Tyrosine Kinase Inhibitors Resistant Cell Lines Research Articles

Abstract 1829: Brusatol overcomes tyrosine kinase inhibitors resistance in myeloid leukemia cells by inhibiting translation of fast turnover protein cyclin D1 and Mcl-1

Abstract Myeloid leukemia is characterized with fast proliferation and apoptosis evasion, which are controlled by mutant or activated tyrosine kinase signalings. Tyrosine kinase inhibitors (TKI) targeting BCR-ABL in chronic myeloid leukemia (CML) and FLT3-ITD in acute myeloid leukemia (AML) significantly improved the therapy, but not cure, due to the general resistance. Overactivated mTOR protein translation pathway is believed to be one of the mechanisms causing resistance and that targeting protein translation should be one effective approach to overcome resistance. Brusatol is a quassinoid extracted from the fruit of Brucea javanica and an inhibitor of protein translation. We tested its antiproliferation and apoptosis induction in a variety of AML and CML cell lines as well as in TKI resistant cell lines. Brusatol effectively inhibits the growth of seven leukemia cell lines with GI50 values less than 20 nM. Brusatol induces apoptosis in AML cell lines and induces G0/G1 phase arrest in CML cell lines. Brusatol decreases the levels of fast turnover protein cyclin D1 and Mcl-1. In FLT3-ITD inhibitor sorafenib resistant MOLM-13/Sor cell line brusatol exhibits similar growth inhibition and apoptosis induction with the parental MOLM-13 cells, associated with inhibition of Mcl-1 and FLT3-ITD proteins. In BCR-ABL inhibitor imatinib resistant K562/STI cell line brusatol is effective as in parental K562 cells, associated with inhibition of cyclin D1 and BCR-ABL proteins. Brusatol at 2 mg/kg effectively inhibit tumor growth of K562 xenografts without causing toxicity to normal organs. These data indicate that brusatol inhibits oncogenic protein translation and has potential to be used to treat TKI resistant leukemia. Citation Format: Huiming Hua, Ying Yang, Guangfuxin Lin, Jingyi Zhang, Samuel Waxman, Yongkui Jing. Brusatol overcomes tyrosine kinase inhibitors resistance in myeloid leukemia cells by inhibiting translation of fast turnover protein cyclin D1 and Mcl-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1829.

Genome‑wide expression and methylation analyses reveal aberrant cell adhesion signaling in tyrosine kinase inhibitor‑resistant CML cells

Although chronic myeloid leukemia (CML) can be effectively treated using BCR-ABL1 kinase inhibitors, resistance due to kinase alterations or to BCR-ABL1 independent mechanisms remain a therapeutic challenge. For the latter, the underlying mechanisms are widely discussed; for instance, gene expression changes, epigenetic factors and alternative signaling pathway activation. In the present study, in vitro-CML cell models of resistance against the tyrosine kinase inhibitors (TKIs) imatinib (0.5 and 2 µM) and nilotinib (0.1 µM) with biological replicates were generated to identify novel mechanisms of resistance. Subsequently, genome-wide mRNA expression and DNA methylation were analyzed. While mRNA expression patterns differed largely between biological replicates, there was an overlap of 71 genes differentially expressed between cells resistant against imatinib or nilotinib. Moreover, all TKI resistant cell lines demonstrated a slight hypermethylation compared with native cells. In a combined analysis of 151 genes differentially expressed in the biological replicates of imatinib resistance, cell adhesion signaling, in particular the cellular matrix protein fibronectin 1 (FN1), was significantly dysregulated. This gene was also downregulated in nilotinib resistance. Further analyses showed significant FN1-downregulation in imatinib resistance on mRNA (P<0.001) and protein level (P<0.001). SiRNA-mediated FN1-knockdown in native cells reduced cell adhesion (P=0.02), decreased imatinib susceptibility visible by higher Ki-67 expression (1.5-fold, P=0.04) and increased cell number (1.5-fold, P=0.03). Vice versa, recovery of FN1-expression in imatinib resistant cells was sufficient to partially restore the response to imatinib. Overall, these results suggested a role of cell adhesion signaling and fibronectin 1 in TKI resistant CML and a potential target for novel strategies in treatment of resistant CML.

Activating JAK-mutations confer resistance to FLT3 kinase inhibitors in FLT3-ITD positive AML in vitro and in vivo.

An important limitation of FLT3 tyrosine kinase inhibitors (TKIs) in FLT3-ITD positive AML is the development of resistance. To better understand resistance to FLT3 inhibition, we examined FLT3-ITD positive cell lines which had acquired resistance to midostaurin or sorafenib. In 6 out of 23 TKI resistant cell lines we were able to detect a JAK1 V658F mutation, a mutation that led to reactivation of the CSF2RB-STAT5 pathway. Knockdown of JAK1, or treatment with a JAK inhibitor, resensitized cells to FLT3 inhibition. Out of 136 patients with FLT3-ITD mutated AML and exposed to FLT3 inhibitor, we found seven different JAK family mutations in six of the cases (4.4%), including five bona fide, activating mutations. Except for one patient, the JAK mutations occurred de novo (n = 4) or displayed increasing variant allele frequency after exposure to FLT3 TKI (n = 1). In vitro each of the five activating variants were found to induce resistance to FLT3-ITD inhibition, which was then overcome by dual FLT3/JAK inhibition. In conclusion, our data characterize a novel mechanism of resistance to FLT3-ITD inhibition and may offer a potential therapy, using dual JAK and FLT3 inhibition.

