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Reversal of oncogene transformation and suppression of tumor growth by the novel IGF1R kinase inhibitor A-928605.

Pappano WN, Jung PM, Meulbroek JA, Wang YC, Hubbard RD, Zhang Q, Grudzien MM, Soni NB, Johnson EF, Sheppard GS, Donawho C, Buchanan FG, Davidsen SK, Bell RL, Wang J - BMC Cancer (2009)

Bottom Line: A-928605 is a selective IGF1R inhibitor that is able to abrogate activation of the pathway both in vitro and in vivo.This novel compound dosed as a single agent is able to produce significant growth inhibition of neuroblastoma xenografts in vivo.A-928605 is also able to provide additive effects when used in combination with clinically approved agents directed against EGFR in non-small cell lung and human pancreatic tumor models.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064, USA. bill.pappano@abbott.com

ABSTRACT

Background: The insulin-like growth factor (IGF) axis is an important signaling pathway in the growth and survival of many cell and tissue types. This pathway has also been implicated in many aspects of cancer progression from tumorigenesis to metastasis. The multiple roles of IGF signaling in cancer suggest that inhibition of the pathway might yield clinically effective therapeutics.

Methods: We describe A-928605, a novel pyrazolo [3,4-d]pyrimidine small molecule inhibitor of the receptor tyrosine kinases (IGF1R and IR) responsible for IGF signal transduction. This compound was first tested for its activity and selectivity via conventional in vitro kinome profiling and cellular IGF1R autophosphorylation. Additionally, cellular selectivity and efficacy of A-928605 were analyzed in an IGF1R oncogene-addicted cell line by proliferation, signaling and microarray studies. Finally, in vivo efficacy of A-928605 was assessed in the oncogene-addicted cell line and in a neuroblastoma model as a single agent as well as in combination with clinically approved therapeutics targeting EGFR in models of pancreatic and non-small cell lung cancers.

Results: A-928605 is a selective IGF1R inhibitor that is able to abrogate activation of the pathway both in vitro and in vivo. This novel compound dosed as a single agent is able to produce significant growth inhibition of neuroblastoma xenografts in vivo. A-928605 is also able to provide additive effects when used in combination with clinically approved agents directed against EGFR in non-small cell lung and human pancreatic tumor models.

Conclusion: These results suggest that a selective IGF1R inhibitor such as A-928605 may provide a useful clinical therapeutic for IGF pathway affected tumors and warrants further investigation.

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A-928605 abrogates modifications of IGF pathway signaling proteins in vitro and is efficacious in the CD8-IGF1R xenograft flank model in vivo. A, One hour of 1 mM A-928605 treatment in vector control and CD8-IGF1R cells results in inhibition of phosphorylation of the IGF1R cytotail and the immediate downstream pathway effectors AKT and ERK1/2. Cyclin D1 is a transcriptional target of the IGF signaling pathway and is therefore not affected by this one hour treatment. B, 24-hours of 1 mM A-928605 treatment in vector control and CD8-IGF1R cells results in a significant decrease in Cyclin D1 expression as seen here and in the microarray results (Figure 3). C, Beginning on day 17 post tumor cell injection, mice were dosed intraperitoneally with A-928605 at 50 mg/kg, twice daily (b.i.d)., or its vehicle for 10 days. At the end of the dosing schedule, the %T/C (A-928605 vs. vehicle) was 21 (P < 0.00001).
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Figure 4: A-928605 abrogates modifications of IGF pathway signaling proteins in vitro and is efficacious in the CD8-IGF1R xenograft flank model in vivo. A, One hour of 1 mM A-928605 treatment in vector control and CD8-IGF1R cells results in inhibition of phosphorylation of the IGF1R cytotail and the immediate downstream pathway effectors AKT and ERK1/2. Cyclin D1 is a transcriptional target of the IGF signaling pathway and is therefore not affected by this one hour treatment. B, 24-hours of 1 mM A-928605 treatment in vector control and CD8-IGF1R cells results in a significant decrease in Cyclin D1 expression as seen here and in the microarray results (Figure 3). C, Beginning on day 17 post tumor cell injection, mice were dosed intraperitoneally with A-928605 at 50 mg/kg, twice daily (b.i.d)., or its vehicle for 10 days. At the end of the dosing schedule, the %T/C (A-928605 vs. vehicle) was 21 (P < 0.00001).

