Development Of Tyrosine Kinase Inhibitors Resistance Research Articles

Relationship between Oxidative Stress and Imatinib Resistance in Model Chronic Myeloid Leukemia Cells.

Chronic myeloid leukemia (CML) develops due to the presence of the BCR-ABL1 protein, a target of tyrosine kinase inhibitors (TKIs), such as imatinib (IM), used in a CML therapy. CML eradication is a challenge due to developing resistance to TKIs. BCR-ABL1 induces endogenous oxidative stress leading to genomic instability and development of TKI resistance. Model CML cells susceptible or resistant to IM, as well as wild-type, non-cancer cells without the BCR-ABL1 protein were treated with IM, hydrogen peroxide (H2O2) as a model trigger of external oxidative stress, or with IM+H2O2. Accumulation of reactive oxygen species (ROS), DNA damage, activity of selected antioxidant enzymes and glutathione (GSH), and mitochondrial potential (MMP) were assessed. We observed increase in ROS accumulation in BCR-ABL1 positive cells and distinct levels of ROS accumulation in IM-susceptible cells when compared to IM-resistant ones, as well as increased DNA damage caused by IM action in sensitive cells. Depletion of GSH levels and a decreased activity of glutathione peroxidase (GPx) in the presence of IM was higher in the cells susceptible to IM. IM-resistant cells showed an increase of catalase activity and a depletion of MMP. BCR-ABL1 kinase alters ROS metabolism, and IM resistance is accompanied by the changes in activity of GPx, catalase, and alterations in MMP.

Epigenetic Alterations in Renal Cell Cancer With TKIs Resistance: From Mechanisms to Clinical Applications.

The appearance of tyrosine kinase inhibitors (TKIs) has been a major breakthrough in renal cell carcinoma (RCC) therapy. Unfortunately, a portion of patients with TKIs resistance experience disease progression after TKIs therapy. Epigenetic alterations play an important role in the development of TKIs resistance. Current evidence suggests that epigenetic alterations occur frequently in RCC patients with poor response to TKIs therapy, and modulation of them could enhance the cytotoxic effect of antitumor therapy. In this review, we summarize the currently known epigenetic alterations relating to TKIs resistance in RCC, focusing on DNA methylation, non-coding RNAs (ncRNAs), histone modifications, and their interactions with TKIs treatment. In addition, we discuss application of epigenetic alteration analyses in the clinical setting to predict prognosis of patients with TKIs treatment, and the potential use of epigenetics-based therapies to surmount TKIs resistance.

Abstract A08: Genetic drug resistance screen identifies LZTR1 as regulator of RAS ubiquitination and signaling

