Mohammed Khurshed

Radioprotection of IDH1-Mutated Cancer Cells by the IDH1-Mutant Inhibitor AGI-5198

Isocitrate dehydrogenase 1 (IDH1) is mutated in various types of human cancer to IDH1(R132H), a structural alteration that leads to catalysis of α-ketoglutarate to the oncometabolite D-2-hydroxyglutarate. In this study, we present evidence that small-molecule inhibitors of IDH1(R132H) that are being developed for cancer therapy may pose risks with coadministration of radiotherapy. Cancer cells heterozygous for the IDH1(R132H) mutation exhibited less IDH-mediated production of NADPH, such that after exposure to ionizing radiation (IR), there were higher levels of reactive oxygen species, DNA double-strand breaks, and cell death compared with IDH1 wild-type cells. These effects were reversed by the IDH1(R132H) inhibitor AGI-5198. Exposure of IDH1 wild-type cells to D-2-hydroxyglutarate was sufficient to reduce IDH-mediated NADPH production and increase IR sensitivity. Mechanistic investigations revealed that the radiosensitivity of heterozygous cells was independent of the well-described DNA hypermethylation phenotype in IDH1-mutated cancers. Thus, our results argue that altered oxidative stress responses are a plausible mechanism to understand the radiosensitivity of IDH1-mutated cancer cells. Further, they offer an explanation for the relatively longer survival of patients with IDH1-mutated tumors, and they imply that administration of IDH1(R132H) inhibitors in these patients may limit irradiation efficacy in this setting.

Study protocol of a phase IB/II clinical trial of metformin and chloroquine in patients with IDH1-mutated or IDH2-mutated solid tumours

Introduction High-grade chondrosarcoma, high-grade glioma and intrahepatic cholangiocarcinoma are aggressive types of cancer with a dismal outcome. This is due to the lack of effective treatment options, emphasising the need for novel therapies. Mutations in the genes IDH1 and IDH2 (isocitrate dehydrogenase 1 and 2) occur in 60% of chondrosarcoma, 80% of WHO grade II–IV glioma and 20% of intrahepatic cholangiocarcinoma. IDH1/2-mutated cancer cells produce the oncometabolite D-2-hydroxyglutarate (D-2HG) and are metabolically vulnerable to treatment with the oral antidiabetic metformin and the oral antimalarial drug chloroquine. Methods and analysis We describe a dose-finding phase Ib/II clinical trial, in which patients with IDH1/2-mutated chondrosarcoma, glioma and intrahepatic cholangiocarcinoma are treated with a combination of metformin and chloroquine. Dose escalation is performed according to a 3+3 dose-escalation scheme. The primary objective is to determine the maximum tolerated dose to establish the recommended dose for a phase II clinical trial. Secondary objectives of the study include (1) determination of pharmacokinetics and toxic effects of the study therapy, for which metformin and chloroquine serum levels will be determined over time; (2) investigation of tumour responses to metformin plus chloroquine in IDH1/2-mutated cancers using CT/MRI scans; and (3) whether tumour responses can be measured by non-invasive D-2HG measurements (mass spectrometry and magnetic resonance spectroscopy) of tumour tissue, serum, urine, and/or bile or next-generation sequencing of circulating tumour DNA (liquid biopsies). This study may open a novel treatment avenue for IDH1/2-mutated high-grade chondrosarcoma, glioma and intrahepatic cholangiocarcinoma by repurposing the combination of two inexpensive drugs that are already approved for other indications. Ethics and dissemination This study has been approved by the medical-ethical review committee of the Academic Medical Center, Amsterdam, The Netherlands. The report will be submitted to a peer-reviewed journal. Trial registration number This article was registered at ClinicalTrials.gov identifier (NCT02496741): Pre-results.

IDH1/2 mutations sensitize acute myeloid leukemia to PARP inhibition and this is reversed by IDH1/2-mutant inhibitors

Purpose: Somatic mutations in IDH1/2 occur in approximately 20% of patients with myeloid neoplasms, including acute myeloid leukemia (AML). IDH1/2MUT enzymes produce D-2-hydroxyglutarate (D2HG), which associates with increased DNA damage and improved responses to chemo/radiotherapy and PARP inhibitors in solid tumor cells. Whether this also holds true for IDH1/2MUT AML is not known. Experimental Design: Well-characterized primary IDH1MUT, IDH2MUT, and IDH1/2WT AML cells were analyzed for DNA damage and responses to daunorubicin, ionizing radiation, and PARP inhibitors. Results: IDH1/2MUT caused increased DNA damage and sensitization to daunorubicin, irradiation, and the PARP inhibitors olaparib and talazoparib in AML cells. IDH1/2MUT inhibitors protected against these treatments. Combined treatment with a PARP inhibitor and daunorubicin had an additive effect on the killing of IDH1/2MUT AML cells. We provide evidence that the therapy sensitivity of IDH1/2MUT cells was caused by D2HG-mediated downregulation of expression of the DNA damage response gene ATM and not by altered redox responses due to metabolic alterations in IDH1/2MUT cells. Conclusions: IDH1/2MUT AML cells are sensitive to PARP inhibitors as monotherapy but especially when combined with a DNA-damaging agent, such as daunorubicin, whereas concomitant administration of IDH1/2MUT inhibitors during cytotoxic therapy decrease the efficacy of both agents in IDH1/2MUT AML. These results advocate in favor of clinical trials of PARP inhibitors either or not in combination with daunorubicin in IDH1/2MUT AML. Clin Cancer Res; 24(7); 1705–15. ©2018 AACR.

