Protein regulation in multiple myeloma

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Gene expression profiling previously defined 7 molecularly distinct multiple myeloma (MM)subgroups associated with clinical behavior. The MMSET/FGFR3 subgroup, harboring the t(4;14) translocation, and the MAF subgroup including t(14;16), t(14,20) and 8q24.3, were associated with the shortest progression-free and overall survival. Recently, we demonstrated that MM cell lines of the MMSET/FGFR3 (MS) and MAF (MF) subgroups are dependent on MEK signaling for growth and survival. Inhibition of mitogen-activated protein (MAP) kinase pathway, by genetic or pharmacologic means, specifically decreased expression of MAF and its target genes. In addition, MEK inhibition enhances myeloma cell apoptosis in the presence of standard therapeutic agents due to the effects on the bone marrow microenvironment, suggesting that MEK inhibitors could kill cells resistant to conventional therapies. As part of our ongoing phase II multi-center clinical trial testing the MEK inhibitor AZD6244 in patients with relapsed or refractory MM, we conducted extensive molecular profiling for a subset of patients, to characterize the underpinnings of MEK inhibition and its correlation with clinical outcomes.To date, of the 37 patients enrolled on trial, 12 patients consented to correlative sampling,including bone marrow aspirates performed at baseline and day 2 after initiation of AZD6244 treatment (75 mg twice daily, continuously on a 28 day cycle). CD138+ cells were purified using magnetic beads (Miltenyi) and subjected to RNA, DNA and protein extraction following standard procedures. qRT-PCR and RT-PCR was performed to detect the expression level of MAF genes (c-MAF, MAF-B, MAF-A ) and the presence of the JH-MMSET hybrid respectively. Nanoproimmunoassay was used to assess the level of total BIM, total and phospho-MEK1/2 and ERK1/2 pre- and post-treatment. Pyrosequencing was performed to detect the presence of mutations in RAS and BRAF genes. At this time, 11 patients have been molecularly characterized and included in this interim analysis.We found 3 (27%), 2 (18%) and 1 (9%) patients with a KRAS, NRAS, and BRAF mutation,respectively. Based on 8 patients analyzed to date, qRT-PCR showed that 4 (50%) overexpressed c-MAF or MAF-B genes; results from the MAF-A analysis are currently pending. RTPCR detected the presence of JH-MMSET hybrid in one patient. At this time, we have detailed clinical data for 8 patients: one patient (MMSET) had a very good partial remission and the duration of response (DOR) was 8 months; one patient (MAF-B) had a partial remission with a DOR of 6 months; and two patients (one MAF-B, one with pending results) had stable disease with DORs of 5+ and 13 months. In these patients, high levels of phospho-ERK1/2 were detected at baseline, and were dramatically reduced after AZD6244 exposure. Also, MEK inhibition resulted in increased levels of total ERK1/2 and MEK1/2 as well as pro-apoptotic BIM. In subanalysis, CD138+ cells obtained from an extramedullar plasmacytoma in the patient carrying the MMSET translocation were treated in vitro with AZD6244; this resulted in undetectable levels of phospho-ERK1/2 post drug exposure.Among this group of relapsed MM patients, refractory to several lines of therapy and withmolecular evidence for myeloma cells being MEK dependent, we found AZD6244 to result in durable responses (up to 13 months). Our novel results provide molecular and clinical insight on MEK inhibition in MEK-dependent myeloma tumor cells, and support development of rational drug allocation in MM.