Effective tumor cell abrogation via Venetoclax-mediated BCL-2 inhibition in KMT2A-rearranged acute B-lymphoblastic leukemia

Abstract

Dysregulation of the intrinsic BCL-2 pathway-mediated apoptosis cascade is a common feature of hematological malignancies including acute B-lymphoblastic leukemia (B-ALL). The KMT2A-rearranged high-risk cytogenetic subtype is characterized by high expression of antiapoptotic protein BCL-2, likely due to the direct activating binding of KMT2A fusion proteins to the BCL2 gene. The BCL-2 inhibitor venetoclax (VEN) has proven great clinical value in other blood cancers, however, data on B-ALL is sparse and past studies have not so far described the effects of VEN on gene and protein expression profiles. Using cell lines and patient-derived in vivo xenograft models, we show BCL-2 pathway-mediated apoptosis induction and decelerated tumor cell counts in KMT2A-rearranged B-ALL but not in other cytogenetic subtypes. VEN treatment of cell line- and patient-derived xenografts reduced blast frequencies in blood, bone marrow, and spleen, and tumor cell doubling times were increased. Growth rates are further correlated with VEN concentrations in blood. In vitro incubation with VEN resulted in BCL-2 dephosphorylation and targeted panel RNA sequencing revealed reduced gene expression of antiapoptotic pathway members BCL2, MCL1, and BCL2L1 (BCL-XL). Reinforced translocation of BAX proteins towards mitochondria induced caspase activation and cell death commitment. Prolonged VEN application led to upregulation of antiapoptotic proteins BCL-2, MCL-1, and BCL-XL. Interestingly, the extrinsic apoptosis pathway was strongly modulated in SEM cells in response to VEN. Gene expression of members of the tumor necrosis factor signaling cascade was increased, resulting in canonical NF-kB signaling. This possibly suggests a previously undescribed mechanism of BCL-2-independent and NF-kB-mediated upregulation of MCL-1 and BCL-XL. In summary, we herein prove that VEN is a potent option to suppress tumor cells in KMT2A-rearranged B-ALL in vitro and in vivo. Possible evasion mechanisms, however, must be considered in subsequent studies.

Combined BCL-2 and PI3K/AKT Pathway Inhibition in KMT2A-Rearranged Acute B-Lymphoblastic Leukemia Cells

“…Previous studies by others and our group have shown that prolonged VEN incubation induces BCL-2, MCL-1, and BCL-xL anti-apoptotic protein expression [ 3 , 22 ]. Combination approaches should therefore counteract these mechanisms to synergistically tip the balance toward apoptosis induction [ 12 ].…”

“…The human B-ALL cell lines SEM and RS4;11 were obtained from DSMZ (Braunschweig, Germany) and cultivated as previously described [ 3 ]. A control for authenticity (cell surface flow cytometry) and mycoplasma contamination was performed at regular intervals.…”

“…SEM and RS4;11 cells were incubated with inhibitors for 48 h and processed as previously described [ 3 ].…”

“…For flow-cytometric apoptosis assessment, cells were incubated with VEN and PI3K/AKT inhibitors for 72 h, treated as previously described [ 3 ], and analyzed using the FACSCalibur device (Becton Dickinson, Heidelberg, Germany). Data were evaluated using FACSuite software (Becton Dickinson, version 1.0.6.5230).…”

“…To address the insufficient response, novel therapeutical approaches based on molecular characteristics are needed. Recent studies by us and others depicted that KMT2A -r ALL is characterized by elevated expression of the anti-apoptotic protein BCL-2 [ 2 , 3 , 4 ]. Increased BCL-2 expression results in a disbalance favoring apoptosis avoidance and mediates chemoresistance [ 5 , 6 ].…”