Chemoresistance In Glioblastoma Research Articles

Abstract 7207: Targeting hippo signaling pathway to overcome chemoresistance in glioblastoma

Abstract The study aims to investigate whether the Hippo signaling pathway contributes to temozolomide chemoresistance in glioblastoma, and to validate whether the combinational treatment of temozolomide and YAP inhibitor is effective against glioblastoma. Temozolomide-sensitive and temozolomide-resistant U87 and U251 GBM human cell lines were previously established. Temozolomide-resistant cells were maintained in low-dose temozolomide. Here, we investigate whether Hippo signaling contributes to temozolomide chemoresistance. By modulating the Hippo signaling pathway through YAP gene knockdown, the outcome on cell proliferation was studied by cell viability assay (in vitro) and mouse orthotopic xenograft (in vivo). Finally, the therapeutic effect of YAP inhibitor is studied in vitro and in vivo respectively. Our data showed that YAP is overexpressed in temozolomide-resistant glioblastoma cells. Nuclear translocation of YAP (which becomes its active state) is increased in temozolomide-resistant cells. In fact, YAP inhibition show decreased cell viability and higher susceptibility to temozolomide. In addition, mice with tumor injection of the YAP knockdown cells reduced tumor size with temozolomide treatment. Similarly, YAP inhibitor resensitizes temozolomide-resistant cells to temozolomide. Combinational treatment of YAP inhibitor and temozolomide is effective in shrinking tumor size in mouse xenograft models. In conclusion, our data suggest dysregulated Hippo signaling pathway contributes to temozolomide chemoresistance, thus targeting the Hippo signaling pathway resensitizes GBM cells to temozolomide. This research provides pre-clinical evidence on using YAP inhibitor for combinational therapy with temozolomide for the treatment of glioblastoma. Citation Format: Cheuk Lun Ethan Wong, Mei Yee Karrie Kiang, Ka Kit Gilberto Leung. Targeting hippo signaling pathway to overcome chemoresistance in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7207.

Abstract 7204: Therapeutic potential of targeting autophagy to overcome chemoresistance in glioblastoma

Abstract Glioblastoma (GBM) is the most malignant subtype of brain tumor in adults, characterized by relentless recurrence and an overall survival of approximately 18 months. Temozolomide (TMZ) has been the basis for current standard of care and represents the control-arm in GBM trials. Despite the use of TMZ having shown promising results in improving patient survival, drug resistance and tumor relapse is almost inevitable. Current literature has provided strong evidence on autophagy modulation as an adjuvant therapy to cytotoxic treatments. By using in vitro and in vivo GBM models, combination regimen with TMZ and an autophagy activator significantly enhanced the cytotoxic effect in tumors that were resistant to chemotherapy. Combined treatment readily induced a mortal autophagy flux in chemo-resistant GBM cells resulted from its high intrinsic autophagy. Our findings suggest that treatment resistant tumors are more sensitive to autophagy activation when compared to treatment-naive tumors. As they often exhibit higher basal level of autophagy after long-term chemotherapy, a mortal autophagy flux can be readily achieved. In conclusion, combination therapy of a cytotoxic drug with autophagy modulator is emerging as a novel therapeutic strategy in the adjuvant setting for cancer patients. Citation Format: Karrie Mei Yee Kiang, Gilberto Ka Kit Leung. Therapeutic potential of targeting autophagy to overcome chemoresistance in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7204.

Abstract 3286: Synergistic impact of long non-coding RNA TUG1 depletion in overcoming temozolomide chemoresistance in glioblastoma

