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PDF file - 151KB, Activity of AZD9291 and metabolites against cellular phospho-HER2 assays.

PDF file - 235KB, Activity of AZD9291 and metabolites against rare EGFR mutations using cellular phosphorylation assays.

PDF file - 294KB, Phenotype and phosphorylation assay data for AZD9291 against lung cancer associated HER2 mutation.

PDF file - 107KB, Mass Spectrometry study showing covalent binding of AZD9291 to cysteine 797 in EGFR T790M.

PDF file - 312KB, Efficacy of AZD9291 in H3255, PC-9VanR and A431 xenograft models. Weight loss data for chronically dosed AZD9291 in PC-9 and H1975 xenograft models.

PDF file - 153K, Figure to support screening for antibodies

PDF file - 470KB, Effect of AZD9291 and AZ5104 in transgenic models of EGFR-TKI sensitizing (C/L858R and C/ex19del) and T790M resistant (C/L+T) lung cancer.

PDF file - 98K

PDF file - 182KB, Pharmacokinetic analysis of AZD9291, AZ5104 and AZ7550 total plasma concentrations vs time following single oral dose of 25mg/kg AZD9291 in mice.

The Notch signaling pathway has been implicated in cell fate determination and differentiation in many tissues. Accumulating evidence points toward a pivotal role in blood vessel formation, and the importance of the Delta-like ligand (Dll) 4-Notch1 ligand–receptor interaction has been shown in both physiological and tumor angiogenesis. Disruption of this interaction leads to a reduction in tumor growth as a result of an increase in nonfunctional vasculature leading to poor perfusion of the tumor. MEDI0639 is an investigational human therapeutic antibody that targets Dll4 to inhibit the interaction between Dll4 and Notch1. The antibody cross-reacts to cynomolgus monkey but not mouse species orthologues. In vitro MEDI0639 inhibits the binding of Notch1 to Dll4, interacting via a novel epitope that has not been previously described. Binding to this epitope translates into MEDI0639 reversing Notch1-mediated suppression of human umbilical vein endothelial cell growth in vitro. MEDI0639 administration resulted in stimulation of tubule formation in a three-dimensional (3D) endothelial cell outgrowth assay, a phenotype driven by disruption of the Dll4-Notch signaling axis. In contrast, in a two-dimensional endothelial cell–fibroblast coculture model, MEDI0639 is a potent inhibitor of tubule formation. In vivo, MEDI0639 shows activity in a human endothelial cell angiogenesis assay promoting human vessel formation and reducing the number of vessels with smooth muscle actin-positive mural cells coverage. Collectively, the data show that MEDI0639 is a potent modulator of Dll4-Notch signaling pathway. Mol Cancer Ther; 11(8); 1650–60. ©2012 AACR.

PDF file - 89KB, Additional biochemical and cellular profiling data for AZD9291 and metabolites.

The PI3K pathway is commonly activated in breast cancer, with PI3K-AKT pathway inhibitors used clinically. However, mechanisms that limit or enhance the therapeutic effects of PI3K-AKT inhibitors are poorly understood at a genome-wide level. Parallel CRISPR screens in 3 PTEN-null breast cancer cell lines identified genes mediating resistance to capivasertib (AKT inhibitor) and AZD8186 (PI3Kβ inhibitor). The dominant mechanism causing resistance is reactivated PI3K-AKT-mTOR signalling, but not other canonical signalling pathways. Deletion of TSC1/2 conferred resistance to PI3Kβi and AKTi through mTORC1. However, deletion of PIK3R2 and INPPL1 drove specific PI3Kβi resistance through AKT. Conversely deletion of PIK3CA, ERBB2, ERBB3 increased PI3Kβi sensitivity while modulation of RRAGC, LAMTOR1, LAMTOR4 increased AKTi sensitivity. Significantly, we found that Mcl-1 loss enhanced response through rapid apoptosis induction with AKTi and PI3Kβi in both sensitive and drug resistant TSC1/2 null cells. The combination effect was BAK but not BAX dependent. The Mcl-1i + PI3Kβ/AKTi combination was effective across a panel of breast cancer cell lines with PIK3CA and PTEN mutations, and delivered increased anti-tumor benefit in vivo. This study demonstrates that different resistance drivers to PI3Kβi and AKTi converge to reactivate PI3K-AKT or mTOR signalling and combined inhibition of Mcl-1 and PI3K-AKT has potential as a treatment strategy for PI3Kβi/AKTi sensitive and resistant breast tumours.

