Stat3 Probes that Target Breast Cancer Stem Cells

描述（由申请人提供）：常规化疗最初在控制和逆转肿瘤生长方面有效。然而，残留的癌症总是会重新生长。我们最近从配对的人乳腺癌样本中获得的数据表明，标准治疗消除了分裂的子细胞，同时富集了残留的肿瘤中的CD 44？/低推定的乳腺“癌症干细胞”（CSC），其具有在乳腺球（MS）培养物中自我更新的能力，并且在异种移植物移植后产生肿瘤。我们已经确定了这些CD 44内的癌症干细胞签名？/低，MS形成细胞，具有Stat 3。使用虚拟配体筛选（VLS），我们开发了领先的化学探针，靶向Src同源（SH）2结构域的Stat 3和竞争性和选择性抑制Stat 3激活。使用最具活性的先导化合物（Cpd 188）和Life Chemicals化合物库进行3-D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Stat 3与其磷酸肽配体结合的抑制。在39种化合物中，除了6种化合物外，所有化合物都具有可测量的IC 50，其中17种化合物的IC 50值等于或小于Cpd 188。一种化合物（Cpd 188 -15）在MS形成抑制试验中的IC 50值比Cpd 188低一个log。我们已经开发了2个高度集中的特异性目标，以直接测试Stat 3的小分子抑制剂可以特异性靶向乳腺癌干细胞的假设。 AIM 1.开发结合亲和力和抑制活性增加的第三代Stat 3化学探针。我们建议使用最活跃的第二代探针，Cpd 188 -15，作为支架用于制造第三代探针。计划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，并围绕磺酰氯和酰胺的直接合成磺酰胺。每种新型磺酰胺化合物将在SPR试验中进行检查，在高通量荧光显微镜（HTFM）试验中，将测试IL-6刺激的细胞质至核易位的抑制作用及其对Stat 3与Stat 1的选择性。 AIM 2.在临床前MS试验和人类异种移植模型中确定Stat 3通路的抑制是否可以改善现有的癌症治疗。我们将使用MS形成抑制试验和人乳腺癌异种移植模型，确定最活跃的第三代探针对Stat 3的抑制是否会改善体外和体内常规治疗的疗效。 除了确定Stat 3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以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。 公共卫生相关性：一小部分乳腺癌细胞对常规治疗无效，导致疾病复发。该提案概述了旨在开发针对这些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与目前的乳腺癌治疗方法相结合，从而防止癌症复发并导致治愈。

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.