School of Medicine

Immune-suppressive tumor microenvironments (TMEs) limit the impact of checkpoint blockade in many cancers by restricting the infiltration and activation of CD8⁺ T, CD4⁺ T, and NK cells. Utilizing murine models of head and neck squamous cell carcinoma, we demonstrated that intratumoral (IT) delivery of CXCL10 drives tumor elimination and inhibits recurrence not only by recruiting these cells but by enhancing their antitumoral functions and stunting angiogenesis. CD8⁺ T cells also display enhanced activation, tumor-antigen specificity, and decreased T cell exhaustion. Despite administration of CXCL10 into tumors, CD8⁺ and CD4⁺ T cells show enhanced presence and proliferation in tumor-draining lymph nodes (TdLNs), consistent with T cell priming and trafficking between tumors and TdLNs. Together, the data suggest that CXCL10 promotes a mutually reinforcing feedback loop that reprograms the TME toward an immunologically responsive antitumoral state. Combining IT-CXCL10 and anti-PD-1 further increased tumor clearance, indicating that CXCL10-driven reprogramming of the TME lowers barriers to effective checkpoint blockade.

Alpha B-crystallin (CryAB) is a small heat-shock protein highly expressed in cardiac tissue, where it functions as a molecular chaperone that helps prevent protein aggregation, particularly under stress conditions. A missense mutation in CryAB (R120G) causes autosomal dominant cardiomyopathy in humans and is characterized by extensive protein aggregation in cardiomyocytes. To better understand the pathogenic mechanisms underlying CryAB^R120G-associated cardiomyopathy, appropriate in vivo models are essential. Genetic mouse models are valuable tools for investigating disease pathogenesis and evaluating potential therapeutic strategies. In this study, we characterized a homozygous CryAB^R120G knock-in (KI) mouse model to assess the impact of this mutation on cardiac function. CryAB^R120G KI mice exhibited no overt changes in cardiac structure and function up to 12 months of age, with minimal changes in cardiac and proteotoxic stress markers, except for an increased atrial natriuretic peptide expression at 12 months. Protein quality control pathways remained largely unchanged. Although mitochondrial respiration was normal in young CryAB^R120G KI mice, it was reduced at 12 months of age. Despite the presence of insoluble protein aggregates, homozygous CryAB^R120G KI mice did not develop overt structural or functional cardiomyopathy through 12 months of age. These findings indicate that, within the age range examined, the CryAB^R120G KI model does not reproduce the overt cardiomyopathic phenotype associated with the CRYAB^R120G mutation in patients.

During cell stress, endoplasmic reticulum autophagy (ER-phagy) receptors remodel the ER by sequestering membrane proteins (cargo) into autophagosomes for degradation. The conserved ER-phagy receptor, Atg40, contains a motif that binds to Atg8 and a reticulon homology domain that is needed for vacuolar/lysosomal delivery. Cargo capture, however, requires the Atg40 binding partner Lst1/SEC24C. To address whether lipids regulate cargo capture during ER-phagy, we analyzed autophagy in neutral lipid-deficient cells. Unexpectedly, we found that Atg40 was delivered to the vacuole in autophagosomes without Lst1/SEC24C or cargo in mutant cells. Lipidomic analysis revealed changes in the ratio of phosphatidylethanolamine to phosphatidylcholine in the neutral lipid-deficient cells that are predicted to alter ER membrane bendability. Our findings imply that phospholipids control cargo sequestration by regulating receptor-cargo coupling at autophagic sites.

The structure of the catalytic subunit of cAMP-dependent protein kinase (PKA-C), a prototype for the protein kinase superfamily, laid the foundation for the development of targeted kinase inhibitors. Here we describe the structure and biophysical characterization of a PKA-C complex with BLU0588, a small PKA-selective inhibitor. The high-resolution crystal structure not only captures the inhibitor's unusual T-shaped geometry, but also shows how the four rings of BLU0588 serve as surrogates for ATP's adenosine and phosphate-organizing sites. Each site contains two subsites. BLU0588's planar azaindole and pyridine rings, which are buried beneath the glycine-rich loop in a hydrophobic shell at the base of the active site cleft, fill the adenine and ribose subsites. In contrast, BLU0588's indane and pyrrolidine rings fill the phosphate-organizing site. The indane ring occupies the α/β-phosphate organizing site while the pyrrolidine ring fills the Mg/γ-phosphate organizing site. The structure also shows how BLU0588 nucleates an open but stable conformation of the entire hydrophobic architecture of the N- and C-lobes. In addition to potently blocking phosphoryl transfer activity, BLU0588 also abolishes the synergistic high-affinity binding of the physiological pseudosubstrate inhibitor, protein kinase inhibitor. The residence time of BLU0588, measured by surface plasmon residence, contributes to its picomolar affinity and is distinct from H89, a commonly used but more promiscuous PKA inhibitor. These molecular insights provide a valuable framework for dissecting the organization of the active site cleft as well as different strategies for the rational design of more potent and selective kinase inhibitors in general.

