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High School Student with a Traditionally Underrepresented Background

HS Poster

Poster 41 - Citrate Synthase is Required for the Initiation of PTEN-Deficient T-Cell Acute Lymphoblastic Leukemogenesis, but not PTEN-Deficient Myeloproliferative Neoplasms

Saturday, October 26, 2024
10:00 AM - 12:00 PM EST
Location: Exhibit Hall E, F & G

Introduction: The “Warburg Effect” marks the metabolic tendencies of cancer cells to primarily utilize aerobic glycolysis over oxidative phosphorylation. However, a comprehensive understanding of the interplay between altered tricarboxylic acid (TCA) cycle activity and mitochondrial dysfunction in the initiation and progression of cancer remains elusive. Particularly in T-cell acute lymphoblastic leukemia (T-ALL), an aggressive hematologic malignancy derived from immature T-cell progenitors, the role of aerobic glycolysis in leukemogenesis warrants further investigation. The PTEN gene is one of the most frequently-mutated/functionally-inactivated onco-suppressors in various cancers, and PTEN mutations account for 16% percent of all T-ALL cases. In this research, I wanted to investigate how changes to aerobic respiration would initiate and relate to the leukemogenesis of PTEN-deficient T-ALL and therefore contradict the Warburg effect, through the removal of the citrate synthase gene, a catalytic metabolite of the TCA cycle.

Materials and

Methods: In this research, Mx1-Cre conditional knockout of citrate synthase (CS), a catalytic metabolite of the TCA cycle, and PTEN, an oncosuppressor often linked to T-ALL, were employed to evaluate T-ALL in wild-type, single knockout (PTEN -/-) and double knockout (CS -/-; PTEN -/-) mouse models. Cre-mediated excision on conditional knockout mice was used due to superiority as a mechanism in studying malignant hematopoiesis; the mice were created to express either Mx1 +/+ (Wild Type) or Mx1 Cre/+ (knockout). All mice were injected with five intraperitoneal injections of 60 μg polyinosinic:polycytidylic acid (pIpC) every other day at 7 weeks of age to induce deletion and were analyzed at 10-12 weeks of age. All three sets of mice were then dissected for select tissue regions and bound to fluorescent antibody cocktails to differentiate lymphocytes for cytometry. First, I conducted both tissue composition analysis with a scale and cell count machine, and supplemental western blotting to ensure that the deletion of citrate synthase was successful. Trichloroacetic acid and acetone were used for the precipitation of proteins, 112.5 μL solubilization buffer was used for protein resuspension, and 37.5 μL Laemmli buffer was used for SDS gel electrophoresis protein isolation. Finally, lymphocytes associated with T-ALL development were analyzed through flow cytometry of the thymus, bone marrow, blood, and spleen cell samples. FlowJo quantitative population comparison gating was used to differentiate between particular cell types, and Microsoft Excel was used for significance testing with T-tests between cell populations of wild type, PTEN knockout, and PTEN/CS knockout.

Results, Conclusions, and Discussions: In the western blots, all five protein samples show vinculin-HRP (control) presence at the expected 124 kDa molecular weight while only the wild-type protein samples show citrate synthase protein presence at the expected 45 kDa molecular weight, indicating successful deletion of CS in the knockout mice. In tissue composition analysis, the abnormal weight of the PTEN-deficient thymus and spleen suggests cancerous development has occurred, but cytometry is needed to identify specific pathophysiology. The reduced cell counts in double knockout samples are associated with a reduced ability for cells to proliferate through aerobic processes. In flow cytometry, aberrant levels of T-ALL-associated lymphocytes amongst solely PTEN-deficient samples, yet a return to normalcy with CS-deficiency, indicated the requirement of citrate synthase for PTEN-deficient T-ALL progression. Individual t-tests on CD3, CD4, and CD8 branch T-cells, amongst other T-ALL-associated lymphocytes, confirm significance in reduced leukemogenesis (p-value < 0.01). Amongst specific T-cells, cell levels of DPCD3+, CD3+CD4+CD8+, and DPCD3- indicate normalcy among WT and PTEN-/CS- samples and irregularity in PTEN- samples, illuminating that proliferation of cancer is only able with aerobic pathways and the presence of citrate synthase. However, analysis of myeloid cells indicates insufficient blockade of PTEN-deficient myeloproliferative neoplasms in double knockout samples, presumably from impaired red blood cell maintenance post-CS deletion. Additionally, the data suggests stunted normal T-cell development, which is a downside of the study. Consequently, the deletion of citrate synthase impedes the ability of PTEN-deficient T-cell acute lymphoblastic leukemia to form, but not PTEN-deficient myeloproliferative neoplasms. However, cancer cells may activate other compensatory pathways when the TCA cycle is inhibited, implying that the scope of the experiment needs to be extended further. In the future, we can further investigate what other metabolites are now fueling the initiation and progression of myeloproliferative neoplasms if glucose is no longer the main contributor to cancer metabolism with the removal of citrate synthase. This research demonstrates that the sequential context achieved via citrate synthase deletion undermines leukemogenesis and contrasts pre-existing ideals about the metabolic tendencies of cancer cells, therefore implying the dual reliance of cancer proliferation on both aerobic and anaerobic metabolic processes.

Acknowledgements (Optional): Thank you to the Children's Research Institute at UT Southwestern Medical Center for allowing me to intern in your labs and providing me with ample background to conduct my experiments.

References (Optional):