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Table S1. Pharmacokinetic parameters of encorafenib and alpelisib in patients at steady state (cycle 2 day 1). Table S2. Criteria for defining dose-limited toxicities. Supplementary Figure 1. Radiological images of response for a patient treated with the dual-combination therapy of encorafenib and cetuximab. Supplementary Figure 2. Time on study by response for patients treated with the dual-combination therapy of encorafenib and cetuximab and patients treated with the triple-combination therapy of encorafenib, alpelisib, and cetuximab.

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(A) Average body weight of mice treated with each BRAF inhibitor in the experiment depicted in Figure 1C; (B) Encorafenib plasma exposure in mice following the last dose from the experiment depicted in Figure 1C. To estimate daily exposure on the BID regimen, multiply the AUC x 2. Encorafenib is 98.6% protein bound in mouse plasma; (C) Gastric hyperplasia and hyperkeratosis observed in the forestomach of mice treated with BRAFi. Hematoxylin and eosin stains of forestomach sections from mice following 14 days of treatment with vehicle, encorafenib, dabrafenib, or vemurafenib from the study depicted in Figure 1C.

Biochemical and cellular potency of encorafenib, dabrafenib, and vemurafenib in biochemical and cell-based assays. Purified BRAF V600E kinase domain was used in the biochemical experiments, using kinase-dead MEK1 as a substrate and a phosphorylated MEK1 (Ser217/221) alpha-screen readout. A375 cells were incubated with inhibitors for 3 hours to determine the phosphorylated ERK (pERK) half maximal effective concentration (EC50) and 72 hours to determine the proliferation EC50

Purpose: Encorafenib, a selective BRAF inhibitor (BRAFi), has a pharmacologic profile that is distinct from that of other clinically active BRAFis. We evaluated encorafenib in a phase I study in patients with BRAFi treatment-naïve and pretreated BRAF-mutant melanoma.Experimental Design: The pharmacologic activity of encorafenib was first characterized preclinically. Encorafenib monotherapy was then tested across a range of once-daily (50–700 mg) or twice-daily (75–150 mg) regimens in a phase I, open-label, dose-escalation and -expansion study in adult patients with histologically confirmed advanced/metastatic BRAF-mutant melanoma. Study objectives were to determine the maximum tolerated dose (MTD) and/or recommended phase II dose (RP2D), characterize the safety and tolerability and pharmacokinetic profile, and assess the preliminary antitumor activity of encorafenib.Results: Preclinical data demonstrated that encorafenib inhibited BRAF V600E kinase activity with a prolonged off-rate and suppressed proliferation and tumor growth of BRAF V600E–mutant melanoma models. In the dose-escalation phase, 54 patients (29 BRAFi-pretreated and 25 BRAFi-naïve) were enrolled. Seven patients in the dose-determining set experienced dose-limiting toxicities. Encorafenib at a dose of 300 mg once daily was declared the RP2D. In the expansion phase, the most common all-cause adverse events were nausea (66%), myalgia (63%), and palmar–plantar erythrodysesthesia (54%). In BRAFi-naïve patients, the overall response rate (ORR) and median progression-free survival (mPFS) were 60% and 12.4 months [95% confidence interval (CI), 7.4–not reached (NR)]. In BRAFi-pretreated patients, the ORR and mPFS were 22% and 1.9 months (95% CI, 0.9–3.7).Conclusions: Once-daily dosing of single-agent encorafenib had a distinct tolerability profile and showed varying antitumor activity across BRAFi-pretreated and BRAFi-naïve patients with advanced/metastatic melanoma. Clin Cancer Res; 23(18); 5339–48. ©2017 AACR.

Supplementary Figure 1, Table 1 from De novo Discovery of a γ-Secretase Inhibitor Response Signature Using a Novel In vivo Breast Tumor Model

Notch pathway signaling plays a fundamental role in normal biological processes and is frequently deregulated in many cancers. Although several hypotheses regarding cancer subpopulations most likely to respond to therapies targeting the Notch pathway have been proposed, clinical utility of these predictive markers has not been shown. To understand the molecular basis of γ-secretase inhibitor (GSI) sensitivity in breast cancer, we undertook an unbiased, de novo responder identification study using a novel genetically engineered in vivo breast cancer model. We show that tumors arising from this model are heterogeneous on the levels of gene expression, histopathology, growth rate, expression of Notch pathway markers, and response to GSI treatment. In addition, GSI treatment of this model was associated with inhibition of Hes1 and proliferation markers, indicating that GSI treatment inhibits Notch signaling. We then identified a pretreatment gene expression signature comprising 768 genes that is significantly associated with in vivo GSI efficacy across 99 tumor lines. Pathway analysis showed that the GSI responder signature is enriched for Notch pathway components and inflammation/immune-related genes. These data show the power of this novel in vivo model system for the discovery of biomarkers predictive of response to targeted therapies, and provide a basis for the identification of human breast cancers most likely to be sensitive to GSI treatment. [Cancer Res 2009;69(23):8949–57]

Introduction: Cinrebafusp alfa is a first-in-class bispecific antibody-Anticalin® fusion protein that targets HER2 and the costimulatory receptor 4-1BB, leading to enhanced activation of T cells in the tumor, while avoiding liver toxicity. In a Phase 1 monotherapy study, cinrebafusp alfa was well tolerated and showed single agent activity in patients with HER2-positive malignancies. Based on pharmacokinetics (PK), pharmacodynamics and clinical efficacy data, a loading dose of 18mg/kg Q2W in cycle 1 followed by 8mg/kg Q2W in subsequent cycles as maintenance dose was chosen for the Phase 2 study. Methods: This Phase 2 study enrolled patients with metastatic gastric/gastroesophageal junction cancer and confirmed HER2-high status (IHC 3+ or IHC 2+ with HER2/neu gene amplification) who had received one or two prior lines of treatment. Patients received cinrebafusp alfa in combination with ramucirumab and paclitaxel. Primary endpoint was objective response rate (ORR), and key secondary endpoints included safety profile, PK, and immunogenicity. Summary of data: 5 patients were enrolled before enrollment ceased for reasons unrelated to safety or efficacy profile. As of the cut-off date (19-Dec-2022), 5 out of 5 patients achieved a partial response (PR) as best overall response with tumor lesion shrinkage ranging between 35% and 66%. Two of the PRs have been confirmed, one PR is unconfirmed, and 2 patients with currently unconfirmed PRs are continuing to receive treatment. In total, 2 of 5 patients discontinued treatment due to disease progression after 140 and 113 days (patient discontinued cinrebafusp alfa after 42 days but continued on ramucirumab and paclitaxel); 3 patients remain on treatment. Median duration of response was 3.8 months at time of data cut-off. The most frequent treatment emergent adverse events (TEAEs) included Grade 1/2 fatigue (4 patients) and Grade 1-3 diarrhea (4 patients). Infusion related reactions (Grade 2/3) were seen in 1 patient, leading eventually to discontinuation of cinrebafusp alfa. With regards to prior treatment history, all patients received trastuzumab and chemotherapy. 2 patients also received trastuzumab deruxtecan while 4 patients previously received anti-PD1 therapy. Conclusions: The combination of cinrebafusp alfa with ramucirumab and paclitaxel was safe and tolerated in the 5 patients treated. Preliminary activity of cinrebafusp alfa in combination with ramucirumab and paclitaxel demonstrated a high response rate indicating that the combination can elicit clinical responses in patients who have progressed on trastuzumab deruxtecan or checkpoint inhibitor regimens. Citation Format: Geoffrey Ku, Jeeyun Lee, Kayti Aviano, Tim Demuth, Laura-Carolin Hasenkamp, Shane A. Olwill. Combination of cinrebafusp alfa with ramucirumab and paclitaxel is well tolerated and elicits encouraging clinical activity in patients with HER2-positive gastric/gastroesophageal junction (GEJ) adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT154.

