Imatinib-resistant Cell Lines Research Articles

Anti-KIT monoclonal antibody inhibits imatinib-resistant gastrointestinal stromal tumor growth

Gastrointestinal stromal tumor (GIST) is the most common sarcoma of the gastrointestinal tract and arises from the interstitial cells of Cajal. It is characterized by expression of the receptor tyrosine kinase CD117 (KIT). In 70-80% of GIST cases, oncogenic mutations in KIT are present, leading to constitutive activation of the receptor, which drives the proliferation of these tumors. Treatment of GIST with imatinib, a small-molecule tyrosine kinase inhibitor, inhibits KIT-mediated signaling and initially results in disease control in 70-85% of patients with KIT-positive GIST. However, the vast majority of patients eventually develop resistance to imatinib treatment, leading to disease progression and posing a significant challenge in the clinical management of these tumors. Here, we show that an anti-KIT monoclonal antibody (mAb), SR1, is able to slow the growth of three human GIST cell lines in vitro. Importantly, these reductions in cell growth were equivalent between imatinib-resistant and imatinib-sensitive GIST cell lines. Treatment of GIST cell lines with SR1 reduces cell-surface KIT expression, suggesting that mAb-induced KIT down-regulation may be a mechanism by which SR1 inhibits GIST growth. Furthermore, we also show that SR1 treatment enhances phagocytosis of GIST cells by macrophages, indicating that treatment with SR1 may enhance immune cell-mediated tumor clearance. Finally, using two xenotransplantation models of imatinib-sensitive and imatinib-resistant GIST, we demonstrate that SR1 is able to strongly inhibit tumor growth in vivo. These results suggest that treatment with mAbs targeting KIT may represent an alternative, or complementary, approach for treating GIST.

Activity of AT13387, a novel, non-ansamycin inhibitor of heat shock protein 90, against gastrointestinal stromal tumors (GIST).

105 Background: AT13387 is a second-generation potent, novel non-ansamycin HSP90 inhibitor (Kd 0.71nM). The majority of GIST tumors are characterized by activating mutations of c-KIT, an HSP90 client protein. Secondary resistance mutations within c-KIT limit clinical responses to TKIs. The dependence of c-KIT and its mutated forms on HSP90 suggests that HSP90 inhibition may be a valuable treatment option for imatinib-sensitive and resistant clones. In vitro, AT13387 inhibited the proliferation of imatinib-sensitive (GIST882, GIST-T1) and imatinib-resistant (GIST430, GIST48) cell lines. In vivo, AT13387 demonstrated anti-tumor activity in the imatinib-sensitive (GIST-PSW) and imatinib-resistant (GIST430) xenograft models. Induction of HSP70, depletion of phospho-c-KIT and inhibition of c-KIT signaling were observed in both models. Combination treatment of imatinib and AT13387 in the GIST430 model was well tolerated and significantly enhanced tumor growth inhibition over either monotherapies. Methods: In a completed phase I study, AT13387 was administered IV over 1 hour twice weekly or weekly of a 28-day cycle in a standard 3+3 dose-escalation design. The primary endpoint was to determine the MTD; secondary endpoints included PK, PD, safety and tolerability. Results: The PK exposures were dose-dependent and linear. AT13387 was well tolerated on both schedules. DLTs included primarily G2 AEs of GI toxicities, fatigue and infusion site reactions. The once weekly RP2D was determined to be 260 mg/m2. HSP70 induction was 2–7 fold at higher doses. A total of 7 GIST subjects were enrolled. An objective and durable PR was observed in one subject and 2 SDs at 8, 7 and 11 months, respectively. The PR subject demonstrated molecular resistance to kinase inhibitor treatment in the c-KIT gene prior to initiation of AT13387 therapy in two resected lesions by harboring the same activating c-KIT deletion in exon 11 and two separate TKI resistance mutations in exon 17. Conclusions: Overall, these results suggest AT13387 is a promising agent in GIST, including TKI-resistant c-Kit positive GIST. AT13387 is currently being evaluated in combination with imatinib in an ongoing phase I/II study. Clinical trial information: NCT01294202.

