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Glioblastoma is the most malignant form of gliomas and is a highly infiltrative while non-metastatic tumor of the central nervous system. Patients with glioblastoma have a poor prognosis of 15 months median survival after diagnosis. Promising results were reported in recent clinical trial regarding glioblastoma treatment with chimeric antigen receptor (CAR) T therapy. The lab has previously developed five novel scFvs targeting IL13R⍺2, a tumor-associated antigen in glioblastoma, and integrated them into the second-generation CAR. We named them, 10CAR, 27CAR, 55CAR, 75CAR and 117CAR. The ex vivo cytotoxicity and proliferation assay demonstrated that the 117CAR T construct has the best functionality, while 27CAR T construct has a poor functionality compared to the rest of the constructs. FACS analysis was performed to check the CAR expression in different constructs. 27CAR T cells showed the lowest surface CAR expression and 117CAR T cells displayed the highest out of five constructs. 27CAR T cells were also activated more without stimulation compared to other constructs. We selected out 27CAR and 117CAR T cells for the further investigation to understand the attribution of the discrepancy between 27CAR and 117CAR T cells.   We observed a larger cellular size for 27CAR T cells compared to the rest constructs in flowcytometry analysis, which is usually associated with activation. IFN-γ production of all constructs without target cells stimulation were detected to examine the activation state of different constructs. We observed the highest IFN-γ production in 27CAR T cells without stimulation. These results together indicate that a potent antigen-independent activation or, in other words, tonic signaling is present in 27CAR T cell. The tonic signaling further leads to an early exhaustive phenotype of 27CAR T cells, that is not present in 117CAR T cells. Removing the endodomain of CAR rescued the antigen-independent activation and early exhaustion of 27CAR T cells. The surface and total CAR expression of 27CAR and 117CAR T cells were determined by flowcytometry. 27CAR T cells presented a lower expression of both surface and total CAR. A significantly lower percentage of total CAR on the surface indicates the internalization of CARs in 27CAR T cells. Removing the intracellular domain of 27CAR did not restore the surface expression of CAR. 27CAR and 117CAR differ in four CDRs of scFv, CDR1, 2,3 in the heavy chain and CDR3 in the light chain. We replaced all the amino acids differing between these two constructs with alanine in a CDR-by-CDR manner and obtained five alanine substitution constructs. We then analyzed the CAR expression in Jurkat cells, and we found that the trafficking of CAR to the surface was significantly improved by mutating the CDR2 in the heavy chain or CDR3 in the light chain. Moreover, when the two CDRs were replaced simultaneously, almost all transduced cells expressed CAR, as was the case of cells transduced with 117CAR.   To summarize, the tonic signaling induced by higher tendency of clustering of 27scFv results in the antigen-independent activation and early exhaustion of 27CAR T cells. By removing the endodomain of 27CAR, we abrogated the phenomenon. Further, CDR2 in heavy and CDR3 in light chain in 27scFv are responsible for the impaired trafficking of CAR to the surface.

Epstein-Barr virus (EBV) is one of the most ubiquitous human viruses, with over 95% of the adult population harboring lifelong latent EBV infection in a small fraction of their B- lymphocytes. EBV is known to cause lymphoproliferative disorders and is associated with Hodgkin’s lymphoma, Burkitt’s lymphoma and Nasopharyngeal carcinoma. However, in most cases, the approach to EBV-positive lymphomas does not differ from EBV-negative lymphomas of the same histology. Latent membrane protein 1 (LMP1) is the major transforming protein of EBV and is critical for EBV-induced B-cell transformation in vitro. LMP1 activates several epigenetic regulators to modify host gene expression, including the chromatin-modifying enzyme Poly(ADP- ribose) polymerase 1, or PARP1. We have determined that LMP1 can activate PARP1 to increase hypoxia-inducible factor 1-alpha (HIF-1α)-dependent gene expression, leading to a change in host cell metabolism indicative of a ‘Warburg effect’ (aerobic glycolysis). This subsequently provides a proliferative advantage to LMP1-expressing cells. The LMP1-induced increase in HIF-1α-dependent gene expression, alteration of cellular metabolism, and accelerated cellular proliferation can be offset with the PARP inhibitor olaparib. We further determined that LMP1 can induce metabolic changes in the form of fatty acid synthesis. Ectopic expression of LMP1 and EBV-mediated B-cell growth transformation induced fatty acid synthase (FASN) and increased lipid droplet formation. FASN is a crucial lipogenic enzyme responsible for de novo biogenesis of fatty acids in transformed cells. FASN can be stabilized at the protein level through interaction with USP2a, a ubiquitin-specific protease that de-ubiquitinates FASN leading to its overexpression. We have evidence to suggest that following ectopic expression of LMP1 and EBV-mediated B-cell growth transformation, USP2a is increased, leading to stabilization of FASN at the post-translational level. Furthermore, following dosing with C75, an inhibitor of lipogenesis, we observed preferential killing of LMP1-expressing B cells. Our findings demonstrate that EBV-mediated B-cell growth transformation, partly through LMP1, leads to induction of FASN, fatty acids and lipid droplet formation, possibly pointing to a reliance on lipogenesis. In summary, targeting PARP1 activity and lipogenesis programs may be an effective strategy in the treatment of LMP1+ EBV-associated malignancies. / Cancer Biology & Genetics