Preclinical studies of ABT-199
The dose-limiting severe thrombocytopenia from ABT-263/navitoclax quenched the enthusiasm for further clinical development of this compound. Preclinical studies of ABT-737 also revealed decreased platelet survival [
61]. A seminal study by Mason et al. [
62] had previously highlighted the importance of BCL-X
L in platelet survival, its role in the pro-apoptotic activity of BAK, and a gradual reduction in BCL-X
L levels culminating in the apoptosis of senescent platelets. The term
molecular clock was used to describe this temporal switch from anti-apoptosis to pro-apoptosis [
62]. Therefore, BCL-X
L inhibition could speed up this molecular clock and lead to decreased platelet survival—the mechanism implicated in ABT-737/ABT-263-induced thrombocytopenia.
The need to develop a BCL-2 inhibitor sparing BCL-X
L and platelets sparked the discovery and development of ABT-199 (venetoclax) [
63]. X-ray complex-based structures determined the presence of high-affinity interactions in two hydrophobic pockets in the three-dimensional structure of anti-apoptotic BCL-2 proteins. These hot spots, named P2 and P4, bind to the 1-chloro-4-(4,4-dimethylcyclohex-1-enyl)benzene and the thiophenyl moiety of navitoclax, respectively. Along with the hydrophobic interactions, electrostatic interactions between arginine residues (on anti-apoptotic proteins) and aspartate residues (on pro-apoptotic molecules) were also underscored. Through reverse engineering of navitoclax, structural modifications were made so that a new molecule was created to have similar hydrophobic interaction but different electrostatic interaction with Arg103 (specific to BCL-2 since BCL-X
L has Glu96). This led to the discovery of BCL-2 binding molecule, ABT-199.
ABT-199 (venetoclax) represents the first-in-class, selective, oral BCL-2 inhibitor sparing platelets [
63]. It showed sub-nanomolar affinity to BCL-2 (
K
i < 0.010 nM) with antitumor activity against non-Hodgkin’s lymphoma (NHL) [
63], CLL [
64], and acute leukemias [
65,
66] in vitro. In vivo mouse xenograft studies showed activity against aggressive (Myc+) lymphomas [
67] as well as acute leukemia [
68].
BCL-2 over-expression plays a central role in follicular lymphoma (FL). In vitro studies were done to characterize the effects of ABT-199 in the t(14;18)+ FL cell lines WSU-FSCCL and FC-TxFL2 and in primary FL cells [
69]. JNK kinase phosphorylation and inactivation as well as substantial decrease of mitochondrial potential in FC-TxFL2 cells were induced by ABT-199. Similar expression levels were seen in the anti-apoptotic (such as BCL-2, BCL-X
L, MCL-1) and pro-apoptotic proteins (such as BAX, PUMA, BAK, BAD, NOXA, Bid, Bok) in both cell lines. No significant change was seen in these proteins upon ABT-199 treatment. In this study, the ABT-199 resistant cell line was established. Increased levels of MCL-1 and elevated phosphorylation of BCL-2 on T56 and of AKT on S473 were demonstrated. In addition, increased autophagy was shown in comparison to parental cells. It was also demonstrated in this study that the combination of two epigenetic agents, decitabine and vorinostat, was able to overcome ABT-199 resistance. Even though ABT-199 should be theoretically active in FL as a BH3 mimetic, activity in patients with FL has not been as dramatic as has been observed for CLL. Acquired resistance developed easily in the in vivo study in the xenograft model, which may explain the relatively higher failure possibility in FL than in CLL.
ABT-199 was studied in combinations with tyrosine kinase inhibitors (TKIs), including imatinib, nilotinib, dasatinib, or ponatinib, in cells from six patients with blast-crisis chronic myeloid leukemia (CML) [
70]. All six patients were resistant to TKIs, three of them with T315I mutation. This study further revealed in a CML mouse model that ABT-199 alone or in combination was better than nilotinib in eradicating CML stem cells in vivo.
A combination of ibrutinib and ABT-199 was also studied in mantle cell lymphoma (MCL) and CLL [
71]. The ibrutinib and ABT-199 combination substantially induced apoptosis of primary cells from MCL and CLL patients compared to each single agent alone (combo: 23 %, ibrutinib: 3.8 %, ABT-199: 3.0 %). When BCL-2 and BTK target genes as well as emergent genes were characterized using a protein-protein network interaction model, synergistic activation of apoptosis genes of p53 and BIM was revealed. Little off-target effect was seen with the combination nor with individual drugs on normal peripheral T cells [
71].
Selinexor (KPT-330), a first-in-class selective inhibitor of nuclear export (SINE) agent, has been shown to have antiproliferative and pro-apoptotic activities against MCL and other cancer cells [
72‐
76]. A combination of ABT-199 and selinexor was studied in MCL cell lines [
77]. The combination demonstrated synergistic antiproliferative effects through inhibition of mTOR signaling, downregulation of ribosomal biosynthesis, and induction of mitochondria-mediated apoptosis.
MCL-1, an anti-apoptotic protein, is one of the main targets of homoharringtonine (HHT) and omacetaxine, both of which have been in clinical applications [
78,
79]. To test the dual inhibition of BCL-2 and MCL-1, HHT and ABT-199 were combined and tested in seven diffuse large B-cell lymphoma cell lines [
80]. The study confirmed decreased expression of MCL-1 protein in all the cell lines. It was also demonstrated that high expression of BCL-2 positively correlated with sensitivity to ABT-199, irrespective of expression levels of BCL-X
L and MCL-1. BCL-2 and BCL-X
L expression levels negatively correlated with sensitivity to HHT. However, the expression level of MCL-1 did not correlate with sensitivity to HHT.