Abstract Small-molecule tyrosine kinase inhibitor (TKI)-based treatment of chronic myeloid leukemia (CML), directed by the presence of the Philadelphia chromosome (Ph+) encoded BCR-ABL tyrosine kinase, is a paradigm of targeted cancer therapy. However, the development of TKI resistance limits the long-term success of these therapeutics. We used a genetic screening approach in the near-haploid CML cell line KBM-7 to identify six genes whose individual loss of function led to TKI drug resistance. We investigated in detail the role of the leucine zipper-like transcription regulator 1 (LZTR1) gene, as it was mechanistically enigmatic despite its involvement in many human developmental and oncologic diseases including glioblastoma, schwannomatosis and Noonan syndrome. We found that LZTR1 inactivation led to enhanced MAPK pathway activity and reduced TKI sensitivity of CML cells in a RAS-dependent way. LZTR1 missense mutations identified in human diseases failed to revert the loss-of-function phenotype. Knockdown of the LZTR1 orthologue CG3711 in Drosophila led to an increase in ectopic wing vein formation that could be rescued by impairment of RAS gene function, confirming the epistatic relationship and suggesting an evolutionary conserved role for LZTR1 in RAS regulation. Endogenous LZTR1 protein associated with the four main RAS isoforms KRAS4A, KRAS4B, NRAS and HRAS in proximity biotinylation proteomic and immunoprecipitation experiments in a manner that for KRAS required farnesylation and palmitoylation. Ectopic expression of LZTR1 along with all four RAS proteins led to their ubiquitination, compatible with the proposed role of LZTR1 as a cullin (CUL)-3 E3 ligase adaptor. Loss of LZTR1 led to reduced ubiquitination of endogenous KRAS and its increased abundance as well as enhanced localization at the plasma membrane. Together with the work on the role of LZTR1 in diseases driven by the dysregulation of RAS ubiquitination and signaling (Steklov, Pandolfi, Baietti et al., Anna Sablina lab, KU Leuven; see separate abstract), we propose that LZTR1 acts as a conserved regulator of RAS ubiquitination and MAPK pathway activation, providing a mechanistic explanation to its genetic involvement across a variety of human pathologies. Citation Format: Johannes W. Bigenzahn, Giovanna M. Collu, Felix Kartnig, Melanie Pieraks, Gregory I. Vladimer, Leonhard X. Heinz, Vitaly Sedlyarov, Fiorella Schischlik, Astrid Fauster, Manuele Rebsamen, Katja Parapatics, Vincent A. Blomen, André C. Müller, Georg E. Winter, Robert Kralovics, Thijn R. Brummelkamp, Marek Mlodzik, Giulio Superti-Furga. Genetic drug resistance screen identifies LZTR1 as regulator of RAS ubiquitination and signaling [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A08.

IA33 Mechanisms of Small-Cell Lineage Transformation in Resistance to Targeted Therapies

EGFR tyrosine kinase inhibitors (TKIs) are highly effective for tumors with EGFR mutations. However, resistance to these compounds remains a major issue, with the most frequent mechanism including the acquisition of a secondary mutation in EGFR (T790M) (1), followed by amplification of the hepatocyte growth factor receptor (MET) gene (2) and mutations in BRAF and PIK3CA genes (3,4). Epithelial–mesenchymal transition (EMT) and lineage transformation are less frequent but also prevalent, with up to 15% of cases with acquired resistance to first- and second-generation EGFR TKIs demonstrating histologic change from lung adenocarcinoma (LUAD) to small-cell lung cancer (SCLC) (4). Histologic plasticity as a mechanism of resistance is becoming increasingly prominent as other resistant mechanisms can now be successfully targeted (5). Currently, as with de novo SCLC, conventional platinum doublet chemotherapy is the standard of care for patients with treatment-induced SCLC. Unfortunately, this treatment often produces an incomplete and nondurable response followed by inevitable relapse within months, leading to poor patient outcomes (6). Thus, this mechanism of resistance will represent a major barrier towards the success of third-generation TKIs, and new strategies to prevent this lineage shift or to treat SCLC transformed tumors are urgently needed. Despite the increasing clinical importance, the biologic pathways regulating LUAD to SCLC transformation are poorly understood. Assessment of clinical samples has revealed that EGFR-mutant tumors universally lose EGFR protein expression upon SCLC transformation, despite still harboring EGFR mutation (7). Furthermore, the mutation spectrum of these transformed cases includes inactivation of the tumor suppressors RB and p53 in nearly all cases, mirroring de novo SCLC (7). However, accumulating experimental evidence has demonstrated that while necessary, dual inactivation of RB and p53 is not sufficient to cause SCLC lineage transformation in EGFR-mutated LUAD, suggesting that additional factors are required (7). MYC amplification and PIK3CA mutation have been proposed to potentially cooperate with RB/p53 loss to facilitate transformation (8), and specific epigenetic regulators may also provide the appropriate context for lineage reprogramming to occur. Despite this, no in vitro or in vivo models of SCLC transformation in EGFR TKI resistance have been developed, making it difficult to comprehensively explore the molecular events driving this lineage shift. Interestingly, there are clear differences between LUAD and SCLC regarding EGFR expression and gene alterations in MAPK pathway including EGFR/KRAS mutations: EGFR is usually not expressed (9) and EGFR/KRAS mutations are extremely rare in SCLC (10); in contrast, EGFR/KRAS play crucial roles in LUAD biology, including regulating differentiation in addition to proliferation (11). To date, however, no clear explanation has been given for these differences. We have recently shown that activation of MAPK signaling in SCLC leads to suppression of the neuroendocrine phenotype—including downregulation of the transcription factors NEUROD1, INSM1, BRN2, and ASCL1—and transformation to an NSCLC-like state (12). Using this model system, we have begun to elucidate the key transcription factors and epigenetic changes that drive SCLC to NSCLC transformation in the hope that the same processes will also be involved in the clinically relevant scenario: SCLC transformation from EGFR mutant LUAD during TKI resistance. We suggest that only EGFR-mutant LUADs that do not reactivate MAPK signaling through secondary EGFR mutations or alterations in parallel kinase pathways (i.e., MET) during development of TKI resistance will be able to undergo SCLC lineage transformation, and that RB/p53 loss and epigenetic plasticity provide the permissive context in which this transformation can occur. Greater understanding of lineage transformation in LUAD will provide important insights in terms of managing outcomes of patients undergoing targeted therapy and offer new avenues towards treatment of TKI resistant tumors.

