2015年肿瘤治疗资料集中贴

AACR Annual Meeting 2015
Abstract Number:        2827
       
Presentation Title:        Discovery of AP26113, a potent, orally active inhibitor of anaplastic lymphoma kinase and clinically relevant mutants
       
Presentation Time:        Monday, Apr 20, 2015, 3:05 PM - 3:20 PM
       
Location:        Room 113, Pennsylvania Convention Center
       
Author Block:        Wei-Sheng Huang, Feng Li, Lisi Cai, Yongjin Xu, Sen Zhang, Scott D. Wardwell, Yaoyu Ning, Anna Kohlmann, Tianjun Zhou, Emily Y. Ye, Xiaotian Zhu, Narayana I. Narasimhan, Tim Clackson, Victor M. Rivera, David Dalgarno, William C. Shakespeare. ARIAD Pharmaceuticals, Inc., Cambridge, MA
       
Abstract Body:        Phosphorus, despite its abundance in the human body, is rarely found in drug molecules, with clinical utility limited to a few phosphonic or bisphosponic acid-based medicines and several phosphonate or phosphate-containing prodrugs. Concerns about poor cell penetration, low oral bioavailability, or biological instability have limited application of these functional classes in drug design. In our efforts to discover pharmaceuticals with novel functionality, we introduced a neutral, stable phosphine oxide moiety as a unique hydrogen-bond acceptor in the design of anaplastic lymphoma kinase (ALK) inhibitors. ALK is a receptor tyrosine kinase first identified as a chromosomal rearrangement (NPM-ALK fusion gene) in anaplastic large cell lymphoma (ALCL) and subsequently detected as an alternate fusion oncogene (EML4-ALK) in a subset of non-small cell lung cancers (NSCLC). Crizotinib, the first approved ALK inhibitor, has demonstrated impressive clinical benefit in EML4-ALK (ALK+) NSCLC patients. Drug resistance, however, emerges rapidly and point mutations within the kinase domain have been identified as a major resistance mechanism. With the specific objective to identify more potent ALK inhibitors with pan-inhibitory activity against crizotinib-resistant ALK mutants, we designed and tested a series of phosphine oxide-based compounds culminating in the identification of the clinical candidate AP26113. AP26113 exhibited 10-fold greater potency than crizotinib against ALK-positive ALCL and NSCLC cell lines, and effectively inhibited clinically relevant crizotinib-resistant mutants. AP26113 displayed ~100-fold increased selectivity for ALK-positive cells over ALK-negative cell lines while maintaining selectivity over insulin receptor tyrosine kinase receptors. Consistent with the in vitro profile, AP26113 demonstrated oral efficacy in multiple ALK+ mouse models including Karpas-299 (ALCL), H3122 (NSCLC), and Ba/F3 cells expressing crizotinib resistant mutants including G1269S and L1196M. Finally, AP26113 exhibited excellent drug-like properties including high cell permeability and solubility, moderate cross-species protein binding, and inactivity toward major cytochrome P450 iso-enzymes and hERG ion channels. Medicinal chemistry efforts leading to the discovery of this potent pan-ALK inhibitor, including design strategy, chemical series evolution, and DMPK optimization will be presented. AP26113 is currently in a global phase 2 registration trial (NCT02094573) in patients with locally advanced or metastatic NSCLC who test positive for the ALK oncogene and were previously treated with crizotinib.

Abstract Number:        781
       
Presentation Title:        The potent ALK inhibitor AP26113 can overcome mechanisms of resistance to first- and second-generation ALK TKIs in preclinical models
       
Presentation Time:        Sunday, Apr 19, 2015, 1:00 PM - 5:00 PM
       
Location:        Section 32
       
Poster Board Number:        13
       
Author Block:        Sen Zhang, Sara Nadworny, Scott D. Wardwell, Lindsey Eichinger, Biplab Das, Emily Y. Ye, J. Graeme Hodgson, Victor M. Rivera. ARIAD Pharmaceuticals, Inc., Cambridge, MA
       
