Single-cell lineage analysis reveals genetic and epigenetic interplay in glioblastoma drug resistance.

BACKGROUND: Tumors can evolve and adapt to therapeutic pressure by acquiring genetic and epigenetic alterations that may be transient or stable. A precise understanding of how such events contribute to intratumoral heterogeneity, dynamic subpopulations, and overall tumor fitness will require experimental approaches to prospectively label, track, and characterize resistant or otherwise adaptive populations at the single-cell level. In glioblastoma, poor efficacy of receptor tyrosine kinase (RTK) therapies has been alternatively ascribed to genetic heterogeneity or to epigenetic transitions that circumvent signaling blockade. RESULTS: We combine cell lineage barcoding and single-cell transcriptomics to trace the emergence of drug resistance in stem-like glioblastoma cells treated with RTK inhibitors. Whereas a broad variety of barcoded lineages adopt a Notch-dependent persister phenotype that sustains them through early drug exposure, rare subclones acquire genetic changes that enable their rapid outgrowth over time. Single-cell analyses reveal that these genetic subclones gain copy number amplifications of the insulin receptor substrate-1 and substrate-2 (IRS1 or IRS2) loci, which activate insulin and AKT signaling programs. Persister-like cells and genomic amplifications of IRS2 and other loci are evident in primary glioblastomas and may underlie the inefficacy of targeted therapies in this disease. CONCLUSIONS: A method for combined lineage tracing and scRNA-seq reveals the interplay between complementary genetic and epigenetic mechanisms of resistance in a heterogeneous glioblastoma tumor model.

A Potent Blood-Brain Barrier-Permeable Mutant IDH1 Inhibitor Suppresses the Growth of Glioblastoma with IDH1 Mutation in a Patient-Derived Orthotopic Xenograft Model.

Gliomas are the second most common primary brain tumors in adults. They are treated with combination therapies, including surgery, radiotherapy, and chemotherapy. There are currently limited treatment options for recurrent gliomas, and new targeted therapies need to be identified, especially in glioblastomas, which have poor prognosis. Isocitrate dehydrogenase (IDH) mutations are detected in various tumors, including gliomas. Most patients with IDH mutant glioma harbor the IDH1R132H subtype. Mutant IDH catalyzes the conversion of α-ketoglutarate to the oncometabolite 2-hydroxyglutarate (2-HG), which induces aberrant epigenetic status and contributes to malignant progression, and is therefore a potential therapeutic target for IDH mutant tumors. The present study describes a novel, orally bioavailable selective mutant IDH1 inhibitor, DS-1001b. The drug has high blood-brain barrier (BBB) permeability and inhibits IDH1R132H. Continuous administration of DS-1001b impaired tumor growth and decreased 2-HG levels in subcutaneous and intracranial xenograft models derived from a patient with glioblastoma with IDH1 mutation. Moreover, the expression of glial fibrillary acidic protein was strongly induced by DS-1001b, suggesting that inhibition of mutant IDH1 promotes glial differentiation. These results reveal the efficacy of BBB-permeable DS-1001b in orthotopic patient-derived xenograft models and provide a preclinical rationale for the clinical testing of DS-1001b in recurrent gliomas.

Selective inhibition of mutant IDH1 by DS-1001b ameliorates aberrant histone modifications and impairs tumor activity in chondrosarcoma.

Chondrosarcoma is the second most common malignant bone tumor. It is characterized by low vascularity and an abundant extracellular matrix, which confer these tumors resistance to chemotherapy and radiotherapy. There are currently no effective treatment options for relapsed or dedifferentiated chondrosarcoma, and new targeted therapies need to be identified. Isocitrate dehydrogenase (IDH) mutations, which are detected in ~50% of chondrosarcoma patients, contribute to malignant transformation by catalyzing the production of 2-hydroxyglutarate (2-HG), a competitive inhibitor of α-ketoglutarate-dependent dioxygenases. Mutant IDH inhibitors are therefore potential novel anticancer drugs in IDH mutant tumors. Here, we examined the efficacy of the inhibition of mutant IDH1 as an antitumor approach in chondrosarcoma cells in vitro and in vivo, and investigated the association between the IDH mutation and chondrosarcoma cells. DS-1001b, a novel, orally bioavailable, selective mutant IDH1 inhibitor, impaired the proliferation of chondrosarcoma cells with IDH1 mutations in vitro and in vivo, and decreased 2-HG levels. RNA-seq analysis showed that inhibition of mutant IDH1 promoted chondrocyte differentiation in the conventional chondrosarcoma L835 cell line and caused cell cycle arrest in the dedifferentiated JJ012 cell line. Mutant IDH1-mediated modulation of SOX9 and CDKN1C expression regulated chondrosarcoma tumor progression, and DS-1001b upregulated the expression of these genes via a common mechanism involving the demethylation of H3K9me3. DS-1001b treatment reversed the epigenetic changes caused by aberrant histone modifications. The present data strongly suggest that inhibition of mutant IDH1 is a promising therapeutic approach in chondrosarcoma, particularly for the treatment of relapsed or dedifferentiated chondrosarcoma.

