Emmanuelle Passegué

AP-1 in mouse development and tumorigenesis.

Genetically modified mice have provided important insights into the biological functions of the dimeric transcription factor complex AP-1. Extensive analyses of mice and cells with genetically modified Fos or Jun proteins provide novel insights into the physiological functions of AP-1 proteins. Using knock-out strategies it was found that some components, such as c-Fos, FosB and JunD are dispensable, whereas others, like c-Jun, JunB and Fra-1 are essential in embryonic development and/or in the adult organism. Besides the specific roles of AP-1 proteins in developmental processes, we are beginning to obtain a better molecular understanding of the cell-context dependent function of AP-1 in cell proliferation and apoptosis, in bone biology as well as in multistep tumorigenesis.

JunB Deficiency Leads to a Myeloproliferative Disorder Arising from Hematopoietic Stem Cells

The AP-1 transcription factor JunB is a transcriptional regulator of myelopoiesis. Inactivation of JunB in postnatal mice results in a myeloproliferative disorder (MPD) resembling early human chronic myelogenous leukemia (CML). Here, we show that JunB regulates the numbers of hematopoietic stem cells (HSC). JunB overexpression decreases the frequency of long-term HSC (LT-HSC), while JunB inactivation specifically expands the numbers of LT-HSC and granulocyte/macrophage progenitors (GMP) resulting in chronic MPD. Further, we demonstrate that junB inactivation must take place in LT-HSC, and not at later stages of myelopoiesis, to induce MPD and that only junB-deficient LT-HSC are capable of transplanting the MPD to recipient mice. These results demonstrate a stem cell-specific role for JunB in normal and leukemic hematopoiesis and provide experimental evidence that leukemic stem cells (LSC) can reside at the LT-HSC stage of development in a mouse model of MPD.

JunB suppresses cell proliferation by transcriptional activation of p16INK4a expression

A role for the transcription factor JunB in proliferation control was investigated in genetically modified mouse fibroblasts. Increased JunB expression induced high levels of the cyclin‐dependent kinase inhibitor p16INK4a, leading to premature senescence in primary cells and reduced proliferation in 3T3 cells, whereas lack of JunB expression results in decreased p16 levels. Furthermore, JunB‐mediated p16 induction in 3T3 cells completely abolished cyclin D‐associated kinase activity, resulting in reduced pRb hyperphosphorylation and G1‐phase extension. Moreover, three AP1‐like binding sites were identified in the p16 promoter through which JunB directly activates p16 transcription. Elevated JunB expression in 3T3 cells also inhibited Ras‐ and Src‐mediated transformation and tumour growth in vivo. The suppressive effect of JunB on cell proliferation was shown to be dependent on p16 since it did not occur in INK4a−/− fibroblasts that lack both p16 and p19ARF. These results demonstrate that p16 is a direct transcriptional target gene of JunB and identify JunB as a negative regulator of cell proliferation.

Chronic Myeloid Leukemia with Increased Granulocyte Progenitors in Mice Lacking JunB Expression in the Myeloid Lineage

The functions of JunB during myelopoiesis were studied in vivo. Transgenic mice specifically lacking JunB expression in the myeloid lineage (junB(-/-)Ubi-junB mice) develop a transplantable myeloproliferative disease eventually progressing to blast crisis, which resembles human chronic myeloid leukemia. Similarly, mice reconstituted with ES cell-derived junB-/- fetal liver cells also develop a myeloproliferative disease. In both cases, the absence of JunB expression results in increased numbers of granulocyte progenitors, which display enhanced GM-CSF-mediated proliferation and extended survival, associated with changes in the expression levels of the GM-CSFalpha receptor, the anti-apoptotic proteins Bcl2 and Bclx, and the cell cycle regulators p16(INK4a) and c-Jun. Importantly, ectopic expression of JunB fully reverts the immature and hyperproliferative phenotype of JunB-deficient myeloid cells. These results identify JunB as a key transcriptional regulator of myelopoiesis and a potential tumor suppressor gene.

MLL-GAS7 transforms multipotent hematopoietic progenitors and induces mixed lineage leukemias in mice

A specific association with mixed lineage leukemias suggests that MLL oncoproteins may selectively target early multipotent hematopoietic progenitors or stem cells. We demonstrate here that a representative MLL fusion protein, MLL-GAS7, impairs the differentiation and enhances the in vitro growth of murine hematopoietic cells with multipotent features. The multilineage differentiation potential of these cells was suggested by their immuno-phenotypes and transcriptional programs and confirmed by their ability to induce three pathologically distinct leukemias in mice, including an acute biphenotypic leukemia (ABL) that recapitulates the distinctive hallmark features of many MLL-associated leukemias in humans. This experimental modeling of ABL in mice highlights its origin from multipotential progenitors that arrest at a bipotential stage specifically targeted or induced by MLL oncogenes.

