Juanita C. Limas

Background: The cohesin complex is essential for proper chromosome structure and gene expression. Defects in cohesin subunits and regulators cause changes in cohesin complex dynamics and thereby alter three-dimensional genome organization. However, the molecular mechanisms that drive cohesin localization and function remain poorly understood. Results: In this study, we observe that loss of WIZ causes changes to cohesin localization that are distinct from loss of the known WIZ binding partner G9a. Whereas loss of WIZ uniformly increases cohesin levels on chromatin at known binding sites and leads to new, ectopic cohesin binding sites, loss of G9a does not. Ectopic cohesin binding on chromatin after the loss of WIZ occurs at regions that are enriched for activating histone modifications and transcription factors motifs. Furthermore, loss of WIZ causes changes in cohesin localization that are distinct from those observed by loss of WAPL, the canonical cohesin unloading factor. Conclusions: The evidence presented here suggests that WIZ can function independently from its previously identified role with G9a and GLP in heterochromatin formation. Furthermore, while WIZ limits the levels and localization pattern of cohesin across the genome, it appears to function independently of WAPL-mediated cohesin unloading.

Cyclin E/CDK2 drives cell cycle progression from G1 to S phase. Despite the toxicity of cyclin E overproduction in mammalian cells, the cyclin E gene is overexpressed in some cancers. To further understand how cells can tolerate high cyclin E, we characterized non-transformed epithelial cells subjected to chronic cyclin E overproduction. Cells overproducing cyclin E, but not cyclins D or A, briefly experienced truncated G1 phases followed by a transient period of DNA replication origin underlicensing, replication stress, and impaired proliferation. Individual cells displayed substantial intercellular heterogeneity in cell cycle dynamics and CDK activity. Each phenotype improved rapidly despite high cyclin E–associated activity. Transcriptome analysis revealed adapted cells down-regulated a cohort of G1-regulated genes. Withdrawing cyclin E from adapted cells only partially reversed underlicensing indicating that adaptation is at least partly non-genetic. This study provides evidence that mammalian cyclin E/CDK inhibits origin licensing indirectly through premature S phase onset and provides mechanistic insight into the relationship between CDKs and licensing. It serves as an example of oncogene adaptation that may recapitulate molecular changes during tumorigenesis.

The onset of COVID‐19, coupled with the finer lens placed on systemic racial disparities within our society, has resulted in increased discussions around mental health. Despite this, mental health struggles in research are still often viewed as individual weaknesses and not the result of a larger dysfunctional research culture. Mental health interventions in the science, technology, engineering, and mathematics (STEM) academic community often focus on what individuals can do to improve their mental health instead of focusing on improving the research environment. In this paper, we present four aspects of research that may heavily impact mental health based on our experiences as research scientists: bullying and harassment; precarity of contracts; diversity, inclusion, and accessibility; and the competitive research landscape. Based on these aspects, we propose systemic changes that institutions must adopt to ensure their research culture is supportive and allows everyone to thrive.

Cyclin E/CDK2 drives cell cycle progression from G1 to S phase. Cyclin E overproduction is toxic to mammalian cells, although the gene encoding cyclin E ( CCNE1 ) is overexpressed in some cancers. To gain insight into how cancer cells tolerate high cyclin E, we extensively characterized non-transformed epithelial cells throughout a time course of chronic cyclin E overproduction. Cells overproducing human cyclin E, but not cyclin D or cyclin A, briefly experienced truncated G1 phases, then endured a transient period of DNA replication origin underlicensing, replication stress, and severely impaired proliferation. Individual cells displayed substantial intercellular heterogeneity in cell cycle dynamics and CDK activity. Each phenotype improved rapidly despite maintaining high cyclin E-associated activity. Transcriptome analysis revealed that adapted cells downregulated a cohort of G1-regulated genes. Withdrawing cyclin E induction only partially reversed the intermediate licensing phenotype of adapted cells indicating that adaptation is at least partly independent of mutations. This study provides evidence that mammalian cyclin E/CDK inhibits origin licensing by an indirect mechanism through premature S phase onset and provides further insight into the relationship between CDK activity and licensing in mammals. It serves as an example of specific oncogene adaptation that may recapitulate molecular changes during tumorigenesis.

Inhibitors based on a 3-acylaminoindazole scaffold were synthesized to yield potent dual AAK1/BMP2K inhibitors. Optimization furnished a small molecule chemical probe (SGC-AAK1-1, 25) that is potent and selective for AAK1/BMP2K over other NAK family members, demonstrates narrow activity in a kinome-wide screen, and is functionally active in cells. This inhibitor represents one of the best available small molecule tools to study the functions of AAK1 and BMP2K.

Successful genome duplication is required for cell proliferation and demands extraordinary precision and accuracy. The mechanisms by which cells enter, progress through, and exit S phase are intense areas of focus in the cell cycle and genome stability fields. Key molecular events in the G1 phase of the cell division cycle, especially origin licensing, are essential for pre‐establishing conditions for efficient DNA replication during the subsequent S phase. If G1 events are poorly regulated or disordered, then DNA replication can be compromised leading to genome instability, a hallmark of tumorigenesis. Upon entry into S phase, coordinated origin firing and replication progression ensure complete, timely, and precise chromosome replication. Both G1 and S phase progressions are controlled by master cell cycle protein kinases and ubiquitin ligases that govern the activity and abundance of DNA replication factors. In this short review, we describe current understanding and recent developments related to G1 progression and S phase entrance and exit with a particular focus on origin licensing regulation in vertebrates.

