Chris Liang

Insights into ALK-driven cancers revealed through development of novel ALK tyrosine kinase inhibitors

Aberrant forms of the anaplastic lymphoma kinase (ALK) have been implicated in the pathogenesis of multiple human cancers, where ALK represents a rational therapeutic target in these settings. In this study, we report the identification and biological characterization of X-376 and X-396, two potent and highly specific ALK small molecule tyrosine kinase inhibitors (TKIs). In Ambit kinome screens, cell growth inhibition studies, and surrogate kinase assays, X-376 and X-396 were more potent inhibitors of ALK but less potent inhibitors of MET compared to PF-02341066 (PF-1066), an ALK/MET dual TKI currently in clinical trials. Both X-376 and X-396 displayed potent antitumor activity in vivo with favorable pharmacokinetic and toxicity profiles. Similar levels of drug sensitivity were displayed by the three most common ALK fusion proteins in lung cancer (EML4-ALK variants E13;A20, E20;A20, and E6b;A20) as well as a KIF5B-ALK fusion protein. Moreover, X-396 could potently inhibit ALK kinases engineered with two point mutations associated with acquired resistance to PF-1066, L1196M, and C1156Y, when engineered into an E13;A20 fusion variant. Finally, X-396 displayed synergistic growth inhibitory activity when combined with the mTOR inhibitor rapamycin. Our findings offer preclinical proof-of-concept for use of these novel agents to improve therapeutic outcomes of patients with mutant ALK-driven malignancies.

Upregulation of MAPK Negative Feedback Regulators and RET in Mutant ALK Neuroblastoma: Implications for Targeted Treatment

Purpose: Activating ALK mutations are present in almost 10% of primary neuroblastomas and mark patients for treatment with small-molecule ALK inhibitors in clinical trials. However, recent studies have shown that multiple mechanisms drive resistance to these molecular therapies. We anticipated that detailed mapping of the oncogenic ALK-driven signaling in neuroblastoma can aid to identify potential fragile nodes as additional targets for combination therapies. Experimental Design: To achieve this goal, transcriptome profiling was performed in neuroblastoma cell lines with the ALKF1174L or ALKR1275Q hotspot mutations, ALK amplification, or wild-type ALK following pharmacologic inhibition of ALK using four different compounds. Next, we performed cross-species genomic analyses to identify commonly transcriptionally perturbed genes in MYCN/ALKF1174L double transgenic versus MYCN transgenic mouse tumors as compared with the mutant ALK-driven transcriptome in human neuroblastomas. Results: A 77-gene ALK signature was established and successfully validated in primary neuroblastoma samples, in a neuroblastoma cell line with ALKF1174L and ALKR1275Q regulable overexpression constructs and in other ALKomas. In addition to the previously established PI3K/AKT/mTOR, MAPK/ERK, and MYC/MYCN signaling branches, we identified that mutant ALK drives a strong upregulation of MAPK negative feedback regulators and upregulates RET and RET-driven sympathetic neuronal markers of the cholinergic lineage. Conclusions: We provide important novel insights into the transcriptional consequences and the complexity of mutant ALK signaling in this aggressive pediatric tumor. The negative feedback loop of MAPK pathway inhibitors may affect novel ALK inhibition therapies, whereas mutant ALK induced RET signaling can offer novel opportunities for testing ALK-RET oriented molecular combination therapies. Clin Cancer Res; 21(14); 3327–39. ©2015 AACR.

