The BCL-2 inhibitor venetoclax combined with hypomethylating agents or low-dose cytarabine represents an important new therapy for older or unfit patients with acute myeloid leukemia (AML). We analyzed 81 patients receiving these venetoclax-based combinations to identify molecular correlates of durable remission, response followed by relapse (adaptive resistance), or refractory disease (primary resistance). High response rates and durable remissions were typically associated with NPM1 or IDH2 mutations, with prolonged molecular remissions prevalent for NPM1 mutations. Primary and adaptive resistance to venetoclax-based combinations was most commonly characterized by acquisition or enrichment of clones activating signaling pathways such as FLT3 or RAS or biallelically perturbing TP53. Single-cell studies highlighted the polyclonal nature of intratumoral resistance mechanisms in some cases. Among cases that were primary refractory, we identified heterogeneous and sometimes divergent interval changes in leukemic clones within a single cycle of therapy, highlighting the dynamic and rapid occurrence of therapeutic selection in AML. In functional studies, FLT3 internal tandem duplication gain or TP53 loss conferred cross-resistance to both venetoclax and cytotoxic-based therapies. Collectively, we highlight molecular determinants of outcome with clinical relevance to patients with AML receiving venetoclax-based combination therapies.

The BCL2 family plays important roles in acute myeloid leukemia (AML). Venetoclax, a selective BCL2 inhibitor, has received FDA approval for the treatment of AML. However, drug resistance ensues after prolonged treatment, highlighting the need for a greater understanding of the underlying mechanisms. Using a genome-wide CRISPR/Cas9 screen in human AML, we identifi ed genes whose inactivation sensitizes AML blasts to venetoclax. Genes involved in mitochondrial organization and function were signifi cantly depleted throughout our screen, including the mitochondrial chaperonin CLPB. We demonstrated that CLPB is upregulated in human AML, it is further induced upon acquisition of venetoclax resistance, and its ablation sensitizes AML to venetoclax. Mechanistically, CLPB maintains the mitochondrial cristae structure via its interaction with the cristae-shaping protein OPA1, whereas its loss promotes apoptosis by inducing cristae remodeling and mitochondrial stress responses. Overall, our data suggest that targeting mitochondrial architecture may provide a promising approach to circumvent venetoclax resistance. SIGNIFICANCE: A genome-wide CRISPR/Cas9 screen reveals genes involved in mitochondrial biological processes participate in the acquisition of venetoclax resistance. Loss of the mitochondrial protein CLPB leads to structural and functional defects of mitochondria, hence sensitizing AML cells to apoptosis. Targeting CLPB synergizes with venetoclax and the venetoclax/azacitidine combination in AML in a p53-independent manner.

Three-dimensional (3D) chromatin architectural differences can influence the integrity of topologically associating domains (TADs) and rewire specific enhancer-promoter interactions, impacting gene expression and leading to human disease. Here, we investigate the 3D chromatin architecture in T cell acute lymphoblastic leukemia (T-ALL) using primary human leukemia specimens and its dynamic responses to pharmacological agents. Systematic integration of matched in situ Hi-C, RNA-seq and CTCF ChIP-seq datasets revealed widespread differences in intra-TAD chromatin interactions and TAD boundary insulation in T-ALL. Our studies identify and focus on a TAD “fusion” event associated with absence of CTCF-mediated insulation, enabling direct interactions between the MYC promoter and a distal super-enhancer. Moreover, our data also demonstrate that small molecule inhibitors targeting either oncogenic signal transduction or epigenetic regulation can alter specific 3D interactions found in leukemia. Overall, our study highlights the impact, complexity and dynamic nature of 3D chromatin architecture in human acute leukemia.

