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PDF file - 84K, Correlation of AZD5363 activity in cell lines from different tumor types with RAS, PIK3CA and PTEN status.

Supplementary Materials text. Supplementary text to support supplementary data.

Supplementary Tables 1-3 and Figure 1-7. Supp Table 1 Summary of cell line origins and verification. Supp Table 2 Summary of antibodies used in all experimental analysis Supp Fig 1 AZD8186 inhibits PI3K pathway in PTEN null but not PTEN WT PIKC3A MT cells Supp Fig 2 AZD8186 inhibits LPA, EGF and rac dependent Ser473 AKT phosphorylation in serum starved prostate and breast cancer cells Supp Fig 3 Western blot analysis of showing induction of FOXO3a association with the chromatin fraction following treatment with AZD8186 Supp Fig 4 AZD8186 inhibits proliferation in a subset of cancer cell lines Supp Table 3 GI50 of all cell lines showing sensitivity to AZD8186 Supp Fig 5 Western blot protein analysis of PTEN status across a panel of breast and prostate cell lines Supp Fig 6 Pathway biomarker suppression in PC3 and HID28 tumours Supp Fig 7 AZD8186 selectively suppresses pathway biomarkers in tumour versus lung

PDF file - 81K, Correlation of AZD5363 activity in cell lines from different tumor types with PIK3CA and PTEN mutation status.

PDF file - 88K, Quantification of cleaved caspase 3 activity in HCC-1187 xenografts following chronic dosing with 5 mg/kg once weekly docetaxel (taxotere), 150 mg/kg bid AZD5363, or a combination of these two agents.

PDF file - 139K, AZD5363 has a predominantly anti-proliferative mechanism of action but induces cell death in BT474c, LNCaP and PC346C-Flut1 cells in vitro, and in BT474c xenografts following a high, intermittent dosing schedule in vivo.

PDF file - 117K, AZD5363 inhibits S6 phosphorylation in TSC1 -/-, TSC2 low-expressing RT4 bladder cancer cells.

AKT is a key node in the most frequently deregulated signaling network in human cancer. AZD5363, a novel pyrrolopyrimidine-derived compound, inhibited all AKT isoforms with a potency of 10 nmol/L or less and inhibited phosphorylation of AKT substrates in cells with a potency of approximately 0.3 to 0.8 μmol/L. AZD5363 monotherapy inhibited the proliferation of 41 of 182 solid and hematologic tumor cell lines with a potency of 3 μmol/L or less. Cell lines derived from breast cancers showed the highest frequency of sensitivity. There was a significant relationship between the presence of PIK3CA and/or PTEN mutations and sensitivity to AZD5363 and between RAS mutations and resistance. Oral dosing of AZD5363 to nude mice caused dose- and time-dependent reduction of PRAS40, GSK3β, and S6 phosphorylation in BT474c xenografts (PRAS40 phosphorylation EC50 ∼ 0.1 μmol/L total plasma exposure), reversible increases in blood glucose concentrations, and dose-dependent decreases in 2[18F]fluoro-2-deoxy-d-glucose (18F-FDG) uptake in U87-MG xenografts. Chronic oral dosing of AZD5363 caused dose-dependent growth inhibition of xenografts derived from various tumor types, including HER2+ breast cancer models that are resistant to trastuzumab. AZD5363 also significantly enhanced the antitumor activity of docetaxel, lapatinib, and trastuzumab in breast cancer xenografts. It is concluded that AZD5363 is a potent inhibitor of AKT with pharmacodynamic activity in vivo, has potential to treat a range of solid and hematologic tumors as monotherapy or a combinatorial agent, and has potential for personalized medicine based on the genetic status of PIK3CA, PTEN, and RAS. AZD5363 is currently in phase I clinical trials. Mol Cancer Ther; 11(4); 873–87. ©2012 AACR.

Supplementary Fig S7 describes western blot and RTPCR of PARG in modified shPARG cells.

Patients with metastatic pancreatic ductal adenocarcinoma (PDAC) have an average survival of less than 1 year, underscoring the importance of evaluating novel targets with matched targeted agents. We recently identified that poly (ADP) ribose glycohydrolase (PARG) is a strong candidate target due to its dependence on the pro-oncogenic mRNA stability factor HuR (ELAVL1). Here, we evaluated PARG as a target in PDAC models using both genetic silencing of PARG and established small-molecule PARG inhibitors (PARGi), PDDX-01/04. Homologous repair–deficient cells compared with homologous repair–proficient cells were more sensitive to PARGi in vitro. In vivo, silencing of PARG significantly decreased tumor growth. PARGi synergized with DNA-damaging agents (i.e., oxaliplatin and 5-fluorouracil), but not with PARPi therapy. Mechanistically, combined PARGi and oxaliplatin treatment led to persistence of detrimental PARylation, increased expression of cleaved caspase-3, and increased γH2AX foci. In summary, these data validate PARG as a relevant target in PDAC and establish current therapies that synergize with PARGi.Significance:PARG is a potential target in pancreatic cancer as a single-agent anticancer therapy or in combination with current standard of care.

Supplementary Data from AZD8931, an Equipotent, Reversible Inhibitor of Signaling by Epidermal Growth Factor Receptor, ERBB2 (HER2), and ERBB3: A Unique Agent for Simultaneous ERBB Receptor Blockade in Cancer

Supplementary Table 1 describes BRCA and KRAS status of PDX models and IC50 of PDDX-001 and olaparib in PDAC/PDX lines

Purpose: To test the hypothesis that simultaneous, equipotent inhibition of epidermal growth factor receptor (EGFR; erbB1), erbB2 (human epidermal growth factor receptor 2), and erbB3 receptor signaling, using the novel small-molecule inhibitor AZD8931, will deliver broad antitumor activity in vitro and in vivo.Experimental Design: A range of assays was used to model erbB family receptor signaling in homodimers and heterodimers, including in vitro evaluation of erbB kinase activity, erbB receptor phosphorylation, proliferation in cells, and in vivo testing in a human tumor xenograft panel, with ex vivo evaluation of erbB phosphorylation and downstream biomarkers. Gefitinib and lapatinib were used to compare the pharmacological profile of AZD8931 with other erbB family inhibitors.Results: In vitro, AZD8931 showed equipotent, reversible inhibition of EGFR (IC50, 4 nmol/L), erbB2 (IC50, 3 nmol/L), and erbB3 (IC50, 4 nmol/L) phosphorylation in cells. In proliferation assays, AZD8931 was significantly more potent than gefitinib or lapatinib in specific squamous cell carcinoma of the head and neck and non–small cell lung carcinoma cell lines. In vivo, AZD8931 inhibited xenograft growth in a range of models while significantly affecting EGFR, erbB2, and erbB3 phosphorylation and downstream signaling pathways, apoptosis, and proliferation.Conclusions: AZD8931 has a unique pharmacologic profile providing equipotent inhibition of EGFR, erbB2, and erbB3 signaling and showing greater antitumor activity than agents with a narrower spectrum of erbB receptor inhibition in specific preclinical models. AZD8931 provides the opportunity to investigate whether simultaneous inhibition of erbB receptor signaling could be of utility in the clinic, particularly in the majority of solid tumors that do not overexpress erbB2. Clin Cancer Res; 16(4); 1159–69

In a consanguineous pedigree in which a severe type of osteogenesis imperfecta was segregating as an autosomal recessive trait, analysis of genetic markers for both collagen I structural loci COL1A1 and COL1A2 showed that the phenotype was unlinked to either locus.