BET protein proteolysis targeting chimera (PROTAC) exerts potent lethal activity against mantle cell lymphoma cells

Abstract

Bromodomain extraterminal protein (BETP) inhibitors transcriptionally repress oncoproteins and nuclear factor-κB (NF-κB) target genes that undermines the growth and survival of mantle cell lymphoma (MCL) cells. However, BET bromodomain inhibitor (BETi) treatment causes accumulation of BETPs, associated with reversible binding and incomplete inhibition of BRD4 that potentially compromises the activity of BETi in MCL cells. Unlike BETi, BET-PROTACs (proteolysis-targeting chimera) ARV-825 and ARV-771 (Arvinas, Inc.) recruit and utilize an E3-ubiquitin ligase to effectively degrade BETPs in MCL cells. BET-PROTACs induce more apoptosis than BETi of MCL cells, including those resistant to ibrutinib. BET-PROTAC treatment induced more perturbations in the mRNA and protein expressions than BETi, with depletion of c-Myc, CDK4, cyclin D1 and the NF-κB transcriptional targets Bcl-xL, XIAP and BTK, while inducing the levels of HEXIM1, NOXA and CDKN1A/p21. Treatment with ARV-771, which possesses superior pharmacological properties compared with ARV-825, inhibited the in vivo growth and induced greater survival improvement than the BETi OTX015 of immune-depleted mice engrafted with MCL cells. Cotreatment of ARV-771 with ibrutinib or the BCL2 antagonist venetoclax or CDK4/6 inhibitor palbociclib synergistically induced apoptosis of MCL cells. These studies highlight promising and superior preclinical activity of BET-PROTAC than BETi, requiring further in vivo evaluation of BET-PROTAC as a therapy for ibrutinib-sensitive or -resistant MCL.

Figure 1.. Compared to OTX015 which induces BRD4 expression, treatment with BET PROTAC causes efficient and sustained depletion of BET proteins including BRD4 in cultured and primary MCL cells.

A. MCL Mino cells were treated with the indicated concentration of OTX015, ARV-825, or ARV-771 for 18 hours. At the end of treatment, cell lysates were prepared and immunoblot analyses were conducted. The expression levels of β-Actin in the cell lysates served as the loading control. The horizontal graphs beneath the Western blots indicate densitometry analysis conducted on three separate experiments. * indicates values significantly greater (p<0.05) in the OTX015-treated cells compared to the control cells. *** indicates values significantly less (P < 0.0001) in the ARV-825 and ARV-771-treated cells compared to control cells. B. Mino cells were treated with the indicated concentrations of OTX015, ARV-825 or ARV-771 for 24 hours and half the cells were collected and snap frozen in liquid nitrogen. The remaining cells were washed three times in serum-free RPMI-1640 media to remove the drug (drug washout, WO) and re-plated in complete media containing no drug for an additional 24 hours. Following this, total cell lysates were prepared and immunoblot analyses were conducted as indicated. The expression levels of β-Actin in the cell lysates served as the loading control. C. Primary MCL cells (n=3) were treated with the indicated concentration of OTX015, ARV-825, or ARV-771 for 18 hours. At the end of treatment, cell lysates were prepared and immunoblot analyses were conducted. The expression levels of β-Actin in the cell lysates served as the loading control. The horizontal graphs beneath the Western blots indicate densitometry analysis conducted on three separate experiments. * indicates values significantly greater (p<0.05) in the OTX015-treated cells compared to the control cells. *** indicates values significantly less (P < 0.0001) in the ARV-825 and ARV-771-treated cells compared to control cells. * p< 0.05 *** P< 0.0001

Figure 2.. Treatment with BET PROTAC ARV-825 or ARV-771 induces greater apoptosis than BETi OTX015 in cultured MCL cells.

A-B. Mino and Z138 cells were treated with the indicated concentrations of OTX015, ARV-825 or ARV-771 for 48 hours. The % of annexin V-positive, apoptotic cells was determined by flow cytometry. Columns, mean of three experiments; Bars, standard of the mean. C. Primary MCL cells were treated with the indicated concentrations of OTX015, ARV-825 or ARV-771 for 48 hours. The % of propidium iodide-positive, non-viable cells was determined by flow cytometry. Columns, mean of seven primary samples; Bars, standard of the mean. ‡ indicates non-viable cell values significantly greater in OTX015 treated cells than control cells (p < 0.05). † indicates non-viable cell values that are significantly greater in ARV-825-treated cells than OTX015-treated cells (p< 0.05). * indicates non-viable cell values that are significantly greater in ARV-771-treated cells than OTX015-treated cells (p< 0.05).

