Mechanism Of TKI Resistance Research Articles

Targeting PKCδ as a Therapeutic Strategy against Heterogeneous Mechanisms of EGFR Inhibitor Resistance in EGFR-Mutant Lung Cancer

Multiple mechanisms of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been identified in EGFR-mutant non-small cell lung cancer (NSCLC); however, recurrent resistance to EGFR TKIs due to the heterogeneous mechanisms underlying resistance within a single patient remains a major challenge in the clinic. Here, we report a role of nuclear protein kinase Cδ (PKCδ) as a common axis across multiple known TKI-resistance mechanisms. Specifically, we demonstrate that TKI-inactivatedEGFR dimerizes with other membrane receptors implicated in TKI resistance to promote PKCδ nuclear translocation. Moreover, the level of nuclear PKCδ is associated with TKI response in patients. The combined inhibition of PKCδ and EGFR induces marked regression of resistant NSCLC tumors with EGFR mutations.

P3.08-18 Changing Resistance Mechanisms in Rebiopsies of ALK-Positive NSCLC During Multiple Lines of Therapy: ALK/BRAF-Mutations Followed By EMT

4-7% of NSCLC patients harbor the oncogenic ALK-rearrangements and may obtain clinical benefit from different ALK-inhibitors. Despite long remissions, resistance inevitably appears in alternative dominating forms. We present a case of a 38-year old male with metastatic ALK-positive NSCLC. The patient received four consecutive ALK-TKIs and two lines of chemotherapy in-between. Significant clinical benefit was observed during different ALK-TKIs, as well as resistant ALK- and BRAF-mutations, and rapid progression shortly before death associated with phenotypical changes related to epithelial-mesenchymal transition (EMT). The diagnostic biopsy from cervical lymph nodes showed ALK-positive adenocarcinoma-metastasis with fusion variant 2 of EML4-ALK rearrangement. To identify TKI-resistance mechanisms during treatment, 4 rebiopsies from new metastatic lesions were analyzed histologically and by IHC, FISH and NGS. Additionally, two liquid biopsies were performed. The treatment was initiated by Crizotinib. After 10 months, the 1st rebiopsy from new hepatic metastases showed a new p.C1156Y-mutation.The patient then received Cisplatin/Vinorelbine, progressed after two cycles and received 3rd line Ceritinib. The 2nd rebiopsy from new metastasis appearing 8 months later displayed persistence of ALK-fusion and p.C1156Y, but also new p.D1203N ALK- and p.V600E BRAF–mutations. Therapy was changed to 4th line Alectinib. A 3rd rebiopsy taken 4 months later from progressing sites revealed persisting ALK-fusion without p.C1156Y and p.D1203N mutations, and still p.V600E BRAF-mutation. No mutations were detected in plasma cfDNA 3 months later. Due to mixed response Pemetrexed was added and the patient had regression for a year. At subsequent progression a 4th rebiopsy from new lesions revealed maintained ALK-fusion without any ALK- or BRAF-mutations. Histology (appearance of spindle cells) and IHC (strong positivity for the mesenchymal marker Vimentin, loss of the epithelial marker E-cadherin) revealed phenotypical changes related to EMT. A liquid biopsy did not show any DNA-mutations either. Meanwhile, 6th line Lorlatinib was initiated without response. The patient died 34 months after initial diagnosis. Following the treatment course of this case we observed heterogeneous and transitional mechanisms of resistance to different ALK-TKIs, such as secondary ALK-mutations, BRAF-mutation, and phenotypical changes consistent with EMT. This resistance heterogeneity suggests a continuously changing state of the disease. Sequential use of different ALK-TKIs and combination therapies may allow long responses with satisfactory quality of life, however phenotypical EMT-related changes remain a significant hurdle for TKI-based therapy that may explain lack of response to 3rd generation’s ALK-TKI, despite preserved ALK-positive status of the tumor.

P3.01-007 Heterogeneous Resistance Mechanisms in Rebiopsies from EGFR-Mutated NSCLC: Transformation to SCLC; FGFR3 and T790M Mutations

Patients with epidermal growth factor receptor (EGFR) gene-mutated NSCLC initially show substantial clinical benefit from EGFR Tyrosine-Kinase Inhibitors (TKIs), but will ultimately develop resistance with a median progression-free survival of 9-15 months. However, the type and timing of TKI-resistance cannot be predicted and several mechanisms may occur simultaneously/subsequently during TKI-treatment. We present a patient case with advanced non-small cell lung cancer (NSCLC) of adenocarcinoma subtype (ADC) with EGFR-mutation and Erlotinib-treated Different mechanisms of TKI-resistance were detected in tumor biopsies during treatment time. The patient was a 49 year-old, previously healthy, Caucasian male with metastatic ADC. A diagnostic biopsy from hepatic metastasis, cytology from metastatic pleural effusion at first progression during TKI-therapy and a biopsy from new liver metastasis at second progression were analyzed by histology, immunohistochemistry and targeted next-generation sequencing (NGS) of hot-spot mutations in 50 cancer-related genes (Ion AmpliSeq Cancer Hotspot Panel v.2, Ion Torrent, Thermo Fisher Scientific). CT-scans revealed tumor in the right upper lobe with mediastinal infiltration and multiple pulmonary and hepatic metastases, stage T4N2M1b. Diagnostic liver biopsy revealed ADC (mucin-producing, CK7- and TTF1-positive epithelial acinar structures), which concomitantly harbored an EGFR exon 19-mutation (p.E746_A750delELREA) and a previously unreported 2 bp microdeletion in the fibroblast growth factor receptor 3 (FGFR3; p.D785fs*31) gene. The patient received first-line Erlotinib but progressed after 7 weeks with metastatic pleural effusion, in which transformation to small cell lung cancer (SCLC) and maintenance of EGFR-mutation and FGFR3-mutation was identified. The progression was treated with standard Carboplatin-Etoposide regimen for SCLC together with Erlotinib continuation. The second progression 7 months later was a new liver-metastasis with persistence of the original EGFR- and FGFR3-mutated ADC-phenotype and additional emergence of the Erlotinib-resistant T790M EGFR-mutation. The patient rapidly deteriorated and deceased. Thus, in this advanced EGFR-mutated NSCLC rapid onset and heterogeneous mechanisms of TKI-resistance occurred at different times of metastatic disease: 1. Concomitant FGFR3-mutation prior to and during TKI-treatment as potential intrinsic resistance-mechanism; 2. Transformation to SCLC at 1st progression during TKI-therapy; 3. Acquisition of T790M EGFR-mutation at 2nd progression. This suggests a continuous variation of TKI-resistant cells’ genetic and phenotypic behavior. Therefore, re-biopsies are important to provide the current status of the disease and better define subsequent treatment options. “Liquid biopsies” may potentially help identify heterogeneous genetic resistance-mechanisms; however assessment of mechanisms such as SCLC-transformation needs tissue biopsies.

