The USP7-TRIM27 axis mediates non-canonical PRC1.1 function and is a druggable target in leukemia

In an attempt to unravel functionality of the non-canonical PRC1.1 Polycomb complex in human leukemogenesis, we show that USP7 and TRIM27 are integral components of PRC1.1. USP7 interactome analyses show that PRC1.1 is the predominant Polycomb complex co-precipitating with USP7. USP7 inhibition results in PRC1.1 disassembly and loss of chromatin binding, coinciding with reduced H2AK119ub and H3K27ac levels and diminished gene transcription of active PRC1.1-controlled loci, whereas H2AK119ub marks are also lost at PRC1 loci. TRIM27 and USP7 are reciprocally required for incorporation into PRC1.1, and TRIM27 knockdown partially rescues USP7 inhibitor sensitivity. USP7 inhibitors effectively impair proliferation in AML cells in vitro, also independent of the USP7-MDM2-TP53 axis, and MLL-AF9-induced leukemia is delayed in vivo in human leukemia xenografts. We propose a model where USP7 counteracts TRIM27 E3 ligase activity, thereby maintaining PRC1.1 integrity and function. Moreover, USP7 inhibition may be a promising new strategy to treat AML patients

Towards identification and targeting of Polycomb signaling pathways in leukemia

The development of leukemia is a multistep process that can be caused by multiple genetic and epigenetic changes which affect normal growth and differentiation of hematopoietic stem and progenitor cells and ultimately lead to full leukemic transformation. Despite most leukemia patients initially achieving successful remission after intensive treatment, the persistence of a rare population of chemotherapy-resistant leukemic stem cells (LSCs) can result in relapse of disease in a relatively large cohort of patients. The studies presented in this thesis were aimed to identify Polycomb signaling pathways in leukemia and whether they can be used to target and eradicate LSCs. Our data suggest an essential role for non-canonical PRC1.1 in controlling distinct gene sets involved in unique cell biological processes required for the maintenance of leukemic cells. We identified that USP7 is part of non-canonical PRC1.1 and its enzymatic activity is critically important to maintain complex integrity and function. Targeting of PRC1.1 strongly reduced cell proliferation of (primary) leukemic cells in vitro and delayed leukemogenesis in vivo. Next, we aimed to obtain insights into the mechanisms by which PRC1.1 might affect transcriptional control in leukemic cells. We observed that PRC1.1 is associated with restrictive and permissive chromatin states, indicating that transcriptional control is a complicated multifactorial process and that beyond PRC1.1 other regulators play clearly important roles as well. Future work will focus on the underlying mechanisms and cross-talk with chromatin regulators and transcriptional machinery. Furthermore, we established a human leukemia mouse model which allowed us to study timing of gene knockdown on the efficacy of leukemia treatment. These findings suggested that it is critical to study gene function during the development of leukemia to find potential new targets for leukemia treatment

Mitochondrial Dysfunction in Human Leukemic Stem/Progenitor Cells upon Loss of RAC2

<div><p>Leukemic stem cells (LSCs) reside within bone marrow niches that maintain their relatively quiescent state and convey resistance to conventional treatment. Many of the microenvironmental signals converge on RAC GTPases. Although it has become clear that RAC proteins fulfill important roles in the hematopoietic compartment, little has been revealed about the downstream effectors and molecular mechanisms. We observed that in BCR-ABL-transduced human hematopoietic stem/progenitor cells (HSPCs) depletion of RAC2 but not RAC1 induced a marked and immediate decrease in proliferation, progenitor frequency, cobblestone formation and replating capacity, indicative for reduced self-renewal. Cell cycle analyses showed reduced cell cycle activity in RAC2-depleted BCR-ABL leukemic cobblestones coinciding with an increased apoptosis. Moreover, a decrease in mitochondrial membrane potential was observed upon RAC2 downregulation, paralleled by severe mitochondrial ultrastructural malformations as determined by automated electron microscopy. Proteome analysis revealed that RAC2 specifically interacted with a set of mitochondrial proteins including mitochondrial transport proteins SAM50 and Metaxin 1, and interactions were confirmed in independent co-immunoprecipitation studies. Downregulation of SAM50 also impaired the proliferation and replating capacity of BCR-ABL-expressing cells, again associated with a decreased mitochondrial membrane potential. Taken together, these data suggest an important role for RAC2 in maintaining mitochondrial integrity.</p></div

shRAC2 from "Mitochondrial dysfunction in human leukemic stem/progenitor cells upon loss of RAC2"

