Abstract A157: Multi-kinase inhibitor H89 enhances activity of pseudomonas exotoxin-based immunotoxins targeting acute lymphoblastic leukemia
Authors: Mueller Fabian , Liu Xiu Fen , Wayne Alan S. , Pastan Ira 
Published: May 2016

Abstract
Objective: The Pseudomonas exotoxin (PE)-based anti-CD22 recombinant immunotoxin (RIT) Moxetumomab pasudotox (HA22) has a response rate of 86% in hairy cell leukemia (Kreitman et al 2012), but only about 30% of children with multiply relapsed ALL respond to HA22 treatment (Wayne et al AACR 2014). We attempted to improve activity against B-cell malignancies with a modified immunotoxin, LMB-11, which is missing domain II, has an Fab instead of a dsFv, and has 7 mutations in domain III. These modifications have been associated with reduced side effects, prolonged half-life, diminished immunogenicity, and better responses in in a Burkitt lymphoma animal model (Bera et al 2014). Activity of HA22 and LMB-11 on ALL cells varies significantly in vitro and in vivo (Müller et al AACR 2015). With the goal to improve activity against ALL, we combined CD22-targeting RITs with the multi-kinase inhibitor H89. H89 inhibits PKA (135 nM) and other kinases such as S6K1 (80 nM), MSK1 (120 nM), and ROCK2 (270 nM) (Davies et al 2000).
Methods: In vitro cytotoxicity (IC50) was determined by WST-8 assay, and cell death by Annexin V-PE/7-AAD flow cytometry. An in vitro screen for H89 targets was performed by Reaction Biology Corp.
Results: 10 uM H89 specifically enhanced activity of LMB-11 or HA22 10- fold on KOPN-8; activity of controls were unchanged. H89 also increased RIT activity by 5 to 10-fold on the ALL cell lines REH, SEM, HAL-01, and Nalm-6 and on 4 primary ALL samples.
H89 did not change the rates of RIT-internalization or of furin-cleavage in KOPN-8. However, the time to ADP-ribosylate EF2 and arrest protein synthesis was shortened from 10 to 4 h with H89. We tested phosphorylation states of downstream targets in H89 treated KOPN-8 cells. Phosphorylation level of the PKA targets p-CREB or p-ATF2 were not reduced by H89. A high baseline phosphorylation of rpS6 or GSK-3β at the S6K1 target site was decreased to undetectable levels 15 minutes after addition of H89 to KOPN-8 and MCL1 fell by 50% within 15 minutes. In KOPN-8, a low dose of 5 ng/ml LMB-11 or 10 uM H89, respectively lowered MCL1 but did not induce apoptosis; MCL1 was undetectable and PARP-cleavage induced when H89 and LMB-11 were combined.
The more specific S6K1 inhibitor PF-4708671 showed only 2-fold enhancement of RIT-induced cytotoxicity. The ROCK2 inhibitor Stemolecule similarly enhanced cytotoxicity 2-fold. MSK-1 inhibition or PKA-inhibition as well as PKA activation did not alter RIT activity. We performed a kinase screen on 359 kinases of which 21 were inhibited more than 90% by H89. None of the additionally identified kinases showed enhancement of RIT activity once blocked with relevant inhibitors. Despite combinations of small molecule inhibitors (e.g. ROCK2 and S6K1; GSK-3β and ROCK2), we were not able to mimic the 10-fold enhancement of RIT-induced cytotoxicity observed with H89.
Conclusion: H89 strongly enhances CD22-targeting RITs against ALL. The increase in activity was in part due to inhibition of S6K1 and ROCK2 and was reflected by a faster onset of ADP-ribosylation of EF2 indicating improved trafficking of RITs through the various intracellular compartments. H89 and RIT lowered MCL1 levels by a distinct mechanism and synergistically induced intrinsic apoptosis. Further pre-clinical testing to assess in vivo efficacy of this combination is ongoing.
Citation Format: Fabian Mueller, Xiu Fen Liu, Alan S. Wayne, Ira Pastan. Multi-kinase inhibitor H89 enhances activity of pseudomonas exotoxin-based immunotoxins targeting acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A157.