Small Molecule PRIMA-1 ^met Sensitizes Waldenstrom Macroglobulinemia Cells To Apoptosis and Displays Synergistic Cytotoxicity With Bortezomib

Waldenstrom macroglobulinemia (WM) is a lymphoplasmacytic lymphoma characterized by heterogeneous infiltration of bone marrow and IgM monoclonal gammopathy. WM constitutes 1-2% of haematological malignancies and patients exhibit diverse outcome. Given the current therapies WM remains incurable with high rate of relapse, therefore, the quest for finding a more effective drug continues. PRIMA-1^met is a small molecule with the ability to restore wild type conformation to mutant P53 and to activate wild type P53. PRIMA-1^met has shown significant anti-tumour activity both in vitro and in vivo by inducing apoptosis in several human cancers including haematological malignancies and is currently under phase I/II clinical trials. To date, the effects of PRIMA-1^met have not been explored in WM. Here we examined the antitumor activity of PRIMA-1^met alone and in combination with dexamethasone or bortezomib in WM.

WM cell lines, BCWM-1(Wild type P53) and MWCL-1 (mutant P53), were used for these studies. Cellular proliferation, viability, migration and percentage of cells undergoing apoptosis in WM cells treated with PRIMA-1^met or DMSO control were evaluated using trypan blue, MTT assay, Boyden chamber assay and Annexin-V staining methods respectively. The status of key role players in apoptosis and cell cycle regulators were examined following treatment with PRIMA-1^met using qRT-PCR and Western blot analyses. Cell viability was also investigated by combining PRIMA-1^metwith either dexamethasone or bortezomib in WM cells by MTT assay.

Both BCWM-1 and MWCL-1 cells showed significant decrease in cell viability after exposure to escalating doses of PRIMA-1^met with observed IC₅₀ of 30 μM in BCWM-1 cells and 28 μM in MWCL-1 cells after 48 hours. Two primary WM patients’ samples exposed to increasing concentration of PRIMA-1^met demonstrated significant decrease in cell viability with IC₅₀ of 30 and 10 μM after 48 hours. Importantly, peripheral and bone marrow mononuclear cells of 3 healthy donors were subjected to PRIMA-1^met and no significant cytotoxicity was observed. Both cell lines showed an increase in apoptosis after PRIMA-1^met treatment in a dose-dependent manner compared to control DMSO treatment. In BCWM-1, cell proliferation, measured by counting viable cells after 7 days, was significantly inhibited by PRIMA-1^met compared to DMSO control (∼54% fewer cells when treated with 25 μM PRIMA-1^met, p<0.05). Decrease in cell migration by as much as 28% at 25 μM compared to DMSO control (p<0.001) was also observed after 8 hours of treatment with PRIMA-1^met. Expressions of P53 and its downstream target p21 were elevated in PRIMA-1^met treated cells compared to DMSO control. In addition, qRT-PCR studies of 50 genes associated with apoptosis also demonstrated enhanced expression of pro-apoptotic markers such as PUMA, Noxa, caspase 8 and 9, BIM and BAD, which were further confirmed for their protein expressions by Western blot analysis. Finally, PRIMA-1^met (25 μM) was found to exert synergistic cytotoxic effect in combination with dexamethasone (2 μM) in MWCL-1 cells (Combination index (CI)=0.5). PRIMA-1^met (25 μM) also displayed synergism with bortezomib (3 μM) in its cytotoxicity toward BCWM-1 cells (CI=0.8).

Our results demonstrate that PRIMA-1^met induces apoptosis irrespective of P53 status in WM cells. It also provides the preclinical framework for evaluation of PRIMA-1^met either alone or in combination with bortezomib or dexamethasone as a novel therapeutic approach for the treatment of WM patients.

Chen:Roche: Honoraria; Johnson & Johnson: Consultancy, Research Funding; Lundbeck: Consultancy; Celgene: Consultancy, Research Funding; GlaxoSmithKline: Research Funding.