Protein regulation in multiple myeloma

基因表达谱先前定义了与临床行为相关的7个分子不同的多发性骨髓瘤（MM）亚组。包含t（4;14）易位的MMSET/FGFR3亚组和包含t（14;16）、t（14,20）和8q24.3的MAF亚组与最短的无进展生存期和总生存期相关。最近，我们证明MMSET/FGFR3 （MS）和MAF （MF）亚群的MM细胞系依赖MEK信号来生长和存活。通过遗传或药理学手段抑制丝裂原活化蛋白激酶途径，特异性地降低MAF及其靶基因的表达。此外，由于MEK抑制剂对骨髓微环境的影响，在标准治疗剂存在的情况下，MEK抑制剂可以增强骨髓瘤细胞的凋亡，这表明MEK抑制剂可以杀死对常规治疗产生抗性的细胞。作为我们正在进行的MEK抑制剂AZD6244在复发或难治性MM患者中的II期多中心临床试验的一部分，我们对一部分患者进行了广泛的分子分析，以表征MEK抑制的基础及其与临床结果的相关性。迄今为止，在参加试验的37名患者中，12名患者同意进行相关采样，包括在基线和开始AZD6244治疗后第2天进行骨髓抽吸（75 mg，每天两次，连续28天周期）。使用磁珠（Miltenyi）纯化CD138+细胞，并按照标准程序提取RNA、DNA和蛋白质。采用qRT-PCR和RT-PCR分别检测MAF基因（c-MAF、MAF- b、MAF- a）的表达水平和JH-MMSET杂交株的存在。采用纳米免疫分析法评估处理前后总BIM、总mek1 /2和磷酸化ERK1/2水平。采用焦磷酸测序检测RAS和BRAF基因突变的存在。目前，11例患者已被分子表征并纳入中期分析。我们分别发现3例（27%）、2例（18%）和1例（9%）患者存在KRAS、NRAS和BRAF突变。基于迄今为止分析的8例患者，qRT-PCR显示4例（50%）c-MAF或MAF-B基因过表达；maa分析的结果正在等待中。RTPCR检测到1例患者存在JH-MMSET杂交。目前，我们有8例患者的详细临床数据：1例患者（MMSET）有非常好的部分缓解，反应持续时间（DOR）为8个月；1例患者（MAF-B）部分缓解，DOR为6个月；2例患者（1例MAF-B， 1例有待结果）病情稳定，DORs分别为5+和13个月。在这些患者中，在基线时检测到高水平的phospho-ERK1/2，并且在暴露于AZD6244后显着降低。此外，MEK抑制导致总ERK1/2和MEK1/2水平升高以及促凋亡BIM。在亚分析中，从携带MMSET易位的患者的髓外浆细胞瘤中获得的CD138+细胞用AZD6244体外处理；这导致药物暴露后磷酸化erk1 /2水平检测不到。在这组多发性骨髓瘤复发患者中，对几种治疗方法都有难治性，并且骨髓瘤细胞对MEK有依赖的分子证据，我们发现AZD6244可以产生持久的反应（长达13个月）。我们的新结果为MEK依赖性骨髓瘤细胞的MEK抑制提供了分子和临床见解，并为MM的合理药物分配提供了支持。

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.