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Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the US. The majority (~85%) of GISTs possess gain-of-function mutations in KITor PDGFRA, causing constitutive activation of the kinase receptor. GIST management has been transformed by the identification of tumor driver mutations leading to unprecedented disease control of advanced GIST with the introduction of imatinib mesylate (IM). Despite IM’s efficacy, most patients experience primary and/or secondary resistance within 2 y of treatment. Additional therapies and methods to optimize screening of novel approaches in preclinical studies are warranted. Clinically, treatment efficacy is typically assessed using Response Evaluation Criteria In Solid Tumors (RECIST) guidelines or Choi criteria. Both require a period of time on therapy before changes indicative of response can be observed. In addition, neither informs directly about cell death. We evaluated the use of molecular imaging technology in an animal model using near-infrared (NIR) imaging probes together with three-dimensional fluorescence molecular tomography (FMT) for assessing therapeutic response and ultimately optimizing our understanding of the biologic effects of these agents. We determined the potential of NIR probes (PSVueTM794 and cell-penetrating KcapQ647) for detecting distinct markers of apoptosis and compare this to tumor size measured by MRI in response to IM treatment in GIST-T1 xenografts. Our studies revealed statistically significant increases in apoptosis due to IM treatment using both probes as early as 24 h post IM treatment which was confirmed by IHC. Molecular imaging will allow for faster and more effective screening of novel therapies in preclinical GIST models.

The majority of gastrointestinal stromal tumors (GISTs) are characterized by oncogenic gain-of-function mutations in the receptor tyrosine kinase (RTK) c-KIT with a minority in PDGFRa. Therapy for GISTs has been revolutionized by the use of the selective tyrosine kinase inhibitor imatinib mesylate (IM). For the subset (~10-15%) of GISTs that lack oncogenic mutations in these receptors, the genetic changes driving tumorigenesis are unknown. We recently reported that the gene encoding the insulin-like growth factor 1 receptor (IGF-1R) is amplified in a subset of GISTs, and the IGF-1R protein is over-expressed in WT and pediatric GISTs. In this report we present a more complete picture of the involvement of components of the insulin-like growth factor-signaling pathway in the pathogenesis of GISTs. We also discuss how the IGF pathway may provide additional molecular targets for the treatment of GISTs that respond poorly to IM therapy.

Nbs1, a member of the Mre11-RAD50-Nbs1 complex, is phosphorylated by ATM, the product of the ataxia-telangiectasia mutated gene and a member of the phosphatidylinositol 3-kinase–related family of serine-threonine kinases, in response to DNA double-strand breaks (DSBs) to regulate DNA damage checkpoints. Here we show that BCR/ABL stimulated Nbs1 expression by induction of c-Myc–dependent transactivation and protection from caspase-dependent degradation. BCR/ABL-related fusion tyrosine kinases (FTKs) such as TEL/JAK2, TEL/PDGFβR, TEL/ABL, TEL/TRKC, BCR/FGFR1, and NPM/ALK as well as interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) also stimulated Nbs1 expression. Enhanced ATM kinase–dependent phosphorylation of Nbs1 on serine 343 (S343) in response to genotoxic treatment was detected in leukemia cells expressing BCR/ABL and other FTKs in comparison to normal counterparts stimulated with IL-3, GM-CSF, and SCF. Expression of Nbs1-S343A mutant disrupted the intra–S-phase checkpoint, decreased homologous recombinational repair (HRR) activity, down-regulated XIAP expression, and sensitized BCR/ABL-positive cells to cytotoxic drugs. Interestingly, inhibition of Nbs1 phosphorylation by S343A mutant enhanced the antileukemia effect of the combination of imatinib and genotoxic agent.

