摘要:PDZD2对稳态造血至关重要,其37-kDa分泌产物sPDZD2在AML中作为可溶性肿瘤抑制因子发挥作用

IF 11.5 Q1 HEMATOLOGY Blood Cancer Discovery Pub Date : 2023-05-01 DOI:10.1158/2643-3249.aml23-a37
Marlise L Guerrero Schimpf, Courtney C Sparger, Hsuan-Ting Huang, Dean Wade, Maria E. Figueroa
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We confirmed sPDZD2 secretion by normal CD34+ HSPC and detected lower levels in AMLs. In addition, treatment of a panel of AML cell lines (n=9) and primary human AML cells (n=4) with recombinant sPDZD2 (r-sPDZD2; dose: 100nM-300nM/day) led to growth inhibition, upregulation of differentiation markers (CD15/CD11b) and cell cycle arrest in G0/G1. To identify signaling pathways downstream of sPDZD2, we treated MV4-11 cells with 100nM r-sPDZD2 and performed phospho-kinase protein array followed by immunoblot. In vitro treatment with r-sPDZD2 led to inhibition of CREB(S133) phosphorylation, as well as increased GSK3b(S9), RSK1/2(S221/S227) and p53(S46) phosphorylation. In order to determine whether PDZD2 plays a role in normal hematopoiesis we performed in vitro differentiation in CRISPR-edited human HSPCs. We used a Cy3-labeled recombinant Cas9 protein and specific single guide RNAs (gRNAs) targeted against PDZD2. Cy3-positive cells were then used for in vitro myeloid or erythroid differentiation, and cell surface markers were evaluated by flow-cytometry analysis over a period of 11 days. While PDZD2-edited cells (PDZD2 KO) displayed a minor defect on myeloid differentiation due to insufficient upregulation of CD15, erythropoiesis appears to be more compromised, with a significant failure to adequately upregulate both CD235a and CD7. To determine the role of Pdzd2 in vivo, we took advantage of a Pdzd2Gt/Gt gene-trap knock-out mouse model and performed peripheral blood (PB) and BM analysis. PB counts at 8-weeks of age showed anemia, with a significant decrease in both red blood cell counts and hemoglobin levels, an observation compatible with the impaired erythropoiesis seen in vitro after PDZD2 KO in human HSPCs. In addition, we observed a trend to decreased MEPs and short-term HSCs, in the BM of 12-week-old mice (n=3), though the small size of the cohort prevented robust statistical analysis. 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引用次数: 0

