The eleven-nineteen leukemia (ENL) protein family, composed of ENL and AF9, is a common component of 3 transcriptional modulators: AF4–ENL–P-TEFb complex (AEP), DOT1L-AF10-ENL complex (referred to as the DOT1L complex) and polycomb-repressive complex 1 (PRC1). Each complex associates with chromatin via distinct mechanisms, conferring different transcriptional properties including activation, maintenance, and repression. The mixed-lineage leukemia (MLL) gene often fuses with ENL and AF10 family genes in leukemia. However, the functional interrelationship among those 3 complexes in leukemic transformation remains largely elusive. Here, we have shown that MLL-ENL and MLL-AF10 constitutively activate transcription by aberrantly inducing both AEP-dependent transcriptional activation and DOT1L-dependent transcriptional maintenance, mostly in the absence of PRC1, to fully transform hematopoietic progenitors. These results reveal a cooperative transcriptional activation mechanism of AEP and DOT1L and suggest a molecular rationale for the simultaneous inhibition of the MLL fusion–AF4 complex and DOT1L for more effective treatment of MLL-rearranged leukemia.

Myeloid malignancies withDDX41mutations are often associated with bone marrow failure and cytopenia before overt disease manifestation. However, the mechanisms underlying these specific conditions remain elusive. Here, we demonstrate that loss of DDX41 function impairs efficient RNA splicing, resulting in DNA replication stress with excess R-loop formation. Mechanistically, DDX41 binds to the 5′ splice site (5′SS) of coding RNA and coordinates RNA splicing and transcriptional elongation; loss of DDX41 prevents splicing-coupled transient pausing of RNA polymerase II at 5ʹSS, causing aberrant R-loop formation and transcription-replication collisions. Although the degree of DNA replication stress acquired in S phase is small, cells undergo mitosis with under-replicated DNA being remained, resulting in micronuclei formation and significant DNA damage, thus leading to impaired cell proliferation and genomic instability. These processes may be responsible for disease phenotypes associated withDDX41mutations.

Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) is a new pathological entity with poor outcomes in T cell ALL (T-ALL) that is characterized by a high incidence of loss-of-function mutations in polycomb repressive complex 2 (PRC2) genes. We generated a mouse model of ETP-ALL by deleting Ezh2, one of the PRC2 genes, in p53-null hematopoietic cells. The loss of Ezh2 in p53-null hematopoietic cells impeded the differentiation of ETPs and eventually induced ETP-ALL–like disease in mice, indicating that PRC2 functions as a bona fide tumor suppressor in ETPs. A large portion of PRC2 target genes acquired DNA hypermethylation of their promoters following reductions in H3K27me3 levels upon the loss of Ezh2, which included pivotal T cell differentiation–regulating genes. The reactivation of a set of regulators by a DNA-demethylating agent, but not the transduction of single regulator genes, effectively induced the differentiation of ETP-ALL cells. Thus, PRC2 protects key T cell developmental regulators from DNA hypermethylation in order to keep them primed for activation upon subsequent differentiation phases, while its insufficiency predisposes ETPs to leukemic transformation. These results revealed a previously unrecognized epigenetic switch in response to PRC2 dysfunction and provide the basis for specific rational epigenetic therapy for ETP-ALL with PRC2 insufficiency.

Once disseminated tumor cells (DTCs) arrive at a metastatic organ, they remain there, latent, and become seeds of metastasis. However, the clonal composition of DTCs in a latent state remains unclear. Here, we applied high-resolution DNA barcode tracking to a mouse model that recapitulated the metastatic dormancy of head and neck squamous cell carcinoma (HNSCC). We found that clones abundantly circulated peripheral blood dominated DTCs. Through analyses of multiple barcoded clonal lines, we identified specific subclonal population that preferentially generated homotypic circulating tumor cell (CTC) clusters and dominated DTCs. Despite no notable features under static conditions, this population significantly generated stable cell aggregates that were resistant to anoikis under fluid shear stress (FSS) conditions in an E-cadherin-dependent manner. Our data from various cancer cell lines indicated that the ability of aggregate-constituting cells to regulate cortical actin-myosin dynamics governed the aggregates’ stability in FSS. The CTC cluster-originating cells were characterized by the expression of a subset of E-cadherin binding factors enriched with actin cytoskeleton regulators. Furthermore, this expression signature was associated with locoregional and metastatic recurrence in HNSCC patients. These results reveal a biological selection of tumor cells capable of generating FSS-adaptive CTC clusters, which leads to distant colonization.