Genetic and Epigenetic Mechanisms of Tki Resistance

Genetic and Epigenetic Mechanisms of Tki Resistance

Circulating tumor DNA profiling to reveal heterogeneity of EGFR-TKI resistance mechanisms in lung adenocarcinoma patients.

11527 Background: EGFR-TKI therapy has significantly improved prognosis of NSCLC patients with EGFR sensitive mutation. However, almost all patients ultimately develop PD while receiving TKI treatment. Circulating tumor DNA (ctDNA) is promising as a minimally-invasive liquid biopsy for comprehensive analysis of molecular abnormalities. Methods: A total of 254 advanced lung adenocarcinoma patients with signs of EGFR-TKI resistance were enrolled in the study. ctDNA was analyzed using next-generation sequencing based ER-Seq method, which enables simultaneously assess single-nucleotide variants, insertions/deletions, rearrangements, and somatic copy-number alterations across 59 genes. Results: ctDNA profiling was possible for all patients, 172 patients had ≥ 1 ctDNA alteration(s). Median number of plasma somatic mutations was 2, predominantly located in EGFR and TP53, with MET, ERBB2 and PIK3CA followed. Of that, 30.6% of mutations detected in ctDNA were at a frequency below 1%. In exploring the mechanisms of TKI-resistance, we found TKI-sensitizing mutations were not detected in plasma of 138 patients (54.3%). Known mechanisms such as EGFR T790M/C797S mutation, activating mutations of PI3K- AKT- mTOR signaling, amplification of MET, activating mutation / amplification of ERBB2, activating mutation of KRAS, BRAF or mutations in EGFR EX20 other than T790M/C797S were identified in 59, 16, 8, 7, 3, 2, and 2 patients respectively. T790M/C797S was detected in 50.8% of patients with plasma positive for TKI-sensitizing mutations. Of note, C797S was only detected in patients treated with AZD9291. EGFR amplification were identified in 15 patients, though whether it would result in TKI-resistance was still controversial. Co-occurrence of resistance mechanisms were observed in 22 patients including 13 patients without TKI-sensitizing mutations. Conclusions: There was a high frequency of inter and intra-patient heterogeneity of resistance mechanisms after EGFR TKI therapy. ctDNA can be used as a ‘liquid biopsy’ to facilitate the broad exploration of potential resistance mechanisms.

Small cell lung cancer transformation and the T790M mutation: A case report of two acquired mechanisms of TKI resistance detected in a tumor rebiopsy and plasma sample of EGFR-mutant lung adenocarcinoma.

The present study describes the case of a 45-year-old man diagnosed with metastatic lung adenocarcinoma, which harbored a deletion within exon 19 of the epidermal growth factor receptor (EGFR) gene. The patient was subsequently treated with gefitinib (250 mg/day orally from May 2013 to March 2014), but developed acquired resistance to the drug following 11 months of treatment. Tumor burden molecular analysis was performed on a tumor rebiopsy and plasma sample, and histological analysis was also performed on the tumor rebiopsy. A small cell transformation retaining the original EGFR mutation was detected in the tumor rebiopsy, while the T790M mutation together with the activating ex19del mutation were identified only in the plasma sample. The patient was treated with cytotoxic chemotherapy (off-label schedule with epirubicin 80 mg/mq and paclitaxel 160 mg/mq every 21 days for 6 cycles) and radiation (50.4 Gy administered in 28 fractions of 1.8 Gy once daily for 5.5 weeks) specific for small cell lung cancer, and may also have benefitted from treatment with a third generation T790M-specific EGFR-TKI. To better describe the mechanisms of resistance to TKI inhibitors and to optimize therapeutic regimens, the simultaneous analysis of tumor biopsies and circulating tumor DNA should be considered.

