Protective autophagy elicited by RAF→MEK→ERK inhibition suggests a treatment strategy for RAS-driven cancers

Abstract

Pancreatic ductal adenocarcinoma (PDA) was responsible for ~ 44,000 deaths in the United States in 2018 and is the epitome of a recalcitrant cancer driven by a pharmacologically intractable oncoprotein, KRAS1-4. Downstream of KRAS, the RAF→MEK→ERK signaling pathway plays a central role in pancreatic carcinogenesis5. However, paradoxically, inhibition of this pathway has provided no clinical benefit to patients with PDA6. Here we show that inhibition of KRAS→RAF→MEK→ERK signaling elicits autophagy, a process of cellular recycling that protects PDA cells from the cytotoxic effects of KRAS pathway inhibition. Mechanistically, inhibition of MEK1/2 leads to activation of the LKB1→AMPK→ULK1 signaling axis, a key regulator of autophagy. Furthermore, combined inhibition of MEK1/2 plus autophagy displays synergistic anti-proliferative effects against PDA cell lines in vitro and promotes regression of xenografted patient-derived PDA tumors in mice. The observed effect of combination trametinib plus chloroquine was not restricted to PDA as other tumors, including patient-derived xenografts (PDX) of NRAS-mutated melanoma and BRAF-mutated colorectal cancer displayed similar responses. Finally, treatment of a patient with PDA with the combination of trametinib plus hydroxychloroquine resulted in a partial, but nonetheless striking disease response. These data suggest that this combination therapy may represent a novel strategy to target RAS-driven cancers.

Conflict of interest statement

Competing Interests Statement:

Dr. McMahon served on external advisory boards for Novartis (June 2017), Genentech (December 2017) and Merck (June 2018). Dr. McMahon is the recipient of research funding from Pfizer, through a grant peer-reviewed, co-funded and awarded by the Melanoma Research Alliance.

Figures

Ext. Figure 1.. Flow cytometry analysis of autophagic flux reporter with autophagy inhibitors and inducers.

a-e: Autophagic flux was assessed by flow cytometry in Mia-PaCa2AFR cells following 48 hours treatment with control, chloroquine (CQ), SAR-405, temsirolimus, or trametinib. Experiments were repeated three times with similar results. f: Autophagic flux was assessed by fluorescent imaging in Mia-PaCa2AFR cells following 48 hours treatment with control, chloroquine (CQ), VPS34i (SAR-405), or trametinib. Experiments were repeated three times with similar results.

Ext. Figure 2.. Inhibition of RAS→RAF→MEK→ERK signaling pathway induces autophagic flux (AF) as seen by p62 degradation and LC3 conversion in pancreatic cancer cells.

a & b: Cell lysates prepared from Mia-PaCa2 (a) or BxPC3 (b) cells treated with 0.1–100 nM of trametinib for 48 hours were analyzed by immunoblotting for the phosphorylation (p) or total (t) abundance of ERK1/2, p62, LC3, or actin as indicated. Experiments were repeated three times with similar results. c: Cell lysates prepared from Mia-PaCa2 cells treated with ARS-853 (KRASG12Ci), SCH772984 (ERKi), or cobimetinib (MEKi) for 48 hours were analyzed by immunoblotting for the phosphorylation (p) or total (t) abundance of ERK1/2, p62, LC3, or actin as indicated. Experiments were repeated three times with similar results. d: Cell lysates prepared from Mia-PaCa2AFR cells transiently expressing exogenous ULK1 WT, ULKM92A (dominant negative), AMPK WT, or AMPKK45R (dominant negative) were analyzed by immunoblotting for ULK1, AMPK, or actin as indicated. Experiments were repeated three times with similar results. e: Cell lysates prepared from Mia-PaCa2AFR cells lentivirally transduced with shRNAs targeting LKB1 or scrambled control were analyzed by immunoblotting for LKB1 or actin as indicated. Experiments were repeated three times with similar results.

Ext. Figure 3.. Trametinib and chloroquine are synergistically cytotoxic in vitro.

