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Supplementary Materials and Methods; Supplementary Figure S1. Binding kinetics of hedgehog antibodies to human and mouse hedgehog proteins using surface plasmon resonance. Supplementary Figure S2. Hedgehog ligands stimulate mGLI1 reporter activity and osteoblast differentiation in a dose-dependent manner. Supplementary Figure S3. Selective inhibition of mGLI1 or mPTC1 induction in C3H10T1/2 cells by hedgehog antibodies. Supplementary Figure S4. MEDI-5304 inhibits GLI1 expression in Colo205 xenograft stromal cells but not tumor cells. Supplementary Figure S5. Hedgehog pathway component RNA expression in primary pancreatic tumor explant model P479 and effects of MEDI-5304 on tumorspheres derived from pancreatic explant model 947. Supplementary Figure S6. Pharmacokinetics of MEDI-5304 in rats. Supplementary Figure S7. Exploratory toxicology of MEDI-5304 in rats. Supplementary Figure S8. Pharmacokinetics of MEDI-5304 from preliminary toxicology study in cynomolgus monkeys. Supplementary Figure S9. Pharmacodynamics of MEDI-5304 in cynomolgus monkeys.

Supplementary material and tables

Supplemental Figure Legends

QC analysis

Supplementary Figure 2

Cox proportional hazards analyses

Purpose:Preclinical studies show that adavosertib, a WEE1 kinase inhibitor, sensitizes TP53-mutant cells to chemotherapy. We hypothesized that adavosertib, plus chemotherapy, would enhance efficacy versus placebo in TP53-mutated ovarian cancer.Patients and Methods:Following safety run-in, this double-blind phase II trial (NCT01357161) randomized women with TP53-mutated, platinum-sensitive ovarian cancer to oral adavosertib (225 mg twice daily for 2.5 days/21-day cycle) or placebo, plus carboplatin (AUC5) and paclitaxel (175 mg/m2), until disease progression or for six cycles. The primary endpoints were progression-free survival (PFS) by enhanced RECIST v1.1 [ePFS (volumetric)] and safety. Secondary/exploratory objectives included PFS by RECIST v1.1 (single dimension), objective response rate, overall survival, and analysis of tumor gene profile versus sensitivity to adavosertib.Results:A total of 121 patients were randomized to adavosertib (A+C; n = 59) and placebo (P+C; n = 62) plus chemotherapy. Adding adavosertib to chemotherapy improved ePFS [median, 7.9 (95% confidence interval (CI), 6.9–9.9) vs. 7.3 months (5.6–8.2); HR 0.63 (95% CI, 0.38–1.06); two-sided P = 0.080], meeting the predefined significance threshold (P < 0.2). Clinical benefit was observed following A+C for patients with different TP53 mutation subtypes, identifying possible response biomarkers. An increase in adverse events was seen with A+C versus P+C: greatest for diarrhea (adavosertib 75%; placebo 37%), vomiting (63%; 27%), anemia (53%; 32%), and all grade ≥3 adverse events (78%; 65%).Conclusions:Establishing an optimal strategy for managing tolerability and identifying specific patient populations most likely to benefit from treatment may increase clinical benefit. Future studies should consider additional adavosertib doses within the chemotherapy treatment cycle and the potential for maintenance therapy.

PFS and OS of PAM50 and OncotypeDx risk categories

Supplementary Figure 3

Purpose: Fulvestrant is an estrogen receptor (ER) antagonist and an approved treatment for metastatic estrogen receptor–positive (ER+) breast cancer. With the exception of ER levels, there are no established predictive biomarkers of response to single-agent fulvestrant. We attempted to identify a gene signature of response to fulvestrant in advanced breast cancer.Experimental Design: Primary tumor samples from 134 patients enrolled in the phase III CONFIRM study of patients with metastatic ER+ breast cancer comparing treatment with either 250 mg or 500 mg fulvestrant were collected for genome-wide transcriptomic analysis. Gene expression profiling was performed using Affymetrix microarrays. An exploratory analysis was performed to identify biologic pathways and new signatures associated with response to fulvestrant.Results: Pathway analysis demonstrated that increased EGF pathway and FOXA1 transcriptional signaling is associated with decreased response to fulvestrant. Using a multivariate Cox model, we identified a novel set of 37 genes with an expression that is independently associated with progression-free survival (PFS). TFAP2C, a known regulator of ER activity, was ranked second in this gene set, and high expression was associated with a decreased response to fulvestrant. The negative predictive value of TFAP2C expression at the protein level was confirmed by IHC.Conclusions: We identified biologic pathways and a novel gene signature in primary ER+ breast cancers that predicts for response to treatment in the CONFIRM study. These results suggest potential new therapeutic targets and warrant further validation as predictive biomarkers of fulvestrant treatment in metastatic breast cancer. Clin Cancer Res; 22(23); 5755–64. ©2016 AACR.