Efficacy of OR21, a Novel Oral Demethylating Agent in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disease induced by t(9;22)(q34;q11) translocation. The prognosis of patients with CML has dramatically improved since tyrosine kinase inhibitors (TKIs) were introduced, and recent studies show that approximately 40 to 50 % of CML patients achieved deep molecular response (DASISION and ENESTnd trial) within several years. However, therapeutic options for patients with CML who are resistant for TKIs are limited. Besides BCR-ABL kinase domain mutation, somatic mutations associated with epigenetic gene alteration (e.g. TET, DNMT3A) are reportedly involved in TKI resistance and disease progression. Thus, we investigated the efficacy of DNA demethylating agents in CML. Azacitidine (AZA) and decitabine (DAC), currently available as DNA demethylating agents, have low bioavailability for oral administration because they are easily degraded by cytidine deaminase. Then, we are developing a novel demethylating agent, OR21, with possible oral absorbability as a prodrug of DAC. In vivo analysis using cynomolgus monkeys, the area under the curve of DAC after intraduodenally administration of DAC (1.5mg/kg, 6.6µmol/kg) or OR21 (2.25mg/kg, 6.6µmol/kg) was 0.01 µM·h, 0.298 µM·h respectively, indicating OR21 have high oral absorbability. To assess the demethylating activity of OR21 for CML, we performed western blot analysis and bisulfite pyrosequencing assay to measure LINE1 methylation, using CML cell-lines (K562, BV173). OR21 decreased DNMT1 protein level as a result of demethylating and LINE1 methylation in K562 and BV173 comparable to DAC. Next, we performed cell growth inhibition and cell apoptosis assay after 72 hours exposure of OR21 to assess anti-tumor effect in vitro. OR21 inhibited cell growth comparable to DAC in CML cell-lines (K562, BV173, KCL22, MYL) in a dose-dependent manner. Notably, OR21 inhibited the cell growth and apoptosis against BV173 with an extremely low concentration (IC50; 5nM) than that of AZA (IC50; 122nM) via significant accumulation of reactive oxygen species. Whereas, OR21 weakly induced cell apoptosis against K562. OR21 induced G2/M phase cell-cycle arrest in CML cell-lines except for BV173 via pRb downregulation. These results indicated mechanisms of anti-tumor effect in OR21 were induction of cell apoptosis (BV173) or cell cycle arrest (K562, KCL22, MYL). Because of the different mechanism of action, we assessed whether OR21 and TKIs combination can enhance the anti-tumor effect of CML. We investigated the combination effects of OR21 with imatinib (IM) or dasatinib (DAS) in K562. Combination index values at IC80 calculated by Calucusyn software showed 0.642±0.129 (with IM), 1.182±0.2 (with DAS), respectively. Accordingly, OR21 combined with TKIs showed at least additive or synergistic effect for K562. TKI resistance, which can be associated with somatic mutations leading to epigenetic gene alteration or loss of function of p53, is an obstacle for molecular remission in patients with CML, thus we examined the effects of OR21 in TKI resistant cell lines or efficacy on p53 mutational status. OR21 inhibited the cell growth in IM-resistant cell-line MYL-R, a derivative of MYL, which had overexpression of Lyn, and Ba/F3 BCR-ABLT315I, which exogenously expressed Bcr-Abl (T315I), indicating OR21 could overcome TKI resistance in CML. OR21 or cytarabine did not enhance cell apoptosis against K562, MYL and KCL22 (p53 deficient or mutant cell lines) combination with nutlin-3a (MDM-2 inhibitor), while increased cell apoptosis was observed in BV173 (p53 wild type) treated with cytarabine and nutlin-3a, but not with OR21 and nutlin-3a. These results suggested the effects of OR21 did not depend on p53 mutational status. Finally, we used a mouse xenograft model to evaluate anti-tumor effect of OR21 in vivo. BALB/c Rag-2/JAK3 double-deficient (BRJ) mice were injected intravenously via tail vein with 5 ×106 BV173 cells. OR21 were administered at a dose of 2.7mg/kg (equivalent to DAC 1.0mg/kg in AUC) and PBS (vehicle) twice weekly. OR21 significantly prolonged survival in a xenograft mice model (median 35 days vs not reached, P&lt;0.01). In conclusion, a novel orally available demethylating agent OR21 is effective for CML cells including TKI resistant clones. The efficacy and safety of OR21 for CML is expected to be verified by early-phase clinical trials. Disclosures Kamachi: Ohara Pharmaceutical Co.: Research Funding. Ureshino:OHARA Pharmaceutical Co.: Research Funding. Yoshida:OHARA Pharmaceutical Co., Ltd.: Research Funding. Kurahashi:Ohara Pharmaceutical Co.: Employment. Watanabe:Ohara Pharmaceutical Co.: Research Funding. Okada:Japan Agency for Medical Research and Development: Research Funding; Bristol-Myers Squibb: Research Funding. Kimura:Ohara Pharmaceutical Co.: Research Funding; Novartis: Honoraria, Research Funding.

The Role of Hypoxia on ABL Tyrosine Kinase Inhibitor Resistant Chronic Myeloid Leukemia Cells

Introduction: ABL tyrosine kinase inhibitors (TKIs) improved outcomes for patients with chronic myeloid leukemia (CML) and Philadelphia chromosome (Ph)-positive leukemia, however, some patients are still resistance to ABL TKIs. One of the most common mechanisms involves point mutations in the kinase domain of BCR-ABL1, however, mechanisms of intrinsic resistance without point mutation of ABL kinase domain are not fully understood. Moreover, ABL TKIs cannot cure the Ph-positive leukemia patients because of leukemia stem cells in the bone marrow niche. Therefore, new approach against leukemia stem cells may improve the outcome of Ph-positive leukemia patients. Hypoxia is an important component of the bone marrow microenvironment. Because oxygen tension plays a key role in driving normal hematopoiesis, leukemia stem cells may be maintained in hypoxic areas of the bone marrow. Materials and methods: In this study, we established ABL TKI-resistant in vitro cell line models (K562 imatinib-R, K562 nilotinib-R, K562 dasatinib-R, K562 ponatinib-R and Ba/F3 T315I). We investigated gene expression profiles in cultured ABL TKI resistant cells and parental cell line, K562 in normoxia and hypoxia condition by DNA microarray. Results: We first investigated gene expression profiles in cultured K562 cells in hypoxia condition. We found gene expression of insulin-like growth factor 1 (IGF1) was increased K562 cells in hypoxia condition by DNA microarray. We next examined ABL TKI resistant cell lines (K562 imatinib-R, K562 nilotinib-R, K562 dasatinib-R, K562 ponatinib-R) in this study. We could not detect the BCR-ABL point mutation in ABL TKI resistant cells. We found gene expression of insulin-like growth factor 1 (IGF1) receptor (IGF1R) was increased ABL TKI resistant K562 cells. IGF1R gene amplification was confirmed by RT-PCR analysis. IGF is tightly regulated by six related IGF-binding proteins (IGFBPs). One of IGFBP, IGFBP5 is related to imatinib sensitivity and resistant in chronic myeloid leukemia (CML) patients (GSE12211). In hypoxia condition, several IGFBPs were also increased in ABL TKI resistant cells. IGF cause intracellular signaling that ultimately results in cellular growth and proliferation. Thus, we initially examined whether addition of IGF1R inhibition could enhance ABL TKIs sensitivity. One of IGF1R inhibitor, linsitinib was inhibited ABL TKI resistant cells and parental cell line, K562 in hypoxia condition. ABL TKI resistant cell lines were more sensitive against linsitinib. Combined treatment of ABL TKI resistant cells and K562 cells with ABL TKIs and linsitinib caused more cytotoxicity than each drug alone in hypoxia condition. Caspase 3/7 activity and cellular cytotoxicity was also increased after ABL TKIs and linsitnib treatment. In the colony formation method, the number of cell colonies were also reduced in hypoxia condition. Intracellular ATP levels have been implicated in vitro as a determinant of cell death by apoptosis. The concentrations of intracellular ATP were reduced after ABL TKIs and linsitinib. We next blocked IGF1R function by small interfering RNA (siRNA). SiRNA transfected cells were reduced cellular proliferation. We also found drug sensitivity of the cells to the imatinib was increased compared to mock-transfected cells. Apoptotic cells and caspase 3/7 activity were increased after imatinib treatment in siRNA transfected cells. Conclusion: The IGF1 pathway is involved in Ph-positive leukemia cells in hypoxia condition and ABL TKI resistant in CML cells. We also provide the promising clinical relevance as a candidate drug for treatment of residual leukemia cells in bone marrow niche which is in hypoxia condition. Disclosures No relevant conflicts of interest to declare.

