Significance: Strong evidence supports the tumor immune landscape as a determinant of patient responses to immunotherapy. Readily available therapies, including radiation, are being investigated as modifying agents with immune checkpoint blockade. However, little is known regarding radiotherapy’s impact within the tumor microenvironment and intratumoral T cell repertoires of patients, leaving critical gaps in the guided design of clinical protocols. In this study, samples from renal cell carcinoma (RCC) patients underwent high-throughput analysis to reveal transcriptional immune activation and increased clonality in irradiated tumors. Analysis across longitudinal blood samples showed that tumor-enriched clonotypes undergo phases of peripheral expansion and contraction following radiation. Collectively, these findings demonstrate that radiotherapy remodels intratumoral T cell responses and support refined sequencing of combination strategies in RCC.

Signaling mucins are evolutionarily conserved regulators of signal transduction pathways. The signaling mucin Msb2p regulates the Cdc42p-dependent mitogen activated protein kinase (MAPK) pathway that controls filamentous growth in yeast. Cleavage and release of the glycosylated inhibitory domain of Msb2p is required for MAPK activation. We show here that proteolytic processing of Msb2p was induced by under-glycosylation of its extracellular domain. Cleavage of under-glycosylated Msb2p required the unfolded protein response (UPR), a quality control (QC) pathway that operates in the endoplasmic reticulum (ER). The UPR regulator Ire1p, which detects mis-folded / under-glycosylated proteins in the ER, controlled Msb2p cleavage by regulating transcriptional induction of Yps1p, the major protease that processes Msb2p. Accordingly, the UPR was required for differentiation to the filamentous cell type. Cleavage of Msb2p occurred in conditional trafficking mutants that trap secretory cargo in the endomembrane system. Processed Msb2p was delivered to the PM, and its turnover by the ubiquitin ligase Rsp5p and ESCRT attenuated the filamentous growth pathway. We speculate that QC pathways may broadly regulate signaling glycoproteins and their cognate pathways by recognizing altered glycosylation patterns that can occur in response to extrinsic cues.

Intismeran autogene (intismeran; formerly V940 or mRNA-4157) is an mRNA-based individualized neoantigen therapy. We report 5-year outcomes of intismeran plus pembrolizumab from the phase 2b KEYNOTE-942 study ( NCT03897881 ). Eligible patients with resected stage IIIB‒IV cutaneous melanoma were randomized 2:1 to receive 9 doses of intramuscular intismeran 1 mg Q3W plus 18 doses of intravenous pembrolizumab 200 mg Q3W or 18 doses of intravenous pembrolizumab 200 mg Q3W. Primary endpoint was recurrence-free survival (RFS); secondary endpoints included distant metastasis-free survival (DMFS) and safety. Five-year analyses were descriptive. Among 157 randomized patients (intismeran plus pembrolizumab, n=107; pembrolizumab, n=50), median planned follow-up at data cutoff (December 15, 2025) was 60.3 (range, 50.5‒76.4) months. Intismeran plus pembrolizumab continued to prolong RFS (HR, 0.510; 95% CI, 0.294‒0.887) and DMFS (0.411; 0.200‒0.843), with a favorable trend in overall survival (0.471; 0.165‒1.345) versus pembrolizumab. Safety profile continued to be manageable, with no new safety signals. Intismeran plus pembrolizumab was associated with increased T-cell receptor clonality and novel clonotypes versus pembrolizumab; greater novel clone expansion was observed in patients without versus with recurrence in the combination arm. After 5 years’ follow-up, intismeran plus pembrolizumab demonstrated sustained, durable treatment benefits versus pembrolizumab alone in resected high-risk melanoma.

Significance: We identify a new role for bud-site–selection proteins outside of their established roles in regulating growth site determination, as components of a surveillance pathway that monitors spatial position during intrinsic and extrinsic morphogenetic stress and regulates a Cdc42p- and MAPK-dependent response.

Filamentous growth is a fungal morphogenetic response that is critical for virulence in some fungal species. Many aspects of filamentous growth remain poorly understood. We have identified an aspect of filamentous growth in the budding yeast Saccharomyces cerevisiae and the human pathogen Candida albicans where cells behave collectively to invade surfaces in aggregates. These responses may reflect an extension of normal filamentous growth, as they share the same signaling pathways and effector processes. Aggregate responses may involve cooperation among individual cells, because aggregation was stimulated by cell adhesion molecules, secreted enzymes, and diffusible molecules that promote quorum sensing. Our study may provide insights into the genetic basis of collective cellular responses in fungi. The study may have ramifications in fungal pathogenesis, in situations where collective responses occur to promote virulence.

Clustered regularly interspaced short palindromic repeats (CRISPR) are a revolutionary tool based on a bacterial acquired immune response system. CRISPR has gained widespread use for gene editing in a variety of organisms and is an increasingly valuable tool for basic genetic research, with far‐reaching implications for medicine, agriculture, and industry. This lab is based on the premise that upper division undergraduate students enrolled in a Life Sciences curriculum must become familiar with cutting edge advances in biotechnology that have significant impact on society. Toward this goal, we developed a new hands‐on laboratory exercise incorporating the use of CRISPR‐Cas9 and homology directed repair (HDR) to edit two well‐characterized genes in the budding yeast,Saccharomyces cerevisiae. The two genes edited in this exercise, Adenine2 (ADE2) and Sterile12 (STE12) affect metabolic and developmental processes, respectively. Editing the premature stop codons in these genes results in clearly identifiable phenotypes that can be assessed by students in a standard laboratory course setting. Making use of this basic eukaryotic model organism facilitates a laboratory exercise that is inexpensive, simple to organize, set up, and present to students. This exercise enables undergraduate students to initiate and follow‐up on all stages of the CRISPR gene editing process, from identification of guide RNAs, amplification of an appropriate HDR fragment, and analysis of mutant phenotypes. The organization of this protocol also allows for easy modification, providing additional options for editing any expressed genes within the yeast genome to produce new mutations, or recovery of existing mutants to wild type. © 2018 International Union of Biochemistry and Molecular Biology, 46(6):592–601, 2018.