Danial-1C-Project Summary & Goals
One Carbon Metabolism in Molecular Subsets of DLBCL
Brief background on one carbon metabolism as it relates to this project

1-carbon pool refers to 1-carbon residues available for metabolic reactions through S-adenosyl methionine
(SAM) and folate [mainly through active forms of folate such as tetrahydrofolate (THF)]. SAM and THF
compounds are key for reactions that involve the transfer of methyl groups (DNA/histone methylation,
protein modification, nucleotide synthesis, etc). SAM is also important for membrane phospholipids and
creatine synthesis (Fig. 1).

This pathway is linked to glucose, serine, and glycine metabolism. For example, serine conversion to glycine
through SHMT (serine hydroxyl-methyl transferase), transfers a methyl group to THF to generate MTHF.
MTHF is an important one methyl carrier for a variety of reaction (Fig. 2). SHMT activity is also the primary
source of intracellular glycine. Serine required for this reaction can be either taken up from the media or it
can be synthesized de novo from glucose (Fig. 2).

Several studies in the past 3 years have indicated important roles for these pathways in various tumors (see
PMID:24451681, PMID:22628656. PMID:24813884, PMID:23242140, PMID:21760589, PMID: 22509023).

One carbon metabolism is known to be compartmentalized between mitochondria and the cytosol though
specific enzymes that are restricted to these compartments, and dedicated transporters that may separate
mitochondrial and cytosolic pools of one carbon intermediates. However, to our knowledge, there is no
context in which a functional distinction between the mitochondrial compartment and the cytosolic
compartment has been observed in cancer cells.
Fig. 1 (From the 2013 review
by Locasale): Nutrient sources
involving amino acids are
either imported (green) or
synthesized de novo and enter
one-carbon metabolism. Onecarbon metabolism can be
viewed as two pathways,
comprising the folate cycle and
the methionine cycle. Nutrients
are processed through these
metabolic cycles. Multiple
outputs can be generated
(orange
boxes),
including
nucleotides, proteins, lipids,
reducing power and substrates
for methylation reactions.
Why are we interested in mitochondrial one carbon metabolism?

Several mitochondrial enzymes for one carbon metabolism are quantitatively enriched in OxPhos-DLBCLs by
our most recent iTRAQ analysis, including SHMT2, MTHFDL1, and MTHFD2 (Fig. 2). SHMT activity is
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Danial-1C-Project Summary & Goals
compartmentalized through activities of a cytosolic (SHMT1) and mitochondrial isoform (SHMT2). SHMT1
and 2 have high sequence homology and are encoded by 2 separate genes.