Correlation between IHC and transcript level by microarray

Supplementary Figure 1

PFS and RORP classifier

Purpose: Preclinical studies show that adavosertib, a WEE1 kinase inhibitor, sensitizes TP53-mutant cells to chemotherapy. We hypothesized that adavosertib, plus chemotherapy, would enhance efficacy versus placebo in TP53-mutated ovarian cancer. Patients and Methods: Following safety run-in, this double-blind phase II trial (NCT01357161) randomized women with TP53-mutated, platinum-sensitive ovarian cancer to oral adavosertib (225 mg twice daily for 2.5 days/21-day cycle) or placebo, plus carboplatin (AUC5) and paclitaxel (175 mg/m2), until disease progression or for six cycles. The primary endpoints were progression-free survival (PFS) by enhanced RECIST v1.1 [ePFS (volumetric)] and safety. Secondary/exploratory objectives included PFS by RECIST v1.1 (single dimension), objective response rate, overall survival, and analysis of tumor gene profile versus sensitivity to adavosertib. Results: A total of 121 patients were randomized to adavosertib (A+C; n = 59) and placebo (P+C; n = 62) plus chemotherapy. Adding adavosertib to chemotherapy improved ePFS [median, 7.9 (95% confidence interval (CI), 6.9–9.9) vs. 7.3 months (5.6–8.2); HR 0.63 (95% CI, 0.38–1.06); two-sided P = 0.080], meeting the predefined significance threshold (P < 0.2). Clinical benefit was observed following A+C for patients with different TP53 mutation subtypes, identifying possible response biomarkers. An increase in adverse events was seen with A+C versus P+C: greatest for diarrhea (adavosertib 75%; placebo 37%), vomiting (63%; 27%), anemia (53%; 32%), and all grade ≥3 adverse events (78%; 65%). Conclusions: Establishing an optimal strategy for managing tolerability and identifying specific patient populations most likely to benefit from treatment may increase clinical benefit. Future studies should consider additional adavosertib doses within the chemotherapy treatment cycle and the potential for maintenance therapy.

Background Emerging evidence supports combining immune checkpoint inhibitors (ICIs) with conventional or targeted therapies to enhance ICI antitumour activity and broaden the spectrum of patients who respond to ICIs. Here, we present the safety and preliminary efficacy of ramucirumab, an anti-VEGFR2 IgG1, plus durvalumab, an anti–PD-L1 IgG1, in previously treated patients with advanced non–small-cell lung cancer (NSCLC), gastric/gastro-oesophageal junction adenocarcinoma (gastric/GEJ), or hepatocellular carcinoma (HCC). Patients and methods A 25-centre, phase Ia/b single-arm, non-randomised, multi-cohort study was undertaken in patients with advanced/metastatic disease, Eastern Cooperative Oncology Group performance status, 0–1, progression on prior therapy, no prior ramucirumab or immunotherapy and any PD-L1 status. Patients received ramucirumab (10 mg/kg) plus durvalumab (1125 mg) intravenously Q3W (NSCLC), or ramucirumab (8 mg/kg) plus durvalumab (750 mg) Q2W (gastric/GEJ, HCC). Results Phase Ia treatment was found safe for phase Ib expansion; final enrolment was NSCLC (n = 28), gastric/GEJ (n = 29), HCC (n = 28). Grade ≥3 treatment–related adverse events occurred in 32.1%, 37.9% and 42.9% of patients, respectively. The most common were fatigue (35.7%), hypertension (34.5%) and diarrhoea (28.6%), respectively. Two patients died owing to an adverse event; one was treatment-related (hepatitis acute, HCC cohort). Objective response rate was 11% for NSCLC and HCC and 21% for gastric/GEJ. Median progression-free survival and overall survival were, respectively, 2.7 and 11 months in NSCLC; 2.6 and 12.4 months in gastric/GEJ; 4.4 and 10.7 months in HCC, with more prolonged survival in patients with high PD-L1 expression. Conclusion Ramucirumab/durvalumab exhibited manageable safety. The combination showed antitumour activity in all cohorts, particularly in patients with high PD-L1 expression.

Preclinical studies show that adavosertib, a WEE1 kinase inhibitor, sensitizesFollowing safety run-in, this double-blind phase II trial (NCT01357161) randomized women withA total of 121 patients were randomized to adavosertib (A+C;Establishing an optimal strategy for managing tolerability and identifying specific patient populations most likely to benefit from treatment may increase clinical benefit. Future studies should consider additional adavosertib doses within the chemotherapy treatment cycle and the potential for maintenance therapy.

Genomic profiling of tumours in patients in clinical trials enables rapid testing of multiple hypotheses to confirm which genomic events determine likely responder groups for targeted agents. A key challenge of this new capability is defining which specific genomic events should be classified as 'actionable' (that is, potentially responsive to a targeted therapy), especially when looking for early indications of patient subgroups likely to be responsive to new drugs. This Opinion article discusses some of the different approaches being taken in early clinical development to define actionable mutations, and describes our strategy to address this challenge in early-stage exploratory clinical trials.

Cancer cells are capable of sphere formation (transformation) through reactive oxygen species (ROS) and glycolysis shift. Transformation is linked to tumorigenesis and therapy resistance, hence targeting regulators of ROS and glycolysis is important for cancer therapeutic candidates. Here, we demonstrate that Smac mimetic AZ58 in combination with tumour necrosis factor-α (TNF-α) was able to inhibit the production of ROS, inhibit glycolysis through Pim-1 kinase-mediated Ser-112 phosphorylation of BAD, and increase depolarization of mitochondria. We also identified mitochondrial isoforms of Pim-1 kinase that were targeted for degradation by AZ58 in combination with TNF-α or AZ58 in combination with Fas ligand (FasL) plus cycloheximide (CHX) through caspase-3 to block transformation. Our study demonstrates that Smac mimetic in combination with TNF-α is an ideal candidate to target Pim-1 expression, inhibit ROS production and to block transformation from blebbishields.