Abstract Body:        Background:
Disease progression in anaplastic lymphoma kinase-positive (ALK+) non-small cell lung cancer (NSCLC) patients (pts) treated with the first-generation tyrosine kinase inhibitor (TKI) crizotinib, or the second-generation inhibitors ceritinib and alectinib, is often associated with secondary resistance mutations in ALK (such as G1202R, linked with resistance to all 3 TKIs in pts), or relapse in the brain. AP26113 is a highly potent ALK inhibitor with promising anti-tumor activity in pts with crizotinib-resistant disease. Here we broadly evaluate AP26113’s ability to overcome mechanisms of resistance to first- and second-generation ALK inhibitors, in preclinical models (including brain cancer models), and describe a novel methodology to compare in vitro potency, clinical pharmacokinetic parameters, and clinical efficacy.
Methods:
Ba/F3 cells were engineered to express native EML4-ALK or 17 mutants resistant to crizotinib, ceritinib, and/or alectinib in clinical or preclinical studies. IC50s for all TKIs were compared to “clinically effective” plasma concentrations (Ceff) of each inhibitor, which were derived using average steady-state levels in pts, and corrected for the functional effects of protein binding in vitro. In vivo studies were performed in mice, in which Ba/F3 cells with mutant or native EML4-ALK were injected subcutaneously, or a NSCLC line with EML4-ALK (H2228) was injected into the brain.
Results:
The Ceff of crizotinib was only ~2-fold above the IC50 for native ALK, suggesting that crizotinib may be susceptible to mutants that inhibit binding by only a few-fold. Indeed, 8/10 mutants previously associated with clinical resistance to crizotinib were found to have IC50s that exceed the Ceff. Similar analyses revealed mutants with IC50s that exceeded the Ceff for ceritinib (e.g. L1198F) and alectinib (e.g. I1171N), with G1202R being the most recalcitrant mutant overall. In contrast, the Ceff of AP26113 was >20-fold above the IC50 for native ALK, and exceeded the IC50 for all 17 mutants, including G2102R. In vivo, among all 4 TKIs tested (at doses effective against native ALK), only AP26113 demonstrated significant efficacy against G1202R-mutant ALK (88% tumor growth inhibition). Finally, compared to crizotinib, AP26113 markedly enhanced survival of mice bearing brain tumors.
Conclusion:
AP26113 inhibits all 17 clinically and preclinically observed crizotinib-, ceritinib-, and/or alectinib-resistant ALK mutants tested in vitro, has potent effects on the recalcitrant G1202R mutant in vivo and has activity in an orthotopic brain model. These results suggest AP26113 may effectively address a broad range of resistance mechanisms identified for other ALK TKIs. AP26113 is currently in a global phase 2 registration trial in patients with locally advanced or metastatic ALK+ NSCLC who were previously treated with crizotinib (NCT02094573).
重点翻译：AP26113在EML4-ALK的细胞株Ba/F3实验结果显示，它对17种alk突变均有良好抑制效果，包括L1196M 突变（crizotinib的耐药突变），L1198F突变（ceritinib的耐药突变），I1171N（alectinib的耐药突变），G1202R突变（crizotinib、ceritinib、alectinib的耐药突变）。

