ROS1 Inhibitor Research Articles

Abstract 764: Rational combination of PF-06463922 (next-generation ALK inhibitor) with PI3K pathway inhibitors overcomes ALKi resistance in EML4-ALK+ NSCLC models

Abstract Crizotinib (PF-02341066) is a small molecule tyrosine kinase inhibitor of ALK, ROS1 and c-MET that is approved in over 70 countries for the treatment of ALK fusion positive non-small cell lung cancer (ALK+ NSCLC). Crizotinib achieved robust objective response rates of approximately 60% in ALK+ NSCLC and significantly improved progression free survival compared to chemotherapy. The emergence of secondary mutations within the ALK kinase domain or the activation of compensatory signaling pathways in crizotinib and other ALKi refractory tumors prompted searches for next generation of ALKi active against resistance mutations as single agents or in combination with other treatments. Such effort led to our recent discovery of PF-06463922, an ALK/ROS1 inhibitor with greatly improved ALK potency, brain penetration, and broad spectrum activity against all known clinical ALKi-resistant mutations. PF-06463922 is being tested in a Phase I clinical trial in both ALK+ and ROS1 fusion positive NSCLC in treatment naive or ALKi relapsed patients. In our current preclinical study, we explored rational combination strategies to further improve the efficacy of PF-06463922 in ALKi resistant cells or tumors. Our results show that compared to PF-06463922 alone, the combination of this compound with PI3K pathway inhibitors, such as PF-05212384 (PI3K/mTOR), GDC0941 (pan-PI3K) or GDC0032 (beta-sparing) leads to more robust anti-proliferative activity in vitro and greater duration of efficacy in vivo in the ALKi resistant models. These PI3K pathway inhibitors also partially overcome EGF or HGF ligand-induced resistance to PF-06463922. Interestingly, in addition to AKT signaling, both compounds inhibit ERK signaling as well, which may be essential for their enhancement of PF-06463922 cell activity or tumor efficacy in combination settings. Studies are ongoing to identify optimal partners for PF-06463922 combination using isoform selective PI3Ki, AKTi and mTORi. We are also exploring the breadth of efficacy of this combination in overcoming resistance to crizotinib, PF-06463922 or other ALKi. The findings provide important evidence that will help define the clinical development path for PF-06463922. This research effort may ultimately lead to more effective approaches to treat ALKi refractory patients in the clinic. Citation Format: Ping Wei, Ming Qiu, Nathan Lee, Joan Cao, Hui Wang, Konstantinos Tsaparikos, Conglin Fan, Timothy Sargis, Justine Lam, Maruja E. Lira, Goldie Lui, James Hardwick, Valeria Fantin, Paul Rejto, Tod Smeal. Rational combination of PF-06463922 (next-generation ALK inhibitor) with PI3K pathway inhibitors overcomes ALKi resistance in EML4-ALK+ NSCLC models. [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 764. doi:10.1158/1538-7445.AM2015-764

Abstract 5390: The TRK inhibitor RXDX-101 enhances the efficacy of temozolomide and irinotecan in a xenograft model of neuroblastoma

Abstract Purpose: Neuroblastoma (NB) is one of the most common and deadly solid tumors of childhood. The Trk family of neurotrophin receptors plays an important role in clinical behavior of NBs. Overexpression of TrkB and its ligand, BDNF, is associated with poor prognosis. We wanted to determine if RXDX-101, an oral pan-TRK, ROS1 and ALK inhibitor, would be effective in our NB xenograft model, either alone or in combination with conventional chemotherapy. Experimental Design: We tested the in vitro effects of RXDX-101 as a single agent, or in combination with the chemotherapeutic agents irinotecan and temozolomide (Irino-TMZ), using a subclone of the SH-SY5Y NB cell line transfected with TrkB. We also examined in vivo growth inhibition of TrkB-expressing NB xenografts with RXDX-101 alone or in combination with Irino-TMZ. Results: RXDX-101 significantly inhibited growth of TrkB-expressing NB cells in vitro. Enhanced in vitro inhibition was observed when RXDX-101 was used in combination with Irino-TMZ. Single agent therapy with RXDX-101 resulted in significant tumor growth inhibition compared to control animals [p<0.0001 for event-free survival (EFS)]. The addition of RXDX-101 to Irino-TMZ also significantly improved the EFS of animals compared to vehicle or Irino-TMZ treated animals (p<0.0001 for combination vs. control, p = 0.0012 for combination vs. Irino-TMZ). Conclusions: We show that RXDX-101 inhibits growth of TrkB expressing NB cells in vitro and in vivo. Furthermore, RXDX-101 cotreatment enhanced the efficacy of conventional chemotherapy in our NB xenograft model. Our data suggest that RXDX-101 has potential for incorporation in clinical trials for NB and other Trk expressing tumors. Citation Format: Radhika Iyer, Rebecca L. Golden, Koumudi Naraparaju, Jamie L. Croucher, Peng Guan, Gang Li, Zachary Hornby, Garrett M. Brodeur. The TRK inhibitor RXDX-101 enhances the efficacy of temozolomide and irinotecan in a xenograft model of neuroblastoma. [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 5390. doi:10.1158/1538-7445.AM2015-5390

Widespread Renal Polycystosis Induced by Crizotinib

With the widespread availability of biological antitumor drugs, the current scene of chemotherapies is changing. New chemotherapy agents, such as crizotinib, an inhibitor of anaplastic lymphoma kinase (ALK) and ROS1, usually used in pretreated advanced ALK-positive non-small-cell lung carcinoma, are more often used, and a description of the onset of side effects with suggestions for their management could be of interest for physicians. We describe a case of diffuse and aggressive renal polycystosis induced by crizotinib, which regressed after therapy, which could be of interest considering its wide extension and disappearance after the end of treatment. We also suggest some considerations from the literature and from the case reported that could be helpful in the management of this condition, which is known to be caused by crizotinib treatment.

