Thursday, January 06, 2011

Oncogenesis Via Altered Enzyme Specificity, Part II

As promised in the EZH2 story, there is another story of cancer-causing mutations tuning an enzyme in an interesting way. It's also a great story of how multiple high-throughput methods can create and exploit an entirely new angle on cancer. I'll try to do a good job on this, but I'm lucky enough to have as regular readers of this space several of the authors who are referenced here, which should enable any egregious errors on my part to be flagged. I'm also trying to tell the main thread of the story as I can see it, and apologize in advance for getting priorities of discovery incorrect. I'm relying on final publication dates for organizing the timeline, which is certainly not a perfect strategy.

First, we have to go back just over two years ago to the late summer and early fall of 2008. Two different groups reported on initial cancer genomics investigations of glioblastoma, a devasting type of brain tumor. Both groups used PCR to amplify targets for Sanger sequencing. One group looked at a focused set of genes in 91 tumor samples; the other group looked at many fewer samples (22) but at most known protein-coding exons.

Now this is the sort of decision which was critical: given a particular sequencing budget, do you sequence a lot of targets in a few patients or a select set in more patients? Given we know a lot of oncogenes and tumor suppressors, there is a logic to the focused search. But this was a case where the broad sweep paid off.

What the broad sweep found, but was not included in the focused search, were mutations in the gene for IDH1, a key enzyme in the citric acid cycle. A rapid follow-up study confirmed the recurrent presence of IDH1 mutations in glioblastoma and also mutations in IDH2, another gene encoding a homologous enzyme.

IDH1 and IDH2 encode isocitrate dehydrogenase, a key enzyme in the citric acid cycle, which is also known as the Krebs cycle or tricarboxylic acid cycle (TCA). This set of metabolic reactions is often shown near the center of a large metabolic diagram as a big circle, which is very appropriate. This set of reactions is central to aerobic energy generation as well as the creation of various useful metabolic intermediates. The normal activity of IDH is

Isocitrate + NADP+ <=> 2-Oxoglutarate + CO2 + NADPH + H+

As with all reactions of the TCA, this reaction is reversible; under some conditions some cells will convert 2-oxoglutarate to isocitrate. Also keep in mind that a common synonym for 2-oxoglurate is alpha-ketoglutarate or aKG.

Now the influence of primary metabolism on cancer is a hot topic -- again. Back in the 1920s Otto Warburg earned a Nobel prize for the observation that tumors seem to rely on anerobic glycolysis more than their normal cousins. The field was pretty cold for a long while but lately it has gotten new interest, including a number of startup companies trying to develop cancer therapeutics. During my last job interruption, I consulted for one of these (Agios), though I have no ongoing financial interest in the company.

A striking observation is the pattern of the mutations: in each case a single arginine residue is mutated, though to multiple possible amino acids. However, these are not equiprobable. In the current COSMIC, there are 1859 reported IDH1 mutations -- and 1468 (78.97%) of those are R132H. Only a handful of mutations have been found outside R132. IDH2 has two hotspot sites, R140 and R172

Now, what are these mutations doing? What is special about IDH? The first attempt to answer this was published in April 2009 and came to the conclusion that the mutant enzyme is less effective at binding its substrate and generating the product alpha ketoglutarate. Furthermore, the mutant enzyme was proposed to poison the wild type copy by the formation of inactive heterodimers. Finally, this was proposed to activate the important HIF1 transcription factor, which regulates a number of tumor-promoting pathways. So in this view of the world, IDH1/2 are tumor suppressors inactivated in glioblastoma.

The part that was unsatisfying about this explanation is that it failed to explain why IDH1/2 mutations are so focused. In general, many mutations can destroy enzymatic function, so tumor suppressor enzymes generally show a diffuse mutation pattern. It is dangerous to think we can think through such biochemical puzzles, but it did mean the solution to the puzzle wasn't a clear winner.

A very different explanation was provided by the group from Agios, published at the end of 2009. Using high-throughput metabolite profiling, their startling discovery is that the IDH1 mutations result in higher levels of 2-hydroxyglutarate (2HG), a compound structurally-related to the normal IDH product alpha-ketoglutarate.They confirmed that the mutant enzyme is no longer capable of driving the normal reaction, but that it now catalyzes an analogue of the reverse reaction which uses the aKG and NADPH generated by the wild-type enzyme to generate 2HG. Heterodimers appeared to be capable of both reactions, raising the possibility that heterodimers enable very efficient production of 2HG through the coupling of the two enzymatic activities. Structural studies supported this explanation, and finally increased 2HG levels could be detected in glioblastoma samples mutant for IDH1, but not those wild-type for IDH1.

