Reprogrammed mRNA translation drives resistance to therapeutic targeting of ribosome biogenesis

Abstract

Elevated ribosome biogenesis in oncogene-driven cancers is commonly targeted by DNA-damaging cytotoxic drugs. Our previous first-in-human trial of CX-5461, a novel, less genotoxic agent that specifically inhibits ribosome biogenesis via suppression of RNA polymerase I (Pol I) transcription, revealed single-agent efficacy in refractory blood cancers. Despite this clinical response, patients were not cured. In parallel, we demonstrated a marked improvement in the in vivo efficacy of CX-5461 in combination with PI3K/AKT/mTORC1 pathway inhibitors. Here, we reveal the molecular basis for this improved efficacy observed in vivo, which is associated with specific suppression of translation of mRNAs encoding regulators of cellular metabolism. Importantly, acquired resistance to this cotreatment is driven by translational rewiring that results in dysregulated cellular metabolism and induction of a cAMP-dependent pathway critical for the survival of blood cancers including lymphoma and acute myeloid leukemia. Our studies thus identify key molecular mechanisms underpinning the response of blood cancers to selective inhibition of ribosome biogenesis and define metabolic vulnerabilities that will facilitate the rational design of more effective regimens for Pol I-directed therapies.

Keywords: RNA Polymerase I inhibitor; cAMP-EPAC1/2 pathway; hematological cancers; metformin; ribosome biogenesis and function.

Figure EV1. In vivo transcriptome‐wide analysis of cellular response to ribosome‐targeting therapy

1. A, B

   Flow cytometry analysis of inguinal lymph node cells that are (A) GFP‐positive and propidium iodide‐positive and (B) GFP‐positive and propidium iodide‐negative isolated from C57BL/6 mice with transplanted Eμ‐Myc lymphoma cells treated as indicated for 2 h. 10,000 viable cells of the correct morphology were collected for each sample. Graphs represent mean ± SEM of n = 6.

2. C, D

   Quantitation of (C) phosphorylated RPS6 levels normalized to total RPS6 and (D) p53 protein levels normalized to actin in Fig 1A. Graphs represent mean ± SEM of n = 6.

3. E, F

   Enrichment analysis by MetaCore^® GeneGO of genes in “translation up” and “translation down” categories identified by anota2seq analysis comparing lymph node cells isolated from mice in (E) everolimus single‐agent treatment group or (F) CX‐5461 single‐agent treatment group with those isolated from mice in vehicle group (n = 6). “Ratio” is the value obtained by dividing the number of genes in an indicated molecular process that is found in our data with the number of genes curated in MetaCore^®'s database. “Direction” visualizes the percentage of genes associated with indicated pathways that are up (red) or down (blue).

Data information: (A–D) Data were analyzed by one‐way ANOVA. ns, not significant, P ≥ 0.05; *P ≤ 0.05; ****P ≤ 0.0001.Source data are available online for this figure.

Figure 1. Acute inhibition of ribosome synthesis and function in vivo reduces translation of components of the translational machinery and decreases the abundance of polysome‐associated, energy metabolism‐related mRNAs

1. Western analysis for on‐target effects for everolimus (EV, P‐RPS6) and CX‐5461 (p53). Each lane represents equal amounts of protein from lymph node tissue isolated from a single mouse that received drug vehicles (everolimus vehicle: 1% methylcellulose; CX‐5461 vehicle: 25 mM NaH₂PO₄; V/V; mouse #1–6), 5 mg/kg everolimus (EV; mouse #7–12), 35 mg/kg CX‐5461 (mouse #13–18), or both drugs (CX‐5461 + EV; mouse #19–24) for 2 h (n = 6 per treatment group). Actin was used as a loading control.

2. A schematic illustration of the polysome profiling analysis workflow: Cytoplasmic lysate was layered on top of a linear 10–40% sucrose gradient, ultracentrifuged (222,228 g, 2¼ h at 4°C using SW41Ti rotor), and fractionated using the Foxy Jr Fraction Collector with constant monitoring of absorbance at 260 (A260) nm by an ISCO UA‐6 Absorbance Detector. Fractions (one fraction per minute: 800 μl per tube) corresponding to polysomal mRNAs that were bound by four or more ribosomes were pooled and analyzed by RNA‐seq followed by data analysis using anota2seq or limma.

