The ALK inhibitor ASP3026 eradicates NPM-ALK+ T-cell anaplastic large-cell lymphoma in vitro and in a systemic xenograft lymphoma model

NPM-ALK⁺ T-cell anaplastic large-cell lymphoma (ALCL) is an aggressive type of cancer. Standard treatment of NPM-ALK⁺ ALCL is CHOP polychemotherapy. Although patients initially respond favorably to CHOP, resistance, relapse, and death frequently occur. Recently, selective targeting of ALK has emerged as an alternative therapeutic strategy. ASP3026 is a second-generation ALK inhibitor that can overcome crizotinib resistance in non-small cell lung cancer, and is currently being evaluated in clinical trials of patients with ALK⁺ solid tumors. However, NPM-ALK⁺ ALCL patients are not included in these trials. We studied the effects of ASP3026 on NPM-ALK⁺ ALCL cell lines in vitro and on systemic lymphoma growth in vivo. ASP3026 decreased the viability, proliferation, and colony formation, as well as induced apoptotic cell death of NPM-ALK⁺ ALCL cells. In addition, ASP3026 significantly reduced the proliferation of 293T cells transfected with NPM-ALK mutants that are resistant to crizotinib and downregulated tyrosine phosphorylation of these mutants. Moreover, ASP3026 abrogated systemic NPM-ALK⁺ ALCL growth in mice. Importantly, the survival of ASP3026-treated mice was superior to that of control and CHOP-treated mice. Our data suggest that ASP3026 is an effective treatment for NPM-ALK⁺ ALCL, and support the enrollment of patients with this lymphoma in the ongoing clinical trials.     

The tyrosine kinase NPM-ALK, associated with anaplastic large cell lymphoma, binds the intracellular domain of the surface receptor CD30 but is not activated by CD30 stimulation

The heterogenous group of anaplastic large cell lymphomas (ALCLs) is characterized by expression of the Ki-1/CD30 antigen, a member of the tumor necrosis factor receptor superfamily. About 40 to 50% of cases diagnosed as ALCL contain a specific chromosomal rearrangement, t(2;5)(p23;q35), resulting in expression of the chimeric tyrosine kinase NPM-ALK. As NPM-ALK-positive lymphomas define a distinct subtype within the group of ALCL, the chimeric protein might be responsible for certain pathogenetic and clinicopathologic characteristics. To better elucidate the function of NPM-ALK, we investigated a possible mechanism for regulation of its activity. We demonstrate that NPM-ALK specifically binds to the intracellular domain of the cytokine receptor CD30. In vitro binding assays revealed that the ALK portion of NPM-ALK mediates interaction of the two proteins. Stimulation of the CD30 receptor by cross-linking with immobilized anti-CD30 antibody results in complete growth inhibition of Karpas 299, an NPM-ALK-positive ALCL cell line, but does not alter proliferation of HDLM-2, a Hodgkin's lymphoma-derived cell line lacking t(2;5). Western blot analysis of coimmunoprecipitated CD30 and NPM-ALK proteins from stimulated Karpas 299 cells showed that the interaction of the proteins is not modified by stimulation. Activation of CD30 neither enhanced NPM-ALK activity measured by autophosphorylation of the chimeric tyrosine kinase nor phosphorylation of phospholipase C-gamma, an NPM-ALK substrate. We conclude that NPM-ALK is not stimulated by CD30 activation, but exists as a constitutively hyperactivated protein. Interaction with CD30 may extend the subcellular localization of NPM-ALK to the microenvironment of membrane-associated proteins.     

RNAi阻断NPM-ALK基因表达及对大细胞间变性淋巴瘤细胞的影响(英文)

目的：应用RNA干扰技术抑制大细胞间变性淋巴瘤细胞系(Karpas299)中NPMALK融合基因表达,观察其对肿瘤细胞生长的影响。方法：针对NPMALK融合位点设计两个siRNA序列siRNAI与siRNAII,经PCR反应构建含U6启动子siRNA正义和反义线性表达载体,通过脂质体转染Karpas299细胞,应用实时荧光定量RTPCR、Westernblot检测siRNA片段对NPMALKmRNA和蛋白表达的抑制作用,MTT、Hoechst荧光染色检测siRNA对肿瘤细胞生长的影响。结果：siRNAI可导致NPMALKmRNA下降约75%(P<0.05),转染72h后可导致蛋白表达下降;转染siRNAII细胞NPMALKmRNA下降为35%(P<0.05),但蛋白水平无明显改变。转染siRNAI的细胞可抑制Karpas299细胞的增殖和诱导凋亡发生,siRNAII则无明显的抑制增殖和诱导凋亡作用。结论：含有针对NPMALK融合位点特异siRNA序列的U6表达载体,可特异地抑制NPMALK基因mRNA和蛋白的表达,并能抑制大细胞间变性淋巴瘤肿瘤细胞株Karpas299细胞的增殖,导致肿瘤细胞凋亡增加,提示NPMALK融合基因的异常表达与大细胞间变性淋巴瘤形成密切相关,为研究NPMALK基因功能和大细胞间变性淋巴瘤基因靶向治疗提供了新策略。     