STARTRK-1: Phase 1/2a study of entrectinib, an oral Pan-Trk, ROS1, and ALK inhibitor, in patients with advanced solid tumors with relevant molecular alterations.

2596 Background: Entrectinib (formerly RXDX-101), a potent and selective small molecule inhibitor of TrkA/B/C, ROS1, and ALK kinases, has demonstrated early clinical activity when orally administered intermittently under fasting conditions (ASCO 2014). In this abstract, we report a companion Phase 1/2a trial of entrectinib administered daily in the fed state. Methods: Pts with advanced solid tumors with molecular alterations in NTRK1/2/3, ROS1 or ALK were treated daily with entrectinib in the fed state. Pts with asymptomatic untreated brain metastases were allowed; pts may have received prior TKI therapy. Endpoints include safety, RP2D, PK, and tumor response Results: Entrectinib was well tolerated across all dose levels. No DLTs or significant safety issues were reported from any cohort. Of 15 pts treated, the majority reported only G1 or G2 AEs. 3 pts reported > G3 AEs (each unrelated to entrectinib). 1 SAE (unrelated) has been reported. Entrectinib was readily absorbed with median Tmax of 4 hr at steady state. Steady state was reached within 7 days of dosing and the estimated t1/2 was approximately 17 hrs. There were dose proportional exposure increases with moderate accumulation (~2x or less) at steady state. 15 pts with various molecular alterations identified by local lab testing were treated in the following dose cohorts (mg/m2): 100 (N = 5), 200 (N = 5), 400 (N = 5). Summary of alterations of enrolled pts: NTRK (point mutation = 5; amplification = 1; rearrangement = 1); ROS1 (SNP = 1); ALK (SNP = 2; ampl = 1; rearr = 4). 3 pts with ALK-rearranged NSCLC failed at least 2 prior ALK inhibitors. No pts with ROS1 rearrangements have been treated. No objective responses observed to date. Conclusions: Entrectinib, administered daily in fed condition, is well tolerated. In STARTRK-1, there have been no responses at doses ≤ 200 mg/m2 (400 mg/m2 cohort recently opened). Doses ≥ 400 mg/m2 were associated with responses in the ALKA-372-001 Phase 1 study. Dose escalation will continue until the RP2D is identified. Thereafter, Phase 2a cohort expansion will begin enrolling pts with NTRK1/2/3, ROS1, or ALK molecular alterations to assess antitumor activity. Clinical trial information: NCT02097810.

Alka-372-001: First-in-human, phase I study of entrectinib – an oral pan-trk, ROS1, and ALK inhibitor – in patients with advanced solid tumors with relevant molecular alterations.

2517 Background: Entrectinib (formerly RXDX-101) is a potent and selective oral small molecule inhibitor of the TrkA/B/C, ROS1, and ALK kinases. Schedule A (fasted, 4d on/3d off for 3wks, 1 wk rest) demonstrated significant antitumor activity (ASCO 2014). This abstract reports completion of Sch A and two other ongoing dosing schedules (B and C). Methods: Pts with advanced solid tumors with molecular alterations in TrkA, ROS1 or ALK were treated in Sch B (QD) or Sch C (4d on/3d off), both in fed state. Results: 31 pts were treated in Sch A (N=19), B (N=6), or C (N=6). In Sch A, doses > 800 mg/m2 did not increase exposure significantly. Thus, accrual in Sch A was closed. Various dose levels (mg/m2) were explored in Sch B [200 (n=3); 400 (n=3)] and Sch C [400 (n=3); 800 (n=3)]. 7 pts had objective responses: 6 PR, 1CR (see Table). We are the first to report clinical activity of a Trk inhibitor in a pt with NTRK1+ (encoding TrkA) CRC. Of 7 ROS1-rearranged evaluable/measurable NSCLC pts, 4 have had an objective response (ORR: 57%), with a median duration of 6+ months. Moreover, in ROS1 pts treated at ≥ 400 mg/m2, the ORR was 80% (4 of 5 pts). Entrectinib is well tolerated. The majority of pts reported G1/ G2 AEs. 13 pts reported ≥ G3 AEs. Asthenia and muscle weakness were possibly related ≥ grade 3 AEs (both reversible). No DLTs have been reported to date. In Sch A, entrectinib was readily absorbed and exposures increased dose-proportionally up to 800 mg/m2. In fed state, entrectinib exposures were approx. 2x compared to fasted state. Conclusions: In this trial with entrectinib administered in 3 different dosing schedules, significant antitumor response was observed in pts with relevant molecular alterations, notably ROS1-rearranged NSCLC at doses ≥ 400 mg/m2/day and the only NTRK1 rearranged pt treated to date. Accrual in Sch B and C continues until RP2D is achieved, followed by dedicated studies in selected tumor types. Clinical trial information: NCT02097810.Objective responses. Tumor type (alteration) Sch/Dose (mg/m2) Best Response Duration of Response (cycles) NSCLC (ALK) A/800 PR 10 NSCLC (ROS1) A/1200 PR 14+ C/400 PR 7+ C/400 CR 5+ B/400 PR 3+ CRC (NTRK1) A/1600 PR 3 Neuroblastoma (ALK) A/800 PR 9

Molecular Changes Associated with Acquired Resistance to Crizotinib in ROS1-Rearranged Non-Small Cell Lung Cancer.

