CDK4/6 inhibitor-mediated cell overgrowth triggers osmotic and replication stress to promote senescence.

Abnormal increases in cell size are associated with senescence and cell cycle exit. The mechanisms by which overgrowth primes cells to withdraw from the cell cycle remain unknown. We address this question using CDK4/6 inhibitors, which arrest cells in G0/G1 and are licensed to treat advanced HR+/HER2- breast cancer. We demonstrate that CDK4/6-inhibited cells overgrow during G0/G1, causing p38/p53/p21-dependent cell cycle withdrawal. Cell cycle withdrawal is triggered by biphasic p21 induction. The first p21 wave is caused by osmotic stress, leading to p38- and size-dependent accumulation of p21. CDK4/6 inhibitor washout results in some cells entering S-phase. Overgrown cells experience replication stress, resulting in a second p21 wave that promotes cell cycle withdrawal from G2 or the subsequent G1. We propose that the levels of p21 integrate signals from overgrowth-triggered stresses to determine cell fate. This model explains how hypertrophy can drive senescence and why CDK4/6 inhibitors have long-lasting effects in patients.

Plasmin activity promotes amyloid deposition in a transgenic model of human transthyretin amyloidosis.

Cardiac ATTR amyloidosis, a serious but much under-diagnosed form of cardiomyopathy, is caused by deposition of amyloid fibrils derived from the plasma protein transthyretin (TTR), but its pathogenesis is poorly understood and informative in vivo models have proved elusive. Here we report the generation of a mouse model of cardiac ATTR amyloidosis with transgenic expression of human TTRS52P. The model is characterised by substantial ATTR amyloid deposits in the heart and tongue. The amyloid fibrils contain both full-length human TTR protomers and the residue 49-127 cleavage fragment which are present in ATTR amyloidosis patients. Urokinase-type plasminogen activator (uPA) and plasmin are abundant within the cardiac and lingual amyloid deposits, which contain marked serine protease activity; knockout of α2-antiplasmin, the physiological inhibitor of plasmin, enhances amyloid formation. Together, these findings indicate that cardiac ATTR amyloid deposition involves local uPA-mediated generation of plasmin and cleavage of TTR, consistent with the previously described mechano-enzymatic hypothesis for cardiac ATTR amyloid formation. This experimental model of ATTR cardiomyopathy has potential to allow further investigations of the factors that influence human ATTR amyloid deposition and the development of new treatments.

EML4-ALK V3 oncogenic fusion proteins promote microtubule stabilization and accelerated migration through NEK9 and NEK7.

EML4-ALK is an oncogenic fusion present in ∼5% of non-small cell lung cancers. However, alternative breakpoints in the EML4 gene lead to distinct variants of EML4-ALK with different patient outcomes. Here, we show that, in cell models, EML4-ALK variant 3 (V3), which is linked to accelerated metastatic spread, causes microtubule stabilization, formation of extended cytoplasmic protrusions and increased cell migration. EML4-ALK V3 also recruits the NEK9 and NEK7 kinases to microtubules via the N-terminal EML4 microtubule-binding region. Overexpression of wild-type EML4, as well as constitutive activation of NEK9, also perturbs cell morphology and accelerates migration in a microtubule-dependent manner that requires the downstream kinase NEK7 but does not require ALK activity. Strikingly, elevated NEK9 expression is associated with reduced progression-free survival in EML4-ALK patients. Hence, we propose that EML4-ALK V3 promotes microtubule stabilization through NEK9 and NEK7, leading to increased cell migration. This represents a novel actionable pathway that could drive metastatic disease progression in EML4-ALK lung cancer.

Mitotic phosphorylation by NEK6 and NEK7 reduces the microtubule affinity of EML4 to promote chromosome congression.

EML4 is a microtubule-associated protein that promotes microtubule stability. We investigated its regulation across the cell cycle and found that EML4 was distributed as punctate foci along the microtubule lattice in interphase but exhibited reduced association with spindle microtubules in mitosis. Microtubule sedimentation and cryo-electron microscopy with 3D reconstruction revealed that the basic N-terminal domain of EML4 mediated its binding to the acidic C-terminal tails of α- and ß-tubulin on the microtubule surface. The mitotic kinases NEK6 and NEK7 phosphorylated the EML4 N-terminal domain at Ser144 and Ser146 in vitro, and depletion of these kinases in cells led to increased EML4 binding to microtubules in mitosis. An S144A-S146A double mutant not only bound inappropriately to mitotic microtubules but also increased their stability and interfered with chromosome congression. In addition, constitutive activation of NEK6 or NEK7 reduced the association of EML4 with interphase microtubules. Together, these data support a model in which NEK6- and NEK7-dependent phosphorylation promotes the dissociation of EML4 from microtubules in mitosis in a manner that is required for efficient chromosome congression.

EML proteins in microtubule regulation and human disease.

The EMLs are a conserved family of microtubule-associated proteins (MAPs). The founding member was discovered in sea urchins as a 77-kDa polypeptide that co-purified with microtubules. This protein, termed EMAP for echinoderm MAP, was the major non-tubulin component present in purified microtubule preparations made from unfertilized sea urchin eggs [J. Cell Sci. (1993) 104: , 445-450; J. Cell Sci. (1987) 87: (Pt 1), 71-84]. Orthologues of EMAP were subsequently identified in other echinoderms, such as starfish and sand dollar, and then in more distant eukaryotes, including flies, worms and vertebrates, where the name of ELP or EML (both for EMAP-like protein) has been adopted [BMC Dev. Biol. (2008) 8: , 110; Dev. Genes Evol. (2000) 210: , 2-10]. The common property of these proteins is their ability to decorate microtubules. However, whether they are associated with particular microtubule populations or exercise specific functions in different microtubule-dependent processes remains unknown. Furthermore, although there is limited evidence that they regulate microtubule dynamics, the biochemical mechanisms of their molecular activity have yet to be explored. Nevertheless, interest in these proteins has grown substantially because of the identification of EML mutations in neuronal disorders and oncogenic fusions in human cancers. Here, we summarize our current knowledge of the expression, localization and structure of what is proving to be an interesting and important class of MAPs. We also speculate about their function in microtubule regulation and highlight how the studies of EMLs in human diseases may open up novel avenues for patient therapy.

Septicaemia models using Streptococcus pneumoniae and Listeria monocytogenes: understanding the role of complement properdin.

Streptococcus pneumoniae and Listeria monocytogenes, pathogens which can cause severe infectious disease in human, were used to infect properdin-deficient and wildtype mice. The aim was to deduce a role for properdin, positive regulator of the alternative pathway of complement activation, by comparing and contrasting the immune response of the two genotypes in vivo. We show that properdin-deficient and wildtype mice mounted antipneumococcal serotype-specific IgM antibodies, which were protective. Properdin-deficient mice, however, had increased survival in the model of streptococcal pneumonia and sepsis. Low activity of the classical pathway of complement and modulation of FcγR2b expression appear to be pathogenically involved. In listeriosis, however, properdin-deficient mice had reduced survival and a dendritic cell population that was impaired in maturation and activity. In vitro analyses of splenocytes and bone marrow-derived myeloid cells support the view that the opposing outcomes of properdin-deficient and wildtype mice in these two infection models is likely to be due to a skewing of macrophage activity to an M2 phenotype in the properdin-deficient mice. The phenotypes observed thus appear to reflect the extent to which M2- or M1-polarised macrophages are involved in the immune responses to S. pneumoniae and L. monocytogenes. We conclude that properdin controls the strength of immune responses by affecting humoral as well as cellular phenotypes during acute bacterial infection and ensuing inflammation.