Abstract 1599: Epithelial mesenchymal transition and its role in TKI resistant NSCLC cell lines

Abstract NSCLC cells acquire resistance to EGFR and c-Met TKIs after prolonged use. Our studies indicate that resistance maybe due to upregulation of alternative signaling pathways such as Wnt and mTOR. We have found that activation of Wnt/β-Catenin is also associated with EMT which results in loss of cell adhesion properties and gain of motility and invasiveness. To understand the mechanism of TKI resistance in NSCLC cells with wild type EGFR, we have developed and used H2170-P (parental) cells and the TKI resistant H2170-ER (erlotinib resistant) and H2170-SR (SU11274 resistant) cells. We aim to study EMT and determine if inhibition of β-Catenin, a key regulator of transcription by siRNA or inhibition of ZEB-1 a transcriptional repressor of cell adhesion proteins by inducing mir-200a will help overcome the TKI resistance in NSCLC cells. Using immunoblotting, we observed modulations in key EMT-related proteins in H2170-ER cells which showed upregulation of ZEB-1, N-cadherin, active beta-catenin, Vimentin, PRMT1 and ZO1 by 1.8, 2.4, 2, 1.8, 2.6 and 2 fold, respectively, and downregulation of E-cadherin (1.8 fold) as compared to H2170-P cells. Similar results were observed for H2170-SR cells. These results were verified using qPCR where we show that β-catenin (3.4-3.2 fold) and N-cadherin (2-1.9 fold) have increased gene expression while E-cadherin (1.7-2.4 fold) has a decreased gene expression in TKI resistant H2170-ER and SR cells when compared to H2170-P cells. miR-200a induction in H2170-ER cells showed significant downregulation of ZEB-1 (3 fold) at 72 hr and an upregulation of E-cadherin (2 fold) when compared to the mock transfected cells. Morphological changes indicative of EMT were detected using immunofluorescence with Vimentin and E-Cadherin antibodies, which displayed upregulation of Vimentin filaments (2 fold) and downregulation of E-Cadherin (3 fold) in H2170-ER and H2170-SR cells when compared to H2170-P cells. We then conducted experiments where we suppressed ZEB-1 by inducing miR-200a in TKI resistant cells. The immunoblotting results suggested recovery of E-Cadherin, and downregulation of ZEB-1 and N-Cadherin in TKI resistant cells. We also observed increased sensitivity towards erlotinib and SU11274 by 20-25%. Additionally, we observed decrease in levels of β-Catenin and upon siRNA knockdown of β-Catenin, suppression of levels of ZEB-1. This indicates a direct correlation between nuclear accumulation of β-Catenin and occurrence of EMT in TKI resistant cells by increase in expression of ZEB-1. Our results indicate that increased activation of Wnt/β-Catenin pathway in the TKI resistant NSCLC cells is due to the EMT. In NSCLC patients, L858R and T790M mutations are associated with TKI resistance which could be responsible for inducing EMT. We are further studying cell lines with EGFR mutations to determine their role in induction of EMT this may provide clinicians with novel targets to overcome TKI resistance in NSCLC patients. Citation Format: Ichwaku Rastogi, Tsatsral Iderzorig, Gagan Chhabra, Gregory M. Botting, Andrew Webb, Brad Foster, Brian Webb, Marie Nlend, Neelu Puri. Epithelial mesenchymal transition and its role in TKI resistant NSCLC cell lines. [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 1599.

Small activating ribonucleic acid reverses tyrosine kinase inhibitor resistance in epidermal growth factor receptor‐mutant lung cancer by increasing the expression of phosphatase and tensin homolog

BackgroundEpidermal growth factor receptor‐tyrosine kinase inhibitors (TKI‐EGFRs) present a new prospect for the treatment of lung cancer. However, in clinical application, the majority of patients become TKI resistant within a year. More and more studies have shown that a loss of phosphatase and tensin homolog (PTEN) expression is associated with TKI resistance. An alternative method of upregulating PTEN expression may reverse TKI resistance.MethodsWe designed five candidate small activating ribonucleic acids (saRNAs) to target PTEN, and transfected them into H‐157 cells to screen out functional saRNA. We used reverse transcriptase‐polymerase chain reaction and Western blot to evaluate the effect of saRNA to PTEN expression. We then analyzed the growth and apoptosis of cells transfected with saRNA under the treatment of TKI to investigate whether saRNAs can reverse TKI resistance by upregulating PTEN expression.ResultsThe functional saRNA we designed could upregulate PTEN expression. The H‐157 cells transfected with saRNA grew slower in the presence of TKI drugs than the cells that were not transfected with saRNA. The apoptosis rate was also obviously higher.ConclusionsOur study proves that loss of PTEN expression is an important mechanism of TKI resistance. It is possible to control TKI resistance by upregulating PTEN expression using RNA activation technology.

Anti-Leukemic Activity of Axitinib Against Cells Harboring the BCR-ABL Point Mutation

