Glioblastoma (GBM) is the most common and deadliest malignancy of the brain. Despite decades of intense research, there has been little change to the overall survival of patients with GBM. Our laboratory recently identified the actin-binding protein advillin (AVIL) as being overexpressed, oncogenic, and necessary for tumorigenesis in GBM. Here, we further examined AVIL expression in GBMs and found that it was enriched across molecular subtypes and states, including GBM stem cells and temozolomide-resistant samples. In contrast, we found that AVIL was scarcely expressed in normal human brain tissue. In addition, Avil knockout in mice had no adverse effects, suggesting that there may be a wide therapeutic window for therapies targeting AVIL. Using high-throughput small-molecule screening, we identified a direct inhibitor of AVIL that bound to the protein and also blocked AVIL binding to its substrate, actin. It induced a transcriptome profile similar to that of AVIL silencing by siRNA and caused down-regulation of FOXM1 and LIN28B, two known downstream targets of AVIL. Moreover, it exhibited selectivity toward tumor cells, sparing astrocytes and neural stem cells in vitro. In vivo, we found that the compound readily crosses the blood-brain barrier and could be delivered orally. We then demonstrated efficacy in five GBM mouse models without evidence of side effects. In summary, we have identified an efficacious first-in-class compound targeting an oncogene in GBM. Further optimization of the molecule may offer an effective therapeutic intervention for GBM.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third deadliest cancer in the United States, with a five-year survival rate of only 13%, and is projected to become the second leading cause of cancer-related mortality by 2030. Despite advances in personalized medicine, therapeutic options for PDAC remain extremely limited, underscoring the need for new, clinically actionable targets. In 2008, Kelly et al. utilized a phage-display-based functional proteomics platform that identified plectin, a cytolinker protein normally restricted to the cytoplasm, as aberrantly mislocalized to the plasma membrane of PDAC cells. This cell-surface form of plectin (CSP) has since been detected across multiple malignancies, including PDAC, ovarian carcinoma, and cholangiocarcinoma, while remaining cytoplasmic in normal tissues. Functional studies using an anti-CSP monoclonal antibody (mAb) revealed that CSP supports pro-tumorigenic behaviors such as proliferation, migration, and invasion. A recent early-phase clinical trial (NCT05074472) evaluating an anti-CSP mAb in CSP-positive cancers demonstrated that CSP-targeted therapy is both safe and clinically feasible. To further resolve the molecular and immunologic roles of CSP, we analyzed transcriptomic data from human PDAC bulk and single-cell RNA-sequencing datasets. CSP-high tumors exhibited suppression of pro-inflammatory immune pathways and reduced infiltration of cytotoxic immune populations—hallmarks of the immune-cold PDAC microenvironment. Anti-CSP mAb treatment in vivo promoted infiltration of CD4+ and CD8+ T cells, reduced tumor burden, and generated a durable effector-memory response capable of preventing tumor re-challenge. Collectively, these findings provide the first direct evidence that CSP functions as an immune suppressor in PDAC. Moreover, therapeutic blockade of CSP can restore anti-tumor immunity in this notoriously immunoresistant cancer, offering a promising avenue for personalized treatment strategies for patients with historically few effective options. Citation Format: Cody L. Wolf, Roxanne K. Ruiz, Sokchea Khou, Robert Cornelison, Edward Stelow, Karl M. Kowalewski, Matthew J. Lazzara, Amanda Poissonnier, Timothy N. Bullock, Lisa M. Coussens, Kimberly A. Kelly. Cell surface plectin as a master regulator of tumor-immune interactions in pancreatic cancer [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 6582.

