Bmi1 deficient neural stem cells have increased Integrin dependent adhesion to self-secreted matrix

Background

Neural cells deficient for Polycomb group (PcG) protein Bmi1 are impaired in the formation and differentiation of high grade glioma, an incurable cancer of the brain. It was shown that mechanisms involved in cell adhesion and migration were specifically affected in these tumors.

Methods

Using biochemical and cell biological approaches, we investigated the adhesive capacities of Bmi1;Ink4a/Arf deficient primary neural stem cells (NSCs).

Results

Bmi1;Ink4a/Arf deficient NSCs have altered expression of Collagen-related genes, secrete increased amounts of extracellular matrix, and exhibit enhanced cell–matrix binding through the Beta-1 Integrin receptor. These traits are independent from the well described role of Bmi1 as repressor of the Ink4a/Arf tumor suppressor locus.

Conclusion

In addition to proliferative processes, Bmi1 controls the adhesive capacities of primary NSCs by modulating extracellular matrix secretion.

General significance

Since PcG protein Bmi1 is important for both normal development and tumorigenesis, it is vital to understand the complete network in which this protein acts. Whereas it is clear that control of Ink4a/Arf is a major Bmi1 function, there is evidence that other downstream mechanisms exist. Hence, our novel finding that Bmi1 also governs cell adhesion significantly contributes to our understanding of the PcG proteins.     

Genetic evidence for functional dependency of p18Ink4c on Cdk4

The INK4 family of cyclin-dependent kinase (CDK) inhibitors negatively regulates cyclin D-dependent CDK4 and CDK6 and induces the growth-suppressive function of Rb family proteins. Mutations in the Cdk4 gene conferring INK4 resistance are associated with familial and sporadic melanoma in humans and result in a wide spectrum of tumors in mice, suggesting that INK4 is a major regulator of CDK4. Mice lacking the Cdk4 gene exhibit various defects in many organs associated with hypocellularity, whereas loss of the p18(Ink4c) gene results in widespread hyperplasia and organomegaly. To genetically test the notion that the function of INK4 is dependent on CDK4, we generated p18; Cdk4 double-mutant mice and examined the organs and tissues which developed abnormalities when either gene is deleted. We show here that, in all organs we have examined, including pituitary, testis, pancreas, kidney, and adrenal gland, hyperproliferative phenotypes associated with p18 loss were canceled. The double-mutant mice exhibited phenotypes very close to or indistinguishable from that of Cdk4 single-mutant mice. Mice lacking p27(Kip1) develop widespread hyperplasia and organomegaly similar to those developed by p18-deficient mice. The p27; Cdk4 double-mutant mice, however, displayed phenotypes intermediate between those of p27 and Cdk4 single-mutant mice. These results provide genetic evidence that in mice p18(Ink4c) and p27(Kip1) mediate the transduction of different cell growth and proliferation signals to CDK4 and that p18(Ink4c) is functionally dependent on CDK4.     

Mice lacking cyclin-dependent kinase inhibitor p19Ink4d show strain-specific effects on male reproduction

p19(Ink4d) is a member of the INK4 family of cyclin-dependent kinase inhibitors, which are important negative regulators of the G1-phase cyclin-dependent kinases CDK4 and CDK6. On a mixed C57BL/6 x 129P2/OlaHsd background, mice deficient for p19(Ink4d) exhibited defects in male reproductive function including testicular atrophy, alteration in serum follicle stimulating hormone, qualitative increase in germ cell apoptosis, and delayed kinetics of meiotic prophase markers (Zindy et al., 2001. Mol Cell Biol 21:3244-3255; Zindy et al., 2000. Mol Cell Biol 20:372-378). In this study, a quantitative assessment of these aspects of reproductive capacity demonstrated relatively mild deficits in p19(Ink4d-/-) males compared to controls. These effects did not dramatically worsen in older males although some seminiferous tubule defects were observed. Following marker-assisted backcrossing into the C57BL/6 background, p19(Ink4d-/-) males did not display defects in testis weights, sperm numbers, serum FSH, germ cell apoptosis, or kinetics of selected meiotic prophase markers. These studies indicate that a reduction in Ink4 family function by the loss of p19(Ink4d) is sufficient to induce mild reproductive defects in male mice with a mixed genetic background, but not in the C57BL/6 genetic background.     

