Acute depletion of HOXA9 reveals its direct role in cis-elements regulation and 3D genome organization during Acute Myeloid Leukemia

Abstract In normal hematopoiesis, HOXA9 maintains early hematopoietic progenitors and is silenced during differentiation. Failure to silence HOXA9 blocks hematopoietic differentiation and leads to leukemia. In acute myeloid leukemia (AML), it is overexpressed in over 70% of cases with diverse genetic alternations and is a strong predictor of poor prognosis. Our previous work has demonstrated that disruption of lineage-specific transcription factors can drive leukemogenesis by reorganizing the 3D genome structure and regulatory networks critical for cell fate determination. As a pioneer factor in AML, HOXA9 is essential for leukemic transformation, and its leukemogenic activity is markedly enhanced by co-overexpression with its partner MEIS1. However, the specific regulatory mechanisms underlying HOXA9-driven leukemia progression, and its role in establishing oncogenic regulatory networks that drive AML pathogenesis, remain poorly understood. To address this, we developed a primary cell system co-expressing HOXA9 and MEIS1, combined with a PROTAC-based degron strategy for rapid and inducible degradation of HOXA9 protein. This approach avoids complex genetic alterations and enables precise, temporal dissection of HOXA9 function under controlled conditions. We isolated hematopoietic stem and progenitor cells (HSPCs) from the bone marrow of C57BL/6J mice and transduced with a single retroviral construct co-expressing FKBP12F36V degron-tagged HOXA9 and MEIS1 (dA9M). Treatment of these transduced cells with dTAGV-1 resulted in rapid and complete degradation of HOXA9 within 1 hour. Importantly, dTAGV-1 treatment led to reduced cell viability of dA9M cells, but had no effect on HSPCs transformed by non-degron-tagged HOXA9 and MEIS1. To precisely detect HOXA9-mediated transcriptional, epigenetic, and higher-order chromatin changes in AML, we collected 3 biological replicates at 0, 1.5, 6, 12, 24, 48, and 96 hours after dTAGV-1 treatment. Integrating transcriptomic and epigenomic profiles, we classified 3 regulatory clusters: immediate loss, early decrease, and late decrease, based on chromatin accessibility changes upon HOXA9 depletion. Chromatin accessibility alterations at early stages (beginning at 1.5 hours) comprised 2 clusters. The first cluster included immediate loss regions, which exhibited chromatin accessibility loss at 1.5 hours with exclusive enrichment for HOXA9 motifs. The second cluster consisted of early decrease regions enriched for HOXA9 along with differentiation associated transcription factors. Overall, at early stages, genes involved in leukemic stem cell self-renewal and myeloid differentiation pathways were broadly downregulated. To directly measure the immediate transcriptional consequences of HOXA9 loss, we performed SLAM-seq (thiol(SH)-linked alkylation for the metabolic sequencing of RNA) at 1.5 hours after dTAGV-1 treatment which revealed a rapid and significant decrease in the transcription rates of 308 genes. This result confirmed that their downregulation was a direct consequence of HOXA9 depletion, establishing them as immediate transcriptional targets of HOXA9. To determine the functional relevance of these genes, we further validated that 42 of them are critical for leukemia cell survival through both in vivo and in vitro screening. Late stage alterations emerged at 48 hours as the third cluster, which was enriched for CTCF and cohesin components including SMC3 and RAD21. These epigenomic changes affected genes involved in chromatin organization and cell cycle regulation, thereby constraining leukemic cell proliferation. At the level of 3D genome organization, HOXA9 depletion induced widespread reorganization events across multiple scales, consistent with the associated epigenetic changes. Notably, loss of HOXA9 led to chromatin compaction, disruption of topologically associating domain (TAD) structures, and reshaping of enhancer promoter interactions. In particular, SOX4 expression was linked to disrupted enhancer-promoter interactions at a TAD boundary. Together, our study provides a higher order chromatin regulatory network mediated by HOXA9 in AML. Furthermore, these results demonstrate that HOXA9 governs specific transcriptional programs and 3D genome organization in AML and thus provides potential targets for disrupting the core regulatory mechanisms that sustain the disease.