MODELING AND THERAPEUTIC TARGETING OF ENDOGENOUS IDH1-MUTANT GLIOMA

Somatic mutations in Isocitrate Dehydrogenase 1 (IDH1) are frequent in low grade and progressive gliomas. IDH1 mutant tumors are phenotypically characterized by the increased production of 2-hydroxyglutarate (2-HG) from α-ketoglutarate. 2-HG is an “oncometabolite” that competitively inhibits α-KG dependent dioxygenases. The inhibition of these dioxygenases causes widespread cellular changes including abnormal hypermethylation of genomic DNA and suppression of cellular differentiation. Recent investigations of malignant gliomas have identified additional genetic and chromosomal abnormalities that cluster with IDH1 mutations into two molecularly distinct subgroups. The astrocytic subgroup is defined by frequent mutations in ATRX, TP53 and displays alternative lengthening of telomeres. The second subgroup with oligodendrocytic morphology has frequent mutations in CIC or FUBP1, and is linked to co-deletion of the 1p/19q arms. These mutations reflect the development of two distinct molecular pathways representing the majority of IDH1 mutant gliomas. Unfortunately, due to the scarcity of endogenously derived IDH1 mutant models, opportunities to evaluate therapies targeted to the mutation or its consequences have been limited. Here we report the generation of an endogenous IDH1 mutant anaplastic astrocytoma model. This novel tumor model expresses the IDH1 (R132H) mutant protein, grows rapidly in vivo, produces 2-HG, exhibits DNA hypermethylation and harbors concurrent mutations in TP53, CDKN2A and ATRX. Furthermore, this model has a similar histopathology as the original patient tumor, and exhibits an alternative lengthening of telomeres phenotype. Using this in vivo model, we have demonstrated the preclinical efficacy and mechanism of action of the FDA approved demethylating drug 5-azacytidine. Long term administration of 5-azacytidine resulted in reduction of DNA methylation of promoter loci, induction of glial differentiation, reduction of cell proliferation and a significant reduction in tumor growth. Tumors regressed by 14 weeks and subsequently showed no signs of re-growth at 7 weeks despite discontinuation of therapy. These results suggest that demethylating agents might be useful for the clinical management of patients with IDH-mutant gliomas

MODELING AND THERAPEUTIC TARGETING OF ENDOGENOUS IDH1-MUTANT GLIOMA

Somatic mutations in Isocitrate Dehydrogenase 1 (IDH1) are frequent in low grade and progressive gliomas. IDH1 mutant tumors are phenotypically characterized by the increased production of 2-hydroxyglutarate (2-HG) from α-ketoglutarate. 2-HG is an “oncometabolite” that competitively inhibits α-KG dependent dioxygenases. The inhibition of these dioxygenases causes widespread cellular changes including abnormal hypermethylation of genomic DNA and suppression of cellular differentiation. Recent investigations of malignant gliomas have identified additional genetic and chromosomal abnormalities that cluster with IDH1 mutations into two molecularly distinct subgroups. The astrocytic subgroup is defined by frequent mutations in ATRX, TP53 and displays alternative lengthening of telomeres. The second subgroup with oligodendrocytic morphology has frequent mutations in CIC or FUBP1, and is linked to co-deletion of the 1p/19q arms. These mutations reflect the development of two distinct molecular pathways representing the majority of IDH1 mutant gliomas. Unfortunately, due to the scarcity of endogenously derived IDH1 mutant models, opportunities to evaluate therapies targeted to the mutation or its consequences have been limited. Here we report the generation of an endogenous IDH1 mutant anaplastic astrocytoma model. This novel tumor model expresses the IDH1 (R132H) mutant protein, grows rapidly in vivo, produces 2-HG, exhibits DNA hypermethylation and harbors concurrent mutations in TP53, CDKN2A and ATRX. Furthermore, this model has a similar histopathology as the original patient tumor, and exhibits an alternative lengthening of telomeres phenotype. Using this in vivo model, we have demonstrated the preclinical efficacy and mechanism of action of the FDA approved demethylating drug 5-azacytidine. Long term administration of 5-azacytidine resulted in reduction of DNA methylation of promoter loci, induction of glial differentiation, reduction of cell proliferation and a significant reduction in tumor growth. Tumors regressed by 14 weeks and subsequently showed no signs of re-growth at 7 weeks despite discontinuation of therapy. These results suggest that demethylating agents might be useful for the clinical management of patients with IDH-mutant gliomas