The cell cycle is canonically described as a series of four consecutive phases: G1, S, G2, and M. In single cells, the duration of each phase varies, but the quantitative laws that govern phase durations are not well understood. Using time‐lapse microscopy, we found that each phase duration follows an Erlang distribution and is statistically independent from other phases. We challenged this observation by perturbing phase durations through oncogene activation, inhibition of DNA synthesis, reduced temperature, and DNA damage. Despite large changes in durations in cell populations, phase durations remained uncoupled in individual cells. These results suggested that the independence of phase durations may arise from a large number of molecular factors that each exerts a minor influence on the rate of cell cycle progression. We tested this model by experimentally forcing phase coupling through inhibition of cyclin‐dependent kinase 2 (CDK2) or overexpression of cyclin D. Our work provides an explanation for the historical observation that phase durations are both inherited and independent and suggests how cell cycle progression may be altered in disease states.

Cell cycle phase transitions are tightly orchestrated to ensure efficient cell cycle progression and genome stability. Interrogating these transitions is important for understanding both normal and pathological cell proliferation. By quantifying the dynamics of the popular FUCCI reporters relative to the transitions into and out of S phase, we found that their dynamics are substantially and variably offset from true S phase boundaries. To enhance detection of phase transitions, we generated a new reporter whose oscillations are directly coupled to DNA replication and combined it with the FUCCI APC/C reporter to create “PIP-FUCCI”. The PIP degron fusion protein precisely marks the G1/S and S/G2 transitions; shows a rapid decrease in signal in response to large doses of DNA damage only during G1; and distinguishes cell type-specific and DNA damage source-dependent arrest phenotypes. We provide guidance to investigators in selecting appropriate fluorescent cell cycle reporters and new analysis strategies for delineating cell cycle transitions.

Glioblastoma (GBM) is the most common adult primary brain tumor. Multimodal treatment is empiric and prognosis remains poor. Recurrent PIK3CA missense mutations (PIK3CA^mut) in GBM are restricted to three functional domains: adaptor binding (ABD), helical, and kinase. Defining how these mutations influence gliomagenesis and response to kinase inhibitors may aid in the clinical development of novel targeted therapies in biomarker-stratified patients. We used normal human astrocytes immortalized via expression of hTERT, E6, and E7 (NHA). We selected two PIK3CA^mut from each of 3 mutated domains and induced their expression in NHA with (NHA^RAS) and without mutant RAS using lentiviral vectors. We then examined the role of PIK3CA^mut in gliomagenesis in vitro and in mice, as well as response to targeted PI3K (PI3Ki) and MEK (MEKi) inhibitors in vitro. PIK3CA^mut, particularly helical and kinase domain mutations, potentiated proximal PI3K signaling and migration of NHA and NHA^RAS in vitro. Only kinase domain mutations promoted NHA colony formation, but both helical and kinase domain mutations promoted NHA^RAS tumorigenesis in vivo. PIK3CA^mut status had minimal effects on PI3Ki and MEKi efficacy. However, PI3Ki/MEKi synergism was pronounced in NHA and NHA^RAS harboring ABD or helical mutations. PIK3CA^mut promoted differential gliomagenesis based on the mutated domain. While PIK3CA^mut did not influence sensitivity to single agent PI3Ki, they did alter PI3Ki/MEKi synergism. Taken together, our results demonstrate that a subset of PIK3CA^mut promote tumorigenesis and suggest that patients with helical domain mutations may be most sensitive to dual PI3Ki/MEKi treatment.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor. Prognosis remains poor despite multimodal therapy. Developing alternative treatments is essential. Drugs targeting kinases within the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) effectors of receptor tyrosine kinase (RTK) signaling represent promising candidates. We previously developed a non-germline genetically engineered mouse model of GBM in which PI3K and MAPK are activated via Pten deletion and Kras^G12D in immortalized astrocytes. Using this model, we examined the influence of drug potency on target inhibition, alternate pathway activation, efficacy, and synergism of single agent and combination therapy with inhibitors of these 2 pathways. Efficacy was then examined in GBM patient-derived xenografts (PDX) in vitro and in vivo. PI3K and mitogen-activated protein kinase kinase (MEK) inhibitor potency was directly associated with target inhibition, alternate RTK effector activation, and efficacy in mutant murine astrocytes in vitro. The kinomes of GBM PDX and tumor samples were heterogeneous, with a subset of the latter harboring MAPK hyperactivation. Dual PI3K/MEK inhibitor treatment overcame alternate effector activation, was synergistic in vitro, and was more effective than single agent therapy in subcutaneous murine allografts. However, efficacy in orthotopic allografts was minimal. This was likely due to dose-limiting toxicity and incomplete target inhibition. Drug potency influences PI3K/MEK inhibitor–induced target inhibition, adaptive kinome reprogramming, efficacy, and synergy. Our findings suggest that combination therapies with highly potent, brain-penetrant kinase inhibitors will be required to improve patient outcomes.