Computational Molecular Pathology Research for Cancer Diagnostics and Biomarkers

在旨在将癌症治疗方法转化为临床应用的研究中，在开发治疗方法方面取得了进展。在47个MM细胞系中进行了高通量药物筛选，并对药物反应进行了硅Huber稳健回归分析，发现了43种潜在的协同组合。我们假设有效的组合可以降低MYC表达并增强p16活性。6种组合共同降低MYC蛋白，在MM中经常过表达，也共同增加p16表达，在MM中经常下调。在蛋白酶体抑制剂耐药和敏感的MM细胞系中，观察到最高组合的活力协同降低，而成纤维细胞则不受影响。三种组合显著延长了晚期MM的可移植ras驱动的同种异体移植模型的生存期，与人类高风险/难治性骨髓瘤密切相关，并降低了体外治疗的患者细胞的活力。这些组合下调的常见遗传通路促进细胞周期转变，而上调最多的通路涉及tgf - β /SMAD信号传导。这些临床前数据确定了用于评估耐药MM的潜在有用药物组合，并揭示了联合药物敏感性的潜在机制。粘膜黑色素瘤联合靶向治疗的药物开发工作也在继续。人和犬粘膜黑色素瘤之间的临床和病理相关性是实质性的，狗的自发性自然发生的粘膜黑色素瘤的相对较高的发病率为预测建模提供了一个有希望的机会。与人类皮肤黑色素瘤相比，犬类和人类粘膜黑色素瘤似乎都不常见BRAF、NRAS和c-kit突变，尽管两者都具有AKT和MAPK信号激活。人类和犬粘膜黑色素瘤的基因组景观呈现高度多样化，通常缺乏与皮肤黑色素瘤相关的复发热点突变。尽管仍有很多有待确定，但有证据表明，Ras/MAPK和/或PI3K/AKT/mTOR信号通路激活在两种物种中都很常见，可能是治疗干预的靶点。通过这项研究，从PI3K/mTOR抑制剂文库中选择了mTORC1/2抑制剂sapanisertib与MEK抑制剂合作；后者的临床前疗效先前已被证明用于犬粘膜黑色素瘤。两药联合的药代动力学研究取得进展。这种组合是一种合理的方法，可以解决两种途径之间的相互交叉激活，这可能是耐药性的一个来源。因此，在没有肿瘤的实验犬中，研究了口服给药进行血浆药代动力学（PK）分析和对双TORC1/2抑制剂sapanisertib和MEK抑制剂trametinib联合用药的耐受性，以评估其在平行途径靶向中的潜在应用。12只狗被分成三个相等的组，接受联合或单一药物治疗。在单次给药和17天重复给药后，通过临床观察、血液学、血清生化、凝血和尿液分析监测动物的PK。单剂量曲美替尼（0.025 mg/kg）经二甲亚砜磺化处理后，其平均最大浓度（Cmax）为0.64 ng/mL[18%变异系数（CV）]，中位时间至最大浓度（Tmax）为1.5 h (hr)，平均浓度-时间曲线下面积（AUC）为16.8 hr*ng/mL (14%CV)，与沙巴尼替尼单用或合用时相似。延长的半衰期使舒美替尼在每日剂量下的血浆蓄积量达到人体的3-4倍。曲美替尼PK反映了之前狗的调节数据，而暴露接近一些已发表的人类值，但通常不是所有患者。单次给药0.1 mg/kg后，犬血浆中单药sapanisertib[平均Cmax 26.3 ng/mL (21%CV)，中位Tmax 2.0 hr，平均AUC 248 hr*ng/mL (41%CV)]与人类治疗试验中的水平相似；而与曲美替尼（一种已知的细胞色素P450 CYP3A4诱导剂）联合使用时，犬sapanisertib暴露减少。两种药物均未观察到性别差异。重复服用其中一种或两种药物的副作用可能包括体重减轻、消化不良和皮肤变色。尽管临床实验室分析显示药物引起的急性期炎症、蛋白尿和网状红细胞减少，但不需要干预的轻微变化，但联合用药是耐受的，没有剂量限制毒性。在狗身上使用这种组合的短期结果似乎为临床试验评估提供了转化的希望，以针对犬和可能的人类黑色素瘤，以及其他具有一种或两种信号转导途径激活的癌症。

In research aimed to translate approaches to cancer treatment intended for clinical application, progress was made in developing therapeutic approaches. A high-throughput drug screen in 47 MM cell lines and in silico Huber robust regression analysis of drug responses revealed 43 potentially synergistic combinations. We hypothesized that effective combinations would reduce MYC expression and enhance p16 activity. Six combinations cooperatively reduced MYC protein, frequently over-expressed in MM and also cooperatively increased p16 expression, frequently downregulated in MM. Synergistic reductions in viability were observed with top combinations in proteasome inhibitor-resistant and sensitive MM cell lines, while sparing fibroblasts. Three combinations significantly prolonged survival in a transplantable Ras-driven allograft model of advanced MM closely recapitulating high-risk/refractory myeloma in humans and reduced viability of ex vivo treated patient cells. Common genetic pathways similarly downregulated by these combinations promoted cell cycle transition, whereas pathways most upregulated were involved in TGFbeta/SMAD signaling. These preclinical data identify potentially useful drug combinations for evaluation in drug-resistant MM and reveal potential mechanisms of combined drug sensitivity. Work also continued on drug development for combined targeted therapy for mucosal melanomas. Clinical and pathological correlates between human and canine mucosal melanomas are substantial, and the relatively greater incidence of spontaneous naturally occurring mucosal melanoma in dogs represents a promising opportunity