Stephanie Grabow

XIAP discriminates between type I and type II FAS-induced apoptosis

FAS (also called APO-1 and CD95) and its physiological ligand, FASL, regulate apoptosis of unwanted or dangerous cells, functioning as a guardian against autoimmunity and cancer development. Distinct cell types differ in the mechanisms by which the ‘death receptor’ FAS triggers their apoptosis. In type I cells, such as lymphocytes, activation of ‘effector caspases’ by FAS-induced activation of caspase-8 suffices for cell killing, whereas in type II cells, including hepatocytes and pancreatic β-cells, caspase cascade amplification through caspase-8-mediated activation of the pro-apoptotic BCL-2 family member BID (BH3 interacting domain death agonist) is essential. Here we show that loss of XIAP (X-chromosome linked inhibitor of apoptosis protein) function by gene targeting or treatment with a second mitochondria-derived activator of caspases (SMAC, also called DIABLO; direct IAP-binding protein with low pI) mimetic drug in mice rendered hepatocytes and β-cells independent of BID for FAS-induced apoptosis. These results show that XIAP is the critical discriminator between type I and type II apoptosis signalling and suggest that IAP inhibitors should be used with caution in cancer patients with underlying liver conditions.

Membrane-bound Fas ligand only is essential for Fas-induced apoptosis

Fas ligand (FasL), an apoptosis-inducing member of the TNF cytokine family and its receptor, Fas, are critical for shutdown of chronic immune responses1-3 and prevention of autoimmunity4,5. Accordingly, mutations in their genes cause severe lymphadenopathy and autoimmune disease in mice6,7 and humans8,9. FasL function is regulated by deposition in the plasma membrane and metalloprotease-mediated shedding10,11. We generated gene-targeted mice that selectively lack either secreted FasL (ΔsFasL) or membrane-bound FasL (ΔmFasL) to resolve which of these forms is required for cell killing and to explore their hypothetical non-apoptotic activities. Mice lacking sFasL (FasLΔs/Δs) appeared normal and their T cells readily killed target cells, whereas T cells lacking mFasL (FasLΔm/Δm) could not kill cells through Fas activation. FasLΔm/Δm mice developed lymphadenopathy and hyper-gammaglobulinaemia, similar to FasLgld/gld mice, which express a mutant form of FasL that cannot bind Fas, but surprisingly, (on a C57BL/6 background) FasLΔm/Δm mice succumbed to SLE-like autoimmune kidney destruction and histiocytic sarcoma, diseases that occur only rarely and considerably later in FasLgld/gld mice. These results demonstrate that mFasL is essential for cytotoxic activity and constitutes the guardian against lymphadenopathy, autoimmunity and cancer whereas excess sFasL appears to promote autoimmunity and tumorigenesis through non-apoptotic activities.Fas ligand (FasL), an apoptosis-inducing member of the TNF cytokine family, and its receptor Fas are critical for the shutdown of chronic immune responses and prevention of autoimmunity. Accordingly, mutations in their genes cause severe lymphadenopathy and autoimmune disease in mice and humans. FasL function is regulated by deposition in the plasma membrane and metalloprotease-mediated shedding. Here we generated gene-targeted mice that selectively lack either secreted FasL (sFasL) or membrane-bound FasL (mFasL) to resolve which of these forms is required for cell killing and to explore their hypothesized non-apoptotic activities. Mice lacking sFasL (FasLΔs/Δs) appeared normal and their T cells readily killed target cells, whereas T cells lacking mFasL (FasLΔm/Δm) could not kill cells through Fas activation. FasLΔm/Δm mice developed lymphadenopathy and hyper-gammaglobulinaemia, similar to FasLgld/gld mice, which express a mutant form of FasL that cannot bind Fas, but surprisingly, FasLΔm/Δm mice (on a C57BL/6 background) succumbed to systemic lupus erythematosus (SLE)-like autoimmune kidney destruction and histiocytic sarcoma, diseases that occur only rarely and much later in FasLgld/gld mice. These results demonstrate that mFasL is essential for cytotoxic activity and constitutes the guardian against lymphadenopathy, autoimmunity and cancer, whereas excess sFasL appears to promote autoimmunity and tumorigenesis through non-apoptotic activities.

Targeting of MCL-1 kills MYC-driven mouse and human lymphomas even when they bear mutations in p53

The transcriptional regulator c-MYC is abnormally overexpressed in many human cancers. Evasion from apoptosis is critical for cancer development, particularly c-MYC-driven cancers. We explored which anti-apoptotic BCL-2 family member (expressed under endogenous regulation) is essential to sustain c-MYC-driven lymphoma growth to reveal which should be targeted for cancer therapy. Remarkably, inducible Cre-mediated deletion of even a single Mcl-1 allele substantially impaired the growth of c-MYC-driven mouse lymphomas. Mutations in p53 could diminish but not obviate the dependency of c-MYC-driven mouse lymphomas on MCL-1. Importantly, targeting of MCL-1 killed c-MYC-driven human Burkitt lymphoma cells, even those bearing mutations in p53. Given that loss of one allele of Mcl-1 is well tolerated in healthy tissues, our results suggest that therapeutic targeting of MCL-1 would be an attractive therapeutic strategy for MYC-driven cancers.

