The Expression of Paks and Its Clinical Significance in T-Cell Lymphoblastic Lymphoma

Background:

T-cell lymphoblastic lymphoma (T-LBL) is a highly aggressive non-Hodgkin’s lymphoma with poor prognosis and lacks of standard treatment approaches. PAK2, a member of the p21 activated kinase (PAKs) family, is a component of the gene-expression-based classifier which made great contributions to prognostic prediction of T-LBL(Cai et al. Leukemia 2020). This study aims to analyze the expression of PAKs in human T-LBL cell lines and tissues to clarify its clinical significance and evaluate the therapeutic activity of PAKs inhibitor in T-LBL.

Methods:

The mRNA and protein expression levels of PAKs in human T-lymphoid cell lines (H9) and T-LBL cell lines (Jurkat, SUP-T1 and CCRF-CEM) were detected by quantitative real-time PCR and western blot, respectively. We retrospectively collected 69 Formalin-fixed, Paraffin-embedded (FFPE) samples and corresponding clinical information from T -LBL patients between September 2000 and May 2015 at Sun Yat-sen University Cancer Center (SYSUCC). Affymetrix Human Gene 2.0 ST microarray (Thermo Fisher Scientific, Waltham, MA, USA) was used to detect the expression of PAKs. Mann-Whitney U test was used for comparing the expression differences between relapsed and non-relapsed patients. Cumulative relapse-free survival (RFS) time was calculated using the Kaplan-Meier method (Expression level higher than the upper quartile (P75) was defined as PAK1 or PAK 2 high expression. High expression (median as cutoff point) of both PAK1 and PAK2 was defined as PAK1/2 high expression). Pearson’s chi-square test and Fisher’s exact test were used to compare the distribution of clinical variables. Correlations between PAK1, PAK2 and NOTCH1 were evaluated using Spearman’s correlation coefficient. Two PAK inhibitors, PF3758309 (PF) and FRAX597, were used to block PAK kinase activity pharmacologically. Cell viability was determined using the viability assay kit CCK-8. Cell cycle and cell apoptosis were analyzed by flow cytometry. All statistical analyses were performed using SPSS 24.0 software (SPSS, Armonk, NY, USA) or GraphPad Prism 8.0 (GraphPad, La Jolla, CA, USA). A P value < 0.05 was considered significant. The study protocol was approved by the Institutional Review Board of SYSUCC.

Results:

The mRNA and protein expression levels of PAK1 in T-LBL cell lines of Jurkat, SUP-T1 and CCRF-CEM cell lines were significantly higher than those in T-lymphoid cell lines H9 cell line (P<0.05) (Figure 1). Of the 69 T-LBL patients, 44 (63.8%) were male and the median age at diagnosis was 30 years (range: 16–44). The majority (72.5%) of the patients received acute lymphoblastic leukemia (ALL)-type chemotherapy regimens. The PAK2 mRNA expression level of 32 patients with relapsed disease was significantly higher than that of 37 cases without relapse (P=0.012), and no difference was found in mRNA expression of PAK1, 3, 4, 7(Figure 2). Patients with high PAK1 and PAK2 expression had significantly shorter median RFS than those with low PAK1 and PAK2 expression (PAK1 mRFS 31.5 months vs not reached(NR), HR=3.001, P=0.028; PAK2 mRFS 25.7 months vs NR, HR=3.981, P=0.027)(Figure 3). Patients with high PAK1/2 mRNA expression experienced earlier relapse than patients with low PAK1/2 mRNA expression (mRFS 26.0 months vs NR, HR=2.721, P=0.032)(Figure 3). PAK1/2 mRNA expression was found to be associated with hemoglobin concentration, Ki-67 expression, pleural and pericardia effusion, bone marrow involvement and chemotherapy response (P<0.05)(Table 1). Further, the PAK1 mRNA was correlated with the expression of NOTCH1, which is frequently mutated in T-LBL (r=0.5716, P<0.0001)(Figure 4). The PAK inhibitors PF and FRAX597 demonstrated strong anti-tumor activity in vitro (Table 2). Both inhibitors suppressed cell proliferation in a time- and dose-dependent manner (Figure 5). Both inhibitors induced cell cycle arrest in the G1/0 phase, accompanied by corresponding S phase reductions in all tested cells(Figure 6). The synergistic effect between PAK inhibitor PF and doxorubicin was also observed (Figure 7). Besides, PF could inhibit the phosphorylation of PAK1/2, Cyclin D1 and NF-κB in Jurkat cell line(Figure 8).

Conclusions:

PAK1 and PAK2 play certain roles in the occurrence and recurrence of T-LBL, and their potential as novel biomarkers deserves further exploring. Our results underscore the potential of PAK inhibitor as effective target therapy for T-LBL.