葡萄糖6-磷酸脱氢酶调控细胞周期蛋白E1和细胞周期蛋白依赖性激酶2促进细胞增殖及其在肾透明细胞癌中的预后价值

肾透明细胞癌(clear cell renal cell carcinoma,ccRCC)是一种转移率高、预后差的细胞代谢性疾病,对其有效诊疗及预后分子标志物的研究十分重要。葡萄糖6-磷酸脱氢酶(glucose 6-phosphatedehydrogenase, G6PD)在ccRCC中高表达,并提示患者不良预后,其促进ccRCC细胞增殖的分子机制有待进一步揭示。本研究发现,降低G6PD可抑制细胞周期G₁/S期转化并显著抑制ccRCC细胞增殖。G6PD可在细胞水平调控G₁/S期转化及增殖相关因子Cyclin D1,CDK4,CDK6,Cyclin E1和CDK2基因表达。TCGA数据库分析结果表明,ccRCC 中Cyclin D1,Cyclin E1 和 CDK2的mRNA 水平显著升高,而CDK4表达无明显差异,CDK6表达却显著降低。相关性分析结果显示,G6PD与Cyclin D1呈显著负相关(P<0.0001),G6PD与CDK4,CDK6之间无显著相关性(P>0.05),G6PD与Cyclin E1(P<0.0001)以及CDK2(P<0.05)显著正相关。进一步免疫组化检测结果表明,Cyclin E1和 CDK2在ccRCC肿瘤组织中表达显著升高。生存预后分析结果显示,Cyclin D1高表达提示ccRCC患者整体预后更为良好,CDK4和CDK6表达水平在ccRCC患者总生存率预测中无意义;而Cyclin E1和CDK2高表达均可提示ccRCC患者预后不良。进一步细胞水平检测发现,Cyclin E1、CDK2表达降低可显著逆转G6PD促进ccRCC细胞增殖的能力。综上,与增殖相关因子Cyclin D1,CDK4和CDK6相比,G6PD有可能通过促进Cyclin E1和CDK2表达升高而发挥促进 ccRCC肿瘤细胞增殖的作用,并且这3者的异常高表达有望成为ccRCC患者不良预后的独立生存预测因素。     

The neural stem cell fate determinant TLX promotes tumorigenesis and genesis of cells resembling glioma stem cells

A growing body of evidence indicates that deregulation of stem cell fate determinants is a hallmark of many types of malignancies. The neural stem cell fate determinant TLX plays a pivotal role in neurogenesis in the adult brain by maintaining neural stem cells. Here, we report a tumorigenic role of TLX in brain tumor initiation and progression. Increased TLX expression was observed in a number of glioma cells and glioma stem cells, and correlated with poor survival of patients with gliomas. Ectopic expression of TLX in the U87MG glioma cell line and Ink4a/Arf-deficient mouse astrocytes (Ink4a/Arf^-/- astrocytes) induced cell proliferation with a concomitant increase in cyclin D expression, and accelerated foci formation in soft agar and tumor formation in in vivo transplantation assays. Furthermore, overexpression of TLX in Ink4a/Arf^-/- astrocytes inhibited cell migration and invasion and promoted neurosphere formation and Nestin expression, which are hallmark characteristics of glioma stem cells, under stem cell culture conditions. Our results indicate that TLX is involved in glioma stem cell genesis and represents a potential therapeutic target for this type of malignancy.     

