Combination of PI3K and PDPK1 Inhibitors Is Highly Effective in Mantle Cell Lymphoma

Introduction: Mantle cell lymphoma (MCL), is a distinct lymphoma subtype with an aggressive clinical course and a median survival of 3-5 years. New emerging strategies include especially inhibitors of the PI3K/Akt/mTOR pathway which is constitutively activated in MCL and plays a critical role in tumor growth and survival. In the present study we analysed four different PI3K-Inhibitors (IPI-145, CAL101, A66 and TGX221) targeting different isoforms (PI3Kδ/γ, PI3Kδ, PI3Kα and PI3Kβ) for their effectiveness in MCL.

Methods: MCL cell lines (Z-138, Mino-1, Granta-519, Jeko-1, Rec-1, Maver-1) were exposed to different PI3K-Inhibitors (IPI-145, CAL101, A66 and TGX221) with or without murine feeder layer (M210B4). The effect of drugs was evaluated by cell count (trypan-blue staining), cell metabolism (WST-assay or ONE-Glo^TMLuciferase assay for luciferized MCL cell lines), cell cycle (FACS) and apoptosis (Annexin V PE/7-AAD staining). Subsequently, combinations of PI3K-inhibitors and other target molecules (PDPK1: BX912, OSU03012, GSK2334470) were analysed. Western blot and mRNA analyses were performed after exposure to various inhibitors of the PI3K/Akt/mTOR pathway and correlated to sensitivity of cell lines. Finally, efficiency of drug combinations were confirmed in primary patient samples.

Results: The PI3K inhibitor IPI145 (8,2%-62,4%) was most effective in MCL cell lines followed by A66 (0%-41,7%), TGX221 (0%-33,8%) and CAL101 (0%-28,2%) suggesting a higher efficiency by inhibiting multiple isoforms of PI3K. In patient samples IPI145 revealed also the highest cytotoxicity followed by CAL101, TGX221 and A66 (72,4%; 67,9%; 67,1% and 43,7%, respectively). Comparing various combinations of PI3K-inhibitors in MCL cell lines the simultaneous inhibition of α (A66) and δ (CAL101) isoforms achieved the highest reduction of cell count (65,2%). However, combined inhibition of both of PI3K δ and PDPK1 (BX912), led to a more efficient G2 arrest and higher rate of apoptosis due to the simultaneous dephosphorylation of members of the mTOR- and MAPK-pathways. Accordingly, the observed cytotoxicity was even higher (59%-87%) than for dual inhibition of PI3K (23%-60%). Currently, Western blot analyses are being expanded to further identify the differential mechanisms of PI3K inhibitor combinations.

In addition, the presence of a murine feeder layer enhanced the effect of PI3K inhibitors in MCL cells nidated into the feederlayer because of higher levels of Akt phosphorylation possibly due to interaction with the microenvironment. Accordingly, this effect was not detectable in the supernatant cells.

Conclusion: Based on our comparative analysis of PI3K inhibitors targeting of a and d isoforms appears to be the most efficient mechanism in MCL. However, this impact could be further increased by simultaneous inhibition of PDPK1. Western blot results suggest that these results are due to the inhibition of complementary pathways. Interestingly, the murine feeder layer enhanced the effect of PI3K inhibitors emphasizing the role of the microenvironment in the molecular mechanisms of targeted strategies.