Jennifer Jaques

MLL-AF9-mediated immortalization of human hematopoietic cells along different lineages changes during ontogeny

The MLL–AF9 fusion gene is associated with aggressive leukemias of both the myeloid and lymphoid lineage in infants, whereas in adults, this translocation is mainly associated with acute myeloid leukemia. These observations suggest that differences exist between fetal and adult tissues in terms of the ‘cell of origin’ from which the leukemia develops. Here we show that depending on extrinsic cues, human neonatal CD34+ cells are readily immortalized along either the myeloid or lymphoid lineage upon MLL–AF9 expression and give rise to mainly lymphoid leukemia in immunocompromised mice. In contrast, immortalization of adult bone marrow CD34+ cells is more difficult to achieve and is myeloid-biased, even when MLL–AF9 is expressed in purified hematopoietic stem cells (HSCs). Transcriptome analysis identified enrichment of HSC but not progenitor gene signatures in MLL–AF9-expressing cells. Although not observed in adult cells, neonatal cells expressing MLL–AF9 were enriched for gene signatures associated with poor prognosis, resistance to chemotherapeutic agents and MYC signaling. These results indicate that neonatal cells are inherently more prone to MLL–AF9-mediated immortalization than adult cells and suggest that intrinsic properties of the cell of origin, in addition to extrinsic cues, dictate lineage of the immortalized cell.

The TAK1-NF-κB axis as therapeutic target for AML

Development and maintenance of leukemia can be partially attributed to alterations in (anti)-apoptotic gene expression. Genome-wide transcriptome analyses revealed that 89 apoptosis-associated genes were differentially expressed between patient acute myeloid leukemia (AML) CD34(+) cells and normal bone marrow (NBM) CD34(+) cells. Among these, transforming growth factor-β activated kinase 1 (TAK1) was strongly upregulated in AML CD34(+) cells. Genetic downmodulation or pharmacologic inhibition of TAK1 activity strongly impaired primary AML cell survival and cobblestone formation in stromal cocultures. TAK1 inhibition was mainly due to blockade of the nuclear factor κB (NF-κB) pathway, as TAK1 inhibition resulted in reduced levels of P-IκBα and p65 activity. Overexpression of a constitutive active variant of NF-κB partially rescued TAK1-depleted cells from apoptosis. Importantly, NBM CD34(+) cells were less sensitive to TAK1 inhibition compared with AML CD34(+) cells. Knockdown of TAK1 also severely impaired leukemia development in vivo and prolonged overall survival in a humanized xenograft mouse model. In conclusion, our results indicate that TAK1 is frequently overexpressed in AML CD34(+) cells, and that TAK1 inhibition efficiently targets leukemic stem/progenitor cells in an NF-κB-dependent manner.

Modeling BCR-ABL and MLL-AF9 leukemia in a human bone marrow-like scaffold based xenograft model

Although NOD-SCID IL2Rγ−/− (NSG) xenograft mice are currently the most frequently used model to study human leukemia in vivo, the absence of a human niche severely hampers faithful recapitulation of the disease. We used NSG mice in which ceramic scaffolds seeded with human mesenchymal stromal cells were implanted to generate a human bone marrow (huBM-sc)-like niche. We observed that, in contrast to the murine bone marrow (mBM) niche, the expression of BCR-ABL or MLL-AF9 was sufficient to induce both primary acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL). Stemness was preserved within the human niches as demonstrated by serial transplantation assays. Efficient engraftment of AML MLL-AF9 and blast-crisis chronic myeloid leukemia patient cells was also observed, whereby the immature blast-like phenotype was maintained in the huBM-sc niche but to a much lesser extent in mBM niches. We compared transcriptomes of leukemias derived from mBM niches versus leukemias from huBM-like scaffold-based niches, which revealed striking differences in the expression of genes associated with hypoxia, mitochondria and metabolism. Finally, we utilized the huBM-sc MLL-AF9 B-ALL model to evaluate the efficacy of the I-BET151 inhibitor in vivo. In conclusion, we have established human niche models in which the myeloid and lymphoid features of BCR-ABL+ and MLL-AF9+ leukemias can be studied in detail.

Inhibition of autophagy as a treatment strategy for p53 wild-type acute myeloid leukemia

Here we have explored whether inhibition of autophagy can be used as a treatment strategy for acute myeloid leukemia (AML). Steady-state autophagy was measured in leukemic cell lines and primary human CD34+ AML cells with a large variability in basal autophagy between AMLs observed. The autophagy flux was higher in AMLs classified as poor risk, which are frequently associated with TP53 mutations (TP53mut), compared with favorable- and intermediate-risk AMLs. In addition, the higher flux was associated with a higher expression level of several autophagy genes, but was not affected by alterations in p53 expression by knocking down p53 or overexpression of wild-type p53 or p53R273H. AML CD34+ cells were more sensitive to the autophagy inhibitor hydroxychloroquine (HCQ) than normal bone marrow CD34+ cells. Similar, inhibition of autophagy by knockdown of ATG5 or ATG7 triggered apoptosis, which coincided with increased expression of p53. In contrast to wild-type p53 AML (TP53wt), HCQ treatment did not trigger a BAX and PUMA-dependent apoptotic response in AMLs harboring TP53mut. To further characterize autophagy in the leukemic stem cell-enriched cell fraction AML CD34+ cells were separated into ROSlow and ROShigh subfractions. The immature AML CD34+-enriched ROSlow cells maintained higher basal autophagy and showed reduced survival upon HCQ treatment compared with ROShigh cells. Finally, knockdown of ATG5 inhibits in vivo maintenance of AML CD34+ cells in NSG mice. These results indicate that targeting autophagy might provide new therapeutic options for treatment of AML since it affects the immature AML subfraction.

