HMGA1-ETV5 Axis Is Essential for Relapsed B-Cell Leukemia and Provides Novel Therapeutic Targets

Introduction: Despite significant advances in therapy for pediatric B-cell acute lymphoblastic leukemia (pB-ALL), relapse remains the leading cause of death in children with cancer and mechanisms driving relapse remain elusive. The High Mobility Group A1 (HMGA1) gene encodes HMGA1 chromatin regulators which are highly expressed in hematopoietic stem cells and diverse malignancies where they portend poor outcomes (Li & Kim et al, Blood 2022). In murine models, Hmga1 overexpression in lymphoid cells drives leukemic transformation. HMGA1 is also overexpressed in leukemic blasts with highest levels at relapse in a pB-ALL cohort (Roy et al, Leuk Lymphoma 2013). Together, these findings suggest that HMGA1 is required for leukemogenesis and drives relapse through epigenetic reprogramming. We therefore sought to: 1) test the hypothesis that HMGA1 is required for leukemogenesis in preclinical models of relapsed pB-ALL, and, 2) identify targetable mechanisms mediated by HMGA1 in pB-ALL.

Methods: To elucidate HMGA1 function and downstream transcriptional networks, we silenced gene expression by CRISPR/Cas9 or shRNAs targeting 2 sequences per gene in cell lines derived from relapsed pB-ALL (REH cells harboring a TEL-AML1 fusion and 697 cells with an E2A-PBX1 fusion). We assessed leukemogenic properties in vitro and with mouse models. To dissect molecular mechanisms, we performed RNAseq, ATACseq, ChIPseq and applied in silico pathway analysis. In human pB-ALL samples, we assessed gene expression via bulk and single cell RNAseq. Connectivity Map (CMAP) was applied to identify drugs to target HMGA1 networks.

Results:Strikingly,HMGA1 is overexpressed in 3 independent pB-ALL cohorts with highest levels at relapse. HMGA1 silencing (CRISPR/Cas9 or shRNA) in relapsed pB-ALL cell lines (REH, 697) disrupts proliferation, decreasing the frequency of cells in S Phase, enhances apoptosis, and impairs clonogenicity. Moreover, HMGA1 depletion decreased leukemic engraftment (spleen, bone marrow) and prolonged survival in mice following intravenous injection of pB-ALL cells (697, REH). Further, leukemic cells that ultimately engraft show increased HMGA1 expression relative to the pool of injected cells with HMGA1 silencing, suggesting that escape from HMGA1 silencing is required for leukemic cell engraftment and expansion. Integrated analysis of RNA/ChIP/ATACseq revealed transcriptional networks governed by HMGA1 that regulate proliferation (G2M checkpoint, E2F, Mitotic spindle), RAS signaling, and ETV5 (ETS variant 5 transcription factor) targets. Given its association with other forms of aggressive ALL, we focused on the ETV5 gene. Surprisingly, silencing ETV5 (CRISPR or shRNA) recapitulates most anti-leukemia phenotypes observed with HMGA1 depletion whereas restoring ETV5 expression partially rescues anti-leukemia phenotypes in pB-ALL cells with HMGA1 silencing, increasing clonogenicity in vitro and leukemic engraftment in vivo. HMGA1 occupies AT-rich sequences within the ETV5 promoter (-0.3 kb), enhances chromatin accessibility, and recruits active histone marks (H3K27Ac, H3K4me3/1). In reporter assays, HMGA1 transactivates ETV5 promoter expression. By CMAP, the histone deacetylase (HDAC) inhibitor, Vorinostat, is predicted to target HMGA1-ETV5 networks. Notably, HMGA1 depletion synergizes with Vorinostat to disrupt proliferation in vitro while delaying leukemogenic engraftment and prolonging survival in mouse recipients of pB-ALL cells. Most importantly, HMGA1 and ETV5 are co-expressed and up-regulated in primary pB-ALL blasts by bulk RNA analyses. Further, scRNA seq of bone marrow from pB-ALL samples harboring the TEL-AML1 fusion reveal that HMGA1 and ETV5 are up-regulated and co-expressed in the cell clusters enriched for leukemic blasts, indicating that the HMGA1-ETV5 pathway is relevant in pediatric B-ALL.

Conclusions: Here, we discovered a novel epigenetic program whereby HMGA1 up-regulates ETV5 and stem cell transcriptional networks in relapsed pB-ALL. Mechanistically, HMGA1 induces ETV5 networks by binding to chromatin and recruiting active histone marks to transactivate ETV5 expression. Further, the HMGA1-ETV5 axis can be targeted by epigenetic drugs (HDAC inhibitors) that synergize with HMGA1 depletion. These findings reveal HMGA1 as a key epigenetic switch in relapsed pB-ALL and promising therapeutic target to treat or prevent relapse.

No relevant conflicts of interest to declare.