Deregulation and epigenetic modification of BCL2-family genes cause resistance to venetoclax in hematologic malignancies.

The BCL2 inhibitor venetoclax has been approved to treat different hematological malignancies. Because there is no common genetic alteration causing resistance to venetoclax in chronic lymphocytic leukemia (CLL) and B-cell lymphoma, we asked if epigenetic events might be involved in venetoclax resistance. Therefore, we employed whole-exome sequencing, methylated DNA immunoprecipitation sequencing, and genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 screening to investigate venetoclax resistance in aggressive lymphoma and high-risk CLL patients. We identified a regulatory CpG island within the PUMA promoter that is methylated upon venetoclax treatment, mediating PUMA downregulation on transcript and protein level. PUMA expression and sensitivity toward venetoclax can be restored by inhibition of methyltransferases. We can demonstrate that loss of PUMA results in metabolic reprogramming with higher oxidative phosphorylation and adenosine triphosphate production, resembling the metabolic phenotype that is seen upon venetoclax resistance. Although PUMA loss is specific for acquired venetoclax resistance but not for acquired MCL1 resistance and is not seen in CLL patients after chemotherapy-resistance, BAX is essential for sensitivity toward both venetoclax and MCL1 inhibition. As we found loss of BAX in Richter's syndrome patients after venetoclax failure, we defined BAX-mediated apoptosis to be critical for drug resistance but not for disease progression of CLL into aggressive diffuse large B-cell lymphoma in vivo. A compound screen revealed TRAIL-mediated apoptosis as a target to overcome BAX deficiency. Furthermore, antibody or CAR T cells eliminated venetoclax resistant lymphoma cells, paving a clinically applicable way to overcome venetoclax resistance.

Actionable perturbations of damage responses by TCL1/ATM and epigenetic lesions form the basis of T-PLL.

T-cell prolymphocytic leukemia (T-PLL) is a rare and poor-prognostic mature T-cell malignancy. Here we integrated large-scale profiling data of alterations in gene expression, allelic copy number (CN), and nucleotide sequences in 111 well-characterized patients. Besides prominent signatures of T-cell activation and prevalent clonal variants, we also identify novel hot-spots for CN variability, fusion molecules, alternative transcripts, and progression-associated dynamics. The overall lesional spectrum of T-PLL is mainly annotated to axes of DNA damage responses, T-cell receptor/cytokine signaling, and histone modulation. We formulate a multi-dimensional model of T-PLL pathogenesis centered around a unique combination of TCL1 overexpression with damaging ATM aberrations as initiating core lesions. The effects imposed by TCL1 cooperate with compromised ATM toward a leukemogenic phenotype of impaired DNA damage processing. Dysfunctional ATM appears inefficient in alleviating elevated redox burdens and telomere attrition and in evoking a p53-dependent apoptotic response to genotoxic insults. As non-genotoxic strategies, synergistic combinations of p53 reactivators and deacetylase inhibitors reinstate such cell death execution.

Is there an acceleration of the CpG transition rate during the mammalian radiation?

MOTIVATION: In this article we build a model of the CpG dinucleotide substitution rate and use it to challenge the claim that, that rate underwent a sudden mammalian-specific increase approximately 90 million years ago. The evidence supporting this hypothesis comes from the application of a model of neutral substitution rates able to account for elevated CpG dinucleotide substitution rates. With the initial goal of improving that model's accuracy, we introduced a modification enabling us to account for boundary effects arising by the truncation of the Markov field, as well as improving the optimization procedure required for estimating the substitution rates. RESULTS: When using this modified method to reproduce the supporting analysis, the evidence of the rate shift vanished. Our analysis suggests that the CpG-specific rate has been constant over the relevant time period and that the asserted acceleration of the CpG rate is likely an artifact of the original model.

Exponential decay of GC content detected by strand-symmetric substitution rates influences the evolution of isochore structure.