Periarteriolar Glioblastoma Stem Cell Niches Express Bone Marrow Hematopoietic Stem Cell Niche Proteins

In glioblastoma, a fraction of malignant cells consists of therapy-resistant glioblastoma stem cells (GSCs) residing in protective niches that recapitulate hematopoietic stem cell (HSC) niches in bone marrow. We have previously shown that HSC niche proteins stromal cell–derived factor-1α (SDF-1α), C-X-C chemokine receptor type 4 (CXCR4), osteopontin (OPN), and cathepsin K (CatK) are expressed in hypoxic GSC niches around arterioles in five human glioblastoma samples. In HSC niches, HSCs are retained by binding of SDF-1α and OPN to their receptors CXCR4 and CD44, respectively. Protease CatK cleaves SDF-1α to release HSCs out of niches. The aim of the present study was to reproduce the immunohistochemical localization of these GSC markers in 16 human glioblastoma samples with the addition of three novel markers. Furthermore, we assessed the type of blood vessels associated with GSC niches. In total, we found seven GSC niches containing CD133-positive and nestin-positive GSCs as a single-cell layer exclusively around the tunica adventitia of 2% of the CD31-positive and SMA-positive arterioles and not around capillaries and venules. Niches expressed SDF-1α, CXCR4, CatK, OPN, CD44, hypoxia-inducible factor-1α, and vascular endothelial growth factor. In conclusion, we show that GSC niches are present around arterioles and express bone marrow HSC niche proteins.

In silico gene expression analysis reveals glycolysis and acetate anaplerosis in IDH1 wild-type glioma and lactate and glutamate anaplerosis in IDH1 -mutated glioma

Hotspot mutations in isocitrate dehydrogenase 1 (IDH1) initiate low-grade glioma and secondary glioblastoma and induce a neomorphic activity that converts α-ketoglutarate (α-KG) to the oncometabolite D-2-hydroxyglutarate (D-2-HG). It causes metabolic rewiring that is not fully understood. We investigated the effects of IDH1 mutations (IDH1MUT) on expression of genes that encode for metabolic enzymes by data mining The Cancer Genome Atlas. We analyzed 112 IDH1 wild-type (IDH1WT) versus 399 IDH1MUT low-grade glioma and 157 IDH1WT versus 9 IDH1MUT glioblastoma samples. In both glioma types, IDH1WT was associated with high expression levels of genes encoding enzymes that are involved in glycolysis and acetate anaplerosis, whereas IDH1MUT glioma overexpress genes encoding enzymes that are involved in the oxidative tricarboxylic acid (TCA) cycle. In vitro, we observed that IDH1MUT cancer cells have a higher basal respiration compared to IDH1WT cancer cells and inhibition of the IDH1MUT shifts the metabolism by decreasing oxygen consumption and increasing glycolysis. Our findings indicate that IDH1WT glioma have a typical Warburg phenotype whereas in IDH1MUT glioma the TCA cycle, rather than glycolytic lactate production, is the predominant metabolic pathway. Our data further suggest that the TCA in IDH1MUT glioma is driven by lactate and glutamate anaplerosis to facilitate production of α-KG, and ultimately D-2-HG. This metabolic rewiring may be a basis for novel therapies for IDH1MUT and IDH1WT glioma.

Three-dimensional histochemistry and imaging of human gingiva

In the present study, 3D histochemistry and imaging methodology is described for human gingiva to analyze its vascular network. Fifteen human gingiva samples without signs of inflammation were cleared using a mixture of 2-parts benzyl benzoate and 1-part benzyl alcohol (BABB), after being immunofluorescently stained for CD31, marker of endothelial cells to visualize blood vessels in combination with fluorescent DNA dyes. Samples were imaged in 3D with the use of confocal microscopy and light-sheet microscopy and image processing. BABB clearing caused limited tissue shrinkage 13 ± 7% as surface area and 24 ± 1% as volume. Fluorescence remained intact in BABB-cleared gingiva samples and light-sheet microscopy was an excellent tool to image gingivae whereas confocal microscopy was not. Histochemistry on cryostat sections of gingiva samples after 3D imaging validated structures visualized in 3D. Three-dimensional images showed the vascular network in the stroma of gingiva with one capillary loop in each stromal papilla invading into the epithelium. The capillary loops were tortuous with structural irregularities that were not apparent in 2D images. It is concluded that 3D histochemistry and imaging methodology described here is a promising novel approach to study structural aspects of human gingiva in health and disease.

