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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 phosphorylation of Rad51 S14 and compromises HR in glioblastoma cellsA. (left) Representative immunoblots of U87MG parental (p) and U87MG EGFRvIII (vIII) cells for pS14 Rad51, Rad51, and Ku86. asyn, asynchronized cells; R-8 h, synchronized cells released from DTB for 8 h. (right) pS14Rad51 was normalized to the total Rad51 after correcting for protein loading using Ku86 level. **, p = 0.0026; *, p = 0.045. B. (left) Representative immunoblot of whole U87MG EGFRvIII cell lysate following 24 h treatment with control or 10 nM BI2536. (right) Quantitative densitometric assessment as above described. **, p = 0.0018.C. (left) Schematic summary of the DR-GFP assay. (right) Percentage of GFP-positive cells detected by FACS using U2OS DR-GFP and U87MG DR-GFP cells, separately. GFP-positive cells were scored after treatment with BI2536 (25 nM) or control for 24 h. **, p = 0.00056 and 0.00057 respectively. D. The effects of RAD51 and BRCA2 knockdown in U87MG parental and U87MG EGFRvIII cells. Cells were transfected with the various siRNAs for 24 h and re-plated overnight. BI2536 (25 nM) or control were then added. Clonogenic survivals were scored after additional 14 days. All results were shown as mean±SD.
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Figure 3: BI2536 inhibits phosphorylation of Rad51 S14 and compromises HR in glioblastoma cellsA. (left) Representative immunoblots of U87MG parental (p) and U87MG EGFRvIII (vIII) cells for pS14 Rad51, Rad51, and Ku86. asyn, asynchronized cells; R-8 h, synchronized cells released from DTB for 8 h. (right) pS14Rad51 was normalized to the total Rad51 after correcting for protein loading using Ku86 level. **, p = 0.0026; *, p = 0.045. B. (left) Representative immunoblot of whole U87MG EGFRvIII cell lysate following 24 h treatment with control or 10 nM BI2536. (right) Quantitative densitometric assessment as above described. **, p = 0.0018.C. (left) Schematic summary of the DR-GFP assay. (right) Percentage of GFP-positive cells detected by FACS using U2OS DR-GFP and U87MG DR-GFP cells, separately. GFP-positive cells were scored after treatment with BI2536 (25 nM) or control for 24 h. **, p = 0.00056 and 0.00057 respectively. D. The effects of RAD51 and BRCA2 knockdown in U87MG parental and U87MG EGFRvIII cells. Cells were transfected with the various siRNAs for 24 h and re-plated overnight. BI2536 (25 nM) or control were then added. Clonogenic survivals were scored after additional 14 days. All results were shown as mean±SD.

Mentions: PLK1 has been shown to phosphorylate Rad51 at serine residue 14 (pS14 Rad51) in HeLa and 293T cells to facilitate HR [26]. Given that PLK1 inhibition resulted in an increased level of DNA damage in EGFRvIII expressing glioblastoma cells, we next tested whether the PLK1-mediated Rad51 phosphorylation played a significant role in glioblastomas. The level of pS14 Rad51 was approximately 3-4-fold higher in U87MG EGFRvIII cells relative to U87MG parental cells in synchronized cell populations (Figure 3A). This level was suppressed by treatment with BI2536 (Figure 3B). BI2536 treatment also reduced the efficiency of HR by approximately 50% in the established DR-GFP assay [27] in both U87MG glioblastoma and U2OS osteosarcoma cells (Figure 3C). Taken together, these results suggest that PLK1 counteracts excessive DNA damage accumulation by promoting HR in EGFRvIII expressing glioblastoma cells. Consistent with this interpretation, siRNAs against Rad51 and BRCA2, two genes essential for HR [26], caused significantly higher toxicity in U87MG EGFRvIII cells relative to U87MG cells. Silencing Rad51 or BRCA2 did not further enhance the cytotoxic effect of BI2536 (Figure 3D and Supplemental Figure 3).