<p>BCR-ABL and shRNA double-transduced cells expanded in stromal co-cultures for 7 days were harvested, pelleted and embedded in EPON, and ultrathin sections were analyzed by STEM. Large area scans of high resolution were acquired allowing evaluation of the ultrastructure of mitochondria in large number of control cells or shRAC2-transduced cells, representative images of the abnormalities in the structure of mitochondria are shown.10 nm pixel size screenshots from www.nanotomy.org.</p

RAC1 and RAC2 interact with distinct sets of proteins.

<p>K562 cells were transduced with retroviral constructs containing Avi-tagged RAC1 (Avi-RAC1) or RAC2 (Avi-RAC2) and BirA biotin ligase, or BirA-only control construct (BirA) sorted, and streptavidin-based pull-down assay was performed. Bound fractions were then used for mass spectrometry analysis to identify RAC1- or RAC2-specific interaction partners (experimental setup shown in A). (B) Efficiency of the pull-down assay was assessed by Western blot detecting Avi-tagged RAC1 and RAC2. T: total cell lysate fraction; B: bound fraction; NB: non-bound fraction. (C) Mass spectrometry analysis identified 335 RAC1-specific and 229-RAC2-specific peptides using 90% protein/90% peptide confidence cut-off. (D) Cytospins of GFP-RAC1- or GFP-RAC2-transduced TF-1 cells were analyzed for RAC localization. Syto62 was used to stain the nucleus.</p

RAC2 interaction with mitochondrial transport proteins is required for the long-term expansion of human BCR-ABL-expressing HSPCs.

<p>(A) Schematic representation of the mitochondrial transport complexes and their function. (B) K562 cells transduced with retroviral constructs containing Avi-tagged RAC2 (Avi-RAC2) and BirA biotin ligase, or BirA-only control construct (BirA) were sorted, and streptavidin-based pull-down assay was performed. Conversely, immunoprecipitation with anti-SAM50 antibody was performed. Alternatively, K562 cells were transduced with lentiviral GFP-tagged RAC2 (GFP-RAC2) and pull-down assay using GFP-affinity beads was performed. Empty GFP vector-transduced cells were used as a control. Efficiency of pull-down assay was assessed by Western blot detecting either SAM50 or Metaxin 1 (Streptavidin and GFP pull-down), or GFP (SAM50 pull-down). T: total cell lysate fraction; B: bound fraction; NB: non-bound fraction. (C) K562 cells transduced with the control scrambled shRNA vector (shSCR) or with the SAM50-targeting shRNA vector (shSAM50) were sorted and used for Western blot analysis to determine SAM50 protein levels. The quantification of protein expression normalized to control is indicated above each lane. (D) CB CD34⁺ stem/progenitor cells were double-transduced with BCR-ABL and either control shSCR or with shSAM50. 5*10³ double-transduced cells were sorted per group and plated on MS5 stromal cells. Cultures were demi-depopulated on indicated days for analysis and replated where indicated. Cumulative cell growth is shown for two representative experiments of 4 independent experiments. (E) 5*10³ BCR-ABL and shSCR or shSAM50 double-transduced cells were sorted and plated on MS5 stromal cells. Suspension cells were harvested after 14 days of co-culture and stained with DilC to measure changes in mitochondrial membrane potential. Representative FACS plots are shown. (F) Quantification of FACS measurements as described in panel (E) represented as changes in MFI relative to control is shown below. Average of four independent experiments is shown with standard deviation. * <i>P</i><0.05.</p