Acute myeloid leukemia (AML) is a heterogeneous malignancy of the bone marrow associated with poor outcomes and limited treatment options available to patients. Recent developments have demonstrated that patient stratification based on disease classification (e.g. somatic mutations) allows for selective treatment regimens and greatly improved outcomes. AML patients can also be stratified based upon the heterogeneous immunophenotype of surface antigen expression on leukemic blasts and stem cells. Here we present data identifying a sub-population of AML patients showing expression of both CD33 and CD7 on their tumour cells, using mass cytometry. This combination of antigens is disease-specific, and is not expressed on healthy haematopoietic cells in patients. We developed a bispecific antibody–drug conjugate (ADC) targeting both CD7 and CD33 and demonstrate that these bispecific ADCs are cytotoxic to AML cells in vitro. Importantly, the anti-CD33/CD7 bispecific ADCs are more selective than single-antigen targeting ADCs and safely discriminate tumour from healthy cells (either myeloid or lymphoid). These anti-CD33/CD7 bispecific ADCs are well tolerated and selectively target AML cells in pre-clinical in vivo studies in mice. This study presents the first proof-of-principle for targeting specific and unique combinations of surface antigens on tumour cells with potential to overcome observed toxicities with current ADCs.

Many chemotherapeutics suffer from poor aqueous solubility and tissue selectivity. Distearoylphosphatidylethanolamine-poly(ethylene glycol) (DSPE-PEG) micelles are a promising formulation strategy for the delivery of hydrophobic anticancer drugs. However, storage and in vivo instability restrict their use. The aim of this study was to prepare mixed micelles, containing a novel polymer, lipoic acid-chitosan-poly(ethylene glycol) (LACPEG), and DSPE-PEG, to overcome these limitations and potentially increase cancer cell internalisation. Drug-loaded micelles were prepared with a model tyrosine kinase inhibitor and characterized for size, surface charge, stability, morphology, drug entrapment efficiency, cell viability (A549 and PC-9 cell lines), in vivo biodistribution, ex vivo tumor accumulation and cellular internalisation. Micelles of size 30-130nm with entrapment efficiencies of 46-81% were prepared. LACPEG/DSPE-PEG mixed micelles showed greater interaction with the drug (condensing to half their size following entrapment), greater stability, and a safer profile in vitro compared to DSPE-PEG micelles. LACPEG/DSPE-PEG and DSPE-PEG micelles had similar entrapment efficiencies and in vivo tumor accumulation levels, but LACPEG/DSPE-PEG micelles showed higher tumor cell internalisation. Collectively, these findings suggest that LACPEG/DSPE-PEG mixed micelles provide a promising platform for tumor delivery of hydrophobic drugs.

// Matthew J. Martin 1, 2 , Cath Eberlein 1 , Molly Taylor 1, 2 , Susan Ashton 1 , David Robinson 1, 2 , Darren Cross 1, 2 1 AstraZeneca Oncology, Innovative Medicines, Alderley Park, Macclesfield, Cheshire, United Kingdom 2 AstraZeneca Oncology, Innovative Medicines, CRUK Cambridge Institute, Cambridge, United Kingdom Correspondence to: Matthew J. Martin, email: matthew.j.martin@astrazeneca.com Keywords: non-small cell lung cancer, signal transduction, drug resistance, metabolism, pre-clinical models of drug efficacy Received: March 30, 2016 Accepted: October 31, 2016 Published: November 16, 2016 ABSTRACT Metabolic plasticity is an emerging hallmark of cancer, and increased glycolysis is often observed in transformed cells. Small molecule inhibitors that target driver oncogenes can potentially inhibit the glycolytic pathway. Osimertinib (AZD9291) is a novel EGFR tyrosine kinase inhibitor (TKI) that is potent and selective for sensitising (EGFRm) and T790M resistance mutations. Clinical studies have shown osimertinib to be efficacious in patients with EGFRm/ T790M advanced NSCLC who have progressed after EGFR-TKI treatment. However experience with targeted therapies suggests that acquired resistance may emerge. Thus there is a need to characterize resistance mechanisms and to devise ways to prevent, delay or overcome osimertinib resistance. We show here that osimertinib suppresses glycolysis in parental EGFR-mutant lung adenocarcinoma lines, but has not in osimertinib-resistant cell lines. Critically, we show osimertinib treatment induces a strict dependence on mitochondrial oxidative phosphorylation (OxPhos), as OxPhos inhibitors significantly delay the long-term development of osimertinib resistance in osimertinib-sensitive lines. Accordingly, growth conditions which promote a less glycolytic phenotype confer a degree of osimertinib resistance. Our data support a model in which the combination of osimertinib and OxPhos inhibitors can delay or prevent resistance in osimertinib-naive tumour cells, and represents a novel strategy that warrants further pre-clinical investigation.

A novel series of covalent inhibitors of EGFR (epidermal growth factor receptor) kinase was discovered through a combination of subset screening and structure-based design. These compounds preferentially inhibit mutant forms of EGFR (activating mutant and T790M mutant) over wild-type EGFR in cellular assays measuring EGFR autophosphorylation and proliferation, suggesting an improved therapeutic index in non-small cell lung cancer patients would be achievable relative to established EGFR inhibitors. We describe our design approaches, resulting in the identification of the lead compound 5, and our efforts to develop an understanding of the structure-activity relationships within this series. In addition, strategies to overcome challenges around metabolic stability and aqueous solubility are discussed. Despite limitations in its physical properties, 5 is orally bioavailable in mice and demonstrates pronounced antitumor activity in in vivo models of mutant EGFR-driven cancers.

