Dasatinib-resistant Cells Research Articles

Abstract 5820: Chloroquine inhibited Rab11 mediated E-cadherin transport in collective colon cell migration in Dasatinib resistant cells

Abstract In has been demonstrated epithelial mesenchymal transition (EMT) is related to E-cadherin expression level. Recently, studies have been demonstrated the collective cancer cell migration which expresses high E-cadherin is a powerful mechanism of metastasis. Therefore, the role of E-cadherin in cancer metastasis is still not clear. Endocytosis was shown to regulate E-cadherin turnover on the membrane and the function of lysosome is also associated with cell vesicle trafficking. The past studies indicated Src can regulate vesicle trafficking. In this study, HT29 colon cancer cells were treated with Src inhibitor, Dasatinib (DST), to investigate the role of lysosome in regulating E-cadherin dynamics through vesicle trafficking in collective cell migration. In the result, the recycling protein Rab11 and lysosome marker Lamp1 were found localized with E-cadherin in the cell-cell contacts, however, Dasatinib sensitive (DSTS) cells showed less colocalization of Rab11 with E-cadherin. When cells were treated with lysosome inhibitor chloroquine, Rab11 were accumulated in the cell-cell contacts and reduced collective cell migration in Dasatinib resistant (DSTR) cells. These results suggest lysosome inhibition can suppress Rab11 mediated vesicle trafficking of E-cadherin transport for collective cell migration, and chloroquine has therapeutic potential in colon cancer treatment. Citation Format: Xiang-Ling Hou, Hui Min Koo, Chia-Wen Lin, Chien-Chi Huang, Yu-Chi Tseng, Kai-Yu Tseng, Wei-Ting Chao. Chloroquine inhibited Rab11 mediated E-cadherin transport in collective colon cell migration in Dasatinib resistant cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5820.

Activation of ERK1/2 by MOS and TPL2 leads to dasatinib resistance in chronic myeloid leukaemia cells.

The development of BCR::ABL1 tyrosine kinase inhibitors (TKIs), such as dasatinib, has dramatically improved survival in cases of chronic myeloid leukaemia (CML). However, the development of resistance to BCR::ABL1 TKIs is a clinical problem. BCR::ABL1 TKI resistance is known to have BCR::ABL1-dependent or BCR::ABL1-independent mechanisms, but the mechanism of BCR::ABL1 independence is not well understood. In the present study, we investigated the mechanism of BCR::ABL1-independent dasatinib resistance. The expression and activation level of genes or proteins were evaluated using array CGH, real time PCR, or western blot analysis. Gene expression was modulated using siRNA-mediated knockdown. Cell survival was assessed by using trypan blue dye method. We found that dasatinib-resistant K562/DR and KU812/DR cells did not harbour a BCR::ABL1 mutation but had elevated expression and/or activation of MOS, TPL2 and ERK1/2. In addition, MOS siRNA, TPL2 siRNA and trametinib resensitized dasatinib-resistant cells to dasatinib. Moreover, expression levels of MOS in dasatinib non-responder patients with CML were higher than those in dasatinib responders, and the expression of TPL2 tended to increase in dasatinib non-responder patients compared with that in responder patients. Our results indicate that activation of ERK1/2 by elevated MOS and TPL2 expression is involved in dasatinib resistance, and inhibition of these proteins overcomes dasatinib resistance. Therefore, MOS, TPL2 and ERK1/2 inhibitors may be therapeutically useful for treating BCR::ABL1-independent dasatinib-resistant CML.

MEK inhibition overcomes resistance to EphA2-targeted therapy in uterine cancer

BackgroundOur pilot clinical study of EphA2 inhibitor (dasatinib) plus paclitaxel and carboplatin showed interesting clinical activity in endometrial cancer with manageable toxicity. However, the underlying mechanisms of dasatinib resistance in uterine cancer are unknown. Here, we investigated potential mechanisms underlying resistance to EphA2 inhibitors in uterine cancer and examined the anti-tumor activity of EphA2 inhibitors alone and in combination with a MEK inhibitor. MethodsWe evaluated the antitumor activity of EphA2 inhibitors plus a MEK inhibitor using in vitro and in vivo orthotopic models of uterine cancer. ResultsEphA2 inhibitor induced MAPK in dasatinib-resistant uterine cancer cells (HEC-1A and Ishikawa) and BRAF/CRAF heterodimerization in HEC-1A cells. EphA2 inhibitor and trametinib significantly increased apoptosis in cancer cells resistant to EphA2 inhibitors compared with controls (p < 0.01). An in vivo study with the orthotopic HEC-1A model showed significantly greater antitumor response to combination treatment compared with dasatinib alone (p < 0.01). Combination treatment increased EphrinA1 and BIM along with decreased pMAPK, Jagged 1, and c-MYC expression in dasatinib-resistant cells. In addition, Spearman analysis using the TCGA data revealed that upregulation of EphA2 was significantly correlated with JAG1, MYC, NOTCH1, NOTCH3 and HES1 expression (p < 0.001, r = 0.25–0.43). Specifically, MAP3K15 and the NOTCH family genes were significantly related to poor clinical outcome in patients with uterine cancer. ConclusionsThese findings indicate that the MAPK pathway is activated in dasatinib-resistant uterine cancer cells and that EphrinA1-mediated MEK inhibition overcomes dasatinib resistance. Dual targeting of both EphA2 and MEK, combined with chemotherapy, should be considered for future clinical development.