Abstract Activating mutations in GNAQ and GNA11 (GNAQ oncogenes) are found in ∼93% of uveal melanoma (UVM) and 4% of skin cutaneous melanoma (SKCM), where they act as driver oncogenes. UVM is the most common primary cancer of the eye in adults, affecting more than 2,500 patients each year in the US alone, nearly 50% of whom will die from liver metastasis. To date, there are limited effective therapeutic options to prevent or treat UVM metastatic disease (mUVM), which typically also fails to respond to immunotherapies. By combining synthetic biology approaches, CRISPR/Cas9 genome-wide screens, and high-throughput chemogenetic drug screening, our team has revealed that classical and novel non-canonical GNAQ signaling circuits converge to promote UVM growth, survival, metastasis, and treatment resistance. Ultimately, elucidating GNAQ oncogenic signaling networks may reveal system vulnerabilities that can be exploited to develop new precision therapies for mUVM. In this regard, we have recently shown that darovasertib acts as a dual PKC-PKN inhibitor and exhibits the highest activity among thousands of drugs tested. Darovasertib has demonstrated encouraging activity in UVM patients, and clinical trials using darovasertib as a single agent in primary UVM lesions and in combination with crizotinib in mUVM are currently ongoing. However, few patients achieve complete responses, and tumors often progress due to the acquisition of resistance mechanisms. We aim to identify new targets that can overcome resistance to darovasertib. RNA-seq analysis revealed that long-term treatment with darovasertib increased the expression of YAP-target genes, and we hypothesized that YAP/TEAD activation may contribute to darovasertib resistance. Indeed, expression of an active YAP mutant (YAP2-5SA) or LATS1/2 inhibition was sufficient to induce darovasertib resistance in UVM cells. In turn, knockdown of YAP or TEAD, or the expression of a doxycycline-induced TEAD inhibitor (TEADi) peptide, increases darovasertib-induced apoptosis. Remarkably, co-targeting with small molecule TEADi decreases the expression of darovasertib-induced YAP targets and acts synergistically to reduce cell viability and increase UVM cell death. Ongoing studies are now exploring the preclinical benefit of combining darovasertib with small-molecule TEADi for the treatment of human UVM tumor xenografts in mice. Emerging evidence will be presented supporting that the Hippo YAP/TEAD pathway represents an adaptive mechanism of resistance to darovasertib treatment, and that the combination of TEADi with darovasertib may prevent the development of treatment resistance, thereby increasing the depth and duration of the anti-tumor response in UVM. Citation Format: Rodolfo Daniel Cervantes-Villagrana, Elena Sofia Cardenas Alcoser, Kuniaki Sato, Simone Lubrano, Tomohiko Ishikawa, Andrew E. Aplin, J. Silvio Gutkind. Targeting Hippo/YAP-TEAD increases the antitumor activity of darovasertib in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1837.

Head and neck squamous cell carcinoma (HNSCC) is a significant cause of morbidity and mortality worldwide, with limited treatment options for patients with locally advanced disease. CD47 immune checkpoint inhibitors have been used to block the CD47/SIRPa interaction that inhibits antigen-presenting cell phagocytosis, thereby enhancing antigen presentation to cytotoxic T-cells, and have shown promise in combination with anti-PD1 immunotherapy in tumors, including recurrent/metastatic HNSCC. We found that CD47 expression is associated with poor prognosis in HNSCC and explored the anti-tumor activity of an anti-CD47 fusion protein in combination with anti-PD1 and lymphatic-sparing radiotherapy in a locally advanced HNSCC model. In the 4MOSC1 syngeneic HPV-negative HNSCC mouse model, ALX301 (an engineered CD47-blocking SIRPα fusion for murine models) induced complete tumor regression when combined with anti-PD-1, and produced a partial tumor response as a monotherapy. An anti-PD1 immune checkpoint inhibitor in a CD47-null tumor background led to complete tumor regression confirming a key role for CD47 in tumor immunity. ALX301 treated mice demonstrated increased MHC-II expression on dendritic cells within the tumor and upregulation of CD86 co-stimulatory molecule on dendritic cells within the tumor, sentinel lymph nodes, and contralateral lymph nodes. Combination ALX301 and anti-PD1 treatment in an anti-PD1 resistant 4MOSC2 model demonstrated significant tumor regression, enhanced survivability, improved response with neoadjuvant radiotherapy, and greater retention of CD8 + T-cells within the tumor microenvironment. Notably, T-cell receptor sequencing revealed increased shared clonality between the tumor and sentinel lymph nodes of ALX301 treated mice. These data demonstrate that a combination of CD47 blockade and anti-PD1 therapy enhances tumor antigen presentation and immune cell infiltration, while further improving anti-tumor responses in combination with tumor-targeted radiotherapy. This study provides support for the rational design of combinatorial immunoradiotherapy, using anti-CD47 inhibitors and anti-PD1 therapy, in a clinical trial targeting locally advanced HPV-negative HNSCC.

