161 - Targeting Treatment-resistant Medulloblastoma

Medulloblastoma (MB) is the most common childhood malignant brain tumor. Although current treatment strategies have improved survival, they cause debilitating long-term sequelae and lack benefit in relapsed disease where the prognosis is grim. Novel therapies are urgently needed to improve outcomes in MB.

Aberrant Sonic Hedgehog (SHH) signaling has been implicated as the driver of aggressive proliferation in ~30% of MBs. Poor outcomes in SHH-MBs have prompted the evaluation of SHH-targeting agents in their treatment but these have had limited success - likely due to resistance from activation of alternate oncogenic pathways (e.g. Ras-MAPK). These pathways increase glycolysis, in part by increasing activity of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) to produce fructose-2,6-bisphosphate (F26BP) which activates a key glycolytic enzyme, 6-phosphofructo-1-kinase. We have found that the PFKFB4 enzyme is highly expressed in patient-derived SHH-MB and that silencing PFKFB4 in SHH-MB cells suppressed glycolysis and proliferation in normoxia and, more markedly, in hypoxia, indicating a critical role for PFKFB4 in SHH-MB growth, particularly in hypoxia. To evaluate the function of PFKFB4 in treatment-resistant SHH-MB, we modeled resistance by prolonged exposure of SHH-MB cells to an SHH inhibitor then evaluated their tumor-associated phenotypes.

Objective:

We hypothesize that PFKFB4 is critical for survival and growth of treatment-resistant SHH-MB and that targeted inhibition of PFKFB4 will significantly decrease their proliferation and survival.

Design/Methods:

Cell counts were examined by microscopy and anchorage independence by soft agar assays. Glycolysis was examined using 3H-glucose and apoptosis by Annexin V/propidium iodide flow cytometry. All assays were repeated ≥3 times.

Results:

We compared SHH-inhibitor resistant (SHH-R) and sensitive (SHH-S) MB cells and observed higher glycolysis, proliferation and anchorage-independent growth in SHH-R relative to SHH-S cells. We found that SHH-R cells had higher PFKFB4 expression than SHH-S cells and therefore evaluated effects of a novel PFKFB4 inhibitor (FB4i) on the cells. We found that SHH-R cells showed decreased survival and, to explore the underlying mechanism, examined effects of FB4i on apoptosis and found that SHH-R cells exhibited higher apoptosis than SHH-S MB cells, with greater effects in hypoxia.

Conclusion(s):

Taken together, our data indicate that targeting PFKFB4 may prove an effective therapeutic strategy in treatment-resistant SHH-MB and strongly support the examination of PFKFB4 inhibitors in these tumors.