Dietary methionine starvation impairs acute myeloid leukemia progression.

Targeting altered tumor cell metabolism might provide an attractive opportunity for patients with acute myeloid leukemia (AML). An amino acid dropout screen on primary leukemic stem cells and progenitor populations revealed a number of amino acid dependencies, of which methionine was one of the strongest. By using various metabolite rescue experiments, nuclear magnetic resonance-based metabolite quantifications and 13C-tracing, polysomal profiling, and chromatin immunoprecipitation sequencing, we identified that methionine is used predominantly for protein translation and to provide methyl groups to histones via S-adenosylmethionine for epigenetic marking. H3K36me3 was consistently the most heavily impacted mark following loss of methionine. Methionine depletion also reduced total RNA levels, enhanced apoptosis, and induced a cell cycle block. Reactive oxygen species levels were not increased following methionine depletion, and replacement of methionine with glutathione or N-acetylcysteine could not rescue phenotypes, excluding a role for methionine in controlling redox balance control in AML. Although considered to be an essential amino acid, methionine can be recycled from homocysteine. We uncovered that this is primarily performed by the enzyme methionine synthase and only when methionine availability becomes limiting. In vivo, dietary methionine starvation was not only tolerated by mice, but also significantly delayed both cell line and patient-derived AML progression. Finally, we show that inhibition of the H3K36-specific methyltransferase SETD2 phenocopies much of the cytotoxic effects of methionine depletion, providing a more targeted therapeutic approach. In conclusion, we show that methionine depletion is a vulnerability in AML that can be exploited therapeutically, and we provide mechanistic insight into how cells metabolize and recycle methionine.

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.

Protein quality control in the nucleolus safeguards recovery of epigenetic regulators after heat shock.

Maintenance of epigenetic modifiers is of utmost importance to preserve the epigenome and consequently appropriate cellular functioning. Here, we analyzed Polycomb group protein (PcG) complex integrity in response to heat shock (HS). Upon HS, various Polycomb Repressive Complex (PRC)1 and PRC2 subunits, including CBX proteins, but also other chromatin regulators, are found to accumulate in the nucleolus. In parallel, binding of PRC1/2 to target genes is strongly reduced, coinciding with a dramatic loss of H2AK119ub and H3K27me3 marks. Nucleolar-accumulated CBX proteins are immobile, but remarkably both CBX protein accumulation and loss of PRC1/2 epigenetic marks are reversible. This post-heat shock recovery of pan-nuclear CBX protein localization and reinstallation of epigenetic marks is HSP70 dependent. Our findings demonstrate that the nucleolus is an essential protein quality control center, which is indispensable for recovery of epigenetic regulators and maintenance of the epigenome after heat shock.

Non-canonical PRC1.1 Targets Active Genes Independent of H3K27me3 and Is Essential for Leukemogenesis.

Polycomb proteins are classical regulators of stem cell self-renewal and cell lineage commitment and are frequently deregulated in cancer. Here, we find that the non-canonical PRC1.1 complex, as identified by mass-spectrometry-based proteomics, is critically important for human leukemic stem cells. Downmodulation of PRC1.1 complex members, like the DNA-binding subunit KDM2B, strongly reduces cell proliferation in vitro and delays or even abrogates leukemogenesis in vivo in humanized xenograft models. PRC1.1 components are significantly overexpressed in primary AML CD34(+) cells. Besides a set of genes that is targeted by PRC1 and PRC2, ChIP-seq studies show that PRC1.1 also binds a distinct set of genes that are devoid of H3K27me3, suggesting a gene-regulatory role independent of PRC2. This set encompasses genes involved in metabolism, which have transcriptionally active chromatin profiles. These data indicate that PRC1.1 controls specific genes involved in unique cell biological processes required for leukemic cell viability.

Conservation of inner nuclear membrane targeting sequences in mammalian Pom121 and yeast Heh2 membrane proteins.

Endoplasmic reticulum-synthesized membrane proteins traffic through the nuclear pore complex (NPC) en route to the inner nuclear membrane (INM). Although many membrane proteins pass the NPC by simple diffusion, two yeast proteins, ScSrc1/ScHeh1 and ScHeh2, are actively imported. In these proteins, a nuclear localization signal (NLS) and an intrinsically disordered linker encode the sorting signal for recruiting the transport factors for FG-Nup and RanGTP-dependent transport through the NPC. Here we address whether a similar import mechanism applies in metazoans. We show that the (putative) NLSs of metazoan HsSun2, MmLem2, HsLBR, and HsLap2ß are not sufficient to drive nuclear accumulation of a membrane protein in yeast, but the NLS from RnPom121 is. This NLS of Pom121 adapts a similar fold as the NLS of Heh2 when transport factor bound and rescues the subcellular localization and synthetic sickness of Heh2ΔNLS mutants. Consistent with the conservation of these NLSs, the NLS and linker of Heh2 support INM localization in HEK293T cells. The conserved features of the NLSs of ScHeh1, ScHeh2, and RnPom121 and the effective sorting of Heh2-derived reporters in human cells suggest that active import is conserved but confined to a small subset of INM proteins.

