Mcl-1 is essential for the survival of plasma cells.

The long-term survival of plasma cells is entirely dependent on signals derived from their environment. These extrinsic factors presumably induce and sustain the expression of antiapoptotic proteins of the Bcl-2 family. It is uncertain whether there is specificity among Bcl-2 family members in the survival of plasma cells and whether their expression is linked to specific extrinsic factors. We found here that deletion of the gene encoding the antiapoptotic protein Mcl-1 in plasma cells resulted in rapid depletion of this population in vivo. Furthermore, we found that the receptor BCMA was needed to establish high expression of Mcl-1 in bone marrow but not spleen plasma cells and that establishing this survival pathway preceded the component of plasma cell differentiation that depends on the transcriptional repressor Blimp-1. Our results identify a critical role for Mcl-1 in the maintenance of plasma cells.

BCL-XL exerts a protective role against anemia caused by radiation-induced kidney damage.

Studies of gene-targeted mice identified the roles of the different pro-survival BCL-2 proteins during embryogenesis. However, little is known about the role(s) of these proteins in adults in response to cytotoxic stresses, such as treatment with anti-cancer agents. We investigated the role of BCL-XL in adult mice using a strategy where prior bone marrow transplantation allowed for loss of BCL-XL exclusively in non-hematopoietic tissues to prevent anemia caused by BCL-XL deficiency in erythroid cells. Unexpectedly, the combination of total body γ-irradiation (TBI) and genetic loss of Bcl-x caused secondary anemia resulting from chronic renal failure due to apoptosis of renal tubular epithelium with secondary obstructive nephropathy. These findings identify a critical protective role of BCL-XL in the adult kidney and inform on the use of BCL-XL inhibitors in combination with DNA damage-inducing drugs for cancer therapy. Encouragingly, the combination of DNA damage-inducing anti-cancer therapy plus a BCL-XL inhibitor could be tolerated in mice, at least when applied sequentially.

Acute myeloid leukemia requires Hhex to enable PRC2-mediated epigenetic repression of Cdkn2a.

Unlike clustered HOX genes, the role of nonclustered homeobox gene family members in hematopoiesis and leukemogenesis has not been extensively studied. Here we found that the hematopoietically expressed homeobox gene Hhex is overexpressed in acute myeloid leukemia (AML) and is essential for the initiation and propagation of MLL-ENL-induced AML but dispensable for normal myelopoiesis, indicating a specific requirement for Hhex for leukemic growth. Loss of Hhex leads to expression of the Cdkn2a-encoded tumor suppressors p16(INK4a) and p19(ARF), which are required for growth arrest and myeloid differentiation following Hhex deletion. Mechanistically, we show that Hhex binds to the Cdkn2a locus and directly interacts with the Polycomb-repressive complex 2 (PRC2) to enable H3K27me3-mediated epigenetic repression. Thus, Hhex is a potential therapeutic target that is specifically required for AML stem cells to repress tumor suppressor pathways and enable continued self-renewal.

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.

Anti-apoptotic Mcl-1 is essential for the development and sustained growth of acute myeloid leukemia.

Acute myeloid leukemia (AML) frequently relapses after initial treatment. Drug resistance in AML has been attributed to high levels of the anti-apoptotic Bcl-2 family members Bcl-x(L) and Mcl-1. Here we report that removal of Mcl-1, but not loss or pharmacological blockade of Bcl-x(L), Bcl-2, or Bcl-w, caused the death of transformed AML and could cure disease in AML-afflicted mice. Enforced expression of selective inhibitors of prosurvival Bcl-2 family members revealed that Mcl-1 is critical for survival of human AML cells. Thus, targeting of Mcl-1 or regulators of its expression may be a useful strategy for the treatment of AML.

Enhanced stability of Mcl1, a prosurvival Bcl2 relative, blunts stress-induced apoptosis, causes male sterility, and promotes tumorigenesis.

