Protein Synthesis Rates Regulate Tumor-Initiating Potential and Chemoresistance in Multiple Myeloma

Introduction: Multiple myeloma (MM) remains incurable despite improved remissions with novel agents. Relapse eventually occurs in the form of drug-resistant disease that carries a dismal prognosis. Relapse is dependent on stem cell functions such as chemoresistance and self-renewal for the maintenance of clonogenic potential over time. A better understanding of the drivers of these functions may lead to novel therapies for relapsed disease. Although MM is prototypically characterized by antibody secreting plasma cells, disease progression has been correlated with loss of protein production and clonal dominance of undifferentiated plasma cell precursors. We hypothesized that low protein translation rates in MM may enhance key stem cell functions, thus exposing a novel therapeutic opportunity for patients with advanced disease.

Methods and Results: MM cell lines were treated with cycloheximide (CHX), a small molecule inhibitor of ribosomal elongation, to determine whether modulating protein translation rates directly impacts sensitivity of MM cells to chemotherapy. At sub-lethal doses, CHX inhibited protein translation by ³⁵S-methionine incorporation in MM cells (NCI-H929 and MM1.S), but increased the relative IC₅₀ of cells to bortezomib, dexamethasone, and melphalan in cell viability assays (range 1.5-5-fold, p-value <0.05). EIF5, a translation initiation complex subunit that can also inhibit translation, was upregulated in response of cells to bortezomib as measured by qRT-PCR (100-fold in NCI-H929, 30-fold in MM1.S). Consistently, forced over-expression of EIF5 in these cell lines significantly inhibited protein translation (range 2.5-3-fold, p-value <0.05), and, similar to CHX, also increased the relative IC₅₀ for multiple chemotherapeutic agents (range 1.8-3-fold, p<0.05). We also found EIF5 to be enriched in progenitor-like, CD138 negative cells in MM cell lines (2.1-2.6-fold, p<0.05) and patient samples (1.5-7.3-fold), which correlated with decreased protein translation rates in these cells (7.4-13.6-fold, p<0.05). Modulation of protein translation rates can influence stem cell functions in normal hematopoiesis, and so we next tested the impact of inhibiting protein translation on MM tumor stem cell functions. In MM cell lines, over-expression of EIF5, or treatment with CHX, significantly enhanced clonogenic growth in vitro as determined in methylcellulose-based colony forming cell assays (2.1-4-fold, p<0.05). Moreover, in a limiting dilution assay, EIF5 expression enhanced tumor initiating cell (TIC) frequency in MM1.S cells that were subcutaneously implanted into immunodeficient mice (TIC frequency of 1/4358 for control vs. 1/262for EIF5 expressing cells; p-value = 0.000003). To explore whether enhancing protein translation has the opposite effect, thus being of possible therapeutic value, we exposed cells to L-leucine, an amino acid that can stimulate protein translation rates when given in excess. L-leucine treatment of MM cell lines significantly decreased colony formation as a single agent (1.5-2-fold, p<0.05).

Conclusion: We have shown that protein translation rates are linked to chemoresistance and stem cell functions in MM. Modulating protein translation is a novel therapeutic opportunity for patients with relapsed disease. Conversely, therapies that inhibit protein synthesis rates may paradoxically lead to poor outcomes.