Abstract Glioblastoma (GBM) stands out as the most prevalent and aggressive primary brain tumor in adults, marked by a high mortality rate. Standard therapy involves temozolomide (TMZ) chemotherapy, an alkylating agent. However, most cases develop resistance to TMZ, necessitating the development of new treatment options to overcome TMZ-resistant GBM. Long non-coding RNAs (lncRNAs) constitute a class of RNA molecules that, despite not encoding proteins, play pivotal roles in regulating diverse cellular processes. Overexpression of specific lncRNAs in cancer is linked to crucial aspects of cancer progression, such as cell proliferation, apoptosis, and metastasis. Taurine upregulated gene 1 (TUG1) is a highly expressed lncRNA in GBM, playing a crucial role in preventing excessive DNA replication stress and associated DNA damage (Nat Commun 2023).This study aimed to investigate the combined effects of TUG1 depletion and TMZ in TMZ-resistant GBM. First, we established a TMZ-resistant U251MG cell line, U251MG-R, characterized by a low DNA methylation level of the O6-methylguanine-DNA methyltransferase (MGMT) promoter. We observed that TMZ increased DNA replication stress in the U251MG-R. Depleting TUG1 by using antisense oligonucleotide (TUG1-ASO) sensitized U251MG-R cells to TMZ. Treatment with TMZ or TUG1-ASO alone inhibited cell proliferation, with the most significant inhibition observed when both were combined. Next, we employed a xenograft mouse model to evaluate the efficacy of intravenous TUG1-ASO treatment coupled with a tumor-specific drug delivery system (antiTUG1-DDS). The combination of antiTUG1-DDS with TMZ displayed the most effective suppression of tumor growth compared to either TMZ or antiTUG1-DDS alone. Targeted therapy aimed at TUG1 may be a promising approach for overcoming TMZ resistance in GBM. Citation Format: Hidekazu Kito, Yuji Kibe, Miho Suzuki, Keiko Shinjo, Yutaka Kondo. Synergistic impact of long non-coding RNA TUG1 depletion in overcoming temozolomide chemoresistance in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3286.

ARF4-mediated retrograde trafficking as a driver of chemoresistance in GBM.

Cellular functions hinge on the meticulous orchestration of protein transport, both spatially and temporally. Central to this process is retrograde trafficking, responsible for targeting proteins to the nucleus. Despite its link to many diseases, the implications of retrograde trafficking in glioblastoma (GBM) are still unclear. To identify genetic drivers of TMZ resistance, we conducted comprehensive CRISPR-knockout screening, revealing ADP-ribosylation factor 4 (ARF4), a regulator of retrograde trafficking, as a major contributor. Suppressing ARF4 significantly enhanced TMZ sensitivity in GBM patient-derived xenograft (PDX) models, leading to improved survival rates (p<0.01) in both primary and recurrent lines. We also observed that TMZ exposure stimulates ARF4-mediated retrograde trafficking. Proteomics analysis of GBM cells with varying levels of ARF4 unveiled the influence of this pathway on EGFR signaling, with increased nuclear trafficking of EGFR observed in cells with ARF4 overexpression and TMZ treatment. Additionally, spatially-resolved RNA-sequencing of GBM patient tissues revealed substantial correlations between ARF4 and crucial nuclear EGFR (nEGFR) downstream targets, such as MYC, STAT1, and DNA-PK. Decreased activity of DNA-PK, a DNA repair protein downstream of nEGFR signaling that contributes to TMZ resistance, was observed in cells with suppressed ARF4 levels. Notably, treatment with DNA-PK inhibitor, KU57788, in mice with a recurrent PDX line resulted in prolonged survival (p<0.01), highlighting the promising therapeutic implications of targeting proteins reliant on ARF4-mediated retrograde trafficking. Our findings demonstrate that ARF4-mediated retrograde trafficking contributes to the development of TMZ resistance, cementing this pathway as a viable strategy to overcome chemoresistance in GBM.

Neovascularization directed by CAVIN1/CCBE1/VEGFC confers TMZ-resistance in glioblastoma