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

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.

Nonredundant and locus-specific gene repression functions of PRC1 paralog family members in human hematopoietic stem/progenitor cells.

The Polycomb group (PcG) protein BMI1 is a key factor in regulating hematopoietic stem cell (HSC) and leukemic stem cell self-renewal and functions in the context of the Polycomb repressive complex 1 (PRC1). In humans, each of the 5 subunits of PRC1 has paralog family members of which many reside in PRC1 complexes, likely in a mutually exclusive manner, pointing toward a previously unanticipated complexity of Polycomb-mediated silencing. We used an RNA interference screening approach to test the functionality of these paralogs in human hematopoiesis. Our data demonstrate a lack of redundancy between various paralog family members, suggestive of functional diversification between PcG proteins. By using an in vivo biotinylation tagging approach followed by liquid chromatography-tandem mass spectrometry to identify PcG interaction partners, we confirmed the existence of multiple specific PRC1 complexes. We find that CBX2 is a nonredundant CBX paralog vital for HSC and progenitor function that directly regulates the expression of the cyclin-dependent kinase inhibitor p21, independently of BMI1 that dominantly controls expression of the INK4A/ARF locus. Taken together, our data show that different PRC1 paralog family members have nonredundant and locus-specific gene regulatory activities that are essential for human hematopoiesis.

The combination of valproic acid and lithium delays hematopoietic stem/progenitor cell differentiation.

Despite increasing knowledge on the regulation of hematopoietic stem/progenitor cell (HSPC) self-renewal and differentiation, in vitro control of stem cell fate decisions has been difficult. The ability to inhibit HSPC commitment in culture may be of benefit to cell therapy protocols. Small molecules can serve as tools to manipulate cell fate decisions. Here, we tested 2 small molecules, valproic acid (VPA) and lithium (Li), to inhibit differentiation. HSPCs exposed to VPA and Li during differentiation-inducing culture preserved an immature cell phenotype, provided radioprotection to lethally irradiated recipients, and enhanced in vivo repopulating potential. Anti-differentiation effects of VPA and Li were observed also at the level of committed progenitors, where VPA re-activated replating activity of common myeloid progenitor and granulocyte macrophage progenitor cells. Furthermore, VPA and Li synergistically preserved expression of stem cell-related genes and repressed genes involved in differentiation. Target genes were collectively co-regulated during normal hematopoietic differentiation. In addition, transcription factor networks were identified as possible primary regulators. Our results show that the combination of VPA and Li potently delays differentiation at the biologic and molecular levels and provide evidence to suggest that combinatorial screening of chemical compounds may uncover possible additive/synergistic effects to modulate stem cell fate decisions.

Genetic and epigenetic alterations that drive leukemic stem cell self-renewal.

Acute myeloid leukemia has emerged as a paradigm for the concept of the cancer stem cell. This hypothesis presumes that the disease is maintained by a rare population of leukemia-initiating stem cells which have acquired genetic or epigenetic changes. It is most likely that a single (epi)genetic event will not be sufficient to cause leukemia, but that a number of sequential events are required. Similar to normal hematopoietic stem cells, both intrinsic as well as extrinsic factors that arise from the bone marrow niche, provide essential cues that regulate cell fate decisions such as leukemic stem cell self-renewal and differentiation. In this chapter, we will review the current understanding of genetic and epigenetic abnormalities that underlie the process of leukemic transformation, and will discuss which events potentially co-operate to induce leukemia.

BMI1 collaborates with BCR-ABL in leukemic transformation of human CD34+ cells.

The major limitation for the development of curative cancer therapies has been an incomplete understanding of the molecular mechanisms driving cancer progression. Human models to study the development and progression of chronic myeloid leukemia (CML) have not been established. Here, we show that BMI1 collaborates with BCR-ABL in inducing a fatal leukemia in nonobese diabetic/severe combined immunodeficiency mice transplanted with transduced human CD34(+) cells within 4-5 months. The leukemias were transplantable into secondary recipients with a shortened latency of 8-12 weeks. Clonal analysis revealed that similar clones initiated leukemia in primary and secondary mice. In vivo, transformation was biased toward a lymphoid blast crisis, and in vitro, myeloid as well as lymphoid long-term, self-renewing cultures could be established. Retroviral introduction of BMI1 in primary chronic-phase CD34(+) cells from CML patients elevated their proliferative capacity and self-renewal properties. Thus, our data identify BMI1 as a potential therapeutic target in CML.