Figure 3.. Treatment with BET PROTAC ARV-825 perturbs more mRNA expressions than OTX015 in MCL cells.

Mino cells were treated with 500 nM of ARV-825 or OTX015 for 4 hours. Cells were treated independently and represent biologic triplicates. At the end of treatment, total RNA was isolated and utilized for RNA-Seq analyses. A. The heatmaps show the number of mRNAs with a ≥ 2-fold change (up or down) relative to the untreated Mino cells. B. The graph shows the log2 fold-change for selected target gene expressions. All p-values were less than 0.05. C. Venn diagram representing the expression signature overlap in down and upregulated genes cells following treatment with ARV-825 and OTX015 in Mino cells.

Figure 4.. Treatment with BET PROTAC ARV-825 or ARV-771 or BET inhibitor OTX015 depletes the mRNA expression levels of MYC, BTK, BCL2, CDK6 and NFkB target genes in MCL cells.

A-B. Mino cells were treated with the indicated concentration of ARV-825, ARV-771 or OTX015 for 8 hours. At the end of treatment, total RNA was isolated and reverse transcribed. The resulting cDNA was used for real-time, quantitative PCR analysis. The relative mRNA expression of each target was normalized to GAPDH and compared to the untreated cells.

Figure 5.. Treatment with BET PROTAC causes greater and more sustained depletion of BRD4 target genes than OTX015 in MCL cells

A. Mino and Z138 cells were treated with the indicated concentrations of OTX015, ARV-825 or ARV-771 for 18 hours. At the end of treatment, cells were harvested and lysed. The resulting cell lysates were utilized for immunoblot analyses. The expression of β-Actin in the cell lysates served as the loading control. B. Mino cells were treated with the indicated concentrations of OTX015, ARV-825 or ARV-771 for 24 hours and half the cells were harvested. The remaining cells were washed and re-plated in media containing no drug for an additional 24 hours (WO). Immunoblot analyses were conducted as indicated. The expression levels of β-Actin in the cell lysates served as the loading control. C. Primary MCL cells were treated with OTX015, ARV-825 or ARV-771, as indicated, for 18 hours. Immunoblot analyses were conducted as indicated. The expression levels of β-Actin in the cell lysates served as the loading control.

Figure 6.. In vivo activity of OTX015 and BET PROTAC ARV-771 against MCL Z138 xenografts.

A. NSG mice were implanted with luciferase-expressing Z138 cells and monitored for 7 days. Mice were treated with vehicle, OTX015 or ARV-771 for 7 days and imaged with a Xenogen camera. Two representative mice are shown for each treatment group. The boxplot shows the mean luminescence flux from the 5 mice in each treatment group +/− S.E.M. B. Kaplan-Meier plot of the in vivo activity of OTX015 or ARV-771 against MCL Z138/Luc xenografts in NSG mice. Significance was determined by Mantle-Cox Rank Sum test. C. Immunoblot analyses of Z138/Luciferase cells in the spleen and bone marrow of NSG mice following 1 week of treatment with OTX015 or ARV-771, as indicated.

Figure 7.. Synergistic lethal activity of BETi or BET PROTAC-based combinations with ibrutinib, venetoclax (ABT-199) or palbociclib in cultured and primary MCL cells.

A-B. Mino and JeKo-1 cells were treated with OTX015 (dose range: 100–2000 nM), or ARV-771 (dose range: 10–250 nM) and ibrutinib (dose range: 0.5–10 μM), or venetoclax (ABT-199) (dose range: 50–500 nM) at a constant ratio for 48 hours. Apoptotic cells were determined by flow cytometry. Median dose effect and isobologram analyses were performed using Compusyn. Combination index (CI) values less than 1.0 indicate a synergistic interaction of the two agents in the combination. C. Combination index values in Mino/Persister cells treated with OTX015 or ARV-771 and venetoclax (as above) or palbociclib (dose range: 1–10 μM) for 48 hours. D. PD CD19+ MCL cells were treated with OTX015 (dose range: 50–1000 nM) or ARV-771 (dose range: 10–500 nM) and ibrutinib (dose range: 0.5–10 μM), venetoclax (ABT-199) (dose range: 50–500 nM), or palbociclib (dose range: 500–5000 nM) at a constant ratio for 48 hours. Non-viable cells were determined by propidium iodide staining followed by flow cytometry. Median dose effect and isobologram analyses were performed. Combination index (CI) values less than 1.0 indicate a synergistic interaction of the two agents in the combination.