AP26113对G1202R也有作用啊，那真的是2.5代了

Abstract Number:        759
Presentation Title:        Metformin enhances the benefit of ALK inhibitors in ALK translocation-positive non-small cell lung cancer cells
Presentation Time:        Sunday, Apr 19, 2015, 1:00 PM - 5:00 PM
Location:        Section 31
Poster Board Number:        21
Author Block:        Mi Young Kim1, Geum Ock Kim1, Dong Hoon Shin2, Tae Min Kim3, Jin-Soo Kim1. 1Boramae Medical Center, Seoul, Korea, Republic of; 2National Cancer Center, Seoul, Korea, Republic of; 3Seoul National University Hospital, Seoul, Korea, Republic of
Abstract Body:        Background: Crizotinib is an effective tyrosine kinase inhibitor (TKI) against non-small cell lung cancers (NSCLCs) with anaplastic lymphoma receptor tyrosine kinase (ALK) translocation. Recently, the second generation ALK inhibitors, including alectinib (CH5424802) and ceritinib (LDK378) are actively investigated in clinical trials and show strong anti-tumor effect both in crizotinib-naïve and refractory NSCLC patients with ALK translocation. Unfortunately, the efficacy of these ALK inhibiting TKIs is limited due to primary and acquired resistance to these agents. Metformin is a widely used antidiabetic drug and shows promising anti-tumor effect in several cancers. Here, we define a therapeutic synergism between ALK inhibitors and metformin.
Material and Methods: NSCLC cells with ALK translocation-positive (ALK+) and crizotinib-sensitive H2228 and H3122, and ALK+, crizotinib-resistant SNU2535 and ALK translocation-negative H226 were evaluated. We assessed response of these NSCLC cells to crizotinib, alectinib and ceritinib alone or in combination with metformin by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay and anchorage independent growth in soft agar. Combination indices (Cis) were calculated by CalcusynTM (Biosoft, Cambridge, UK). Western blots were performed to assess biochemical changes, especially those related with ALK signaling components.
Results: The combination of ALK inhibitors with metformin induced a strong anti-proliferative effect only in ALK+ cell lines (CI of crizotinib and metformin: 0.31 - 0.79, CI of alectinib and metformin: 0.40 - 0.86, CI of ceritinib and metformin: 0.38 - 0.65). Alectinib and ceritinib effectively inhibited growth of crizotinib-resistant SNU2535, while crizotinib did not. Notably, synergistic effects of these ALK inhibitors and metformin were also shown in SNU2535 cells. Compared to ALK inhibitor alone, the combination of ALK inhibitor plus metformin resulted in decreased phosphorylation of ALK and downstream targets, such as AKT or ERK, both in crizotinib-sensitive and resistant ALK+ cells.
Conclusion: Collectively, these data support that ALK inhibitors and metformin are synergistic in ALK+ NSCLC cells. Resistance to crizotinib as well as alectinib and ceritinib might be overcome by addition of metformin.
重点翻译：体外ALK突变肿瘤细胞株实验显示，1、2代ALK抑制剂联合二甲双胍有协同增强作用，甚至可以克服ALK耐药。

Abstract Number:        761
Presentation Title:        Changing the paradigm for treating drug resistance in NSCLC: Novel combinations of AZD6094, a selective MET inhibitor, and an irreversible, selective (EGFRm/T790M) EGFRTKI, AZD9291
Presentation Time:        Sunday, Apr 19, 2015, 1:00 PM - 5:00 PM
Location:        Section 31
Poster Board Number:        23
Author Block:        Celina D'Cruz1, Evan Barry1, Ryan Henry1, Lillian Castriotta1, Alwin Schuller1, Garry Beran2, Susan Ashton2, Cath Eberlein2, Corinne Reimer1, Melanie Frigault1, Michael Zinda1, Darren Cross3, Stephen Fawell1. 1AstraZeneca, Waltham, MA; 2AstraZeneca, Alderley Park, United Kingdom; 3AstraZeneca, Cambridge, United Kingdom
Abstract Body:        Aberrant receptor tyrosine kinase (RTK) signaling is a well-documented driver of disease onset and progression across myriad cancer types, where the MET RTK contributes to tumor progression, maintenance and resistance to targeted therapies. Here we explore the therapeutic potential of AZD6094, a highly potent and selective MET inhibitor, in EGFR mutant (EGFRm) non-small cell lung cancer (NSCLC). Although many EGFRm NSCLC patients receiving first-line EGFR Tyrosine Kinase Inhibitors (TKI)
benefit from therapy initially, the majority of patients will acquire resistance in 9-14 months1,2. Of this patient population, ~10-15% of patients with emerging resistance to early generation EGFRm-TKI will have MET amplification3. Using xenograft models (HCC827) of resistance to erlotinib or gefitinib, both first-generation EGFRm-TKI, we assessed the efficacy of AZD6094 in models with varying copy number gain for MET. We demonstrate that the combination of AZD6094 with gefitinib, or AZD9291, an irreversible, selective (EGFRm/T790M) EGFR TKI, results in tumor growth inhibition (TGI) of >100% in 3 models (HCC827-ER1, PCS030 clone 1 and 2)4, suggesting that the combination is necessary and sufficient to address acquired resistance due to MET gene amplification. Moreover, we explore efficacy of AZD6094 in models representative of resistance to first-line treatment with EGFRm-TKIs, harboring MET amplification and T790M EGFR mutations. In NCI-H820 xenografts (EGFRm/T790M/MET), we demonstrate for the first time that combining MET and EGFRm/T790M TKIs (AZD6094 with AZD9291) induces tumor regressions (TGI% >100%, 94% regressions) and the loss of palpable tumors in 5/7 animals as compared to AZD9291 (TGI 48%) or AZD6094 treatment alone (TGI >100%). Pharmacodynamic analysis of tumor lysates demonstrated potent and durable inhibition of pMET in all AZD6094 treatment groups. Due to the clinical importance of understanding acquired resistance to targeted TKIs, we then generated a model of resistance to AZD6094 in MET-amplified NSCLC NCI-H1993 cells and analyzed several resistant clones (H1993R). Interestingly, MET phosphorylation remains strongly inhibited in AZD6094-treated H1993R cells, while EGFR protein
expression is upregulated and leads to co-dependency between both pathways. Enhanced expression and phosphorylation of EGFR, as well as AKT, MEK and ERK activation were commonly observed in H1993R cells. Taken together, our data support the
potential of AZD6094 as a novel combination therapy for MET-driven NSCLC in the context of EGFRm TKI resistance, and highlight the clinical relevance of EGFR and MET signaling in the context of emerging TKI resistance mechanisms and coordinated pathways.
重点摘要：体外EGFR突变肿瘤细胞株实验显示，Met抑制剂AZD6094联合易瑞沙可以对付Met扩增的EGFR突变，AZD6094联合AZD9291可以对付EGFRm/T790M/MET类型。

