Nilotinib-resistant Cells Research Articles

Investigating the potential therapeutic role of targeting STAT3 for overcoming drug resistance by regulating energy metabolism in chronic myeloid leukemia cells.

Objective(s):STATs are one of the initial targets of emerging anti-cancer agents due to their regulatory roles in survival, apoptosis, drug response, and cellular metabolism in CML. Aberrant STAT3 activity promotes malignancy, and acts as a metabolic switcher in cancer cell metabolism, contributing to resistance to TKI nilotinib. To investigate the possible therapeutic effects of targeting STAT3 to overcome nilotinib resistance by evaluating various cellular responses in both sensitive and nilotinib resistant CML cells and to test the hypothesis that energy metabolism modulation could be a mechanism for re-sensitization to nilotinib in resistant cells.Materials and Methods:By using RNAi-mediated STAT3 gene silencing, cell viability and proliferation assays, apoptotic analysis, expressional regulations of STAT mRNA transcripts, STAT3 total, pTyr705, pSer727 protein expression levels, and metabolic activity as energy metabolism was determined in CML model K562 cells, in vitro.Results:Targeting STAT3 sensitized both parental and especially nilotinib resistant cells by decreasing leukemic cell survival; inducing leukemic cell apoptosis, and decreasing STAT3 mRNA and protein expression levels. Besides, cell energy phenotype was modulated by switching energy metabolism from aerobic glycolysis to mitochondrial respiration in resistant cells. RNAi-mediated STAT3 silencing accelerated the sensitization of leukemia cells to nilotinib treatment, and STAT3-dependent energy metabolism regulation could be another underlying mechanism for regaining nilotinib response.Conclusion:Targeting STAT3 is an efficient strategy for improving the development of novel CML therapeutics for regaining nilotinib response, and re-sensitization of resistant cells could be mediated by induced apoptosis and regulation in energy metabolism.

Effect of dual inhibition of histone deacetylase and phosphatidylinositol-3 kinase in Philadelphia chromosome-positive leukemia cells.

ABL tyrosine kinase inhibitors (TKIs) have demonstrated potency in the treatment of chronic myeloid leukemia (CML) patients. However, resistance to ABL TKIs can develop in CML patients due to BCR-ABL point mutations. Furthermore, CUDC-907 is an oral inhibitor of class I phosphoinositide 3-kinase (PI3K) as well as class I and II histone deacetylase (HDAC) enzymes. In this study, we evaluated the effect of combination therapy of CUDC-907 and ABL TKIs, using BCR-ABL-positive cell lines and primary samples. CUDC-907 treatment for 72h resulted in cell growth inhibition. Over the same period, an increase in histone acetylation and both caspase three and poly (ADP-ribose) polymerase (PARP) enzyme activity was observed. When ABL TKI treatment and CUDC-907 treatment were combined, significantly greater cytotoxicity was observed. Moreover, combined oral therapy with ponatinib (20mg/kg/day) and CUDC-907 (30mg/kg/day) greatly inhibited tumor growth compared to each drug alone. Lastly, CUDC-907 treatment also inhibited the growth of Ba/F3 ponatinib-resistant cells, K562 nilotinib-resistant cells, and T315I mutant primary samples. Taken together, our results indicate that administration of CUDC-907, a dual PI3K and HDAC inhibitor, may be an effective strategy against ABL TKI-resistant cells, including cells harboring the T315I mutation. Moreover, CUDC-907 may enhance the cytotoxic effects of ABL TKI when a combined treatment strategy is used against Philadelphia chromosome-positive leukemia cells.

ABCC6 plays a significant role in the transport of nilotinib and dasatinib, and contributes to TKI resistance in vitro, in both cell lines and primary patient mononuclear cells.

ATP Binding Cassette family efflux proteins ABCB1 and ABCG2 have previously been demonstrated to interact with Tyrosine Kinase Inhibitors (TKIs); however, evidence for the interaction of other potentially relevant drug transporters with TKIs is lacking. Through Taqman transporter array technology we assessed the impact of nilotinib on mRNA expression of ABC transporters, with ABCC6 identified as a transporter of interest. Additionally, increased expression of ABCC6 mRNA was observed during in vitro development of nilotinib resistance in BCR-ABL1-expressing cell lines. K562 cells exposed to gradually increasing concentrations of nilotinib (to 2 μM) expressed up to 57-fold higher levels of ABCC6 mRNA when compared with control cells (p = 0.002). Analogous results were observed in nilotinib resistant K562-Dox cells (up to 33-fold higher levels of ABCC6, p = 0.002). IC50 experiments were conducted on patient mononuclear cells in the absence and presence of three ABCC6 inhibitors: indomethacin, probenecid and pantoprazole. Results demonstrated that all three inhibitors significantly reduced nilotinib IC50 (p<0.001) indicating ABCC6 is likely involved in nilotinib transport. Cell line data confirmed these findings. Similar results were obtained for dasatinib, but not imatinib. Combined, these studies suggest that nilotinib and dasatinib are likely substrates of ABCC6 and to our knowledge, this is the first report of ABCC6 involvement in TKI transport. In addition, ABCC6 overexpression may also contribute to nilotinib and dasatinib resistance in vitro. With nilotinib and dasatinib now front line therapy options in the treatment of CML, concomitant administration of ABCC6 inhibitors may present an attractive option to enhance TKI efficacy.