Hypoxia-Like Signatures Induced by BCR-ABL Potentially Alter the Glutamine Uptake for Maintaining Oxidative Phosphorylation

The Warburg effect is probably the most prominent metabolic feature of cancer cells, although little is known about the underlying mechanisms and consequences. Here, we set out to study these features in detail in a number of leukemia backgrounds. The transcriptomes of human CB CD34+ cells transduced with various oncogenes, including BCR-ABL, MLL-AF9, FLT3-ITD, NUP98-HOXA9, STAT5A and KRASG12V were analyzed in detail. Our data indicate that in particular BCR-ABL, KRASG12V and STAT5 could impose hypoxic signaling under normoxic conditions. This coincided with an upregulation of glucose importers SLC2A1/3, hexokinases and HIF1 and 2. NMR-based metabolic profiling was performed in CB CD34+ cells transduced with BCR-ABL versus controls, both cultured under normoxia and hypoxia. Lactate and pyruvate levels were increased in BCR-ABL-expressing cells even under normoxia, coinciding with enhanced glutaminolysis which occurred in an HIF1/2-dependent manner. Expression of the glutamine importer SLC1A5 was increased in BCR-ABL+ cells, coinciding with an increased susceptibility to the glutaminase inhibitor BPTES. Oxygen consumption rates also decreased upon BPTES treatment, indicating a glutamine dependency for oxidative phosphorylation. The current study suggests that BCR-ABL-positive cancer cells make use of enhanced glutamine metabolism to maintain TCA cell cycle activity in glycolytic cells.

Identification of USP7 as an essential component to maintain integrity and function of non-canonical PRC1.1 in leukemia

Acute myeloid leukemia (AML) is a highly heterogeneous disease in which genetic and epigenetic changes disturb regulatory mechanisms controlling stem cell fate and maintenance. AML still remains difficult to treat, in particular in poor risk AML patients carrying TP53 mutations. Here, we identify the deubiquitinase USP7 as an integral member of non-canonical PRC1.1 and show that targeting of USP7 provides an alternative therapeutic approach for AML. USP7 inhibitors effectively induced apoptosis in (primary) AML cells, also independent of the USP7-MDM2-TP53 axis, whereby survival of both the cycling as well as quiescent populations was affected. MLL-AF9-induced leukemia was significantly delayed in vivo in human leukemia xenografts. We previously showed that non-canonical PRC1.1 is critically important for leukemic stem cell self-renewal, and that genetic knockdown of the PRC1.1 chromatin binding component KDM2B abrogated leukemia development in vitro and in vivo [1]. Here, by performing KDM2B interactome studies in TP53mut cells we identify that USP7 is an essential component of PRC1.1 and is required for its stability and function. USP7 inhibition results in disassembly of the PRC1.1 complex and consequently loss of binding to its target loci. Loss of PRC1.1 binding coincided with reduced H2AK119ub and H3K27ac levels and diminished gene transcription, whereas H3K4me3 levels remained unaffected. Our studies highlight the diverse functions of USP7 and link it to Polycomb-mediated epigenetic control. USP7 inhibition provides an efficient therapeutic approach for AML, also in the most aggressive subtypes with mutations in TP53. Key points USP7 is a therapeutic target in leukemia, including poor risk TP53mut AML. USP7 is an essential component of non-canonical PRC1.1 and is required for its stability and function.Polycomb proteins are essential epigenetic regulators of gene transcription. KDM2B, the chromatin-binding moiety of non-canonical PRC1.1, is critically important for human leukemias. Here, we investigated the complete interactome of KDM2B in human leukemic cells and identified that the deubiquitinase USP7 is an essential component of PRC1.1 and required for its stability and function. USP7 inhibition results in disassembly of the PRC1.1 complex and consequently loss of binding to its target loci. PRC1.1 can be associated with active loci and loss of PRC1.1 binding coincided with loss of H2AK119ub, reduced H3K27ac levels and reduced gene transcription, whereas H3K4me3 levels remained unaffected. Survival was reduced in (primary) acute myeloid leukemia cells in both cycling as well as quiescent populations upon USP7 inhibition, also independent of the USP7-MDM2-p53 axis. Finally, we evaluated the efficacy of USP7 inhibition in vivo and find that progression of MLL-AF9-induced leukemia is delayed, although in a niche-dependent manner.