Abstract Number:        768
Presentation Title:        A kinome-wide siRNA screen identifies modifiers of sensitivity to the EGFR T790M-targeted tyrosine kinase inhibitor (TKI), AZD9291, in EGFR mutant lung adenocarcinoma
Presentation Time:        Sunday, Apr 19, 2015, 1:00 PM - 5:00 PM
Location:        Section 31
Poster Board Number:        30
Author Block:        Eiki Ichihara1, Joshua A. Bauer2, Pengcheng Lu3, Fei Ye3, Darren Cross4, William Pao1, Christine M. Lovly1. 1Vanderbilt University School of Medicine, Nashville, TN; 2Vanderbilt Institute of Chemical Biology High-Throughput Screening Facility, Nashville, TN; 3Vanderbilt University Medical Center, Nashville, TN; 4AstraZeneca Oncology Innovative Medicines, United Kingdom
Abstract Body:        New epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) that are targeted against EGFR T790M, such as AZD9291, are among the most promising agents for the treatment of EGFR mutant lung adenocarcinomas, as they can overcome a key mechanism of acquired resistance to first or second generation EGFR-TKIs. Emerging clinical data indicate that these drugs achieve partial responses in ~60% of patients and demonstrate variable duration of benefit (Yang J et al. Ann Oncol 2014;25 (suppl 4): iv149). However, most responding patients are expected to develop eventually progressive disease. Mechanisms of resistance for this class of mutant specific EGFR TKIs are only beginning to be elucidated.
To identify modifiers of sensitivity to AZD9291 in EGFR mutant lung adenocarcinoma, we performed a kinome-wide short-interfering (si) RNA screen using clonal PC-9/BRc1 (EGFR exon19 deletion/T790M) cells and siRNA triplicates directed against 714 phylogenetically-related kinases (GE Dharmacon). The cells were derived from PC-9 cells chronically exposed to afatinib (Chmielecki J et al. Sci Transl Med. 2011;3:90ra59). The screen was performed in the absence and presence of AZD9291 (5 nM). Among the top 20 hits, siRNAs against the serine threonine kinases, MAPK1 (ERK2) and BRAF, sensitized cells to AZD9291. Both genes encode products involved in the RAS-MAP kinase signaling pathway. Confirming the significance of these hits, combinations of AZD9291 and the MEK inhibitor, selumetinib, were more effective at inhibiting cell growth than AZD9291 alone. Consistent with these findings, we found that ERK1/2 is readily dephosphorylated upon exposure to AZD929, however, ERK1/2 becomes re-phosphorylated after continuous exposure to AZD9291 for 96 hours. These data suggest that reactivation of ERK signaling may provide an early escape mechanism mediating drug resistance. ERK reactivation after AZD9291 treatment and stronger growth inhibition by AZD9291 + selumetinib combination therapy were also seen in multiple other EGFR mutant lines. The SRC kinase is known to activate RAS-MAP kinase pathway signaling, and pharmacologic or genetic ablation of SRC partially inhibited ERK reactivation, suggesting that SRC is involved in this escape mechanism.
In conclusion, through a kinome wide siRNA screen, we identified that gene products in the MAP kinase signaling pathway modify sensitivity to AZD9291. Such sensitivity may be associated with ERK re-phosphorylation within 96h of drug treatment. Collectively, these data suggest rational drug combinations that could be used to forestall resistance to AZD9291. Additional hits from the screen are currently under investigation.
This study is supported by AstraZeneca Oncology Innovative Medicines, National Institutes of Health (NIH) NCI grants R01-CA121210, P01-CA129243, U54-CA143798, and the Uehara Memorial Foundation.