Efficacy of a combination therapy targeting CDK4/6 and autophagy in a mouse xenograft model of t(8;21) acute myeloid leukemia

One of the most frequent cytogenetic abnormalities in acute myeloid leukemia (AML) is t(8;21). Although patients with t(8;21) AML have a more favorable prognosis than other cytogenetic subgroups, relapse is still common and novel therapeutic approaches are needed. A recent study showed that t(8;21) AML is characterized by CCND2 deregulation and that co-inhibition of CDK4/6 and autophagy induces apoptosis in t(8;21) AML cells. In this study, we examined the in vivo effects of co-inhibiting CDK4/6 and autophagy. We used a mouse model in which t(8;21)-positive Kasumi-1 cells were subcutaneously inoculated into NOD/Shi-scid IL2Rg^null mice. The mice were treated with the autophagy inhibitor chloroquine (CQ), a CDK4/6 inhibitor (either abemaciclib or palbociclib), or a CDK4/6 inhibitor plus CQ. After 20 days of treatment, tumor volume was measured, and immunostaining and transmission electron microscopy observations were performed. There was no change in tumor growth in CQ-treated mice. However, mice treated with a CDK4/6 inhibitor plus CQ had significantly less tumor growth than mice treated with a CDK4/6 inhibitor alone. CDK4/6 inhibitor treatment increased the formation of autophagosomes. The number of single-strand DNA-positive (apoptotic) cells was significantly higher in the tumors of mice treated with a CDK4/6 inhibitor plus CQ than in mice treated with either CQ or a CDK4/6 inhibitor. These results show that CDK4/6 inhibition induces autophagy, and that co-inhibition of CDK4/6 and autophagy induces apoptosis in t(8;21) AML cells in vivo. The results suggest that inhibiting CDK4/6 and autophagy could be a novel and promising therapeutic strategy in t(8;21) AML.     

Losses of both products of the Cdkn2a/Arf locus contribute to asbestos-induced mesothelioma development and cooperate to accelerate tumorigenesis

The CDKN2A/ARF locus encompasses overlapping tumor suppressor genes p16(INK4A) and p14(ARF), which are frequently co-deleted in human malignant mesothelioma (MM). The importance of p16(INK4A) loss in human cancer is well established, but the relative significance of p14(ARF) loss has been debated. The tumor predisposition of mice singly deficient for either Ink4a or Arf, due to targeting of exons 1α or 1β, respectively, supports the idea that both play significant and nonredundant roles in suppressing spontaneous tumors. To further test this notion, we exposed Ink4a(+/-) and Arf(+/-) mice to asbestos, the major cause of MM. Asbestos-treated Ink4a(+/-) and Arf(+/-) mice showed increased incidence and shorter latency of MM relative to wild-type littermates. MMs from Ink4a(+/-) mice exhibited biallelic inactivation of Ink4a, loss of Arf or p53 expression and frequent loss of p15(Ink4b). In contrast, MMs from Arf(+/-) mice exhibited loss of Arf expression, but did not require loss of Ink4a or Ink4b. Mice doubly deficient for Ink4a and Arf, due to deletion of Cdkn2a/Arf exon 2, showed accelerated asbestos-induced MM formation relative to mice deficient for Ink4a or Arf alone, and MMs exhibited biallelic loss of both tumor suppressor genes. The tumor suppressor function of Arf in MM was p53-independent, since MMs with loss of Arf retained functional p53. Collectively, these in vivo data indicate that both CDKN2A/ARF gene products suppress asbestos carcinogenicity. Furthermore, while inactivation of Arf appears to be crucial for MM pathogenesis, the inactivation of both p16(Ink4a) and p19(Arf) cooperate to accelerate asbestos-induced tumorigenesis.     

CDK4/6 inhibition suppresses tumour growth and enhances the effect of temozolomide in glioma cells

