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J. Gao et al.
to THZ531. Western blotting revealed that, in contrast to THZ531-sensitive lines, the resistant REC-1 cells were refractory to THZ531 treatment and THZ531 failed to change the expression of MCL-1, MYC, and 4EBP1, PARP cleavage as well as all Ser2 phosphorylation of RNAPII (Figure 4B). Next, to investigate the cellular pathways responsible for THZ531 resistance, we analyzed the tran- scriptome changes after THZ531 treatment in REC-1. As illustrated in Figure 4C, in contrast to cells that were responsive to THZ531, THZ531 treatment for 24 h at a dose (100 nM) that was highly potent in THZ531-sensi- tive lines did not induce gene expression changes in REC- 1. We then compared the differential gene expression between THZ531-sensitive cell lines (Z138, Jeko-1) and a THZ531-resistant one (REC-1). Among drug transport genes identified by RNA-sequencing, the level of MDR1 transcript was dramatically higher in REC-1 cells than in Z138 and Jeko-1 cells (Figure 4D). Moreover, quantitative reverse transcriptase polymerase chain reaction analysis revealed greater MDR1 abundance in REC-1 cells (Online Supplementary Figure S4A). To study the functional role of MDR1 in resistance to THZ531, we tested whether inhi- bition of MDR1 by its specific inhibitor (tariquidar) could overcome THZ531 resistance. As shown in Figure 4E, tariquidar overcame THZ531 resistance and synergized with THZ531 in the suppression of cell viability in REC- 1 cells. In addition, we developed two cell lines, KARPAS- 422-THZ-R and Maver-1-THZ-R (Online Supplementary Figure S4B), with resistance to THZ531: “acquired” by chronic exposure to escalating doses of THZ531 over 3 months. In line with cells with de novo drug resistance, the THZ531-resistant KARPAS-422 and Maver-1 cells exhib- ited higher MDR1 expression at both the mRNA and pro- tein levels (Online Supplementary Figure S4C). Cell viability of both cell lines was dramatically reduced when exposed to the combination of THZ531 with tariquidar (Figure 4F). Together, these data support that MDR1 upregulation drives THZ531 resistance in MCL and other aggressive B- cell lymphomas.
EZH2 inhibitors restored sensitivity to THZ531 in THZ531-resistant cells by competing with THZ531 for MDR1
To define the molecular determinants that drive resist- ance to THZ531, reverse this resistance and enhance THZ531 activity in THZ531-resistant cell lines, we per- formed drug screen assays on REC-1 against a set of epi- genetic modifiers and kinase inhibitors in the presence or absence of THZ531 as an “anchor” in our drug screen platform. Using AUC to quantify the effect and potency of each single and combination treatment, we identified the inhibitors that enhanced THZ531 activity and ranked the combination potency by the differential killing effect between drug combinations and single inhibitors. Inhibitors found to enhance the potency of THZ531 included MEK, BTK, mTOR and EZH2 inhibitors. Among them, the drug screen assay identified that inhibitors tar- geting EZH2 in combination with THZ531 had the great- est increased potency against REC-1 cells (Figure 5A, Online Supplementary Figure S5A). Indeed, cell viability assays revealed that the combination of EZH2 inhibitors (GSK343 or UNC1999) with THZ531 had synergistic effects against cell survival in REC-1 cells (Figure 5B). Mechanistically, western blots revealed that neither EZH2 inhibitor nor THZ531 alone had an effect on Ser2
phosphorylation of RNAPII, p-p70S6K, p-4EBP1, MCL-1 or PARP cleavage (Figure 5C, Online Supplementary Figure S5B). However, the combination of either EZH2 inhibitor with THZ531 triggered dramatic decreases of Ser2 phos- phorylation of RNAPII, p-p70S6K, p-4EBP1 and MCL-1 levels, and significantly increased PARP cleavage (Figure 5C, Online Supplementary Figure S5B). Consistently, syner- gistic effects of THZ531 combined with GSK343 or UNC1999 on cell viability were also observed in the THZ531-resistant cell lines KARPAS-422-THZ-R and Maver-1-THZ-R (Online Supplementary Figure S5C).
Next, to investigate the mechanisms by which EZH2 inhibition reinstated THZ531 sensitivity in resistant cells, we examined changes in MDR1 protein expression upon EZH2 inhibitor treatment in THZ531-resistant cells. As shown in Figure 5D, although treatments with any single agent had no effect on MDR1 protein expression, EZH2 inhibitor significantly sensitized REC-1 cells to THZ531 treatment, as measured by PARP cleavage. In contrast, EZH2 knockdown through shRNA showed that EZH2 downregulation had no effect on basal MDR1 expression and failed to reverse THZ531-induced downstream sig- naling via RNAPII-Ser2, MCL-1 and MYC in REC-1 cells (Online Supplementary Figure S5D). These data suggest that the potency enhancing effects of GSK343 and UNC1999 were not due to biological EZH2 regulation on either MDR1 or THZ531 signaling. Thus, to further reveal the underlying mechanism of MDR1 modulation by GSK343 and UNC1999, we performed P-gP-Glo assays, which served to elucidate whether GSK343 and UNC1999 are direct MDR1 inhibitors or substrates in REC-1, KARPAS- 422-THZ-R and Maver-1-THZ-R cells. GSK343 and UNC1999 were confirmed not to be direct inhibitors, but strong MDR1 substrates, comparable to verapamil (Figure 5E). Thus, the data support that EZH2 inhibitors GSK343 and UNC1999 function by competitively binding to MDR1.
Activation of MEK-ERK and PI3K-AKT-mTOR pathways contribute to MDR1 upregulation in THZ531-resistant cells
We next systematically tested for synergistic combina- tions of THZ531 with EZH2 inhibitors in the sensitive as well as resistant cells to validate the combination as an effective strategy to overcome and block emergent resist- ance in primary samples. Importantly, in the innately THZ531-resistant primary samples, treatment with EZH2 inhibitors sensitized the cells to THZ531 (Figure 6A), strengthening the rationale to combine THZ531 and EZH2 inhibitors in the upfront setting.
Given that the drug screen on REC-1 cells showed inhibitors of MEK and AKT sensitized REC-1 to THZ531 treatment, we hypothesized that activation of the MEK- ERK and PI3-AKT-mTOR pathways in REC-1 regulated MDR1 expression and affected the efficacy of THZ531. As shown in Figure 6B, inhibition of MEK with trame- tinib and PI3K-AKT with either AZD8055 or BEZ235 induced a significant decrease of the abundance of MDR1 protein in REC-1 cells. Accordingly, combined treatment of THZ531 with trametinib, AZD8055 or BEZ235 all induced a synergistic effect on REC-1 cell viability, as measured by MTT assays (Figure 6C). Overall, these data imply that activation of MEK-ERK and PI3K-AKT-mTOR pathways contributes to MDR1 upregulation in THZ531- resistant cells.
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