2528 Background: A Phase 1b study (NCT02572687) was conducted to examine the combined effects of Ram (anti VEGFR2) and Durva (anti PD-L1). Methods: Patients (pts) with previously-treated, advanced NSCLC (Cohort [CH] A), G/GEJ adenocarcinoma (CH B), HCC (CH C), ECOG PS 0-1, and no prior Ram or IO therapy, received Ram (10 mg/kg) + Durva (1125 mg) Q3W (CH A) or Ram (8 mg/kg) + Durva (750 mg) Q2W (CH B, C). Primary objective was to assess safety; efficacy was also examined. PD-L1 expression of tumor cells (TC) +/- immune cells (IC) in pretreatment tumor biopsies were assessed using SP263 immunohistochemistry. “High” PD-L1 is ≥25% TC for NSCLC and ≥25% TC or IC for G/GEJ, HCC. Results: CH A, B and C enrolled pts with ECOG PS 1 (%) of 43, 66, 68; and average of 2, 2, 1 prior regimens, respectively. The most common grade 3/4 treatment-emergent adverse events (AE) are hypertension (HTN) (14.3, 17.2, 17.9%), anemia (3.6, 24.1, 21.4%), and fatigue (10.7, 10.3, 10.7%). Grade 3/4 AEs of special interest ( > 5% total pts) for Ram: HTN, bleeding events (3.6, 10.3, 10.7%), Venous thromboembolic events (0, 10.3, 7.1%); for Durva: increase in lipase (10.7, 3.4, 10.6%) and AST (3.6, 3.4, 17.9 %). Data from CH B,C suggest a trend toward increased efficacy in pts with high PD-L1 expressing tumors. Conclusions: Ram + Durva generated no unexpected toxicities and demonstrated antitumor activity. Results in pts with high PD-L1 HCC and G/GEJ cancer warrant further evaluation. Clinical trial information: NCT02572687. [Table: see text]

A series of dimeric compounds based on the AVPI motif of Smac were designed and prepared as antagonists of the inhibitor of apoptosis proteins (IAPs). Optimization of cellular potency, physical properties, and pharmacokinetic parameters led to the identification of compound 14 (AZD5582), which binds potently to the BIR3 domains of cIAP1, cIAP2, and XIAP (IC50 = 15, 21, and 15 nM, respectively). This compound causes cIAP1 degradation and induces apoptosis in the MDA-MB-231 breast cancer cell line at subnanomolar concentrations in vitro. When administered intravenously to MDA-MB-231 xenograft-bearing mice, 14 results in cIAP1 degradation and caspase-3 cleavage within tumor cells and causes substantial tumor regressions following two weekly doses of 3.0 mg/kg. Antiproliferative effects are observed with 14 in only a small subset of the over 200 cancer cell lines examined, consistent with other published IAP inhibitors. As a result of its in vitro and in vivo profile, 14 was nominated as a candidate for clinical development.

Purpose: Fulvestrant is an estrogen receptor (ER) antagonist and an approved treatment for metastatic estrogen receptor–positive (ER+) breast cancer. With the exception of ER levels, there are no established predictive biomarkers of response to single-agent fulvestrant. We attempted to identify a gene signature of response to fulvestrant in advanced breast cancer. Experimental Design: Primary tumor samples from 134 patients enrolled in the phase III CONFIRM study of patients with metastatic ER+ breast cancer comparing treatment with either 250 mg or 500 mg fulvestrant were collected for genome-wide transcriptomic analysis. Gene expression profiling was performed using Affymetrix microarrays. An exploratory analysis was performed to identify biologic pathways and new signatures associated with response to fulvestrant. Results: Pathway analysis demonstrated that increased EGF pathway and FOXA1 transcriptional signaling is associated with decreased response to fulvestrant. Using a multivariate Cox model, we identified a novel set of 37 genes with an expression that is independently associated with progression-free survival (PFS). TFAP2C, a known regulator of ER activity, was ranked second in this gene set, and high expression was associated with a decreased response to fulvestrant. The negative predictive value of TFAP2C expression at the protein level was confirmed by IHC. Conclusions: We identified biologic pathways and a novel gene signature in primary ER+ breast cancers that predicts for response to treatment in the CONFIRM study. These results suggest potential new therapeutic targets and warrant further validation as predictive biomarkers of fulvestrant treatment in metastatic breast cancer. Clin Cancer Res; 22(23); 5755–64. ©2016 AACR.

A series of structurally unique Smac mimetics that act as antagonists of inhibitor of apoptosis proteins (IAPs) has been discovered. While most previously described Smac mimetics contain the proline ring (or a similar cyclic motif) found in Smac, a key feature of the compounds described herein is that this ring has been removed. Despite this, compounds in this series potently bind to cIAP1 and elicit the expected phenotype of cIAP1 inhibition in cancer cells. Marked selectivity for cIAP1 over XIAP is observed for these compounds, which is attributed to a slight difference in the binding groove between the two proteins and the resulting steric interactions with the inhibitors. XIAP binding can be improved by constraining the inhibitor so that these unfavorable steric interactions are minimized.

