Accumulation of Mad2–Cdc20 complex during spindle checkpoint activation requires binding of open and closed conformers of Mad2 in Saccharomyces cerevisiae

The spindle assembly checkpoint (SAC) coordinates mitotic progression with sister chromatid alignment. In mitosis, the checkpoint machinery accumulates at kinetochores, which are scaffolds devoted to microtubule capture. The checkpoint protein Mad2 (mitotic arrest deficient 2) adopts two conformations: open (O-Mad2) and closed (C-Mad2). C-Mad2 forms when Mad2 binds its checkpoint target Cdc20 or its kinetochore receptor Mad1. When unbound to these ligands, Mad2 folds as O-Mad2. In HeLa cells, an essential interaction between C- and O-Mad2 conformers allows Mad1-bound C-Mad2 to recruit cytosolic O-Mad2 to kinetochores. In this study, we show that the interaction of the O and C conformers of Mad2 is conserved in Saccharomyces cerevisiae. MAD2 mutant alleles impaired in this interaction fail to restore the SAC in a mad2 deletion strain. The corresponding mutant proteins bind Mad1 normally, but their ability to bind Cdc20 is dramatically impaired in vivo. Our biochemical and genetic evidence shows that the interaction of O- and C-Mad2 is essential for the SAC and is conserved in evolution

Short-term treatment with multi-drug regimens combining BRAF/MEK-targeted therapy and immunotherapy results in durable responses in Braf-mutated melanoma

Targeted and immunotherapy regimens have revolutionized the treatment of advanced melanoma patients. Despite this, only a subset of patients respond durably. Recently, combination strategies of BRAF/MEK inhibitors with immune checkpoint inhibitor monotherapy (α-CTLA-4 or α-PD-1) have increased the rate of durable responses. Based on evidence from our group and others, these therapies appear synergistic, but at the cost of significant toxicity. We know from other treatment paradigms (e.g. hematologic malignancies) that combination strategies with multi-drug regimens (\u3e4 drugs) are associated with more durable disease control. To better understand the mechanism of these improved outcomes, and to identify and prioritize new strategies for testing, we studied several multi-drug regimens combining BRAF/MEK targeted therapy and immunotherapy combinations in a Braf-mutant murine melanoma model (BrafV600E/Pten−/−). Short-term treatment with α-PD-1 and α-CTLA-4 monotherapies were relatively ineffective, while treatment with α-OX40 demonstrated some efficacy [17% of mice with no evidence of disease, (NED), at 60-days]. Outcomes were improved in the combined α-OX40/α-PD-1 group (42% NED). Short-term treatment with quadruplet therapy of immunotherapy doublets in combination with targeted therapy [dabrafenib and trametinib (DT)] was associated with excellent tumor control, with 100% of mice having NED after combined DT/α-CTLA-4/α-PD-1 or DT/α-OX40/α-PD-1. Notably, tumors from mice in these groups demonstrated a high proportion of effector memory T cells, and immunologic memory was maintained with tumor re-challenge. Together, these data provide important evidence regarding the potential utility of multi-drug therapy in treating advanced melanoma and suggest these models can be used to guide and prioritize combinatorial treatment strategies

In Vivo Functional Platform Targeting Patient-Derived Xenografts Identifies WDR5-Myc Association as a Critical Determinant of Pancreatic Cancer

SummaryCurrent treatment regimens for pancreatic ductal adenocarcinoma (PDAC) yield poor 5-year survival, emphasizing the critical need to identify druggable targets essential for PDAC maintenance. We developed an unbiased and in vivo target discovery approach to identify molecular vulnerabilities in low-passage and patient-derived PDAC xenografts or genetically engineered mouse model-derived allografts. Focusing on epigenetic regulators, we identified WDR5, a core member of the COMPASS histone H3 Lys4 (H3K4) MLL (1–4) methyltransferase complex, as a top tumor maintenance hit required across multiple human and mouse tumors. Mechanistically, WDR5 functions to sustain proper execution of DNA replication in PDAC cells, as previously suggested by replication stress studies involving MLL1, and c-Myc, also found to interact with WDR5. We indeed demonstrate that interaction with c-Myc is critical for this function. By showing that ATR inhibition mimicked the effects of WDR5 suppression, these data provide rationale to test ATR and WDR5 inhibitors for activity in this disease