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells are enzymes and their activity is heavily regulated at many levels, including the transcriptional, mRNA stability, translational, post-translational and functional level. This complex regulation means that conventional quantitative or imaging assays, such as quantitative mRNA experiments, Western Blots and immunohistochemistry, yield incomplete information regarding the ultimate activity of enzymes, their function and/or their subcellular localization. Quantitative enzyme cytochemistry and histochemistry (i.e., metabolic mapping) show in-depth information on in situ enzymatic activity and its kinetics, function and subcellular localization in an almost true-to-nature situation. We describe a protocol to detect the activity of dehydrogenases, which are enzymes that perform redox reactions to reduce cofactors such as NAD(P)+ and FAD. Cells and tissue sections are incubated in a medium that is specific for the enzymatic activity of one dehydrogenase. Subsequently, the dehydrogenase that is the subject of investigation performs its enzymatic activity in its subcellular site. In a chemical reaction with the reaction medium, this ultimately generates blue-colored formazan at the site of the dehydrogenases activity. The formazans absorbance is therefore a direct measure of the dehydrogenases activity and can be quantified using monochromatic light microscopy and image analysis. The quantitative aspect of this protocol enables researchers to draw statistical conclusions from these assays. Besides observational studies, this technique can be used for inhibition studies of specific enzymes. In this context, studies benefit from the true-to-nature advantages of metabolic mapping, giving in situ results that may be physiologically more relevant than in vitro enzyme inhibition studies. In all, metabolic mapping is an indispensable technique to study metabolism at the cellular or tissue level. The technique is easy to adopt, provides in-depth, comprehensive and integrated metabolic information and enables rapid quantitative analysis.

Cysteine cathepsins B, X and K expression in peri-arteriolar glioblastoma stem cell niches

Glioblastoma (GBM) is the most lethal brain tumor also due to malignant and therapy-resistant GBM stem cells (GSCs) that are localized in protecting hypoxic GSC niches. Some members of the cysteine cathepsin family of proteases have been found to be upregulated in GBM. Cathepsin K gene expression is highly elevated in GBM tissue versus normal brain and it has been suggested to regulate GSC migration out of the niches. Here, we investigated the cellular distribution of cathepsins B, X and K in GBM tissue and whether these cathepsins are co-localized in GSC niches. Therefore, we determined expression of these cathepsins in serial paraffin sections of 14 human GBM samples and serial cryostat sections of two samples using immunohistochemistry and metabolic mapping of cathepsin activity using selective fluorogenic substrates. We detected cathepsins B, X and K in peri-arteriolar GSC niches in 9 out of 16 GBM samples, which were defined by co-expression of the GSC marker CD133, the niche marker stromal-derived factor-1α (SDF-1α) and smooth muscle actin as a marker for arterioles. The expression of cathepsin B and X was detected in stromal cells and cancer cells throughout the GBM sections, whereas cathepsin K expression was more restricted to arteriole-rich regions in the GBM sections. Metabolic mapping showed that cathepsin B, but not cathepsin K is active in GSC niches. On the basis of these findings, it is concluded that cathepsins B, X and K have distinct functions in GBM and that cathepsin K is the most likely GSC niche-related cathepsin of the three cathepsins investigated.

Development of calcium bodies in Hylonsicus riparius (crustacea: Isopoda)

Calcium bodies are internal epithelial sacs found in terrestrial isopods of the family Trichoniscidae that contain a mineralized extracellular matrix that is deposited and resorbed in relation to the molt cycle. Calcium bodies in several trichoniscids are filled with bacteria, the function of which is currently unknown. The woodlouse Hyloniscus riparius differs from other trichoniscids in that it possesses two different pairs of calcium bodies, the posterior pair being filled with bacteria and the anterior pair being devoid of bacteria. We explored the development of these organs and bacterial colonization of their lumen during the postmarsupial development with the use of optical clearing and whole-body confocal imaging of larval and juvenile stages. Our results show that calcium bodies are formed as invaginations of the epidermis in the region of intersegmental membranes during the postmarsupial development. The anterior pair of calcium bodies is generated during the first postmarsupial manca stage, whereas the posterior calcium bodies first appear in juveniles and are immediately colonized by bacteria, likely through a connection between the calcium body lumen and the body surface. Mineral is deposited in calcium bodies as soon as they are present.