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Orthogonal targeting of EGFRvIII expressing glioblastomas through simultaneous EGFR and PLK1 inhibition.

Shen Y, Li J, Nitta M, Futalan D, Steed T, Treiber JM, Taich Z, Stevens D, Wykosky J, Chen HZ, Carter BS, Becher OJ, Kennedy R, Esashi F, Sarkaria JN, Furnari FB, Cavenee WK, Desai A, Chen CC - Oncotarget (2015)

Bottom Line: Accordingly, PLK1 inhibition enhanced the cytotoxic effects of the DNA damaging agent, temozolomide (TMZ).Although BI2536 significantly augmented the anti-neoplastic effect of EGFR inhibitors in the Ink4a/Arf(-/-) EGFRvIII model, durable response was not achieved until TMZ was added.Our results suggest that optimal therapeutic effect against glioblastomas requires a "multi-orthogonal" combination tailored to the molecular physiology associated with the target cancer genome.

View Article: PubMed Central - PubMed

Affiliation: Center for Theoretical and Applied Neuro-Oncology, Moores Cancer Center, Division of Neurosurgery, University of California San Diego, La Jolla, CA, USA.

ABSTRACT
We identified a synthetic lethality between PLK1 silencing and the expression of an oncogenic Epidermal Growth Factor Receptor, EGFRvIII. PLK1 promoted homologous recombination (HR), mitigating EGFRvIII induced oncogenic stress resulting from DNA damage accumulation. Accordingly, PLK1 inhibition enhanced the cytotoxic effects of the DNA damaging agent, temozolomide (TMZ). This effect was significantly more pronounced in an Ink4a/Arf(-/-) EGFRvIII glioblastoma model relative to an Ink4a/Arf(-/-) PDGF-β model. The tumoricidal and TMZ-sensitizing effects of BI2536 were uniformly observed across Ink4a/Arf(-/-) EGFRvIII glioblastoma clones that acquired independent resistance mechanisms to EGFR inhibitors, suggesting these resistant clones retain oncogenic stress that required PLK1 compensation. Although BI2536 significantly augmented the anti-neoplastic effect of EGFR inhibitors in the Ink4a/Arf(-/-) EGFRvIII model, durable response was not achieved until TMZ was added. Our results suggest that optimal therapeutic effect against glioblastomas requires a "multi-orthogonal" combination tailored to the molecular physiology associated with the target cancer genome.

No MeSH data available.


