Abstract
BackgroundPediatric patients with high-risk neuroblastoma (HR NB) often fail to respond to upfront intensive multimodal therapy. Tumor-acquired suppression of apoptosis contributes to therapy resistance. Many HR NB tumors depend on the anti-apoptotic protein Bcl-2 for survival, through Bcl-2 sequestration and inhibition of the pro-apoptotic protein, Bim. Bcl-2 dependent xenografts derived from aggressive human NB tumors are cured with a combination of cyclophosphamide and ABT-737, a Bcl-2/Bcl-XL/Bcl-w small molecule antagonist. The oral analogue to ABT-737, Navitoclax (ABT-263), clinically causes an immediate drop in peripheral platelet counts as mature platelets depend on Bcl-xL for survival. This led to the creation of a Bcl-2 selective inhibitor, ABT-199 (Venetoclax). A Phase I trial of ABT-199 in CLL showed remarkable antitumor activity and stable patient platelet counts. Given Bcl-XL does not play a role in HR NB survival, we hypothesized that ABT-199 would be equally potent against HR NB.MethodsCytotoxicity and apoptosis were measured in human derived NB cell lines exposed to ABT-199 combinations. Co-Immunoprecipitation evaluated Bim displacement from Bcl-2, following ABT-199. Murine xenografts of NB cell lines were grown and then exposed to a 14-day course of ABT-199 alone and with cyclophosphamide.ResultsBcl-2 dependent NB cell lines are exquisitely sensitive to ABT-199 (IC50 1.5–5 nM) in vitro, where Mcl-1 dependent NBs are completely resistant. Treatment with ABT-199 displaces Bim from Bcl-2 in NB to activate caspase 3, confirming the restoration of mitochondrial apoptosis. Murine xenografts of Mcl-1 and Bcl-2 dependent NBs were treated with a two-week course of ABT-199, cyclophosphamide, or ABT-199/cyclophosphamide combination. Mcl-1 dependent tumors did not respond to ABT-199 alone and showed no significant difference in time to tumor progression between chemotherapy alone or ABT-199/cyclophosphamide combination. In contrast, Bcl-2 dependent xenografts responded to ABT-199 alone and had sustained complete remission (CR) to the ABT-199/cyclophosphamide combination, with one recurrent tumor maintaining Bcl-2 dependence and obtaining a second CR after a second course of therapy.ConclusionHR NB patients are often thrombocytopenic at relapse, raising concerns for therapies like ABT-263 despite its HR NB tumor targeting potential. Our data confirms that Bcl-2 selective inhibitors like ABT-199 are equally potent in HR NB in vitro and in vivo and given their lack of platelet toxicity, should be translated into the clinic for HR NB.
Highlights
Pediatric patients with high-risk neuroblastoma (HR NB) often fail to respond to upfront intensive multimodal therapy
IMR5, a MYCN amplified HR NB cell line characterized as Mantle cell lymphoma 1 (Mcl-1) dependent [17], was completely
Given our previous data implicates Bim is the key death effector in HR NB, we evaluated whether ABT-199 induces apoptosis in NB by displacing Bim from B-cell Lymphoma 2 (Bcl-2)
Summary
Pediatric patients with high-risk neuroblastoma (HR NB) often fail to respond to upfront intensive multimodal therapy. The oral analogue to ABT-737, Navitoclax (ABT-263), clinically causes an immediate drop in peripheral platelet counts as mature platelets depend on Bcl-xL for survival. Chemotherapy induces the intrinsic pathway of apoptosis that is tightly regulated by mitochondrial resident Bcl-2 family proteins whose structure is comprised of one to four highly conserved Bcl-2 homology (BH) domains [1]. Within this family Bcl-2 proteins with multiple BH domains can be either anti-apoptotic (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, Bfl-1/A1) or pro-apoptotic (Bax, Bak) and a subset containing only the BH3 domain are all pro-apoptotic (Bim, Bid, Puma, Bad, Bik, etc.) [2]. A cancer dependent on Bcl-2 for survival, such as chronic lymphocytic leukemia (CLL), has an abundance of Bcl-2 that actively sequesters the BH3 protein, Bim, making the cancer cell “primed to die” in the event that other cellular stressors activate additional BH3 proteins to overcome the Bcl-2 binding capacity within the cell [3]
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