MUC1 Inhibition Overcomes Chemotherapy Resistance in Acute Myeloid Leukemia

Chemotherapy is not curative for the majority of patients with acute myeloid leukemia (AML) due to the presence of leukemia stem cells (LSCs) and the emergence of other clonal populations that exhibit resistance to cytotoxic therapy. Understanding the pathways responsible for the development of chemotherapy resistance is critical for developing novel strategies that more effectively target AML. We have previously demonstrated that the MUC1 oncogene is expressed on AML cells including LSCs. MUC1 is a heterodimeric glycoprotein where the MUC1-C subunit functions as an oncoprotein. Importantly in AML, MUC1-C facilitates the nuclear translocation of active β-catenin necessary for downstream effectors including survivin, a negative regulator of apoptosis. Recent data has demonstrated that survivin inhibition is crucial in conferring susceptibility to chemotherapeutic agents in a leukemia model. In the current study, we sought to examine the effect of MUC1-C inhibition on survivin levels and the sensitivity of leukemic cells to cytotoxic chemotherapy.

To assess the effect of MUC1-mediated signaling on survivin expression, MUC1-C was silenced using a lentiviral shRNA hairpin against MUC1-C in two AML cell lines, MOLM-14 and THP-1. Silencing of MUC1-C was confirmed by flow cytometric and western blot analyses and resulted in the downregulation of β-catenin and its target, survivin, at both the protein and mRNA level. In contrast, MUC1-C overexpression led to increased survivin expression.

The role of MUC1-C as a mediator of resistance to cytotoxic chemotherapy was assessed. A stable MUC1-C gene knockdown of the AML cell line, MOLM-14, was generated using CRISPR/Cas9 technology. The MOLM-14 CRISPR and MOLM-14 wild-type (WT) cell lines were independently treated with increasing doses of the cytotoxic chemotherapeutic agent, cytarabine (Ara-C 50-1000 nM). The MOLM-14 CRISPR cell line demonstrated reduced cell viability utilizing an ATP-based luminescence assay (CTG, Promega) as compared to the MOLM-14 WT cell line at 72 hours (14% vs. 32%) and 96 hours (6% vs. 28%) after treatment with Ara-C. The results demonstrate that MUC1-C confers resistance to chemotherapy, and that the loss of MUC1-C in leukemic blasts significantly increases AML susceptibility to cytotoxic chemotherapy.

Next, we investigated if the functional inhibition of MUC1-C would increase the sensitivity of AML to Ara-C. A novel cell-penetrating peptide, GO-2O3, binds to the MUC1-C subunit and blocks MUC1-C homodimerization and function. Two AML cell lines, MOLM-14 and MV4-11, were treated with increasing doses of Ara-C (25-1000nM) and GO-2O3 (1-5uM) to establish dose-dependent cytotoxicity curves. Based on the cytotoxicity curves, doses of Ara-C (50, 100, 125 nM) and GO-2O3 (1.0, 1.5, 2.0 uM) were selected for combination therapy.  Analysis at 48 hours utilizing CTG demonstrated statistically significant synergy validated by the combination index (CI) calculated through CompuSyn [MV4-11 (0.54) and MOLM-14 (0.86)] where CI values < 0.90 indicate drug synergy. These results were confirmed in both the MOLM-14 and MV4-11 cell lines by staining for Annexin V/PI with FACS analysis after 48 hours of treatment. In MOLM-14, apoptosis and necrosis were noted as follows: no treatment (1.5%, 0.6%), GO-2O3 (3.7%, 6%), Ara-C (12%, 9.8%), and the combination (17%, 34%). In MV4-11, apoptosis and necrosis were noted as follows: no treatment (2.1%, 0.9%), GO-2O3 (4%, 6%), Ara-C (17.3%, 11.2%), and the combination (14%, 40%). The functional inhibition of MUC1-C in combination with Ara-C resulted in both decreased cell viability and increased cell death as compared to either agent alone.

In conclusion, the data demonstrates that MUC1 expression on AML cells plays a critical role in conferring resistance to chemotherapy. Via its effector, survivin, MUC1-C inhibition renders leukemia cells more susceptible to cytotoxic injury in synergy with Ara-C. A clinical trial evaluating the combination of Ara-C and GO-203 in patients with relapsed AML is planned.