Acquisition of resistance to temozolomide (TMZ) poses a significant challenge in glioblastoma (GBM) therapy. Neovascularization, a pivotal process in tumorigenesis and development, remains poorly understood in its contribution to chemoresistance in GBMs. This study unveils aberrant vascular networks within TMZ-resistant (TMZ-R) GBM tissues and identifies the extracellular matrix (ECM) protein CCBE1 as a potential mediator. Through in vivo and in vitro experiments involving gain and loss of function assessments, we demonstrate that high expression of CCBE1 promotes hyper-angiogenesis and orchestrates partial endothelial-to-mesenchymal transition (EndMT) in human microvascular endothelial cells (HCMEC/d3) within GBM. This is likely driven by VEGFC/Rho signaling. Intriguingly, CCBE1 overexpression substantially fails to promote tumor growth, but endows resistance to GBM cells in a vascular endothelial cell-dependent manner. Mechanically, the constitutive phosphorylation of SP1 at Ser101 drives the upregulation of CCBE1 transcription in TMZ resistant tumors, and the excretion of CCBE1 depends on caveolae associated protein 1 (CAVIN1) binding and assembling. Tumor cells derived CCBE1 promotes VEGFC maturation, activates VEGFR2/VEGFR3/Rho signaling in vascular endothelial cells, and ultimately results in hyper-angiogenesis in TMZ-R tumors. Collectively, the current study uncovers the cellular and molecular basis of abnormal angiogenesis in a chemo resistant microenvironment, implying that curbing CCBE1 is key to reversing TMZ resistance.

FBXO7 Confers Mesenchymal Properties and Chemoresistance in Glioblastoma by Controlling Rbfox2-Mediated Alternative Splicing.

Mesenchymal glioblastoma (GBM) is highly resistant to radio-and chemotherapy and correlates with worse survival outcomes in GBM patients; however, the underlying mechanism determining the mesenchymal phenotype remains largely unclear. Herein, it is revealed that FBXO7, a substrate-recognition component of the SCF complex implicated in the pathogenesis of Parkinson's disease, confers mesenchymal properties and chemoresistance in GBM by controlling Rbfox2-mediated alternative splicing. Specifically, FBXO7 ubiquitinates Rbfox2 Lys249 through K63-linked ubiquitin chains upon arginine dimethylation at Arg341 and Arg441 by PRMT5, leading to Rbfox2 stabilization. FBXO7 controls Rbfox2-mediated splicing of mesenchymal genes, including FoxM1, Mta1, and Postn. FBXO7-induced exon Va inclusion of FoxM1 promotes FoxM1 phosphorylation by MEK1 and nuclear translocation, thereby upregulates CD44, CD9, and ID1 levels, resulting in GBM stem cell self-renewal and mesenchymal transformation. Moreover, FBXO7 is stabilized by temozolomide, and FBXO7 depletion sensitizes tumor xenografts in mice to chemotherapy. The findings demonstrate that the FBXO7-Rbfox2 axis-mediated splicing contributes to mesenchymal transformation and tumorigenesis, and targeting FBXO7 represents a potential strategy for GBM treatment.

Inhibition of autophagy and induction of glioblastoma cell death by NEO214, a perillyl alcohol-rolipram conjugate

ABSTRACT Glioblastoma (GBM) is the most aggressive primary brain tumor, exhibiting a high rate of recurrence and poor prognosis. Surgery and chemoradiation with temozolomide (TMZ) represent the standard of care, but, in most cases, the tumor develops resistance to further treatment and the patients succumb to disease. Therefore, there is a great need for the development of well-tolerated, effective drugs that specifically target chemoresistant gliomas. NEO214 was generated by covalently conjugating rolipram, a PDE4 (phosphodiesterase 4) inhibitor, to perillyl alcohol, a naturally occurring monoterpene related to limonene. Our previous studies in preclinical models showed that NEO214 harbors anticancer activity, is able to cross the blood-brain barrier (BBB), and is remarkably well tolerated. In the present study, we investigated its mechanism of action and discovered inhibition of macroautophagy/autophagy as a key component of its anticancer effect in glioblastoma cells. We show that NEO214 prevents autophagy-lysosome fusion, thereby blocking autophagic flux and triggering glioma cell death. This process involves activation of MTOR (mechanistic target of rapamycin kinase) activity, which leads to cytoplasmic accumulation of TFEB (transcription factor EB), a critical regulator of genes involved in the autophagy-lysosomal pathway, and consequently reduced expression of autophagy-lysosome genes. When combined with chloroquine and TMZ, the anticancer impact of NEO214 is further potentiated and unfolds against TMZ-resistant cells as well. Taken together, our findings characterize NEO214 as a novel autophagy inhibitor that could become useful for overcoming chemoresistance in glioblastoma. Abbreviations: ATG: autophagy related; BAFA1: bafilomycin A1; BBB: blood brain barrier; CQ: chloroquine; GBM: glioblastoma; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MGMT: O-6-methylguanine-DNA methyltransferase; MTOR: mechanistic target of rapamycin kinase; MTORC: MTOR complex; POH: perillyl alcohol; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TMZ: temozolomide