MINI01.02: Response and Plasma Genotyping from Phase I/II Trial of Ensartinib (X-396) in Patients (pts) with ALK+ NSCLC: Topic: Medical Oncology

MINI ORAL SESSIONS SATURDAY, SEPTEMBER 24, 2016 MINI01.01 Whole Body and Intracranial Efficacy of Ceritinib in ALK-inhibitor Naïve Patients with ALK+ NSCLC and Brain Metastases: Results of ASCEND 1 and 3 Topic: Medical Oncology Alice T. Shaw, David R. Spigel, Daniel S.-W. Tan, Dong-Wan Kim, Ranee Mehra, Sergey Orlov, Keunchil Park, Chong-Jen Yu, Tony Mok, Makoto Nishio, Giorgio Scagliotti, Santosh Sutradhar, Dajana Cesic, Enriqueta Felip Massachusetts General Hospital, Boston/United States of America, Sarah Cannon Research Institute, Nashville, TN/United States of America, National Cancer Centre Singapore, Singapore/Singapore, Seoul National University Hospital, Seoul/Korea, Republic of, Fox Chase Cancer Center, Philadelphia, PA/United States of America, St. Petersburg State Medical University, St. Petersburg/Russian Federation, Innovative Cancer Medicine Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul/ Korea, Republic of, National Taiwan University Hospital, Taipei City/Taiwan, Chinese University of Hong Kong, Sha Tin/China, Japanese Foundation for Cancer Research, Tokyo/Japan, University of Torino, Torino/ Italy, Novartis Pharmaceuticals Corporation, East Hanover, NJ/United States of America, Vall d’Hebron University, Barcelona/Spain Background: Here we present efficacy outcomes in ALKrearranged (ALK+) NSCLC patients with baseline (BL) brain metastases (BM) treated with the selective oral ALKi ceritinib in the ASCEND-1 (phase 1; NCT01283516) and ASCEND-3 (phase 2; NCT01685138) trials. Methods: ALKi-naive patients with ALK+ NSCLC and stable BL BM received ceritinib 750 mg/day. Efficacy analyses (by blinded independent review committee [BIRC]) assessed whole body responses for ASCEND-1 and -3 according to RECIST 1.0 and 1.1 criteria, respectively. Pooled intracranial responses were evaluated by BIRC (ASCEND-1, retrospectively; ASCEND-3, prospectively) in patients with measureable BL BM (RECIST 1.1). Results: Of 26 and 50 ALKi-naïve patients with BL BM enrolled in ASCEND-1 and -3, respectively, 88.5% and Journal of Thoracic Oncology Vol. 11 No. 11S: S256-S269 100% had prior chemotherapy and 57.7% and 54.0% had prior brain radiotherapy (RT); median times from prior RT to first ceritinib dose were 4.6 and 2.7 months. Ceritinib showed whole body and intracranial efficacy (Table). The most common AEs (ASCEND-1; ASCEND-3) were nausea (84.6%; 78.0%), diarrhea (92.3%; 76.0%) and vomiting (76.9%; 72.0%); 46 patients (ASCEND-1: 19; ASCEND-3: 27) had dose reductions and 4 patients (ASCEND-1: 3; ASCEND-3: 1) discontinued due to AEs. Conclusion: Clinically meaningful whole body and intracranial activity with an acceptable tolerability profile were observed in ALKi-naïve patients with ALK+ NSCLC and BL BM treated with ceritinib. MINI01.02 Response and Plasma Genotyping from Phase I/II Trial of Ensartinib (X-396) in Patients (pts) with ALK+ NSCLC Topic: Medical Oncology Leora Horn, Heather Wakelee, Karen L. Reckamp, George Blumenschein Jr., Jeffrey R. Infante, Corey A. Carter, Saiama N. Waqar, Joel W. Neal, Kimberly Harrow, Jon P. Gockerman, Gary Dukart, Chris Liang, James L. Gibbons, Jennifer Hernandez, Tera Newman-Eerkes, Lee Lim, Christine M. Lovly Vanderbilt University Medical Center, Nashville, TN/United States of America, Stanford Cancer Institute, Stanford, CA/United States of America, City of Hope Comprehensive Cancer Center, Duarte/United States of America, Department of Thoracic/Head & Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX/United States of America, Sarah Cannon Research Institute, Nashville, TN/United States of America, Walter Reed National Military Medical Center, Germantown, MD/ November 2016 Abstracts S257 United States of America, Washington University in St. Louis, St. Louis, MO/United States of America, Xcovery Holding Company, LLC, Palm Beach Gardens, FL/United States of America, Novella Clinical, Morrisville, NC/United States of America, Xcovery Holding Company, LLC, Palm Beach Gardens/United States of America, Resolution Bio, Bellevue, WA/United States of America Background: Ensartinib (X-396) is a novel, potent anaplastic lymphoma kinase (ALK) small molecule tyrosine kinase inhibitor (TKI) with additional activity against