The B-cell CLL/lymphoma 2 (Bcl2) relative Myeloid cell leukemia sequence 1 (Mcl1) is essential for cell survival during development and for tissue homeostasis throughout life. Unlike Bcl2, Mcl1 turns over rapidly, but the physiological significance of its turnover has been unclear. We have gained insight into the roles of Mcl1 turnover in vivo by analyzing mice harboring a modified allele of Mcl1 that serendipitously proved to encode an abnormally stabilized form of Mcl1 due to a 13-aa N-terminal extension. Although the mice developed normally and appeared unremarkable, the homozygous males unexpectedly proved infertile due to defective spermatogenesis, which was evoked by enhanced Mcl1 prosurvival activity. Under unstressed conditions, the modified Mcl1 is present at levels comparable to the native protein, but it is markedly stabilized in cells subjected to stresses, such as protein synthesis inhibition or UV irradiation. Strikingly, the modified Mcl1 allele could genetically complement the loss of Bcl2, because introduction of even a single allele significantly ameliorated the severe polycystic kidney disease and consequent runting caused by Bcl2 loss. Significantly, the development of c-MYC-induced acute myeloid leukemia was also accelerated in mice harboring that Mcl1 allele. Our collective findings reveal that, under certain circumstances, the N terminus of Mcl1 regulates its degradation; that some cell types require degradation of Mcl1 to induce apoptosis; and, most importantly, that rapid turnover of Mcl1 can serve as a tumor-suppressive mechanism.

Reversible growth factor dependency and autonomy during murine myelomonocytic leukemia induced by oncogenes.

When murine fetal liver cells were transduced with either of the human acute myeloid leukemia fusion oncogenes MLL-ENL or MLL-AF9 and then transplanted to irradiated recipient mice, myelomonocyte leukemias rapidly developed from the transplanted cells. Analysis of initial events following transduction showed that both oncogenes immediately induced a wide range of enhanced proliferative states, the most extreme of which could generate continuous lines of cells. Maturation defects accompanied the enhanced proliferative states. At all times, the transformed cells exhibited complete dependency on hematopoietic growth factors, particularly GM-CSF and IL-3. Myelomonocytic leukemic cells from primary or transplanted mice formed colonies in semisolid agar. The large majority were dependent on hematopoietic growth factors, but a low frequency of autonomous colonies was also detected. Unexpectedly, reculture of autonomous leukemic colonies generated large numbers of growth factor-dependent clonogenic progeny. Similarly, transplanted clonal autonomous leukemic cells produced leukemias containing a majority of factor-dependent cells. Conversely, recultures of factor-dependent colonies in vitro always produced small numbers of autonomous colonies among the dependent progeny. The reversible relationship between factor dependency and autonomy is surprising because autonomy would have been presumed to represent the final, irreversible, leukemic state.

Targeting acute myeloid leukemia by dual inhibition of PI3K signaling and Cdk9-mediated Mcl-1 transcription.

Resistance to cell death is a hallmark of cancer and renders transformed cells resistant to multiple apoptotic triggers. The Bcl-2 family member, Mcl-1, is a key driver of cell survival in diverse cancers, including acute myeloid leukemia (AML). A screen for compounds that downregulate Mcl-1 identified the kinase inhibitor, PIK-75, which demonstrates marked proapoptotic activity against a panel of cytogenetically diverse primary human AML patient samples. We show that PIK-75 transiently blocks Cdk7/9, leading to transcriptional suppression of MCL-1, rapid loss of Mcl-1 protein, and alleviation of its inhibition of proapoptotic Bak. PIK-75 also targets the p110α isoform of PI3K, which leads to a loss of association between Bcl-xL and Bak. The simultaneous loss of Mcl-1 and Bcl-xL association with Bak leads to rapid apoptosis of AML cells. Concordantly, low Bak expression in AML confers resistance to PIK-75-mediated killing. On the other hand, the induction of apoptosis by PIK-75 did not require the expression of the BH3 proteins Bim, Bid, Bad, Noxa, or Puma. PIK-75 significantly reduced leukemia burden and increased the survival of mice engrafted with human AML without inducing overt toxicity. Future efforts to cotarget PI3K and Cdk9 with drugs such as PIK-75 in AML are warranted.