In adults, glioma is the most commonly occurring and invasive brain tumour. For malignant gliomas, the current advanced chemotherapy includes TMZ (temozolomide). However, a sizeable number of gliomas are unyielding to TMZ, hence, giving rise to an urgent need for more efficient treatment choices. Here, we report that cyclin‐dependent kinases 4 (CDK4) is expressed at significantly high levels in glioma cell lines and tissues. CDK4 overexpression enhances colony formation and proliferation of glioma cells and extends resistance to inhibition of TMZ‐mediated cell proliferation and induction of apoptosis. However, CDK4 knockdown impedes colony formation and cell proliferation, and enhances sensitivity of glioma cells to TMZ. The selective inhibition of CDK4/6 impedes glioma cell proliferation and induces apoptotic induction. The selective inhibitors of CDK4/6 may enhance glioma cell sensitivity to TMZ. We further showed the possible role of RB phosphorylation mediated by CDK4 for its oncogenic function in glioma. The growth of glioma xenografts was inhibited in vivo, through combination treatment, and corresponded to enhanced p‐RB levels, reduced staining of Ki‐67 and enhanced activation of caspase 3. Therefore, CDK4 inhibition may be a favourable strategy for glioma treatment and overcomes TMZ resistance.     

p19Ink4d Is a Tumor Suppressor and Controls Pituitary Anterior Lobe Cell Proliferation

Pituitary tumors develop in about one-quarter of the population, and most arise from the anterior lobe (AL). The pituitary gland is particularly sensitive to genetic alteration of genes involved in the cyclin-dependent kinase (CDK) inhibitor (CKI)–CDK-retinoblastoma protein (Rb) pathway. Mice heterozygous for the Rb mutation develop pituitary tumors, with about 20% arising from the AL. Perplexingly, none of the CKI-deficient mice reported thus far develop pituitary AL tumors. In this study, we show that deletion of p19^Ink4d (p19), a CKI gene, in mice results in spontaneous development of tumors in multiple organs and tissues. Specifically, more than one-half of the mutant mice developed pituitary hyperplasia or tumors predominantly in the AL. Tumor development is associated with increased cell proliferation and enhanced activity of Cdk4 and Cdk6 and phosphorylation of Rb protein. Though Cdk4 is indispensable for postnatal pituitary cell proliferation, it is not required for the hyperproliferative pituitary phenotype caused by p19 loss. Loss of p19 phosphorylates Rb in Cdk4^−/− pituitary AL cells and mouse embryonic fibroblasts (MEFs) and rescues their proliferation defects, at least partially, through the activation of Cdk6. These results provide the first genetic evidence that p19 is a tumor suppressor and the major CKI gene that controls pituitary AL cell proliferation.     

Activated K‐Ras and INK4a/Arf deficiency promote aggressiveness of pancreatic cancer by induction of EMT consistent with cancer stem cell phenotype

Pancreatic ductal adenocarcinoma (PDAC) is one of the most frequently diagnosed cancers and the fourth leading cause of cancer‐related death in the United States, suggesting that there is an urgent need to design novel strategies for achieving better treatment outcome of patients diagnosed with PDAC. Our previous study has shown that activation of Notch and NF‐κB play a critical role in the development of PDAC in the compound K‐Ras^G12D and Ink4a/Arf deficient transgenic mice. However, the exact molecular mechanism by which mutated K‐Ras and Ink4a/Arf deficiency contribute to progression of PDAC remains largely elusive. In the present study, we used multiple methods, such as real‐time RT‐PCR, Western blotting assay, and immunohistochemistry to gain further mechanistic insight. We found that the deletion of Ink4a/Arf in K‐Ras^G12D expressing mice led to high expression of PDGF‐D signaling pathway in the tumor and tumor‐derived cell line (RInk‐1 cells). Furthermore, PDGF‐D knock‐down in RInk‐1 cells resulted in the inhibition of pancreatosphere formation and down‐regulation of EZH2, CD44, EpCAM, and vimentin. Moreover, we demonstrated that epithelial–mesenchymal transition (EMT) was induced in the compound mice, which is linked with aggressiveness of PDAC. In addition, we demonstrated that tumors from compound transgenic mice have higher expression of cancer stem cell (CSC) markers. These results suggest that the acquisition of EMT phenotype and induction of CSC characteristics could be linked with the aggressiveness of PDAC mediated in part through the activation of PDGF‐D, signaling. J. Cell. Physiol. 228: 556–562, 2013. © 2012 Wiley Periodicals, Inc.     