Introduction: The BCR-ABL1 fusion gene is a causative oncogene in chronic myeloid leukemia (CML) and 30-50% of acute lymphoblastic leukemia cases. Although ABL tyrosine kinase inhibitors (ABL TKI) such as imatinib have improved CML treatment, such therapies cannot cure patients with Philadelphia chromosome (Ph)-positive leukemia because of leukemia stem cells. Moreover, some patients develop BCR-ABL point mutations and become resistant to ABL TKI therapy. In particular, the ABL kinase domain mutation T315I is resistant to imatinib and second-generation ABL TKIs (e.g., nilotinib, dasatinib, and bostinib). Accordingly, this mutation is often found in patients with TKI-resistant disease. A third-generation ABL TKI, ponatinib, was recently developed. Ponatinib is a potent oral tyrosine kinase inhibitor that affects both unmutated and mutated BCR-ABL; it is effective against T315I mutant cells and has been approved for TKI-resistant or intolerant CML and Ph-positive ALL patients.Recently, the vascular endothelial growth factor receptor (VEGFR) inhibitor axitinib was found to exhibit anti-leukemic activity against T315I-mutant disease. Axitinib is an orally active and potent TKI of VEGFRs 1, 2, and 3 and inhibits BCR-ABL1, especially the T315I variant, via a distinct binding conformation.Materials and methods: In this study, we investigated whether axitinib could suppress ponatinib-resistant compound mutant cells harboring the T315I mutation and primary samples.Results: A 72 h axitinib treatment inhibited the growth of Ba/F3 T315I cells (Figure 1A). Immunoblot analysis of axitinib-treated cells revealed dose-dependent decreases in BCR-ABL, the downstream molecule CrkL, and ribosomal S6 protein phosphorylation and increases in caspase 3 and Poly (ADP-ribose) polymerase (PARP) activity. Ponatinib and axitinib also induced apoptosis, significantly increased caspase activity, and reduced Akt activity. In contrast, clinically available concentrations of axitinib did not inhibit the growth of ponatinib-resistant Ba/F3 cells. Immunoblot analysis revealed that BCR-ABL, Crk-L, and S6 kinase phosphorylation were not inhibited by axitinib or ponatinib. Similarly, no increase in caspase activity or decrease in Akt activity was observed following axitinib treatment, and neither ponatinib nor axitinib affected apoptosis in these cells. We next evaluated primary T315I-mutant and ponatinib-resistant compound mutant samples. Axitinib potently inhibited the growth of T315I mutant primary cells in a dose-dependent manner. Immunoblot analysis further revealed reduced Crk-L and S6 kinase phosphorylation after axitinib or ponatinib treatment. In contrast, the growth of ponatinib-resistant primary cells was not affected by ponatinib or axitinib. Immunoblotting revealed that neither ponatinib nor axitinib affected the phosphorylation of Crk-L and S6 kinase in ponatinib-resistant cells.Conclusion: In CML, ABL TKI resistance is frequently caused by ABL kinase domain mutations. The T315I mutation is resistant to all ABL TKIs except ponatinib. Although axitinib, which is currently being investigated for efficacy in patients with Ph-positive T315I-mutant leukemia, induced apoptosis in T315I-mutant cells, it was ineffective against cells with a compound mutation including T315I. Current evidence to direct the management of ABL TKI-resistant disease, particularly those harboring T315I and compound mutations, is limited. New molecular-targeted drugs and an understanding of ABL TKI resistance mechanisms are required to apply an appropriate therapeutic approach. DisclosuresNo relevant conflicts of interest to declare.

Abstract 347: Mechanism of c-Met TKI resistance and role of epithelial mesenchymal transition in melanoma

Abstract Various tyrosine kinase inhibitors (TKI’s) are approved for the treatment of melanoma, but their efficacies are limited due to development of drug resistance. To analyze the resistance to c-Met inhibitors we used two melanoma cell lines: MU which has mutated BRAF (V600E +ve) and RU which has wild type BRAF. These cell lines were also made resistant to c-Met inhibitor, SU11274 by growing them in progressively increasing concentrations of drug. In earlier studies we observed activation of Wnt pathway in both of the aforementioned cell lines indicating the role of active β-Catenin in transcription and hence cell proliferation. β-Catenin levels when localized and accumulated in the nucleus binds to TCF/LEF and activates transcription. The level of β-Catenin in the cytosol is regulated by the adherens junction proteins (E-Cadherin) along with the Wnt pathway. Increased levels of β-Catenin might be a possible result of loss of E-Cadherin which is related to epithelial mesenchymal transition. During epithelial mesenchymal transition (EMT) cells transition from epithelial (attached, non-migratory) to mesenchymal (unattached and migratory) cells which promotes metastasis. We examined the role of EMT in the development of resistance in the c-Met TKI resistant melanoma cell lines. We quantified the β-Catenin activity using luciferase assay and observed a 5 fold upregulation in the SU11274 resistant MU cells when compared to MU parental cells in the presence of HGF. Additionally, we performed immunoblotting, where we observed modulation in EMT related proteins in MU and RU parental and resistant cells. E-Cadherin, ZO-1, Claudin-1 and Slug were found to be downregulated 1.5 - 2.7 fold, 1.5 - 2.0 fold, 2 - 9 fold and 1.5 - 2 fold respectively and N-Cadherin and β-Catenin were found to be upregulated 1.8 - 5.3 and 2.4 - 3.7 fold respectively in the SU11274 resistant MU cells in the presence and absence of HGF and SU11274 treatments. Similarily we observed upregulation of β-Catenin by 1.7 fold and downregulation of E-Cadherin and ZO-1 by 2 - 3.5 fold and 6 - 9 fold respectively in SU11274 resistant RU cells when compared to RU parental cells in the presence and absence of HGF and SU11274 treatments. Further studies are being carried to understand the role of EMT in the development of this acquired resistance. These results may help in development of novel therapies for melanoma patients or may help in increasing the efficacy and beneficence from the currently available therapeutics. Citation Format: Supriya Rajanna, Ichwaku Rastogi, Sunil Palani, Neelu Puri. Mechanism of c-Met TKI resistance and role of epithelial mesenchymal transition in melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 347. doi:10.1158/1538-7445.AM2015-347

Abstract 1608: Profiling c-Met and EGFR kinase inhibitor resistance pathways in non-small lung cancer cells

