Abstract Growth Differentiation Factor 15 (GDF15) is a stress-induced cytokine upregulated in several cancers, including pancreatic cancer. Its most well-known role is signaling through GFRAL in the brain to induce an anorexia/cachexia syndrome. Outside the brain, GDF15 acts as an immunosuppressor. In cancer, GDF15 is thought to leverage this function to protect tumors from immune surveillance. Recently, much attention has been drawn to the relationship between GDF15 and T cell activity, as blocking GDF15 has been shown to enhance responses to anti-PD-1 therapies. Interestingly, the only known receptor for GDF15 is GFRAL, whose expression is restricted to a subpopulation of neurons located in the brainstem. As GDF15 emerges as a target for enhancing immune cell activity, the role of GFRAL in this relationship remains underexplored in animal models of cancer. The purpose of this study is to evaluate the necessity of GFRAL in mediating the immunosuppressive activity of GDF15 with relevance to pancreatic ductal adenocarcinoma (PDAC), the most aggressive form of pancreatic cancer. To test this, T cells were isolated from the whole spleen of immunocompetent mice and activated in the presence of conditioned media (CM) from murine KPC PDAC cells (PdxCre; Kras+/G12D; Trp53fl/fl). In culture, we find that CM containing GDF15 suppressed the proliferation of CD4 and CD8 T cells. qRT-PCR confirmed that T cells lack GFRAL expression, suggesting that GDF15-mediated suppression of T cell activity does not require its canonical receptor. Additionally, KPC CM equally inhibited the proliferation of T cells isolated from GFRAL+/+ and GFRAL−/- mice. We previously showed that GDF15 is required for early-stage development of pancreatic cancer in mice. To determine if this function of GDF15 is mediated through GFRAL, KPC cells were orthotopically injected into the pancreas of immunocompetent GFRAL+/+ and GFRAL−/- mice. No significant differences in tumor burden or overall survival were observed between GFRAL+/+ and GFRAL−/- mice at the experimental endpoint. In addition, both GFRAL+/+ and GFRAL−/- mice exhibited similar myeloid and lymphoid immune responses to tumor implantation compared to non-tumor controls. Moreover, expression of the T cell exhaustion marker PD-1 remained consistent between groups. These results suggest that GDF15 modulates the tumor microenvironment independently of its sole known receptor, GFRAL. Further, disruption of the GDF15-GFRAL signaling axis does not alter the immune cell landscape or enhance T cell expansion. These findings provide valuable insights into the immunomodulatory function of GDF15 in PDAC and the possible distinct roles that the GDF15-GFRAL signaling axis has in regulating anorexia/cachexia vs tumorigenesis. Citation Format: Emma Crockett Funk, Abasi-ama Udeme, Cynthia F. Wright, Jenna Schwesig, Michael C. Ostrowski, Teresa A. Zimmers, Leonidas G. Koniaris, David Wang, Denis C. Guttridge. GFRAL is not required for PDAC tumor development and TME immune cell suppression [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 4999.

Outcome data from 6 National Institutes of Health-funded Postbaccalaureate Research Education Programs (PREPs) in the Mid-Atlantic region were combined to give a multi-institutional perspective on their scholars' characteristics and progress through biomedical research training. The institutions hosting these programs were Johns Hopkins University School of Medicine, the Medical University of South Carolina, the University of Maryland School of Medicine, the University of North Carolina at Chapel Hill, Virginia Commonwealth University, and Virginia Polytechnic Institute and State University. The authors summarize the institutional pathways, demographics, undergraduate institutions, and graduate institutions for a total of 384 PREP scholars who completed the programs by June 2021. A total of 228 (59.4%) of these PREP scholars identified as Black or African American, 116 (30.2%) as Hispanic or Latinx, and 269 (70.0%) as female. The authors found that 376 of 384 scholars (97.9%) who started PREP finished their program, 319 of 376 (84.8%) who finished PREP matriculated into PhD or MD/PhD programs, and 284 of 319 (89.0%) who matriculated have obtained their PhD or are successfully making progress toward their PhD.