Abstract Number:        764
Presentation Title:        Rational combination of PF-06463922 (next-generation ALK inhibitor) with PI3K pathway inhibitors overcomes ALKi resistance in EML4-ALK+ NSCLC models
Presentation Time:        Sunday, Apr 19, 2015, 1:00 PM - 5:00 PM
Location:        Section 31
Poster Board Number:        26
Author Block:        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. Pfizer, Inc., San Diego, CA
Abstract Body:       
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.
重点翻译：ALK耐药模型的实验显示PF-06463922联合PI3K抑制剂可以增强药性和延长有效时间。

Abstract Number:        793
Presentation Title:        Insulin-like growth factor 1 (IGF1R)/insulin receptor (INSR) inhibitory activity of rociletinib (CO-1686) and its metabolites in nonclinical models
Presentation Time:        Sunday, Apr 19, 2015, 1:00 PM - 5:00 PM
Location:        Section 32
Poster Board Number:        25
Author Block:        Andrew D. Simmons, Sarah Jaw-Tsai, Henry J. Haringsma, Andrew Allen, Thomas C. Harding. Clovis Oncology, San Francisco, CA
Abstract Body:        Rociletinib (CO-1686) is a novel, oral, targeted irreversible inhibitor of the cancer-causing mutant forms of EGFR currently being studied for the treatment of NSCLC. Rociletinib was designed to spare wild-type EGFR signaling. Heavily-pretreated T790M+ patients treated with rociletinib at 500 or 625mg BID demonstrated a 67% objective response rate (n=56, Soria et al., ENA 2014). Adverse events (AEs) typical of wild-type EGFR inhibition, such as cutaneous toxicities, have not been observed. The most frequent AE in patients dosed with rociletinib is hyperglycemia (32% all grades/14% grade 3), which is typically managed with oral hypoglycemic therapy. Rociletinib does not directly play a role in hyperglycemia based on kinase and cellular profiling, toxicology studies performed in Sprague-Dawley rats and beagle dogs, and an oral glucose tolerance test (OGTT) in Sprague-Dawley rats. We examined the hypothesis that hyperglycemia may result from a metabolite of rociletinib. Metabolite profiling from in vitro hepatocyte incubations, as well as the analysis of plasma samples from healthy subjects and NSCLC patients, revealed three metabolites of interest referenced according to their protonated molecular ion: M460, M502, and M544. Metabolites M460 and M502 are present in higher levels in humans than in rats and dogs, whereas M544 levels are comparable between human and rat. In patients the levels of M502 are at least 5-fold greater than that of M460. All 3 metabolites demonstrated limited potency against EGFR, T790M EGFR, and HER2. Expanded cellular profiling revealed that M460 and M502 have 2-3 fold and 3-7 fold greater potency against INSR and IGF1R, respectively, as compared to rociletinib, whereas metabolite M544 demonstrated similar or reduced potency. Metabolite profiling in rats suggests that M502 is rapidly converted to M544 by acetylation, thus a rat OGTT was performed with a single dose of M502 at 1000 mg/kg to reach exposure levels comparable to those observed in rociletinib treated NSCLC patient samples. Metabolite M502 caused significant elevations in post-prandial glucose and insulin excursion. Taken together, the increased potency of M502 towards IGF1R and INSR, its absolute exposure in humans and the rat OGTT results suggest that M502 is likely to play a causative role in the hyperglycemia observed in patients. Additional data on the clinical implications of these findings, as well as the role of IGF1R pathway activation in resistance to EGFR inhibitors, will be presented.

