J de Boer

Crosstalk among Bcl-2 family members in B-CLL: seliciclib acts via the Mcl-1/Noxa axis and gradual exhaustion of Bcl-2 protection.

Seliciclib (R-roscovitine) is a cyclin-dependent kinase inhibitor in clinical development. It triggers apoptosis by inhibiting de novo transcription of the short-lived Mcl-1 protein, but it is unknown how this leads to Bax/Bak activation that is required for most forms of cell death. Here, we studied the effects of seliciclib in B-cell chronic lymphocytic leukemia (B-CLL), a malignancy with aberrant expression of apoptosis regulators. Although seliciclib-induced Mcl-1 degradation within 4 h, Bax/Bak activation occurred between 16 and 20 h. During this period, no transcriptional changes in apoptosis-related genes occurred. In untreated cells, prosurvival Mcl-1 was engaged by the proapoptotic proteins Noxa and Bim. Upon drug treatment, Bim was quickly released. The contribution of Noxa and Bim as a specific mediator of seliciclib-induced apoptosis was demonstrated via RNAi. Significantly, 16 h after seliciclib treatment, there was accumulation of Bcl-2, Bim and Bax in the ‘mitochondria-rich’ insoluble fraction of the cell. This suggests that after Mcl-1 degradation, the remaining apoptosis neutralizing capacity of Bcl-2 is gradually overwhelmed, until Bax forms large multimeric pores in the mitochondria. These data demonstrate in primary leukemic cells hierarchical binding and crosstalk among Bcl-2 members, and suggest that their functional interdependence can be exploited therapeutically.

In focus: MLL-rearranged leukemia

The molecular mechanisms underlying oncogenesis in leukemias associated with rearrangement of the Mixed Lineage Leukemia (MLL) gene have received a considerable amount of attention over the last two decades. In this review we will focus on recent studies, published over the past year, that reveal new insights into the multi-protein complexes formed by MLL and MLL fusion proteins, the role of epigenetic deregulation in MLL fusion function, downstream transcriptional target genes, the importance of the leukemia cell of origin, the role played by microRNAs, cooperating mutations and the implications that recent research has for the therapy of MLL-rearranged leukemia.

Tnfaip8 is an essential gene for the regulation of glucocorticoid-mediated apoptosis of thymocytes

Glucocorticoids have significant immunoregulatory actions on thymocytes and T cells and act by binding and activating cytosolic glucocorticoid receptors, which translocate to the nucleus and control gene expression through binding to specific response elements in target genes. Glucocorticoids promote cell death by activating an apoptotic program that requires transcriptional regulation. We set out to identify genes that are crucial to the process of glucocorticoid-mediated thymocyte apoptosis. Freshly isolated murine primary thymocytes were treated with dexamethasone, mRNA isolated and used to screen DNA microarrays. A set of candidate genes with upregulated expression was identified and selected members assayed in reconstituted fetal thymic organ culture (FTOC). Fetal liver-derived hematopoietic progenitor cells (HPCs) were infected with retroviruses expressing individual genes then used to repopulate depleted fetal thymic lobes. Reconstituted FTOCs expressing the gene Tnfaip8 were treated with dexamethasone and shown to be greatly sensitized to dexamethasone. Retrovirus-mediated RNA interference was applied to knock down Tnfaip8 expression in HPCs and these were used to reconstitute FTOCs. We observed that downregulating the expression of Tnfaip8 alone was sufficient to effectively protect thymocytes against glucocorticoid-induced apoptosis. We propose that Tnfaip8 is crucial in regulating glucocorticoid-mediated apoptosis of thymocytes.

Autophagy limits proliferation and glycolytic metabolism in acute myeloid leukemia.

Decreased autophagy contributes to malignancies; however, it is unclear how autophagy has an impact on tumor growth. Acute myeloid leukemia (AML) is an ideal model to address this as (i) patient samples are easily accessible, (ii) the hematopoietic stem and progenitor cells (HSPC) where transformation occurs is well characterized and (iii) loss of the key autophagy gene Atg7 in HSPCs leads to a lethal pre-leukemic phenotype in mice. Here we demonstrate that loss of Atg5 results in an identical HSPC phenotype as loss of Atg7, confirming a general role for autophagy in HSPC regulation. Compared with more committed/mature hematopoietic cells, healthy human and mouse HSPCs displayed enhanced basal autophagic flux, limiting mitochondrial damage and reactive oxygen species in this long-lived population. Taken together, with our previous findings these data are compatible with autophagy-limiting leukemic transformation. In line with this, autophagy gene losses are found within chromosomal regions that are commonly deleted in human AML. Moreover, human AML blasts showed reduced expression of autophagy genes and displayed decreased autophagic flux with accumulation of unhealthy mitochondria, indicating that deficient autophagy may be beneficial to human AML. Crucially, heterozygous loss of autophagy in an MLL–ENL model of AML led to increased proliferation in vitro, a glycolytic shift and more aggressive leukemias in vivo. With autophagy gene losses also identified in multiple other malignancies, these findings point to low autophagy, providing a general advantage for tumor growth.