MET, ABL, Axl, EPHA2, LTK, ROS1 and SLK. We report data on the ALK TKI-naïve and crizotinib (C)-resistant NSCLC pts treated with ensartinib. Clinical trial information: NCT01625234 Methods: In this multicenter expansion study, pts with ALK+NSCLCwere treatedwith ensartinib 225mgdaily on a 28-day schedule. Pts had measurable disease, ECOG PS 0-1, and adequate organ function. Untreated brain metastases (CNS) and leptomeningeal disease were allowed. Next Generation Sequencing (NGS) was performed on plasma samples collected at baseline and on study and compared with central tissue results (FISH/IHC). All pts were assessed for response to therapy using RECIST 1.1 and for adverse events (AEs) using CTCAE version 4.03. Results: 83 pts (51% female) have been enrolled. Median age 54 (20-79) years, 64% ECOG PS 1. Of 42 ALK+ NSCLC pts evaluable for response; partial response (PR) was achieved in 25 pts (60%) and stable disease (SD) in 6 pts (14%). In the C-naïve pts (n 1⁄4 9), PRs were observed in 7 pts (77%). In the 22 pts with prior C but no other ALK TKI, 16 pts (73%) achieved PR and 4 (18%) SD. In the 10 pts who had received two or more prior ALK TKIs, there were 2 PR, 2 SD (40% DCR). CNS responses (53% PR) have been observed in both C-naïve and C-resistant pts. Plasma and tissue genotyping were available on 27 pts (26 ALK+ and 1 ALK-). ALK was detected in plasma in 16 pts, all of whom had a response to therapy. 2 pts with PD were tissue +ve and plasma -ve. 9 plasma samples were unevaluable. Serial sequencing demonstrated a decrease in ALK in pts responding and an increase at the time of progression. The most common drug-related AEs ( 20% of pts) included rash (53%), nausea (33%), vomiting (27%), fatigue (22%), and pruritus (23%). Most AEs were Grade (G) 1-2. The G3 treatment-related AEs in 2 pts were rash (9 pts), pruritus (3 pts), and fatigue (2 pts). Conclusion: Ensartinib is generally well-tolerated and induces response in both C-naïve and C-resistant ALK+ NSCLC pts, as well as pts with CNS disease. Plasma sequencing appears to be promising to select pts for therapy and monitor for response and development of resistance. MINI01.03 Phase (Ph) I Study of the Safety and Efficacy of the cMET Inhibitor Capmatinib (INC280) in Patients with Advanced cMET+ NSCLC Topic: Medical Oncology Todd M. Bauer, Martin Schuler, Rossana Berardi, Wan-Teck Lim, Robin Van Geel, Maja De Jonge, Analia Azaro, Maya Gottfried, Ji-Youn Han, Dae Ho Lee, Mira Wollner, David Hong, Arndt Vogel, Angelo Delmonte, Alexander Krohn, Yong Zhang, Matthew Squires, Monica Giovannini, Mikhail Akimov, Dong-Wan Kim Sarah Cannon Research Institute, Tennessee Oncology, PLCC, Nashville, TN/United States of America, West German Cancer Center, University Hospital Essen, Essen/Germany, Università Politecnica delle Marche, Ancona/Italy, National Cancer Center Singapore, Singapore/Singapore, Netherlands Cancer Institute, Amsterdam/Netherlands, Erasmus MC Cancer Center, Rotterdam/Netherlands, Vall d’Hebron University Hospital, Barcelona/Spain, Oncology Institute of Meir Medical Center, Tel-Aviv/ Israel, National Cancer Center, Seoul/Korea, Republic of, Asan Medical Center, University of Ulsan College of Medicine Seoul, Seoul/Korea, Republic of, Rambam Health Care Campus, Haif/Israel, MD Anderson Cancer Center, Houston, TX/United States of America, Hannover Medical School, Hannover/Germany, Istituto Romagnolo per lo Studio e la cura dei Tumori IRCCS, Meldola/Italy, University Medical Center Freiburg, Freiburg/Germany, Novartis Pharmaceuticals Corporation, East Hanover, NJ/United States of America, Novartis Pharma AG, Basel/Switzerland, Seoul National University Hospital, Seoul/Korea, Republic of Background: cMET dysregulation occurs in 3e10% of NSCLC and is a negative prognostic factor. INC280 is a highly selective cMET inhibitor with preclinical and clinical antitumor activity. The dose-escalation part of this ongoing Ph I study (NCT01324479) included a cMET-dysregulated NSCLC expansion group. Preliminary efficacy was seen in NSCLC pts with high cMET expression, prompting the addition of a second expansion group of pts with EGFRwt, high cMETexpressing NSCLC. We report here on both NSCLC groups. Methods: The primary objective of this Ph I study was met; INC280 RP2D was established at 400 mg BID in a tablet formulation. Secondary objectives included

