Abstract
Introduction: Double hit DLBCL with concurrent rearrangement of c-Myc and BCL-2 as well as double expressor DLBCL with concomitant overexpression of c-Myc and BCL-2 are characterized to have very poor outcomes in the following standard R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) therapy as well as short survival in progressing clinical courses. In DLBCL patients, about 20% to 30% of patients have shown the dual c-Myc and BCL-2 aberrance. Thus, several new drugs are being developed to achieve a durable response in double hit and double expressor DLBCL. The phase I study in patients with RR DLBCL using Venetoclax monotherapy which is a BCL-2 inhibitor, showed a disappointing objective response [CR:4/34(12%), PR:2/34(6%)]. This result shows that a combined approach involving a c-Myc controlling agent and BCL-2 inhibitor may be required to overcome the poor response of RR DLBCL, especially double hit and double expressor DLBCL. BR101801 is a first-in-class, oral, dual target inhibitor of DNA-PK and PI3Kδ. The combined inhibition of dual targets tightly regulates the protein stability of c-Myc independent of genetic alterations such as translocation, amplification, and overexpression. The purpose of this study is to investigate the functional significance of BR101801 on the growth inhibition and c-Myc regulation in double hit and double expressor DLBCL cell lines.
Methods: Westernblot of protein expression levels was analyzed with the human DLBCL cell lines that were treated with BR101801, PI3Kδ-specific inhibitors (Idelalisib, TGR-1202) and DNA-PK inhibitor (NU-7441) or DMSO for 24 hr. After treating SU-DHL-4 cell with cyclohexamide (CHX, 20 ug/ml), and BR101801 (0.5 μM) alone or in combination, the lysates was analyzed with the westernblot. Cells (5x103/well) were incubated with various concentrations of compounds and the cell viability was determined after 72 hr by the WST-8 assay. Cell lines were classified as c-Myc translocation (DOHH2, SU-DHL-6, SU-DHL-10, OCI-LY-18), c-Myc amplification (SU-DHL-4, OCI-LY-3), and c-Myc expressor (U2932). Cell lines additionally showed BCL-2 alteration such as translocation (DOHH2, SU-DHL-4, SU-DHL-6, OCI-LY-18) and amplification (OCI-LY-3, U2932).
Results: BR101801 showed potent inhibitory activity on c-Myc protein stability whereas no activity was found in Idelalisib, TGR-1202, or NU7441 (Figure 1A; SU-SHL-4, 1B; SU-DHL-6). Using CHX to block de novo protein synthesis on SU-DHL-4 cells, the increased rate of c-Myc degradation by GSK3β regulation was observed (Figure 1C). SU-DHL-10 cell lines harboring c-Myc alterations exhibited no sensitivity to the PI3Kδ inhibitor such as Idelalisib (IC50≒4.4 μM) and TGR-1202 (IC50>10 μM) and DNA-PK inhibitor such as NU7441 (IC50≒1.1 μM). However, the combination of Idelalisib and NU7441 resulted in 200-fold (IC50≒ 0.02 uM) growth inhibition (Figure 1D), which was comparable to the effect of BR101801. SU-DHL-6 cells showed low sensitivity to Venetoclax alone with IC50≒0.16 μM. However, the IC50 value of Venetoclax at 0.1, 1, 10 nM of BR101801 was decreased to 0.12 uM, 0.003 uM and < 0.001 uM (11 pM), respectively in a dose-dependent manner (Figure 1E).
Conclusions: In this study, the rationale of co-targeting of PI3Kδ and DNA-PK in c-Myc regulation and growth inhibition were demonstrated. The dual targets of DNA-PK and PI3Kδ were essential to control the protein stability of c-Myc in DLBCL cell lines harboring various c-Myc alterations. In conclusion, the potent effect of BR101801 with Venetoclax in DLBCL cells harboring alterations on both c-Myc and BCL-2 suggests a mechanistic rationale as a breakthrough treatment for RR DLBCL patients. To ensure translation to humans, the study on the efficacy of BR101801 in primary cells isolated from double hit and double expressor as well as RR DLBCL patients is currently conducted.
No relevant conflicts of interest to declare.
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