Mia-PaCa2 cells, BxPC3 and PDX220 cells were treated for 48–96 as indicated with trametinib and chloroquine and analyzed for cell viability by ATPlite assay. Synergy scores were generated utilizing Combenefit Software. Experiments were repeated four times with similar results.

Ext. Figure 4.. Treatment of pancreatic tumors with trametinib and chloroquine results in decreased pERK and increased p62 abundance respectively.

Representative images of immunohistochemical analysis of sections of PDX 227 tumors that were treated with 1. vehicle (Control), 2. trametinib; 3. chloroquine or; 4. the combination of both agents. Sections were stained with H&E or with antisera against pERK1/2 or p62 as indicated. Experiments were repeated four times with similar results. Scale bar is 500 μM located in the bottom right of the upper left panel and is consistent for all images.

Ext. Figure 5.. Treatment of orthopically xenografted pancreatic tumors with trametinib and hydroxychloroquine demonstrates regression consistent with subcutaneous xenografts.

a: PDX220 tumors were orthotopically transplanted and after 3 weeks were imaged via FDG-PET/CT for baseline. They were then treated with trametinib, hydroxychloroquine or trametinib plus hydroxychloroquine for 2 weeks prior to re-imaging. n=3 for control; n=2 for HCQ; n=3 for trametinib; n=2 for trametinib+hydroxychloroquine. b & c: Quantification of total lesion glycolysis (b) and % change (c) for individual tumors within each treatment group.

Ext. Figure 6.. Regression of established NRAS driven melanoma tumors by combined inhibition of MEK1/2 plus chloroquine.

a. The growth of NRAS-mutated melanoma (NCI515677) PDX was assessed over 21 days in mice treated with: 1. vehicle (Control), 2. trametinib (1mg/kg), 3. chloroquine (25mg/kg) or; 4. the combination of both agents at the aforementioned doses as indicated. n=4 for all treatment groups except combination of both agents n=5. Center values are the mean; statistical testing was performed by two-sided t-test; ***p<0.001 vs. control; tttp<0.001 vs. trametinib. Error bars represent SD. b-e. The percentage weight change of HCI-Mel002 NRAS-mutated PDX was assessed over 21 days in mice treated with: b. vehicle (Control), c. trametinib (1mg/kg), d. chloroquine (50mg/kg) or; e. the combination of both agents at the aforementioned doses as indicated. However, side-effects of facial rash and hair loss were noted.

Ext. Figure 7.. Lack of autophagy induction by MEK1/2 inhibition results in resistance to combined trametinib and chloroquine treatment.

a: Cell lysates prepared from two suitably manipulated KRASG12D/TP53Null mouse lung cancer-derived cell lines, SC196 or SC274 treated with 100nM of trametinib were analyzed by immunoblotting for the phosphorylation (p) or total (t) abundance of ERK1/2, p62, LC3, or actin as indicated. Experiments were repeated three times with similar results. b: SC196 and SC274 KRASG12D/TP53Null mouse lung cancer cells were treated for 48 hours respectively with trametinib and chloroquine and analyzed for cell viability by ATPlite assay. Synergy scores were generated utilizing Combenefit Software. Experiments were repeated four times with similar results. c & d: Autophagic flux was measured in SC196AFR (c) or SC274AFR (d) following treatment with 0.1–1000nM trametinib for 48 hours.. n=3; center values are the mean; statistical testing was performed by two-sided t-test of control high (red) versus experimental high; ***p<0.001 vs. control. Error bars represent SD. e & f: The growth of SC196 (e) or SC274 (f) KRASG12D/TP53Null mouse lung cancer derived tumors in xenografted mice treated with: 1. vehicle (Control), 2. trametinib (1mg/kg), 3. chloroquine (50mg/kg) or; 4. the combination of both agents was assessed over ~15 days as indicated. n=10 for all treatment groups.. Center values are the mean; statistical testing was performed by two-sided t-test;***p<0.001 vs. control; tttp<0.001 vs. trametinib. Error bars represent SD.