The distribution of guanine and cytosine nucleotides throughout a genome, or the GC content, is associated with numerous features in mammals; understanding the pattern and evolutionary history of GC content is crucial to our efforts to annotate the genome. The local GC content is decaying toward an equilibrium point, but the causes and rates of this decay, as well as the value of the equilibrium point, remain topics of debate. By comparing the results of 2 methods for estimating local substitution rates, we identify 620 Mb of the human genome in which the rates of the various types of nucleotide substitutions are the same on both strands. These strand-symmetric regions show an exponential decay of local GC content at a pace determined by local substitution rates. DNA segments subjected to higher rates experience disproportionately accelerated decay and are AT rich, whereas segments subjected to lower rates decay more slowly and are GC rich. Although we are unable to draw any conclusions about causal factors, the results support the hypothesis proposed by Khelifi A, Meunier J, Duret L, and Mouchiroud D (2006. GC content evolution of the human and mouse genomes: insights from the study of processed pseudogenes in regions of different recombination rates. J Mol Evol. 62:745-752.) that the isochore structure has been reshaped over time. If rate variation were a determining factor, then the current isochore structure of mammalian genomes could result from the local differences in substitution rates. We predict that under current conditions strand-symmetric portions of the human genome will stabilize at an average GC content of 30% (considerably less than the current 42%), thus confirming that the human genome has not yet reached equilibrium.

Parameter estimation in ordinary differential equations for biochemical processes using the method of multiple shooting.

In silico investigations by simulating dynamical models of biochemical processes play an important role in systems biology. If the parameters of a model are unknown, results from simulation studies can be misleading. Such a scenario can be avoided by estimating the parameters before analysing the system. Almost all approaches for estimating parameters in ordinary differential equations have either a small convergence region or suffer from an immense computational cost. The method of multiple shooting can be situated in between of these extremes. In spite of its good convergence and stability properties, the literature regarding the practical implementation and providing some theoretical background is rarely available. All necessary information for a successful implementation is supplied here and the basic facts of the involved numerics are discussed. To show the performance of the method, two illustrative examples are discussed.

Mixing properties of the Rössler system and consequences for coherence and synchronization analysis.

Cross-spectral and synchronization analysis of two independent, identical chaotic Rössler systems suggest a coupling although there is no interaction. This spuriously detected interaction can either be explained by the absence of mixing or by finite size effects. To decide which alternative holds the phase dynamics is studied by a model of the fluctuations derived from the system's equations. The basic assumption of the model is a diffusive character for the system which corresponds to mixing. Comparison of theoretical properties of the model with empirical properties of the Rössler system suggests that the system is mixing but the rate of mixing appears to be rather low.

On studentising and blocklength selection for the bootstrap on time series.

For independent data, non-parametric bootstrap is realised by resampling the data with replacement. This approach fails for dependent data such as time series. If the data generating process is at least stationary and mixing, the blockwise bootstrap by drawing subsamples or blocks of the data saves the concept. For the blockwise bootstrap a blocklength has to be selected. We propose a method for selecting the optimal blocklength. To improve the finite size properties of the blockwise bootstrap, studentised statistics is considered. If the statistic can be represented as a smooth function model this studentisation can be approximated efficiently. The studentised blockwise bootstrap method is applied for testing hypotheses on medical time series.

Variations in substitution rate in human and mouse genomes.

We present a method to quantify spatial fluctuations of the substitution rate on different length scales throughout genomes of eukaryotes. The fluctuations on large length scales are found to be predominantly a consequence of a coarse-graining effect of fluctuations on shorter length scales. This is verified for both the mouse and the human genome. We also found that the relative standard deviation of fluctuations in substitution rate is about a factor three smaller in mouse than in human. The method allows furthermore to determine time-resolved substitution rate maps of the genomes, where the corresponding autocorrelation functions quantify the velocity of spatial chromosomal reorganization.

Drosophila APC2 and Armadillo participate in tethering mitotic spindles to cortical actin.

Proper positioning of mitotic spindles ensures equal allocation of chromosomes to daughter cells. This often involves interactions between spindle and astral microtubules and cortical actin. In yeast and Caenorhabditis elegans, some of the protein machinery that connects spindles and cortex has been identified but, in most animal cells, this process remains mysterious. Here, we report that the tumour suppressor homologue APC2 and its binding partner Armadillo both play roles in spindle anchoring during the syncytial mitoses of early Drosophila embryos. Armadillo, alpha-catenin and APC2 all localize to sites of cortical spindle attachment. APC2-Armadillo complexes often localize with interphase microtubules. Zeste-white 3 kinase, which can phosphorylate Armadillo and APC, is also crucial for spindle positioning and regulates the localization of APC2-Armadillo complexes. Together, these data suggest that APC2, Armadillo and alpha-catenin provide an important link between spindles and cortical actin, and that this link is regulated by Zeste-white 3 kinase.

Wingless can't fly so it hitches a ride with dynein.

