Dual Targeting PAK4 and NAMPT As a Novel Therapeutic Approach for Aggressive Non-Hodgkin's Lymphoma

Aggressive non-Hodgkin's lymphomas (NHLs) [diffuse large b-cell lymphoma (DLBCL), grade 3b follicular lymphoma, and mantle cell lymphoma (MCL)] have dismal cure rates, especially in the relapsed/refractory setting, substantiating the need for novel treatment strategies to improve outcomes. Here we show that p21-activated kinase 4 (PAK4) and nicotinamide phosphoribosyltransferase (NAMPT) are essential for lymphoma subsistence and dual-targeting PAK4-NAMPT by the small molecule inhibitor KPT-9274 (orally bioavailable phase 1 compound), previously shown to be cancer cell-selective, acts by energy depletion, inhibiting cell proliferation and eventual apoptosis as a reasonable treatment strategy for aggressive NHLs.

To demonstrate PAK4-NAMPT is essential for lymphoma survival, we showed increased PAK4 expression in primary NHL tissue (n=94) than in normal lymph nodes (n=8) (Fig1A). Lymphoma cell lines were sensitive to the PAK4/NAMPT dual inhibitor KPT-9274 (induced apoptosis with an EC ₅₀ of 95.17 nM in WSU-DLCL2 and 13.9 nM in WSU-FSCCL cells). Similar results were obtained with positive controls PAK4 [PF-3578309] and NAMPT specific inhibitors [FK-866]. RT-PCR confirmed PAK4 and NAMPT down-expression in KPT-9274 treated WSU-FSCCL cells. Western blot of WSU-DLCL2 and WSU-FSCCL cells treated with KPT-9274 showed significant reductions in BCL-2 and enhanced cleaved-Caspase-3 and cleaved-PARP expression.

We then assessed the impact of KPT-9274 on the cellular ATP and NAD pool since NAMPT inhibition can suppress both and induce cell death. NAD/NADH and ATP levels decreased in both cell lines [WSU-DLCL2 & WSU-FSCCL], with increasing KPT-9274 concentrations (Fig.1B&1C). We then showed KPT-9274 to synergize with the standard of care chemotherapy cyclophosphamide, vincristine, and adriamycin used for NHL management (decreased IC25 and IC50 in several combination treatments).

In lymphoma xenografts, mice with transplanted WSU-DLCL2 fragments showed ~50% reduction in tumor volume (p<0.01) after treatment with KPT-9274 (Fig.1D) without any significant change in body weight (Fig.1E). RT-PCR of extracted RNA from residual tumors (n=3) showed statistically significant inhibition of PAK4, BCL-2, and the PAK4 target β-catenin. Furthermore, caspase 9 activation was also observed. Western blots confirmed decreased p-PAK4 and increased cleaved caspase 9 expressions. In the WSU-FSCCL systemic model, KPT-9274 treatment improved animal survival (> 150-day increase in host life span compared to control and 3/6 mice were cured) (Fig.1F). After day 150, the remaining mice were dissected to confirm cures. Blood smears drawn from mice post three weeks of KPT-9274 treatment showed a significant reduction of circulating WSU-FSCCL cells validating its efficacy systemically (Fig.1G).

Since exogenous niacin can regenerate NAD regulated by NAMPT in a rate-limiting step, the impact of niacin co-dosing with KPT-9274 was evaluated on tumor growth using the Z-138 MCL tumor xenograft model in nude mice. Tumor-bearing mice were treated with vehicle, KPT-9274 (200 mg/kg BID), niacin (30 mg/kg), or a combination of KPT-9274/niacin. All groups that received KPT-9274 treatment showed reductions in tumor size, while the vehicle control and niacin alone groups showed a rapid increase in tumor volume. Both KPT-9274 and combo groups showed a significant decrease in tumor growth compared to control (p=0.0002); however, there were no statistically significant differences between the KPT-9274 and the KPT-9274/niacin combo (Fig.1H). Similarly, in another MCL xenograft model, KPT-9274 resulted in a statistically significant reduction in JeKo-1 tumors compared to control (p = 0.0059). Niacin did not affect the tumor growth suppression induced by KPT-9274 treatment. No animals died in this study. Thus, supplementing niacin with KPT-9274 may alleviate the adverse effect without compromising the efficacy of the treatment. Also, KPT-9274 treated Z-138 residual tumors showed a reduction in PAK4, GEF-H1, paxillin, vinculin, cyclin D1, Dvl2, and Ki67 and enhancement in apoptosis by IHC staining (Fig.1I).

The anti-tumor potential of KPT-9274 ± niacin in several aggressive lymphoma models strongly supports its efficacy in this setting. It strengthens our phase I study design to evaluate safety and tolerability and anti-tumor activity in patients with advanced solid malignancies or NHL (NCT02702492).

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