Abstract 119: The influence of CD44/HA signaling on CML survival and resistance to tyrosine kinase inhibitor treatment

Abstract The development of tyrosine kinase inhibitors (TKIs) such as imatinib has greatly improved the prognosis and survival of patients diagnosed with chronic myeloid leukemia (CML). However, many CML patients relapse due to the presence of minimal residual disease and the development of TKI resistance. It is unknown exactly what causes the relapse, but based on research from our laboratory we hypothesize that hyaluronic acid (HA) produced by fibroblasts in the microenvironment of the bone marrow provide for a protective environment preventing the tumor cells from therapy-induced cell death or senescence. The purpose of this study is to determine the influence that HA and its binding to its receptor (CD44) has on CML survival and resistance development. Initially, we examined the effect that stromal cell derived and/or exogenously added HA has on CML resistance to imatinib. Cell survival, apoptosis, HA production, and signaling events elicited by the interactions of HA with CD44 was examined using methods such as ELISA, western blotting, and MTT analysis. Furthermore, we examined the potential therapeutic use of compounds known to alter HA production and/or signaling for the treatment of CML. Our study demonstrates increased cell survival when the tumor cells are co-cultured with fibroblasts or have exogenous HA added to them after imatinib treatment. Additionally, we show that knocking out hyaluronic acid synthases reduces this protective effect. This data supports our hypothesis and we believe it could help lead to improved therapeutic treatment for CML. Note: This abstract was not presented at the meeting. Citation Format: Kylie Humphries, Olga Uchakina, Bryan Hostetler, Robert McKallip. The influence of CD44/HA signaling on CML survival and resistance to tyrosine kinase inhibitor treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 119.

Correction: EGFR kinase domain mutation positive lung cancers are sensitive to intrapleural perfusion with hyperthermic chemotherapy (IPHC) complete treatment.

[This corrects the article DOI: 10.18632/oncotarget.6491.].

PLIN2 confers gefitinib resistance by inhibiting cell apoptosis via activation of EGFR/AKT/survivin in PC9R cells