Association Study of Germline Variants in CCNB1 and CDK1 with Breast Cancer Susceptibility,Progression, and Survival among Chinese Han Women

The CCNB1 and CDK1 genes encode the proteins of CyclinB1 and CDK1 respectively, which interact with each other and are involved in cell cycle regulation, centrosome duplication and chromosome segregation. This study aimed to investigate whether the genetic variants in these two genes may affect breast cancer (BC) susceptibility, progression, and survival in Chinese Han population using haplotype-based analysis. A total of ten tSNPs spanning from 2kb upstream to 2kb downstream of these genes were genotyped in 1204 cases and 1204 age-matched cancer-free controls. The haplotype blocks were determined according to our genotyping data and linkage disequilibrium (LD) status of these SNPs. For CCNB1, rs2069429 was significantly associated with increased BC susceptibility under recessive model (OR=2.352, 95%CI=1.480-3.737), so was the diplotype TAGT/TAGT (OR=1.947 95%CI=1.154-3.284, P=0.013). In addition, rs164390 was associated with Her2-negative BC. For CDK1, rs2448343 and rs1871446 were significantly associated with decreased BC risk under dominant models, so was the haplotype ATATT. These two SNPs also showed a dose-dependent effect on BC susceptibility. Using stratified association analysis, we found that women with the heterozygotes or minor allele homozygotes of rs2448343 had much less BC susceptibility among women with BMI<23. In CDK1, three closely located SNPs, rs2448343, rs3213048 and rs3213067, were significantly associated with tumor’s PR status: the heterozygotes of rs2448343 were associated with PR-positive tumors, while the minor allele homozygotes of rs3213048 and heterozygotes of rs3213067 were associated with PR-negative BC tumors. In survival analysis, rs1871446 was associated with unfavorable event-free survival under recessive model, so was the CDK1 diplotype ATATG/ATATG, which carried the minor allele homozygote of rs1871446. Our study indicates that genetic polymorphisms of CCNB1 and CDK1 are related to BC susceptibility, progression, and survival in Chinese Han women. Further studies need to be performed in other populations as an independent replication to verify these results.     

A High-Throughput In Vitro Drug Screen in a Genetically Engineered Mouse Model of Diffuse Intrinsic Pontine Glioma Identifies BMS-754807 as a Promising Therapeutic Agent

Diffuse intrinsic pontine gliomas (DIPGs) represent a particularly lethal type of pediatric brain cancer with no effective therapeutic options. Our laboratory has previously reported the development of genetically engineered DIPG mouse models using the RCAS/tv-a system, including a model driven by PDGF-B, H3.3K27M, and p53 loss. These models can serve as a platform in which to test novel therapeutics prior to the initiation of human clinical trials. In this study, an in vitro high-throughput drug screen as part of the DIPG preclinical consortium using cell-lines derived from our DIPG models identified BMS-754807 as a drug of interest in DIPG. BMS-754807 is a potent and reversible small molecule multi-kinase inhibitor with many targets including IGF-1R, IR, MET, TRKA, TRKB, AURKA, AURKB. In vitro evaluation showed significant cytotoxic effects with an IC₅₀ of 0.13 μM, significant inhibition of proliferation at a concentration of 1.5 μM, as well as inhibition of AKT activation. Interestingly, IGF-1R signaling was absent in serum-free cultures from the PDGF-B; H3.3K27M; p53 deficient model suggesting that the antitumor activity of BMS-754807 in this model is independent of IGF-1R. In vivo, systemic administration of BMS-754807 to DIPG-bearing mice did not prolong survival. Pharmacokinetic analysis demonstrated that tumor tissue drug concentrations of BMS-754807 were well below the identified IC₅₀, suggesting that inadequate drug delivery may limit in vivo efficacy. In summary, an unbiased in vitro drug screen identified BMS-754807 as a potential therapeutic agent in DIPG, but BMS-754807 treatment in vivo by systemic delivery did not significantly prolong survival of DIPG-bearing mice.     