Ensartinib (X-396) in ALK-Positive Non–Small Cell Lung Cancer: Results from a First-in-Human Phase I/II, Multicenter Study

Purpose: Evaluate safety and determine the recommended phase II dose (RP2D) of ensartinib (X-396), a potent anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI), and evaluate preliminary pharmacokinetics and antitumor activity in a first-in-human, phase I/II clinical trial primarily in patients with non–small cell lung cancer (NSCLC). Patients and Methods: In dose escalation, ensartinib was administered at doses of 25 to 250 mg once daily in patients with advanced solid tumors; in dose expansion, patients with advanced ALK-positive NSCLC were administered 225 mg once daily. Patients who had received prior ALK TKI(s) and patients with brain metastases were eligible. Results: Thirty-seven patients enrolled in dose escalation, and 60 enrolled in dose expansion. The most common treatment-related toxicities were rash (56%), nausea (36%), pruritus (28%), vomiting (26%), and fatigue (22%); 23% of patients experienced a treatment-related grade 3 to 4 toxicity (primarily rash and pruritus). The maximum tolerated dose was not reached, but the RP2D was chosen as 225 mg based on the frequency of rash observed at 250 mg without improvement in activity. Among the ALK-positive efficacy evaluable patients treated at ≥200 mg, the response rate (RR) was 60%, and median progression-free survival (PFS) was 9.2 months. RR in ALK TKI-naïve patients was 80%, and median PFS was 26.2 months. In patients with prior crizotinib only, the RR was 69% and median PFS was 9.0 months. Responses were also observed in the central nervous system, with an intracranial RR of 64%. Conclusions: Ensartinib was active and generally well tolerated in patients with ALK-positive NSCLC. Clin Cancer Res; 24(12); 2771–9. ©2018 AACR.

P1.44 (also presented as PD2.02): Phase I/II Trial of X-396, A Novel ALK Inhibitor, in Patients With ALK+ NSCLC: Track: Advanced NSCLC