Bcl-2, Bcl-x(L), and Bcl-w are not equivalent targets of ABT-737 and navitoclax (ABT-263) in lymphoid and leukemic cells.

The BH3-mimetic ABT-737 and an orally bioavailable compound of the same class, navitoclax (ABT-263), have shown promising antitumor efficacy in preclinical and early clinical studies. Although both drugs avidly bind Bcl-2, Bcl-x(L), and Bcl-w in vitro, we find that Bcl-2 is the critical target in vivo, suggesting that patients with tumors overexpressing Bcl-2 will probably benefit. In human non-Hodgkin lymphomas, high expression of Bcl-2 but not Bcl-x(L) predicted sensitivity to ABT-263. Moreover, we show that increasing Bcl-2 sensitized normal and transformed lymphoid cells to ABT-737 by elevating proapoptotic Bim. In striking contrast, increasing Bcl-x(L) or Bcl-w conferred robust resistance to ABT-737, despite also increasing Bim. Cell-based protein redistribution assays unexpectedly revealed that ABT-737 disrupts Bcl-2/Bim complexes more readily than Bcl-x(L)/Bim or Bcl-w/Bim complexes. These results have profound implications for how BH3-mimetics induce apoptosis and how the use of these compounds can be optimized for treating lymphoid malignancies.

Current issues in mouse genome engineering.

The mouse is the foremost vertebrate experimental model because its genome can be precisely and variously engineered. Now that the mouse genome has been sequenced and annotated, the task of mutating each gene is feasible, and an international cooperation is providing mutated embryonic stem cells and mice as readily available resources. Because these resources will change biomedical research, decisions about their nature will have far-reaching effects. It is therefore timely to consider topical issues for mouse genome engineering, such as the background genotype; homologous, site-specific and transpositional recombination; conditional mutagenesis; RNA-mediated interference; and functional genomics with embryonic stem cells.

MLL2 is required in oocytes for bulk histone 3 lysine 4 trimethylation and transcriptional silencing.

During gametogenesis and pre-implantation development, the mammalian epigenome is reprogrammed to establish pluripotency in the epiblast. Here we show that the histone 3 lysine 4 (H3K4) methyltransferase, MLL2, controls most of the promoter-specific chromatin modification, H3K4me3, during oogenesis and early development. Using conditional knockout mutagenesis and a hypomorph model, we show that Mll2 deficiency in oocytes results in anovulation and oocyte death, with increased transcription of p53, apoptotic factors, and Iap elements. MLL2 is required for (1) bulk H3K4me3 but not H3K4me1, indicating that MLL2 controls most promoters but monomethylation is regulated by a different H3K4 methyltransferase; (2) the global transcriptional silencing that preceeds resumption of meiosis but not for the concomitant nuclear reorganization into the surrounded nucleolus (SN) chromatin configuration; (3) oocyte survival; and (4) normal zygotic genome activation. These results reveal that MLL2 is autonomously required in oocytes for fertility and imply that MLL2 contributes to the epigenetic reprogramming that takes place before fertilization. We propose that once this task has been accomplished, MLL2 is not required until gastrulation and that other methyltransferases are responsible for bulk H3K4me3, thereby revealing an unexpected epigenetic control switch amongst the H3K4 methyltransferases during development.

The preleukemic state of mice reconstituted with Mixl1-transduced marrow cells.