Although ROS1-rearranged non-small cell lung cancer (NSCLC) is sensitive to crizotinib, development of resistance is inevitable. Here, we identified molecular alterations in crizotinib-resistant tumors from two NSCLC patients with the CD74-ROS1 rearrangement, and in HCC78 cells harboring SLC34A2-ROS1 that showed resistance to crizotinib (HCC78CR cells). ROS1 kinase domain mutations were examined in fresh tumor tissues from two NSCLC patients and HCC78CR1-3 cells by direct sequencing. Ba/F3 cells expressing ROS1 secondary mutations were constructed to evaluate resistance to crizotinib. An upregulated pathway was identified using phospho-receptor tyrosine kinase array, EGFR signaling antibody array, and RNA sequencing (RNA-seq). Cell proliferation and ROS1 downstream signaling pathways were compared between HCC78 and HCC78CR1-3 cells. The ROS1 G2032R mutation was identified in crizotinib-resistant tumors from one patient. Furthermore, HCC78CR1 and CR2 cells harbored a novel ROS1 L2155S mutation (73.3% and 76.2%, respectively). ROS1 G2032R and L2155S mutations conferred resistance to crizotinib in Ba/F3 cells. Evidence of epithelial-to-mesenchymal transition with downregulated E-cadherin and upregulated vimentin was observed in HCC78CR1-2 cells and in the other patient. RNA-seq and EGFR signaling antibody array revealed that the EGFR pathway was significantly upregulated in HCC78CR3 versus HCC78 cells. Cells with the ROS1 mutation and upregulated EGFR were sensitive to foretinib, an inhibitor of c-MET, VEGFR2, and ROS1 and irreversible EGFR tyrosine kinase inhibitors plus crizotinib, respectively. Molecular changes associated with acquired crizotinib resistance in ROS1-rearranged NSCLC are heterogeneous, including ROS1 tyrosine kinase mutations, EGFR activation, and epithelial-to-mesenchymal transition.

ROS1 amplification mediates resistance to gefitinib in glioblastoma cells.

Glioblastoma (GBM) is the most aggressive brain tumor in adults and remains incurable despite multimodal intensive treatment regimens. The majority of GBM tumors show a mutated or overexpressed EGFR, however, tumors treated with tyrosine kinase inhibitors (TKIs) will inevitably recur highlighting the need to identify signalling pathways involved in GBM resistance to these drugs. To this end, we treated GBM cells that overexpress EGFR with increasing concentrations of gefitinib and isolated resistant clones. These resistant clones were subject to RNAseq and the expression of several genes was found to be upregulated. These genes are mainly tyrosine kinase receptors and include ROS1, DDR1 and PDGFRA and are known to control several downstream targets of EGFR. The upregulation of ROS1 and DDR1 was confirmed at the protein level by western blot. Treatment with a potent and highly specific pyrazole ROS1 inhibitor in ROS1 overexpressing clones led to a sensitization of these cells to low concentrations of gefitinib. Combined treatment with gefitinib and ROS1 inhibitor induces massive cell death by apoptosis following a prolonged S phase cell cycle arrest. Our current study led to the discovery of alternative pathways used by GBM cells to evade cell death following treatment with gefitinib and identifies new therapeutic targets to prevent GBM cell resistance to the drug.

Abstract P4-01-21: Analysis and single-cell retrieval of circulating tumor cells to monitor treatment response and assess genotype in triple-negative breast cancer

Abstract Introduction: We used a high-recovery rare cell analysis and single-cell picking system to enrich, visualize, and isolate circulating tumor cells (CTCs) for genomic analysis from the blood of patients with advanced triple-negative breast cancer (TNBC) undergoing treatment with cisplatin as part of a study to intensively characterize TNBC. CTCs were evaluated regularly during treatment to monitor CTC burden and characteristics that could be associated with treatment response or disease progression, and perform single-cell mutational analysis to inform clinical decision making. Methods: Patients were enrolled in the study at the University of Washington Center for Cancer Innovation after informed consent for participation in investigation of their disease, including molecular analysis of multiple biopsies of accessible tumor. CTCs were evaluated prior to treatment and tracked longitudinally. Density-based enrichment of blood cells was performed using the AccuCyte tube, float and collector system. Collected cells were processed and applied to microscopic slides. Fluorescently labeled antibodies to cytokeratin, CD45 and EpCAM, and a nuclear dye were applied to samples using an automated slide stainer. Slides were scanned on a digital microscope and candidate CTCs identified using image analysis software. CTCs were verified by appropriate morphology and expression of epithelial and nuclear stains without CD45 expression. Other antibodies used to characterize cells included Her2, EGFR, and Ki-67. A mutation hypothesized to lead to the activation of ROS1 was identified in the cancer cells isolated from the bone marrow of one patient. CTCs were picked using our integrated semi-automated system and evaluated for the ROS1 variant using whole genome amplification followed by nested PCR and Sanger sequencing. Results: Seven patients have been enrolled to date. At least 1 CTC/mL has been found in all patients. Pre-treatment CTC levels in the patient with the ROS1 mutation were extremely high (1500/mL). One week after treatment, CTC levels spiked to more than 5000/mL. CTC counts then dropped exponentially to 9/mL after 4 months. CTC clusters and Ki-67 positive cells also decreased during therapy. Treatment with cisplatin was discontinued in this patient due to toxicity and progression, and CTC levels increased to nearly 9000/mL over 4 months. The ROS1 mutation was found in approximately 50% of individually picked CTCs before treatment with crizotinib, a ROS1 inhibitor. A second patient was found to have somatic loss of BRCA1, and was therefore treated with the PARP inhibitor, veliparib. CTC levels increased during veliparib treatment up to 13/mL. The same patient was subsequently treated with ponatinib, an FGFR inhibitor, based on the identification of two linked somatic missense mutations involving FGFR2 (S252W and Y375C). After beginning ponatinib, CTCs fell to undetectable levels. Conclusions: Analysis of CTCs may provide a non-invasive measure of cancer progression/response and the molecular evolution of tumor cells in patients with TNBC. Single-cell CTC retrieval after slide-based immunofluorescent visualization is compatible with whole genome amplification and sequencing methods. Citation Format: Arturo Ramirez, Daniel Campton, Elisabeth Mahen, Sibel Blau, Anthony Blau, Eric Kaldjian, Jackie Stilwell. Analysis and single-cell retrieval of circulating tumor cells to monitor treatment response and assess genotype in triple-negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-21.