PRS-344/S095012 is a bispecific antibody-Anticalin® fusion protein targeting PD-L1 and 4-1BB. It is designed to block the PD-1/PD-L1 axis and localize 4-1BB co-stimulation to PD-L1-positive tumor microenvironment (TME) or tumor draining lymph nodes with the aim of maximizing antitumor immunity and increasing the therapeutic window of 4-1BB monoclonal antibodies (mAbs). Multiple in vitro assays have shown that PRS-344/S095012 inhibits PD-1/PD-L1 signaling while simultaneously activating 4-1BB signaling on T cells. This resulted in a synergistic effect on both pathways leading to anti-tumor immune responses. PRS-344/S095012 presents mAb-like pharmacokinetics in vivo and non-clinical mouse models demonstrated dose-dependent efficacy in anti-PD-L1 refractory tumors. In the higher dose range, complete tumor regression was achieved in PD-L1 high and PD-L1 low expressing xenograft mouse models. This first-in-human (FIH), phase 1/2, open-label, multi center, dose escalation and cohort expansion study is designed to determine the safety and tolerability of intravenously administered PRS-344/S095012 in patients with advanced and/or metastatic solid tumors (NCT05159388). Patients with histologically confirmed diagnosis of unresectable, locally advanced, or metastatic solid tumors for which standard treatment options are not available, no longer effective, or not tolerated are eligible. Patients must have Eastern Cooperative Oncology Group (ECOG) status 0 or 1, RECIST 1.1 measurable disease and should have documented progression on prior therapy. Prior therapy with 4-1BB agonists is prohibited. Phase 1 is a dose escalation guided by a Bayesian Logistic Regression Model and a 28-day dose limiting toxicities (DLT) period. Primary objectives are safety and tolerability of PRS-344/S095012; secondary objectives include exploration of antitumor activity and evaluation of pharmacokinetics. Pharmacodynamics and comprehensive biomarker program will be explored. Additional patients may be enrolled to backfill cohort(s)to further explore pharmacokinetic/pharmacodynamic signals. Phase 2 is an expansion with primary objective of anti-tumor activity. First patient was dosed in November 2021 and the study is planned to enroll at sites in Belgium, Spain and Australia. Citation Format: Christiane Jungels, Nuria Kotecki, Emiliano Calvo, Elena Garralda, Timothy Price, Xiaojiang Zahn, Atif Abbas, Lisa Mahnke, Winrich Rauschning, Aizea Morales-Kastresana, Lucia Pattarini, Birgit Bossenmaier, Alix Scholer-Dahirel, Tim Demuth, Julie Legrande. Study of PRS-344/S095012 a PD-L1/4-1BB bispecific antibody-Anticalin®-fusion in patients with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT255.

Background: For patients (pts) with HER2-overexpressing metastatic gastric cancer, trastuzumab + chemotherapy +/- pembrolizumab is a standard first-line option but only provides an incremental overall survival (OS) benefit vs chemotherapy. Anticalin® proteins are recombinant human proteins based on lipocalins. Cinrebafusp alfa, a first-in-class bispecific antibody-Anticalin fusion protein, targets HER2 and the co-stimulatory immune receptor 4-1BB on T cells. In a previous Phase 1 study cinrebafusp alfa monotherapy was generally well tolerated and showed deep and durable responses in patients with HER2-positive gastrointestinal malignancies at doses of 8mg/kg Q2W and 18mg/kg Q2W. Significant induction of plasma 4-1BB as well as increase of CD8+ cells was observed in on-treatment tumor biopsies at active dose levels (Piha-Paul, SITC 2020). Based on pharmacokinetics (PK), pharmacodynamics (PD) and clinical efficacy data, a phase 2 dose of 18mg/kg Q2W in C1 followed by 8mg/kg Q2W maintenance was chosen. Methods: This is a global, open-label, multicenter, two-arm phase 2 trial of cinrebafusp alfa in patients with metastatic gastric or gastroesophageal junction cancer. Arm 1 is enrolling patients with HER2 high (Immunohistochemistry (ICH) 3+ or IHC 2+ with HER2/neu gene amplification) disease. Pts who have received one prior treatment regimen for metastatic disease, including HER2-directed therapy such as trastuzumab are eligible. Pts will receive cinrebafusp alfa in combination with ramucirumab and paclitaxel. Arm 2 is enrolling patients with HER2 low (IHC 1+ or 2+ without HER2/neu gene amplification) disease. Pts who have received at least one prior treatment regimen for metastatic disease are eligible. Pts will receive cinrebafusp alfa in combination with tucatinib. After a run-in consisting of 3 pts in each arm, an additional 17 patients will be enrolled in each arm. For Arm 1, an additional 40 patients may be enrolled after a futility analysis has been conducted. Treatment will continue until disease progression, unacceptable toxicity, or consent withdrawal. Primary endpoint is confirmed overall response rate per RECIST 1.1 and key secondary endpoints are duration of response, progression free survival, overall survival, safety, PK, and immunogenicity. Recruitment is ongoing. Approximately 10 sites in 3 countries in US, Asia and Europe are expected to participate. Citation Format: Sarina A. Piha-Paul, Mukul Gupta, Do-Youn Oh, Yeul Hong Kim, Sun Young Rha, Yoon-Koo Kang, Marc Diez Garcia, Tania Fleitas Kanonnikoff, Virginia Arrazubi Arrula, Kayti Aviano, Tim Demuth, Geoffrey Ku. A phase 2, multi-center, open-label study of cinrebafusp alfa (PRS-343) in combination with ramucirumab and paclitaxel in patients with HER2-positive gastric or gastroesophageal junction (GEJ) adenocarcinoma and in combination with tucatinib in patients with HER2 low gastric or gastroesophageal junction (GEJ) adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT122.

Purpose: Encorafenib, a selective BRAF inhibitor (BRAFi), has a pharmacologic profile that is distinct from that of other clinically active BRAFis. We evaluated encorafenib in a phase I study in patients with BRAFi treatment-naïve and pretreated BRAF-mutant melanoma.Experimental Design: The pharmacologic activity of encorafenib was first characterized preclinically. Encorafenib monotherapy was then tested across a range of once-daily (50–700 mg) or twice-daily (75–150 mg) regimens in a phase I, open-label, dose-escalation and -expansion study in adult patients with histologically confirmed advanced/metastatic BRAF-mutant melanoma. Study objectives were to determine the maximum tolerated dose (MTD) and/or recommended phase II dose (RP2D), characterize the safety and tolerability and pharmacokinetic profile, and assess the preliminary antitumor activity of encorafenib.Results: Preclinical data demonstrated that encorafenib inhibited BRAF V600E kinase activity with a prolonged off-rate and suppressed proliferation and tumor growth of BRAF V600E–mutant melanoma models. In the dose-escalation phase, 54 patients (29 BRAFi-pretreated and 25 BRAFi-naïve) were enrolled. Seven patients in the dose-determining set experienced dose-limiting toxicities. Encorafenib at a dose of 300 mg once daily was declared the RP2D. In the expansion phase, the most common all-cause adverse events were nausea (66%), myalgia (63%), and palmar–plantar erythrodysesthesia (54%). In BRAFi-naïve patients, the overall response rate (ORR) and median progression-free survival (mPFS) were 60% and 12.4 months [95% confidence interval (CI), 7.4–not reached (NR)]. In BRAFi-pretreated patients, the ORR and mPFS were 22% and 1.9 months (95% CI, 0.9–3.7).Conclusions: Once-daily dosing of single-agent encorafenib had a distinct tolerability profile and showed varying antitumor activity across BRAFi-pretreated and BRAFi-naïve patients with advanced/metastatic melanoma. Clin Cancer Res; 23(18); 5339–48. ©2017 AACR.