Introduction: Epacadostat, a potent and highly selective oral inhibitor of the indoleamine 2,3- dioxygenase 1 (IDO1) enzyme, plus durvalumab, an anti-PD-L1 antibody, is being studied in patients with advanced solid tumors (NCT02318277). Phase 1 preliminary safety data for the overall population and efficacy data for patients with advanced pancreatic cancer (PC) are reported as of 29 Oct 2017 data cutoff.Methods: Adult patients with PC, melanoma, NSCLC, or SCCHN were enrolled in the 3+3 dose-escalation phase 1. Prior treatment with immune checkpoint inhibitors (in unapproved indications) or IDO inhibitors was not allowed. Patients received epacadostat (25, 50, 75, 100, or 300 mg twice daily [BID]) plus durvalumab (3 or 10 mg/kg every 2 weeks [Q2W]). Safety was assessed in patients receiving ≥1 treatment dose. Response was measured in evaluable patients (≥1 postbaseline scan or discontinued/died before the first scan) per modified RECIST v1.1.Results: Thirty-four patients were enrolled in phase 1. Median age (range) was 68 (46-84) years; most patients were male (62%) and had an ECOG PS of 1 (82%). There was 1 dose-limiting toxicity (grade 3 rash requiring systemic steroids; epacadostat 300 mg BID plus durvalumab 10 mg/kg Q2W) during the 42-day observation period. The most common (≥10%) treatment-related adverse events (TRAEs) were fatigue (32%), pruritus (15%), diarrhea, nausea, and rash (12% each). Grade ≥3 TRAEs occurring in >1 patient included fatigue and rash (n=3 [9%] each). Five patients (15%) discontinued because of TRAEs (grade 1 pneumonitis, grade 2 diarrhea, grade 2 subarachnoid hemorrhage, grade 2 peripheral edema, and grade 3 dyspnea). There were no TRAEs leading to death.Fifteen patients with PC were enrolled across multiple dose levels. Median age (range) was 66 (46-72) years, 67% had liver metastases, and 87% had an ECOG PS of 1. Fourteen patients had received ≥1 prior therapy. PD-L1 expression test results were available in 7/15 patients: 2 had detectable (≥1%) staining of tumor cells; 5 did not. No responses were observed among patients with PC; the disease control rate was 27% (4 SD) per RECIST v1.1 (1 of 4 patients with SD discontinued treatment because of clinical progression). The median duration of disease control was 156 days (95% CI, 91-219).Epacadostat exposure was consistent with previous reports, except in patients with PC where somewhat lower peak exposures (Cmax) were observed.Conclusions: Epacadostat plus durvalumab was generally well tolerated in patients with advanced cancers; the safety profile was consistent with previous reports of durvalumab and epacadostat as monotherapies. In unselected patients with PC, no objective responses were observed; a phase 2 expansion for PC was not conducted. Epacadostat 100 and 300 mg BID are being evaluated in the ongoing phase 2 expansions, including patients with NSCLC, SCCHN, and urothelial carcinoma. Citation Format: Aung Naing, John D. Powderly, Gerald Falchook, Benjamin Creelan, John Nemunaitis, Jose Lutzky, Adi Diab, Judy S. Wang, Naomi Laing, Michelle Niewood, Xiaohua Gong, Gongfu Zhou, Manish Patel. Epacadostat plus durvalumab in patients with advanced solid tumors: preliminary results of the ongoing, open-label, phase I/II ECHO-203 study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT177.

92 Background: Angiogenesis and immunosuppression are hallmarks of tumor growth. This global phase 1 trial evaluates the combination of R (anti-VEGFR2) and D (anti-PD-L1) in pts with G/GEJ by simultaneously targeting these two processes. Methods: This ongoing, multi-cohort, phase 1a/b trial (NCT02572687) enrolled pts with confirmed G/GEJ adenocarcinoma with prior progression on 1 or 2 lines of systemic therapy, measurable disease, ECOG PS 0-1, and baseline tumor tissue. PD-L1 expression was assessed using the SP-263 IHC; MSI status was determined using PCR. Enrolled pts received R (8 mg/kg IV) and D (750 mg IV) every two weeks on a 28-day cycle. Primary objective was to assess safety and tolerability of R+D; preliminary efficacy was also examined. Results: As of 26-May-2017, 29 G/GEJ adenocarcinoma pts were treated. The median age was 55 y; 69% were male; 66% had ECOG PS of 1; 48% had PD-L1 ≥25% expression in tumor or immune cells, 3.5% were MSI-high; and 72% received study treatment as second line for advanced disease. Median duration of treatment was 2.5 mo for R and 3.0 mo for D. Treatment-emergent adverse events (TEAEs) occurred in 29 (100%) pts and 21 (72%) pts experienced grade 3/4 TEAEs, while treatment-related AEs (TRAE) occurred in 24 (83%) pts; none resulted in treatment discontinuation. Ten (35%) pts had grade 3 TRAEs, and no grade 4 or 5. All grade TRAEs occurring in ≥10% of pts were hypertension (34%), fatigue (31%), headache (24%), diarrhea (21%), pyrexia (10%) and decreased appetite (10%). Five pts (17%) reported a serious adverse event related to study treatment. Preliminary efficacy data (RECIST v1.1) showed 5 of 29 pts (17%) achieved a confirmed PR. Only 1 responding pt was MSI high. The overall response rate for pts with PD-L1 ≥25% was 36%. Median PFS was 2.6 mo (95% CI, 1.45 to 6.28). As of data cut-off, 6 pts (21%) remain on treatment. Conclusions: R+D generated no unexpected toxicity, and demonstrated antitumor activity in pts with previously treated advanced G/GEJ adenocarcinoma. Clinical trial information: NCT02572687.

Activity-guided fractionation of a chloroform-soluble extract of the leaves of Ormosia sumatrana, using a proteasome inhibition assay, led to the isolation of a new A-type proanthocyanidin derivative, 3'-O-cinnamoylprocyanidin A-2 (1), and a new cerebroside, sumatranoside (2). The structures of these two isolates were determined as epicatechin-(2 beta--O--7',4 beta--8')-epicatechin-3'-O-cinnamate (1) and 1-O-(beta-d-glucopyranosyl)-(2S,3S,4R)-2N-[(2'R)-2'-hydroxy-tetracosanoyl]-9Z-octadecene-1,3,4-triol (2), respectively, by spectroscopic and chemical methods. Sumatranoside (2) exhibited proteasome inhibitory activity with an IC(50) value of 30 microM.