Abstract Number:        668
Presentation Title:        Phosphoproteomic characterization of selumetinib action in KRAS mutant lung cancer
Presentation Time:        Sunday, Apr 19, 2015, 1:00 PM - 5:00 PM
Location:        Section 28
Poster Board Number:        12
Author Block:        Jae-Young Kim, Eric A. Welsh, Bin Fang, Fumi Kinose, John M. Koomen, Eric B. Haura. Moffitt Cancer Center & Research Institute, Tampa, FL
Abstract Body:        In the field of lung cancer therapeutics, KRAS-mutant lung cancer is a significant problem due to limited therapeutic options and poor prognosis. Pathway inhibition of the RAS-driven RAF-MEK-ERK pathway using small-molecule kinase inhibitors has been a key focus for treating cancers driven by oncogenic RAS; however, significant clinical responses are lacking. Our initial experiments assessed the efficacy of the clinical MEK inhibitor AZD6244 (selumetinib) in a panel of non-small cell lung cancer cell lines harboring oncogenic KRAS mutations. None of the cell lines showed pronounced apoptosis nor did we observe significant reduction in cell viability in nano-molar range (<1 μM). Given that cancer cells can rapidly rewire signaling cascades to adapt to loss of survival signaling, we hypothesized that a phosphoproteomics approach would provide further insight into adaptive signaling alteration associated with AZD6244. To this aim, we assessed global phosphoproteome change after AZD6244 treatment (1 μM, 24hr) in A427 and A549 lung adenocarcinoma cell lines harboring oncogenic KRAS mutations. We identified 7,730 unique phosphopeptides (corresponding to 3,137 unique phosphoproteins) after removing low-confidence phosphopeptides. We defined phosphopeptide significantly altered by AZD6244 as one that has had quantity changed by two-fold, corresponding to one standard deviation of fold change within the data. This resulted in 882 increased and 831 decreased phosphopeptides after MEK inhibition. As expected, AZD6244 decreased ERK phosphorylation and induced feedback increase of MEK and B- and C-RAF phosphorylation. In addition, we observed that AZD6244 increased tyrosine phosphorylation (Y1172) and decreased serine phosphorylation (S1045) of EGFR, which are involved in kinase activation and receptor trafficking, respectively. We also found reduced inhibitory serine phosphorylation of MET (S985), suggesting MEK inhibition could lead to MET activation by relieving inhibitory serine phosphorylation. These observations raise the possibility that MEK inhibition leads to feedback activation of RTKs, as well as its downstream activation (e.g., wild-type RAS). Notably, AZD6244 increased phosphorylation of KSR-1, a scaffolding protein required for assembly of MAPK signaling complex as well as phosphorylation of GEF-H1, a recently reportedly novel regulator of KSR-1 required for proper activation of RAS-driven MAPK activation. Collectively, our phosphoproteomics results indicate altered phosphorylation of a series of signaling proteins in MAPK signaling cascade, presumably contributing feedback activation of RAF, MEK and ERK reactivation following MEK inhibition. We are currently characterizing the importance of GEF-H1 and KSR-1 in lung cancer cell growth and survival and their role in adaptive signaling change associated with MEK inhibition. Updated work will be presented.

Abstract Number:        685
Presentation Title:        Mechanisms of intrinsic and acquired resistance to third generation inhibitors in EGFR mutant NSCLC
Presentation Time:        Sunday, Apr 19, 2015, 1:00 PM - 5:00 PM
Location:        Section 28
Poster Board Number:        29
Author Block:        Matt J. Niederst. MGH/Harvard Medical School, Charlestown, MA
Abstract Body:        Acquired resistance is a fundamental problem limiting the efficacy of tyrosine kinase inhibitors (TKIs) in EGFR mutant NSCLC. In over half of the cases of acquired resistance to the first generation inhibitors gefitinib and erlotinib, a secondary mutation to EGFR, T790M, promotes resistance by preventing these drugs from fully suppressing the receptor. The recent development of next generation EGFR TKIs has proven to be a major breakthrough in the treatment of these patients. In clinical trials testing the third generation EGFR inhibitors CO-1686 and AZD9291, response rates of greater than 50% in T790M+ patients have been reported. While these drugs hold a great deal of promise, they too will be limited, initially by intrinsic resistance, and eventually by acquired resistance. To study intrinsic resistance in this setting, we have developed a panel of cell lines derived from biopsies of T790M+ erlotinib-resistant patients, tested their sensitivity to third generation inhibitors and identified biomarkers that predict a lack of response. To study acquired resistance, we have generated resistance in these cells lines in vitro and derived additional models directly from patient biopsies following progression on the clinical trial. Using a combination of functional, sequencing, and drug screening approaches we have begun to identify resistance mechanisms to third generation inhibitors.

