Radiotherapy and radio‐sensitization in H3 K27M ‐mutated diffuse midline gliomas

Abstract Background H3 K27M mutated diffuse midline gliomas (DMGs) are extremely aggressive and the leading cause of cancer‐related deaths in pediatric brain tumors with 5‐year survival <1%. Radiotherapy is the only established adjuvant treatment of H3 K27M DMGs; however, the radio‐resistance is commonly observed. Methods We summarized current understandings of the molecular responses of H3 K27M DMGs to radiotherapy and provide crucial insights into current advances in radiosensitivity enhancement. Results Ionizing radiation (IR) can mainly inhibit tumor cell growth by inducing DNA damage regulated by the cell cycle checkpoints and DNA damage repair (DDR) system. In H3K27M DMGs, the aberrant genetic and epigenetic changes, stemness genotype, and epithelial‐mesenchymal transition (EMT) disrupt the cell cycle checkpoints and DDR system by altering the associated regulatory signaling pathways, which leads to the development of radio‐resistance. Conclusions The advances in mechanisms of radio‐resistance in H3 K27M DMGs promote the potential targets to enhance the sensitivity to radiotherapy.


| INTRODUC TI ON
Glioma is one of the most common brain malignancies. Although the integrative therapies including surgeries and chemoradiotherapies are applied in clinical treatment, the outcomes of glioma patients are unsatisfied. 1 Table 1). Current clinical management of H3 K27M -mutated DMGs include surgical resections and post-behavior of H3 K27M -mutated DMGs, 8,9 as well as the anatomical adjacent with most important brain structures such as thalamus, brainstem and medulla oblongata, the total resection of tumors is impractical. [8][9][10][11][12] Thus, the adjuvant therapies are essential to prevent tumor recurrence and metastasis in H3 K27M DMGs. Although the resistance of first-line temozolomide has commonly observed in H3 K27M DMGs due to the universal O6-methylguanine-DNA methyltransferase (MGMT) promotor unmethylation and intact blood-brain barrier (BBB). 13,14 Radiotherapy, as a standard and efficient strategy, can significantly improve the life qualities of H3 K27M DMGs with 70%-80% of patients obtaining temporary symptom relief and increased survival. [15][16][17][18] Ionizing radiation (IR) can inhibit tumor growth by inducing DNA damage directly or through reactive oxygen species (ROS). 19 The cell cycle checkpoints and DNA damage repair (DDR) system are essential for regulating the process. 20 In H3 K27M DMGs, the aberrant genetic and epigenetic changes, stemness genotype, and epithelialmesenchymal transition (EMT) disrupt the cell cycle checkpoints and DDR system by altering the associated regulatory signaling pathways, 9,[21][22][23] which could lead to the development of radioresistance. In clinical, a large proportion of H3 K27M DMGs relapses within 7 months after radiotherapy. 4,24 To improve the sensitivity of tumor cells to radiation, extensive investigations have been conducted to understand the mechanisms of radioresistance and discover the approach to enhance the radiosensitivity including modulating radiotherapy doses and fractions and a combination of immunotherapy and chemotherapy. Here, we provide crucial insights into the molecular responses of H3 K27M DMGs to radiotherapy and current advances in radiosensitivity enhancement.

| Histones and cancers
Histones including four core proteins H3, H4, H2A and H2B are the major structural components of chromatins which play important roles in gene expression and epigenetic modification. 25,26 The protruding N-terminal amino acid tail of a histone is subject to various post-translational modifications (PTMs) such as methylation, acetylation, phosphorylation, ubiquitylation, and SUMOylation. [27][28][29][30][31] Histone PTMs can active or decrease gene transcription through a local structural alteration of chromatin conducted by the interactions between histones and DNA. 32 The alterations of histones are demonstrated to be associated with various cancers such as gliomas, sarcomas, head and neck cancers and carcinosarcomas. 33,34 Of note, "oncohistones", the cancer-associated histone mutations, are highly specific in certain type of cancers. For instance, H3 K27M and H3 G34R/V mutations are specifically detected in brain tumors, H3 K36M and H3 G34W/L are mostly detected in bone cancers, while H1 mutations are commonly found in lymphomas. 35

| Molecular mechanisms and signaling transduction in H3 K27M tumor
In DMGs, H3 K27M mutation can induce a global reduction of H3K27me3, an epigenetic mark to H3 indicating the trimethylation of lysine 27 on histone H3 protein, which is associated with transcriptional silencing of the key genes regulating cell differentiation via the formation of heterochromatic regions. [43][44][45] H3K27me3 is catalyzed by the histone methyltransferase enhancer of zeste homolog 2 (EZH2), a subunit of polycomb repressive complex 2 (PRC2), which is the central modulator of H3K27me3-decorated facultative heterochromatin. 46,47 The crystal structure analysis suggests that K27M-mutant H3 can bind directly to the active SET domain of EZH2 with 16-fold higher affinity than wildtype H3 leading to the sequestration of EZH2 and further impairment of PRC2 methyltransferase activity and the reduction of H3K27me3. 4,15,48,49 In addition, H3 K27M mutation can decrease the auto-methylation of EZH2/PRC2 which is required for histone methyltransferase activity, resulting the detention of H3K27me2 to H3K27me3. 50,51 Another epigenetic mark to H3 in H3 K27M mutation cells is that the increased acetylation of H3K27 (H3K27ac), which is considered to be a super-enhancer to promote gene transcription involved in stem cell differentiation and tumorigenesis 45,52 (Figure 1).

