Abstract B021: LIG1 deficiency increases sensitivity to NAMPT inhibitors in pediatric extracranial sarcomas

Abstract

Pediatric extracranial sarcomas represent a heterogeneous group of malignancies with limited therapeutic options and poor outcomes for relapsed or refractory disease. Nicotinamide phosphoribosyl-transferase (NAMPT) is the rate-limiting enzyme in the NAD+ salvage biosynthesis pathway, and its inhibition has emerged as a promising therapeutic strategy in cancers due to increased metabolic demands and, subsequently, high turnover rate of NAD+ levels. However, the success of NAMPT inhibitors (NAMPTi) in clinical trials with adult patients has been limited due to dose-limiting toxicities and the lack of an appropriate biomarker. Our recent study has shown that depleting NAD+ by inhibiting NAMPT resulted in significant anti-tumor activity in pediatric rhabdomyosarcoma, but more work is needed to improve responses to NAMPTi. NAD+ is an essential cofactor for PARP enzymes, which are central mediators of the DNA damage repair and response (DDR). Therefore, we hypothesize that the DNA repair landscape modulates the level of PARP activity and, consequently, the rate of NAD+ consumption, thereby creating distinct metabolic vulnerability states under NAMPT inhibition. To test this, we utilized a targeted CRISPR screening library encompassing 355 DDR genes and screened for dependencies associated with FK866, a 1st generation NAMPTi. Interestingly, loss of DNA Ligase 1 (LIG1), an enzyme responsible for sealing Okazaki fragments during DNA replication and ligating single-strand break (SSB) repair intermediates, emerged as a sensitizer to NAMPTi. Orthogonally validating this finding, we performed LIG1 knockdown (KD) in-vitro and treated them with increasing doses of NAMPTi. Impressively, LIG1 KD induced a 6-to-8-fold increase in sensitivity to NAMPTi across pediatric sarcoma cell lines. This was further corroborated in colony formation assays where LIG1 KD resulted in significantly less colony accumulation than non-targeting control at the same concentration of NAMPTi. We propose that LIG1 deficiency leads to the pathological accumulation of unsealed Okazaki fragments and persistent SSBs at the replication fork. These unligated DNA breaks serve as potent PARP activators, driving hyperactivation of PARP and accelerated NAD+ consumption. NAD+ levels were depleted upon LIG1 KD and further reduced upon NAMPTi treatment. This context of enhanced NAD+ consumption by PARP in conjunction with NAMPTi effectively collapses NAD+ levels below the threshold required to maintain viability, resulting in synthetic lethality. Taken together, our data suggests LIG1 deficiency as a novel and actionable biomarker of NAMPTi sensitivity. Furthermore, these findings support the rational development of combination strategies pairing NAMPTi with agents that promote DNA damage for pediatric patients with extracranial sarcomas.