The Pathophysiological Significance and Therapeutic Targeting of PPM1D-Mediated Signaling in Mantle Cell Lymphoma

Introduction Mantle cell lymphoma (MCL) is a distinct type of non-Hodgkin lymphoma genetically characterized by balanced t(11:14) translocation and cyclin D1 overexpression. A great majority of patients with MCL remain incurable. The selection of chemoresistant subclones occurs during therapies, leading to relapse of MCL. Many chemotherapeutic agents induce DNA damage and activate the tumor suppressor p53. Recently, non-genotoxic p53 activators like MDM2 (master regulator of p53) inhibitors and XPO1 (nuclear exporter for p53) inhibitors have entered clinical trials. p53 stabilization, however, has not always led to p53-mediated transcriptional activation in MCL. PPM1D is a serine/threonine phosphatase that negatively regulates key DNA damage response proteins, such as p53, p38 MAPK, histone H2A.X, and ATM. PPM1D has been thought to be an oncoprotein that inhibits p53. PPM1D overexpression or amplification has been reported in various cancers, including lung, breast, kidney and ovarian cancers. We investigated the pathophysiological significance of PPM1D and its therapeutic targeting by the novel PPM1D inhibitor GSK2830371 (GSK) (Nat Chem Biol 2014) in MCL.

RESULTS Oncomine-based analyses indicated increased PPM1D mRNA levels in MCL cells compared with their normal counterpart B-lymphocytes. PPM1D mRNA levels positively correlated with CCND1 (Cyclin D1) mRNA levels (r = 0.33, P = 0.0014; n = 92) and with proliferation signature averages (r = 0.54, P < 0.0001; n = 92) in a series of MCL samples. Increased PPM1D expression at diagnosis was itself associated with a poorer prognosis in MCL patients (median overall survival of 3.9 years and 1.4 years for cases in the lowest and highest PPM1D expression tertiles, respectively; P = 0.0047). PPM1D levels in MCL were as high as those in aggressive lymphomas including Burkitt's lymphoma and diffuse large B-cell lymphoma, and were significantly higher than those in indolent lymphomas including chronic lymphocytic leukemia/small lymphocytic lymphoma (P = 0.0076) and follicular lymphoma (P = 0.011). Eight MCL (three p53 wild-type (WT) and five mutant (MUT)) cell lines were exposed to GSK. GSK inhibited the cell growth, being more prominent in p53 WT cells (48.3 ± 9.8% versus 14.8 ± 4.7% growth inhibition, P = 0.036). In sensitive cells, GSK caused a significant loss of mitochondrial membrane potential in addition to increased annexin V positivity, indicating apoptosis induction. Stable p53-specific shRNA-expressing cells were generated in p53 WT Z-138 and JVM-2 cells, with > 85% knockdown efficiency. p53 knockdown cells were less susceptible to GSK than control cells, indicating that GSK utilizes p53-mediated signaling to eradicate MCL cells. This idea is supported by the Western blot data that GSK treatment increased total and phosphorylated p53 levels and those of p53 targets p21 and PUMA. Basal and GSK-induced levels of PPM1D and its target proteins NFkB-p65, p38 MAPK, and histone H2A.X did not predict MCL cell sensitivity to GSK. GSK and the MDM2 inhibitor Nutlin-3a acted synergistically in p53 WT MCL cells. Importantly, GSK sensitized MCL cells to bortezomib (BTZ) and doxorubicin (DOX) irrespective of p53 mutational status. BTZ and DOX are clinically active agents against MCL. In p53 WT cells, GSK/BTZ and GSK/DOX combinations potently activated p53-mediated apoptosis signaling. In p53 MUT cells, combination treatment did not activate p53 signaling. Instead, p38 signaling appeared to be actively involved in the GSK/BTZ lethality, as the selective p38 inhibitor SB203580 significantly attenuated bortezomib- and GSK/bortezomib-induced lethality. Activation of p38 MAPK has been found to cause MCL cell death. SB203580 did not protect MCL cell from doxorubicin- or GSK/doxorubicin-induced lethality.

CONCLUSION PPM1D inhibition increases levels of phosphorylated p53 at Ser15 in a non-genotoxic manner, which enhances p53-mediated transcription in MCL cells. In addition, PPM1D inhibition shows anti-lymphoma effects in p53 MUT cells, partially through activation of p38 signaling. Although a single-agent activity of GSK was modest against MCL cells, especially against those with mutant p53, its p53-independent potentiation effect on BTZ and DOX may support the use of PPM1D inhibitors as part of a combination therapeutic strategy for MCL. Collectively, PPM1D inhibition may offer a novel therapeutic strategy for MCL.