摘要

PDZ结构域蛋白2 (PDZD2)在髓系恶性肿瘤中通过高甲基化几乎普遍沉默。PDZD2编码一个300kDa的蛋白,它被切割成一个37kDa的蛋白,分泌到组织微环境中(sPDZD2)。鉴于sPDZD2已被报道在实体肿瘤中作为肿瘤抑制因子发挥作用,并且在AML中几乎普遍缺失,我们假设PDZD2是正常造血所必需的,sPDZD2在造血系统中作为可溶性肿瘤抑制因子发挥作用。为了确定sPDZD2是否分泌到骨髓(BM)微环境中,我们使用ELISA分析了原代aml和健康供者BM血浆中sPDZD2的水平。我们证实了正常CD34+ HSPC分泌sPDZD2,并在AMLs中检测到较低的水平。此外,用重组sPDZD2 (r-sPDZD2;剂量:100nM-300nM/天)导致G0/G1期生长抑制、分化标志物(CD15/CD11b)上调和细胞周期阻滞。为了确定sPDZD2下游的信号通路,我们用100nM r-sPDZD2处理MV4-11细胞,并进行磷酸化激酶蛋白阵列和免疫印迹。体外用r-sPDZD2处理可抑制CREB(S133)磷酸化,并增加GSK3b(S9)、RSK1/2(S221/S227)和p53(S46)磷酸化。为了确定PDZD2是否在正常造血中发挥作用,我们对crispr编辑的人造血干细胞进行了体外分化。我们使用cy3标记的重组Cas9蛋白和靶向PDZD2的特异性单导rna (gRNAs)。然后将cy3阳性细胞用于体外髓系或红细胞分化,并在11天的时间内通过流式细胞术分析评估细胞表面标记物。虽然PDZD2编辑的细胞(PDZD2 KO)由于CD15上调不足而在髓系分化上表现出轻微缺陷,但红细胞生成似乎受到更大的损害,CD235a和CD7都无法充分上调。为了确定Pdzd2在体内的作用,我们利用Pdzd2Gt/Gt基因陷阱敲除小鼠模型并进行外周血(PB)和BM分析。8周龄的PB计数显示贫血,红细胞计数和血红蛋白水平均显著下降,这与人造血干细胞PDZD2 KO后体外红细胞功能受损的观察结果一致。此外,我们在12周龄小鼠的BM中观察到MEPs和短期hsc减少的趋势(n=3),尽管队列规模较小,无法进行稳健的统计分析。总之,我们的发现首次揭示了PDZD2在造血过程中以前未被认识到的作用。在体外和体内,HSPC中PDZD2的缺失导致红细胞分化受损。此外,我们的研究结果证实了sPDZD2在AML中的可溶性肿瘤抑制作用,类似于在实体肿瘤中看到的作用。重要的是,我们进一步证明了重组sPDZD2作为AML新治疗方法的潜在作用。引用格式:Marlise L Guerrero Schimpf, Courtney C Sparger, Hsuan-Ting Huang, Dean Wade, Maria E. Figueroa。PDZD2对于稳态造血至关重要,其37 kda分泌产物sPDZD2在AML中作为可溶性肿瘤抑制因子发挥作用[摘要]。摘自:AACR特别会议论文集:急性髓性白血病和骨髓增生异常综合征;2023年1月23-25日;费城(PA): AACR;血癌发现[j]; 2009;4(3 -增刊):摘要nr A37。
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Abstract A37: PDZD2 is essential for steady-state hematopoiesis and its 37-kDa secreted product, sPDZD2, functions as a soluble tumor suppressor in AML
Abstract PDZ domain-containing protein 2 (PDZD2) is almost universally silenced through hypermethylation in myeloid malignancies. PDZD2 encodes for a 300kDa protein that it is cleaved into a 37kDa protein that is secreted to the tissue microenvironment (sPDZD2). Given that sPDZD2 has been reported to function as a tumor suppressor in solid tumors and its almost universal loss in AML, we hypothesized that PDZD2 is required for normal hematopoiesis and that sPDZD2 functions as a soluble tumor suppressor in the hematopoietic system. To determine if sPDZD2 is secreted to the bone marrow (BM) microenvironment we analyzed sPDZD2 levels in BM plasma isolated from primary AMLs and healthy donors using ELISA. We confirmed sPDZD2 secretion by normal CD34+ HSPC and detected lower levels in AMLs. In addition, treatment of a panel of AML cell lines (n=9) and primary human AML cells (n=4) with recombinant sPDZD2 (r-sPDZD2; dose: 100nM-300nM/day) led to growth inhibition, upregulation of differentiation markers (CD15/CD11b) and cell cycle arrest in G0/G1. To identify signaling pathways downstream of sPDZD2, we treated MV4-11 cells with 100nM r-sPDZD2 and performed phospho-kinase protein array followed by immunoblot. In vitro treatment with r-sPDZD2 led to inhibition of CREB(S133) phosphorylation, as well as increased GSK3b(S9), RSK1/2(S221/S227) and p53(S46) phosphorylation. In order to determine whether PDZD2 plays a role in normal hematopoiesis we performed in vitro differentiation in CRISPR-edited human HSPCs. We used a Cy3-labeled recombinant Cas9 protein and specific single guide RNAs (gRNAs) targeted against PDZD2. Cy3-positive cells were then used for in vitro myeloid or erythroid differentiation, and cell surface markers were evaluated by flow-cytometry analysis over a period of 11 days. While PDZD2-edited cells (PDZD2 KO) displayed a minor defect on myeloid differentiation due to insufficient upregulation of CD15, erythropoiesis appears to be more compromised, with a significant failure to adequately upregulate both CD235a and CD7. To determine the role of Pdzd2 in vivo, we took advantage of a Pdzd2Gt/Gt gene-trap knock-out mouse model and performed peripheral blood (PB) and BM analysis. PB counts at 8-weeks of age showed anemia, with a significant decrease in both red blood cell counts and hemoglobin levels, an observation compatible with the impaired erythropoiesis seen in vitro after PDZD2 KO in human HSPCs. In addition, we observed a trend to decreased MEPs and short-term HSCs, in the BM of 12-week-old mice (n=3), though the small size of the cohort prevented robust statistical analysis. In summary, our findings are the first to shed light on a previously unrecognized role for PDZD2 in hematopoiesis. Loss of PDZD2 in HSPC resulted in impaired erythroid differentiation both in vitro and in vivo. Moreover, our findings validate a soluble tumor suppressor role for sPDZD2 in AML, similar to that seen in solid tumors. Importantly, we further demonstrate a potential role for recombinant sPDZD2 as a novel therapeutic approach for AML. Citation Format: Marlise L Guerrero Schimpf, Courtney C Sparger, Hsuan-Ting Huang, Dean Wade, Maria E. Figueroa. PDZD2 is essential for steady-state hematopoiesis and its 37-kDa secreted product, sPDZD2, functions as a soluble tumor suppressor in AML [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A37.
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来源期刊
CiteScore
12.70
自引率
1.80%
发文量
139
期刊介绍: The journal Blood Cancer Discovery publishes high-quality Research Articles and Briefs that focus on major advances in basic, translational, and clinical research of leukemia, lymphoma, myeloma, and associated diseases. The topics covered include molecular and cellular features of pathogenesis, therapy response and relapse, transcriptional circuits, stem cells, differentiation, microenvironment, metabolism, immunity, mutagenesis, and clonal evolution. These subjects are investigated in both animal disease models and high-dimensional clinical data landscapes. The journal also welcomes submissions on new pharmacological, biological, and living cell therapies, as well as new diagnostic tools. They are interested in prognostic, diagnostic, and pharmacodynamic biomarkers, and computational and machine learning approaches to personalized medicine. The scope of submissions ranges from preclinical proof of concept to clinical trials and real-world evidence. Blood Cancer Discovery serves as a forum for diverse ideas that shape future research directions in hematooncology. In addition to Research Articles and Briefs, the journal also publishes Reviews, Perspectives, and Commentaries on topics of broad interest in the field.
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