Abstract A025-PR001: The potential of BCL-xL degradation as a strategy to eliminate chemotherapy-resistant neuroblastoma persister cells

Abstract BACKGROUND: We recently showed that tumor-intrinsic and -extrinsic activation of NFκB is a major mediator of high-risk neuroblastoma (HRNB) chemoresistance (Grossmann et al. , Cancer Discovery, ‘24). The anti-apoptotic protein BCL-xL is a downstream effector of the NFκB pathway and is upregulated in HRNB persister cells after chemotherapy. While showing efficacy, BCL-xL small molecule inhibitor development has been limited by on-target off-tumor thrombocytopenia. DT-2216 (DT), a BCLxL PROteolysis TArgeting Chimera (PROTAC), mitigates this toxicity by exploiting the E3 ligase VHL, expressed at low levels in platelets. AIMS: To validate NFκΒ-mediated BCL-xL overexpression as a HRNB chemoresistance mechanism, and exploit this therapeutic vulnerability to eradicate persister cells using DT. METHODS: Isogenic BCL-xLhigh and BCL-xL low cell lines were generated using an overexpression vector and a doxycycline-inducible CRISPRi system. Six human HRNB cell lines were treated with topotecan/cyclophosphamide (topo/cpm) and DT. DC50 and IC50 values were determined via immunoblots and cell viability assays over 5 days. Five chemotherapeutics were combined with DT in 6x6 dose-response matrices. Zero Interaction Potency (ZIP) scores were calculated using R software. Two Patient-Derived Xenograft (PDX) HRNB flank models in CB17-SCID mice were treated with two subsequent cycles of topo/cpm +/- low-dose DT (15mg/kg) for 6 weeks. Three PDXs were treated with vehicle or escalating DT doses (15mg/kg, 22. 5mg/kg) for 2 weeks. Intratumoral BCL-XL degradation was determined by immunoblots. Complete blood counts were measured weekly. RESULTS: BCL-xL overexpression and knock-down significantly (p0. 05) enhanced and reduced cell viability following chemotherapy, respectively. Combination of high-dose (IC50) topo/cpm and 500nM-1μM DT facilitated the eradication of persister cells in vitro. DT demonstrated synergy with all tested chemotherapeutics across multiple cell lines. The ZIP scores were higher (ZIP10) around the chemotherapy IC50 and DT DC50, indicating a clinically-relevant range-specific therapeutic synergy. However, DT monotherapy at 15mg/kg twice weekly showed incomplete degradation of BCL-xL at day 7 (55%, 37-70%) and 14 (79%, 43-112%) and no effect on tumor growth across three PDXs. Combining DT with topo/cpm in two PDXs did not lead to significant enhancement of chemotherapy anti-tumor efficacy. Escalating DT doses to 22. 5 mg/kg led to further BCL-xL depletion at day 7 (44%, 27-61%) and day 14 (49%, 26-73%) across three models. Dose-dependent thrombocytopenia was the only observed side effect, with platelet counts (x10⁵/uL) of 15 10-18, 4 2-10, 2 1-4 in vehicle, 15mg/kg, 22. 5mg/kg arms, respectively. CONCLUSIONS: BCL-xL mediates chemoresistance in HRNB. Combination of DT and chemotherapy is synergistic in vitro. DT showed incomplete BCL-xL degradation in vivo at the dose and schedule tested here, which may be required for anti-tumor efficacy. Ongoing efforts may provide insight into de novo or acquired mechanisms of DT resistance. Citation Format: Matteo Calafatti, Emily Mycek, Dana Al-halawani, Josh Kalna, David Groff, Esther Coronado Mondragón, Patel Khushbu, Mark Gerelus, Jarrett Lindsay, Kyabeth Torres-rodriguez, Gregory Connelly, Smita Matkar, Sajid Khan, Zhengyu Wang, Hong Yu Li, Jaime Font de Mora, Yael P Mossé, Daohong Zhou, Liron Grossmann, John M Maris. The potential of BCL-xL degradation as a strategy to eliminate chemotherapy-resistant neuroblastoma persister cells abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85 (18Suppl₂): Abstract nr A025-PR001.