Dual HDAC and PI3K Inhibitor, CUDC-907 Alone or in Combination with ABL Tyrosine Kinase Inhibitor Against ABL Tyrosine Kinase Inhibitor Resistant Leukemia Cells

Introduction: Chronic myeloid leukemia (CML) is characterized by the t(9:22) translocation known as the Philadelphia chromosome (Ph). ABL tyrosine kinase inhibitor (TKI), imatinib and second-generation ABL TKIs, nilotinib and dasatinib have demonstrated the potency against CML patients. However, resistance to ABL TKI can develop in CML patients due to BCR-ABL point mutations. Moreover, ABL TKIs do not eliminate the leukemia stem cells (LSCs). Therefore, new approach against BCR-ABL mutant cells and LSCs may improve the outcome of Ph-positive leukemia patients. In eukaryotic cells, histone acetylation/deacetylation is important in transcriptional regulation. Chromatin acetylation is controlled by the opposing effects of two families of enzymes: histone acetyltransferases (HAT) and histone deacetylases (HDACs). Deregulation of HDAC activity may be a cause of malignant disease in humans. Phosphoinositide 3-kinase (PI3K) pathway also regulates cell metabolism, proliferation and survival. Furthermore, aberrant activation of PI3K signaling pathway has been shown to be important in initiation maintenance of human cancers. CUDC-907 is an oral inhibitor of class I PI3K as well as class I and II HDAC enzymes. CUDC-907 is currently being investigated in a pivotal phase 1 clinical trial against hematological malignancies such as malignant lymphoma. We suggested that CUDC-907 mediated inhibition PI3K and HDAC activity and in combination with ABL TKIs may abrogate the proliferation and survival of Ph-positive leukemia cells including T315I mutation and ABL TKI resistant.Materials and methods: In this study, we investigated the combination therapy with a CUDC-907 and an ABL TKIs (imatinib, nilotinib and ponatinib) by using the BCR-ABL positive cell line, K562, murine Ba/F3 cell line which was transfected with T315I mutant, nilotinib resistant K562 and ponatinib resistant Ba/F3 cells and primary samples.Results: The treatment of imatinib, nilotinib and ponatinib exhibits cell growth inhibition partially against K562 cells in the presence of feeder cell (HS-5). We found that mRNA of PI3K subunit is significantly increased after a co-culture with HS-5 in K562 and primary CD34 positive CML samples. 72 h treatment of CUDC-907 exhibits cell growth inhibition and induced apoptosis against K562 cells in a dose dependent manner. We examined the intracellular signaling after treatment of CUDC-907. Phosphorylation of JNK, histone acetylation and activity of caspase 3, poly (ADP-ribose) polymerase (PARP) was increased. Anti-apoptotic protein, Mcl-1 was decreased in a dose dependent. We next investigated the efficacy between imatinib and CUDC-907 by using these cell line. Combined treatment of K562 cells with imatinib and CUDC-907 caused significantly more cytotoxicity than each drug alone. Caspase activity was increased and Akt activity was reduced. Phosphorylation of BCR-ABL, Crk-L was reduced and cleaved PARP was increased after imatinib and CUDC-907 treatment. We investigated the CUDC-907 activity against T315I positive cells. CUDC-907 potently induced cell growth inhibition of Ba/F3 T315I cells in a dose dependent manner. Combined treatment of Ba/F3 T315I cells with ponatinib and CUDC-907 caused significantly more cytotoxicity than each drug alone. Caspase activity was increased and Akt activity was reduced after ponatinib and CUDC-907 treatment. To assess the activity of ponatinib and CUDC-907, we performed to test on tumor formation in mice. We injected nude mice subcutaneously with Ba/F3 T315I mutant cells. A dose of 20 mg/kg/day p.o of ponatinib and 30 mg/kg/day p.o of CUDC-907 inhibited tumor growth and reduced tumor volume compared with control mice. The treatments were well tolerated with no animal health concerns observed. We also found that the treatment of CUDC-907 exhibits cell growth inhibition against Ba/F3 ponatinib resistant cells, K562 nilotinib resistant cells, T315I mutant primary samples and CD34 positive CML samples.Conclusion: These results indicated that administration of the dual PI3K and HDAC inhibitor, CUDC-907 may be a powerful strategy against ABL TKI resistant cells including T315I mutation and enhance cytotoxic effects of ABL TKI against those Ph-positive leukemia cells. DisclosuresNo relevant conflicts of interest to declare.

Anti-Leukemic Activity of Phosphoinositide 3-Kinase Inhibitor, Copanlisib in ABL Tyrosine Kinase Resistant Leukemia Cells