Crizotinib therapy for advanced lung adenocarcinoma and a ROS1 rearrangement: results from the EUROS1 cohort.

Approximately 1% of lung adenocarcinomas are driven by oncogenic ROS1 rearrangement. Crizotinib is a potent inhibitor of both ROS1 and ALK kinase domains. In the absence of a prospective clinical trial in Europe, we conducted a retrospective study in centers that tested for ROS1 rearrangement. Eligible patients had stage IV lung adenocarcinoma, had ROS1 rearrangement according to fluorescent in situ hybridization, and had received crizotinib therapy through an individual off-label use. Best response was assessed locally using RECIST (version 1.1). All other data were analyzed centrally. We identified 32 eligible patients. One patient was excluded because next-generation sequencing was negative for ROS1 fusion. Median age was 50.5 years, 64.5% of patients were women, and 67.7% were never-smokers. Thirty patients were evaluable for progression-free survival (PFS), and 29 patients were evaluable for best response. We observed four patients with disease progression, two patients with stable disease, and objective response in 24 patients, including five complete responses (overall response rate, 80%; disease control rate, 86.7%). Median PFS was 9.1 months, and the PFS rate at 12 months was 44%. No unexpected adverse effects were observed. Twenty-six patients received pemetrexed (either alone or in combination with platinum and either before or after crizotinib) and had a response rate of 57.7% and a median PFS of 7.2 months. Crizotinib was highly active at treating lung cancer in patients with a ROS1 rearrangement, suggesting that patients with lung adenocarcinomas should be tested for ROS1. Prospective clinical trials with crizotinib and other ROS1 inhibitors are ongoing or planned.

Cabozantinib overcomes crizotinib resistance in ROS1 fusion-positive cancer.

ROS1 rearrangement leads to constitutive ROS1 activation with potent transforming activity. In an ongoing phase I trial, the ALK tyrosine kinase inhibitor (TKI) crizotinib shows remarkable initial responses in patients with non-small cell lung cancer (NSCLC) harboring ROS1 fusions; however, cancers eventually develop crizotinib resistance due to acquired mutations such as G2032R in ROS1. Thus, understanding the crizotinib-resistance mechanisms in ROS1-rearranged NSCLC and identification of therapeutic strategies to overcome the resistance are required. The sensitivity of CD74-ROS1-transformed Ba/F3 cells to multiple ALK inhibitors was examined. Acquired ROS1 inhibitor-resistant mutations in CD74-ROS1 fusion were screened by N-ethyl-N-nitrosourea mutagenesis with Ba/F3 cells. To overcome the resistance mutation, we performed high-throughput drug screening with small-molecular inhibitors and anticancer drugs used in clinical practice or being currently tested in clinical trials. The effect of the identified drug was assessed in the CD74-ROS1-mutant Ba/F3 cells and crizotinib-resistant patient-derived cancer cells (MGH047) harboring G2032R-mutated CD74-ROS1. We identified multiple novel crizotinib-resistance mutations in the ROS1 kinase domain, including the G2032R mutation. As the result of high-throughput drug screening, we found that the cMET/RET/VEGFR inhibitor cabozantinib (XL184) effectively inhibited the survival of CD74-ROS1 wild-type (WT) and resistant mutants harboring Ba/F3 and MGH047 cells. Furthermore, cabozantinib could overcome all the resistance by all newly identified secondary mutations. We developed a comprehensive model of acquired resistance to ROS1 inhibitors in NSCLC with ROS1 rearrangement and identified cabozantinib as a therapeutic strategy to overcome the resistance.

Development of Aseptic Renal Abscess in a Patient with Non-Small-Cell Lung Cancer with ALK Translocation during Crizotinib Treatment

Background: Crizotinib is a tyrosine kinase inhibitor of ALK, MET and ROS1. In a safety database trial, it was suggested an association of Crizotinib with the development of renal cyst in patients with non-small-cell lung cancer (NSCLC). Aim: To report an uncommon side effect of Crizotinib in a patient with NSLC. Case Presentation: We report the case of a 68-year-old woman with NSCLC who developed bilateral progressive aseptic renal abscesses during Crizotinib treatment. Conclusion: Further studies may be necessary to determinate the risk of renal cyst development and the management of these complications.

Activation of RAS family members confers resistance to ROS1 targeting drugs.

