B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with TCF3-PBX1 fusion 
gene expression has constitutively elevated levels of Wnt16b and ROR1 (receptor 
tyrosine kinase-like orphan receptor), a ligand and a receptor from the Wnt 
signaling pathway, respectively. Although survival rate is usually high after 
the initial chemotherapy, many TCF3-PBX1 BCP-ALL patients relapse and 
subsequently develop treatment resistance, resulting in poor prognosis. Here, we 
aimed to investigate the molecular signaling associated with Wnt16b and ROR1 
overexpression in TCF3-PBX1 cell lines and primary samples, and to identify 
effective treatment options via ROR1 targeting. We detected higher ROR1 
expression on TCF3-PBX1 leukemic cells even at a later stage of patient relapse, 
providing a strong rationale for the use of ROR1-targeted therapy. We found that 
Wnt5a-ROR1 signaling enhances proliferation of TCF3-PBX1 cells via RhoA/Rac1 
GTPases activation and STAT3 upregulation. Wnt16b also activated the RhoA/Rac1 
signaling cascade suggesting the activation of a non-canonical Wnt pathway in 
TCF3-PBX1 cells. Wnt16 could interact with ROR1 but not in TCF3-PBX1 cells, 
suggesting that Wnt5a is the ligand signaling via ROR1 in TCF3-PBX1 cells. By 
high throughput drug-sensitivity testing of TCF3-PBX1 cells before and after 
ROR1 knockdown we found that targeting ROR1 significantly improves the 
therapeutic efficacy of Bcl-2 family inhibitors venetoclax and navitoclax, and 
this synergism was confirmed ex vivo using a drug-resistant primary sample from 
a relapsed TCF3-PBX1 patient. Our work underlines a new type of targeted 
combination therapy that could be clinically advantageous for patients with 
TCF3-PBX1 BCP-ALL.