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 phosphorylation of Rad51 S14 and compromises HR in glioblastoma cellsA. (left) Representative immunoblots of U87MG parental (p) and U87MG EGFRvIII (vIII) cells for pS14 Rad51, Rad51, and Ku86. asyn, asynchronized cells; R-8 h, synchronized cells released from DTB for 8 h. (right) pS14Rad51 was normalized to the total Rad51 after correcting for protein loading using Ku86 level. **, p = 0.0026; *, p = 0.045. B. (left) Representative immunoblot of whole U87MG EGFRvIII cell lysate following 24 h treatment with control or 10 nM BI2536. (right) Quantitative densitometric assessment as above described. **, p = 0.0018.C. (left) Schematic summary of the DR-GFP assay. (right) Percentage of GFP-positive cells detected by FACS using U2OS DR-GFP and U87MG DR-GFP cells, separately. GFP-positive cells were scored after treatment with BI2536 (25 nM) or control for 24 h. **, p = 0.00056 and 0.00057 respectively. D. The effects of RAD51 and BRCA2 knockdown in U87MG parental and U87MG EGFRvIII cells. Cells were transfected with the various siRNAs for 24 h and re-plated overnight. BI2536 (25 nM) or control were then added. Clonogenic survivals were scored after additional 14 days. All results were shown as mean±SD.
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Figure 3: BI2536 inhibits phosphorylation of Rad51 S14 and compromises HR in glioblastoma cellsA. (left) Representative immunoblots of U87MG parental (p) and U87MG EGFRvIII (vIII) cells for pS14 Rad51, Rad51, and Ku86. asyn, asynchronized cells; R-8 h, synchronized cells released from DTB for 8 h. (right) pS14Rad51 was normalized to the total Rad51 after correcting for protein loading using Ku86 level. **, p = 0.0026; *, p = 0.045. B. (left) Representative immunoblot of whole U87MG EGFRvIII cell lysate following 24 h treatment with control or 10 nM BI2536. (right) Quantitative densitometric assessment as above described. **, p = 0.0018.C. (left) Schematic summary of the DR-GFP assay. (right) Percentage of GFP-positive cells detected by FACS using U2OS DR-GFP and U87MG DR-GFP cells, separately. GFP-positive cells were scored after treatment with BI2536 (25 nM) or control for 24 h. **, p = 0.00056 and 0.00057 respectively. D. The effects of RAD51 and BRCA2 knockdown in U87MG parental and U87MG EGFRvIII cells. Cells were transfected with the various siRNAs for 24 h and re-plated overnight. BI2536 (25 nM) or control were then added. Clonogenic survivals were scored after additional 14 days. All results were shown as mean±SD.
Mentions: PLK1 has been shown to phosphorylate Rad51 at serine residue 14 (pS14 Rad51) in HeLa and 293T cells to facilitate HR [26]. Given that PLK1 inhibition resulted in an increased level of DNA damage in EGFRvIII expressing glioblastoma cells, we next tested whether the PLK1-mediated Rad51 phosphorylation played a significant role in glioblastomas. The level of pS14 Rad51 was approximately 3-4-fold higher in U87MG EGFRvIII cells relative to U87MG parental cells in synchronized cell populations (Figure 3A). This level was suppressed by treatment with BI2536 (Figure 3B). BI2536 treatment also reduced the efficiency of HR by approximately 50% in the established DR-GFP assay [27] in both U87MG glioblastoma and U2OS osteosarcoma cells (Figure 3C). Taken together, these results suggest that PLK1 counteracts excessive DNA damage accumulation by promoting HR in EGFRvIII expressing glioblastoma cells. Consistent with this interpretation, siRNAs against Rad51 and BRCA2, two genes essential for HR [26], caused significantly higher toxicity in U87MG EGFRvIII cells relative to U87MG cells. Silencing Rad51 or BRCA2 did not further enhance the cytotoxic effect of BI2536 (Figure 3D and Supplemental Figure 3).

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