Asymmetric RNA localization is required for many developmental processes in a wide range of organisms. For example, wingless and pair-rule transcripts are localized to the apical membrane of polarized cells. It has been unclear, however, if this localization is important for biological activity and, in addition, how the transcripts are transported. Two recent studies (1,2) have identified cis-elements and trans-acting factors that are required for the asymmetric localization of mRNAs. Correct localization is shown to be required for biological activity, and a mechanism of RNA transport involving the microtubule motor dynein has been revealed.

Wnt signaling: the naked truth?

Frizzled receptors can activate two alternative signal transduction pathways: the canonical Wnt pathway or the planar cell polarity pathway. Recent studies of the Naked cuticle protein suggest a mechanism for the inactivation of the canonical pathway and concomitant activation of the planar cell polarity pathway.

Activated armadillo/beta-catenin does not play a general role in cell migration and process extension in Drosophila.

Human beta-catenin and its fly homolog Armadillo are best known for their roles in cadherin-based cell-cell adhesion and in transduction of Wingless/Wnt signals. It has been hypothesized that beta-catenin may also regulate cell migration and cell shape changes, possibly by regulating the microtubule cytoskeleton via interactions with APC. This hypothesis was based on experiments in which a hyperstable mutant form of beta-catenin was expressed in MDCK cells, where it altered their migratory properties and their ability to send out long cellular processes. We tested the generality of this hypothesis in vivo in Drosophila. We utilized three model systems in which cell migration and/or process extension are known to play key roles during development: the migration of the border cells during oogenesis, the extension of axons in the nervous system, and the migration and cell process extension of tracheal cells. In all cases, cells expressing activated Armadillo were able to migrate and extend cell processes essentially normally. The one alteration from normal involved an apparent cell fate change in certain tracheal cells. These results suggest that only certain cells are affected by activation of Armadillo/beta-catenin, and that Armadillo/beta-catenin does not play a general role in inhibiting cell migration or process extension.

Cadherin sequences that inhibit beta-catenin signaling: a study in yeast and mammalian cells.

Drosophila Armadillo and its mammalian homologue beta-catenin are scaffolding proteins involved in the assembly of multiprotein complexes with diverse biological roles. They mediate adherens junction assembly, thus determining tissue architecture, and also transduce Wnt/Wingless intercellular signals, which regulate embryonic cell fates and, if inappropriately activated, contribute to tumorigenesis. To learn more about Armadillo/beta-catenin's scaffolding function, we examined in detail its interaction with one of its protein targets, cadherin. We utilized two assay systems: the yeast two-hybrid system to study cadherin binding in the absence of Armadillo/beta-catenin's other protein partners, and mammalian cells where interactions were assessed in their presence. We found that segments of the cadherin cytoplasmic tail as small as 23 amino acids bind Armadillo or beta-catenin in yeast, whereas a slightly longer region is required for binding in mammalian cells. We used mutagenesis to identify critical amino acids required for cadherin interaction with Armadillo/beta-catenin. Expression of such short cadherin sequences in mammalian cells did not affect adherens junctions but effectively inhibited beta-catenin-mediated signaling. This suggests that the interaction between beta-catenin and T cell factor family transcription factors is a sensitive target for disruption, making the use of analogues of these cadherin derivatives a potentially useful means to suppress tumor progression.

Abelson kinase regulates epithelial morphogenesis in Drosophila.

Activation of the nonreceptor tyrosine kinase Abelson (Abl) contributes to the development of leukemia, but the complex roles of Abl in normal development are not fully understood. Drosophila Abl links neural axon guidance receptors to the cytoskeleton. Here we report a novel role for Drosophila Abl in epithelial cells, where it is critical for morphogenesis. Embryos completely lacking both maternal and zygotic Abl die with defects in several morphogenetic processes requiring cell shape changes and cell migration. We describe the cellular defects that underlie these problems, focusing on dorsal closure as an example. Further, we show that the Abl target Enabled (Ena), a modulator of actin dynamics, is involved with Abl in morphogenesis. We find that Ena localizes to adherens junctions of most epithelial cells, and that it genetically interacts with the adherens junction protein Armadillo (Arm) during morphogenesis. The defects of abl mutants are strongly enhanced by heterozygosity for shotgun, which encodes DE-cadherin. Finally, loss of Abl reduces Arm and alpha-catenin accumulation in adherens junctions, while having little or no effect on other components of the cytoskeleton or cell polarity machinery. We discuss possible models for Abl function during epithelial morphogenesis in light of these data.

Evidence for functional differentiation among Drosophila septins in cytokinesis and cellularization.

