy in pts with RR solid tumor, including lymphoma. Methods: Adult pts received oral AZD5153 QD/BID to determine the MTD. During dose escalation, a continual reassessment model was used to estimate toxicity and all final decisions were made by the Safety Review Committee. PK and PD were characterized using standard methods. Results: As of 1 Nov 2018, 28 pts (78.6% female, median age 66.5 y) were treated in 7 cohorts: 2 mg QD (3 pts), 5 mg QD (3 pts), 10 mg QD (3 pts), 10 mg BID (5 pts), 15 mg BID (4 pts), 20 mg BID (7 pts), and 30 mg QD (3 pts). Treatment was ongoing in 8 pts at data cut-off. Safety findings showed 50% of pts experienced treatment-related AEs. 25% of pts experienced treatment-related Grade ≥3 AEs, which were thrombocytopenia and fatigue (7.1% each), and anemia, diarrhea, and platelet count decreased (3.6% each). SAEs were observed in 25% of pts; none of the SAEs was attributable to AZD5153 alone. Dose-limiting toxicities of thrombocytopenia (1 pt) and diarrhea with herpetic rash leading to discontinuation (1 pt) occurred at 20 mg BID. 53.6% of pts discontinued due to disease progression. Total median treatment duration was 1.3 mo (range up to 8.9 mos). Dose proportional increase in Cmax and AUC were observed across the dose range tested. Tmax ranged from 0.5 to 3 h and t1/2 was 6 h. Dose-dependent changes in expression of target genes (eg, HEXIM1, HIST2H2BF, CD274, and CCR2) and platelet counts were observed in the peripheral blood. Conclusions: AZD5153 monotherapy is safe and tolerated at doses up to 30 mg QD and 15 mg BID. Linear increase in PK was observed. Additional safety and efficacy updates will be reported at the annual meeting. Clinical trial information: NCT03205176.

Acute myeloid leukemia (AML) is an aggressive, heterogeneous malignancy. AML patients whose disease relapses on chemotherapy or are unfit for aggressive induction regimens have limited therapeutic options. Many patients benefit from the combination of venetoclax (BCL2i) and a hypomethylating agent (HMA) but this regimen is rarely curative. The addition of novel agents could provide improved benefit for relapsed/refractory patients. To identify such regimens, we screened a panel of 10 AML cell lines with combinations of venetoclax and novel targeted agents. The agents used spanned multiple mechanisms of action (e.g. DNA damage response, kinase signaling, pro-apoptotic agents) and are all in early clinical development. Cells were treated for 72hrs and viability was assessed by CellTiter-Glo. In several of the cell lines that were insensitive or partially sensitive to venetoclax (OCI-AML3, KG1a, MonoMac6, THP1), combinations with inhibitors of MCL1 (AZD5991), AURKB (AZD2811), and BRD4 (AZD5153) showed synergistic activity (Loewe synergy score >5, growth inhibition > 180%) (Table 1). We next asked if these combinations were active in patient-derived xenograft (PDX) models of AML. We established an ex vivo co-culture assay using the HS-5 bone marrow stromal cell line. AML PDX cells were isolated from mouse spleens and plated in 96-well format in direct co-culture with HS-5 cells or in HS-5-derived conditioned media. Cells were treated with three doses of each monotherapy and three doses of fixed ratio combination. Replicate screens using cells from individual mice on different days confirmed data were reproducible (r2=0.687) across animals engrafted with the same PDX. Drug response was similar between conditioned media and direct co-culture assays (r2=0.81). Venetoclax sensitivity varied across PDX models ex vivo. Notably, 2/5 PDX models screened (DFAM-68555 and DFAM-10360) were insensitive to both venetoclax and the combination of venetoclax + 5-azacytidine (HMA) ex vivo. Both models were established from untreated/1L patients and harbor TP53 mutations. Combination treatments did not add additional benefit over venetoclax monotherapy in the DFAM-10360 model. However, in DFAM-68555, AZD5153, AZD5991, and AZD2811 showed improved activity over venetoclax alone (67%, 54%, and 67% vs. 26% decrease in viability for venetoclax alone, respectively). Since combination strategies will likely be most impactful in patients refractory to or relapsed after venetoclax, we chose this venetoclax