MARCKS Confers Resistance to Velcade Through Skp2/p27-Mediated Pathway in Multiple Myeloma

Abstract 985

Despite advances in clinical care, multiple myeloma (MM) remains an almost universally fatal disease. Velcade, a novel proteasome inhibitor represents a promising new clinical strategy for relapsed and refractory MM. However, only 30–40% of MM patients respond to this treatment. In order to gain insight into the mechanism of velcade resistance, we examined the differentially expressed proteins between 8226S (velcade sensitive) and 8226R5 (velcade resistant) myeloma cell lines using iTRAQ mass spectrometry (MS). The MS based multiple-reaction-monitoring technique (MRM) was then used to independently verify the quantitative differences of the protein expression levels between the two cell lines. We identified several cancer specific proteins to be highly differentially expressed between 8226S and 8226R5 by iTRAQ mass spectrometry. Of particular interest is Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS), a PKC substrate that has been implicated in conferring drug resistance to ovarian carcinoma cells. Differential expression of MARCKS between sensitive and resistant cells was verified by MRM and then confirmed by RT-PCR and Western blot analysis. MARCKS over-expression was also found in additional MM cell lines as well as patient samples that were resistant to velcade. Knockdown of MARCKS through siRNA strategies enhanced velcade sensitivity by increasing apoptosis in the velcade treated resistant cell line 8226R5. Moreover, inhibition of PKC, the upstream activator of MARCKS by enzastaurin significantly blocked phosphorylation of MARCKS leading to a caspase dependent apoptosis in the 8226R5 cells. In addition, enzastaurin enhanced velcade proteosome inhibitory effect and rendered the 8226R5 cells more sensitive to velcade treatment. Combination of velcade with enzastaurin showed a synergistic cytotoxic response in MM cells compared to either drug used alone. Furthermore, knockdown of MARCKS or inhibition of MARCKS phosphorylation by enzastaurin led to a decrease in Skp2 expression and an increase in p27 and p21, as well as a reduction of cyclin E and CDK2, resulting in G1/S cell cycle arrest and an increase in apoptosis in MM cells. Taken together our results suggest that phosphorylated MARCKS plays an important role in velcade resistance in MM and as such may be used as an important therapeutic target. In addition, MARCKS may be a biomarker to select patients that would most benefit from patient specific treatment.