The lysine-specific demethylase 1 (LSD1) regulates hematopoietic stem cell differentiation and has been identified as a therapeutic target in hematological disorders. LSD1 demethylates mono and dimethylated histones 3 at lysine 4 and 9. In addition, it acts as a scaffold for the formation of chromatin-modifying complexes that regulates the transcription of myeloid-lineage-specific genes in complex with GFI1, a transcriptional repressor. While both enzymatic and non-enzymatic functions of LSD1 have been well defined, the relative importance of these two functions in hematopoiesis remains incompletely understood. Here, we investigated the contribution of enzymatic and non-enzymatic functions of LSD1 to myelopoiesis. We show that myeloid differentiation is independent of the enzymatic functions of LSD1 but requires the non-enzymatic, scaffolding function, which directs GFI1 binding to target sequences. In the absence of the LSD1 protein, GFI1 DNA binding is diminished, and myeloid cell differentiation arrests at an immature, myelomonocytic-like cell stage, which overexpresses Prtn3. We provide functional data implicating Prtn3 as an effector of the stem cell expansion and myeloid maturation block caused by the loss of LSD1.

Cellular composition and spatial architecture of the bone marrow niche play a critical role in regulating both normal and malignant hematopoiesis. Although flow cytometry enables rapid analysis of hematopoietic and niche cell composition, it requires single-cell dissociation, thereby disrupting the spatial context. In contrast, immunofluorescence imaging preserves tissue architecture but is limited in the number of markers that can be simultaneously detected. Recent advances in multiplex staining technologies now permit visualization of numerous antigens within a single tissue section, providing a more comprehensive view of the bone marrow microenvironment. Despite the central role of murine models in studying hematopoiesis, validated multiplex panels for analyzing murine bone marrow remain limited. This is particularly true for formalin-fixed, paraffin-embedded (FFPE) sections, where challenges such as high autofluorescence and tissue damage during antigen retrieval place substantial demands on tissue preparation. In this study, we optimized tissue preparation for staining murine FFPE bone marrow and developed an efficient multiplex immunohistochemistry panel for detecting hematopoietic stem cell-containing populations within their native niche architecture. Applying our panel to FFPE bone marrow sections from Jak2^V617F mice revealed alterations in the megakaryocytic niche, demonstrating the utility of this approach for characterizing disease-associated spatial changes within the hematopoietic microenvironment.

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic disorders characterized by the abnormal proliferation of myeloid cells. In addition to the main driver mutations in JAK2, MPL, and CALR, the transcription factor nuclear factor erythroid 2 (NFE2) has emerged as a key contributor to MPN pathophysiology. NFE2 expression is elevated in the majority of MPN patients, and augmented NFE2 activity in hematopoietic stem cells is sufficient to induce an MPN phenotype with spontaneous leukemic transformation in murine models. Moreover, NFE2 mutations, found in a subset of MPN patients, augment NFE2 activity and are associated with a markedly increased risk of progression to acute myeloid leukemia (AML). However, the molecular mechanism by which NFE2 mutations cause leukemogenesis is not understood. Here, we demonstrate that the E3 ubiquitin ligase ITCH mediates proteasomal degradation of wild-type (wt) NFE2 in HEK-293T cells. A gain-of-function truncation mutant, NFE2-226aa, retains the capacity to interact with ITCH but is no longer degraded. Rather, NFE2-226aa protects wt NFE2 from ITCH-dependent degradation, resulting in enhanced NFE2 activity.

Elevated activity of transcription factor NFE2 is sufficient to cause leukemic transformation in absence of a classical leukemic driver. However, the molecular mechanism promoting NFE2-driven leukemogenesis is not known. Here we report a previously unrecognized role for NFE2 as a key regulator of the oxidative stress response in leukemic cells. Through a comprehensive analysis of NFE2 genomic occupancy and its effect on chromatin accessibility and transcription, we demonstrate that NFE2 regulates glutathione homeostasis as well as the expression of central detoxifying enzymes. NFE2 constituted one of the highest scoring gene dependencies in MLL-AF9-transformed primary leukemic cells under redox stress, substantially more significant than the universally recognized redox regulator NRF2. Consequently, NFE2 knockdown sensitized leukemic cells to GSH depletion, ferroptosis induction, as well as to cytarabine treatment. Deriving an NFE2 RedOx score, we demonstrate that increased NFE2 activity constitutes an independent predictor of inferior outcome in AML patients at diagnosis.