// Meijia Chang 1, 3, * , Jie Hu 1, 3, * , Tao Fang 2, * , Jing Li 1, 3 , Yuanlin Song 1, 3 , Chunxue Bai 1, 3, 4 and Jian Zhou 1, 3 1 Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, Shanghai, China 2 Department of Oncology, Shengli Oilfield Central Hospital, Dongying, China 3 Shanghai Respiratory Research Institute, Shanghai, China 4 State Key Laboratory of Respiratory Disease, Guangzhou Medical University, Guangzhou, China * These authors contributed equally to this work Correspondence to: Jian Zhou, email: zhou.jian@fudan.edu.cn Chunxue Bai, email: bai.chunxue@zs-hospital.sh.cn Keywords: non-small cell lung cancer; PLIN2; gefitinib; apoptosis; TKI resistance Received: November 01, 2017      Accepted: January 04, 2018      Published: January 10, 2018 ABSTRACT Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the first-line drugs for advanced non-small cell lung cancer (NSCLC) patients with activating EGFR mutations. However, the development of TKI resistance is a major clinical challenge. Here we show that the adipophilin/adipose differentiation-related protein perilipin (PLIN)2 was upregulated in gefitinib-resistant PC9R cells; PLIN2 knockdown in PC9R cells conferred gefitinib resistance by inhibiting cancer cell apoptosis in vitro and in vivo and by inducing mitochondrial dysfunction and caspase activation. PLIN2 was also shown to activate EGFR/AKT/survivin signalling and was overexpressed in gefitinib-resistant cells in clinical samples. These results indicate that PLIN2 confers gefitinib resistance in lung cancer by inhibiting apoptosis and activating the EGFR/AKT/survivin pathway, and is thus a potential therapeutic target in EGFR TKI-resistant NSCLC patients.

Abstract 4741: Targeting tumor growth in a medullary thyroid carcinoma mouse model using a combinatorial treatment with a tyrosine kinase inhibitor and histone deacetylase inhibitor

Abstract Neuroendocrine tumors are rare forms of cancers arising from hormone-producing cells that are scattered throughout the body. Medullary thyroid carcinoma (MTC) originates from the thyroid gland parafollicular cells. Treatment options for progressive, metastatic MTC patients are limited to the recently FDA-approved tyrosine kinase inhibitors (TKIs), Vandetanib and Cabozantinib, which target multiple receptor tyrosine kinases, including VEGFR2. However, the efficacy of TKIs is limited and development of TKI resistance is common. Here we examined, the effect of a combinatorial drug therapy using the TKI, Nintedanib, and the histone deacetylase (HDAC) inhibitor, Romidepsin, on MTC growth in the NSE/p25-gfp bi-transgenic mouse line, an inducible MTC mouse model (see Pozo et al. 2013). The TKI- and HDAC inhibitors were administered intraperitoneally for 3 weeks and tumor growth progression was monitored using a T2 weighted magnetic resonance imaging on a 7 Tesla system. Tumor tissues were analyzed for oncogenic signaling pathways by immunoblotting and immunohistochemistry. We find that co-administration of Nintedanib and Romidepsin stops tumor growth and increases the number of necrotic foci within the tumor tissue. Interestingly, Nintedanib treatment alone reduced tumor vascularization as shown by decreased density of the vascular marker, CD31, but did not stop parafollicular cell proliferation. Taken together, these results suggest that a combinatorial drug therapy with a TKI- and an HDAC inhibitor may be a more efficient strategy to target MTC progression and overcome recurrence. Citation Format: Karine Pozo, Stefan Zahler, Chunfeng Tan, Keisuke Ishimatsu, Masaya Takahashi, James Bibb. Targeting tumor growth in a medullary thyroid carcinoma mouse model using a combinatorial treatment with a tyrosine kinase inhibitor and histone deacetylase inhibitor. [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 4741.

EGFR kinase domain mutation positive lung cancers are sensitive to intrapleural perfusion with hyperthermic chemotherapy (IPHC) complete treatment.

Lung cancer is the global leading cause of cancer-related deaths. A significant portion of lung cancer patients harbor kinase domain mutations in the epidermal growth factor receptor (EGFR). While EGFR tyrosine kinase inhibitors (TKI) effectively shrink tumors harboring mutant EGFR, clinical efficacy is limited by the development of TKI resistance. Effective alternatives are desperately needed in clinic for treating EGFR kinase domain mutation positive lung cancer. In our clinic in treating M1a lung cancer patients through intrapleural perfusion with hyperthermic chemotherapy (IPHC) followed by cycles of systemic chemotherapy (we termed this procedure IPHC complete treatment, IPHC-CT), we found dramatic tumor shrinkage in mutant EGFR-positive patients. We further confirmed the sensitivity of EGFR mutation-positive lung cancer cell lines derived from patients to HC (hyperthermic chemotherapy) treatment. We found that hyperthermia promoted accumulation of cisplatin in lung cancer cells. Hyperthermia and cisplatin synergistically downregulated the EGFR protein level, leading to quenching of signal from EGFR and induction of apoptosis. Our work therefore showed IPHC-CT is an effective treatment for EGFR kinase domain mutation positive lung cancer patients.