Introduction: Chronic myelogenous leukemia (CML) is characterized by cytogenetic aberration (Philadelphia chromosome: Ph) and chimeric oncoprotein BCR-ABL. ABL tyrosine kinase inhibitor (TKI) therapy such as imatinib, nilotinib and dasatinib has improved the survival of Ph-positive leukemia patients. Although impressive clinical responses are obtained in the majority of CML patients, increasing evidence of acquired resistance to TKIs have been documented in clinically. Moreover, ABL TKIs cannot eradicate leukemia stem cells, thus, TKIs do not appear to lead to a cure the diseases. Therefore, new approach against BCR-ABL mutant cells and leukemia stem cells may improve the outcome of Ph-positive leukemia patients. Phosphoinositide 3-kinase (PI3K) pathway regulates cell metabolism, proliferation and survival. Furthermore, aberrant activation of PI3K signaling pathway has been shown to be important in initiation maintenance of human cancers. Copanlisib, also known as BAY80-6946 is a PI3K inhibitor with potential antineoplastic activity. Copanlisib is being investigated in a pivotal phase 2 clinical trial against hematological malignancies such as malignant lymphoma. We hypothesized that targeting PI3K, in combination with ABL TKI, would result in enhanced therapeutic activity in Ph-positive leukemia cells including T315I mutation and ABL TKI resistant.Materials and methods: We investigated the combination therapy with a copanlisib and an ABL TKIs (imatinib, nilotinib and ponatinib) by using the BCR-ABL positive cell line, K562, murine Ba/F3 cell line which was transfected with T315I mutant, nilotinib resistant K562, ponatinib resistant Ba/F3 cells and primary sample.Results: 72 h treatment of copanlisib exhibits cell growth inhibition against K562 cells in a dose dependent manner. The treatment of imatinib, nilotinib and ponatinib exhibits cell growth inhibition partially against K562 cells co-cultured with feeder cell line, HS-5. We found that the treatment of copanlisib abrogated the protective effects of HS-5 in K562 cells. We examined the intracellular signaling after treatment of copanlisib. High concentration of copanlisib reduced the phosphorylation of BCR-ABL and Crk-L. Activity of caspase 3 and poly (ADP-ribose) polymerase (PARP) was increased. We next investigated the efficacy between ABL TKI and copanlisib by using these cell line. Phosphorylation of BCR-ABL, Crk-L and Akt was reduced after imatinib and copanlisib treatment. We investigated the copanlisib activity against T315I positive cells. Copanlisib potently induced cell growth inhibition of Ba/F3 T315I cells. Combined treatment of Ba/F3 T315I cells with ponatinib and copanlisib caused significantly more cytotoxicity than each drug alone. Phosphorylation of BCR-ABL and Crk-L was reduced and cleaved PARP was increased after ponatinib and copanlisib treatment. To assess the activity of copanlisib and ponatinib, we performed to test on CML tumor formation in mice. We injected nude mice subcutaneously with Ba/F3 T315I mutant cells. A dose of 20 mg/kg/day p.o of ponatinib and 6 mg/kg/three times per week i.p of copanlisib inhibited tumor growth and reduced tumor volume compared with control mice. The treatments were well tolerated with no animal health concerns observed. We also found that the treatment of copanlisib exhibits cell growth inhibition against Ba/F3 ponatinib resistant cells, K562 nilotinib resistant cells and primary sample.Conclusion: These results indicated that administration of the PI3K inhibitor, copanlisib may be a powerful strategy against ABL TKI resistant cells including T315I mutation and enhance cytotoxic effects of ABL TKI against those Ph-positive leukemia cells. DisclosuresOhyashiki:Bristol: Honoraria, Research Funding.

Inhibition of PI3K/mTOR Overcomes Nilotinib Resistance in BCR-ABL1 Positive Leukemia Cells through Translational Down-Regulation of MDM2

Chronic myeloid leukemia (CML) is a cytogenetic disorder resulting from formation of the Philadelphia chromosome (Ph), that is, the t(9;22) chromosomal translocation and the formation of the BCR-ABL1 fusion protein. Tyrosine kinase inhibitors (TKI), such as imatinib and nilotinib, have emerged as leading compounds with which to treat CML. t(9;22) is not restricted to CML, 20-30% of acute lymphoblastic leukemia (ALL) cases also carry the Ph. However, TKIs are not as effective in the treatment of Ph+ ALL as in CML. In this study, the Ph+ cell lines JURL-MK2 and SUP-B15 were used to investigate TKI resistance mechanisms and the sensitization of Ph+ tumor cells to TKI treatment. The annexin V/PI (propidium iodide) assay revealed that nilotinib induced apoptosis in JURL-MK2 cells, but not in SUP-B15 cells. Since there was no mutation in the tyrosine kinase domain of BCR-ABL1 in cell line SUP-B15, the cells were not generally unresponsive to TKI, as evidenced by dephosphorylation of the BCR-ABL1 downstream targets, Crk-like protein (CrkL) and Grb-associated binder-2 (GAB2). Resistance to apoptosis after nilotinib treatment was accompanied by the constitutive and nilotinib unresponsive activation of the phosphoinositide 3-kinase (PI3K) pathway. Treatment of SUP-B15 cells with the dual PI3K/mammalian target of rapamycin (mTOR) inhibitor BEZ235 alone induced apoptosis in a low percentage of cells, while combining nilotinib and BEZ235 led to a synergistic effect. The main role of PI3K/mTOR inhibitor BEZ235 and the reason for apoptosis in the nilotinib-resistant cells was the block of the translational machinery, leading to the rapid downregulation of the anti-apoptotic protein MDM2 (human homolog of the murine double minute-2). These findings highlight MDM2 as a potential therapeutic target to increase TKI-mediated apoptosis and imply that the combination of PI3K/mTOR inhibitor and TKI might form a novel strategy to combat TKI-resistant BCR-ABL1 positive leukemia.