Abstract 2085: Treatment of human melanoma cells with dasatinib requires inactivation of both mTOR and MAPK pathways to achieve high cell eradication efficacy

Abstract Advanced stages of melanoma are resilient to therapy and new strategies for the treatment are required. About 60% of melanomas harbor the mutated BRAF oncogene, which activate the MAPK pathway. This BRAF driver mutation can be targeted by a small molecule drug vemurafenib. However, most of patients develop resistance during treatment and even drug addiction is acquired. The nonreceptor SRC tyrosine kinase is highly expressed and deregulated in melanoma and can be efficiently inhibited by the inhibitor dasatinib, an anticancer drug which is already used for the treatment of some tumor types. SRC does not have its own canonical pathway but is capable of activating and augmenting different signaling routes in cancer cells. We found that melanoma tumor cell lines analysed were generally resistant to dasatinib irrespective of BRAF or NRAS mutational status. The exception was the cell line Hbl which was very sensitive even to low dasatinib doses and Hbl cells were completely eradicated by 10 nM of dasatinib in several days. The sensitivity of Hbl to dasatinib was dependent on the drug ability to inhibit completely both the MAPK and AKT/mTOR signaling pathways. Dasatinib was capable of inhibiting SRC activation resulting in the repression of AKT activation in Hbl cells. AKT phosphorylation at Ser473 was fully abolished and Thr308 was severely attenuated through inhibiting SRC by dasatinib in Hbl cells. These two phosphorylations are required for AKT activity. MAPK pathway (ERK1/2 phosphorylation) was also inhibited in Hbl cells. Dasatinib also inhibited the phosphorylation of SRC at Tyr416 (a hallmark of SRC activity) in Hbl cells and, surprisingly, also in other dasatinib-resistant melanoma cells. Importantly, although SRC was dephosphorylated at Tyr416, dasatinib was unable to inactivate AKT and MAPK pathway (ERK1/2) in all other melanoma cells. Molecular changes closely correlated with proliferation assays. Our data indicate that the combination therapy of melanoma with dasatinib would bring benefit to patients only by blocking both MAPK and AKT signaling pathways in melanoma cells. Altogether, more molecular knowledge of the effects of the powerful anticancer drug dasatinib is required in order to be used for the treatment of melanoma. This work was supported by the Institutional research project PROGRESQ25 from Charles University Prague. Citation Format: Jiri Vachtenheim, Jiri Réda, Kateřina Vlčková, Lubica Ondrušová. Treatment of human melanoma cells with dasatinib requires inactivation of both mTOR and MAPK pathways to achieve high cell eradication efficacy [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 2085.

Abstract 1820: Overcoming drug resistance in NSCLC with SRC inhibitor

Abstract Lung cancer accounts for about 27 percent of all cancer deaths in the United States. Approximately 85 percent of lung cancers are non-small cell type (NSCLC) and, when treated with both chemotherapy and targeted therapies, have a five-year overall survival rate of approximately 16 percent. These statistics clearly emphasize the need for developing treatment methodologies more effective than the current standard of care. Here, we evaluated the efficacy of combined inhibition of SRC and AXL in NSCLC.Dasatinib, a BCR/abl and src family tyrosine kinase inhibitor (TKI) is a drug that has a potential to be used against both EGFR and KRAS mutated lung cancer patients, alone (phase II clinical trial) and in combination with cetuximab (in phase III clinical trial), osimertinib and erlotinib (both in phase II clinical trials) by exploiting its SRC inhibition capabilities. However, NSCLC patients treated with dasatinib often develop resistance and is the major reason for the low median survival in stage IV patients. Recently, AXL pathway has been shown to be upregulated in Dasatinib resistant cells causing activation of EMT, AKT and survival pathways. It has been suggested that auto-phosphorylation of DDR2 and subsequent activation of SHP2/RAS/AKT pathway as a result of AXL upregulation is one of the major reasons for failure to induce apoptosis in cells during dasatinib resistance. Therefore, we examined whether inhibition of AXL resensitized TKI resistant NSCLC to dasatinib. Our results show that AXL knockdown can resensitize cells to dasatinib. IC50 values decreased significantly following AXL inhibition. We have also demonstrated that nanoparticle-mediated AXL knockdown also produces similar resensitization. The details of the study will be presented. Citation Format: Soumavo Mukherjee, Dhananjay Suresh, Ajit P. Zambre, Anandhi Upendran, Raghuraman Kannan. Overcoming drug resistance in NSCLC with SRC inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1820.

Sustained activation of the AKT/mTOR and MAP kinase pathways mediate resistance to the Src inhibitor, dasatinib, in thyroid cancer.

