Hironori Matsunaga

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.

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.

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.

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.

Abstract LB-252: IDH mutations are promising targets for acute myeloid leukemia

Mutations in isocitrate dehydrogenase (IDH) 1 and 2 are frequently observed in acute myeloid leukemia (AML), glioma, and many other cancers. While wild-type IDHs convert isocitrate to α-ketoglutarate (α-KG), mutant IDHs convert α-KG to oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-KG-dependent dioxygenases, such as TETs, histone demethylases, EGLNs, and other enzymes. Because the role of mutant IDH is not necessary for normal cells, inhibitors directed against mutant IDH are not expected to have the side effects as those of anti-cancer agents. To determine whether mutant IDH enzymes are valid targets for cancer therapy, we created a mouse model of mutant IDH-dependent AML. Previously, the IDH mutation alone was shown to be insufficient for the induction of AML, and IDH mutations occur simultaneously with mutations in other genes such as NPM, DNMT3A, and FLT3. In accordance with these observations, we found that NPM+/- hematopoietic progenitor cells transduced with IDH2/R140Q, NPMc, DNMT3A/R882H, and FLT3/ITD cooperatively induced AML in a mouse model. However, when only three of these mutant genes were transduced, myeloproliferative neoplasms (MPNs) rather than AML was more frequently induced and their onset was delayed in any combinations of the mutant genes. These results clearly indicate that all four mutations are necessary for the efficient induction of AML. By using a combination of AML model mice with cre-loxp, we conditionally deleted IDH2/R140Q from AML mice, which blocked 2-HG production and resulted in the loss of leukemia stem cells. Accordingly, the progression of AML was significantly delayed. Because IDH mutations and TET2 mutations are mutually exclusive in AML, the inhibition of TET-mediated conversion of 5mC to 5hmC is considered one of the main roles of mutant IDH. We found that IDH2/R140Q decreased the level of 5hmC and the expression of differentiation-inducing genes, including Ebf1, Spib and Pax5. Gene expression analysis revealed that IDH2/R140Q activated the hypoxia pathway and the expression of Meis1. These results indicate that the function of IDH2 mutation is critical for the development and maintenance of AML stem cells, and that mutant IDHs are promising targets for anticancer therapy. Based on these findings, we developed potent and specific inhibitors of mutant IDH1 and tested their effects in the mutant IDH1-dependent AML mouse model, created by introducing four mutant genes including mutant IDH1. The 2HG level was promptly and dramatically decreased in AML cells soon after treatment with the mutant IDH1 inhibitors, and the number of leukemia cells was reduced after a 4-week treatment. These results indicate that IDH1 mutant inhibitors are effective for the treatment for AML. Citation Format: Yoko Ogawara, Hironori Matsunaga, Takahiko Seki, Yukino Machida, Kazushi Araki, Issay Kitabayashi. IDH mutations are promising targets for acute myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-252. doi:10.1158/1538-7445.AM2015-LB-252