BackgroundPancreatic adenocarcinoma (PDAC) is an abysmal disease with poor clinical outcomes, largely due to limited life-extending treatments. Notably, PDAC displays a T cell-suppressive tumor microenvironment, and the underlying molecular mechanisms that lead to this phenotype remain poorly understood.MethodsUtilizing the TCGA-PAAD dataset, tumor samples were separated by PLEC expression to evaluate patient survival, and pathway analyses associated with increased tumorigenesis. Evaluation of immune infiltration and subsequent immune deconvolution was performed using tidyestimate and CIBERSORTx R packages and immunohistochemistry (IHC) from human PDAC samples was performed to analyze PLEC expression and immune cell infiltration. Single-cell RNA-seq (scRNA-seq) analysis from 229 PDAC patients was analyzed to investigate signaling dynamics and immune cell infiltration in PLECHigh patients. Functional validation was provided using a monoclonal antibody (mAb) against cell surface plectin (CSP) in two murine PDAC models to examine changes in tumor growth and immune cell subset abundance.ResultsOur studies revealed that high plectin expression results in an overall worse survival associated with activation of pro-tumorigenic pathways and decreased anti-tumor immune signature in PDAC patients. Analysis via GSEA indicates PLECHigh patients display an aggressive phenotype and suppressed pro-inflammatory signaling pathways. Immune ESTIMATE scores were significantly decreased in PLECHigh patients, and IHC and scRNA-seq analysis revealed that PLECHigh tumors display a decrease in anti-tumor CD8+ T cells. In vivo analyses using an anti-CSP mAb revealed a reduction in tumor growth kinetics compared to IgG control corresponding with a significant increase in proliferating and activated cytotoxic CD8+ T cells. Anti-CSP-mediated tumor suppression was inhibited when CD8+ T cells were depleted, indicating that anti-CSP treatment is contingent on cytotoxic T cell functionality.DiscussionOur findings identify plectin as a biomarker of aggressive disease in PDAC, with high plectin expression associated with decreased T cell infiltration, and anti-CSP treatment reinstates antitumor immunity and decreases tumor volume in vivo. These findings suggest that plectin is a novel therapeutic target with the potential to enhance immune responses in PDAC and improve patient outcomes.

Abstract Rhabdomyosarcoma (RMS) is a prevalent pediatric soft-tissue cancer with a survival rate below 27% for high-risk patients. Despite significant genetic differences among RMS subtypes, frontline therapy remains largely uniform and ineffective for metastatic and recurrent cases. After decades of research, targeted therapies are currently unavailable, underscoring the urgent need for novel targets and effective treatment strategies. Our previous published studies identified AVIL as a novel, bona fide RMS oncogene. AVIL encodes a calcium-regulated, actin-binding protein critical for actin cytoskeleton organization. While its expression in healthy adult tissue is limited, AVIL is aberrantly upregulated in RMS and correlates with poor patient prognosis. AVIL silencing significantly reduces cell proliferation and migration, induces cell death in vitro, and prevents tumor formation in vivo. Our recent studies reveal that AVIL functions upstream of the transcriptional coactivator YAP, a key regulator of proliferation, survival, and stemness that is frequently hyperactivated in RMS. Through integrated transcriptomic, biochemical, and imaging analyses, we discovered that AVIL promotes YAP nuclear translocation, enhances its transcriptional activity, and increases cellular sensitivity to YAP pathway blockade. Conversely, genetic silencing of AVIL disrupts YAP signaling and restores the expression of myogenic differentiation markers, implicating AVIL’s role in maintaining the undifferentiated, proliferative state of RMS cells. Functionally, AVIL-high RMS cells exhibit enhanced response to pharmacologic YAP inhibition, while AVIL depletion diminishes this effect, underscoring a mechanistic dependency of AVIL-driven oncogenic programs on YAP signaling. In vivo, administration of a YAP inhibitor to patient-derived RMS xenografts with high AVIL expression significantly reduces tumor incidence compared with vehicle-treated controls. Together, these findings uncover a previously unrecognized mechanism by which AVIL modulates YAP activity in RMS, revealing a therapeutically actionable axis that sustains tumor growth and impedes myogenic differentiation. Citation Format: Martyna Glowczyk-Gluc, Robert Cornelison, Zhongqiu Xie, Julie Fanburg-Smith, Robin D. LeGallo, Eyas Alzayadneh, Hui Li. AVIL’s dependency on YAP as a key mediator of oncogenic activity offers a therapeutic opportunity in rhabdomyosarcoma [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 633.

Chimeric RNAs resulting from intergenic splicing represent a distinct mechanism for transcriptome expansion. To explore the role of this previously unidentified layer of the transcriptome in sex-specific immunity, we analyzed RNA sequencing data from 425 blood samples and identified a female-specific chimeric RNA, UBA1-CDK16, which was further validated in more than 1200 blood samples. This chimeric RNA forms via cis-splicing between two adjacent X-linked parental genes, UBA1 and CDK16, despite both being expressed in both sexes. We demonstrated that a female-specific chromatin loop at the UBA1-CDK16 junction sites facilitates the intergenic splicing. Evolutionary analysis revealed that UBA1-CDK16 became female specific in humans through at least two independent paths. Functional studies suggested that UBA1-CDK16 is enriched in the myeloid lineage and may regulate myeloid cell development. Notably, its abnormal expression in female patients with COVID-19 correlates with altered neutrophil counts, highlighting its potential role in the disease progression.