New targeted therapies are needed for advanced thyroid cancer. Our lab has shown that Src is a key mediator of tumorigenic processes in thyroid cancer. However, single-agent Src inhibitors have had limited efficacy in solid tumors. In order to more effectively target Src in the clinic, our lab has previously generated four thyroid cancer cell lines that are resistant to dasatinib through gradual dose escalation. We further tested two additional Src inhibitors and shown the dasatinib-resistant (DasRes) cells exhibit cross-resistance to saracatinib, but are sensitive to bosutinib, suggesting that unique off-targets of bosutinib play an important role in mediating sensitivity to bosutinib. To identify the kinases targeted by dasatinib and bosutinib, we utilized an unbiased compound centric chemical proteomics screen. We identified 33 kinases that were enriched in the bosutinib pull down. Using the STRING database to map protein-protein interactions of the unique bosutinib targets, we identified a signaling axis which included mTOR, FAK, and MEK. Inhibition of the mTOR, MEK, and Src/FAK nodes simultaneously was the most effective at reducing cell growth and survival. Overall, these studies have identified key mediators of Src inhibitor resistance, and show that targeting these signaling nodes are necessary for anti-tumor efficacy.

Quantitative phosphoproteomic analysis of acquired cancer drug resistance to pazopanib and dasatinib

Acquired drug resistance impacts the majority of patients being treated with tyrosine kinase inhibitors (TKIs) and remains a key challenge in modern anti-cancer therapy. The lack of clinically effective therapies to overcome resistance represents an unmet need. Understanding the signalling that drives drug resistance will facilitate the development of new salvage therapies to treat patients with secondary TKI resistance. In this study, we utilise mass spectrometry to characterise the global phosphoproteomic alterations that accompany the acquisition of resistance to two FDA-approved TKIs, pazopanib and dasatinib, in the A204 rhabdoid tumour cell line. Our analysis finds that only 6% and 9.7% of the quantified phosphoproteome is altered upon the acquisition of pazopanib and dasatinib resistance, respectively. Pazopanib resistant cells display elevated phosphorylation in cytoskeletal regulatory pathways while dasatinib resistant cells show an upregulation of the insulin receptor/IGF-1R signalling pathway. Drug response profiling rediscovers several previously reported vulnerabilities associated with pazopanib and dasatinib resistance and identifies a new dependency to the second generation HSP90 inhibitor NVP-AUY-922. This study provides a useful resource detailing the candidate signalling determinants of acquired TKI resistance; and reveals a therapeutic approach of inhibiting HSP90 function as a means of salvage therapy to overcome pazopanib and dasatinib resistance. SignificancePazopanib and dasatinib are tyrosine kinase inhibitors (TKIs) approved for the treatment of multiple cancer types. Patients who are treated with these drugs are prone to the development of drug resistance and consequently tumour relapse. Here we use quantitative phosphoproteomics to characterise the signalling pathways which are enriched in cells that have acquired resistance to these two drugs. Furthermore, targeted drug screens were used to identify salvage therapies capable of overcoming pazopanib and dasatinib resistance. This data advances our understanding of the mechanisms of TKI resistance and highlights candidate targets for cancer therapy.

In Vitro Modeling of Ph-like ALL Fusions Identifies Novel Kinase-Domain Mutations As Mode of TKI-Resistance - Implications for Targeted Therapy

