035: Potential Epigenetic Opioid-Chemotherapy Drug Interactions

Statement of Purpose, Innovation or Hypothesis: Leukemia is characterized by aberrant cytosine methylation (5mC) that alters the regulation of tumor suppressors or apoptotic programs. DNA methylation inhibitors (DNMTi) are standard treatment for adults with acute myeloid leukemia (AML) and children with relapsed AML. However, DNMTi resistance universally develops. New information suggests drug interactions between DNMTi and routinely used opioids are likely. Opioids at nanomolar (clinical) concentrations act on the DNA de-methylase epigenetic regulator family (TET) which catalyzes conversion of 5mC to 5-hydroxymethylcytosine (5hmC). While aberrant TET activity is well established with chemoresistance in AML, evidence of opioids acting on TET is new. The consequences of opioid-induced changes to DNA 5mC-to-5hmC on the cancer cell killing activity of DNMTi is not understood. We hypothesize that opioids could change the activity of DNMTi chemotherapy.

Description of Methods & Materials: Cell lines/CRISPR/culture: Philadelphia chromosome-positive (Ph⁺) chronic myeloid leukemia (CML; K562), Ph⁺ acute lymphocytic leukemia (ALL; SUP-B15), T cell-acute lymphocytic leukemia (T-ALL; CCRF-CEM), and acute myeloid leukemia (AML; HL-60, Kasumi-1, THP-1, and U937). K562 OPRM1-/- cells were generated using CRISPR-Cas9 and GFP-tagged mu-opioid receptor was reintroduced to generate stable rescue cell lines. Viability and apoptosis data were generated using a validated high-throughput assay (CellTiter-Glo; Caspase-Glo 3/7). Changes in global DNA hydroxymethylation (5hmC) were assessed using a DNA 5hmC ELISA. TET gene and protein expression were assessed using qRT-PCR and chemiluminescent western blot (TET, OPRM1, GFP). respectively. Data and statistical analyses were performed in R (version 4.2.0), Prism 10.2.1, and SynergyFinder 3.0 (synergyfinder.fimm.fi). All experiments were performed in ≥3 independent experiments with ≥4 replicates per data point.

Data & Results: At clinical concentrations, opioids (morphine, fentanyl, oxycodone) and a synthetic opioid peptide, stimulated leukemic cell growth in all tested cell lines, except THP1. Naloxone, an opioid receptor antagonist, enhanced leukemic cell growth including in the presence of full mu-opioid receptor agonists. Morphine, fentanyl, and oxycodone increased resistance to molecularly targeted inhibitors (imatinib, dasatinib, venetoclax, azacitidine) and conventional cytotoxic (cytarabine, daunorubicin) chemotherapy in ALL, AML, and CML cells. The highest degree of resistance was with DNMTi (azacitidine) (Figure 1). When comparing WT (or OPRM1 rescue lines) and OPRM1-/- cells, resistance to frontline therapy for Ph⁺ CML (imatinib/dasatinib) as well as DNMTi was substantially higher in WT where the mu-opioid receptor was intact (p < 0.0001). K562 OPRM1-/- had ~30% less TET2 compared to WT. Additionally, TET2 gene expression increased upon opioid treatment (morphine 24h) but not for OPRM1-/- (p < 0.05). Opioid treatment and the absence of OPRM1 shifted expression of subcellular isoforms of TET2. In WT K562, opioid treatment reduced expression of the ‘cytoplasmic’ TET2 isoform ~12% but increased the TET2 ‘nuclear’ isoform ~54% (39% increase in OPRM1-/-). Changes in DNA 5hmC content varied by cell line and opioid type but were most pronounced with fentanyl (K562; p=0.026, U937; p=0.002). Among leukemic cell lines, basal TET expression differed substantially.

Interpretation, Conclusion or Significance: Recently it was discovered that morphine and naloxone can bind and inhibit TET at nanomolar concentrations, stimulating neural stem cell proliferation. Our findings confirm that opioids can engage mu-opioid receptor-dependent and -independent pro-growth pathways and influence chemotherapeutic activity, likely via TET-mediated epigenetic changes.

Disclosures: The authors have no conflicts or disclosures to report.

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