Combination Of AZD6244 Research Articles

Abstract B40: Evaluation of the combination of AZD2014 and olaparib in pancreatic cancer cells

Abstract The PI3K/Akt/mTOR pathway is frequently activated in human pancreatic ductal adenocarcinoma (PDAC) and in mouse models of Kras-driven pancreatic cancer. In addition, 20-30% of pancreatic cancers may have “unstable” genomes, associated with somatic or germline BRCA mutations, mutations in genes involved in DNA maintenance (eg. PALB2, ATM) or genes not previously associated with DNA maintenance. We therefore evaluated the efficacy of the mTORC1/2 inhibitor AZD2014 and olaparib, as single agents and in combination in vitro in human (MIA PaCa-2 and Capan-1) and murine (K8484: KRASG12D; p53R172H; Pdx1-cre (KPC)) PDAC cell lines. We also assessed the effect of AZD2014 in combination with gemcitabine, and the triple agent combination. Drug-induced cytotoxicity and cell proliferation were evaluated in 96-well plates, using the high-throughput sulforhodamine B (SRB) assay. The efficacy of 2-drug combinations was assessed by treating cells with serial dilutions of the compounds in an 8X8 grid format. Synergy was assessed using the Bliss Independence model, as implemented in Combenefit software (http://www.cruk.cam.ac.uk/research-groups/jodrell-group/combenefit). Western blotting showed that phosphorylation of Akt, NDRG1, S6 ribosomal protein and 4EBP1 were suppressed by AZD2014. AZD2014 inhibited the growth of all 3 cell lines, with GI50s in the nanomolar range. At the highest AZD2014 concentration, the growth of all 3 cell lines was inhibited at least 70%. Olaparib was ineffective as a single agent in K8484 and MIA PaCa-2 cells, but reassuringly inhibited the growth of Brca2 deficient Capan-1 cells. Synergy was observed in K8484 cells, and mild synergy in MIA PaCa-2 cells, when AZD2014 was combined with gemcitabine. No synergy was observed with the combination of AZD2014 and olaparib in any of the cell lines, although some additivity was observed in Capan-1 cells. When the 3 drugs were combined, higher olaparib concentrations increased cytotoxicity at lower concentrations of AZD2014, however this did not provide any additional benefit, compared to the combination of AZD2014 and gemcitabine at higher concentrations. Although the combination of AZD2014 and olaparib did not exhibit synergistic activity, AZD2014 showed efficacy as a single agent, and with gemcitabine is a promising combination that warrants further investigation in patients with pancreatic cancer. Further work is required to understand the mechanism of action of this combination, which may also reveal other promising targets to combine with inhibition of mTORC1/2. This study was funded by the Cancer Research UK New Agents Committee, with compounds supplied by AstraZeneca. Citation Format: Aarthi Gopinathan, Frances M. Richards, Sabina Cosulich, Bristi Basu, Duncan I. Jodrell.{Authors}. Evaluation of the combination of AZD2014 and olaparib in pancreatic cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B40.

Abstract PR10: Exploiting the G2-M cell cycle checkpoint dependency in small cell lung cancer (SCLC) using pharmacological inhibitors of CHK1 and WEE1

Abstract Background: Small cell lung cancer (SCLC), the most lethal form of lung cancer, is characterized by early metastasis and rapid development of drug resistance. SCLC is characterized by ubiquitous loss of TP53 and RB1 and mutually exclusive amplification of the MYC oncogene (CMYC, MYCN, MYCL). The loss of TP53 is thought to render SCLC cells deficient at the G1-S cell cycle checkpoint, thus making these cells completely dependent on the G2-M checkpoint for DNA repair, mainly governed by CHK1 and WEE1. CHK1 is a vital serine-threonine kinase regulating the G2-M checkpoint upon DNA damage. The WEE1 kinase is a regulator of CDK1/2 and inhibition of WEE1 leads to aberrant DNA replication and premature mitosis. In this study we investigated the effect of CHK1 (LY2606368) and WEE1 (AZD1775) inhibitors as therapeutic targets for SCLC. Methods: Protein and gene expression was determined by immunoblot and real-time-RT-PCR respectively. Effect of the drugs on cell viability was determined by CellTiter-Glo. Efficacy of LY2606368 in H69 (chemosensitive) and H69/CR (chemoresistant) models was further determined by in vivo flank tumor regression analysis. The effect of LY2606368 monotherapy was also tested in a spontaneous SCLC genetically engineered mouse model (GEMM) model. The mechanistic studies were conducted by immunoblot analysis, apoptosis assay and flow cytometry. Pre and post-treatment cell lysates were analyzed by reverse phase protein array (RPPA) for biomarker exploration. Result: Higher expression of CHK1 and WEE1 was observed in SCLC patient tumor samples compared to normal lung (p<0.001) and in SCLC cells as compared to NSCLC cells (p<0.05). LY2606368 showed efficacy in both primary and acquired cisplatin resistant in vitro and in vivo models. LY2606368 treatment showed striking single agent activity in a spontaneous GEMM model of SCLC. Proteomic analysis identified an association between LY2606368 sensitivity and high levels of the CMYC protein. The level of CMYC decreased upon LY2606368 treatment (p<0.001) while pro-apoptotic pathways increased (p<0.05). CHK1 was a top marker of cisplatin sensitivity and treatment of cells with LY2606368 reduced activation of the MEK/MAPK pathway which we show as markers of de novo cisplatin resistance. LY2606368 treatment caused accumulation of cells in the sub-G1 phase and increased cell death. WEE1 inhibitor AZD1775 treatment decreased cell viability (IC50<100nM in 70% SCLC cells) in majority of SCLC cells. We identified AXL (receptor tyrosine kinase) and phospho-S6K as markers of AZD1775 resistance and treatment with AXL inhibitor, TP0903, resensitized the cells to AZD1775. Cell cycle analysis revealed increased apoptosis upon treatment of AZD1775 in combination with TP0903. Western blot analysis of pre and post AZD1775 treated samples revealed sustained activation of mTOR pathway in AZD1775 primary resistant lines. Pre-treatment of the cells with the mTOR inhibitor everolimus sensitized SCLC cells to AZD1775 by causing downregulation of AKT/mTOR pathway. Significance: The study establishes CHK1 and WEE1 as therapeutic targets in SCLC, a disease for which the standard of care has remained unchanged for >30 years. Together, these results strongly indicate the potency of G2-M cell cycle checkpoint inhibitors as effective targeted therapies in both chemosensitive and chemoresistant patient population. The CHK1 inhibitor, LY2606368, and the WEE1 inhibitor, AZD1775, are both in current Phase 1 clinical trials. The correlative biomarker data from this study will not only indicate the subset of the patient population who are most likely to respond to these treatments, but it will also guide the selection of most effective combination strategies with CHK1 and WEE1 inhibition. This abstract is also being presented as Poster A25. Citation Format: Triparna Sen, Pan Tong, Catherine Allison Stewart, Sandra Cristea, You Hong-Fan, Bonnie S. Glisson, Don L. Gibbons, Julien Sage, Jing Wang, Lauren A. Byers. Exploiting the G2-M cell cycle checkpoint dependency in small cell lung cancer (SCLC) using pharmacological inhibitors of CHK1 and WEE1. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr PR10.