Efficacy Of The Polo-Like Kinase Inhibitor, Rigosertib Alone Or In Combination With ABL Tyrosine Kinase Inhibitor Against BCR-ABL-Positive Leukemia Cells

Chronic myeloid leukemia (CML) is characterized by the t(9:22) translocation known as the Philadelphia chromosome (Ph). ABL tyrosine kinase inhibitor (TKI), imatinib and second-generation ABL TKIs, nilotinib and dasatinib have demonstrated the potency against CML patients. However, resistance to ABL TKI can develop in CML patients due to BCR-ABL point mutations. Moreover, ABL TKIs do not eliminate the leukemia stem cells (LSCs). These leukemia cells are contained within a niche in the bone marrow and are often impervious to current treatments. Therefore, new approach against BCR-ABL mutant cells and LSCs may improve the outcome of Ph-positive leukemia patients. Polo-like kinases (PLK) are the family of serine threonine kinases and essential for mitosis. PLK is also critical regulator of cell cycle progression and DNA damage response. One of the PLK and phosphoinositide 3-kinase (PI3K) inhibitor, rigosertib (ON 01910.Na) is a novel synthetic benzyl styryl sulfone that is cytotoxic against a variety of human tumor cell lines. Rigosertib is currently being investigated in a pivotal phase 3 clinical trial against hematological malignancies such as myelodysplastic syndromes (MDS). We suggested that rigosertib mediated inhibition PLK and PI3K activity and in combination with ABL TKIs may abrogate the proliferation and survival of Ph-positive leukemia cells including T315I mutation and ABL TKI resistant. In this study, we investigated the combination therapy with a rigosertib and an ABL TKIs (imatinib, nilotinib and ponatinib) by using the BCR-ABL positive cell line, K562, murine Ba/F3 cell line with T315I mutant, nilotinib resistant K562 and ponatinib resistant Ba/F3 cells. 72 h treatment of rigosertib exhibits cell growth inhibition and induced apoptosis against K562 cells in a dose dependent manner. The treatment of imatinib, nilotinib and ponatinib exhibits cell growth inhibition partially against K562 cells in the presence of feeder cell (HS-5) conditioned media. We found that the treatment of rigosertib abrogated the protective effects of HS-5 conditioned media in K562 cells. We examined the intracellular signaling after treatment of rigosertib. Phosphorylation of BCR-ABL, Crk-L was not reduced. However, activity of caspase 3 was increased. We next investigated the efficacy between imatinib and rigosertib by using these cell line. Combined treatment of K562 cells with imatinib and rigosertib caused significantly more cytotoxicity than each drug alone. Phosphorylation of BCR-ABL, Crk-L was reduced and cleaved PARP and γH2A.X phosphorylation was increased after imatinib and rigosertib treatment. Anti-apoptotic protein, Mcl-1 was also decreased. We also found the phosphorylation of histone H3 was increased after rigosertib treatment suggested that the cells arrested in G2/M phase. We investigated the rigosertib activity against T315I positive cells. Rigosertib potently induced cell growth inhibition of Ba/F3 T315I cells. Combined treatment of Ba/F3 T315I cells with ponatinib and rigosertib caused significantly more cytotoxicity than each drug alone.We next investigated by using ponatinib resistant Ba/F3 cells and nilotinib resistant K562 cells. In the ponatinib resistant cell lines, IC50 of ponatinib was up to 200 nM. BCR-ABL triple point mutations (T315I, E255K and Y253H) were detected by direct sequence analysis. The treatment of rigosertib exhibits cell growth inhibition against Ba/F3 ponatinib resistant cells and K562 nilotinib resistant cells. These results indicated that administration of the PLK and PI3K inhibitor, rigosertib may be a powerful strategy against ABL TKI resistant cells and enhance cytotoxic effects of ABL TKI against those Ph-positive leukemia cells. Disclosures:Ohyashiki:Novartis: Honoraria, Research Funding.

Inhibition Of PI3K/mTOR Overcomes Nilotinib Resistance In BCR-ABL1 Positive Leukemia Cells

Expression of the Philadelphia chromosome (Ph), i.e., the t(9;22) chromosomal translocation and the formation of the BCR-ABL1 fusion protein, is the hallmark of chronic myeloid leukemia (CML). Tyrosine kinase inhibitors (TKI), such as imatinib and nilotinib, have emerged as leading compounds to treat CML. Translocation t(9;22) does not only occur in CML, 20-30% of acute lymphoblastic leukemia (ALL) are also found to carry the Ph. However, TKIs are not as effective in the treatment of Ph+ ALL as in CML. In this study, the Ph+ cell lines JURL-MK2 and SUP-B15 were used to investigate TKI resistance mechanisms and sensitization of Ph+ tumor cells to TKI treatment. The annexin V/PI (propidium iodide) assay revealed that nilotinib induced apoptosis in JURL-MK2 cells, but not in SUP-B15 cells. There was no mutation in the tyrosine kinase domain of BCR-ABL1 in both cell lines. SUP-B15 cells were not generally irresponsive to TKI, evidenced by dephosphorylation of the BCR-ABL1 downstream target GAB2 (Grb-associated binder-2). The resistance to apoptosis after nilotinib treatment was accompanied by the constitutive and nilotinib irresponsive activation of the phosphoinositide 3-kinase (PI3K) pathway. Treatment of SUP-B15 with the dual PI3K/mammalian target of rapamycin (mTOR) inhibitor BEZ235 alone induced apoptosis in a low percentage of cells, combination of nilotinib and BEZ235 led to a synergistic effect. Caspase 3 and PARP (poly ADP ribose polymerase) cleavage confirmed that apoptosis was induced after combined treatment. The main role of PI3K/mTOR inhibitor BEZ235 and the reason for apoptosis in the nilotinib-resistant cells was the block of the translational machinery, leading to the rapid down-regulation of anti-apoptotic protein MDM2 (human homolog of the murine double minute 2, hMDM2). The current findings suggest that MDM2 may be a therapeutic target to increase TKI-mediated apoptosis and that the combination of PI3K/mTOR dual inhibitor and TKI inhibitor might turn out to be a novel strategy for TKI-resistant BCR-ABL1 positive leukemia. Disclosures:No relevant conflicts of interest to declare.

Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells

Because a substantial number of patients with chronic myeloid leukemia acquire resistance to ABL tyrosine kinase inhibitors (TKIs), their management remains a challenge. Ponatinib, also known as AP24534, is an oral multi-targeted TKI. Ponatinib is currently being investigated in a pivotal phase 2 clinical trial. In the present study, we analyzed the molecular and functional consequences of ponatinib against imatinib- or nilotinib-resistant (R) K562 and Ba/F3 cells. The proliferation of imatinib- or nilotinib-resistant K562 cells did not decrease after treatment with imatinib or nilotinib. Src family kinase Lyn was activated. Point mutation Ba/F3 cells (E334V) were also highly resistant to imatinib and nilotinib. Treatment with ponatinib for 72h inhibited the growth of imatinib- and nilotinib-resistant cells. The phosphorylation of BCR-ABL, Lyn, and Crk-L was reduced. This study demonstrates that ponatinib has an anti-leukemia effect by reducing ABL and Lyn kinase activity and this information may be of therapeutic relevance.

Abstract 4095: Paradoxical activation of the RAF-MEK-ERK pathway in response to nilotinib induces synthetic lethality with MEK inhibition in head and neck cancer cells.

Abstract Cancers of the head and neck are difficult to treat because both the therapy and the tumor impact essential functions such as speech and swallowing and they can significantly alter facial appearance. The RAS/RAF/MEK/ERK cascade is one of the major signaling pathways that leads to head and neck squamous cell carcinoma (HNSCC) cell survival. There are three RAS genes (HRAS, KRAS, and NRAS) and together, they are mutated in about 30% of human cancers. RAS can also be activated by upstream signaling. Paradoxically RAF inhibition induces RAF-dimerization and causes subsequent activation of MEK/ERK pathway in presence of activated RAS in melanoma. Similarly the ABL inhibitor nilotinib acts as weak inhibitor of RAF kinase and also induces RAF-dimerization and subsequent activation of MEK/ERK in the presence of activated RAS in leukemia and pancreatic cancer cells. Nilotinib synergizes with MEK inhibitition to kill nilotinib-resistant leukemia cells (Abl T351I). We hypothesized that nilotinib and the MEK inhibitor MEK162 would be synergistic in HNSCC due to the frequent activation of RAS via upstream growth factor receptors. RAS mutations are relatively uncommon in HNSCC. We screened 60 validated HNSCC cell lines through sequenome analysis and we found three HNSCC cell lines with activating HRAS mutations (G12D and Q61L) [∼5%]. We found that nilotinib induces significant RAF dimerization and subsequent activation of MEK-ERK pathway irrespective of RAS mutational status. We next investigated how paradoxical MEK/ERK pathway activation affected the growth of HNSCC cells. In this context we treated a panel of HNSCC cell lines harboring WT and mutated HRAS with nilotinib and MEK162. Nilotinib alone had nearly no effect on cell viability and MEK162 had a moderate effect on some HNSCC lines. The combination of nilotinib and MEK162 synergized to inhibit the growth of HNSCC cells independent of RAS mutational status. The drug combination led to an accumulation of cells in G1 and a significant reduction in cell proliferation as measured with BrDU incorporation and colony formation. HNSCC cells with mutant HRAS undergo apoptosis in presence of both the drugs. In contrast none of these drugs induce senescence in HNSCC cell lines. Here we show that in HNSCC, nilotinib as single agent drives the paradoxical activation of RAF-MEK-ERK pathway in RAS mutation-independent manner and we uncovered a synthetic lethal pathway that can be used treat HNSCC. Animal studies are planned. This work was supported by University of Texas SPORE in Head and Neck Cancer P50 CA097007. Citation Format: Banibrata Sen, Tuhina Mazumdar, Shaohua Peng, Katherine S. Hale, Bonnie S. Glisson, Faye M. Johnson. Paradoxical activation of the RAF-MEK-ERK pathway in response to nilotinib induces synthetic lethality with MEK inhibition in head and neck cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4095. doi:10.1158/1538-7445.AM2013-4095

Mechanisms responsible for nilotinib resistance in human chronic myeloid leukemia cells and reversal of resistance

