ROR1 Protein Expression Is Regulated By UHRF1 in t(1;19) Pre B-Cell ALL
Authors: Chow Marilynn , MacManiman Jason D. , Bicocca Vincent T. , Gao Lina , Alumkal Joshi A. , Chang Bill H. , Tyner Jeffrey W. 
Published: October 2019

Abstract
Introduction: Acute lymphoblastic leukemia is the most common form of pediatric cancer, and a translocation between chromosomes 1 and 19 represents one of the most frequent cytogenetic abnormalities, accounting for ~5% of ALL cases. The 1;19 rearrangement results in a fusion between the E2A and PBX1 genes, and pre B-ALL cells carrying the E2A-PBX1 fusion are consistently arrested at an intermediate stage of B cell lineage development in which the cells express a functional pre B-cell receptor (pre-BCR). We have previously shown that receptor orphan tyrosine kinase receptor 1 (ROR1) is highly expressed on t(1;19) pre B-ALL blasts and required for their viability, even though ROR1 is not directly regulated by either the E2A-PBX1 fusion or the pre-BCR. Moreover, ROR1 exhibits compensatory signaling cross-talk with the pre-BCR such that maximal killing of t(1;19) pre B-ALL cells is achieved by combining silencing of ROR1 with siRNA and inhibition of the pre-BCR with kinase inhibitors such as dasatinib. However, clinical employment of this strategy is hampered as ROR1 is a pseudokinase whose biology is poorly understood and no ROR1 small-molecule inhibitors currently exist. Therefore, it is critical to advance our understanding of the upstream regulatory mechanisms responsible for ROR1 expression to uncover new therapeutic strategies for t(1;19) pre B-cell ALL.
Methods: To identify potential regulators of ROR1 expression, we designed two siRNA panels targeting transcription factors predicted to bind the ROR1 locus or an array of chromatin remodeling factors and epigenetic regulators. Pre B-ALL cell lines that are positive or negative for the 1;19 rearrangement were screened with these siRNA panels and any gene targets required for the viability of t(1;19) pre B-cell ALL cells but not control cells lines were considered candidate regulators of ROR1 expression. Candidates were validated for the capacity to regulate ROR1 expression by qRT-PCR and immunoblot.
Results: Screening of the t(1;19) pre B-cell ALL cell line, RCH-ACV, with our siRNA panels targeting transcription factors and chromatin remodeling/epigenetic regulators identified four potential regulators of ROR1 that were required for viability of RCH-ACV cells but not our negative control cell line, REH (t(12;21)-positive). Among these four candidates was UHRF1, which interacts with histone and DNA methyltransferases. Interestingly, silencing of UHRF1 by siRNA led to reduced ROR1 protein levels, but had no significant impact on ROR1 mRNA expression. Since one primary function of UHRF1 is to regulate DNA methylation, we hypothesized that UHRF1 regulates ROR1 protein expression through an intermediate gene whose transcription is impacted by UHRF1-mediated DNA methylation. To test this hypothesis, RCH-ACV cells were treated with the RNA synthesis inhibitor actinomycin D 48 hours after silencing UHRF1. This resulted in a transient rescue of ROR1 protein levels, indicating that UHRF1 regulation of ROR1 protein depends on the transcription of an intermediate gene whose gene product subsequently regulates ROR1 protein levels.
Conclusions: Data from siRNA screens and subsequent validation experiments suggest that UHRF1 functions to regulate ROR1 protein levels, but not mRNA expression. This is intriguing due to the primary role of UHRF1 as a modulator of DNA and histone methylation. The observed rescue of ROR1 protein with actinomycin D suggests that UHRF1 regulation of ROR1 protein occurs through an undefined intermediary whose locus is methylated in a UHRF1-dependent manner and whose gene product modulates ROR1 protein levels. Inhibitors of additional stages of gene transcription, translation, and stabilization are currently being tested to continue characterizing UHRF1-dependent regulation of ROR1. Cumulatively, this work has identified a novel regulatory mechanism of ROR1 protein expression by UHRF1 in t(1;19) pre B-cell ALL. In addition to advancing our understanding of ROR1 biology, this regulatory mechanism suggests a new therapeutic strategy to target ROR1-expressing cancers.

Disclosures
Tyner: Incyte: Research Funding; Aptose Biosciences: Research Funding; Janssen Pharmaceuticals: Research Funding; Constellation Pharmaceuticals: Research Funding; Array Biopharma: Research Funding.