Abstract CT035: A dose escalation trial of the WEE1 inhibitor AZD1775, gemcitabine, and concurrent radiation in locally advanced pancreatic cancer

Abstract Background: Gemcitabine based chemoradiation for locally advanced pancreatic cancer (LAPC) is safe and effective, although most patients remain unresectable and freedom from local progression is not ensured. Targeting cell cycle checkpoints may enhance the efficacy of chemoradiation. Tumor cells commonly have an abnormal G1 checkpoint due to mutations in p53 or its pathway making them reliant on the G2 checkpoint to arrest and repair DNA damage after radiation. In our preclinical studies, WEE1 inhibition with AZD1775 abrogates the G2 checkpoint and sensitizes pancreatic cancer cell lines and xenografts to gemcitabine and radiation. Additionally, AZD1775 attenuates homologous recombination and promotes replication stress in cancer cells. Given these preclinical findings, we designed a phase I dose escalation study of the WEE1 inhibitor AZD1775 in combination with gemcitabine and radiation in patients with LAPC (NCT02037230). Methods: The primary objective of this phase I study is to determine the maximum tolerated dose (MTD) of AZD1775 combined with gemcitabine and radiation in patients with LAPC. Secondary objectives include estimation of the efficacy of this regimen at the target dose and to determine if WEE1 is inhibited by AZD1775 at or below its target dose in surrogate tissues. Protocol therapy consists of the administration of AZD1775 and gemcitabine at an assigned dose level in accordance with a Time-to-Event Continual Reassessment Method (TITE-CRM). Study treatment consists of four 21 day cycles. Gemcitabine is given on day 1 and day 8, and AZD1775 on days 1, 2 and 8, 9 of each cycle. Cycles 2 and 3 are administered with concurrent radiation, 52.5 Gy in 25 daily fractions to gross disease using volumetric modulated arc therapy to spare normal tissues. Following cycle 3, subjects have a 3 week break prior to cycle 4. The MTD will be determined by the development of dose limiting toxicities within the first 105 days of therapy with a target dose limiting toxicity rate of 25%. Tumor assessment using CT or MRI is performed at baseline, 1 month after radiation, and approximately every 3 months afterwards, with response characterized per RECIST. Blood samples obtained at baseline and after cycles 1, 2, and 4 will be used for correlative studies on circulating tumor cells and tumor derived exosomes. Target accrual is 36 patients; to date, 16 patients have been enrolled. Dose Escalation SchemaRadiation (Gy)Gemcitabine(mg/m⁁2)AZD1775 (mg)Level -252.5800100Level -152.51000100Level 052.51000125Level 152.51000150Level 252.51000175 Citation Format: Kyle C. Cuneo, Meredith Morgan, Matthew J. Schipper, Jonathan Maybaum, Mary U. Feng, Mahmoud Al-Hawary, Diane M. Simeone, Leah D. Olson, Vaibhav Sahai, Mark Zalupski, Theodore S. Lawrence. A dose escalation trial of the WEE1 inhibitor AZD1775, gemcitabine, and concurrent radiation in locally advanced pancreatic 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 CT035.

Abstract 337: Genetic analysis of tumors from a phase II trial evaluating AZD1775, carboplatin and paclitaxel in patients with TP53-mutant ovarian cancer

Abstract 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.

PAXIP1 Potentiates the Combination of WEE1 Inhibitor AZD1775 and Platinum Agents in Lung Cancer.

The DNA damage response (DDR) involves a complex network of signaling events mediated by modular protein domains such as the BRCA1 C-terminal (BRCT) domain. Thus, proteins that interact with BRCT domains and are a part of the DDR constitute potential targets for sensitization to DNA-damaging chemotherapy agents. We performed a pharmacologic screen to evaluate 17 kinases, identified in a BRCT-mediated interaction network as targets to enhance platinum-based chemotherapy in lung cancer. Inhibition of mitotic kinase WEE1 was found to have the most effective response in combination with platinum compounds in lung cancer cell lines. In the BRCT-mediated interaction network, WEE1 was found in complex with PAXIP1, a protein containing six BRCT domains involved in transcription and in the cellular response to DNA damage. We show that PAXIP1 BRCT domains regulate WEE1-mediated phosphorylation of CDK1. Furthermore, ectopic expression of PAXIP1 promotes enhanced caspase-3-mediated apoptosis in cells treated with WEE1 inhibitor AZD1775 (formerly, MK-1775) and cisplatin compared with cells treated with AZD1775 alone. Cell lines and patient-derived xenograft models expressing both PAXIP1 and WEE1 exhibited synergistic effects of AZD1775 and cisplatin. In summary, PAXIP1 is involved in sensitizing lung cancer cells to the WEE1 inhibitor AZD1775 in combination with platinum-based treatment. We propose that WEE1 and PAXIP1 levels may be used as mechanism-based biomarkers of response when WEE1 inhibitor AZD1775 is combined with DNA-damaging agents. Mol Cancer Ther; 15(7); 1669-81. ©2016 AACR.

Phase ib trial of dose-escalating AZD1775 in combination with concurrent radiation and cisplatin for intermediate and high risk head and neck squamous cell carcinoma.

TPS6106Background: Patients with significant tobacco histories (intermediate risk) and those who are HPV negative (high risk) have modest to poor outcomes and have been shown to have more disruptiv...

PATRIOT: A phase I study to assess the tolerability, safety and biological effects of a specific ataxia telangiectasia and Rad3-related (ATR) inhibitor (AZD6738) as a single agent and in combination with palliative radiation therapy in patients with solid tumours.

