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Atypical teratoid/rhabdoid tumors (AT/RT) and diffuse intrinsic pontine glioma (DIPG) are incurable pediatric brain tumors. Developing new targets and novel therapeutics are urgently needed. We have previously shown that multiple primary brain tumors and cell lines express increased amounts of the epigenetic modifier high mobility group AT-hook 2 (HMGA2). HMGA2 is a DNA-binding oncoprotein that regulates transcription during normal embryogenesis and in cancer stem cells. Targeting HMGA2 using short hairpins significantly decreased AT/RT and glioma growth and increased survival of xenografted mice. We hypothesized that pharmacological inhibition of HMGA proteins using DNA minor-groove binding drugs like quinacrine will decrease AT/RT and DIPG growth due to displacement of HMGA proteins from the DNA. We used quinacrine in ten patient-derived cell lines: five AT/RT (BT37, CHLA-05, CHLA-06, BT-12, CHLA-266) and five DIPG (JHHDIPG1, SUDIPGXIII, JHHDIPG16A, SF7761, HSJD-007). Quinacrine has been used in millions of humans to treat malaria and parasitic infections, has a well-known safety profile and can penetrate the brain. Using quinacrine fluorescence as a surrogate, we can achieve therapeutically efficacious micromolar concentration of quinacrine in the mouse and zebrafish brain after oral administration without overt toxicity. In both tumor cell lines, quinacrine causes a dose-dependent reduction in growth (MTS) and proliferation (BrdU) compared to vehicle-treated cells (P<0.01). Treatment of both tumor lines with quinacrine significantly increased apoptosis (cleaved caspase-3 and cleaved PARP) compared to control cells (P<0.01). Quinacrine had no effect on growth of normal hindbrain cells. Our results suggest that minor groove binding drugs like quinacrine are a viable potential treatment strategy for these lethal tumors. Ongoing studies include validating the in vivo efficacy of quinacrine using zebrafish and mouse models of AT/RT and DIPG. Future studies are aimed at investigating the mechanism of quinacrine in these devastating tumors. Citation Format: Harpreet Kaur, Huizi Guo, Peter Green, Sepehr Akhtarkhavari, Smit Shah, Charles G. Eberhart, Eric H. Raabe. Targeting the fatal pediatric brain tumors AT/RT and DIPG with the DNA binding agent quinacrine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2888.

AimsAnecdotal reports have suggested increased soft tissue calcification in individuals with long-term exposures to high blood glucose. The association of costal cartilage calcification (CCC), a reliably quantifiable marker obtainable from non-contrast cardiac computed tomography (CT) with cumulative fasting blood glucose (FBG) exposure, is unknown. In this study, we aimed to determine the association between quantified CCC and cumulative glucose exposure using non-contrast coronary artery calcium (CAC) scoring computed tomography (CT) images in the Multi-Ethnic Study of Atherosclerosis (MESA).MethodsThe volume of bilateral CCC was quantified in high-density pixels (threshold of Hounsfield Unit>180) using the CAC scoring CT images acquired in the 5th MESA exam. Prior long-term cumulative exposure to FBG was calculated by area under the FBG-time curve over ten years before the time of the CT exam.ResultsA total of 2,305 participants (mean age: 69, female/male: 1.3) were included in this study. The median CCC volume was lower in females than males (1158 mm3 [IQR: 1751] vs. 3054 mm3 [3851], p<0.001). In cross-sectional analysis, quantified CCC was associated with FBG (9% increase per SD) and HbA1c (7% increase per SD) at the CT exam only in female participants after adjustment for age, race, BMI, and glomerular filtration rate. Only in female participants, quantified CCC was also associated with prior cumulative FBG (3% increase per decile change). In the subgroup of females with zero CAC scores, the adjusted CCC was still associated with FBG (13% increase per SD) at the time of CT exam and with prior cumulative FBG exposure (4% increase per decile change) before the CT exam.ConclusionsThe CCC, a reliably quantified marker in non-contrast cardiac CT, is associated with 10-year cumulative FBG exposure only in female participants, even those with zero CAC.