MEK 1/2 Inhibitor U0126 Reversed Imatinib Resistance in IM-Resistant K562R

Abstract 4914 ObjectiveTo study the anti-leukemic activity of MEK inhibitor U0126 alone or in combination with imatinib and explored the reversing mechanism to imatinib resistance in imatinib resistant K562R cell line. MethodsCytotoxicity of drug was detected by the MTT assay in the IM-sensitive cell line K562 and IM-resistant cell line K562R. Western Blot assay were employed to examine the expression of p-cAbl, p-Lyn, p-STAT5, ERK signaling pathway (p-cRaf, p-MEK, p-ERK), PI3K/AKT/mTOR signaling pathway(p-AKT, p-mTOR, p-4EBP1) and the apoptosis related proteins (Bax, Bcl2). The apoptosis rates were analyzed by Annexin V/PI double staining flow cytometry assay. The levels of lyn and erk1/2 gene were assayed by RT-PCR. Results1. BCR-ABL-independent activation of Lyn and ERK1/2 may be the IM resistant mechanism in K562R cell line.The IC50 value of K562R cell line(1. 505±0. 459 μmol/L) inhibited by imatinib for 72 hours was higher than the values of K562(0. 159±0. 032μmol/L) and the Resistant Fold(RF)was 9. 465. Western Blot assays showed that p-Lyn, p-MEK and p-ERK of ERK pathway, p-mTOR and p-4EPB1 were over-expressed in K562R cell line relative to K562 cell line. However, the levels of p-cAbl, p-STAT5, p-Raf, p-AKT of PI3K/AKT/mTOR were similar in the K562R and K562 cell lines. The treatment of IM could reduce the expression of p-cAbl and p-MEK, but not that of p-Lyn, p-ERK, p-mTOR, p-4EBP1, and even up-regulate the p-Lyn, p-ERK, p-mTOR. The expression of Bax, Bcl2 and the apoptosis rates were the similar in both cell lines.2. MEK1/2 inhibitor U0126 could reverse the IM resistance in K562R cell line.MTT assay showed single-agent U0126 is more sensitive to K562R than K562. The IC50 values of the two cell lines were 34. 235±5. 658 μmol/L and 85. 824±4. 474 μmol/L respectively. The combination of imatinib and U0126 markedly enhanced inhibitory effect as measured by MTT assay in K562R cell line, combination with 10μmol/L U0126, the IC50 values of IM was 0. 134±0. 059μmol/L, which reduced to 8. 9% of single IM treatment.3. The reversing mechanism of U0126 to imatinib resistance in K562R cell line.Western Blot showed single IM up-regulated the p-Lyn and p-ERK, while U0126 reduced the expression of them, the combination of the IM and U0126 could synergisticly reduce the p-Lyn expression and neutralize the up-regulation of p-ERK caused by IM single agent. Single IM also up-regulated the p-mTOR in K562R while U0126 reduced it, single IM or U0126 had no influence on p-4EBP1 in K562R, the combination of the two drugs could synergisticly reduce the p-4EBP1 and neutralize the up-regulation of p-mTOR caused by IM single agent. IM but not U0126could reduce p-cAbl and the combination of the two was more effective than IM treatment. IM alone or combination with U0126 could not regulate p-STAT5 expression in K562R. RT-PCR showed that neither IM treatment nor its combination with U0126 could change the level of lyn and erk1, 2 gene in the cell lines. Conclusions1. BCR-ABL-independent activation of Lyn and ERK1/2 involved in IM resistance mechanism in IM-resistant K562R cell line. Imatinib alone could up-regulated the expression of the p-Lyn, p-ERK, p-mTOR in K562R cell line. MEK1/2 inhibitor U0126 could reverse the IM resistance by reducing the expression of the p-Lyn, p-ERK, p-mTOR, p-4EBP1 of IM-resistant K562R cell line, and the combination of U0216 and Imatinib could synergisticly depress up-regulation of the p-Lyn, p-ERK, p-mTOR and p-4EBP1 caused by IM.Grant support: National Natural Science Foundation of China (No. 30770912), Foundation of the Science & Technology Department of Sichuan Province (No. 2008SZ0017). Disclosures:No relevant conflicts of interest to declare.

STAT3 Inhibition Synergizes with BCR-ABL1 Inhibition to Overcome Kinase-Independent TKI Resistance in Chronic Myeloid Leukemia (CML)