Related in: MedlinePlus

BI2536 inhibits tumor growth of EGFR inhibitor-resistant murine Ink4a/Arf(−/−) EGFRvIII clones and sensitizes them to the DNA damaging agent TMZA. (upper) Clonogenic survival of murine Ink4a/Arf(−/−) EGFRvIII cells (parental) and established EGFR inhibitor resistant cells (erlotinib-resistant E4, E5 and gefitinib-resistant G1, G5, G12, GR-1, GR-7, GR-11) with BI2536 treatment. (lower) Representative colony formation images. B. (upper) Tumor growth curve of the subcutaneous GR-7 allografts. Nude mice bearing established GR-7 tumors in the flank were treated with control, TMZ (for 3 days starting treatment at Day 10 after implantation), BI2536 (starting at Day 13), or combined (T+B) (starting with TMZ at Day 10, then with BI2536 at Day 13). Mean tumor volume±SD are shown in 5-6 mice per group. (lower) Typical tumors isolated from each group. C. (left) Survival curve of intracranial G12 allografts bearing mice. The mice were treated with control, TMZ (starting at Day 10), BI2536 (starting at Day 13), or combined (T+B) (starting with TMZ at Day 10, then with BI2536 at Day 13) in 5-6 mice per group. (right) p values derived from survival comparisons. *, p < 0.05; **, p < 0.01; ***, p < 0.001. D. Tumor growth of the subcutaneous Ink4a/Arf(−/−) EGFRvIII allografts. Nude mice bearing established Ink4a/Arf(−/−) EGFRvIII tumors in the flank were treated as indicated in Methods. T, TMZ; B, BI2536; G, Gefitinib. Mean tumor volume±SD are shown in 5-6 mice per group. E. Schematic representation of “multi-orthogonal” approach. Upper panel: EGFRvIII expressing glioblastomas adapt to EGFR inhibition (EGFRi) by activation of alternative oncogenic signaling cascade, such as ones mediated by the urokinase receptor (uPAR). Other resistance mechanisms involving activation of cytoplasmic proteins, such as Src, have also been reported [42]. Despite the change in oncogenic signaling, the intrinsic physiological “architecture” of the transformed cells and the cellular dependence on DDR remain largely unaltered. Red arrow: EGFRvIII signaling. Yellow arrow: EGFR inhibition induced up-regulation of uPAR signaling. Bottom panel: As such, simultaneous inhibition of DDR and EGFR inhibition impose independent and parallel selection against glioblastoma cells. The therapeutic efficacy of the regimen is further magnified induction of additional DNA damage by temozolomide (TMZ), a DNA alkylating agent and the standard-of-care chemotherapy for glioblastomas.
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Figure 7: BI2536 inhibits tumor growth of EGFR inhibitor-resistant murine Ink4a/Arf(−/−) EGFRvIII clones and sensitizes them to the DNA damaging agent TMZA. (upper) Clonogenic survival of murine Ink4a/Arf(−/−) EGFRvIII cells (parental) and established EGFR inhibitor resistant cells (erlotinib-resistant E4, E5 and gefitinib-resistant G1, G5, G12, GR-1, GR-7, GR-11) with BI2536 treatment. (lower) Representative colony formation images. B. (upper) Tumor growth curve of the subcutaneous GR-7 allografts. Nude mice bearing established GR-7 tumors in the flank were treated with control, TMZ (for 3 days starting treatment at Day 10 after implantation), BI2536 (starting at Day 13), or combined (T+B) (starting with TMZ at Day 10, then with BI2536 at Day 13). Mean tumor volume±SD are shown in 5-6 mice per group. (lower) Typical tumors isolated from each group. C. (left) Survival curve of intracranial G12 allografts bearing mice. The mice were treated with control, TMZ (starting at Day 10), BI2536 (starting at Day 13), or combined (T+B) (starting with TMZ at Day 10, then with BI2536 at Day 13) in 5-6 mice per group. (right) p values derived from survival comparisons. *, p < 0.05; **, p < 0.01; ***, p < 0.001. D. Tumor growth of the subcutaneous Ink4a/Arf(−/−) EGFRvIII allografts. Nude mice bearing established Ink4a/Arf(−/−) EGFRvIII tumors in the flank were treated as indicated in Methods. T, TMZ; B, BI2536; G, Gefitinib. Mean tumor volume±SD are shown in 5-6 mice per group. E. Schematic representation of “multi-orthogonal” approach. Upper panel: EGFRvIII expressing glioblastomas adapt to EGFR inhibition (EGFRi) by activation of alternative oncogenic signaling cascade, such as ones mediated by the urokinase receptor (uPAR). Other resistance mechanisms involving activation of cytoplasmic proteins, such as Src, have also been reported [42]. Despite the change in oncogenic signaling, the intrinsic physiological “architecture” of the transformed cells and the cellular dependence on DDR remain largely unaltered. Red arrow: EGFRvIII signaling. Yellow arrow: EGFR inhibition induced up-regulation of uPAR signaling. Bottom panel: As such, simultaneous inhibition of DDR and EGFR inhibition impose independent and parallel selection against glioblastoma cells. The therapeutic efficacy of the regimen is further magnified induction of additional DNA damage by temozolomide (TMZ), a DNA alkylating agent and the standard-of-care chemotherapy for glioblastomas.

Mentions: We determined whether glioblastoma cells with acquired resistance to EGFR inhibitors maintain cell states with oncogenic stress that require PLK1 as a compensatory mechanism. To test this hypothesis, we characterized the effect of BI2536 on a panel of 8 murine Ink4a/Arf(−/−) EGFRvIII clones selected for their resistance to EGFR inhibitors. The parental Ink4a/Arf(−/−) EGFRvIII clone was sensitive to EGFR inhibitors. These parental cells were cultured in the presence of either Gefitinib (clones G1, G5, G12, GR-1, GR-7 and GR-11) or Erlotinib (clones E4 and E5) to select for acquired EGFR inhibitor resistance [34]. These clones utilize distinct EGFR independent-signaling pathways [34]. Remarkably, all resistant clones remain uniformly sensitive to BI2536 in vitro at concentrations comparable to that required to ablate the parental Ink4a/Arf(−/−) EGFRvIII cells (Figure 7A). This effect is specific to EGFRvIII expressing cells, as evidenced by the lack of cytotoxic effect of the BI2536 (5 nM) toward the Ink4a/Arf(−/−) astrocytes (Figure 1D).