Phenotyping of AML patient samples shows that CD33, a myeloid cell marker, and CD7, a lymphoid cell marker, are specifically co-expressed in 25% of AML cases. Development of a bispecific antibody drug conjugate (ADC) dependent on co-expression of CD7 and CD33 may lead to an ADC with better tumor-cell selectivity than the anti-CD33 ADC, gemtuzumab ozogamicin. Anti-CD7 and -CD33 Fabs were expressed in HEK293 cells, and affinities assessed using surface plasmon resonance. Chemically cross-linked anti-CD7, anti-CD33 Bi-Fabs were produced using click chemistry via an individual cysteine residue in each CH1 domain. Bi-Fabs were conjugated to valine-citrulline-monomethylauristatin E (vcMMAE) and maleimidocaproyl monomethylauristatin F (mcMMAF) at native cysteine residues. Bi-Fab drug conjugates were assessed for cytotoxicity against HNT-34 and Kasumi-3 cells (CD7+CD33+), and assessed for selectivity against primary human T cells and myeloid progenitor cells, using monospecific Bi-Fab and gemtuzumab drug conjugates with equivalent drug-antibody ratios as controls. The activity of the lead Bi-Fab, BVX130-mcMMAF, was assessed in a subcutaneous (s.c.) xenograft model in SCID mice implanted with HNT-34 cells. Animals (n=10/group) received BVX-130-mcMMAF (10mg/kg twice weekly i.v. for 4 weeks), cytarabine at its maximum tolerated dose (20mg/kg, 5 days on 2 days off i.p. for 3 weeks), or vehicle control for 4 weeks. The wild-type Fabs had Kd values of 3.6nM (anti-CD7 Fab) and 1.6 nM (anti-CD33 Fab) for their respective antigens. The resulting anti-CD7, anti-CD33 Bi-Fab MMAE conjugate (BVX100-vcMMAE) was highly cytotoxic to HNT-34 and Kasumi-3 cells with IC50 values of 0.20±0.04nM and 0.20±0.03nM respectively, compared to IC50 values of 1.3nM and 1.2nM for gemtuzumab MMAE. BVX100-vcMMAE had 5-fold lower cytotoxicity towards Jurkat cells (CD7+CD33−) and >10-fold margins relative to MV4.11 and SHI-1 (CD7−CD33+) cells whereas corresponding homo-dimeric Bi-Fabs were non-selective. Affinity reduction of the CD7 Fab resulted in Bi-Fab drug conjugates with similar activity and improved selectivity. One such Bi-Fab, BVX130mcMMAF, had no measurable cytotoxicity to resting T-cells, and did not inhibit colony formation in a GM-CFU assay, at concentrations up to 10nM. Treatment of mice carrying s.c. HNT-34 tumors with BVX130-mcMMAF resulted in Tumor Growth Inhibition (TGI) values of 100% (98% regression, p Anti-CD7, anti-CD33 Bi-Fab drug conjugates are highly cytotoxic to CD7+CD33+ cells in vitro and in vivo, and have substantially greater selectivity than monospecific drug conjugates for such cells, supporting the development of bispecific antibody drug conjugates targeting CD7 and CD33 for the treatment of CD7+/CD33+ AML. Citation Format: Richard Bethell, Cath Eberlein, Victoria Pollard, Theonie Georgiou, George Orphanides, Lorraine Mooney, Jane Kendrew, Tiffany Daniels. Bispecific antibody drug conjugates targeting CD7 and CD33 for the treatment of acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2887.

First-generation EGFR tyrosine kinase inhibitors (EGFR TKI) provide significant clinical benefit in patients with advanced EGFR-mutant (EGFRm+) non–small cell lung cancer (NSCLC). Patients ultimately develop disease progression, often driven by acquisition of a second T790M EGFR TKI resistance mutation. AZD9291 is a novel oral, potent, and selective third-generation irreversible inhibitor of both EGFRm+ sensitizing and T790M resistance mutants that spares wild-type EGFR. This mono-anilino–pyrimidine compound is structurally distinct from other third-generation EGFR TKIs and offers a pharmacologically differentiated profile from earlier generation EGFR TKIs. Preclinically, the drug potently inhibits signaling pathways and cellular growth in both EGFRm+ and EGFRm+/T790M+ mutant cell lines in vitro, with lower activity against wild-type EGFR lines, translating into profound and sustained tumor regression in EGFR-mutant tumor xenograft and transgenic models. The treatment of 2 patients with advanced EGFRm+ T790M+ NSCLC is described as proof of principle. Significance: We report the development of a novel structurally distinct third-generation EGFR TKI, AZD9291, that irreversibly and selectively targets both sensitizing and resistant T790M+ mutant EGFR while harboring less activity toward wild-type EGFR. AZD9291 is showing promising responses in a phase I trial even at the first-dose level, with first published clinical proof-of-principle validation being presented. Cancer Discov; 4(9); 1046–61. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 973