3. Enrichment analysis by MetaCore^® GeneGO of genes in “translation up” (red) and “translation down” (blue) categories identified by anota2seq analysis comparing lymph node cells isolated from mice in CX‐5461 + EV treatment group with the V/V group (n = 6). “Ratio” values were obtained by dividing the number of significant genes in our data assigned to a molecular process by the total number of genes in the process in MetaCore^®'s database.

4. Genes implicated in “Translation: initiation” and “Translation: Elongation‐Termination” processes based on Fig 1C and Fig EV1E. log₂FC: log₂ fold change; FDR: false discovery rate (adjusted P value).

5. Activity levels of key biological processes involved in cellular growth, proliferation, and metabolism based on single sample gene set enrichment analysis (ssGSEA) of indicated comparisons. Percentage increase (red) or decrease (blue) in the activity levels of key biological processes involved in cellular growth, proliferation, and metabolism based on ssGSEA of indicated comparisons. Data were obtained from n = 6 mice per treatment group.

Source data are available online for this figure.

Figure EV2. Metformin improves the efficacy of CX‐5461 as a single agent or in combination with Everolimus in vitro

1. A

   Propidium iodide (PI) exclusion analysis of Eμ‐Myc lymphoma cells treated with CX‐5461 in the presence and absence of metformin for 48 h. Graphs represent mean ± SEM of n = 3 experiments, and data were analyzed by one‐way ANOVA.

2. B

   PI exclusion analysis of Eμ‐Myc lymphoma cells treated with CX‐5461 + EV in the presence and absence of metformin as indicated for 48 h. Graphs represent mean ± SEM of n = 3 experiments, and data were analyzed by Student's t‐test.

3. C–F

   PI exclusion analysis of (C) MV4‐11, (D) SHI‐1, (E) SKM‐1, and (F) THP‐1 human acute myeloid leukemia cell lines in response to treatments with CX‐5461, everolimus (EV), and metformin as indicated for 72 h. Graphs represent mean ± SEM of n = 4–6, and data were analyzed by two‐way ANOVA.

Data information: ns, not significant, P ≥ 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.Source data are available online for this figure.

Figure 2. The early passage Eμ‐Myc lymphoma cells exhibit altered drug response and distinct metabolic profiles

1. The early passage Eμ‐Myc lymphoma cells used in this study, which were established from mice that were drug‐naïve (“CTRL” cell lines), previously treated with everolimus (“EV” cell lines) or CX‐5461 (“CX” cell lines) alone, or combination of both (“CMB” cell lines). Harvest day indicates the number of days post‐transplantation of lymphoma cells.

2. Cell viability analysis of the indicated early passage Eμ‐Myc lymphoma cells treated with indicated compound(s) for 48 h as determined by Beckman^® Coulter counter. Graphs represent mean ± SEM of n = 3. Data were analyzed by two‐way ANOVA.

3. Hierarchical clustering analysis of metabolomics data obtained from gas chromatography (GC)–mass spectrometry (MS) analysis of early passage cell lines (n = 5–6).

4. Steady‐state abundance of indicated metabolites (fold‐over CTRL cells; FDR ≤ 0.05; n = 5–6).

5. Intracellular ATP levels determined using the liquid chromatography–mass spectrometry in CTRL, EV, CX, and CMB early passage cell lines. Graphs represent mean ± SEM of n = 3.

6. Basal extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) in the early passage cell lines determined using the Seahorse XF96 Extracellular Flux Analyzer; graphs represent mean ± SEM of 6–8 technical replicates for each biological replicate (n = 3).

7. Propidium iodide (PI) exclusion analysis of early passage CX‐5461-everolimus combination therapy‐resistant (CMB), CX‐5461-resistant (CX), and drug‐naïve (CTRL) lymphoma cells treated with indicated concentrations of metformin for 48 h. Graphs represent mean ± SEM of n = 3.

8. CellTiterGLO^®‐based assay measuring cellular ATP levels of the CX-5461‐resistant (CX) and CX-5461‐everolimus combination therapy‐resistant (CMB) cells treated with metformin as indicated for 48 h. Graphs represent mean ± SEM of n = 3.

9. PI analysis of CTRL and CMB cells treated with CX‐5461 and everolimus in the presence and absence of metformin for 48 h as indicated. Graphs represent mean ± SEM of n = 3.

10. PI analysis of CTRL and CX cells treated with CX‐5461 in the presence and absence of metformin for 48 h as indicated. Graphs represent mean ± SEM of n = 3.