Background: The purpose of the study is to report our updated results using Gamma Knife Radiosurgery (GKS) for the management of brain metastases from NSCLC in an unselected group of patients. Method: This is a retrospective review of 616 patients (336 males and 280 females) who were treated with GKS independently of primary status from October 1993 to April 2016. The rationale of treatment was to improve survival and quality of life. Ages ranged from 19 to 91 years, with a median age of 64 years. A total of 1085 procedures were performed. Doses ranged from 12 to 24 Gy, mean minimum dose delivered was 15.5Gy. Seventy five patients of 615 had tumors retreated. Results: The median overall survival for the entire group was 6.6 months, with 14.2 months for 25% of the patients and 56.5 months for 5% of them by Kaplan Meier Survival Analysis. Survival at 1, and 5 years are 28%, 4.2% respectively. The median follow-up was from 2 months to 276 months. Overall local control by lesion was 95%. Thirty four out of 486 evaluable documented deaths were due to progression of brain metastases. The other 411 documented deaths were due to progression of disease unrelated to brain metastases. Our longest surviving patient is currently alive 21 years after treatment with GKS to 15 tumors in 3 procedures with local control up-to this date. There was no radiation-induced dementia. Only 3% developed radiation necrosis diagnosed both pathological and by imaging studies. Conclusion: Our results continue to show excellent local control associated with prolonged survival and a low risk of neurological death in spite of advanced stage disease. Number of lesions should not be a contraindication for Radiosurgery in NSCLC. Our report confirms the fact that for patients with NSCLC whole brain radiations should be reserved for late and extensive stage brain disease and or after failure from SRS. GKS provides high local control regardless of the number of lesions or presence of extra cranial disease. We also demonstrated in our retrospective analysis that re-treatment is feasible and safe.

Abstract CT088: Phase I/II trial of X-396, a novel anaplastic lymphoma kinase (ALK) inhibitor, in patients with ALK+ non-small cell lung cancer (NSCLC)

Background: X-396 is a novel, potent anaplastic lymphoma kinase (ALK) small molecule tyrosine kinase inhibitor (TKI) with additional activity against MET, ABL, Axl, EPHA2, LTK, ROS1 and SLK. It has demonstrated significant anti-tumor activity in both ALK TKI-naive and crizotinib-resistant models of ALK fusion-positive NSCLC. Methods: In this multicenter phase I/II study, patients (pts) with advanced solid tumors were enrolled in the phase I dose escalation portion of the study and given X-396 on a continuous 28-day schedule (NCT01625234). Doses from 25 up to 250 mg once daily were evaluated and 225mg was selected for further evaluation in the phase II expansion. Patients in this phase were required to have ALK+ NSCLC and measurable disease. Cohorts included pts who were 1) ALK TKI-naive, 2) pts who progressed on prior crizotinib and had not received a 2 nd generation ALK TKI, 3) pts who progressed on a 2 nd generation ALK TKI (may also have received crizotinib), 4) pts with untreated or recurrent central nervous system (CNS) metastases, and 5) pts with leptomeningeal disease. All pts were assessed for adverse events (AEs) using CTCAE version 4.03, response to therapy was assessed using RECIST 1.1. Results: As of the December 09, 2015 data cutoff, 57 pts (31 men, 26 women) have been enrolled. Median age is 56 (20-79) years, the majority of patients had ECOG performance status 1 (67%). The most common drug-related AEs included rash (49%), nausea (28%), vomiting (25%), and fatigue (23%). Most AEs were Grade (G) 1-2. The G3 treatment-related AEs were rash (7 pts), fatigue (1 pt), decreased appetite (1 pt), dehydration (1 pt), pruritus (1 pt), and face edema (1 pt). In particular, no G3 treatment-related gastrointestinal toxicity or liver enzyme elevation has been reported. To date, 27 ALK+ NSCLC pts treated at doses ? 200 mg are evaluable for response; partial response (PR) was achieved in 19 pts (70%) and stable disease (SD) in 2 pts (7%). In the crizotinib-naive pts (n = 8), responses were observed in 7 pts (88%). In the 12 pts with prior crizotinib but no other ALK TKI, 10 pts (83%) achieved PR and 1 (8%) SD. CNS responses have been observed in both crizotinib naive and crizotinib resistant pts. The median duration of treatment in the 27 evaluable ALK+ pts is 16+ weeks, with the longest being 128+ weeks. Conclusion: X-396 is well-tolerated and induces responses in both crizotinib-naive and crizotinib-resistant ALK+ NSCLC pts, as well as patients with CNS lesions. Enrollment is ongoing in the expansion cohorts. Citation Format: Christine M. Lovly, Jeffrey R. Infante, George R. Blumenschein, Karen Reckamp, Heather Wakelee, Corey A. Carter, Saiama N. Waqar, Joel Neal, Jon P. Gockerman, Gary Dukart, Kimberly Harrow, Chris Liang, James J. Gibbons, Leora Horn. Phase I/II trial of X-396, a novel anaplastic lymphoma kinase (ALK) inhibitor, in patients with ALK+ non-small cell lung cancer (NSCLC). [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 CT088.