Undifferentiated embryonic cell transcription factor 1 regulates ESC chromatin organization and gene expression.

Previous reports showed that embryonic stem (ES) cells contain hyperdynamic and globally transcribed chromatin-properties that are important for ES cell pluripotency and differentiation. Here, we demonstrate a role for undifferentiated embryonic cell transcription factor 1 (UTF1) in regulating ES cell chromatin structure. Using chromatin immunoprecipitation-on-chip analysis, we identified >1,700 UTF1 target genes that significantly overlap with previously identified Nanog, Oct4, Klf-4, c-Myc, and Rex1 targets. Gene expression profiling showed that UTF1 knock down results in increased expression of a large set of genes, including a significant number of UTF1 targets. UTF1 knock down (KD) ES cells are, irrespective of the increased expression of several self-renewal genes, Leukemia inhibitory factor (LIF) dependent. However, UTF1 KD ES cells are perturbed in their differentiation in response to dimethyl sulfoxide (DMSO) or after LIF withdrawal and display increased colony formation. UTF1 KD ES cells display extensive chromatin decondensation, reflected by a dramatic increase in nucleosome release on micrococcal nuclease (MNase) treatment and enhanced MNase sensitivity of UTF1 target genes in UTF1 KD ES cells. Summarizing, our data show that UTF1 is a key chromatin component in ES cells, preventing ES cell chromatin decondensation, and aberrant gene expression; both essential for proper initiation of lineage-specific differentiation of ES cells.

UTF1 is a chromatin-associated protein involved in ES cell differentiation.

Embryonic stem (ES) cells are able to grow indefinitely (self-renewal) and have the potential to differentiate into all adult cell types (pluripotency). The regulatory network that controls pluripotency is well characterized, whereas the molecular basis for the transition from self-renewal to the differentiation of ES cells is much less understood, although dynamic epigenetic gene silencing and chromatin compaction are clearly implicated. In this study, we report that UTF1 (undifferentiated embryonic cell transcription factor 1) is involved in ES cell differentiation. Knockdown of UTF1 in ES and carcinoma cells resulted in a substantial delay or block in differentiation. Further analysis using fluorescence recovery after photobleaching assays, subnuclear fractionations, and reporter assays revealed that UTF1 is a stably chromatin-associated transcriptional repressor protein with a dynamic behavior similar to core histones. An N-terminal Myb/SANT domain and a C-terminal domain containing a putative leucine zipper are required for these properties of UTF1. These data demonstrate that UTF1 is a strongly chromatin-associated protein involved in the initiation of ES cell differentiation.

DNA damage stabilizes interaction of CSB with the transcription elongation machinery.

The Cockayne syndrome B (CSB) protein is essential for transcription-coupled DNA repair (TCR), which is dependent on RNA polymerase II elongation. TCR is required to quickly remove the cytotoxic transcription-blocking DNA lesions. Functional GFP-tagged CSB, expressed at physiological levels, was homogeneously dispersed throughout the nucleoplasm in addition to bright nuclear foci and nucleolar accumulation. Photobleaching studies showed that GFP-CSB, as part of a high molecular weight complex, transiently interacts with the transcription machinery. Upon (DNA damage-induced) transcription arrest CSB binding these interactions are prolonged, most likely reflecting actual engagement of CSB in TCR. These findings are consistent with a model in which CSB monitors progression of transcription by regularly probing elongation complexes and becomes more tightly associated to these complexes when TCR is active.

When machines get stuck--obstructed RNA polymerase II: displacement, degradation or suicide.

The severe hereditary progeroid disorder Cockayne syndrome is a consequence of a defective transcription-coupled repair (TCR) pathway. This special mode of DNA repair aids a RNA polymerase that is stalled by a DNA lesion in the template and ensures efficient DNA repair to permit resumption of transcription and prevent cell death. Although some key players in TCR, such as the Cockayne syndrome A (CSA) and B (CSB) proteins have been identified, the exact molecular mechanism still remains illusive. A recent report provides new unexpected insights into TCR in yeast. The identification and characterisation of a novel protein co-purifying with the yeast homologue of CSB (Rad26) imposes reassessment of our current understanding of TCR in yeast. What about humans?