尼达尼布联合治疗方案
1.药物简介
尼达尼布（Nintedanib，BIBF 1120，Ofev）：
尼达尼布是德国勃林格殷格翰公司研发的三联血管激酶抑制剂，可同时阻断血管内皮生长因子受体（VEGFR） 、血小板源性生长因子受体（PDGFR）以及成纤维细胞生长因子受体（FGFR）的信号转导通路。美国食品和药物管理局（FDA）于2014年10月15日批准了Ofev(Nintedanib)对于特发性肺纤维化（IPF）的治疗。
FDA批准尼达尼布基于来自一项II期临床试验 （TOMORROW研究）以及两项III期临床试验（INPULSIS&#8482;-1研究和 INPULSIS&#8482;-2研究的结果）。
临床试验证实尼达尼布可使绝大多数IPF患者人群获益于疾病进展的减缓，表现为患者的肺功能降幅减少50%，其中也包括处于疾病早期阶段的患者（用力肺活量[FVC]占预计值百分比>90%）、高分辨率计算机体层扫描（HRCT）未检出蜂窝肺病变和/或合并肺气肿的患者。尼达尼布是首个在两项具有相同研究设计的III期临床试验中均达到主要终点的IPF靶向治疗药物。尼达尼布还可显著降低经过仲裁判定的IPF急性加重的发生风险。IFP急性加重可显著影响疾病进程，导致肺部疾病恶化的一系列事件。在因IPF急性加重而住院的患者中，有半数的人会在住院期间死亡。
关于特发性肺纤维化
特发性肺纤维化（IPF）是一种慢性、进行性、具有严重致残性、最终可致命的肺部疾病，迄今为止针对这种疾病的治疗选择非常有限。在全球范围内，每十万人中，就有14至43人罹患IPF。IPF的特征性病变是肺组织的进行性瘢痕形成和肺功能的进行性下降。瘢痕组织的形成被称为纤维化。随着时间的进展，肺组织由于瘢痕形成而不断增厚和变硬，肺脏摄取氧气并将其传输到血液中的能力不断丧失，从而导致机体的重要脏器无法获得充足的氧气。最终，IPF患者会出现气促和咳嗽，并经常难以从事日常体力活动。
IPF 患者较普通人群更易合并肺癌。IPF 合并肺癌者多见于男性、年龄＞60 岁和吸烟患者。临床症状并无特异性，主要为 IPF 的本身表现，包括活动后呼吸困难、干咳等，部分患者可有肺癌症状，如痰中带血、声音嘶哑等。肺癌病人经过化疗、放疗和靶向药治疗均可能会导致IPF，引起慢性肺纤维化，或者出现IPF 急性加重（AEIPF）。

1.2药代动力学
尼达尼布半衰期为9.5小时，在进食条件下口服给药后约2至4小时到达最大血浆浓度，给药后一周实现稳态血浆浓度。P-gp或CYP3A4抑制剂共同给药可能增加尼达尼布血药浓度。吸烟会影响血药浓度。
1.3副作用       
最常见不良反应(≥5%)是：腹泻，恶心，腹痛，呕吐，肝酶升高，食欲减退，头痛，体重减轻，和高血压。
出血的风险：在临床试验中，用尼达尼布治疗患者报道出血事件10%和用安慰剂治疗患者7%。
胃肠道穿孔：。在临床试验中，用尼达尼布治疗患者报道胃肠道穿孔 0.3%与之比较安慰剂治疗患者0例。
心脏毒性：在临床试验中，用尼达尼布治疗患者报道动脉血栓栓塞事件 2.5%和安慰剂治疗患者 0.8%。心肌梗死是动脉血栓栓塞事件最常见不良反应，尼达尼布治疗患者发生1.5%与之比较安慰剂治疗患者0.4%。尼达尼布不会导致QT间期延长。
当治疗患者处于较高心血管风险包括已知冠状动脉疾病谨慎使用尼达尼布。发生急性心肌梗死特征或症状患者中考虑中断治疗。
肝酶和胆红素升高：肝问题的任何症状：皮肤或眼睛转为黄色，尿呈暗或棕色（茶色），胃右侧疼痛，出血或比正常更易瘀伤，嗜睡等。