Abstract CT151: CNS activity of ensartinib in ALK-positive non-small cell lung cancer patients

Background: Ensartinib (X-396) is a novel, potent anaplastic lymphoma kinase (ALK) small molecule tyrosine kinase inhibitor (TKI) with additional activity against MET, ABL, Axl, EPHA2, LTK, ROS1 and SLK. In animal studies, brain concentration of ensartinib in mice given at the therapeutic dose was 4 times higher than the IC50 for growth inhibition of ALK positive cells in vitro. Ensartinib was significantly more effective than crizotinib at inhibiting the intracranial growth of the SH-SY5Y neuroblastoma model harboring the F1174L mutation. We subsequently evaluated the CNS activity of ensartinib in patients with ALK positive non-small cell lung cancer (NSCLC). Methods: In this multicenter phase I/II study, patients (pts) with advanced solid tumors were enrolled and given ensartinib orally on a continuous 28-day schedule (NCT01625234). Doses from 25 mg up to 250 mg once daily were evaluated and 225 mg was selected for further evaluation in the phase II expansion. We report on patients enrolled with CNS metastases (with or without systemic disease) who were ALK TKI naive or had received prior crizotinib or a second generation ALK TKI. Patients with untreated asymptomatic CNS metastases were allowed to enroll. All pts were assessed for adverse events using CTCAE version 4.03, and response to therapy was assessed using RECIST 1.1. Patients with only CNS disease had to have at least one measurable target lesion ≥ 3 mm in diameter. Results: As of the December 13, 2016 data cutoff, 26 pts with ALK+ NSCLC and baseline CNS metastases have been treated at ≥ 200 mg. Of the 26 pts, 13 pts had baseline target lesions (8 of which also had non-target lesions) and 13 pts had baseline non-target lesions only. Median age is 52 (21-72) years and the majority of patients had ECOG performance status of 1 (69%). CNS responses have been observed in both ALK TKI naive pts and pts that received prior crizotinib. In the 13 pts with baseline target CNS lesions, intracranial response was achieved in 9 pts (69%), including 1 CR, and 4 pts (31%) had SD, a 100% disease control rate. In the 13 pts with non-target baseline lesions only, 1 CR was achieved and 8 pts had SD. All 3 (100%) ALK TKI naive pts with baseline target lesions achieved CR or PR, and 5/8 pts (62%) that received prior crizotinib only and had baseline target lesions responded. The median duration of intracranial response in the 10 pts who responded (9 with target lesions, 1 with non-target lesions only) is 5.8+ months, with the longest duration being 24 months. Conclusions: Our clinical findings support the preclinical results that the use of ensartinib at doses generally well-tolerated in the clinic may result in favorable therapeutic outcomes in pts with ALK+ NSCLC with baseline CNS metastases. The ongoing phase III eXalt3 study will assess CNS response rate and time to CNS progression in pts receiving first-line ensartinib vs crizotinib (NCT02767804 and NCT01625234). Citation Format: Leora Horn, Karen L. Reckamp, Sandip Patel, George Blumenschein, Joel W. Neal, Barbara Gitlitz, Saiama Waqar, Geoffrey Oxnard, Christina Brzezniak, Gary Dukart, Fenlai Tan, Kimberly Harrow, Chris Liang, James Gibbons, Heather A. Wakelee. CNS activity of ensartinib in ALK-positive non-small cell lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT151. doi:10.1158/1538-7445.AM2017-CT151

