Abstract 5967: Blockade of Wnt5A/ROR1 signaling as cancer stem cell targeting therapy for metastatic prostate cancer
Authors: Jamieson Christina A.M. , Murtadha Jamillah , Oh Christopher S. , Muldong Michelle T. , Etemadfard Niloofar , Koutouan Evodie , Yoon Young E. , Lee Sanghee , Kwon Oh , Oh Jong J. , Kim Min S. , Willert Karl , Cacalano Nicholas A. , Jamieson Catriona H.M. , Gaasterland Terry , Mckay Rana R. , Kane Christopher J. , Kulidjian Anna A. , Prussak Charles E. 
Published: March 2024

Abstract
BACKGROUND– Wnt5A and its non-canonical Wnt receptor, ROR1, have emerged as a promising new signaling pathway target for lethal, metastatic prostate cancer. Wnt5A has emerged as a significant marker of poor prognosis in circulating tumor cells (CTCs) of metastatic castration resistant prostate cancer (mCRPC) patients. ROR1 is expressed in lethal types of mCRPC, especially neuroendocrine prostate cancer (NEPC). A therapeutic ROR1 antibody, Zilovertamab, has been clinically tested in chronic lymphocytic leukemia (CLL) and metastatic breast cancer and shown to be safe. We sought to investigate Zilovertamab-based therapies for metastatic prostate cancer in pre-clinical model systems.
HYPOTHESIS – Wnt5A/ROR1 signaling may mediate a cancer stem cell program which makes cells resistant to therapies which target the cell cycle and proliferation. Blocking ROR1 may reveal vulnerabilities which sensitize cancer cells to chemotherapies like docetaxel.
METHODS– ROR1 expression was determined using RNASeq, qRT-PCR, FACS, IHC and Western blotting. ROR1 signaling was blocked using the anti-ROR1 therapeutic antibody, Zilovertamab, or CRISPRCas9 ROR1 knock out. Cell growth was measured using an Incucyte real time imaging system. Stem cell activity was assessed in 3D organoids. Cell cycle analysis was performed in live cells in 2D cultures and 3D organoids using the Fucci2BL bicistronic Fluorescent, Ubiquitination-based Cell Cycle Indicator reporter system. PDX PCSD13 tumor growth was measured via calipers and IVIS. RNASeq was performed on tumors.
RESULTS– We showed that ROR1 was expressed at high levels in mCRPC cell lines: PC3, DU145, and in the bone metastatic prostate cancer PDX: PCSD13. CRISPR/Cas9 Knock out of ROR1 in PC3 and DU145 cells showed increased sensitivity to docetaxel inhibition of proliferation in vitro in 2D real time Incucyte proliferation assays and in 3D organoids. Organoid formation was significantly reduced in ROR1 KO cells. Cells expressing the FUCCI live cell cycle tracker showed docetaxel led to G2 arrest and ROR1 signaling inhibition increased efficacy of docetaxel induced cell cycle arrest. Treatment of PCSD13 PDX in vivo with Zilovertamab plus docetaxel synergistically increased tumor growth inhibition in vivo and modulated cancer stem cell and cell cycle expression profiles.
CONCLUSIONS– Cancer stem cells represent the fundamental precursors from which all diverse tumor subpopulations evolve. Thus, therapeutic targeting of these tumor initiating stem/progenitor cells may prevent the evolutionary diversification of a tumor and overcome a critical clinical barrier to cancer treatment. We showed synergistic response in our PDX and PCs cell line models to Zilovertamab plus docetaxel. We are now conducting a phase 1b clinical trial with zilovertamab plus docetaxel in patients with metastatic CRPC (CirmD, NCT05156905, PI R Mckay).
Citation Format: Christina A.M. Jamieson, Jamillah Murtadha, Christopher S. Oh, Michelle T. Muldong, Niloofar Etemadfard, Evodie Koutouan, Young E. Yoon, Sanghee Lee, Oh Kwon, Jong J. Oh, Min S. Kim, Karl Willert, Nicholas A. Cacalano, Catriona H.M. Jamieson, Terry Gaasterland, Rana R. Mckay, Christopher J. Kane, Anna A. Kulidjian, Charles E. Prussak. Blockade of Wnt5A/ROR1 signaling as cancer stem cell targeting therapy for metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5967.