Nbs1, a member of the Mre11-RAD50-Nbs1 complex, is phosphorylated by ATM, the product of the ataxia-telangiectasia mutated gene and a member of the phosphatidylinositol 3-kinase–related family of serine-threonine kinases, in response to DNA double-strand breaks (DSBs) to regulate DNA damage checkpoints. Here we show that BCR/ABL stimulated Nbs1 expression by induction of c-Myc–dependent transactivation and protection from caspase-dependent degradation. BCR/ABL-related fusion tyrosine kinases (FTKs) such as TEL/JAK2, TEL/PDGFβR, TEL/ABL, TEL/TRKC, BCR/FGFR1, and NPM/ALK as well as interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) also stimulated Nbs1 expression. Enhanced ATM kinase–dependent phosphorylation of Nbs1 on serine 343 (S343) in response to genotoxic treatment was detected in leukemia cells expressing BCR/ABL and other FTKs in comparison to normal counterparts stimulated with IL-3, GM-CSF, and SCF. Expression of Nbs1-S343A mutant disrupted the intra–S-phase checkpoint, decreased homologous recombinational repair (HRR) activity, down-regulated XIAP expression, and sensitized BCR/ABL-positive cells to cytotoxic drugs. Interestingly, inhibition of Nbs1 phosphorylation by S343A mutant enhanced the antileukemia effect of the combination of imatinib and genotoxic agent.

BCR/ABL-positive leukemia cells accumulated more replication-dependent DNA double-strand breaks (DSBs) than normal counterparts after treatment with cisplatin and MMC, as assessed by pulse field gel electrophoresis (PFGE) and neutral comet assay. In addition, leukemia cells could repair these lesions more efficiently than normal cells and eventually survive genotoxic treatment. Elevated levels of drug-induced DSBs in leukemia cells were associated with higher activity of ATR kinase, and enhanced phosphorylation of histone H2AX on serine 139 (γ-H2AX). γ-H2AX eventually started to disappear in BCR/ABL cells, while continued to increase in parental cells. In addition, the expression and ATR-mediated phosphorylation of Chk1 kinase on serine 345 were often more abundant in BCR/ABL-positive leukemia cells than normal counterparts after genotoxic treatment. Inhibition of ATR kinase by caffeine but not Chk1 kinase by indolocarbazole inhibitor, SB218078 sensitized BCR/ABL leukemia cells to MMC in a short-term survival assay. Nevertheless, both caffeine and SB218078 enhanced the genotoxic effect of MMC in a long-term clonogenic assay. This effect was associated with the abrogation of transient accumulation of leukemia cells in S and G2/M cell cycle phases after drug treatment. In conclusion, ATR - Chk1 axis was strongly activated in BCR/ABL-positive cells and contributed to the resistance to DNA cross-linking agents causing numerous replication-dependent DSBs.

Growing evidence indicate that ABL kinase inhibitors may need partner drugs to cure BCR/ABL-positive leukemias. Genotoxic drugs have been successfully combined with imatinib mesylate to increase its anti-leukemia activity in vitro. Although BCR/ABL-positive cells may accumulate even higher levels of DNA damage in comparison to their normal counterparts the former cells repair the lesions more proficiently and eventually survive. Therefore, targeting the mechanisms responsible for survival of leukemia cells after genotoxic treatment may increase the chances to eradicate BCR/ABL-positive leukemias. Nbs1, a member of the Rad50/Mre11/Nbs1 complex, is phosphorylated by ATM on Serine 343 (S343) in response to DNA double strand breaks (DSBs) to regulate intra-S and G2/M cell cycle checkpoints and DNA repair. Here we show that BCR/ABL and other fusion tyrosine kinases (FTKs) such as TEL/ABL, TEL/JAK2, TEL/PDGFßR, TEL/TRKC, BCR/FGFR, and NPM/ALK, stimulate Nbs1 expression by protection from caspase-dependent degradation and induction of c-Myc-dependent transactivation. Downregulation of Nbs1 in BCR/ABL positive cells using siRNA increased their sensitivity to mitomycin C (MMC). Enhanced phosphorylation of Nbs1 on S343 (pNbs1) was detected by Western analysis in BCR/ABL-positive leukemia cells (CD34+ CML patient cells and leukemic cell lines) treated with various cytotoxic drugs (MMC, hydroxyurea = HU, cisplatin - CPL) in comparison to normal counterparts. This effect is associated with increased ATM kinase activity in BCR/ABL cells treated with MMC. In addition, immunofluoresence studies demonstrated an increase of the pNbs1 nuclear foci in BCR/ABL cells after MMC treatment in comparison to parental counterparts. DNA damage-dependent enhancement of pNbs1 appears to be a broad phenomenon because it was also detected in MMC-treated tumor cells expressing other FTKs. The radioresistant DNA synthesis (RDS) assay showed that MMC-treated CML patient cells and BCR/ABL-transformed cell lines displayed an inhibition of DNA synthesis associated with transient accumulation of the cells in S phase, indicating an intact intra-S phase checkpoint. Expression of the Nbs1-S343A phosphorylation-less mutant downregulated pNbs1 and disrupted intra-S phase checkpoint resulting in reduced accumulation of BCR/ABL leukemia cells in S phase after MMC treatment. This effect was associated with an increase of the sensitivity of leukemia cells to genotoxic treatment (MMC, HU, CPL). A combinatorial strategy was employed targeting enhanced Nbs1 phosphorylation and the deregulated BCR/ABL tyrosine kinase activity, using the Nbs1-S343A phosphorylation-less mutant and a sub-optimal concentration of STI571 eliminating ~50% of leukemia cells, respectively. Targeting both BCR/ABL kinase activity and Nbs1 phosphorylation in combination significantly sensitizes B/A-positive cells to MMC treatment, nearly eradicating all leukemia cells.