The ROS1 tyrosine kinase is activated in lung cancer as a consequence of chromosomal rearrangement. Although high response rates and disease control have been observed in lung cancer patients bearing rearranged ROS1 tumors (ROS1+) treated with the kinase inhibitor crizotinib, many of these patients eventually relapse.To identify mechanisms of resistance to ROS1 inhibitors we generated resistant cells from HCC78 lung cancer cells bearing the SLC34A2-ROS1 rearrangement. We found that activation of the RAS pathway in the HCC78 cell model, due to either KRAS/NRAS mutations or to KRAS amplification, rendered the cells resistant to ROS1 inhibition. These cells were cross-resistant to different ROS1 inhibitors, but sensitive to inhibitors of the RAS signaling pathway. Interestingly, we identified focal KRAS amplification in a biopsy of a tumor from a patient that had become resistant to crizotinib treatment.Altogether our data suggest that the activation of members of the RAS family can confer resistance to ROS1 inhibitors. This has important clinical implications as: (i) RAS genetic alterations in ROS1+ primary tumors are likely negative predictors of efficacy for targeted drugs and (ii) this kind of resistance is unlikely to be overcome by the use of more specific or more potent ROS1 targeting drugs.

Convergent Mutations and New Kinase Fusions Lead to Oncogenic STAT3 Activation in Anaplastic Large Cell Lymphoma

Introduction. If the role of ALK chimeras is well known in the pathogenesis of ALK+ Anaplastic Large Cell Lymphoma (ALCL), the mechanisms leading to the transformation of ALK- ALCL remain elusive. Combining Whole Exome Sequencing (WES), Copy Number Variation analysis and RNAseq, we provide a comprehensive characterization of different driving genetic alterations leading to the constitutive activation of STAT3 in ALK- ALCL.Methods. The discovery panel was composed of 23 ALCL (5 ALK+ and 18 ALK). The frequency of JAK/STAT3 mutations was investigated by Sanger and deep sequencing analyses in an independent panel of 158 ALCL (88 ALK-, 26 ALK+ ALCL and 44 cALCL) and 67 PTCL. RNAseq analysis was performed in a total of 23 ALCL (18 ALK- and 5 ALK+ ALCL). Functional tests were performed by transfection/transduction of different cells lines (STAT3 -/- MEF, HEK-293T, SUPM2 TTA and SUPM2 S3S). Validation studies were executed via Western Blot, and using cells viability analyses, Luciferase Assay, 2D and soft agar colony assays.Results. WES of paired normal/tumoral DNA identified a broad number of missense mutations (n=752) with 54 genes mutated in at least two different samples, without any preferential chromosomal distribution. We focused on recurrent activating mutations in JAK1 and STAT3, known to have relevant biological activity, demonstrating that 20% of nodal ALK- ALCL and cutaneous ALCL displayed mutations in either one of them and that 38% of JAK1 or STAT3 mutants showed convergent lesions, with a single case harboring L910P JAK1, G1097D/V JAK1 and Y640F STAT3 mutations. Missense STAT3 mutations occurred within the SH2 domain (Y640F, N647I, D661Y and A662V), meanwhile recurrent JAK1 mutants targeted the kinase domain (G1097D/V). The forced expression of JAK1 mutants in MEF cells led to the constitutive activation of pSTAT3, a phenotype enhanced by co-expression of JAK1 and STAT3 mutants and linked to colony formation. The cell growth of single and double mutant MEF was efficiently controlled in vitro by JAK1/2 and Hsp90 inhibitors (ruxolitinib and PUH71). Similarly, the treatment of mice bearing a JAK1 and STAT3 mutated ALK- ALCL Patient Derived Tumorgraft (PDT) efficiently controlled the lymphoma cell growth. Next we proved that the transcriptome of reconstituted JAK1 or STAT3 mutated STAT3 MEF -/- cells showed a hyperactive STAT3 signaling pathway and the up-regulation of multiple transcription factors. Among STAT3 associated genes, we discovered ATF3 and its known transcriptional regulated genes. The relationship between pSTAT3 and ATF3 was strengthened by a Gene Set enrichment Analysis and by their down-regulation in NPM-ALK cells after genomic or pharmacological knockdowns (KD) of ALK signaling. Moreover, we showed that the protein levels of pSTAT3 and ATF3 concordantly decreased in NPM-ALK Karpas 299 cells treated with an ALK inhibitor (CEP28122) or after IL-2 starvation of NK/T-cells . The analysis of a large cohort of Peripheral T-cell lymphoma confirmed the preferential expression of “bona fide” STAT3 genes in ALK- (30%) and in ALK+ ALCL; data confirmed by immunohistochemistry approach. Because JAK1 and/or STAT3 mutations did not recapitulate all pSTAT3+ ALK- ALCL, we performed a massive parallel RNA sequencing in search of other lesions in this pathway. This approach recurrent in-frame fusions combining transcription/repressor factors (NFkB2 or NCOR2) with a tyrosine kinase (ROS1 or TYK2) were identified. These chimera (NFkB2/ROS1, NFkB2/TYK2, NCOR2/ROS1, PABP4/TYK2) were proven to have both transcriptional and enzymatic activities, and to be oncogenic, a phenotype that could be abrogated by a novel ROS1 inhibitor (JNJ-ROS1i-A). Lastly, we proved that NFkB2-ROS1 rescued the phenotype of NPM-ALK cells after ALK signaling ablation but not after STAT3 knock-down.Conclusions. Our data demonstrate that a subset of ALK- ALCLs displays the constitutive activation of JAK/STAT3 pathway via multiple and alternative mechanisms (single and convergent somatic mutations and translocations), which could be significantly abrogated by specific inhibitors (i.e. JAK1/2 and ROS1). The central role of STAT3 and the possibility of using novel target strategies provide new avenues in the treatment of ALK- ALCL patients, as suggested by our preclinical ALCL PDT model. DisclosuresRosenwald:Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties.

Oxido-reductive regulation of vascular remodeling by receptor tyrosine kinase ROS1.