MCL-1 but not BCL-XL is critical for the development and sustained expansion of thymic lymphoma in p53-deficient mice.

Apoptosis plays a role in normal lymphopoiesis and lymphoid malignancies. Pro-survival MCL-1 is essential for survival of T-cell progenitors, BCL-XL for immature thymocytes, and BCL-2 for mature T cells. Conversely, little is known about the regulators that are required for the survival of T-cell lymphomas. We used constitutive and conditionally gene-targeted mice to investigate which pro-survival BCL-2 family member is required for the sustained survival of thymic lymphomas initiated by loss of p53. Constitutive loss of a single Mcl-1 allele delayed tumor onset. In contrast, lymphomas emerging in p53(-/-) mice in which Mcl-1 could be conditionally deleted had been selected for retention of MCL-1 expression. In contrast, complete loss of BCL-XL had no impact on lymphoma development in p53(-/-) mice. These results demonstrate that thymic lymphomas elicited by loss of p53 must arise from cancer-initiating cells that require MCL-1 for their survival. Acute deletion of both Mcl-1 alleles abrogated the expansion of p53(-/-) lymphomas in mice, whereas inducible loss of BCL-XL had little impact. This reveals that MCL-1 is essential for the sustained survival of these malignant cells and suggests that targeting MCL-1 may be an attractive strategy for the treatment of T-cell lymphoma.

Endogenous Bcl-xL is essential for Myc-driven lymphomagenesis in mice

Impaired apoptosis is a cancer hallmark, and some types of lymphomas and other cancers harbor mutations that directly affect key cell death regulators, such as Bcl-2 family members. However, because the majority of tumors seem to lack such mutations, we are examining the hypothesis that tumorigenesis can be sustained at least initially by the normal expression of specific endogenous pro-survival Bcl-2 family members. We previously demonstrated that the lymphomagenesis in Εμ-myc transgenic mice, which constitutively overexpress the c-Myc oncoprotein in B-lymphoid cells and develop pre-B and B-cell lymphomas, does not require endogenous Bcl-2. In striking contrast, we report here that loss in these mice of its close relative Bcl-x(L) attenuated the pre-neoplastic expansion of pro-B and pre-B cells otherwise driven by c-Myc overexpression, sensitized these cells to apoptosis and ablated lymphoma formation. Remarkably, even loss of a single bcl-x allele delayed the lymphomagenesis. These findings identify Bcl-x(L) as a prerequisite for the emergence of c-Myc-driven pre-B/B lymphoma and suggest that BH3 mimetic drugs may provide a prophylactic strategy for c-Myc-driven tumors.

Antagonism between MCL-1 and PUMA governs stem/progenitor cell survival during hematopoietic recovery from stress

Understanding the critical factors that govern recovery of the hematopoietic system from stress, such as during anticancer therapy and bone marrow transplantation, is of clinical significance. We investigated the importance of the prosurvival proteins myeloid cell leukemia-1 (MCL-1) and B-cell lymphoma-extra large (BCL-XL) in stem/progenitor cell survival and fitness during hematopoietic recovery from stress. Loss of a single Mcl-1 allele, which reduced MCL-1 protein levels, severely compromised hematopoietic recovery from myeloablative challenge and following bone marrow transplantation, whereas BCL-XL was dispensable in both contexts. We identified inhibition of proapoptotic p53 upregulated modulator of apoptosis (PUMA) as the key role of MCL-1 in both settings, with Mcl-1(+/-);Puma(-/-) mice completely protected from the deleterious effects of loss of 1 Mcl-1 allele. These results reveal the molecular mechanisms that govern cell survival during hematopoietic recovery from stress.

Prosurvival Bcl-2 family members reveal a distinct apoptotic identity between conventional and plasmacytoid dendritic cells

Significance Dendritic cells (DCs) are pivotal for immune responses as they present antigens to T cells. Different DC subsets have different functions and are associated with different diseases. For example, plasmacytoid DCs (pDCs) produce type 1 interferons and are associated with the autoimmune disease, systemic lupus erythematosus. Understanding control of survival/apoptosis in different DC subsets may not only provide a molecular basis for their homeostasis but also guide therapeutic intervention of immunopathology. We revealed that two major DC subsets (pDCs and conventional DCs) express distinct BCL-2 family proteins at different levels and this correlated with their survival requirements. Accordingly, clinically applicable antagonist drugs killed the appropriate DC subsets, informing on the future use of these compounds for treating immune-mediated damage. Dendritic cells (DCs) are heterogeneous, comprising subsets with functional specializations that play distinct roles in immunity as well as immunopathology. We investigated the molecular control of cell survival of two main DC subsets: plasmacytoid DCs (pDCs) and conventional DCs (cDCs) and their dependence on individual antiapoptotic BCL-2 family members. Compared with cDCs, pDCs had higher expression of BCL-2, lower A1, and similar levels of MCL-1 and BCL-XL. Transgenic overexpression of BCL-2 increased the pDC pool size in vivo with only minor impact on cDCs. With a view to immune intervention, we tested BCL-2 inhibitors and found that ABT-199 (the BCL-2 specific inhibitor) selectively killed pDCs but not cDCs. Conversely, genetic knockdown of A1 profoundly reduced the proportion of cDCs but not pDCs. We also found that conditional ablation of MCL-1 significantly reduced the size of both DC populations in mice and impeded DC-mediated immune responses. Thus, we revealed that the two DC types have different cell survival requirements. The molecular basis of survival of different DC subsets thus advocates the antagonism of selective BCL-2 family members for treating diseases pertaining to distinct DC subsets.

