Improving Localized Radiotherapy for Glioblastoma via Small Molecule Inhibition of KIF11.

Glioblastoma, IDH-wild type (GBM) is the most common and lethal malignant primary brain tumor. Standard of care includes surgery, radiotherapy, and chemotherapy with the DNA alkylating agent temozolomide (TMZ). Despite these intensive efforts, current GBM therapy remains mainly palliative with only modest improvement achieved in overall survival. With regards to radiotherapy, GBM is ranked as one of the most radioresistant tumor types. In this study, we wanted to investigate if enriching cells in the most radiosensitive cell cycle phase, mitosis, could improve localized radiotherapy for GBM. To achieve cell cycle arrest in mitosis we used ispinesib, a small molecule inhibitor to the mitotic kinesin, KIF11. Cell culture studies validated that ispinesib radiosensitized patient-derived GBM cells. In vivo, we validated that ispinesib increased the fraction of tumor cells arrested in mitosis as well as increased apoptosis. Critical for the translation of this approach, we validated that combination therapy with ispinesib and irradiation led to the greatest increase in survival over either monotherapy alone. Our data highlight KIF11 inhibition in combination with radiotherapy as a new combinatorial approach that reduces the overall radioresistance of GBM and which can readily be moved into clinical trials.

Establishment and characterization of two novel patient-derived lines from canine high-grade glioma.

High-grade glioma is an aggressive cancer that occurs naturally in pet dogs. Canine high-grade glioma (cHGG) is treated with radiation, chemotherapy or surgery, but has no curative treatment. Within the past eight years, there have been advances in our imaging and histopathology standards as well as genetic charactereization of cHGG. However, there are only three cHGG cell lines publicly available, all of which were derived from astrocytoma and established using methods involving expansion of tumour cells in vitro on plastic dishes. In order to provide more clinically relevant cell lines for studying cHGG in vitro, the goal of this study was to establish cHGG patient-derived lines, whereby cancer cells are expanded in vivo by injecting cells into immunocompromized laboratory mice. The cells are then harvested from mice and used for in vitro studies. This method is the standard in the human field and has been shown to minimize the acquisition of genetic alterations and gene expression changes from the original tumour. Through a multi-institutional collaboration, we describe our methods for establishing two novel cHGG patient-derived lines, Boo-HA and Mo-HO, from a high-grade astrocytoma and a high-grade oligodendroglioma, respectively. We compare our novel lines to G06-A, J3T-Bg, and SDT-3G (traditional cHGG cell lines) in terms of proliferation and sensitivity to radiation. We also perform whole genome sequencing and identify an NF1 truncating mutation in Mo-HO. We report the characterization and availability of these novel patient-derived lines for use by the veterinary community.

The small molecule drug CBL0137 increases the level of DNA damage and the efficacy of radiotherapy for glioblastoma.

Glioblastoma (GBM) is an incurable brain tumor with inevitable recurrence. This is in part due to a highly malignant cancer stem cell (CSC) subpopulation of tumor cells that is particularly resistant to conventional treatments, including radiotherapy. Here we show that CBL0137, a small molecule anti-cancer agent, sensitizes GBM CSCs to radiotherapy. CBL0137 sequesters the FACT (facilitates chromatin transcription) complex to chromatin, resulting in cytotoxicity preferentially within tumor cells. We show that when combined with radiotherapy, CBL0137 inhibited GBM CSC growth and resulted in more DNA damage in the CSCs compared to irradiation or drug alone. Using an in vivo subcutaneous model, we showed that the frequency of GBM CSCs was reduced when tumors were pretreated with CBL0137 and then exposed to irradiation. Survival studies with orthotopic GBM models resulted in significantly extended survival for mice treated with combinatorial therapy. As GBM CSCs contribute to the inevitable recurrence in patients, targeting them is imperative. This work establishes a new treatment paradigm for GBM that sensitizes CSCs to irradiation and may ultimately reduce tumor recurrence.

An interphase pool of KIF11 localizes at the basal bodies of primary cilia and a reduction in KIF11 expression alters cilia dynamics.