Angioplasty and stenting is the primary treatment for flow-limiting atherosclerosis; however, this strategy is limited by pathological vascular remodeling. Using a systems approach, we identified a role for the network hub gene glutathione peroxidase-1 (GPX1) in pathological remodeling following human blood vessel stenting. Constitutive deletion of Gpx1 in atherosclerotic mice recapitulated this phenotype of increased vascular smooth muscle cell (VSMC) proliferation and plaque formation. In an independent patient cohort, gene variant pair analysis identified an interaction of GPX1 with the orphan protooncogene receptor tyrosine kinase ROS1. A meta-analysis of the only genome-wide association studies of human neointima-induced in-stent stenosis confirmed the association of the ROS1 variant with pathological remodeling. Decreased GPX1 expression in atherosclerotic mice led to reductive stress via a time-dependent increase in glutathione, corresponding to phosphorylation of the ROS1 kinase activation site Y2274. Loss of GPX1 function was associated with both oxidative and reductive stress, the latter driving ROS1 activity via s-glutathiolation of critical residues of the ROS1 tyrosine phosphatase SHP-2. ROS1 inhibition with crizotinib and deglutathiolation of SHP-2 abolished GPX1-mediated increases in VSMC proliferation while leaving endothelialization intact. Our results indicate that GPX1-dependent alterations in oxido-reductive stress promote ROS1 activation and mediate vascular remodeling.

Abstract 4745: Discovery of potent and selective Ros1 inhibitors with a unique DFG-out binding mode

Abstract Chromosomal rearrangements resulting in oncogenic fusion proteins containing the ROS1 receptor tyrosine kinase have been described in subsets of a variety of human malignancies including non-small-cell lung cancer (NSCLC), cholangiocarcinoma, and glioblastoma multiforme. Promising clinical responses have been observed in patients bearing tumors with ROS1 fusions treated with Xalkori, an ALK/MET kinase inhibitor that also inhibits Ros1. However, resistance has been observed and a ROS1 kinase domain mutation, G2032R, was identified in a ROS1 fusion positive NSCLC patient who developed resistance to Xalkori treatment. Clinical experience with other receptor tyrosine kinase inhibitors suggests that additional resistance mutations are likely to arise, highlighting the need for therapeutic agents that can overcome this type of resistance. We identified a novel chemical series of potent and selective Ros1 inhibitors with a unique DFG-out binding mode. The structure of human Ros1 in a complex with a ligand from this chemical class was confirmed by X-ray crystallography. Here we describe the structure-activity relationships and synthesis route for this chemical series. A representative compound from this series inhibited isolated recombinant Ros1 kinase activity with an IC50 of approximately 30 nM. This compound inhibited less than 6% of kinases in a panel of 400 at 1 μM concentration. Growth of Ba/F3 cells engineered to express Ros1 autophosphorylation in HCC78 NSCLC cells that harbor a SLC34A2-ROS1 fusion were inhibited at similar concentrations as the isolated protein. This activity translated into potent Ba/F3-Ros1 tumor growth inhibition in mice. This compound was also active on Ba/F3 cells containing Ros1 with a mutation in the gatekeeper residue, L2026M, in cell growth assays in vitro and tumor growth in vivo. Furthermore, this unique binding mode provides scope for activity on additional Ros1 mutations that confer resistance to Xalkori, such as the clinically relevant G2032R mutation. The results shown here describe a novel chemical series with a unique binding mode that has potential for activity in Ros1 driven tumors with mutations that confer resistance to Xalkori. Citation Format: Laurence Mevellec, Berthold Wroblowski, Ron Gilissen, Sophie Descamps, Elisabeth Pasquier, Christophe Adelinet, Marine Bourgeois, Guillaume Mercey, Matthieu Jeanty, Thierry Jousseaume, Aurélie Luguern, Javier Astray Gandara, Said Akzinnay, Etienne Daras, Inge Boeckx, Nele Van Slycken, Mariette Bekkers, Jeroen Van De Ven, Tinne Verhulst, Lieven Meerpoel, Jorge Vialard. Discovery of potent and selective Ros1 inhibitors with a unique DFG-out binding mode. [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 4745. doi:10.1158/1538-7445.AM2014-4745

Screening for the FIG-ROS1 fusion in biliary tract carcinomas by nested PCR.

ROS1 rearrangements have been detected in a variety of tumors and are considered as suitable targets of anticancer therapies. We developed a new, quick, specific, and sensitive PCR test to screen for the FIG-ROS1 fusion and applied it to a series of Italian patients with bile duct carcinoma (BTC). Formalin-fixed, paraffin-embedded tissues, derived from 65 Italian BTC patients, and six cell lines were analyzed by nested PCR to investigate the prevalence of a previously reported FIG-ROS1 fusion. The specificity and sensitivity of nested PCR were investigated in FIG-ROS1 positive U118MG cells in reconstitution experiments with peripheral blood mononuclear cells. We found that six out of 65 (9%) BTC patients were positive for the FIG-ROS1 fusion, comprising two out of 14 (14%) gallbladder carcinoma (GBC) patients and four out of 25 (16%) extrahepatic cholangiocarcinoma (ECC) patients. None of the 26 intrahepatic cholangiocarcinoma cases harbored the FIG-ROS1 fusion. All the cell lines were negative for this variant. In conclusion, 14-16% of GBC and ECC were positive for FIG-ROS1. This may have clinical implications, since these patients will potentially benefit from the treatment with specific ROS1 inhibitors.

Cancers broncho-pulmonaires réarrangés pour ALK: comment assurer une tolérance optimale du crizotinib en pratique clinique?

