SHP1 Loss Augments DLBCL Cellular Response to Ibrutinib: A Candidate Predictive Biomarker

Background

B-cell receptor (BCR) signaling plays a key role in B-cell non-Hodgkin lymphoma (NHL). Protein tyrosine kinases such as LYN, SYK and BTK are essential in the activation of BCR leading to B-cell survival, proliferation and differentiation. SHP1 is a cytosolic protein tyrosine phosphatase functioning as a negative regulator of the BCR signaling cascade. It dephosphorylates and counteracts the activities of the tyrosine kinases, including CD79A/CD79B, SYK and BLNK (Curr. Opin. Immunol.12, 307).

Diffuse large B-cell lymphoma (DLBCL) is the most common type of aggressive NHL. DLBCL demonstrates heterogeneity in clinical presentation, genomic alterations and cell signaling. BCR signature has been identified in a subset of DLBCL (Monti Blood 105, 1851). Chronic active BCR plays a particular role in the subtype of activated B-cell like (ABC) DLBCL by cell-of-origin subclassification. Based on the success of ibrutinib in other B-cell NHL, clinical trials of ibrutinib in DLBCL was conducted and the initial results showed a 37% response rate in ABC-lymphomas vs 5% in GCB-DLBCL (Wilson Nat Med 21, 922). However, a large phase 3 clinical trial of ibrutinib-containing regimen in >800 non-GCB patients did not find any substantial difference in response between those received ibrutinib vs placebo. Thus, biomarker based on cell-of-origin did not seem to help stratify patients as expected (Younes JCO 27, 1285). New predictive biomarkers are needed to identify patients who likely benefit from ibrutinib or other BCR-directed therapeutic agents.

We hypothesized that SHP1 may play a role in defining the level of the activity of the BCR pathway. Suppressed SHP1 expression in various types of NHL has been reported. We hypothesized that lack of SHP1 may contribute to the hyperactivity of BCR in DLBCL; and tumors with low SHP1 may respond better to ibrutinib. In this work, we set out to explore the relationship between SHP1 & ibrutinib sensitivity in DLBCL.

Methods and Results

We first reviewed the published literature on SHP1 in NHL using a meta-analysis to provide a quantitative and thus unbiased analysis. Seventeen articles were selected based on criteria including inclusion of primary tumors with normal benign controls. The analysis of the SHP1 protein expression by immunohistochemistry (IHC) revealed that lack of SHP1 expression is found in 178 of 242 (73.6%) NHL tissues vs. 32 of 264 (12%) normal/benign controls. The odds ratio derived by the meta-analysis was 119.58 with a 95% confidence interval of 37.60-380.15, suggesting loss of SHP1 is strongly associated with NHL including DLBCL. Likewise, the analysis of the methylation studies revealed a strong association between SHP1 promoter hypermethylation and NHL (OR 69.46, 95% CI 12.32-391.74).

To further understand the range of variability of SHP1 expression in subtypes of DLBCL tumors, we constructed a tissue microarray with 48 DLBCL tumors and 12 cell lines. By immunohistochemistry, we found a wide range of variability with regards to the expression levels and no substantial difference between the GCB and non-GCB subtypes.

Using 12 cell lines together with resting B-cells from normal donors, we identified a strong reverse linear correlation between SHP1 protein expression and promoter methylation (r = 0.85, P=0.0002) suggesting that promoter hypermethylation may be responsible for reduced SHP1 expression in DLBCL cells.

Using CRISPR, we knocked out SHP1 in both GCB and ABC cell lines. The phosphorylation of early BCR components, BTK, SYK and LYN, were increased in the SHP1 knockout clones compared to the wild type ones. The results demonstrate that SHP1 depletion results in increased proximal BCR signaling activity. Moreover, the knock out clones demonstrated increased sensitivity of ibrutinib. We further showed that re-introduction of SHP1 vector to the knockout clones via stable lentiviral transduction restored normal sensitivity of cells to ibrutinib.

Conclusions

Together, our results show that SHP1 expression is suppressed in a substantial fraction of DLBCL tumors regardless of cell-of-origin subclassifications. Using genetic approaches, we demonstrated that loss of SHP1 increases BCR signaling activity and sensitizes tumor cells to the inhibition by ibrutinib. Our results suggest that loss of SHP1 may be used as an alternative biomarker to cell-of-origin to identify patients who potentially benefit from ibrutinib treatment.