Abstract Tyrosine kinase inhibitors (TKIs) against c-MET and EGFR have positive therapeutic effects in a subset of Non-Small Cell Lung Cancer (NSCLC) patients; however, their clinical efficacies are limited due to the development of TKI resistance. To determine mechanisms of TKI resistance, two NSCLC model cell lines H2170 and H358 were made resistant to the c-Met/EGFR TKIs SU11274, erlotinib or a combination of both by exposing the cells to progressively increasing concentrations of inhibitor. Initial characterization of these cell lines using phospho-specific antibodies showed that phospho-c-Met was found to be downregulated 4- and 1.5- fold in SU11274-resistant (SR) H2170 and SR H358 cells, respectively. Phospho-EGFR was found to be constitutively autophosphorylated (upregulated 19-fold) in erlotinib-resistant (ER) H2170 cells, but downregulated 6-fold in ER H358 cells. Interestingly, c-Met/EGFR downstream signaling proteins p-mTOR, p-p70S6K and p-ERK were found to be 2-4-fold upregulated, 2-fold upregulated and 2-5-fold upregulated, respectively, in ER H2170 and H358 cells. Phospho-p70S6K was also found to be upregulated 20-fold in SR H2170 cells. These results indicate that alternative signaling pathways may confer TKI resistance in these cell lines. To identify which signaling pathways may be responsible for TKI resistance, H358 parental and resistant cell lines were analyzed by mass spectrometry (MS). Differences in protein expression in cell lines treated with and without EGF and/or erlotinib were measured using higher multiplexing Thermo Scientific™ Tandem Mass Tag™ (TMT™) reagents. More than 1500 protein groups were identified and quantified using a Thermo Scientific™ Orbitrap™ Fusion™ platform with HCD-MS2 and MS3 fragmentation methods. Protein expression in ER H358 cells showed a marked increase in expression of DNA replication proteins, transcription factors, apoptosis inhibitors and some Ras-associated signal transduction pathways. To further investigate the mechanism of TKI resistance, phospho-peptide enrichment was also used in combination with TMT quantitation to profile changes in protein phosphorylation state. Overall, the combination of phospho-peptide enrichment coupled with higher multiplexing TMT and high resolution MS analysis enabled deeper global profiling of TKI resistance signaling pathways compared to previous studies without enrichment. Citation Format: Ryan D. Bomgarden, Ryan Jacobs, Jason Fong, David Moravec, Gregory M. Botting, Michael Blank, Rosa I. Viner, John C. Rogers, Neelu Puri. Profiling c-Met and EGFR kinase inhibitor resistance pathways in non-small lung cancer cells. [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 1608. doi:10.1158/1538-7445.AM2014-1608

The BIM Deletion Polymorphism: A Paradigm Of a Permissive Interaction Between Germline and Acquired TKI Resistance Factors In Chronic Myeloid Leukemia

Germline polymorphisms and tumor-specific genetic mutations together contribute to the behavior of human cancers, including the response to therapy. However, few specific models allow for the detailed study of how inherited and acquired genetic factors interact to cause clinical drug resistance, nor how their interaction can be prevented or overcome. We recently reported a germline deletion polymorphism in the BIM gene that was sufficient to mediate intrinsic resistance to targeted therapies in cancer, including the example of imatinib in chronic myeloid leukemia (CML) (Ng et al. Nature Medicine, 2012). Mechanistically, the deletion polymorphism led to the generation of BIM splice forms that lacked the pro-apoptotic BH3 domain, and that were incapable of activating apoptosis in response to targeted therapy. Clinically, we found that CML patients with the deletion polymorphism experienced inferior responses to imatinib. Furthermore, within the cohort of resistant patients harboring the BIM deletion polymorphism, we found that the majority developed cross-resistance to second or third generation TKI (nilotinib, dasatinib, and bosutinib), and that TKI resistance-conferring BCR-ABL kinase domain mutations were less frequent in patients with the BIM deletion polymorphism than those without. These observations suggested that the BIM deletion polymorphism cooperates with both BCR-ABL-independent and -dependent TKI resistance mechanisms to produce broad resistance to TKI.We generated K562 cells with and without the BIM deletion polymorphism that were resistant to imatinib by using the method described by Mahon et al (Blood, 2000). We found that deletion-containing cells exhibited 3- to 4-fold increased viability as compared to wildtype cells, and this was due to reduced apoptosis in the deletion-containing cells. We then compared the panel of wildtype and deletion-containing cells for the development of known TKI resistance mechanisms. The table below summarizes our findings. Importantly, we also found that, in a number of cells, both ERK and LYN activation occurred independently of BCR-ABL.Known TKI resistance mechanismWildtypeHeterozygous for BIM deletion polymorphismHomozygous for BIM deletion polymorphismSomatic mutation in BCR-ABL kinase domain--G250EBCR-ABL gene amplification--+++LYN activation+++++ERK activation+++-++Next, we tested the ability of second-generation TKI, including dasatinib and nilotinib, to overcome resistance induced by long-term culture. We also determined if the addition of ABT-737, a BH3-mimetic, to the TKI could overcome resistance in our cell lines. First, we found that prolonged incubation with imatinib induced cross-resistance to both nilotinib and dasatinib, a result that reproduced our observation in patients with the BIM deletion polymorphism. Next, we found that a combination of second generation TKI, especially dasatinib, and ABT-737 induced maximal cell death in cells with the BIM deletion polymorphism. Based on the information generated from our cell lines, we evaluated the viability of TKI-resistant primary CML cells when treated with TKI with and without ABT-737. In TKI-resistant primary CML cells, regardless of their BIM deletion status, maximal reduction in cell viability was observed when both TKI and ABT-737 were used.We conclude that: 1. The BIM deletion polymorphism is permissive for the acquisition of somatic events that mediate both BCR-ABL-dependent and -independent mechanisms of TKI resistance. 2. The permissive action of the BIM deletion polymorphism is associated with reduced apoptosis, and likely accounts for the ability of BIM deletion polymorphism-containing CML cells to acquire secondary resistance-conferring events. 3. The combined use of TKI with ABT-737 represents a potential therapeutic strategy to overcome TKI resistance in CML patients with and without the BIM deletion polymorphism. 4. In our cell line system, dasatinib with ABT-737 induced maximum cell death. Finally, our results demonstrate that germline and acquired resistance factors can interact to produce resistance to targeted therapies in cancer, and that cell line systems allow us to both elucidate these mechanisms as well as devise therapeutic strategies to overcome such resistance. Disclosures:Chuah:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria.

