Inhibition of the monocarboxylate transporter MCT1 by AZD3965 results in an increase in glycolysis in human tumor cell lines and xenografts. This is indicated by changes in the levels of specific glycolytic metabolites and in changes in glycolytic enzyme kinetics. These drug-induced metabolic changes translate into an inhibition of tumor growth in vivo. Thus, we combined AZD3965 with fractionated radiation to treat small cell lung cancer (SCLC) xenografts and showed that the combination provided a significantly greater therapeutic effect than the use of either modality alone. These results strongly support the notion of combining MCT1 inhibition with radiotherapy in the treatment of SCLC and other solid tumors.

DNA-PK is an enzyme that is required for proper DNA-repair and is thought to confer radio-resistance in cancer cells. As a consequence, it is a high-profile validated target for new pharmaceutical development. However, no FDA-approved DNA-PK inhibitors have emerged, despite many years of drug discovery and lead optimization. This is largely because existing DNA-PK inhibitors suffer from poor pharmacokinetics. They are not well absorbed and/or are unstable, with a short plasma half-life. Here, we identified the first FDA-approved DNA-PK inhibitor by “chemical proteomics”. In an effort to understand how doxycycline targets cancer stem-like cells (CSCs), we serendipitously discovered that doxycycline reduces DNA-PK protein expression by nearly 15-fold (> 90%). In accordance with these observations, we show that doxycycline functionally radio-sensitizes breast CSCs, by up to 4.5-fold. Moreover, we demonstrate that DNA-PK is highly over-expressed in both MCF7- and T47D-derived mammospheres. Interestingly, genetic or pharmacological inhibition of DNA-PK in MCF7 cells is sufficient to functionally block mammosphere formation. Thus, it appears that active DNA-repair is required for the clonal expansion of CSCs. Mechanistically, doxycycline treatment dramatically reduced the oxidative mitochondrial capacity and the glycolytic activity of cancer cells, consistent with previous studies linking DNA-PK expression to the proper maintenance of mitochondrial DNA integrity and copy number. Using a luciferase-based assay, we observed that doxycycline treatment quantitatively reduces the anti-oxidant response (NRF1/2) and effectively blocks signaling along multiple independent pathways normally associated with stem cells, including STAT1/3, Sonic Hedgehog (Shh), Notch, WNT and TGF-beta signaling. In conclusion, we propose that the efficacy of doxycycline as a DNA-PK inhibitor should be tested in Phase-II clinical trials, in combination with radio-therapy. Doxycycline has excellent pharmacokinetics, with nearly 100% oral absorption and a long serum half-life (18–22 hours), at a standard dose of 200-mg per day. In further support of this idea, we show that doxycycline effectively inhibits the mammosphere-forming activity of primary breast cancer samples, derived from metastatic disease sites (pleural effusions or ascites fluid). Our results also have possible implications for the radio-therapy of brain tumors and/or brain metastases, as doxycycline is known to effectively cross the blood-brain barrier. Further studies will be needed to determine if other tetracycline family members also confer radio-sensitivity.

Cancer cells produce unique heterogeneous vesicles 1 capable of transferring oncogenic material 2,3 to other cells, 4,5 with the potential of modulating a tumor-supportive environment. [6][7][8] We have previously reported the presence of lipid-enriched, membrane-bound subcellular vesicles at the periphery of acute lymphoblastic leukemia (ALL) cell lines. 9,10 We now extend these findings to describe heterogeneous anucleate vesicles released into extracellular fluids in vitro and in vivo by primary B-cell precursor (BCP) ALL blasts and cell lines. Leukemic extracellular vesicles (LEVs) were internalized by stromal cells, and induced a metabolic switch.Extracellular vesicles (EVs) are enclosed in lipid bilayers originating from the cell of origin, released by both normal and cancer cells.1 Here, the BCP cell-specific membrane protein CD19 present within membrane lipid rafts 11 was used to identify the cell of origin of EVs in clinical samples. We directly compared plasma samples from CD19 1 primary BCP-ALL bone marrow aspirates at diagnosis containing .95% malignant blasts with matched remission samples obtained after 28 days of therapy ( Figure 1A LEVs ( Figure 1E). The effect of LEV internalization by BMSCs was investigated in the human mesenchymal stem cell line HS5 14 exposed to LEVs released by the BCP-ALL cell lines SD1 and NALM6. Proliferation and viability assays revealed no significant differences from control ( Figure 2A). Despite a sustained increase in AKT phosphorylation over 24 hours ( Figure 2B), nonsignificant reductions in adenosine triphosphate (ATP) concentrations were observed ( Figure 2C). Next, the 2 major energyproducing pathways of the cell and parameters of metabolism were assessed. At 24 hours, HS5 1 LEVs showed a reduced oxygen consumption rate (OCR) compared with control, were less sensitive to the inhibition of ATP by oligomycin, and did not change OCR when electron transport from ATP generation in the mitochondria was uncoupled ( Figure 2D). Disrupting the electron transport chain (rotenone/antimycin A) reduced OCR to a comparable level in all cells, suggesting that the rate of oxygen consumption from nonmitochondrial sources was comparable. HS5 1 LEV have a significantly reduced spare respiratory capacity, an indicator of a decreased ability to respond to stress or metabolic challenge ( Figure 2D). Overall, these results suggest that uptake of LEVs significantly reduced mitochondrial respiration in recipient stromal cells.In the absence of glucose, HS5 and HS5 1 LEVs had comparable extracellular acidification rates (ECARs) ( Figure 2E). In the presence of glucose, HS5 1 LEVs initiated a sharp increase in ECAR compared with control (;fivefold), suggesting a higher glycolytic rate. Inhibiting ATP synthase increased ECAR in both HS5 1 LEVs and controls, but more sharply in the latter. Following the addition of 2-deoxy-D-glucose, a competitive inhibitor of glycolysis, ECARs returned to base levels in both control and LEV-exposed cells. Thus, in the presence of glucose, LEV-exposed HS5 s...

Following radiation induced DNA damage, several repair pathways are activated to help preserve genome integrity. Double Strand Breaks (DSBs), which are highly toxic, have specified repair pathways to address them. The main repair pathways used to resolve DSBs are Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). Cell cycle phase determines the availability of HR, but the repair choice between pathways in the G2 phases where both HR and NHEJ can operate is not clearly understood. This study compares several in silico models of repair choice to experimental data published in the literature, each model representing a different possible scenario describing how repair choice takes place. Competitive only scenarios, where initial protein recruitment determines repair choice, are unable to fit the literature data. In contrast, the scenario which uses a more entwined relationship between NHEJ and HR, incorporating protein co-localisation and RNF138-dependent removal of the Ku/DNA-PK complex, is better able to predict levels of repair similar to the experimental data. Furthermore, this study concludes that co-localisation of the Mre11-Rad50-Nbs1 (MRN) complexes, with initial NHEJ proteins must be modeled to accurately depict repair choice.