Abstract 2651: MK2206 disrupts cytoplasmic AKT-p21 complex in mantle cell lymphoma: Evidence of p21 coupled cell cycle arrest
Authors: Sarkar Aloke Kumar 
Published: August 2015

Abstract
Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin's lymphomas with poor response to conventional chemotherapy. MCL has several deregulated signaling pathways including PI3K/Akt/mTOR axis. The serine/threonine kinase AKT is a proto-oncogene that has emerged recently as a major focus of target due of its critical regulatory role in diverse cellular processes, including cancer development. We hypothesized that inhibition of AKT activation through an inhibitor, MK2206 or stable knockdown of AKT expression via Lentiviral shAKT would result in cytotoxicity and sensitize MCL cells to chemotherapeutic drug such as bendamustine. MCL cell lines (Jeko-1 and Mino) were treated either with MK2206 (2.5 or 5μM) or bendamustine (15 or 30μM) for 24hr and molecular changes or cell death were evaluated by immunoblot or Annexin-V-PI FACS analysis, respectively. MK2206 treatment significantly reduced pAKTser473 or its target pBADser110 phosphorylation in both cell lines. Further, MK2206 treatment resulted in growth arrest in both Jeko-1 and Mino cells via p21Cip1/WAF1 upregulation. Preliminary studies on immunoprecipitaion with anti-AKT antibody and cell fraction revealed that MK2206 treatment disrupts cytoplasmic AKT-p21 complex and enhanced p21-nuclear PCNA interaction in both Jeko-1 and Mino cells thereby allowing more p21Cip1/WAF1 to accumulate into the nucleus leading to p21 coupled S-phase changes in Jeko-1 (control = 41% vs MK2206 treated = 28%) and Mino (control = 54% vs MK2206 = 39%). Genetic and stable lentiviral knockdown of AKT resulted in similar impact on S-phase progression in Jeko-1 (control = 58% vs shAKT = 34%) and sensitized cells to bendamustine (15μM for 24hr) -induced cell death in Mino (control shRNA = 12%, bendamustine alone = 44%, and shAKT plus bendamustine = 68%, p&lt;0.01). MK2206 did not induce any detectable cell death by itself, however a significant apoptosis was induced when these cells were treated with 30μM bendamustine (p&lt;0.04). MK2206 (2.5μM) treatment in combination with bendamustine (15μM) resulted in significant cell death in both p53 deficient Jeko-1 (40% vs control p&lt;0.01) and p53 proficient Mino (65% vs control p&lt;0.014). Similarly, 5μM MK2206 plus 30μM bendamustine combination was more potent (65% in Jeko-1 and 76% in Mino) compared with either MK2206 (2-5%) or bendamustine alone (20-45%). Western blot analysis revealed induction of intrinsic apoptotic pathway including PARP cleavage, loss of XIAP and activation of caspase-3 cleavage. In conclusion, a novel mechanism of MK2206 is demonstrated where disruption of cytoplasmic AKT-p21 interaction followed by more nuclear p21-PCNA complex leading to p21 coupled growth arrest in MCL cell lines. These studies underscore the importance of AKT inhibition to sensitize apoptotic cell death in combination with standard chemotherapies in difficult to treat MCL.
Citation Format: Aloke Kumar Sarkar. MK2206 disrupts cytoplasmic AKT-p21 complex in mantle cell lymphoma: Evidence of p21 coupled cell cycle arrest. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2651. doi:10.1158/1538-7445.AM2015-2651