PAX5 Epigenetically Orchestrates CD58 Transcription and Modulates Blinatumomab Response in Acute Lymphoblastic Leukemia

Blinatumomab is a bi-specific molecule that engages CD3+ T cells with CD19-expressing malignant B cells, thus inducing tumor lysis by T-cell immune response. While large frontline ALL trials of blinatumomab are lacking, existing data strongly indicate substantial inter-patient variability in response and toxicities. Complete remission rate ranges from 36 to 66%, depending on the regimen used and patient characteristics. Moreover, the duration of remission varies among trials, with a significant proportion of patients experiencing relapse or progressive disease following blinatumomab treatment, raising concerns about acquired drug resistance. In particular, mechanisms of resistance in CD19+ relapse remain largely unclear.

Using genome-wide CRISPR screen in B-ALL cell line in vitro, we comprehensively identified leukemia intrinsic factors of blinatumomab sensitivity. In addition to CD19 deletion the loss of CD58 emerged as a top driver for resistance, plausibly compromising the CD2-CD58-mediated interaction between T cells and B-ALL. Genomic editing of the CD58 gene or blocking with anti-CD58 antibody completely abrogated blinatumomab sensitivity across different B-ALL cell lines. Screening 1,639 transcription factor genes, we then identified PAX5 as the key activator of CD58. Interestingly, primary ALL samples with PAX5 P80R mutation also showed complete loss of CD58 expression, based on RNA-seq of 1,988 B-ALL samples across 20 molecular subtypes. To further validate this, we developed isogenic clones of the B-ALL cell line 697, with different PAX5 diplotypes: wildtype/wildtype (WT/WT), wildtype/frameshift (WT/fs), and P80R/frameshift (P80R/fs). P80R/fs and WT/fs clones exhibited significantly lower CD58 expression as compared to the WT/WT clone, and both were markedly resistant to blinatumomab. PAX5 mutation did not influence CD19 expression in these B-ALL cells. By testing a panel of primary ALL samples in vitro, we found that blast harboring the PAX5 P80R mutation has the lowest CD58 expression and was the most resistant to blinatumomab, which was reversed by ectopic expression of CD58.

To delineate the mechanism by which PAX5 mutation affects CD58 suppression in ALL, we comprehensively profiled epigenomic marks within the CD58 locus in 697 cells with WT/fs, P80R/fs, and WT/WT genotypes. Using ChIP-seq, we identified PAX5 binding sites within Intron 1 of the CD58 genes. By contrast, PAX5 binding at this locus was decreased in WT/fs cells and completely eliminated in the P80R/fs clones, suggesting a loss-of-function effect of the P80R mutation. This region was also characterized by H3K27ac and H3K4me3 marks, signifying its enhancer activity, which was also lost in P80R/fs cells. ATAC-seq profiling of these samples showed a similar pattern of PAX5-dependent open chromatin status. CRISPR interference showed that blocking the genomic sequence encompassing this enhancer resulted in significant downregulation of CD58 expression. PAX5 binding was also observed in the CD19 promoter region, but P80R mutation had only modest effects on histone modification and chromatin accessibility at this locus.

Finally, we explored the effects of CD58 loss on T cell activation. T cell cocultured with CD58^KO Nalm6 clone showed a substantial decrease in activation and proliferation, as compared to that cocultured with parental Nalm6. Defects of activation and proliferation were also observed in T cells cocultured with PAX5 P80R/fs 697 clone as compared to those with parental 697. Using RNA-seq, we identified 111 genes that were highly expressed in T cells cocultured with parental (relative to those with CD58^KO Nalm6). Among these hits, there was significant enrichment of genes involved in oxygen metabolisms, immune response, and cytokine signaling pathways, indicating the loss of CD58 abolished blinatumomab-induced T cell activation. ATAC-seq of these samples further confirmed the findings of RNA-seq studies.

In conclusion, our study identified the critical role of CD58-mediated interaction between B-ALL and T cells for blinatumomab sensitivity in this type of leukemia, pointing to potential strategies for genomics-guided blinatumomab treatment individualization.

Konopleva:Cellectis: Consultancy, Other: Grant support, Research Funding; Calithera: Other: Grant Support, Research Funding; Ablynx: Other: Grant support, Research Funding; Agios: Other: grant support, Research Funding; Ascentage: Other: grant support, Research Funding; AstraZeneca: Other: grant support, Research Funding; Rafael Pharmaceutical: Other: grant support, Research Funding; Sanofi: Other: grant support, Research Funding; Novartis: Patents & Royalties, Research Funding; Eli Lilly: Consultancy, Patents & Royalties, Research Funding; Reata Pharmaceuticals: Current equity holder in private company, Patents & Royalties; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kisoji: Consultancy, Honoraria; Amgen: Consultancy; Forty-Seven: Consultancy, Honoraria, Other: Grant support; Stemline Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffman La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grant support, Research Funding; Genentech: Consultancy, Other: grant support, Research Funding; AbbVie: Consultancy, Other: grant support, Research Funding. Inaba:Servier: Other: Grants and Personal Fees; Amgen: Other: Grants and Personal Fees; Incyte: Other: Grants; JAZZ: Other: Personal Fees; Chugai: Other: Personal Fees. Pui:Novartis: Other: Data monitoring committee; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees. Yang:Takeda: Research Funding.