Preclinical evidence suggests that concomitant BRAF and EGFR inhibition leads to sustained suppression of MAPK signaling and suppressed tumor growth in BRAFV600E colorectal cancer models. Patients with refractory BRAFV600-mutant metastatic CRC (mCRC) were treated with a selective RAF kinase inhibitor (encorafenib) plus a monoclonal antibody targeting EGFR (cetuximab), with (n = 28) or without (n = 26) a PI3Kα inhibitor (alpelisib). The primary objective was to determine the maximum tolerated dose (MTD) or a recommended phase II dose. Dose-limiting toxicities were reported in 3 patients receiving dual treatment and 2 patients receiving triple treatment. The MTD was not reached for either group and the phase II doses were selected as 200 mg encorafenib (both groups) and 300 mg alpelisib. Combinations of cetuximab and encorafenib showed promising clinical activity and tolerability in patients with BRAF-mutant mCRC; confirmed overall response rates of 19% and 18% were observed and median progression-free survival was 3.7 and 4.2 months for the dual- and triple-therapy groups, respectively.Significance: Herein, we demonstrate that dual- (encorafenib plus cetuximab) and triple- (encorafenib plus cetuximab and alpelisib) combination treatments are tolerable and provide promising clinical activity in the difficult-to-treat patient population with BRAF-mutant mCRC. Cancer Discov; 7(6); 610–9. ©2017 AACR.See related commentary by Sundar et al., p. 558.This article is highlighted in the In This Issue feature, p. 539

γ-Secretase mediates amyloid production in Alzheimer's disease (AD) and oncogenic activity of Notch. γ-Secretase inhibitors (GSIs) are thus of interest for AD and oncology. A peripheral biomarker of Notch activity would aid determination of the therapeutic window and dosing regimen for GSIs, given toxicities associated with chronic Notch inhibition. This study examined the effects of GSI MK-0752 on blood and hair follicle transcriptomes in healthy volunteers. The effects of a structurally diverse GSI on rhesus blood and hair follicles were also compared. Significant dose-related effects of MK-0752 on transcription were observed in hair follicles, but not blood. The GSI biomarker identified in follicles exhibited 100% accuracy in a clinical test cohort, and was regulated in rhesus by a structurally diverse GSI. This study identified a translatable, accessible pharmacodynamic biomarker of GSI target engagement and provides proof of concept of hair follicle RNA as a translatable biomarker source.

Preclinical evidence suggests that concomitant BRAF and EGFR inhibition leads to sustained suppression of MAPK signaling and suppressed tumor growth in BRAF V600E colorectal cancer (CRC) models. Patients with refractory BRAF V600–mutant metastatic CRC (mCRC) were treated with a selective RAF kinase inhibitor (encorafenib) plus a monoclonal antibody targeting EGFR (cetuximab), with (n = 28) or without (n = 26) a PI3K-alpha inhibitor (alpelisib). The primary objective was to determine the maximum tolerated dose (MTD) or a recommended phase 2 dose. Dose-limiting toxicities were reported in three patients receiving dual- and two patients receiving triple-treatment. The MTD was not reached for either group and the Phase 2 doses were selected as 200 mg encorafenib (both groups) and 300 mg alpelisib. Combinations of cetuximab and encorafenib show promising clinical activity and tolerability in patients with BRAF-mutant mCRC; confirmed overall response rates of 19% and 18% were observed, and median progression-free survival was 3.7 and 4.2 months, for the dual- and triple-therapy groups, respectively.

Purpose Aberrant Notch signaling has been implicated in the pathogenesis of many human cancers. MK-0752 is a potent, oral inhibitor of γ-secretase, an enzyme required for Notch pathway activation. Safety, maximum-tolerated dose, pharmacokinetics (PKs), pharmacodynamics, and preliminary antitumor efficacy were assessed in a phase I study of MK-0752. Patients and Methods MK-0752 was administered in three different schedules to patients with advanced solid tumors. Hair follicles were collected at higher dose levels to assess a gene signature of Notch inhibition. Results Of 103 patients who received MK-0752, 21 patients received a continuous once-daily dosing at 450 and 600 mg; 17 were dosed on an intermittent schedule of 3 of 7 days at 450 and 600 mg; and 65 were dosed once per week at 600, 900, 1,200, 1,500, 1,800, 2,400, 3,200, and 4,200 mg. The most common drug-related toxicities were diarrhea, nausea, vomiting, and fatigue. PKs (area under the concentration-time curve and maximum measured plasma concentration) increased in a less than dose proportional manner, with a half-life of approximately 15 hours. Significant inhibition of Notch signaling was observed with the 1,800- to 4,200-mg weekly dose levels, confirming target engagement at those doses. One objective complete response and an additional 10 patients with stable disease longer than 4 months were observed among patients with high-grade gliomas. Conclusion MK-0752 toxicity was schedule dependent. Weekly dosing was generally well tolerated and resulted in strong modulation of a Notch gene signature. Clinical benefit was observed, and rational combination trials are currently ongoing to maximize clinical benefit with this novel agent.

Blocking Notch signaling in pancreatic cancer promotes hypoxia and cell death., Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease that is refractory to medical intervention. Notch pathway antagonism has been shown to prevent pancreatic preneoplasia progression in mouse models, but potential benefits in the setting of an established PDA tumor have not been established. We demonstrate that the gamma secretase inhibitor MRK003 effectively inhibits intratumoral Notch signaling in the KPC mouse model of advanced PDA. Although MRK003 monotherapy fails to extend the lifespan of KPC mice, the combination of MRK003 with the chemotherapeutic gemcitabine prolongs survival. Combination treatment kills tumor endothelial cells and synergistically promotes widespread hypoxic necrosis. These results indicate that the paucivascular nature of PDA can be exploited as a therapeutic vulnerability, and the dual targeting of the tumor endothelium and neoplastic cells by gamma secretase inhibition constitutes a rationale for clinical translation.

Background: New strategies to enhance endocrine therapy (ET) efficacy and/or overcome resistance by targeting key survival pathways are needed. Preclinical data indicate that unwanted effects of ET include reactivation of the Notch pathway, critical for breast tumor initiating (stem) cells. Notch inhibition with gamma secretase inhibitors (GSI) enhances tamoxifen (tam) efficacy in xenografts, but impact of GSI+ET in human breast cancer (BC) is unknown. Our objective was to add short exposure of the GSI MK-0752 to ongoing tam or letrozole (let) in the presurgical window to assess feasibility, safety and biomarker/pathway impact in a 20-patient (pt) pilot study (ClinTrials.gov NCT00756717). We previously evaluated several biomarkers in the first cohort, which showed promise with Notch and proliferation inhibition. We present new results adding the final cohort, plus additional biomarkers and microarray analyses. Methods: Pts with early stage ER+ BC received 25 days (d) of ET. MK-0752 was added d15 (350 mg PO 3d on, 4d off, 3d on) with definitive surgery d25. Core biopsies were done at baseline, d14 and d25, with qRT-PCR for Notch-related and other genes critical to stem cell renewal/proliferation. Gene expression levels after GSI (d25) vs ET alone (d14) were analyzed and d25 changes in all pts combined for each gene were compared. Microarray expression estimates and modeling were performed using dCHip and Red-R, implementing gene-wise comparisons using Limma. Probes were defined as significantly regulated by paired t tests if p ≤ 0.001 for the comparisons of baseline to tam/let and tam/let to tam/let+GSI. Data were exploratory so all probe data were included in the modeling, and no corrections for multiple comparisons were used. Differentially expressed genes were submitted to DAVID for pathway analysis. Results: Of 22 pts accrued, 20 (11 tam, 9 let) were evaluable, meeting accrual goals (2 withdrew before MK-0752); 19 completed therapy to date. Toxicity was minimal. Significant (p Conclusions: Short exposure of MK-0752 added to ET was feasible, well tolerated, and resulted in significant biomarker response in all tumors. MK-0752 favorably modulated proliferation, apoptosis, stem cell and metastasis-related targets, and impacted critical cancer pathways. This suggests potential roles for MK-0752 in optimizing endocrine therapy and overcoming endocrine resistance. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S1-5.

The purpose of this study was to determine the capacity of MK-1775, a potent Wee-1 inhibitor, to abrogate the radiation-induced G2 checkpoint arrest and modulate radiosensitivity in glioblastoma cell models and normal human astrocytes. The radiation-induced checkpoint response of established glioblastoma cell lines, glioblastoma neural stem (GNS) cells, and astrocytes were determined in vitro by flow cytometry and in vivo by mitosis-specific staining using immunohistochemistry. Mechanisms underlying MK-1775 radiosensitization were determined by mitotic catastrophe and γH2AX expression. Radiosensitivity was determined in vitro by the clonogenic assay and in vivo by tumor growth delay. MK-1775 abrogated the radiation-induced G2 checkpoint and enhanced radiosensitivity in established glioblastoma cell lines in vitro and in vivo, without modulating radiation response in normal human astrocytes. MK-1775 appeared to attenuate the early-phase of the G2 checkpoint arrest in GNS cell lines, although the arrest was not sustained and did not lead to increased radiosensitivity. These results show that MK-1775 can selectively enhance radiosensitivity in established glioblastoma cell lines. Further work is required to determine the role Wee-1 plays in checkpoint activation of GNS cells. Mol Cancer Ther; 10(12); 2405–14. ©2011 AACR.