Selective Targeting of Brain Tumors with Gold Nanoparticle-Induced Radiosensitization

Successful treatment of brain tumors such as glioblastoma multiforme (GBM) is limited in large part by the cumulative dose of Radiation Therapy (RT) that can be safely given and the blood-brain barrier (BBB), which limits the delivery of systemic anticancer agents into tumor tissue. Consequently, the overall prognosis remains grim. Herein, we report our pilot studies in cell culture experiments and in an animal model of GBM in which RT is complemented by PEGylated-gold nanoparticles (GNPs). GNPs significantly increased cellular DNA damage inflicted by ionizing radiation in human GBM-derived cell lines and resulted in reduced clonogenic survival (with dose-enhancement ratio of ∼1.3). Intriguingly, combined GNP and RT also resulted in markedly increased DNA damage to brain blood vessels. Follow-up in vitro experiments confirmed that the combination of GNP and RT resulted in considerably increased DNA damage in brain-derived endothelial cells. Finally, the combination of GNP and RT increased survival of mice with orthotopic GBM tumors. Prior treatment of mice with brain tumors resulted in increased extravasation and in-tumor deposition of GNP, suggesting that RT-induced BBB disruption can be leveraged to improve the tumor-tissue targeting of GNP and thus further optimize the radiosensitization of brain tumors by GNP. These exciting results together suggest that GNP may be usefully integrated into the RT treatment of brain tumors, with potential benefits resulting from increased tumor cell radiosensitization to preferential targeting of tumor-associated vasculature.     

Design of a brain-penetrant CDK4/6 inhibitor for glioblastoma

CDK4 and CDK6 are kinases with similar sequences that regulate cell cycle progression and are validated targets in the treatment of cancer. Glioblastoma is characterized by a high frequency of CDKN2A/CCND2/CDK4/CDK6 pathway dysregulation, making dual inhibition of CDK4 and CDK6 an attractive therapeutic approach for this disease. Abemaciclib, ribociclib, and palbociclib are approved CDK4/6 inhibitors for the treatment of HR+/HER2? breast cancer, but these drugs are not expected to show strong activity in brain tumors due to poor blood brain barrier penetration. Herein, we report the identification of a brain-penetrant CDK4/6 inhibitor derived from a literature molecule with low molecular weight and topological polar surface area (MW = 285 and TPSA = 66 Ų), but lacking the CDK2/1 selectivity profile due to the absence of a basic amine. Removal of a hydrogen bond donor via cyclization of the pyrazole allowed for the introduction of basic and semi-basic amines, while maintaining in many cases efflux ratios reasonable for a CNS program. Ultimately, a basic spiroazetidine (cpK_a = 8.8) was identified that afforded acceptable selectivity over anti-target CDK1 while maintaining brain-penetration in vivo (mouse K_p,uu = 0.20–0.59). To probe the potency and selectivity, our lead compound was evaluated in a panel of glioblastoma cell lines. Potency comparable to abemaciclib was observed in Rb-wild type lines U87MG, DBTRG-05MG, A172, and T98G, while Rb-deficient cell lines SF539 and M059J exhibited a lack of sensitivity.     

Dual growth arrest pathways in astrocytes and astrocytic tumors in response to Raf-1 activation

Normal human fibroblasts have been shown to undergo a p16(Ink4a)-associated senescence-like growth arrest in response to sustained activation of the Ras/Raf/MEK/ERK pathway. We noted a similar p16(Ink4a)-associated, senescence-like arrest in normal human astrocytes in response to expression of a conditional form of Raf-1. While HPV16 E7-mediated functional inactivation of the p16(Ink4a)/pRb pathway in astrocytes blocked the p16(Ink4a)-associated growth arrest in response to activation of Raf-1, it also revealed a second p21(Cip1)-associated, senescence-associated, beta-galactosidase-independent growth arrest pathway. Importantly, the p21(Cip1)-associated pathway was present not only in normal astrocytes but also in p53-, p14(ARF)-, and p16(Ink4a)/pRb-deficient high grade glioma cells that lacked the p16(Ink4a)-dependent arrest mechanism. These results suggest that normal human cells have redundant arrest pathways, which can be activated by Raf-1, and that even tumors that have dismantled p16(Ink4a)-dependent growth arrest pathways are potentially regulated by a second p21(Cip1)-dependent growth arrest pathway.     