Elevated S100A8/S100A9 expression causes glucocorticoid resistance in MLL-rearranged infant acute lymphoblastic leukemia

MLL-rearranged acute lymphoblastic leukemia (ALL) in infants is characterized by a poor clinical outcome and resistance to glucocorticoids (for example, prednisone and dexamethasone). As both the response to prednisolone in vitro and prednisone in vivo are predictive for clinical outcome, understanding and overcoming glucocorticoid resistance remains an essential step towards improving prognosis. Prednisolone-induced apoptosis depends on glucocorticoid-evoked Ca2+ fluxes from the endoplasmic reticulum towards the mitochondria. Here, we demonstrate that in MLL-rearranged infant ALL, over-expression of S100A8 and S100A9 is associated with failure to induce free-cytosolic Ca2+ and prednisolone resistance. Furthermore, we demonstrate that enforced expression of S100A8/S100A9 in prednisolone-sensitive MLL-rearranged ALL cells, rapidly leads to prednisolone resistance as a result of S100A8/S100A9 mediated suppression of prednisolone-induced free-cytosolic Ca2+ levels. In addition, the Src kinase inhibitor PP2 markedly sensitized MLL-rearranged ALL cells otherwise resistant to prednisolone, via downregulation of S100A8 and S100A9, which allowed prednisolone-induced Ca2+ fluxes to reach the mitochondria and trigger apoptosis. On the basis of this novel mechanism of prednisolone resistance, we propose that developing more specific S100A8/S100A9 inhibitors may well be beneficial for prednisolone-resistant MLL-rearranged infant ALL patients.

Dysregulation of the HIF pathway due to VHL mutation causing severe erythrocytosis and pulmonary arterial hypertension.

To the editor: Hereditary erythrocytosis can be caused by mutations in genes involved in the hypoxia-inducible factor (HIF) pathway.[1][1]–[3][2] For example, Chuvash polycythemia is caused by an R200W substitution in the von Hippel–Lindau protein (VHL).[1][1] There is increasing evidence

The E2A-HLF oncogenic fusion protein acts through Lmo2 and Bcl-2 to immortalize hematopoietic progenitors.

The oncogenic fusion protein E2A–HLF is a chimeric transcription factor that arises from the t(17;19) translocation in childhood B-cell acute lymphoblastic leukemias (B-precursor ALL) and is associated with very poor outcome. We show that retroviral-mediated expression of E2A–HLF alone is sufficient to immortalize primary lymphoid progenitors. We identify Lmo2 and Bcl-2 as direct target genes downstream of E2A–HLF. We use real-time PCR analysis to show that LMO2 and BCL-2 expression is preferentially upregulated both in biopsy material from t(17;19) B-precursor ALL patients and lymphoid cell lines derived from t(17;19) leukemias. Co-expression of Lmo2 and Bcl-2 was sufficient to immortalize lymphoid progenitor cells resulting in a similar phenotype to that induced by E2A–HLF alone. Both shRNA-mediated knockdown of Lmo2 expression and pharmacological inhibition of BCL-2 function in E2A–HLF immortalized cells severely compromised their viability. These data suggest that both Lmo2 and Bcl-2 are required for the action of E2A–HLF in leukemogenesis.

The AAA(+) ATPase RUVBL2 is a critical mediator of MLL-AF9 oncogenesis

The most frequent chromosomal translocations in pediatric acute myeloid leukemia affect the 11q23 locus and give rise to mixed lineage leukemia (MLL) fusion genes, MLL-AF9 being the most prevalent. The MLL-AF9 fusion gene has been shown to induce leukemia in both mouse and human models. In this study, we demonstrate that leukemogenic activity of MLL-AF9 requires RUVBL2 (RuvB-like 2), an AAA+ ATPase family member that functions in a wide range of cellular processes, including chromatin remodeling and transcriptional regulation. Expression of RUVBL2 was dependent on MLL-AF9, as it increased upon immortalization of human cord blood-derived hematopoietic progenitor cells with the fusion gene and decreased following loss of fusion gene expression in conditionally immortalized mouse cells. Short hairpin RNA-mediated silencing experiments demonstrated that both the immortalized human cells and the MLL-AF9-expressing human leukemia cell line THP-1 required RUVBL2 expression for proliferation and survival. Furthermore, inhibition of RUVBL2 expression in THP-1 cells led to reduced telomerase activity and clonogenic potential. These data were confirmed with a dominant-negative Walker B-mutated RUVBL2 construct. Taken together, these data suggest the possibility of targeting RUVBL2 as a potential therapeutic strategy for MLL-AF9-associated leukemia.

Targeting acute myeloid leukemia by drug-induced c-MYB degradation

Despite advances in our understanding of the molecular basis for particular subtypes of acute myeloid leukemia (AML), effective therapy remains a challenge for many individuals suffering from this disease. A significant proportion of both pediatric and adult AML patients cannot be cured and since the upper limits of chemotherapy intensification have been reached, there is an urgent need for novel therapeutic approaches. The transcription factor c-MYB has been shown to play a central role in the development and progression of AML driven by several different oncogenes, including mixed lineage leukemia (MLL)-fusion genes. Here, we have used a c-MYB gene expression signature from MLL-rearranged AML to probe the Connectivity Map database and identified mebendazole as a c-MYB targeting drug. Mebendazole induces c-MYB degradation via the proteasome by interfering with the heat shock protein 70 (HSP70) chaperone system. Transient exposure to mebendazole is sufficient to inhibit colony formation by AML cells, but not normal cord blood-derived cells. Furthermore, mebendazole is effective at impairing AML progression in vivo in mouse xenotransplantation experiments. In the context of widespread human use of mebendazole, our data indicate that mebendazole-induced c-MYB degradation represents a safe and novel therapeutic approach for AML.

NPM-MLF1 synergizes with Npm haploinsufficiency to enhance myeloid progenitor activity.

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