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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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Related in: MedlinePlus

A composite transcription signature is induced by CD8-IGF1R over-expression and A-928605 treatment of the transformed cellsA, Expression profile of 1860 Affymetrix probe sets that were differentially expressed between CD8-IGF1R and vector control cells and between CD8-IGF1R vehicle and A-928605-treated cells by at least 1.5-fold and with a 5% FDR. Three independent plates, each represented by a column on the heat map, were analyzed from the vehicle-treated vector control, A-928605-treated vector control, vehicle-treated CD8-IGF1R, A-928605-treated CD8-IGF1R cell groups. Genes exhibiting decreased expression in CD8-IGF1R relative to vector control cells represent classes I (579 transcripts) and II (162 transcripts). Class III (968) and IV (151) transcripts exhibited increased expression in CD8-IGF1R relative to vector control cells. B, Relative expression level of key class I and III genes for each treatment group relative to vehicle-treated vector control cells. All vehicle treated vector control cells have gene expression normalized to 1.
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Figure 3: A composite transcription signature is induced by CD8-IGF1R over-expression and A-928605 treatment of the transformed cellsA, Expression profile of 1860 Affymetrix probe sets that were differentially expressed between CD8-IGF1R and vector control cells and between CD8-IGF1R vehicle and A-928605-treated cells by at least 1.5-fold and with a 5% FDR. Three independent plates, each represented by a column on the heat map, were analyzed from the vehicle-treated vector control, A-928605-treated vector control, vehicle-treated CD8-IGF1R, A-928605-treated CD8-IGF1R cell groups. Genes exhibiting decreased expression in CD8-IGF1R relative to vector control cells represent classes I (579 transcripts) and II (162 transcripts). Class III (968) and IV (151) transcripts exhibited increased expression in CD8-IGF1R relative to vector control cells. B, Relative expression level of key class I and III genes for each treatment group relative to vehicle-treated vector control cells. All vehicle treated vector control cells have gene expression normalized to 1.

Mentions: The multiple members of the IGF axis, the lack of known receptor activating mutations and the ligand-dependent nature of IGF signal transduction make this pathway difficult to model both in vitro and in vivo. To bypass these inherent complications, we utilized a previously published technique where fusion of the extracellular sequence of the human T-cell antigen CD8α to the cytoplasmic domain of receptor tyrosine kinases leads to constitutively active receptor kinases that can act as oncogenes [23-25]. CD8α extracellular peptides are known to form dimers [26], and when fused to the cytoplasmic tail of a receptor tyrosine kinase this dimerization results in the kinase domains being brought into proximity and allows constitutive activation. NIH-3T3 cells with over-expressed CD8-IGF1R were generated and the resulting cell line has a strongly constitutively phosphorylated IGF1R cytotail (Figure 3A). After transfection and selection, the surviving CD8-IGF1R positive cells had morphology suggestive of oncogenic transformation by the chimeric protein (see Additional File 1: Supplemental Figure S1A). Treatment of the CD8-IGF1R cell line with increasing amounts of A-928605 produced a dose-dependent inhibition of IGF1R phosphorylation near its known cellular phosphorylation IC50 value of ~100 nM (Figure 3B). So while the CD8-IGF1R chimeric kinase is constitutively activated, this activation can be effectively reversed by intervention of the ATP-competitive inhibitor A-928605. This can also be seen at the morphological level as increasing concentrations of A-928605 in the CD8-IGF1R cells results in a reversal of their transformed phenotype (see Additional File 1: Supplemental Figure S1B). A-928605 and six current clinical therapeutic kinase inhibitors were subsequently tested for their ability to inhibit cellular proliferation in the NIH-3T3 CD8-IG1R and vector control cell lines. While there is little effect of A-928605 in the vector control cell line at concentrations sufficient for the inhibition of cellular IGF1R (Figure 3C), it is a potent inhibitor of cellular proliferation of the CD8-IGF1R cells with an IC50 of less than 0.3 μM (Figure 3D), indicating that these transformed cells now rely on IGF1R signaling to proliferate. It is interesting to note the switch in signaling dependence between the vector control and CD8-IGF1R lines. Dasatinib (ABL and SRC inhibitor) is a potent inhibitor of the vector control NIH-3T3 cells, but has no effect on the CD8-IGF1R line at concentrations up to 10 μM and none of the other known kinase inhibitors tested showed anti-proliferative activities towards the transformed cells at concentrations below 10 μM. A-928605 is also capable of completely inhibiting the anchorage-independent growth of the transformed NIH-3T3 CD8-IGF1R line when grown in soft agar (see Additional File 2: Supplemental Figure S2). In total, these data suggest that A-928605 can effectively inhibit cellular IGF1R kinase activity and reverse the effects of IGF1R-dependent transformation.