KIF11 is a homotetrameric kinesin that peaks in protein expression during mitosis. It is a known mitotic regulator, and it is well-described that KIF11 is necessary for the formation and maintenance of the bipolar spindle. However, there has been a growing appreciation for non-mitotic roles for KIF11. KIF11 has been shown to function in such processes as axon growth and microtubule polymerization. We previously demonstrated that there is an interphase pool of KIF11 present in glioblastoma cancer stem cells that drives tumor cell invasion. Here, we identified a previously unknown association between KIF11 and primary cilia. We confirmed that KIF11 localized to the basal bodies of primary cilia in multiple cell types, including neoplastic and non-neoplastic cells. Further, we determined that KIF11 has a role in regulating cilia dynamics. Upon the reduction of KIF11 expression, the number of ciliated cells in asynchronously growing populations was significantly increased. We rescued this effect by the addition of exogenous KIF11. Lastly, we found that depleting KIF11 resulted in an increase in cilium length and an attenuation in the kinetics of cilia disassembly. These findings establish a previously unknown link between KIF11 and the dynamics of primary cilia and further support non-mitotic functions for this kinesin.

Hyperphosphorylation of CDH1 in Glioblastoma Cancer Stem Cells Attenuates APC/CCDH1 Activity and Pharmacologic Inhibition of APC/CCDH1/CDC20 Compromises Viability.

Glioblastoma (GBM) is the most common and lethal primary brain tumor and remains incurable. This is in part due to the cellular heterogeneity within these tumors, which includes a subpopulation of treatment-resistant cells called cancer stem-like cells (CSC). We previously identified that the anaphase-promoting complex/cylosome (APC/C), a key cell-cycle regulator and tumor suppressor, had attenuated ligase activity in CSCs. Here, we assessed the mechanism of reduced activity, as well as the efficacy of pharmacologically targeting the APC/C in CSCs. We identified hyperphosphorylation of CDH1, but not pseudosubstrate inhibition by early mitotic inhibitor 1 (EMI1), as a major mechanism driving attenuated APC/CCDH1 activity in the G1-phase of the cell cycle in CSCs. Small-molecule inhibition of the APC/C reduced viability of both CSCs and nonstem tumor cells (NSTCs), with the combination of proTAME and apcin having the biggest impact. Combinatorial drug treatment also led to the greatest mitotic arrest and chromosomal abnormalities. IMPLICATIONS: Our findings demonstrate how the activity of the APC/CCDH1 tumor suppressor is reduced in CSCs and also validates small-molecule inhibition of the APC/C as a promising therapeutic target for the treatment of GBM.

A Role for Hypocretin/Orexin in Metabolic and Sleep Abnormalities in a Mouse Model of Non-metastatic Breast Cancer.

We investigated relationships among immune, metabolic, and sleep abnormalities in mice with non-metastatic mammary cancer. Tumor-bearing mice displayed interleukin-6 (IL-6)-mediated peripheral inflammation, coincident with altered hepatic glucose processing and sleep. Tumor-bearing mice were hyperphagic, had reduced serum leptin concentrations, and enhanced sensitivity to exogenous ghrelin. We tested whether these phenotypes were driven by inflammation using neutralizing monoclonal antibodies against IL-6; despite the reduction in IL-6 signaling, metabolic and sleep abnormalities persisted. We next investigated neural populations coupling metabolism and sleep, and observed altered activity within lateral-hypothalamic hypocretin/orexin (HO) neurons. We used a dual HO-receptor antagonist to test whether increased HO signaling was causing metabolic abnormalities. This approach rescued metabolic abnormalities and enhanced sleep quality in tumor-bearing mice. Peripheral sympathetic denervation prevented tumor-induced increases in serum glucose. Our results link metabolic and sleep abnormalities via the HO system, and provide evidence that central neuromodulators contribute to tumor-induced changes in metabolism.

Mammary Tumors Induce Central Pro-inflammatory Cytokine Expression, but Not Behavioral Deficits in Balb/C Mice.

Breast cancer survivors are more likely to develop mood disorders and cognitive deficits than women in the general population. Previous studies suggest that peripheral tumors elicit central pro-inflammatory cytokine production, in turn leading to depression and cognitive deficits. In the current study, two cohorts of female Balb/C mice received bilateral orthotopic injections of syngeneic 67NR, 4T07, or 4T1cells (1 × 105 cells per injection) to induce mammary tumors. Approximately three weeks later, learned fear (via fear conditioning) or depressive-like behavior (via tail suspension and forced swim test) was assessed. Proinflammatory cytokine levels were increased in the serum (IL-1ß, TNFα, IFNγ) and livers (IL-1ß, IL-6, TNFα) of mice with 4T07 or 4T1 tumors compared to 67NR tumors and the vehicle control. IL-1ß was increased in both the hippocampus and cortex of mice injected with 4T07 or 4T1 cell lines relative to the other treatment groups. However, mammary tumors had no effect on hippocampal doublecortin + and did not alter depressive-like behavior or learned fear. These data demonstrate that similarly sized tumors can produce differential immune responses and that tumor-induced central pro-inflammatory cytokine production can exist in the absence of depressive-like behavior or cognitive deficits.