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Novel receptor tyrosine kinase targeted combination therapies for imatinib-resistant gastrointestinal stromal tumors (GIST).

Mahadevan D, Theiss N, Morales C, Stejskal AE, Cooke LS, Zhu M, Kurtzman D, Swart R, Ong E, Qi W - Oncotarget (2015)

Bottom Line: GIST882 and GIST430/654 cells have an IC50 0.077 and 0.59 µM to IM respectively.GIST48 have an IC50 0.66 µM to IM, 0.91 µM to amuvatinib [AMU] and 0.67 µM to erlotinib (Erl).Synergistic combinations: GIST882, AMU + Erl (CI 0.20); IM + AMU (CI 0.50), GIST430/654, IM + afatinib (CI 0.39); IM + AMU (CI 0.42), GIST48, IM + afatinib (CI 0.03); IM + AMU (CI 0.04); AMU + afatinib (CI 0.36); IM + Erl (CI 0.63).

View Article: PubMed Central - PubMed

Affiliation: West Cancer Center/University of Tennessee Health Science Center (UTHSC), Memphis, TN.

ABSTRACT

Background: c-Kit/α-PDGFR targeted therapies are effective for gastrointestinal stromal tumors (GIST), but, >50% develop drug resistance.

Methods: RTK expression (c-Kit, c-Met, AXL, HER-1, HER-2, IGF-1R) in pre-/post-imatinib (IM) GIST patient samples (n=16) and 4 GIST cell lines were examined for RTK inhibitor activity. GIST-882 cells were cultured in IM every other day, cells collected (1 week to 6 months) and analyzed by qRT-PCR and Western blotting.

Results: Immunohistochemistry pre-/post-IM demonstrated continued expression of c-Kit and HER1, while a subset expressed IGF-1R, c-Met and AXL. In GIST cells (GIST-882, GIST430/654, GIST48) c-Kit, HER1 and c-Met are co-expressed. Acute IM over-express c-Kit while chronic IM, lose c-Kit and HER-1 in GIST882 cells. GIST882 and GIST430/654 cells have an IC50 0.077 and 0.59 µM to IM respectively. GIST48 have an IC50 0.66 µM to IM, 0.91 µM to amuvatinib [AMU] and 0.67 µM to erlotinib (Erl). Synergistic combinations: GIST882, AMU + Erl (CI 0.20); IM + AMU (CI 0.50), GIST430/654, IM + afatinib (CI 0.39); IM + AMU (CI 0.42), GIST48, IM + afatinib (CI 0.03); IM + AMU (CI 0.04); AMU + afatinib (CI 0.36); IM + Erl (CI 0.63).

Conclusion: Targeting c-Kit plus HER1 or AXL/c-Met abrogates IM resistance in GIST.

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Related in: MedlinePlus

Quantitative RT-PCR of GIST 882-R Cells(A). Quantitative RT-PCR of Receptor Tyrosine Kinases (c-Kit, c-Met, AXL, IGF-1R and HER-1) after treatment of GIST882 cells with IM (0.5μM) every other day for 3, 4, 5 and 6 months and (B). Western Blotting for c-Kit and EGFR down-regulation measured at 3, 4, 5 and 6 months in the presence of IM (0.5μM). Beta-actin protein was used as a loading control.
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Figure 3: Quantitative RT-PCR of GIST 882-R Cells(A). Quantitative RT-PCR of Receptor Tyrosine Kinases (c-Kit, c-Met, AXL, IGF-1R and HER-1) after treatment of GIST882 cells with IM (0.5μM) every other day for 3, 4, 5 and 6 months and (B). Western Blotting for c-Kit and EGFR down-regulation measured at 3, 4, 5 and 6 months in the presence of IM (0.5μM). Beta-actin protein was used as a loading control.

Mentions: In medium to long term culture of GIST882 cells treated with IM (0.5μM), showed a significant decrease in the c-Kit message from 27-fold at 3 months to 4-fold at 6 months compared to control (Figure 3A). This observation was confirmed by c-Kit protein level which decreased by ~50% at 4 months (compared to 3 months) and complete loss at 5 and 6 months (Figure 3B) consistent with our prior observations [1]. HER1 expression also diminished at 4 months of IM therapy (Figure 3B). The ‘control, 6 months’ (Figure 3B) are GIST882 cells grown without IM with the media changed every other day. The GIST882 cells were not split during the course of the study in order to maintain continuity between the samples and reached 100% confluency prior to the 6 month termination of the experiment. These GIST cells do not express c-Kit, most likely, due to cell cycle arrest. RT-PCR for AXL showed dramatic increases in message from 120 to 340-fold compared to control (Figure 3A). However, c-Met showed an increase message from 14-fold (3, 4 months) to 18 to 22-fold (5, 6 months) compared to control (Figure 3A). In contrast, no changes in mRNA levels were observed for IGF-1R and HER-1 with duration of treatment (Figure 3A).