Growing evidence indicate that ABL kinase inhibitors may need partner drugs to cure BCR/ABL-positive leukemias. Genotoxic drugs have been successfully combined with imatinib mesylate to increase its anti-leukemia activity in vitro. Although BCR/ABL-positive cells may accumulate even higher levels of DNA damage in comparison to their normal counterparts the former cells repair the lesions more proficiently and eventually survive. Therefore, targeting the mechanisms responsible for survival of leukemia cells after genotoxic treatment may increase the chances to eradicate BCR/ABL-positive leukemias. Nbs1, a member of the Rad50/Mre11/Nbs1 complex, is phosphorylated by ATM on Serine 343 (S343) in response to DNA double strand breaks (DSBs) to regulate intra-S and G2/M cell cycle checkpoints and DNA repair. Here we show that BCR/ABL and other fusion tyrosine kinases (FTKs) such as TEL/ABL, TEL/JAK2, TEL/PDGFβR, TEL/TRKC, BCR/FGFR, and NPM/ALK, stimulate Nbs1 expression by protection from caspase-dependent degradation and induction of c-Myc-dependent transactivation. Downregulation of Nbs1 in BCR/ABL positive cells using siRNA increased their sensitivity to mitomycin C (MMC). Enhanced phosphorylation of Nbs1 on S343 (pNbs1) was detected by Western analysis in BCR/ABL-positive leukemia cells (CD34+ CML patient cells and leukemic cell lines) treated with various cytotoxic drugs (MMC, hydroxyurea = HU, cisplatin - CPL) in comparison to normal counterparts. This effect is associated with increased ATM kinase activity in BCR/ABL cells treated with MMC. In addition, immunofluoresence studies demonstrated an increase of the pNbs1 nuclear foci in BCR/ABL cells after MMC treatment in comparison to parental counterparts. DNA damage-dependent enhancement of pNbs1 appears to be a broad phenomenon because it was also detected in MMC-treated tumor cells expressing other FTKs. The radioresistant DNA synthesis (RDS) assay showed that MMC-treated CML patient cells and BCR/ABL-transformed cell lines displayed an inhibition of DNA synthesis associated with transient accumulation of the cells in S phase, indicating an intact intra-S phase checkpoint. Expression of the Nbs1-S343A phosphorylation-less mutant downregulated pNbs1 and disrupted intra-S phase checkpoint resulting in reduced accumulation of BCR/ABL leukemia cells in S phase after MMC treatment. This effect was associated with an increase of the sensitivity of leukemia cells to genotoxic treatment (MMC, HU, CPL). A combinatorial strategy was employed targeting enhanced Nbs1 phosphorylation and the deregulated BCR/ABL tyrosine kinase activity, using the Nbs1-S343A phosphorylation-less mutant and a sub-optimal concentration of STI571 eliminating ~50% of leukemia cells, respectively. Targeting both BCR/ABL kinase activity and Nbs1 phosphorylation in combination significantly sensitizes B/A-positive cells to MMC treatment, nearly eradicating all leukemia cells.