Whole Exome Sequencing Reveals That DNMT3A and ASXL1 Mutation Are Involved in the Development of Tyrosine Kinase Inhibitor Resistance in Chronic Myeloid Leukemia Patients without ABL1 Tyrosine Kinase Domain Mutation

BACKGROUND:Tyrosine kinase inhibitor (TKI) resistance is the most relevant event during the treatment of chronic myeloid leukemia (CML), which correlates with high risk of treatment failure, disease progression and death, explaining half of treatment failed CML patients. However, the remaining half with TKI resistance does not show any ABL1 tyrosine kinase domain (TKD) mutation indicating the presence of alternative pathogenic pathways behind TKI resistance. Thus we hypothesized that the novel mutation besides ABL1-TKD mutation occurs during the development of TKI resistance. Using whole exome sequencing, we screened 13 pairs of CML cases with TKI resistance, but without ABL1-TKD mutation. The present study attempts to: 1) explore novel mutation(s) developing TKI resistance to CML treatment and 2) validate the somatic variants in an independent cohort of CML patients (n=100).METHODS:Thirteen CML cases with TKI resistance but not having ABL1 TKD mutation were included prospectively. Reason for TKI resistance includes Loss of MCyR (n=7) with (n=2) or without additional cytogenetic abnormality (ACA; n=5), progression to blastic crisis (n=3), development of ACA (n=1), development of clonal evolution in Ph neg clone (n=1), primary cytogenetic resistance (n=1). TKI resistance were demonstrated to imatinib (n=12), dasatinib (n=5), nilotinib (n=4) or ponatinib (n=2). The latest treatment includes ponatinib (n=3), dasatinib (n=8) alone (n=4), with smoothen inhibitor (n=2), or with after systemic chemotherapy (n=2), omacetaxine (n=1), and nilotinib (n=1). Disease stage at the time of exome sequencing was chronic phase (n=10) or blastic crisis (n=3). Germline and tumor samples at the time of TKI resistance were compared using whole exome sequencing (Illumina TruSeq kit, HiSeq 2000). Targeted sequencing for selected variants was performed to validate the result. All patients were confirmed the absence of ABL1-TKD mutations using Sanger sequencing.RESULTS:1) Exome sequencing (Illumina Truseq kit) was performed as per the manufacturer's protocol using an Illumina HiSeq 2000 sequencer. DNA from buccal mucosa was used as a control for variant calling. Exome sequencing reads processing includes mapping to human genome hg19, marking PCR duplicates, realignment of indels, fixing mate information, and discard the reads with more than 2 mismatches to increase the true positive rate. In the end, we have on-target-coverage of 57x. Lastly, 72% of target positions are mapped more than 30x.2) One hundred nineteen somatic variants were identified in 13 patients in 108 genes. Among them 5 genes have variants in multiple patients including DNMT3A (n=3), ASXL1 (n=2), NPIPB5 (n=2), ATXN3 (n=2) and EFEMP1 (n=2) . We also found at least 1 mutation in well-known driver genes in 6 patients (6/13 = 46%).3) Three out of 4 patients with ACA carry variants at least one of DNMT3A (n=2), ASXL1 (n=2), and SETBP1 (n=1). Also, 2 out of 3 cases progressed to blastic crisis demonstrate variants in DNMT3A (n=1) and IDH1 (n=1).4) Interestingly, in one patient, exome sequencing reveals ABL1-TKD mutation (T315I), which was not detected at the initial screening by Sanger sequencing.5) The result of targeted sequencing in an independent cohort of CML patients (n=100) will be presented in the annual meeting of American Society of Hematology in Dec 2015.CONCLUSION:Our study suggest that DNMT3A and ASXL1 mutations seem to be the driver mutations involved in the development of TKI resistance/progression, independent of ABL1-TKD mutation. Also, exome sequencing can detect ABL1-TKD mutations including T315I prior to be detected by initial Sanger sequencing. DisclosuresLipton:Ariad: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding. Kim:Bristol-Myers Squibb: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding.