PIK3CB as a potential target to regulate chemosensitivity in glioblastoma.

e14051 Background: Glioblastoma (GBM) is the most common primary central nervous system malignancy but exhibits universally poor outcomes in part due to chemoresistance. Temozolomide (TMZ) can significantly improve overall survival, but resistance mechanisms limit its consistent efficacy. The phosphoinositide 3 kinase (PI3K) pathway is involved in multiple TMZ resistance mechanisms and is highly druggable; however, pan-inhibition of its four grossly homologous but functionally distinct catalytic subunits (PIK3CA/B/D/G encode p110α/β/δ/γ, respectively) was unsuccessful clinically. Therefore, by understanding how the individual PI3K subunits regulate TMZ sensitivity, a more selective treatment strategy can be developed to combat chemoresistance. Methods: Differential expression of PI3K subunit genes was assessed using RNA sequencing (RNAseq) results obtained from public databases (e.g., DepMap, The Cancer Tissue Genome Atlas) for GBM cell lines and patient tumors expressing high or low O6-methylguanine-DNA-methyltransferase (MGMT), a DNA repair enzyme highly implicated in TMZ resistance. PI3K protein levels were assessed using immunohistochemistry of gliomas available at The Human Protein Atlas (THPA). PI3K gene expression was correlated with PI3K pathway activation, quantified as protein kinase B phosphorylation (pAKT). PI3K mRNAs and pAKT were then correlated with the half maximal inhibitory concentrations (IC50s) of TMZ in GBM cell lines to evaluate for a relationship between PI3K gene expression, PI3K pathway activity, and TMZ sensitivity. Finally, hazard ratios were used to compare PI3K gene expression in patient tumors with survival and response to chemotherapy to determine if expression was predictive of prognosis and/or chemosensitivity. Results: PIK3CB was the only PI3K gene enriched in both GBM cell lines and tumors, demonstrating significantly higher expression than all other PI3K genes in GBM (P &lt; 0.0001), independent of MGMT levels. PIK3CB expression remained the highest even after controlling for GBM risk factors such as tumor recurrence, isocitrate dehydrogenase (IDH) mutations, molecular subtypes, or patient gender/age differences. Levels of the p110β protein were also significantly higher than those of other PI3K catalytic subunits (P &lt; 0.001). PIK3CB expression was positively correlated with pAKT levels (P &lt; 0.05), but only in cell lines and tumors with low MGMT activity. Both PIK3CB expression and pAKT levels correlated positively with TMZ IC50s (P &lt; 0.05) in MGMT-deficient GBMs, suggesting an inverse relationship between PIK3CB and TMZ sensitivity. Finally, better prognosis and significant survival benefit with chemotherapy were both associated with lower levels of PIK3CB in MGMT-deficient patients. Conclusions: PIK3CB may selectively regulate chemoresistance in GBM, suggesting a role for p110β selective inhibition to improve chemosensitivity.

Overcoming temozolomide resistance in glioblastoma through selective inhibition of phosphoinositide 3-kinase catalytic subunit.

