Methods Transduction: SMOC-2 knockdown cell lines were prepared by transduction with lentivirus (Figure 1) encoding SMOC-2 specific shRNA. To garner virus, low-passage HEK293T cells were transfected with pLKO.1 plasmids encoding 5 individual SMOC-2 shRNAs. Cells were then transduced with this viral supernatant supplemented with polybrene. The virus containing media was then removed after one hour and the cells were resuspended in RPMI 10% FBS media. After 2 days, the cells incubated with GFP viral supernatant were checked under a fluorescence microscope to ascertain if transduction was successful (Figure 6).

The drug rituximab is frequently used to treat lymphoma due to its detrimental effects on malignant B cells1. It binds specifically to the CD20 antigen present on the surface of normal and malignant B cells and induces lysis through several possible mechanisms: apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and inhibition. In CDC, the C1q binds the antibody and this binding triggers the complement cascade, which leads to the formation of the membrane attack complex (MAC) resulting in subsequent cell lysis. We have used public information on genotype and gene expression data to analyze associations between loci/genes and drug response2. Our multitier analysis strategy indicated SMOC-2 as a gene of interest. This gene encodes a SPARC-like matricellular protein that promotes matrix assembly. Expression array technology identified SPARC as a marker of poor prognosis, very often associated with most aggressive tumors in the vast majority of human cancer types3. Even though there is a significant amount of evidence that SPARC can play in tumor growth, not many experiments have been performed that explore SMOC-2’s role in cancer growth.

Results

Discussion

References

•  In this study, we used several genetic approaches including association, linkage and gene expression to observe important genetic factors in monoclonal antibody sensitivity and discovered a role for rituximab drug sensitivity mediated by SMOC-2.

•  A new, more specific antibody will be used to further assess the knockdown efficiency of the SMOC-2 shRNAs.

•  We have shown that SMOC-2 expression seems to affect complement-dependent cytotoxicity induced by monoclonal antibody treatment, but actual expression levels must be verified.

•  The full mechanism through which SMOC-2 expression leads to altered anti-CD20 antibody susceptibility will require additional studies.

•  Since SMOC-2 is a component of complement-dependent cell death by anti-CD20 monoclonal antibodies, exploring additional newly developed anti-CD20 antibodies may further test the hypotheses generated in this study.

•  Understanding the role of SMOC-2 in monoclonal antibody treatment could potentially be used in determining more logical and cost-efficient treatments to combat lymphoma.

1.  Weiner, G. J. 2010. Rituximab: mechanism of action. Seminars in hematology. Vol. 47. No. 2. WB Saunders.

2.  Jack, J. et al. 2016. Gene Expression and Linkage Analysis Implicates CBLB as a Mediator of Rituximab Resistance. In review.

3.  Benedetti, L. G. et al. 2012. Role of the matricellular protein SPARC in breast tumor growth and metastatic dissemination. Cancer Research. 72.8 Supplement:1417-1417.

Resistance to Rituximab Conferred by SMOC-2 Suppression in DLBCLs Dhanesh Binda2, Sadia Salahud Din2,3, Kristy Richards2,3

2Department of Biomedical Sciences, Cornell University, Ithaca, NY, USA; 3Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

Acknowledgments This work was supported by a Mentored Research Scholar Grant in Applied and Clinical Research (MSRG-12-086-01-TBG) from the American Cancer Society, and summer research was supported by the Pre Professional Program and the Office of Academic Diversity Initiatives of Cornell University.

Figure 1. The mechanism of shRNA

Figure 2. The mechanism of CDC Figure 3. CDC Assay after 24 hours

Figure 4. Manhattan Plot for Rituximab, Ofatumumab, and Multivariate GWAS. The negative log transform of p-values for three GWAS are given for 2.1 million SNPs. Nominal p-values are provided for all cases except any significant associations (-log10(p) > 6; blue threshold line above). For all significant associations, the p-values are adjusted for multiple comparisons via permutation testing.

Table 1. Summary of Peak P-values Found in Rituximab and Ofatumumab GWAS. Chromosome location, ID, genotype distribution, and nearby genes are given for each SNP. Nominal p-values are given along with p-values adjusted for multiple comparisons via permutation testing. Peak associations are marked “Yes” for significance if the negative log transform of the adjusted p-value is greater than 6.

Abstract: Antibody therapy involves administering antibodies that have been specifically made to target an antigen on a cancer cell and either directly kill the cell or signal other immune cells to destroy it. Because rituximab is one of the top selling monoclonal antibodies that provide substantial benefit to patients with B-cell malignancies, it is important to understand any potential mechanism of resistance. Since these resistance mechanisms are difficult to study in non-human models, we used in vitro studies linking genotype to phenotype via Genome-Wide Association study (GWAS) and identified a Single Nucleotide Polymorphism (SNP) near SMOC-2, an extracellular matrix protein, that might be involved in rituximab sensitivity. In order to observe if the expression of SMOC-2 affects rituximab sensitivity, malignant B cells with a SMOC-2 knockdown were tested in an in vitro Complement Dependent Cytotoxicity (CDC) assay. Gene knockdown was performed using shRNAs via RNA interference introduced with lentiviral transduction. After performing CDC assays on several Diffuse Large B-Cell Lymphoma (DLBCL) SMOC-2 knockdown cell lines, the HBL-1 cell line exhibited the most significant change in sensitivity to rituximab, i.e., knockdowns were resistant. However, Western Blot analysis yielded inconclusive evidence about the knockdown of the SMOC-2 gene, perhaps due to a poorly specific antibody. A new antibody will be used to further assess the knockdown efficiency of the SMOC-2 shRNAs. Nevertheless, this study demonstrates the utility of genome-wide mapping to discover novel biological mechanisms of potential clinical advantage. Understanding the mechanisms of intrinsic and acquired resistance to drugs in cancer therapy may enable the use of patient genotypes to determine which anti-CD20 antibody (rituximab, ofatumumab, or obinutuzumab) would be most effective or allow interventions to restore rituximab sensitivity in patients.

Introduction

CDC Assay: CDC Assays (Figures 2 and 3) were then used to test whether the SMOC-2 knockdowns exhibited altered sensitivity to rituximab. HBL-1 SMOC-2 knockdown cells were plated at 1 × 104 cells in a 96-well plate in a total volume of 100µl media containing 25% pooled human serum with or without 10 µg/ml rituximab. Proliferation was measured using Alamar Blue and was expressed relative to samples without antibody.

Results

Figure 5. GFP Expression of HBL-1 Cells indicating successful transduction.

Figure 6. CDC Assay results for HBL-1 SMOC-2 Knockdowns. Data suggests that cells are resistant to rituximab when the SMOC-2 gene is suppressed.

Figure 7. Western Blot for HBL-1 SMOC-2 Knockdowns – Resistant Phenotype. Western Blot with lysates of cells with rituximab resistant phenotype. There is no noticeable and difference between the HBL-1 and knockdown (KD) bands, possibly due to a poorly specific antibody.

0

10

20

30

40

50

60

70

80

90

CELLS PLKO 43

cells

+ se

rum

+ r

tx /

cells

+ se

rum

HBL-1 (Post-Selection)

24 hr

Western Blot: Western blotting was performed using SMOC-2 (H-53), a rabbit polyclonal antibody, and anti-b-actin was used as a load control. Anti-mouse IgG peroxidase secondary antibodies were used for beta actin and Anti-rabbit IgG HRP linked H+L secondary antibodies were used for SMOC-2. ECL Prime detection reagent was used to visualize immunoreactive bands.

___________________

SMOC-2 Predicted Size – 54kD

Beta – Actin Predicted Size – 42kD