The septins are a conserved family of proteins that are involved in cytokinesis and other aspects of cell-surface organization. In Drosophila melanogaster, null mutations in the pnut septin gene are recessive lethal, but homozygous pnut mutants complete embryogenesis and survive until the pupal stage. Because the completion of cellularization and other aspects of early development seemed likely to be due to maternally contributed Pnut product, we attempted to generate embryos lacking the maternal contribution in order to explore the roles of Pnut in these processes. We used two methods, the production of germline clones homozygous for a pnut mutation and the rescue of pnut homozygous mutant flies by a pnut(+) transgene under control of the hsp70 promoter. Remarkably, the pnut germline-clone females produced eggs, indicating that stem-cell and cystoblast divisions in the female germline do not require Pnut. Moreover, the Pnut-deficient embryos obtained by either method completed early syncytial development and began cellularization of the embryo normally. However, during the later stages of cellularization, the organization of the actin cytoskeleton at the leading edge of the invaginating furrows became progressively more abnormal, and the embryos displayed widespread defects in cell and embryo morphology beginning at gastrulation. Examination of two other septins showed that Sep1 was not detectable at the cellularization front in the Pnut-deficient embryos, whereas Sep2 was still present in normal levels. Thus, it is possible that Sep2 (perhaps in conjunction with other septins such as Sep4 and Sep5) fulfills an essential septin role during the organization and initial ingression of the cellularization furrow even in the absence of Pnut and Sep1. Together, the results suggest that some cell-division events in Drosophila do not require septin function, that there is functional differentiation among the Drosophila septins, or both.

A screen for mutations that suppress the phenotype of Drosophila armadillo, the beta-catenin homolog.

During development signaling pathways coordinate cell fates and regulate the choice between cell survival or programmed cell death. The well-conserved Wingless/Wnt pathway is required for many developmental decisions in all animals. One transducer of the Wingless/Wnt signal is Armadillo/beta-catenin. Drosophila Armadillo not only transduces Wingless signal, but also acts in cell-cell adhesion via its role in the epithelial adherens junction. While many components of both the Wingless/Wnt signaling pathway and adherens junctions are known, both processes are complex, suggesting that unknown components influence signaling and junctions. We carried out a genetic modifier screen to identify some of these components by screening for mutations that can suppress the armadillo mutant phenotype. We identified 12 regions of the genome that have this property. From these regions and from additional candidate genes tested we identified four genes that suppress arm: dTCF, puckered, head involution defective (hid), and Dpresenilin. We further investigated the interaction with hid, a known regulator of programmed cell death. Our data suggest that Wg signaling modulates Hid activity and that Hid regulates programmed cell death in a dose-sensitive fashion.

Cell biology. Travel bulletin--traffic jams cause tumors.

All animal cells have a polarity, that is, different proteins are clustered in distinct domains of the plasma membrane and these regions carry out different jobs. As Peifer discusses in a lively Perspective, new work (Bilder et al.) identifies some of the molecular characters that direct proteins to their different cellular destinations.

The canonical Wg and JNK signaling cascades collaborate to promote both dorsal closure and ventral patterning.

Elaboration of the Drosophila body plan depends on a series of cell-identity decisions and morphogenetic movements regulated by intercellular signals. For example, Jun N-terminal kinase signaling regulates cell fate decisions and morphogenesis during dorsal closure, while Wingless signaling regulates segmental patterning of the larval cuticle via Armadillo. wingless or armadillo mutant embryos secrete a lawn of ventral denticles; armadillo mutants also exhibit dorsal closure defects. We found that mutations in puckered, a phosphatase that antagonizes Jun N-terminal kinase, suppress in a dose-sensitive manner both the dorsal and ventral armadillo cuticle defects. Furthermore, we found that activation of the Jun N-terminal kinase signaling pathway suppresses armadillo-associated defects. Jun N-terminal kinase signaling promotes dorsal closure, in part, by regulating decapentaplegic expression in the dorsal epidermis. We demonstrate that Wingless signaling is also required to activate decapentaplegic expression and to coordinate cell shape changes during dorsal closure. Together, these results demonstrate that MAP-Kinase and Wingless signaling cooperate in both the dorsal and ventral epidermis, and suggest that Wingless may activate both the Wingless and the Jun N-terminal kinase signaling cascades.

Teaching tumour suppressors new tricks.

Examination of the tumour suppressor adenomatous polyposis coli (APC) has shown that it may be multifunctional. Recent work has demonstrated dynamic interactions of APC with the microtubule cytoskeleton, supporting the idea that APC has an important function in cell migration.