Cross-cohort gut microbiome associations with immune checkpoint inhibitor response in advanced melanoma

The composition of the gut microbiome has been associated with clinical responses to immune checkpoint inhibitor (ICI) treatment, but there is limited consensus on the specific microbiome characteristics linked to the clinical benefits of ICIs. We performed shotgun metagenomic sequencing of stool samples collected before ICI initiation from five observational cohorts recruiting ICI-naive patients with advanced cutaneous melanoma (n = 165). Integrating the dataset with 147 metagenomic samples from previously published studies, we found that the gut microbiome has a relevant, but cohort-dependent, association with the response to ICIs. A machine learning analysis confirmed the link between the microbiome and overall response rates (ORRs) and progression-free survival (PFS) with ICIs but also revealed limited reproducibility of microbiome-based signatures across cohorts. Accordingly, a panel of species, including Bifidobacterium pseudocatenulatum, Roseburia spp. and Akkermansia muciniphila, associated with responders was identified, but no single species could be regarded as a fully consistent biomarker across studies. Overall, the role of the human gut microbiome in ICI response appears more complex than previously thought, extending beyond differing microbial species simply present or absent in responders and nonresponders. Future studies should adopt larger sample sizes and take into account the complex interplay of clinical factors with the gut microbiome over the treatment course

Fecal Microbiota Transplantation Controls Murine Chronic Intestinal Inflammation by Modulating Immune Cell Functions and Gut Microbiota Composition

Different gastrointestinal disorders, including inflammatory bowel diseases (IBD), have been linked to alterations of the gut microbiota composition, namely dysbiosis. Fecal microbiota transplantation (FMT) is considered an encouraging therapeutic approach for ulcerative colitis patients, mostly as a consequence of normobiosis restoration. We recently showed that therapeutic effects of FMT during acute experimental colitis are linked to functional modulation of the mucosal immune system and of the gut microbiota composition. Here we analysed the effects of therapeutic FMT administration during chronic experimental colitis, a condition more similar to that of IBD patients, on immune-mediated mucosal inflammatory pathways. Mucus and feces from normobiotic donors were orally administered to mice with established chronic Dextran Sodium Sulphate (DSS)-induced colitis. Immunophenotypes and functions of infiltrating colonic immune cells were evaluated by cytofluorimetric analysis. Compositional differences in the intestinal microbiome were analyzed by 16S rRNA sequencing. Therapeutic FMT in mice undergoing chronic intestinal inflammation was capable to decrease colonic inflammation by modulating the expression of pro-inflammatory genes, antimicrobial peptides, and mucins. Innate and adaptive mucosal immune cells manifested a reduced pro-inflammatory profile in FMT-treated mice. Finally, restoration of a normobiotic core ecology contributed to the resolution of inflammation. Thus, FMT is capable of controlling chronic intestinal experimental colitis by inducing a concerted activation of anti-inflammatory immune pathways, mechanistically supporting the positive results of FMT treatment reported in ulcerative colitis patients

The Mad1/Mad2 Complex as a Template for Mad2 Activation in the Spindle Assembly Checkpoint

AbstractBackground: The spindle assembly checkpoint (SAC) imparts fidelity to chromosome segregation by delaying anaphase until all sister chromatid pairs have become bipolarly attached. Mad2 is a component of the SAC effector complex that sequesters Cdc20 to halt anaphase. In prometaphase, Mad2 is recruited to kinetochores with the help of Mad1, and it is activated to bind Cdc20. These events are linked to the existence of two distinct conformers of Mad2: a closed conformer bound to its kinetochore receptor Mad1 or its target in the checkpoint Cdc20 and an open conformer unbound to these ligands.Results: We investigated the mechanism of Mad2 recruitment to the kinetochore during checkpoint activation and subsequent transfer to Cdc20. We report that a closed conformer of Mad2 constitutively bound to Mad1, rather than Mad1 itself, is the kinetochore receptor for cytosolic open Mad2 and show that the interaction of open and closed Mad2 conformers is essential to sustain the SAC.Conclusions: We propose that closed Mad2 bound to Mad1 represents a template for the conversion of open Mad2 into closed Mad2 bound to Cdc20. This simple model, which we have named the “Mad2 template” model, predicts a mechanism for cytosolic propagation of the spindle checkpoint signal away from kinetochores