Abstract 31In CML, TKI resistance in the absence of BCR-ABL1 kinase mutations is mechanistically unclear. Since extrinsic signals from the bone marrow (BM) microenvironment protect CML cells from TKIs in a STAT3-dependent manner (Bewry et al. 2008; Traer et al. 2011), we hypothesized that overt resistance may occur when CML cells maintain intrinsic STAT3 activation in the absence of extrinsic signals. We also asked whether combined targeting of STAT3 and BCR-ABL1 could produce synthetic lethality to overcome TKI resistance. To model extrinsic BCR-ABL1 kinase-independent resistance, we grew CML cell lines and CMLCD34+ progenitor cells from newly diagnosed patients in the presence of conditioned medium (CM) derived from HS-5 BM stromal cells. To model intrinsic resistance, we used the imatinib-resistant K562R and AR230R cell lines. Our models of TKI resistance proliferate in 1.0–2.5 μM imatinib and exhibit pSTAT3Y705 activation despite suppression of BCR-ABL1 kinase activity.To investigate the role of pSTAT3Y705 in TKI resistance, we used shRNA-mediated knockdown (shSTAT3), a dominant-negative mutant (dnSTAT3), and pharmacologic STAT3 inhibitors developed in our laboratories to block STAT3 activity. Compared to scrambled controls in the presence of HS-5 CM, shSTAT3 reduced colony formation (33.8% reduction, p<0.008) and increased apoptosis (17.9% increase, p<0.03) of parental K562 cells following imatinib exposure, thereby abolishing the protective effect of extrinsic BM-derived factors. In contrast, shSTAT3 had no effect on cells grown in regular medium. In intrinsic resistance, shSTAT3 reduced clonogenicity of K562R cells by 58.3% (p<0.0004) and AR230R cells by 62.2% (p<0.002) in the presence of 1.0 μM imatinib, with no effect on TKI-sensitive controls. A dnSTAT3 mutant lacking the C-terminal activation domain also impaired the clonogenicity of K562R and AR230R cells by 36.1% (p<0.003) and 50.0% (p<0.004), respectively. Thus, STAT3 plays a functional role in mediating intrinsic and extrinsic TKI resistance in BCR-ABL1-expressing cell lines.Preliminary immunofluorescence data indicates that pSTAT3Y705 levels are higher in drug-resistant CML CD34+CD38− stem cells than in CD34+CD38+ progenitor cells, suggesting a potential role for pSTAT3Y705 in TKI resistance of primitive CML stem cells. To investigate whether pharmacologic STAT3 inhibition in combination with TKIs could overcome resistance, we tested a library of compounds that interfere with STAT3 dimerization. The most selective of these inhibitors, S3I-201.1066 (SF1-066), was biochemically shown to disrupt STAT3 dimerization and transcriptional activation. Compared to cells treated with 1.0 μM imatinib alone, addition of 10 μM SF1-066 impaired the clonogenicity of K562R cells by 68.6% (p<0.03) and AR230R cells by 72.5% (p<0.0004), with no effect on TKI-sensitive controls. When CMLCD34+ progenitor cells from newly diagnosed patients (n=4) were cultured in the presence of HS-5 CM and treated with the SF1-066/imatinib combination, clonogenicity following drug exposure was reduced by 45.2% (p<0.002) compared to imatinib. Therefore, pharmacologic inhibition of STAT3 and BCR-ABL1 overcomes intrinsic and extrinsic TKI resistance in both CML cell lines and CMLCD34+ progenitor cells.We next developed a series of 14 second-generation STAT3 inhibitors and tested them using a luciferase reporter harboring sequential STAT3 cis-inducible elements to measure endogenous STAT3 activity in AR230R cells. Two lead compounds, BP2-047 and BP3-163, reduced luciferase activity by 69.2% (p<3.6×10−6) and 51.8% (p<8.6×10−6), respectively, with little effect on cells expressing a scrambled reporter. Selected inhibitors then provided scaffolds for the synthesis of focused libraries with enhanced potency and selectivity. Immunofluorescence data revealed that two of these inhibitors, BP5-087 and BP5-088, resulted in cytoplasmic accumulation of pSTAT3Y705 in CMLCD34+ progenitors grown in HS-5 CM, consistent with a block of dimerization. Screening and validation of third-generation STAT3 inhibitors are underway and will be reported.Altogether, our data identifies STAT3 as a universal target in TKI-resistant CML cells, and suggests that simultaneous pharmacologic inhibition of STAT3 and BCR-ABL1 may be a suitable therapeutic strategy for treatment of patients with TKI resistance despite inhibition of BCR-ABL1 kinase activity. Disclosures:No relevant conflicts of interest to declare.

All tyrosine kinase inhibitor-resistant chronic myelogenous cells are highly sensitive to Ponatinib

The advent of tyrosine kinase inhibitor (TKI) therapy has considerably improved the survival of patients suffering chronic myelogenous leukemia (CML). Indeed, inhibition of BCR-ABL by imatinib, dasatinib or nilotinib triggers durable responses in most patients suffering from this disease. Moreover, resistance to imatinib due to kinase domain mutations can be generally circumvented using dasatinib or nilotinib, but the multi-resistant T315I mutation that is insensitive to these TKIs, remains to date a major clinical problem. In this line, ponatinib (AP24534) has emerged as a promising therapeutic option in patients with all kinds of BCR-ABL mutations, especially the T315I one. However and surprisingly, the effect of ponatinib has not been extensively studied on imatinib-resistant CML cell lines. Therefore, in the present study, we used several CML cell lines with different mechanisms of resistance to TKI to evaluate the effect of ponatinib on cell viability, apoptosis and signaling. Our results show that ponatinib is highly effective on both sensitive and resistant CML cell lines, whatever the mode of resistance and also on BaF3 murine B cells carrying native BCR-ABL or T315I mutation. We conclude that ponatinib could be effectively used for all types of TKI-resistant patients.

1482PD - Dasatinib First-Line Treatment in Gastrointestinal Stromal Tumors. a Multicenter Phase II Trial of the SAKK (SAKK 56/07)

