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

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 (p<0.05) 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 (ZIP>10) 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 (x10^5/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(18_Suppl_2):Abstract nr A025-PR001.