IntroductionTreatment-resistant acute lymphoblastic leukemia (ALL) remains a significant clinical issue. Recently, genomic profiling has identified a new subtype of high-risk ALL termed Philadelphia-chromosome-like (Ph-like) ALL, associated with a poor outcome1. Ph-like ALL has a gene expression profile similar to Ph+ (BCR-ABL1+) ALL, characterized by the presence of fusion genes converging on kinase and cytokine signaling pathways. These pathways have been shown to be targetable both in vitro and in case reports by tyrosine kinase inhibitors (TKIs). Despite well-documented efficacy profiles, it is known from TKI-use in chronic myeloid leukemia (CML) andPh+ ALL that resistance is likely, resulting in relapse. Our study aims to model and understand mechanisms of TKI-resistance inPh-like ALL, informing future therapeutic strategies that may avert or overcome resistance, potentially improving patient outcomes.MethodsThree Ph-like ALL lines were generated via retroviral-transduction from plasmids of fusion genes identified in patient cohorts (RANBP2-ABL1, SSBP2-CSF1R and PAX5-JAK2, a kind gift from C. Mullighan)2 into Ba/F3 pro-B cells. Transformation was confirmed via growth of cells in the absence of IL-3. Cells were tested for sensitivity to a panel of TKIs (imatinib, dasatinib, ponatinib, ruxolitinib and BMS-911543) via Annexin-V/7-AAD flow-cytometry and western blotting of downstream effector proteins. Drug resistance was generated through exposure of cells to incrementally increasing concentrations of TKIs over a period of 3-6 months, and cell death LD50 determined byAnnexin-V/7-AAD. Sanger sequencing of the 3-prime partner gene of each fusion was performed to identify the emergence of any kinase-domain mutations.ResultsBa/F3 Ph-like cells demonstrated sensitivity to TKIs at clinically relevant doses (RANBP2-ABL1: 1 μM imatinib, 5 nM dasatinib & 5 nM ponatinib; SSBP2-CSF1R: 1 μM imatinib, 6 nM dasatinib; PAX5-JAK2: 1 μM ruxolitinib & 2 μM BMS-911543). This correlated with decreased levels of relevant downstream signaling proteins including p-Stat5, p-Erk and p-CrkL. TKI-resistant Ph-like ALL lines were tolerant to a significantly higher concentration of TKIs compared to control (RANBP2-ABL1: 10 μM imatinib, 200 nM dasatinib & 200 nM ponatinib; SSBP2-CSF1R: 10 μM imatinib, 200 nM dasatinib; PAX5-JAK2: 10μMruxolitinib & 10μM BMS-911543; Table 1).Sequencing analysis revealed that Ba/F3 RANBP2-ABL1 imatinib and dasatinib resistant cells acquired the clinically significant ABL1 T315I (c.944C>T) kinase-domain mutation, which was ultimately targetable using the third-generation TKI ponatinib (LD50: 25 nM). An ABL1 E255K (c.763G>A) and c-terminus deletion was discovered in the ponatinib-resistant line. In Ba/F3 SSBP2-CSF1R cells, a novel CSF1R L785M (c.2566C>A) mutation was identified in imatinib-resistant cells whereas a deletion spanning the SSBP2-CSF1R breakpoint was acquired in the dasatinib-resistant line. A JAK2 Y931C (c.3286A>G) point mutation previously associated with resistance to ATP-competitive inhibitors was acquired in Ba/F3 PAX5-JAK2ruxolitinib and BMS-911543 resistant lines.ConclusionIn vitro modeling of Ph-like ALL resistance has identified novel kinase domain mutations and deletions that may arise as a result of targeted TKI therapy. In addition, previously identified mutations (T315I and E255K) were also identified. Detection of these mutations is important because alterations in drug-binding regions are known to result in significantly reduced TKI sensitivity, leading clinically to relapse3. This study describes an in vitro platform that can be utilized to inform future clinical approachesincluding the development of rational therapeutic approaches (and/or combination therapies) to avert resistance inPh-like ALL cases treated with rationally targeted therapies.Abbreviations:ABL1 - Abelson tyrosine protein kinase 1CSF1R - Colony stimulating factor 1 receptorJAK2 - Janus kinase 2PAX5 - Paired box 5RANBP2 - RAN-binding protein 2SSBP2 - Single-stranded DNA binding protein 2

Abstract 4802: PB1, a DDR2 inhibitor with antitumor activity in preclinical models of squamous cell carcinoma and KRAS-mutated adenocarcinoma of the lung

Abstract Introduction: Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase (RTK) activated by collagen I and identified as an oncogenic driver in squamous cell carcinoma of the lung (SCC) (1). DDR2 mutations have been reported in 4% of SCC patients and at lower frequencies in lung adenocarcinoma, gastric, breast and brain tumors (2). In addition, DDR2 is expressed during epithelial to mesenchymal transition (EMT) (3) and acts as an upstream activator of SHP2. SHP2 in turn is a key signaling effector which leads to activation of multiple signaling pathways (4). DDR2 has recently emerged as a potential therapeutic target in DDR2-mutated tumors and drugs such as dasatinib are being tested in this setting. Results and discussion: PB1 is a small organic molecule with a simple synthesis pathway that showed strong inhibitory activity against wild type (wt) and mutated purified DDR2 protein. The compound was subsequently tested in a panel of tumor cell lines from different origins and histologies. PB1 showed good antiproliferative activity: IC50&amp;lt;1 μM in (i) SCC cell lines with DDR mutations such as H2286 or H520, (ii) SCC lung tumor cell lines wt for DDR2, and (iii) some lung adenocarcinoma cell lines with KRAS mutations such as A549. Remarkably, the two cell lines tested carrying the DDR2 activating mutation I638F were extremely sensitive to PB1, with IC50 in the nanomolar range. The compound also showed good activity against cells harboring the DDR2 L239R mutation. We also generated two dasatinib-resistant cells lines from the H2286 and H520 cell lines. Cells had IC50s for dasatinib over 25 uM but remained sensitive to PB1, with IC50s of 0.6 and 5.92, respectively. Finally, in non tumor cell lines, no toxicity was observed with PB1 at concentrations tested (up to 50 μM), indicating a good therapeutic index. Western blotting was used to study the effects of PB1 on DDR2, ERK and AKT activation in H2286 and A549 cells. The compound strongly inhibited FBS-induced phosphorylation of the three proteins. Conclusion: PB1 shows significant antitumor activity in preclinical models of mutated and wt DDR2 in SCC of the lung and some KRAS mutated adenocarcinoma cell lines. PB1 is also active against tumor cells with secondary resistance to dasatinib.