In an adenosine triphosphate (ATP)-dependent process, the hSWI/SNF chromatin remodeling complex functions to alter chromatin structure, thereby regulating transcription factor access to DNA. In addition to interactions with transcription factors and recognition of acetylated histone residues, the chromatin remodeling activity of hSWI/SNF has also been shown to respond to a variety of cell signaling pathways. Our results demonstrate a novel interaction between the serine/threonine kinase Akt and members of the hSWI/SNF chromatin remodeling complex. Activation of Akt in HeLa cells resulted in its association with hSWI/SNF subunits: INI1, BAF155 and BAF170, as well as actin. BAF155 became preferentially recognized by an antibody that detects phosphorylated Akt substrates upon activation of Akt, suggesting that BAF155 may be an in vivo target for phosphorylation by Akt. Glutathione-S-transferase (GST) pulldown experiments demonstrated that INI1 and BAF155 were both capable of directly interacting with Akt. Finally, in vitro kinase assays provided additional evidence that BAF155 and potentially INI1 are substrates for Akt phosphorylation. These data provide the first evidence that Akt signaling may modulate function of the hSWI/SNF complex.

In an adenosine triphosphate (ATP)-dependent process, the hSWI/SNF chromatin remodeling complex functions to alter chromatin structure, thereby regulating transcription factor access to DNA. In addition to interactions with transcription factors and recognition of acetylated histone residues, the chromatin remodeling activity of hSWI/SNF has also been shown to respond to a variety of cell signaling pathways. Our results demonstrate a novel interaction between the serine/threonine kinase Akt and members of the hSWI/SNF chromatin remodeling complex. Activation of Akt in HeLa cells resulted in its association with hSWI/SNF subunits: INI1, BAF155 and BAF170, as well as actin. BAF155 became preferentially recognized by an antibody that detects phosphorylated Akt substrates upon activation of Akt, suggesting that BAF155 may be an in vivo target for phosphorylation by Akt. Glutathione- S -transferase (GST) pulldown experiments demonstrated that INI1 and BAF155 were both capable of directly interacting with Akt. Finally, in vitro kinase assays provided additional evidence that BAF155 and potentially INI1 are substrates for Akt phosphorylation. These data provide the first evidence that Akt signaling may modulate function of the hSWI/SNF complex.

This chapter describes a protocol that allows accurate in vitro transcription of vaccinia virus late genes. In this method, extracts are made from vaccinia virus-infected cells and used as enzyme sources to produce mRNAs from plasmid templates containing late gene promoter sequences.

The INI1 gene, which encodes a functionally uncharacterized protein component of the hSWI/SNF chromatin remodeling complex, is often mutated or deleted in malignant rhabdoid tumor (MRT). Two isoforms of INI1, that differ by the variable inclusion of nine amino acids, potentially are produced by differential RNA splicing. To determine the effect of the two INI1 isoforms on cell growth, INI1‐devoid (MRT) and INI1‐expressing cell lines were transfected separately with mammalian expression vectors or transduced with adenoviruses. Transfection of the short form of INI1 into either INI1‐deficient or expressing cell lines resulted in complete suppression of cell growth in colony formation assays. The longer splice variant induced moderate to severe growth suppression of MRT cells, but had a far milder effect on non‐MRT cells. Transduction of MRT cells with adenoviruses expressing either isoform of INI1 led to a dramatic change in morphology, growth suppression, and cell cycle arrest. Furthermore, senescence‐associated proteins were up‐regulated after transduction, while levels of proteins implicated in cell cycle progression were down‐regulated. Adenoviral delivery of INI1 into a non‐MRT cell line, however, had no demonstrable effect on any of these parameters. These results support the genetic evidence that INI1 is a tumor suppressor gene gone awry in MRT cells, and also suggest that delivery of the INI1 gene to MRT cells by adenoviruses may lead to a more effective treatment of this highly aggressive malignancy. © 2003 Wiley‐Liss, Inc.