Mentions: Microarray analysis was performed to assess the possible molecular mechanisms underlying the IGF pathway-dependent transformation as well as the effects of IGF axis inhibition by A-928605 in the NIH-3T3 cell lines. Cells were treated with 1 μM of compound or DMSO control and grown under normal conditions for 24 hours. RNA was isolated, and the microarray profiles were analyzed for differential gene expression at a 5% False Discovery Rate (FDR) [22]. A comparison of the vehicle-treated CD8-IGF1R line with the vehicle-treated NIH-3T3 vector control line revealed large gene expression changes including 5,968 transcripts that showed significant changes (5% FDR, fold change > 1.5) split evenly between up- and down-regulation, of which approximately 2,931 were up regulated and 3,037 were down-regulated (Figure 4A). Ingenuity pathway analysis (IPA) of these genes indicated that the constitutive IGF signaling leads to an up-regulation in both mitogenic and anti-apoptotic pathways at the transcriptional level. There is also a significant quantitative increase in connective tissue and extracellular-matrix messenger RNAs including Tgfβ1 (2.4-fold) and Vegfa (4.3-fold), aiding in the ability of the cells to grow anchorage independently and as tumors (vide infra). As a result of the constitutive activation of the IGF axis, the mRNAs for the ligand Igf2, the native receptor Igf1r, and IGF binding proteins Igfbp5 and Igfbp7 were down-regulated, while Insig2, Igfbp4 and Igfbp6 were up-regulated, suggesting an attempt at a compensatory down-regulation of the IGF axis by the cell (see Additional File 3: Supplemental Table 1). Several transcriptional changes in the insulin/IGF pathway observed in these CD8-IGF1R positive cells, such as decreases in the insulin receptor substrate proteins, Irs1 and Irs2, have been reported for cells treated with IGF1 [27].


Reversal of oncogene transformation and suppression of tumor growth by the novel IGF1R kinase inhibitor A-928605.

Pappano WN, Jung PM, Meulbroek JA, Wang YC, Hubbard RD, Zhang Q, Grudzien MM, Soni NB, Johnson EF, Sheppard GS, Donawho C, Buchanan FG, Davidsen SK, Bell RL, Wang J - BMC Cancer (2009)

