The FACT complex interacts with the E3 ubiquitin ligase Psh1 to prevent ectopic localization of CENP-A.

Centromere identity and its epigenetic maintenance require the incorporation of a histone H3 variant called CENP-A at centromeres. CENP-A mislocalization to ectopic sites may disrupt chromatin-based processes and chromosome segregation, so it is important to uncover the mechanisms by which this variant is exclusively localized to centromeres. Here, we identify a role for the conserved chromatin-modifying complex FACT (facilitates chromatin transcription/transactions) in preventing budding yeast CENP-A(Cse4) mislocalization to euchromatin by mediating its proteolysis. The Spt16 subunit of the FACT complex binds to Psh1 (Pob3/Spt16/histone), an E3 ubiquitin ligase that targets CENP-A(Cse4) for degradation. The interaction between Psh1 and Spt16 is critical for both CENP-A(Cse4) ubiquitylation and its exclusion from euchromatin. We found that Psh1 cannot efficiently ubiquitylate CENP-A(Cse4) nucleosomes in vitro, suggesting that additional factors must facilitate CENP-A(Cse4) removal from chromatin in vivo. Consistent with this, a Psh1 mutant that cannot associate with FACT has a reduced interaction with CENP-A(Cse4) in vivo. Together, our data identify a previously unknown mechanism to maintain centromere identity and genomic stability through the FACT-mediated degradation of ectopically localized CENP-A(Cse4).

Interleukin-21 engineering enhances NK cell activity against glioblastoma via CEBPD.

Glioblastoma (GBM) is an aggressive brain cancer with limited therapeutic options. Natural killer (NK) cells are innate immune cells with strong anti-tumor activity and may offer a promising treatment strategy for GBM. We compared the anti-GBM activity of NK cells engineered to express interleukin (IL)-15 or IL-21. Using multiple in vivo models, IL-21 NK cells were superior to IL-15 NK cells both in terms of safety and long-term anti-tumor activity, with locoregionally administered IL-15 NK cells proving toxic and ineffective at tumor control. IL-21 NK cells displayed a unique chromatin accessibility signature, with CCAAT/enhancer-binding proteins (C/EBP), especially CEBPD, serving as key transcription factors regulating their enhanced function. Deletion of CEBPD resulted in loss of IL-21 NK cell potency while its overexpression increased NK cell long-term cytotoxicity and metabolic fitness. These results suggest that IL-21, through C/EBP transcription factors, drives epigenetic reprogramming of NK cells, enhancing their anti-tumor efficacy against GBM.

CD28 Costimulation Augments CAR Signaling in NK Cells via the LCK/CD3ζ/ZAP70 Signaling Axis.

Multiple factors in the design of a chimeric antigen receptor (CAR) influence CAR T-cell activity, with costimulatory signals being a key component. Yet, the impact of costimulatory domains on the downstream signaling and subsequent functionality of CAR-engineered natural killer (NK) cells remains largely unexplored. Here, we evaluated the impact of various costimulatory domains on CAR-NK cell activity, using a CD70-targeting CAR. We found that CD28, a costimulatory molecule not inherently present in mature NK cells, significantly enhanced the antitumor efficacy and long-term cytotoxicity of CAR-NK cells both in vitro and in multiple xenograft models of hematologic and solid tumors. Mechanistically, we showed that CD28 linked to CD3ζ creates a platform that recruits critical kinases, such as lymphocyte-specific protein tyrosine kinase (LCK) and zeta-chain-associated protein kinase 70 (ZAP70), initiating a signaling cascade that enhances CAR-NK cell function. Our study provides insights into how CD28 costimulation enhances CAR-NK cell function and supports its incorporation in NK-based CARs for cancer immunotherapy. Significance: We demonstrated that incorporation of the T-cell-centric costimulatory molecule CD28, which is normally absent in mature natural killer (NK) cells, into the chimeric antigen receptor (CAR) construct recruits key kinases including lymphocyte-specific protein tyrosine kinase and zeta-chain-associated protein kinase 70 and results in enhanced CAR-NK cell persistence and sustained antitumor cytotoxicity.

BATF is a major driver of NK cell epigenetic reprogramming and dysfunction in AML.

Myelodysplastic syndrome and acute myeloid leukemia (AML) belong to a continuous disease spectrum of myeloid malignancies with poor prognosis in the relapsed/refractory setting necessitating novel therapies. Natural killer (NK) cells from patients with myeloid malignancies display global dysfunction with impaired killing capacity, altered metabolism, and an exhausted phenotype at the single-cell transcriptomic and proteomic levels. In this study, we identified that this dysfunction was mediated through a cross-talk between NK cells and myeloid blasts necessitating cell-cell contact. NK cell dysfunction could be prevented by targeting the αvß-integrin/TGF-ß/SMAD pathway but, once established, was persistent because of profound epigenetic reprogramming. We identified BATF as a core transcription factor and the main mediator of this NK cell dysfunction in AML. Mechanistically, we found that BATF was directly regulated and induced by SMAD2/3 and, in turn, bound to key genes related to NK cell exhaustion, such as HAVCR2, LAG3, TIGIT, and CTLA4. BATF deletion enhanced NK cell function against AML in vitro and in vivo. Collectively, our findings reveal a previously unidentified mechanism of NK immune evasion in AML manifested by epigenetic rewiring and inactivation of NK cells by myeloid blasts. This work highlights the importance of using healthy allogeneic NK cells as an adoptive cell therapy to treat patients with myeloid malignancies combined with strategies aimed at preventing the dysfunction by targeting the TGF-ß pathway or BATF.