Background: Numb-mediated regulation of Notch signaling pathway might play a causative role in early breast cancer. Little information is available on whether Numb expression differs according to tumor characteristics including hormone receptor status. We investigated Numb expression status in archival tissue from primary tumor of 197 breast cancer patients identified within the 2004 cohort of the Registry of the Danish Breast Cancer Cooperative Group's (DBCG), where patients were treated according to the guidelines of the DBCG. Materials and Methods: Numb expression was measured by immunohistochemistry method, and Numb(-) was defined as Numb immunoreactivity present in less than 10% of the invasive tumor cells. Chi-square test was used to assess the correlation between Numb(-) and hormone receptor status. Results: The median age was 57 years (range, 26-98), 32% were premenopausal. 82% were ductal, 9% lobular, 9% others. 10% were grade I, 41% grade II, 47% grade III, 2% non-graded. 70% were node positive. 60% were ER+, 41% PR+, and 24% were HER2+ by IHC and FISH. 58% were operated by mastectomy, 42% by lumpectomy. The adjuvant treatment comprised Tamoxifen (36%), cyclophosphamide/methotrexate/fluorouracil (CEF, 19%), CEF+Tamoxifen (11%), and 22% were missing treatment information. 49% relapsed and 3% metastasized during 10 year of follow up. Overall, 10% (19 out of 197) of patients were Numb(-). Numb expression was significantly associated with hormone status, with a Numb(-) frequency of 24% (9/37) among ER+/PR-/HER2-, 9% (6/65) among ER+/PR+/HER2-, 6% (3/47) among HER+ (1/16 among ER+/PR+/HER2+ and 2/31 among ER-/PR-/HER2+), and 2% (1/47) among ER-/PR-/HER2- patients (P Discussion: Our results suggested Numb(-) is correlated with hormone status, with the higher frequency of Numb(-) among ER+/PR-/HER2- patients, and lower frequency among other groups, particularly the triple negative group. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-12-09.

Background: Breast tumor initiating cells (TIC) use Notch receptors/ligands with other pathways for self renewal, resulting in tumor proliferation and progression. We showed that Notch inhibition with gamma secretase inhibitors (GSI) potentiates the effects of tamoxifen (tam) in xenografts (Rizzo et al. Cancer Res 2008). It is unknown whether GSIs plus endocrine therapy result in modulation of Notch and other proliferation markers in human breast cancer. Our objective was to add short exposure of the GSI MK-0752 to ongoing tam or letrozole (letr) during the presurgical window to determine 1) feasibility, 2) safety/tolerance, and 3) impact on biomarkers. We report the initial cohort of this pilot study (ClinTrials. gov NCT00756717). Methods: Patients (pts) with early stage ERα + breast cancer were treated with 25 days of tam or letr. On day 15 MK-0752 was added to endocrine therapy (350 mg orally 3 days on, 4 days off, 3 days on), with definitive surgery day 25. Formalin fixed, paraffin embedded biopsies were obtained at baseline, day 14 and final surgery, with histologic confirmation of tumor content >50% and RNA extraction by standard methods. Q-PCR was done for Notch1, Notch3, Notch4, Deltex, Jagged1, c-myc, HEY1, HEY2, HES1, PS2, C-Myc, Cyclin A2, NOXA (pro-apoptotic protein), Ki67, Dicer-1, RPL13 (internal control). Ct averages for 3 replicates were used and mRNA levels were calculated by the 2ΔΔCt method. Baseline gene expression levels were used as comparators for days 14 and 25 levels in each pt. The first cohort of 10 pts was analyzed to determine if enough signals were present to justify expanding the cohort at this dose to 20 pts and possibly test a second cohort on an alternate MK-0752 dose/schedule. Results: The initial cohort of 10 pts completed all therapy (4 tam, 6 letr), all biopsies and definitive surgery on schedule. One other pt withdrew prior to starting MK-0752 due to hypertension. Toxicity was minimal: grade 1 periorbital edema/cough, nausea, and axillary paresthesias in 1 pt each; grade 1 facial rash, 2 pts; and grade 2 fatigue, 1 pt. There was no diarrhea or surgical complications. Significant changes occurred in molecular marker levels after MK-0752 plus tam/letr (day 25) vs. end of tam/letr alone (day 14) as follows: Ki67 mRNA decreased in 9/10 pts; Notch4 decreased, 10/10; NOXA increased, 6/10; and Notch1 decreased, 6/10. Other markers showed inter-individual variations and will be presented, along with results of the global gene expression profiling (in progress). Conclusions: The addition of a short exposure of the GSI MK-0752 to ongoing endocrine therapy was feasible, safe, and well tolerated in pts with ERα + early breast cancer prior to definitive surgery. It results in anti-proliferative and pro-apoptotic effects at the molecular level. Notch4, which plays a key role in breast TIC, was the most consistent molecular marker of response in this setting. This suggests a potential anti-TIC effect of this combination and a role in overcoming endocrine resistance. Accrual to the expanded cohort is underway. If findings are confirmed, the second study with alternate MK-0752 dose/schedule may commence. Funding: Swim Across America, Inc. (clinical trial costs); Merck (drug supply, profiling) Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD05-12.

Notch pathway signaling plays a fundamental role in normal biological processes and is frequently deregulated in many cancers. Although several hypotheses regarding cancer subpopulations most likely to respond to therapies targeting the Notch pathway have been proposed, clinical utility of these predictive markers has not been shown. To understand the molecular basis of γ-secretase inhibitor (GSI) sensitivity in breast cancer, we undertook an unbiased, de novo responder identification study using a novel genetically engineered in vivo breast cancer model. We show that tumors arising from this model are heterogeneous on the levels of gene expression, histopathology, growth rate, expression of Notch pathway markers, and response to GSI treatment. In addition, GSI treatment of this model was associated with inhibition of Hes1 and proliferation markers, indicating that GSI treatment inhibits Notch signaling. We then identified a pretreatment gene expression signature comprising 768 genes that is significantly associated with in vivo GSI efficacy across 99 tumor lines. Pathway analysis showed that the GSI responder signature is enriched for Notch pathway components and inflammation/immune-related genes. These data show the power of this novel in vivo model system for the discovery of biomarkers predictive of response to targeted therapies, and provide a basis for the identification of human breast cancers most likely to be sensitive to GSI treatment. [Cancer Res 2009;69(23):8949–57]

Invasion is a defining hallmark of glioblastoma multiforme, just as metastasis characterizes other high-grade tumors. Glial tumors invariably recur due to the regrowth of invasive cells, which are unaffected by standard treatment modalities. Drivers of glioma invasion include autocrine signals propagated by secreted factors that signal through receptors on the tumor. These secreted factors are able to diffuse through the peritumoral stroma, thereby influencing parenchymal cells that surround the tumor mass. Here we describe various autocrine motility factors that are expressed by invasive glioma cells and explore the effects that they may have on normal cells present in the path of invasion. Conversely, normal brain parenchymal cells secrete ligands that can stimulate receptors on invasive glioma cells and potentially facilitate glioma invasion or create a permissive microenvironment for malignant progression. Parallel observations have been made for solid tumors of epithelial origin, in which parenchymal and stromal cells either support or suppress tumor invasion. Most autocrine and paracrine interactions involved in glioma invasion constitute known signaling systems in stages of central nervous system development that involve the migration of precursor cells that populate the developing brain. Key paracrine interactions between glioma cells and the brain microenvironment can influence glioma pathobiology and therefore contribute to its poor prognosis. Current therapies for glioma that could have an impact on paracrine communication between tumors and normal cells are discussed. We suggest that cells in the normal brain parenchyma be considered as potential targets for adjuvant therapies to control glioma growth because such cells are less likely to develop resistance than glioma cells.

Glioblastoma multiforme is the most common and lethal primary malignant brain tumor. Although considerable progress has been made in technical proficiencies of surgical and radiation treatment for brain tumor patients, the impact of these advances on clinical outcome has been disappointing, with median survival time not exceeding 15 months. Over the last 30 years, no significant increase in survival of patients suffering from this disease has been achieved. A fundamental source of the management challenge presented in glioma patients is the insidious propensity of tumor invasion into distant brain tissue. Invasive tumor cells escape surgical removal and geographically dodge lethal radiation exposure and chemotherapy. Recent improved understanding of biochemical and molecular determinants of glioma cell invasion provide valuable insight into the underlying biological features of the disease, as well as illuminating possible new therapeutic targets. These findings are moving forward to translational research and clinical trials as novel antiglioma therapies.