The following data indicate that the protein-level enrichment of these mitochondrial enzymes is functionally
relevant in OxPhos-DLBCLs (also see the ppt file attached with this summary):
1. Knocking down SHMT2 is selectively toxic to OxPhos-DLBCLs while SHMT1 knockdown does not
change their viability. Comparing the outcome of SHMT2 vs SHMT1 knockdown in this case
helps us ask which compartment (mitohondria vs cytosol) is predominantly contributing to
survival. The viability of non-OxPhos DLBCLs is not affected by SHMT1/2 depletion.
2. Particular reliance on glycine (the byproduct of the SHMT reaction) can also be deduced
independently by amino acid deprivation studies that showed OxPhos-DLBCL survival is highly
sensitive to glycine deprivation.
3. Cell death in response to SHMT2 knockdown can be rescued by multiple different downstream
metabolites of this reaction (glycine, methionine, SAM).
4. In surveying which biosynthetic outputs of the one carbon metabolism pathway (Orange blocks
in Fig. 1) are specifically dependent on SHMT2, we have learned that DNA methylation
(measured by methyl cytosine levels) and histone modification (measured by combinatorial
histone modifications using the Broad platform) are specifically affected by SHMT2 knockdown.
Importantly, some of these changes in DNA and histone modifications in SHMT2 knockdowns
can be rescued with glycine.
What are the questions we want to answer?
The data described in 1-4 above suggest that the contribution of SHMT2 to the methyl/one carbon pool is of
particular significance in OxPhos –DLBCLs compared to BCRs. To understand what can account for this, we want
to know:
1. If there are specific metabolite signatures associated with SHMT2 activity compared to SHMT1
in DLBCL subtypes that can give us clues (metabolomics excel file)
2. If the pattern of changes we see with combinatorial histone modifications (histone marks excel
file) we see with SHMT2 knockdown is associated with specific gene expression programs
(microarray data file).
The big picture
The answer to the above questions may help explain why OxPhos-DLBCLs are selectively dependent on SHMT2
for survival. In addition, this can teach us about a potential mitochondrial retrograde signal that initiates from
mitochondrial one carbon pathway and affects selective nuclear events through histone modification. This idea
is has enormous discovery potential for 2 reasons (big picture significance):
1. Mitochondrial retrograde signaling is a very hot topic in metabolism and there are very few
examples of mitochondrial retrograde signals regulating nuclear events.
2. Of the recent tumor metabolism papers that highlighted the importance of serine/glycine or one
carbon metabolism in different tumor types, none so far showed a connection with epigenetics.
If we are successful, this could be a first example.
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Danial-1C-Project Summary & Goals
What is in the files I sent you and what kind of comparisons would be initially helpful?
Microarray-The sample ID/key for microarray data is provided in a separate excel file named
“Danial_sampleIDs_microarray, and the actual array data are in a zip file.
Experimental set up: We knockdown SHMT2 in 2 OxPhos (Toledo and Ly4) and 1 non-OxPhos (Ly3). We were
somewhat limited by the number of lines we could analyzed for both cost reasons and the fact that few are
know to have EZH mutations, which we wanted to avoid. We used the Human 2.0- ST chip. As explained in the
key, the cell line notations are T, L4 and L3, and for each cell lines we have the following conditions:
Control knockdown x 3 independent repeat marked as C1, C 2, C3 (for example TC1 is Toledo control 1 etc)
SHMT2 knockdown x3 independent repeat marked as SH1, SH2, SH3 (for example L3SH3 is Ly3 SHMT2
knockdown 3 etc)
Suggested analysis:
My suggestion is to analyze this data set first. We want to know:
1. What genes are differentially expressed when SHMT2 is knocked down
2. Which SHMT2-dependent changes are specific for OxPhos vs non-OxPhos samples
Metabolomics-In this excel file, the tabs of interest are norm TS (normalized data) and Key, which explains the
way the samples were labeled.
Experimental set up: We knocked down SHMT1 and SHMT2 separately in 3 OxPhos (Toledo, Pfeiffer and Ly4)
and 3 non-OxPhos (Ly3, U2932, D4). As explained in the key tab, the cell line notations are T, P, L4, L3, U, D4)
and for each cell line, we have measured 155 metabolites on Clary Clish’s platform in the following conditions:
Control knockdown x 3 independent repeat marked as Cntrl 1, Cntrl 2, Cntrl 3
SHMT2 knockdown x3 independent repeat marked as SH2-1, SH2-2, SH2-3
SHMT1 knockdown x3 independent repeat marked as SH1-1, SH1-2, SH1-3
Suggested analysis:
I suggest doing 1 and 2 below first, then 3:
1. Control OxPhos vs control non-OxPhos-this will reveal basal metabolite differences in the two
subsets
2. Control vs SHMT2 in each subtype-this will help focus on metabolite changes that are specific to
SHMT2 knock down. We predict some of these will also be selective for OxPhos vs non-OxPhos
cells.
3. Comparison of SHMT2 vs SHMT1 in each subtype
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Danial-1C-Project Summary & Goals
Fig. 2 Key pathways of one-carbon metabolism (From Stanley & Ribeiro, TCB review 2014): Folatederived THF can be metabolized in the cytosol or in mitochondria by the indicated enzymes. THF is
converted to mTHF (methylene-tetrahydrofolate) in a cycle that is coupled to the conversion of serine to
glycine. Serine, a major one-carbon donor for the synthesis of mTHF, can be imported from the
extracellular milieu, synthesized from glycine, or from glucose-derived carbons. Glycine can be generated
from serine or from threonine. In addition to nucleotide biosynthesis, One-carbon metabolism involving
mTHF supports nucleotide biosynthesis, as well as methionine and SAM production.
Abbreviations: SHMT1 – Serine hydroxymethyltransferase 1, SHMT2 – Serine hydroxymethyltransferase 2,
MTHFDL1 – 10-formyl-tetrahydrofolate synthetase , MTHFD1 – Methylenetetrahydrofolate
dehydrogenase 1, MTHFD2 – Methylenetetrahydrofolate dehydrogenase 2, PSP – Phosphoserine
phosphatase, TDH – Threonine dehydrogenase, PSAT1 – Phosphoserine aminotransferase, PHGDH – 3Phosphoglycerate dehydrogenase, mTHF – Methylene-tetrahydrofolate, THF – Tetrahydrofolate, SAM – SAdenosyl-methionine, G6PDH – Glucose-6-phosphate dehydrogenase, PRPP – Phosphoribosyl
pyrophosphate.
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