| R ADIOTHER APIE S AND MECHANIS MS
Radiotherapy serves as cornerstone in the adjuvant treatment of H3 K27M DMGs, which significantly improve the clinical outcomes in 70%-80% of patients. [16][17][18] Currently, the recommend total dose setting of radiotherapy is 54-60 Gy with a daily fraction of 1.8-2.0 Gy over 6 weeks. 70 Mechanically, radiation mainly inhibits tumor growth by inducing DNA damage. 19 The damaged DNA would trigger tumor cell death once failure in the repairment. However, DNA damage detection and repair are complicated regulated by intricate intracellular and extracellular networks. Under certain circumstances, tumor cells can exhibit radio-resistance due to cell-intrinsic mechanisms and microenvironment resulting treatment failure and recurrence. 71,72 Thus, understanding the mechanisms underlying radio-resistance and developing mechanism-based radio sensitization to enhance radiotherapy responses would improve the outcomes of glioma patients. A set of sophisticated and highly regulated cellular processes can be triggered by the exposure of radiation. Among these, the cell cycle checkpoints and DDR system are the main components coordinated by complex signal transduction. 73,74 Mechanically, the sensors recognizing the radiation induced DNA damage can initiate the cascade activation of a series of kinases involved in the cell cycle checkpoints and DDR networks to complete DNA repairs.

| Cell cycle checkpoints
Cell cycle checkpoints consisting of G1, intra-S, G2 and spindle phase act as surveillance mechanisms and regulators to monitor the major events among the process of cell cycle (such as DNA damage). Once aberrant events are detected by the checkpoints during segregation at mitosis, a pulse would be placed to arrest the cell cycle which allows to provide a time for DNA repair in order to accurately passage bioinformation into daughter cells and maintain the genomic stability.
For example, G1 phase checkpoint assure the damaged DNA cannot enter S phase, and G2 checkpoint could prevent the passing of abnormal DNA to future generations. 75 The dysfunction of cell cycle checkpoints or failure in the repairment would trigger cell death eventually.
cycle and proliferation to avoid cells death or senescence presenting radiotherapy resistance. Thus, targeting the checkpoints could be a promising strategy of enhancing radio-sensitivity. 89

| Signaling pathways regulate DNA damage repair
DDR process receives regulations from multiple intra-and extracellular signalings. 105 RTKs and their signaling pathways including PI3K/AKT/mTOR and MEK/ERK can regulate DDR by interacting with the components in both HR and NHEJ, such as PCNA, ATM, and DNA-PKcs. 106,107 For example, in DSBs, AKT can activate Rad51 and DNA-PKcs to facilitate DNA damage repair. 108 mTOR is able to mediate DDR by regulating RNF168 ubiquitination and P53. 109,110 In addition, mTOR can increase FA complementation group D2 (FANCD2) to promote ATM-CHKs checkpoints by activating NF-κB pathway. 111,112 MEK/ERK pathway has been shown to interfere with cell cycle checking points and DDR by enhancing ATM and ATR activation [113][114][115] (Figure 5).

| R AD I O -RE S IS TAN CE AND CURRENT R AD I O -S EN S ITIZ ATI ON S TR ATEG IE S
In H3 K27M DMGs, the genetic and epigenetic abnormalities could disrupt the cell cycle checkpoints and DDR system by directly or indirectly altering the associated regulatory signaling pathways. 9,[21][22][23]72,116 Which results in the common development of radio-resistance.
Thus, current strategies of enhancing radiosensitivity of H3 K27M DMGs mainly focus on the amendment of the H3 K27M epigenic and genetic alterations and the aberrant cell cycle checkpoints and DDR system ( Table 2), as well as the combination therapies with immunotherapy and chemotherapy.

| Targeting cell cycle checkpoints and DDR system
Cell cycle checkpoints and DDR intertwined signaling networks can arrest cell cycles, identify, and repair DNA damages induced by radiation. Targeting the components of cell cycle checkpoints and DDR is one of the main strategies to rescue radio-resistance.
In H3 K27M DMGs, the abnormalities of G1/S checkpoint regulators such as CDK4/6 and cyclinD and G2/S regulators such as Wee1, PLK1 were commonly detected. [121][122][123][124] Although the block of CDK4/6 could result in G1 arrest which potentially decrease the sensitivity of radiotherapy, CDK4/6 inhibition alone or following radiotherapy have been suggested to significantly reduce tumor ATM deletion is proved to enhance tumor radiosensitivity in p53deficient DIPG, 134 and its inhibitor AZD1390 is currently involved in a clinical trial of the brain cancer patients underwent radiation therapy (NCT03423628). In addition, the enzymes such as BMI1 and PARP1 are hyperactive in DIPG. 135,136 The inhibition of BMI1 could sensitize the DIPG cells response to radiotherapy via inhibiting damaged DNA ends resection in HR. 135 PARP1 is important for the recognition of SSBs and recruitment of XRCC1. 105 The inactivation of PARP1 exhibits enhanced radiosensitivity in DIPGs. 137,138 However, a phase I/II study of veliparib combined with chemoradiotherapy in younger patients with newly diagnosed DIPG could not improve the the survial. 139

