Newly Synthesized Indolylmalemide PDA-66 and Its Derivates Induce Antiproliferative Effects In Acute Lymphoblastic Leukemia
Authors: Roolf Catrin , Kretzschmar Christin , Langhammer Tina-Susann , Sekora Anett , Pews-Davtyan Anahit , Beller Matthias , Rolfs Arndt , Freund Mathias , Junghanss Christian 
Published: October 2019

Abstract
The PI3K/Akt pathway is dysregulated is some acute lymphoblastic leukemias (ALL) and might therefore serve as therapeutic target. Indolylmaleimides exhibit inhibitory potencies against different protein kinases -like glycogen synthase kinase 3 (GSK3β) or protein kinase c- influencing thereby several cellular processes. Recently, it was demonstrated that PDA-66, a newly synthesized indolylmalemide based on the well known GSK3β inhibitor SB-216763, hinders microtubule polymerization in human neuronal progenitor and neuroblastoma cells. GSK3β is a downstream substrate of the PI3K/Akt and Wnt pathways and is often deregulated in tumor tissues. Herein, we investigated the effects of PDA-66 and its derivates (PDA-66E und PDA-377) on B and T-lymphoblastic leukemia cells.

Methods
B- and T-ALL cell lines (SEM, RS4;11, REH, Jurkat, MOLT-4 and CEM) were incubated for 72 h with increasing concentrations (0.1 µM-5.0 µM) of PDA-66, PDA-66E, PDA-377 and comparatively analyzed to SB-216763. To evaluate the effect of each substance WST-1 assay, cell proliferation, cell cycle analyses as well as apoptosis rates were determined. Activities of indolylmalemides were analyzed by GSK3β kinase assay. Detection of key molecules of Wnt and PI3K/Akt signaling pathway was performed using Western blot.

Results
PDA-66 and derivates inhibited proliferation and metabolism of ALL cells significantly in a dose dependent manner. Interestingly, all PDA derivates showed a stronger inhibitory effect on proliferation than SB-216763 but the inhibitory effect on GSK3β kinase was lower than SB-216763. Antiproliferative effects of PDA-66 were studied in more detail. The incubation of 1 µM PDA-66 led to condensation of chromatin in the nucleus, karyorrhexis and an increasing amount of vacuoles after 48 h of treatment. PDA-66 influenced the cell cycle distribution of ALL cell lines differently. While RS4;11 and MOLT-4 cells were arrested in G2 phase, SEM cells remained increasingly in G0/1 phase. After 48 h all PDA-66 treated cell lines showed a significant increase in apoptosis compared to control cells (SEM: 2.1 ± 0.9 % to 10.5 ± 1.3 %; RS4;11: 2.5 ± 0.7 % to 7.4 ± 1.1 %; Jurkat: 3.8 ± 0.6 % to 8.3 ± 1.9 %; MOLT-4: 3.7 ± 1.2 % to 16.3 ± 5.1 %). Apoptosis of ALL cells was initiated by cleavage of caspase 3 and 7 and Poly (ADP-ribose) polymerase (PARP). Furthermore, with increasing concentration of PDA-66 a decrease of pGSK3βSer9 was observed after 4 h in SEM cells. However, no influence on the total form of β-catenin was detectable. Nevertheless, there was an influence of PDA-66 on the expression of 4EBP-1 and p4EBP-1Ser65. SEM, RS4;11 and Jurkat cells showed a decrease of the phosphorylated as well as the total form of 4EBP-1 after an incubation of 4 and 24 h.
In conclusion, our results demonstrate that the newly synthesized indolymalemides have pronounced antiproliferative effects in ALL cells. In particular, the indolymalemide PDA-66 should be further investigated concerning it’s clinical efficiency as well as well as it's intracellular ways of action.

Disclosures:
No relevant conflicts of interest to declare.