Nbs1, a member of the DNA repair/checkpoint activation complex Mre11/Rad50/Nbs1, is phosphorylated by ATM in response to the presence of DNA double strand breaks (DSBs) resulting in the activation of the S phase checkpoint. Here we show that the BCR/ABL tyrosine kinase, as well as growth factors (IL-3, GM-CSF), stimulate the expression of Mre11 and Nbs1, but not Rad50. This effect is dependent on the kinase activity of BCR/ABL protecting Mre11 and Nbs1 from caspase-dependent, but not proteasome-dependent degradation. Cells expressing the BCR/ABL kinase are resistant to chemotherapeutic agents, including mitomycin C (MMC). Western analysis showed enhanced phosphorylation of Nbs1 on Serine 343 in MMC-treated BCR/ABL leukemia cells (CML patient cells and leukemic cell lines) in comparison to normal cells in the presence of growth factors. Immunofluoresence studies demonstrated an increase of γ-H2AX nuclear foci (DSBs indicator) and pNbs1 nuclear foci in BCR/ABL cells after MMC treatment in comparison to parental counterparts. In addition, BCR/ABL-positive cells displayed a higher percentage of colocalization of γ-H2AX and p-Nbs1 implicating the presence of p-Nbs1 at the DSBs. Clonogenic assays performed after down regulation of Nbs1 in BCR/ABL positive cells using siRNA showed increased sensitivity to MMC. Specifically, the expression of the Nbs1-S343A phosphorylation-less mutant also decreased resistance in BCR/ABL cells to MMC. The radioresistant DNA synthesis (RDS) assay showed that MMC-treated CML patient cells, BCR/ABL-transformed cell lines and normal counterparts displayed an inhibition of DNA synthesis associated with transient accumulation of the cells in S phase. Expression of Nbs1-S343A mutant caused a significant decrease in the accumulation of BCR/ABL leukemia cells in S phase after MMC treatment, whereas cells transfected with both the empty construct and wild-type Nbs1 displayed S phase accumulation. Surprisingly, Nbs1-S343A mutant did not affect the ability of normal cells to accumulate in S phase in response to MMC. Altogether, we hypothesize that enhanced phosphorylation of Nbs1 on S343 leads to increased resistance to genotoxic agents in BCR/ABL leukemia cells by prolonging the S phase checkpoint and allowing longer time for the repair of excessive DNA damage.

BCR/ABL-positive leukemia cells accumulated more replication-dependent DNA double-strand breaks (DSBs) than normal counterparts after treatment with cisplatin and mitomycin C, as assessed by pulse field gel electrophoresis (PFGE) and neutral comet assays. In addition, leukemia cells could repair these lesions more efficiently than normal cells and eventually survive genotoxic treatment. BCR/ABL leukemia cells displayed higher activity of ATR kinase, which is stimulated by replication-dependent DSBs. ATR phosphorylates histone H2AX on serine 139 (γ-H2AX) on megabase-length fragments near DSB sites. In accordance with PFGE and comet assay, BCR/ABL-positive leukemia cells accumulated more γ-H2AX protein and γ-H2AX nuclear foci than normal counterparts during 6h after genotoxic treatment. γ-H2AX started to disappear in BCR/ABL cells, while continued to increase in parental cells. Altogether, these results support the hypothesis that higher kinase activity of ATR in BCR/ABL leukemia cells results from their response to elevated levels of drug-induced replication-dependent DSBs, and that more efficient repair is associated with better survival. More abundant expression and prolonged phosphorylation on serine 345 of the Chk1 kinase, an important ATR kinase downstream effector and cell cycle regulator, was detected in leukemia cell lines and CML patient cells after genotoxic treatment. Interestingly, inhibition of ATR kinase by caffeine, but not Chk1 kinase by indolocarbazole inhibitor SB-218078 sensitized BCR/ABL leukemia cells to mitomycin C. In conclusion, although ATR - Chk1 axis appeared strongly activated in BCR/ABL leukemia cells responding to DNA cross-linking agents causing numerous replication-dependent DSBs, its function in drug resistance may be disassembled downstream of the Chk1 kinase.