ABSTRACT Introduction First line imatinib has improved the outcome of patients (pts) with gastrointestinal stromal tumor (GIST), but primary and secondary resistance remain a problem. Dasatinib is a second generation tyrosine kinase inhibitor (TKI) of bcr-abl, src-family kinases and a number of other oncogenic kinases including kit. Dasatinib has shown activity against imatinib-resistant cell lines in vitro. 18F-fluorodeoxyglucose positron-emission-tomography (FDG-PET) measures tumor metabolic activity for early response assessment, which might be of particular use in the clinical trial setting. Methods This two-stage phase II trial investigated dasatinib in patients with histologically proven, FDG-PET positive, TKI-naive GIST. Dasatinib starting dose was 2x70mg/day. Response evaluation was done by serial CT and FDG-PET using EORTC PET response criteria (Young et al. 1999). PET was reviewed centrally. Elective surgery was allowed after 6 months of trial treatment. Primary endpoint was response (CR + PR) by FDG-PET after one month of dasatinib. Results 47 of planned 52 pts have been enrolled from December 2007 to November 2011, when the trial was terminated due to slow accrual. 43 pts were eligible. Median age was 61 years, 24 pts were male, 19 female. 30 pts had a performance status of 0 and thirteen of 1. Mutational data are available for 28 pts so far. 20 had a KIT exon 11, one a KIT exon 9 mutation, and 7 were wild-type. At a median follow-up of 12.4 months, 15 pts were still on treatment. Pts went off trial for elective surgery (n = 6), progression (n = 13), toxicity (n = 4), other reasons (n = 2) and three patients died. 5% of pts experienced G4, and 38% of pts G3 toxicity, which were most often gastrointestinal or pulmonary. 28 pts had their dose reduced or interrupted. The primary endpoint, FDG-PET response rate (CR + PR) at 4 weeks was 72% (14 CR, 17 PR, 7 SD, 3 PD, 2 not evaluable). The response rate in patients with a KIT Exon 11 mutation was 80%, in wild-type patients 57%. Median progression-free survival is 11.1 months and median overall survival not yet reached. Conclusions Dasatinib shows promising efficacy in TKI-naive pts with FDG-PET positive GIST. Disclosure M. Montemurro: Speakers fees and travel grants - Bayer, Pfizer, Novartis. P. Rutkowski: Consultant - BMS, Novartis, MSD Speakers fees and travel grants - Novartis, Pfizer, MSD, BMS, Roche. R. von Moos: Consultant/Advisor - AMGEN, Novartis, Roche, BMS, Pfizer, Merck Speakers fees - Roche, Amgen institutional Research Funds - AMGEN Expert Testimony - AMGEN. All other authors have declared no conflicts of interest.

Bcr-Abl Amplification Plays a Major Role in Resistance to Tyrosine Kinase Inhibitors in K-562 Cell Line

An emerging problem in patients with chronic myeloid leukemia (CML) is increasing resistance to tyrosine kinase inhibitors (TKIs). To determine genetic and cellular mechanisms involved in the development of resistance to TKIs, nine imatinib-resistant cell lines were derived from K- 562 cell line followed by testing of drug sensitivity, multidrug resistance proteins and cytogenetic studies. In imatinib-resistant cell lines cross-resistance to daunorubicin, etoposide and cytarabine were observed whereas sensitivity to dasatinib, nilotinib, cyclophpsphamide, bortezomib and busulfan was preserved. Treatment with imatinib decreased PGP and LRP expression, however it did not significantly influence MRP1 expression. Amount of signals in FISH analysis from ABL, BCR and from fusion genes (BCR-ABL or ABL-BCR) was mostly higher in imatinib-resistant cell lines in comparison to parental K-562 cell line. We concluded that BCR-ABL amplification but not cellular sensitivity is the major mechanisms of resistance in K-562 cell line.

‘Big’-Insulin-Like Growth Factor–II Signaling Is an Autocrine Survival Pathway in Gastrointestinal Stromal Tumors

New treatment targets need to be identified in gastrointestinal stromal tumors (GISTs) to extend the treatment options for patients experiencing failure with small-molecule tyrosine kinase inhibitors, such as imatinib. Insulin-like growth factor (IGF)-II acts as an autocrine factor in several tumor types by binding to IGF receptor type 1 (IGF-1R) and/or the insulin receptor (IR) isoform A. The aim of the present study was to investigate the putative role of unprocessed pro-IGF-II, called 'big'-IGF-II, in GISTs. The imatinib-sensitive GIST882 and imatinib-resistant GIST48 cell lines secrete high levels of big-IGF-II as demonstrated by ELISA and Western blotting analyses. IR isoform A mRNA and protein expression, but not that of IGF-1R, was found in these KIT mutant cell lines and in KIT and platelet-derived growth factor receptor α-mutant GIST specimens. Down-regulation of either big-IGF-II or IR affected AKT and MAPK signaling and reduced survival in both cell lines. Disruption of big-IGF-II signaling in combination with imatinib had additive cytotoxic effects on GIST882 cells. IGF-II mRNA as determined by in situ hybridization was present in 91% of 60 primary GISTs. Immunohistochemical analysis of big-IGF-II protein expression was associated with moderate- to high-risk tumors compared with tumors with a lower risk classification (P < 0.028). Our data put forth the big-IGF-II/IR isoform A axis as an autocrine survival pathway and potential therapeutic target in GISTs.

Dasatinib first-line treatment in gastrointestinal stromal tumors: A multicenter phase II trial of the SAKK (SAKK 56/07).

