Abstract
Abstract 2560
The mammalian orphan receptor tyrosine kinase-1 (ROR1) is expressed in a wide-variety of tissues during early embryonic development. By the late stages of embryogenesis the expression of this developmentally important protein is greatly diminished. Although not expressed in the tissues of post-partum animals, the ROR1 protein is expressed on neoplastic cells in chronic lymphocytic leukemia (CLL), some B-cell malignancies, and a variety of different carcinomas. We examined for expression of ROR1 in primary acute myeloid leukemia (AML) cells harvested from marrow aspirates and their normal counterparts by whole transcriptome paired-end RNA sequencing and by flow-cytometric analyses. These studies revealed selective expression of ROR1 in 62 (35%) of 179 AML samples examined. Many of these samples were found to have cells that co-expressed ROR1 and CD34, suggesting that ROR1 was present on the self-renewing leukemia stem-cell population, which resides in the marrow niche and potentially accounts for resistance to many cytotoxic drugs used in therapy. We investigated the activity of a chimeric anti-ROR1 mAb found effective in clearing CLL cells (UC99961) on AML expansion, growth, and renewal in a leukemia-stem-cell supportive niche assay. Mouse marrow cells lines SL/SL and M2–10B4 (transfected to produce hSCF,hIL3 and hIL3, hG-CSF respectively) were mixed 1:1 after mitomycin-C treatment, and used as a SLM2 stromal monolayer. CD34+ cells were selected from ROR1-positive (n=6) or negative (n=4) AML primary samples. As a normal control, CD34+ cells from cord blood (CB) were used (CB, n=3). In some experiments CD34+ cells were transfected with a GLP-lentivirus prior to co-culture. At the initiation of the co-culture, 10–50 μg/ml of the chimeric anti-ROR-1 mAb (UC99961) or control hIgG were added to the cultures. Two weeks after co-culture initiation, both stromal attached and floating cells were collected and their survival investigated by colony forming assay in methylcellulose. The UC99961 mAb was not cytotoxic to CB or ROR1-negative AML samples. In contrast, the UC99961 mAb provided a dose-dependent inhibition of colony formation for all ROR-1-positive AML samples examined. These results demonstrate the in vitro anti-leukemic specificity of this anti-ROR1 mAb in down-regulating AML stem and progenitor cell populations, without effecting normal CD34+ stem cells. To analyze the effect of ROR1 ligation on AML stem cell populations exclusively, AML self-renewal assays (2-ry colonies) were performed. In these studies, ROR1–positive AML samples were divided based on their response to mAb treatment. Half of the samples (n=3; 50%) demonstrated statistically significant (up to 90%) dose-dependent decreases in colony formation. However, another half was non-responsive and no correlation was found between ROR1 expression on leukemia CD34+ cells and response to anti-ROR1 mAb treatment in the self-renewal assays. Again UC99961 mAb treatment did not negatively impact CD34+ cells from CB or ROR1-negative AML, confirming the specificity and selective toxicity of the mAb for ROR1+ AML stem cells. These studies reveal selective expression of ROR1 on leukemia-stem-cells of large subset of AML patients. Furthermore, this study demonstrates that an anti-ROR1 mAb (UC99961) can inhibit survival and self-renewal in LSC supportive niche assays. Targeted ROR1 inhibition may represent a vital component of therapeutic strategies aimed at eradicating therapeutically recalcitrant malignant stem cells in AML and potentially other refractory cancer-stem-cell-driven malignancies.
Disclosures:
No relevant conflicts of interest to declare.
Bortezomib suppresses self‐renewal and leukemogenesis of leukemia stem cell by NF‐ĸB‐dependent inhibition of CDK6 in MLL‐rearranged myeloid leukemia
