Autophagy and RAS signaling: Clinical implications

By Christina Towers, PhD

The cellular recycling process known as autophagy is currently being targeted in over 60 clinical trials focused on treating different types of cancer¹. To date, the only autophagy-targeted agents used in patients are late stage autophagy inhibitors that target the lysosome, including chloroquine (CQ) and hydroxychloroquine (HCQ). Over the last decade a host of studies performed in both cancer cell lines and mouse models have indicated that autophagy plays a tumor-promotional role in established tumors. Nonetheless, initial results from the first wave of trials have shown lackluster results when these drugs are used as single agents, however, there have been modest results seen when used in combination with other targeted agents. While there are a host of reasons these drugs might be underperforming in clinical trials, including a lack of specificity, mechanisms or resistance, and a lack of biomarkers – one clear shortcoming of the field is an inadequate understanding of autophagy dependence. It is still unclear which tumor types or sub-types may be most sensitive to autophagy-targeted agents.

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There is evidence, however, that tumors addicted to the RAS-RAF-MEK-ERK signaling pathway have extremely high levels of basal autophagy compared to other oncogene drivers. This may be especially true in pancreatic ductal adenocarcinoma (PDAC) where greater than 95% of PDAC tumors harbor a RAS activating mutation^2,3. Moreover, genetically engineered mouse models of PDAC have shown exquisite sensitivity to genetic autophagy inhibition⁴. These results led to the hypothesis that RAS pathway activation may be activating autophagy as a pro-survival mechanism.

Autophagy antibody pack (NB910-94159) for analysis of various significant autophagy targets includes sample sizes for antibodies targeting LC3 (NB100-2220SS), Beclin1 (NB110-87318SS), ATG5 (NB110-53818SS), ATG9A (NB110-56893SS), and ATG16L1 (NB110-60928SS). (Left) Lysates of HeLa parental cell line and LC3B knockout HeLa cell line (KO) untreated (-) or treated (+) with 50 uM Chloroquine for 18 hours. PVDF (Polyvinylidene difluoride) membrane was probed with 0.5 ug/mL of Rabbit Anti-LC3B Polyclonal Antibody (NB100-2220) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (HAF008). A specific band was detected for LC3B at a molecular weight of approximately 15 kDa (as indicated) in the parental HeLa cell line, but is not detectable in the knockout HeLa cell line. GAPDH is shown as a loading control. This experiment was conducted under reducing conditions. (Right) Analysis of a FFPE tissue section of human breast cancer using Beclin 1 antibody (NB110-87318) at 1:300 dilution, followed by a HRP labeled secondary antibody and DAB. Nuclei of the cells were counterstained with hematoxylin. The signal is strongest in a subset of stromal cells, likely the cancer associated fibroblasts.

It was therefore unexpected when Kinsey et al⁵ and Bryant et al⁶ published in the same issue of Nature Medicine in 2019 that inhibition of the RAS-RAF-MEK-ERK signaling pathway actually causes a robust increase in autophagic flux. Both groups utilized the mCherry-GFP-LC3 tandem construct to quantify autophagic flux across a panel or PDAC cell lines after either genetic or pharmacological inhibition of the pathway. These results indicate the relationship between the RAS-RAF-MEK-ERK signaling pathway and autophagy pathway may be more complex. The two groups go on to show that RAS pathway inhibition, at any stage in the pathway, synergizes with the clinically used autophagy inhibitors CQ and HCQ.  Importantly, Kinsley et al show these findings extend beyond PDAC to other RAS addicted tumors including NRAS-mutant melanoma and BRAF-mutant colon cancer.

LC3B was detected in immersion fixed Chloroquine treated HeLa cells (left) but was not detected in LC3B knockout HeLa cells (right) using rabbit anti-human LC3B polyclonal antibody (NB100-2220) at 0.3 ug/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated anti-Rabbit IgG Secondary Antibody (NL004) (red) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm

These results could have important implications clinically. Indeed, Kinsley et al presents a case study with a PDAC patient who was refractory to all standard-of-care therapy.  Four months of treatment with escalating doses of hydroxychloroquine and the MEK inhibitor, trametinib, led to a dose dependent decrease in the PDAC blood born cancer agent, CA19-9, by 95% and a corresponding 50% reduction in tumor burden. Previously, similar case studies in BRAF-mutant brain cancer patients have also indicated synergistic affects after treatment with autophagy inhibitors in combination with Ras pathway inhibition⁷. Together, these studies indicate that co-targeting with RAS pathway inhibitors and autophagy inhibitors may improve overall patient outcomes, particularly in tumors addicted to the RAS-RAF-MEK-ERK signaling pathway.

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Christina Towers, PhD   
University of Colorado (AMC)
Dr. Towers studies the roles of autophagy, apoptosis and cell death in cancer.

References

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