Multidrug resistance remains a significant obstacle to successful chemotherapy. The ability to determine the possible resistance mechanisms and surmount the resistance is likely to improve chemotherapy. Nilotinib is a very effective drug in the treatment of imatinib-sensitive or -resistant patients. Although very successful hematologic and cytogenetic responses have been obtained in nilotinib-treated patients, in recent years cases showing resistance to nilotinib have been observed. We aimed to examine the mechanisms underlying nilotinib resistance and to provide new targets for the treatment of chronic myeloid leukemia (CML). There was an up-regulation of antiapoptotic BCR/ABL, GCS and SK-1 genes and MRP1 transporter gene and down-regulation of apoptotic Bax and CerS1 genes in nilotinib-resistant cells. There was no mutation in the nilotinib-binding region of BCR/ABL in resistant cells. Inhibiton of GCS and SK-1 restored nilotinib sensitivity. Targeting the proteins that are involved in nilotinib resistance in addition to the inhibition of BCR/ABL could be a better method of treatment in CML.

Macromolecular Changes in Nilotinib Resistant K562 Cells; an In vitro Study by Fourier Transform Infrared Spectroscopy

Nilotinib is a second generation tyrosine kinase inhibitor which is used in both first and second line treatment of chronic myeloid leukemia (CML). In the present work, the effects of nilotinib resistance on K562 cells were investigated at the molecular level using Fourier transform infrared (FT-IR) spectroscopy. Human K562 CML cells were exposed to step-wise increasing concentrations of nilotinib, and sub-clones of K562 cells resistant to 50nM nilotinib were generated and referred to as K562/NIL-50 cells. Antiproliferative effects of nilotinib were determined by XTT cell proliferation assay. Changes in macromolecules in parental and resistant cells were studied by FT-IR spectroscopy. Nilotinib resistance caused significant changes which indicated increases in the level of glycogen and membrane/lipid order. The amount of unsaturated lipids increased in the nilotinib resistant cells indicating lipid peroxidation. The total amount of lipids did not change significantly but the relative proportion of cholesterol and triglycerides altered considerably. Moreover, the transcriptional status decreased but metabolic turn-over increased as revealed by the FT-IR spectra. In addition, changes in the proteome and structural changes in both proteins and the nucleus were observed in the K562/NIL-50 cells. Protein secondary structural analyses revealed that alpha helix structure and random coil structure decreased, however, anti-parallel beta sheet structure, beta sheet structure and turns structure increased. These results indicate that the FT-IR technique provides a method for analyzing drug resistance related structural changes in leukemia and other cancer types.

Abstract PL04-02: Ras and Raf signaling: From biology to therapeutics

Abstract The protein kinase BRAF is mutated in about 50% of human melanomas and the small G-protein NRAS is mutated in another 20% of cases. We have developed mouse models of melanoma driven by oncogenic BRAF, NRAS and KRAS. These studies have established that acquisition of BRAF, NRAS or KRAS mutations can be founder events in melanomagenesis and we are currently using these models to investigate gene-gene and gene-environment interactions in cutaneous melanoma. We are also investigating other targets of BRAF in melanoma cells and have shown that BRAF drives melanoma cell migration and invasion by down-regulating expression of the cyclic GMP phosphodiesterase PDE5A. 2011 was a landmark year in melanoma research with the registration of vemurafenib and ipilimumab, drugs that show clinical benefit in patients with advanced disease. Critically, BRAF inhibitory drugs achieve objective clinical responses in about 80% of melanomas that harbour mutations in BRAF. Surprisingly however, these drugs also drive paradoxical activation of CRAF in melanoma cells that harbour mutations in NRAS, or in cells in which RAS is activated due to increased upstream signalling. This is due to the induction of BRAF/CRAF heterodimers and CRAF homodimers and importantly, activation of the pathway by this paradox can drive tumourigenesis in some circumstances. We have used a two-stage skin carcinogenesis model to show that BRAF inhibitors are not tumour promoters pre se, but rather that they work by accelerating the growth of tumours carrying pre-existing mutations in HRAS. Thus, we conclude that BRAF inhibitors appear to accelerate the inevitable in susceptible patients, but do not induce these lesions de novo. Notably, concurrent administration of a MEK inhibitor with a BRAF drug blocks the development of these skin lesions, providing a rationale for combining BRAF and MEK inhibitors in melanoma patients. Unexpectedly, we have also found that the ABL/CKIT inhibitor nilotinib also drives paradoxical activation of the RAF pathway because it possesses weak off-target activity against BRAF and CRAF. The consequence of this paradoxical activation is that nilotinib provides a survival advantage to chronic myeloid leukaemia (CML) cells that are resistant to nilotinib, but also provides an Achilles heel to these cells because they become dependent on MEK signalling in the presence of nilotinib. Thus, MEK inhibitors cooperate with nilotinib to drive synthetic lethality in nilotinib-resistant CML cells. Thus, paradoxical activation of the RAF proteins by small molecular weight drugs can have important consequences to patient responses, creating specific clinical problems in some circumstances, but therapeutic opportunities in others. 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 PL04-02. doi:1538-7445.AM2012-PL04-02

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.