Hematopoietic stem cells (HSCs) are able to both self-renew and differentiate. However, how individual HSC makes the decision between self-renewal and differentiation remains largely unknown. Here we report that ablation of the key epigenetic regulator Uhrf1 in the hematopoietic system depletes the HSC pool, leading to hematopoietic failure and lethality. Uhrf1-deficient HSCs display normal survival and proliferation, yet undergo erythroid-biased differentiation at the expense of self-renewal capacity. Notably, Uhrf1 is required for the establishment of DNA methylation patterns of erythroid-specific genes during HSC division. The expression of these genes is enhanced in the absence of Uhrf1, which disrupts the HSC-division modes by promoting the symmetric differentiation and suppressing the symmetric self-renewal. Moreover, overexpression of one of the up-regulated genes, Gata1, in HSCs is sufficient to phenocopy Uhrf1-deficient HSCs, which show impaired HSC symmetric self-renewal and increased differentiation commitment. Taken together, our findings suggest that Uhrf1 controls the self-renewal versus differentiation of HSC through epigenetically regulating the cell-division modes, thus providing unique insights into the relationship among Uhrf1-mediated DNA methylation, cell-division mode, and HSC fate decision.Uhrf1 | HSCs | epigenetic regulation | cell-division mode | cell fate decision

The transcription factor interferon regulatory factor 4 (IRF4) was originally found to be preferentially expressed in lymphoid cells and to be required for the function, differentiation, and homeostasis of both mature T and B lymphocytes. Recent studies have indicated that IRF4 is also involved in early B-cell development. However, the role of IRF4 in intrathymic T-cell development remains unknown. In this study, we show that IRF4 is upregulated in TCR-signaled thymocytes and is predominantly expressed in CD4 singlepositive (SP), but not in CD8 SP, cells. T-cell-specific overexpression of IRF4 impaired the generation and maturation of CD8 SP thymocytes. Further analysis revealed that IRF4 selectively bound to the distal promoter region of Runx3 and repressed its transcription, probably through the deacetylation of histones H3 and H4 in intermediate CD4 [1][2][3]. This process is accompanied by new gene expression programs. One apparent change that occurs is the silencing of CD4 or CD8 expression in DP thymocytes [4,5]. Previous research has revealed that transcription factors c-Myb, Gata3, Tox, and cKrox (also known as Thpok or Zbtb7b) act as CD4 lineage-specifying factors [6][7][8][9][10][11][12][13], whereas Runx3 is required for CD8 lineage differentiation [14,15]. Runx3 has dual functions during CD8 lineage differentiation, where it can act as a repressor by binding to the cis-regulatory silencer elements of the Cd4 and Zbtb7b gene locus and as an activator by promoting CD8 lineage-related gene expression [15][16][17][18]. However, the mechanisms by which these lineage-specifying factors are regulated remain largely unknown.Interferon regulatory factor (IRF) family members are transcription factors that are composed of a DNA-binding domain and a regulatory domain that regulate a series of immune-related à These authors have contributed equally to this work. 3198genes [19,20]. The functions of IRF include a number of distinct roles in biological processes, such as pathogen response, cytokine signaling, cell growth regulation, and hematopoietic development [19,20]. IRF4 (also known as Pip, ICSAT, or LSIRF) is a lymphocyte-restricted member of the IRF family of transcription factors that bind to the interferon-stimulated response element (ISRE; consensus sequence: A / G NGAAANNGAAACT) and interferon-g-activated sequence element (GAS; consensus sequence: TTTNCNNNAA) [19,[21][22][23]. IRF4 can act as either a transcriptional activator or a repressor, which is determined by the DNAbinding motif on specific promoters as well as interaction with distinct transcription factors [22]. IRF4 is not only critical for lymphocyte maturation, homeostasis, and differentiation, including Th2, Th17, and B-lymphocyte differentiation, but is also important for cytotoxic, antitumor, and humoral responses [19,20,[24][25][26]. As a transcription factor, IRF4 is required for early B-cell development and regulates a series of B-cell-specific genes in activated B cells and myeloma, such as the immunoglobulin light chain gene [19,27]....