Protein Kinase Inhibitor H89 Enhances the Activity of Pseudomonas Exotoxin A-Based Immunotoxins

Abstract

HA22 (Moxetumomab pasudotox) is a recombinant immunotoxin (RIT), composed of an anti-CD22 Fv fused to a truncated portion of Pseudomonas exotoxin A. HA22 is in clinical trials to treat patients with hairy cell leukemia and acute lymphoblastic leukemia (ALL). LMB-11 is an improved variant of HA22 with reduced immunogenicity, has a longer half-life in the blood and high activity in vitro and in a Burkitt lymphoma model in vivo Searching for RIT enhancing combination therapies, we found the protein kinase A inhibitor H89 to enhance LMB-11 and HA22 activity 5- to 10-fold on ALL cell lines and on patient-derived ALL samples. In addition, H89 increased the activity of mesothelin-targeting RITs SS1P (38-fold) and RG7787 (7-fold) against the cervical cancer cell line KB31. Unexpectedly we found that the enhancement by H89 was not because of inhibition of protein kinase A; it was partially recapitulated by inhibition of S6K1, which led to inactivation of its downstream targets rpS6 and GSK3β, resulting in a fall in MCL1 levels. H89 increased the rate of ADP-ribosylation of eukaryotic elongation factor 2, enhancing the arrest of protein synthesis and the reduction of MCL1 in synergy with the RIT. In summary, H89 increased RIT activity by enhancing the two key events: ADP-ribosylation of eEF2 and reduction of MCL1 levels. Significant enhancement was seen with both CD22- and mesothelin-targeting RITs, indicating that H89 might be a potent addition to RIT treatment of CD22-positive ALL and mesothelin-expressing solid tumors. Mol Cancer Ther; 15(5); 1053-62. ©2016 AACR.

Figure 1. H89 specifically enhanced CD22-targeting RITs

A–E. KOPN-8 cells were incubated with 10 μM H89 or vehicle for 1 hour, then treated with LMB-11 (A), HA22 (B), LMB-31 (C), RG7787 (D) or Cycloheximide (CHX) (E) at indicated concentrations. For F, the ALL cell lines HAL-01, NALM-6, SEM or REH were treated with 10 μM H89 or vehicle for 1 hour after which LMB-11 was added at indicated concentrations. Cell viability was measured 72 hours later by WST8 assay, normalized to untreated control, and IC₅₀ determined using non-linear regression.

Figure 2. H89 enhanced arrest of protein synthesis, but not RIT uptake or furin cleavage

A. KOPN-8 cells were incubated with 10 μM H89 or vehicle (Con) for 1 hour and then treated with LMB-11-Alexa647 for the indicated times at 37°C. Surface-bound LMB-11 molecules were stripped off before analysis by flow cytometry. Untreated and unstained cells defined background signal (neg). B. KOPN-8 cells were treated with 10 μM H89 for 1 hour. HA22 was added at 1 μg/ml, incubated on ice for 30 minutes to allow for saturate binding, and then incubated at 37°C for the indicated times. Total cell lysates were analyzed with anti-PE by western blot. PE35 is the furin-cleaved catalytically active fragment of HA22. C. KOPN-8 cells were treated with 10 μM H89 or vehicle (Con) for 1 hour after which LMB-11 was added. Rate of protein synthesis was determined 16 hours later by incorporation of ³H-leucine. Values represent averages of three reads and are background subtracted absolute counts per minute (CPM).

Figure 3. H89 increased ADP-ribosylation of eEF2 and rate of apoptosis

A. KOPN-8 cells were treated with 10 μM H89 for 1 hour, then, 200 ng/ml of LMB-11 was added for the indicated times before cell lysis, followed by Western Blot analysis for Mcl-1, cleaved PARP, and actin. Not yet modified eEF2 in the cell lysates was labeled in a cell free reaction with biotinylated ADP by LMB-11 as described in methods. B. KOPN-8 cells were treated with 10 μM H89 or vehicle for 1 hour, then left untreated or treated with the indicated concentrations of LMB-11 for 24 (left panel) or 48 hours (right panel), respectively. Cells were then stained with Annexin V-PE and 7-AAD. Living cells were defined as double negative and blotted as percentage of all cells. Standard errors of mean were generated from three independent experiments.

Figure 4. PKA regulators did not affect LMB-11 activity; H89 inhibited S6K1 activity

A–B. KOPN-8 cells were treated with indicated concentrations of PKI (A) or Dibutyl-cAMP (B) for 1 hour and then with indicated concentrations of LMB-11 for 72 hours. Cell viability was measured by WST8 assays. C. KOPN-8 cells were treated with 10 μM H89 or vehicle (Con) for the indicated times. Total cell lysates were analyzed by Western Blot for anti-phospho-S6-Kinase 1 (p-S6K1(T389)), anti-total S6K1, anti-p-CREB(S133)/p-ATF-1, anti-p-ribosomal protein S6 (p-rpS6(S240/244)), anti-total rpS6, p-GSK3β(S9), anti-total GSK3β, anti-MCL1 and Actin.

Figure 5. H89 enhanced anti-mesothelin RIT-induced cytotoxicity against KB31 cells

A–B. KB31 cells were treated with 10 μM H89 for 1 hour. Then, indicated concentrations of SS1P (A) or RG7787 (B) were added. Cell viability was determined by WST8 assay. C. KB31 cells were plated on day 0, grown overnight in 6-well plates and on day 1 simultaneously treated with the combination of 20 ng/ml RG7787 and 15 μM H89 or vehicle. Images were taken at the day of treatment (Day1) and on days 3 and 4.

Figure 6. H89 enhanced activity of RIT on patient-derived ALL cells

Four patient-derived ALL samples were propagated in mice, extracted from murine spleen, and viably frozen. Thawed samples were treated with 15 μM H89 for 1 hour. Then, LMB-11 (A) or HA22 (B) was added at indicated concentrations. After 72 hours, cells were stained with anti-human CD19-FITC, Annexin V-PE and 7-AAD and analyzed by flow cytometry. ALL was defined as hu-CD19-positive. Percent living ALL cells were determined (negative for Annexin V-PE and 7-AAD) and normalized to RIT and H89 untreated control.