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830 EBF1 Activity Controls Leukemia Sensitivity to Inotuzumab Ozogamicin

Program: Oral and Poster Abstracts
Type: Oral
Session: 605. Molecular Pharmacology and Drug Resistance: Lymphoid Neoplasms: Mechanisms of Therapy Resistance in Lymphoma
Hematology Disease Topics & Pathways:
Lymphoid Leukemias, ALL, Diseases, Lymphoid Malignancies
Monday, December 9, 2024: 3:00 PM

Carolin S. Escherich, MD1*, Zhenhua Li, PhD2, Kelly R. Barnett, PhD3*, Yizhen Li, PhD3*, Megan Walker, PhD3*, Wenjian Yang, PhD2*, Xin Huang, PhD3*, Jiyang Yu, PhD3*, Wendy Stock, MD4, Elisabeth Paietta, PhD5*, Marina Konopleva6, Steven M. Kornblau, MD7, Mark R. Litzow, MD8, Hiroto Inaba, MD, PhD3, Ching-Hon Pui, MD3, Mignon L. Loh, MD, PhD9, Daniel Savic, PhD3 and Jun J. Yang, PhD3

1St. Jude Children’S Research Hospital, Memphis, TN
2Department of Pharmacy and Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, TN
3St. Jude Children's Research Hospital, Memphis, TN
4University of Chicago, Chicago, IL
5Department of Oncology, Montefiore Medical Center, Bronx, NY
6Montefiore Einstein Comprehensive Cancer Center, Albert Einstein College of Medicine, Bronx, NY
7Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX
8Division of Hematology, Mayo Clinic, Rochester, MN
9Ben Towne Center for Childhood Cancer Research, Seattle Children’s Hospital, Fred Hutch Cancer Center, University of Washington, Seattle, WA

Background: Inotuzumab Ozogamicin (InO) is an antibody-cytotoxin conjugate with high activity in B-cell acute lymphoblastic leukemia (B-ALL). InO targets the B-cell surface protein CD22, which is expressed in most B-ALL cases, albeit with great variability in expression levels. Emerging evidence suggest that this variation is associated with the stage of leukemia differentiation arrest [PMID: 21348573]. However, the exact relationship between CD22 expression and leukemia sensitivity to InO, particularly in the context of leukemia developmental state, remains unclear. Furthermore, our understanding of the molecular mechanisms regulating CD22 transcription in B-ALL is still incomplete.

Methods: To comprehensively investigate the genomic basis of variability in leukemia CD22 expression and thus B-ALL sensitivity to InO, we performed a multi-omic characterization of 196 primary human B-ALL samples, using whole transcriptome sequencing (RNAseq), assay for transposase-accessible chromatin with sequencing (ATAC-seq), coupled with ex vivo profiling of InO sensitivity. Additionally, genomic editing studies were performed in Nalm6 and REH B-ALL cell lines, and patient derived xenograft (PDX) models were used for in vitro and in vivo validations.

Results: Applying digital cytometry to leukemia bulk RNAseq data to infer the B-ALL developmental state, we found that blast cells with early Pre-pro-B-like signature had low levels of CD22 expression and were resistant to InO. As B-cell differentiation is controlled by stage-specific transcription factors (TFs), we performed a targeted CRISPR library screen of 1,639 TF genes and identified EBF1 as a key activator of CD22 expression (FDR=7.1x10-4). In primary B-ALL samples, EBF1 expression was significantly correlated with CD22 expression (R=0.69, P<0.0001) and leukemia response to InO (measured as LC50, R=-0.42, P<0.0001). In addition, TF footprint analysis of leukemia ATAC-seq data identified EBF1 as the most differentially activated TF comparing InO-sensitive and -resistant B-ALL cases (P=8x10-174). Blocking the EBF1 binding sites within the CD22 promoter by CRISPR interference resulted in a 50-fold and 49-fold decrease in cell surface CD22 expression, and a 22-fold and 25.7-fold increase in InO LC50 for Nalm6 and REH, respectively, confirming the direct impact of EBF1 on CD22 transcription and InO sensitivity.

Because BCR::ABL1-positive ALL showed the greatest variability in InO sensitivity among all B-ALL subtypes, we further investigate the role of the EBF1/CD22 axis on InO sensitivity within this subtype. Unsupervised clustering of gene expression profiles revealed two distinct groups, namely BCR::ABL1 ALL with multilineage (BCR::ABL1-M) or lymphoid only (BCR::ABL1-L) involvement [PMID: 38153913]. Compared to BCR::ABL1-L, BCR::ABL1-M displayed markedly reduced CD22 expression (P=8.3×10-11) and resistance to InO (P=1.9×10-4), likely due to the lower expression and frequent somatic alteration of EBF1. Finally, we established PDX models for representative cases of BCR::ABL1-M and BCR::ABL1-L. Strikingly, InO treatment did not affect leukemia progression in the BCR::ABL1-M PDX model, whereas the BCR::ABL1-L PDX model achieved complete remission and has remained leukemia free to date.

Conclusion: Collectively, these data demonstrate the direct impact of EBF1 on CD22 expression during B-cell development, and explain the heterogeneity of InO sensitivity, even within the same molecular B-ALL subtype.

Disclosures: Stock: Adaptive: Consultancy, Honoraria; Kura: Research Funding; Kura, Servier, Newave, Adaptive, Jazz, Asofarma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Konopleva: Curis: Consultancy; Adaptive: Consultancy; MEI Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Servier: Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees; Auxenion GmbH: Membership on an entity's Board of Directors or advisory committees; Legend Biotech: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: clinical trials, Research Funding; Intellisphere: Speakers Bureau; Menarini Group: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Other: clinical trials; Vincerx: Consultancy; Sanofi Aventis: Consultancy; Klondike Biopharma: Research Funding; Syndax: Membership on an entity's Board of Directors or advisory committees; Dark Blue Therapeutics: Membership on an entity's Board of Directors or advisory committees. Litzow: Abbvie: Research Funding; Amgen: Research Funding, Speakers Bureau; Actinium: Research Funding; Astellas: Research Funding; Pluristem: Research Funding; Sanofi: Research Funding; Beigene: Speakers Bureau; Biosight: Other: Data Safety Monitoring Committee. Inaba: Jazz: Consultancy; Servier: Consultancy, Research Funding; Incyte: Research Funding.

*signifies non-member of ASH