Targeting BCL-XL to Overcome Anti-CD20 Antibodies and Venetoclax Resistance in Pre-Clinical Model of Diffuse Large B Cell Lymphoma

Standard systemic chemo-immunotherapy cures a significant number of diffuse large B-cell lymphoma (DLBCL) patients, but 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 or chimeric antigen receptor anti-CD19 T-cell therapy respectively. For patients who failed salvage therapy have a 12% long-term survival and the median survival is only 6.3 months at best. Venetoclax, a second generation of BCL-2 inhibitors, was found to be clinically active in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) leading to its approval by the Federal Drug Administration (FDA). However, venetoclax activity in aggressive B-cell lymphoma is very limited. Moreover, acquired resistance to venetoclax is becoming an obstacle in the clinical setting, stressing the need to identify other druggable anti-apoptotic biomarkers associated with therapy resistance and poor clinical outcomes.

Using r/r DLBCL cell lines we generated in our lab, our group reported that dysregulation of apoptotic molecules, up-regulation of PI3K-Akt-mTOR pathway and abnormal mitotic cell cycle arrest led to increased level of BCL-xL in relapsed/refractory (r/r) DLBCL. BCL-xL is a member of the BH3 domain containing Bcl-2 family proteins localizing in the outer mitochondrial membrane, functioning as an antiapoptotic protein. Our previous data showed over-expression of BCL-xL RNA levels were associated with a shorter OS. Immunohistochemistry also confirmed that overexpression of BCL-xL protein level was co-related to the worse PFS and OS in the same r/r DLBCL patients (N=21). Our findings were observed prior to the incorporation of CART-19 therapy and other novel agents commonly used in the third line setting such as polatuzumab vedotin, tafasitamab-cxix or loncastuximab tesirine-lpyl.

To explore the molecular mechanisms of BCL-xL in r/r DLBCL, we found resistant cell lines have a less expression level of BCL-xL compared to the sensitive cell lines. But the binding complex of BCL-xL and BIM was more abundant in resistant cell lines by immunoprecipitation and western blot. This less sequestered BIM was available in resistant cells to facilitate its pro-apoptosis function. Therefore, we generated a stable BCL-xL knockout (KO) DLBCL cell lines by CRISPR-Cas9 Gene Editing system. Silencing of BCL-xL increased the antitumor activity of venetoclax, as well as the complement mediated cytotoxicity of anti-CD20 antibodies (rituximab, obinotuzumab, and ofatumumab) in vitro. Blockage BCL-xL by small inhibitor induced caspase dependent cell death in a dose- and time-dependent manner in DLBCL and rituximab resistant cell lines tested. Furthermore, in lymphoma xenograft mouse models, silencing of BCL-xL potentiated the anti-tumor activity of rituximab and venetoclax. SCID mice bearing BCL-xL KO Raji cells exhibited a prolonged survival following therapy with rituximab and venetoclax when compared to SCID bearing non-target control Raji cells treated with the same combination (median survival time of 39 days vs 28 days, p<0.05).

The data generated here will give us solid preclinical evidence to support the clinical testing of B cell lymphoma specific BCL-xL inhibitor in patients with r/r lymphoma, both as a single agent and in rational combinations.

Torka:Genentech: Consultancy; ADC Therapeutics: Consultancy; Lilly USA: Consultancy; TG Therapeutics: Consultancy; Epizyme: Consultancy; Targeted Oncology, Physician Education Review: Honoraria.