e14045 Background: Glioblastoma is one of medicine's most difficult-to-treat cancers due to its inevitable resistance to chemotherapies, such as temozolomide (TMZ). Tumor resistance to TMZ often comes from overactivation of phosphoinositide 3-kinase (PI3K), making this enzyme an ideal anti-cancer drug target; however, in practice, non-selective targeting of PI3K has resulted in undesirable clinical outcomes. Therefore, it is necessary to explore each subunit of PI3K to determine their effect on chemoresistance in order to develop therapeutics to overcome said resistance. PI3K has four homologous yet functionally distinct catalytic subunits: p110α, p110β, p110δ, and p110γ. Preliminary in silico analysis of each subunit has demonstrated that p110β is more highly expressed in glioblastoma than the other subunits. Here we report that TMZ resistance in glioblastoma is driven primarily by activation of p110β. Methods: Glioblastoma cell lines with high p110β levels (SF295 and U87MG) and low p110β levels (A172 and LN229) were cultured. CRISPR-Cas9 was used to knockout p110α, p110β, or p110δ in cultured cell lines. The viability of cultured cells was measured using an MTS viability assay. Newly cultured wild-type cells were treated with IC50 doses of selective p110β inhibitor TGX-221 and TMZ. Mice bearing SF295 xenograft tumors were treated with DMSO, TGX-221, and/or TMZ. A Bliss model of independence was used to assess the association between TGX-221 and TMZ with synergistic and antagonistic patterns measured using excess over Bliss (EOB) scores. Results: There was a significant reduction in viability among SF295 p110β-knockout cells (p &lt; 0.001) and an additive cytotoxic effect following treatment with TMZ. The LN229 p110β-knockout cells treated with TMZ did not reduce cell viability. Similarly, no substantial declines in viability were observed in the p110α- or p110δ-knockout cells before or after TMZ treatment in either cell line. In newly cultured wild-type p110β-high and p110β-low glioblastoma cell lines, TGX-221 or TMZ alone were insufficient to inhibit cell viability; however, when combined, TGX-221 and TMZ synergistically decreased cell survival with an EOB of 45.5% in U87MG cells and 26.3% in SF295 cells. No additive effects were detected in the p110β-low cell lines following TGX-221 and TMZ treatment. Additionally, TGX-221 combined with TMZ resulted in a drastic reduction in xenograft tumor growth, which was significantly greater than TGX-221 (p &lt; 0.02) or TMZ (p &lt; 0.003) treatment alone. Conclusions: Inhibition of p110β via CRISPR-Cas9 knockout or pharmaceutical blockade restores TMZ sensitivity in p110β-high glioblastoma cells and xenograft glioblastoma tumors. These results demonstrate the varying importance of PI3K catalytic subunits and necessitate further exploration into p110β as a potential drug target to overcome chemoresistance in glioblastoma.

Temozolomide Resistance in Glioblastoma by NRF2: Protecting the Evil

The transcription factor NRF2 is constitutively active in glioblastoma, a highly aggressive brain tumor subtype with poor prognosis. Temozolomide (TMZ) is the primary chemotherapeutic agent for this type of tumor treatment, but resistance to this drug is often observed. This review highlights the research that is demonstrating how NRF2 hyperactivation creates an environment that favors the survival of malignant cells and protects against oxidative stress and TMZ. Mechanistically, NRF2 increases drug detoxification, autophagy, DNA repair, and decreases drug accumulation and apoptotic signaling. Our review also presents potential strategies for targeting NRF2 as an adjuvant therapy to overcome TMZ chemoresistance in glioblastoma. Specific molecular pathways, including MAPKs, GSK3β, βTRCP, PI3K, AKT, and GBP, that modulate NRF2 expression leading to TMZ resistance are discussed, along with the importance of identifying NRF2 modulators to reverse TMZ resistance and develop new therapeutic targets. Despite the significant progress in understanding the role of NRF2 in GBM, there are still unanswered questions regarding its regulation and downstream effects. Future research should focus on elucidating the precise mechanisms by which NRF2 mediates resistance to TMZ, and identifying potential novel targets for therapeutic intervention.

STAT3-mediated upregulation of LINC00520 contributed to temozolomide chemoresistance in glioblastoma by interacting with RNA-binding protein LIN28B