Erlotinib as a salvage treatment for patients with advanced non-small cell lung cancer after failure of gefitinib treatment: Is there a rationale for a clinical trial?

e19099 Background: Patients (pts) with advanced NSCLC and sensitizing EGFR mutations who initially respond to gefitinib or erlotinib eventually develop acquired resistance to the TKIs. Our goal was to determine the effects of erlotinib 150 mg/d in EGFR mutated pts resistant to gefitinib 250 mg/d, because the EGFR TKI erlotinib is given at a higher biologically active dose than gefitinib. Methods: Retrospective review of 5 EGFR mutated (exon 19 deletions) patients that were given gefitinib and subsequently erlotinib. Results: All pts responded to gefitinib with median progression-free survival of 13 months (95% confidence interval, 4-16). After gefitinib resistance, 60% (3 of 5) of these pts displayed progressive disease while on erlotinib with progression-free survival of 2 months (95% confidence interval, 2-3). These pts acquired the T790M mutation. 2 gefitinib-resistant pts with the acquired L858R-L747S EGFR, which in vitro is sensitive to achievable serum concentrations of erlotinib 150 mg/d, achieved a partial response to erlotinib. In literature doesn’t exist a prospective study about the stratification of pts ordered by PS, age, gender, smoker/non-smoker, which could test the efficiency of the erlotinib after gefitinib in pts with different EGFR mutations. We only have at our disposal few studies control-case in non-selected pts which show a potential efficiency of erlotinib after the failure of the gefitinib, nevertheless, without an evident increase of the overall survival. Conclusions: In EGFR mutated tumors resistant to gefitinib 250 mg/d, a switch to erlotinib 150 mg/d does not lead to responses in most pts. These findings are consistent with preclinical models, because the common mechanisms of TKI resistance (T790M and MET amplification) in vitro are not inhibited by clinically achievable doses of gefitinib or erlotinib. Alternative strategies to overcome TKI resistance must be evaluated.

Abstract 2450: Either pan class I PI3K inhibitor or PI3K alpha selective inhibitor sensitizes head and neck squamous cell carcinoma cells to Erlotinib, an EGFR TKI.

Abstract The epidermal growth factor receptor (EGFR) is overexpressed in 90% of head and neck squamous cell carcinoma (HNSCC), and is correlated with poor prognosis. EGFR is therefore a potential therapeutic target for HNSCC. However, most HNSCC patients do not respond well to small molecular EGFR inhibitors. One of the possible mechanisms of EGFR TKI resistance is compensatory collateral activation of PI3K/Akt pathway. This study sought to explore whether NVP-BKM120, a selective pan class I PI3 kinase inhibitor, and NVP-BYL719, a novel PI3 kinase alpha selective inhibitor, may reverse the resistance of HNSCC cells toward erlotinib, an EGFR TKI. Cells were treated with the PI3K inhibitors, either BKM120 or BYL719, alone, erlotinib alone, or in combination, and viability was measured at 72 hours with MTT assay. Combination effect was evaluated by median effect analysis. Synergism, indicated by a combination index (CI) value <1, were seen in all five cell lines (SAS, HSC3, UMCC1, CA922 and FaDu) studied. By immunoblotting analysis, combination of Erlotinib and either PI3K inhibitor resulted in dowregulation of p-EGFR and p-AKT expression in SAS cells. When SAS and HSC3 cells were treated with erlotinib in combination with either BKM120 or BYL719, an additional increase in the subG1 population by flow cytometry analysis were observed, indicating an increase of apoptotic cell death. Our data suggest that additional PI3K blockade might be an effective strategy to overcome resistance of HNSCC cells toward EGFR TKIs, and the beneficial effect is similar between pan Class I PI3K inhibitor and PI3K alpha selective inhibitor. (The study was supported by the grant of DOH101-TD-B-111-001). Citation Format: Ming Gao, Yen-Shen Lu, Li-Ping Hsiao, Suz Wen Chen, Ann-Lii Cheng. Either pan class I PI3K inhibitor or PI3K alpha selective inhibitor sensitizes head and neck squamous cell carcinoma cells to Erlotinib, an EGFR TKI. [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 2450. doi:10.1158/1538-7445.AM2013-2450

Abstract 5653: Novel targets mediating resistance to EGFR/c-Met tyrosine kinase inhibitors in NSCLC.