Glioma cells that migrate out of the main tumor mass into normal brain tissue contribute to the failure of most gliomas to respond to treatment. Treatments that target migratory glioma cells may enhance the therapeutic response. Multiple lines of evidence suggest that suppression of apoptosis accompanies activation of the migratory phenotype. Here, we determine whether migration and apoptosis are consistently linked in glioma cells and whether manipulation of migration influences cytotoxic therapy-induced apoptosis. Camptothecin and Trail-induced apoptosis were decreased 2-5-fold in actively migrating glioma cells relative to migration-restricted cells. Consistent with a mechanistic link between migration and apoptosis, the dose-response for stimulation of migration on laminin was inversely proportional to apoptosis induction. Treatment of glioma cells with migration inhibitors alone had little effect on basal rates of apoptosis and had little effect on Trail-induced or camptothecin-induced apoptosis in migration-restricted cells. By contrast, migration inhibitors increased camptothecin and Trail-induced apoptosis in actively migrating glioma cells. Migrating glioma cells have increased amounts of phosphorylated Akt and its downstream substrate glycogen synthase kinase-3 relative to migration restricted cells. Treatment of migrating cells with a specific inhibitor of phosphoinositide 3-kinase (PI3-K), LY294002, blocked the phosphorylation of Akt and increased the sensitivity to apoptosis. LY294002 had no effect on the migration of restricted cells. This suggests that migrating glioma cells activate the PI3-K survival pathway, protecting migrating cells from apoptosis. Taken together, these data provide support for a link between migration and apoptosis in glioma cells. In addition, evidence indicates that treatment with migration inhibitors, while not affecting apoptosis-induction in migration-restricted cells, can sensitize migrating glioma cells to cytotoxic agents.

Microarray analysis of complementary DNA (cDNA) allows large-scale, comparative, gene expression profiling of two different cell populations. This approach has the potential for elucidating the primary transcription events and genetic cascades responsible for increased glioma cell motility in vitro and invasion in vivo. These genetic determinants could become therapeutic targets. We compared cDNA populations of a glioma cell line (G112) exposed or not to a motility-inducing substrate of cell-derived extracellular matrix (ECM) proteins using two sets of cDNA microarrays of 5,700 and 7,000 gene sequences. The data were analyzed considering the level and consistency of differential expression (outliers) and whether genes involved in pathways of motility, apoptosis, and proliferation were differentially expressed when the motility behavior was engaged. Validation of differential expression of selected genes was performed on additional cell lines and human glioblastoma tissue using quantitative RT-PCR. Some genes involved in cell motility, like tenascin C, neuropilin 2, GAP43, PARG1 (an inhibitor of Rho), PLCy, and CD44, were over expressed; other genes, like adducin 3y and integrins, were down regulated in migrating cells. Many key cell cycle components, like cyclin A and B, and proliferation markers, like PCNA, were strongly down regulated on ECM. Interestingly, genes involved in apoptotic cascades, like Bcl-2 and effector caspases, were differentially expressed, suggesting the global down regulation of proapoptotic components in cells exposed to cell-derived ECM. Overall, our findings indicate a reduced proliferative and apoptotic activity of migrating cells. cDNA microarray analysis has the potential for uncovering genes linking the phenotypic aspects of motility, proliferation, and apoptosis.

A new migration assay, the time-lapse individual cell migration assay (TIM-assay), was developed, which allows the observation of cells over 24 h under controlled conditions. Using this technique, the migratory behavior of 8 human glioblastoma cell lines in vitro was studied. Special features are simultaneous documentation of migratory parameters of individual cells, i.e., migration velocities and migration paths of individual cells. Migration velocity for cell populations of the same cell line ranged from 0 to 24 microm/h. The migration paths were examined for being directional. Two thirds of all cells showed directional migration. Migration paths were further classified according to visual judgements for being linear, oscillating or mixed. The migration index had a mean of 91%. The presented TIM-assay allows the assessment of several new parameters. that may be useful to identify subgroups of gliomas with different biological characteristics.

Givinostat, a histone-deacetylase inhibitor (HDACi), inhibits proliferation of cells bearing the JAK2 V617F mutation and has shown significant activity with good tolerability in patients with chronic myeloproliferative neoplasms (MPN). In this multicentre, open-label, phase II study, 44 patients with polycythaemia vera (PV), unresponsive to the maximum tolerated doses (MTD) of hydroxycarbamide (HC), were treated with Givinostat (50 or 100 mg/d) in combination with MTD of HC. The European LeukaemiaNet response criteria were used to assess the primary endpoint after 12 weeks of treatment. Complete or partial response was observed in 55% and 50% of patients receiving 50 or 100 mg of Givinostat, respectively. Control of pruritus was observed in 64% and 67% of patients in the 50 and 100 mg groups, respectively. The combination of Givinostat and HC was well tolerated: eight patients (18%) discontinued, four in each treatment arm; grade 3 adverse events were reported in one patient (4·5%) in each treatment arm. The combined use of Givinostat and HC was safe and clinically effective in HC-unresponsive PV patients.

3544Background: BRAFm CRC carries a poor prognosis, with survival of 5–6 months after failure of first-line therapy. Reactivation of epidermal growth factor receptor (EGFR) signaling with BRAF inhibition and uninhibited PI3K signaling may explain the limited efficacy of BRAF inhibitor monotherapy in patients (pts) with BRAFm CRC. This study investigated the BRAF inhibitor ENCO and the anti-EGFR antibody CETUX with or without the PI3Kα inhibitor ALP (BYL719) in pts with advanced BRAFm CRC. Methods: In this ongoing phase 1b/randomized phase 2 study (ClinicalTrials.gov: NCT01719380), phase 1b did not identify a maximum tolerated dose for either combination. Based on general tolerability of the triplet, the ENCO dose for phase 2 was chosen to be the same in both arms. In phase 2, pts with advanced BRAFm CRC failing ≥ 1 prior therapy were randomized 1:1 to a doublet (ENCO 200 mg PO QD and CETUX per label) or triplet (ENCO, CETUX, and ALP 300 mg PO QD). Progression-free survival (PFS) was the primary endpoint. ...

Molecular targeted therapy can potentially provide more effective treatment for patients with high-grade gliomas. Notch and Akt are notable target molecules as they play important roles in a variety of cellular processes, such as regeneration, differentiation, proliferation, migration, and invasion. Here, we assessed the therapeutic possibility of inhibiting Notch and Akt in gliomas using the clinically available, selective small molecule inhibitors MRK003 and MK-2206. We evaluated their efficacy individually and as a combination therapy in U251 and U87 glioma cell lines. We confirmed that MK-2206 effectively inhibits Akt phosphorylation in a dose-dependent manner, whereas MRK003 inhibits Notch signaling and Akt phosphorylation. Both MRK003 and MK-2206 significantly inhibited cell growth, migration, and invasion in a dose-dependent manner. Akt dephosphorylation was enhanced by combination therapy with MRK003 and MK-2206. However, the effect of combination treatment did not exceed that of MK-2206 monotherapy in proliferation assay. Inhibition of invasion, further enhanced by combination therapy, correlated with increased Akt inactivation. In summary, combination therapy with MRK003 and MK-2206 may be effective for inhibiting invasion but not proliferation.

To identify rational therapeutic combinations with cytarabine (Ara-C), we developed a high-throughput, small-interference RNA (siRNA) platform for myeloid leukemia cells. Of 572 kinases individually silenced in combination with Ara-C, silencing of 10 (1.7%) and 8 (1.4%) kinases strongly increased Ara-C activity in TF-1 and THP-1 cells, respectively. The strongest molecular concepts emerged around kinases involved in cell-cycle checkpoints and DNA-damage repair. In confirmatory siRNA assays, inhibition of WEE1 resulted in more potent and universal sensitization across myeloid cell lines than siRNA inhibition of PKMYT1, CHEK1, or ATR. Treatment of 8 acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic myeloid leukemia (CML) cell lines with commercial and the first-in-class clinical WEE1 kinase inhibitor MK1775 confirmed sensitization to Ara-C up to 97-fold. Ex vivo, adding MK1775 substantially reduced viability in 13 of 14 AML, CML, and myelodysplastic syndrome patient samples compared with Ara-C alone. Maximum sensitization occurred at lower to moderate concentrations of both drugs. Induction of apoptosis was increased using a combination of Ara-C and MK1775 compared with using either drug alone. WEE1 is expressed in primary AML, ALL, and CML specimens. Data from this first siRNA-kinome sensitizer screen suggests that inhibiting WEE1 in combination with Ara-C is a rational combination for the treatment of myeloid and lymphoid leukemias.