Data information: (G, H) Data were analyzed by Student's t‐test. (E, I, J) Data were analyzed by one‐way ANOVA. ns, not significant, P ≥ 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.Source data are available online for this figure.

Figure EV3. In vitro and in vivo characterization of the early passage cell lines

1. A

   Kaplan–Meier curve of C57BL/6 mice transplanted with the CX-5461‐resistant CX #39 early passage Eμ‐Myc lymphoma cells treated with CX‐5461 (35 mg/kg every 3 days; n = 5–6). Survival curves of drug‐naïve cells were actual data reproduced from Devlin et al, 2016.

2. B

   Kaplan–Meier curve of C57BL/6 mice transplanted with CX‐5461 + EV‐resistant CMB #9 early passage cells treated with CX‐5461 (35 mg/kg every 3 days) and everolimus (5 mg/kg daily; n = 5–6). Survival curves of drug‐naïve cells were actual data reproduced from Devlin et al, 2016.

3. C

   Western blot analysis of markers for the on‐target effect of 20 nM everolimus treatment for 3 h, specifically phosphorylation of RPS6 at Serine 240/244 residues and total RPS6 (n = 3). Actin was used as a loading control.

4. D–J

   Synthesis rates of 47S pre‐rRNAs in (D) drug‐naïve (CTRL), (E) everolimus‐resistant (EV), (F) CX-5461‐resistant (CX), and (G) CX‐5461 + EV‐resistant (CMB) cell lines in response to 3‐h 50 nM CX‐5461 treatment were determined by ³²P‐orthophosphate “pulse” labeling. Synthesis rate of 47S pre‐rRNAs in (H) CX and (I) CMB cell lines in response to 10‐h 50 nM CX‐5461 was determined by ³²P‐orthophosphate “pulse” labeling and (J) its quantitation (n = 3). (D–I) 28S rRNA abundance was used as a loading control. EtBr: ethidium bromide. Data were analyzed by Student's t‐test. Graphs represent mean ± SEM of n = 3. *P ≤ 0.05. ***P ≤ 0.001.

5. K

   Western blot analysis for p53 abundance in response to 50 nM CX‐5461 treatment for 3 h. Actin was used as a loading control (n = 2–3).

Source data are available online for this figure.

Figure 3. cAMP‐dependent pathway mediates resistance to CX‐5461‐everolimus cotreatment

1. Enrichment analysis by GeneGO MetaCore^® of the polysomal RNA‐seq data comparing CX‐5461 + EV‐resistant (CMB) and drug‐naïve cells (CTRL; n = 3; false discovery rate (FDR) ≤ 0.05; fold change (FC) ≥ 1.5 or ≤ ‐1.5).

2. Abundance of intracellular 3'5’‐cyclic AMP as measured by a liquid chromatography (LC)–mass spectrometry (MS) analysis. Graphs represent mean ± SEM of n = 3 (5–6 technical replicates each). Data were analyzed by one‐way ANOVA. CTRL vs. EV, P = 0.8951; CTRL vs. CX, P = 0.0871; CTRL vs. CMB, P = 0.0012.

3. Western analysis of EPAC1 and EPAC2 abundance, as well as active GTP‐bound RAP1 levels in the indicated early passage cells during their log‐phase growth period (n = 3). Actin and total RAP1 were used as loading controls. Quantitations of the blots are shown in Appendix Fig S3A–C.

4. Propidium iodide (PI) exclusion assays of CMB cells treated with CX‐5461 and everolimus as indicated in the presence or absence of a selective PKA inhibitor H89 for 48 h.

5. PI exclusion analysis of early passage drug‐naïve (CTRL) lymphoma cells treated with CX‐5461 and everolimus in the presence or absence of selective PKA activator 6‐Bnz-cAMP for 48 h.

6. PI exclusion analysis of the CMB cells treated with CX‐5461 and everolimus as indicated in the presence or absence of EPAC1 inhibitor CE3F4 or EPAC2 inhibitor ESI‐05 for 48 h.

7. PI exclusion analysis of early passage drug‐naïve (CTRL) cells treated with CX‐5461 and everolimus in the presence or absence of the selective EPAC activator 8‐pCPT-2‐O-Me-cAMP for 48 h.