Around the same time, another key thread entered the story. Several attempts to identify IDH mutations in other cancers had been made, and while a few had been found there wasn't an obvious cancer with a high frequency of mutations. But, the second acute myelogenous leukemia complete genome sequenced by the Wash U group identified an IDH1 mutation and went on to confirm recurrence of IDH1 mutations in just under 10% of AML samples assayed. Now a second tumor type showed IDH recurrence. Further studies identified IDH2 mutations as well in this disease and confirmed that IDH-mutant leukemias accumulate 2HG.

So now we have an odd mutation pulling and interesting trick of changing the reaction specificity of a metabolic enzyme and showing up repeatedly in two very different cancers. But why is this odd metabolite valuable to the cancer? That is where the latest paper comes in. Published last month, it demonstrated a number of features. First, leukemias mutant for IDH1 or IDH2 show a distinctive DNA methylation profile, one which is not specific for which enzyme is mutated. This methylation profile also shows a greater degree of methylation than most other AML samples. Second, the RNA expression profiles for these tumors is not quite as highly clustered. Third, expression of mutant IDH enzymes in cell lines raises the amount of 5-methylcytosine in their DNA.

The big clue uncovered is that IDH1/2 mutations are not only mutually exclusive, they are also strictly exclusive with another recurrent mutation in AML, those inactivating the enzyme TET2. More strikingly, TET2's enzymatic role appears to be the first step in demethylating DNA -- and TET2 requires alpha ketoglutarate! Indeed, co-expression of TET2 and IDH1 mutant (R132H) reduced the degree of formation of the TET2 product (5-hydroxy-methylC) vs. TET2 + wild-type IDH1. Furthermore, TET2-mutant leukemias actually show a similar methylation profile as IDH1/2-mutant leukemias.

How does this drive leukemogenesis? Looking at the differentially-methylated sites in IDH1/2 mutant AMLs versus other AMLs, an enrichment for motifs associated with the transcription factors GATA1/2 and EVI1, both known to be important in myeloid differentiation. 40% of the genes in the IDH1/2 signature are known targets of GATA2 and 19% direct targets of GATA1. Furthermore, GATA1 was hypermethylated in their patient cohort, suggesting two levels of suppression of this pathway. Finally, mutant IDH expression or loss of TET2 function was shown to generate more cells with stem-like characteristics, a hallmark of leukemias. In particular the oncogenic kinase c-KIT showed higher expression; mutational activation of c-KIT characterizes yet another subset of AML.

So in just over two years, we've gone from high-throughput sequencing finding a curious recurrent mutation, to a novel oncogenic modification of metabolism and now a mechanistic explanation of how this drives leukemias. I've left out a lot of other literature using these mutations to guide better prognosis in cancers and the identification of recurrence of IDH mutations in some other tumor types, notably thyroid tumors. Curiously, another set of thyroid tumors appear to be wild-type for IDH1/2 (at least in the hotspot) but have elevated levels of 2HG. Germline IDH2 mutations have also been identified in a subset of patients with abnormal levels of 2HG. Some patients have inactivating mutations in a different gene, succinic semialdehyde dehydrogenase; will this show up as mutant in yet another set of cancers?

So what next? Ideally the clinical value of these findings would go beyond simply staging patients. There are hints that some chemotherapies may perform better or worse in the context of these mutations. Ideally, therapies directed at inhibiting the mutant IDH activity (whilst sparing the wild-type activity) will be developed. The higher expression of c-KIT in IDH1/2 and TET2 mutant AMLs may suggest the use of c-KIT inhibitors. Certainly one suggestion is to look in other IDH1/2 mutant tumors and in 2HG-elevated IDH1/2 wild-type tumors for distinctive hypermethylation. With larger and larger mutational datasets, more mutations may be found which are clearly mutually exclusive with IDH mutations (exclusion with NPM has also been observed in leukemia); such findings could lead to identifying further genes affecting genome methylation.
Figueroa ME, Abdel-Wahab O, Lu C, Ward PS, Patel J, Shih A, Li Y, Bhagwat N, Vasanthakumar A, Fernandez HF, Tallman MS, Sun Z, Wolniak K, Peeters JK, Liu W, Choe SE, Fantin VR, Paietta E, Löwenberg B, Licht JD, Godley LA, Delwel R, Valk PJ, Thompson CB, Levine RL, & Melnick A (2010). Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation. Cancer cell, 18 (6), 553-67 PMID: 21130701

1 comment:

Sai said...

Great. Very fascinating story for Biochemists