Abstract P5-05-08: Sensitivity to c-Met inhibition is increased in dasatinib resistant TNBC cells

Abstract Pre-clinical models TNBC cells have demonstrated sensitivity to the multi-targeted Src kinase inhibitor dasatinib, however clinical trials with single agent dasatinib showed limited efficacy in unselected populations. Trials of dasatinib in combination with chemotherapy are ongoing. In order to study potential mechanisms of resistance to dasatinib in TNBC we established a cell line model of acquired dasatinib resistance (231-DasB). The dasatinib resistant cell line (231-DasB) was developed by constant exposure to incrementally increasing concentrations of dasatinib, from 200 nM to 500 nM over a period of 13 weeks.Cell proliferation was measured by acid phosphatase assay after 5 day treatment with SRC inhibitors (dasatinib, PD180970), EGFR inhibitors (gefitinib and neratinib), chemotherapy drugs (carboplatin, docetaxel and doxorubicin) and a c-Met inhibitor (CpdA, Amgen). P values were calculated using the Student’s T-Test (2 tailed with unequal variance). Expression and phosphorylation of c-Met and Src was measured by immunoblotting and multiplex magnetic bead assays carried out on a MAGPIX® Instrument. Following approximately 3 months exposure to dasatinib, 231-DasB cells were resistant to dasatinib with IC50 &amp;gt; 5μM compared to 0.04 ± 0.001 µM in MDA-MB-231. 231-DasB cells also showed resistance (2.2-fold) to the Src kinase inhibitor PD180970 [Table 1]. 231-DasB cells showed small but statistically significantly increases in sensitivity to docetaxel and doxorubicin. Table 1: Sensitivity to chemotherapy and targeted therapies in the MDA-MB-231 and 231-DasB cells.DrugMDA-MB-231231DasBp valuePD180970 IC50 (µM)0.40 ± 0.040.87 ± 0.070.003CpdA IC50 (µM)&amp;gt;102.1 ± 0.10.0001Docetaxel IC50 (nM)1.9 ± 0.11.2 ± 0.10.003Doxorubicin IC50 (nM)138.6 ±1.097.2 ±6.60.007Carboplatin IC50 (µM)13.2 ± 0.811.5 ± 1.30.147Gefitinib IC50 (µM)23.1 ± 2.220.9 ± 2.10.289Neratinib (% growth @ 10µM)94.5 ± 2.095.9 ± 7.60.834 No significant change in sensitivity to carboplatin or to the EGFR inhibitors gefitinib and neratinib was observed. However, the 231-DasB cells demonstrated a significant increase in sensitivity to the c-Met inhibitor, CpDA, with an IC50 value of 2.1 ± 0.1 µM compared to an IC50 greater than 10 µM in the parental MDA-MB-231 cells. Treatment of MDA-MB-231 cells with dasatinib (100 nM) blocked phosphorylation of Src kinase. In contrast, dasatinib treatment (100 nM) did not decrease p-Src levels in the 231-DasB cells. p-Met levels were significantly increased in 231-DasB cells relative to MDA-MB-231. Treatment with 2 µM CpdA decreased p-Met and p-Src in both 231-DasB and MDA-MB-231 cells. Other key receptor tyrosine kinases (EGFR, HER2, HER3, HER4, IGFIR and IR) show no significant changes in phosphorylation in 231-DasB cells compared to MDA-MB-231. Constitutive activation of p-Src through increased c-Met signalling may be a potential mechanism of resistance and suggests that combined treatment with dasatinib and a c-Met inhibitor may block the development of acquired resistance. Citation Format: Patricia Gaule, Brendan Corkery, John Crown, Micheal J Duffy, Norma O' Donovan. Sensitivity to c-Met inhibition is increased in dasatinib resistant TNBC cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-05-08.

Abstract A73: AMPK activation mediates dasatinib-induced EGFR degradation and apoptosis in head and neck cancer

Abstract Head and neck squamous cell carcinoma (HNSCC) is a worldwide disease with aggressive course and dismal outcome. Dasatinib, a Bcr-abl and Src in hibitor, has been approved clinically for CML and shown activities against solid tumors in vitro. In our recent work, degradation of epidermal growth factor receptor (EGFR) plays a role in dasatinib-induced apoptosis in HNSCC cells. We further explored the mechanism of this event. ER stress was induced by dasatinib and played a role in dasatinib-EGFR degradation. C-cbl activation was induced by ER stress and shown to mediate ER-stress induced EGFR degradation. In addition, AMPK activation was induced by dasatinib, which was proved to be involved in dasatinib-induced ER stress and EGFR degradation. Activation of AMPK with metformin not only enhanced dasatinib-induced growth inhibition and apoptosis but also sensitized dasatinib-resistant cells. Furthermore, xenograft model showed that metformin potentiated anti-cancer effect of dasatinib through activation of AMPK and ER stress. Our results disclose that AMPK-dependent ER stress plays a crucial role in the anti-cancer effect of dasatinib in HNSCC and further activation of AMPK by metformin might enhance dasatinib efficacy. Citation Format: Yu-Chin Lin Lin, Meng-Hsuan Wu, Tzu-Tang Wei, Yun-Chieh Lin, Ching-Chow Chen. AMPK activation mediates dasatinib-induced EGFR degradation and apoptosis in head and neck cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A73. doi:10.1158/1538-7445.CHTME14-A73