A-928605 abrogates modifications of IGF pathway signaling proteins in vitro and is efficacious in the CD8-IGF1R xenograft flank model in vivo. A, One hour of 1 mM A-928605 treatment in vector control and CD8-IGF1R cells results in inhibition of phosphorylation of the IGF1R cytotail and the immediate downstream pathway effectors AKT and ERK1/2. Cyclin D1 is a transcriptional target of the IGF signaling pathway and is therefore not affected by this one hour treatment. B, 24-hours of 1 mM A-928605 treatment in vector control and CD8-IGF1R cells results in a significant decrease in Cyclin D1 expression as seen here and in the microarray results (Figure 3). C, Beginning on day 17 post tumor cell injection, mice were dosed intraperitoneally with A-928605 at 50 mg/kg, twice daily (b.i.d)., or its vehicle for 10 days. At the end of the dosing schedule, the %T/C (A-928605 vs. vehicle) was 21 (P < 0.00001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: A-928605 abrogates modifications of IGF pathway signaling proteins in vitro and is efficacious in the CD8-IGF1R xenograft flank model in vivo. A, One hour of 1 mM A-928605 treatment in vector control and CD8-IGF1R cells results in inhibition of phosphorylation of the IGF1R cytotail and the immediate downstream pathway effectors AKT and ERK1/2. Cyclin D1 is a transcriptional target of the IGF signaling pathway and is therefore not affected by this one hour treatment. B, 24-hours of 1 mM A-928605 treatment in vector control and CD8-IGF1R cells results in a significant decrease in Cyclin D1 expression as seen here and in the microarray results (Figure 3). C, Beginning on day 17 post tumor cell injection, mice were dosed intraperitoneally with A-928605 at 50 mg/kg, twice daily (b.i.d)., or its vehicle for 10 days. At the end of the dosing schedule, the %T/C (A-928605 vs. vehicle) was 21 (P < 0.00001).
Mentions: Microarray analysis was performed to assess the possible molecular mechanisms underlying the IGF pathway-dependent transformation as well as the effects of IGF axis inhibition by A-928605 in the NIH-3T3 cell lines. Cells were treated with 1 μM of compound or DMSO control and grown under normal conditions for 24 hours. RNA was isolated, and the microarray profiles were analyzed for differential gene expression at a 5% False Discovery Rate (FDR) [22]. A comparison of the vehicle-treated CD8-IGF1R line with the vehicle-treated NIH-3T3 vector control line revealed large gene expression changes including 5,968 transcripts that showed significant changes (5% FDR, fold change > 1.5) split evenly between up- and down-regulation, of which approximately 2,931 were up regulated and 3,037 were down-regulated (Figure 4A). Ingenuity pathway analysis (IPA) of these genes indicated that the constitutive IGF signaling leads to an up-regulation in both mitogenic and anti-apoptotic pathways at the transcriptional level. There is also a significant quantitative increase in connective tissue and extracellular-matrix messenger RNAs including Tgfβ1 (2.4-fold) and Vegfa (4.3-fold), aiding in the ability of the cells to grow anchorage independently and as tumors (vide infra). As a result of the constitutive activation of the IGF axis, the mRNAs for the ligand Igf2, the native receptor Igf1r, and IGF binding proteins Igfbp5 and Igfbp7 were down-regulated, while Insig2, Igfbp4 and Igfbp6 were up-regulated, suggesting an attempt at a compensatory down-regulation of the IGF axis by the cell (see Additional File 3: Supplemental Table 1). Several transcriptional changes in the insulin/IGF pathway observed in these CD8-IGF1R positive cells, such as decreases in the insulin receptor substrate proteins, Irs1 and Irs2, have been reported for cells treated with IGF1 [27].

Bottom Line: A-928605 is a selective IGF1R inhibitor that is able to abrogate activation of the pathway both in vitro and in vivo.This novel compound dosed as a single agent is able to produce significant growth inhibition of neuroblastoma xenografts in vivo.A-928605 is also able to provide additive effects when used in combination with clinically approved agents directed against EGFR in non-small cell lung and human pancreatic tumor models.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064, USA. bill.pappano@abbott.com

ABSTRACT

Background: The insulin-like growth factor (IGF) axis is an important signaling pathway in the growth and survival of many cell and tissue types. This pathway has also been implicated in many aspects of cancer progression from tumorigenesis to metastasis. The multiple roles of IGF signaling in cancer suggest that inhibition of the pathway might yield clinically effective therapeutics.

Methods: We describe A-928605, a novel pyrazolo [3,4-d]pyrimidine small molecule inhibitor of the receptor tyrosine kinases (IGF1R and IR) responsible for IGF signal transduction. This compound was first tested for its activity and selectivity via conventional in vitro kinome profiling and cellular IGF1R autophosphorylation. Additionally, cellular selectivity and efficacy of A-928605 were analyzed in an IGF1R oncogene-addicted cell line by proliferation, signaling and microarray studies. Finally, in vivo efficacy of A-928605 was assessed in the oncogene-addicted cell line and in a neuroblastoma model as a single agent as well as in combination with clinically approved therapeutics targeting EGFR in models of pancreatic and non-small cell lung cancers.

Results: A-928605 is a selective IGF1R inhibitor that is able to abrogate activation of the pathway both in vitro and in vivo. This novel compound dosed as a single agent is able to produce significant growth inhibition of neuroblastoma xenografts in vivo. A-928605 is also able to provide additive effects when used in combination with clinically approved agents directed against EGFR in non-small cell lung and human pancreatic tumor models.

Conclusion: These results suggest that a selective IGF1R inhibitor such as A-928605 may provide a useful clinical therapeutic for IGF pathway affected tumors and warrants further investigation.

Show MeSH
Related in: MedlinePlus