Silencing TG2 in Acute Myeloid Leukemia. Possible Role of TG2 in Microenvironment-Mediated Chemoresistance

Background: Tissue transglutaminase (TG2) is a multi-domain, multi-functional enzyme with diverse biological functions including calcium-dependent posttranslational modification of proteins, extracellular matrix formation, integrin-mediated signaling, and signal transduction. In many cancer types, increased TG2 expression has been associated with malignancy and resistance to chemotherapy. Previous studies using reverse phase proteomic arrays (RPPA) on 511 AML samples indicated in many cases that TG2 expression was elevated in samples from patients with relapsed AML when compared to samples collected at AML diagnosis (PMID: 23576428). In addition, elevated TG2 expression correlated with increased expression of proteins involved in cell-adhesion and the regulation of apoptosis. These findings suggest that the expression of TG2, in combination with other makers, may be suitable to predict survival outcome in AML patients and that targeting TG2 might sensitize AML cells to standard chemotherapy.

Results: Our preliminary data demonstrated that the expression of TG2 mRNA, as determined by cDNA array analysis and validated by qRT-PCR, is induced (>30 fold increase) in OCI-AML3 leukemia cells when co-cultured with normal donor-derived Bone Marrow-derived Mesenchymal Stromal Cells (BM-MSC) in hypoxic conditions (1% pO₂) for 48hs. Increase in mRNA correlated with an increase in protein level as determined by Western Blot. In order to test the hypothesis that the expression of TG2 in leukemia cells is related to resistance to chemotherapy and poor prognosis we down-regulated or over-expressed TG2 by lentiviral transduction with TG2-shRNA or TG2-over-expressing vector, respectively, in OCI-AML3 cells. Our in vitro studies showed that although the over-expression of TG2 in OCI-AML3 cells did not have a significant impact on resistance to chemotherapy, down-regulation of TG2 had a moderate effect on sensitizing cells to AraC (13% increase in apoptosis; P = 0.0006). To further investigate these findings we performed in vivo experiments in NGS mice engrafted with OCI-AML3 stably-transduced with either scramble-shRNA or TG2-shRNA. After confirmation of engraftment by bioluminescence imaging, leukemia-bearing mice were treated with a combination of AraC and doxorubicin. Consistent with the in vitro data, the down-regulation of TG2 rendered OCI-AML3 cells more susceptible to chemotherapy and prolonged the survival of leukemia-bearing mice suggesting a role for TG2 in resistance to chemotherapy (median survival of mice transplanted with scramble-shRNA cells, 44 days; compared to mice transplanted with TG2-shRNA cells, 51 days; P = 0.0027).

Conclusions: Our data indicate that TG2 is up-regulated in response to microenvironmental signals like hypoxia and cell-cell contact with stromal cells. Silencing of TG2 in AML cells sensitized cells to chemotherapy in vitro and in vivo and extended the survival of the leukemia-bearing mice suggesting that targeting TG2 in combination with standard chemotherapy in AML could be developed into a novel therapeutic approach.