PF400 ANTI-LEUKEMIA EFFECT OF DISULFIRAM AND ASCORBATE IN ABL TYROSINE KINASE INHIBITOR RESISTANT LEUKEMIA CELLS

Background: ABL tyrosine kinase inhibitors (TKIs) such as imatinib improved outcomes for patients with Philadelphia chromosome (Ph)-positive leukemia patients, however, some patients are still resistance to ABL TKIs. One of the most common mechanisms involves point mutations in the kinase domain of BCR-ABL1, however, mechanisms of intrinsic resistance without point mutation of ABL kinase domain are not fully understood. Therefore, new approach against ABL TKI resistant cells may improve the outcome of Ph-positive leukemia patients. Aims: Because disulfiram (DSF), which is used to treat alcohol dependence in clinically, has been reported to have anti-tumor effects in various cancers, DSF may be the promising therapeutic value in ABL TKI resistant leukemia cells. Methods: In this study, we established ABL TKI resistant in vitro cell line models. We also investigated whether DSF could suppress Ph-positive leukemia cells including T315I mutation and ABL TKI resistant cells. Results: Drug repositioning and repurposing can be an important part of any drug discovery. In the drug screening system, the alcohol-aversion drug disulfiram, which is already approved for the treatment of alcohol dependence, is selected the candidate drug for ABL TKI resistant cells. Therefore, we examined DSF efficacy by using Ph-positive leukemia cells. We found that DSF treatment for 72 h decreased cell viability in a dose-dependent manner. We also found that DSF inhibited the proliferation of ABL TKI resistant cells (K562 ponatinib-R, K562 nilotinib-R) and T315I mutant Ba/F3 cells. Vitamin C (ascorbate) has also been well documented to reduce the incidence of malignancies in humans. We next investigated the efficacy of DSF and ascorbate against ABL TKI resistant and T315I mutant Ba/F3 cells. DSF and ascorbate treatment induced cellular growth inhibition compared with each drug alone. Most strikingly, cell death started in ABL TKI resistant cell lines within 3 to 6 h of treatment, indicating a first response to DSF and ascorbate lesions, to cell fate decision. Apoptotic cells and cellular cytotoxicity was also increased after DSF and ascorbate treatment. In the Colony Formation method, the number of cell colonies are also reduced. We next investigated the signaling pathway. Nuclear factor-kappaB (NF-kB) is also implicated in cancer development. DSF and ascorbate inhibited constitutive NF-kB activity. Adenosine triphosphate (ATP) is the most important source of energy for intracellular reactions. Intracellular ATP levels drastically decreased after DSF and ascorbate treatment. Because mitochondria generate ATP and participate in signal transduction and cellular pathology and cell death. The quantitative analysis of JC-1 stained cells changed mitochondrial membrane potential (Δψm) in cell death induced by DSF and ascorbate on T315I mutant Ba/F3 cells. The in vivo efficacies of DSF and ascorbate were evaluated in a mouse xenograft model. DSF and ascorbate inhibited the growth of T315I mutant Ba/F3 cells in vivo more than the control vehicle did. We also found that co-treatment with DSF and ascorbate increased mouse survival. Combination treatment with DSF and ascorbate was also well tolerated and no animal death in treated mice. Summary/Conclusion: The results of our study indicate that the DSF and ascorbate may be a powerful strategy against ABL TKI resistant cells including T315I mutation and provide the promising clinical relevance as a candidate drug for treatment of ABL TKI resistant leukemia patients.

Abstract A139: Mechanism of treatment-induced drug resistance in lung cancer

Abstract Objectives: Lung cancer is the number one cause of cancer-related deaths. About 20% of all non-small cell lung cancer (NSCLC) patients are expected to harbor an Epidermal Growth Factor Receptor (EGFR) activating mutation. First- and second-generation EGFR tyrosine kinase inhibitors (TKIs) are clinically approved to treat advanced NSCLC patients. However, despite the initial clinical responses to these EGFR-targeted therapies, long-term efficacy is not possible because acquired drug resistance hampers the effectiveness of these therapies. Even with gradually increasing knowledge, many routes to resistance have yet to be discovered. We found that a polymeric nanofibrous scaffold platform established in our laboratory allows growth of three-dimensional (3D) tumor-like aggregates (tumoroids), which resemble in vivo tumors. We hypothesize that 3D scaffold will allow us to elucidate EGFR TKI resistance mechanisms and help design more efficient treatment strategies to block resistance. Methods: To investigate drug resistance to EGFR TKIs, we have developed lapatinib-resistant human lung cancer cell lines as models for de novo drug resistance. To derive drug-resistant (DR) cells, three human lung cancer cell lines with different EGFR-activating mutations were grown in static concentrations of lapatinib for up to 30 days. The drug sensitivity of the parental and DR cells on both the monolayer and 3D scaffold was determined by testing a panel of standard-of-care chemotherapeutics along with EGFR TKIs. Mass spectroscopy was used to determine the relative levels of certain key proteins being expressed in the H1975 and lapatinib DR-H1975 cell lines. Expression of intriguing proteins found in the mass spec data was confirmed and validated in our EGFR TKI resistant models using other methods, including Western blotting and mRNA transcript analysis. Inhibitors, as well as siRNA, of selected proteins were then used to determine if these proteins played a role in EGFR TKI resistance. Results: A comparison of the drug sensitivity showed that parental cells were at least 3-fold more sensitive to the EGFR TKIs compared to the DR cells. The sensitivity to EGFR TKIs was further decreased when DR cells were cultured on our fibrous 3D scaffold. EGFR TKI resistant cells were also resistant to other anticancer agents, such as taxol and gemcitabine. Data mining the significantly differentially expressed proteins list generated by the mass spectroscopic analysis revealed that the protein expression is skewed in lapatinib DR-H1975 cell line as compared to the H1975 cell line. Proteins involved in all aspects of homeostasis including metabolism, biosynthesis, and oxidative regulation were significantly upregulated in the lapatinib-resistant cell line. We are currently working to determine if all the EGFR TKI resistant cell lines are producing similar mechanisms of resistance. Conclusions: Studying the cellular effects of acquired EGFR TKI resistance, as well as the mechanism through which this resistance is maintained, can help to better understand how lung cancer cells become drug resistant to EGFR TKIs and help strategize potential ways of overcoming this resistance. Research supported by: R01CA152005, HHSN261201400022C, HHSN261201400028C. Citation Format: Mark C. Howell, Ryan Green, Rajesh Nair, Stanley Stevens, Jit Banerjee, Shyam Mohapatra, Subhra Moahpatra. Mechanism of treatment-induced drug resistance in lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A139.