Orthogonal targeting of EGFRvIII expressing glioblastomas through simultaneous EGFR and PLK1 inhibition.

Shen Y, Li J, Nitta M, Futalan D, Steed T, Treiber JM, Taich Z, Stevens D, Wykosky J, Chen HZ, Carter BS, Becher OJ, Kennedy R, Esashi F, Sarkaria JN, Furnari FB, Cavenee WK, Desai A, Chen CC - Oncotarget (2015)

BI2536 inhibits tumor growth of EGFR inhibitor-resistant murine Ink4a/Arf(−/−) EGFRvIII clones and sensitizes them to the DNA damaging agent TMZA. (upper) Clonogenic survival of murine Ink4a/Arf(−/−) EGFRvIII cells (parental) and established EGFR inhibitor resistant cells (erlotinib-resistant E4, E5 and gefitinib-resistant G1, G5, G12, GR-1, GR-7, GR-11) with BI2536 treatment. (lower) Representative colony formation images. B. (upper) Tumor growth curve of the subcutaneous GR-7 allografts. Nude mice bearing established GR-7 tumors in the flank were treated with control, TMZ (for 3 days starting treatment at Day 10 after implantation), BI2536 (starting at Day 13), or combined (T+B) (starting with TMZ at Day 10, then with BI2536 at Day 13). Mean tumor volume±SD are shown in 5-6 mice per group. (lower) Typical tumors isolated from each group. C. (left) Survival curve of intracranial G12 allografts bearing mice. The mice were treated with control, TMZ (starting at Day 10), BI2536 (starting at Day 13), or combined (T+B) (starting with TMZ at Day 10, then with BI2536 at Day 13) in 5-6 mice per group. (right) p values derived from survival comparisons. *, p < 0.05; **, p < 0.01; ***, p < 0.001. D. Tumor growth of the subcutaneous Ink4a/Arf(−/−) EGFRvIII allografts. Nude mice bearing established Ink4a/Arf(−/−) EGFRvIII tumors in the flank were treated as indicated in Methods. T, TMZ; B, BI2536; G, Gefitinib. Mean tumor volume±SD are shown in 5-6 mice per group. E. Schematic representation of “multi-orthogonal” approach. Upper panel: EGFRvIII expressing glioblastomas adapt to EGFR inhibition (EGFRi) by activation of alternative oncogenic signaling cascade, such as ones mediated by the urokinase receptor (uPAR). Other resistance mechanisms involving activation of cytoplasmic proteins, such as Src, have also been reported [42]. Despite the change in oncogenic signaling, the intrinsic physiological “architecture” of the transformed cells and the cellular dependence on DDR remain largely unaltered. Red arrow: EGFRvIII signaling. Yellow arrow: EGFR inhibition induced up-regulation of uPAR signaling. Bottom panel: As such, simultaneous inhibition of DDR and EGFR inhibition impose independent and parallel selection against glioblastoma cells. The therapeutic efficacy of the regimen is further magnified induction of additional DNA damage by temozolomide (TMZ), a DNA alkylating agent and the standard-of-care chemotherapy for glioblastomas.
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Figure 7: BI2536 inhibits tumor growth of EGFR inhibitor-resistant murine Ink4a/Arf(−/−) EGFRvIII clones and sensitizes them to the DNA damaging agent TMZA. (upper) Clonogenic survival of murine Ink4a/Arf(−/−) EGFRvIII cells (parental) and established EGFR inhibitor resistant cells (erlotinib-resistant E4, E5 and gefitinib-resistant G1, G5, G12, GR-1, GR-7, GR-11) with BI2536 treatment. (lower) Representative colony formation images. B. (upper) Tumor growth curve of the subcutaneous GR-7 allografts. Nude mice bearing established GR-7 tumors in the flank were treated with control, TMZ (for 3 days starting treatment at Day 10 after implantation), BI2536 (starting at Day 13), or combined (T+B) (starting with TMZ at Day 10, then with BI2536 at Day 13). Mean tumor volume±SD are shown in 5-6 mice per group. (lower) Typical tumors isolated from each group. C. (left) Survival curve of intracranial G12 allografts bearing mice. The mice were treated with control, TMZ (starting at Day 10), BI2536 (starting at Day 13), or combined (T+B) (starting with TMZ at Day 10, then with BI2536 at Day 13) in 5-6 mice per group. (right) p values derived from survival comparisons. *, p < 0.05; **, p < 0.01; ***, p < 0.001. D. Tumor growth of the subcutaneous Ink4a/Arf(−/−) EGFRvIII allografts. Nude mice bearing established Ink4a/Arf(−/−) EGFRvIII tumors in the flank were treated as indicated in Methods. T, TMZ; B, BI2536; G, Gefitinib. Mean tumor volume±SD are shown in 5-6 mice per group. E. Schematic representation of “multi-orthogonal” approach. Upper panel: EGFRvIII expressing glioblastomas adapt to EGFR inhibition (EGFRi) by activation of alternative oncogenic signaling cascade, such as ones mediated by the urokinase receptor (uPAR). Other resistance mechanisms involving activation of cytoplasmic proteins, such as Src, have also been reported [42]. Despite the change in oncogenic signaling, the intrinsic physiological “architecture” of the transformed cells and the cellular dependence on DDR remain largely unaltered. Red arrow: EGFRvIII signaling. Yellow arrow: EGFR inhibition induced up-regulation of uPAR signaling. Bottom panel: As such, simultaneous inhibition of DDR and EGFR inhibition impose independent and parallel selection against glioblastoma cells. The therapeutic efficacy of the regimen is further magnified induction of additional DNA damage by temozolomide (TMZ), a DNA alkylating agent and the standard-of-care chemotherapy for glioblastomas.
Mentions: We determined whether glioblastoma cells with acquired resistance to EGFR inhibitors maintain cell states with oncogenic stress that require PLK1 as a compensatory mechanism. To test this hypothesis, we characterized the effect of BI2536 on a panel of 8 murine Ink4a/Arf(−/−) EGFRvIII clones selected for their resistance to EGFR inhibitors. The parental Ink4a/Arf(−/−) EGFRvIII clone was sensitive to EGFR inhibitors. These parental cells were cultured in the presence of either Gefitinib (clones G1, G5, G12, GR-1, GR-7 and GR-11) or Erlotinib (clones E4 and E5) to select for acquired EGFR inhibitor resistance [34]. These clones utilize distinct EGFR independent-signaling pathways [34]. Remarkably, all resistant clones remain uniformly sensitive to BI2536 in vitro at concentrations comparable to that required to ablate the parental Ink4a/Arf(−/−) EGFRvIII cells (Figure 7A). This effect is specific to EGFRvIII expressing cells, as evidenced by the lack of cytotoxic effect of the BI2536 (5 nM) toward the Ink4a/Arf(−/−) astrocytes (Figure 1D).