| Targeting RTKs and downstream signaling pathways
RTKs and their downstream pathways regulate the cell cycle checkpoints and DDR process involving in the response of tumor cells to F I G U R E 5 RTKs and downstream signaling associated with radio-resistance in H3 K27M -mutated DMGs. Yellow ovals indicate the molecules enhancing radiosensitivity in clinical and investigation. radiotherapy and radio-resistance in DMGs. 22,23,72 Although the clinical benefit of targeting RTKs such as EGFR and vascular endothelial growth factor receptor (VEGFR) in combination with radiotherapy are still not conclusive in H3 K27M DMGs or DIPGs. 9,56,[164][165][166][167][168][169][170] The inhibition of their downstream PI3K/AKT/mTOR and MEK/ERK pathways can significantly sensitize glioma cells to radiotherapy exhibiting increased cell apoptosis and reduced cancer stemness. [171][172][173][174][175][176][177] The mTOR inhibitor TAK228 has been suggested to induce cell apoptosis and enhance radiosensitivity in DIPG. 176 In addition, combined targeting strategy is under estimation. A study of dual inhibition of PI3K and HDACs in DIPGs shows an increased radiosensitivity though abrogating NFκB and Forkhead box M1 (FOXM1) mediated in DNA damage responses. 119,178,179 FOXM1 is a transcription factor regulating the expression of cell cycle genes essential for DNA replication and mitosis, and DNA damage repair. 178

Accumulating evidence suggests that cancer stem cells (CSCs) in
DMGs impact the sensitivity of radiotherapies. 177,180,181 CSCs are a small subpopulation of cells within tumors with capabilities of self-renewal, differentiation, and tumorigenicity. The stemness of CSCs regulated by intrinsic factors such as cell survival and self-renewal pathways [182][183][184][185][186][187][188][189] and extrinsic causes such as hypoxia microenvironment [190][191][192][193][194] allow to activate cell cycle check points and DDR to decrease the DNA damage from radiation and develop radio-resistance. 182 Notch pathway is considered to be an essential self-renewal signaling to maintain CSCs stemness in H3 K27M gliomas, while its inhibitor, MRK003, presents an enhanced irradiation-induced apoptosis of tumor cells. 195 Recently, targeting mitochondrial metabolism and tumor hypoxia microenvironment are suggested to effectively ameliorate radio-resistance in CSCs in highgrade gliomas. 196 EMT is commonly involved in tumor initiation, invasion and metastasis in a broad set of cancers, which presents a transition of epithelial cells to mesenchymal cells with the lost function of cell-cell junctions and cell polarity. [197][198][199][200][201][202][203] EMT requires a robust reprogramming of gene expression which is associated with maintenance of cancer stemness leading chemo-and radio-resistance. [204][205][206][207] programs are regulated by multiple pathways including JAK/STAT pathway, TGFβ/SMAD, NF-κB, PI3K/AKT signaling, and Notch to explore the synergetic mechanism on radiation combined with immunotherapy.
As for chemotherapy, the combination of temozolomide (TMZ) and radiotherapy is recommended in H3 K27M DMGs. However, the clinical of outcomes are not satisfied due to the unmethylation of the O 6 -methylguanine-DNA methyltransferase (MGMT) associated facilitation of DNA damage repair, and relatively intact blood-brain barrier (BBB). 13,14,[236][237][238][239][240][241][242] Recently, the development of nanotechnologies allows to reformulate TMZ to enhance therapeutic efficiency with the evasion of MGMT enzyme. 243 In addition, pulsed ultrasound and convection-enhanced delivery (CED) have been applied to temporally disrupt the BBB and increase the concentration of drug delivery in tumor area, as a result, to enhance the sensitivity of chemotherapies and radiotherapy. 244,245

| CON CLUS IONS
The clinical treatment of H3 K27M DMGs remains challenging.
Beyond maximal safe tumor resection, adjuvant radiotherapy is reliable treatment to improve patients' outcomes. However, the genetic and epigenetic alterations and signaling dysregulation participating in cell cycle checkpoints and DDR in H3 K27M DMGs frequently associates with radio-resistance resulting in unsat-

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
No new data were created.

I N S TITUTI O N A L R E V I E W B OA R D S TATE M E NT
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I N FO R M E D CO N S E NT S TATE M E NT
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