insensitive model to prioritize in vivo. To confirm the translatability of these findings, we designed a pilot in vivo study using DFAM-68555. Mice were randomized to receive vehicle, venetoclax + HMA, or venetoclax + AZD5153 when peripheral blood disease reached ~5% (hCD45+hCD33+ cells by flow cytometry). After two weeks of dosing, animals were sacrificed to evaluate disease burden in bone marrow (sternum), spleen, and peripheral blood. The model remained insensitive to venetoclax + HMA in vivo. The combination of AZD5153 with venetoclax decreased disease burden in blood and spleen compared to vehicle (30% and 42% hCD45+CD33+ cells by flow cytometry vs 70% and 95%, respectively) with similar efficacy seen by immunohistochemistry in the bone. Finally, we screened these venetoclax combinations in additional aggressive AML PDX models which were resistant or only partially responsive to venetoclax in vivo. Addition of AZD2811NP and AZD5991 to venetoclax was more effective than venetoclax alone and venetoclax + HMA in the bone marrow. The most active combination varied from model to model. Efficacy screening in additional models is ongoing to further build ex vivo to in vivo translation and prioritize development of specific combinations. Also ongoing is genomic and transcriptomic profiling of these PDXs to identify potential predictive biomarkers of combination activity. In summary, we developed an ex vivo screening platform to test clinically actionable combinations for activity in clinically relevant models. Using this platform and subsequent in vivo efficacy, we identified venetoclax combinations across multiple mechanisms (pro-apoptotic, cell cycle regulation, transcriptional regulation, DNA damage response) with activity in venetoclax-insensitive models. These results suggest potential therapeutic options to explore clinically for AML patients. Disclosures Andersen: AstraZeneca: Employment. Christie:AstraZeneca: Employment. Rosen:Astrazeneca: Employment. Maratea:AstraZeneca: Employment. Hattersley:AstraZeneca: Employment. Travers:AstraZeneca: Employment. Cidado:AstraZeneca: Employment. Pulukuri:AstraZeneca: Employment. Saeh:AstraZeneca: Employment. Clark:AstraZeneca: Employment, Equity Ownership. Reimer:AstraZeneca: Employment. Mettetal:AstraZeneca: Employment.

Aggressive B-cell lymphomas are genetically and clinically heterogeneous. Standard clinical trial designs do not efficiently evaluate the safety and efficacy of multiple drug combinations within the context of underlying molecular biology. The rapid identification of oncogenic driver pathways and development of multiple targeted drugs in lymphomas create many potential combinations. To address this need, we developed a Phase 1 master protocol termed PRISM (NCT03527147) to evaluate multiple targeted therapies alone or in combination for the treatment of relapsed/refractory (R/R) aggressive B-cell lymphoma. Each study arm is conducted in a predefined disease subset with the aim of addressing clinical and translational questions within an overarching protocol. All study arms are open label and not randomized. Enrolment of subjects into a given study arm is based on meeting inclusion/exclusion criteria and available slots. Pertinent inclusion criteria for the master protocol are: (a) a diagnosis of R/R non-Hodgkin lymphoma based on established World Health Organization criteria; (b) ≥1 prior line of therapy for the treatment of current histology, no known curative treatment options available, or the subject is ineligible for potential curative options; (c) the presence of radiographically measurable lymphadenopathy or extranodal lymphoid malignancy and; (d) an ECOG performance status ≤2. Exclusion criteria for the master protocol include: (a) a history of prior malignancy, severe or uncontrolled disease or conditions; (b) use of anti-lymphoma therapy within 14 days of the first dose of study drug and; (c) a requirement for ongoing immunosuppressive therapy. Treatment-specific inclusion/exclusion criteria are also provided (see www.clinicaltrials.gov). As PRISM has multiple study arms, subjects can be simultaneously screened for multiple arms. In each arm, a safety review for dose-limiting toxicity (DLT) is performed