Abstract 1487: Gene expression and linkage analysis implicates CBLB as a mediator of rituximab resistance
Authors: Jack John , Small George , Havener Tammy , Brown Chad , McLeod Howard , Motsinger Alison , Richards Kristy L. 
Published: July 2016

Abstract
Drug resistance remains one of the largest challenges in the curative treatment of cancer. We investigated the problem of monoclonal antibody resistance in vitro in a high throughput, complement-dependent cytotoxic (CDC) assay using immortalized as well as cancerous cell lines. First, the heritability of rituximab and ofatumumab sensitivity was explored using Epstein Barr Virus-immortalized human lymphoblastoid cell lines from two independent sources: a collection of trios (95 samples, 13 trios) from the Centre d’Etude du Polymorphisme Humain and an unrelated collection (486 samples) from the Children's Hospital Oakland Research Initiative. Cell viability in the presence or absence of 10ug/ml rituximab or 10ug/ml ofatumumab treatment was quantitated using AlamarBlue. Heritability was estimated using variance component analysis using MERLIN: H2 = 33.11% (p&lt;0.001) and H2 = 31.11% (p&lt;0.001) for rituximab and ofatumumab, resp. Next, public information on genotype and gene expression was used to analyze associations between loci/genes and drug response with three methods: genome-wide association, linkage, and gene expression analysis. GWAS were performed on the 486 unrelated samples: (a) rituximab responses, (b) ofatumumab responses, and (c) the vector of both responses modeled jointly. There was one suggestive (-log10(P)&gt;6) result across all three analyses; this result for rituximab response occurred within the gene, SMOC2. Next, linkage analysis on the 95 related samples revealed one significant linkage peak on chromosome 12 for rituximab and two significant peaks for ofatumumab, on chromosome 12 and 3. Also, we identified genes whose mRNA expression level was correlated with the degree of either rituximab or ofatumumab sensitivity. Quantitative Significance Analysis of Microarrays revealed 13 genes whose expression was correlated with rituximab sensitivity and 25 genes whose expression was correlated with ofatumumab sensitivity (false discovery rate cutoff&lt;0.001%). CBLB, present on both gene lists, was the only gene that was located in either the chromosome 3 or chromosome 12 linkage peak. As such, CBLB was chosen for further functional validation. We used RNA silencing in several cell line and cancerous models to functionally validate the role of CBLB expression on rituximab resistance. Knockdown of CBLB in cell lines, both LCLs and lymphoma, led to increased resistant to both rituximab and ofatumumab. Additionally, we performed immunofluorescence in CBLB knockdown cells. CD20 localization was altered in cells with CBLB knockdown. Our current work has uncovered new mechanistic insight into the relationship between anti-CD20 antibody susceptibility, CBLB, and CD20, illustrating the power of comprehensive genetic analyses to discover a previously unknown mediator of rituxumab response. Additional studies are ongoing to further elucidate the mechanisms of CBLB-mediated resistance as well as its clinical relevance.
Citation Format: John Jack, George Small, Tammy Havener, Chad Brown, Howard McLeod, Alison Motsinger, Kristy L. Richards. Gene expression and linkage analysis implicates CBLB as a mediator of rituximab resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1487.