Miriam Erlacher

FOXO3a-dependent regulation of Puma in response to cytokine/growth factor withdrawal

Puma is an essential mediator of p53-dependent and -independent apoptosis in vivo. In response to genotoxic stress, Puma is induced in a p53-dependent manner. However, the transcription factor driving Puma up-regulation in response to p53-independent apoptotic stimuli has yet to be identified. Here, we show that FOXO3a up-regulates Puma expression in response to cytokine or growth factor deprivation. Importantly, dysregulated Akt signaling in lymphoid cells attenuated Puma induction upon cytokine withdrawal. Our results suggest that Puma, together with another BH3 only member, Bim, function as FOXO3a downstream targets to mediate a stress response when PI3K/Akt signaling is down-regulated.

Germline CBL mutations cause developmental abnormalities and predispose to juvenile myelomonocytic leukemia

CBL encodes a member of the Cbl family of proteins, which functions as an E3 ubiquitin ligase. We describe a dominant developmental disorder resulting from germline missense CBL mutations, which is characterized by impaired growth, developmental delay, cryptorchidism and a predisposition to juvenile myelomonocytic leukemia (JMML). Some individuals experienced spontaneous regression of their JMML but developed vasculitis later in life. Importantly, JMML specimens from affected children show loss of the normal CBL allele through acquired isodisomy. Consistent with these genetic data, the common p.371Y>H altered Cbl protein induces cytokine-independent growth and constitutive phosphorylation of ERK, AKT and S6 only in hematopoietic cells in which normal Cbl expression is reduced by RNA interference. We conclude that germline CBL mutations have developmental, tumorigenic and functional consequences that resemble disorders that are caused by hyperactive Ras/Raf/MEK/ERK signaling and include neurofibromatosis type 1, Noonan syndrome, Costello syndrome, cardiofaciocutaneous syndrome and Legius syndrome.

Puma cooperates with Bim, the rate-limiting BH3-only protein in cell death during lymphocyte development, in apoptosis induction.

The physiological role of B cell lymphoma 2 (Bcl-2) homology 3–only proteins has been investigated in mice lacking the individual genes identifying rate-limiting roles for Bim (Bcl-2–interacting mediator of cell death) and Puma (p53–up-regulated modulator of apoptosis) in apoptosis induction. The loss of Bim protects lymphocytes from apoptosis induced by cytokine deprivation and deregulated Ca++ flux and interferes with the deletion of autoreactive lymphocytes and the shutdown of immune responses. In contrast, Puma is considered the key mediator of p53-induced apoptosis. To investigate the hypothesis that Bim and Puma have overlapping functions, we generated mice lacking both genes and found that bim−/−/puma−/− animals develop multiple postnatal defects that are not observed in the single knockout mice. Most strikingly, hyperplasia of lymphatic organs is comparable with that observed in mice overexpressing Bcl-2 in all hemopoietic cells exceeding the hyperplasia observed in bim−/− mice. Bim and Puma also have clearly overlapping functions in p53-dependent and -independent apoptosis. Their combined loss promotes spontaneous tumorigenesis, causing the malignancies observed in Bcl-2 transgenic mice, but does not exacerbate the autoimmunity observed in the absence of Bim.

p14-MP1-MEK1 signaling regulates endosomal traffic and cellular proliferation during tissue homeostasis.

The extracellular signal-regulated kinase (ERK) cascade regulates proliferation, differentiation, and survival in multicellular organisms. Scaffold proteins regulate intracellular signaling by providing critical spatial and temporal specificity. The scaffold protein MEK1 (mitogen-activated protein kinase and ERK kinase 1) partner (MP1) is localized to late endosomes by the adaptor protein p14. Using conditional gene disruption of p14 in mice, we now demonstrate that the p14–MP1-MEK1 signaling complex regulates late endosomal traffic and cellular proliferation. This function its essential for early embryogenesis and during tissue homeostasis, as revealed by epidermis-specific deletion of p14. These findings show that endosomal p14–MP1-MEK1 signaling has a specific and essential function in vivo and, therefore, indicate that regulation of late endosomal traffic by extracellular signals is required to maintain tissue homeostasis.

A hypomorphic mouse model of dystrophic epidermolysis bullosa reveals mechanisms of disease and response to fibroblast therapy

Dystrophic epidermolysis bullosa (DEB) is a severe skin fragility disorder associated with trauma-induced blistering, progressive soft tissue scarring, and increased risk of skin cancer. DEB is caused by mutations in type VII collagen. In this study, we describe the generation of a collagen VII hypomorphic mouse that serves as an immunocompetent animal model for DEB. These mice expressed collagen VII at about 10% of normal levels, and their phenotype closely resembled characteristics of severe human DEB, including mucocutaneous blistering, nail dystrophy, and mitten deformities of the extremities. The oral blistering experienced by these mice resulted in growth retardation, and repeated blistering led to excessive induction of tissue repair, causing TGF-beta1-mediated contractile fibrosis generated by myofibroblasts and pseudosyndactyly in the extremities. Intradermal injection of WT fibroblasts resulted in neodeposition of collagen VII and functional restoration of the dermal-epidermal junction. Treated areas were also resistant to induced frictional stress. In contrast, untreated areas of the same mouse showed dermal-epidermal separation following induced stress. These data demonstrate that fibroblast-based treatment can be used to treat DEB in a mouse model and suggest that this approach may be effective in the development of clinical therapeutic regimens for patients with DEB.