Targeting autophagy enhances the anti-tumoral action of crizotinib in ALK-positive anaplastic large cell lymphoma

Anaplastic Lymphoma Kinase-positive Anaplastic Large Cell Lymphomas (ALK+ ALCL) occur predominantly in children and young adults. Their treatment, based on aggressive chemotherapy, is not optimal since ALCL patients can still expect a 30% 2-year relapse rate. Tumor relapses are very aggressive and their underlying mechanisms are unknown. Crizotinib is the most advanced ALK tyrosine kinase inhibitor and is already used in clinics to treat ALK-associated cancers. However, crizotinib escape mechanisms have emerged, thus preventing its use in frontline ALCL therapy. The process of autophagy has been proposed as the next target for elimination of the resistance to tyrosine kinase inhibitors. In this study, we investigated whether autophagy is activated in ALCL cells submitted to ALK inactivation (using crizotinib or ALK-targeting siRNA). Classical autophagy read-outs such as autophagosome visualization/quantification by electron microscopy and LC3-B marker turn-over assays were used to demonstrate autophagy induction and flux activation upon ALK inactivation. This was demonstrated to have a cytoprotective role on cell viability and clonogenic assays following combined ALK and autophagy inhibition. Altogether, our results suggest that co-treatment with crizotinib and chloroquine (two drugs already used in clinics) could be beneficial for ALK-positive ALCL patients.     

Correlation of Seven Biological Factors （Hsp90a,p53, MDM2, Bcl-2, Bax,Cytochrome C,and Cleaved caspase3） with Clinical Outcomes of ALK＋ Anaplastic Large-cell Lymphoma

ObjectiveTo explore correlation of seven apoptosis-related proteins (Hsp90a, p53, MDM2, Bcl-2, Bax, Cytochrome C, and Cleaved caspase3) with clinical outcomes of ALK+ anaplastic large-cell lymphoma (ALCL).MethodsUsing immunohistochemistry and immunofluorescence double staining methods, the expressions of these seven apoptosis-associated proteins were studied to clarify their relationship with clinical outcomes of 36 ALK+ and 25 ALK-systemic ALCL patients enrolled between 1996 and 2006. The relationship of these apoptosis-regulating proteins with NPM-ALK status was also evaluated with the tyrosine inhibitor herbimycin A (HA) in vitro by immunocytochemistry, Western blotting and flow cytometric assays.ResultsThe presence of Hsp90α-, MDM2-, Bax-, Cytochrome C, and Cleaved caspase3-positive tumor cells was found significantly different in ALK+ and ALK-ALCLs, which was correlated with highly favorable clinical outcome. The Bcl-2- and p53-positive tumor cells were found in groups of patients with unfavorable prognosis. Inhibition of NPM-ALK by HA could reactivate the p53 protein and subsequent apoptosis-related proteins and therefore induced apoptosis in ALK+ ALCL cells.ConclusionOur results suggest that these seven proteins might be involved in apoptosis regulation and associated with clinical outcome of ALK+ systemic ALCLs. We also reveal a dynamic chain relation that NPM-ALK regulates p53 expression and subsequent apoptosis cascade in ALK+ ALCLs.     

Detection of NPM-ALK DNA rearrangement in CD30 positive anaplastic large cell lymphoma

CD30 positive anaplastic large cell lymphoma (ALCL) is a type of non-Hodgkin's lymphoma associated with a specific chromosome translocation between chromosomes 2 and 5. Recent molecular characterization of the translocation breakpoint has identified a gene fusion between NPM (nucleophosmin) and ALK (anaplastic lymphoma kinase). Using a DNA hybridization technique, the NPM rearrangement was found among 5/5 ALCL samples. We have developed a PCT methodology which has enabled the detection of the NPM-ALK rearrangements amongst seven t(2; 5)(p23; q35) ALCL cases based on a long-range PCR of genomic DNA. The rapidity and robustness of this method may have diagnostic applications for ALCL.