A novel series of small-molecule inhibitors has been developed to target the double mutant form of the epidermal growth factor receptor (EGFR) tyrosine kinase, which is resistant to treatment with gefitinib and erlotinib. Our reported compounds also show selectivity over wild-type EGFR. Guided by molecular modeling, this series was evolved to target a cysteine residue in the ATP binding site via covalent bond formation and demonstrates high levels of activity in cellular models of the double mutant form of EGFR. In addition, these compounds show significant activity against the activating mutations, which gefitinib and erlotinib target and inhibition of which gives rise to their observed clinical efficacy. A glutathione (GSH)-based assay was used to measure thiol reactivity toward the electrophilic functionality of the inhibitor series, enabling both the identification of a suitable reactivity window for their potency and the development of a reactivity quantitative structure-property relationship (QSPR) to support design.

Inhibition of VEGFR-2 signaling reduces angiogenesis and retards tumor growth. Current biotherapeutics that inhibit VEGFR-2 signaling by either sequestering VEGF ligand or inhibiting VEGF binding to VEGFR-2 may be compromised by high VEGF concentrations. Here we describe a biotherapeutic that targets VEGFR-2 signaling by binding to Ig domains 4-7 of VEGFR-2 and therefore has the potential to work independently of ligand concentration. 33C3, a fully human VEGFR-2 antibody, was generated using XenoMouse technology. To elucidate the mechanism of action of 33C3, we have used a number of competition and binding assays. We show that 33C3 binds VEGFR-2 Ig domains 4-7, has no impact on VEGF-A binding to VEGFR-2, and does not compete with an antibody that interacts at the ligand binding site. 33C3 has a high affinity for VEGFR-2 (KD < 1 nmol/L) and inhibits VEGF-A induced phosphorylation of VEGFR-2 with an IC50 of 99 ± 3 ng/mL. In vitro, in a 2D angiogenesis assay, 33C3 potently inhibits both tube length and number of branch points, and endothelial tubule formation in a 3D assay. In vivo, 33C3 is a very effective inhibitor of angiogenesis in both a human endothelial angiogenesis assay and in a human skin chimera model. These data show targeting VEGFR-2 outside of the ligand binding domain results in potent inhibition of VEGFR-2 signaling and inhibition of angiogenesis in vitro and in vivo. Mol Cancer Ther; 10(5); 770–83. ©2011 AACR.

Off-target pharmacology may contribute to both adverse and beneficial effects of a new drug. In vitro pharmacological profiling is often applied early in drug discovery; there are fewer reports addressing the relevance of broad profiles to clinical adverse effects. Here, we have characterized the pharmacological profile of the active metabolite of fostamatinib, R406, linking an understanding of drug selectivity to the increase in blood pressure observed in clinical studies. R406 was profiled in a broad range of in vitro assays to generate a comprehensive pharmacological profile and key targets were further investigated using functional and cellular assay systems. A combination of traditional literature searches and text-mining approaches established potential mechanistic links between the profile of R406 and clinical side effects. R406 was selective outside the kinase domain, with only antagonist activity at the adenosine A3 receptor in the range relevant to clinical effects. R406 was less selective in the kinase domain, having activity at many protein kinases at therapeutically relevant concentrations when tested in multiple in vitro systems. Systematic literature analyses identified KDR as the probable target underlying the blood pressure increase observed in patients. While the in vitro pharmacological profile of R406 suggests a lack of selectivity among kinases, a combination of classical searching and text-mining approaches rationalized the complex profile establishing linkage between off-target pharmacology and clinically observed effects. These results demonstrate the utility of in vitro pharmacological profiling for a compound in late-stage clinical development.