Impact of the combined loss of BOK, BAX and BAK on the hematopoietic system is slightly more severe than compound loss of BAX and BAK.

It is well established that BAX and BAK play crucial, overlapping roles in the intrinsic pathway of apoptosis. Gene targeted mice lacking both BAX and BAK have previously been generated, but the majority of these animals died perinatally. BOK is a poorly studied relative of BAX and BAK that shares extensive amino acid sequence homology to both proteins, but its function remains largely unclear to date. To determine whether BOK plays an overlapping role with BAX and BAK, we utilized a hematopoietic reconstitution model where lethally irradiated wild type mice were transplanted with Bok−/−Bax−/−Bak−/− triple knockout (TKO) fetal liver cells, and compared alongside mice reconstituted with a Bax−/−Bak−/− double knockout (DKO) hematopoietic compartment. We report here that mice with a TKO and DKO hematopoietic system died at a similar rate and much earlier than control animals, mostly due to severe autoimmune pathology. Both TKO and DKO reconstituted mice also had altered frequencies of various leukocyte subsets in the thymus, bone marrow and spleen, displayed leukocyte infiltrates and autoimmune pathology in multiple tissues, as well as elevated levels of anti-nuclear autoantibodies. Interestingly, the additional deletion of BOK (on top of BAX and BAK loss) led to a further increase in peripheral blood lymphocytes, as well as enhanced lymphoid infiltration in some organs. These findings suggest that BOK may have some functions that are redundant with BAX and BAK in the hematopoietic system.

MOZ regulates B-cell progenitors and, consequently, Moz haploinsufficiency dramatically retards MYC-induced lymphoma development

The histone acetyltransferase MOZ (MYST3, KAT6A) is the target of recurrent chromosomal translocations fusing the MOZ gene to CBP, p300, NCOA3, or TIF2 in particularly aggressive cases of acute myeloid leukemia. In this study, we report the role of wild-type MOZ in regulating B-cell progenitor proliferation and hematopoietic malignancy. In the Eμ-Myc model of aggressive pre-B/B-cell lymphoma, the loss of just one allele of Moz increased the median survival of mice by 3.9-fold. MOZ was required to maintain the proliferative capacity of B-cell progenitors, even in the presence of c-MYC overexpression, by directly maintaining the transcriptional activity of genes required for normal B-cell development. Hence, B-cell progenitor numbers were significantly reduced in Moz haploinsufficient animals. Interestingly, we find a significant overlap in genes regulated by MOZ, mixed lineage leukemia 1, and mixed lineage leukemia 1 cofactor menin. This includes Meis1, a TALE class homeobox transcription factor required for B-cell development, characteristically upregulated as a result of MLL1 translocations in leukemia. We demonstrate that MOZ localizes to the Meis1 locus in pre-B-cells and maintains Meis1 expression. Our results suggest that even partial inhibition of MOZ may reduce the proliferative capacity of MEIS1, and HOX-driven lymphoma and leukemia cells.

Physiological restraint of Bak by Bcl-xL is essential for cell survival

Due to the myriad interactions between prosurvival and proapoptotic members of the Bcl-2 family of proteins, establishing the mechanisms that regulate the intrinsic apoptotic pathway has proven challenging. Mechanistic insights have primarily been gleaned from in vitro studies because genetic approaches in mammals that produce unambiguous data are difficult to design. Here we describe a mutation in mouse and human Bak that specifically disrupts its interaction with the prosurvival protein Bcl-xL Substitution of Glu75 in mBak (hBAK Q77) for leucine does not affect the three-dimensional structure of Bak or killing activity but reduces its affinity for Bcl-xL via loss of a single hydrogen bond. Using this mutant, we investigated the requirement for physical restraint of Bak by Bcl-xL in apoptotic regulation. In vitro, Bak(Q75L) cells were significantly more sensitive to various apoptotic stimuli. In vivo, loss of Bcl-xL binding to Bak led to significant defects in T-cell and blood platelet survival. Thus, we provide the first definitive in vivo evidence that prosurvival proteins maintain cellular viability by interacting with and inhibiting Bak.