Caenorhabditis elegans cyclin B3 is required for multiple mitotic processes including alleviation of a spindle checkpoint-dependent block in anaphase chromosome segregation.

The master regulators of the cell cycle are cyclin-dependent kinases (Cdks), which influence the function of a myriad of proteins via phosphorylation. Mitotic Cdk1 is activated by A-type, as well as B1- and B2-type, cyclins. However, the role of a third, conserved cyclin B family member, cyclin B3, is less well defined. Here, we show that Caenorhabditis elegans CYB-3 has essential and distinct functions from cyclin B1 and B2 in the early embryo. CYB-3 is required for the timely execution of a number of cell cycle events including completion of the MII meiotic division of the oocyte nucleus, pronuclear migration, centrosome maturation, mitotic chromosome condensation and congression, and, most strikingly, progression through the metaphase-to-anaphase transition. Our experiments reveal that the extended metaphase delay in CYB-3-depleted embryos is dependent on an intact spindle assembly checkpoint (SAC) and results in salient defects in the architecture of holocentric metaphase chromosomes. Furthermore, genetically increasing or decreasing dynein activity results in the respective suppression or enhancement of CYB-3-dependent defects in cell cycle progression. Altogether, these data reveal that CYB-3 plays a unique, essential role in the cell cycle including promoting mitotic dynein functionality and alleviation of a SAC-dependent block in anaphase chromosome segregation.

Shared decision-making conversations and smoking cessation interventions: critical components of low-dose CT lung cancer screening programs.

Lung cancer remains the world's deadliest cancer despite efforts to decrease smoking rates. Lung cancer screening (LCS) with low-dose computed tomography (LDCT) was shown to reduce lung cancer deaths by 20%. Screening with LDCT comes with potential harms including a high rate of false-positive test results, subsequent follow-up procedures, and radiation exposure. For some patients, the potential benefits associated with screening may be outweighed by the harms. The decision to screen must therefore take into consideration patients' risk of developing lung cancer, comorbidities that may prevent diagnostic procedures or curative surgery, and their values and preferences regarding the benefits and harms of screening. A process called shared decision-making (SDM) is recognized as a crucial feature of LCS. SDM is a patient-centered approach where healthcare providers provide best clinical evidence and then work together with patients to discern if the screening process aligns with the patient's values and preferences. Unfortunately, clinician SDM skills are often of poor quality which can lead to patients making uninformed decisions. Decision support tools that help patients make informed decisions and increase SDM on LCS are available. In 2015, the Centers for Medicare & Medicaid Services issued a coverage memo for LCS that contained an unprecedented requirement: an initial patient counseling and SDM visit with the use of at least one decision aid must occur for screening services to be reimbursed. This review focuses on SDM and suggests ways to increase the prevalence and effectiveness of SDM in LCS programs. Stopping smoking greatly reduces a person's risk for developing lung cancer, and smoking cessation messages in LCS guidelines from major medical organizations and interventions in LCS programs are explored. LCS has come of age; so too has SDM as it is an integral part of LCS programs.

Histone H4 Facilitates the Proteolysis of the Budding Yeast CENP-ACse4 Centromeric Histone Variant.

The incorporation of histone variants into nucleosomes can alter chromatin-based processes. CENP-A is the histone H3 variant found exclusively at centromeres that serves as an epigenetic mark for centromere identity and is required for kinetochore assembly. CENP-A mislocalization to ectopic sites appears to contribute to genomic instability, transcriptional misregulation, and tumorigenesis, so mechanisms exist to ensure its exclusive localization to centromeres. One conserved process is proteolysis, which is mediated by the Psh1 E3 ubiquitin ligase in Saccharomyces cerevisiae (budding yeast). To determine whether there are features of the CENP-A nucleosome that facilitate proteolysis, we performed a genetic screen to identify histone H4 residues that regulate CENP-ACse4 degradation. We found that H4-R36 is a key residue that promotes the interaction between CENP-ACse4 and Psh1 Consistent with this, CENP-ACse4 protein levels are stabilized in H4-R36A mutant cells and CENP-ACse4 is enriched in the euchromatin. We propose that the defects in CENP-ACse4 proteolysis may be related to changes in Psh1 localization, as Psh1 becomes enriched at some 3' intergenic regions in H4-R36A mutant cells. Together, these data reveal a key residue in histone H4 that is important for efficient CENP-ACse4 degradation, likely by facilitating the interaction between Psh1 and CENP-ACse4.