Therapeutic Targeting of MLL Degradation Pathways in MLL-Rearranged Leukemia.

Chromosomal translocations of the mixed-lineage leukemia (MLL) gene with various partner genes result in aggressive leukemia with dismal outcomes. Despite similar expression at the mRNA level from the wild-type and chimeric MLL alleles, the chimeric protein is more stable. We report that UBE2O functions in regulating the stability of wild-type MLL in response to interleukin-1 signaling. Targeting wild-type MLL degradation impedes MLL leukemia cell proliferation, and it downregulates a specific group of target genes of the MLL chimeras and their oncogenic cofactor, the super elongation complex. Pharmacologically inhibiting this pathway substantially delays progression, and it improves survival of murine leukemia through stabilizing wild-type MLL protein, which displaces the MLL chimera from some of its target genes and, therefore, relieves the cellular oncogenic addiction to MLL chimeras. Stabilization of MLL provides us with a paradigm in the development of therapies for aggressive MLL leukemia and perhaps for other cancers caused by translocations.

Regulation of MLL/COMPASS stability through its proteolytic cleavage by taspase1 as a possible approach for clinical therapy of leukemia.

Chromosomal translocations of the Mixed-lineage leukemia 1 (MLL1) gene generate MLL chimeras that drive the pathogenesis of acute myeloid and lymphoid leukemia. The untranslocated MLL1 is a substrate for proteolytic cleavage by the endopeptidase threonine aspartase 1 (taspase1); however, the biological significance of MLL1 cleavage by this endopeptidase remains unclear. Here, we demonstrate that taspase1-dependent cleavage of MLL1 results in the destabilization of MLL. Upon loss of taspase1, MLL1 association with chromatin is markedly increased due to the stabilization of its unprocessed version, and this stabilization of the uncleaved MLL1 can result in the displacement of MLL chimeras from chromatin in leukemic cells. Casein kinase II (CKII) phosphorylates MLL1 proximal to the taspase1 cleavage site, facilitating its cleavage, and pharmacological inhibition of CKII blocks taspase1-dependent MLL1 processing, increases MLL1 stability, and results in the displacement of the MLL chimeras from chromatin. Accordingly, inhibition of CKII in a MLL-AF9 mouse model of leukemia delayed leukemic progression in vivo. This study provides insights into the direct regulation of the stability of MLL1 through its cleavage by taspase1, which can be harnessed for targeted therapeutic approaches for the treatment of aggressive leukemia as the result of MLL translocations.

Not All H3K4 Methylations Are Created Equal: Mll2/COMPASS Dependency in Primordial Germ Cell Specification.

The spatiotemporal regulation of gene expression is central for cell-lineage specification during embryonic development and is achieved through the combinatorial action of transcription factors/co-factors and epigenetic states at cis-regulatory elements. Here, we show that in addition to implementing H3K4me3 at promoters of bivalent genes, Mll2 (KMT2B)/COMPASS can also implement H3K4me3 at a subset of non-TSS regulatory elements, a subset of which shares epigenetic signatures of active enhancers. Our mechanistic studies reveal that association of Mll2's CXXC domain with CpG-rich regions plays an instrumental role for chromatin targeting and subsequent implementation of H3K4me3. Although Mll2/COMPASS is required for H3K4me3 implementation on thousands of loci, generation of catalytically mutant MLL2/COMPASS demonstrated that H3K4me3 implemented by this enzyme was essential for expression of a subset of genes, including those functioning in the control of transcriptional programs during embryonic development. Our findings suggest that not all H3K4 trimethylations implemented by MLL2/COMPASS are functionally equivalent.

Repression of TRIM13 by chromatin assembly factor CHAF1B is critical for AML development.

Acute myeloid leukemia (AML) is an aggressive blood cancer that stems from the rapid expansion of immature leukemic blasts in the bone marrow. Mutations in epigenetic factors represent the largest category of genetic drivers of AML. The chromatin assembly factor CHAF1B is a master epigenetic regulator of transcription associated with self-renewal and the undifferentiated state of AML blasts. Upregulation of CHAF1B, as observed in almost all AML samples, promotes leukemic progression by repressing the transcription of differentiation factors and tumor suppressors. However, the specific factors regulated by CHAF1B and their contributions to leukemogenesis are unstudied. We analyzed RNA sequencing data from mouse MLL-AF9 leukemic cells and bone marrow aspirates, representing a diverse collection of pediatric AML samples and identified the E3 ubiquitin ligase TRIM13 as a target of CHAF1B-mediated transcriptional repression associated with leukemogenesis. We found that CHAF1B binds the promoter of TRIM13, resulting in its transcriptional repression. In turn, TRIM13 suppresses self-renewal of leukemic cells by promoting pernicious entry into the cell cycle through its nuclear localization and catalytic ubiquitination of cell cycle-promoting protein, CCNA1. Overexpression of TRIM13 initially prompted a proliferative burst in AML cells, which was followed by exhaustion, whereas loss of total TRIM13 or deletion of its catalytic domain enhanced leukemogenesis in AML cell lines and patient-derived xenografts. These data suggest that CHAF1B promotes leukemic development, in part, by repressing TRIM13 expression and that this relationship is necessary for leukemic progression.

USP7 Cooperates with NOTCH1 to Drive the Oncogenic Transcriptional Program in T-Cell Leukemia.

PURPOSE: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Chemotherapy treatments show high response rates but have debilitating effects and carry risk of relapse. Previous work implicated NOTCH1 and other oncogenes. However, direct inhibition of these pathways affects healthy tissues and cancer alike. Our goal in this work has been to identify enzymes active in T-ALL whose activity could be targeted for therapeutic purposes. EXPERIMENTAL DESIGN: To identify and characterize new NOTCH1 druggable partners in T-ALL, we coupled studies of the NOTCH1 interactome to expression analysis and a series of functional analyses in cell lines, patient samples, and xenograft models. RESULTS: We demonstrate that ubiquitin-specific protease 7 (USP7) interacts with NOTCH1 and controls leukemia growth by stabilizing the levels of NOTCH1 and JMJD3 histone demethylase. USP7 is highly expressed in T-ALL and is transcriptionally regulated by NOTCH1. In turn, USP7 controls NOTCH1 levels through deubiquitination. USP7 binds oncogenic targets and controls gene expression through stabilization of NOTCH1 and JMJD3 and ultimately H3K27me3 changes. We also show that USP7 and NOTCH1 bind T-ALL superenhancers, and inhibition of USP7 leads to a decrease of the transcriptional levels of NOTCH1 targets and significantly blocks T-ALL cell growth in vitro and in vivo. CONCLUSIONS: These results provide a new model for USP7 deubiquitinase activity through recruitment to oncogenic chromatin loci and regulation of both oncogenic transcription factors and chromatin marks to promote leukemia. Our studies also show that targeting USP7 inhibition could be a therapeutic strategy in aggressive leukemia.