A novel strategy for ABL tyrosine kinase inhibitor resistant ph-chromosome positive leukemia cells

Although ABL tyrosine kinase inhibitors (TKIs) such as imatinib have demonstrated the potency against Philadelphia chromosome (Ph)-positive leukemia patients, resistance to ABL TKI can develop in chronic myeloid leukemia (CML) patients. We hypothesized that ABL TKI resistance may often happen due to additional somatic mutations in the oncogene. We established several TKI-resistant in vitro cell line models. We also investigated model to evaluate the next-generation sequencing (NGS) panel. We established ABL TKI resistant cell lines (K562 imatinib-R, K562 nilotinib-R, K562 dasatinib-R, K562 ponatinib-R, Ba/F3 T315I and Ba/F3 ponatinib-R) in this study. BCR-ABL expression levels were not increased. We could not detect the BCR-ABL point mutation. However, the exon 4 deletion in the BCR-ABL gene was found in K562 ponatinib-R cells. In contrast, compound mutations in BCR-ABL were found in Ba/F3 ponatinib-R cells. We next evaluated the NGS panel (GeneRead DNAseq Targeted Panels V2) to investigate the mutation. We found that several somatic mutations in TET2, FLT3, RB1, TP53, SETBP1, ASXL1, and BCORL1 in parental K562 cells. We also found that additional somatic mutations in K562 imatinib-R (IDH1 and KRAS), K562 dasatinib-R (IDH1) and K562 ponatinib-R (SF3A1). We could not detect additional mutation in K562 nilotinib-R cells. Combined treatment of ABL TKI resistant K562 with imatinib or dasatinib and MEK inhibitor or IDH1 inhibitor caused more cytotoxicity. Because aberrant activation of PI3K signaling pathway and deregulation of HDAC activity may be a cause of malignant disease in humans, we examined the PI3K and HDAC inhibitor in ABL TKI resistant cells. The inhibitor of class I PI3K as well as class I and II HDAC enzymes, CUDC-907 exhibits cell growth inhibition. Our study indicated that leukemia cells have acquired resistance through somatic mutation or exon 4 deletion in the BCR-ABL gene, suggested that individual based investigations may be important to evaluate the ABL TKI resistance. We also provide the promising clinical relevance as a candidate drug for treatment of ABL TKI resistant leukemia patients.

Abstract 3077: Tumor cells with acquired resistance to EGFR inhibitors and overexpression or activation of AXL, MET and FGFR1 are insensitive to single-agent treatment targeting AXL, MET or FGFR

Abstract Background: Aberrant activity of the MET, FGFR1 and AXL receptors has been associated with the development of resistance to first, second and third generation EGFR tyrosine kinase inhibitors (TKI) in EGFR-mutated non-small cell lung cancer (NSCLC) patients. Methods: We obtained 6 resistant lines by treating EGFR-mutated (exon 19), TKI sensitive PC9 cells with increasing concentrations of gefitinib or erlotinib. The p.T790M resistance mutation emerged in two cell lines (GR1, GR4), which remained sensitive to osimertinib, a third generation EGFR TKI. Six new cell lines to resistant to “second line” osimertinib were generated from GR1 and GR4 by exposure to increasing concentrations of the inhibitor. Finally, six more cell lines resistant to “first line” osimertinib were derived from the PC9 parental cells. All resistant cell lines were genotyped for selected genes (including EGFR) and characterized for AXL, MET and FGFR1 expression and activation by Q-RT-PCR, immunohistochemistry and Western blotting. The effects of AXL (BGB324), MET (crizotinib, capmatinib) and FGFR1 (nindetanib) inhibitors on the parental and the 18 resistant cell lines were analyzed by MTT and, in some cases, by colony formation. AXL was stably silenced in some of the resistant cell lines. Results: All cell lines resistant to “first line” gefitinib, erlotinib and osimertinib maintained the exon 19 EGFR sensitizing mutation. In contrast, three of the resistant cell lines to “second line” osimertinib lost the exon 19 and the p.T790M mutations. In two more, the p.T790M dropped to low allelic fractions (1% and 0.03%). Regardless of the EGFR status, AXL overexpression was the most common event related to EGFR TKI resistance in our panel of 18 cell lines, with FGFR1 and MET overexpression or activation as less frequent events. In proliferation assays, the IC50 of the EGFR TKI resistant cell lines for BGB324 (AXL inhibitor) was indistinguishable from the IC50 of the parental, EGFR TKI sensitive cell line. Similar results were obtained in the case of capmatinib, crizotinib (MET inhibitors) and nintedanib (FGFR inhibitor). Stable silencing of AXL on some of the AXL-overexpressing resistant cell lines had no effects in terms of doubling times, morphology of cells or sensitivity to EGFR TKIs. In combination experiments, the effect of BGB and MET inhibitors was found to be additive. Conclusions: In tumor cell line models of acquired resistance to EGFR TKIs, overexpression or activation of AXL, MET and FGFR1 was not associated to sensitivity to single-agent treatment with AXL, MET or FGFR inhibitors. Multitargeted approaches might be more effective in this setting. Citation Format: Jordi Bertran-Alamillo, Miguel Angel Molina-VIla, Cristina Teixidó, Jordi Codony-Servat, Ana Giménez-Capitán, Carles Codony-Servat, Silvia García-Román, Erika Aldeguer, Sonia Rodríguez, Rafael Rosell. Tumor cells with acquired resistance to EGFR inhibitors and overexpression or activation of AXL, MET and FGFR1 are insensitive to single-agent treatment targeting AXL, MET or FGFR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3077. doi:10.1158/1538-7445.AM2017-3077

Abstract 2109: Clonal heterogeneity of EGFR TKI-resistant NSCLC revealed through single-cell dilutional cloning and high-throughput functional screens