Tumour control rates from radiation therapy (RT) are limited by normal tissue toxicities. Novel strategies are required to selectively sensitize tumour cells to radiation-induced DNA damage. The G2 cell cycle checkpoint is an attractive target for this, as normal cells will be protected by their intact G1 checkpoint, which is lost in the majority of cancer cells. ATR is an important mediator of the G2 checkpoint. Preclinical data suggest that ATR inhibition will sensitise to DNA damaging therapies, including RT. This multi-part phase 1 trial aims to assess safety and tolerability and preliminary anti-tumour activity of the ATR inhibitor AZD6738 as monotherapy and in combination with palliative RT, escalating both drug and radiation dose at a dose-fractionation relevant to radical treatment. The design aims to test a novel agent at the earliest stage of clinical development and assess safety in combination with RT, with the aim of moving to a radically-treated population if tolerated. Methods: Participants have advanced solid tumours without standard systemic therapy options. The trial comprises three parts: parts A and B will assess AZD6738 as a single agent in dose escalation to MTD (part A), followed by expansion cohorts enriched for defective DNA damage response (part B). Part C will assess AZD6738 in combination with palliative RT in which participants will receive 20 Gy in 10 fractions, with per cohort escalation of drug dose to monotherapy MTD if tolerated. At the highest tolerated combination dose, the RT dose will be escalated to 30 Gy in 15 fractions. Maintenance AZD6738 post-RT will be tested at the highest tolerated combination dose. The study opened in August 2014. The study is dose escalating in part A and part C has opened and treated its first patient. Part B will open when dose escalation has completed. PATRIOT is sponsored by The Royal Marsden, funded by the Cancer Research UK/AstraZeneca Combinations Alliance and supported by supply of free drug and distribution costs from Astra Zeneca. Clinical trial information: NCT02223923

Abstract OT1-03-13: A phase II, double blind, randomised, placebo-controlled study of the AKT Inhibitor AZD5363 in combination with paclitaxel in triple-negative advanced or metastatic breast cancer (TNBC)(NCT02423603)

Abstract Management of metastatic TNBC remains a challenge. Chemotherapy is the mainstay of treatment but benefits are frequently short-lived with rapid development of resistance. The PI3K/AKT/mTOR pathway has been implicated in many ways in TNBC, making inhibition of AKT an attractive therapeutic target. Based on downstream pathway activation signatures, PI3K pathway activation appears higher in TNBC compared to other molecular subtypes, despite a relatively low percentage of activating PI3K mutations. Alternative means of activating the PI3K pathway have been identified in TNBC, including loss or mutation of PTEN (up to 35%) and INPP4B (up to 30%) and/or amplification of PIK3CA, AKT2 or AKT3, resulting in increased activation of AKT. Induction of AKT by chemotherapy can be an early compensatory mechanism that can be exploited therapeutically to increase the efficacy of chemotherapy. Preclinical TNBC models with activated AKT signalling have been shown to be highly sensitive to AKT inhibitors. AZD5363 is a potent pan-AKT inhibitor with good oral bioavailability. Multiple lines of investigation have demonstrated strong synergistic effects between AKT inhibition and taxane chemotherapy in models of TNBC both in vitro and in vivo, providing rationale for the combination of AZD5363 and paclitaxel in TNBC. PAKT is designed to test the hypothesis that inhibition of AKT will increase the anti-tumour activity of paclitaxel chemotherapy in TNBC. The study will try to characterize those patients who may benefit from this treatment to identify potential predictors of sensitivity. PAKT is an international investigator led and sponsored, double-blind, placebo controlled, randomised phase II trial. Patients are randomised 1:1 to receive paclitaxel weekly (90mg/m2) on days 1,8, and 15 plus AZD5363 (400mgBD) or placebo (400mgBD) on days 2-5, 9-12, 15-19 (28 day treatment cycles). Patients are stratified by the number of metastatic sites and the interval from the end of adjuvant chemotherapy. Treatment is given until disease progression (RECIST 1.1), intolerable toxicity or elective withdrawal. Tumour assessments are carried out every 8 weeks. PAKT enrols patients with histologically documented locally advanced/metastatic TNBC (ER≤Allred2, PR≤Allred2, HER2=0,1+or2+), no prior systemic therapy for advanced TNBC, ECOG PS 0-2 and measurable disease per RECIST v1.1. Patients with brain metastases, significant cardiovascular disease, motor polyneuropathy are excluded. The primary endpoint is progression-free survival. Secondary endpoints are objective response rate, change in tumour size, clinical benefit rate, overall survival, duration of response, and patient reported outcomes. Archival tumour tissue must be available to evaluate potential biomarkers associated with therapeutic response and resistance. PFS will be compared between treatment arms by the stratified log-rank test. HR for disease progression/death will be estimated using a stratified Cox proportional hazards model. Kaplan-Meier methodology will be used to estimate the median PFS for each arm. Approximately 140 patients will be enrolled at ≈65 sites in the UK, France, Hungary, Romania, Georgia & South Korea. Citation Format: Schmid P, Wheatley D, Baird R, Chan S, Abraham J, Tutt A, Kristeleit H, Patel G, Bathakur U, Bishop J, Harper-Wynne C, Sims E, Copson E, Perren T, Stein R, Poole C, Cartwright H, Sarker S-J, Mousa K, Turner N. A phase II, double blind, randomised, placebo-controlled study of the AKT Inhibitor AZD5363 in combination with paclitaxel in triple-negative advanced or metastatic breast cancer (TNBC)(NCT02423603). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT1-03-13.

Abstract B10: Noninvasive pharmacodynamic markers of the dual mTORC1/2 inhibitor AZD2014 in combination with paclitaxel, in cisplatin-resistant ovarian carcinoma xenografts