Abstract 337: Mechanism of TKI resistance and role of epithelial mesenchymal transition in NSCLC

Abstract Current EGFR/c-Met tyrosine kinase inhibitor (TKI) combination therapies are shown to be susceptible to acquired resistance in the majority of NSCLC patients. To understand how cells develop EGFR/c-Met TKI resistance, we used three model NSCLC cell lines: H2170, H3255 and H1975. H2170 cell line express high levels of EGFR and c-Met and are EGFR wild-type. The H3255 cell line is positive for an EGFR TKD mutation, L858R, and is sensitive to initial TKI treatment. The H1975 cell line is positive for two EGFR TKD mutations, T790M and L858R, which confer resistance to erlotinib. Previous studies in our lab have shown that activation of alternative signaling pathways lead to the development of TKI resistance. In the wild type TKI resistant cells, H2170 erlotinib resistant (ER) and H2170 SU11274 resistant(SR), we observed activation of Wnt and mTOR pathway along with increased accumulation of β-catenin in the nucleus. However, in H1975 cells with mutated EGFR (L858R and T790M), we observed activation of only mTOR pathway. β-catenin is associated with Epithelial-Mesenchymal Transition (EMT), during which cells lose tight junction proteins such E-Cadherin and gain expression of transcriptional proteins such as β-catenin and ZEB1, which may lead to aberrant activation of Wnt signaling pathway and increased motility and invasion. Activation of mTOR pathway leads to increased expression of p70S6K and 4E-BP1 which are key translational regulators. We found increased accumulation of active β-catenin in the nucleus by 1.9-2.5 folds and 2.9-3.1 folds in H2170 ER and SR cells, respectively, compared to H2170 parental cells. In H2170 ER cells we observed modulations in key EMT-related proteins, N-Cadherin (2-3.3 fold) and Claudin-1 (2.5-14 fold) were upregulated and Snail was downregulated 1.7-2.5 fold in the presence and absence of erlotinib and EGF, when compared to H2170 parental cells. Similarly in H2170 SR cells we observed upregulation of N-Cadherin (1.3-2.3 fold) and Zeb-1 (1.2-1.4 fold) and downregulation of ZO-1 (1.4-1.9 fold) and E-Cadherin (1.5-2.0 fold) in the presence and absence of HGF and SU11274 when compared to H2170 parental cells. Morphological changes indicative of EMT were detected using immunostaining with Vimentin and E-Cadherin antibodies, which displayed increased Vimentin filaments (2-fold) and loss of E-Cadherin (5-10 fold) in H2170 ER cells when compared to H2170 parental cells. In H1975 cells we observed that proteins related to mTOR signaling pathway such as p-mTOR, p-GSK3-β, p-4EBP1 and p-p70S6K were upregulated 2.5 fold, 4-5 fold, 2.2-2.4 fold, and 4.4-10.4 fold, respectively, when compared to H3255 cells in the presence of erlotinib. Based on these results, we treated H1975 cells with combination of EGFR and mTOR inhibitors. We observed that 1 μM everolimus with 2.5 μM erlotinib synergistically inhibited cell growth by 53%. These studies may provide clinicians with novel targets for improving treatment options for future NSCLC patients. Citation Format: Ichwaku Rastogi, Gregory M. Botting, Andrew Webb, Brian L. Webb, Marie C. Nlend, Neelu Puri. Mechanism of TKI resistance and role of epithelial mesenchymal transition in NSCLC. [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 337. doi:10.1158/1538-7445.AM2015-337

