ROR1 Expression Is Associated with Oncogenic Dedifferentiation in Chronic Lymphocytic Leukemia
Authors: Ghia Emanuela M. , Choi Michael Y. , Widhopf II George F. , Rassenti Laura Z. , Kipps Thomas J. 
Published: October 2019

Abstract
An integrated analysis of transcriptomic signatures applied to almost 12,000 primary human tumors of 33 different cancer types from The Cancer Genome Atlas (TCGA) datasets defined a signature to quantify various degrees of stemness and assigned a stemness index to each tumor included in the analysis. Stemness indices were lowest in normal cells, increased in primary tumors, and were highest in metastatic disease, consistent with the notion that tumor progression generally involves oncogenic dedifferentiation. Higher values for stemness indices were associated with biological processes active in cancer stem cells, which had the highest levels of oncogenic dedifferentiation (Malta TM, et al, Cell 173:338, 2018). We examined whether such oncogenic dedifferentiation stemness indices could distinguish different prognostic subgroups of patients with chronic lymphocytic leukemia (CLL). For this, we performed gene set enrichment analysis (GSEA) on array-based transcriptomes (GSE49896 in Mraz M, et al, Blood 124(1):84, 2014) of 100 CLL samples, that expressed unmutated IGHV (U-IGHV) (N=44) or mutated IGHV (M-IGHV) (N=56). We found that the oncogenic dedifferentiation stemness signature was enriched in CLL cells with U-IGHV relative to that of cases with M-IGHV (FDR q = 0.03). Included in the oncogenic dedifferentiation stemness signature is ROR1, which encodes an oncoembryonic orphan receptor for Wnt5a that can contribute to tumor-cell survival, proclivity to relapse, and cancer metastases (Zhang S, et al, PNAS 111:17266, 2014 and Cui B, et al, Cancer Res 73(12):3649, 2013). Moreover, ROR1 may contribute to disease progression in CLL (Cui B, et al, Blood 128(25):2931, 2016). Hence, we interrogated whether ROR1 expression is associated with the oncogenic dedifferentiation stemness signature. We performed GSEA on the transcriptomes of: 98 CLL cases (Ferreira PG, et al, Genome Research 24:212, 2014) and 448 CLL cases available through GEO database (GSE13204) in Kohlmann A, et al, BJH 142:802, 2008, segregated based on ROR1 expression in ROR1Hi or ROR1Lo. We also performed GSEA on the transcriptome of: 90 CLL cases in GSE13204 dataset segregated into two groups representing the 10% of patients who had CLL cells with highest levels of ROR1 mRNA (ROR1>90%), and the 10% of patients who had CLL cells with lowest levels of ROR1 mRNA (ROR1<10%), 24 CLL cases with leukemic cells either expressing ROR1 (ROR1pos) or with low to negligible ROR1 (ROR1neg)CLL (Cui B, et al, Blood 128(25):2931, 2016), and isolated CD5+B220low splenic leukemia cells that developed in ROR1xTCL1 transgenic (Tg) mice or TCL1 Tg mice (Widhopf II GF, et al, PNAS 111(2):793, 2014). We found that the oncogenic dedifferentiation stemness signature was significantly enriched in ROR1HiCLL in the cohort of 98 CLL cases and in the cohort of 448 CLL cases (FDR q values of 0.02 and <0.001, respectively). In addition, the oncogenic dedifferentiation stemness signature was significantly enriched in ROR1>90% relative to ROR1<10% CLL, ROR1pos relative to ROR1neg CLL, and in the ROR1+ leukemia of ROR1xTCL1 Tg mice relative to ROR1Neg leukemia that develops in TCL1 mice (FDR q values of 0.01, 0.01 and <0.001, respectively). To explore whether ROR1 signaling is involved in inducing oncogenic dedifferentiation, we compared the transcriptomes of matched sets of CLL cells obtained from patients before and after treatment with cirmtuzumab, a humanized monoclonal antibody (mAb) that can inhibit ROR1-signaling (Choi MY, et al, Cell Stem Cell 22:951, 2018). GSEA showed that, compared with pre-treatment CLL cells, the post-treatment leukemia cells had a highly significant reversal of the oncogenic dedifferentiation stemness signatures (FDR q < 0.001). Collectively these findings indicate that oncogenic dedifferentiation stemness signature may be enriched in subgroups of CLL patients that have more aggressive disease and enhanced by ROR1 signaling. Moreover, treatment with agents such as cirmtuzumab that can inhibit ROR1-signaling may reverse the expression of genes associated with stemness and oncogenic dedifferentiation in patients with CLL.

Disclosures
Choi: Pharmacyclics: Consultancy, Research Funding, Speakers Bureau; Genentech: Speakers Bureau; Rigel: Consultancy; Gilead: Speakers Bureau; AbbVie, Inc: Consultancy, Speakers Bureau.