ROR1-Signaling Increases Levels of Mutant TP53 Protein and Cyclin A1 Via Activation of NRF2, Potentially Potentiating Genomic Instability in Chronic Lymphocytic Leukemia Cells with Del(17p)
Authors: Ghia Emanuela M. , Choi Michael Y. , Widhopf II George F. , Kipps Thomas J. 
Published: December 2024

ROR1 is a developmentally-restricted receptor for Wnt5a, which can induce ROR1-signaling leading to expression of target genes induced by activation of ERK1/2, NF-κB, or NRF2 in leukemia cells of patients (pts) with chronic lymphocytic leukemia (CLL). Prior work found that expression levels of ROR1, and expression levels of target genes induced by ROR1-signaling, vary between pts; furthermore, pts with CLL cells with high-level ROR1 (ROR1Hi) were found to have a significantly shorter median time from diagnosis until initial therapy and overall survival than pts with CLL cells with low-levels of ROR1 (ROR1Lo) (Blood 128:2931, 2016). Therapy of pts with CLL with an anti-ROR1 antibody (zilovertamab), which blocks ROR1-signaling, inhibited expression of such target genes and reversed cancer stemness in CLL cells of pts treated in a phase I study (Cell Stem Cell 22:951, 2018). We find 64 genes involved in cell-cycle control also were enriched in ROR1Hi versus ROR1Lo CLL cells (FDR q value of 0.001) or in pre-treatment CLL cells versus CLL cells of the same pts treated with zilovertamab (FDR q value of 0.02). Most pronounced was CCNA1, encoding cyclin A1, which is involved in regulating G1/S and G2/M phase transitions and DNA repair of double-strand breaks. CCNA1 transcriptionally is activated by TP53, and possibly also by TP53 mutants that typically are found in CLL with del(17p) (Mol Cell Biol 24:8917, 2004; Nucleic Acids Res 36:5362, 2008, PathwayNet database). Such mutant forms of TP53 are susceptible to chaperone-mediated degradation unless bound by proteins, like NQO1, that are induced by activation of NRF2, which induces expression of proteins that can protect cells from genotoxic stress. To examine the relationship between NQO1, Cyclin A1, mutant TP53, and ROR1-signaling in leukemia cells with del(17p) with mutant TP53 (Q317fs*29), we used a CLL-cell line, MEC1, which lacks expression of ROR1 but makes high levels of Wnt5a. We also used MEC1Wnt5a-/- cells, in which we knocked out WNT5A via CRISPR-Cas9. MEC1 cells or MEC1Wnt5a-/- cells were transduced with a lentivirus vector encoding ROR1 to generate MEC1-ROR1 cells or MEC1-ROR1Wnt5a-/- cells. MEC1-ROR1 cells had increased expression of NRF2 target genes and cell-cycle control genes compared to MEC1, or MEC1-ROR1Wnt5a-/- cells (FDR q-values of 0.03 and &lt; 0.001, respectively). MEC1-ROR1 cells also had significantly higher levels of NQO1, mutant TP53 protein, and cyclin A1 (p values of 0.005 and 0.001 for NQO1, p values of 0.001 and 0.004 for p53, and p values of 0.003 and 0.001 for cyclin A). Silencing NQO1 with siRNA in MEC1-ROR1 cells significantly reduced expression of NQO1, mutant TP53, and cyclin A1 (p values of 0.02, 0.01 and 0.03, respectively). Similarly, culture of ROR1+ del(17p) CLL cells with Wnt5a increased expression of NQO1, Cyclin A1, and mutant TP53; this effect of Wnt5a could be blocked by concomitant treatment with zilovertamab (p values of 0.01, 0.02 and 0.02, respectively). Because of these findings, we re-interrogated the database on 493 untreated pts with CLL cells characterized for ROR1 protein expression, FISH, and metaphase cytogenetics. We found that cases with del(17p), involving ≥10% of interphase nuclei, or with complex karyotype, were significantly more likely to have ROR1Hi CLL cells than ROR1Lo CLL cells. (Χ2 p values of &lt;0.01 for both), suggesting ROR1-signaling may mitigate the genotoxic stress caused by mutant TP53, thereby enhancing survival of (del17p) CLL and potentially allowing for emergence of mutant subclones with acquired resistance to targeted therapy, e.g. BTK inhibitors (BTKi's). Conversely, concomitant treatment with agents such as zilovertamab that inhibit ROR1-signaling may mitigate the risk of developing drug-resistance to BTKi's or other targeted therapies in pts with del(17p) CLL.