Loss of the BH3-only protein Bmf impairs B cell homeostasis and accelerates γ irradiation–induced thymic lymphoma development

Members of the Bcl-2 protein family play crucial roles in the maintenance of tissue homeostasis by regulating apoptosis in response to developmental cues or exogenous stress. Proapoptotic BH3-only members of the Bcl-2 family are essential for initiation of cell death, and they function by activating the proapoptotic Bcl-2 family members Bax and/or Bak, either directly or indirectly through binding to prosurvival Bcl-2 family members. Bax and Bak then elicit the downstream events in apoptosis signaling. Mammals have at least eight BH3-only proteins and they are activated in a stimulus-specific, as well as a cell type–specific, manner. We have generated mice lacking the BH3-only protein Bcl-2–modifying factor (Bmf) to investigate its role in cell death signaling. Our studies reveal that Bmf is dispensable for embryonic development and certain forms of stress-induced apoptosis, including loss of cell attachment (anoikis) or UV irradiation. Remarkably, loss of Bmf protected lymphocytes against apoptosis induced by glucocorticoids or histone deacetylase inhibition. Moreover, bmf −/− mice develop a B cell–restricted lymphadenopathy caused by the abnormal resistance of these cells to a range of apoptotic stimuli. Finally, Bmf-deficiency accelerated the development of γ irradiation–induced thymic lymphomas. Our results demonstrate that Bmf plays a critical role in apoptosis signaling and can function as a tumor suppressor.

Apoptosis of leukocytes triggered by acute DNA damage promotes lymphoma formation

Apoptosis triggered by p53 upon DNA damage secures removal of cells with compromised genomes, and is thought to prevent tumorigenesis. In contrast, we provide evidence that p53-induced apoptosis can actively drive tumor formation. Mice defective in p53-induced apoptosis due to loss of its proapoptotic target gene, puma, resist gamma-irradiation (IR)-induced lymphomagenesis. In wild-type animals, repeated irradiation injury-induced expansion of hematopoietic stem/progenitor cells (HSCs) leads to lymphoma formation. Puma(-/-) HSCs, protected from IR-induced cell death, show reduced compensatory proliferation and replication stress-associated DNA damage, and fail to form thymic lymphomas, demonstrating that the maintenance of stem/progenitor cell homeostasis is critical to prevent IR-induced tumorigenesis.

Deregulated cell death and lymphocyte homeostasis cause premature lethality in mice lacking the BH3-only proteins Bim and Bmf

BH3 domain-only proteins (BH3-only) proteins are members of the Bcl-2 family that play crucial roles in embryogenesis and the maintenance of tissue homeostasis by triggering apoptotic cell death. The BH3-only protein Bim is critical for developmental apoptosis of lymphocytes, securing establishment of tolerance and for the termination of immune responses. Bim is believed to act in concert with other BH3-only proteins or members of the tumor necrosis factor receptor family in getting rid of unwanted cells. Bmf, a related BH3-only protein, was shown to play a role in B-cell homeostasis and to mediate cell death in response to certain apoptotic triggers, including glucocorticoid, histone deacetylase inhibitors, and overexpression of the c-Myc proto-oncogene. Here we show that Bim and Bmf have overlapping functions during mouse development and coregulate lymphocyte homeostasis and apoptosis in a nonredundant manner. Double deficiency of Bim and Bmf caused more B lymphadenopathy than loss of either BH3-only protein alone, and this was associated with autoimmune glomerulonephritis and a range of malignancies in aged mice. Thus, our results demonstrate that Bim and Bmf act in concert to prevent autoimmunity and malignant disease, strengthening the rational for the development of BH3-only protein mimicking therapeutics for the treatment of such disorders.

TCR signaling inhibits glucocorticoid-induced apoptosis in murine thymocytes depending on the stage of development.

Signaling by either the TCR or glucocorticoid receptor (GR) induces apoptosis in thymocytes. Interestingly, it has been shown previously that hybridoma T cells escape apoptosis induced by either TCR or GR when both of these receptors signal simultaneously. Whether such mutual antagonism is present in primary thymocytes was the subject of the present study. Both glucocorticoids (GC) and anti‐TCR/CD28 (or anti‐CD3/CD28) mAb induced apoptosis in total thymocytes. When these signals were present at the same time, GC‐induced apoptosis was partially inhibited by TCR/CD3 signaling. Costimulation by anti‐CD28 enhanced the inhibitory effects of anti‐CD3 on GC‐induced apoptosis about 30‐fold. However, subset analysis revealed that most cells rescued from GC‐induced apoptosis were mature CD4+ and CD8+ thymocytes, and these cells were resistant to TCR/CD3‐induced apoptosis in the absence of GC. Similar results were obtained with mature splenic CD4+ and CD8+ T cells. TCR/CD3 signaling alone, while inducing apoptosis in CD4+CD8+TCRlow thymocytes, rescued a small subset of CD4+CD8+TCRlow thymocytes from GC‐induced apoptosis. Thus, TCR signaling increasingly reverses GC‐induced apoptosis as thymocyte development progresses. As GC are infinitely present in vivo, these findings support a model wherein TCR signaling may be required to prevent GC‐induced apoptosis both under basal and immune challenging conditions.

Childhood cancer predisposition syndromes—A concise review and recommendations by the Cancer Predisposition Working Group of the Society for Pediatric Oncology and Hematology

Heritable predisposition is an important cause of cancer in children and adolescents. Although a large number of cancer predisposition genes and their associated syndromes and malignancies have already been described, it appears likely that there are more pediatric cancer patients in whom heritable cancer predisposition syndromes have yet to be recognized. In a consensus meeting in the beginning of 2016, we convened experts in Human Genetics and Pediatric Hematology/Oncology to review the available data, to categorize the large amount of information, and to develop recommendations regarding when a cancer predisposition syndrome should be suspected in a young oncology patient. This review summarizes the current knowledge of cancer predisposition syndromes in pediatric oncology and provides essential information on clinical situations in which a childhood cancer predisposition syndrome should be suspected.