Crizotinib (XALKORI™, Pfizer) is a tyrosine kinase inhibitor of ALK, MET, and ROS1, which is currently approved for the second line treatment for ALK-rearranged lung cancer. This work from an expert group, based on the review of the data from the Profile studies, aims to provide practical elements in order to optimize the tolerability of crizotinib. Specific major or frequent side effects of crizotinib are discussed: visual disturbances, cardiac effects, elevated transaminases, and hypogonadism. In the routine practice, patients should be advised about visual disturbances, especially with regard to driving in low brightness. Digestive disorders related to crizotinib are exceptionally persistent or severe. Dietary measures and symptomatic treatments usually control these disorders. It is recommended to perform an electrocardiogram before introduction of crizotinib, to identify prolonged QT interval. Torsades de pointes may produce dizziness or syncope. Hypogonadism should be considered in case of fatigue, decreased libido, and even depression, taking into account that these symptoms may be related to cancer; testosterone serum level should be measured to identify patients that may be eligible to receive a supplementation. Monitoring of liver function tests, including transaminases and bilirubin, is necessary. To conclude, these practical elements are helpful to optimize treatment with crizotinib in patients with ALK-rearranged lung cancer; in the future, academic initiatives should be taken to study these aspects, based on the monitoring of large cohorts of patients treated with crizotinib.

Clinical Activity of Crizotinib in Ros1-Rearranged Non-Small Cell Lung Cancer

ABSTRACT Aim: Chromosomal rearrangements of the ROS1 receptor tyrosine kinase gene define a distinct molecular subset of non-small cell lung cancer (NSCLC), which may be susceptible to therapeutic ROS1 inhibition. Crizotinib is a small molecule tyrosine kinase inhibitor of anaplastic lymphoma kinase (ALK), ROS1 and c-MET. Methods: Fifty patients with advanced NSCLC harboring a ROS1 rearrangement were enrolled into an expansion cohort of the global phase 1 study of crizotinib. Local ROS1 testing was used, including break-apart fluorescence in situ hybridization (FISH) in 47 of 50 cases. Patients were treated with crizotinib at the standard oral dose of 250 mg twice daily, and assessed for safety and response to therapy. The primary endpoint was objective response rate (ORR). Duration of response (DR), progression-free survival (PFS), and overall survival (OS) were estimated using the Kaplan-Meier method. ROS1 fusion partners (if tissue was available) were identified using next generation sequencing (NGS) or reverse-transcriptase-polymerase-chain reaction (RT-PCR) assays. Results: At the time of data cutoff, median follow-up of the 50 ROS1 patients was 16.1 months. The majority of patients (86%) had received at least one prior line of standard therapy for advanced NSCLC. The ORR was 70% (95% CI, 55%, 82%), with 3 complete responses and 32 partial responses. Median DR was 75.9 weeks (95% CI, 61.6, NR). Median PFS was 19.2 months (95% CI, 14.6, NR), with 24 patients (48%) still in follow-up for PFS. Median OS has not been reached; 1-year OS was 85% (95% CI, 71%, 93%). Among 24 tumors tested (21 with NGS and 3 with RT-PCR), 7 different ROS1 fusion partners were identified, including 5 known and 2 novel partner genes. No correlation was observed between type of ROS1 rearrangement and clinical response to crizotinib. The safety profile of crizotinib in ROS1 patients was similar to that seen in patients with ALK-rearranged NSCLC. Conclusions: Crizotinib exhibits robust antitumor activity in patients with advanced ROS1-rearranged NSCLC, with an ORR of 70% and a median PFS of 19.2 months. Responses were durable with a median DR of 75.9 weeks. In this trial ROS1 defines an additional distinct population of NSCLC patients for whom crizotinib is highly effective. Disclosure: A. Shaw: Consultant/SAB: Pfizer, Novartis, Ariad, Chugai, Genentech, Ignyta; S.I. Ou: Advisory boards: Pfizer; Corporate-sponsored research: Pfizer; Y. Bang: Advisory boards: Pfizer; Corporate-sponsored research: Pfizer; B. Solomon: Advisory boards: Pfizer, Novartis, Roche, Clovis Oncology, AstraZeneca, Merck, BMS, Lilly; G.J. Riely: Advisory boards: Ariad, Celgene, Mersana; Corporate-sponsored research: Novartis, Pfizer, Millennium, Roche, GSK, Merck; M. Varella-Garcia: Advisory board: Exelixis; Corporate-sponsored research: Abbott Molecular; Other substantive relationships (Speaker engagement in educational seminars): Abbott Molecular; D. Costa: Other substantive relationships (Consultancy): Pfizer; R. Doebele: Advisory board: Pfizer, Boehringer-Ingelheim; Corporate-sponsored research: Pfizer, Mirati Therapeutics; L.P. Le: Stock ownership: Enzymatics; Other substantive relationships (Consulting with compensation): Enzymatics; Z. Zheng: Stock ownership: Enzymatics; P. Stephenson: Other substantive relationships (Consultancy): Pfizer; S.M. Shreeve: Stock ownership: Johnson and Johnson, Pfizer; Other substantive relationships (Employment): Janssen Pharmaceutical Companies of Johnson and Johnson; L. Tye: Employment and stock ownership: Pfizer; J. Christensen: Stock ownership: Pfizer; K. Wilner: Employment, stock ownership, advisory board and corporate-sponsored research: Pfizer; A. Iafrate: Advisory boards: Chugai, Constellation, Enzymatics; Stock ownership: Enzymatics. All other authors have declared no conflicts of interest.