10033 Background: First line imatinib has improved the outcome of patients (pts) with gastrointestinal stromal tumor (GIST), but primary and secondary resistance remain a problem. Dasatinib is a second generation tyrosine kinase inhibitor (TKI) and inhibits the activity of bcr-abl, src-family kinases along with a number of other oncogenic kinases including kit. In vitro, dasatinib has shown activity against imatinib-resistant cell lines. Routinely, pts diagnosed with GIST and treated with TKIs are monitored by computed tomography (CT). 18F-fluorodeoxyglucose positron-emission-tomography (FDG-PET) measures tumor metabolic activity for early response assessment, which might be of particular use in the clinical trial setting. Methods: This two-stage phase II trial investigated dasatinib in pts with TKI-naïve GIST. Dasatinib starting dose was 2x70mg/day in pts with histologically proven, FDG-PET positive GIST. Response evaluation was done by serial CT and FDG-PET using EORTC PET response criteria (Young et al. 1999). Elective surgery was allowed after 6 months of trial treatment. Primary endpoint was response (CR+PR) by FDG-PET after one month of dasatinib. Results: 47 of planned 52 pts have been enrolled from December 2007 to November 2011, when the trial was terminated due to slow accrual. 43 pts were eligible. Median age was 61 years, 24 pts were male, 19 female and 41 had a performance status of 0 or 1. At a median follow-up of 11.9 months, 20 pts were still on treatment. Pts went off trial for elective surgery (n=6), progression (n=11), toxicity (n=3), and three patients died (one on-drug). 5% of pts experienced G4, and 38% of pts G3 toxicity, which were most often gastrointestinal or pulmonary. 28% had their dose reduced or interrupted. The primary endpoint, FDG-PET response rate (CR+PR) at 4 weeks was 67% (13 CR, 16 PR, 7 SD, 3 PD, 4 not evaluable). Median progression-free survival is 11.1 months and median overall survival not yet reached. Conclusions: Dasatinib shows promising efficacy in TKI-naïve pts with FDG-PET positive GIST. Mutational data will be presented at the meeting.

Abstract 1822: Novel potent inhibitor of Bcr-Abl mutated imatinib resistant chronic myeloid leukemia cell lines

Abstract The hallmark of chronic myeloid leukemia (CML) is the presence of Bcr-Abl oncoprotein that is essential for leukemogenesis and accumulation of neoplastic cells. The Abl tyrosine kinase inhibitor imatinib mesylate (Gleevec) represents the standard treatment for CML. However, a number of CML patients develop imatinib resistance and several mechanisms of resistance have been described. While most mutant forms are sensitive to second-generation tyrosine kinase inhibitors, the T315I mutation remains completely refractory to imatinib, as well as to these agents. Treatment of CML patients harboring T315I mutation is still an unmet medical need in the imatinib and post-imatinib era. We have identified P2745, a novel orally bioavailable small molecule that is effective in imatinib resistant mutations and particularly T315I mutation form to address this unmet medical need. In vitro P2745 showed potent cytotoxicity (IC50 0.8-5 μM) in twelve Bcr-Abl mutated imatinib resistant cell lines including T315I compared to wild type Bcr-Abl cell lines. P2745 did not show cytotoxicity upto 30 μM in normal fibroblasts (MRC-5 and WI-38) and normal human peripheral blood mononuclear cells thus indicating specificity towards cancerous cells. P2745 inhibited pCrkLTyr207 in imatinib-resistant CML cell lines accompanied with down regulation of Bcr-Abl protein levels. Additionally, it up regulated tumor suppressor protein SMAR-1 which resulted in negative regulation TGF-β/SMAD-dependent pathway as demonstrated by low phosphorylated levels of SMAD2 and SMAD3, two key players implicated in the preservation of the malignant progenitor population, and partly responsible for the resistance to treatments targeting Bcr-Abl. P2745 also induced significant apoptosis in imatinib resistant cell lines (Y253F, T315I and E255V) but not in normal MRC-5 cell line. In addition, like imatinib it induced 80-90 % apoptosis in cells derived from CML patients. In vivo studies demonstrated significant (p&amp;lt;0.001), dose dependent efficacy in imatinib resistant T315I cells xenograft in SCID mice. It also demonstrated significant in vivo efficacy in Bcr-Abl wild type imatinib sensitive K-562 (p&amp;lt;0.05) and Ba/F3 (p&amp;lt;0.001) xenografts in SCID mice. PK-PD evaluation in imatinib resistant xenograft experiments indicated that at the efficacious dose, P2745 modulated proteins responsible for CML pathogenesis (60-80 %), which correlates with the levels of P2745 in the tumors. This unique mechanism of action makes P2745 a promising clinical candidate for CML patients harboring T315I mutation. 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 1822. doi:1538-7445.AM2012-1822

Knock-down of Kaiso induces proliferation and blocks granulocytic differentiation in blast crisis of chronic myeloid leukemia