AbstractSpinal cord injury (SCI) initiates a complex cascade of chemical and biophysical phenomena that result in tissue swelling, progressive neural degeneration, and formation of a fluid‐filled cavity. Previous studies show fluid pressure above the spinal cord (supraspinal) is elevated for at least 3 days after injury and contributes to a phase of damage called secondary injury. Currently, it is unknown how fluid forces within the spinal cord itself (interstitial) are affected by SCI and if they contribute to secondary injury. We find spinal interstitial pressure increases from −3 mmHg in the naive cord to a peak of 13 mmHg at 3 days post‐injury (DPI) but relatively normalizes to 2 mmHg by 7 DPI. A computational fluid dynamics model predicts interstitial flow velocities up to 0.9 μm/s at 3 DPI, returning to near baseline by 7 DPI. By quantifying vascular leakage of Evans Blue dye after a cervical hemi‐contusion in rats, we confirm an increase in dye infiltration at 3 DPI compared to 7 DPI, suggestive of higher fluid velocities at the time of peak fluid pressure. In vivo expression of the apoptosis marker caspase‐3 is strongly correlated with regions of interstitial flow at 3 DPI, and exogenously enhancing interstitial flow exacerbates tissue damage. In vitro, we show overnight exposure of neuronal cells to low pathological shear stress (0.1 dynes/cm2) significantly reduces cell count and neurite length. Collectively, these results indicate that interstitial fluid flow and shear stress may play a detrimental role in post‐traumatic neural degeneration.

Efficient nucleic acid extraction and purification are crucial for cellular and molecular biology research, yet they pose challenges for large-scale clinical RNA sequencing and PCR assays. Here, we present BLADE-R, a magnetic bead-based protocol that simplifies the process by combining cellular lysis and nucleic acid binding into a single step, followed by a unique on-bead rinse for nuclease-free separation of genomic DNA and RNA. The Agilent TapeStation and RT-qPCR analyses show that RNA extracted from HEK293T cell line using BLADE-R outperforms the TRIzol protocol in terms of time and cost. RNA sequencing reveals no differences in sequence quality or gene count variance between samples processed with BLADE-R and those processed with TRIzol followed by RNA kit clean-up. Additionally, BLADE-R outperformed TRIzol in RNA extraction from frozen tissue and whole blood samples, as confirmed by RT-qPCR. Our protocol can be adapted to a 96-well plate format, enabling RNA purification of up to 96 human blood samples in less time than a single-sample traditional extraction. Using BLADE-R in this format, we confirmed minimal well-to-well contamination in RNA purification, cDNA synthesis, and PCR. Therefore, our novel BLADE-R protocol, suitable for both low and high-throughput formats, is effective even in limited-resource settings for preparing clinical samples for PCR and sequencing assays. Thus, our new BLADE-R technique works well even in low-resource environments to prepare clinical samples for PCR and sequencing experiments. It can be adapted for both low- and high-throughput formats.

RNA processing mechanisms, such as alternative splicing and RNA editing, have been recognized as critical means to expand the transcriptome. Chimeric RNAs formed by intergenic splicing provide another potential layer of RNA diversification. By analyzing a large set of RNA-Seq data and validating results in over 1,200 blood samples, we identified UBA1-CDK16 , a female-specific chimeric transcript. Intriguingly, both parental genes, are expressed in males and females. Mechanistically, UBA1-CDK16 is produced by cis-splicing between the two adjacent X-linked genes, originating from the inactive X chromosome. A female-specific chromatin loop, formed between the junction sites, facilitates the alternative splicing of its readthrough precursor. This unique chimeric transcript exhibits evolutionary conservation, evolving to be female-specific from non-human primates to humans. Furthermore, our investigation reveals that UBA1-CDK16 is enriched in the myeloid lineage and plays a regulatory role in myeloid differentiation. Notably, female COVID-19 patients who tested negative for this chimeric transcript displayed higher counts of neutrophils, highlighting its potential role in disease pathogenesis. These findings support the notion that chimeric RNAs represent a new repertoire of transcripts that can be regulated independently from the parental genes, and a new class of RNA variance with potential implications in sexual dimorphism and immune responses.