Metabolic Shift In Hypoxia-Adapted CML Cells and Mechanisms Of Acquired Dasatinib Resistance Uncovered By Proteomic iTRAQ Profiling

Hypoxia is one of the essential components of the leukemia bone marrow (BM) microenvironment that promotes leukemia cell homing, survival and chemoresistance (Benito, PlosOne 2011). Tyrosine kinase inhibitors (TKIs) do not eradicate the total mass of chronic myeloid leukemia (CML) cells including primitive and quiescent cells. Persistence of CML cells in the hypoxic BM niche after cessation of TKI therapy may result in disease relapse. We have reported that hypoxia adapted CML cells acquire TKI resistance associated with higher glyoxalase-1 (Glo-1) enzyme activity which detoxifies methylglyoxal, a cytotoxic by-product of glycolysis (Takeuchi, Cell Death Differ 2010).In this study, we employed a proteomic approach based on isobaric tags for relative and absolute quantification (iTRAQ, Applied Biosystems) to investigate additional molecular mechanisms of CML adaptation to hypoxia and acquired resistance to dasatinib under hypoxic conditions. The specific pathway alterations were identified by KEGG(Kyoto University) and MetaCore (GeneGo). We utilized two hypoxia-adapted (HA) subclones of CML cell lines, KCL22-HA and KBM5-HA cells, which were selected over a month under 1.0 % oxygen culture conditions. The growth rate of both HA-CML cell lines was slower than that of the corresponding parental cells (ratio of incremental increase in cell numbers, 0.39 for KCL22-HA/KCL22, 0.54 for KBM5-HA/KBM5 at 48hrs). Although parental KCL22 cells were sensitive to dasatinib, KCL22-HA cultured under hypoxia acquired resistance to dasatinib (IC50: KCL22 0.1 nM, KCL22-HA >20nM, at 48hrs by MTT). In contrast, dasatinib induced more prominent cell growth inhibition in KBM5-HA cells cultured under hypoxia compared to KBM5 parental cells (IC50: KBM5 1.3 nM, KBM-5/HA 0.3 nM). Dasatinib effectively downregulated the phosphorylation levels of Stat5 and ERK in parental KCL22 and KBM5 cells. Notably, the baseline levels of p-Stat5 and p-ERK were markedly diminished in both KCL22-HA and KBM5-HA cells. We next performed iTRAQ proteomic analysis and detected more than 1,300 proteins in each cell type. Comparison of the basal proteome of KCL22 vs KCL22-HA and KBM5 vs KBM5-HA cells showed differential expression of 54 proteins in KCL22 isogenic cells (37 upregulated, 17 downregulated) and of 159 proteins in KBM5 cells (56 upregulated, 103 downregulated). These alterations included consistent activation of glycolysis and gluconeogenesis pathway (p<0.0001) along with increased ATP synthase (p=0.02) and hypoxia induced HIF1 activation (p<0.0001) in both cells adapted to grow under hypoxia. In dasatinib-resistant KCL-HA cells, these changes were partially reversed by dasatinib which affected expression of 19 proteins (5 up-regulated / 14 down-regulated) with significant decrease in oxidative phosphorylation (p=0.01), ATP synthase (p=0.02), spliceosome (p=0.02) and Cytochrome C (p=0.04). On the contrary, in dasatinib-sensitive KBM5-HA cells dasatinib upregulated 296 proteins including 39 apoptogenic proteins such as Cytochrome C (p<0.001) and apoptosis related mitochondrial chaperones HSP10 and HSP60 (p<0.001), along with further upregulation of the enzymes involved in the glycolytic energy production (p<0.001) and stimulation of nuclear mRNA splicing via spliceosome (p<0.001), known to play a crucial role in the control of gene expression of the apoptosis-regulating genes. We observed the consistent upregulation of glycolysis and increases of ribosome and spliceosome after dasatinib treatment in dasatinib-sensitive parental KCL22 and KBM5 cells. Interestingly, dasatinib-resistant KBM5-STI cells harboring T315 Bcr-Abl mutation exhibited significantly lower levels of baseline gluconeogenesis (p<0.0001) and translation regulation of translation initiation pathways (p<0.0001) compared to the parental KBM5 cells.In summary, these findings demonstrate the dramatic metabolic changes in hypoxia adjusted CML cells and indicate that hypoxia adapted / dasatinib resistant CML cells repress their stimulated glycolic metabolic state and oxidative phosphorylation along with spliceosomal mRNA splicing after dasatinib exposure, which allow them to enter quiescent stage and escape from apoptosis. Altogether these data indicate novel mechanisms of acquired bone marrow microenvironment-mediated resistance to TKI in CML. Disclosures:Tabe:Bristol-Myers Squibb: Research Funding.

