Targeting flavin-containing enzymes eliminates cancer stem cells (CSCs), by inhibiting mitochondrial respiration: Vitamin B2 (Riboflavin) in cancer therapy

Abstract

Here, we performed high-throughput drug-screening to identify new non-toxic mitochondrial inhibitors. This screening platform was specifically designed to detect compounds that selectively deplete cellular ATP levels, but have little or no toxic side effects on cell viability. Using this approach, we identified DPI (Diphenyleneiodonium chloride) as a new potential therapeutic agent. Mechanistically, DPI potently blocks mitochondrial respiration by inhibiting flavin-containing enzymes (FMN and FAD-dependent), which form part of Complex I and II. Interestingly, DPI induced a chemo-quiescence phenotype that potently inhibited the propagation of CSCs, with an IC-50 of 3.2 nano-molar. Virtually identical results were obtained using CSC markers, such as CD44 and CD24. We further validated the effects of DPI on cellular metabolism. At 10 nM, DPI inhibited oxidative mitochondrial metabolism (OXPHOS), reducing mitochondrial driven ATP production by >90%. This resulted in a purely glycolytic phenotype, with elevated L-lactate production. We show that this metabolic inflexibility could be rapidly-induced, after only 1 hour of DPI treatment. Remarkably, the mitochondrial inhibitory effects of DPI were reversible, and DPI did not induce ROS production. Cells maintained in DPI for 1 month showed little or no mitochondrial activity, but remained viable. Thus, it appears that DPI behaves as a new type of mitochondrial inhibitor, which maintains cells in a state of metabolic-quiescence or “suspended animation”. In conclusion, DPI treatment can be used to acutely confer a mitochondrial-deficient phenotype, which we show effectively depletes CSCs from the heterogeneous cancer cell population. These findings have significant therapeutic implications for potently targeting CSCs, while minimizing toxic side effects. We also discuss the possible implications of DPI for the aging process. Interestingly, previous studies in C. elegans have shown that DPI prevents the accumulation of lipofuscin (an aging-associated hallmark), during the response to oxidative stress. Our current results are consistent with data showing that flavins (FAD, FMN and/or Riboflavin) are auto-fluorescent markers of i) increased mitochondrial “power” (OXPHOS) and ii) elevated CSC activity. Finally, we believe that DPI is one of the most potent and highly selective CSC inhibitors discovered to date. Therefore, our current findings suggest a new impetus to create novel analogues of i) DPI (Diphenyleneiodonium chloride) and ii) DPI-related compounds (Diphenyliodonium chloride), using medicinal chemistry, to optimize this very promising and potent anti-CSC activity. We propose to call these new molecules “Mitoflavoscins”. For example, DPI is ∼30 times more potent than Palbociclib (IC-50 = 100 nM), which is an FDA-approved CDK4/6 inhibitor, that broadly targets proliferation in any cell type, including CSCs.