Local and systemic control of multiple myeloma colonization and growth by MMP-13

摘要 尽管医学上取得了进步，但多发性骨髓瘤仍然是一种致命的疾病。支撑其实现的机制 骨髓瘤在局部骨微环境中进展，并通过骨髓瘤定植骨骼以 产生多处疼痛的溶骨性病变，亟待解决。基质金属蛋白酶(MMPs) 是通过细胞因子和生长因子调节肿瘤-骨相互作用的关键调节因子 生物利用度/活性来自我们团队的新数据已经确定，在人类的疾病样本中， 基质金属蛋白酶-13在骨髓瘤细胞和骨微环境细胞中均高度表达， 造骨成骨细胞。体内分析表明，当基质金属蛋白酶-13从宿主中被基因消融时 总体存活率显著增加，骨髓瘤发病率也随之降低 骨病。我们还证实了骨髓瘤来源的外切体可以促进骨骼的定植。 骨髓瘤和骨髓瘤中，均富含基质金属蛋白酶-13。此外，一种新的高选择性基质金属蛋白酶-13抑制剂的初步数据 显着限制骨髓瘤的体外和体内生长，强调基质金属蛋白酶-13活性在推动 多发性骨髓瘤。根据这些初步发现，我们假设基质金属蛋白酶-13参与了多发性 骨髓瘤进展。我们将通过以下几个方面验证我们的假设：1)定义肿瘤和成骨细胞来源的作用 基质金属蛋白酶-13在多发性骨髓瘤进展中的作用我们将使用CRISPR/cDNA过表达方法来 利用Cre重组酶驱动的特异性启动子调控骨髓瘤细胞系中基质金属蛋白酶-13的表达 目的：消除成骨细胞基质金属蛋白酶-13的表达。然后我们将测试是否存在基质金属蛋白酶-13 在一个或两个间隔内导致骨髓瘤生长和相关的骨病 活体模型(5TGM1和U266)。我们还将通过前期工作探索基质金属蛋白酶-13的作用机制 指向对转化生长因子β(转化生长因子β)活性的调节。2)确定骨髓瘤的作用 衍生的外切体和特定的外切体基质金属蛋白酶-13在疾病的骨骼定植中。我们还将 检查胞外基质金属蛋白酶-13是否可以识别阴燃的多发性骨髓瘤患者(n=200) 利用蛋白质组学技术进展为活动性疾病。3)确定选择性基质金属蛋白酶-13的疗效 应用CD138分离的骨髓瘤细胞抑制多发性骨髓瘤活性 在一种新的体外高通量平台中诊断的患者和临床相关的体内模型 疾病。根据预期的结果，询问基质金属蛋白酶-13在多发性骨髓瘤进展中的作用将 揭示了一些新的见解，从而为选择性基质金属蛋白酶-13的翻译提供了强有力的理论基础 由于骨骼受限，我们预测的临床抑制剂的非靶向效应将是有限的 基质金属蛋白酶-13的表达。

SUMMARY Despite medical advances, multiple myeloma remains a fatal disease. The mechanisms underpinning how myeloma progresses in the local bone microenvironment and through which myeloma colonizes the skeleton to generate multiple painful osteolytic lesions needs to be addressed urgently. Matrix metalloproteinases (MMPs) are key regulators of tumor-bone interaction via the regulation of cytokine and growth factor bioavailability/activity. Emerging data from our group has identified that in human specimens of the disease, MMP-13 is highly expressed by both the myeloma cells and the cells of the bone microenvironment namely, bone building osteoblasts. In vivo analyses show that when MMP-13 is genetically ablated from the host compartment there is a significant increase in overall survival and a concomitant decrease in myeloma induced bone disease. We also have identified that myeloma derived exosomes can promote the skeletal colonization of myeloma and, are rich in MMP-13. Further, preliminary data with a novel highly selective MMP-13 inhibitor significantly limits myeloma growth in vitro and in vivo underscoring the role for MMP-13 activity in driving multiple myeloma. Based on these preliminary findings we hypothesize that MMP-13 contributes to multiple myeloma progression. We will test our hypothesis by; 1) Defining the role of tumor and osteoblast derived MMP-13 in multiple myeloma progression. We will use CRISPR/cDNA overexpression approaches to manipulate the levels of MMP-13 in myeloma cell lines while using specific Cre-recombinase driven promoters to eliminate MMP-13 expression by osteoblasts. We will then test whether the presence or absence of MMP-13 in one or both compartments contributes to myeloma growth and associated bone disease in two separate in vivo models (5TGM1 and U266). We will also explore MMP-13 mechanisms of action with preliminary work pointing to regulation of transforming growth factorβ (TGFβ) activity. 2) Determining the role of myeloma derived exosomes and specifically exosomal MMP-13 in the skeletal colonization of the disease. We will also examine whether exosomal MMP-13, can identify smoldering multiple myeloma patients (n=200) at high-risk of progression to active disease using proteomic techniques. 3) Identifying the efficacy of a selective MMP-13 inhibitor in limiting multiple myeloma viability using CD138 isolated myeloma cells obtained from newly diagnosed patients in a novel ex vivo high throughput platform and clinically relevant in vivo models of the disease. Based on the anticipated results, interrogating the role MMP-13 in multiple myeloma progression will reveal a number of novel insights thereby providing a strong rationale for the translation of selective MMP-13 inhibitors to the clinic that we predict would have limited off-target effects due to the restricted skeletal expression of MMP-13.