The Novel CDK9/CDK4/6/PI3K Triple Inhibitor LCI139 for the Treatment of MYC-Driven Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an aggressive B cell malignancy with characteristic t(11;14)(q13;q32) translocation juxtaposing IGH and CCND1 gene loci. This chromosomal rearrangement leads to Cyclin D1 overexpression and cell cycle dysregulation at the G1/S phase transition. MCL is generally diagnosed at advanced stages and follows an aggressive course of disease. Although chemotherapy-based combination regimens are initially effective, most patients relapse with a median survival of only 2.5-5 years. While novel targeted therapeutics have shown promising activity in relapsed/refractory patients, inevitable relapse associated with progressively declining efficacy and increasing resistance to single agent targeted therapy is a major concern. To overcome this barrier, we designed in-silico based a novel multitarget small molecule inhibitor LCI139 which simultaneously blocks three oncogenic targets: cyclin-dependent kinase 9 (CDK9), CDK4/6, and phosphatidylinositol-3 kinase (PI3K). LCI139 is a potent CDK9/CDK4/6/PI3K inhibitor with IC₅₀s of 0.000039µM (CDK9), 0.0015µM/0.0036µM (CDK4/6) 0.070µM (p110α), 0.461µM (p110γ), and 0.214 (p110δ), as determined by cell free assay alpha screens.

Dose-dependent screening of LCI139 against a panel of MCL cell lines revealed robust cytotoxic effects with maximal IC₅₀ of 86.67 nM in JeKo-1 cells, 64.8 nM in Mino cells, and 202.5 nM in Rec-1 cells. Compared with clinical single agent CDK9 inhibitor AZD4573, LCI139 is less toxic to normal human stromal cells (HS-5), whereas both LCI139 and AZD4573 have no significant cytotoxic effect on normal human foreskin fibroblast cells (Hs68). LCI139 treatment at various concentrations for 24hrs induced significant apoptotic response in two MCL cell lines, JeKo-1 (p<0.001 DMSO vs LCI139 100 nM, p<0.0001 DMSO vs LCI139 200 nM) and Mino (p<0.05 DMSO vs LCI139 100 nM, p<0.0001 DMSO vs LCI139 200 nM) as determined by Annexin V/7-AAD apoptosis staining. Similarly, LCI139-induced apoptotic cell death was further confirmed by increasing cl-PARP protein levels compared to control as assessed by Western blot analysis. LCI139 induced robust G2/M cell cycle arrest in JeKo-1 cells and G1 arrest in Mino cells.

The mechanism of action of pan-CDK inhibitors has been associated with downregulation of the anti-apoptotic protein MCL-1 through inhibition of transcriptional CDKs. Our results show that short-term exposure of LCI139 (1hr and 3hr) significantly suppressed MCL-1 and MYC mRNA expression in JeKo-1 (p<0.00001 DMSO vs LCI139) and Mino cells (p<0.00001 DMSO vs LCI139). LCI139 suppressed pRpb1 (Ser2), MCL-1 and cMYC protein expression in IgM-stimulated JeKo-1 and Mino cells in a dose-dependent manner. Treatment with LCI139 had no significant effect on pAKT (Ser473) in Jeko-1 cells, whereas pAKT (Ser473) was completely abolished in Mino cells at 500nM of LCI139. Mechanistically, our findings also demonstrate that LCI139 decreased MCL-1 and cMYC protein stability and reduced half-life as accessed by cycloheximide chase assay. This decrease in cMYC stability is associated with decreased of cMYC phosphorylation at Ser62. Adaptive kinome remodeling via drug-induced transcriptional reprogramming is a known contributor to ibrutinib resistant (IR) MCL. RNA-sequencing analysis of JeKo-1 vs Jeko-1 IR cells revealed a total of 3468 differentially expressed genes (FC > 1.5 and FDR < 0.05) where 110 kinases display altered expression in JeKo-1 IR cells. Finally, we show that LCI139 overcomes chronic ibrutinib resistance by decreasing viability in JeKO-1 IR and Mino IR cells with an IC₅₀ of 79 nM and 89 nM, respectively. Therefore, the multitarget small molecule inhibitor LCI139 has superior potency against IR MCL cell lines and overcomes ibrutinib-resistance at nanomolar doses.