Author Correction: Clear and transparent nanocrystals for infrared-responsive carrier transfer.

The original version of this Article omitted the fourth author Taizo Yoshinaga, who is from the 'Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Japan'. Consequently, the third sentence of the Author Contributions, 'M.S. and M.K. synthesized the ITO NCs and ITO/semiconductor oxides' was revised to 'M.S., M.K. and T.Y. synthesized the ITO NCs and ITO/semiconductor oxides'. This has been corrected in both the PDF and HTML versions of the Article.

Clear and transparent nanocrystals for infrared-responsive carrier transfer.

Infrared-light-induced carrier transfer is a key technology for 'invisible' optical devices for information communication systems and energy devices. However, clear and colourless photo-induced carrier transfer has not yet been demonstrated in the field of photochemistry, to the best of our knowledge. Here, we resolve this problem by employing short-wavelength-infrared (1400-4000 nm) localized surface plasmon resonance-induced electron injection from indium tin oxide nanocrystals to transparent metal oxides. The time-resolved infrared measurements visualize the dynamics of the carrier in this invisible system. Selective excitation of localized surface plasmon resonances causes hot electron injection with high efficiency (33%) and long-lived charge separation (~ 2-200 µs). We anticipate our study not only provides a breakthrough for plasmonic carrier transfer systems but may also stimulate the invention of state-of-the-art invisible optical devices.

Near infrared light induced plasmonic hot hole transfer at a nano-heterointerface.

Localized surface plasmon resonance (LSPR)-induced hot-carrier transfer is a key mechanism for achieving artificial photosynthesis using the whole solar spectrum, even including the infrared (IR) region. In contrast to the explosive development of photocatalysts based on the plasmon-induced hot electron transfer, the hole transfer system is still quite immature regardless of its importance, because the mechanism of plasmon-induced hole transfer has remained unclear. Herein, we elucidate LSPR-induced hot hole transfer in CdS/CuS heterostructured nanocrystals (HNCs) using time-resolved IR (TR-IR) spectroscopy. TR-IR spectroscopy enables the direct observation of carrier in a LSPR-excited CdS/CuS HNC. The spectroscopic results provide insight into the novel hole transfer mechanism, named plasmon-induced transit carrier transfer (PITCT), with high quantum yields (19%) and long-lived charge separations (9.2 µs). As an ultrafast charge recombination is a major drawback of all plasmonic energy conversion systems, we anticipate that PITCT will break the limit of conventional plasmon-induced energy conversion.

Nature-Inspired, Highly Durable CO2 Reduction System Consisting of a Binuclear Ruthenium(II) Complex and an Organic Semiconductor Using Visible Light.

A metal-free organic semiconductor of mesoporous graphitic carbon nitride (C3N4) coupled with a Ru(II) binuclear complex (RuRu') containing photosensitizer and catalytic units selectively reduced CO2 into HCOOH under visible light (λ > 400 nm) in the presence of a suitable electron donor with high durability, even in aqueous solution. Modification of C3N4 with Ag nanoparticles resulted in a RuRu'/Ag/C3N4 photocatalyst that exhibited a very high turnover number (>33000 with respect to the amount of RuRu'), while maintaining high selectivity for HCOOH production (87-99%). This turnover number was 30 times greater than that reported previously using C3N4 modified with a mononuclear Ru(II) complex, and by far the highest among the metal-complex/semiconductor hybrid systems reported to date. The results of photocatalytic reactions, emission decay measurements, and time-resolved infrared spectroscopy indicated that Ag nanoparticles on C3N4 collected electrons having lifetimes of several milliseconds from the conduction band of C3N4, which were transferred to the excited state of RuRu', thereby promoting photocatalytic CO2 reduction driven by two-step photoexcitation of C3N4 and RuRu'. This study also revealed that the RuRu'/Ag/C3N4 hybrid photocatalyst worked efficiently in water containing a proper electron donor, despite the intrinsic hydrophobic nature of C3N4 and low solubility of CO2 in an aqueous environment.

IDH2 and NPM1 Mutations Cooperate to Activate Hoxa9/Meis1 and Hypoxia Pathways in Acute Myeloid Leukemia.

