ROR1 Can Interact With TCL1 and Enhance Leukemogenesis In Eµ-TCL1 Transgenic Mice
Authors: Widhopf George F. , Cui Bing , Ghia Emanuela M. , Chen Liguang , Messer Karen , Shen Zhouxin , Briggs Steven P. , Croce Carlo M. , Kipps Thomas J. 
Published: October 2019

Abstract
ROR1 is an onco-embryonic antigen found on chronic lymphocytic leukemia (CLL) B cells, but not on normal adult tissues. We generated B6 transgenic (Tg) mice with human ROR1 regulated by the murine Ig promoter/enhancer (ROR1 Tg). Such animals had B-cell-restricted expression of ROR1 and infrequently developed ROR1+/CD5+/B220low leukemia resembling human CLL at ≥15 months of age. The leukemia cells of these animals had phenotypic features in common with those of the leukemia that originates in B6 Eµ-TCL1-Tg (TCL1) mice, which develop a CLL-like disease at ≥ 9 months of age. However, in contrast to human CLL, the leukemia that develops in TCL1 Tg mice does not express ROR1, indicating that expression of this antigen is not necessary for leukemogenesis. However, in immune-precipitation and mass spectrometry studies we found that ROR1 could complex with TCL1 in human CLL cells. TCL1 is a proto-oncogene product that can serve as a co-activator of AKT. To investigate whether expression of ROR1 could affect the biology of the leukemia that develops in TCL1 Tg mice, we crossed our ROR1 Tg animals with TCL1-Tg mice. Progeny with both transgenes (ROR1xTCL1) developed CD5+/B220low B-cell leukemia at a significantly younger median age than did littermates with either transgene alone. ROR1xTCL1 leukemia B-cells had higher levels of pAKT than TCL1 leukemia-cells and expressed high-levels of human ROR1, which we also found could complex with TCL1. Exploratory subnetwork analyses of transcriptome microarray data on isolated leukemia cells using Ingenuity Pathway network tools revealed 51 subnetworks that were expressed at different levels between the two types of leukemia, 21 of which had z scores in excess of 0.8, and an associated functional annotation with a false-discovery-rate (FDR) of less than 0.05. This analysis implied that ROR1xTCL1 leukemia cells had higher expression of subnetworks implicated in embryonic and tumor-cell proliferation, but lower expression of subnetworks involved in cell-cell adhesion or cell-death, than did TCL1 leukemia-cells. ROR1xTCL1 leukemia-cells also had higher proportions of Ki-67-positive cells, lower proportions of cells undergoing spontaneous apoptosis, and produced more aggressive disease upon adoptive transfer than TCL1 leukemia-cells.
We examined the activity of two different mouse anti-human ROR1 mAbs, D10 and 4A5, which bind to distinct non-overlapping epitopes of ROR1, as assessed in cross-blocking studies. Treatment of ROR1xTCL1 leukemia cells with D10 in vitro resulted in reduced expression of pAKT within 1 hour after addition of the antibody to the leukemia cells, an effect that was not apparent in control Ig or 4A5-treated cells. We treated ROR1 Tg mice engrafted with CD5+/B220low/ROR1+ leukemia cells with intravenous injections of D10, 4A5, or control mouse IgG (mIgG), at 10 mg/kg. At five weeks, mice given D10 in one representative experiment had significantly fewer CD5+/B220low leukemia cells (2.4 ± 1.0 x106, n=3) in the blood than mice that received mIgG (2.0 ± 0.3 x107, n=3, p=0.032), whereas the number of leukemia cells in the blood of mice given 4A5 (1.6 ± 0.3 x107, n=3, p>0.05) was not significantly different than that of mice treated with mIgG. Furthermore, mice that received CD5+/B220low/ROR1+ B cells and that were treated with D10 had significantly smaller spleens than mice that were treated with mIgG or 4A5. Although D10 was effective in inhibiting the engraftment of ROR1xTCL1 leukemia cells, administration of either anti-ROR1 mAb had no effect on the endogenous non-leukemia (CD5-/B220Hi/ROR1+) B cells or T cells, or on engraftment of leukemia cells from TCL1 Tg mice, which do not express ROR1. Our data demonstrate that ROR1 accelerates progression of TCL1 leukemia and may be a target for therapy of patients with CLL.

Disclosures:
No relevant conflicts of interest to declare.