Abstract Background: The PI3K/AKT/mTOR pathway is known to regulate glucose metabolism, proliferation and apoptosis, hence the inhibition of mTOR is expected have impact on these important mechanisms of tumor growth and survival. Studies of cancer metabolism following mTOR inhibition reported to date have only been carried out on allosteric mTOR inhibitors which selectively inhibit the activity of mTORC1. The metabolic changes associated with dual mTORC1 and 2 inhibitions remain unknown. AZD2014 is a dual mTORC1/2 inhibitor. The aims of this study are to (i) examine the effects on tumor metabolism following mTORC1/2 inhibition alone and in combination with paclitaxel, (ii) to develop a non-invasive pharmacodynamic (PD) biomarker for tumor response. Methods: We investigated the effects of AZD2014 alone (A: 50mg/kg/3 days/week po), paclitaxel alone (P: 20mg/kg/1 day/week ip) and the combination of AZD2014 and paclitaxel (A+P: same doses) on tumor growth, signal transduction (pSer473-AKT, pSer240/244-S6) and apoptosis (cleaved PARP) in a cisplatin resistant ovarian (A2780CisR) carcinoma xenograft model following 2 weeks of treatment. Metabolic profiles of A2780CisR tumor extracts after 2 weeks of treatment were also measured by high resolution in vitro 1H- and 31P-MRS. In order to develop early non-invasive PD markers of tumor response, in vivo 1H-MRS before and after 3 days of treatment were also performed. Results: Growth and molecular response- Following two weeks of treatment, tumor volumes of the A2780CisR xenografts increased by 280±100% in A+P (p<0.05; compared to vehicle-controls (V)), 420±160% in A (not significant compared to V (ns)), 730±180% in P (ns) and 560±20% in V. Reduced pSer473-AKT and pSer240/244-S6 protein levels and increased cleaved-PARP expression were observed in A- and A+P-treated xenografts consistent with mTORC1/2 inhibition and induction of apoptosis, respectively. In vitro 1H- and 31P-MRS of tumor extracts- Similar significant increases in branched-chain amino acids (p<0.03), glutamine (p<0.002), tyrosine (p<0.0001), phenylalanine (p<0.02) and glucose (p<0.03) were found in A- and A+P-treated tumors when compared with V. Significant decreases in phosphocholine (PC, p<0.002), glycero-phosphocholine (GPC, p<0.002), phosphoethanolamine (PE, p<0.02) and ATP (p<0.003), together with reduced expression of choline kinase, were only seen in A+P-treated tumors. An increase in threonine (p<0.03) was only observed in P- and A+P-treated tumors compared with V. In vivo 1H-MRS- The reduction in phospholipid metabolites (PC, GPC and PE) following A+P treatment was confirmed in vivo with a significant decrease in the ratio of total choline/water signal (p=0.04) observed at the early time-point of 3 days of treatment. Conclusions: A+P is effective in preclinical ovarian tumors, with additive growth inhibition and increased apoptosis. Very few metabolic changes were observed in P-treated tumors compared with V. The non-phospholipid changes in the metabolic profiles of A- and A+P-treated tumors are consistent with previous findings where similar metabolic profiles were found following mTOR inhibition (1). A+P treatment also caused decreases in PC (associated with reduced choline kinase expression) and other choline-related metabolites and these phospholipid changes may have potential as surrogate non-invasive markers for determining tumor response following AZD2014 and paclitaxel combination treatment. (1) Lin et al. Cell Symposia: Angiogenesis, Metabolic Regulation, and Cancer Biology in association with VIB, Belgium 2012. This work is supported by the CR-UK and EPSRC Cancer Imaging Centre in association with the MRC and Department of Health (England) grants C1060/A10334 and C1060/A16464, NHS funding to the NIHR Biomedical Research Centre. MOL is an NIHR Senior Investigator. Citation Format: Anne-Christine LF Wong Te-Fong, Efthymia Papaevangelou, Martin O. Leach, Udai Banerji, Yuen-Li Chung. Noninvasive pharmacodynamic markers of the dual mTORC1/2 inhibitor AZD2014 in combination with paclitaxel, in cisplatin-resistant ovarian carcinoma xenografts. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B10.

Combined Targeting of JAK2 with a Type II JAK2 Inhibitor and mTOR with a TOR Kinase Inhibitor Constitutes Synthetic Activity in JAK2-Driven Ph-like Acute Lymphoblastic Leukemia

Background and rationale: Philadelphia chromosome-like acute lymphoblastic leukemia ("Ph-like ALL") is a subtype of high-risk B-precursor ALL (B-ALL), which carries a high risk of relapse with conventional chemotherapy(Roberts et al, N Engl J Med. 2014). Rearrangements in CRLF2, leading to overexpression of cytokine receptor for thymic stromal lymphopoietin (TSLP), are present in approximately 50% of Ph-like ALL and are associated with hyperactive JAK/STAT and PI3K/mTOR signaling (Harvey et al, Blood 2010;Tasian et al, Blood 2014).In addition,JAK2 fusion proteins, such as PAX5-JAK2 represent a novel class of JAK2-driven cellular transformation in B-ALL (Dagmar et al, Blood 2015). Our prior studies in Ph+ B-ALL established that combining tyrosine kinase inhibitors (TKIs) with second generation ATP-competitive mTOR kinase inhibitors (TOR-KIs) provides greater anti-leukemia efficacy compared to TKIs in Ph+ ALL (Janeset al, Nat. Med. 2013). In this study, we investigated anti-leukemia efficacy and intracellular signaling networks upon combination of type I or type II JAK2 inhibitors and TOR-KIs in JAK2-driven Ph-like ALL models.Methods. The human B-precursor Ph-like ALL cell lines MUTZ5 (which harborsIGH-CRLF2 translocation and JAK2 R683G mutation), MHH-CALL-4 (IGH-CRLF2 translocation and JAK2 I682F),Reh (ETV6-RUNX1 B-precursor ALL cell line)and mouse Arf-null PAX5-JAK2-MIG + IK6-MIR(IL7-dependent primary Arf-/- pre-B cells expressing the dominant negative Ikaros isoform IK6 with PAX5-JAK2 fusion protein) were studied. Signal transduction inhibitors (STIs): JAK2 type I inhibitor ruxolitinib and type II inhibitor NVP-BBT594 (Andraos et al., Cancer Discovery 2012); allosteric mTOR inhibitor rapamycin or mTOR-KI AZD2014. Effects on intracellular signaling were determined using phospho-flow cytometry and Westernblot analysis. Anti-leukemia effects were quantified using CellTiter-Glo viability assay and annexin V flow cytometry.Results. In vitro stimulation of CRLF2-rearranged cells with TSLP robustly induced JAK/STAT signaling (Fig 1D). JAK2 inhibition with ruxolitinib or BBT594 efficiently inhibited TLSP-induced STAT5, AKT, ERK and S6 activation, yet failed to affect4E-BP1 activation. The TOR-KI AZD2014 but not rapamycin fully inhibited phosphorylation of 4E-BP1, consistent with efficient inhibition of TORC1, and caused profound cell cycle arrest and growth inhibition of Ph-like cells. Combination of ruxolitinib and AZD2014 further inhibited cell proliferation, yet did not induce apoptotic cell death. Recent studies indicate persistence of JAK2-mutated cells upon chronic exposure to type I JAK2 inhibitors, through an adaptive resistance mechanism involving JAK2 heterodimerization and reactivation of JAK-STAT signaling (Koppikar et al., Nature 2012). We therefore compared the in vitro efficacy of ruxolitinib and BBT594, a type II JAK2 inhibitor that retains the ability to bind inactive JAK2 in Ph-like ALL cells. In MUTZ-5 but not in MHH-CALL-4 cells, ruxolitinib increased JAK2 activation loop phosphorylation (p-JAK2-Tyr1008) despite suppression of p-STAT5; in contrast, BBT594 diminished bothp-JAK2 and p-STAT5 in both cell lines. Unexpectedly, BBT594 induced apoptotic cell death in all JAK2-driven Ph-like ALL cell lines MUTZ5, MHH-CALL-4 and Arf-null PAX5-JAK2+IK6, but not in REH cells. Combination of BBT594 with AZD2014 further inhibited phosphorylation of JAK2, AKT, 4E-BP1 and eIF4E, and synergistically induced apoptosis and reduced cell viability in Ph-like ALL cell lines(combination index: MUTZ5, 0.71; MHH-CALL-4, 0.57; Arf-nullPAX5-JAK2+ IK6, 0.81). Of importance, BBT594 and AZD2014 combination induced apoptosis in five JAK2-mutant Ph-like ALL xenograft primary samples.In summary, these results suggest that efficient blockade of JAK2/STAT5 with type II JAK2 inhibitors translates into cell death of mutant JAK2-driven Ph-like ALL cells. Furthermore, concomitant blockade of TORC1 signaling with TOR-KI reduces B-ALL cell proliferation through potent inhibition of 4E-BP1 and causes synthetic activity, providing avenues for novel rationally designed combinatorial regimens in this subset of Ph-like B-ALL. The in vivo studies to test these hypotheses are ongoing using patient-derived xenografts. [Display omitted] DisclosuresJabbour:Pfizer: Consultancy, Research Funding. Tasian:Incyte: Consultancy; Gilead: Research Funding. Mullighan:Amgen: Honoraria, Speakers Bureau; Cancer Science Institute: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria; Loxo Oncology: Research Funding. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