A considerable number of glioblastoma (GBM) patients developed drug resistance to Temozolomide (TMZ) during chemotherapy, resulting in therapeutic failure and tumor recurrence. However, the exact mechanism of TMZ chemoresistance in GBM is still poorly clarified. As a novel identified lncRNA, LINC00520 was located on chromosome 14 and overexpressed in multiple human cancers. This study was designed and conducted to investigate the role and underlying mechanism of LINC00520 in GBM chemoresistance to TMZ. The qRT-PCR assay demonstrated that LINC00520 was significantly overexpressed in TMZ-sensitive and/or TMZ-resistant GBM cells (P < 0.001). The silencing of LINC00520 markedly reduced the cell viability, suppressed colony formation, induced cell apoptosis and G1/S phase arrest in TMZ-resistant cells (P < 0.001). In contrast, overexpression of LINC00520 conferred TMZ-resistant phenotype of GBM cells in vitro (P < 0.001). The orthotopic xenograft model was established and the results indicated that the volume of tumor xenografts in vivo was markedly inhibited by TMZ treatment after the silencing of LINC00520 (P < 0.001). Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay revealed a strong affinity of transcription factor STAT3 to the promoter regions of LINC00520, suggesting that STAT3 mediated the aberrant expression of LINC00520 in GBM. Further experiments demonstrated that LINC00520 could interact with RNA-binding protein LIN28B to inhibit autophagy and reduce DNA damage, thereby contributing to TMZ chemoresistance in GBM. These findings suggested that STAT3/LINC00520/LIN28B axis might be a promising target to improve TMZ chemoresistance of GBM.

Genome-scale CRISPR Cas9a knockout screen reveals genes that control glioblastoma susceptibility to the alkylating agent temozolomide

ABSTRACT Glioblastoma is the most fatal of all primary human brain tumors, with a 14-month median survival. The mainstay therapy for this tumor involves temozolomide, surgery, radiotherapy, and tumor-treating electric field. Cancer resistance to commonly available chemotherapeutics remains a major challenge in glioblastoma patients receiving treatment and unfavorably impacts their overall survival and outcome. However, the lack of progress in this area could be attributed to the lack of tools to probe unbiasedly at the genome-wide level the coding and non-coding elements’ contribution on a large scale for factors that control resistance to chemotherapeutics. Understanding the mechanisms of resistance to chemotherapeutics will enable precision medicine in the treatment of cancer patients. CRISPR Cas9a has emerged as a functional genomics tool to study at the genome level the factors that control cancer resistance to drugs. Recently, we used genome-wide CRISPR-Cas9a screen to identify genes responsible for glioblastoma susceptibility to etoposide. We extended our inquiry to understanding genes that control glioblastoma response to temozolomide using the genome-scale CRISPR. This study shows that the unbiased genome-wide loss of function approach can be applied to discover genes that influence tumor resistance to chemotherapeutics and contribute to chemoresistance in glioblastoma.

Overcoming chemoresistance in glioblastoma by fluvastatin via prenylation-dependent inhibition of Ras signaling.

The resistance of glioblastoma to chemotherapy remains a significant clinical problem. Targeting alternative pathways such as protein prenylation is known to be effective against many cancers. Fluvastatin is a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl- CoA (HMG-CoA) reductase, thereby inhibits prenylation. We demonstrate that fluvastatin alone effectively inhibits proliferation and induces apoptosis in multiple human glioblastoma cell lines. The combination index analysis shows that fluvastatin acts synergistically with common chemotherapy drugs for glioblastoma: temozolomide and irinotecan. We further show that fluvastatin acts on glioblastoma through inhibiting prenylation-dependent Ras activation. The combination of fluvastatin and low dose temozolomide resulted in remarkable inhibition of glioblastoma tumor in mice throughout the whole treatment duration without causing toxicity. Such combinatorial effects provide the basis for utilizing these FDA-approved drugs as a potential clinical approach in overcoming resistance and improving glioblastoma treatment.

De novo purine biosynthesis is a major driver of chemoresistance in glioblastoma.

Glioblastoma is a primary brain cancer with a near 100% recurrence rate. Upon recurrence, the tumour is resistant to all conventional therapies, and because of this, 5-year survival is dismal. One of the major drivers of this high recurrence rate is the ability of glioblastoma cells to adapt to complex changes within the tumour microenvironment. To elucidate this adaptation's molecular mechanisms, specifically during temozolomide chemotherapy, we used chromatin immunoprecipitation followed by sequencing and gene expression analysis. We identified a molecular circuit in which the expression of ciliary protein ADP-ribosylation factor-like protein 13B (ARL13B) is epigenetically regulated to promote adaptation to chemotherapy. Immuno-precipitation combined with liquid chromatography-mass spectrometry binding partner analysis revealed that that ARL13B interacts with the purine biosynthetic enzyme inosine-5'-monophosphate dehydrogenase 2 (IMPDH2). Further, radioisotope tracing revealed that this interaction functions as a negative regulator for purine salvaging. Inhibition of the ARL13B-IMPDH2 interaction enhances temozolomide-induced DNA damage by forcing glioblastoma cells to rely on the purine salvage pathway. Targeting the ARLI3B-IMPDH2 circuit can be achieved using the Food and Drug Administration-approved drug, mycophenolate mofetil, which can block IMPDH2 activity and enhance the therapeutic efficacy of temozolomide. Our results suggest and support clinical evaluation of MMF in combination with temozolomide treatment in glioma patients.