Despite recent advances in therapy, chronic lymphocytic leukaemia (CLL) remains incurable and new treatment strategies are therefore urgently required. Inhibitor of apoptosis proteins (IAPs) are over-expressed in CLL, suggesting both a role in disease pathogenesis and the potential for therapeutic targeting. To explore these questions, we evaluated the effects on primary CLL cells of AZD5582, a novel potent and selective inhibitor of IAPs. AZD5582 at nanomolar concentrations induced extensive degradation of cIAP-1 and cIAP-2, but minimally of X chromosome-linked IAP (XIAP). However, these effects of AZD5582 produced little or no direct cytotoxicity, nor did they sensitize CLL cells to p53-dependent killing by fludarabine or p53-independent killing by dexamethasone. In contrast, AZD5582 significantly enhanced apoptosis induced by the death receptor (DR) agonist tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Importantly, killing by TRAIL plus AZD5582 was independent of adverse prognostic features including TP53 deletion which is strongly associated with chemoresistance in CLL. Coculture experiments involving transfected mouse fibroblasts expressing human CD40L (CD154) to mimic the effect of T cells at sites of tissue involvement showed that CD40 stimulation almost completely prevented the killing of CLL cells by TRAIL plus AZD5582 despite up-regulating TRAIL receptors 1 and 2. In conclusion, our findings confirm the rate-limiting, upstream involvement of IAPs in the extrinsic but not intrinsic apoptotic pathway of CLL cells and suggest that drug combinations that simultaneously activate DRs and inhibit IAPs may have therapeutic potential in patients with CLL who have failed T-cell-depleting chemotherapy.

The hedgehog pathway has been implicated in the tumorigenesis, tumor progression, and metastasis of numerous human cancers. We generated the first fully human hedgehog antibody MEDI-5304 and characterized its antitumor activity and preclinical toxicology. MEDI-5304 bound sonic hedgehog (SHH) and Indian hedgehog (IHH) with low picomolar affinity and neutralized SHH and IHH activity in cellular mGLI1 reporter assays. The antibody inhibited transcription of hedgehog target genes and osteoblast differentiation of C3H10T1/2 cells. We evaluated the activity of MEDI-5304 in vivo in model systems that allowed us to evaluate two primary hypotheses of hedgehog function in human cancer, paracrine signaling between tumor and stromal cells and cancer stem cell (CSC) self-renewal. MEDI-5304 displayed robust pharmacodynamic effects in stromal cells that translated to antitumor efficacy as a single agent in an HT-29/MEF coimplantation model of paracrine hedgehog signaling. MEDI-5304 also improved responses to carboplatin in the HT-29/MEF model. The antibody, however, had no effect as a single agent or in combination with gemcitabine on the CSC frequency or growth of several primary pancreatic cancer explant models. These findings support the conclusion that hedgehog contributes to tumor biology via paracrine tumor-stromal signaling but not via CSC maintenance or propagation. Finally, the only safety study finding associated with MEDI-5304 was ondontodysplasia in rats. Thus, MEDI-5304 represents a potent dual hedgehog inhibitor suitable for continued development to evaluate efficacy and safety in human patients with tumors harboring elevated levels of SHH or IHH. Mol Cancer Ther; 13(2); 386–98. ©2013 AACR.

Cisplatin-based chemotherapy is considered the gold standard for patients with advanced bladder cancer. However, despite initial response, many patients will relapse; therefore, novel salvage treatment strategies are desperately needed. Herein, we studied a mechanism based treatment combination using a Smac mimetic with standard chemotherapy. Using a panel of 10 urothelial cancer cell lines, we exposed them to a combination of gemcitabine, cisplatin, and a Smac mimetic. Sensitivity was determined using a DNA fragmentation assay. We determined that three cell lines (UMUC-3, UMUC-13, and RT4v6) were considered sensitive to the combination of gemcitabine and cisplatin and an additional three cell lines were sensitized to gemcitabine and cisplatin with the addition of the Smac mimetic (UMUC-6, UMUC-12, and UMUC-18). We next explored the constitutive expression of selected members of the IAP family (XIAP, cIAP-1, cIAP-2, and Survivin), the BCL family (BCL-2, BCLXL, and BAX) and Smac using gene expression profiling and western blotting. We determined that RNA and protein expression of SMAC, selected members of the IAP family and members of the BCL family did not correlate to drug sensitivity. Lastly, using an in vivo mouse model, we determined that treatment with the Smac mimetic in combination with gemcitabine and cisplatin resulted in increased apoptosis, decreased microvessel density and decreased cellular proliferation. This novel treatment strategy may be effective in patients with advanced urothelial carcinoma and warrants further investigation.

Platelet-derived growth factor receptor α (PDGFRα) is a receptor tyrosine kinase that promotes cell survival and is expressed in both the tumor and the stromal components of human cancers. We have developed a fully human monoclonal antibody, MEDI-575, that selectively binds to human PDGFRα with high affinity, with no observable affinity for murine PDGFRα. To more fully characterize the role of PDGFRα in the regulation of tumor stroma, we evaluated the in vivo antitumor effects of MEDI-575 in tumor-bearing severe combined immunodeficient (SCID) mice and in genetically altered SCID mice expressing human PDGFRα in place of murine PDGFRα. We used the Calu-6 non-small cell lung cancer model because it lacks an in vitro proliferative response to PDGFRα activation. Antitumor activity was observed when the study was performed in mice expressing the human receptor, but no activity was observed in the mice expressing the murine receptor. Immunohistologic analysis of the tumors from mice expressing human PDGFRα showed a highly significant reduction in stromal fibroblast content and only minor changes in tumor proliferative index in tumors exposed to MEDI-575 compared with the results seen in vehicle-treated tumors or in tumors from mice expressing murine PDGFRα. Additional in vitro studies indicated that exposure of primary cancer-associated fibroblasts to MEDI-575 can directly affect proliferation and key signaling pathways in these cells. These results highlight the potential for observing antitumor activity with MEDI-575 through modulation of the stromal component of tumors and confirm that the PDGFRα pathway can play a role in maintaining a tumor microenvironment conducive to tumor growth.

