The efficacy of a ketogenic diet in preclinical models of IDH wild-type and IDH mutant glioma

Infiltrative gliomas are the most common neoplasms arising in the brain, and remain largely incurable despite decades of research. A subset of these gliomas contains mutations in isocitrate dehydrogenase 1 (IDH1) or, less commonly, IDH2 (together called “IDHmut”). These mutations alter cellular biochemistry, and IDHmut gliomas are generally less aggressive than IDH wild-type (IDHwt) gliomas. Some preclinical studies and clinical trials have suggested that a ketogenic diet (KD), characterized by low-carbohydrate and high-fat content, may be beneficial in slowing glioma progression. However, not all studies have shown promising results, and to date, no study has addressed whether IDHmut gliomas might be more sensitive to KD. The aim of the current study was to compare the effects of KD in preclinical models of IDHwt versus IDHmut gliomas. In vitro, simulating KD by treatment with the ketone body β-hydroxybutyrate had no effect on the proliferation of patient-derived IDHwt or IDHmut glioma cells. Likewise, a cycling KD, wherein mice alternated between KD and a standard diet (SD), had no effect on the in vivo growth of patient-derived IDHwt or IDHmut gliomas, even though the cycling KD did result in persistently elevated circulating ketones. Furthermore, KD conferred no survival benefit in mice engrafted with Sleeping-Beauty transposase-engineered IDHmut glioma. These data suggest that neither IDHwt nor IDHmut gliomas are particularly responsive to KD.


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ABSTRACT Infiltrative gliomas are the most common neoplasms arising in the brain, and remain largely 23 incurable despite decades of research. A subset of these gliomas contains mutations in isocitrate 24 dehydrogenase 1 (IDH1) or, less commonly, IDH2 (together called "IDH mut "). These mutations 25 alter cellular biochemistry, and IDH mut gliomas are generally less aggressive than IDH wild-type 26 (IDH wt ) gliomas. Some preclinical studies and clinical trials have suggested that a ketogenic diet 27 (KD), characterized by low-carbohydrate and high-fat content, may be beneficial in slowing glioma 28 progression. However, not all studies have shown promising results, and to date, no study has 29 addressed whether IDH mut gliomas might be more sensitive to KD. The aim of the current study 30 was to compare the effects of KD in preclinical models of IDH wt versus IDH mut gliomas. In vitro,

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simulating KD by treatment with the ketone body β-hydroxybutyrate had no effect on the 32 proliferation of patient-derived IDH wt or IDH mut glioma cells. Likewise, a cycling KD, wherein mice 33 alternated between KD and a standard diet (SD), had no effect on the in vivo growth of patient-34 derived IDH wt or IDH mut gliomas, even though the cycling KD did result in persistently elevated 35 circulating ketones. Furthermore, KD conferred no survival benefit in mice engrafted with

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Sleeping-Beauty transposase-engineered IDH mut glioma. These data suggest that neither IDH wt 37 nor IDH mut gliomas are particularly responsive to KD. INTRODUCTION

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Diffusely infiltrative gliomas strike over 17,000 people in the United States per year [1].

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The vast majority of these tumors recur and progress, despite advancements in surgery, 41 chemotherapy, radiotherapy, and immunotherapy. In 20-39 year-olds, gliomas are the 2 nd most 42 common cause of cancer death in men, and are the 5 th most common cause in women [1]. The 43 most common type of primary brain cancer in adults is diffusely infiltrative glioma, and the most 44 common subtype of infiltrative glioma, glioblastoma (GBM), is unfortunately also the most lethal.

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Despite great advances in treating many other kinds of cancer, the median survival of GBM

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In vitro proliferation 119 Cells were plated in 24 well plates at a concentration of 5x10 4 cells per well in triplicate.

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To mimic the low glucose environment characteristic of physiological ketosis, a formulation of 122 DMEM (Corning) with 1.0 g/L of glucose was tested alongside the normal glucose concentration 123 of 4.5 g/L. Plates were trypsinized at specific time points, and live cells were counted via trypan 124 blue exclusion using a BioRad TC20 Automated Cell Counter. Absolute cell counts were used to 126 assay, since the latter uses mitochondrial metabolism as a marker of cell viability, and we sought 127 to avoid any potential confounding effects of culture conditions on mitochondria.

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In vivo studies All animals were fed standard rodent chow (standard diet, or SD) for 3 days following flank 148 or intracerebral engraftment before being randomized to either remain on SD ad libitum, or given 152 KD group were kept on KD for one week, followed by SD for one week, and so on, in order to 153 prevent obesity and reduce midlife mortality. Mice were still allowed to feed ad libitum, in order to 154 reduce potentially confounding effects of weight loss from calorie-restricted diets and more 155 effectively maintain plasma ketone levels, as was described by others [13,18]. At the end of each 156 week, blood from the saphenous vein was collected to test circulating metabolite levels using the

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First, we examined the effects of a ketogenic-like diet on cultured IDH1 wt and IDH1 mut 178 patient-derived cancer cell lines under the following conditions: (i) normal basal glucose (NG) of 179 4.5 g/L; (ii) low glucose (LG) of 1.0 g/L; (iii) NG with 10 mM β-hydroxybutyrate (BHB); (iv) LG with 180 BHB (Figure 1). Both pairs of IDH1 wt and IDH1 mut cell types responded similarly, with slightly 181 attenuated proliferation in LG medium, but no effect of BHB in either NG or LG medium, as 182 indicated by two-way ANOVA analyses (

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Next, we evaluated the ability of a cycling KD (see "Methods") to induce ketosis in mice 207 without tumors. Over the first 5 weeks that mice were on KD, blood glucose levels were similar to 208 control mice on SD, even during weeks in which KD was implemented (Figure 2A). Interestingly, 209 blood glucose rose 15% in KD mice after week 6 (147.8 mg/dl in KD versus 128.0 mg/dl in SD,

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P=0.0053). Ketones, in contrast, increased by 112.5% after just one week on KD compared to   (Figure 4). Among mice maintained on SD, those engrafted with IDH1 mut 240 NPAC1 cells survived 20% longer than mice engrafted with IDH1 wt NPA cells (median survival