Abstract While EGFR tyrosine kinase inhibitors (TKIs) can elicit significant tumor shrinkage in the clinic, drug resistance inevitably ensues, likely due to a persistent subpopulation. T790M mutation is a common resistance mechanism found in 60% of tumors progressing on TKI. The aim of this study is to uncover novel mechanisms of gefitnib-resistant (GR) NSCLC by selecting TKI naïve PC9 single cell clones (SCC) differing in stemness and vulnerabilities to gefitinib (G), and inducing resistance through sublethal drug exposures. A high throughput drug screen was performed in 4 SCC to discover alternate therapies to target T790M negative GR clones. 231 SCC of PC9 were isolated and confirmed to be T790M negative (0.3% sensitivity) using Agena SABER assay. All clones were characterized by their vulnerability to G (viable after 48 hour exposure to 0.1uM) and ALDH1 activity using the ALDEFLUOR assay and flow cytometry. Four “diverse” clones (stratified according to ALDH1 and sensitivity to TKI) were selected to generate GR cell lines. Agglomerative hierarchical clustering of differentially expressed genes revealed that the clone exhibiting extreme low survival and ALDH1 signature clusters significantly apart from the rest. Subsequent iterations of this experiment (in total 5 per clone) revealed unique (and mostly stochastic) patterns of acquiring T790M across all clones. GR clones were then subjected to mutational analysis using the Ion AmpliSeq™ Colon and Lung Cancer Panel v1, and showed that 11/20 (55%) PC9 SCC GR lines have acquired the T790M mutation. T790M positive (AF range: 2.4 - 41.0%) GR lines were sensitive to Afatinib and WZ4002, a 2nd and 3rd generation EGFR TKI, with EGFR1086 phosphorylation being completely absent in all non-responding lines. T790M negative GR lines further acquired NRAS Q61K, G12D mutation and KRAS Q22K mutation. A high throughput drug screen with kinase inhibitor library from Selleck Chemicals (n = 273 kinases, n = 349 anticancer library) confirmed the selectivity of MEK inhibitors, as well as identified a subset of T790M-ve lines that are sensitive to Aurora kinase inhibitors. Our study highlights the potential for pEGFR to complement T790M status as a selection biomarker, particularly where low tumor cell concentration may yield false negative results. Taken together, we have uncovered potential therapeutically tractable subsets of T790M negative EGFR TKI resistant cell lines. pEGFR may identify a subset of T790M negative tumors that remain driven by and may be an alternate selection biomarker to 2nd/3rd gen TKI. Combinational approaches with MEK and aurora kinase inhibitors could be further explored in T790M negative/ pEGFR negative tumors. Citation Format: Dawn Pingxi Lau, Shivaji Rikka, Hui Sun Leong, Gek San Tan, Eleni G Christodoulou, Sai Sakktee Krisna, Shen Yon Toh, Xue Lin Kwang, Fui Teen Chong, Audrey Ann Liew, Tony Kiat Hon Lim, DasGupta Ramanuj, N Gopalakrishna Iyer, Daniel Shao-Weng Tan. Clonal heterogeneity of EGFR TKI-resistant NSCLC revealed through single-cell dilutional cloning and high-throughput functional screens. [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 2109.

Inhibition of Aurora kinase B is important for biologic activity of the dual inhibitors of BCR-ABL and Aurora kinases R763/AS703569 and PHA-739358 in BCR-ABL transformed cells.

ABL tyrosine kinase inhibitors (TKI) like Imatinib, Dasatinib and Nilotinib are the gold standard in conventional treatment of CML. However, the emergence of resistance remains a major problem. Alternative therapeutic strategies of ABL TKI-resistant CML are urgently needed. We asked whether dual inhibition of BCR-ABL and Aurora kinases A-C could overcome resistance mediated by ABL kinase mutations. We therefore tested the dual ABL and Aurora kinase inhibitors PHA-739358 and R763/AS703569 in Ba/F3- cells ectopically expressing wild type (wt) or TKI-resistant BCR-ABL mutants. We show that both compounds exhibited strong anti-proliferative and pro-apoptotic activity in ABL TKI resistant cell lines including cells expressing the strongly resistant T315I mutation. Cell cycle analysis indicated polyploidisation, a consequence of continued cell cycle progression in the absence of cell division by Aurora kinase inhibition. Experiments using drug resistant variants of Aurora B indicated that PHA-739358 acts on both, BCR-ABL and Aurora Kinase B, whereas Aurora kinase B inhibition might be sufficient for the anti-proliferative activity observed with R763/AS703569. Taken together, our data demonstrate that dual ABL and Aurora kinase inhibition might be used to overcome ABL TKI resistant CML.

Increasing Expression Of The Efflux Transporter ABCB1 May Predispose CML Cells To Overt TKI Resistance

Tyrosine kinase inhibitors (TKIs) result in excellent responses in most Chronic Myeloid Leukemia (CML) patients. However, up to 35% of patients treated with imatinib (IM) exhibit resistance and more recently nilotinib (NIL) and dasatinib (DAS) resistance have also been observed. Mutations in the BCR-ABL kinase domain (KD) are the main cause of secondary TKI resistance. Other mechanisms include overexpression of BCR-ABL, LYN and ABCB1. Predicting patients with susceptibility to mutation development and disease progression is crucial, thus we investigated the kinetics of TKI resistance emergence in vitro and in vivo.ABCB1 is implicated in TKI efflux hence we postulated that overexpression of ABCB1 leads to reduced intracellular TKI concentrations, resulting in inferior inhibition of Bcr-Abl predisposing cells to resistance development. Accordingly, 3 CML blast crisis (BC) cell lines (K562, K562-Dox, KU812) were cultured in increasing concentrations of IM to 2 μM, NIL to 2 μM and DAS to 200 nM until we observed overt resistance defined as a significant increase in survival in cytotoxicity assays and p-Crkl dependent IC50. Mechanisms of resistance were investigated in cell line intermediates: BCR-ABL, ABCB1 and LYN mRNA expression levels were determined by RT-PCR and KD mutation sequencing was performed.In our TKI resistant cell lines (Table 1), an increase in ABCB1 mRNA was the initial change observed prior to the development of additional resistance mechanisms (KD mutations, ABCB1 BCR-ABL and LYN overexpression). Interestingly, in 4/6 cells lines ABCB1 mRNA reduced to basal levels or below following establishment of these additional resistance mechanisms.ABCB1 levels were assessed in 37 de novo CML patients treated with IM who achieved major molecular response (MMR) compared with patients who progressed to BC, lost MMR or developed KD mutations. ABCB1 levels were determined in blood at diagnosis and following therapy (selected patients summarized in Table 2). A sustained >2 fold rise in ABCB1 was observed prior to disease progression in 3/3 patients and in 13/16 patients who did not achieve MMR. Importantly, the same was not observed in patients who achieved MMR (1/6 patients). The fold change of ABCB1 mRNA at day 22 vs diagnosis in patients achieving MMR was significantly different to that in patients not achieving MMR (p=0.004). ABCB1 increased by >2 fold post therapy and decreased following mutation development in 3/12 patients, confirming observations made in vitro, while 6/12 patients demonstrated sustained increase in ABCB1 post mutation similar to results observed in progression patients. ABCB1 mRNA did not change during therapy in 3/12 patients with mutations. While we recognize the majority of cells present in patients who achieve MMR are normal rather than leukemic, it is important to note that in patients who do not achieve MMR, ABCB1 expression increases in the remaining leukemic cells.We conclude ABCB1 overexpression acts as an initial mediator of resistance, providing a favorable environment for development of further resistance. Sustained increased levels of ABCB1 may contribute to disease progression and lack of response to IM. Additionally, ABCB1 may serve as a prognostic indicator (eg: level at day 22) and potentially assist in development of treatment strategies using TKIs in combination with other medications to enhance intracellular TKI concentration. Disclosures:Hughes:Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL: Research Funding. White:Novartis: Research Funding; BMS: Research Funding, Speakers Bureau; Ariad: Research Funding; CSL: Research Funding.