Quantitative phosphoproteomics revealed interplay between Syk and Lyn in the resistance to nilotinib in chronic myeloid leukemia cells

AbstractIn this study, we have addressed how Lyn kinase signaling mediates nilotinib-resistance by quantitative phospho-proteomics using Stable Isotope Labeling with Amino acid in Cell culture. We have found an increased tyrosine phosphorylation of 2 additional tyrosine kinases in nilotinib-resistant cells: the spleen tyrosine kinase Syk and the UFO family receptor tyrosine kinase Axl. This increased tyrosine phosphorylation involved an interaction of these tyrosine kinases with Lyn. Inhibition of Syk by the inhibitors R406 or BAY 61-3606 or by RNA interference restored the capacity of nilotinib to inhibit cell proliferation. Conversely, coexpression of Lyn and Syk were required to fully induce resistance to nilotinib in drug-sensitive cells. Surprisingly, the knockdown of Syk also strongly decreased tyrosine phosphorylation of Lyn and Axl, thus uncovering interplay between Syk and Lyn. We have shown the involvement of the adaptor protein CDCP-1 in resistance to nilotinib. Interestingly, the expression of Axl and CDCP1 were found increased both in a nilotinib-resistant cell line and in nilotinib-resistant CML patients. We conclude that an oncogenic signaling mediated by Lyn and Syk can bypass the need of Bcr-Abl in CML cells. Thus, targeting these kinases may be of therapeutic value to override imatinib or nilotinib resistance in CML.

Dasatinib preferentially induces apoptosis by inhibiting Lyn kinase in nilotinib-resistant chronic myeloid leukemia cell line

Nilotinib is approved for treatment of newly diagnosed chronic myeloid leukemia (CML) and it is shown superiority over imatinib in first-line treatment for patients of CML. In this study, we established a nilotinib-resistant cell line, K562NR, and evaluated the resistance to nilotinib and efficacy of dasatinib. We found activation of Lyn plays a dominant role in survival of the nilotinib-resistant cell line. We found dasatinib induces the apoptosis of nilotinib-resistant cells and inhibits Lyn kinase activity. This novel nilotinib-resistant CML cell line may help to explore novel therapy for CML.

Quantitative Phosphoproteomics Identified a New Syk-Lyn-Axl Signalling Pathway Involved In Resistance to Nilotinib In Chronic Myeloid Leukemia Cells.

AbstractAbstract 3376Nilotinib has been developed to overcome resistance to imatinib, the first line treatment of chronic myeloid leukemia (CML). To anticipate resistance to nilotinib, we generate nilotinib resistant CML cell lines in vitro to characterize mechanisms and signaling pathways that may contribute to resistance. Among the different mechanisms of resistance identified, the overexpression of the Src-kinase Lyn was involved in resistance both in vitro, in a K562 cell line (K562-rn), and in vivo, in nilotinib-resistant CML patients. To characterize how Lyn mediates resistance, we performed a phosphoproteomic study using SILAC (Stable Isotope Labelling with Amino acid in Cell culture). Quantification and identification of phosphotyrosine proteins in the nilotinib resistant cells point out two tyrosine kinases, the spleen tyrosine kinase Syk and the UFO receptor Axl. The two tyrosine kinase Syk and Axl interact with Lyn as seen by coimmunopreciptation. Syk is phosphorylated on tyrosine 323 and 525/526 in Lyn dependent manner in nilotinib resistant cells. The inhibition of Syk tyrosine kinase by R406 or BAY31-6606 restores sensitivity to nilotinib in K562-rn cells. In parallel, the inhibition of Syk expression by ShRNA in K562-rn cells abolishes Lyn and Axl phosphorylation and then interaction between Lyn and Axl leading to a full restoration of nilotinib efficacy. In the opposite, the coexpression of Lyn and Syk in nilotinib sensitive K562 cells induced resistance to nilotinib whereas a Syk kinase dead mutant did not. These results highlight for the first time the critical role of Syk in resistance to tyrosine kinase inhibitors in CML disease emphasizing the therapeutic targeting of this tyrosine kinase. Moreover, Axl, which is already a target in solid tumor, will be also an interesting pathway to target in CML. Disclosures:No relevant conflicts of interest to declare.

Tyrosine Kinase Proteins profiling of Nilotinib Resistant Chronic Myelogenous Leukemia Cells Unravels a Tyrosine Kinase-Mediated Bypass.

Abstract 2175Poster Board II-152Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in Chronic Myeloid Leukemia (CML) and in Bcr-Abl positive Acute Lymphoblastic Leukemia. Whereas imatinib, a selective inhibitor of Bcr-Abl tyrosine kinase, is now used in frontline therapy for CML, second generation inhibitors such as nilotinib or dasatinib have been developed for the treatment of imatinib-resistant or –intolerant disease. We have shown that one of the mechanisms of resistance to nilotinib is an increasing expression of the p53/56 Lyn kinase, both at mRNA and protein level in cell lines. This result was confirmed in vivo in nilotinib-resistant CML patients (Mahon et al. Cancer Res., 2008, 68(23):9809-16.). To elucidate Lyn mediated-nilotinib resistance, a phosphoproteomic study was performed by Stable Isotope Labelling with Amino acid in Cell culture (SILAC) which highlights the potential role of downstream tyrosine kinases. Among different candidate proteinsThe Spleen tyrosine kinase Syk and the UFO family receptor tyrosine kinase Axl were the most relevant in the nilotinib resistant cell line as compared to the sensitive counterpart. Syk hyperphosphorylation was confirmed in the nilotinib resistant cell line using western blot at least on tyrosine residues Y323 and Y525/526, two critical tyrosine residues respectively involved in Lyn-mediated Syk phosphorylation and autophosphorylation-associated Syk activation. Lyn interacts with Syk as detected in Syk immunoprecipitates in nilotinib resistant cells. Furthermore, Syk-Lyn interaction is inhibited by dasatinib suggesting the requirement of Lyn kinase activity and Syk phosphorylation. Targeting Syk expression in nilotinib resistant cells by siRNA or tyrosine kinase activity by pharmacological inhibitors leads respectively to a partial (35%) or to a full restoration of nilotinib sensitivity. Moreover, the identification of Axl by SILAC is correlated to a 9 fold increase of its level of expression in the resistant cell line and the inhibition of Axl tyrosine kinase activity decreases proliferation of both nilotinib sensitive and resistant CML cells. All together these results disclose a new pathway for tyrosine kinase inhibitors resistance in CML involving at least the two Lyn downstream tyrosine kinases Syk and Axl. Disclosures:Mahon:Amgen: Honoraria; Novartis Pharma: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria.