FK506 confers chemosensitivity to anticancer drugs in glioblastoma multiforme cells by decreasing the expression of the multiple resistance-associated protein-1

Malignant rhabdoid tumor (MRT) is a rare and highly aggressive neoplasm of young children. MRT is characterized by inactivation of integrase interactor 1 (INI1). Cyclin-dependent kinase 4 (CDK4), which acts downstream of INI1, is required for the proliferation of MRT cells. Here we investigated the effects of PD 0332991 (PD), a potent inhibitor of CDK4, against five human MRT cell lines (MP-MRT-AN, KP-MRT-RY, G401, KP-MRT-NS, KP-MRT-YM). In all of the cell lines except KP-MRT-YM, PD inhibited cell proliferation >50%, (IC₅₀ values 0.01 to 0.6 μM) by WST-8 assay, and induced G1-phase cell cycle arrest, as shown by flow cytometry and BrdU incorporation assay. The sensitivity of the MRT cell lines to PD was inversely correlated with p16 expression (r = 0.951). KP-MRT-YM cells overexpress p16 and were resistant to the growth inhibitory effect of PD. Small interfering RNA against p16 significantly increased the sensitivity of KP-MRT-YM cells to PD (p < 0.05). These results suggest that p16 expression in MRT could be used to predict its sensitivity to PD. PD may be an attractive agent for patients with MRT whose tumors express low levels of p16.     

Cancer Stem Cell Chemotherapeutics Assay for Prospective Treatment of Recurrent Glioblastoma and Progressive Anaplastic Glioma: A Single-Institution Case Series

BACKGROUND: Chemotherapy-resistant cancer stem cells (CSC) may lead to tumor recurrence in glioblastoma (GBM). The poor prognosis of this disease emphasizes the critical need for developing a treatment stratification system to improve outcomes through personalized medicine. METHODS: We present a case series of 12 GBM and 2 progressive anaplastic glioma cases from a single Institution prospectively treated utilizing a CSC chemotherapeutics assay (ChemoID) guided report. All patients were eligible to receive a stereotactic biopsy and thus undergo ChemoID testing. We selected one of the most effective treatments based on the ChemoID assay report from a panel of FDA approved chemotherapy as monotherapy or their combinations for our patients. Patients were evaluated by MRI scans and response was assessed according to RANO 1.1 criteria. RESULTS: Of the 14 cases reviewed, the median age of our patient cohort was 49 years (21–63). We observed 6 complete responses (CR) 43%, 6 partial responses (PR) 43%, and 2 progressive diseases (PD) 14%. Patients treated with ChemoID assay-directed therapy, in combination with other modality of treatment (RT, LITT), had a longer median overall survival (OS) of 13.3 months (5.4-NA), compared to the historical median OS of 9.0 months (8.0–10.8 months) previously reported. Notably, patients with recurrent GBM or progressive high-grade glioma treated with assay-guided therapy had a 57% probability to survive at 12 months, compared to the 27% historical probability of survival observed in previous studies. CONCLUSIONS: The results presented here suggest that the ChemoID Assay has the potential to stratify individualized chemotherapy choices to improve recurrent and progressive high-grade glioma patient survival.Importance of the StudyGlioblastoma (GBM) and progressive anaplastic glioma are the most aggressive brain tumor in adults and their prognosis is very poor even if treated with the standard of care chemoradiation Stupp's protocol. Recent knowledge pointed out that current treatments often fail to successfully target cancer stem cells (CSCs) that are responsible for therapy resistance and recurrence of these malignant tumors. ChemoID is the first and only CLIA (clinical laboratory improvements amendment) -certified and CAP (College of American Pathologists) -accredited chemotherapeutic assay currently available in oncology clinics that examines patient's derived CSCs susceptibility to conventional FDA (Food and Drugs Administration) -approved drugs. In this study we observed that although the majority of our patients (71.5%) presented with unfavorable prognostic predictors (wild type IDH-1/2 and unmethylated MGMT promoter), patients treated with ChemoID assay-directed therapy had an overall response rate of 86% and increased median OS of 13.3 months compared to the historical median OS of 9.1 months (8.1–10.1 months) previously reported [1] suggesting that the ChemoID assay may be beneficial in personalizing treatment strategies.     