Abstract Combination therapy of c-Met/EGFR TKIs against c-Met/EGFR, while initially effective, has been shown to be susceptible to acquired resistance. To establish a mechanism of TKI resistance, the NSCLC model cell lines H2170 and H358 were made resistant to the c-Met/EGFR TKIs SU11274, erlotinib, or a combination by exposing the cells to progressively increasing concentrations of inhibitors. Phospho-Met (Tyr1003) was found to be downregulated 4- and 1.5- fold in H2170 and H358 cells, respectively. Phospho-EGFR was found to be upregulated in H2170 cells and downregulated in H358 cells. Interestingly, no significant differences were observed in c-Met/EGFR downstream signaling proteins, indicating that alternative signaling pathways such as the canonical Wnt pathway may confer TKI resistance. To determine differences in Wnt signaling between parental and resistant cell lines, immunoblotting was performed. In the H2170 erlotinib resistant (H2170-ER) cell line, a 5- and 6-fold increase in phospho-LRP5/6 as well as a 2- and 3-fold increase in total-LRP5/6 was observed in the presence or absence of erlotinib, respectively. Furthermore, H2170-ER cells exhibited a 3- and 16-fold increase in NKD2 as well as a 2.5- and 6-fold increase in Wnt3A in the presence or absence of EGF, respectively. A 2- to 2.5-fold increase was also observed in DVL2/3 in the absence of EGF. However, the Wnt pathway did not seem to have a significant role in H358 cells. Since EGFR and Wnt signaling are known to exhibit crosstalk, activation of the Wnt pathway may stimulate activation of EGFR causing resistance. This is suggested as upregulation of proteins involved in the Wnt pathway was seen only in the H2170 cell line. These results were confirmed by treating H2170 cells with the Wnt inhibitor XAV939. MTT analysis indicated that parental cells showed no significant response to XAV939, however, resistant cell lines showed a 2-fold decrease in viability when compared to a c-Met/EGFR TKI combination treatment. We propose a new treatment modality with Wnt inhibitors which have not yet entered clinical testing. To identify new targets for NSCLC, stable isotope labeling by heavy/light amino acids in cell culture (SILAC) coupled with mass spectroscopy was used. Analysis revealed more than 200 proteins modulated greater than 1.5-fold, six of which have significant potential to influence cell growth and survival based on their biological roles in NSCLC. These proteins include total-β-catenin, TPR, syntenin, HMGB2, TOM34 and AIF. We are currently using tandem mass tagging (TMT) to further investigate additional modulated proteins. Proteins of interest identified by this method include TPD52, erbB2, MESDC2, PIN1, PEA15 and NRAS. Proteins confirmed to be upregulated/activated in resistant cell lines will be further investigated as targets which induce drug resistance in NSCLC. Citation Format: Ryan Jacobs, Jason Fong, David Moravec, Greg Botting, Ryan Bomgarden, John Rogers, Rosa Viner, Michael Blank, Neelu Puri. Novel targets mediating resistance to EGFR/c-Met tyrosine kinase inhibitors in NSCLC. [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 5653. doi:10.1158/1538-7445.AM2013-5653 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Inhibition of Autophagy in Combination with Ponatinib or Dual PI3K/mTOR Inhibition to Improve Treatment Response for Both Bcr-Abl Dependent and Independent Mechanisms of TKI-Resistance in CML

Inhibition of Autophagy in Combination with Ponatinib or Dual PI3K/mTOR Inhibition to Improve Treatment Response for Both Bcr-Abl Dependent and Independent Mechanisms of TKI-Resistance in CML

SY14-4 - Acquired Resistance to ALK Tyrosine Kinase Inhibitors

ABSTRACT The ALK tyrosine kinase inhibitor crizotinib has demonstrated clinical efficacy in ALK-rearranged NSCLC patients and is currently under additional phase III clinical development as both initial and second-line therapy for advanced ALK-rearranged NSCLC. However, their efficacy will ultimately be limited by the development of acquired drug resistance. Recent studies have identified and studied two mechanisms of ALK TKI resistance based on evidence from a crizotinib-treated ALK-positive lung cancer patient and in a cell line generated from the resistant tumor. To date four secondary mutations, all identified from crizotinib-treated ALK-rearranged tumor patients, have been reported. These mutations either involve the ‘gatekeeper’ residue (L1196) or sites away from crizotinib binding (F1174L, L1152R and C1156Y). The mechanistic basis for how the different mutations lead to crizotinib resistance is not fully understood. The L1196 mutation may create a steric hindrance for crizotinib binding, whereas the F1174L mutation likely promotes the active conformation of ALK, thus disfavoring crizotinib binding which preferentially binds the inactive conformation of ALK. The other mechanism was activation of EGFR signaling as a bypass signaling mechanisms that contributes to ALK inhibitor resistance. Finally, we discuss the strategies that are underway to clinically overcome acquired drug resistance.

Abstract 278: Regulation of constitutive Met phosphorylation in breast cancer by EGFR

Abstract Breast cancer remains a major health problem, despite advances in detection, diagnosis, and treatment. Four genomic subtypes of breast cancer have been identified: luminal A and B, HER2 positive, and basal type breast cancers. Three of these subtypes help to define the proper targeted therapeutic as breast cancers of luminal subtypes tend to be estrogen receptor positive and treatable with tamoxifen while breast cancers of HER2 positive subtype are treatable with trastuzumab. The fourth subtype, the basal type breast cancers, has a poor prognosis and has yet to have a molecular target identified. One molecule highly expressed in basal type breast cancers is epidermal growth factor receptor (EGFR). However, targeting EGFR kinase activity with small molecule kinase inhibitors has yet to show efficacy in the treatment of breast cancers. Recently, a panel of EGFR expressing breast cancer cell lines was characterized as sensitive or resistant to EGFR kinase inhibitors. One mechanism of resistance to EGFR kinase inhibitors was found to be the constitutive activation of Met, another molecule associated with basal type breast cancers. Here, the mechanism of constitutive Met activation in EGFR tyrosine kinase inhibitor resistant breast cancer cells is explored. Previously, constitutive Met activation was found to associate with acquired EGFR inhibitor resistance in lung cancer via genetic amplification of Met. In the EGFR TKI resistant breast cancer cells with constitutive Met phosphorylation, Met was not amplified or significantly overexpressed. In addition, constitutive Met phosphorylation was found to occur independent of ligand (hepatocyte growth factor (HGF)) stimulation, as HGF was not expressed in the breast cancer cell lines and inhibiting HGF association with Met via an inhibitory antibody failed to reduce Met phosphorylation. These data suggest a ligand-independent mechanism for Met constitutive phosphorylation. Interestingly, ligand-independent Met activation can be mediated by Met association with other membrane molecules including integrins, tetraspanins and receptor tyrosine kinases, including EGFR. Co-immunoprecipitation studies demonstrated that EGFR and Met co-associate and that this association occurs independent of EGFR or Met kinase activities. In addition, Met phosphorylation was reduced with EGFR TKIs as well as with downregulation of EGFR expression. Taken together, these data suggest that one potential mechanism of EGFR TKI resistance in breast cancer cells is the association and transphosphorylation of Met by EGFR. 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 278.

