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Proteolysis targeting chimeric molecules in MM
ARV 825 induces cell cycle arrest and apoptosis in multiple myeloma cells
Cell cycle analysis of MM cells was performed in the presence of various concentrations of ARV 825 for 48 h compared to control cells. The drug increased the G1 phase and decreased the S and G2/M phases in MM cells (Figure 4A). Flow cytometric analysis of KMS11 and KMS28BM MM cells showed a marked dose-dependent increase in the percentage of apoptotic cells (apoptotic cells defined as Annexin V+ and PI+) after treatment with various concentrations of ARV 825 for 48 h (Figure 4B). For example, ARV 825 (10, 20 and 40 nM) led to 10%, 30% and 50% of apoptotic KMS11 cells, respectively. ARV 825 at 100, 200 and 400 nM produced 26%, 31% and 34%, respectively, of apoptotic KMS28BM cells.
Small molecule inhibitors which are synergistic with ARV 825
We performed high-throughput small-molecule inhibitor screen (panel of 170 drugs, FDA-approved or in clinical trial) to identify novel anti-MM compounds that may have synergistic activity with ARV 825. IC50s were determined for each compound, both alone and in combi- nation with ARV 825. ARV 825 relatively sensitive KMS11 and relatively resistant KMS28BM MM cells were exam- ined. Of the 170 tested drugs, 60 are shown in Online Supplementary Figure S3A. Combination of ARV 825 and cediranib (VEGFR inhibitor), crenolanib (PDGFRα/b and FLT3 inhibitor), GSK 1904529A (IGF-1R inhibitor), mote- sanib (VEGFR1/2/3 inhibitor), and LY3023414 (dual PI3K/mTOR inhibitor) produced synergistic growth inhibitory activity against both KMS11 and KMS28BM. Additional confirmation of synergistic effect of these five promising small molecules on 8226 cells was performed [Combination Index (CI) <1] (Figure 5). Selinexor (CRM1 inhibitor), gilteritinib (FLT3/AXL inhibitor), LY333531 (PKCb1 and 2 inhibitor), IGC003 (CBP/EP300 inhibitor), ruxolitinib (JAK inhibitor) produced synergistic growth inhibitory activity against either KMS11 or KMS28BM cells (Online Supplementary Figure S3B). The CI analysis of 10 of these synergistic small molecule inhibitors with ARV 825 is shown in Online Supplementary Table S3.
We also performed combination of MZ1 with promis- ing small molecules (cediranib, Crenolanib, GSK 1904529A, motesanib and LY3023414). Each combination showed synergistic activity with MZ1 inhibiting growth of both KMS11 and KMS28BM MM cells. However, com- bination of MZ1 with LY3023414 has only synergistic effect against KMS11 but not KMS28BM cells (Figure 6). The CI analysis of these promising small molecule inhibitors with MZ1 is shown in Online Supplementary Table S4. A list of inhibitors is provided in Online Supplementary Table S5.
Transcriptome analysis showed MYC is significantly down-regulated by ARV 825 in multiple myeloma cells
We examined the effect of ARV 825 on mRNA expres- sion of MM cells (KMS11) by RNA sequencing. Heatmaps (Figure 7A) displayed the top 20 down-regulated and up- regulated transcripts of KMS11 MM cells following treat- ment of the cells with 20 nM ARV 825 for 8 h. ARV 825 markedly down-regulated CCR1, RGS1, MYB, and MYC. RNA sequencing data were further verified using quanti- tative RT-PCR for 9 selected genes (FJX1, ZNF8, SSTR3, CCR1, MYB, NRROS, MYC, RGS1 and DOK4) in KMS11
(Figure 7B) and KMS28BM cells (Online Supplementary Figure S2A). Furthermore, Gene-Set Enrichment Analysis (GSEA) indicated the robust downregulation of function- ally-defined MYC targets following ARV 825 treatment of KMS11 MM cells (Online Supplementary Figure S2B and C). Primers for qRT-PCR are listed in Online Supplementary Table S6.
ARV 825 inhibited multiple myeloma growth in vivo Anti-proliferative effect of ARV 825 was examined in vivo against MM xenografts growing in SCID-Beige mice. Two weeks after injection, the MM cells were easily observed by bioluminescence imaging, after which, mice (n=9 per group) were randomly assigned to receive either ARV 825 (5 mg/kg) dissolved in 200 μL of vehicle daily intraperitoneally or 200 μL of vehicle alone. ARV 825 sig- nificantly slowed tumor growth in experimental mice compared to control mice receiving vehicle, as measured by bioluminescence (Figure 8A and B) at days 7, 14, 21 and 28. Importantly, ARV 825 treatment significantly pro- longed the murine OS compared to vehicle-treated mice (Figure 8C). ARV 825 treated mice maintained normal activity and insignificant weight loss compared to diluent control mice (Online Supplementary Figure S4A). The IC50 of ARV 825 using normal mouse BM cells (2x105
cells/well) is 500 nM (Online Supplementary Figure S4B). Discussion
Despite the major advances in the treatment of MM made over the last decade, disease management still remains challenging as most patients either do not achieve a complete remission or eventually relapse. Bortezomib and lenalidomide have become a part of standard manage- ment. Auto-transplants are often also given; nevertheless, patients are rarely cured. New targeted therapeutic strate- gies are needed. Next generation BET inhibitor ARV 825 degrades bromodomains. We found BRD 2 and BRD 4 were profoundly depleted, consistent with previous reports of PROTAC for other malignancies.7,9,10,16 Previous studies have shown a fusion of JQ1 and thalidomide (dBET6) has significant potency against MM.17 We also showed that ARV 825 leads to significant growth inhibi- tion of myeloma cells in liquid culture, clonogenic assay and, most importantly, in a xenograft model. Flow cyto- metric analysis showed that ARV 825 induced apoptosis and G0/G1 cell cycle arrest of these cells in vitro.
We demonstrated that both ARV 825 and MZ1 have promising activity against MM cells. ARV 825 induced degradation of BET proteins via CRBN E3 ligase. Importantly, we found a positive correlation of intracellu- lar levels of CRBN and their sensitivity to ARV 825. CRBN expression is prominent in hematologic malignancies, including MM. Response to immunomodulatory drugs is clinically correlated with expression of CRBN.18 Loss of function of CRBN causes resistance to dBET6 by perturb- ing dBET-mediated BRD 4 degradation.17 We postulate that levels of CRBN will serve as a predictive biomarker for cellular responsiveness to ARV 825. Indeed, two pairs of isogenic cells, one of each pair resistant to lenalidomide (KMS11 res and MM1S res), had very low expression of CRBN. Genetically either silencing or over-expressing CRBN decreased and increased, respectively, the sensitiv- ity of an MM cell line to growth inhibition by ARV 825. In
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