Efficacy of chemotherapy plus gefitinib treatment in advanced non-small-cell lung cancer patients following acquired resistance to gefitinib

Non-small-cell lung cancer (NSCLC) may exhibit oncogene addiction in patients who benefited from prior treatment with epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs). Preclinical data suggested that EGFR addiction persists after the development of TKI resistance, leading many clinicians to continue TKI treatment along with chemotherapy. However, this strategy has not been adequately evaluated in clinical practice. Patients who benefited from gefitinib followed by acquired resistance to this drug were reviewed in the Zhejiang Cancer Hospital. Patients were included if they received chemotherapy and gefitinib following failure of prior gefitinib treatment. A total of 26 patients were included in the present study. Six patients (23.1%) exhibited a partial response (PR), 13 (50%) achieved stable disease (SD) and 7 (26.9%) had progressive disease (PD) during the chemotherapy and gefitinib treatment. The disease control rate (DCR) was 73.1% and the median progression-free survival (PFS) was 4.6 months [95% confidence interval (CI): 3.8–5.4]. The toxicities associated with gefitinib and chemotherapy were generally acceptable. In conclusion, continued concurrent gefitinib and chemotherapy may be a valuable strategy, with acceptable and well-tolerated toxicity. However, this treatment requires further investigation.

Effective Elimination of CML Progenitor and Stem Cells Through Combination of α-CD123 Antibody-Dependent Cell-Mediated Cytotoxicity and Tyrosine Kinase Inhibitor Treatment

AbstractAbstract 32Since the introduction of tyrosine kinase inhibitor (TKI) therapy overall survival and complete molecular response rates in chronic phase chronic myeloid leukemia (CP-CML) patients have significantly improved. However, leukemic stem cells (LSCs) and progenitor cells persist and are thought to be responsible for disease progression, development of TKI resistance and disease recurrence after stopping TKI therapy. Protection by cytokines, such as IL-3 and GM-CSF, provides a potential mechanism of LSC resistance. While in acute myeloid leukemia (AML) monoclonal antibody (mAb) targeting of IL-3 receptor α (CD123), a recognized marker for AML LSCs, has been studied in vitro and in vivo, similar investigations have not been undertaken in CML to date. CSL362 is a genetically-engineered form of the specific blocking mAb 7G3 optimized for Fc receptor binding to achieve maximal antibody-dependent cell-mediated cytotoxicity (ADCC) capacity. Here we investigate the expression of CD123 in CD34+ progenitors and CD34+CD38− LSCs, isolated from CP- and blast crisis (BC) - CML patients, and study the benefits of targeting those cells by CSL362 alone and in combination with TKIs.Flow cytometry analysis established significantly elevated expression of CD123 on CD34+CD38− cells from CP-CML (53.0 ± 5.8 %, n=16, p=0.003) and BC-CML (73.2 ± 6.7 %, n=18, p<0.001) patients compared to normal donors (20.3 ± 4.2 %, n=8), with clear increases in CD123 expression with disease progression in matched samples (n=2). Subsequent assessment of apoptosis, colony forming unit (CFU-GM) and long-term culture-initiating cell (LTC-IC) potential confirmed the ability of CSL362 to block IL-3-mediated rescue of TKI-induced cell death. However, in the presence of other cytokines, likely found in the physiological bone marrow microenvironment, this effect was lost.We also demonstrate by lactate dehyrogenase release and clonogenic assays that CML CD34+ cell numbers are significantly reduced, in a dose-dependent manner, by CSL362-induced ADCC employing NK cells from healthy donors (42.4 ± 8.1 % lysis, n=3, and CFU-GM decreased to 30 ± 10.8 % of controls, n=5, p=0.003). In keeping with this, flow cytometry analysis revealed specific elimination of CP- and BC-CML CD123+ CD34+CD38− cells (from 42.9 % to 18.6 %, n=5, p=0.004, and from 71 % to 35.3 %, n=3, p=0.044, respectively). Importantly, autologous CML patient NK cells, collected after achievement of major molecular response, also mediate CSL362-dependent cytotoxicity similar to allogeneic healthy donor NK cells as indicated by equivalent numbers of remaining CFUs (28 ± 6.7 % vs. 34.9 ± 3.4 %, n=5, Fig. A). We further have evidence to suggest preferential elimination of CML over normal LTC-ICs (30.3 ± 9.9 % vs. 62.6 ± 11.2 % remaining, n=3, p=0.096) in the autologous setting. Of clinical importance, the combination of Nilotinib and CSL362 resulted in a significantly greater reduction in CFUs (additive effect) when compared to either agent alone (Fig. B).Taken together these data suggest that selective ADCC-mediated lysis, likely the major mode of action of CSL362 in vivo, efficiently eliminates CML progenitor and stem cells. Promising results evaluating CSL362/TKI combination treatments, with the expectation to further enhance specificity for leukemic while sparing normal progenitor and stem cells as indicated from preliminary experiments, warrant further studies. [Display omitted] Disclosures:Nievergall:CSL Ltd: Research Funding. White:BMS: Research Funding; CSL Ltd: Research Funding; Novartis Oncology: Honoraria, Research Funding. Ramshaw:CSL Ltd: Research Funding. Busfield:CSL Ltd: Employment. Vairo:CSL Ltd: Employment. Lopez:CSL Ltd: Research Funding. 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 Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Ltd: Research Funding. Hiwase:CSL Ltd: Research Funding.