Murine granulocytic cells, in becoming leukemic, need to acquire enhanced self-generation and a capacity for autocrine growth stimulation. Mice transplanted with bone marrow cells transduced with the Mixl1 homeobox gene develop a very high frequency of myeloid leukemia derived from the transduced cells. Preleukemic mice contained a high frequency of transduced clonogenic granulocytic cells. They exhibited an abnormally high capacity for self-replication and could generate immortalized granulocytic cell lines that remained absolutely dependent on either GM-CSF or IL-3 and were not leukemic. Organs from mice repopulated by marrow cells transduced either with Mixl1 or the control murine stem cell virus vector exhibited a capacity to produce IL-3 in vitro, activity being highest with the lungs, marrow, bladder, and thymus. Supporting evidence for the in vivo production of IL-3 was the frequent development of mast cells in the marrow. Overexpression of Mixl1 appears capable of inducing an abnormal self-renewal capacity in granulocytic precursors. Aberrant production of IL-3 was not present in the continuous Mixl cell lines and was therefore not in itself likely to be a leukemogenic change but it could support the enhanced survival and proliferation of the Mixl1 granulocytic populations until a final leukemogenic mutation occurs in them.

Increased apoptosis and skewed differentiation in mouse embryonic stem cells lacking the histone methyltransferase Mll2.

Epigenetic regulation by histone methyltransferases provides transcriptional memory and inheritable propagation of gene expression patterns. Potentially, the transition from a pluripotent state to lineage commitment also includes epigenetic instructions. The histone 3 lysine 4 methyltransferase Mll2/Wbp7 is essential for embryonic development. Here, we used embryonic stem (ES) cell lines deficient for Mll2 to examine its function more accurately. Mll2-/- ES cells are viable and retain pluripotency, but they display cell proliferation defects due to an enhanced rate of apoptosis. Apoptosis was not relieved by caspase inhibition and correlated with decreased Bcl2 expression. Concordantly, Mll2 binds to the Bcl2 gene and H3K4me(3) levels are reduced at the binding site when Mll2 is absent. In vitro differentiation showed delays along representative pathways for all three germ layers. Although ectodermal delays were severe and mesodermal delays persisted at about three days, endodermal differentiation seemed to recover and overshoot, concomitant with prolonged Oct4 gene expression. Hence, Mll2 is not required for ES cell self-renewal or the complex changes in gene expression involved in lineage commitment, but it contributes to the coordination and timing of early differentiation decisions.

Blood pressure changes after renal denervation are more pronounced in women and nondiabetic patients: findings from the Austrian Transcatheter Renal Denervation Registry.

OBJECTIVES: Three recently published sham-controlled studies proved the efficacy of renal denervation (RDN) in hypertensive patients. The study presented here analyzed a nationwide multicentre registry database to clarify which patient subgroups benefit most from radiofrequency RDN. METHODS: This is a post hoc analysis from the multicentre Austrian Transcatheter Renal Denervation Registry hosted by the Austrian Society of Hypertension. We correlated change of SBP after RDN to sex and presence/absence of comorbidities. Univariable correlation and multiple linear regression analyses were performed. RESULTS: Two hundred and ninety-one patients (43% women, median age 64 years) undergoing RDN between April 2011 and September 2014 were included in this analysis. Mean baseline ambulatory 24 h BP (systolic/diastolic) was 150 ±â€Š18/89 ±â€Š14 mmHg and mean baseline office BP was 170 ±â€Š16/94 ±â€Š14 mmHg.After RDN, mean ambulatory 24 h BP reduction was 9 ±â€Š19/6 ±â€Š16 mmHg. The following features were associated with a good response to RDN: high baseline systolic ambulatory BP, high baseline diastolic office BP, female sex, absence of diabetes mellitus, and absence of peripheral artery disease. Multivariable analysis identified female sex and absence of diabetes mellitus as strongest predictors for ambulatory BP reduction, although those groups had the lowest baseline ambulatory BP. DISCUSSION: Ambulatory BP reductions after RDN were substantially more pronounced in female and in nondiabetic patients despite lower baseline BP. It is concluded that in terms of efficacy female patients and nondiabetic patients might benefit more from RDN.

