Activation of the endomitotic spindle assembly checkpoint and thrombocytopenia in Plk1-deficient mice.

Polyploidization in megakaryocytes is achieved by endomitosis, a specialized cell cycle in which DNA replication is followed by aberrant mitosis. Typical mitotic regulators such as Aurora kinases or Cdk1 are dispensable for megakaryocyte maturation, and inhibition of mitotic kinases may in fact promote megakaryocyte maturation. However, we show here that Polo-like kinase 1 (Plk1) is required for endomitosis, and ablation of the Plk1 gene in megakaryocytes results in defective polyploidization accompanied by mitotic arrest and cell death. Lack of Plk1 results in defective centrosome maturation and aberrant spindle pole formation, thus impairing the formation of multiple poles typically found in megakaryocytes. In these conditions, megakaryocytes arrest for a long time in mitosis and frequently die. Mitotic arrest in wild-type megakaryocytes treated with Plk1 inhibitors or Plk1-null cells is triggered by the spindle assembly checkpoint (SAC), and can be rescued in the presence of SAC inhibitors. These data suggest that, despite the dispensability of proper chromosome segregation in megakaryocytes, an endomitotic SAC is activated in these cells upon Plk1 inhibition. SAC activation results in defective maturation of megakaryocytes and cell death, thus raising a note of caution in the use of Plk1 inhibitors in therapeutic strategies based on polyploidization regulators.

Cdk4 and Cdk6 cooperate in counteracting the INK4 family of inhibitors during murine leukemogenesis.

Cdk4 and Cdk6 are related protein kinases that bind d-type cyclins and regulate cell-cycle progression. Cdk4/6 inhibitors are currently being used in advanced clinical trials and show great promise against many types of tumors. Cdk4 and Cdk6 are inhibited by INK4 proteins, which exert tumor-suppressing functions. To test the significance of this inhibitory mechanism, we generated knock-in mice that express a Cdk6 mutant (Cdk6 R31C) insensitive to INK4-mediated inhibition. Cdk6(R/R) mice display altered development of the hematopoietic system without enhanced tumor susceptibility, either in the presence or absence of p53. Unexpectedly, Cdk6 R31C impairs the potential of hematopoietic progenitors to repopulate upon adoptive transfer or after 5-fluorouracil-induced damage. The defects are overcome by eliminating sensitivity of cells to INK4 inhibitors by introducing the INK4-insensitive Cdk4 R24C allele, and INK4-resistant mice are more susceptible to hematopoietic and endocrine tumors. In BCR-ABL-transformed hematopoietic cells, Cdk6 R31C causes increased binding of p16(INK4a) to wild-type Cdk4, whereas cells harboring Cdk4 R24C and Cdk6 R31C are fully insensitive to INK4 inhibitors, resulting in accelerated disease onset. Our observations reveal that Cdk4 and Cdk6 cooperate in hematopoietic tumor development and suggest a role for Cdk6 in sequestering INK4 proteins away from Cdk4.

Integrated phenotypic and activity-based profiling links Ces3 to obesity and diabetes.

Phenotypic screening is making a comeback in drug discovery as the maturation of chemical proteomics methods has facilitated target identification for bioactive small molecules. A limitation of these approaches is that time-consuming genetic methods or other means are often required to determine the biologically relevant target (or targets) from among multiple protein-compound interactions that are typically detected. Here, we have combined phenotypic screening of a directed small-molecule library with competitive activity-based protein profiling to map and functionally characterize the targets of screening hits. Using this approach, we identify carboxylesterase 3 (Ces3, also known as Ces1d) as a primary molecular target of bioactive compounds that promote lipid storage in adipocytes. We further show that Ces3 activity is markedly elevated during adipocyte differentiation. Treatment of two mouse models of obesity-diabetes with a Ces3 inhibitor ameliorates multiple features of metabolic syndrome, illustrating the power of the described strategy to accelerate the identification and pharmacologic validation of new therapeutic targets.

Thrombocytopenia-associated mutations in Ser/Thr kinase MASTL deregulate actin cytoskeletal dynamics in platelets.

MASTL, a Ser/Thr kinase that inhibits PP2A-B55 complexes during mitosis, is mutated in autosomal dominant thrombocytopenia. However, the connections between the cell-cycle machinery and this human disease remain unexplored. We report here that, whereas Mastl ablation in megakaryocytes prevented proper maturation of these cells, mice carrying the thrombocytopenia-associated mutation developed thrombocytopenia as a consequence of aberrant activation and survival of platelets. Activation of mutant platelets was characterized by hyperstabilized pseudopods mimicking the effect of PP2A inhibition and actin polymerization defects. These aberrations were accompanied by abnormal hyperphosphorylation of multiple components of the actin cytoskeleton and were rescued both in vitro and in vivo by inhibiting upstream kinases such as PKA, PKC, or AMPK. These data reveal an unexpected role of Mastl in actin cytoskeletal dynamics in postmitotic cells and suggest that the thrombocytopenia-associated mutation in MASTL is a pathogenic dominant mutation that mimics decreased PP2A activity resulting in altered phosphorylation of cytoskeletal regulatory pathways.