for predictive modeling. Both canine and human mucosal melanomas appear to harbor BRAF, NRAS and c-kit mutations uncommonly, compared to human cutaneous melanomas, although both species share AKT and MAPK signaling activation. The genomic landscapes of human and canine mucosal melanoma appear highly diverse and generally lack recurring hotspot mutations associated with cutaneous melanomas. Although much remains to be determined, evidence indicates that Ras/MAPK and/or PI3K/AKT/mTOR signaling pathway activations are common in both species and may represent targets for therapeutic intervention. Through this research sapanisertib, an mTORC1/2 inhibitor, was selected from a PI3K/mTOR inhibitor library to collaborate with MEK inhibition; the latter preclinical efficacy was demonstrated previously for canine mucosal melanoma. Progress was made in the pharmacokinetics of the two-drug combination. The combination is a rational approach to address the reciprocal cross activation between the two pathways, which can be a source of drug resistance. Consequently, oral dosing for plasma pharmacokinetic (PK) analysis and tolerability to a combination of sapanisertib, a dual TORC1/2 inhibitor, and trametinib, a MEK inhibitor, was evaluated in nontumor-bearing laboratory dogs for its potential application in parallel pathway targeting. Twelve dogs, divided into three equal cohorts, received either the combination or single agents. Animals were monitored for PK following single dose and 17-day repeat dosing, and by clinical observations, hematology, serum biochemistry, coagulation studies and urinalyses. A single trametinib dose (0.025 mg/kg), sulfated as dimethyl sulfoxide which enhanced its absorption, reached mean maximum concentration (Cmax) 0.64 ng/mL [18% coefficient of variation (CV)] at a median time to maximum concentration (Tmax) of 1.5 h (hr), and mean area under the concentration-time curve (AUC) 16.8 hr*ng/mL (14%CV), which were similar when given alone or in combination with sapanisertib. A prolonged half-life afforded 3-4-fold plasma accumulation of trametinib with daily dosing, analogous to humans. Trametinib PK mirrored previous regulatory data in dogs, while exposure approximated some published human values but generally not all patients. Sapanisertib-alone in canine plasma following single 0.1 mg/kg dose [mean Cmax 26.3 ng/mL (21%CV), median Tmax 2.0 hr, and mean AUC 248 hr*ng/mL (41%CV)] resembled levels in human therapeutic trials; whereas canine sapanisertib exposure was reduced when combined with trametinib, a known cytochrome P450 CYP3A4 inducer. Sex differences were not observed for either drug. Side effects upon repeat dosing with either or both drugs may include body weight loss, maldigestion, and cutaneous discoloration. The combination was tolerated without dose limiting toxicity, although clinical laboratory analyses revealed drug-induced acute-phase inflammation, proteinuria, and decreased blood reticulocytes, mild changes not necessitating intervention. Short-term results in dogs with this combination would appear to hold translational promise for clinical trial evaluation to target canine and possibly human melanoma, as well as other cancers having one or both signal transduction pathway activations.