Cells release vesicles that serve important roles in long-range signaling and intercellular communication. These vesicles are released not just in response to stress, inflammation, injury, and chemoresistance, but also during... Cells release vesicles that serve important roles in long-range signaling and intercellular communication. These vesicles are released not just in response to stress, inflammation, injury, and chemoresistance, but also during homeostatic regulation. A particular class of vesicles called ectosomes or microparticles are released by the outward budding of the plasma membrane, a process which requires both the detachment of the membrane from the cortex and the exposure of negatively charged, curvature-inducing lipids such as phosphatidylserine from the inner leaflet to the outer leaflet. In this work, we develop a biophysical model that accounts for the interaction between these different factors. Using our model, we predict how linker properties influence outward budding of the plasma membrane and identify conditions that can promote or inhibit membrane curvature generation. These findings provide insight into the fundamental mechanisms underlying microparticle formation, elucidating the basic biology of this critical process. Further, these mechanistic insights may inspire techniques for inhibiting microparticles where they are harmful, such as chemoresistant drug efflux by tumor cells.

INTRODUCTION: Despite sepsis having growing awareness nationally, efforts to reduce the public health impact of sepsis have lagged. Although there are known pathophysiologic mechanisms and preventive strategies, sepsis is rarely approached as a predictable or preventable condition. Predicting who will develop sepsis in patients with infection still remains a challenge. This study examined modifiable and nonmodifiable risk factors associated with patients initially discharged home with an infection and had future sepsis-related admissions within 7 days of the index Emergency Department (ED) visit. METHODS: We conducted a multi-center retrospective cohort analysis of adults presenting to two university hospital EDs. The inclusion criteria encompassed adult patients who were discharged from the ED at their index visit with discharge diagnosis (ICD 10-CM code) of pneumonia, urinary tract infection (UTI), and/or cellulitis and who returned for hospital admission within 7 days of the index visit due to sepsis, severe sepsis without septic shock, and/or septic shock. Using multivariate regression, risk factors that predict return sepsis admission within 7 days of ED index visit were evaluated, and a 7-day return sepsis admission model was constructed. The predictive power of the model was measured by c-statistic. RESULTS: Among 10,179 unique ED patients, return sepsis admissions within 7 days occurred in 113 visits (1.11 % of discharged patients). Statistically significant risk factors among patients with infection associated with subsequent sepsis admission in the chosen model were Cardiovascular Disease (OR 2.07 95 % CI 1.26-3.42), Hypertension (OR 2.21 95 % CI 1.37-3.56), Chronic Kidney Disease (OR 1.80 95 % CI 1.11-2.91), Cancer (OR 2.22 95 % CI 1.43-3.45), Male (OR 1.67 95 % CI 1.13-2.45), arriving in an ambulance (vs. walk in OR 2.55 95 % CI 1.46-4.44), higher heart rate (OR 1.29 95 % CI 1.16-1.45), and higher temperature (OR 1.23 95 % CI 1.05-1.45), Hyperlipidemia was protective (OR 0.56 95 %CI 0.34-0.91). The c-statistic of our chosen model was 0.77 (95 % CI 0.73-0.81). The Hosmer-Lemeshow test for our logistic regression model resulted in a chi-square value of 7.23 with 8 degrees of freedom with a p-value of 0.51. This suggests that our model fits the data well. CONCLUSION: Our findings may be used to risk stratify and guide outpatient disposition decisions for ED patients with infection and to determine which patients need to be more closely monitored in the outpatient setting following ED discharge.

Programmes that support diversity, equity and inclusion (DEI) in science are under attack in the USA. Data indicate that diversity in the scientific workforce increases creativity and success in tackling challenging problems. Loss of promising talent supported by these programmes will substantially weaken our research capacity, limit innovation and substantially reduce discoveries important for driving scientific advancements.

Mitochondria are multifunctional organelles that are important for many different cellular processes, including energy production and biosynthesis of fatty acids, haem and iron–sulfur clusters. Mitochondrial dysfunction leads to a disruption in these processes, the generation of excessive reactive oxygen species, and the activation of inflammatory and cell death pathways. The consequences of mitochondrial dysfunction are particularly harmful in energy-demanding organs such as the heart. Loss of terminally differentiated cardiomyocytes leads to cardiac remodelling and a reduced ability to sustain contraction. Therefore, cardiomyocytes rely on multilayered mitochondrial quality control mechanisms to maintain a healthy population of mitochondria. Mitochondrial chaperones protect against protein misfolding and aggregation, and resident proteases eliminate damaged proteins through proteolysis. Irreparably damaged mitochondria can also be degraded through mitochondrial autophagy (mitophagy) or ejected from cells inside vesicles. The accumulation of dysfunctional mitochondria in cardiomyocytes is a hallmark of ageing and cardiovascular disease. This accumulation is driven by impaired mitochondrial quality control mechanisms and contributes to the development of heart failure. Therefore, there is a strong interest in developing therapies that directly target mitochondrial quality control in cardiomyocytes. In this Review, we discuss the current knowledge of the mechanisms involved in regulating mitochondrial quality in cardiomyocytes, how these pathways are altered with age and in disease, and the therapeutic potential of targeting mitochondrial quality control pathways in cardiovascular disease.