after 6 subjects have completed the protocol-defined DLT window. Further enrolment will only proceed in that arm if ≤1 subject experiences a DLT (Figure 1). The sample size for each respective arm is determined based on prior clinical/experimental data on anticipated/clinically meaningful activity of each drug combination. This determines a minimally acceptable response and a desirable response. For each arm, a futility analysis occurs after approximately 10 sequentially enrolled subjects. An arm is considered futile if there is <10% probability for the overall response rate (ORR) to be above the desirable response. A final analysis after approximately 21 enrolled subjects will determine whether the treatment should be studied further. The primary criterion for success is set as having >80% chance for the response rate to be above the minimally acceptable response. The study endpoints include safety, ORR, duration of response, progression-free survival, overall survival, and standard pharmacokinetic parameters. Exploratory analyses include in depth translational studies employing peripheral blood and tumor tissue collected at screening and during treatment. These investigations aim to discover predictive biomarkers, identify the molecular correlates of response based on known genetic subtypes, investigate pharmacodynamic and pathway changes and define the depth of response using assays for measurable residual disease (MRD). Exploratory translational endpoints may inform additional biomarker selection strategies for future arms of the PRISM study. All study arms within PRISM to date have combined acalabrutinib, a highly selective BTK inhibitor, with additional targeted agents in subjects with R/R diffuse large B-cell lymphoma. The mechanism of action of the drugs combined with acalabrutinib are as follows: 1. AZD9150 is a 16-nucleotide antisense oligonucleotide designed to target and down-regulate expression of human STAT3 mRNA; administered intravenously. 2. AZD6738 is an inhibitor of ATR; administered orally. 3. Hu5F9-G4 is an anti-CD47 antibody and rituximab is an anti-CD20 antibody; both administered intravenously. 4. AZD5153 is a BRD4 inhibitor; administered orally. In summary, PRISM is a unique platform protocol designed to efficiently evaluate targeted agents in R/R aggressive B-cell lymphoma with an emphasis on comprehensive translational and molecular investigations. Disclosures Izumi: AstraZeneca: Equity Ownership; Acerta Pharma: Employment, Equity Ownership, Patents & Royalties: Acalabrutinib patents. Hamdy:AstraZeneca: Equity Ownership; Acerta Pharma: Employment, Equity Ownership, Patents & Royalties: Acalabrutinib patents. Arkenau:Acerta Pharma: Research Funding. de Vos:Portola Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy; Verastem: Consultancy. Reagan:Kite, A Gilead Company: Consultancy; Curis: Consultancy; Seattle Genetics: Research Funding. Zinzani:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy; Verastem: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Eusapharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Portola: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Servier: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sandoz: Membership on an entity's Board of Directors or advisory committees; Immune Design: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Therapeutics: Honoraria, Speakers Bureau. Davies:BioInvent: Research Funding; ADCT Therapeutics: Honoraria, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Research Funding; Karyopharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Research Funding; Pfizer: Honoraria, Research Funding; Acerta Pharma: Honoraria, Research Funding; MorphoSys AG: Honoraria, Membership on an entity's Board of Directors or advisory committees. Pagel:AstraZeneca: Consultancy; Pharmacyclics, Inc.: Consultancy. Vose:Legend Pharmaceuticals: Honoraria; Acerta Pharma: Honoraria, Other: Grants, Research Funding; Bristol-Meyers Squibb Company: Research Funding; Celgene Corporation: Research Funding; Incyte Corporation: Research Funding; Kite Pharma: Honoraria, Other: Grants, Research Funding; Novartis: Research Funding; Seattle Genetics: Research Funding; AbbVie: Consultancy, Honoraria; Epizyme: Consultancy, Honoraria. Bitman:Acerta Pharma: Employment; AstraZeneca: Equity Ownership. Brock:Acerta Pharma: Employment; AstraZeneca: Equity Ownership. Clark:AstraZeneca: Employment, Equity Ownership. Frigault:Acerta Pharma: Employment; AstraZeneca: Employment, Equity Ownership. Ware:Acerta Pharma: Employment; Astrazeneca: Employment, Equity Ownership. Yang:Acerta Pharma: Employment; AstraZeneca: Equity Ownership. Staudt:Nanostring: Patents & Royalties. Flinn:TG Therapeutics, Trillum Therapeutics, Abbvie, ArQule, BeiGene, Curis, FORMA Therapeutics, Forty Seven, Merck, Pfizer, Takeda, Teva, Verastem, Gilead Sciences, Astra Zeneca (AZ), Juno Therapeutics, UnumTherapeutics, MorphoSys, AG: Research Funding; Acerta Pharma, Agios, Calithera Biosciences, Celgene, Constellation Pharmaceuticals, Genentech, Gilead Sciences, Incyte, Infinity Pharmaceuticals, Janssen, Karyopharm Therapeutics, Kite Pharma, Novartis, Pharmacyclics, Portola Pharmaceuticals: Research Funding; AbbVie, Seattle Genetics, TG Therapeutics, Verastem: Consultancy; TG Therapeutics, Trillum Therapeutics, Abbvie, ArQule, BeiGene, Curis, FORMA Therapeutics, Forty Seven, Merck, Pfizer, Takeda, Teva, Verastem, Gilead Sciences, Astra Zeneca (AZ), Juno Therapeutics, UnumTherapeutics, MorphoSys, AG: Research Funding; F. Hoffmann-La Roche Ltd: Research Funding. OffLabel Disclosure: acalabrutinib in DLBCL

BRD4 is a transcriptional co-activator functioning to recruit regulatory complexes to acetylated chromatin. A subset of High-grade Serous Ovarian Cancer (HGSOC) patients are typified by focal, recurrent BRD4 gene amplifications. Despite previously described cancer dependencies, it is unclear whether BRD4 amplification events are oncogenic in HGSOC. We find that physiologically relevant levels of expression of BRD4 isoforms in non-transformed ovarian cells result in cellular transformation. Transcriptional profiling of BRD4-transformed ovarian cells, and BRD4-amplified HGSOC patient samples revealed shared expression patterns, including enriched MYC, and E2F1 gene signatures. Furthermore, we demonstrate that a novel BET inhibitor, AZD5153, is highly active in BRD4-amplified patient derived xenografts and uncover Neuregulin-1 as a novel BRD4 effector. Experiments involving Neuregulin-1 inhibition and exogenous addition, demonstrate Neuregulin-1 as necessary and sufficient for BRD4-mediated transformation. This study demonstrates the oncogenic potential of BRD4 amplification in cancer and establishes BRD4-amplified HGSOC as a potential patient population that could benefit from BET inhibitors.

Mcl-1, a member of the Bcl/Mcl family, is a key protein involved in evasion of apoptosis in a wide variety of tumors. Its amplification and overexpression have also been implicated in innate and acquired resistance to anticancer drugs. Mcl-1 is capable of preventing induction of apoptosis, both by binding and inactivating the pro-apoptotic executioner Bcl-2 protein, Bak, as well as by sequestering other pro-apoptotic BH3-only proteins such as Bim and Noxa. AZD5991 is a rationally designed macrocycle with sub-nanomolar affinity for Mcl-1. It demonstrates all the hallmarks of a true Mcl-1 inhibitor: 1. potent, selective, and rapid apoptosis in Mcl-1-dependent cell lines (e.g., GI 50 as low as 10 nM in multiple myeloma cell lines); 2. loss of activity upon overexpression of Bcl-xL or siRNA-mediated knockout of Bak; 3. Mcl-1:Bak complex disruption as demonstrated by co-immunoprecipitation. AZD5991 is active in vivo, with complete (100%) tumor regression demonstrated in several mouse xenograft models after a single tolerated dose. We have also demonstrated synergistic in vivo efficacy in combination with standard-of-care agents. Analysis of ex vivo activity in primary samples from leukemia patients indicates that a high percentage of leukemia patients should respond to drug treatment, which supports our plan for a phase I trial of AZD5991 in patients with hematologic cancers. Citation Format: Alexander W. Hird, J. Paul Secrist, Ammar Adam, Matthew A. Belmonte, Eric Gangl, Frank Gibbons, David Hargreaves, Jeffrey W. Johannes, Stephen L. Kazmirski, Jason G. Kettle, Stephen E. Kurtz, Michelle L. Lamb, Martin J. Packer, Bo Peng, Craig R. Stewart, Jeffrey W. Tyner, Wenzhan Yang, Qing Ye, XiaoLan Zheng, Edwin A. Clark. AZD5991: A potent and selective macrocyclic inhibitor of Mcl-1 for treatment of hematologic cancers [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 DDT01-02. doi:10.1158/1538-7445.AM2017-DDT01-02