Generation of stable PDX derived cell lines using conditional reprogramming

Abstract Efforts to develop effective cancer therapeutics have been hindered by a lack of clinically predictive preclinical models which recapitulate this complex disease. Patient derived xenograft (PDX) models have emerged as valuable tools for translational research but have several practical limitations including lack of sustained growth in vitro. In this study, we utilized Conditional Reprogramming (CR) cell technology- a novel cell culture system facilitating the generation of stable cultures from patient biopsies- to establish PDX-derived cell lines which maintain the characteristics of the parental PDX tumor. Human lung and ovarian PDX tumors were successfully propagated using CR technology to create stable explant cell lines (CR-PDX). These CR-PDX cell lines maintained parental driver mutations and allele frequency without clonal drift. Purified CR-PDX cell lines were amenable to high throughput chemosensitivity screening and in vitro genetic knockdown studies. Additionally, re-implanted CR-PDX cells proliferated to form tumors that retained the growth kinetics, histology, and drug responses of the parental PDX tumor. CR technology can be used to generate and expand stable cell lines from PDX tumors without compromising fundamental biological properties of the model. It offers the ability to expand PDX cells in vitro for subsequent 2D screening assays as well as for use in vivo to reduce variability, animal usage and study costs. The methods and data detailed here provide a platform to generate physiologically relevant and predictive preclinical models to enhance drug discovery efforts

Pan genome of the phytoplankton Emiliania underpins its global distribution

Coccolithophores have influenced the global climate for over 200 million years1. These marine phytoplankton can account for 20 per cent of total carbon fixation in some systems2. They form blooms that can occupy hundreds of thousands of square kilometres and are distinguished by their elegantly sculpted calcium carbonate exoskeletons (coccoliths), rendering themvisible fromspace3.Although coccolithophores export carbon in the form of organic matter and calcite to the sea floor, they also release CO2 in the calcification process. Hence, they have a complex influence on the carbon cycle, driving either CO2 production or uptake, sequestration and export to the deep ocean4. Here we report the first haptophyte reference genome, from the coccolithophore Emiliania huxleyi strain CCMP1516, and sequences from 13 additional isolates. Our analyses reveal a pan genome (core genes plus genes distributed variably between strains) probably supported by an atypical complement of repetitive sequence in the genome. Comparisons across strains demonstrate thatE. huxleyi, which has long been considered a single species, harbours extensive genome variability reflected in different metabolic repertoires. Genome variability within this species complex seems to underpin its capacity both to thrive in habitats ranging from the equator to the subarctic and to form large-scale episodic blooms under a wide variety of environmental conditions

Conversion of ATP to adenosine by CD39 and CD73 in multiple myeloma can be successfully targeted together with adenosine receptor A2A blockade

Background PD1/PDL1-directed therapies have been unsuccessful for multiple myeloma (MM), an incurable cancer of plasma cells in the bone marrow (BM). Therefore, other immune checkpoints such as extracellular adenosine and its immunosuppressive receptor should be considered. CD39 and CD73 convert extracellular ATP to adenosine, which inhibits T-cell effector functions via the adenosine receptor A2A (A2AR). We set out to investigate whether blocking the adenosine pathway could be a therapy for MM.Methods Expression of CD39 and CD73 on BM cells from patients and T-cell proliferation were determined by flow cytometry and adenosine production by Liquid chromatograpy-mass spectrometry (HPCL/MS). ENTPD1 (CD39) mRNA expression was determined on myeloma cells from patients enrolled in the publicly available CoMMpass study. Transplantable 5T33MM myeloma cells were used to determine the effect of inhibiting CD39, CD73 and A2AR in mice in vivo.Results Elevated level of adenosine was found in BM plasma of MM patients. Myeloma cells from patients expressed CD39, and high gene expression indicated reduced survival. CD73 was found on leukocytes and stromal cells in the BM. A CD39 inhibitor, POM-1, and an anti-CD73 antibody inhibited adenosine production and reduced T-cell suppression in vitro in coculture of myeloma and stromal cells. Blocking the adenosine pathway in vivo with a combination of Sodium polyoxotungstate (POM-1), anti-CD73, and the A2AR antagonist AZD4635 activated immune cells, increased interferon gamma production, and reduced the tumor load in a murine model of MM.Conclusions Our data suggest that the adenosine pathway can be successfully targeted in MM and blocking this pathway could be an alternative to PD1/PDL1 inhibition for MM and other hematological cancers. Inhibitors of the adenosine pathway are available. Some are in clinical trials and they could thus reach MM patients fairly rapidly