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UBC Theses and Dissertations

Functional consequences of transmembrane protein 30A (TMEM30A) inactivating mutations in diffuse large B-cell lymphoma Baticados, Abigail Morales


Novel recurrent inactivating mutations and copy number loss of transmembrane 30A protein (TMEM30A) were identified in 347 de novo DLBCL cases. Specifically, TMEM30A loss of function (LOF) mutations were discovered to co-occur with hemizygous deletion or copy neutral LOH in 80% of the subset of cases with TMEM30A alteration. Furthermore, patients with biallelic inactivation of TMEM30A had superior treatment outcomes compared to patients with WT TMEM30A. Presently, the role of TMEM30A in lymphomagenesis remains elusive. Therefore, this study aimed to clarify its biological function and significance in DLBCL. DOHH-2, a human DLBCL cell line was genetically modified to create several mutant clones with TMEM30A LOF. Specifically, a stable TMEM30A knockdown (KD) and biallelic TMEM30A CRISPR knockout (KO) and control TMEM30A non-silenced (NS) cells were created. All experimental cells including DOHH-2 wildtype (WT) were subjected to serum deprivation and treatment with cytotoxic agents. The rate of apoptosis and viability of the cells were measured using flow cytometry (FCM) assays and cleaved caspase 3 (Casp-3) immunoassays. TMEM30A active cells (WT and NS) showed significantly higher (P< 0.01 and P< 0.05) rates of apoptosis compared to the mutant TMEM30A-LOF (KO and KD) cells at 24h post-serum starvation, respectively. Similarly, increased cell death was observed to be significant (P< 0.01) in control cells compared to the mutant cells at 96h serum starvation, confirmed by results of Casp-3 immunoassays, FCM assays, and densitometry analyses. (P< 0.001). Restoring TMEM30A expression in KO cells showed a reversal in phenotype in which restored cells became more sensitive to serum deprivation. Conversely, cytotoxicity experiments demonstrated that cell survival significantly (P< 0.01) decreases in KO compared to WT cells after treatment with zeocin, a radiomimetic drug that induces DNA double-strand breaks. These findings suggest that TMEM30A inactivation plays multiple roles in the presence of environmental stress. Future studies that will clarify the mechanisms underlying increased resistance to nutrient deprived conditions and increased chemo-sensitivity observed in DLBCL cells with TMEM30A inactivation may allow the identification of novel therapeutic approaches for the treatment of B-cell lymphomas.

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