PAK4-NAMPT Dual Inhibition Sensitizes Pancreatic Neuroendocrine Tumors to Everolimus

Abstract

Metastatic pancreatic neuroendocrine tumors (PNET) remain an unmet clinical problem. Chronologic treatment in PNETs includes observation (watchful protocol), surgery, targeted therapy, and chemotherapy. However, increasing evidence illustrates that the outcomes of targeted therapeutic options for the treatment of advanced PNETs show minimal response. The FDA-approved mTOR inhibitor everolimus does not shrink these tumors. It only delays disease progression in a subset of patients, while a significant fraction acquires resistance and shows disease progression. Thus, there is a need for more effective targeted approaches to sensitize PNETs to everolimus for better treatment outcomes. Previously, we showed that mTOR regulator p21 activated kinase 4 (PAK4) and nicotinamide adenine dinucleotide biosynthesis enzyme nicotinamide phosphoribosyl transferase (NAMPT) were aberrantly expressed in PNET tissue and promoted everolimus resistance. In this report, we demonstrate that PAK4-NAMPT dual inhibitor KPT-9274 can synergize with everolimus (growth inhibition, colony suppression, and glucose uptake assays). KPT-9274-everolimus disrupted spheroid formation in multiple PNET models. Molecular analysis showed alteration of mTORC2 through downregulation of RICTOR as a mechanism supporting synergy with everolimus in vitro KPT-9274 suppressed β-catenin activity via inhibition of PAK4, highlighting the cross-talk between Rho GTPases and Wnt signaling in PNETs. KPT-9274, given at 150 mg/kg in combination with sub-MTD everolimus (2.5 mg/kg), significantly suppressed two PNET-derived xenografts. These studies bring forward a well-grounded strategy for advanced PNETs that fail to respond to single-agent everolimus.

Trial registration: ClinicalTrials.gov NCT00932126 NCT02702492.

©2021 American Association for Cancer Research.

Figures

Figure 1:. KPT-9274 in combination with everolimus dramatically inhibits the growth of PNET cell lines in vitro.

[A-C] Colony formation assay showing that KPT-9274 plus everolimus promotes a long-term inhibition of PNET cell line in vitro. [D] Caspase3/7 activity assay performed according to the manufacturers’ protocol. The assay shows activation of the mechanism of apoptosis 72 hours post-treatment with the indicated drugs. Caspase activity was enhanced in the combination treatment. The result is representative of three independent experiments. [E, F] BON-1 and QGP-1 spheroids, KPT-9274 in combination with everolimus significantly inhibit the growth and the number PNET spheroids in vitro. [G] NAD Cell Titer-Glo assay was performed according to the manufacturer’s protocol. The graphs show a statically significant reduction of the NAD pool after treatment with the indicated drugs. FK866 was used as a positive control. The reduction of NAD in the control treatment was mainly due to the effect of KPT-9274 rather than everolimus. RFU, relative fluorescence unit; *P<0.05, **P<0.01, and *** P<0.005). Each graph represents 3 independent experiments.

Figure 2:. KPT-9274 downregulates the expression of PAK4 and NAMPT in BON-1 and QGP-1.

[A, B, and D] 50000 BON-1 and 100000 QGP-1 cells were grown in 100 mm Petri dishes and exposed for 72 hours to the shown concentrations of each small molecule inhibitor as described in the methods section. 50 μg of protein lysates were resolved on a 12% SDS-PAGE following by western blot comparing the expression level of PAK4, NAMPT, NAPRT-1. β-TUBULIN was used as an internal control. [C, E, and F] RT-qPCR comparing the expression level of PAK4, NAMPT, and NAPRT-1 in PNET cell line BON-1 and QGP-1. Here, we used mRNA samples with RIN# greater than 2. Each expression level was normalized with actin mRNA. Each graph represents 3 independent experiments.

Figure 3:. WNT/β-CATENIN regulates glucose uptake in PNETs cells.

50000 BON-1 and 100000 QGP-1 cells were grown in 100 mm Petri dishes and exposed for 24 hours to the shown concentrations of the small molecule inhibitor KPT-9274 as described in the methods section. [4A, and 4B] represent RT-qPCR comparing the expression level of β-CATENIN, WNT in PNET cell lines post-treatment with increasing concentration of KPT-9274. Each expression level was normalized with actin mRNA. We used samples with RNA integrity numbers greater than 2. Each graph represents 3 independent experiments. [4C, and 4D] Glucose uptake in PNET cells 24 hours post-treatment with KPT-9274 (solid, dashed and dottle lines represent untreated, apigenin treated and KPT-9274 treated cells respectively), each condition was done in triplicate.

Figure 4:. Inhibition of mTORC2 by KPT-9274 promote synergy with everolimus in PNET cell lines.

[A, B, C, D, and E] RT-qPCR comparing the expression level of PI3K, Akt, mTOR, RICTOR, and RAPTOR in PNET cell line QGP-1 post-treatment with increasing concentration of KPT-9274. Each expression level was normalized with actin mRNA. Each graph represents 3 independent experiments. [F] BON-1 and QGP-1 cells were grown in 100 mm Petri dishes and exposed to the shown concentrations of KPT-9274 as described in the methods section. 50 μg of proteins were resolved on a 10% PAGE following by western blot comparing the expression level of pmTOR, mTOR, pP70S6K, P70S6K, RICTOR, RAPTOR. β-TUBULIN was used as an internal control.

Figure 5:. KPT-9274-everolimus shrink BON-1 and QGP-1 growth in vivo.

BON-1 and QGP-1 cells were grown as subcutaneous xenografts in ICR-SCID mice. [A, and D] Gross visualization of excised tumors. [B, and E] Graphical representation of tumor weight post-treatment. [C, and F] Animal body weight at the indicated days during the treatment. Five mice per group were used in the QGP-1 experiment and eight mice per group were used in the BON-1 experiment. Treatment in the BON-1 experiment was extended to 12 weeks to track eventual toxicity. [A] In the control group, one mouse developed malocclusion and was euthanized; in the KPT-9274 single-agent group, one mouse did not develop tumors (*** representing P<0.02).

Figure 6:. Mechanism of action of KPT-9274 in PNET cells.

mTORC2 regulates resistance to everolimus in NET cells. Everolimus inhibits mTORC1 and its downstream effectors 4EBP-1 and P70S6K by preventing mTORC1 to interact with its intracellular receptor FKBP12 which induces mTORC2 to phosphorylate AKT thus promoting PI3K/AKT/mTOR signaling. KPT-9274 inhibits PAK4 which downregulates RICTOR then alters the activation of mTORC2. Inhibition of PAK4 also downregulates β-CATENIN. Inhibition of NAMPT reduces the cellular level of NAD and ATP. All the latter events promote PNETs shrinkage.