Design, Synthesis, and Biological Activity of 1,2,3-Triazolobenzodiazepine BET Bromodomain Inhibitors.

A number of diazepines are known to inhibit bromo- and extra-terminal domain (BET) proteins. Their BET inhibitory activity derives from the fusion of an acetyl-lysine mimetic heterocycle onto the diazepine framework. Herein we describe a straightforward, modular synthesis of novel 1,2,3-triazolobenzodiazepines and show that the 1,2,3-triazole acts as an effective acetyl-lysine mimetic heterocycle. Structure-based optimization of this series of compounds led to the development of potent BET bromodomain inhibitors with excellent activity against leukemic cells, concomitant with a reduction in c-MYC expression. These novel benzodiazepines therefore represent a promising class of therapeutic BET inhibitors.

Site-specific recombinatorics: in situ cellular barcoding with the Cre Lox system.

BACKGROUND: Cellular barcoding is a recently developed biotechnology tool that enables the familial identification of progeny of individual cells in vivo. In immunology, it has been used to track the burst-sizes of multiple distinct responding T cells over several adaptive immune responses. In the study of hematopoiesis, it revealed fate heterogeneity amongst phenotypically identical multipotent cells. Most existing approaches rely on ex vivo viral transduction of cells with barcodes followed by adoptive transfer into an animal, which works well for some systems, but precludes barcoding cells in their native environment such as those inside solid tissues. RESULTS: With a view to overcoming this limitation, we propose a new design for a genetic barcoding construct based on the Cre Lox system that induces randomly created stable barcodes in cells in situ by exploiting inherent sequence distance constraints during site-specific recombination. We identify the cassette whose provably maximal code diversity is several orders of magnitude higher than what is attainable with previously considered Cre Lox barcoding approaches, exceeding the number of lymphocytes or hematopoietic progenitor cells in mice. CONCLUSIONS: Its high diversity and in situ applicability, make the proposed Cre Lox based tagging system suitable for whole tissue or even whole animal barcoding. Moreover, it can be built using established technology.

Impact of loss of BH3-only proteins on the development and treatment of MLL-fusion gene-driven AML in mice.

Inhibition of the apoptosis pathway controlled by opposing members of the Bcl-2 protein family plays a central role in cancer development and resistance to therapy. To investigate how pro-apoptotic Bcl-2 homology domain 3 (BH3)-only proteins impact on acute myeloid leukemia (AML), we generated mixed lineage leukemia (MLL)-AF9 and MLL-ENL AMLs from BH3-only gene knockout mice. Disease development was not accelerated by loss of Bim, Puma, Noxa, Bmf, or combinations thereof; hence these BH3-only proteins are apparently ineffectual as tumor suppressors in this model. We tested the sensitivity of MLL-AF9 AMLs of each genotype in vitro to standard chemotherapeutic drugs and to the proteasome inhibitor bortezomib, with or without the BH3 mimetic ABT-737. Loss of Puma and/or Noxa increased resistance to cytarabine, daunorubicin and etoposide, while loss of Bim protected against cytarabine and loss of Bmf had no impact. ABT-737 increased sensitivity to the genotoxic drugs but was not dependent on any BH3-only protein tested. The AML lines were very sensitive to bortezomib and loss of Noxa conveyed significant resistance. In vivo, several MLL-AF9 AMLs responded well to daunorubicin and this response was highly dependent on Puma and Noxa but not Bim. Combination therapy with ABT-737 provided little added benefit at the daunorubicin dose trialed. Bortezomib also extended survival of AML-bearing mice, albeit less than daunorubicin. In summary, our genetic studies reveal the importance of Puma and Noxa for the action of genotoxics currently used to treat MLL-driven AML and suggest that, while addition of ABT-737-like BH3 mimetics might enhance their efficacy, new Noxa-like BH3 mimetics targeting Mcl-1 might have greater potential.