BackgroundKaiso protein has been identified as a new member of the POZ-ZF subfamily of transcription factors that are involved in development and cancer. There is consistent evidence of the role of Kaiso and its involvement in human tumorigenesis but there is no evidence about its role in hematopoietic differentiation or establishment of chronic myeloid leukemia (CML). We used, normal K562 cell line, established from a CML patient in blast crisis, and imatinib-resistant K562 cell line, to investigate the specific distribution of Kaiso and their contribution to the cell differentiation status of the blast crisis of CML (CML-BP).ResultsWe found cytoplasmic expression of Kaiso, in K562 cells and patients, confirmed by immunofluorescence, immunohistochemistry and western blot of cytoplasmic protein fraction. Kaiso was weakly expressed in the imatinib-resistant K562 cell line confirmed by immunofluorescence and western blot. The cytoplasmic expression of Kaiso was not modified when the K562 cells were treated for 16 h with imatinib 0.1 and 1 μM. In our study, small interfering RNA (siRNA) was introduced to down regulate the expression of Kaiso and p120ctn in K562 cell line. Kaiso and p120ctn were down regulated individually (siRNA-Kaiso or siRNA-p120ctn) or in combination using a simultaneous co-transfection (siRNA-Kaiso/p120ctn). We next investigated whether knockdown either Kaiso or p120ctn alone or in combination affects the cell differentiation status in K562 cells. After down regulation we analyzed the expression of hematopoietic cell differentiation and proliferation genes: SCF, PU-1, c-MyB, C/EBPα, Gata-2 and maturation markers of hematopoietic cells expressed in the plasma membrane: CD15, CD11b, CD33, CD117. The levels of SCF and c-MyB were increased by 1000% and 65% respectively and PU-1, Gata-2 and C/EBPα were decreased by 66%, 50% and 80% respectively, when Kaiso levels were down regulated by siRNA. The results were similar when both Kaiso and p120ctn were down regulated by siRNA. The increased expression of SCF and decreased expression of GATA-2 could be responsible by the higher cell viability detected in K562 cells double knock-down of both Kaiso and p120ctn. Finally, we studied the effect of knock-down either Kaiso or p120ctn, alone or in combination on CD15, CD11b, CD33 and Cd117 expression. Using siRNA approach a reduction of 35%, 8% and 13% in CD15, CD33 and CD117 levels respectively, were achieved in all transfections, when compared to scrambled knock-down cells.ConclusionThese results suggest that both Kaiso and p120ctn, contributes to maintaining the differentiated state of the K562 cells and similar to other cancers, cytoplasmic localization of Kaiso is related to a poor prognosis in CML-BP. By the broad and profound effects on the expression of genes and markers of hematopoietic differentiation produced by Kaiso knock-down, these findings reveal Kaiso as a potential target for selective therapy of CML.

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.

Overexpression of P-glycoprotein induces acquired resistance to imatinib in chronic myelogenous leukemia cells

Imatinib, a breakpoint cluster region (BCR)-Abelson murine leukemia (ABL) tyrosine kinase inhibitor (TKI), has revolutionized the treatment of chronic myelogenous leukemia (CML). However, development of multidrug resistance (MDR) limits the use of imatinib. In the present study, we aimed to investigate the mechanisms of cellular resistance to imatinib in CML. Therefore, we established an imatinib-resistant human CML cell line (K562-imatinib) through a stepwise selection process. While characterizing the phenotype of these cells, we found that K562-imatinib cells were 124.6-fold more resistant to imatinib than parental K562 cells. In addition, these cells were cross-resistant to second- and third-generation BCR-ABL TKIs. Western blot analysis and reverse transcription-polymerase chain reaction(RT-PCR) demonstrated that P-glycoprotein (P-gp) and MDR1 mRNA levels were increased in K562-imatinib cells. In addition, accumulation of [14C]6-mercaptopurine (6-MP) was decreased, whereas the ATP-dependent efflux of [14C] 6-MP and [3H]methotrexate transport were increased in K562-imatinib cells. These data suggest that the overexpression of P-gp may play a crucial role in acquired resistance to imatinib in CML K562-imatinib cells.

BCR-ABL- and Ras-independent activation of Raf as a novel mechanism of Imatinib resistance in CML

Although the BCR-ABL tyrosine kinase inhibitor Imatinib has undoubtedly revolutionized the therapy of chronic myeloid leukaemia (CML), acquired drug resistance remains a common problem in CML therapy. Resistance often arises from second-line mutations in BCR-ABL or overexpression of the BCR-ABL protein but in ~20% of CML cases resistance mechanisms do not involve altered BCR-ABL function. Imatinib-resistant CML cell lines have been widely used for comparative proteome/genome-wide expression screens in order to decipher resistance mechanisms but a clearcut molecular mechanism or molecular player in BCR-ABL-independent resistance to Imatinib has not yet evolved from those studies. Here, we report the identification of a novel mechanism for Imatinib resistance in CML cells with unaltered BCR-ABL function. Pharmacological analysis evidenced a constitutive, Imatinib-insensitive activation of the Erk-MAPK pathway in resistant cells. A systematic analysis of pathway constituents illustrated that Ras-GTP accumulation remained fully sensitive to Imatinib but c-Raf activity from serum-fed cultures was largely resistant to the drug's action. Sequencing excluded mutations in either B-Raf or c-Raf as the origin of resistance, indicating that a functional alteration in the regulation of c-Raf activity was responsible for this effect. Collectively, these findings highlight a novel mechanism of acquired Imatinib resistance based on the BCR-ABL and Ras-independent constitutive activation of the Erk-MAPK pathway through activated c-Raf, which could prove helpful for a better functional classification of the causes of Imatinib resistance in CML.