Deciphering the Role of Autophagy in Treatment of Resistance Mechanisms in Glioblastoma.

Autophagy is a process essential for cellular energy consumption, survival, and defense mechanisms. The role of autophagy in several types of human cancers has been explicitly explained; however, the underlying molecular mechanism of autophagy in glioblastoma remains ambiguous. Autophagy is thought to be a “double-edged sword”, and its effect on tumorigenesis varies with cell type. On the other hand, autophagy may play a significant role in the resistance mechanisms against various therapies. Therefore, it is of the utmost importance to gain insight into the molecular mechanisms deriving the autophagy-mediated therapeutic resistance and designing improved treatment strategies for glioblastoma. In this review, we discuss autophagy mechanisms, specifically its pro-survival and growth-suppressing mechanisms in glioblastomas. In addition, we try to shed some light on the autophagy-mediated activation of the cellular mechanisms supporting radioresistance and chemoresistance in glioblastoma. This review also highlights autophagy’s involvement in glioma stem cell behavior, underlining its role as a potential molecular target for therapeutic interventions.

TRIM31 enhances chemoresistance in glioblastoma through activation of the PI3K/Akt signaling pathway.

Temozolomide (TMZ) resistance is a complication of treatment of glioma, and new strategies are urgently required to overcome chemoresistance in glioma cells. In the present study, it was demonstrated that tripartite motif-containing 31 (TRIM31) was abnormally upregulated in glioma tissues and cell lines compared with normal samples. Furthermore, the role of TRIM31 was assessed by overexpressing and knocking down its expression. Overexpression of TRIM31 increased cell viability, increased TMZ IC50 values and inhibited apoptosis in A172 and U251 cells; whereas overexpression of TRIM31 decreased the expression of the apoptosis-associated protein p53. Knockdown of TRIM31 increased apoptosis in cells treated with TMZ. Additionally, the mechanisms by which TRIM31 affected glioma cells treated with TMZ were determined. Overexpression of TRIM31 increased phosphorylation of AKT and inhibiting the PI3K/AKT signaling pathway abolished the increase in cell viability and decreased phospho-Akt protein expression in TRIM31 overexpressing A172 cells treated with TMZ. Together, the findings suggest that TRIM31 may be a potentially novel target for glioma chemotherapy.

NUSAP1 potentiates chemoresistance in glioblastoma through its SAP domain to stabilize ATR

NUSAP1, which is a microtubule-associated protein involved in mitosis, plays essential roles in diverse biological processes, especially in cancer biology. In this study, NUSAP1 was found to be overexpressed in GBM tissues in a grade-dependent manner compared with normal brain tissues. NUSAP1 was also highly expressed in GBM patients, dead patients, and GBM cells. In addition, NUSAP1 was found to participate in cell proliferation, apoptosis, and DNA damage in GBM cells. Ataxia telangiectasia and Rad3-related protein (ATR) are a primary sensor of DNA damage, and ATR is also a promising target in cancer therapy. Here, we found that NUSAP1 positively regulated the expression of ATR. Mechanistically, NUSAP1 suppressed the ubiquitin-dependent proteolysis of ATR. The SAP (SAF-A/B, Acinus, and PIAS) domain is a common motif of many SUMO (small ubiquitin-like modifier) E3 ligases, and this domain is involved in substrate recognition and ligase activity. This study further demonstrated that the SAP domain of NUSAP1 promoted the sumoylation of ATR, and thereby antagonized the ubiquitination of ATR. These results suggest that NUSAP1 stabilizes ATR by sumoylation. Moreover, NUSAP1 potentiated chemotherapeutic resistance to temozolomide (TMZ) and doxorubicin (DOX) through its SAP domain. Overall, this study indicates that NUSAP1 is a promising therapeutic target in GBM.

