An unexpected protein interaction promotes drug resistance in leukemia

Abstract

The overall survival of patients with acute myeloid leukemia (AML) is poor and identification of new disease-related therapeutic targets remains a major goal for this disease. Here we show that expression of MPP1, a PDZ-domain-containing protein, highly correlated with ABCC4 in AML, is associated with worse overall survival in AML. Murine hematopoietic progenitor cells overexpressing MPP1 acquired the ability to serially replate in methylcellulose culture, a property crucially dependent upon ABCC4. The highly conserved PDZ-binding motif of ABCC4 is required for ABCC4 and MPP1 to form a protein complex, which increased ABCC4 membrane localization and retention, to enhance drug resistance. Specific disruption of this protein complex, either genetically or chemically, removed ABCC4 from the plasma membrane, increased drug sensitivity, and abrogated MPP1-dependent hematopoietic progenitor cell replating in methylcellulose. High-throughput screening identified Antimycin A as a small molecule that disrupted the ABCC4-MPP1 protein complex and reversed drug resistance in AML cell lines and in primary patient AML cells. In all, targeting the ABCC4-MPP1 protein complex can lead to new therapies to improve treatment outcome of AML, a disease where the long-term prognosis is poor.

Fig. 1

The PDZ-domain protein MPP1 is highly expressed in AML, associates with poor AML survival, and interacts with the ABC transporter, ABCC4. a Venn diagram depicting approach used to identify PDZ proteins, in AML, that correlate with ABCC4 (left) and MPP1 expression among human cancers (right). b A heatmap of mRNA expression data depicting the relative level of MPP1 and ABCC4 in pediatric AML patients. c Immunoblot analysis of ABCC4 and MPP1 protein levels in AML cell lines. d Kaplan–Meier survival curve of AML patients with MPP1 expression above or below the median. e Model depicting amino acids of the MPP1 PDZ-domain interacting with the amino acids ETAL of the ABCC4-PDZ motif. Consensus sequence of the PDZ motif predicted to interact with MPP1 PDZ domain. f Purified MPP1 protein was incubated with purified ABCC4, subsequently various amounts of a small competitor peptide harboring either the ABCC4-PDZ motif or one that lacks the ABCC4-PDZ motif (ABCC4ΔC) were added to the complex. Following immunoprecipitation the amount of MPP1 in the complex was determined by immunoblot analysis (n = 3)

Fig. 2

MPP1 promotes hematopoietic progenitor cell (HPC) replating and only requires the PDZ domain to bind ABCC4. a (left panel) HPC were transduced with retroviral plasmids containing cDNAs for either MPP1-ires-CFP or MYCN-ires-YFP, purified by FAC sorting and plated in methycellulose medium containing IL3, IL6, SCF, and EPO. Colonies were counted after 7 days growth and serially replated (right panel). HPC from either Abcc4^+/+ or Abcc4^−/− were transduced with MPP1 as described above, FAC sorted and replated in methylcellulose serially. b Immunoblot analysis of lysates from the AML cell line MO7e following immunoprecipitation of either ABCC4 or MPP1. SN supernatant. IP immunoprecipate. c ABCC4 expression plasmids either containing the PDZ motif or lacking the PDZ-motif (ABCC4ΔC) with an MPP1 expression vector. d Diagram depicting the protein domains of MPP1. e Coomassie-stained polyacrylamide gel of purified full-length MPP1 and purified sub-domains. f Incubation of purified MPP1 fragments with MO7e lysates, followed by immunoprecipitation and probing the immunoprecipitate with an antibody against ABCC4 (n = 3, bars represent SEM). g A proximity-ligation assay shows ABCC4 and MPP1 interact in MO7e cells (red). Experiments were replicated three times; bars represent SEM