Purpose: Investigate the efficacy and pharmacodynamic effects of MK-1775, a potent Wee1 inhibitor, in both monotherapy and in combination with gemcitabine (GEM) using a panel of p53-deficient and p53 wild-type human pancreatic cancer xenografts. Experimental Design: Nine individual patient-derived pancreatic cancer xenografts (6 with p53-deficient and 3 with p53 wild-type status) from the PancXenoBank collection at Johns Hopkins were treated with MK-1775, GEM, or GEM followed 24 hour later by MK-1775, for 4 weeks. Tumor growth rate/regressions were calculated on day 28. Target modulation was assessed by Western blotting and immunohistochemistry. Results: MK-1775 treatment led to the inhibition of Wee1 kinase and reduced inhibitory phosphorylation of its substrate Cdc2. MK-1775, when dosed with GEM, abrogated the checkpoint arrest to promote mitotic entry and facilitated tumor cell death as compared to control and GEM-treated tumors. MK-1775 monotherapy did not induce tumor regressions. However, the combination of GEM with MK-1775 produced robust antitumor activity and remarkably enhanced tumor regression response (4.01-fold) compared to GEM treatment in p53-deficient tumors. Tumor regrowth curves plotted after the drug treatment period suggest that the effect of the combination therapy is longer-lasting than that of GEM. None of the agents produced tumor regressions in p53 wild-type xenografts. Conclusions: These results indicate that MK-1775 selectively synergizes with GEM to achieve tumor regressions, selectively in p53-deficient pancreatic cancer xenografts. Clin Cancer Res; 17(9); 2799–806. ©2011 AACR.

To reveal molecular drivers of glioma invasion, two distinct glioblastoma (GBM) cell phenotypes (invading cells and tumor core cells) were collected from 19 GBM specimens using laser capture microdissection. Isolated RNA underwent whole human genome expression profiling to identify differentially expressed genes. Pathway enrichment analysis highlighted the bidirectional receptor/ligand tyrosine kinase system, EphB/ephrin-B, as the most tightly linked system to the invading cell phenotype. Clinical relevance of ephrin-B genes was confirmed in a clinically annotated expression data set of 195 brain biopsy specimens. Levels of ephrin-B1 and -B2 mRNA were significantly higher in GBM (n = 82) than in normal brain (n = 24). Kaplan-Meier analysis demonstrated ephrin-B2, but not ephrin-B1, expression levels were significantly associated with short term survival in malignant astrocytomas (n = 97, p = 0.016). In human brain tumor specimens, the production and phosphorylation of ephrin-B2 were high in GBM. Immunohistochemistry demonstrated ephrin-B2 localization primarily in GBM cells but not in normal brain. A highly invasive glioma cell line, U87, expressed high levels of ephrin-B2 compared with relatively less invasive cell lines. Treatment with EphB2/Fc chimera further enhanced migration and invasion of U87 cells, whereas treatment with an ephrin-B2 blocking antibody significantly slowed migration and invasion. Forced expression of ephrin-B2 in the U251 cell line stimulated migration and invasion in vitro and ex vivo, concomitant with tyrosine phosphorylation of ephrin-B2. These results demonstrate that high expression of ephrin-B2 is a strong predictor of short-term survival and that ephrin-B2 plays a critical role in glioma invasion rendering this signaling pathway as a potential therapeutic target.

Background Glioblastoma multiforme (GBM) is the most common primary intracranial tumor and despite recent advances in treatment regimens, prognosis for affected patients remains poor. Active cell migration and invasion of GBM cells ultimately lead to ubiquitous tumor recurrence and patient death. To further understand the genetic mechanisms underlying the ability of glioma cells to migrate, we compared the matched transcriptional profiles of migratory and stationary populations of human glioma cells. Using a monolayer radial migration assay, motile and stationary cell populations from seven human long term glioma cell lines and three primary GBM cultures were isolated and prepared for expression analysis. Results Gene expression signatures of stationary and migratory populations across all cell lines were identified using a pattern recognition approach that integrates a priori knowledge with expression data. Principal component analysis (PCA) revealed two discriminating patterns between migrating and stationary glioma cells: i) global down-regulation and ii) global up-regulation profiles that were used in a proband-based rule function implemented in GABRIEL to find subsets of genes having similar expression patterns. Genes with up-regulation pattern in migrating glioma cells were found to be overexpressed in 75% of human GBM biopsy specimens compared to normal brain. A 22 gene signature capable of classifying glioma cultures based on their migration rate was developed. Fidelity of this discovery algorithm was assessed by validation of the invasion candidate gene, connective tissue growth factor (CTGF). siRNA mediated knockdown yielded reduced in vitro migration and ex vivo invasion; immunohistochemistry on glioma invasion tissue microarray confirmed up-regulation of CTGF in invasive glioma cells. Conclusion Gene expression profiling of migratory glioma cells induced to disperse in vitro affords discovery of genomic signatures; selected candidates were validated clinically at the transcriptional and translational levels as well as through functional assays thereby underscoring the fidelity of the discovery algorithm.

To gain insight into brain tumor invasion, experiments are conducted on multicellular tumor spheroids grown in collagen gel. Typically, a radius of invasion is reported, which is obtained by human measurement. We present a simple, heuristic algorithm for automated invasive radii estimation (AIRE) that uses local fluctuations of the image intensity. We then derive an analytical expression relating the image graininess to the cell density for a model imaging system. The result agrees with the experiment up to a multiplicative constant and thus describes a novel method for estimating the cell density from bright-field images.

Glioblastoma multiforme (GBM) is inherently invasive, and it is from the invasive cell population that the tumor recurs. The GBM invasion transcriptome reveals over-expression of various autocrine factors that could act as motility drivers, such as autotaxin (ATX). Some of these factors could also have paracrine roles, modulating the behavior of cells in the peri-tumoral brain parenchyma. ATX generates lysophosphatidic acid (LPA), which signals through LPA receptors expressed by GBM as well as in astrocytes, oligodendrocytes (ODC) and microglia; their activation manifest cell specific effects. ATX stimulates invasion of GBM cells in vitro and ex vivo invasion assays. ATX activity enhances GBM adhesion in cells expressing the LPA1 receptor, as well as stimulating rac activation. GBM secreted ATX can also have paracrine effects: ATX activity results in reduced ODC adhesion. ODC monolayer invasion showed that U87 and U251 GBM cells expressing ATX invaded through an ODC monolayer significantly more than cells depleted of ATX or cells expressing inactive ATX, suggesting that GBM cells secreting ATX find ODCs less of a barrier than cells that do not express ATX. Secreted factors that drive GBM invasion can have autocrine and paracrine roles; one stimulates GBM motility and the other results in ODC dis-adhesion.