Abstract The bromodomain and extraterminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Pharmacological targeting of BRD4 bromodomains by small molecule inhibitors has proven to be an effective means to disrupt aberrant transcriptional programs critical for tumor growth and/or survival. Herein, we report AZD5153, a potent, selective, and orally available BET/BRD4 bromodomain inhibitor possessing a bivalent binding mode. Unlike previously described monovalent inhibitors, AZD5153 ligates two bromodomains in BRD4 simultaneously. The enhanced avidity afforded through bivalent binding translates into increased cellular and antitumor activity in preclinical hematologic tumor models. In vivo administration of AZD5153 led to tumor stasis or regression in multiple xenograft models of acute myeloid leukemia, multiple myeloma, and diffuse large B-cell lymphoma. The relationship between AZD5153 exposure and efficacy suggests that prolonged BRD4 target coverage is a primary efficacy driver. AZD5153 treatment markedly affects transcriptional programs of MYC, E2F, and mTOR. Of note, mTOR pathway modulation is associated with cell line sensitivity to AZD5153. Transcriptional modulation of MYC and HEXIM1 was confirmed in AZD5153-treated human whole blood, thus supporting their use as clinical pharmacodynamic biomarkers. This study establishes AZD5153 as a highly potent, orally available BET/BRD4 inhibitor and provides a rationale for clinical development in hematologic malignancies. Mol Cancer Ther; 15(11); 2563–74. ©2016 AACR.

Proteins of the bromodomain and extraterminal (BET) family, in particular bromodomain-containing protein 4 (BRD4), are of great interest as biological targets. BET proteins contain two separate bromodomains, and existing inhibitors bind to them monovalently. Here we describe the discovery and characterization of probe compound biBET, capable of engaging both bromodomains simultaneously in a bivalent, in cis binding mode. The evidence provided here was obtained in a variety of biophysical and cellular experiments. The bivalent binding results in very high cellular potency for BRD4 binding and pharmacological responses such as disruption of BRD4-mediator complex subunit 1 foci with an EC50 of 100 pM. These compounds will be of considerable utility as BET/BRD4 chemical probes. This work illustrates a novel concept in ligand design-simultaneous targeting of two separate domains with a drug-like small molecule-providing precedent for a potentially more effective paradigm for developing ligands for other multi-domain proteins.

Lung cancer is the most common cause of cancer death globally with a significant, unmet need for more efficacious treatments. The receptor tyrosine kinase MET has been implicated as an oncogene in numerous cancer subtypes, including non-small cell lung cancer (NSCLC). Here we explore the therapeutic potential of savolitinib (volitinib, AZD6094, HMPL-504), a potent and selective MET inhibitor, in NSCLC. In vitro, savolitinib inhibits MET phosphorylation with nanomolar potency, which correlates with blockade of PI3K/AKT and MAPK signaling as well as MYC down-regulation. In vivo, savolitinib causes inhibition of these pathways and significantly decreases growth of MET-dependent xenografts. To understand resistance mechanisms, we generated savolitinib resistance in MET-amplified NSCLC cell lines and analyzed individual clones. We found that upregulation of MYC and constitutive mTOR pathway activation is a conserved feature of resistant clones that can be overcome by knockdown of MYC or dual mTORC1/2 inhibition. Lastly, we demonstrate that mechanisms of resistance are heterogeneous, arising via a switch to EGFR dependence or by a requirement for PIM signaling. This work demonstrates the efficacy of savolitinib in NSCLC and characterizes acquired resistance, identifying both known and novel mechanisms that may inform combination strategies in the clinic.

