Since the findings above showed that vemurafenib treatment caused these drug- tolerant lines to induce AMPK signaling and autophagy, we next examined whether this induction was contributing to their ability to survive and tolerate BRAF inhibition. To address this, we treated YUKSI cells with chloroquine (CQ)28 or bafilomycin (Baf), which block autophagy by inhibiting autophagosome clearance (Number 4a). Both CQ and Baf strongly sensitized YUKSI cells to vemurafenib (Figure 4b; Supplementary Number 4A). identify a CGS 21680 HCl key mechanism of tolerance to Ras-Raf pathway inhibitors and suggest that obstructing either AMPK or autophagy in combination with these targeted inhibitors CGS 21680 HCl could increase tumor regression and decrease the probability of eventual recurrence. Intro The Ras (rat sarcoma viral oncogene)-Raf pathway is frequently activated in human being cancers through mutations in Ras or its downstream effector, BRAF (v-Raf murine sarcoma viral oncogene homolog B). Given the central part that it takes on in traveling tumorigenesis, the Ras-Raf pathway has become a KR1_HHV11 antibody major focus for the development of targeted treatments. Although several strategies for inhibiting this pathway are becoming explored, probably the most successful strategy to date offers been to develop targeted inhibitors of oncogenic forms of the BRAF protein. This has led to the development of vemurafenib and additional targeted inhibitors that specifically inhibit the oncogenic forms of BRAF.1, 2, 3 Vemurafenib and additional targeted medicines significantly extend patient survival, but all tumors eventually develop resistance after a median time of 5C8 weeks.3, 4 Tumors that develop resistance are able to maintain MAPK phosphorylationa downstream measure of Ras-Raf pathway signalingeven in the presence of vemurafenib.5, 6, 8, 9, 10, 11 Although all tumors eventually develop resistance and bring back MAPK phosphorylation, they display significant differences in their initial responses to treatment. Vemurafenib causes some tumors to completely regress, most tumors only regress partially, if at all. Importantly, these variations in initial response cannot be explained by variations in the degree to which vemurafenib inhibits Ras-Raf pathway signaling as gauged by MAPK phosphorylation.5, 12, 13, 14 These observations in CGS 21680 HCl individuals look like analogous to observations that have been made with populations of cancer cells in tradition: inhibition of the Ras pathway causes rapid apoptosis in some cancer cell lines (habit), whereas other lines do not undergo apoptosis but rather survive pathway inhibition (tolerance).15 The phenomenon of drug tolerance can be distinguished from resistance because only the latter is associated with re-activation of Ras pathway signaling in the presence of the targeted inhibitor. Since resistant clones can only arise if some malignancy cells survive the initial drug treatment, inhibiting the pathways that confer tolerance could improve patient outcomes by eliminating the reservoir of cells without which resistance would be unable to develop. However, while several mechanisms of resistance have been reported,5, 6, 7, 8, 9, 10, 11 how cells develop tolerance to Ras-Raf pathway inhibitors is not well recognized. Mutations in the Ras-Raf pathway provide a competitive advantage by enabling tumor cells to increase their glucose uptake in low nutrient conditions.16, 17 Consistent with this, vemurafenib and other inhibitors of Ras-Raf signaling reduce glucose uptake by cancer cells, which can be measured through positron emission tomography imaging with the glucose analog fluorodeoxyglucose.13, 18 It seems plausible that malignancy cells might cope with this quick reduction in glucose by activating some of the same pathways that normal cells use to adapt to the effects of nutrient starvation. In normal cells, nutrient starvation causes the activation of AMP-activated protein kinase (AMPK), which is a sensor of the cellular AMP:ATP percentage.19 Upon its activation, AMPK stimulates an internal scavenging program, termed autophagy, which provides essential nutrients by breaking down cellular components.20, 21 When autophagy is inhibited, cells are unable to survive even short bouts of nutrient starvation.22, 23 In this study, we examined whether autophagy might also promote tumor drug tolerance by enabling malignancy cells to survive the starvation provoked by inhibition of the Ras-Raf pathway. Results Ras-Raf pathway inhibitors activate AMPK signaling in malignancy cells with Ras pathway mutations To examine whether inhibitors of the Ras pathway were provoking a starvation response, we assessed AMPK activation after 48 h of inhibitor treatment, inside a panel of malignancy cell lines with mutations in either Ras or Braf. Treatment with vemurafenib caused a dose-dependent increase in the phosphorylation of AMPK in two melanoma lines (YUKSI, YUSIK) and a colon cancer collection (HT29) with BRAFV600 mutations (Number 1a). Treatment with vemurafenib also improved the phosphorylation of ULK1, a downstream target of AMPK (Number 1a).20, 24 Like a control, vemurafenib treatment did not increase.