Figure 1.. Inhibition of RAS→RAF→MEK→ERK signaling pathway induces autophagic flux (AF) in pancreatic cancer cells

a: Pancreatic cancer cells expressing an autophagic flux reporter (AFR) were generated by ectopic expression of a chimeric fusion protein comprised of mCherry-EGFP-LC3 in which the increased ratio of red:green fluorescence assessed by flow cytomtery is indicative of elevated AF. b: Autophagic flux was assessed by flow cytometry in Mia-PaCa2AFR cells following 48 hours treatment with various pharmacological inhibitors (CQ 20 uM or SAR-405 10 uM) or inducers (temsirolimus 10 uM or trametinib 100 nM) of autophagy. n=3; center values are the mean; statistical testing was performed by two-sided t-test of control high (red) versus experimental high; ***p<0.001 vs. control (0 nM/uM). Error bars represent SD. c-h: Mia-PaCa2AFR, BxPC3AFR or PDX220AFR (derived from a human pancreatic cancer PDX) cells were treated for 48 hours with inhibitors of KRASG12C (ARS-853), ERK1/2 (SCH772984), or MEK1/2 (trametinib or cobimetinib) with autophagic flux assessed by flow cytometry. n=3; center values are the mean; statistical testing was performed by two-sided t-test of control high (red) versus experimental high; ***p<0.001, **p<0.01, *p<0.05 vs. control (0 nM/uM). Error bars represent SD. i & j: Cell lysates prepared from Mia-PaCa2 treated with 1–100nM trametinib for 48 hours (a) or PDX220 derived cells treated with 100nM of trametinib over a time course (b), were analyzed by immunoblotting for the phosphorylation (p) or total (t) abundance of ERK1/2, p62, LC3, LKB1 (pS428), AMPK (pT172), ULK1 (pS555) or actin as indicated. Experiments were repeated three times with similar results. k: Schematic model of the proposed mechanism by which inhibition of RAS→RAF→MEK→ERK signaling may elicit autophagic flux in pancreatic cancer cells. l & m: Autophagic flux was assessed by flow cytometry in Mia-PaCa2AFR cells transiently expressing exogenous ULK1 WT, ULK M92A (dominant negative) (d), AMPK WT, or AMPKK45R (dominant negative) (e) and treated with 1–100nM of trametinib for 48 hours. n=3; center values are the mean; statistical testing was performed by two-sided t-test of matched treatment control high (red) versus matched dominant negative treatment high or matched treatment WT versus matched dominant negative treatment high; ***p<0.001 vs. matched treatment control; tttp<0.001 vs. matched treatment WT. Error bars represent SD. n: Autophagic flux was assessed by flow cytometry in Mia-PaCa2AFR cells stably expressing shRNAs targeting LKB1 or scrambled control and treated with 1–100nM of trametinib for 48 hours. n=3; center values are the mean; statistical testing was performed by two-sided t-test; ***p<0.001 vs. matched treatment scrambled control. Error bars represent SD.

Figure 2.. Trametinib and chloroquine are synergistically cytotoxic in vitro.

a: Mia-PaCa2, BxPC3 and PDX220 cells were treated for 48–96 as indicated with trametinib and chloroquine and analyzed for cell viability by ATPlite assay. Synergy graphs were generated utilizing Combenefit Software. Experiments were repeated 4 times with similar results. b-d: Mia-PaCa2, BxPC3 and PDX220 cells were treated for 48 hours as indicated with vehicle (Control; DMSO), trametinib 100nM, chloroquine (CQ) 20μM or trametinib plus chloroquine and analyzed for cell viability by CytoxRed assay using an Incucyte Microscope. n=3; center values are the mean; statistical testing was performed by one-way ANOVA; p