Efficacy of Ponatinib Against ABL Tyrosine Kinase Inhibitor Resistant Leukemia Cells

Abstract 4413Dasatinib (Sprycel®) and nilotinib (Tasigna®) have each shown superior efficacy as front line treatment for patients with chronic myeloid leukemia (CML)-chronic phase (CP) in comparison with imatinib. Dasatinib and nilotinib are also used for the treatment of CML patients resistant or intolerant to imatinib therapy. However, a substantial number of patients are acquired resistance to nilotinib or dasatinib, the management of CML following the development of ABL tyrosine kinase inhibitor (TKI) resistance remains a challenge. Ponatinib, also known as AP24534, is an oral, the multi-targeted TKI. Ponatinib is currently being investigated in a pivotal phase 2 clinical trial (PACE trial) in patients with resistant or intolerant CML and Philadelphia positive acute lymphoblastic leukemia (Ph+ ALL). However, the molecular and functional consequences of ponatinib against ABL TKI resistant cells are not fully known. In this study, we investigated the ponatinib efficacy by using the BCR-ABL positive cell line, K562 and ABL TKI resistant (K562 imatinib resistant (K562IR), K562 nilotinib resistant (K562NR), K562 dasatinib resistant (K562DR) cells and murine Ba/F3 cell line which was transfected BCR-ABL random mutation, and established the new imatinib and nilotinib resistant Ba/F3 BCR-ABL point mutant (E334V) cells. We first examined the cell proliferation by using resistant cell lines. The proliferation of K562IR and K562 NR and K562DR did not decrease after imatinib (10 μM) or nilotinib (2 μM) or dasatinib (1 μM) treatment compared with parental cell line, K562. The BCR-ABL kinase domain mutation was not found. Point mutant Ba/F3 cell (E334V) was also highly resistant to imatinib (IC50: 15μM) and nilotinib (IC50: 7.5μM). We next examined the intracellular signaling by using these cell lines. Phosphorylation of BCR-ABL and Crk-L was not decreased by ABL TKIs in K562IR, K562NR and Ba/F3 BCR-ABL point mutant cells (E334V). We found the one of src family kinase, Lyn was activated in K562IR and K562NR cells. Co-treatment src kinase inhibitor, PP2 and imatinib or nilotinib significantly reduced the cell proliferation of K562IR and K562NR cells. We also found the phosphorylation of Lyn was reduced and poly (ADP-ribose) polymerase (PARP) was activated. We next examined the efficacy of ponatinib against imatinib and nilotinib resistant cell lines. 72 hours treatment of ponatinib exhibits cell growth inhibition against K562 (IC50: 0.02nM), K562IR (IC50: 15nM), and K562NR (IC50: 3.5nM) cells. We also found the phosphorylation of BCR-ABL, Lyn and Crk-L was reduced and PARP was activated after ponatinib treatment. We next examined the imatinib and nilotinib resistant Ba/F3 cells with point mutant (E334V). We found the cell proliferation was significantly decreased after ponatinib treatment (IC50: 3nM). We also found the phosphorylation of BCR-ABL, Crk-L was reduced and PARP was activated after ponatinib treatment. We next investigated the ponatinib activity against dasatinib resistant cells. We found K562DR cells were highly resistant to ponatinib. IC50 was 400nM. These results suggest that the expression and protein activation signatures identified in this study provide insight into the mechanism of resistance to ABL TKIs. We also demonstrate ponatinib has anti-leukemia effect by reducing ABL and Lyn kinase activity and development of ponatinib resistance and suggests that this information may be of therapeutic relevance. Disclosures:No relevant conflicts of interest to declare.

Development of Candidate Genomic Markers to Select Breast Cancer Patients for Dasatinib Therapy

Patient selection is important for targeted therapies, yet phase I/II trials are often underpowered for developing predictors of drug response. The goal of this research was to define genomic predictors for dasatinib that could be prospectively tested in early-phase clinical trials. Gene expression profiles of dasatinib-sensitive and dasatinib-resistant cell lines (n = 23) were compared to develop a dasatinib-sensitivity index (modified DS index). A Src pathway activity index (revised Src index) was defined using genes induced by the Src transfection of mammary epithelial cells and was optimized to be reproducible across cell lines and human specimens. A dasatinib target index was devised using the weighted sum of 19 kinases that bind to dasatinib with variable affinity. The performance of these prediction models was assessed in independent cell lines with known dasatinib sensitivity. The feasibility of applying these genomic tests to human samples was evaluated on 133 biopsies of primary breast cancers. The modified DS index showed 90% accuracy in independent breast cancer cell lines (n = 12) and the target index, but not the revised Src index signature, also distinguished dasatinib-sensitive and dasatinib-resistant cells (P = 0.0024). The genomic predictors showed acceptable reproducibility in replicate cell line and human gene expression data. When all three predictors were applied to the same 133 patient samples, the predictors identified different patient subsets as potentially sensitive. We defined three conceptually different potential predictors of dasatinib response that were reproducible across cell lines and human data. These candidate markers are being tested in a clinical trial to determine their utility.