Rapid Inflammation in Mice Lacking Both SOCS1 and SOCS3 in Hematopoietic Cells.

The Suppressors of Cytokine Signalling (SOCS) proteins are negative regulators of cytokine signalling required to prevent excess cellular responses. SOCS1 and SOCS3 are essential to prevent inflammatory disease, SOCS1 by attenuating responses to IFNγ and gamma-common (γc) cytokines, and SOCS3 via regulation of G-CSF and IL-6 signalling. SOCS1 and SOCS3 show significant sequence homology and are the only SOCS proteins to possess a KIR domain. The possibility of overlapping or redundant functions was investigated in inflammatory disease via generation of mice lacking both SOCS1 and SOCS3 in hematopoietic cells. Loss of SOCS3 significantly accelerated the pathology and inflammatory disease characteristic of SOCS1 deficiency. We propose a model in which SOCS1 and SOCS3 operate independently to control specific cytokine responses and together modulate the proliferation and activation of lymphoid and myeloid cells to prevent rapid inflammatory disease.

Eliminating Legionella by inhibiting BCL-XL to induce macrophage apoptosis.

Human pathogenic Legionella replicate in alveolar macrophages and cause a potentially lethal form of pneumonia known as Legionnaires' disease(1). Here, we have identified a host-directed therapeutic approach to eliminate intracellular Legionella infections. We demonstrate that the genetic deletion, or pharmacological inhibition, of the host cell pro-survival protein BCL-XL induces intrinsic apoptosis of macrophages infected with virulent Legionella strains, thereby abrogating Legionella replication. BCL-XL is essential for the survival of Legionella-infected macrophages due to bacterial inhibition of host-cell protein synthesis, resulting in reduced levels of the short-lived, related BCL-2 pro-survival family member, MCL-1. Consequently, a single dose of a BCL-XL-targeted BH3-mimetic therapy, or myeloid cell-restricted deletion of BCL-XL, limits Legionella replication and prevents lethal lung infections in mice. These results indicate that repurposing BH3-mimetic compounds, originally developed to induce cancer cell apoptosis, may have efficacy in treating Legionnaires' and other diseases caused by intracellular microbes.

MCL-1 is required throughout B-cell development and its loss sensitizes specific B-cell subsets to inhibition of BCL-2 or BCL-XL.

Pro-survival BCL-2 family members protect cells from programmed cell death that can be induced by multiple internal or external cues. Within the haematopoietic lineages, the BCL-2 family members BCL-2, BCL-XL and MCL-1 are known to support cell survival but the individual and overlapping roles of these pro-survival BCL-2 proteins for the persistence of individual leukocyte subsets in vivo has not yet been determined. By combining inducible knockout mouse models with the BH3-mimetic compound ABT-737, which inhibits BCL-2, BCL-XL and BCL-W, we found that dependency on MCL-1, BCL-XL or BCL-2 expression changes during B-cell development. We show that BCL-XL expression promotes survival of immature B cells, expression of BCL-2 is important for survival of mature B cells and long-lived plasma cells (PC), and expression of MCL-1 is important for survival throughout B-cell development. These data were confirmed with novel highly specific BH3-mimetic compounds that target either BCL-2, BCL-XL or MCL-1. In addition, we observed that combined inhibition of these pro-survival proteins acts in concert to delete specific B-cell subsets. Reduced expression of MCL-1 further sensitized immature as well as transitional B cells and splenic PC to loss of BCL-XL expression. More markedly, loss of MCL-1 greatly sensitizes PC populations to BCL-2 inhibition using ABT-737, even though the total wild-type PC pool in the spleen is not significantly affected by this drug and the bone marrow (BM) PC population only slightly. Combined loss or inhibition of MCL-1 and BCL-2 reduced the numbers of established PC >100-fold within days. Our data suggest that combination treatment targeting these pro-survival proteins could be advantageous for treatment of antibody-mediated autoimmune diseases and B-cell malignancies.