E. J. Rellinger1, B. T. Craig1, J. Qiao1, K. Kim1, D. H. Chung1 1Vanderbilt University Medical Center,Pediatric Surgery,Nashville, TN, USA
Introduction: Neuroblastoma (NB) is a heterogeneous pediatric solid tumor with diverse outcomes ranging from spontaneous regression to clinical progression despite our most aggressive therapies. MYCN-amplifications are common with high-risk NB, a designation that portends <50% survival. Myc-driven tumors are ‘glucose addicts’ relying predominantly upon glycolysis for energy production even in the presence of adequate oxygen, a phenomenon known as the Warburg effect. Lactate dehydrogenase is the terminal enzyme of glycolysis. One of its isoforms, lactate dehydrogenase A (LDHA), is preferentially expressed in Myc-driven tumors and avidly converts pyruvate to lactate. FX11 [3-dihydroxy-6-methyl-7-(phenylmethyl)-4-propylnaphthalene-1-carboxylic acid] is a gossypol derivative that selectively inhibits the LDHA isoform and demonstrates preclinical efficacy in C-myc driven pancreatic cancer and lymphomas models. However, the efficacy of FX11 in N-Myc driven NBs is unknown. We hypothesized that MYCN-amplified NBs would be susceptible to LDHA inhibition using FX11.
Methods: We first evaluated the protein expression of LDHA in twelve of our NB cell lines (eight MYCN-amplified and four MYCN-single copy). Two MYCN-amplified cell lines (BE(2)-C and IMR-32) and two MYCN-single copy cell lines (SK-N-SH and SK-N-AS) were used to evaluate the effects of FX11 on NB growth. Cell viability was examined using a tetrazolium-based assay with validation completed by manual cell counts. Immunoblotting for PARP and caspase 3 cleavage products was our measure of apoptosis induction. Lactate concentration was quantified from culture supernatants using a colorimetric assay.
Results: LDHA was ubiquitously expressed in both MYCN-amplified and MYCN-single copy NB cell lines. Treatment with FX11 (10 μM) significantly decreased the levels of lactate at 6 h, validating that FX11 effectively blocks lactate production in NB cells. Both tetrazolium-based measures and manual cell counts demonstrated that FX11 decreased cell viability of NB cells after three days of treatment in our MYCN-amplified cell lines (BE(2)-C and IMR-32) and four days for MYCN-single copy cell lines (SK-N-SH and SK-N-AS). These time courses coincided with induction of PARP and Caspase 3 cleavage in MYCN-amplified cell lines. Notably, no Caspase 3 products were detectable with FX11 treatment in MYCN-single copy cell lines (Fig.).
Conclusion: FX11 has comparable in vitro efficacy in MYCN–amplified NBs to those previously reported in C-Myc driven pancreatic and lymphoma models. LDHA blockade successfully induced apoptosis in our MYCN-amplified tumors. In vivo trials are now underway to evaluate the preclinical efficacy of FX11 in MYCN-amplified NBs.