TTT-3002 Is Active Against Relapsed/Refractory AML Patient Bone Marrow With FLT3 TKI-Resistance Mutations At Residue D835

Over 35% of acute myeloid leukemia (AML) patients harbor a constitutively activating mutation in FLT3, either internal tandem duplication (ITD) or point mutations (FLT3/PM). FLT3/ITD mutations in particular confer a poor prognosis, and thus several FLT3 tyrosine kinase inhibitors (TKIs) have been developed and are currently in clinical trials. However, many have failed due to insufficient achievement of FLT3 kinase inhibition in vivo. This is thought to be due to insufficient potency, reduced activity against FLT3/PM, and/or selection for resistance-conferring point mutations in FLT3/ITD. Therefore, the search for novel FLT3 TKIs that overcome some of these resistance mechanisms that result in persistent FLT3 activation is necessary to improve the cure rate for this disease. We have previously reported on TTT-3002, a novel TKI that possesses the most potent activity against FLT3 phosphorylation, with IC50s of 100-250pM. Here we characterize the activity of this compound against TKI-resistant AML.The phenomenon of drug resistance associated with selection of FLT3 point mutations is being observed with increasing frequency as higher levels of inhibition by FLT3 TKIs are achieved in clinical trials of FLT3/ITD AML patients. In order to predict the ability of TTT-3002 to successfully treat some of these resistance mutations, we used site-directed mutagenesis to generate a series of FLT3/ITD TKI resistant cell lines. We directly compared the activity of TTT-3002 to other FLT3 TKIs currently in clinical trials including CEP-701, sorafenib, AC220 and PKC412. TTT-3002 remained the most potent inhibitor of all six FLT3/ITD TKI resistance mutants screened, with viability IC50 values of less than 1nM (with the single exception of Ba/F3-G697R/ITD, IC50=11nM). It was thus able to effectively inhibit FLT3 with TKI resistance mutations against which other TKIs are ineffective. Therefore, this compound's broad spectrum of activity against FLT3/ITD TKI resistance mutations may enable it to successfully treat FLT3/ITD AML patients who have become resistant to other FLT3 TKIs.To examine this directly, human AML samples were obtained from patients that developed resistance to sorafenib or AC220 while on clinical trials and presented with a dual D835/ITD mutation or D835 point mutation alone at the time of relapse. These blasts were evaluated for their ex vivo sensitivity to TTT-3002, sorafenib and AC220, with a diagnostic FLT3/ITD+ patient sample for comparison. Encouragingly, we observed significant effects on proliferation, induction of apoptosis, and cell signaling when cells were treated with TTT-3002. AC220 was moderately active against FLT3/ITD+ blasts and one patient sample with a D835/ITD mutation, and sorafenib was active against only the diagnostic FLT3/ITD sample, but none of the three relapse samples. Therefore, TTT-3002 maintains activity against human AML patient samples that are resistant in clinical trials to the FLT3 TKIs sorafenib and AC220.To account for the patterns of drug resistance observed for different FLT3 TKIs in our study, we modeled the positions of staurosporine-like (TTT-3002, CEP-701 and PKC412) and sorafenib-like inhibitors (sorafenib and AC220) bound to FLT3. Examination of amino-acid substitutions in FLT3 known to confer resistance to TKIs from clinical trials shows that substituting arginine for glycine at position 697 likely leads to steric clashes with FLT3 TKIs. It has been proposed that the F691 residue forms a stabilizing π-π stacking interaction with AC220, and thus a mutation to a Leu accounts for reduced binding affinity for this TKI. TTT-3002 is predicted to bind FLT3 without making direct contact with the F691 residue, and thus is unaffected by mutations at this site accounting for its continued potency. The closer proximity of F691 and N676 to sorafenib compared to staurosporine likely underlies the trend for substitutions at these sites to confer resistance to sorafenib-like but not staurosporine-like inhibitors.In summary, TTT-3002 has the potential for increased clinical applicability due to its ability to target not only FLT3/ITD and FLT3/PM AML patients but also patients who have developed resistance to other FLT3 TKIs through the selection of resistance mutations within the FLT3/ITD allele. These preclinical studies further support the entry of TTT-3002 into human clinical trials. Disclosures:Levis:Ambit: Consultancy.

FLT3-ITD Interacts with and Phosphorylates IL-3β, and JAK1/2 Dependent IL-3β Activation Bypasses FLT3-ITD in FLT3 Kinase Independent Inhibitor Resistance in Vitro: Evidence for the Significance of IL-3β for FLT3-ITD Dependent Oncogeneic Signaling in AML.

