MOMA-313, an inhibitor of the DNA polymerase theta helicase domain, prevents BRCA reversions in preclinical models

MOMA-313, an inhibitor of the DNA polymerase theta helicase domain, prevents BRCA reversions in preclinical models Giulia Bottoni, Anthony Tubbs, Kelly McGlynn, Vinny Motwani, Michael Reutershan, Timothy Guzi, Erica Evans, Peter Hammerman and Jordan Krall MOMA Therapeutics, Cambridge, Massachusetts, USA 249 Single-strand breaks are a primary source of Polθ-mediated DNA repair in HR-deficient cells 2026 AACR Annual Meeting DNA single-strand breaks are a potential source of clinical BRCA reversions MOMA-313 is a small molecule inhibitor of DNA polymerase theta (Polθ) in development with a PARP inhibitor to drive deeper and more durable responses in homologous recombination (HR)-deficient tumors.1 Polθ repairs DNA double-strand breaks (DSBs) by annealing flanking regions of DNA microhomology, a process called theta-mediated end-joining (TMEJ), and the inhibition of Polθ synergizes with PARP inhibition in HR-deficient tumors models.1,2 Clinical resistance to PARP inhibitors is predominantly caused by BRCA reversions – secondary mutations that restore BRCA function – that often display the hallmarks of TMEJ.3,4 It has therefore been hypothesized that inhibiting Polθ may extend the duration of PARP inhibitor responses by preventing or delaying the emergence of BRCA reversions. However, due to a limited mechanistic understanding of BRCA reversion mutations, this hypothesis has not been experimentally addressed. Here, we use genetic tools to investigate the likely mechanism by which BRCA reversions are generated. Specifically, we ask whether single-strand breaks (SSBs) – the most common form of endogenous DNA damage – initiate mutagenic DNA repair in HR-deficient settings, and whether Polθ-driven TMEJ is a prominent repair mechanism that can generate BRCA reversions following these DNA lesions. BACKGROUND Acute 2-ended break: Uncommon End-resected DNA: repaired by Polθ/TMEJ DSB (1-ended): repaired by HR proteinsDNA single-strand break: Frequent Fork progression: stalled by HR proteins Blunt DNA: repaired by NHEJ –or– • Acute 2-ended double-strand breaks (DSBs) are uncommon under normal cellular conditions • Can be repaired by multiple DNA repair pathways depending on end resection and cell cycle stage • Single-strand breaks (SSBs) occur frequently in most cells • Persistent SSBs can be converted to DSBs during DNA replication • HR prevents and/or repairs replication-associated DSBs • Polθ/TMEJ performs repair in the absence of functional HR Replication machinery NHEJ TMEJ Homologous recombination DNA double-strand breaks can arise by distinct mechanisms Repair of 2-ended DSBs can be quantitatively monitored using amplicon sequencing WT KO 0 10 20 30 40 50 Deletion Size (bp) WT KO 0 2 4 6 8Microhomology (bp) WT KO 0 2 4 6 8Microhomology (bp) WT KO 0 10 20 30 40 50 Deletion Size (bp) WT KO 0 50 100 14.1% 0.3% Mutagenic DNA repair [% of total amplicons] BRCA2: Mutagenic repair of SSBs (Cas9D10A) is observed only in HR-deficient cells and contains hallmarks of TMEJ Mutations (% of amplicons) Deletion Size (bp) Flanking Microhomology 6.43 32 AGGC 1.99 17 CATCC 1.11 37 AGAAGG 1.01 16 GGGAG 0.96 32 G 0.89 20 AAGG 0.66 28 GG 0.58 9 GGGA 0.43 23 CGG 0 10 20 0.3% 11.0% Mutagenic DNA repair [% of total amplicons] 0 50 100 150 Deletion Size (bp) 0 2 4 6 8 10 Microhomology (bp) MOMA-313 prevents the mutagenic repair of targeted SSBs in HR-deficient Capan-1 cells • Mutagenic repair of Cas9D10A-derived SSBs is observed in HR-deficient BRCA2-KO cells • Data suggest that in the absence of HR, SSBs are converted to DSBs during DNA replication • Large deletion and microhomology size of DNA repair products suggests a dominant role for Polθ/TMEJ in the repair of these DNA lesions when generated in an HR-deficient setting • DNA repair products measured following the introduction of a targeted 2-ended DSB using wild-type Cas9 • The size of DNA deletions and flanking DNA microhomology (MH) provide insight into contributions of distinct DNA repair processes DNA single-strand breaks generate BRCA2 reversions that are Polθ-dependent and can be inhibited by MOMA-313 0.001 0.01 0.1 1 10 100 0 50 100 [olaparib], µM Cell Viability [% of DMSO] sgAAVS1 (control) sgBRCA2 #17 (reversion) DNA SSBs initiate secondary mutations in BRCA2 and restore the wild-type reading frame HYPOTHESIS: Single-strand breaks proximal to pathogenic BRCA mutations