Insights into the Genetic Signature Associated with Cell Death Induced by an Antibody-Avidin Fusion Protein Specific for Transferrin Receptor.
Authors: Helguera Gustavo , Rodriguez Jose A. , Daniels Tracy R. , Diaz-Cano David Casero , Pellegrini Matteo , Penichet Manuel L. 
Published: October 2019

Abstract
We have previously developed an anti-human transferrin receptor IgG3-avidin fusion protein (anti-hTfR IgG3-Av) that exhibits a potent anti-proliferative/pro-apoptotic activity against malignant B cells. This effect has been attributed, at least in part, to the internalization and rapid degradation of the hTfR by anti-hTfR IgG3-Av, resulting in lethal iron deprivation. However, in a panel of eight malignant B cells treated with anti-hTfR IgG3-Av we observed varying levels of sensitivity among the cell lines, where IM-9 (a human lymphoblastoid cell line) showed high sensitivity, while U266 (a human multiple myeloma cell line) was significantly resistant; although both cell lines express high levels of hTfR. To elucidate the mechanisms behind resistance and sensitivity of these human malignant B cells we conducted a global gene expression comparison treating the two cell lines for 0, 1, 3, 9, and 24 hours with anti-hTfR IgG3-Av or buffer control. Samples of total RNA were collected in duplicate at each time point and gene expression levels analyzed using HumanRef-8 v2 Expression BeadChips (Illumina Inc. San Diego, CA). Microarray data were preprocessed using quantile normalization and the difference in levels of gene expression was established using Bayesian statistics. Out of 8,095 array probes detected at time zero in both IM-9 and U266 cell lines, 167 had a greater than four-fold significant difference (regularized bayesian t-test p<0.05). With the genes selected by this global comparison, we performed a gene ontology analysis to identify genes associated with functional processes that may be involved in the differential sensitivity to anti-hTfR IgG3-Av. We found that genes facilitating apoptotic signaling through RAS, MAPK, and P53 (such as PIK3CD and CBLB) were expressed at significantly higher levels in IM-9 compared to U266, while genes involved in cell cycle including the cyclin-dependent growth regulator CDKN2C and a number of core histones were more highly expressed in U266 compared to IM-9. Of all the genes present in both cell lines, a time course study of 24 hour treatment with anti-hTfR IgG3-Av identified 74 genes in IM-9 cells and 10 genes in U266 cells with significant changes (p<0.05) at greater than 1.5-fold different expression levels compared to buffer control treated cells. IM-9 cells treated with anti-hTfR IgG3-Av showed increased expression of a number of apoptosis related genes including GADD45A, IKIP, DDB2, and TP53I3 at 24 hours. In contrast, U266 cells treated with anti-hTfR IgG3-Av showed decreased expression of genes involved in apoptosis induction and cell death such as PLEKHF1, OKL38, and CDC14B. Although further studies are needed to validate the expression changes, these results illustrate the complexity of the mechanism responsible of cell death induced by anti-hTfR IgG3-Av, and suggest that the molecular pathway involving p53 may be associated with cell death in the sensitive cell line. These studies are expected to pave the way to fully elucidate the mechanisms responsible for cell death induced by anti-hTfR IgG3-Av and the identification of a genetic signature responsible for resistance to this therapeutic.