Premature mitotic entry induced by ATR inhibition potentiates olaparib inhibition-mediated genomic instability, inflammatory signaling, and cytotoxicity in BRCA2-deficient cancer cells

Poly(ADP-ribose) polymerase (PARP) inhibitors are selectively cytotoxic in cancer cells with defects in homologous recombination (HR) (e.g., due to BRCA1/2 mutations). However, not all HR-deficient tumors efficiently respond to PARP inhibition and often acquire resistance. It is therefore important to uncover how PARP inhibitors induce cytotoxicity and develop combination strategies to potentiate PARP inhibitor efficacy in HR-deficient tumors. In this study, we found that forced mitotic entry upon ATR inhibition potentiates cytotoxic effects of PARP inhibition using olaparib in BRCA2-depleted and Brca2 knockout cancer cell line models. Single DNA fiber analysis showed that ATR inhibition does not exacerbate replication fork degradation. Instead, we find ATR inhibitors accelerate mitotic entry, resulting in the formation of chromatin bridges and lagging chromosomes. Furthermore, using genome-wide single-cell sequencing, we show that ATR inhibition enhances genomic instability of olaparib-treated BRCA2-depleted cells. Inhibition of CDK1 to delay mitotic entry mitigated mitotic aberrancies and genomic instability upon ATR inhibition, underscoring the role of ATR in coordinating proper cell cycle timing in situations of DNA damage. Additionally, we show that olaparib treatment leads to increased numbers of micronuclei, which is accompanied by a cGAS/STING-associated inflammatory response in BRCA2-deficient cells. ATR inhibition further increased the numbers of cGAS-positive micronuclei and the extent of cytokine production in olaparib-treated BRCA2-deficient cancer cells. Altogether, we show that ATR inhibition induces premature mitotic entry and mediates synergistic cytotoxicity with PARP inhibition in HR-deficient cancer cells, which involves enhanced genomic instability and inflammatory signaling

A synthetic lethal screen identifies HDAC4 as a potential target in MELK overexpressing cancers

Maternal embryonic leucine zipper kinase (MELK) is frequently overexpressed in cancer, but the role of MELK in cancer is still poorly understood. MELK was shown to have roles in many cancer-associated processes including tumor growth, chemotherapy resistance, and tumor recurrence. To determine whether the frequent overexpression of MELK can be exploited in therapy, we performed a high-throughput screen using a library of Saccharomyces cerevisiae mutants to identify genes whose functions become essential when MELK is overexpressed. We identified two such genes: LAG2 and HDA3. LAG2 encodes an inhibitor of the SCF ubiquitin-ligase complex, while HDA3 encodes a subunit of the HDA1 histone deacetylase complex. We find that one of these synthetic lethal interactions is conserved in mammalian cells, as inhibition of a human homolog of HDA3 (HDAC4) is synthetically toxic in MELK overexpression cells. Altogether, our work identified a novel potential drug target for tumors that overexpress MELK

The H3.3K27M oncohistone affects replication stress outcome and provokes genomic instability in pediatric glioma

While comprehensive molecular profiling of histone H3.3 mutant pediatric high-grade glioma has revealed extensive dysregulation of the chromatin landscape, the exact mechanisms driving tumor formation remain poorly understood. Since H3.3 mutant gliomas also exhibit high levels of copy number alterations, we set out to address if the H3.3K27M oncohistone leads to destabilization of the genome. Hereto, we established a cell culture model allowing inducible H3.3K27M expression and observed an increase in mitotic abnormalities. We also found enhanced interaction of DNA replication factors with H3.3K27M during mitosis, indicating replication defects. Further functional analyses revealed increased genomic instability upon replication stress, as represented by mitotic bulky and ultrafine DNA bridges. This co-occurred with suboptimal 53BP1 nuclear body formation after mitosis in vitro, and in human glioma. Finally, we observed a decrease in ultrafine DNA bridges following deletion of the K27M mutant H3F3A allele in primary high-grade glioma cells. Together, our data uncover a role for H3.3 in DNA replication under stress conditions that is altered by the K27M mutation, promoting genomic instability and potentially glioma development

Identification of Two Protein-Signaling States Delineating Transcriptionally Heterogeneous Human Medulloblastoma