initiate BRCA reversions • SSBs → replication-associated 1-ended DSBs→ Polθ-mediated repair (TMEJ) • TMEJ restores BRCA reading frame → MOMA-313 inhibits BRCA reversions to prevent PARPi resistance 1 4 14 16 17 0 10 20 30 40 50 0.0% 0.0% 0.0% 0.3% 6.2% BRCA2 reversions [% of total reads] sgRNA #: 1 4 14 16 17 0 10 20 30 40 50 7.3% 9.4% 5.4% 6.1% 18.0% Mutagenic DNA repair [% of total amplicons] sgRNA #: 1 4 14 16 17 0 10 20 30 40 50 0.0% 0.3% 0.0% 24.4% 29.6% BRCA2 reversions [% of total reads] sgRNA #: BRCA2 WT (restored) BRCA2 mutant Vinculin Capan-1 parental + sg #17 + olaparib (35 d) Approach: Generate targeted SSBs near the pathogenic BRCA2 mutation in Capan-1 cells to assess the frequency of BRCA reversions and role of Polθ/TMEJ in their generation 1 4 14 16 17 0 10 20 30 40 50 60 70 80 2.3% 9.2% 0.7% 44.0% 52.7% BRCA2 reversions [% of total reads] sgRNA #: Total Mutations (Day 7) BRCA2 Reversions (Day 7) BRCA2 Reversions (Day 35) - olaparib selection - BRCA2 Reversions (Day 35) - No selective pressure - BRCA2 reversions from SSBs are functional and provide olaparib resistance SSBs are a likely initiating event in the generation of BRCA reversion mutations • SSBs are a common source of DNA damage that initiates mutagenic repair in HR-deficient settings • SSBs proximal to a pathogenic BRCA2 mutation generate functional BRCA reversions causing PARP inhibitor resistance MOMA-313 prevents BRCA reversions initiated by single- strand DNA breaks in HR-deficient cells • MOMA-313 broadly and efficiently prevents mutagenic repair initiated by SSBs in HR-deficient cells • Data collectively indicate that Polθ-mediated repair following SSBs is a likely source of clinical BRCA reversions • MOMA-313 in combination with a PARP inhibitor may prevent resistance and extend the duration of clinical responses WT KO 0 50 100 98.2% 98.6% Mutagenic DNA repair [% of total amplicons] BRCA2: MOMA-313 broadly prevents repair of SSBs and inhibits the formation of BRCA2 reversions TMEJ is the dominant pathway driving mutagenic repair of DNA lesions initiated by SSBs in HR-deficient cells Studies suggest a mechanism for the generation of BRCA reversions and a pharmacologic approach to prevention DMSO MOMA-313 DMSO MOMA-313 DMSO MOMA-313 Reversions enriched to 50% allelic fraction sg #17 - + - + - + - + - + 0 10 20 30 40 Mutagenic DNA repair [% of total amplicons] 4 bp 5 bp 2 bp 3 bp 0 bp 1 bp MOMA-313 (250 nM) Microhomology sg #1 sg #4 sg #14 sg #16 sg #17 -89% -95% -100%-89% -74% 0 5 10 BRCA2 reversions [% of total amplicons] -74% DMSO MOMA -313 All DNA repair products BRCA Reversions (sg #17) MH > 2 bp Likely TMEJ MH < 2 bp Likely NHEJ The majority of SSB-initiated products are deletions with flanking DNA microhomology MOMA-313 efficiently inhibits nearly all repair products including those causing BRCA reversions 1 Krall, J. et al., Cancer Res. 85 (8_Supplement_1): 1749 (2025). 2 Ramsden, D. A. et al., Nat. Rev. Mol. Cell Biol. 23, 125–140 (2022). 3 Pettitt, S. J. et al., Cancer Discov. 10, 1475–1488 (2020). 4 Tobalina, L. et al., Ann. Oncol. 32, 103-112 (2021). Most deletions contain flanking DNA microhomology, suggesting Polθ-mediated repair Figure adapted from Ref 3. BRCA reversions are commonly caused by secondary, frame-restoring deletions Olaparib sensitivity – cell viability 1. DNA single-strand breaks create a specific reliance on Polθ in HR- deficient cells and are a likely source of mutagenic DNA repair 2. Polθ-mediated repair following single-strand DNA lesions proximal to pathogenic BRCA2 mutations generates functional BRCA reversions 3. The Polθ inhibitor MOMA-313 inhibits mutagenic DNA repair initiated by SSBs and prevents BRCA reversions in a preclinical model 4. Preclinical data support the hypothesis that MOMA-313 will inhibit clinical BRCA reversions to extend the duration of PARP inhibitor response CONCLUSIONS HYPOTHESIS: DNA single-strand breaks are common and create a specific reliance on Polθ-mediated repair in HR-deficient cells In the absence of HR, persistent SSBs generate replication-associated DSBs that are predominantly repaired by Polθ Experimental Approach: 1. Use Cas9WT and Cas9D10A (nickase) to generate targeted DSBs (2-ended) and SSBs, respectively 2. Introduce SSBs and DSBs into a BRCA2-isogenic cell line pair derived from DLD1 cells 3. Monitor DNA repair products using targeted amplicon sequencing to identify context-dependent repair products

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