Melanoma is an aggressive form of skin cancer whose progression is influenced by the tumor microenvironment (TME), containing immune cells and stromal cells. Autophagy, a crucial cellular process that recycles old or damaged cell components, is heightened in the TME. Previous studies have shown that preventing autophagy induction in tumor-supporting cells suppresses tumor growth in mice. This study investigates which cell types within the TME are critical for autophagy-dependent tumor growth. Atg7, an essential gene for autophagy, was knocked out using a Cre-lox gene editing system in order to disrupt autophagy and create autophagy deficiency within a specific cell population. Bone marrow transplants were used to create variations of mice with autophagy-deficient host endogenous and immune cells and evaluated for tumor progression. Results show that an autophagy-deficient transplanted immune system is sufficient to reduce melanoma tumor growth, indicating that immune cell autophagy plays a key role in melanoma progression. These findings highlight immune cell autophagy as a potential therapeutic target, with future studies needed to evaluate effectiveness in human patients.

Principal component analysis (PCA) has become an integral component of the scRNA-seq data processing pipeline for its ability to shrink high-dimensional datasets down to the axes of maximal transcriptional variation. PCA performs best on datasets with relatively normally distributed gene expression, where the mean is a stable measure of central tendency. Composition of such datasets produces oval-shaped distributions in gene space that are well approximated by orthogonal axes of maximal variance. However, our analysis of scRNA-seq datasets revealed that underlying data distributions differ from the standard assumptions used by PCA. For instance, we found that many scRNA-seq datasets do not feature normally distributed gene expression, and are instead heavily right-skewed. This results in covariation patterns that differ significantly from the cases where PCA provides a good summary of the data. In particular, we found that negative correlations between gene expression do not have the expected oval shape, but rather a “mutual exclusion” behavior. We developed a statistical test for this mutal exclusivity, and found multiple instances of mutually exclusive distributions where we had expected to see negatively correlated, oval-shaped ones. These findings suggest caution when using PCA on scRNA-seq datasets due to incompatibility between actual expression distributions and the assumptions underlying PCA regarding linear covariance structure.

SLC6A8 (sodium and chloride dependent creatine transporter 1) deficiency is a rare, X-linked disorder that presents in males before the age of three. Patients with SLC6A8 deficiency experience creatine brain deficiency which results in mental retardation, speech delay, and epilepsy. SLC6A8 is a ubiquitous transporter protein for creatine, an endogenous energy metabolite crucial for cellular metabolism. Creatine is unable to cross the blood brain barrier (BBB) without a protein transporter. Prior treatments of SLC6A8 deficiency include creatine supplementation; however, this has been unsuccessful in prevention or repair of these neurological symptoms due to the lack of a functioning SLC6A8 transporter. Thus, we delivered the SLC6A8 human codon-optimized transgene using adeno-associated viruses (AAV) in a mutant SLC6a8 murine model at one month of age. We have identified two serotypes, F and PHP.eB, which could package this vector. SLC6a8 mutant mice were injected with one of the two serotypes, which packaged a codon-optimized SLC6a8 transgene and either the CMV early enhancer/ chicken β actin (CAG) or elongation factor 1ɑ short (EFS) promoters. Analysis will include brain, heart and muscle creatine quantification; gene expression quantification via RT-qPCR; SLC6a8 RNA localization via in situ hybridization; and protein localization via immunohistochemistry. If one of the AAV serotypes proves successful, a new gene treatment could relieve neurological symptoms of affected patients and replenish brain creatine levels.

Bloom syndrome (BSyn) is a rare autosomal recessive disorder caused by mutations in the BLM gene, impairing helicase activity and defective DNA repair. It is characterized by short stature, photosensitivity, immunodeficiency, and a significantly increased risk of early-onset cancers, especially leukemia and lymphoma, compared to the general population. BSyn patients are hypersensitive to conventional cancer therapies, highlighting the need for alternative strategies. The lab previously demonstrated that Blm mice had increased sensitivity to radiation and decreased survival, compared to wildtype (WT) mice. If Blm mice were co-housed with WT mice after radiation, their survival was significantly improved. Thus, we hypothesized microbiome differences may be harnessed to improve post-irradiation survival. In this study, we demonstrated that fecal microbiota transplantation (FMT) in a Blm mouse model reduces DNA damage and improves survival compared to controls. Irradiated and non-irradiated Blm mice exhibited altered gut microbiome composition and short-chain fatty acid (SCFA) profiles relative to WT mice. Single-cell gel electrophoresis revealed decreased DNA damage in Blm mice after FMT treatment. Ongoing studies investigate acetic acid supplementation, a major SCFA, in Blm mice to assess its role in reducing DNA damage. Understanding the relationship between the gut microbiome and genomic stability in BSyn may provide insight into improved therapies and clinical outcomes.