The LMO2-LDB1-TAL1 Complex Regulates Transcription Networks in AML Relapse

Relapsed acute myeloid leukemia (AML) remains a clinical challenge with patients suffering from poor clinical outcomes. To date, studies have not found specific recurrent somatic events that explain disease relapse in most patients. We have previously shown that epigenetic heterogeneity may contribute to transcriptional dysregulation and disease progression in AML but the specific transcriptional programs that drive disease relapse have not been identified.

To characterize the transcriptional landscapes in relapsed AML, we analyzed multi-omics molecular profiles from two cohorts of patient-matched diagnostic and relapse AML specimens (cohorts I: n = 29 and II: n = 30). We identified 5,416 differentially expressed genes (DEGs) between diagnosis and relapse in cohort I. The majority of these DEGs did not associate with DNA methylation changes at their respective promoters. Unsupervised clustering of the DEGs identified two transcriptomic subtypes characterized by a subset of DEGs that were disparate in direction of expression change in the two subtypes. These subtypes were not significantly associated with specific somatic events or evolution patterns between diagnosis and relapse. Similar differential expression patterns and patient clusters were observed in Cohort II. We utilized Binding Analysis for Regulation of Transcription (PMID: 29608647) to computationally predict transcriptional regulators (TRs) that might regulate the disparate gene expression patterns observed. The expression patterns of the top 30 TRs predicted for the disparate genes associated with clinical outcomes in the TCGA and BEAT AML patient cohorts (Advanced Expression Survival Analysis [PMID: 31607216]; p < 0.02).

Functional annotation of the top TRs predicted for the disparate genes was notable for transcription factors (TFs) known to regulate hematopoiesis, including members of the LIM domain Only 2 (LMO2) multi-subunit complex (TAL transcription Factor 1 [TAL1], GATA binding protein 1 [GATA1], and LIM domain binding protein 1 [LDB1]). A mouse xenograft model of AML relapse was used to generate gene expression data from the human malignant cells. DEGs were identified between engrafted and post-chemotherapy treated AML cells. BART analysis on the DEGs also identified LMO2/LDB1/LMO2 complex members as potential TRs. The LMO2/LDB1/TAL1 complex is known to function in hematopoiesis and has been shown to have an oncogenic role in T-cell acute lymphoblastic leukemia, where the complex has been shown to enforce a stem cell phenotype in T-cell progenitor cells. However, a functional role for it in relapsed AML has not been defined. We hypothesized that this complex is an oncogenic TR in AML relapse.

Analysis of DepMap (https://depmap.org/portal) results in AML cell lines identified LMO2-dependent cells. A subset of these cell lines harbored high expression of TAL1, suggesting coordinated regulation. To confirm interactions between LMO2/LDB1/TAL1 complex members, we performed immunoprecipitation for LMO2 and LDB1 followed by tandem mass spectrometry analysis of binding proteins in HEL and K562 cells. TAL1, Transcription Factor 12, and other known complex members co-purified with LMO2 and LDB1. Loss of function experiments targeting LMO2, TAL1, and LDB1 in these AML cell lines altered gene expression and reduced growth (t-test p < 0.05). Functional analyses of the DEGs identified in the in vitro experiments suggested that loss of function associated with downregulation of cell cycle checkpoints, MYC, and MYC target genes (Normalized enrichment score [NES] < -1.5, q < 0.05). Accordingly, we observed upregulation of cell cycle and MYC gene sets in the patient-matched AML specimens with increased TAL1 expression at relapse (NES > 1.5, q < 0.05). Importantly, overall changes in expression acquired with LMO2 and LDB1 loss of function inversely correlated with DEGs in relapsed patients with higher TAL1 expression (NES < -1.5, q < 0.05).

Our data from primary AML specimens and functional analyses in AML cell lines supports an essential role for the LMO2/LDB1/TAL1 complex, especially in AML relapsed disease. The macromolecular protein complex plays a role in the maintenance of hematopoietic stem cells, a role that may be co-opted in AML. Targeting the complex or its downstream effectors could be a novel therapeutic consideration for relapsed AML patients.

Dillon:Jazz: Other: Consultancy and educational events (paid to institution); Abbvie, Amgen: Other: Research support (paid to institution); Abbvie, Astellas, Pfizer: Consultancy, Other: Educational events. Levine:Mana: Membership on an entity's Board of Directors or advisory committees; Auron: Membership on an entity's Board of Directors or advisory committees; Bridge Medicines: Consultancy; Syndax: Consultancy; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Bridge Bio: Consultancy; Jubilant: Membership on an entity's Board of Directors or advisory committees; Anovia: Consultancy; Kurome: Membership on an entity's Board of Directors or advisory committees; Qiagen: Membership on an entity's Board of Directors or advisory committees; Bakx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Membership on an entity's Board of Directors or advisory committees; Epiphanes: Membership on an entity's Board of Directors or advisory committees; Imago: Consultancy; Ajax: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Zentalis: Membership on an entity's Board of Directors or advisory committees; Scorpion: Membership on an entity's Board of Directors or advisory committees. Melnick:Astra Zeneca: Research Funding; Janssen Global Advisory: Membership on an entity's Board of Directors or advisory committees; Treeline Biosciences: Consultancy; Ipsen (formerly Epizyme): Consultancy, Research Funding; Daiichi Sankyo: Consultancy; Exsciencia AI Ltd.: Consultancy. Neuberg:Madrigal Pharmaceutical: Current equity holder in publicly-traded company.