Co-Targeting of BCL2 with Venetoclax and MCL1 with S63845 Is Synthetically Lethal In Vivo in Relapsed Refractory Mantle Cell Lymphoma with Complex Karyotype Changes
Authors: Klener Pavel , Trněný Marek , Andera Ladislav , Nahacka Zuzana , Klanova Magdalena , Karolova Jana , Svaton Michael , Havranek Ondrej , Soukup Jan , Zemanova Zuzana , Tuskova Diana , Pokorna Eva , Forsterova Kristina , Vockova Petra , Fronkova Eva , Prukova Dana 
Published: October 2019

Abstract
Introduction
Mantle cell lymphoma (MCL) is an aggressive subtype of B-cell non-Hodgkin lymphomas characterized by (over)expression of BCL2 and good sensitivity to a small molecule BCL2 inhibitor venetoclax. In the present study we analyzed molecular mechanisms of venetoclax resistance in MCL cells, and tested strategies to overcome it based on concurrent targeting of BCL2 a MCL1.
Methods
Cell death was determined by flow cytometry using Annexin-V/PI staining. Establishment of MCL cell clones with knock-down or transgenic overexpression of MCL1, BIM and NOXA, western blotting, immunohistochemistry of formalin-fixed paraffin-embedded tissue sections, and immunoprecipitation experiments were carried out as previously described (Klanova et al, Clin Cancer Res, 2016). All PDXs were derived in our laboratory from patients with relapsed MCL. All PDX were confirmed by NGS to keep majority of somatic mutations with the primary MCL cells from which they were derived. Samples were sequenced using SureSelectXT Human All Exon V6+UTR (Agilent Technologies, Santa Clara, CA) on the NextSeq 500 (Illumina, San Diego, CA) instrument according to manufacturer's protocols. Experimental therapies were implemented using NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice purchased from Jackson Laboratory (Bar Harbor, Maine, USA). Therapy was initiated when all mice developed palpable subcutaneous tumors (= day 1, D1). Venetoclax (VTX) and S63845 were from MedchemExpress, carfilzomib (CFZ) was from Charles University General hospital pharmacy. Carfilzomib (4 mg / kg) was administered intravenously (IV) on days 1 and 6. Venetoclax (40 mg / kg) was given by oral gavage on days 1, 2, 3, 6 and 7. S63845 (25 mg / kg) was administered IV on days 1, 2, 3, 6 and 7. Tumor volumes were calculated using the following formula: π / 6 × tumor length × width × height.
Results
By transgenic overexpression or shRNA-mediated knock-down we confirmed key roles of proapoptotic proteins BIM and NOXA in mediating venetoclax-induced cell death in MCL. We demonstrated that both BIM and NOXA are differentially expressed between MCL cell lines on one side, and primary MCL cells and patient-derived xenograft (PDX) cells on the other side. First, NOXA protein is significantly overexpressed in most MCL cell lines. Second, biallelic deletions of BIM harbored by three commonly used MCL cell lines (JEKO-1, MINO and Z138), and previously reported to be present in approx. 30% of MCL patients, were not found in primary MCL cells. As a consequence, vast majority of the in vitro data was implemented on venetoclax-sensitive cell lines HBL2 and MAVER-1, whose patterns of expression of BCL2, MCL1, BIM and NOXA are similar to primary MCL cells. We demonstrated that MCL1, another key anti-apoptotic protein, plays an essential role in mediating resistance to venetoclax. First, MCL1 functions as a buffer for BIM released from BCL2 upon binding of venetoclax thereby preventing activation of BAX and induction of apoptosis. Second, marked upregulation of MCL1 protein was associated with acquired resistance to venetoclax in two most sensitive MCL cell lines HBL2 and MAVER-1. Based on the in vitro data we proposed two experimental treatment strategies that co-targeted MCL1 (along with inhibition of BCL2 with venetoclax): a direct blockage with a highly specific small molecule MCL1 inhibitor S63845, and an indirect blockage achieved by proteasome inhibitor carfilzomib that upregulates the proapoptotic protein NOXA that specifically binds and blocks MCL1. The combination of venetoclax and S63845 demonstrated synthetic lethality in vivo inducing the longest "remissions" of MCL bearing mice (i.e. temporary disappearance of subcutaneous MCL tumors) using a panel of four different PDXs derived from patients with relapsed / refractory MCL with complex karyotype changes (Figure 1). The combination of carfilzomib and venetoclax was far less effective, and at the same time more toxic suggesting functional blockage of MCL1 induced by overexpressed NOXA is either incomplete or insufficient.
Conclusions
Our data strongly support investigation of venetoclax in combination with S63845 as an innovative proapoptotic treatment strategy for chemoresistant MCL patients with adverse cytogenetics in the clinical grounds.
Figure 1 Figure 1.

Disclosures
Trněný: Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Gilead: Honoraria; Morphosys: Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Abbvie: Honoraria, Research Funding; F. Hoffman-La Roche Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory board, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Sandoz: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Advisory board.