SFK-inhibitor enhancement of ATRA-mediated differentiation of APL

项目摘要/摘要 视黄酸信号传导在正常骨髓细胞发育中起关键作用。异常信号传导 导致骨髓成熟停滞和持续的细胞增殖。这在急性早幼粒细胞性白血病中是例证。 白血病（APL），其中视黄酸受体（RAR）的突变融合蛋白抑制视黄酸- 依赖转录。在APL中，药理剂量的全反式维甲酸（ATRA）克服了抑制作用， RAR依赖性转录，导致胚细胞的终末分化。ATRA的引入 虽然这一疗法改变了APL的治疗，但高达30%的老年和“高危”患者最终复发并死于 这种疾病。目前的联合药物治疗是有毒的。我们提出了一种新的策略，以提高 全反式维甲酸治疗APL的疗效我们已经证明Src家族激酶（SFKs）在细胞内是组成性激活的。 APL和负调控RAR介导的基因表达。这表明了一个模型，其中激活 SFK沉默内源RAR以帮助RAR靶基因的转录抑制。我们预测 SFK抑制剂将增强ATRA激活RAR的活性并增加APL的分化。的确， 我们发现，使用泛SFK抑制剂PP 1和PP 2抑制SFK导致ATRA-1的增强。 诱导分化达沙替尼是FDA批准的SFK抑制剂，其作用方式与PP 1相似。 和PP 2抑制剂，并显著增强ATRA诱导的髓样分化。根据这些发现， 我们假设SFK抑制剂将增强ATRA在APL治疗中的活性。为了验证这一 假设，我们将首先验证我们在一组冷冻保存的库细胞中的观察结果，这些细胞来自患有 标准和高危APL我们将在APL小鼠模型中测试达沙替尼和ATRA的组合， 评估发展APL的时间、复发的时间和肿瘤负荷的降低程度。我们还将测试 ATRA和达沙替尼在ATRA耐药小鼠模型中的作用。ATRA本身并不能根除APL 白血病起始细胞（LIC）是负责复发：我们将评估的影响， 全反式维甲酸加SFK抑制剂我们建议ATRA加达沙替尼的组合将被证明是 更有效并导致更长期的缓解。我们还将确定SFK 调节RAR活性，这将识别其他靶向途径。这些研究旨在发展一种新的 药物类别的组合，以改变APL的治疗，其毒性低于当前方案。两 达沙替尼和全反式维甲酸是FDA批准的口服制剂，其性质允许快速翻译 把我们的发现送到诊所

PROJECT SUMMARY/ABSTRACT Retinoic acid signaling plays a key role in normal myeloid bone marrow cell development. Aberrant signaling leads to myeloid maturation arrest and continued cell proliferation. This is exemplified in Acute Promyelocytic Leukemia (APL), in which a mutated fusion protein of the receptor for retinoic acid (RAR) inhibits retinoic acid- dependent transcription. In APL, pharmacologic doses of all-trans retinoic acid (ATRA) overcome the inhibition of RAR-dependent transcription, leading to terminal differentiation of the blasts. The introduction of ATRA has transformed the treatment of APL, yet up to 30% of elderly and “high-risk” patients ultimately relapse and die of this disease. Current combination drug treatments are toxic. We propose a novel strategy to enhance the effectiveness of ATRA in APL. We have shown that Src family kinases (SFKs) are constitutively activated in APL and negatively regulate gene expression mediated by RAR. This suggests a model in which activated SFKs silence the endogenous RAR to aid in transcriptional repression of RAR-target genes. We predict that SFK inhibitors will enhance the activity of ATRA to activate RAR and increase differentiation in APL. Indeed, we found that inhibition of SFKs using the pan-SFK inhibitors PP1 and PP2 resulted in enhancement of ATRA- induced differentiation. Dasatinib, an FDA-approved inhibitor of SFKs, acted in a similar fashion to the PP1 and PP2 inhibitors, and dramatically enhanced ATRA-induced myeloid differentiation. Based on these findings, we hypothesize that SFK-inhibitors will enhance the activity of ATRA in therapies for APL. To test this hypothesis, we will first validate our observations in a panel of cryopreserved banked cells from patients with both standard and high-risk APL. We will test the combination of dasatinib and ATRA in an APL mouse model, assessing time to develop APL, time to relapse, and degree of decrease in tumor burden. We will also test the effects of ATRA and dasatinib in an ATRA-resistant mouse model. ATRA alone does not eradicate the APL leukemia initiating cell (LIC) that is responsible for relapse: we will assess the effects of the combination of ATRA plus SFK-inhibitors on the LIC. We propose that the combination of ATRA plus dasatinib will prove to be more efficacious and result in longer-term remissions. We will also determine the mechanism by which SFKs modulate RAR activity, which will identify other targetable pathways. These studies aim to develop a novel combination of drug classes to transform treatment of APL with less toxicity than current regimens. Both dasatinb and ATRA are FDA-approved, orally-administered agents, properties that will allow ready translation of our findings into the clinic.