Background: AZD1775 (formerly MK-1775) is a selective inhibitor of WEE1 kinase, which has been shown to sensitize TP53-mutant cancer cells to genotoxic agents such as platinum-based chemotherapies. Mutation of TP53 abrogates the G1/S checkpoint in cells, which may enhance their dependency on the G2/M checkpoint and WEE1 kinase activity for control of the cell cycle and effective repair of DNA damage. The results from a randomized Phase II trial (NCT01357161) evaluating the effects of adding AZD1775 to carboplatin and paclitaxel in patients with TP53-mutant ovarian cancer showed an increase in progression-free survival (PFS) for the AZD1775 arm versus placebo (enhanced RECIST: median PFS 34.14 vs 31.86 weeks; HR = 0.63, 80% CI: 0.45-0.89, P = 0.080; RECIST 1.1: median PFS 42.86 vs 34.86 weeks; HR = 0.55, 80% CI 0.39-0.79, P = 0.030; Oza et al, ASCO 2015). We investigated whether particular genetic factors were associated with an increased response to the combination of AZD1775 and chemotherapy in this trial. Methods: A retrospective analysis was performed to determine whether any specific subtype of TP53 alteration was associated with a greater response to the AZD1775 combination compared with placebo. In addition, next-generation sequencing (NGS) was performed on archival tumors from a subset of patients who provided consent in an effort to identify additional genetic alterations that may predict increased clinical benefit following the addition of AZD1775 to chemotherapy. Results: TP53 data were available for 136 patients (15 patients from an initial open-label safety run-in and 121 patients from the randomized trial) and 133 patients were evaluable for response. Fifty-five patients provided additional consent for NGS of tumor samples. The genetic aberrations observed in the NGS subset of tumors from this trial were heterogeneous, and the total mutational load in cancer-related genes was also variable across tumors. Although the small number of patients with a tumor BRCA mutation limited the statistical power of the comparison between randomized arms, the median PFS was longer in those patients treated with AZD1775 (53.86 weeks, 95% CI 24.43-66.57) versus placebo (45.86 weeks, 95% CI 35.71-55.86) in this BRCA-mutated subgroup. TP53 subgroup analyses showed a similar benefit for patients with missense mutations compared to those with splice site, nonsense and frameshift TP53 mutations. The heterogeneity of the G1/S checkpoint gene aberrations limited the statistical power of the subgroup analysis, but specific genes that warrant further investigation were identified. Conclusions: These results highlight a number of potential candidate genes for increased response to AZD1775 and chemotherapy compared with chemotherapy alone. Citation Format: Naomi Laing, Zhongwu Lai, J. Carl Barrett, Mark J. O’Connor, Amit M. Oza, David Lawrence. Genetic analysis of tumors from a phase II trial evaluating AZD1775, carboplatin and paclitaxel in patients with TP53-mutant ovarian cancer. [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 337.

TPS2608Background: Many cancers are associated with DNA repair and cell cycle mutations that result in G1/S checkpoint deficiencies and high levels of endogenous damage and replication stress. This can lead to dependence on WEE1 kinase, which provides an important G2/M checkpoint, allowing repair of DNA damage prior to mitosis. AZD1775 is a highly selective, small-molecule WEE1 inhibitor being developed for the treatment (tx) of advanced solid tumors. It has previously shown single-agent activity in patients (pts) with BRCA-1/2 mutations (Do, JCO 2015). We described initial safety and efficacy of the dose escalation portion of an open Phase Ib study (NCT02482311) of AZD1775 in pts with refractory solid tumors, and a dose of AZD1775 175 mg PO BID was identified (submitted to AACR 2016). Here we describe the expansion portion of this study in 6 matched cohorts: BRCA-1/2-mutant and wildtype (WT) ovarian cancer (OvC); cyclin-E (CCNE1)-amplified and non-amplified triple-negative breast cancer (TNBC); and any o...

Apoptosis culminates in secondary necrosis due to lack of ATP. Cancer stem cells form spheres after apoptosis by evoking the blebbishield emergency program. Hence, determining how blebbishields avoid secondary necrosis is crucial. Here we demonstrate that N-Myc and VEGFR2 control transformation from blebbishields, during which oligomers of K-Ras, p27, BAD, Bax, and Bak boost glycolysis to avoid secondary necrosis. Non-apoptotic cancer cells also utilize oligomers to boost glycolysis, which differentiates the glycolytic function of oligomers from their apoptotic action. Smac mimetic in combination with TNF-α or TRAIL but not in combination with FasL abrogates transformation from blebbishields by inducing secondary necrosis. Thus blebbishield-mediated transformation is dependent on glycolysis, and Smac mimetics represent potential candidates to abrogate the blebbishield emergency program.

Despite the widespread use of rituximab, a chimeric monoclonal antibody with demonstrated efficacy in the treatment of non-Hodgkin's lymphomas, there is a recognized need to develop new agents with improved efficacy. Towards this end, using XenoMouse technology, a fully human IgG1 anti-CD20 monoclonal antibody was generated. This antibody, denoted mAb 1.5.3, evoked enhanced pro-apoptotic activity in vitro, as compared to rituximab, in the Ramos lymphoma cell line. Also, mAb 1.5.3 mediated both complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) similar to rituximab in human B-lymphoma lines. Interestingly, mAb 1.5.3 demonstrated superior ADCC compared to rituiximab when FcgammaRIIIa F/F allotype donors were profiled and superior cytolytic activity across multiple human B-lymphoma and chronic B-cell leukemia lines in an in vitro whole blood assay. Furthermore, mAb 1.5.3 exhibited enhanced anti-tumor activity in Ramos, Daudi, and Namalwa tumour xenograft models. Lastly, mAb 1.5.3 produced a superior B-cell depletion profile in lymph node organs and bone marrow as compared to rituximab in a primate pharmacodynamic (PD) model. These findings underscore the potential of mAb 1.5.3 to exhibit improved clinical activity in the treatment of B-cell malignancies compared to rituximab.

