B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive hematologic malignancy characterized by the overproduction of malignant B-lymphoblasts in the bone marrow. It is the most common pediatric cancer and the second leading cause of cancer death among children. Standard therapies, including drugs such as dexamethasone and vincristine, achieve remission in approximately 90% of cases, but 10% of patients exhibit resistance. Furthermore, standard therapies result in significant short- and long-term toxicities. Thus, alternative treatment strategies are needed. FHD-286, a BRG1/BRM ATPase inhibitor currently in clinical trials for acute myeloid leukemia (AML), is a potential candidate for improving B-ALL therapy by targeting chromatin remodeling dependencies and reducing reliance on less tolerable chemotherapies. Our study evaluates the efficacy of FHD-286 in combination with dexamethasone and vincristine, hypothesizing that the combination may overcome treatment resistance in B-ALL. We tested these combinations across genetically diverse B-ALL cell lines. We treated the cell lines with varying doses of each drug alone and in combination and, after 3 to 5 days, assessed cell counts and apoptosis using Annexin V staining. Compared to vincristine or dexamethasone alone, when combined with FHD-286, we observed an increase in apoptosis. After three days of treatment, we detected a significant decrease in cell count, while after five days, cell viability dropped, suggesting that the drug combination may induce both cell cycle arrest and followed by apoptosis over time. Notably, FHD-286 demonstrated effectiveness in KMT2A-rearranged B-ALL, a high-risk subtype prone to relapse, while also demonstrating potent effects in non-KMT2A-rearranged B-ALL. Our findings suggest that FHD-286 enhances therapeutic efficacy in B-ALL when combined with current standard treatments, offering a potential strategy to overcome resistance and reduce chemotherapy toxicity across multiple leukemia subtypes.

Stress-related factors can have a direct impact on cancer biology and patient outcomes. Exposure to a stressor leads to the sympathetic nervous system (SNS) activating downstream signaling pathways that impact cancer-related processes; SNS activity can be measured with heart rate variability (HRV). Low HRV indicates less ‘autonomic flexibility’ and has been associated with poor health outcomes, while high HRV has been associated with better health outcomes. Psychosocial factors such as resilience, stress, and social support are important for adolescents and young adults (AYAs), but the relationship between psychosocial factors and HRV is unknown. The goal of this study is to examine changes in HRV among AYAs with cancer during a qualitative interview about psychosocial factors. Eligible participants were 12-24 years old within six months of initial cancer diagnosis and undergoing treatment at Seattle Children’s Hospital. Once enrolled, participants wore an HRV sensor while participating in a 1:1 semi-structured interview querying topics including stress, resilience, and social support. I used a commonly reported HRV metric, the standard deviation of normal-to-normal intervals (SDNN) to quantify HRV. I defined baseline HRV as the first 5 minutes of the interview, reactive HRV as 5 minutes at the midpoint of the interview and recovered HRV as the last 5 minutes of the interview. I compared baseline HRV to reactive HRV and recovered HRV. I expect to find that both reactive HRV and recovered HRV are lower (‘worse’) than baseline HRV. Results from this study can give insight on the impact psychosocial factors have on the biomarkers of stress in AYAs with cancer.

Colorectal cancer (CRC) is the second leading cause of cancer-related mortality worldwide. In CRC and several other cancers, chromosomal rearrangements lead to the fusion of the kinase domain of TRK, which normally regulates neuronal survival and proliferation, with the oligomerization domains of other proteins. This produces a constitutively active fusion protein that drives cancer by hyperactivating pro-survival and proliferative signaling. An exemplary recurring genetic alteration is the fusion of TPM3 and TRKA (TPM3-TRKA) that drives CRC. The importance of TRK fusions in cancer has led to the development of several TRK inhibitors targeting its kinase activity. However, despite short-term benefits to patients, the current FDA-approved TRK inhibitors are susceptible to off-target effects that lead to toxicity and resistance mutations that limit effectiveness. To overcome these limitations, the goal of my project is to apply a novel therapeutic modality to target TRK fusions, known as targeted protein degradation. I hypothesized that degradation of TRK fusions would compromise their kinase and scaffolding functions and decrease oncogenic signaling. To degrade TRK fusions, we designed a library of heterobifunctional molecules called proteolysis-targeting chimeras (PROTACs). These small molecules engage with a TRK fusion protein and recruit an E3 ubiquitin ligase to ubiquitinate it, causing its degradation by the cellular ubiquitin-proteasome system. To evaluate our PROTACs, I used immunoblotting to monitor the level of TPM3-TRKA in KM12 cells, a CRC cell line. I performed dose-responses and time-courses to identify a highly potent PROTAC (10 nM dose) that completely degrades TPM3-TRKA within two hours. Degradation was maintained for at least 24 hours and led to sustained downregulation of signaling. In the future, we will compare the efficacy of our PROTACs to inhibitors in CRC. In summary, we have developed PROTACs targeting TRK fusions, which will serve as a promising new therapeutic modality for CRC.

