BCL-Xl Expression Is Druggable Biomarker Associated with a Poor Clinical Outcome in Relapsed/Refractory Diffuse Large B-Cell Lymphoma (DLBCL)

Background: Although first-line chemo-immunotherapy cures a significant number of diffuse large B-cell lymphoma (DLBCL) patients, long term survival is only observed in 30% or 41% of patients treated with second-line therapy followed with high dose chemotherapy and autologous stem cell support (HDC-ASCS) or chimeric antigen receptor anti-CD19 T-cell therapy (CART-19) respectively. The use of third-line therapy with chemotherapy drugs or current available small molecule inhibitors for relapsed/refractory (rel/ref) diffuse large B-cell lymphoma (DLBCL) is associated with a poor response rate and a median survival of 6.3 months, stressing the need to identify druggable biomarkers associated with therapy resistance and poor clinical outcomes. Methods: To this end, we identified patients with rel/ref DLBCL that failed at least 2 prior lines of therapy treated at our Institute. Demographic, clinical and pathological characteristics were identified. Archived pathological material from the biopsy performed at the second relapse/progression was obtained for each patient. Using the Nanostring nCounter Analysis System (Nanostring Technologies, Seattle, WA), gene expression profiling (GEP) was performed for each sample as previously described using a custom designed code set containing 770 genes. Analysis and normalization of the raw Nanostring data was performed using nSolver Analysis Software v1.1. Raw counts were normalized to internal levels of 7 reference genes: CNOT2, GAPDH, HPRT1, PHGDH, SUMO2, SYS1 and WDR45L. A background count level was estimated using the average count of the 8 negative control probes in every reaction plus 2 standard deviations. Correlation between clinical outcomes (response rate to third line therapy, progression free-survival [PFS] and overall survival [OS]) and IPI score, clinical characteristics and GEP. Subsequently and based on the GEP results, we evaluated the expression of Bcl-XL and other Bcl-2 family proteins in a panel of DLBCL cell lines. Immuno-precipitation (IP) studies were conducted to determine protein-protein interaction between Bcl-XL and other Bcl-2 related proteins. In addition, cell lines were exposed to A1331852, a novel Bcl-XL inhibitor at different doses alone or in combination with chemotherapy drugs. Cell viability was evaluated using Presto Blue assay and IC₅₀ values were calculated using the GraphPad Prism6 software. Results: A total of 53 rel/ref DLBCL were identified that received third line therapy at our Institute. The median age was 59yrs. The median PFS and OS was 23 months and 21 months respectively. Over-expression of BCL-XL RNA levels was associated with a shorter OS (P=0.015, HR=1.17). Pre-clinically higher levels of Bcl-XL as determined by western blotting were found in lymphoma cell lines resistant to chemotherapy agents in vitro. IP studies demonstrated a strong interaction between Bcl-XL and BIM in chemotherapy resistant cell lines. In vitro exposure of DLBCL cell lines to A1331852 resulted in dose- and time-dependent cell death. Moreover, synergistic activity was observed when A1331852 was combined with chemotherapy drugs (doxorubicin, vincristine and etoposide). Conclusion: Our data suggests that high expression level of BCLxL was associated with inferior prognosis in the refractory and/or relapsed DLBCL patients. Inhibition of Bcl-XL results in cell death of rituximab-chemotherapy resistant lymphoma cell lines and potentiates the anti-tumor activity of chemotherapy agents. Selective targeting of Bcl-XL may be a novel therapeutic strategy for rel/ref aggressive B-cell lymphoma.