NOTCH blockade combined with radiation therapy and temozolomide prolongs survival of orthotopic glioblastoma - PubMed (original) (raw)

. 2016 Jul 5;7(27):41251-41264.

doi: 10.18632/oncotarget.9275.

Henry King 2, Venus Sosa Iglesias 1, Patrick V Granton 1 3, Lydie M O Barbeau 1, Stefan J van Hoof 1, Arjan J Groot 1, Roger Habets 1, Jos Prickaerts 4, Anthony J Chalmers 5, Daniëlle B P Eekers 6, Jan Theys 1, Susan C Short 2, Frank Verhaegen 1, Marc Vooijs 1

Affiliations

NOTCH blockade combined with radiation therapy and temozolomide prolongs survival of orthotopic glioblastoma

Sanaz Yahyanejad et al. Oncotarget. 2016.

Abstract

Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults. The current standard of care includes surgery followed by radiotherapy (RT) and chemotherapy with temozolomide (TMZ). Treatment often fails due to the radiation resistance and intrinsic or acquired TMZ resistance of a small percentage of cells with stem cell-like behavior (CSC). The NOTCH signaling pathway is expressed and active in human glioblastoma and NOTCH inhibitors attenuate tumor growth in vivo in xenograft models. Here we show using an image guided micro-CT and precision radiotherapy platform that a combination of the clinically approved NOTCH/γ-secretase inhibitor (GSI) RO4929097 with standard of care (TMZ + RT) reduces tumor growth and prolongs survival compared to dual combinations. We show that GSI in combination with RT and TMZ attenuates proliferation, decreases 3D spheroid growth and results into a marked reduction in clonogenic survival in primary and established glioma cell lines. We found that the glioma stem cell marker CD133, SOX2 and Nestin were reduced following combination treatments and NOTCH inhibitors albeit in a different manner. These findings indicate that NOTCH inhibition combined with standard of care treatment has an anti-glioma stem cell effect which provides an improved survival benefit for GBM and encourages further translational and clinical studies.

Keywords: RO4929097 NOTCH inhibitor; glioblastoma; glioma stem cells; image guidance radiotherapy; temozolomide.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. NOTCH signaling in GBM cell lines

(A and C) mRNA expression of the NOTCH receptors, ligands and target genes in GBM cell lines were determined by qRT-PCR in U87MG-Luc2 and E2 cells. (B and D) mRNA expression of NOTCH target genes (HES1, HEY1 and HEY2) reduced after treatment with different concentrations of clinically available GSI RO4929097 as determined by qRT-PCR. Values were normalized to Actin. Error bars indicate SEM. Asterisk indicates significance (*P < 0.05, **P < 0.01, ns: not significant).

Figure 2

Figure 2. Effect of NOTCH inhibition combined with TMZ and RT on proliferation and clonogenicity in vitro

(A–B) Proliferation analysis of U87MG-Luc2 and E2 cells after indicated treatments. (C–D) Survival fraction of U87MG-Luc2 and E2 cells upon indicated treatments following radiation. Error bars indicate SEM.

Figure 3

Figure 3. Effect of NOTCH inhibition combined with TMZ and RT on 3D spheroid growth in vitro

(A) Schematic of the 3D spheroid assay treatment schedules. (B–E) Individual spheroid growth in U87 cells was imaged 3 ×/week and the volume was calculated after indicated treatments till time to reach 20× starting volume. Spheroid growth demonstrates delays upon different treatment combinations compared with DMSO as vehicle control. Error bars indicates SEM. Asterisk indicates significance (*p < 0.05, **p < 0.01 and ***p < 0.001, ns: not significant).

Figure 4

Figure 4. Radiation treatment set-up and the resulting dose volume histograms

(A–C) Visualization of the tumor in the brain from different planes (Sagittal, Axial and coronal) and applied parallel-opposed radiation beams to target the tumor. (D) Resulting DVHs of the tumors after 8 Gy irradiation. Red line shows the average of the DVHs.

Figure 5

Figure 5. Effect of NOTCH inhibition combined with TMZ and RT in orthotopic U87MG-Luc2 glioblastoma in vivo

(A) Schematic of the treatment schedules in vivo. (B) Tumor growth delay upon indicated treatments measured by BLI signal intensity for mice in each treatment group and the end point was when the BLI signal intensity reached to 10× intensity of the starting treatment day. (C) Kaplan-meier survival curve indicates number of days mice survived post-treatment. End-point was assessed based on the neurological sign and weight loss. (D–E) mRNA expression of the HES1 and CD133 in tumor samples treated with GSI vs. control was determined by qRT-PCR. Values were normalized to Actin. Error bars indicates SEM. Asterisk indicates significance (*p < 0.05, **p < 0.01 and ***p < 0.001, ns: not significant).

Figure 6

Figure 6. Effect of NOTCH inhibition combined with TMZ and RT on CD133, SOX2 and Nestin expression

(A–B) The expression of CD133 marker upon indicated treatments in U87MG-Luc2 and E2 cells was measured by flow cytometry. Graphs indicate the quantification of the relative CD133 expression normalized to IgG control upon indicated treatments. DAPI was used for the live cell staining. Concentration used in all cases was 10 μM GSI and 10 μM TMZ. (C–D) mRNA expression of the SOX2 stem cell marker in U87MG-Luc2 and E2 cells upon indicated treatments was measured by qRT-PCR. E) Protein expression of SOX2, Nestin and active form of NOTCH1 (NICD1) in E2 and U87 cells upon indicated treatments was analyzed by Western blotting. Actin and Lamin A/C serve as loading controls. Concentration used in all cases was 10 μM GSI and 10 μM TMZ. Error bars indicate SEM. Asterisk indicates significance (*p < 0.05, **p < 0.01 and ***p < 0.001, ns: not significant).

References

    1. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes AA, Marosi C, Bogdahn U, Curschmann J, Janzer RC, Ludwin SK, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 2005;352:987–996. - PubMed
    1. Friedman HS, McLendon RE, Kerby T, Dugan M, Bigner SH, Henry AJ, Ashley DM, Krischer J, Lovell S, Rasheed K, Marchev F, Seman AJ, Cokgor I, et al. DNA mismatch repair and O6-alkylguanine-DNA alkyltransferase analysis and response to Temodal in newly diagnosed malignant glioma. J Clin Oncol. 1998;16:3851–3857. - PubMed
    1. Hegi ME, Liu L, Herman JG, Stupp R, Wick W, Weller M, Mehta MP, Gilbert MR. Correlation of O6-methylguanine methyltransferase (MGMT) promoter methylation with clinical outcomes in glioblastoma and clinical strategies to modulate MGMT activity. J Clin Oncol. 2008;26:4189–4199. - PubMed
    1. Cahill DP, Levine KK, Betensky RA, Codd PJ, Romany CA, Reavie LB, Batchelor TT, Futreal PA, Stratton MR, Curry WT, Iafrate AJ, Louis DN. Loss of the mismatch repair protein MSH6 in human glioblastomas is associated with tumor progression during temozolomide treatment. Clin Cancer Res. 2007;13:2038–2045. - PMC - PubMed
    1. Yip S, Miao J, Cahill DP, Iafrate AJ, Aldape K, Nutt CL. MSH6 Mutations Arise in Glioblastomas during Temozolomide Therapy and Mediate Temozolomide Resistance. Clin Cancer Res. 2013;19:4543–4544. - PMC - PubMed

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