The Role of UNG2 Degradation in Antibody Maturation

Somatic hypermutation (SHM) and class-switch recombination (CSR), are two molecular
processes that are central to antibody maturation in mammals. A murine protein, mFAM72A, is
expressed in germinal center B lymphocytes and plays a key role in both these processes. It
interacts with the nuclear form of the DNA repair protein, mUNG2, and is required for optimal
SHM and CSR. It causes inhibition of the enzymatic activity of the latter protein and promotes its
degradation through a proteosome-dependent pathway. The enzyme activation-induced
deaminase (AID) is essential for both SHM and CSR, and converts cytosines in DNA to uracil
creating U•G mispairs. The uracils are processed by two distinct DNA repair pathways. The
uracils may be excised by mUNG2 creating abasic sites that are processed by the base-
excision repair machinery or the U•G mispairs may be recognized by a non-canonical mismatch
repair (ncMMR) process that is not linked to replication. Together these repair pathways cause
base substitution mutations and strand breaks that promote SHM and CSR. ncMMR works only
when U•G pairs created by AID persist in the immunoglobulin genes and recent studies strongly
suggest that mFAM72A helps with the persistence of U•G pairs through interference with
mUNG2 stability and activity. However, it is unclear why the degradation of mUNG2 is
necessary when the binding of mFAM72A to mUNG2 in itself causes substantial inhibition of
enzymatic activity of mUNG2. It is possible that the degradation of mUNG2 is necessary to
reduce the nuclear concentration of this protein changing its association with other proteins that
are inhibitory towards SHM and CSR. To test whether the inhibition of UNG activity is sufficient
for optimal SHM and CSR, we will synthesize a family of known chemical inhibitors of
mammalian UNG and test them in the murine cell culture model for CSR, CH12F3 cells. These
inhibitors bind within the active site of the enzyme, have micromolar to submicromolar IC50 and
are able to inhibit the enzyme inside cells. We will also synthesize proteolysis-targeting chimera
(PROTAC) versions of these inhibitors that should cause degradation of mUNG2. We will treat
mFAM72A knockout (KO) CH12F3 cells with different concentrations of these inhibitors or
PROTACs and determine the frequency of isotype switching from IgM to IgA in these cells. If
inhibition of enzymatic activity of mUNG2 is sufficient for optimal CSR then CSR frequency
should initially increase with increasing concentration of inhibitor reaching a maximum similar to
that found in FAM72A+/+ cells. However, if degradation of mUNG2 is essential for optimal CSR,
the PROTAC treatment- but not inhibitor treatment- should achieve maximal CSR. Following
inhibitor and PROTAC treatment, we will also monitor mutations in the 5'Sµ region of CH12F3
genome as a proxy for SHM. Additionally, we will use a newly constructed RASH-1 cell line as a
model for SHM. This RAMOS-derived human cell line contains an inducible AID gene and SHM
in its genome can be conveniently monitored as loss of GFP fluorescence. mFAM72A promotes
higher frequency of hypermutations in the variable region of the IGH gene in the B lymphocyte
genome and hence we expect that the drug treatments will increase the SHM frequency. If the
maximum SHM frequency is achieved using inhibitors alone, we will conclude that inhibition of
UNG2 activity is sufficient for optimal SHM.

Grant Number: 5R21AI180183-02
NIH Institute/Center: NIH
Principal Investigator: ASHOK BHAGWAT