AbstractAbstract 2423Activating FLT3 mutations are found in 30% of AML patients. Internal tandem duplication (ITD) mutations are most common, and are associated with poor prognosis. FLT3 tyrosine kinase inhibitors (TKI) display limited activity in FLT3 mutant AML. Most patients experience primary or secondary TKI resistance. Only single cases of FLT3 kinase-dependent TKI resistance by secondary FLT3-ITD kinase domain mutations were reported. We therefore examined the mechanism of FLT3-ITD kinase-independent TKI resistance using an in vitro model. FLT3-ITD cell lines resistant to PKC412 or Sorafenib in 50% of the cases did not harbor secondary FLT3-ITD kinase domain mutations. However, 30% of these lines displayed a persistent phosphorylation of JAK2, IL-3βc and STAT5, while FLT3 phosphorylation was suppressed, indicating that these cell lines bypassed FLT3-ITD by activation of IL-3β by JAK2. Strikingly, TKI sensitive FLT3-ITD cells also displayed IL-3β phosphorylation, which in contrast to TKI resistant cells was JAK2 independent and was suppressed upon FLT3 TKI treatment. Expression of FLT3-ITD in IL-3β and JAK2 deficient g2A cells was sufficient for IL-3β phosphorylation. Co-IP experiments indicated a direct interaction of IL-3β and FLT3-ITD in TKI-sensitive and -resistant cells, which did not require interaction of IL-3β with IL-3a. This indicates that FLT3-ITD “physiologically” uses IL-3β as signaling intermediate. Mapping experiments showed that FLT3-ITD binding occurs in the membrane proximal region of IL-3β, independent of 8 tyrosines present in the cytoplasmic part. In search of the kinase bypassing FLT3-ITD in TKI-resistant cell lines, we identified an activating JAK1 mutation V658F in all TKI resistant FLT3-ITD cell lines displaying phosphorylation of JAK2, IL-3β and STAT5. JAK1 V658F is homologous to JAK2 V617F present in PV, phosphorylates IL-3β and STAT5, and confers TKI resistance in FLT3-ITD positive cells, which could be overcome by the JAK1/2 inhibitor Ruxolitinib. In summary, these data indicate that IL-3β not only mediates “physiological” FLT3-ITD dependent STAT5 activation, but also serves as a signaling module mediating JAK1/2-dependent TKI resistance. Thus, JAK inhibitors might represent an attractive therapeutic option in addition to FLT3 inhibitors in FLT3-ITD positive AML. Disclosures:Duyster:Novartis: Honoraria, Speakers Bureau. von Bubnoff:Novartis: Honoraria, Research Funding, Speakers Bureau.

Abstract 915: Effect of AZD1480 on lung tumor in transgenic mice carrying activating EGFR mutation

Abstract Signal transducer and activator of transcription 3 (STAT3) plays a major role in inducing and maintaining a pro-carcinogenic inflammatory microenvironment and is reported to be a critical mediator of the oncogenic effects of epidermal growth factor receptor (EGFR) mutations. STAT3 activation is mediated through the Janus family kinases (JAK) and SRC. JAK1/2 inhibitors such as AZD1480 suppress activation of STAT3 in human solid tumor cell lines and xenografts. Furthermore, they show anticancer effects in various cancer cell lines and xenograft models, including lung cancer. However, the effect of the JAK inhibitors on non-small cell lung cancer harboring EGFR mutation remains unclear. PC-9 is a lung adenocarcinoma cell line harboring exon19 EGFR deletion mutation. Four EGFR tyrosine kinase inhibitors-resistant cell lines, RPC-9, PC-9/Van-R, PC-9/ER3 and PC-9/GR, were established from PC-9 in our laboratory and the resistant mechanisms were mainly caused by a secondary mutation of T790M, JAK2 overexpression or ERK reactivation. Growth inhibition was measured by MTT assays. The drug concentration required to inhibit the growth of tumor cells by 50% (IC50) for 96 h exposure was used to evaluate the effectiveness of AZD1480. Protein expression was determined by Western blotting. The IC50s were 2.4 μM for PC-9, 1.7 μM for RPC-9, 1.2 μM for PC-9/Van-R, 1.2 μM for PC-9/ER3 and 1.6 μM for PC-9/GR. Thus, no cross-resistance was observed. Phosphorylated JAK2 and phosphorylated STAT3 were suppressed by treatment with AZD1480. We generated transgenic mice expressing the delE748-A752 mutant of mouse Egfr, which corresponded to the delE746-A750 mutant of human EGFR. In the absence of treatment, the transgenic mice developed adenocarcinoma at 6 to 7 weeks of age and died due to tumor progression at 13 to 18 weeks. To investigate the effect of AZD1480 on lung tumors induced by an activating EGFR mutation, the transgenic mice were treated with oral AZD1480 (30 mg/kg/day) or vehicle alone from 7 to 10 weeks of age. The number of lung tumors (long axis exceeding 1 mm) ± SE in the AZD1480-treated group and control group was 0.37 ± 0.18 and 2.25 ± 0.53 (t-test, p&amp;lt;0.001), respectively. Immunohistochemistry and Western blotting showed that phosphorylation of STAT3 was suppressed during AZD1480 treatment. To evaluate the efficacy of AZD1480 on survival in EGFR transgenic mice, AZD1480 (30 mg/kg/day, n = 13) or vehicle (n = 13) was administered orally from 7 weeks of age. The median survival time in the AZD1480-treated group (217 days) was significantly longer than that in the control group (106 days) (logrank test, p&amp;lt;0.0001). In conclusion, AZD1480 may be effective for treatment of lung tumors induced by an activating EGFR mutation and may overcome the resistance to EGFR-tyrosine kinase inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 915. doi:1538-7445.AM2012-915

Abstract 4689: Inhibition of IL-6/Jak/Stat3 signaling: a novel and potent targeting therapy for lung cancer

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of death in the United States. A fraction of these tumors harbor somatic activating mutations in the epidermal growth factor receptor (EGFR) leading to constitutive activation of the kinase. Targeting this pathway using a class of tyrosine kinase inhibitors (TKI) such as gefitinib or erlotinib induces significant initial responses in patients with NSCLC expressing activating EGFR mutations, however, disease progression invariably occurs in part due to the outgrowth of cancers with EGFR TKI resistant secondary mutations. There are many ongoing attempts to overcome this problem, but the current efforts are mostly focused on EGFR itself and their effectiveness remains inconclusive. The interleukin-6 (IL-6) /Jak/Stat3 signaling pathway is critical not only for inflammation and immunity, but also for oncogenesis. Stat3 is persistently activated in many human cancers, promoting tumorigenesis through the regulation of genes important for proliferation, apoptosis, invasion, angiogenesis and suppression of anti-tumor immunity. We previously determined that Stat3 is activated in mutant EGFR expressing NSCLC cell lines and primary tumors in part through autocrine upregulation of the IL-6 gene that activates the gp130/Jak signaling. In our current report, we studied the in vivo effect of inhibiting the IL-6/Jak/Stat3 signaling in EGFR TKI-resistant models of NSCLC. Our data demonstrated that Jak inhibition by a novel orally available Jak inhibitor markedly repressed in vivo growth of 3 different TKI resistant NSCLC cell lines as well as a TKI sensitive cell line. Immunohistochemical analyses of these samples revealed reduced proliferation and angiogenesis in Jak inhibitor treated tumors. In addition, administration of an IL-6R blocking antibody to xenograft tumors of a TKI resistant NSCLC cell line also achieved significant reduction in tumor growth compared to controls. We further tested the potential role of IL-6 in a transgenic murine model of TKI-resistant, mutant EGFR mediated lung cancer. Our results demonstrated mice lacking IL-6 still developed lung tumors but at a much slower rate of tumor progression compared to IL-6 expressing littermates. Additional examinations showed that mice lacking IL-6 developed tumors with papillary features compared to IL-6 expressing littermates whereas most of the tumors were of the more advanced, solid/pleiomorphic subtype. IL-6 deficient lung tumors had decreased proliferation and angiogenesis compared to tumors from wildtype littlermates. In summary, our findings lead us to hypothesize that the IL-6/Jak pathway should be considered as a novel therapeutic target for this disease, especially TKI resistant NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4689. doi:10.1158/1538-7445.AM2011-4689