Figure 3.. Tumor cell autonomous inhibition of autophagy cooperates with MEK1/2 inhibition to elicit regression of xenografted pancreatic tumors

a: Mia-PaCa2AFR cells, engineered to express a doxycycline-regulated dominant-negative (DN) form of ATG4B (Mia-PaCa2AFR TetI-ATG4BDN) were treated with trametinib in the absence or presence of doxycycline with autophagic flux measured by flow cytometry. n=3; center values are the mean; statistical testing was performed by two-sided t-test of control high (red) versus experimental high; ***p<0.001 vs. trametinib treatment alone. Error bars represent SD. b: Immunoblot analysis of the expression of the autophagy indicator proteins p62 and LC3 in Mia-PaCa2AFR TetI-ATG4BDN treated with trametinib, doxycycline (to induce ATG4BDN expression) or both agents. This was repeated three times with similar results. c: The growth of xenografted tumors of Mia-PaCa2AFR/TetI-ATG4BDN cells was assessed over 20 days in mice treated with: 1. vehicle (Control) n=11; 2. Trametinib n=11; 3. Doxycycline n=10 or; 4. the combination of both agents n=12. Center values are the mean; statistical testing was performed by two-sided t-test; ***p<0.001 vs. control; tttp<0.001 vs. trametinib. Error bars represent SD. d: Representative images of immunohistochemical analysis of sections of xenografted Mia-PaCa2AFR TetI-ATG4BDN tumors that were treated with 1. vehicle (Control), 2. trametinib; 3. doxycycline or; 4. the combination of both agents. Sections were stained with H&E or with antisera against pERK1/2, ATG4B or p62 as indicated. Scale bar is 500 μM located in the bottom right of the upper left panel and is consistent for all images. e & f: The growth of tumor xenografts of Mia-PaCa2 (a) or BxPC3 (b) cells over ~60 days in mice treated with: 1. vehicle (Control); 2. trametinib (1mg/kg); 3. chloroquine (50mg/kg), or; 4. the combination of both agents at the aforementioned doses were assessed as indicated. Mia-PaCa2: control n=5, trametinib n=6, chloroquine n=5, combination of both agents n=4. BxPC3: n=6 for all treatment groups. Center values are the mean; statistical testing was performed by two-sided t-test; ***ptttp<0.001 vs. trametinib. Error bars represent SD. g & h: The growth of two pancreatic cancer patient derived xenografts (PDX220 or PDX227) in mice treated with: 1. vehicle (Control), 2. trametinib (1mg/kg), 3. hydroxychloroquine (40mg/kg in PDX220), chloroquine (50mg/kg in PDX227); 4. gemcitabine plus abraxane or; 5. the combination of trametinib plus CQ/HCQ at the aforementioned doses were assessed over ~30–40 days as indicated. PDX220: control n=6, trametinib n=5, hydroxychloroquine n=5, combination of both agents n=4, gemcitabine plus abraxane n=6. PDX227: n=5 for all groups except for gemcitabine plus abraxane n=6. Center values are the mean; statistical testing was performed by two-sided t-test; ***ptttp<0.001 vs. trametinib, xxxp<0.001, xxp<0.01 vs. gemcitabine plus abraxane. Error bars represent SD. i & j: The growth of NRAS-mutated melanoma (HCI-Mel002) PDX or a BRAF-mutated colorectal cancer PDX (HCI-CRC004) was assessed over 18–21 days in mice treated with: 1. vehicle (Control), 2. trametinib (1mg/kg), 3. chloroquine (50mg/kg) or; 4. the combination of both agents at the aforementioned doses as indicated. HCI-Mel002: control n=5, trametinib n=5, chloroquine n=4, combination of both agents n=4. HCI-CRC004: n=5 for all groups except combination of both agents n=4. Center values are the mean; statistical testing was performed by two-sided t-test; **p<0.01, *p<0.05 vs. control; tttp<0.001 vs. trametinib. Error bars represent SD.

Figure 4.. Treatment of a pancreatic cancer patient with trametinib plus hydroxychloroquine (T/HCQ) lead to a reduction in tumor marker cancer antigen 19–9 (CA19–9) and overall tumor burden.

a: The patient’s blood-borne CA19–9 tumor marker was measured periodically throughout the the entire clinical course and is annotated with the dates and treatments administered. b, c, d & e: CT imaging 2 days after starting (b, d) the 2mg trametinib (q.d.) plus 1200mg (600mg b.i.d.) dosing of HCQ (which had been started with lower doses of HCQ two weeks previously) and two months post (c & e). The recurrent pancreatic bed lesion is dramatically reduced in size (comparing panel b to c), while the metastatic lesions in the liver are largely resolved (comparing panels b to c and d to e).