BCL6-Dependent Negative Regulation of Cell Cycle Checkpoint Regulators Enables Drug-Resistance in Ph+ Acute Lymphoblastic Leukemia.

Abstract 765BCR-ABL1 tyrosine kinase inhibitors (TKI) are widely used for the treatment of patients with Ph+ ALL and CML. To elucidate mechanisms of TKI-resistance in Ph+ ALL, we studied gene expression changes of a set of 11 primary cases of Ph+ ALL in response to TKI-treatment (16 hours; 2 μmol/l Imatinib). Sorting genes based on the ratio of gene expression values in the presence vs the absence of TKI-treatment, the BCL6 gene consistently ranked #1 in this analysis. As confirmed by quantitative RT-PCR and Western blotting, BCL6 is upregulated by 60- to 90-fold in response to TKI-treatment of Ph+ ALL cells. The BCL6 transcription factor functions as a protooncogene in germinal center (GC)-derived B cell lymphomas. We focused our analysis on BCL6, because it functions a transcriptional repressor of p53 and other cell cycle check point regulators and thereby protects GC-derived B cell lymphoma cells from apoptosis. Of note, BCL6 protein levels in TKI-treated Ph+ ALL cells were as high as in GC-derived B cell lymphoma. We hypothesize that dramatic upregulation of the BCL6 gene in response to TKI-treatment represents a defense mechanism of Ph+ ALL to evade cell death induced by activation of p53 and cell cycle checkpoint regulators. We tested the role of BCL6 in Ph+ ALL in a genetic loss-of-function experiment: To this end, B cell precursors from bone marrow of BCL6+/+ and BCL6−/− mice were transformed with BCR-ABL1. Upon treatment with 1 mmol/l Imatinib for three days, viability of BCL6−/− BCR-ABL1 ALL cells (0.4% ± 0.2%) was lower by two log orders compared to BCL6+/+ leukemia cells (46.0% ± 8.2%; p=0.001). Consistent with our hypothesis that p53 and other cell cycle checkpoint regulators are transcriptional targets of BCL6 in Ph+ ALL, we observed in a comprehensive gene expression analysis that mRNA levels of p53, p21, Arf and p27 were significantly higher in BCL6−/− compared to BCL6+/+ and BCR-ABL1 ALL cells (confirmed by quantitative RT-PCR). To identify target genes of the BCL6 transcriptional repressor, we performed a ChIP-chip analysis for BCL6 both in Ph+ ALL and GC-derived B cell lymphoma. Among the 1,235 target genes of BCL6 in Ph+ ALL, 736 were shared targets with GC-derived B cell lymphoma and 499 were exclusive for Ph+ ALL. These findings suggest that the function of BCL6 in Ph+ ALL does not replicate all aspects of its function in GC-B cell lymphoma. Single locus ChIP analysis confirmed strong recruitment of BCL6 to the promoter regions of the p53, p21 (CDKN1A), Arf (CDKN2A) and p27 (CDKN1B) genes in human Ph+ ALL cells after TKI-treatment. In a genetic loss-of-function experiment, we tested whether transcriptional suppression of p53, p21, Arf and p27 represents a major function of BCL6-upregulation in response to TKI-treatment in Ph+ ALL. To this end, we measured sensitivity of wildtype and Arf−/−, p53−/−, p21−/− and p27−/− BCR-ABL1 ALL cells to either Imatinib alone or to a combination of Imatinib and a novel BCL6 peptide inhibitor (RI-BPI). TKI-treatment alone had approximately the same effect in wildtype BCR-ABL1 ALL cells compared to Arf−/−, p53−/−, p21−/− and p27−/− counterparts. However, when TKI-treatment was combined with BCL6 inhibition (5 μmol/l RI-BPI), the viability of Arf−/−, p53−/−, p21−/− and to lesser degree, p27−/−BCR-ABL1 ALL cells was significantly higher than survival of the respective wildtype control leukemia. These findings confirm that transcriptional suppression of Arf, p53 and p21 (to lesser extent p27) represents an important characteristic of BCL6-mediated drug-resistance in response to TKI-treatment.We next tested the potential therapeutic usefulness of concomitant TKI-treatment and BCL6 peptide inhibition: To this end, 2 × 106 BCR-ABL1 ALL cells were luciferase-labeled and injected into sublethally irradiated NOD/SCID mice. Mice were treated six times between days 7 and 21 after leukemia cell injection with either the BCR-ABL1 kinase inhibitor Nilotinib (75 mg/kg) alone or a combination of Nilotinib and RI-BPI (20 mg/kg). Addition of RI-BPI resulted in significantly prolonged median survival (29 days) compared to Nilotinib alone (22 days; 12 mice per group; p=0.004). We conclude that BCL6-mediated transcriptional repression of p53 and other cell cycle checkpoint regulators represents a novel and critical mechanism of drug-resistance in Ph+ ALL. Combination of TKI-treatment with BCL6 peptide inhibition represents a new promising approach to target drug-resistance in Ph+ ALL. Disclosures:No relevant conflicts of interest to declare.