Purpose: We evaluated the in vitro sensitivity of four malignant rhabdoid tumor (MRT) cell lines to six chemotherapeutic agents: 5-fluororuacil, vincristine, carboplatin, doxorubicin, etoposide, and paclitaxel. We also sought to determine whether a defect in the p53 signaling pathway may contribute to the pronounced drug resistance of MRT. Methods: MRT cells were treated with various concentrations of each drug and the effects on DNA synthesis were quantified using a thymidine incorporation assay. In addition, the effect of various concentrations of doxorubicin on cell growth was evaluated in all four cell lines. Functionality of the p53 pathway was evaluated by incubating cells with carboplatin or doxorubicin and monitoring the effects on the levels of the p53, p21WAF1/CIP1, and MDM 2 proteins by Western blot analyses. Results: Vincristine (EC50 0.5–2.9 nM) and doxorubicin (EC50 1.9–5.7 nM) were found to be most effective in inhibiting proliferation and were within clinically relevant concentrations. However, only doxorubicin exhibited cytotoxicity (EC50 2.4–13.1 nM), whereas vincristine and the other drugs tested were cytostatic. Interestingly, all four cell lines had remarkably similar dose response curves to all drugs tested, despite the fact that they were derived from different patients and arose in different tissues. When challenged with DNA-damaging drugs, p53 and the downstream effectors, p21WAF1/CIP1 and MDM 2 were upregulated. Conclusions: These studies indicate that the p53 pathway is functional and responsive to DNA-damaging drugs, and does not likely contribute to the drug resistance of MRT. The in vitro sensitivity of MRT cells to doxorubicin suggests that it may be a clinically important agent for the treatment of MRT.

Vaccinia virus gene expression is temporally regulated, and three gene classes have been identified: early, intermediate, and late. Several virus-encoded proteins and an activity designated VLTF-X are required for maximum transcription in vitro of a template containing a late promoter. VLTF-X is present in both cytoplasmic and nuclear extracts prepared from uninfected mammalian cells and co-purifies with a late promoter DNA-binding activity. Here, extensive purification of VLTF-X has revealed that heterogeneous nuclear ribonucleoproteins A2/B1 and RBM3 co-purified with in vitro late transcription stimulation. Overexpression and purification of these proteins fromEscherichia coli demonstrated that they both complemented for VLTF-X activity in in vitro transcription reactions. These studies identify two host cell factors potentially contributing to poxvirus replication in vivo.

ABSTRACT We have previously described a vaccinia virus late transcription factor, VLTF-X, which we found to be present in cells at early and late times in infection. In this study, transcription complementation assays were used to demonstrate that VLTF-X activity is also present in virion extracts and in the cytoplasm of uninfected HeLa cells. Mobility shift assays performed on various VLTF-X preparations revealed that a late promoter DNA-binding activity cochromatographed and cosedimented with VLTF-X activity. Competition experiments demonstrated that this binding was specific for the late promoter region of the probe and that late transcription was dramatically reduced by an oligonucleotide that blocked factor-DNA complex formation but was only minimally affected by an oligonucleotide that did not inhibit complex formation. These results suggest that a cellular factor may participate in vaccinia virus late transcription. These findings also confirm the requirement for VLTF-X and distinguish it from any of the previously described vaccinia virus late transcription factors, which have all been mapped to the viral genome. Finally, these studies also suggest that the biochemical role for VLTF-X may be in late promoter recognition.

Malignant rhabdoid tumor (MRT) is a rare, enigmatic childhood cancer characterized by extreme aggressiveness and resistance to chemotherapy, To understand better the origin of the tumor and the mechanisms by which it develops and resists treatment, five cell lines were established from patients presenting with MRT (two renal and three extrarenal tumors). All of the cell lines display the light microscopic and ultrastructural features, as well as the variable immunohistochemical profile, characteristic of MRT. All are capable of forming tumors in nude mice. Three of the cell lines have detectable abnormalities of chromosome 22: one a t(22, 22) unbalanced translocation and two others a loss of heterozygosity of polymerase chain reaction-based microsatellite markers. Northern blot analysis showed that overexpression of the c-myc message was a consistent characteristic of the five MRTs evaluated, Although mutations of the p53 gene were not detectable by sequence analysis, all of the cell lines showed nuclear accumulation of the p53 protein by an immunocytochemical analysis in a minority of the cells. This result suggests that dysfunction in a p53-dependent apoptotic pathway might play a role in the multiple drug resistance phenotype of these tumors.

A factor designated VLTF-X is required to support vaccinia virus late transcription in vitro. It has been found that a late promoter DNA binding activity cochromatographs and cosediments with VLTF-X activity. Current experiments show that VLTF-X activity is present in a variety of uninfected mammalian cell types and is indistinguishable from that recovered from infected cells based upon several criteria. VLTF-X activity from both sources displays the same purification profile over phosphocellulose and DNA affinity resins and has the same sedimentation coefficient. In addition, the factors purified from both infected and uninfected cells form protein-DNA complexes of identical electrophoretic mobility in the presence of vaccinia virus late promoter-containing DNA. The affinity of these factors for the late promoter probes is identical and late promoter-specific based on competition experiments. Moreover, VLTF-X purified from both sources bound to late promoter-containing DNA in the presence or absence of MgCl2 and ATP and formed complexes resistant to heat inactivation. These experiments offer proof that vaccinia virus factor VLTF-X is a host cell protein that supports transcription of the viral late genes.

