IKZF1 Deletion Confers Resistance to CAR19 in r/r B-ALL Via CD19 Intron Retention

In recent years, CD19-directed chimeric antigen receptor T-cell (CAR19) therapy has provided a promising outcome for hematological malignancies, but a considerable number of patients ultimately have progressive disease. Our previous study also found that tumor intrinsic IKZF1 deletion (IKZF1del) conferred an inferior event-free survival (EFS) in refractory and/or refractory (r/r) B-ALL patients receiving CAR19 therapy, with 1-year EFS of 27.8 ± 12.4%, compared with 53.6 ± 6.1% of the IKZF1wt group (P=0.011). [28th EHA oral presentation; 2023] However, the underlying impact of tumor intrinsic IKZF1del on the resistance to CAR19 therapy remains elusive. Previous studies have emphasized the importance of IKZF1 in the process of B cell development, as well as the abnormal alternative splicing of CD19 in CAR19 resistance. herein we presumed that IKZF1del mediated CAR19 resistance via CD19 introns retention. Firstly, we performed RNA-Seq analysis using bone marrow mononuclear cells from patients newly diagnosed with B-ALL. A total of 113 patients (IKZF1del (37 of 113, 32.7%) vs. IKZF1WT (76 of 113, 67.3%)) was collected to analyze the frequency of CD19 introns retention (IR). Expectedly, we found that the average frequency of IR was higher in the IKZF1Del group, compared with the IKZF1WT group. Furthermore, we conducted a protein-structure prediction, which showed a premature termination codon in CD19-IR2, and caused a truncated CD19 protein with a dys-function. And the contact of CAR scFv structure (FMC63) and target antigen was predicted, suggesting an unstable binding region of FMC63 and CD19-IR2 structure, which yielded a lower affinity of -8.4 kcal/mol, compared to -11.4 kcal/mol in the structure of CAR and wildtype CD19. Additionally, to verify whether tumor intrinsic IKZF1Del mediated CAR19 resistance via an unstable contact structure, we generated Nalm6 leukemic cells with IKZF1 deletion subtype (Nalm6_IK6). Nalm6_IK6 cells showed a higher level of non-functional IK6 subtype and lower lever of functional IK1 subtype, and the expression of CD19-IR2 was higher in Nalm6_IK6 cells. Then we tested their avidity to CAR19 cells using a single-cell avidity measurement technology, z-MOVI, which determined the precise binding force between the effector and target cells. Inspiring, after a 5-minute co-culture period, Nalm6_IK6 cells showed a significantly lower binding capability followed by acoustic force exposure compared to Nalm6_WT cells. The cytotoxic capacity of CAR19 to these tumor cells was also investigated. Notably, after a 24-hour co-culture period, CAR19 cells showed weaker lytic activity of Nalm6_IK6 cells than Nalm6_WT cells. In conclusion, herein we suggested that tumor intrinsic IKZF1del conferred an affinity-dependent resistance to CAR19 via CD19 introns retention. These data not only enriched the field of how tumor suppressor gene-IKZF1del mediated CAR19 immunotherapy resistance, but offered a theoretical basis for optimal strategy to these IKZF1del r/r B-ALL patients.