INTRODUCTION: Osimertinib is a third-generation, mutant-selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) recently approved for the treatment of T790M-positive EGFR-mutant lung cancer. The mutant-selective nature of osimertinib improves its tolerability by limiting its inhibition of wild-type EGFR compared to first- and second-generation EGFR inhibitors, making it a good candidate for rationally-designed combination therapies. One such combination, osimertinib plus the MEK inhibitor selumetinib (AZD6244; ARRY-142886), is currently being studied as part of the phase 1b TATTON trial (NCT02143466). Previous work by our group demonstrated that selumetinib may delay or reverse resistance to osimertinib in some cases; however, we anticipate that resistance to this combination will ultimately develop in patients. Here, we investigate acquired resistance to osimertinib and selumetinib combination therapy in vitro and in vivo. DESIGN: In cells sensitive to osimertinib plus selumetinib (hereafter referred to as combination-sensitive cells), ERK phosphorylation was monitored via Western blot over the course of a 7-day treatment with 200 nM osimertinib plus 1 µM selumetinib. To detect RAS-GTP, an active RAS precipitation was performed in combination-sensitive and combination-resistant cell lines following treatment with osimertinib. In combination-resistant cells, pharmacologic inhibitors of various MAPK pathway components were used to assess whether they could restore potency. Western blot analysis was used to confirm on-target effects. Lastly, an analysis of differences in gene expression between four isogenic sets of combination-sensitive and combination-resistant cell lines is currently ongoing. RESULTS: In cell lines that are sensitive to growth inhibition by the combination of osimertinib plus selumetinib, we observed complete re-activation of the MAPK pathway after 5 days of continuous exposure to both inhibitors. Additionally, RAS activity was elevated in combination-resistant cell lines, and these cells remained sensitive to the ERK inhibitor SCH772984. Likewise, the addition of a pan-RAF inhibitor restored the growth inhibitory effects of osimertinib plus selumetinib in combination-resistant cells, suggesting that incomplete inhibition of MAPK is responsible for resistance in these cells. Furthermore, an alternative MEK inhibitor, trametinib, was efficacious in combination with osimertinib in cell lines that were resistant to osimertinib plus selumetinib. CONCLUSION: These data identify a potential mechanism of resistance to a combination therapy that is currently being tested in the clinic. Specifically, these data demonstrate that re-activation of the MAPK pathway is a mechanism of resistance to osimertinib plus selumetinib and that this pathway is still targetable in combination-resistant cells. Citation Format: David Westover, Catherine B. Meador, Eiki Ichihara, Hayden F. Byrd, Cath Eberlein, Yingjun Yan, Darren A. Cross, Christine M. Lovly. Sustained MAPK activation as a mechanism of resistance to osimertinib plus selumetinib in models of EGFR-mutant cancer [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 3160. doi:10.1158/1538-7445.AM2017-3160

Vascular Endothelial Growth Factor Receptor (VEGFR) mediated signalling drives angiogenesis. This is predominantly attributed to the activity of VEGFR-2 following binding of VEGF-A. Whether other members of the VEGFR and ligand families such as VEGFR-1 and its ligand Placental Growth Factor (PlGF) can also contribute to developmental and pathological angiogenesis is less clear. We explored the function of PlGF in VEGF-A dependent angiogenesis using an in vitro co-culture assay in which endothelial cells are cultured on a fibroblast feeder layer. In the presence of 2% FS MCDB media (containing limited growth factors) in vitro endothelial tube formation is driven by endogenous angiogenic stimuli which are produced by the fibroblast and endothelial cells. Under these conditions independent sequestration of either free VEGF-A or PlGF with polyclonal and monoclonal antibodies inhibited tube formation suggesting that both ligands are required to drive an angiogenic response. Endothelial tube formation could only be driven within this assay by the addition of exogenous VEGF-A, VEGF-E or VEGF-A/PlGF heterodimer, but not by PlGF alone, implying that activation of either VEGFR-2/VEGFR-1 heterodimers or VEGFR-2 homodimers were responsible for eliciting an angiogenic response directly, but not VEGFR-1 homodimers. In contrast to results obtained with an endogenous angiogenic drive, sequestration of PlGF did not affect endothelial tube formation when the assay was driven by 1 ng/ml exogenous VEGF-A. These data suggest that although neutralising PlGF can be shown to reduce endothelial tube formation in vitro, this effect is only observed under restricted culture conditions and is influenced by VEGF-A. Such data questions whether neutralising PlGF would have a therapeutic benefit in vivo in the presence of pathological concentrations of VEGF-A.