TPS5608 Background: WEE1 is DNA damage cell-cycle checkpoint protein that inactivates cyclin-dependent kinase 1 (CDC2) and is overexpressed in a variety of malignancies. Pts with deficient p53 expr...

Introduction: Several multi-gene expression based assays have been developed to assess the prognosis and predict response to endocrine treatments in early stage hormone receptor positive (HR+) breast cancer. Although a significant number of patients with metastatic ER+ disease will not respond to endocrine treatments, molecular assays to predict response in this setting are limited. In addition, tissue specimens of metastatic lesions for molecular studies are not always available. In this study we sought to identify a molecular profile in the primary tumors of patients who developed disease recurrence that could predict response to endocrine treatment in metastatic disease. Methods: We used the primary breast tumor samples from a subgroup of patients participating in the randomized phase III CONFIRM trial, which compared 500mg versus 250mg of fulvestrant in post-menopausal women with HR+ advanced breast cancer. Formalin- fixed paraffin embedded tumors were collected from 130 of the participants and were centrally reviewed for ER, PR, HER2 and Ki67. RNA was sufficient for gene expression profiling in 112 of the cases using the NuGEN Ovation FFPE WTA System and Affymetrix HTA 2.0 GeneChip. The majority of the patients in this analysis developed metastatic disease during adjuvant endocrine treatment (N=55) or had de-novo metastatic disease (N=39) versus relapse after adjuvant treatment (N=18). The association between gene expression and progression free survival (PFS) was investigated using a multivariate Cox proportional hazard model adjusting for statistically significant clinicopatholgical factors. In addition we performed pathway-level analysis and evaluated the PAM50 subtype predictor and Risk of Relapse (ROR) score. Results: The median PFS was 8 months in our cohort. HER2 level by immunohistochemistry above 1+, high PR level, defined as Allred score of above 6, and Ki67 of above 50% were significantly associated with PFS and were included in the multivariate model. Dose of fulvestrant was not associated with PFS in this cohort. We identified a signature of 25 genes that is inversely associated with PFS on fulvestrant treatment (FDR 20%). When compared to other published datasets of breast cancer tumors, these genes are enriched in tumors with poor outcome and triple negative cancers. Pathway analysis revealed an association between activation of the EGFR pathway and decreased PFS (P=0.01). PAM50 subtypes varied with the luminal subtype being the most common (65%) and were generally concordant with the clinical subtype. However, we did not detect a significant trend between PAM50 subtype or ROR score and PFS or overall survival. Conclusions: In this cohort of patients with early and de-novo metastatic disease we identified a gene signature in the primary tumors that is associated with decreased response to fulvestrant treatment in metastatic disease. This signature warrants further validation to determine it’s predictive value and potential to assist in treatment decision making for patients with HR+ metastatic disease. Citation Format: Rinath M Jeselsohn, William T Barry, Jin Zhao, Gilles Buchwalter, Cristina Guarducci, Ilenia Migliaccio, Chiara Biagioni, Martina Bonechi, Naomi Laing, Yuri Rukazenkov, Eric P Winer, Myles Brown, Angelo Di Leo, Luca Malorni. TransCONFIRM: The correlative analysis of breast tumors from patients with advanced hormone receptor positive disease identifies a genetic signature associated with decreased benefit from single agent fulvestrant [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S1-01.

Synthesis and in vitro characterization of novel, lactam boronic acid based, selective, and rapidly reversible inhibitor 14 of the 20S-proteasome is presented.

A tetrahedral intermediate is the prominent feature of the generally accepted mechanism for aspartate transcarbamoylase. We have synthesized N-pyrophosphoryl-L-aspartate as a charged analogue of the postulated intermediate. Surprisingly, its affinity for the enzyme from Escherichia coli was substantially lower than that of the previously known inhibitor phosphonoacetyl-L-aspartate which contained a trigonal carbonyl group. Similar results were obtained with the corresponding mercaptosuccinate derivatives. We also tested a number of new pyrophosphate analogues as inhibitors. Our results cast doubt on some aspects of the current model for the mechanism of this enzyme.