Diffuse Intrinsic Pontine Glioma (DIPG) is an incurable, invasive and aggressive pediatric brain tumor. Identifying molecular markers that regulate tumor growth and invasion are needed for developing efficient treatment strategies. LIN28B is a stem cell factor expressed during normal fetal development and re-expressed in cancer cells. We had previously shown that LIN28A, another family member of LIN28 proteins, regulates invasion and tumorigenicity in adult high grade gliomas. We observed increased LIN28B expression in patient-derived DIPG neurosphere cell lines using western blotting. We hypothesized that LIN28B promotes proliferation and prevents apoptosis in DIPG. Using two different lentiviral transduced short hairpin RNAs (shRNA), we suppressed LIN28B protein levels in DIPG neurospheres, as confirmed by western blotting. DIPG neurospheres that have been treated with LIN28B shRNA showed reduced proliferation as measured by BrdU incorporation (P<0.01) and increased apoptosis as measured by cleaved caspase-3 (CC-3) expression (P<0.01). To determine the molecular mechanism of LIN28B-mediated phenotypes in DIPG, we studied the canonical downstream effector of LIN28B called HMGA2, which is a DNA-binding protein that functions as a transcriptional regulator. Using western blotting, we found decreased HMGA2 protein levels in DIPG neurospheres infected with LIN28B shRNA. Taken together, our results suggest that LIN28B is important for promoting DIPG cell proliferation and preventing apoptotic cell death. Additionally, we also found that LIN28B regulates HMGA2 expression in DIPG neurospheres. Future studies will focus on expanding our understanding of the molecular mechanisms of LIN28B-regulated malignancy in DIPG. Citation Format: Huizi Guo, Sepehr Akhtarkhavari, Charles G. Eberhart, Harpreet Kaur, Eric H. Raabe. The stem cell factor LIN28B regulates proliferation and apoptosis in diffuse intrinsic pontine glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3868. doi:10.1158/1538-7445.AM2017-3868

Diffuse intrinsic pontine glioma (DIPG) is an invasive, incurable and aggressive pediatric brain tumor found in the brainstem. Improved understanding of the biology of DIPG tumors is urgently needed to develop novel treatments. Our previous studies have shown that DIPG tumors and cell lines express high levels of the DNA-binding stem cell factor high mobility group AT-hook 2 (HMGA2).. Targeting HMGA2 using lentiviral shRNA decreased DIPG cell invasion, proliferation and increased apoptosis in our studies. We hypothesized that inhibiting HMGA2 using DNA minor groove binding drugs like quinacrine would decrease DIPG proliferation and increase apoptotic cell death. We used three DIPG cell lines (JHHDIPG1, JHHDIPG16A, and SUDIPG13) to test the effect of quinacrine. Quinacrine has traditionally been used as an anti-malarial drug and is known to strongly bind to DNA. We used BrdU incorporation as a measure of proliferation and expression of cleaved caspase-3 (CC-3) as a measure of apoptosis. Treatment of DIPG cells with quinacrine showed a dose dependent increase in cell death (CC-3 expression) from 3uM to 20uM, with the most effective doses being from 3uM to 5uM. In all three DIPG cell lines, treatment with 1uM to 5uM quinacrine showed a significant reduction in cell proliferation (BrdU) from 3uM to 5uM compared to vehicle-treated cells (P<0.0001). Additionally, treatment of DIPG cell lines with 1uM to 5uM quinacrine showed a significant increase in apoptotic cell death (CC-3 expression) compared to vehicle control cells from 3uM to 5uM (P<0.001). Our data suggests that minor groove binding agents like quinacrine could be effective therapies for this lethal brain tumor. Our future studies are aimed at testing the efficacy of quinacrine in inhibiting DIPG tumorigenicity and elucidating the mechanism of action. Citation Format: Huizi Guo, Harpreet Kaur, Sepehr Akhtarkhavari, Charles G. Eberhart, Eric H. Raabe. The minor groove binding agent quinacrine inhibits growth and increases apoptotic death in diffuse intrinsic pontine glioma tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4850.