Although astrocytic brain tumors do not metastasize systemically, during tumorigenesis glioma cells adopt an invasive phenotype that is poorly targeted by conventional therapies; hence, glioma patients die of recurrence from the locally invasive tumor population. Our work is aimed at identifying and validating novel therapeutic targets and biomarkers in invasive human gliomas. Transcriptomes of invasive glioma cells relative to stationary cognates were produced from a three-dimensional spheroid in vitro invasion assay by laser capture microdissection and whole human genome expression microarrays. Qualitative differential expression of candidate invasion genes was confirmed by quantitative reverse transcription-PCR, clinically by immunohistochemistry on tissue microarray, by immunoblotting on surgical specimens, and on two independent gene expression data sets of glial tumors. Cell-based assays and ex vivo brain slice invasion studies were used for functional validation. We identify mitogen-activated protein kinase (MAPK) kinase 3 (MKK3) as a key activator of p38 MAPK in glioma; MKK3 activation is strongly correlated with p38 activation in vitro and in vivo. We further report that these members of the MAPK family are strong promoters of tumor invasion, progression, and poor patient survival. Inhibition of either candidate leads to significantly reduced glioma invasiveness in vitro. Consistent with the concept of synthetic lethality, we show that inhibition of invasion by interference with these genes greatly sensitizes arrested glioma cells to cytotoxic therapies. Our findings therefore argue that interference with MKK3 signaling through a novel treatment combination of p38 inhibitor plus temozolomide heightens the vulnerability of glioma to chemotherapy. [Mol Cancer Ther 2007;6(4):1212–22]

Background: Although BRAF V600-mutated (BRAFm) colorectal carcinoma (CRC) does not generally respond to BRAF inhibitors, results of preclinical studies suggest strong synergistic activity between a BRAF inhibitor and an EGFR inhibitor or a PI3K inhibitor. The aims of this study were to establish safety and preliminary activity of combination therapy with encorafenib (ENC), a highly selective BRAF V600 inhibitor, the EGFR monoclonal antibody cetuximab (CTX), ± the α-specific PI3K inhibitor alpelisib (ALP) in patients (pts) with BRAFm CRC; and assess the potential clinical significance of biomarkers and microsatellite instability (MSI) status by genomic analysis of tumor samples. Methods: In this phase I study, the maximum tolerated dose/recommended phase II dose (RP2D) was determined and safety, tolerability, and anti-tumor activity of ENC + CTX ± ALP were evaluated, in pts with advanced BRAFm/KRAS wild-type CRC. Archival or fresh screening tumor samples were analyzed for somatic mutations and copy number aberrations to evaluate potential correlation with response. Results: Fifty-four pts were enrolled in the dose-escalation phase; 26 in the dual arm (ENC + CTX) and 28 in the triple arm (ENC + ALP + CTX). RP2D for each treatment arm was established as 200 mg ENC QD + CTX weekly (dual arm) + 300 mg ALP once daily (triple arm). The most common grade 3/4 treatment-related adverse events were fatigue and hypophosphatemia in the dual arm (8% each), and nausea and hypophosphatemia in the triple arm (4% each). Hyperglycemia was observed in one patient (3.8%) in the dual arm and 8 patients (29%) in the triple arm. Overall response rates (≥ partial response [PR]) for the dual and triple arms were 23% (1 CR, 4 PR, and 1 unconfirmed (u) PR) and 32% (4 PR and 5 uPR), respectively. Median progression-free survival was 3.7 and 4.3 months, respectively. Biomarker analysis was performed on 21 tumor samples. Several genetic alterations occurred with similar frequency between patient samples and the FoundationOne database (PIK3CA, TP53, APC). Specifically, the PIK3CA mutation and PTEN alterations were found in 24% and 38% of patient samples, respectively; however, these alterations did not appear to be correlated with response. Non-MSI-high status was associated with a lower PFS for the dual arm (2.8 vs 3.7 months), but not the triple arm (4.4 vs 4.3 months). Updated biomarker analyses, including MSI status, will be presented. Conclusions: These results indicate that both ENC + CTX and ENC + CTX + ALP are well tolerated with promising antitumor activity in pts with advanced BRAFm CRC who failed at least one prior therapy. Additional biomarker analyses will help to clarify the clinical significance of genetic alterations, particularly with MSI status and the PI3K pathway. Enrollment in the phase II portion of the study is ongoing. Citation Format: Jan H. M. Schellens, Robin van Geel, Johanna C. Bendell, Anna Spreafico, Martin Schuler, Takayuki Yoshino, Jean-Pierre Delord, Yasuhide Yamada, Martijn P. Lolkema, Jason E. Faris, Ferry A. L. M. Eskens, Sunil Sharma, Rona Yaeger, Heinz-Josef Lenz, Zev A. Wainberg, Emin Avsar, Arkendu Chatterjee, Savina Jaeger, Tim Demuth, Josep Tabernero. Final biomarker analysis of the phase I study of the selective BRAF V600 inhibitor encorafenib (LGX818) combined with cetuximab with or without the α-specific PI3K inhibitor alpelisib (BYL719) in patients with advanced BRAF-mutant colorectal cancer. [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 CT136. doi:10.1158/1538-7445.AM2015-CT136

Glioblastoma, the most malignant form of brain cancer, is responsible for 23% of primary brain tumors and has extremely poor outcome. Confounding the clinical management of glioblastomas is the extreme local invasiveness of these cancer cells. The mechanisms that govern invasion are poorly understood. To gain insight into glioblastoma invasion, we conducted experiments on the patterns of growth and dispersion of U87 glioblastoma tumor spheroids in a three-dimensional collagen gel. We studied two different cell lines, one with a mutation to the EGFR (U87ΔEGFR) that is associated with increased malignancy, and one with an endogenous (wild-type) receptor (U87WT). We developed a continuum mathematical model of the dispersion behaviors with the aim of identifying and characterizing discrete cellular mechanisms underlying invasive cell motility. The mathematical model quantitatively reproduces the experimental data, and indicates that the U87WT invasive cells have a stronger directional motility bias away from the spheroid center as well as a faster rate of cell shedding compared to the U87ΔEGFR cells. The model suggests that differences in tumor cell dispersion may be due to differences in the chemical factors produced by cells, differences in how the two cell lines remodel the gel, or different cell-cell adhesion characteristics.

Human brain tumors continue to benefit from ongoing investigation of the molecular genetic changes that explain gliomagenesis, malignant progression, patient outcome, and possibly therapeutic responsiveness. Optimal utilization of current therapies may be gained by using molecular genetic markers of glial tumors, but meaningful advances in improved patient treatment will emerge from new therapies matched to the specific chemo-vulnerabilities of these tumors as addressed by small-molecule agents specifically targeting the points of vulnerability.

Gliomas are the most common intracranial tumors. In the US, approximately 15,000 patients die with glioblastoma per year (CBTRUS 2002). Despite modern diagnostics and treatments the median survival time does not exceed 15 months. However, it has long been observed that after surgical removal, tumors recur predominantly within 1 cm of the resection cavity. This is mainly due to the fact that at the time of surgery, cells from the bulk tumor have already invaded normal brain tissue. Decades ago Matsukado showed that more than 50% of untreated brain tumors had already reached the contralateral hemisphere (J Neurosurg 18: 636-644, 1961). Therefore one of the most important hallmarks of malignant gliomas is their invasive behavior. Dandy already recognized the highly invasive characteristics of this tumor type and performed hemispherectomy in patients with preoperative hemiplegia (J Am Med Assoc 90: 823-825, 1928). Despite his and others' heroic efforts, recurrence was detected as early as 3 months after surgery (Bell, LJ: J Neurosurg 6: 285-293, 1949), leading to the discontinuation of this radical approach. Diffuse gliomas remain a particularly challenging clinical management problem. Over the last 20 years no significant increase in survival of patients suffering from this disease has been achieved. Even drugs directed against newly identified targets like MMPs or angiogenesis-related targets fail to increase survival duration (Tonn, Goldbrunner: Acta Neurochir Suppl 88: 163-167, 2003) Furthermore, anti-angiogenic drugs have been shown to increase glioma invasiveness, finally leading to gliomatosis cerebri. (Lamszus et al.: Acta Neurochir Suppl 88: 169-177, 2003). In this review we focus on the main features which may underlie the invasive phenotype of human gliomas, and offer a biological basis for optimism towards therapeutic advances to come.