BCR-ABL mutants spread resistance to non-mutated cells through a paracrine mechanism

Patients with chronic myeloid leukemia who become resistant to the Abl kinase inhibitor imatinib can be treated with dasatinib. This sequential treatment can lead to BCR-ABL mutations conferring broad resistance to kinase inhibitors. To model the evolution of resistance, we exposed the mouse DA1-3b BCR-ABL(+) leukemic cell line to imatinib for several months, and obtained resistant cells carrying the E255K mutation. We then exposed these cells to dasatinib, and obtained dasatinib-resistant cells with composite E255K+T315I mutations. Subcloning isolated a minor clone also carrying V299L. In co-culture, mutated cells were able to spread resistance to non-mutated cells through overexpression of interleukin 3, activation of MEK/ERK and JAK2/STAT5 pathways, and downregulation of Bim. Even the presence of less than 10% of mutated cells was sufficient to protect non-mutated cells. Blocking JAK2 and MEK1/2 inhibited the protective effect of co-culture. Mutated cells were also sensitive to JAK2 inhibition, but blocking MEK1/2 alone, or in association with kinase inhibitors, had little effect. These data indicate that sequential Abl kinase inhibitor therapy can generate sub-populations of mutated cells, which may coexist with non-mutated cells and protect them through a paracrine mechanism. Targeting JAK2 could eliminate both populations.

Mechanism of Drug Resistance to Dasatinib (BMS-354825) and Imatinib in Chronic Myelogenous Leukemia Cells.

The selective tyrosine kinase inhibitor, imatinib has shown remarkable clinical activity in patients with chronic myelogenous leukemia (CML), however, imatinib does not completely eradicate BCR/ABL-expressing cells, and cells resistant to imatinib develop. It has been reported that mutations in the kinase domain (KD) of BCR/ABL impairs binding of imatinib and causes resistance to it. So, additional therapy is necessary to overcome imatinib resistance in patients with an acute form of CML. The novel BCR/ABL inhibitor, dasatinib (BMS-354825), which A dual inhibitor of both Src and Abl kinases, suppresses the activity of these kinases at subnanomolar concentrations in imatinib resistance cells. Dasatinib is presently being evaluated in a clinical trial in CML patients with imatinib resistance, but drug resistance to dasatinib and imatinib are not fully evaluated. In this study, we used TF-1 BCR/ABL cell lines, which were transfected with the BCR/ABL gene, as well as parental TF-1 cells and K562 cell lines and established dasatinib resistant TF-1 BCR-ABL BMS-R and K562 BMS-R cells and imatinib resistant TF-1 BCR-ABL IM-R and K562 IM-R cells. We show here that dasatinib potently induced apoptosis of TF-1 BCR-ABL and K562 cells in 72 hours treatment. IC50 of dasatinib was 0.75nM (TF-1 BCR/ABL) and 1nM (K562) and IC50 of imatinib was 500nM (TF-1 BCR/ABL) and 750nM (K562). In the resistant cell lines, IC50 of dasatinib was 15μM (TF-1 BCR/ABL BMS-R) and 25μM (K562 BMS-R). TF-1 BCR/ABL BMS-R and K562 BMS-R cells were also resistant to imatinib (IC50: more than 10μM). TF-1 BCR-ABL IM-R and K562 IM-R cells were also resistant to dasatinib (IC50: 7.5 nM (TF-1 BCR/ABL IM-R) and the value was more than 10nM for K562 IM-R). There was no mutation in Abl kinase in these resistant cell lines suggesting that BCR/ABL mutation-independent mechanism is involved in resistance to imatinib and dasatinib. Because the correlation of parameters with defined protein expression patterns and protein signatures would predict disease progression and drug resistant, we investigated the protein expression pattern by using antibody microarray system (Antibody Microarray: Lab Vision Corporation). We found that dasatinib resistant cells had reduced protein levels of BCR/ABL. In TF-1 BCR/ABL BMS-R cells, Zap-70 protein was increased and activated. In contrast, the src kinase, lck, is increased and activated in K562 BMS-R cells. Moreover, mitogen-activated protein kinase (MAPK) and Akt were also activated in K562 BMS-R cells. We also found that src kinases, especially Lyn, as well as MAPK and Akt were activated in K562 IM-R cells. The phosphatase SHP-2 was decreased in dasatinib resistant cell lines. Signal-transducing activators of transcription 5 (STAT5) phosphorylation was reduced in K562 BMS-R cells. 24 hour removal of dasatinib from culture medium of K562 BMS-R led to apoptosis of the cells and activated caspase 3 and poly (ADP-ribose) polymerase (PARP). We also found that the phosphatase SHP-1 was increased after removal of dasatinib. The expression and protein activation signatures identified in this study provide insight into the mechanism of resistance to dasatinib and imatinib. Our study demonstrates development of dasatinib resistance and suggests that this information may be of therapeutic relevance.