IDH1 and IDH2 mutations occur frequently in acute myeloid leukemia (AML) and other cancers. The mutant isocitrate dehydrogenase (IDH) enzymes convert α-ketoglutarate (α-KG) to the oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-KG-dependent dioxygenases. To determine whether mutant IDH enzymes are valid targets for cancer therapy, we created a mouse model of AML in which mice were transplanted with nucleophosmin1 (NPM)(+/-) hematopoietic stem/progenitor cells cotransduced with four mutant genes (NPMc, IDH2/R140Q, DNMT3A/R882H, and FLT3/ITD), which often occur simultaneously in human AML patients. Conditional deletion of IDH2/R140Q blocked 2-HG production and maintenance of leukemia stem cells, resulting in survival of the AML mice. IDH2/R140Q was necessary for the engraftment or survival of NPMc(+) cells in vivo. Gene expression analysis indicated that NPMc increased expression of Hoxa9. IDH2/R140Q also increased the level of Meis1 and activated the hypoxia pathway in AML cells. IDH2/R140Q decreased the 5hmC modification and expression of some differentiation-inducing genes (Ebf1 and Spib). Taken together, our results indicated that IDH2 mutation is critical for the development and maintenance of AML stem-like cells, and they provided a preclinical justification for targeting mutant IDH enzymes as a strategy for anticancer therapy.

A validated quantitative liquid chromatography-tandem quadrupole mass spectrometry method for monitoring isotopologues to evaluate global modified cytosine ratios in genomic DNA.

5-Hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) represent important epigenetic modifications to DNA, and a sensitive analytical method is required to determine the levels of 5hmC in the genomic DNA of tumor cells or cultured cell lines because 5hmC is present at particular low levels in these cells. We have developed a sensitive liquid chromatography-tandem quadrupole mass spectrometric method for quantifying 5-hydroxymethyldeoxycytidine (5hmdC), 5-methyldeoxycytidine (5mdC), and deoxyguanosine (dG) levels using stable isotope labeled internal standards, and used this method to estimate the global level of 2 modified cytosines in genomic DNA prepared from small number of cells. The quantification limits for 5hmdC, 5mdC and dG were 20pM, 2nM and 10nM, respectively. MRM transitions for isotopologue (isotopologue-MRM) were used to quantify the 5mdC and dG levels because of the abundance of these nucleosides relative to 5hmdC. The use of isotopologue-MRM for the abundant nucleosides could also avoid the saturation of the detector, and allow for all three nucleosides to be analyzed simultaneously without the need for the dilution and re-injection of samples into the instrument. The global ratios of modified cytosine nucleosides to dG were estimated following the quantification of each nucleoside in the hydrolysate of genomic DNA. The limit of estimation for the global 5hmC level was less than 0.001% using 200ng of DNA. Using this method, we found that MLL-TET1, which a fusion protein in acute myelogenous leukemia, did not produce 5hmC, but interfered with TET1 activity to produce 5hmC in cells. Our analytical method is therefore a valuable tool for further studies aiming at a deeper understanding of the role of modified cytosine in the epigenetic regulation of cells.

IQGAP1 selectively interacts with K-Ras but not with H-Ras and modulates K-Ras function.

K-Ras is frequently mutated and activated especially in pancreatic cancers. To analyze K-Ras function, we have searched for K-Ras interacting proteins and found IQ motif containing GTPase activating protein 1 (IQGAP1) as a novel K-Ras binding protein. IQGAP1 has been known as a scaffold protein for B-Raf, MEK1/2 and ERK1/2. Here we showed that IQGAP1 selectively formed a complex with K-Ras but not with H-Ras, and recruited B-Raf to K-Ras. We found that IQ motif region of IQGAP1 interacted with K-Ras. Both active and inactive K-Ras interacted with IQGAP1, and effector domain mutants of K-Ras also associated with IQGAP1, indicating that IQGAP1 interacts with K-Ras irrespective of Ras-effectors like B-Raf. We also found that overexpression or knock-down of IQGAP1 affected the interaction between K-Ras and B-Raf, and IQGAP1 overexpression increased ERK1/2 phosphorylation in K-Ras dependent manner in PANC1 cells. Our data suggest that IQGAP1 has a novel mechanism to modulate K-Ras pathway.

IDH1 and IDH2 have critical roles in 2-hydroxyglutarate production in D-2-hydroxyglutarate dehydrogenase depleted cells.

D-2-hydroxyglutaric aciduria (D-2HGA) is a hereditary metabolic disorder characterized by the elevated levels of D-2-hydroxyglutaric acid (D-2HG) in urine, plasma and cerebrospinal fluid. About half of the patients have autosomal recessive mutations in D-2-hydroxyglutarate dehydrogenase (D2HGDH) gene. To analyze the origin of D-2HG in D2HGDH-depleted cells, we used small interfering RNA (siRNA) techniques. We found that knockdown of D2HGDH in MCF7 cells increased the levels of 2HG, mimicking D2HGDH mutant cells. Additional knockdown of isocitrate dehydrogenase 1 (IDH1) or isocitrate dehydrogenase 2 (IDH2) decreased the level of 2HG in D2HGDH knockdown MCF7 cells. Conversely, ectopic expression of IDH1 or IDH2 increased 2HG in MCF7 cells. These results suggest that IDH1 and IDH2 have roles in production of D-2HG in cells.