Aberrant receptor tyrosine kinase (RTK) signaling is a well-documented driver of disease onset and progression across myriad cancer types, where the MET RTK contributes to tumor progression, maintenance and resistance to targeted therapies. Here we explore the therapeutic potential of AZD6094, a highly potent and selective MET inhibitor, in EGFR mutant (EGFRm) non-small cell lung cancer (NSCLC). Although many EGFRm NSCLC patients receiving first-line EGFR Tyrosine Kinase Inhibitors (TKI) benefit from therapy initially, the majority of patients will acquire resistance in 9-14 months1,2. Of this patient population, ∼10-15% of patients with emerging resistance to early generation EGFRm-TKI will have MET amplification3. Using xenograft models (HCC827) of resistance to erlotinib or gefitinib, both first-generation EGFRm-TKI, we assessed the efficacy of AZD6094 in models with varying copy number gain for MET. We demonstrate that the combination of AZD6094 with gefitinib, or AZD9291, an irreversible, selective (EGFRm/T790M) EGFR TKI, results in tumor growth inhibition (TGI) of >100% in 3 models (HCC827-ER1, PCS030 clone 1 and 2)4, suggesting that the combination is necessary and sufficient to address acquired resistance due to MET gene amplification. Moreover, we explore efficacy of AZD6094 in models representative of resistance to first-line treatment with EGFRm-TKIs, harboring MET amplification and T790M EGFR mutations. In NCI-H820 xenografts (EGFRm/T790M/MET), we demonstrate for the first time that combining MET and EGFRm/T790M TKIs (AZD6094 with AZD9291) induces tumor regressions (TGI% >100%, 94% regressions) and the loss of palpable tumors in 5/7 animals as compared to AZD9291 (TGI 48%) or AZD6094 treatment alone (TGI >100%). Pharmacodynamic analysis of tumor lysates demonstrated potent and durable inhibition of pMET in all AZD6094 treatment groups. Due to the clinical importance of understanding acquired resistance to targeted TKIs, we then generated a model of resistance to AZD6094 in MET-amplified NSCLC NCI-H1993 cells and analyzed several resistant clones (H1993R). Interestingly, MET phosphorylation remains strongly inhibited in AZD6094-treated H1993R cells, while EGFR protein expression is upregulated and leads to co-dependency between both pathways. Enhanced expression and phosphorylation of EGFR, as well as AKT, MEK and ERK activation were commonly observed in H1993R cells. Taken together, our data support the potential of AZD6094 as a novel combination therapy for MET-driven NSCLC in the context of EGFRm TKI resistance, and highlight the clinical relevance of EGFR and MET signaling in the context of emerging TKI resistance mechanisms and coordinated pathways. 1. Mok et al. N Engl J Med 2009;361:947-957. 2. Rosell et al. Lancet Oncol 2012;13:239-246. 3. Engelman et al. Science 2007;316:1039-1043. 4. Models from Precos Ltd Citation Format: Celina D'Cruz, Evan Barry, Ryan Henry, Lillian Castriotta, Alwin Schuller, Garry Beran, Susan Ashton, Cath Eberlein, Corinne Reimer, Melanie Frigault, Michael Zinda, Darren Cross, Stephen Fawell. Changing the paradigm for treating drug resistance in NSCLC: Novel combinations of AZD6094, a selective MET inhibitor, and an irreversible, selective (EGFRm/T790M) EGFRTKI, AZD9291. [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 761. doi:10.1158/1538-7445.AM2015-761

First- and second-generation EGFR tyrosine kinase inhibitors (TKIs) are established first line therapies for patients with advanced NSCLC with activating/sensitising mutations in EGFR. Unfortunately, patients ultimately develop disease progression with acquisition of a second-site EGFR T790M mutation in more than half of cases. This has led to the development of third generation EGFR TKIs such as AZD9291 which inhibit both the EGFRm+ and T790M mutations in preclinical models, and are showing activity in patients with TKI-resistant tumors harbouring T790M in Phase I studies. Despite the potential improvements brought by third generation EGFR-TKIs, advanced EGFRm+ tumor cells will still remain highly adaptable, and the inevitability of further resistance will potentially limit the effectiveness of these drugs. As such, the identification of resistance mechanisms to these agents is essential to guide future therapeutic strategies and identify novel:novel combinations. To interrogate resistance to AZD9291, we have generated panels of EGFRm+ cell populations resistant to gefitinib (first generation TKI), and EGFRm+ and EGFRm+/T790M cell populations resistant to afatinib (second generation TKI) and WZ4002 or AZD9291 (third generation TKIs). Subsequently, we have characterized the cell lines using a phenotypic screen to compare the sensitivity of small molecule inhibitors of canonical signaling pathways between the resistant and parental cell populations. In addition we have used a variety of molecular profiling techniques to determine the DNA mutation and copy number status and mRNA expression profile of a panel of cancer associated genes within the resistant cell populations. The effects on cell survival across the range of resistant models by a panel of pathway inhibitors, in combination with the originating TKI, indicated that resistance to the EGFR inhibitors was frequently associated with increased sensitivity to selumetinib (AZD6244; ARRY-142886) (MEK1/2 inhibitor), suggesting that ERK signaling is commonly reactivated to circumvent inhibition of the EGFR pathway. Further, molecular analysis indicated the presence of mutations in NRAS, including a novel E63K mutation, or increased copy number of NRAS or KRAS within 12/25 and 3/9 resistant cell populations representing resistance in EGFRm+ and EGFRm+/T790M settings respectively. Analysis of the functional consequence of the observed RAS modifications confirmed their role in driving the survival of EGFR pathway addicted cells when EGFR signaling is inhibited. Collectively, these data suggest that ERK pathway activation, in particular as a result of increased RAS activation, is frequently associated with acquired resistance to AZD9291 and other EGFR TKI inhibitors. In addition the data suggests that combining AZD9291 with selumetinib could prevent or delay resistance, and therefore potentially drive superior duration of benefit compared to TKI alone. Citation Format: Cath Eberlein, Katie Al-Kadhimi, Sarah Ross, Henry Brown, Paul Fisher, Daniel Stetson, Zhongwu Lai, Kenneth Thress, Brian Dougherty, William Pao, Darren Cross. ERK pathway activation is associated with acquired resistance to AZD9291, a third-generation irreversible inhibitor targeting EGFR sensitizing (EGFRm+) and resistance (T790M) mutations in NSCLC. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr PR05.