Abstract 269: Disruption of lipid rafts sensitizes EGFR expressing breast cancer cells to EGFR inhibitors

Abstract While therapeutic strategies have improved, ∼500,000 women worldwide die from breast cancer each year. Many proteins have been implicated in breast cancer, including the HER family of receptors. Overexpression of epidermal growth factor receptor (EGFR) and one of its family members, HER2, each occur in ∼30% of breast cancers and correlates with poor clinical prognosis. Herceptin, an inhibitor of HER2, has been approved for treatment of breast cancer patients; however small molecule tyrosine kinase inhibitors (TKIs) targeting EGFR have not shown efficacy in breast cancer. To understand why breast cancers do not respond to EGFR tyrosine kinase inhibition, a panel of twenty breast cancer cell lines was characterized for EGFR expression. Thirteen of the twenty cell lines expressed EGFR; seven of which were resistant to EGFR TKIs. Unlike the response of HER2 positive breast cancers to Herceptin, increased expression of EGFR correlated with resistance to EGFR TKIs. One possible mechanism for resistance to EGFR TKIs is that EGFR expression is not required for growth. However, when EGFR expression was knocked down, cell growth was significantly decreased in four of the seven cell lines suggesting that EGFR expression is important for cell growth in a portion of EGFR TKI resistant breast cancers. A second mechanism for resistance to EGFR may be elevated HER2 and HER3 expression as observed in lung cancer. Conversely, in these cell lines, HER2 and HER3 expression levels did not correlate with response to EGFR TKIs. In addition, an EGFR family kinase inhibitor also was unable to abrogate the growth of these cells suggesting that HER2 expression and kinase activity was not a mediator of cell growth in the presence of EGFR TKIs. EGFR is known to localize within many sub-cellular regions including the nucleus, mitochondria, endosomes, and plasma membrane. Localization of EGFR to membrane microdomains (lipid rafts) is known to modulate EGFR signaling. Therefore, a third potential mechanism for EGFR TKI resistance may be localization of EGFR within lipid rafts. Using biochemical raft isolation and immunofluorescence, EGFR was shown to localize to lipid rafts in all four EGFR-dependent, EGFR TKI resistant breast cancer cell lines. To determine if this localization mediates resistance to EGFR TKIs, cholesterol (a main structural component of lipid rafts) was depleted using cholesterol biosynthesis inhibitors. While such inhibitors had no effect on cell growth alone, combinatorial depletion of lipid rafts and inhibition of EGFR-kinase activity resulted in decreased cell growth. Further, the interaction between cholesterol biosynthesis inhibitors and EGFR inhibitors was synergistic. Thus, we have discovered that EGFR expression and localization to lipid rafts correlate with EGFR TKI resistance in breast cancer, and that depletion of lipid rafts can overcome such resistance in EGFR expression-dependent breast cancer cells. 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 269.

Histone Deacetylase Inhibitor Romidepsin Enhances Anti-Tumor Effect of Erlotinib in Non-small Cell Lung Cancer (NSCLC) Cell Lines

Most epidermal growth factor receptor (EGFR) mutant non-small cell lung cancers (NSCLCs) are sensitive to EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib or gefitinib, but many EGFR wild type NSCLCs are resistant to TKIs. In this study, we examined the effects of the histone deacetylase inhibitor, romidepsin, in combination with erlotinib, in NSCLC cell lines and xenografts. For in vitro studies, nine NSCLC cell lines with varying mutation status and histology were treated with erlotinib and romidepsin alone or in combination. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assays were performed to determine the concentration that inhibits 50% (IC50) value of each drug or the combination. For in vivo studies, NCI-H1299 xenografts were inoculated subcutaneously into athymic nude mice. Romidepsin and/or erlotinib were injected intraperitoneally after tumors developed and tumor sizes were measured. We found that romidepsin increased the sensitivity of erlotinib synergistically in all nine NSCLC cell lines including EGFR and KRAS wild type cell lines, KRAS mutant cell lines, and TKI resistant EGFR mutant cell lines. This effect was partially due to enhanced apoptosis. Furthermore, cotreatment of erlotinib and romidepsin inhibited NCI-H1299 xenograft growth in athymic nude mice. These observations support a role for the combination of a histone deacetylase inhibitor and a TKI in the treatment of NSCLCs.

Prolonged Exposure to FLT3 Inhibitors Leads to Resistance Via Activation of Parallel Signaling Pathways.

A number of tyrosine kinase inhibitors (TKI) have been developed to treat a variety of malignancies. However, continuous treatment with TKIs may select for resistant clones as has been seen with Gleevec treatment of CML. To study resistance to TKIs targeting FLT3, a receptor tyrosine kinase that is frequently mutated in AML, we developed resistant human cell lines through prolonged co-culture with FLT3 TKIs. Both FLT3 TKI sensitive and resistant cell lines exhibit inhibition of FLT3 phosphorylation upon FLT3 TKI treatment. However, FLT3 TKI resistant cell lines and primary samples often show continued activation of downstream PI3K/Akt and/or Ras/MEK/MAPK signaling pathways as well as continued expression of genes involved in FLT3-mediated cellular transformation. Inhibition of these pathways restores partial sensitivity to FLT3 TKIs. Mutational screening of FLT3 TKI resistant cell lines and primary samples failed to reveal any mutations in FLT3 or in 100 kinases/phosphatases tested but did reveal activating N-Ras mutations that were not present in the parental FLT3 TKI sensitive cell line. Taken together, these data indicate that FLT3 TKI resistant cells most frequently become FLT3 independent due to activation of parallel signaling pathways that provide compensatory survival / proliferation signals when FLT3 is inhibited. IMC-EB10, an unconjugated monoclonal antibody against FLT3, is still cytotoxic to FLT3 TKI resistant clones in vivo. An approach combining FLT3 TKIs with anti-FLT3 antibodies may prove superior and result in reduced chances of developing resistance.