Downregulation of BMI-1 By the Small Molecule PTC596 Induces Mitochondrial Apoptosis in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma typically associated with the t(11;14) translocation, resulting in overexpression of cyclin D1 (CCND1). Although the clinical approval of the first-in-class Bruton's tyrosine kinase (BTK) inhibitor ibrutinib provides a new treatment option, the great majority of patients with MCL remain incurable. Studies are needed to optimize the role of ibrutinib in MCL therapy and to address ibrutinib resistance. BMI-1 is crucially involved in cancer stem cell self-renewal and maintenance. BMI-1 has been shown to be up-regulated in MCL cells, especially in tumor-initiating side population (SP) cells (Teshima et al. Oncogene 2014). Importantly, BMI-1 levels are higher in refractory/relapsed patients than those at initial diagnosis. PTC-209 and PTC596 are recently developed novel small-molecule selective BMI-1 inhibitors with different modes of action (Kreso et al. Nat Med 2014). PTC-209 inhibits BMI-1 production while PTC596 accelerates BMI-1 degradation. We have reported anti-leukemia effects of the BMI-1 inhibitors PTC-209 and PTC596 (Cancer Sci 2015; Blood Cancer J 2017) in acute myeloid leukemia cell lines and patient-derived leukemia blasts. In this study, we investigated the effects of PTC-209 and PTC596 in MCL. Oncomine analysis showed that BMI1 mRNA levels were higher in MCL cells than in normal B cells. The levels were not specifically associated with CCND1 levels, ATM, CDKN2A or TP53 mutations, or patient prognosis, implying that BMI1 overexpression may be a fundamental abnormality in MCL. PTC-209 and PTC596 inhibited cell growth and induced apoptosis in 8 MCL cell lines in a dose- and time-dependent manner. Their IC₅₀ values (concentration at which cell growth is inhibited by 50%) ranged from 1.5 to 11.2 µM for PTC-209 and from 68 to 340 nM for PTC596. ED₅₀ values (effective concentration inducing 50% cell killing as measured by annexin V positivity) ranged from 2.7 to > 50 µM for PTC-209 and from 150 to 507 nM for PTC596. PTC596 was > 10 times more potent than PTC-209. IC₅₀ and ED₅₀ values of PTC-209 significantly correlated with those of PTC596 [r =0.94 (P= 0.0004) for IC₅₀ and r =0.85 (P= 0.015) for ED₅₀], supporting the idea that their anti-lymphoma activities depend on downregulation of BMI-1. In support of this, high BMI-1 protein levels predicted high susceptibility to PTC596 (r = -0.88; P=0.0039). The anti-lymphoma effects of PTC596 appeared to be p53-independent since IC₅₀ and ED₅₀ values were not significantly different against p53 wild-type and p53 defective cell lines. In addition, stable knockdown of p53 did not significantly affect MCL cell susceptibility to PTC596. Activity of PTC596 was independent of MCL cell susceptibility to ibrutinib. PTC596 induced mitochondrial apoptosis, as evidenced by loss of mitochondrial membrane potential, caspase-3 cleavage, BAX activation and phosphatidylserine externalization in MCL cells. PTC596 effectively decreased SP cells (by 97.3% at 150 nM, 99.8% at 300 nM and >99.9% at 600 nM PTC596) compared to ibrutinib that decreased SP cells by 33% even at 5 µM ibrutinib. Combination of PTC596 with bortezomib or doxorubicin showed additive anti-proliferative and apoptotic effects. The averaged combination index values were approximately 1, ranging from 0.95 to 1.18. Interestingly, PTC596 synergized with ibrutinib in inducing cell death, through modulation of the BCL-2 family protein MCL-1. Collectively, BMI-1 inhibition may offer a novel therapeutic strategy for MCL.