First- and second-generation EGFR TKIs are established first line therapy for patients with NSCLC with activating mutations in EGFR. Unfortunately, patients ultimately develop disease progression with acquisition of a second-site EGFR T790M mutation in more than half of cases. This has led to the development of third generation EGFR TKIs which inhibit both the EGFRm+ and T790M mutations in preclinical models and are showing activity in TKI-resistant patients in Phase I studies. Despite the potential improvements brought by third generation EGFR-TKIs, advanced EGFRm+ tumor cells will still remain highly adaptable and the inevitability of further resistance will limit the effectiveness of these drugs. As such, the identification of resistance mechanisms to these agents is essential to guide future therapeutic strategies and identify novel:novel combinations. To interrogate resistance to AZD9291, we have generated panels of EGFRm+ cell lines resistant to gefitinib (first generation TKI) and EGFRm+ and EGFRm+/T790M cell lines resistant to afatinib and AZD9291 (second and third generation TKIs, respectively). Subsequently, we characterised the cell lines using a variety of molecular profiling techniques including Next Generation Sequencing (NGS), Affymetrix gene expression analysis, and phenotypic profiling following pharmacological modulation by small molecule inhibitors of canonical signaling pathways. The effects on cell survival across the range of resistant models by a panel of pathway inhibitors indicated that resistance to the EGFR inhibitors, in particular AZD9291, is frequently associated with increased sensitivity to selumetinib (AZD6244; ARRY-142886) (MEK1/2 inhibitor), suggesting that ERK signaling is commonly reactivated to circumvent inhibition of the EGFR pathway. Molecular characterisation of the panel of cell lines suggests a range of different resistance mechanisms may be responsible for reactivation of ERK signaling, including a decrease in negative regulators of ERK such as DUSP6 or an epithelial to mesenchymal transition consistent with previous observations. Collectively, these data suggest that combining an EGFR TKI with a MEK inhibitor could prevent or delay resistance and drive superior duration of benefit from these agents. Citation Format: Cath Eberlein, Laura Ratcliffe, Lucy O'Brien, Katie Al-Khadimi, Henry Brown, Paul Fisher, Daniel Stetson, Zhongwu Lai, Gayle Marshall, Claire Barnes, Kenneth Thress, Brian Dougherty, William Pao, Darren Cross. Investigating resistance to AZD9291. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1722. doi:10.1158/1538-7445.AM2014-1722

The first generation EGFR TKIs gefitinib and erlotinib provide significant clinical benefit in patients with advanced lung adenocarcinoma harbouring activating EGFR mutants (EGFRm+), but patients will ultimately develop disease progression due to acquired resistance. Acquisition of the EGFR T790M mutation is the most common mechanism of drug resistance, detected in more than 50% of gefitinib/erlotinib resistant patients. Current therapeutic strategies are limited for advanced lung adenocarcinoma patients with EGFR T790M (EGFRm+/T790M), so this remains a key area of unmet need. AZD9291 (structure to be disclosed at meeting) is an oral, irreversible, third generation, selective inhibitor of both EGFR activating (EGFRm+) and resistance (EGFRm+/T790M) mutations. The mechanistic and functional activity of AZD9291 was characterised in vitro and in vivo across a number of cell lines harbouring various EGFR-mutations or wild type EGFR. Presented data shows AZD9291 potently inhibits EGFR phosphorylation in EGFRm+ (e.g. PC9; 500nM). Consistently, AZD9291 showed significantly more potent inhibition of proliferation in mutant EGFR cell lines compared to wild-type in vitro. In addition, AZD9291 administered once daily orally at 5mg/kg caused profound regression of tumours across EGFRm+ (PC9; 178% growth inhibition) and EGFRm+/T790M (H1975; 119% growth inhibition) tumour models in vivo, after 14 days dosing. Furthermore 5mg/kg AZD9291 was sufficient to cause significant shrinkage of EGFRm+ and EGFRm+/T790M transgenic mouse lung tumours. Tumour growth inhibition was associated with profound inhibition of EGFR phosphorylation and key downstream signaling pathways such as AKT and ERK. Chronic long-term treatment of PC9 and H1975 xenograft tumours with AZD9291 led to a complete and sustained macroscopic response, with no visible tumours after 40 days dosing, and being maintained beyond 100 days. Furthermore, pre-clinical data also indicates that AZD9291 could target tumours that have acquired resistance to the more recently identified HER2-amplification mechanism, thus potentially extending its benefit in TKI resistant patients. Taken together, preclinical data demonstrates that AZD9291 is a potent and effective inhibitor of both EGFR activating (EGFRm+) and resistance (EGFRm+/T790M) mutations whilst sparing wild-type EGFR. These data support the further clinical investigation of AZD9291 in advanced EGFR mutant lung adenocarcinoma. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A109. Citation Format: Darren Cross, Sue Ashton, Caroline Nebhan, Cath Eberlein, M. Raymond V. Finlay, Gareth Hughes, Vivien Jacobs, Martine Mellor, Monica Red Brewer, Catherine Meador, Jonathon Orme, Paula Spitzler, Steve Powell, Amar Rahi, Paula Taylor, Richard A. Ward, Paula Daunt, Anne Galer, Teresa Klinowska, Graham Richmond, William Pao. AZD9291: an irreversible, potent and selective third generation tyrosine kinase inhibitor (TKI) targeting EGFR activating (EGFRm+) and resistance (T790M) mutations in advanced lung adenocarcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A109.

