S55746 is a novel orally active BCL-2 selective and potent inhibitor that impairs hematological tumor growth

Escape from apoptosis is one of the major hallmarks of cancer cells. The B-cell Lymphoma 2 (BCL-2) gene family encodes pro-apoptotic and anti-apoptotic proteins that are key regulators of the apoptotic process. Overexpression of the pro-survival member BCL-2 is a well-established mechanism contributing to oncogenesis and chemoresistance in several cancers, including lymphoma and leukemia. Thus, BCL-2 has become an attractive target for therapeutic strategy in cancer, as demonstrated by the recent approval of ABT-199 (Venclexta™) in relapsed or refractory Chronic Lymphocytic Leukemia with 17p deletion. Here, we describe a novel orally bioavailable BCL-2 selective and potent inhibitor called S55746 (also known as BCL201). S55746 occupies the hydrophobic groove of BCL-2. Its selectivity profile demonstrates no significant binding to MCL-1, BFL-1 (BCL2A1/A1) and poor affinity for BCL-XL. Accordingly, S55746 has no cytotoxic activity on BCL-XL-dependent cells, such as platelets. In a panel of hematological cell lines, S55746 induces hallmarks of apoptosis including externalization of phosphatidylserine, caspase-3 activation and PARP cleavage. Ex vivo, S55746 induces apoptosis in the low nanomolar range in primary Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma patient samples. Finally, S55746 administered by oral route daily in mice demonstrated robust anti-tumor efficacy in two hematological xenograft models with no weight lost and no change in behavior. Taken together, these data demonstrate that S55746 is a novel, well-tolerated BH3-mimetic targeting selectively and potently the BCL-2 protein.


Synthesis of intermediate 2
Step
FL5.12 cells overexpressing BCL-2 or BCL-XL were kindly provided by Drs. A. Letai and L. Boise, respectively and cultured in RPMI 1640 supplemented with 10% Fetal Calf Serum, 2 mM L-glutamine and 10% of IL3-containing WEHI-3 cell culture supernatant. To assess sensitivity to BCL-2 inhibitors, FL5.12 cells were cultured for 24 h in the absence of IL-3 before treatment with BCL-2-inhibitors for an additional 24 h.

THP-1 knock-out BAX/BAK generation
For each round of transduction, lentiviral particles (Sigma Aldrich) were used to transduce 2 × 10 5 cells with a multiplicity of infection (MOI) of 10 for 2 h at 32° C in the presence of 8 µg/ml of polybrene. The virus-containing medium was then replaced with fresh medium and cells were incubated at 37° C for 48 h/72 h before starting selection.
THP-1 cells were first transduced with EF1a-Cas9-2A Neomycin (Sigma Aldrich). 96 h after transduction cells were put under neomycin selection (800 µg/ml, Invitrogen) for 10 days before being sorted as single-cells for the isolation of clones. One clone was then further transduced with pLV-U6g-EGFP-gRNA BAK1 (Sigma Aldrich, gRNA sequence: 5′ GCATGAAGTCGACCACGAAG 3′). 48 h after transduction cells were sorted as single-cells based on the expression of the GFP reporter gene. Cells were then further transduced with pLV-U6g-Puromycin gRNA BAX (Sigma Aldrich, gRNA sequence: 5′ CTGC AGGATGATTGCCGCCG 3′). 96 h after transduction cells were put under puromycin selection (1 µg/ml) for 9 days before being sorted as single-cells for the isolation of clones. BAX and BAK knock-out efficiency on different clones was verified by Western blot.
Cleared lysates (5 µg) were assayed for immunodetection of cleaved PARP and cleaved caspase-3 with MSD Apoptosis Panel Whole Cell Lysate kit (MSD K15102D) in 96-well plates according to manufacturer's instructions, and analyzed on the Sector Image 2400. Control DMSO conditions were set to 100% and fold-activation calculated. For ex vivo experiments, once corrected with the blank, data from treated groups were represented as mean of the fold-increase compared to untreated control group.