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 composite transcription signature is induced by CD8-IGF1R over-expression and A-928605 treatment of the transformed cellsA, Expression profile of 1860 Affymetrix probe sets that were differentially expressed between CD8-IGF1R and vector control cells and between CD8-IGF1R vehicle and A-928605-treated cells by at least 1.5-fold and with a 5% FDR. Three independent plates, each represented by a column on the heat map, were analyzed from the vehicle-treated vector control, A-928605-treated vector control, vehicle-treated CD8-IGF1R, A-928605-treated CD8-IGF1R cell groups. Genes exhibiting decreased expression in CD8-IGF1R relative to vector control cells represent classes I (579 transcripts) and II (162 transcripts). Class III (968) and IV (151) transcripts exhibited increased expression in CD8-IGF1R relative to vector control cells. B, Relative expression level of key class I and III genes for each treatment group relative to vehicle-treated vector control cells. All vehicle treated vector control cells have gene expression normalized to 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2749869&req=5

Figure 3: A composite transcription signature is induced by CD8-IGF1R over-expression and A-928605 treatment of the transformed cellsA, Expression profile of 1860 Affymetrix probe sets that were differentially expressed between CD8-IGF1R and vector control cells and between CD8-IGF1R vehicle and A-928605-treated cells by at least 1.5-fold and with a 5% FDR. Three independent plates, each represented by a column on the heat map, were analyzed from the vehicle-treated vector control, A-928605-treated vector control, vehicle-treated CD8-IGF1R, A-928605-treated CD8-IGF1R cell groups. Genes exhibiting decreased expression in CD8-IGF1R relative to vector control cells represent classes I (579 transcripts) and II (162 transcripts). Class III (968) and IV (151) transcripts exhibited increased expression in CD8-IGF1R relative to vector control cells. B, Relative expression level of key class I and III genes for each treatment group relative to vehicle-treated vector control cells. All vehicle treated vector control cells have gene expression normalized to 1.
Mentions: The multiple members of the IGF axis, the lack of known receptor activating mutations and the ligand-dependent nature of IGF signal transduction make this pathway difficult to model both in vitro and in vivo. To bypass these inherent complications, we utilized a previously published technique where fusion of the extracellular sequence of the human T-cell antigen CD8α to the cytoplasmic domain of receptor tyrosine kinases leads to constitutively active receptor kinases that can act as oncogenes [23-25]. CD8α extracellular peptides are known to form dimers [26], and when fused to the cytoplasmic tail of a receptor tyrosine kinase this dimerization results in the kinase domains being brought into proximity and allows constitutive activation. NIH-3T3 cells with over-expressed CD8-IGF1R were generated and the resulting cell line has a strongly constitutively phosphorylated IGF1R cytotail (Figure 3A). After transfection and selection, the surviving CD8-IGF1R positive cells had morphology suggestive of oncogenic transformation by the chimeric protein (see Additional File 1: Supplemental Figure S1A). Treatment of the CD8-IGF1R cell line with increasing amounts of A-928605 produced a dose-dependent inhibition of IGF1R phosphorylation near its known cellular phosphorylation IC50 value of ~100 nM (Figure 3B). So while the CD8-IGF1R chimeric kinase is constitutively activated, this activation can be effectively reversed by intervention of the ATP-competitive inhibitor A-928605. This can also be seen at the morphological level as increasing concentrations of A-928605 in the CD8-IGF1R cells results in a reversal of their transformed phenotype (see Additional File 1: Supplemental Figure S1B). A-928605 and six current clinical therapeutic kinase inhibitors were subsequently tested for their ability to inhibit cellular proliferation in the NIH-3T3 CD8-IG1R and vector control cell lines. While there is little effect of A-928605 in the vector control cell line at concentrations sufficient for the inhibition of cellular IGF1R (Figure 3C), it is a potent inhibitor of cellular proliferation of the CD8-IGF1R cells with an IC50 of less than 0.3 μM (Figure 3D), indicating that these transformed cells now rely on IGF1R signaling to proliferate. It is interesting to note the switch in signaling dependence between the vector control and CD8-IGF1R lines. Dasatinib (ABL and SRC inhibitor) is a potent inhibitor of the vector control NIH-3T3 cells, but has no effect on the CD8-IGF1R line at concentrations up to 10 μM and none of the other known kinase inhibitors tested showed anti-proliferative activities towards the transformed cells at concentrations below 10 μM. A-928605 is also capable of completely inhibiting the anchorage-independent growth of the transformed NIH-3T3 CD8-IGF1R line when grown in soft agar (see Additional File 2: Supplemental Figure S2). In total, these data suggest that A-928605 can effectively inhibit cellular IGF1R kinase activity and reverse the effects of IGF1R-dependent transformation.

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