16-Hydroxycleroda-3,13-dien-15,16-olide deregulates PI3K and Aurora B activities that involve in cancer cell apoptosis

The PI3K-AKT pathway and Aurora kinase play essential roles in such cellular processes as cell survival, angiogenesis, and differentiation, and are usually expressed at maximum levels during cancer cell proliferation. The present study investigated the effect of the natural compound, 16-hydroxycleroda-3,13-dien-15,16-olide (PL3), on regulating the PI3K-AKT pathway and Aurora B, which led to cancer cell apoptosis. PL3 acts as a PI3K inhibitor by influencing cell survival, signaling transduction, and cell cycle progression. It was observed that PL3 targeted and induced dephosphorylation of the PI3K pathway, degradation of Aurora B and mitotic-related gene expressions, and sequentially shut down the cell cycle. This eventually resulted in cell death. As Aurora B was downregulated, spindle dysfunction and destruction of the G 2/M phase checkpoint resulted in DNA-damaged cells undergoing apoptosis. Moreover, PL3 also resensitized T315I-mutated Bcr-ABL + BA/F3 cells to improve the cytotoxicity of Imatinib in Imatinib-resistant cell line. Taken together, PL3 can perturb the PI3K-AKT pathway and Aurora B resulting in gene silencing and cell cycle disturbance. It was demonstrated that PL3 acted like a novel small-molecule PI3K modulator, thereby potentially contributing to cancer chemotherapy and combination medication.

Abstract 2310: Down-regulating BCL-2 gene expression in a resistant GIST cell line by a RNA G-quadruplex interacting small molecule ligand

Abstract We have previously shown that KIT protein expression in the gastrointestinal tumour (GIST) cell line GIST882 can be down-regulated using a small molecule approach that targets expression at the transcriptional level. The small-molecule naphthalene diimide derivative ND1 developed as a G-quadruplex telomere targeting agent can inhibit telomerase activity and can also reduce c-kit mRNA levels, consistent with a dual G-quadruplex mechanism of action in this cell line. An Imatinib (Gleevec) resistant patient-derived cell line GIST48 expresses high levels of the anti-apoptotic protein bcl-2. The compound ND1 down-regulates the expression of bcl-2 in this resistant cell line, while having no discernable effect on c-kit protein expression levels or telomerase activity, both associated with DNA quadruplex stabilization. Instead we observe that translation of the apoptosis-related protein is impeded, while mRNA levels remain unaffected. The identification of a G-rich quadruplex-forming sequence in the 5’-UTR region of the bcl-2 gene, upstream of the translation start site, lead us to the development of a dual luciferase reporter assay. Utilizing a psiCHECK-2 vector we inserted the G-rich 67 base pair bcl-2 5’-UTR gene sequence upstream of the Renilla luciferase start codon. In vitro experiments have shown that the G-rich sequence can form a stable RNA G-quadruplex and interfere with translation. Our in vivo transfection experiments with ND1 in a GIST cell line shows concentration-dependent inhibition of bcl-2 protein expression. RNA G-quadruplex formation and ligand stabilization may thus be a novel anti-cancer route for the down-regulation of the anti-apoptotic protein bcl-2 in chemo-resistant cancers. 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 2310. doi:10.1158/1538-7445.AM2011-2310

Abstract 4860: Identification of novel epitopes, drug resistance markers and drug resistance mechanism using alternative splicing studies

Abstract Alternative RNA splicing is a key molecular mechanism for the generation of functional protein diversity. Abnormal alternative splicing can occur in cancer, resulting in the production of novel transcript variants. Therefore, development of platform technologies aimed at studying alternative splicing will help to understand the diverse mechanisms by which splicing dysregulation contribute to human disease. The impact of alternative splicing in disease and its regulation by drug actions have opened up new perspectives for target discovery, drug development and drug response monitoring. ExonHit has generated this type of discovery platform, the Genome Wide SpliceArrayTM, and is currently building libraries of alternative splicing events that are deregulated in cancer and in cases of therapy resistance. These libraries can be interrogated to answer questions such as drug response monitoring, specificity and selectivity of drug response, diagnostics for monitoring disease status, progression or relapse. Using ExonHit's SpliceArrayTM, we previously reported a significant contribution of splicing to cancer development and drug response (1). Here, we demonstrated the capability of ExonHit's platforms 1/ to identify novel cell surface epitopes for antibody development and 2/ to study drug resistance. Based on SpliceArray™ microarray analysis, transcripts alternatively spliced were isolated from breast tumor tissues for the epitope study. Different splicing patterns were evidenced in tumoral versus normal tissues and specificity analysis was performed too. Based on combination of statistical analysis and protein knowledge, most relevant events were selected as alternatively spliced transcripts encoding novel amino acid sequence that can be used to target monoclonal antibodies. Finally, QPCR expression analysis validate the specificity of the selected events identified by the probe sets that emerged from the genome-wide splicing analysis. For the drug resistance study, two types of Imatinib-resistant leukemia cell lines were established. Distinct splicing patterns were evidenced using principal component analysis, through statistical analysis of differential splicing and pathway analyses. We also implemented selectivity filters allowing to identify a drug resistance signature and a set of pathways/genes involved in drug resistance. These signatures will need to be confirmed in subsequent studies. Our results demonstrate that alternative RNA splicing offers a currently underexploited source of novel disease targets. Platforms dedicated to studying alternative splicing can be integrated into discovery processes to allow identification of novel targets for drug discovery and we have began to identify genomic expression profiles related to drug treatment that will ultimately help monitoring drug response and clinical trial design. 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 4860. doi:10.1158/1538-7445.AM2011-4860