Inhibition of Thromboxane Synthase Activity Improves Glioblastoma Response to Alkylation Chemotherapy

Thromboxane synthase (TXSA), an enzyme of the arachidonic acid metabolism, is upregulated in human glial tumors and is involved in glioma progression. Here, we analyzed the in vitro and in vivo effects of pharmacological inhibition of TXSA activity on human glioblastoma cells. Furegrelate, a specific inhibitor of TXSA, significantly inhibited tumor growth in an orthotopic glioblastoma model by inducing proapoptotic, antiproliferative, and antiangiogenic effects. Inhibition of TXSA induced a proapoptotic disposition of glioma cells and increased the sensitivity to the chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea, significantly prolonging the survival time of intracerebral glioma-bearing mice. Our data demonstrate that the targeted inhibition of TXSA activity improves the efficiency of conventional alkylation chemotherapy in vivo. Our study supports the role of TXSA activity for the progression of malignant glioma and the potential utility of its therapeutic modulation for glioma treatment.     

Antitumor Activity of a Polypyridyl Chelating Ligand: In Vitro and In Vivo Inhibition of Glioma

Glioblastoma multiforme is an extremely aggressive and invasive form of central nervous system tumor commonly treated with the chemotherapeutic drug Temozolomide. Unfortunately, even with treatment, the median survival time is less than 12 months. 2,9-Di-sec-butyl-1,10-phenanthroline (SBP), a phenanthroline-based ligand originally developed to deliver gold-based anticancer drugs, has recently been shown to have significant antitumor activity in its own right. SBP is hypothesized to initiate tumor cell death via interaction with non-DNA targets, and considering most glioblastoma drugs kill tumors through DNA damage processes, SBP was tested as a potential novel drug candidate against glial-based tumors. In vitro studies demonstrated that SBP significantly inhibited the growth of rodent GL-26 and C6 glioma cells, as well as human U-87, and SW1088 glioblastomas/astrocytomas. Furthermore, using a syngeneic glioma model in mice, in vivo administration of SBP significantly reduced tumor volume and increased survival time. There was no significant toxicity toward nontumorigenic primary murine and human astrocytes in vitro, and limited toxicity was observed in ex vivo tissues obtained from noncancerous mice. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining and recovery assays suggest that SBP induces apoptosis in gliomas. This exploratory study suggests SBP is effective in slowing the growth of tumorigenic cells in the brain while exhibiting limited toxicity to normal cells and tissues and should therefore be further investigated for its potential in glioblastoma treatment.     

Histidine-Rich Glycoprotein Can Prevent Development of Mouse Experimental Glioblastoma

Extensive angiogenesis, formation of new capillaries from pre-existing blood vessels, is an important feature of malignant glioma. Several antiangiogenic drugs targeting vascular endothelial growth factor (VEGF) or its receptors are currently in clinical trials as therapy for high-grade glioma and bevacizumab was recently approved by the FDA for treatment of recurrent glioblastoma. However, the modest efficacy of these drugs and emerging problems with anti-VEGF treatment resistance welcome the development of alternative antiangiogenic therapies. One potential candidate is histidine-rich glycoprotein (HRG), a plasma protein with antiangiogenic properties that can inhibit endothelial cell adhesion and migration. We have used the RCAS/TV-A mouse model for gliomas to investigate the effect of HRG on brain tumor development. Tumors were induced with platelet-derived growth factor-B (PDGF-B), in the presence or absence of HRG. We found that HRG had little effect on tumor incidence but could significantly inhibit the development of malignant glioma and completely prevent the occurrence of grade IV tumors (glioblastoma).