Background - Survivin is a member of the inhibitor of apoptosis protein (IAP) family, and is reported to be overexpressed in a number of human cancers with high unmet medical need including breast, non-small-cell lung (NSCLC), colorectal, gastric, glioblastoma, and pancreatic cancers. Multiple studies have described survivin as a key regulator of tumour cell proliferation, survival and drug resistance, which has led to the advancement of survivin inhibitors, including antisense and small molecule drugs, and cancer vaccines, into clinical testing. In the tumour micro-environment, survivin overexpression has also been reported in tumour blood vessels and conditional deletion of survivin in endothelial cells results in murine embryonic lethality secondary to haemorrhage and defective vascular development. We therefore hypothesised that inhibition of survivin function in endothelial cells could have anti-angiogenic or vascular disruptive effects. Methods and Results - To better understand the role of survivin in primary endothelial cells we designed siRNAs capable of targeting multiple isoforms of human survivin. Using high content imaging assays we demonstrated that the siRNA were highly selective for survivin, inhibited the expression of multiple survivin splice variants, and had potent in vitro effects on endothelial cells. Survivin knockdown resulted in inhibition of cell cycle progression with the formation of enlarged, multi-nucleated cells, and in reduced endothelial cell survival. Survivin knockdown also impaired the formation of tube-like networks in vitro when co-cultured with human fibroblasts, but did not disrupt more established networks. Conclusion These data show an important role for survivin in primary endothelial cells, and suggest that targeted inhibition of survivin may have anti-angiogenic effects, but may not disrupt more established blood vessels. Citation Format: Cath Eberlein, Grace Opoku-Ansah, James Legg, Tim London, Frank Gebhardt, Gareth Davies, Natalie Tigue, Matthew McCourt, Julia White, Darren Cross, Ute Schaeper, Viia E. Valge-Archer. Survivin regulates endothelial cell proliferation, survival and angiogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1616. doi:10.1158/1538-7445.AM2013-1616

Human solid tumours are comprised of epithelial cells interacting with a surrounding stroma or microenvironment. The stroma consists of several cell types including immune cells, endothelial cells and fibroblasts as well as macromolecules constituting the extracellular matrix. It has become clear from both pre-clinical and clinical studies that the tumour stroma plays a critical role in tumour progression and response to therapeutics. TGF-b is one of a number of pivotal regulators of the tumour microenvironment. Among its roles, TGF-b drives the cross-talk between tumour cells and fibroblasts resulting in a cancer associated fibroblast phenotype. This activated CAF state is linked to increased progression and survival of tumour cells and response of tumours to therapeutics. Modeling this cross-talk is challenging however we have developed in vitro assays using tumour cells from a range of tissue types, for example NSCLC, breast, pancreatic and prostate, co-cultured with normal human fibroblasts and assessed cell/cell and cell/extracellular matrix interactions using a range of end-points including -smooth muscle actin and fibronectin. We have further characterized the assays, for NSCLC cell lines, using pharmacologically active agents to modulate canonical and non-canonical TGF-beta signaling pathways and assessed the effects on tumour cell growth, fibroblast activation and secretion of key growth factors, cytokines and proteases. We have also used RNA expression profiling of a selection of genes to investigate signaling pathways activated in tumour and fibroblast co-cultures compared to when the cells are grown as monocultures. We will describe the development of methods to establish and quantify the different end-points explored in tumour and fibroblast co-culture assays and show data using inhibitors to validate these methods with NSCLC cell lines. We will show data from further comparison of monocultures and co-cultures by analysis of RNA and protein in cell lysates and conditioned media samples. The development of more complex in vitro assays with more than a single cell type allows greater insight into the various tumour-stromal interactions that take place in tumours in vivo. This allows early understanding and testing of agents which may provide therapeutic benefit in limiting tumour-stromal interactions and signalling in patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B127.