Bottom Line: Accordingly, PLK1 inhibition enhanced the cytotoxic effects of the DNA damaging agent, temozolomide (TMZ).Although BI2536 significantly augmented the anti-neoplastic effect of EGFR inhibitors in the Ink4a/Arf(-/-) EGFRvIII model, durable response was not achieved until TMZ was added.Our results suggest that optimal therapeutic effect against glioblastomas requires a "multi-orthogonal" combination tailored to the molecular physiology associated with the target cancer genome.

View Article: PubMed Central - PubMed

Affiliation: Center for Theoretical and Applied Neuro-Oncology, Moores Cancer Center, Division of Neurosurgery, University of California San Diego, La Jolla, CA, USA.

ABSTRACT
We identified a synthetic lethality between PLK1 silencing and the expression of an oncogenic Epidermal Growth Factor Receptor, EGFRvIII. PLK1 promoted homologous recombination (HR), mitigating EGFRvIII induced oncogenic stress resulting from DNA damage accumulation. Accordingly, PLK1 inhibition enhanced the cytotoxic effects of the DNA damaging agent, temozolomide (TMZ). This effect was significantly more pronounced in an Ink4a/Arf(-/-) EGFRvIII glioblastoma model relative to an Ink4a/Arf(-/-) PDGF-β model. The tumoricidal and TMZ-sensitizing effects of BI2536 were uniformly observed across Ink4a/Arf(-/-) EGFRvIII glioblastoma clones that acquired independent resistance mechanisms to EGFR inhibitors, suggesting these resistant clones retain oncogenic stress that required PLK1 compensation. Although BI2536 significantly augmented the anti-neoplastic effect of EGFR inhibitors in the Ink4a/Arf(-/-) EGFRvIII model, durable response was not achieved until TMZ was added. Our results suggest that optimal therapeutic effect against glioblastomas requires a "multi-orthogonal" combination tailored to the molecular physiology associated with the target cancer genome.

No MeSH data available.


Related in: MedlinePlus