Stat3 Probes that Target Breast Cancer Stem Cells

描述(申请人提供)：常规化疗最初在控制和逆转肿瘤生长方面是有效的。然而，残留的癌症总是会重新生长。我们最近对人类乳腺癌样本的数据表明，标准治疗消除了子代细胞的分裂，同时丰富了残留肿瘤的CD44？/低假定乳腺癌“癌症干细胞”(CSCs)，这些细胞具有在乳腺(MS)培养中自我更新的能力，并在异种移植后产生肿瘤。我们已经在这些以STAT3为特征的CD44？/Low，MS形成细胞中发现了一种癌症干细胞的特征。利用虚拟配体筛选(VLS)技术，我们开发了针对STAT3的Src同源(SH)2结构域的先导化学探针，并竞争性和选择性地抑制STAT3的激活。使用最活跃的先导化合物(Cpd188)和生命化学品化合物库进行三维药效团分析；通过表面等离子体共振(SPR)测试鉴定的得分最高的化合物对STAT3与其磷肽配体结合的抑制作用。在测试的39种化合物中，除6种外，所有化合物的IC50都可测得，其中17种化合物的IC50值等于或小于Cpd188。一种化合物(Cpd188-15)在MS形成抑制试验中的IC50值比Cpd188低一个对数。我们已经开发了两个高度聚焦的特异性靶点，以直接测试STAT3的小分子抑制剂可以特异性靶向乳腺癌干细胞的假设。目的1.开发第三代(第3代)STAT3化学探针，提高其结合亲和力和抑制活性。我们建议使用最活跃的第二代探针Cpd188-15作为制造第三代探针的支架。计划中的修改是基于对第二代探针进行的结构-活性关系(SAR)分析的结果，并以从磺酰氯和酰胺面板直接合成磺胺为中心。每一种新的磺胺化合物都将在SPR分析、高通量荧光显微镜(HTFM)分析中进行检测，该分析将测试对IL-6刺激的细胞质到核转位的抑制以及它们对STAT3和STAT1的选择性。目的2.在临床前MS试验和人类异种移植模型中，确定抑制STAT3通路是否可以改善现有的癌症治疗方法。我们将使用MS形成抑制试验和人乳腺癌异种移植模型，在体外和体内确定最活跃的第三代探针抑制STAT3是否会改善传统治疗的有效性。除了确定STAT3对乳腺癌干细胞存活和自我更新的贡献外，这些临床前研究还可能为未来临床试验的设计提供信息，并确定抑制干细胞自我更新和治疗耐药途径是否可以改善患者现有的乳腺癌治疗方法。 与公共卫生相关：一小部分乳腺癌细胞对常规治疗没有反应，并导致复发。这项提案概述了旨在开发针对这些治疗耐药细胞的小分子探针的化学和生物学研究。如果这些研究成功，这些小分子探针可能会与目前乳腺癌的治疗方法相结合，从而防止癌症复发并最终治愈。

DESCRIPTION (provided by applicant): Conventional chemotherapy is effective initially in controlling and reversing tumor growth. However, residual cancers will invariably re-grow. Our recent data from paired human breast cancer samples indicates that standard therapy eliminates dividing daughter cells while enriching the residual tumor for CD44?/low putative breast "cancer stem cells" (CSCs) that have the ability to self-renew in mammosphere (MS) cultures, and to give rise to tumors upon xenograft transplantation. We have identified a cancer stem cell signature within these CD44?/low, MS-forming cells that features Stat3. Using virtual ligand screening (VLS), we developed lead chemical probes that target the Src homology (SH) 2 domain of Stat3 and that competitively and selectively inhibited Stat3 activation. 3-D pharmacophore analysis was performed using the most active lead compound (Cpd188) and a Life Chemicals compound library; the top scoring compounds identified were tested for inhibition of Stat3 binding to its phosphopeptide ligand by surface plasmon resonance (SPR). All but six of 39 compounds tested have measurable IC50s, with 17 having IC50 values equal to or less than Cpd188. One compound (Cpd188-15) has an IC50 value in MS formation inhibition assays that is one log lower than Cpd188. We have developed 2 highly focused SPECIFIC AIMS to directly test the hypothesis that small- molecule inhibitors of Stat3 can specifically target breast cancer stem cells. AIM 1. To develop third (3rd) generation Stat3 chemical probes with increased binding affinity and inhibitory activity. We propose using the most active 2nd generation probe, Cpd188-15, as a scaffold for making 3rd generation probes. Modifications planned are based on the results of a structure-activity relationship (SAR) analysis performed on 2nd generation probes and center around the straightforward synthesis of sulfamides from panels of sulfonyl chlorides and amides. Each novel sulfamide compound will be examined in the SPR assay, in a high throughput fluorescence microscopy (HTFM) assay that will test inhibition of IL-6-stimulated cytoplasmic-to-nuclear translocation and for their selectivity for Stat3 vs. Stat1. AIM 2. To determine whether suppression of the Stat3 pathway can improve existing cancer therapies in preclinical MS assays and human xenograft models. We will determine whether Stat3 inhibition by the most active 3rd generation probes will improve efficacy of conventional therapy in vitro and in vivo, using MS formation inhibition assays and human breast cancer xenograft models. In addition to establishing the contribution of Stat3 to breast cancer stem cell survival and self-renewal, these preclinical studies may inform the design of future clinical trials and determine whether suppressing stem cell self-renewal and treatment resistance pathways can improve existing breast cancer therapies in patients. PUBLIC HEALTH RELEVANCE: A small proportion of breast cancer cells fail to respond to the usual treatments and cause relapsing disease. This proposal outlines chemical and biological studies designed to develop small molecule probes that target these therapy-resistant cells. If these studies are successful, these small molecule probes may be combined with current treatments for breast cancer thereby preventing cancer relapse and resulting in a cure.