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The Role of UNG2 Degradation in Antibody Maturation

Award Number: R21AI180183
ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 06/01/2024
PERIOD OF PERFORMANCE END DATE: 04/30/2027

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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.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $0 )
2026	2025	WAYNE STATE UNIVERSITY	5700 CASS AVE STE 4900	DETROIT	MI	48202	WAYNE	USA	Allergy and Infectious Diseases Research	000	2	3/17/2026	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2025 ( Subtotal = $228,200 )
2025	2025	WAYNE STATE UNIVERSITY	5700 CASS AVE STE 4900	DETROIT	MI	48202	WAYNE	USA	Allergy and Infectious Diseases Research	000	2	4/18/2025	NON-COMPETING CONTINUATION	$228,200
														Subtotal = $228,200

Issue Date FY: 2024 ( Subtotal = $215,100 )
2024	2024	WAYNE STATE UNIVERSITY	5700 CASS AVE STE 4900	DETROIT	MI	48202	WAYNE	USA	Allergy and Infectious Diseases Research	000	1	5/29/2024	NEW	$193,590
2024	2024	WAYNE STATE UNIVERSITY	5700 CASS AVE STE 4900	DETROIT	MI	48202	WAYNE	USA	Allergy and Infectious Diseases Research	001	1	9/11/2024	NEW	$21,510
														Subtotal = $215,100

Grand Total All Awards = $443,300

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The Role of UNG2 Degradation in Antibody Maturation

Award Number: R21AI180183

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 06/01/2024

PERIOD OF PERFORMANCE END DATE: 04/30/2027

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