1.3推荐剂量
IPF患者：尼达尼布每天2次，一次150 mg，间隔约12小时与食物服用，特定情况下考虑暂时剂量减低至每天2次，一次100mg。尼达尼布胶囊以乙磺酸钠盐存在（180.6 mg尼达尼布乙磺酸钠盐相当于150mg尼达尼布）。
2 联合治疗方案
2.1尼达尼布联合化疗
2.1.1 尼达尼布联合多西他赛
        基于 LUME-Lung 1临床试验的结果，此项研究证实，与多西他赛单药治疗方案相比，尼达尼布与多西他赛的联合治疗方案可使一线化疗之后的晚期腺癌患者的中位总体生存时间从10.3个月延长至 12.6个月，是首个已被证实可延长腺癌患者总体生存时间超过一年的非小细胞肺癌二线治疗药物。
关于LUME-Lung 1 试验
LUME-Lung 1试验是一项随机、双盲、III期临床试验，此项试验针对尼达尼布联合多西他赛应用于一线治疗后的局部晚期/转移性NSCLC患者的效果与安慰剂联合多西他赛进行了比较。此项试验纳入了来自欧洲、亚洲和南非的1,314 名患者，这些患者随机接受尼达尼布 200 mg每日给药两次联合多西他赛 75mg/m2 每日给药一次的治疗方案（n=655）或安慰剂联合多西他赛的治疗方案（n=659），治疗时间为3周。
优点：相对其它抗血管生成药物，尼达尼布的心脏毒性和出血概率较低，不容易出现反弹现象。另外，阿瓦斯汀治疗无效的病人，尼达尼布可能有效。

2.1.2 尼达尼布联合培美曲塞
        尼达尼布每天2次，一次200 mg，连续服用，培美曲塞500mg/m2每3周。

2.1.3尼达尼布联合依托泊苷（Etoposide VP-16）
        尼达尼布每天2次，一次100-150mg，连续服用，依托泊苷胶囊口服50-75mg每天，吃二周停一周，三周一个疗程。
依托泊苷的优点：非小细胞肺癌和小细胞肺癌的一线化疗药物，相当一部分肺癌病人是混合类型。依托泊苷胶囊为医保药物，口服方便，副作用温和。
2.2尼达尼布联合靶向药
2.2.1 尼达尼布联合阿法替尼
方案：尼达尼布每天2次，一次100-150mg，连续服用，阿法替尼30mg每天或者40mg每天吃一周停一周。
该方案对KRAS突变病人的疗效有待临床实验证实。

2.2.1 尼达尼布联合其它EGFR TKI
方案：尼达尼布150mg每天2次，其它EGFR TKI使用标准剂量。
参考文献：
[1] Phase i study of afatinib (bibw 2992), an erbb family blocker plus nintedanib (bibf 1120), a triple angiokinase inhibitor, in patients with advanced solid tumours.
[2] Combining VEGF(R) and EGFR-targeted agents:the example of nintedanib and afatinib.
[3] Ofev(nintedanib)使用说明书2014年第一版（中文版）
http://blog.sina.com.cn/s/blog_5948305d0102v4tn.html
[4] Lume-lung 2: A multicenter, randomized, double-blind, phase III study of nintedanib plus pemetrexed versus placebo plus pemetrexed in patients with advanced nonsquamous non-small cell lung cancer (NSCLC) after failure of first-line chemotherapy.
http://www.boehringer-ingelheim. ... nna_119913_P327.pdf
[5] LUME-Lung 1: Nintedanib plus chemotherapy extends the life of lung cancer patients with adenocarcinoma.
https://www.boehringer-ingelheim ... _2013_oncology.html
[6] 依托泊苷胶囊
http://www.yuaigongwu.com/forum. ... amp;_dsign=943d88ed