Data information: (D, E, F, G) Data (n = 3) were analyzed by paired one‐way ANOVA. Green triangle: CX‐5461‐everolimus combination therapy‐resistant (CMB) cells clone #8; green square: CMB cells clone #9; green circle: CMB cells clone #31. Blue circle: early passage drug‐naive (CTRL) cells clone #6; blue square: CTRL cells clone #33; blue triangle: CTRL cells clone #38. ns, not significant, P ≥ 0.05; *P ≤ 0.05; **P ≤ 0.01.Source data are available online for this figure.

Figure EV4. Polysome profiling analysis of the early passage Eμ‐Myc lymphoma “CX” cell lines and investigating potential roles of RAPGEF3 (EPAC1) and RAPGEF4 (EPAC2) in human blood cancers

1. A

   Enrichment analysis by GeneGO MetaCore^® of the polysomal RNA‐seq data comparing CX‐5461‐resistant (CX) and drug‐naïve cells (CTRL; n = 3; false discovery rate (FDR) ≤ 0.05; fold change (FC) ≥ 1.5 or ≤ ‐1.5).

2. B, C

   Median mRNA expression levels of (B) RAPGEF3 (mRNA encoding EPAC1 protein) and (C) RAPGEF4 (mRNA encoding EPAC2 protein) generated by gene expression profiling interactive analysis (GEPIA) bioinformatics web tool based on The Cancer Genome Atlas (TCGA) database. LAML: acute myeloid leukemia (tumor, n = 173; normal, n = 70). DLBC: diffuse large B‐cell lymphoma (tumor, n = 47; normal, n = 337). Red: tumor, black: normal. The central band corresponds to the median. The lower and upper hinges correspond to the first and third quartiles (the 25th and 75th percentiles). The upper and lower whiskers extend from the hinges to the largest and smallest values no further than 1.5 * interquartile range from the hinges, respectively. Data points are plotted individually. Data were analyzed by one‐way ANOVA; *P < 0.05. tpm, transcript count per million.

3. D

   Western analysis of EPAC1 and EPAC2 abundance in white blood cell samples (isolated from healthy donors #PMC-15 and #PMC-16) versus MV4‐11, SHI‐1, SKM1, and THP1 human AML cell lines (representative of n = 2).

4. E

   Survival curves of AML patients with low and high EPAC1 (RAPGEF3; n = 27 each; cut‐off: 50%) generated by GEPIA based on TCGA database.

Source data are available online for this figure.

Figure EV5. Inhibiting EPAC1/2 in human acute myeloid leukemia (AML) cell lines and in vivo‐derived Eμ‐Myc lymphoma cell lines

1. A

   Western analysis of the activated, GTP‐bound RAP1 of the indicated human AML cell lines following treatments with the EPAC1 inhibitor CE3F4 and EPAC2 inhibitor ESI‐05 for 72 h (representative of n = 2).

2. B

   CellTiterGLO^®‐based viability assay of MV4‐11, SHI‐1, SKM‐1, and THP‐1 human AML cells treated as indicated for 72 h. Graphs represent mean ± SEM of n = 3. Data were analyzed by one‐way ANOVA.

3. C, D

   Western analysis of PKA activity using the phosphorylated PKA substrate antibody on whole cell extracts isolated from the indicated early passage cell lines treated for 24 h with (C) H89 (n = 3) or (D) 6‐BNZ-cAMP (representatives of n = 3). Actin was used as a loading control.

4. E

   Western analysis of the activated, GTP‐bound RAP1 of the early passage CX‐5461 + EV‐resistant (CMB) cell extracts isolated following treatments with indicated EPAC inhibitors for 24 h (representative of n = 2).

5. F

   Western analysis of activated, GTP‐bound RAP1 using early passage drug‐naive (CTRL) Eμ‐Myc lymphoma cell extracts isolated following treatments with the selective EPAC activator 8‐pCPT-2‐O-Me-cAMP for 24 h (n = 1).

6. G

   Western analysis of the activated, GTP‐bound RAP1 of the early passage CX‐5461-resistant (CX) cell extracts isolated following treatments with indicated EPAC inhibitors for 24 h (n = 1).

7. H

   PI exclusion analysis of the CX cells treated with CX‐5461 as indicated in the presence or absence of EPAC1 inhibitor CE3F4 or EPAC2 inhibitor ESI‐05 for 48 h. Data were analyzed by paired one‐way ANOVA. Graphs represent mean ± SEM of n = 3. Black triangle: CX-5461‐resistant (CX) cells clone #14, black square: CX cells clone #20, black circle: CX cells clone #39.