Trauma to the central and peripheral nervous systems such as traumatic brain injury, spinal cord injury and stroke leads to significant loss of function. This poses a considerable burden for patients as this loss of function often leads to lifelong disabilities due to the limited regenerative capacity of nervous tissue. Historically, astrocytes, the most abundant type of glial cell that resides in the central and peripheral nervous systems, have been thought to predominantly inhibit nerve growth after injury. However, recent advancements have discovered the importance of astrocytes and their integral role in recovery following spinal cord injury. In particular, the manipulation of astrocyte pro- (A1) and anti- (A2) inflammatory phenotypes can provide valuable insights into astrocyte behavior, migration and wound healing potential. A1 and A2 reactive states can be induced through cytokine activation. The combination of interleukin (IL)-1α, tumor necrosis factor (TNF) and complement component 1q (C1q) has shown to exhibit the strongest A1, or neurotoxic phenotype. Interleukin-10 has shown to induce the A2, or neuroprotective phenotype. Research has also shown that A1 reactive astrocytes lose the ability to promote neuron survival, regeneration and outgrowth, and induce the death of neurons and oligodendrocytes. Additionally, the discovery of cell migration effects through tumor-derived matrices supports a novel approach in inducing astrocyte reactivity through matrix activation. Our research describes a methodology to discover and eventually reprogramme astrocyte behavior to promote neuroprotective phenotypes through cellular interaction with matrix glycoproteins. Through a migration scratch assay, we were able to explore the A1- and A2- inducing nature of glioblastoma stem cell (GSC) matrix on astrocytes, specifically the various glycoproteins composing each stem cell matrix as compared to the baseline cytokine activation. After quantifying results, cell migration was found to vary between each GSC line, leading us to infer that GSC glycoproteins promote astrocyte activation. Further quantification through qPCR will allow us to examine the markers within each GSC matrix and confirm which proteins specifically induce A1 and A2 activity. With this knowledge, we will be better able to understand and manipulate astrocyte neuroprotective responses to neural injury, and develop effective therapeutics to promote wound healing. This project was conducted partly in support from the Undergraduate Research at University of Massachusetts Amherst, and funding from the National Institute of Health.

Rhabdomyosarcoma (RMS) is the most common pediatric soft-tissue cancer with a survival rate below 27% for high-risk children despite aggressive multi-modal therapeutic interventions. After decades of research, no targeted therapies are currently available. Therapeutically targeting actin-binding proteins, although promising, has historically been challenging. Recent advances have made this possibility more salient, including our lab's identification of advillin (AVIL), a novel oncogenic actin-binding protein that plays a role in many cytoskeletal functions. AVIL is overexpressed in many RMS cell lines, patient-derived xenograft models, and a cohort of 30 clinical samples of both the alveolar (ARMS) and embryonal (ERMS) subtypes. Overexpression of AVIL in mesenchymal stem cells induces neoplastic transformation both in vitro and in vivo, and reversing overexpression through genetic modulation reverses the transformation. This suggests a critical role of AVIL in RMS tumorigenesis and maintenance. As an actin-binding protein, AVIL would not traditionally be considered a druggable target. This perspective will address the feasibility of targeting differentially expressed actin-binding proteins such as AVIL therapeutically, and how critical cell infrastructure can be damaged in a cancer-specific manner.

Explanation and example analysis pipeline used to quantitate changes in cell size and other parameters. Supplemental Figure 1: (A) Sample images of immunofluorescence of F-actin (red) and DAPI (blue) before and after IC50treatment with CX-5461. Images were taken at random at 200x magnification. Thresholding was performed to identify DAPI stained nuclei using the Mixture of Gaussian (MoG) technique to bin pixels into foreground and background classes. Cytoplasm, marked by F-actin, was segmented using the watershed technique after Sobel transformation followed by Otsu thresholding. All image segmentation and quantitation was performed by CellProfiler. (B) Summary quantitation in representative images of changes to segmented cells' parameters before and after treatment.