To function as a tumor suppressor, BRCA1 (breast cancer 1) must dimerize with BARD1 (BRCA1-associated RING domain protein 1). Due to this critical interaction, loss-of-function BARD1 variants are associated with increased breast and ovarian cancer risk. Genetic testing has identified many rare single-nucleotide variants (SNVs) that cause missense substitutions in BARD1. Currently, 85.6% (1,736 of 2,028) of BARD1 missense SNVs are classified as a variant of uncertain significance (VUS) in ClinVar. A VUS classification prevents clinicians from using genetic test results to guide patient care. Consequently, there is a strong need to functionally assess BARD1 SNVs to help resolve VUS. We applied saturation genome editing (SGE) to functionally assess all possible 12,000 SNVs and 2,300 3-base deletions in BARD1. In SGE, we use CRISPR-Cas9, to edit all possible SNVs into a region of BARD1 in haploid cells. BARD1 is essential for cell growth, therefore, cells edited with loss-of-function variants become depleted from the population. We use DNA sequencing to track which SNVs become depleted from the population after 13 days in culture and are likely loss-of-function. All 14,000 variants have completed the full experimental pipeline. We show that 98% stop-gain, 29.6% splice-region, and 14.3% missense variants are loss of function relative to 1.6% of synonymous/intronic variants. The SGE data also agree strongly with current pathogenic/likely pathogenic and benign/likely benign BARD1 variants in ClinVar. Moreover, I have identified previously known and potential new protein-protein interaction interfaces through mapping our SGE data to the surfaces of BARD1’s structured domains. Ultimately, the functional scores for all BARD1 variants provide key functional evidence needed to reclassify BARD1 VUS and provide new insight into the mechanisms of BARD1 function.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes joint damage, frailty, and potential disability. Its progression is unpredictable, making it difficult to manage in clinical settings. A major challenge in treatment is the lack of reliable clinical indicators or biomarkers to track disease activity and predict long-term outcomes like frailty and joint damage. Growth differentiation factor-15 (GDF-15) has shown promise as a biomarker in other diseases, but its role in RA remains unclear. This study explores whether GDF-15 can predict disease progression, frailty, and joint damage in RA patients. To understand the role of GDF-15 in RA, we measured its levels in both RA patients and healthy individuals using ELISA, which detects specific proteins. We explored how GDF-15 levels are related to disease activity, inflammation, and joint damage. In a group of patients followed for 8 years, we investigated whether GDF-15 levels at diagnosis could predict how the disease might progress. We used various statistical tests to analyze the data. The Mann-Whitney U-test helped compare GDF-15 levels between RA patients and healthy controls, Spearman’s correlation showed the relationship between GDF-15 levels and disease activity, and logistic regression allowed us to evaluate whether GDF-15 levels at diagnosis could predict future RA development. Through this study, we (i) analyzed how GDF-15 levels are linked to disease activity and inflammation in RA, (ii) explored whether measuring GDF-15 levels early on could predict disease progression and (iii) assessed whether GDF-15 could help identify patients at higher risk of developing severe joint damage or other complications. Ultimately, this research could help rheumatologists better understand and predict how RA will progress in patients, leading to more personalized and effective treatments.

Approximately 500 million people worldwide live with osteoporosis, a disease of low bone mineral density (BMD) and bone fragility caused by a disequilibrium between osteoblasts, cells that build bone, and osteoclasts, cells that reabsorb bone. Existing osteoporosis treatments are single-action anti-resorptive or osteoanabolic (bone-promoting) drugs, which make them insufficient for individuals with severe disease or those at high imminent risk for fractures. RANK is a receptor on osteoclastic progenitor cells that, when activated by RANK ligand binding, induces osteoclast formation and spurs the translocation of a transcription factor, NFATc1, into the nucleus, where it initiates RANK transcription. The available literature on the topic has traditionally only acknowledged RANK to be present in osteoclasts. Contrary to this view, our lab recently identified Rank in osteoblasts. Thus, my project examines how Rank acts in osteoblasts to regulate bone formation. I hypothesize that in osteoblasts of developing bone, Rank signaling is regulated by Nfatc1 via a positive feedback loop, similar to what occurs in osteoclasts. Using in situ hybridization chain reaction, I found that nfatc1 is expressed strongly and specifically in the same developing skeletal structures as rank+ osteoblastic cells in 3, 5, 12, and 14 day post-fertilization zebrafish, supporting my hypothesis. My ongoing studies focus on identifying the Rank and Nfatc1 interactions that may promote osteoblast differentiation. I am achieving this by analyzing the skeletal phenotypes of a rank loss-of-function mutant I am crossing with a reporter transgene line that fluoresces when Nfatc1 signaling is activated, as well as analyzing fish chronically subjected to FK506—a pharmacological inhibitor of calcineurin, which is required for NFATc1 translocation. My preliminary data suggest that the proposed positive feedback loop between RANK and NFATc1 is conserved across osteoclasts and osteoblasts, revealing potential targets for dual-action (anti-resorptive and osteoanabolic) osteoporosis therapies.