Venetoclax Resistance in Mantle Cell Lymphoma Is Mediated By BCL-XL and Can be Circumvent By Inhibiting the BH4 Domain of BCL-2
Authors: Steinbrecher Daniela , Seyfried Felix , Tausch Eugen , Bloehdorn Johannes , Jebaraj Billy Michael Chelliah , Debatin Klaus-Michael , Meyer Lüder Hinrich , Döhner Hartmut , Stilgenbauer Stephan , Schneider Christof 
Published: November 2019

Apoptosis is controlled by the expression levels and interplay of pro- and anti-apoptotic BCL-2 family proteins. The specific BCL-2 inhibitor Venetoclax (VEN) showed high efficiency in BCL-2 dependent cancers like chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). Despite its high efficiency in CLL and MCL, refractory disease can develop. BCL-2 mutations have been described to mediate resistance in CLL cases, however these mutations are only found in a proportion of VEN resistant cases and in a fraction of cells. In order to design alternative therapeutic strategies to overcome drug resistance, a better understanding of the mechanisms mediating resistance to VEN is necessary.
VEN-resistant (VEN-R) MCL cell lines (MINO and MAVER-1) were generated by chronic exposure to increasing amounts of VEN (up to 3µM). A significant and stable upregulation of BCL-XL mRNA and protein was seen in the MINO and MAVER-1 resistant cell lines (2 and 4 fold increase in mRNA and 2.6 and 4.5 fold increase in protein, respectively). We used BH3 profiling in combination with VEN treatment for 4h to investigate the differences in anti- and pro-apoptotic signaling in parental and VEN-R cell lines. Additionally, sensitivity to VEN was restored upon shRNA-mediated knockdown of BCL-XL. These results confirmed the importance of BCL-XL upregulation in mediating resistance. Furthermore, we did not detect mutations in BCL-2 upon resistance to VEN via targeted NGS, which is in contrast to results obtained in VEN-R CLL patients (Blombery et al., Cancer Discovery 2019 and Tausch et al., Hematologica 2019).
However, the results obtained by dynamic BH3-profiling (VEN treatment in combination with BH3 Profiling) suggest that increase in BCL-XL is most likely not the only alteration necessary to render cells resistant to VEN. In addition, reduced activation of pro-apoptotic proteins like BAX and BAK might contribute to resistance to VEN.
In order, to investigate if VEN resistance can be overcome by drug mediated inhibition of BCL-XL we used different therapeutic approaches. Combinational treatment with the BCL-XL inhibitor A-1331852 and VEN or the single treatment with Navitoclax, a combined inhibitor of BCL-2, BCL-W and BCL-XL for 48h reduced cell viability in VEN-R MINO and MAVER-1 cell lines. Furthermore, BDA-366, a BH4 domain BCL-2 inhibitor effectively reduced the cell viability after 48h of treatment in a dose dependent manner in both parental and VEN-R cell lines. The binding of BDA-366 to the anti-apoptotic BCL-2 protein leads to a conformational change into a pro-apoptotic molecule by the exposure of the BH3 domain of the protein.
Despite mediating apoptosis in a TP53-independent manner, VEN treatment in CLL has been associated with inferior outcome in the presence of TP53 aberrations. In order to address the role of TP53 dysfunction in mediating resistance to VEN, we generated p53 knock out cell lines (N=2) by CRISPR/Cas9 gene editing. This significantly decreased the sensitivity to VEN compared to p53 WT cell lines. Additionally, the sensitivity to BDA-366 was significantly reduced upon knockout of p53, suggesting an interference of p53 downstream of BCL-2.
Overall, VEN resistance is mediated by a permanent increase in BCL-XL mRNA and protein level in MCL. Importantly, BDA-366, which converts the anti-apoptotic BCL-2 molecule into a BAX-like death molecule, could be a potential alternative treatment strategy for BCL-2 dependent cancers even when resistant to VEN. Despite mediating apoptosis in a p53 independent manner, VEN seems to be less effective in p53 deficient cells, underlining the importance of further investigations of treatment combinations in these groups.

Disclosures
Tausch: Roche: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Speakers Bureau. Döhner:AbbVie, Agios, Amgen, Astellas, Astex, Celator, Janssen, Jazz, Seattle Genetics: Consultancy, Honoraria; AROG, Bristol Myers Squibb, Pfizer: Research Funding; Celgene, Novartis, Sunesis: Honoraria, Research Funding. Stilgenbauer:Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Hoffmann La-Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Other: Travel support; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau. Schneider:Celgene: Other: travel grant.