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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

Western Blotting for c-Kit, c-Met, phosphor-c-Met, and AXL in GIST Patient SamplesGIST patients [1, 2, 4, 7 and 16] analyzed by Western blotting for c-Kit, c-Met, Phospho-c-Met and AXL expression. Patient 1, 2 and 4 had serial specimen's available pre- and post-IM. A piece of frozen tissue was homogenized, lysed with NP-40 lysis buffer and 50 μg total protein was resolved by electrophoresis on a 10% SDS-PAGE. Immunoblotting was performed using anti-c-Kit, anti-c-Met, anti-phospho-c-Met and anti-AXL antibodies, respectively. GAPDH is used as a loading control.
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Figure 2: Western Blotting for c-Kit, c-Met, phosphor-c-Met, and AXL in GIST Patient SamplesGIST patients [1, 2, 4, 7 and 16] analyzed by Western blotting for c-Kit, c-Met, Phospho-c-Met and AXL expression. Patient 1, 2 and 4 had serial specimen's available pre- and post-IM. A piece of frozen tissue was homogenized, lysed with NP-40 lysis buffer and 50 μg total protein was resolved by electrophoresis on a 10% SDS-PAGE. Immunoblotting was performed using anti-c-Kit, anti-c-Met, anti-phospho-c-Met and anti-AXL antibodies, respectively. GAPDH is used as a loading control.

Mentions: In order to ascertain whether the cell culture model recapitulates [1] the human situation, we investigated 5 GIST patients that had progressed on chronic IM therapy and had debulking surgeries as part of their management strategy (Table S1). Based on an expert pathology review, snap frozen active tumors were analyzed by Western blotting for expression of c-Kit, c-Met and AXL along with phosphorylation of c-Met (Figure 2). Patient 1 (c-Kit+) progressed on IM and the biopsy at debulking surgery continued to be c-Kit positive. Patient 1 was treated with AMG706 (a c-Kit/VEGFR SMI, Phase II clinical trial) but continued to have progressive disease. On subsequent biopsy at the second debulking surgery (2007), c-Kit expression was negative by IHC. Similarly, patient 2 (c-Kit+) treated with IM for 2 years had progressive disease and IM dose was increased from 400 mg to 600 mg with stabilization of disease. Due to progressive disease, a biopsy from a debulking surgery showed continued c-Kit positivity. Patient 2 then was started on SM with disease stabilization. However, due to progressive symptomatic disease, another debulking surgical biopsy showed c-Kit negativity by IHC. Patient 7, c-Kit maintains over-expression on IM therapy with no evidence of AXL or c-Met induction. However, patient 7 was resistant to IM and SM, which may be due to acquired secondary mutations. Patient 16, c-Kit expression is lost with IM treatment but there is induction of AXL expression. Patient 4, c-Kit continues to be over-expressed despite IM/SM therapy and there is a modest expression of AXL and c-Met RTKs. The resistance to IM here appears to be mixed (secondary c-Kit mutations and alternative RTK expression). Together these observations support the notion that RTK driven down stream signaling pathways continue to be activated and provide a survival and proliferative advantage in c-Kit negative GIST patients.


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)

Western Blotting for c-Kit, c-Met, phosphor-c-Met, and AXL in GIST Patient SamplesGIST patients [1, 2, 4, 7 and 16] analyzed by Western blotting for c-Kit, c-Met, Phospho-c-Met and AXL expression. Patient 1, 2 and 4 had serial specimen's available pre- and post-IM. A piece of frozen tissue was homogenized, lysed with NP-40 lysis buffer and 50 μg total protein was resolved by electrophoresis on a 10% SDS-PAGE. Immunoblotting was performed using anti-c-Kit, anti-c-Met, anti-phospho-c-Met and anti-AXL antibodies, respectively. GAPDH is used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Western Blotting for c-Kit, c-Met, phosphor-c-Met, and AXL in GIST Patient SamplesGIST patients [1, 2, 4, 7 and 16] analyzed by Western blotting for c-Kit, c-Met, Phospho-c-Met and AXL expression. Patient 1, 2 and 4 had serial specimen's available pre- and post-IM. A piece of frozen tissue was homogenized, lysed with NP-40 lysis buffer and 50 μg total protein was resolved by electrophoresis on a 10% SDS-PAGE. Immunoblotting was performed using anti-c-Kit, anti-c-Met, anti-phospho-c-Met and anti-AXL antibodies, respectively. GAPDH is used as a loading control.
Mentions: In order to ascertain whether the cell culture model recapitulates [1] the human situation, we investigated 5 GIST patients that had progressed on chronic IM therapy and had debulking surgeries as part of their management strategy (Table S1). Based on an expert pathology review, snap frozen active tumors were analyzed by Western blotting for expression of c-Kit, c-Met and AXL along with phosphorylation of c-Met (Figure 2). Patient 1 (c-Kit+) progressed on IM and the biopsy at debulking surgery continued to be c-Kit positive. Patient 1 was treated with AMG706 (a c-Kit/VEGFR SMI, Phase II clinical trial) but continued to have progressive disease. On subsequent biopsy at the second debulking surgery (2007), c-Kit expression was negative by IHC. Similarly, patient 2 (c-Kit+) treated with IM for 2 years had progressive disease and IM dose was increased from 400 mg to 600 mg with stabilization of disease. Due to progressive disease, a biopsy from a debulking surgery showed continued c-Kit positivity. Patient 2 then was started on SM with disease stabilization. However, due to progressive symptomatic disease, another debulking surgical biopsy showed c-Kit negativity by IHC. Patient 7, c-Kit maintains over-expression on IM therapy with no evidence of AXL or c-Met induction. However, patient 7 was resistant to IM and SM, which may be due to acquired secondary mutations. Patient 16, c-Kit expression is lost with IM treatment but there is induction of AXL expression. Patient 4, c-Kit continues to be over-expressed despite IM/SM therapy and there is a modest expression of AXL and c-Met RTKs. The resistance to IM here appears to be mixed (secondary c-Kit mutations and alternative RTK expression). Together these observations support the notion that RTK driven down stream signaling pathways continue to be activated and provide a survival and proliferative advantage in c-Kit negative GIST patients.

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