Glioblastoma (GBM) is a highly aggressive and lethal brain cancer type. PI3K and MAPK inhibitors have been studied preclinically in GBM as monotherapy, but not in combination with radiotherapy, which is a key component of the current standard treatment of GBM. In our study, GBM cell lines and patient representative primary cultures were grown as multicellular spheroids. Spheroids were treated with a panel of small-molecule drugs including MK2206, RAD001, BEZ235, MLN0128, and MEK162, alone and in combination with irradiation. Following treatment, spheroid growth parameters (growth rate, volume reduction, and time to regrow), cell-cycle distribution and expression of key target proteins were evaluated. , the effect of irradiation (3 × 2 Gy) without or with MEK162 (50 mg/kg) was studied in orthotopic GBM8 brain tumor xenografts with endpoints tumor growth and animal survival. The MAPK-targeting agent MEK162 was found to enhance the effect of irradiation as demonstrated by growth inhibition of spheroids. MEK162 downregulated and dephosphorylated the cell-cycle checkpoint proteins CDK1/CDK2/WEE1 and DNA damage response proteins p-ATM/p-CHK2. When combined with radiation, this led to a prolonged DNA damage signal. data on tumor-bearing animals demonstrated a significantly reduced growth rate, increased growth delay, and prolonged survival time. In addition, RNA expression of responsive cell cultures correlated to mesenchymal stratification of patient expression data. In conclusion, the MAPK inhibitor MEK162 was identified as a radiosensitizer in GBM spheroids and in orthotopic GBM xenografts The data are supportive for implementation of this targeted agent in an early-phase clinical study in GBM patients.

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<div>Abstract<p>Glioblastoma (GBM) is a highly aggressive and lethal brain cancer type. PI3K and MAPK inhibitors have been studied preclinically in GBM as monotherapy, but not in combination with radiotherapy, which is a key component of the current standard treatment of GBM. In our study, GBM cell lines and patient representative primary cultures were grown as multicellular spheroids. Spheroids were treated with a panel of small-molecule drugs including MK2206, RAD001, BEZ235, MLN0128, and MEK162, alone and in combination with irradiation. Following treatment, spheroid growth parameters (growth rate, volume reduction, and time to regrow), cell-cycle distribution and expression of key target proteins were evaluated. <i>In vivo</i>, the effect of irradiation (3 × 2 Gy) without or with MEK162 (50 mg/kg) was studied in orthotopic GBM8 brain tumor xenografts with endpoints tumor growth and animal survival. The MAPK-targeting agent MEK162 was found to enhance the effect of irradiation as demonstrated by growth inhibition of spheroids. MEK162 downregulated and dephosphorylated the cell-cycle checkpoint proteins CDK1/CDK2/WEE1 and DNA damage response proteins p-ATM/p-CHK2. When combined with radiation, this led to a prolonged DNA damage signal. <i>In vivo</i> data on tumor-bearing animals demonstrated a significantly reduced growth rate, increased growth delay, and prolonged survival time. In addition, RNA expression of responsive cell cultures correlated to mesenchymal stratification of patient expression data. In conclusion, the MAPK inhibitor MEK162 was identified as a radiosensitizer in GBM spheroids <i>in vitro</i> and in orthotopic GBM xenografts <i>in vivo</i>. The data are supportive for implementation of this targeted agent in an early-phase clinical study in GBM patients. <i>Mol Cancer Ther; 17(2); 347–54. ©2017 AACR</i>.</p><p>See all articles in this <i><a href="http://mct.aacrjournals.org/content/17/2#MCTFocus" target="_blank">MCT Focus</a></i> section, “Developmental Therapeutics in Radiation Oncology.”</p></div>

<p>Supplemental Figure 1. Chemical structures of non-FDA approved compounds used in the Study; Supplemental Figure 2. Effect of drugs on growth and target phosphorylation; Supplemental Figure 3. mTOR inhibition does not show dose dependent Radiosensitization; Supplemental Figure 4. MEK162 + RT abrogate spheroid regrowth; Supplemental Figure 5. MEK162 radiosensitizes GBM8 primary spheroid culture and increases γH2AX levels; Supplemental Figure 6. MEK162 abrogates BrdU washout and increases sub-G1 accumulation of BrdU+ cells; Supplemental Figure 7. MEK162 + RT in vivo; Supplemental Table 1: Antibodies used; Supplemental Table 2: Gene signature MEK+RT response</p>

Radiotherapy remains one of the most effective modalities for anticancer treatment. Boosting the efficacy of radiotherapy is therefore a logical avenue to improve patient survival. We have developed a radiosensitization strategy called ‘induction of mitotic enrichment’. It has long been known that the radiosensitivity of a cell depends to a large extent on the phase of the cell cycle and that especially mitotic cells are especially vulnerable. Enriching the tumor for mitotic cells by arresting them during division prior to each radiotherapy fraction should therefore render the tumor population more sensitive to irradiation. Ideally, induction of mitotic enrichment should be reversible and non-cytotoxic to prevent healthy tissue toxicity and be compatible with clinically applied fractionated radiotherapy regimens. We have now identified an orally available targeted tubulin polymerization inhibitor that can achieve repeated and reversible mitotic enrichment for up to 10 hours prior to radiotherapy, without causing cytotoxicity in vitro or healthy tissue toxicity in vivo. Most importantly, this tubulin inhibitor efficiently radiosensitizes a range of preclinical glioblastoma models in vitro and in vivo and significantly improves survival, but only in a mitotic enrichment setup when given several hours prior to radiotherapy to allow accumulation in mitosis. We have initiated development of mitotic enrichment for GBM, the most common primary malignant brain tumor. Their location and highly aggressive nature renders GBM among the most deadly and devastating of human malignancies. Despite extensive treatment involving surgery and adjuvant chemo-radiotherapy, prognosis is still dismal and novel treatment strategies are urgently needed. Of all available adjuvant therapies, radiotherapy contributes most to extending overall survival. Increasing the efficacy of existing radiotherapeutic regimens therefore offers a logical rationale to improve the survival of GBM patients. We are currently also expanding our preclinical development of mitotic enrichment as a radiosensitization strategy to other mitotic targets and different cancers for which radiotherapy is a mainstay treatment and have thus far achieved promising results in vitro. In parallel, we are preparing a phase 0 trial to demonstrate induction of mitotic enrichment in human GBM. Citation Format: Mark C. de Gooijer, Paul L.G. Slangen, Hilal Çolakoğlu, Ceren H. Çitirikkaya, Amal El Ouazani, Ronak Shah, Gerben R. Borst, Olaf van Tellingen. Mitotic enrichment as an efficient radiosensitization strategy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-003.