Therapeutic relevance of the PP2A-B55 inhibitory kinase MASTL/Greatwall in breast cancer.

PP2A is a major tumor suppressor whose inactivation is frequently found in a wide spectrum of human tumors. In particular, deletion or epigenetic silencing of genes encoding the B55 family of PP2A regulatory subunits is a common feature of breast cancer cells. A key player in the regulation of PP2A/B55 phosphatase complexes is the cell cycle kinase MASTL (also known as Greatwall). During cell division, inhibition of PP2A-B55 by MASTL is required to maintain the mitotic state, whereas inactivation of MASTL and PP2A reactivation is required for mitotic exit. Despite its critical role in cell cycle progression in multiple organisms, its relevance as a therapeutic target in human cancer and its dependence of PP2A activity is mostly unknown. Here we show that MASTL overexpression predicts poor survival and shows prognostic value in breast cancer patients. MASTL knockdown or knockout using RNA interference or CRISPR/Cas9 systems impairs proliferation of a subset of breast cancer cells. The proliferative function of MASTL in these tumor cells requires its kinase activity and the presence of PP2A-B55 complexes. By using a new inducible CRISPR/Cas9 system in breast cancer cells, we show that genetic ablation of MASTL displays a significant therapeutic effect in vivo. All together, these data suggest that the PP2A inhibitory kinase MASTL may have both prognostic and therapeutic value in human breast cancer.

Aurora B Overexpression Causes Aneuploidy and p21Cip1 Repression during Tumor Development.

Aurora kinase B, one of the three members of the mammalian Aurora kinase family, is the catalytic component of the chromosomal passenger complex, an essential regulator of chromosome segregation in mitosis. Aurora B is overexpressed in human tumors although whether this kinase may function as an oncogene in vivo is not established. Here, we report a new mouse model in which expression of the endogenous Aurkb locus can be induced in vitro and in vivo. Overexpression of Aurora B in cultured cells induces defective chromosome segregation and aneuploidy. Long-term overexpression of Aurora B in vivo results in aneuploidy and the development of multiple spontaneous tumors in adult mice, including a high incidence of lymphomas. Overexpression of Aurora B also results in a reduced DNA damage response and decreased levels of the p53 target p21(Cip1) in vitro and in vivo, in line with an inverse correlation between Aurora B and p21(Cip1) expression in human leukemias. Thus, overexpression of Aurora B may contribute to tumor formation not only by inducing chromosomal instability but also by suppressing the function of the cell cycle inhibitor p21(Cip1).

AMPK and PFKFB3 mediate glycolysis and survival in response to mitophagy during mitotic arrest.

Blocking mitotic progression has been proposed as an attractive therapeutic strategy to impair proliferation of tumour cells. However, how cells survive during prolonged mitotic arrest is not well understood. We show here that survival during mitotic arrest is affected by the special energetic requirements of mitotic cells. Prolonged mitotic arrest results in mitophagy-dependent loss of mitochondria, accompanied by reduced ATP levels and the activation of AMPK. Oxidative respiration is replaced by glycolysis owing to AMPK-dependent phosphorylation of PFKFB3 and increased production of this protein as a consequence of mitotic-specific translational activation of its mRNA. Induction of autophagy or inhibition of AMPK or PFKFB3 results in enhanced cell death in mitosis and improves the anti-tumoral efficiency of microtubule poisons in breast cancer cells. Thus, survival of mitotic-arrested cells is limited by their metabolic requirements, a feature with potential implications in cancer therapies aimed to impair mitosis or metabolism in tumour cells.

Identification of regulatory elements that control PPARγ expression in adipocyte progenitors.

Adipose tissue renewal and obesity-driven expansion of fat cell number are dependent on proliferation and differentiation of adipose progenitors that reside in the vasculature that develops in coordination with adipose depots. The transcriptional events that regulate commitment of progenitors to the adipose lineage are poorly understood. Because expression of the nuclear receptor PPARγ defines the adipose lineage, isolation of elements that control PPARγ expression in adipose precursors may lead to discovery of transcriptional regulators of early adipocyte determination. Here, we describe the identification and validation in transgenic mice of 5 highly conserved non-coding sequences from the PPARγ locus that can drive expression of a reporter gene in a manner that recapitulates the tissue-specific pattern of PPARγ expression. Surprisingly, these 5 elements appear to control PPARγ expression in adipocyte precursors that are associated with the vasculature of adipose depots, but not in mature adipocytes. Characterization of these five PPARγ regulatory sequences may enable isolation of the transcription factors that bind these cis elements and provide insight into the molecular regulation of adipose tissue expansion in normal and pathological states.