Aim: Few treatment options exist for pts with BRAF-mutant mCRC. Encorafenib, a potent and selective oral BRAF inhibitor, showed signs of efficacy in pts with BRAF-mutant advanced melanoma in a phase 1 dose-escalation study. Here we present results from pts with BRAF-mutant mCRC enrolled in the dose-expansion phase of the same trial. Methods: Adult pts with nonresectable, advanced mCRC with a BRAF V600 mutation, for whom there was no existing effective therapy, received encorafenib 300 mg (n = 6) or 450 mg (n = 12) once daily. Responses were assessed by investigator per RECIST 1.1. Results: All 18 pts had a BRAF V600E mutation. Median age was 62 y (range, 34-73). Ten pts (55.6%) were female. Median number of prior treatment regimens was 3 (range, 1-8); 1 pt received prior therapy with a BRAF inhibitor (vemurafenib). At the data cutoff (7 Jan 2014), median treatment duration was 11 wks; 4 pts (22.2%) remained on treatment, and 14 (77.8%) discontinued due to progressive disease (PD; n = 10), adverse events (AEs; n = 3), and death (n = 1, not related to study drug). Twelve pts (66.7%) achieved a best response of stable disease (SD). No pt achieved confirmed complete response (CR) or confirmed partial response (PR) by the data cutoff (Table). Progression-free survival (PFS) events occurred in 13 pts (PD, n = 10; death, n = 3). Median PFS was 4.0 mo. The most common AEs of any grade were palmar-plantar erythrodysaesthesia syndrome (66.7%), myalgia (44.4%), and dry skin (44.4%). Three pts had dose-limiting toxicities (arthralgia and myalgia [n = 1]; insomnia and myalgia [n = 1]; bone pain and vomiting [n = 1]; all grade 3), all on encorafenib 450 mg. Encorafenib 300 mg n = 6 Encorafenib 450 mg n = 12 All pts N = 18 Best Overall Response, n (%) _CRa 0 0 0 _PRa 0 0 0 _SD 4 (66.7) 8 (66.7) 12 (66.7) __With unconfirmed CR/PR 1 (16.7) 2 (16.7) 3 (16.7) _PD 1 (16.7) 3 (25.0) 4 (22.2) __With unconfirmed CR/PR 0 2 (16.7) 2 (11.1) _Unknownb 1 (16.7) 1 (8.3) 2 (11.1) Median PFS, mo (95% CI) 2.3 (1.7-7.2) 4.0 (1.8-5.5) 4.0 (1.8-5.6) a CR and PR required confirmation in a second assessment at least 4 wks after the first. b Best response was unknown for 2 pts who achieved SD before 6 wks of treatment. Conclusions: Encorafenib showed modest activity in this difficult-to-treat population, with SD observed in two-thirds of pts. Studies in BRAF-mutant mCRC are ongoing in combination with epidermal growth factor receptor and phosphoinositide 3-kinase inhibitors. Disclosure: M. Hidalgo: has been on a board of directors and/or advisory board for and received research funding from Novartis; R. von Moos: has consulted for Novartis, Roche, GSK, BMS, MSD; received research funding from Amgen, Merck, Roche; received honoraria from Novartis, Roche, GSK, BMS, Sanofi; A. Arance: has received research funding from Roche; has consulted with Roche, BMS, GSK D. Michel, A. Seroutou and T. Demuth: is an employee of Novartis Pharma AG; J. Tabernero: has consulted for Amgen, Imclone, Lilly, Merck KGaA, Millenium, Novartis, Roche, Sanofi, Celgene, Chugai, Taiho and has received honoraria from Amgen, Merck KGaA, Novartis, Roche, Sanofi. All other authors have declared no conflicts of interest.

3514 Background: In contrast to BRAFV600 mutated (BRAFm) advanced melanoma, BRAFm colorectal carcinoma (CRC) does not respond to BRAF inhibitors due to strong feedback activation of the epidermal g...

e13598 Background: Wee1 kinase regulates the G2 checkpoint through phosphorylation of CDC2. MK-1775 is a first-in-class inhibitor of Wee1, and thereby reduces pCDC2 levels relative to CDC2. pCDC2therefore can be used as a target engagement (TE) biomarker to assess activity of MK-1775. This was investigated in a phase I first-in-man clinical trial of MK-1775. Methods: This is a multicenter, open-label, non-randomized phase I dose escalation study in patients with locally advanced or metastatic solid tumors. MK-1775 was administered orally in escalating doses as monotherapy, and either single dose or multi-dose in combination with chemotherapy including cisplatin, carboplatin and gemcitabine. Serial skin biopsies were performed at baseline and either 8, 24 or 48 hours following MK-1775 administration and analyzed by IHC for CDC2 and pCDC2. Based on preclinical efficacy experiments, TE was defined as a decrease of pCDC2 of at least 50% (or fold change > 0.50) from pre- to post-dose MK-1775. Results: To date a total of 176 patients have received at least one dose of MK-1775 either as monotherapy or in combination (single or multi-dose MK-1775) with chemotherapy at doses ranging from 25 mg to 1300 mg to define maximum tolerated dose. Dose dependent decreases in pCDC2 were observed in skin biopsies between pre-dose and post-dose treatment in all treatment groups. TE with monotherapy was achieved at 325 mg. TE with multi-dose MK-1775 in combination with cisplatin and carboplatin were achieved at 125 mg BID and 225 mg BID. Gemcitabinecombination treatment is on-going. In contrast, chemotherapy alone resulted in an increase of pCDC2. Conclusions: Based on preclinical data, in this first-in-man clinical trial of MK-1775, we were able to demonstrate TE required for maximal efficacy at tolerable doses of MK-1775 either as a single agent or in combination with chemotherapy. [Table: see text]

Abstract 229 Introduction: Optimal rational combination targets with Cytarabine (AraC) have not been identified. After the development of a high-throughput, small-interfering RNA (siRNA) platform for suspension cells, we performed the first RNAi sensitizer screen of the human kinome with AraC in myeloid cell lines. Herein we present the final data of this first siRNA-sensitizer screen in leukemias in combination with AraC. Our results include full validation of the selected targets as well as ex vivo data assessing the first in class clinically tested WEE1 inhibitor MK1775 with AraC providing a pre-clinical rationale for using the combination of AraC with WEE1 inhibitors in leukemias. RNAi Screens and siRNA Validation: In siRNA sensitizer screens, we silenced 572 kinases of the human kinome in combination with AraC. Few kinases strongly sensitized to AraC and the targets CHEK1 and the WEE1 related kinase PKMYT1 emerged as the most potent genes whose silencing sensitized leukemia cells to AraC. In secondary siRNA validation experiments, silencing of WEE1 kinase was found to be more potent than silencing of CHEK1, PKMYT1 or other kinases (i.e. ATR, BRCA, AURKB) in combination with AraC treatment. Importantly, the sensitizing activity observed with WEE1 inhibition was universal across all AML cell lines examined, whereas it was not for CHEK1 inhibition. siRNA assays were robust, exhibiting little ( Pharmacological WEE1 Inhibitor Experiments In vitro and Ex vivo: In vitro treatment of eight AML, ALL and CML cell lines with commercial and the first clinically developed WEE1 kinase inhibitor MK1775, demonstrated strong sensitization to AraC up to 97-fold in individual cell lines. Ex vivo, MK1775 substantially reduced viability in 13 of 14 AML, MDS and CML patient samples compared to AraC alone. The optimal dose to achieve maximum sensitization was observed for AraC between ∼10–120 nM and for MK1775 between 200–400 nM. Synergy at these optimal concentrations was confirmed in all ex vivo samples tested for which sufficient data for analysis with CalcuSyn Software was available (n=7): with Combination Indices (CI) ranging from 0.21–0.45. At higher AraC and MK1775 concentrations antagonism was observed both ex vivo and in vitro. Thus, we have also established optimal suggested concentration dose ranges for the currently ongoing design of a clinical trial. Clinically relevant, WEE1 is expressed in primary AML, ALL and advanced CML specimens with a trend towards higher expression in relapsed samples. However, there was no direct correlation with WEE1 mRNA expression or TP53 status. Our initial in vitro experiments further suggest that inhibition of WEE1 with MK1775 in combination with AraC at optimal concentrations was associated with higher degree of caspase 3 cleavage, DNA damage induction as measured by gamma-H2AX, and greatest inhibition of proliferation. Thereby establishing a context of optimal drug concentration ranges with molecular correlates and anti-leukemic activity directly informing design of the clinical trial and correlative biomarkers. In conclusion, data from this first siRNA sensitizer screen of the kinome in leukemias identifies inhibition of WEE1 kinase as the most potent sensitizing target to AraC. Inhibiting WEE1 with pharmacological inhibitors in combination with AraC is a rational treatment strategy for myeloid and lymphoid leukemias that warrants clinical investigation. Furthermore, our RNAi loss-of-function genomics approach has high potential to rapidly identify and validate crucial genes whose inhibition modulate the response to cytotoxic agents. Establishment of the siRNA sensitizer platform allows accelerating the development of new rational combinations for leukemia therapy. Disclosures: Off Label Use: AraC in combination with the experimental Wee1 inhibitor MK1775. Mesa:NS Pharma: Research Funding; Astra Zeneca: Research Funding; SBio: Research Funding; Lilly: Research Funding; Incyte: Research Funding; Celgene: Research Funding. Demuth:Merck and CO: Employment.