Limits...
Combination of the ABL kinase inhibitor imatinib with the Janus kinase 2 inhibitor TG101348 for targeting residual BCR-ABL-positive cells.

Okabe S, Tauchi T, Katagiri S, Tanaka Y, Ohyashiki K - J Hematol Oncol (2014)

Bottom Line: In addition, the combined treatment of CD34-positive primary samples resulted in considerably increased cytotoxicity, decreased Crk-L phosphorylation, and increased apoptosis.We also investigated TG101348 activity against feeder cells and observed that STAT5 phosphorylation, granulocyte macrophage colony-stimulating factor, and interleukin 6 levels decreased, indicating reduced cytokine production in HS-5 cells treated with TG101348.These results showed that JAK inhibitors may enhance the cytotoxic effect of imatinib against residual CML cells and that a combined approach may be a powerful strategy against the stroma-associated drug resistance of Philadelphia chromosome-positive cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: First Department of Internal Medicine, Tokyo Medical University, Shinjuku-ku, Tokyo 160-0023, Japan. okabe@tokyo-med.ac.jp.

ABSTRACT

Background: The ABL kinase inhibitor imatinib is highly effective in treating most, but not all, patients with chronic myeloid leukemia (CML). This is because residual CML cells are generally present in the bone marrow microenvironment and are refractory to imatinib. Hematopoietic cytokine receptor signaling is mediated by Janus kinases (JAKs) and their downstream transcription factor, signal transducer and activator of transcription (STAT). TG101348 (SAR302503) is an oral inhibitor of JAK2.

Methods: We investigated the efficacy of imatinib and TG101348 using the break point cluster region-c-Abelson (BCR-ABL)-positive cell line and primary CML samples wherein leukemia cells were protected by a feeder cell line (HS-5).

Results: Imatinib treatment resulted in partial inhibition of cell growth in HS-5-conditioned medium. Furthermore, combined treatment with imatinib and TG101348 abrogated the protective effects of HS-5-conditioned medium on K562 cells. Phosphorylation of Crk-L, a BCR-ABL substrate, decreased considerably, while apoptosis increased. In addition, the combined treatment of CD34-positive primary samples resulted in considerably increased cytotoxicity, decreased Crk-L phosphorylation, and increased apoptosis. We also investigated TG101348 activity against feeder cells and observed that STAT5 phosphorylation, granulocyte macrophage colony-stimulating factor, and interleukin 6 levels decreased, indicating reduced cytokine production in HS-5 cells treated with TG101348.

Conclusions: These results showed that JAK inhibitors may enhance the cytotoxic effect of imatinib against residual CML cells and that a combined approach may be a powerful strategy against the stroma-associated drug resistance of Philadelphia chromosome-positive cells.

Show MeSH

Related in: MedlinePlus

Effect of TG101348 on HS-5 cells. (A) HS-5 cells were cultured in the presence of different concentrations of TG101348 for 24 h, and total extracts were examined by immunoblotting using anti-phospho STAT5, STAT5, and β-actin Abs. Blots were scanned and optical densities were determined using ImageJ software. (B) HS-5 cells were treated with TG101348 for 24 h and conditioned medium was collected. The relative levels of 36 different cytokines were analyzed using the Human Cytokine Array Kit. The expression of cytokines was determined using ImageJ software. Results are representative of three separate experiments. *p < 0.05, TG101348 treatment vs. control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4012544&req=5

Figure 5: Effect of TG101348 on HS-5 cells. (A) HS-5 cells were cultured in the presence of different concentrations of TG101348 for 24 h, and total extracts were examined by immunoblotting using anti-phospho STAT5, STAT5, and β-actin Abs. Blots were scanned and optical densities were determined using ImageJ software. (B) HS-5 cells were treated with TG101348 for 24 h and conditioned medium was collected. The relative levels of 36 different cytokines were analyzed using the Human Cytokine Array Kit. The expression of cytokines was determined using ImageJ software. Results are representative of three separate experiments. *p < 0.05, TG101348 treatment vs. control.

Mentions: The HS-5 cell line was treated with TG101348 at different concentrations for 24 h (Figure 5). We found that STAT5 phosphorylation was partially reduced after TG101348 treatment using immunoblotting analysis (Figure 5A). We next investigated cytokine levels by assaying HS-5 conditioned medium after 1 day of culture in the presence or absence of TG101348 using the human cytokine array according to the manufacturer’s instructions. We analyzed 36 cytokines simultaneously and evaluated their relative expression levels. We found that GM-CSF, and IL-6 levels decreased considerably after TG101348 treatment compared to that in the untreated conditioned medium (Figure 5B). These results indicate that TG101348 regulates cytokine production in HS-5 cells.