Evidence that Resistance to Nilotinib May Be Due to BCR-ABL, Pgp, or Src Kinase Overexpression

Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in chronic myeloid leukemia (CML) and in Bcr-Abl-positive acute lymphoblastic leukemia. Whereas imatinib, a selective inhibitor of Bcr-Abl tyrosine kinase, is now used in frontline therapy for CML, second-generation inhibitors of Bcr-Abl tyrosine kinase such as nilotinib or dasatinib have been developed for the treatment of imatinib-resistant or imatinib-intolerant disease. In the current study, we generated nilotinib-resistant cell lines and investigated their mechanism of resistance. Overexpression of BCR-ABL and multidrug resistance gene (MDR-1) were found among the investigated mechanisms. We showed that nilotinib is a substrate of the multidrug resistance gene product, P-glycoprotein, using verapamil or PSC833 to block binding. Up-regulated expression of p53/56 Lyn kinase, both at the mRNA and protein level, was found in one of the resistant cell lines and Lyn silencing by small interfering RNA restored sensitivity to nilotinib. Moreover, failure of nilotinib treatment was accompanied by an increase of Lyn mRNA expression in patients with resistant CML. Two Src kinase inhibitors (PP1 and PP2) partially removed resistance but did not significantly inhibit Bcr-Abl tyrosine kinase activity. In contrast, dasatinib, a dual Bcr-Abl and Src kinase inhibitor, inhibited the phosphorylation of both BCR-ABL and Lyn, and induced apoptosis of the Bcr-Abl cell line overexpressing p53/56 Lyn. Such mechanisms of resistance are close to those observed in imatinib-resistant cell lines and emphasize the critical role of Lyn in nilotinib resistance.

Lyn Kinase Overexpression Is One of the Mechanisms of Resistance to Nilotinib In Chronic Myeloid Leukemia

Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in Chronic Myeloid Leukemia (CML) and in Bcr-Abl positive Acute Lymphoblastic Leukemia. Whereas imatinib, a selective inhibitor of Bcr-Abl tyrosine kinase, is now used in frontline therapy for CML, second generation inhibitors of Bcr-Abl tyrosine kinase such as nilotinib or dasatinib have been developed. In the current study, we generated nilotinib-resistant cell lines and investigated their mechanism of resistance. Three nilotinib-resistant cell lines were obtained from the Ph-positive cell lines AR230, LAMA84 and K562. Over expression of the BCR-ABL gene was found in two nilotinib-resistant cell lines and the multidrug resistance gene (MDR-1) was found overexpressed in one of them, i.e, LAMA84 nilotinib resistant cell. The K562/DOX cell line, that displays resistance to several drugs by over expressing Pgp, was resistant to nilotinib, and this was reversed by simultaneous incubation with either verapamil or PSC833 confirming that nilotinib is a substrate of the Pgp. In one nilotinib-resistant cell line (K562-rn), we found over expression of p53/56 Lyn kinase, both at the mRNA and protein level (10- fold), and these cells were compared to their sensitive counterpart. Lyn silencing by siRNA restored sensitivity to nilotinib. Two Src kinase inhibitors (PP1 and PP2) partially restored sensitivity to nilotinib, but did not significantly inhibit Bcr-Abl tyrosine kinase activity. In contrast, dasatinib, an inhibitor of Abl and Src-family kinases, inhibited phosphorylation of both BCR-ABL and Lyn, and induced apoptosis of the Bcr-Abl cell line which over expressed p53/56 Lyn. Of 7 nilotinib-resistant CML patients, failure of nilotinib treatment was accompanied by mutations in Bcr-Abl kinase domain in 2 patients and an increase of Lyn mRNA expression (RQ-PCR) in 2 other patients. As an approach to confirm the involvement of the Lyn signalling pathway in nilotinib-resistance, we have used the Stable Isotope Labeling with Amino acids in Cell culture (SILAC) technique. The tyrosine phosphorylated protein fraction was analyzed by tandem mass spectrometry. Peptide sequencing and quantification in the nilotinib-resistant cell line identified >350 proteins, of which several were hyper-phosphorylated; functional analysis of the different candidates is in progress.In conclusion, mechanisms of resistance to nilotinib in imatinib-resistant cell lines resemble those operating in CML patients, and up-regulated Lyn kinase signalling can play an important role in nilotinib resistance.