Abstract B052: IKAROS mediates antigen escape following CD19- or CD22-targeted immunotherapies in r/r B-ALL
Authors: Domizi Pablo , Sarno Jolanda , Jager Astraea , Merchant Milton , Pacheco Kaithlen , Yamada-Hunter Sean , Rotiroti Maria Caterina , Liu Yuxuan , Baskar Reema , Reynolds Warren , Sworder Brian , Sahaf Bita , Bendall Sean , Mullighan Charles , Alizadeh Ash , Leahy Allison , Myers Regina , Grupp Stephan , Majzner Robbie , Barrett David , Sotillo Elena , Davis Kara 
Published: September 2024

Abstract
CD19 CAR T cell (CART19) therapy has shown impressive results in relapsed or refractory B cell acute lymphoblastic leukemia (r/r B-ALL). However, 30 – 70% of initial responders will eventually relapse with CD19 loss. Early identification of patients that will respond successfully to CART19 therapy vs. those at risk for CD19neg relapse would support clinical decision making and improve patient care. To address this question, we profiled by CyTOF 25 pre-CART19 samples from patients that achieved an ongoing complete remission (CR, N. = 6), underwent CD19neg (n = 11) or CD19pos relapse (n = 4) or were refractory to CART19 (n = 4). We also analyzed paired post-CART19 samples from CD19neg relapse (n = 8), CD19pos relapse (n = 2), and refractory (n = 4) tumors. In pre-CART19 samples, we observed that low levels of transcription factor IKAROS in a subset of CD19pos pro-B like tumor cells correlate with a higher risk for CD19neg relapse. Through single cell CITE-seq of pre-CART19 samples from CR (n = 3) or CD19neg relapsed (n = 3) tumors, we confirmed that patients that underwent CD19neg relapse bear a subset of pro-B like tumor cells with lower expression of IKZF1, IKAROS gene. Although IKZF1 alterations have been associated with poor response to front-line treatment, IKAROS’ involvement in CART19 failure has not been described. To elucidate how IKAROS modulates CD19 surface expression, we performed CyTOF, RNA-seq and ATAC-seq analysis of isogenic IKAROSWT or IKAROSKD B-ALL cell lines. We found that IKAROS regulates several genes associated with mRNA splicing and that IKAROSKD B-ALL cells resemble multipotent progenitor cells, suggesting a loss in their B cell identity. Moreover, we observed that CD19 mRNA preferentially retains intron 10 in IKAROSKD cells. Interestingly, intron 10 was also more retained in CD19neg relapsed leukemia and lymphoma patients after blinatumomab or CART19 treatments. In addition, we observed that IKAROS also modulates CD22 levels. Performing co-cultured experiments, we confirmed that IKAROSlow B-ALL cells express lower CD19 and CD22 levels, thus conferring resistance against CD19-targeted (blinatumomab, 19.BBz or 19.28z CAR T cells), CD22-targeted (22.BBz CAR T cells) and dual CD19/ CD22-targeted (19-22.BBz CAR T cells) immunotherapies. This work demonstrates a novel role for IKAROS in CD19 and CD22 modulation and CD19 and/or CD22 targeted immunotherapies, identifying it as a potential prognostic target. In addition, given the role of IKAROS in lineage specification and chemotherapy resistance, IKAROS mediated splicing regulation could have broader beyond CD19- or CD22-directed immunotherapies.
Citation Format: Pablo Domizi, Jolanda Sarno, Astraea Jager, Milton Merchant, Kaithlen Pacheco, Sean Yamada-Hunter, Maria Caterina Rotiroti, Yuxuan Liu, Reema Baskar, Warren Reynolds, Brian Sworder, Bita Sahaf, Sean Bendall, Charles Mullighan, Ash Alizadeh, Allison Leahy, Regina Myers, Stephan Grupp, Robbie Majzner, David Barrett, Elena Sotillo, Kara Davis. IKAROS mediates antigen escape following CD19- or CD22-targeted immunotherapies in r/r B-ALL [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr B052.