Fig. 3

Peptides that block the ABCC4–MPP1 protein interaction reduce ABCC4 membrane localization and MPP1-driven hematopoietic progenitor replating in methylcellulose. a Representative image of ABCC4 membrane localization in cells transfected with ABCC4-GFP and MPP1 treated with either a cell penetrating peptide harboring the ABCC4-PDZ motif or lacking the PDZ motif (ABCC4ΔC). Blue-wheat germ agglutinin conjugated with Alexa Fluor 647, Red = Calreticuin-RFP, and Green = ABCC4-GFP. b Quantification of ABCC4 localization (n = 20 fields of view; bars represent SEM). c HPC were transduced with retroviral plasmid containing cDNAs for MPP1-ires-CFP or the empty vector, purified by FAC sorting and plated in methycellulose medium containing IL3, IL6, SCF, EPO and supplemented with cell penetrating peptide harboring the ABCC4-PDZ motif or lacking the PDZ-motif (ABCC4ΔC). Colonies were counted after 7 days growth and serially replated. Fresh peptide was added at each replating step (n = 3, bars represent SEM)

Fig. 4

ABCC4 plasma membrane localization depends upon MPP1 as revealed by CRISPR-Cas9 deletion. a HEK293 cells were transfected with a constant amount of an ABCC4 expression vector and various amounts of an MPP1 expression vector. The total amount of DNA was the same in each condition. The upper panel shows a representative immunoblot of lysates from the transfected cells probed for ABCC4, MPP1 and actin. The lower panel depicts the IC50 for 6-mercaptopurine (6MP) for the transfected cells which provides a functional measure of ABCC4 function. b The sensitivity of cells to the non-ABCC4 drug etoposide is not affected by MPP1. c Incubation of cells with the cell penetrating peptide harboring the ABCC4-PDZ motif increases the sensitivity of these cells to 6MP, whereas the peptide lacking the ABCC4-PDZ motif (ABCC4ΔC) is without effect. d MO7e cells are sensitized to cytarabine cytotoxicity when incubated with the ABCC4-PDZ peptide. MO7e cells lacking ABCC4 have no additional sensitivity to cytarabine. e Immunoblot analysis of drug transport protein expression in MO7e cells in which CRISPR-Cas9 gRNAs to either ABCC4 or MPP1 were used to delete either ABCC4, MPP1, or both. f Deletion of either ABCC4, MPP1, or both genes sensitized MO7e cells to 6MP. g Deletion of either ABCC4, MPP1, or both genes did not affect etoposide sensitivity. h Deletion of MPP1 strongly reduces ABCC4 surface expression, but has no effect on Na⁺/K⁺ ATPase. Surf plasma membrane surface, SN supernatant. All experiments were performed in triplicate, bars represent SEM, *P < 0.05

Fig. 5

A high-throughput screen identified Antimycin A as a small molecule that blocks the ABCC4 and MPP1 protein interaction. a Diagram depicts TR-FRET assay screening for small molecule inhibitors of the MPP1 and ABCC4 protein interaction. b TR-FRET produces positive signal only when MPP1 and ABCC4 interact. c Scatterplot of all compounds tested in the TR-FRET assay (Blue = hits, red = negative control, green = positive control). d Dose response of Antimycin A. Structure (left), disruption of protein interaction between MPP1 and ABCC4 (middle) and graphical estimate of the IC₅₀ for Antimycin A (right). e The cytotoxicity profile of Antimycin A is not influenced by ABCC4. All experiments were performed in triplicate, bars represent SEM

Fig. 6

Antimycin A decreases ABCC4 membrane localization and selectively increases drug sensitivity in cell line and primary AML patient samples. a, b Antimycin A (10 nM) specifically sensitizes ABCC4 expressing MO7e cells, but not ABCC4 KO cells, to 6MP (a) and cytarabine (b). c Primary AML samples from three adult patients show increased Annexin V⁺ percentage of cells with combination treatment of Antimycin A and cytarabine vs cytarabine alone. Each value was determined from three technical replicates and the error bars represent the SEM. d Confocal microscopy shows Antimycin A causes ABCC4 to relocalize from the plasma membrane. e Quantification of ABCC4 localization (n = 20). f Surface biotinylation demonstrates that Antimycin A reduces ABCC4 at the plasma membrane. g MPP1-driven hematopoietic progenitor cell replating is attenuated by Antimycin A (10 nM). All experiments were performed in triplicate, bars represent SEM, *P < 0.05