Supplementary Figure 1 from Large-Scale Profiling of Archival Lymph Nodes Reveals Pervasive Remodeling of the Follicular Lymphoma Methylome

Supplementary Figure 1 from Assessment of Automated Image Analysis of Breast Cancer Tissue Microarrays for Epidemiologic Studies

Supplementary Table 6 from Large-Scale Profiling of Archival Lymph Nodes Reveals Pervasive Remodeling of the Follicular Lymphoma Methylome

Supplementary Table 4 from Large-Scale Profiling of Archival Lymph Nodes Reveals Pervasive Remodeling of the Follicular Lymphoma Methylome

Peripheral nerve injury results in loss of motor and sensory function distal to the nerve injury and is often permanent in nerve gaps longer than 5 cm. Autologous nerve grafts (nerve autografts) utilize patients' own nerve tissue from another part of their body to repair the defect and are the gold standard in care. However, there is a limited autologous tissue supply, size mismatch between donor nerve and injured nerve, and morbidity at the site of nerve donation. Decellularized cadaveric nerve tissue alleviates some of these limitations and has demonstrated success clinically. We previously developed an alternative apoptosis-assisted decellularization process for nerve tissue. This new process may result in an ideal scaffold for peripheral nerve regeneration by gently removing cells and antigens while preserving delicate topographical cues. In addition, the apoptosis-assisted process requires less active processing time and is inexpensive. This study examines the utility of apoptosis-decellularized peripheral nerve scaffolds compared to detergent-decellularized peripheral nerve scaffolds and isograft controls in a rat nerve gap model. Results indicate that, at 8 weeks post-injury, apoptosis-decellularized peripheral nerve scaffolds perform similarly to detergent-decellularized and isograft controls in both functional (muscle weight recovery, gait analysis) and histological measures (neurofilament staining, macrophage infiltration). These new apoptosis-decellularized scaffolds hold great promise to provide a less expensive scaffold for nerve injury repair, with the potential to improve nerve regeneration and functional outcomes compared to current detergent-decellularized scaffolds.

Supplementary Figure Legend for Figure 2 from Assessment of Automated Image Analysis of Breast Cancer Tissue Microarrays for Epidemiologic Studies

Supplementary Figure 2, Part 1 from Assessment of Automated Image Analysis of Breast Cancer Tissue Microarrays for Epidemiologic Studies

Purpose Specific gene fusions and their fusion products (chimeric RNA and protein) have served as ideal diagnostic markers and therapeutic targets for cancer. However, few systematic studies for chimeric RNAs have been conducted in neuroendocrine prostate cancer (NEPC). In this study, we explored the landscape of chimeric RNAs in different types of prostate cancer (PCa) cell lines and aimed to identify chimeric RNAs specifically expressed in NEPC. Methods To do so, we employed the RNA-seq data of eight prostate related cell lines from Cancer Cell Line Encyclopedia (CCLE) for chimeric RNA identification. Multiple filtering criteria were used and the candidate chimeric RNAs were characterized at multiple levels and from various angles. We then performed experimental validation on all 80 candidates, and focused on the ones that are specific to NEPC. Lastly, we studied the clinical relevance and effect of one chimera in neuroendocrine process. Results Out of 80 candidates, 15 were confirmed to be expressed preferentially in NEPC lines. Among them, 13 of the 15 were found to be specifically expressed in NEPC, and four were further validated in another NEPC cell line. Importantly, in silico analysis showed that tumor malignancy may be correlated to the level of these chimeric RNAs. Clinically, the expression of TMPRSS2-ERG (e2e4) was elevated in tumor tissues and indicated poor clinical prognosis, whereas the parental wild type transcripts had no such association. Furthermore, compared to the most frequently detected TMPRSS2-ERG form (e1e4), e2e4 encodes 31 more amino acids and accelerated neuroendocrine process of prostate cancer. Conclusions In summary, these findings painted the landscape of chimeric RNA in NEPC and supported the idea that some chimeric RNAs may represent additional biomarkers and/or treatment targets independent of parental gene transcripts.

Significance Rhabdomyosarcoma (RMS) is a common pediatric soft-tissue cancer. There are two main subtypes driven by distinct mechanisms. Currently, there is no targeted therapy for the disease. We found an oncogene AVIL, whose activity is often abnormally up-regulated in RMS. Silencing the gene kills RMS cells and prevents tumor formation in animals; yet, normal control cells are indifferent to the interference. Mechanistically, AVIL activation turns on both oncogene pathways in RMS. These findings support that AVIL may be a good therapeutic target and that targeting AVIL may be a viable approach for both types of RMS.