Our studies in zebrafish define the cellular and molecular differences between human T-LBL and T-ALL, providing for a biological basis for the different clinical presentations of these two T-cell malignancies. The results indicate that aberrant overexpression of BCL2
together with MYC
accelerates the onset of malignant transformation by suppressing Myc-induced apoptosis (Strasser et al., 1990
), while elevated S1P1 and ICAM1 levels promote homotypic cell adhesion through binding to LFA1, associated with a blockade of intravasation and thymic egress. The transformed T-LBL lymphoblasts that are unable to intravasate and undergo hematologic dissemination remain trapped in the thymic region, where they proliferate to the capacity of their local nutrient supply and induce the autophagy program in response to metabolic stress. Conversely, MYC-stimulated lymphoblasts with low levels of BCL2
expression appear to undergo a more protracted multistep transformation process that may involve activation of alternative cell survival programs, as well as molecular pathways that promote dissemination outside of the thymic environment. These T-ALL lymphoblasts rapidly undergo hematologic dissemination to nutrient rich environments throughout the host, thus avoiding metabolic stress and the induction of autophagy.
Thymocytes express a number of adhesion molecules, including N-cadherin, E-cadherin, ICAM1, and LFA1, during specific stages of maturation that are associated with specific functions including thymocyte emigration and intravasation (Petrie and Zuniga-Pflucker, 2007
; Boyd et al., 1988). The regulated expression of ICAM1 controls the balance of homotypic cell-cell adhesion and heterotypic adhesion to vascular endothelial cells, which modulates the intravasation process (Boyd et al., 1988; Guo et al., 2009; Gares and Pilarski, 2000). Recent evidence supports the contribution of S1P1 function to the process of thymocyte intravasation through its regulation of ICAM1 levels (Lin et al., 2007
), and S1P1 agonists such as SEW2874 have been shown to increase S1P1 signaling in the thymus and inhibit mature thymocyte egress (Sanna et al., 2006
). Consistent with these data, we show that T-LBL cases overexpressing BCL-2 have high S1P1 levels mirroring those of immature cortical thymocytes that do not emigrate from the thymus (). The mechanism underlying this association is uncertain, but it does not appear to be solely dependent on the state of thymocyte differentiation, since cases of both T-ALL and T-LBL can present with cell surface markers indicating arrested T-cell development at all maturation stages (Crist et al., 1988
). Our experiments also show that the W146 S1P1 inhibitor reduces homotypic thymocyte cell-cell adhesion and implicate the loss of homotypic cell-cell adhesion in the ability of T-LBL cells to intravasate in our in vivo
transplantation assays. The evidence of elevated S1P1 and ICAM1 expression in human T-LBL cells, together with evidence for S1P1-dependent cell aggregation in vitro
and in vivo
, strongly support a role of homotypic cell adhesion mediated through elevated ICAM1, in regulating T-LBL intravasation and subsequent hematologic dissemination.
Our results suggest that the induction of autophagy is a consequence rather than a cause of the inability of malignant T-lymphoblasts to disseminate in our zebrafish model. First, when zebrafish Myc;Cre;bcl-2 T-LBL cells were cultured in vitro, their survival indicated that their inability to disseminate could not be attributed to their inability to survive outside the thymic niche. Second, inhibitors of autophagy failed to restore the ability of T-LBL cells to disseminate.
While low levels of activated Akt were observed in Myc;Cre;bcl-2 zebrafish with localized T-LBL lymphomas, the Myc;Cre;bcl-2 lymphomas that progressed to T-ALL possessed high levels of phospho-Akt (Ser 473-p-Akt), suggesting that AKT activation provides a mechanism allowing bcl-2-overexpressing cells to disseminate. Furthermore, the expression of a constitutively active form of murine Akt2 (Myr-Akt2) in Myc;Cre;bcl-2 transgenic zebrafish promoted rapid dissemination of the disease while lymphoblasts overexpressing Akt failed to aggregate in vitro, further supporting the association between activated Akt signaling, the loss of cell adhesion and T-ALL dissemination.
Human T-ALL and T-LBL are considered to represent different clinical presentations of the same disease that are often treated with identical treatment regimens. Our studies suggest that different molecular and cell biologic properties may render these diseases uniquely susceptible to different types of targeted therapies. Thus in T-LBL patients, combination of BCL2 and AKT inhibitors could promote lymphoblast death while blocking pathways that lead to lymphoblast escape and dissemination. Such approaches would likely have little efficacy for the majority of patients with T-ALL, who have low levels of BCL2 expression and lack evidence of activation of autophagy. Our studies also suggest that BCL2 levels, AKT phosphorylation, and LC3 and BECLIN1 levels should be carefully analyzed in future clinical trials, to determine whether these biomarkers predict clinical response and implicate pathways for targeted therapy.