ID1 Is Critical for Tumorigenesis and Regulates Chemoresistance in Glioblastoma.

Glioblastoma is the most common primary brain tumor in adults. While the introduction of temozolomide chemotherapy has increased long-term survivorship, treatment failure and rapid tumor recurrence remains universal. The transcriptional regulatory protein, inhibitor of DNA-binding-1 (ID1), is a key regulator of cell phenotype in cancer. We show that CRISPR-mediated knockout of ID1 in glioblastoma cells, breast adenocarcinoma cells, and melanoma cells dramatically reduced tumor progression in all three cancer systems through transcriptional downregulation of EGF, which resulted in decreased EGFR phosphorylation. Moreover, ID1-positive cells were enriched by chemotherapy and drove tumor recurrence in glioblastoma. Addition of the neuroleptic drug pimozide to inhibit ID1 expression enhanced the cytotoxic effects of temozolomide therapy on glioma cells and significantly prolonged time to tumor recurrence. Conclusively, these data suggest ID1 could be a promising therapeutic target in patients with glioblastoma. SIGNIFICANCE: These findings show that the transcriptional regulator ID1 is critical for glioblastoma initiation and chemoresistance and that inhibition of ID1 enhances the effect of temozolomide, delays tumor recurrence, and prolongs survival.

Acquired temozolomide resistance in MGMT-deficient glioblastoma cells is associated with regulation of DNA repair by DHC2.

The acquisition of temozolomide resistance is a major clinical challenge for glioblastoma treatment. Chemoresistance in glioblastoma is largely attributed to repair of temozolomide-induced DNA lesions by O6-methylguanine-DNA methyltransferase (MGMT). However, some MGMT-deficient glioblastomas are still resistant to temozolomide, and the underlying molecular mechanisms remain unclear. We found that DYNC2H1 (DHC2) was expressed more in MGMT-deficient recurrent glioblastoma specimens and its expression strongly correlated to poor progression-free survival in MGMT promotor methylated glioblastoma patients. Furthermore, silencing DHC2, both in vitro and in vivo, enhanced temozolomide-induced DNA damage and significantly improved the efficiency of temozolomide treatment in MGMT-deficient glioblastoma. Using a combination of subcellular proteomics and in vitro analyses, we showed that DHC2 was involved in nuclear localization of the DNA repair proteins, namely XPC and CBX5, and knockdown of either XPC or CBX5 resulted in increased temozolomide-induced DNA damage. In summary, we identified the nuclear transportation of DNA repair proteins by DHC2 as a critical regulator of acquired temozolomide resistance in MGMT-deficient glioblastoma. Our study offers novel insights for improving therapeutic management of MGMT-deficient glioblastoma.

MSI2-TGF-β/TGF-β R1/SMAD3 positive feedback regulation in glioblastoma.

Glioblastoma is the most malignant glioma tumors with inevitable relapse and resistance to chemotherapy; however, the mechanisms driving chemoresistance remain to be fully elucidated. This study is to explore the molecular and cellular mechanisms involving in the chemoresistance of glioblastoma. The expression of musashi (MSI) RNA-binding protein in the tumor tissues and cells of glioblastoma was measured. The effects of MSI2 in epithelial-to-mesenchymal transition (EMT), resistance to temozolomide (TMZ), tumor cell invasion, migration, and proliferation and associated signaling were evaluated. High MSI2 expression was observed in the glioblastoma tissues. Silencing or overexpression of MSI2 significantly affected tumor cells invasion, migration, and proliferation. Silencing of MSI2 expression significantly inhibited O6-methylguanine-DNA methyltransferase (MGMT) expression and tumor growth, and reversed resistance to TMZ in xenograft tumor models. MSI2 expression regulated EMT through activating the transcription factors Snail and the TGFβ R1/SMAD3 signaling. Our study demonstrated a positive feedback loop of MSI2-TGFβ/SMAD3 signaling which activates the EMT and MGMT which may contribute to chemoresistance in glioblastoma. This study also highlights that MSI2 could be a new target for the therapy of glioblastoma.