Targeting Drug-Resistant Myeloma Cells in an Acidic Milieu by Pim Inhibition

Abstract 5011

Myeloma (MM) cells and osteoclasts (OCs) mutually interact in MM bone lesions to confer aggressiveness and drug resistance in MM cells along with the progression of bone destruction. The MM-OC interaction appears to create a highly acidic milieu in bone lesions by proton produced from activated OCs and lactate from proliferating MM cells (the Warburg effect). Tumor acidity is known to cause drug resistance in cancers. As suggested, acidic conditions or cocultures with OCs blunt the cytotoxic effects of anti-MM agents such as doxorubicin. Therefore, the development of innovative modalities is strongly needed to overcome the drug resistance in an acidic microenvironment in MM. We have found that the serine/threonine kinase Pim-2 is over-expressed in MM cells as an anti-apoptotic mediator, and further up-regulated to cause their aggressiveness and drug resistance when cocultured with bone marrow stromal cells or OCs (Leukemia, 2011). In the present study, we aim to clarify the role of Pim-2 in MM cell growth and survival in acidic conditions and the therapeutic impact of Pim inhibition on drug-resistant MM cells. The phosphorylation of 4E-BP1, a substrate of Pim-2, as well as Pim-2 expression were up-regulated in MM cells in media acidified by lactic acid or HCl; the Pim inhibitor SMI-16a preferentially induced MM cell death at pH 6. 8 or 6. 4 rather than at pH7. 4, suggesting Pim-2-dependent MM cell survival in an acidic milieu. In contrast to their quick death at pH 7. 4, substantial numbers of MM cells remained intact at pH6. 8 or lower upon treatment with doxorubicin. Interestingly, Pim inhibition enhanced the cytotoxic activity of doxorubicin against MM cells, which was more marked in acidic conditions. The ABC transporter BCRP is aberrantly over-expressed in drug-resistant MM cells. The intracellular levels of auto-fluorescence emitting doxorubicin and mitoxantron were reduced over time after their passive incorporation into BCRP-expressing RPMI8226 cells. However, the Pim inhibitor SMI-16a at 50 microM substantially restored the intracellular levels of these drugs; the intracellular retention of these drugs by the Pim inhibition was more prominent in acidic conditions, suggesting the correlation of BCRP function with Pim-2 up-regulation by acid. BCRP phosphorylation has been demonstrated to be essential for its functionality. Treatment with SMI-16a reduced BCRP phosphorylation in BCRP-expressing INA6 cells, suggesting Pim-dependent activation of BCRP. The reduction was more obvious at pH6. 8 than at pH7. 4. “Side population (SP)” is regarded as a highly drug-resistant fraction with enhanced BCRP activity, which is considered to contain clonogenic or tumor-initiating cells. The Pim inhibitor SMI-16a minimized the size of SP fractions in RPMI8226 and KMS11 cells; the reduction of SP fractions by the Pim inhibition was also more marked in acidic conditions. Collectively, these results suggest that Pim-2 plays an important role in MM cell survival and drug resistance in an acidic milieu, and that Pim-2 may become an important therapeutic target of MM cells which preferentially gain drug resistance in acidic bone lesions.