448PD - Rxdx-101, an Oral Pan-Trk, Ros1, and Alk Inhibitor, in Patients with Advanced Solid Tumors with Relevant Molecular Alterations

ABSTRACT Aim: RXDX-101 is a small molecule inhibitor of TrkA, TrkB, TrkC, ROS1 and ALK, with high potency and selectivity. RXDX-101 demonstrated potent pharmacological activity in preclinical studies and is potentially first-in-class against the Trk family of kinases. This study aims to determine MTD, PD, PK and anti-tumor activity in patients with advanced cancer and applicable molecular alterations. Methods: Phase 1 dose escalation in patients with advanced solid tumors. RXDX-101 was dosed orally once/day in a 4 day on, 3 day off schedule for 3 weeks, followed by a 7 day rest period, in continuous 28-day cycles. Minimum of 3 patients were enrolled at each dose level. Endpoints include safety, PK, and tumor response by RECIST. Results: 19 patients have been treated at 6 dose levels (100, 200, 400, 800,1200 and 1600 mg/m2). RXDX-101 has been well tolerated to date; the MTD has not been reached. No DLT has been seen at any dose level. The most common AEs (mainly grade 1-2) considered possibly treatment-related included asthenia, paresthesias, nausea and diarrhea. Possibly related grade 3/4 AEs include asthenia and increased lipase. No treatment-related SAEs were observed. A patient with colorectal carcinoma (TrkA+) has a PR and is in cycle 2. Two patients with NSCLC (1 ROS1 + ; 1 ALK+) have also achieved PRs. An additional patient with neuroblastoma (ALK+) has a PR and is currently in cycle 16. Two patients have had prolonged stabilization of their disease and remain on treatment: a patient with NSCLC (ALK+) in cycle 14 and a patient with pancreatic cancer (ROS1+) in cycle 11. PK analysis shows maximum concentrations of RXDX-101 were generally achieved within 2 to 4 hours following dosing and exposure increased in an approximate dose proportional manner up to doses of 800 mg/M2 with minimal accumulation following multiple doses. Mean terminal half-life was 21- 32 hours and steady state reached within 4 days. Conclusions: RXDX-101 has been well tolerated in patients with advanced solid tumors. Early responses in patients with 3 different relevant molecular alterations are promising. PK profile seems suitable for once daily dosing, which is being evaluated. A global phase I/II trial was recently initiated. Disclosure: F.G.M. De Braud: Consultant or Advisory Roles Novartis Compensated Bristol-Myers Squibb; ompensated Roche Compensated Stock Ownership No Honoraria No Research Funding Yes; E. Ardini: Senior Scientist, Nerviano Medical Sciences Employee Compensated; M. Martignoni: CLIOSS Employee, Clinical Leader Compensated; A. Isacchi: Director, Nerviano Medical Sciences Compensated Employee; P. Pearson: Consultant Role Ignyta, Inc. Compensated Stock Ownership Ignyta, Inc.; D. Luo: Ignyta, Inc. Employee Sr. Director, Clinical Operations Compensated; J.L. Freddo: Consultant Role; Member Board of Directors Ignyta, Inc. Compensated Stock Ownership, Ignyta, Inc. Yes; S. Siena: Consultant or Advisory Role Amgen Compensated Bayer Compensated Celgene Compensated Sanofi Compensated Roche Compensated; tock Ownership No Research Funding Bayer. All other authors have declared no conflicts of interest.

152P - Epidermal Growth Factor Receptor Signaling Conferred Acquired Crizotinib Resistance to a Non-Small Cell Lung Cancer Cell Line Harboring the Slc34A2-Ros1 Fusion Gene

ABSTRACT Aim: The ROS1 inhibitor crizotinib produces a remarkable response in patients with non-small cell lung cancer (NSCLC) harboring the ROS1 fusion gene (J Clin Oncol 2013 abstr 8032); however, acquired crizotinib resistance is inevitable, but the mechanism is unclear. Methods: HCC78 cells harboring the SLC34A2-ROS1 gene alteration were used to model acquired crizotinib resistance to explore alternative molecular mechanisms. The resistant cell line (HCC78R) was established from HCC78 cells after continuous exposure to crizotinib for 4 months in vitro. Results: The 50% inhibitory concentration (IC50) of crizotinib in HCC78R cells (4085.9 nM) was 47-fold larger than the IC50 in parental HCC78 cells (85.8 nM) (p = 0.0145). The expression of ROS1 protein was decreased significantly in HCC78R cells, as compared to HCC78 cells. Crizotinib inhibited the phosphorylation of AKT or ERK and downstream ROS1 signaling in HCC78 cells, but did not inhibit this in HCC78R. A receptor tyrosine kinas array showed that there was no increased tyrosine kinase activation in HCC78R cells, as compared to HCC78 cells. Interestingly, however, the phosphorylation of EGFR was maintained under 2,000 nM crizotinib exposure in HCC78R cells, but inhibited in parental HCC78 cells. Therefore, we hypothesize that the EGFR signaling pathway plays a role in HCC78R cells. Expectedly, HCC78R cells were very sensitive to EGFR-TKI in a cell proliferation assay (IC50: 117 nM vs. > 10,000 nM for HCC78R vs. HCC78 cells). Consistently, the addition of EGF to the culture medium rendered the parental HCC78 cells resistant to crizotinib. No activation of EGFR mutations were found in HCC78R cells. Finally, foretinib, a multi-kinase inhibitor of ROS1 and EGFR, effectively inhibited the proliferation of both HCC78R and HCC78 cells. Conclusions: These studies suggest that EGFR signaling plays a role in crizotinib resistance in lung cancer cells harboring ROS1 fusion genes. Disclosure: All authors have declared no conflicts of interest.