New Evidence Supporting Megakaryocyte-Erythrocyte Potential of Flk2/Flt3+ Multipotent Hematopoietic Progenitors

A model of hematopoietic development wherein multipotentiality is conserved until segregation of myeloid and lymphoid potential has recently been challenged, proposing that megakaryocyte/erythrocyte (MegE) potential is lost in Flk2/Flt3-expressing early progenitors. Here, we used sensitive in vivo approaches to quantitatively and kinetically assess the MegE potential of hematopoietic stem cells and various Flk2(+) early progenitors and compared it with the MegE potential of downstream committed myeloid and lymphoid progenitors and with their ability to give rise to mature myelomonocytic and lymphoid cells. We demonstrate that Flk2(+) early progenitors retain MegE potential in vivo both at the population and clonal levels. These results indicate that Flk2 expression by early progenitors is not at the expense of full multipotency and support the current model of hematopoietic development with segregation of myeloid and lymphoid lineages from multipotent progenitors.

JunB can substitute for Jun in mouse development and cell proliferation.

The Jun and JunB components of the AP-1 transcription factor are known to have antagonistic functions. Here we show, by a knock-in strategy and a transgenic complementation approach, that Junb can substitute for absence of Jun during mouse development. Junb can rescue both liver and cardiac defects in Jun-null mice in a manner dependent on gene dosage. JunB restores the expression of genes regulated by Jun/Fos, but not those regulated by Jun/ATF, thereby rescuing Jun-dependent defects in vivo as well as in primary fibroblasts and fetal hepatoblasts in vitro. Thus, the transcriptionally less active JunB has the potential to substitute for Jun, indicating that the spatial and temporal regulation of expression of the transcription factor AP-1 may be more important than the coding sequence of its components.

Leukemic stem cells: Where do they come from?

Leukemias can now be viewed as aberrant hematopoietic processes initiated by rare cancer stem cells, or leukemic stem cells (LSCs) that have maintained or reacquired the capacity for indefinite proliferation through accumulated mutations and/or epigenetic changes. Yet, despite their critical importance, much remains to be learned about the developmental origin of LSCs and the mechanisms responsible for their emergence in the course of the disease. Mouse models of human leukemias have provided a unique system to study the mechanisms influencing LSC generation and function, and were recently used to demonstrate that LSCs can arise from both self-renewing hematopoietic stem cells (HSCs) and committed progenitor populations. This striking finding indicates that LSC identity is largely dictated by the nature of the oncogenic events and by how these events perturb essential processes such as self-renewal, proliferation, differentiation, and survival. Such approaches in the mouse are essential for the basic understanding of leukemogenesis and for the conceptual design of novel therapeutic strategies that could lead to improved treatments for human leukemias.

Leukemia and Leukemic Stem Cells

Leukemias are cancers of the hematopoietic system. Like all cancers, several genetic and epigenetic events aid in the transition from normal to malignant cell. These usually, if not always, include at least: 1) avoidance of programmed cell death from intrinsic signals; 2) acquisition of poorly regulated or unregulated self-renewal capacity; 3) prevention of critical telomere shortening; 4) inhibition of differentiation to an increasing degree as the malignancy progresses; and 5) avoidance of innate and adaptive immune responses that cause the death and/or phagocytosis of tumor cells. Many of these processes are properties of the hematopoietic stem cell (HSC) and are highly regulated, yet the hallmark populations in the most advanced leukemias, e.g., the expanded population of leukemic blasts, are not HSC. The phenotypic identity of the leukemic stem cells (LSC), i.e., the only cells within the leukemia capable of propagating the disease, has not been clearly elucidated. In this speculative review, we have two goals: to discuss the stage of hematopoietic differentiation in which LSC reside, and to begin to understand how recurrent genetic changes, including translocations and inversions, and epigenetic changes, resulting in increased or decreased expression of selected genes, play roles in the above described behaviors of leukemia cells.

Chronic versus acute myelogenous leukemiaA question of self-renewal

Leukemia stem cells are defined as transformed hematopoietic stem cells or committed progenitor cells that have amplified or acquired the stem cell capacity for self-renewal, albeit in a poorly regulated fashion. In this issue of Cancer Cell, Huntly and colleagues report a striking difference in the ability of two leukemia-associated fusion proteins, MOZ-TIF2 and BCR-ABL, to transform myeloid progenitor populations. This rigorous study supports the idea of a hierarchy among leukemia-associated protooncogenes for their ability to endow committed myeloid progenitors with the self-renewal capacity driving leukemic stem cell propagation, and sheds new light on the pathogenesis of chronic and acute myelogenous leukemias.