Abstract 3164: Detection and CAR T-cell directed targeting of inflammation-responsive Wnt pathway receptors on multiple myeloma cancer stem-like cells
Authors: Avsharian Lara C. , Oh Christopher S. , Vora Ashni A. , Fernandez Jayde , Shin Caitlin , Sinha Navyaa , Prussak Charles , Crews Leslie 
Published: June 2022

Abstract
Multiple myeloma (MM) is the second most common hematological malignancy characterized by a malignant expansion of abnormal antibody-producing cells. Despite available treatments, relapsed disease is a common occurrence with patients receiving survival predictions of less than one year. Relapsed MM is often refractory to multiple lines of therapy and occurs due to the uncontrolled regeneration of a population of malignant cells displaying progenitor-cell-like phenotypes. Selectively targeting this cancer stem cell (CSC) population provides an effective way to limit incidence of relapsed disease, improving the prognosis for high-risk patients. In MM, the defining characteristics of CSCs remain poorly understood, though signaling pathways promoting ‘stemness’ that are often deregulated in CSCs, such as interleukin-6 (IL-6) signaling, as well as canonical and non-canonical Wnt signaling are also aberrantly activated in MM. In this context, the central objective of this project was to explore the functionality of two proteins which have been shown to be downstream targets of IL-6/JAK2/STAT3 signaling and potentiate Wnt signaling, the embryonic G-protein coupled receptor LGR4 and the fetal-oncogene tyrosine kinase like orphan receptor-1 (ROR1), as CSC biomarker candidates, and explore the feasibility of CSC targeting therapeutics for MM. Receptor expression was assessed by quantitative PCR and flow cytometry in primary plasma cell dyscrasia patient samples across the spectrum of disease, as well as in human myeloma cell lines (HMCLs). Whole transcriptome sequencing analyses were carried out in HMCLs induced to overexpress LGR4, which enriched genes associated with ion transport and embryonic development. Cell adhesion and extracellular matrix genes were downregulated, along with genes associated with metabolic and mitochondrial pathways. Results of ROR1 gene expression and flow cytometry studies suggest that a subset of MM patient samples harbor a low-frequency ROR1-positive cell population, and expression is enriched in an in vitro model of acquired drug resistance. Overexpression of interferon regulatory factor-4 (IRF4) - a myeloma cell-enriched transcription factor that we recently showed promotes myeloma regeneration - also contributed to increased human ROR1 protein levels. HMCLs with high endogenous ROR1 expression were sensitive to in vitro killing by anti-ROR1 CAR T-cell treatment, which employs an scFv generated from the fully humanized ROR1-specific monoclonal antibody cirmtuzumab, at effector:target cell ratios (ET) as low as 0.33:1. Together, these results suggest a central role for Wnt signaling in MM stem cell phenotypes and activation of embryonic transcriptional pathways, while also paving the way towards development of clinical therapies that target a CSC population in MM.
Citation Format: Lara C. Avsharian, Christopher S. Oh, Ashni A. Vora, Jayde Fernandez, Caitlin Shin, Navyaa Sinha, Charles Prussak, Leslie Crews. Detection and CAR T-cell directed targeting of inflammation-responsive Wnt pathway receptors on multiple myeloma cancer stem-like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3164.