Abstract C60: Pre-clinical efficacy of the ATR inhibitor AZD6738 in combination with the PARP inhibitor olaparib

Abstract The PARP inhibitor olaparib acts through both inhibition of DNA single-strand-break repair and trapping of PARP-DNA complexes creating DNA lesions which cause replication fork stalling and collapsed fork DNA breaks. Cells which have lost BRCA-dependent homologous recombination repair are highly sensitive to olaparib treatment and this has led to its approval in patients with tumors carrying BRCA mutations. In addition, ATM (Ataxia telangiectasia mutated) and ATR (Ataxia telangiectasia mutated and Rad3 related) dependent DNA repair processes are hypothesized to be important survival pathways to PARP inhibitor treatment. In pre-clinical studies cancer cells which have defects in either ATM or ATR have been shown to be sensitive to PARP inhibitors. Here we present data that the orally bioavailable ATR inhibitor AZD6738 (in Phase-I clinial trials) combines synergistically with olaparib leading to cell death and anti-tumour activity in pre-clinical models. The combinations are effective across a panel of gastric and lung cancer cell lines in vitro. In addition results from isogenic ATM−/-/− knockout versus ATM+/+/+FaDu head and neck cancer cell line pairs show enhanced combination activity in ATM knockout cells versus ATM wild-type cells. Studies in vivo show that through intermittent dosing the combination is tolerated while demonstrating significant anti-tumour efficacy and regressions across multiple human patient derived primary explant models. Together, these data support the notion of development of AZD6738 and olaparib combinations for the treatment of ATM-deficient cancers. Citation Format: Alan Lau, Elaine Brown, Andrew Thomason, Rajesh Odedra, Victoria Sheridan, Elaine Cadogan, Shirlian Xu, Andy Cui, Paul R. Gavine, Mark O'Connor. Pre-clinical efficacy of the ATR inhibitor AZD6738 in combination with the PARP inhibitor olaparib. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C60.

Abstract B112: Synergistic anti-lymphoma activity of ibrutinib and dual mTORC1/2 inhibitors in diffuse large B cell lymphoma is maintained in vivo on an intermittent schedule

Abstract Diffuse large B cell lymphoma (DLBCL) is the most common type of aggressive non Hodgkins lymphoma. The Activated B-Cell (ABC) subtype of DLBCL is driven by chronic active BCR signaling and remains the most difficult to treat. Resistant ABC-DLBCL occurs in as many as 40% of patients following treatment with R-CHOP. To identify additional treatment options, we performed a combination screen in DLBCL cell lines using a panel of drugs known to target survival and proliferation pathways in DLBCL, and uncovered a drug combination with highly synergistic activity that included the BTK inhibitor, ibrutinib, with the dual mTORC1/2 inhibitor, AZD2014. Ibrutinib is known to have activity in ABC-DLBCL; in a recent Phase I/II clinical trial of relapsed/refractory ABC-DLBCL, ibrutinib treatment resulted in 55% response rate in patients with B cell receptor mutations (Wilson et al, Nature Medicine 2015). In ABC-DLBCL cell lines, combination of AZD2014 with ibrutinib resulted in cell death, induction of cleaved caspase 3, and potent inhibition of c-myc and p-4EBP1. In vivo, combination of ibrutinib with AZD2014 was well tolerated and resulted in potent anti-tumor activity in OCI-Ly10 that was greater than either agent alone, with greater than 100% tumor growth inhibition as well as synergistic inhibition of p-4EBP1 (Ezell et al, Oncotarget 2014). This work was evaluated using a continuous daily dosing schedule of AZD2014 and ibrutinib. More recently, both continuous and intermittent dosing schedules (2 days on, 5 off) have been explored in patients with other solid tumours and was found to be better tolerated. We evaluated an intermittent schedule of AZD2014 in our preclinical ABC-DLBCL model, OCI-Ly10, in combination with ibrutinib and demonstrated that the synergistic combination activity was maintained, with 60% regression compared to only 36% regression with the daily dosing schedule combination. In addition to p-4EBP1 inhibition, we also observed synergistic inhibition of p-pRAS40, p-AKT, p-NDRG1, c-myc, survivin and HMGSC1. Ongoing preclinical work is focused on understanding whether more significant suppression of relevant pathways was achieved with intermittent compared to continuous dosing. Collectively, our data support a rationale for combining BTK and dual mTORC1/2 inhibitors for an effective and alternative treatment option in ABC-DLBCL. Citation Format: Theresa Proia, Nanhua Deng, Urzsula Polanska, Deborah Lawson, Vasu Sah, Maryann San Martin, Corinne Reimer, Sabina Cosulich. Synergistic anti-lymphoma activity of ibrutinib and dual mTORC1/2 inhibitors in diffuse large B cell lymphoma is maintained in vivo on an intermittent schedule. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B112.