Previously, the in vitro late transcription system of vaccinia virus was resolved into four components: the 17- and 30-kDa products of the A1L and G8R intermediate genes, respectively, the viral DNA-dependent RNA polymerase, and an unmapped factor sedimenting at 32 to 38 kDa. Another protein, the 26-kDa product of the A2L open reading frame was predicted to be a late transcription factor on the basis of a transient-expression assay but was not recognized as being necessary for transcriptional activity in vitro, We now report that both the unmapped factor and the 26-kDa protein are required for transcription from a vaccinia virus late promoter in vitro, Since the 26-kDa protein has now been shown to be a trans-activator of late transcription and it is the product of a known gene, we suggest that it be designated VLTF-3.

Fifty-two cases of malignant fibrous histiocytoma (MFH) were evaluated for amplification of the MDM2 gene, mutation of the P53 gene, accumulation of the P53 gene product, and their relation to disease-free and overall survival. All tests were carried out on formalin-fixed, paraffin-embedded tissue samples. Amplification of the MDM2 gene was detected in 15 of 52 cases (29%). Six of 52 cases (12%) demonstrated abnormalities of the P53 gene. Sequence analysis detected point mutations in four cases and a 1-base pair deletion in one case, whereas differential polymerase chain reaction (dPCR) indicated that the P53 gene had been entirely deleted in one case. Eight of 52 cases (15%) demonstrated staining for the P53 protein in >10% of tumor cells. The presence of MDM2 amplification did not have a significant effect on either disease-free or overall survival. Patients with accumulation of the P53 gene product did not differ in disease-free or overall survival from patients without P53 accumulation. Survival also was not significantly different in patients with genetic aberration in P53. However, when the patients were stratified by histologic grade, the results indicated that patients with alterations in the P53 gene may have shorter overall survival.

Chromatography of RNA polymerase purified from vaccinia virions and from vaccinia virus-infected HeLa cells resulted in the separation of populations active for early and late transcription. An RNA polymerase population immunodepleted for the vaccinia virus H4 gene peptide could support late transcription.

The use of semiquantitative PCR (SQPCR) to assess Pneumocystis carinii pneumonia (PCP) infection and its response to treatment was studied with rats. Groups of eight rats were immunosuppressed with steroids for 3 to 12 weeks. Untreated controls were maintained for the same periods. Three groups of rats were treated with pentamidine, three groups were treated with trimethoprim-sulfamethoxazole, and three groups of rats were tapered from steroids. At various times during suppression, rats from the different groups were sacrificed. At necropsy, lungs were lavaged to obtain bronchoalveolar fluids and then homogenized. Bronchoalveolar fluids and homogenates were assayed by cyst counting and SQPCR. An increase in the SQPCR signal was seen throughout immunosuppression, with a slow decrease upon the withdrawal of steroids and a faster decrease with drug treatment. SQPCR results with lung homogenates and bronchoalveolar fluids strongly correlated with each other and with cyst counts. These results warrant investigation of SQPCR for assessing treatment results of human P. carinii pneumonia infection.

We have resolved the in vitro late transcription system of vaccinia virus into four components consisting of RNA polymerase and three accessory factors. One of these additional factors is a 30-kDa protein which was previously shown to be required for late transcription in vitro and was indirectly shown to be the product of the G8R open reading frame. Another factor, of 17 kDa, was previously identified as a possible late transcription factor by an assay which demonstrated that the gene encoding it, A1L, was required for late gene expression in vivo. The G8R and A1L open reading frames have now been cloned into a baculovirus expression system, and the corresponding proteins have been purified. Both are necessary for late transcription in vitro, confirming that these intermediate genes encode late transcription factors. The third factor has a sedimentation coefficient consistent with a protein of 32 to 38 kDa. Experimental results suggest that this is a previously unidentified factor encoded by a vaccinia virus early gene. The RNA polymerase functioning in this system was purified from vaccinia virus-infected cells; however, it can be complemented by the RNA polymerase which is packaged in virions. The three smaller proteins and RNA polymerase are all necessary, and together are sufficient, for the synthesis of late viral mRNA in vitro.