BCR/ABL-positive leukemia cells accumulated more replication-dependent DNA double-strand breaks (DSBs) than normal counterparts after treatment with cisplatin and mitomycin C, as assessed by pulse field gel electrophoresis (PFGE) and neutral comet assays. In addition, leukemia cells could repair these lesions more efficiently than normal cells and eventually survive genotoxic treatment. BCR/ABL leukemia cells displayed higher activity of ATR kinase, which is stimulated by replication-dependent DSBs. ATR phosphorylates histone H2AX on serine 139 (γ-H2AX) on megabase-length fragments near DSB sites. In accordance with PFGE and comet assay, BCR/ABL-positive leukemia cells accumulated more γ-H2AX protein and γ-H2AX nuclear foci than normal counterparts during 6h after genotoxic treatment. γ-H2AX started to disappear in BCR/ABL cells, while continued to increase in parental cells. Altogether, these results support the hypothesis that higher kinase activity of ATR in BCR/ABL leukemia cells results from their response to elevated levels of drug-induced replication-dependent DSBs, and that more efficient repair is associated with better survival. More abundant expression and prolonged phosphorylation on serine 345 of the Chk1 kinase, an important ATR kinase downstream effector and cell cycle regulator, was detected in leukemia cell lines and CML patient cells after genotoxic treatment. Interestingly, inhibition of ATR kinase by caffeine, but not Chk1 kinase by indolocarbazole inhibitor SB-218078 sensitized BCR/ABL leukemia cells to mitomycin C. In conclusion, although ATR - Chk1 axis appeared strongly activated in BCR/ABL leukemia cells responding to DNA cross-linking agents causing numerous replication-dependent DSBs, its function in drug resistance may be disassembled downstream of the Chk1 kinase.

Nbs1, a member of the DNA repair/checkpoint activation complex Mre11/Rad50/Nbs1, is phosphorylated by ATM in response to the presence of DNA double strand breaks (DSBs) resulting in the activation of the S phase checkpoint. Here we show that the BCR/ABL tyrosine kinase, as well as growth factors (IL-3, GM-CSF), stimulate the expression of Mre11 and Nbs1, but not Rad50. This effect is dependent on the kinase activity of BCR/ABL protecting Mre11 and Nbs1 from caspase-dependent, but not proteasome-dependent degradation. Cells expressing the BCR/ABL kinase are resistant to chemotherapeutic agents, including mitomycin C (MMC). Western analysis showed enhanced phosphorylation of Nbs1 on Serine 343 in MMC-treated BCR/ABL leukemia cells (CML patient cells and leukemic cell lines) in comparison to normal cells in the presence of growth factors. Immunofluoresence studies demonstrated an increase of γ-H2AX nuclear foci (DSBs indicator) and pNbs1 nuclear foci in BCR/ABL cells after MMC treatment in comparison to parental counterparts. In addition, BCR/ABL-positive cells displayed a higher percentage of colocalization of γ-H2AX and p-Nbs1 implicating the presence of p-Nbs1 at the DSBs. Clonogenic assays performed after down regulation of Nbs1 in BCR/ABL positive cells using siRNA showed increased sensitivity to MMC. Specifically, the expression of the Nbs1-S343A phosphorylation-less mutant also decreased resistance in BCR/ABL cells to MMC. The radioresistant DNA synthesis (RDS) assay showed that MMC-treated CML patient cells, BCR/ABL-transformed cell lines and normal counterparts displayed an inhibition of DNA synthesis associated with transient accumulation of the cells in S phase. Expression of Nbs1-S343A mutant caused a significant decrease in the accumulation of BCR/ABL leukemia cells in S phase after MMC treatment, whereas cells transfected with both the empty construct and wild-type Nbs1 displayed S phase accumulation. Surprisingly, Nbs1-S343A mutant did not affect the ability of normal cells to accumulate in S phase in response to MMC. Altogether, we hypothesize that enhanced phosphorylation of Nbs1 on S343 leads to increased resistance to genotoxic agents in BCR/ABL leukemia cells by prolonging the S phase checkpoint and allowing longer time for the repair of excessive DNA damage.