Evaluating ROR1 As a Target in Childhood Leukemia
Authors: Diamanti Paraskevi , Bailey Bethan Kathleen , Iheanacho Obinna Ebere , Nathwani Amit C. , Blair Allison 
Published: November 2019

Disease relapse in childhood acute lymphoblastic leukemia (ALL) patients may occur due to persistence of resistant cells with leukemia propagating ability (LPC). Immunotherapeutic approaches, such as bi-specific T cell engagers (BiTEs) and chimeric antigen receptor (CAR) T cells against CD19, have been shown to successfully treat relapsed ALL. However, such therapies cannot target LPC that lack or have lost expression of CD19. Receptor tyrosine kinase like orphan 1 receptor (ROR1) is a surface antigen, overexpressed in hematological malignancies, including chronic lymphocytic leukemia (CLL) and solid tumors, with limited expression in normal tissues. ROR1 is found in cancer initiating cells and high levels correlated with metastases and poor outcomes in ovarian and breast cancers, making it a promising target for immunotherapy. Preclinical studies using a ROR1-BiTE have shown efficacy against a range of solid cancers and CLL. The aims of this study were to investigate i) ROR1 expression in B cell precursor (BCP)-ALL, T-ALL and respective LPC; ii) the proliferative ability of ROR1+ cells and iii) the effects of induction therapy on ROR1+ cells. Bone marrow (BM) cells from 16 BCP-ALL and 10 T-ALL samples were stained with antibodies against CD19 or CD7, respectively, in addition to ROR1 and CD34. The proportion of ROR1+ cells and the number of antigen binding sites were determined by flow cytometry. These measurements were also assessed in CD34+/CD19+, CD34+/CD19-, CD34-/CD19+, CD34-/CD19- BCP-ALL LPC, CD34+/CD7+, CD34+/CD7-, CD34-/CD7+, CD34-/CD7- T-ALL LPC and compared with normal BM (NBM) cells. ROR1 expression varied amongst the samples studied with higher levels observed in T-ALL cases. The median expression of ROR1 was 8.2% (range 0.4-83%) in T-ALL cases, significantly higher than in BCP-ALL (median 2.4, range 0.01-85%, P=0.03). The number of ROR1 binding sites was also significantly higher in T-ALL cases (median 233, range 93-1238) compared to BCP-ALL (98, range 2-1540) and NBM cells (55, range 30-223, P=0.01). There was no difference in ROR1 expression between low or risk MRD groups in BCP- and T-ALL cases. In LPC subpopulations, ROR1 was only detected in subpopulations expressing the respective lineage markers. Again, higher numbers of binding sites were detected in LPC from T-ALL cases, CD34+/CD7+ (median 130, range 0-948) and CD34-/CD7+ (112, range 0-1148). To investigate proliferative capacity, ALL cells were incubated with cell trace violet (CTV) and monitored in culture for up to 7 days. ROR1- T-ALL cells had lower CTV median fluorescence intensity (MFI), indicating more divisions, decreasing 19 fold by day 7 compared to ROR1+ cells (4 fold decrease). In LPC subpopulations, greatest proliferation was seen in CD34+/CD19-/ROR1- and CD34+/CD19+/ROR1- cells in BCP-ALL with 269 and 177 fold decreases in the CTV MFI, respectively. In T-ALL cases, most cell divisions were observed in CD34+/CD7-/ROR1- cells (100 fold decrease) followed by CD34-/CD7-/ROR1- (49 fold). Proliferative activity was more modest in the ROR1+ subpopulations with a decrease in the CTV MFI ranging from 28 to 4 fold. To determine whether induction therapy had an impact on ROR1+ cells, samples at day 29 and/or week 14 post treatment were investigated in 3 BCP- and 6 T-ALL cases and compared to the immunophenotype at diagnosis. In BCP-ALL, ROR1+ cells were undetectable at day 29 and week 14. In contrast, in 5/6 T-ALL cases ROR1 levels were higher following treatment. In 3 cases, ROR1 expression increased in CD34-/CD7+ LPC at day 29. While in the other 2 cases emergence of ROR1+ populations were observed in CD34+/CD7+ and CD34-/CD7+ cells at week 14, that were not present at diagnosis. In summary, ROR1 was more prevalent in childhood T-ALL and was co-expressed with CD7 in T-ALL and CD19 in BCP-ALL. ROR1- ALL cells had higher short-term proliferative capacity than ROR1+ cells, including known refractory CD34+/CD19-, CD34+/CD7- and CD34-/CD7- LPC. Further long-term functional studies in vitro and in vivo to investigate the role of ROR1 in ALL are ongoing. However, these data together with persistence of ROR1+ T-ALL cells after treatment, indicate that cell cycle specific agents may be ineffective on these cells. Therefore, ROR1 targeted therapy may be beneficial for T-ALL patients.

Disclosures
Nathwani: Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.