Abstract Alterations in the MET oncogene occurs across a broad range of tumor indications. Amplification or mutations in MET lead to increased activity of downstream pathways including PI3K and MAPK, eventually resulting in tumor formation. Several small molecule inhibitors are currently in clinical trials, including the selective inhibitor Savolitinib (HMP-504, Volitinib, AZD6094), which shows single digit nanomolar activity in MET-amplified cell lines. Newly emerging data suggest mutations in MET causing complete skipping of Exon 14 occur in approximately 4% of non-small cell lung cancer (NSCLC), and are more rare in other indications [1, 2]. MET exon 14 skipping mutations were shown to be mutually exclusive from EGFRm, ALK and KRAS and can occur in the context of MET gene amplification [3]. Exon 14 harbors the CBL binding site (Y1003), which is critical for receptor degradation after binding of its ligand, HGF, and suppression of downstream signaling events. Clinical trial results with less potent, pan RTK inhibitors Crizotinib (31nM GI50 vs 3nM for Savolitinib) and Cabozantinib show promising early results, but fall short in long term responses. Therefore, better therapies targeting MET are needed. Human cell line models with Exon 14 deletions are rare. Therefore, we used engineered cell lines to test the effect of Savolitinib on these mutations. To do this, we expressed MET-Y1003F mutants in NIH-3T3 and HEK293T cells. We found that Savolitinib potently inhibited phospho-MET in both models expressing this mutant (100% phospho-MET inhibition). In addition, we tested whether or not Savolitinib could inhibit HGF-dependent signaling and growth of a NSCLC cell line, NCI-H596. In the presence of FBS (10%), Savolitinib had no effect on the growth rate of these cells, however was highly efficient at blocking HGF-dependent growth in the absence of FBS. To test the effect of this mutation in the background of amplification, we also tested the gastric cancer cell line Hs746T, which harbors exon 14 skipping in addition to MET amplification. Savolitinib was highly efficacious at blocking the growth of this cell line. Future studies are aimed at looking at the in vivo effect of Savolitinib targeting exon 14 mutants. These data provide a platform of evidence for using Savolitinib to target exon 14 mutant MET in patients. 1. Paik, P.K., et al., Response to MET inhibitors in patients with stage IV lung adenocarcinomas harboring MET mutations causing exon 14 skipping. Cancer Discov, 2015. 5(8): p. 842-9. 2. Frampton, G.M., et al., Activation of MET via diverse exon 14 splicing alterations occurs in multiple tumor types and confers clinical sensitivity to MET inhibitors. Cancer Discov, 2015. 5(8): p. 850-9. 3. Cancer Genome Atlas Research, N., Comprehensive molecular profiling of lung adenocarcinoma. Nature, 2014. 511(7511): p. 543-50. Citation Format: Evan Barry, Elizabeth Maloney, Ryan Henry, Alexandra Borodovsky, Edwin Clark, Melanie Frigault, Michael Zinda, Celina D’Cruz. Targeting MET Exon 14 mutations with the selective small molecule inhibitor Savolitinib. [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 1150.

Here we report the discovery and optimization of a series of bivalent bromodomain and extraterminal inhibitors. Starting with the observation of BRD4 activity of compounds from a previous program, the compounds were optimized for BRD4 potency and physical properties. The optimized compound from this campaign exhibited excellent pharmacokinetic profile and exhibited high potency in vitro and in vivo effecting c-Myc downregulation and tumor growth inhibition in xenograft studies. This compound was selected as the development candidate, AZD5153. The series showed enhanced potency as a result of bivalent binding and a clear correlation between BRD4 activity and cellular potency.