Abstract LB-A03: AXL as a potential mediator of WEE1 inhibitor resistance in small cell lung cancer (SCLC)

Abstract Background: Wee1 is a protein kinase that plays a key role in regulating the G2 checkpoint and homologous recombination (HR) in response to DNA damage. Recent studies have demonstrated activity of the WEE1 inhibitor, AZD1775 (previously MK1775), in cancers with loss of the tumor suppressor TP53, including colon, pancreatic and breast. Hence, we hypothesize that WEE1 would be an ideal target for monotherapy in small cell lung cancer (SCLC), which is marked by ubiquitous loss of TP53. Moreover, because Wee1 inhibition induces a HR deficient state, dual targeting of Wee1 and PARP (another promising target previously identified in our group and in clinical trial testing) may induce synthetic lethality. In the present study we evaluate the efficacy of AZD1775 alone and with the PARP inhibitor, olaparib, in SCLC and investigate potential predictive biomarkers by proteomic profiling. Experimental procedures: We evaluated the gene expression of WEE1 in SCLC tumor sample (n = 23) compared to normal lung (n = 42). We then treated with AZD1775 +/- olaparib SCLC cell lines (n = 10). Drug sensitivity (IC50) was correlated with baseline expression level of 200 total or phosphorylated proteins measured by reverse phase protein array (RPPA) to identify potential predictive markers. SCLC cell lines with AZD1775 resistance (n = 3) were treated with TP0903 (10, 20, 40, 80nM) in combination with AZD1775 (0.1nM to 1uM) to test the efficacy of AXL co targeting in augmenting WEE1 inhibitor response. Results: SCLC tumors have a significantly higher gene expression level of WEE1 as compared to normal lung (p<0.07×10-6). Single-agent WEE1 or PARP inhibition decreased viability of SCLC cells in a dose-dependent manner with AZD1775 being more potent as a single agent treatment (IC50 <100nM in 70% SCLC cell lines) than olaparib (IC50 <1uM in 20% of SCLC cell lines) in a 6day assay. Combination of AZD1775 with olaparib revealed an additive effect in vitro in 90% of SCLC cell lines. Proteomic analysis revealed an association between WEE1 inhibitor resistance and higher levels of AXL and total S6K (p<0.05 for both). In contrast, WEE1 inhibitor sensitivity was associated with elevated basal expression of PDK1 (p<0.002), treatment of SCLC cells with the AXL inhibitor TP0903 (40nM) re-sensitized cells to AZD1775 (IC50 <10nM) in a 6day assay. Conclusion and significance: The therapeutic options for patients with SCLC are limited and survival is poor. The WEE1 inhibitors are currently in clinical trials for SCLC. However, as with any targeted therapy, primary and secondary drug resistance is an important barrier to clinical benefit which could potentially be addressed with therapeutic combinations. In this study we show the over expression of WEE1 in SCLC and its efficacy as a single agent and in combination with olaparib. Here we also show that the activity of the WEE1 inhibitors might be limited in cancer cells overexpressing of AXL and that AXL inhibition re-sensitized the cells to AZD1775. Our work supports further exploration of the combination of PARP and WEE1 in SCLC and also the possibility of AXL inhibition as a mechanism to overcome WEE1 inhibition resistance in SCLC. Acknowledgement: This work was supported by Uniting Against Lung Cancer grant (LB). Citation Format: Triparna Sen, Pan Tong, Jennifer Hoff, C. Allison Stewart, Jing Wang, Lauren Byers. AXL as a potential mediator of WEE1 inhibitor resistance in small cell lung cancer (SCLC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A03.

Wee-1 Kinase Inhibition Sensitizes High-Risk HPV+ HNSCC to Apoptosis Accompanied by Downregulation of MCl-1 and XIAP Antiapoptotic Proteins.

Although the majority of patients with HPV(+) oropharyngeal cancers have a favorable prognosis, there are some patients with tumors that are resistant to aggressive chemoradiotherapy with unusual patterns of locoregional and systemic recurrences. Therefore, more effective therapies are needed. In this study, we investigated the chemosensitizing efficacy of the selective Wee-1 kinase inhibitor, AZD-1775, in HPV(+) head and neck squamous cell carcinoma (HNSCC). Clonogenic survival assays and an orthotopic mouse model of HPV(+) oral cancer were used to examine the in vitro and in vivo sensitivity of HPV(+) HNSCC cell lines to AZD-1775 in combination with cisplatin, respectively. Cell-cycle analysis, DNA damage (γH2AX), homologous recombination (HR), and apoptosis were examined to dissect molecular mechanisms. We found that AZD-1775 displays single-agent activity and enhances the response of HPV(+) HNSCC cells to cisplatin both in vitro and in vivo. The sensitivity of the HPV(+) HNSCC cells to AZD-1775 alone or in combination with cisplatin was associated with G2 checkpoint abrogation, persistent DNA damage, and apoptosis induction. This finding of AZD-1775 increasing the sensitivity of HPV(+) HNSCC cells to cisplatin through apoptosis was not seen previously in the HPV(-) HNSCC cancer cells and is accompanied by a decreased expression of the antiapoptotic proteins, MCl-1and XIAP, which appear to be cleaved following AZD-1775 treatment. AZD-1775 selectively sensitizes HPV(+) HNSCC cells and orthotopic oral xenografts to cisplatin through apoptosis and support the clinical investigation of AZD-1775 in combination with cisplatin particularly in patients with advanced and recurrent metastatic HPV(+) HNSCC tumors.