Data information: ns, not significant, P ≥ 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.Source data are available online for this figure.

Figure 4. Polysome profiling analysis identifies a link between translational reprogramming and resistance to CX‐5461 and CX‐5461 + everolimus

1. A heatmap illustrating the abundances of polysome‐associated mRNAs that are associated with the mRNA translation/protein synthesis based on the Gene Ontology Resource Database in drug‐naïve (CTRL) or CX-5461‐everolimus combination therapy‐resistant (CMB) cell lines (n = 3). The colors illustrate the expression values of indicated mRNAs that were normalized using voom (red: high expression; blue: low expression). Values represent the median gene expression levels across replicate samples.

2. Polysome profiles demonstrating increased ribosome abundance in CMB cells as compared to CTRL and CX-5461‐resistant (CX) cells (representatives of n = 3).

3. Quantitation of polysome:monosome ratio in the indicated early passage Eμ‐Myc lymphoma cells as determined by measuring the area under the curve using ImageJ software. Graph represents mean ± SEM of n = 3. Data were analyzed by one‐way ANOVA. ns, not significant; **P ≤ 0.01.

4. Length of the 5'UTR of mRNAs that are more actively translated by the CMB cells (as compared to CTRL cells) vs. non‐target mRNAs (n = 3). The central band corresponds to the median. The lower and upper hinges correspond to the first and third quartiles (the 25^th and 75^th percentiles). The upper and lower whiskers extend from the hinges to the largest and smallest values no further than 1.5 * interquartile range from the hinges, respectively. Data beyond the end of the whiskers are plotted individually. One‐sided Wilcoxon tests were used to determine significance.

5. The most significantly enriched motif in the 5'UTR of mRNAs that are more actively translated in the CMB cells compared to CTRL cells based on Multiple Em for Motif Elicitation (MEME) analysis (n = 3).

6. Polysomal abundance of mRNAs (selected based on a nominal P value < 0.01 cut‐off in either the CMB vs. CTRL or CX vs. CTRL as analyzed by limma) encoding components of mitochondrial oxidative phosphorylation based on polysome profiling data. Red denotes upregulation, and blue denotes downregulation (n = 3; genes with an adjusted P value (false discovery rate; FDR) ≤ 0.05 were considered to be significant and are denoted with a black border).

Source data are available online for this figure.

Figure 5. Metformin inhibits cAMP‐EPAC1/2 signaling and resensitizes combination therapy‐resistant early passage Eμ‐Myc lymphoma cells to CX‐5461‐everolimus cotreatment in vivo

1. A

   Western analysis demonstrating the effects of metformin treatment for 48 h on the levels of active GTP‐bound RAP1 in CX and CMB cells (n = 3) and its quantitation.

2. B, C

   Proportion of green fluorescent protein (GFP)‐positive CMB (clone #8) cells in (B) lymph node and (C) spleen of transplanted C57BL/6 mice treated as indicated for 6 h on day 12 post‐transplant. Graphs represent mean ± SEM of six mice per group.

3. D

   Kaplan–Meier curve of C57BL/6 mice transplanted with CX‐5461‐everolimus‐resistant (CMB #8) early passage Eμ‐Myc lymphoma cells treated with vehicles (everolimus vehicle: 1% methylcellulose; CX‐5461/metformin vehicle: 25 mM NaH₂PO₄; n = 6); CX‐5461 (35 mg/kg every twice weekly) and everolimus (5 mg/kg daily; n = 8), metformin (400 mg/kg twice daily; n = 6), or CX‐5461, everolimus and metformin (35 mg/kg twice weekly, 5 mg/kg daily and 400 mg/kg twice daily, respectively; n = 8). Light gray: 5‐day metformin pre‐treatment period, dark gray: treatment period. Data were analyzed by a log‐rank (Mantel–Cox) test. Vehicle vs. CX-5461‐everolimus: P = 0.0006, Vehicle vs. CX-5461‐everolimus‐metformin: P = 0.0001. CX-5461‐everolimus vs. CX-5461‐everolimus‐metformin: P = 0.0003.

Data information: (A) Graphs represent mean ± SEM of n = 3. Data were analyzed by Student's t‐test (A) or one‐way ANOVA (B, C). ns, not significant, P ≥ 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.Source data are available online for this figure.