Combination of the ABL kinase inhibitor imatinib with the Janus kinase 2 inhibitor TG101348 for targeting residual BCR-ABL-positive cells.

Okabe S, Tauchi T, Katagiri S, Tanaka Y, Ohyashiki K - J Hematol Oncol (2014)

Effect of TG101348 on HS-5 cells. (A) HS-5 cells were cultured in the presence of different concentrations of TG101348 for 24 h, and total extracts were examined by immunoblotting using anti-phospho STAT5, STAT5, and β-actin Abs. Blots were scanned and optical densities were determined using ImageJ software. (B) HS-5 cells were treated with TG101348 for 24 h and conditioned medium was collected. The relative levels of 36 different cytokines were analyzed using the Human Cytokine Array Kit. The expression of cytokines was determined using ImageJ software. Results are representative of three separate experiments. *p < 0.05, TG101348 treatment vs. control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4012544&req=5

Figure 5: Effect of TG101348 on HS-5 cells. (A) HS-5 cells were cultured in the presence of different concentrations of TG101348 for 24 h, and total extracts were examined by immunoblotting using anti-phospho STAT5, STAT5, and β-actin Abs. Blots were scanned and optical densities were determined using ImageJ software. (B) HS-5 cells were treated with TG101348 for 24 h and conditioned medium was collected. The relative levels of 36 different cytokines were analyzed using the Human Cytokine Array Kit. The expression of cytokines was determined using ImageJ software. Results are representative of three separate experiments. *p < 0.05, TG101348 treatment vs. control.
Mentions: The HS-5 cell line was treated with TG101348 at different concentrations for 24 h (Figure 5). We found that STAT5 phosphorylation was partially reduced after TG101348 treatment using immunoblotting analysis (Figure 5A). We next investigated cytokine levels by assaying HS-5 conditioned medium after 1 day of culture in the presence or absence of TG101348 using the human cytokine array according to the manufacturer’s instructions. We analyzed 36 cytokines simultaneously and evaluated their relative expression levels. We found that GM-CSF, and IL-6 levels decreased considerably after TG101348 treatment compared to that in the untreated conditioned medium (Figure 5B). These results indicate that TG101348 regulates cytokine production in HS-5 cells.

Bottom Line: In addition, the combined treatment of CD34-positive primary samples resulted in considerably increased cytotoxicity, decreased Crk-L phosphorylation, and increased apoptosis.We also investigated TG101348 activity against feeder cells and observed that STAT5 phosphorylation, granulocyte macrophage colony-stimulating factor, and interleukin 6 levels decreased, indicating reduced cytokine production in HS-5 cells treated with TG101348.These results showed that JAK inhibitors may enhance the cytotoxic effect of imatinib against residual CML cells and that a combined approach may be a powerful strategy against the stroma-associated drug resistance of Philadelphia chromosome-positive cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: First Department of Internal Medicine, Tokyo Medical University, Shinjuku-ku, Tokyo 160-0023, Japan. okabe@tokyo-med.ac.jp.

ABSTRACT

Background: The ABL kinase inhibitor imatinib is highly effective in treating most, but not all, patients with chronic myeloid leukemia (CML). This is because residual CML cells are generally present in the bone marrow microenvironment and are refractory to imatinib. Hematopoietic cytokine receptor signaling is mediated by Janus kinases (JAKs) and their downstream transcription factor, signal transducer and activator of transcription (STAT). TG101348 (SAR302503) is an oral inhibitor of JAK2.

Methods: We investigated the efficacy of imatinib and TG101348 using the break point cluster region-c-Abelson (BCR-ABL)-positive cell line and primary CML samples wherein leukemia cells were protected by a feeder cell line (HS-5).

Results: Imatinib treatment resulted in partial inhibition of cell growth in HS-5-conditioned medium. Furthermore, combined treatment with imatinib and TG101348 abrogated the protective effects of HS-5-conditioned medium on K562 cells. Phosphorylation of Crk-L, a BCR-ABL substrate, decreased considerably, while apoptosis increased. In addition, the combined treatment of CD34-positive primary samples resulted in considerably increased cytotoxicity, decreased Crk-L phosphorylation, and increased apoptosis. We also investigated TG101348 activity against feeder cells and observed that STAT5 phosphorylation, granulocyte macrophage colony-stimulating factor, and interleukin 6 levels decreased, indicating reduced cytokine production in HS-5 cells treated with TG101348.

Conclusions: These results showed that JAK inhibitors may enhance the cytotoxic effect of imatinib against residual CML cells and that a combined approach may be a powerful strategy against the stroma-associated drug resistance of Philadelphia chromosome-positive cells.

Show MeSH
Related in: MedlinePlus