Both monomeric and dimeric SMAC (Second Mitochondria-derived Activator of Caspaces) mimetics acting as IAP (Inhibitor of apoptosis proteins) anatgonists have been reported in the clinic as well as extensively in the literature (1). The first four amino acids in the N-terminal of SMAC (AVPI) are critical for binding to IAP proteins. Reported medicinal chemistry exploration of the AVPI template has primarily consisted of variation to the VPI position in the amino-terminal of the SMAC peptide. Structural illucidation of XIAP bound to IAP inhibitors has revealed a critical role for the alanine with dense hydrogen bonding, electrostatic and hydrophibic complementarity with the protein. To our knowledge, exploration of alanine modifications has been limited and generally led to significant reduction in potency. Using the molecular modeling software SuperStar(2), we investigated the publically available co-crystal structures of Smac-mimetics with cIAP1 and hypothesized that homologating the basic amine might be tolerated. Applying this strategy, we report on the successful transfer of a beta-alanine warhead to a number of monomeric scaffolds. The resulting novel monomers maintained cIAP1/2 potency albeit with a reduction in xIAP potency. We report here the first co-crystal structure of xIAP baculoviral IAP repeat 3 domain (BIR3) with a beta-alanine derived monomer. Examination of the binding site contacts in the co-crystal structure provided further insight into the optimization of the warhead. Herein we describe the synthesis, SAR and SPR of this novel warhead and the discovery of beta-alanine derived pan-IAP inhibitors. We show that the SAR can be transferred to dimers and is invariant to the position of dimerization. We report our efforts to optimize the series and mitigate Cyp3A4 inhibition. This work led to the discovery of AZ7732, a novel dimeric SMAC-mimetic; a pan inhibitor of IAPs (cIAP BIR3 IC50 = 12 nM, XIAP BIR3 IC50 = 13 nM, and XIAP BIR2 IC50 = 30 nM); potent in cells as a single agent (MDA-MB231 cIAP degradation IC50 = 0.2 nM, GI50 = 0.4 nM) and is synergistic in vitro in combination with gemcitabine. AZ7732 has favorable in vivo PK with physical properties suitable for IV dosing. AZ7732 is active in vivo as a single agent. Once weekly dosing in MDA-MB231 led to dose-dependent tumor growth inhibition with stasis achieved at 2.5 mpk, ¼ MTD. In conclusion, Structure-based design and medicinal chemistry efforts have successfully identified novel monomeric and dimeric SMAC mimetics leading to the discovery of a novel in vivo active dimeric pan-IAP inhibitor. (1) Fulda et al, Nat. Rev. Drug Disc., 11, 109-123, 2012. (2) M. L. Verdonk, et al, J. Mol. Biol., 289, 1093-1108, 1999 Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B100. Citation Format: Jamal C. Saeh, Brian Aquila, Daniel Russell, Edward Hennessy, Alex Hird, Melissa Vasbinder, Andrew Ferguson, Bin Yang, Maureen Hattersley, Naomi Laing, Terry MacIntyre, Troy Patterson, Galina Repik, Michael Rooney, Haiyun Wang, Dave Witson, Li Sha, Donald Cook, Paula Lewis, John Lee, Danyang Li, Victor Kamhi, Vibha Oza, Charles Omer. Structure-based design of AZ7732 a novel in vivo active beta-alanine-derived pan-IAP inhibitor. [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 B100.

261 Background: Cisplatin-based chemotherapy is considered the gold standard for patients with advanced bladder cancer. Despite initial response, many patients will relapse; therefore, novel salvage treatment strategies are desperately needed. Herein, we studied a mechanism based treatment combination using a Smac mimetic with standard chemotherapy. Methods: A panel of 10 urothelial cancer cell lines was exposed to gemcitabine and cisplatin, a Smac mimetic, and the combination. Sensitivity was determined using a DNA fragmentation assay. Constitutive expression of select members of the IAP family (XIAP, cIAP-1, cIAP-2, Survivin), the BCL family (BCL-2, BCLXL, BAX) and Smac were evaluated by gene expression profiling and Western blotting. Changes in protein expression under treatment conditions were examined using Western blotting. TNF-a blocking antibody was used to explore the contribution of autocrine TNF-a in cellular death. Lastly, a mouse model of bladder cancer was used to determine the effectiveness of the drug combination. Results: UMUC-3, UMUC-13 and RT4v6 were considered sensitive to the combination of gemcitabine and cisplatin based on an apoptosis assay. Three additional cell lines were sensitized to gemcitabine and cisplatin with the addition of the Smac mimetic (UMUC-6, UMUC-12, UMUC-18). The constitutive RNA and protein expression of SMAC, select members of the IAP family, and members of the BCL family did not correlate to drug sensitivity. Autocrine TNF-a demonstrated a partial contribution to cell death in UMUC-12. In an in vivo mouse model, the Smac mimetic alone and in combination with gemcitabine and cisplatin resulted in decreased tumor volume and increased apoptosis compared to the chemotherapy treatment alone. The combination resulted in decreased cellular proliferation and decreased microvessel density. Conclusions: Smac mimetic is able to augment the activity of gemcitabine and cisplatin based chemotherapy in vitro and in vivo. This may be an effective treatment strategy in patients with urothelial carcinoma and warrants further investigation.

In spite of the widespread use of Rituximab, a chimeric monoclonal antibody with demonstrated efficacy in the treatment of non-Hodgkin’s lymphomas, there is a recognized need to develop fully human antibodies with improved efficacy. Towards this end, using XenoMouse™ technology, a fully human IgG1 monoclonal antibody specific to human CD20 was generated. This antibody, denoted mAb 1.5.3, evoked enhanced pro-apoptotic activity in vitro, as compared to Rituximab, in the Ramos human lymphoma cell line. In addition, mAb 1.5.3 was active in mediating complement dependent cytotoxicity (CDC) and elicited improved antibody-dependent cellular cytotoxicity (ADCC) relative to Rituximab in Ramos, Raji, and Daudi human B-lymphoma lines. To recapitulate various aspects of acquired resistance to Rituximab, as observed in a subpopulation of patients, Rituximab-resistant clones were established from lymphoma lines. Interestingly, mAb 1.5.3 demonstrated superior cytolytic activity against engineered Rituximab-refractory lymphoma clones, as well as across multiple human B-lymphoma and chronic B-cell leukemia lines in an in vitro whole blood assay. Furthermore, mAb 1.5.3 exhibited enhanced anti-tumor activity in Rituximab-sensitive cell lines and -refractory engineered lymphoma clones in vivo. Lastly, mAb 1.5.3 produced a superior B-cell depletion profile in lymph node organs and bone marrow as compared to Rituximab in a primate PD model. In contrast to Rituximab, mAb 1.5.3 is a fully human antibody and is thus anticipated to exhibit a longer serum half-life with minimal immunogenicity following repeated administration. In sum, these results demonstrate the superior anti-tumor activity of mAb 1.5.3 relative to Rituximab and its potential for improved clinical activity in the treatment of B-cell malignancies.