Pediatric high-grade gliomas (pHGG) are highly aggressive brain tumors with limited therapies and extremely poor clinical outcomes. pHGG and many other cancers are often driven by genetic fusion events, which produce oncogenic fusion proteins such as TPM3-NTRK1. NTRK1 regulates signaling pathways promoting cell survival and differentiation. In cancer, NTRK fusions lead to constitutive activation of these pathways, resulting in uncontrolled cell growth. The purpose of my project is to validate TPM3-NTRK1 as a potential drug target for a novel therapeutic strategy known as targeted protein degradation and to clarify the role of TPM3-NTRK1 in cancer signaling. I hypothesize that degrading the TPM3-NTRK1 protein will decrease the activity of downstream signaling pathways that lead to pHGG. To model pharmacological degradation of TPM3-NTRK1, I applied the degradation tag (dTAG) system to rapidly and reversibly degrade a target protein fused with an FKBP12^F36V-tag. In this approach, dTAG molecules bind to the FKBP12^F36V-tag and recruit an E3 ubiquitin ligase to ubiquitinate the target protein, leading to its degradation by the proteasome. In my project, I used Gateway cloning to generate a lentiviral expression plasmid containing FKBP12^F36V-TPM3-NTRK1. I then produced lentiviruses, which I used to transduce NIH/3T3 cells and express FKBP12^F36V-TPM3-NTRK1. To test whether the FKBP12^F36V-TPM3-NTRK1 protein can be successfully degraded, I plan to treat the cells with dTAG molecules and use Western blotting at various time points to evaluate levels of FKBP12^F36V-TPM3-NTRK1 and known proteins involved in downstream signaling pathways, including pERK and pAKT. I expect that samples treated with dTAG molecules will show significantly decreased FKBP12^F36V-TPM3-NTRK1, pERK and pAKT. Importantly, these results will justify the development of small-molecule degraders to target TPM3-NTRK1, a promising therapeutic approach for pHGG and other hard-to-treat cancers. Additionally, a cell line expressing degradable TPM3-NTRK1 will serve as a powerful model system to further explore its role in cancer.

Myeloproliferative neoplasms (MPNs) are blood cancers that can arise from the constitutive activation of the Janus kinase (JAK) and signal transducer and activator of transcription (STAT) pathway. One mutation in this pathway, JAK2^V617F, is especially prevalent among MPN patients. JAK2^V617F alters the JAK2 protein structure so that the kinase is persistently phosphorylated and JAK-STAT signaling is perpetually active. However, the role of JAK2^V617F in MPN development is not well defined and selective therapeutic strategies to target JAK2^V617F remain elusive. To address these challenges, my project’s goal is to selectively degrade the JAK2^V617F protein and evaluate its downstream effects using the Ba/F3 cell system. Ba/F3 cells are interleukin-3 (IL-3) dependent for survival but can become IL-3 independent in the presence of an oncogene. I hypothesize that JAK2^V617F expression in Ba/F3 cells will confer IL-3 independence, while degradation of the mutant protein will revert the cells to their native, IL-3-dependent phenotype. To model pharmacological degradation of JAK2^V617F, I applied the degradation tag (dTAG) system, which harnesses the cellular ubiquitin-proteasome machinery to degrade proteins fused with an FKBP12^F36V tag using dTAG molecules. I first cloned plasmids to lentivirally express FKBP12^F36V-JAK2^V617F in Ba/F3 cells. Following viral transductions, I treated the transduced cells expressing FKBP12^F36V-JAK2^V617F with vehicle control or dTAG molecules to induce degradation. Western blots were used to confirm FKBP12^F36V-JAK2^V617F expression and dose-dependent degradation. To evaluate whether JAK2^V617F confers IL-3 independence, I cultured the Ba/F3 cells expressing FKBP12^F36V-JAK2^V617F without IL-3, treated them with vehicle control or dTAG molecules, and assessed cell viability using CellTiter-Glo luminescence assays. I anticipate IL-3 independent cell survival of the Ba/F3 cells expressing FKBP12^F36V-JAK2^V617F to decrease with increasing dTAG molecule treatment. The results from this project will build a new pipeline for studying oncogenes and contribute to the evaluation of JAK2^V617F degradation as a novel therapeutic approach against MPNs.