Simultaneous perturbation of the MAPK and the PI3K/mTOR pathways does not lead to increased radiosensitization

BackgroundThe mitogen-activated protein kinases (MAPK) and the phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathways are intertwined on various levels and simultaneous inhibition reduces tumorsize and prolonges survival synergistically. Furthermore, inhibiting these pathways radiosensitized cancer cells in various studies. To assess, if phenotypic changes after perturbations of this signaling network depend on the genetic background, we integrated a time series of the signaling data with phenotypic data after simultaneous MAPK/ERK kinase (MEK) and PI3K/mTOR inhibition and ionizing radiation (IR).MethodsThe MEK inhibitor AZD6244 and the dual PI3K/mTOR inhibitor NVP-BEZ235 were tested in glioblastoma and lung carcinoma cells, which differ in their mutational status in the MAPK and the PI3K/mTOR pathways. Effects of AZD6244 and NVP-BEZ235 on the proliferation were assessed using an ATP assay. Drug treatment and IR effects on the signaling network were analyzed in a time-dependent manner along with measurements of phenotypic changes in the colony forming ability, apoptosis, autophagy or cell cycle.ResultsBoth inhibitors reduced the tumor cell proliferation in a dose-dependent manner, with NVP-BEZ235 revealing the higher anti-proliferative potential. Our Western blot data indicated that AZD6244 and NVP-BEZ235 perturbed the MAPK and PI3K/mTOR signaling cascades, respectively. Additionally, we confirmed crosstalks and feedback loops in the pathways. As shown by colony forming assay, the AZD6244 moderately radiosensitized cancer cells, whereas NVP-BEZ235 caused a stronger radiosensitization. Combining both drugs did not enhance the NVP-BEZ235-mediated radiosensitization. Both inhibitors caused a cell cycle arrest in the G1-phase, whereas concomitant IR and treatment with the inhibitors resulted in cell line- and drug-specific cell cycle alterations. Furthermore, combining both inhibitors synergistically enhanced a G1-phase arrest in sham-irradiated glioblastoma cells and induced apoptosis and autophagy in both cell lines.ConclusionPerturbations of the MEK and the PI3K pathway radiosensitized tumor cells of different origins and the combination of AZD6244 and NVP-BEZ235 yielded cytostatic effects in several tumor entities. However, this is the first study assessing, if the combination of both drugs also results in synergistic effects in terms of radiosensitivity. Our study demonstrates that simultaneous treatment with both pathway inhibitors does not lead to synergistic radiosensitization but causes cell line-specific effects.Electronic supplementary materialThe online version of this article (doi:10.1186/s13014-015-0514-5) contains supplementary material, which is available to authorized users.

AZD1775 sensitizes T cell acute lymphoblastic leukemia cells to cytarabine by promoting apoptosis over DNA repair.

While some children with acute lymphoblastic leukemia (ALL) have excellent prognoses, the prognosis for adults and children with T cell ALL is more guarded. Treatment for T-ALL is heavily dependent upon antimetabolite chemotherapeutics, including cytarabine. Targeted inhibition of WEE1 with AZD1775 has emerged as a strategy to sensitize cancer cells to cytarabine and other chemotherapeutics. We sought to determine if this strategy would be effective for T-ALL with clinically relevant anti-leukemia agents. We found that AZD1775 sensitizes T-ALL cells to several traditional anti-leukemia agents, acting synergistically with cytarabine by enhancing DNA damage and apoptosis. In addition to increased phosphorylation of H2AX at serine 139 (γH2AX), AZD1775 led to increased phosphorylation of H2AX at tyrosine 142, a signaling event associated with promotion of apoptosis over DNA repair. In a xenograft model of T-ALL, the addition of AZD1775 to cytarabine slowed leukemia progression and prolonged survival. Inhibition of WEE1 with AZD1775 sensitizes T-ALL to several anti-leukemia agents, particularly cytarabine and that mechanistically, AZD1775 promotes apoptosis over DNA repair in cells treated with cytarabine. These data support the development of clinical trials including AZD1775 in combination with conventional chemotherapy for acute leukemia.

Synergistic interaction between MEK inhibitor and gefitinib in EGFR-TKI-resistant human lung cancer cells.

With the increasing use of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKIs) in patients with advanced non-small cell lung cancer (NSCLC), acquired resistance has become a major clinical problem. A combination of different signaling pathway inhibitors is a promising strategy to overcome this. In the present study, the mitogen-activated protein kinase kinase 1/2 inhibitor, AZD6244, was used in combination with gefitinib to investigate the efficacy of this treatment in NSCLC cell lines, particularly in gefitinib-resistant cells. The EGFR-TKI-sensitive PC-9 (mutant EGFR/wild-type K-Ras) and EGFR-TKI-resistant A549 (wild-type EGFR/mutant K-Ras) human NSCLC cell lines were treated with AZD6244 alone, gefitinib alone or the combination of the two drugs, and the effects were evaluated using cell proliferation assays, with alterations in signaling pathways analyzed by western blotting. It was found that the growth inhibitory effect of combination treatment with gefitinib and AZD6244 was greater than that of gefitinib alone in the EGFR-TKI-resistant A549 cells. Treatment of A549 cells with gefitinib alone reduced the expression level of the activated form of Akt, and the combination of the two drugs showed stronger inhibition of phosphorylated-Akt and phosphorylated-extracellular signal-regulated kinases. The data showed that the combination of AZD6244 and gefitinib exhibited dose-dependent synergism in gefitinib-resistant NSCLC cells. Thus, a preclinical rationale exists for the use of AZD6244 to enhance the efficacy of gefitinib in patients with EGFR-TKI-resistant NSCLC.

Abstract 4719: Targeting interdependent signaling pathways to increase the durability and magnitude of response: promising combination therapy with dual mTORC1/2 inhibitors and CDK4/6 inhibitors

