The BH3-mimetic GX15-070 induces autophagy-dependent cell death of therapy-sensitive and -resistant aggressive B cell non-Hodgkin’s lymphoma (B-NHL)

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The Bcl-2 family of proteins represents a rheostat that controls cellular viability. Interactions between members of BH3-domain containing proteins play an important role in the development, progression and prognosis in various subtypes of B-cell lymphomas. We previously reported that targeting Bcl-2 family proteins with GX15-070, a novel pan-inhibitor of anti-apoptotic Bcl-2 family members, induced time- and dose- dependent cell death of lymphoma pre-clinical models. Moreover, GX15-070 improved rituximab and/or chemotherapy activity in vitro. In an attempt to improve our knowledge of the therapeutic effects of BH3-mimetics, we studied the mechanisms that mediate GX15-070 anti-tumor activity in a panel of therapy-sensitive (TSCL) and therapy-resistant cell lines (TRCL) and primary lymphoma cells isolated from patients with treatment-naïve or refractory/relapsed diffuse large B-cell lymphomas (DLBCL). NHL cell lines and/or primary malignant B-cell cells isolated from DLBCL patients (n=8) were exposed in vitro to GX15-070 (100nM - 1μM) +/- zVAD-fmk. Viability was determined by trypan blue exclusion and/or CellTitre-Glo^® luminescent assay 24 to 72 hours following treatment. PARP cleavage and LC3 conversion were studied by Western blotting. In vitro exposure of TSCL/TRCL to GX15-070 resulted in induction of caspase-independent cell death. The ability of GX15-070 to induce caspase-dependent PARP cleavage varied between patient samples and was not observed in therapy-resistant cell lines (TRCL). Surprisingly, conversion of LC3 was observed in all cell lines and most DLBCL cells tested, suggesting that GX15-070 is a potent inducer of autophagy. To determine if autophagy contributed to GX15-070 activity, autophagy was inhibited by treatment with 3-methyladenine or siRNA-mediated Beclin-1 knock-down. Under conditions where autophagy was inhibited, cell death induced by GX15-070 was significantly reduced. These data suggest that GX15-070-induced cell death is mediated by autophagy in therapy-sensitive and -resistant aggressive B-NHL. In summary, our data suggest that GX15-070 is a novel and potent therapeutic strategy against aggressive B-cell lymphoma. A better understanding in the mechanisms of action of BH3-mimetics, including GX15-070, is necessary for the optimal design of clinical trials for B-cell lymphoma patients. Supported by USPHS grant PO1-CA103985 from the National Cancer Institute.