Abstract 2622: New therapeutic strategies in neuroblastoma: combined targeting of a novel tyrosine kinase inhibitor and liposomal siRNAs against ALK

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The anaplastic lymphoma kinase (ALK), a tyrosine kinase receptor involved in the genesis of several human cancers, is a promising therapeutic target in Neuroblastoma (NB). Many studies on sporadic cases with advanced NB have shown ALK mutated or highly expressed independently of its genetic status (mutated, amplified, wild-type) and its pivotal role in NB growth and survival. As ALK is currently considered a master driver of NB oncogenesis, the use of therapies inhibiting ALK represents a suitable treatment option. The ALK inhibitor Crizotinib was recently approved for the treatment of advanced NSCLC patient. However, as often observed with kinase inhibitors, some patients develop acquired resistance in part due to genetic ALK mutations. To overcome this limitation a selective and more potent ALK inhibitor, X-396, was developed that is currently in phase I clinical trial in adult patients with advanced solid tumors, but it has not been tested in preclinical animal models of NB. We have successfully employed a RNAi-mediated therapeutic approach to selectively knockdown ALK expression by using nanoliposomes entrapping siRNA and coupled with anti-GD2 antibodies to target NB cells. Our data demonstrate for the first time a role of ALK in NB-induced angiogenesis and in tumor invasion. Here, we aimed to test whether the combination of the novel ALK kinase inhibitor X-396 with the liposome-based silencing of ALK could represent a powerful synergistic and or additive effect in NB xenografts to promote long-term survival. In vitro, X-396 showed to be more effective than Crizotinib in inhibiting cell proliferation and in inducing cell death in both LAN-5 (R1275Q ALK mut, Crizotinib-sensitive) and SH-SY5Y (F1174L ALK mut, Crizotinib-resistant) cells. Pharmacokinetic profiles of X-396 after multiple PO administration in NB-xenografts revealed a good bioavailability and a moderate half-life. Biodistribution study clearly indicated that the concentration of X-396 8h post-administration was 35 fold higher in the tumor site than in plasma. X-396 reduced the tumor volume in subcutaneous SH-SY5Y NB-model in a dose-dependent manner and it was more effective than Crizotinib when administered at the same concentration. In orthotopic xenografts of SH-SY5Y and LAN-5 cells, X-396 significantly increased life span. In combination studies, all effects were significantly improved in the mice treated with targeted liposomal formulation of ALK-siRNA and X-396 compared to untreated control mice or mice receiving the single agent treatments. In the latter studies, further enhancement of life span was obtained by increasing the X-396 dose. Moreover, the combined treatment was also proven to be effective in the SH-SY5Y-pseudometastatic model in determining long-term survivors. Our findings provide a rational basis to design innovative molecular-based combination of treatments for clinical application in NB. Citation Format: Daniela Di Paolo, Laura Emionite, George Liu, Michele Cilli, Annarita Di Fiore, Chiara Brignole, Chris Liang, Fabio Pastorino, Jay Gibbons, Mirco Ponzoni, Patrizia Perri. New therapeutic strategies in neuroblastoma: combined targeting of a novel tyrosine kinase inhibitor and liposomal siRNAs against ALK . [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2622. doi:10.1158/1538-7445.AM2014-2622