Abstract Two emerging mechanisms of endocrine resistance in estrogen receptor positive (ER+) breast cancers include the activation of the phosphatidylinositol 3-kinase (PI3K) / mammalian target of rapamycin (mTOR) pathway and the de-coupling of cell cycle control from ER signaling via de-regulation of the cyclin D/cyclin dependent kinase (CDK4/6) pathway. In this study, we hypothesized that combining inhibitors of both pathways using the dual mTORC1 and mTORC2 inhibitor AZD2014 and the CDK4/6 inhibitor palbociclib would elicit an improved tumor response over agents that inhibit either pathway alone. Moreover, we hypothesized that combined inhibition of CDK4/6 and TORC1/2 together with inhibition of ER signaling, would cause a profound anti-tumor effect in breast cancer models. In breast cancer cell lines, the combination of AZD2014 and palbociclib caused a synergistic inhibitory effect on cell growth. These effects occurred under conditions where addition of 300nM AZD2014 resulted in significant blockade on both TORC1 and TORC2 downstream effectors (>80% inhibition of p-AKT, p-S6 and p-4EBP1) as well as significant down-regulation of cyclin D1 levels (>70%). Similarly, inhibition of CDK4/6 using palbociclib (300nM) caused >80% inhibition of p-RB and subsequent cell cycle blockade.The effects observed in breast cancer cell lines were recapitulated in vivo using the MCF7 xenograft model, where tumor regressions (>105%) were observed with the combination. Furthermore, combining AZD2014, palbociclib and fulvestrant in this model also caused tumor regressions.To assess bone marrow tolerability of this combination, we investigated the response of human bone marrow multipotent progenitors (CD34+) in vitro. Palbociclib caused transient cell cycle G1 arrest. The combination of palbociclib with AZD2014 delayed entry into cell cycle, but did not have significant impact on the cell viability (<15%). We hypothesized that inhibition of CDK4/6 in combination with AZD2014 would also significantly affect pathway outputs and the transcriptional events downstream of the transcription factors E2F (e.g. CENPE, CCNA2, CDC6 and E2F1) and ER (e.g. PR). We therefore measured specific gene signatures downstream of these receptors in breast cancer cell lines. The ability to dose AZD2014 intermittently compared with rapalogues, together with its ability to block signaling from both TORC1 and 2, make this compound an ideal candidate for combining with CDK4/6 inhibitors such as palbociclib. Furthermore, addition of anti-hormonal therapies such as fulvestrant to this combination may provide additional benefit to breast cancer patients. Citation Format: Claire Crafter, Jon Curwen, Oona Delpuech, Lenka Oplustilova, Stephen Green, Henry Brown, Cath Trigwell, Sabina Cosulich. Targeting interdependent signaling pathways to increase the durability and magnitude of response: promising combination therapy with dual mTORC1/2 inhibitors and CDK4/6 inhibitors. [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 4719. doi:10.1158/1538-7445.AM2015-4719

Abstract LB-060: A phosphoproteomic comparison of BRAF(V600E) and MKK1/2 inhibition in melanoma

Abstract Inhibitors of oncogenic B-RAF(V600E) and MKK1/2 have yielded remarkable responses in B-RAF(V600E)-positive melanoma patients. However, the efficacy of these inhibitors is limited by the inevitable onset of resistance. Despite the fact that these inhibitors target the same pathway, combination treatment with B-RAF(V600E) and MKK1/2 inhibitors has been shown to improve both response rates and progression-free survival in B-RAF(V600E) melanoma patients. To provide insight into the molecular nature of the combinatorial response, we used quantitative mass spectrometry to characterize the inhibitor-dependent phosphoproteome of human melanoma cells treated with the B-RAF(V600E) inhibitor PLX4032 (vemurafenib) or the MKK1/2 inhibitor AZD6244 (selumetinib). In three replicate experiments, we quantified changes at a total of 23,986 phosphosites on 4,722 proteins. This included 1,317 phosphosites that reproducibly decreased in response to at least one inhibitor. Phosphosites that responded to both inhibitors grouped into networks that included the nuclear pore complex, growth factor signaling, and transcriptional regulators. While the majority of phosphosites were responsive to both inhibitors, we identified 16 sites that decreased only in response to PLX4032, suggesting rare instances where oncogenic B-RAF signaling occurs in an MKK1/2-independent manner. Only two phosphosites were identified that appeared to be uniquely responsive to AZD6244. When cells were treated with the combination of AZD6244 and PLX4032 at subsaturating concentrations (30 nM), responses at nearly all phosphosites were additive. We conclude that AZD6244 does not substantially widen the range of phosphosites inhibited by PLX4032 and that the benefit of the drug combination is best explained by their additive effects on suppressing ERK1/2 signaling. Comparison of our results to another recent ERK1/2 phosphoproteomics study revealed a surprising degree of variability in the sensitivity of phosphosites to MKK1/2 inhibitors in human cell lines, revealing unexpected cell specificity in the molecular responses to pathway activation. Citation Format: Scott Stuart, Natalie Ahn, Stephane Houel, William Old. A phosphoproteomic comparison of BRAF(V600E) and MKK1/2 inhibition in melanoma. [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 LB-060. doi:10.1158/1538-7445.AM2015-LB-060

Abstract B24: A phosphoproteomic comparison of oncogenic BRAF and MKK1/2 inhibition in human melanoma

Abstract The purpose of this study was to use phosphoproteomics to gain insight into the different clinical responses to BRAF inhibition, MKK1/2 inhibition, and combined BRAF/MKK inhibition. Experiments were designed to test whether BRAF inhibition may affect a distinct subset of targets that MKK1/2 inhibition does not and whether BRAF and MKK1/2 inhibitors work additively or synergistically when used in combination. To this end, we employed a SILAC-based method to quantify changes in the phosphoproteome of WM239A melanoma cells treated with a clinically relevant BRAF inhibitor (PLX4032; 10μM) or MEK1/2 inhibitor (AZD6244; 10μM) inhibitor. Phosphopeptides were enriched by titanium dioxide prior to fractionation on an electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) column. In a set of three biological replicate experiments, we quantified changes at 23,986 phosphosites on 4,722 proteins. We identified 1,356 phosphosites that decreased in abundance, and 341 that increased in abundance, in response to treatment with either PLX4032 or AZD6244. The overwhelming majority of phosphosites were responsive to both inhibitors. Of the phosphosites that decreased in abundance, 13 responded only to PLX4032, suggesting that certain targets are regulated by BRAF but not MKK1/2. In contrast, we failed to identify any phosphosites that responded only to AZD6244. To determine whether the therapeutic benefit of combined BRAF and MKK1/2 inhibition may be a result of drug synergy, we treated cells either with a single inhibitor or with a combination of AZD6244 and PLX4032, each at submaximal doses (30 nM). We then compared the magnitude of the responses observed with the combination treatment to those observed for each drug alone. Comparison of observed versus predicted changes at 6,463 phosphosites demonstrated that the primary effect of the two inhibitors is additive. Taken together, we show that the combination of BRAF and MKK1/2 inhibitor leads to predominantly additive interactions, and that the BRAF inhibitor has a potential for regulating phosphorylation events that are not targeted by MKK1/2 inhibitor. Citation Format: Scott Stuart, Stephane Houel, Nan Wang, William Old, Natalie Ahn. A phosphoproteomic comparison of oncogenic BRAF and MKK1/2 inhibition in human melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B24.