Abstract 5364: AURA kinases as markers and targets in GIST

Abstract Mitotic index has been found to be an important prognostic factor in primary gastrointestinal stromal tumor (GIST). By using gene sets related to cell cycle (Proc Natl Acad Sci U S A. 2008; 22;105(3):955-60), we can stratify a group of patients with advanced GIST (J Clin Oncol 2008;26:4100-4108.) into two risk groups with distinct survival. So it is interesting to explore whether genes involved in regulation of cell cycle could be used as a target in treating GIST. Aurora kinase A (AURKA) and B (AURKB) were two important genes in cell cycle regulation. In the same cohort of patients, expression levels of both AURKA and AURKB were found higher in high risk group patients than those in low risk group. By using MTT assay, GIST882, an imatinib-sensitive GIST cell line, was relatively resistant to both AURKA inhibitor MLN8237 and AURKB inhibitor ZM447439 than GIST 48IM, an imatinib-resistant GIST cell line. MLN8237 could induce significant cell cycle arrest in both GIST cell lines, but apoptosis could only be seen in GIST48IM. On the other hand, after ZM447439 treatment, cell cycle inhibition could only be found in GIST48IM, and none of them had apoptosis. Instead, ZM447439 could induce autophagy in both cell lines. We then used immunohistochemistry (IHC) to examine 76 primary GIST patients. High expression of AURKA but not AURKB was more often seen in non-gastric GIST. In univariate analysis, patients with tumors having strong AURKA IHC staining, as well as large tumor size, high mitotic index and NIH high risk group, had significant lower survival than comparative groups. However, in the multivariate analysis, only high mitotic count and high AURKA expression were independent adverse prognostic factors. In conclusion, cell cycle-related genes could be used in predicting prognosis in advanced GIST, and cell cycle regulated genes such as AURKA and AURKB could be used as treatment targets. 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 5364. doi:10.1158/1538-7445.AM2011-5364

Sphingosine kinase 1 overexpression is regulated by signaling through PI3K, AKT2, and mTOR in imatinib-resistant chronic myeloid leukemia cells

As a better understanding of the molecular basis of carcinogenesis has emerged, oncogene-specific cell-signaling pathways have been successfully targeted to treat human malignances. Despite impressive advances in oncogene-directed therapeutics, genetic instability in cancer cells often manifest acquired resistance. This is particularly noted in the use of tyrosine kinase inhibitors therapies and not more evident than for chronic myeloid leukemia. Therefore, it is of great importance to understand the molecular mechanisms affecting cancer cell sensitivity and resistance to tyrosine kinase inhibitors. In this study, we used continuous exposure to stepwise increasing concentrations of imatinib (0.6-1 μM) to select imatinib-resistant K562 cells. Expression of BCR-ABL increased both at RNA and protein levels in imatinib-resistant cell lines. Furthermore, expression levels of sphingosine kinase 1 (SphK1) were increased significantly in resistant cells, channeling sphingoid bases to the SphK1 pathway and activating sphingosine-1-phosphate-dependent tyrosine phosphorylation pathways that include the adaptor protein Crk. The partial inhibition of SphK1 activity by N,N-dimethylsphingosine or expression by small interfering RNA increased sensitivity to imatinib-induced apoptosis in resistant cells and returned BCR-ABL to baseline levels. To determine the resistance mechanism-induced SphK1 upregulation, we used pharmacological inhibitors of the phosphoinositide 3-kinase/AKT/mammalian target of rapamycin signaling pathway and observed robust downmodulation of SphK1 expression and activity when AKT2, but not AKT1 or AKT3, was suppressed. These results demonstrate that SphK1 is upregulated in imatinib-resistant K562 cells by a pathway contingent on a phosphoinositide 3-kinase/AKT2/mammalian target of rapamycin signaling pathway. We propose that SphK1 plays an important role in development of acquired resistance to imatinib in chronic myeloid leukemia cell lines.

Establishment and biological characters of an Imatinib-resistant cell line of gastrointestinal stromal tumor

Objective To establish the Imatinib-resistant model GIST882-R.Methods The intermittent concentration gradient doubling method was used to get Imatinib-resistant cell line GIST882-R.Changes of cell morphology were observed and compared under the optical microscopy.Changes of cell cycle were assayed and compared by using flow cytometer.The growth curvature,doubling time and IC50 were calculated by using methylthiazol tetrazolium (MTT) assay.Gene sequencing was applied to detect DNA mutation.Results (1) Significant changes occurred to the morphologic profiles,such as epithelioid shaped,the increased ratio of nucleolus to cytoplasm,emerging multinucleate cell,increase of granule substances;(2) The cell cycle of GIST882-S and GIST882-R was respectively 20.23% and 17.38% in S phase,68.33% and 72.25% in G0/G1 phase,11.44% and 10.37% in G2/M phase respectively (P＜0.01 );(3) The doubling time of GIST882-S and GIST882-R was (47.10 ±2.01 ) h and (39.56 ± 1.18)h respectively (P ＜0.01 ).IC50 for GIST882-S and GIST882-R was ( 1.549 ±0.366) μmol and (0.136 ±0.025) μmol respectively (P＜ 0.01 ).The resistance of GIST882-R to Imatinib was 11.39 fold of GIST882-S cells;(4) K642E mutation of exon 13 was found in both GIST882-S and GIST882-R.Conclusion The sensitivity of GIST882-R to Imatinib treatment is decreased significantly. Key words: Gastrointestinal stromal tumor; Cell line; Imatinib; Drug resistance