Novel receptor tyrosine kinase targeted combination therapies for imatinib-resistant gastrointestinal stromal tumors (GIST).

Mahadevan D, Theiss N, Morales C, Stejskal AE, Cooke LS, Zhu M, Kurtzman D, Swart R, Ong E, Qi W - Oncotarget (2015)

Quantitative RT-PCR of GIST 882-R Cells(A). Quantitative RT-PCR of Receptor Tyrosine Kinases (c-Kit, c-Met, AXL, IGF-1R and HER-1) after treatment of GIST882 cells with IM (0.5μM) every other day for 3, 4, 5 and 6 months and (B). Western Blotting for c-Kit and EGFR down-regulation measured at 3, 4, 5 and 6 months in the presence of IM (0.5μM). Beta-actin protein was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385828&req=5

Figure 3: Quantitative RT-PCR of GIST 882-R Cells(A). Quantitative RT-PCR of Receptor Tyrosine Kinases (c-Kit, c-Met, AXL, IGF-1R and HER-1) after treatment of GIST882 cells with IM (0.5μM) every other day for 3, 4, 5 and 6 months and (B). Western Blotting for c-Kit and EGFR down-regulation measured at 3, 4, 5 and 6 months in the presence of IM (0.5μM). Beta-actin protein was used as a loading control.
Mentions: In medium to long term culture of GIST882 cells treated with IM (0.5μM), showed a significant decrease in the c-Kit message from 27-fold at 3 months to 4-fold at 6 months compared to control (Figure 3A). This observation was confirmed by c-Kit protein level which decreased by ~50% at 4 months (compared to 3 months) and complete loss at 5 and 6 months (Figure 3B) consistent with our prior observations [1]. HER1 expression also diminished at 4 months of IM therapy (Figure 3B). The ‘control, 6 months’ (Figure 3B) are GIST882 cells grown without IM with the media changed every other day. The GIST882 cells were not split during the course of the study in order to maintain continuity between the samples and reached 100% confluency prior to the 6 month termination of the experiment. These GIST cells do not express c-Kit, most likely, due to cell cycle arrest. RT-PCR for AXL showed dramatic increases in message from 120 to 340-fold compared to control (Figure 3A). However, c-Met showed an increase message from 14-fold (3, 4 months) to 18 to 22-fold (5, 6 months) compared to control (Figure 3A). In contrast, no changes in mRNA levels were observed for IGF-1R and HER-1 with duration of treatment (Figure 3A).

Bottom Line: GIST882 and GIST430/654 cells have an IC50 0.077 and 0.59 µM to IM respectively.GIST48 have an IC50 0.66 µM to IM, 0.91 µM to amuvatinib [AMU] and 0.67 µM to erlotinib (Erl).Synergistic combinations: GIST882, AMU + Erl (CI 0.20); IM + AMU (CI 0.50), GIST430/654, IM + afatinib (CI 0.39); IM + AMU (CI 0.42), GIST48, IM + afatinib (CI 0.03); IM + AMU (CI 0.04); AMU + afatinib (CI 0.36); IM + Erl (CI 0.63).

View Article: PubMed Central - PubMed

Affiliation: West Cancer Center/University of Tennessee Health Science Center (UTHSC), Memphis, TN.

ABSTRACT

Background: c-Kit/α-PDGFR targeted therapies are effective for gastrointestinal stromal tumors (GIST), but, >50% develop drug resistance.

Methods: RTK expression (c-Kit, c-Met, AXL, HER-1, HER-2, IGF-1R) in pre-/post-imatinib (IM) GIST patient samples (n=16) and 4 GIST cell lines were examined for RTK inhibitor activity. GIST-882 cells were cultured in IM every other day, cells collected (1 week to 6 months) and analyzed by qRT-PCR and Western blotting.

Results: Immunohistochemistry pre-/post-IM demonstrated continued expression of c-Kit and HER1, while a subset expressed IGF-1R, c-Met and AXL. In GIST cells (GIST-882, GIST430/654, GIST48) c-Kit, HER1 and c-Met are co-expressed. Acute IM over-express c-Kit while chronic IM, lose c-Kit and HER-1 in GIST882 cells. GIST882 and GIST430/654 cells have an IC50 0.077 and 0.59 µM to IM respectively. GIST48 have an IC50 0.66 µM to IM, 0.91 µM to amuvatinib [AMU] and 0.67 µM to erlotinib (Erl). Synergistic combinations: GIST882, AMU + Erl (CI 0.20); IM + AMU (CI 0.50), GIST430/654, IM + afatinib (CI 0.39); IM + AMU (CI 0.42), GIST48, IM + afatinib (CI 0.03); IM + AMU (CI 0.04); AMU + afatinib (CI 0.36); IM + Erl (CI 0.63).

Conclusion: Targeting c-Kit plus HER1 or AXL/c-Met abrogates IM resistance in GIST.

Show MeSH
Related in: MedlinePlus