Summary: The brain cancer medulloblastoma consists of different transcriptional subgroups. To characterize medulloblastoma at the phosphoprotein-signaling level, we performed high-throughput peptide phosphorylation profiling on a large cohort of SHH (Sonic Hedgehog), group 3, and group 4 medulloblastomas. We identified two major protein-signaling profiles. One profile was associated with rapid death post-recurrence and resembled MYC-like signaling for which MYC lesions are sufficient but not necessary. The second profile showed enrichment for DNA damage, as well as apoptotic and neuronal signaling. Integrative analysis demonstrated that heterogeneous transcriptional input converges on these protein-signaling profiles: all SHH and a subset of group 3 patients exhibited the MYC-like protein-signaling profile; the majority of the other group 3 subset and group 4 patients displayed the DNA damage/apoptotic/neuronal signaling profile. Functional analysis of enriched pathways highlighted cell-cycle progression and protein synthesis as therapeutic targets for MYC-like medulloblastoma. : Using peptide phosphorylation profiling, Zomerman et al. identify two medulloblastoma phosphoprotein-signaling profiles that have prognostic value and are potentially targetable. They find that these profiles extend across transcriptome-based subgroup borders. This suggests that diverse genetic information converges on common protein-signaling pathways and highlights protein-signaling as a unique information layer. Keywords: medulloblastoma, protein-signaling, protein synthesis, MYC, TP53, proteome, phosphoproteom

Genome-wide Analyses Identify KIF5A as a Novel ALS Gene

To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.Peer reviewe

Thresholds of auditory-motor coupling measured with a simple task in musicians and non-musicians : was the sound simultaneous to the key press?

The human brain is able to predict the sensory effects of its actions. But how precise are these predictions? The present research proposes a tool to measure thresholds between a simple action (keystroke) and a resulting sound. On each trial, participants were required to press a key. Upon each keystroke, a woodblock sound was presented. In some trials, the sound came immediately with the downward keystroke; at other times, it was delayed by a varying amount of time. Participants were asked to verbally report whether the sound came immediately or was delayed. Participants’ delay detection thresholds (in msec) were measured with a staircase-like procedure. We hypothesised that musicians would have a lower threshold than non-musicians. Comparing pianists and brass players, we furthermore hypothesised that, as a result of a sharper attack of the timbre of their instrument, pianists might have lower thresholds than brass players. Our results show that non-musicians exhibited higher thresholds for delay detection (180±104 ms) than the two groups of musicians (102±65 ms), but there were no differences between pianists and brass players. The variance in delay detection thresholds could be explained by variance in sensorimotor synchronisation capacities as well as variance in a purely auditory temporal irregularity detection measure. This suggests that the brain’s capacity to generate temporal predictions of sensory consequences can be decomposed into general temporal prediction capacities together with auditory-motor coupling. These findings indicate that the brain has a relatively large window of integration within which an action and its resulting effect are judged as simultaneous. Furthermore, musical expertise may narrow this window down, potentially due to a more refined temporal prediction. This novel paradigm provides a simple test to estimate the temporal precision of auditory-motor action-effect coupling, and the paradigm can readily be incorporated in studies investigating both healthy and patient populations

Correlations between keystroke-sound delay detection and anisochrony (A) and sensorimotor synchronisation accuracy (B).

<p>The dot colour indicates the group: blue for non-musicians, red for pianists and green for brass players.</p

Recognition memory for human motor learning

Motor skill retention is typically measured by asking participants to reproduce previously learned movements from memory. The analog of this retention test (recall memory) in human verbal memory is known to underestimate how much learning is actually retained. Here we asked whether information about previously learned movements, which can no longer be reproduced, is also retained. Following visuomotor adaptation, we used tests of recall that involved reproduction of previously learned movements and tests of recognition in which participants were asked whether a candidate limb displacement, produced by a robot arm held by the subject, corresponded to a movement direction that was experienced during active training. The main finding was that 24 h after training, estimates of recognition memory were about twice as accurate as those of recall memory. Thus, there is information about previously learned movements that is not retrieved using recall testing but can be accessed in tests of recognition. We conducted additional tests to assess whether, 24 h after learning, recall for previously learned movements could be improved by presenting passive movements as retrieval cues. These tests were conducted immediately prior to recall testing and involved the passive playback of a small number of movements, which were spread across the workspace and included both adapted and baseline movements, without being marked as such. This technique restored recall memory for movements to levels close to those of recognition memory performance. Thus, somatic information may enable retrieval of otherwise inaccessible motor memories. © 2021 Elsevier Inc