Chemotherapy with erlotinib or chemotherapy alone in advanced NSCLC with acquired resistance to EGFR tyrosine kinase inhibitors (TKI).

7524 Background: EGFR-mutant NSCLC has an oncogene-addicted phenotype that confers sensitivity to EGFR TKIs. Prior data suggests EGFR addiction persists after development of TKI resistance, leading many clinicians to continue TKI along with chemotherapy (chemo); however, this strategy has not been formally evaluated. Methods: An institutional database was reviewed to identify patients (pts) with advanced NSCLC and acquired resistance to EGFR TKIs by Jackman criteria. Pts were included if they subsequently received chemo. Objective response rate (RR) to chemo/erlotinib or chemo alone was assessed by blinded radiographic review and compared by Fisher’s exact test and multivariable logistic regression. Progression-free survival (PFS) and overall survival (OS) from TKI failure (defined as chemo initiation date) were compared by log-rank test and multivariable Cox analysis. Results: 78 pts were eligible (34 chemo/erlotinib, 44 chemo alone). 70 pts (90%) had a documented EGFR mutation and were on TKI for a median of 15 months (range 4-51); in the 8 pts with unknown genotype the median duration on TKI was 11 months (range 5-16). Baseline characteristics were well balanced except more pts received erlotinib as initial TKI in the chemo/erlotinib group. RR was evaluable in 57 pts and was higher with chemo/erlotinib compared to chemo alone (41% vs 18%; OR 0.31, 95% CI 0.09, 1.04; p=0.08). RR adjusted for chemo regimen and time to TKI failure yielded OR 0.20 (95% CI 0.05, 0.78; p=0.02), favoring chemo/erlotinib. Median PFS was 4.4 months in the chemo/erlotinib group and 4.2 months in the chemo alone group (crude HR=0.84, 95% CI 0.52, 1.38; p=0.50; adjusted HR 0.79, 95% CI 0.48, 1.29; p=0.34). There was no significant difference in OS in the crude or adjusted analyses. Conclusions: This is the first study, to our knowledge, to show that continuation of an EGFR TKI with chemo compared to chemo alone significantly increases the RR in pts with advanced NSCLC and acquired TKI resistance, though we did not observe an impact on PFS or OS. Continued concurrent TKI may be a valuable strategy, particularly for pts with symptomatic progression, when a higher response rate may be beneficial. Further study is warranted.