Abstract 3262: Targeting PI3K-coupled MEK/ERK pathway for therapy in pediatric B-cell acute lymphocytic leukemia.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy diagnosed in children and B cell type is prevalent among pediatric ALL. At present, cytotoxic agents are the major components for chemotherapy against ALL, which are limited by their severe side effects. PI3Kδ was found to be over activated in most B cell type leukemia, and one of its inhibitor CAL-101 (GS-1101) is in active clinical trials for B cell type acute or chronic leukemia with promising outcomes. However, the mechanism of action of PI3Kδ inhibitor against B-ALL needs to be further studied to optimize the therapeutic success. Herein, for the first time we found that blockade of PI3K-coupled MEK/ERK pathway by a novel PI3Kδ selective inhibitor X-370 is essential for its efficacy against B cell acute lymphocytic leukemia. X-370, which was obtained through rational design and screening displayed potent activity against PI3Kδ (IC50=7 nM), while sparing class II/III PI3Ks as well as a panel of protein kinases representative of the human kinome. X-370 selectively blocked constitutively activated and stimuli-induced PI3Kδ signaling among four isoforms of class I PI3Ks. X-370 abrogated both Akt and Erk signaling in human B cell leukemia cells, which is accompanied with induction of cell cycle arrest and apoptosis. Mechanism studies revealed that X-370 obstructed PDK1 from binding to and/ or phosphorylating MEK in a PI3Kδ-dependent manner. Accordingly, forced expression of constitutively activated MEK attenuated the antiproliferative activity of X-370. Furthermore, X-370 preferentially inhibited the survival of primary B-ALL cells containing PI3Kδ-coupled Erk phosphorylation, which were obtained from pediatric B-ALL patients. In summary, with a novel PI3Kδ selective inhibitor X-370, we found that inhibition of PI3Kδ led to abrogation of ERK signaling via an atypical PI3K/ PDK1/ MEK/ Erk signaling cascade. Inhibition of PI3Kδ-coupled Erk by a PI3Kδ inhibitor may predict its efficacy against B cell acute lymphocytic leukemia. These findings support the rationale for clinical testing of PI3Kδ inhibitor for the treatment of pediatric B-ALL and provide insight to optimize therapeutic strategy for such treatment. Citation Format: Xiang Wang, Ben-shang Li, Li-xia Ding, Chris Liang, Jian Ding, Ling-hua Meng. Targeting PI3K-coupled MEK/ERK pathway for therapy in pediatric B-cell acute lymphocytic leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3262. doi:10.1158/1538-7445.AM2013-3262

Abstract 1788: Preclinical development of a selective, potent small molecule ALK inhibitor

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC A chromosomal inversion [inv (2)(p21p23)] resulting in the expression of an oncogenic kinase fusion protein known as EML4-ALK [echinoderm microtubule-associated protein-like 4- anaplastic lymphoma kinase] has been implicated in the pathogenesis of a subset of lung adenocarcinomas. An agent (PF-02341066), originally described as a MET tyrosine kinase inhibitor (TKI) but with “off-target” ALK activity, has demonstrated significant activity in ALK-fusion positive lung cancer patients and is currently in phase II/III clinical trials. In this study, we describe the development and characterization of a novel and highly selective ALK TKI. X-276 (racemic mixture) is an orally bioavailable ATP mimetic that has high selectivity for ALK as assessed by Ambit assays (binding constant 2nM). Treatment of H3122 lung adenocarcinoma cells, which harbor the EML4-ALK E13;A20 fusion product, resulted in potent inhibition of cell growth with an IC50 of 10nM, approximately 10 fold less than the IC50 of H3122 cells treated with a racemic mixture of PF-02341066. Immunoblot analysis confirmed that X-276 inhibited ALK phosphorylation in H3122 cells. Importantly, the IC50 of X-276 in H1993 lung cancer cells, known to be MET-dependent, was >100 fold higher. In addition, the selectivity index of H3122 cells vs. hepatocyte HepG2 cells was >135. Finally, compared to vehicle controls, X-276 delayed the growth of H3122 xenografts in established nude mice by >60%. Collectively, these data show that TKIs can be developed with greater specificity against ALK. Such compounds may lead to the development of ‘second-generation’ ALK TKIs with more potency against ALK-mutant cancers. Future studies will focus on determining the mechanism of acquired resistance to ALK TKIs using these more potent ALK inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1788.