Pub Date : 2023-05-01DOI: 10.1158/2643-3249.aml23-ia09
M. Figueroa
While significant progress has been made to understand the genetic landscape of MDS, less is known about the epigenetic makeup of this disease and how this may impact biology and response to azacitidine (AZA). To address this, we performed genomic, epigenomic and transcriptomic analysis on CD34+ cells from a multicenter cohort of 94 intermediate or higher risk MDS patients treated with AZA who had documented responses. DNA methylation (DNAme) by ERRBS, gene expression (GE) by RNA-seq, mutational profiling, and detailed clinical, cytogenetic and laboratory data were documented. Supervised and unsupervised analyses revealed that aberrant DNAme in MDS is not distributed randomly but rather is highly correlated with disease phenotypes, capturing clinically relevant heterogeneity, beyond what is identified by methodologies used in the past that focused solely on promoter regions. Moreover, this epigenetic information can be harnessed for the development of robust biomarkers predictive of AZA response and integrative approaches combining GE and DNAme data can further improve the predictive performance of these biomarkers, with an AUC score=0.92. Citation Format: Maria E. Figueroa. Epigenomic landscapes of MDS [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 IA09.
虽然在了解MDS的遗传格局方面取得了重大进展，但对这种疾病的表观遗传组成以及它如何影响生物学和对阿扎胞苷(AZA)的反应知之甚少。为了解决这个问题，我们对94名接受AZA治疗的中度或高风险MDS患者的CD34+细胞进行了基因组、表观基因组和转录组分析。ERRBS检测DNA甲基化(DNAme)、RNA-seq检测基因表达(GE)、突变谱分析以及详细的临床、细胞遗传学和实验室数据均被记录下来。监督和非监督分析显示，MDS中的异常DNAme不是随机分布的，而是与疾病表型高度相关，捕获临床相关的异质性，超出了过去仅关注启动子区域的方法所确定的。此外，这种表观遗传信息可以用于开发预测AZA反应的强大生物标志物，并且结合GE和DNAme数据的综合方法可以进一步提高这些生物标志物的预测性能，AUC评分为0.92。引用格式:Maria E. Figueroa。MDS的表观基因组景观[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/ Abstract
{"title":"Abstract IA09: Epigenomic landscapes of MDS","authors":"M. Figueroa","doi":"10.1158/2643-3249.aml23-ia09","DOIUrl":"https://doi.org/10.1158/2643-3249.aml23-ia09","url":null,"abstract":"\u0000 While significant progress has been made to understand the genetic landscape of MDS, less is known about the epigenetic makeup of this disease and how this may impact biology and response to azacitidine (AZA). To address this, we performed genomic, epigenomic and transcriptomic analysis on CD34+ cells from a multicenter cohort of 94 intermediate or higher risk MDS patients treated with AZA who had documented responses. DNA methylation (DNAme) by ERRBS, gene expression (GE) by RNA-seq, mutational profiling, and detailed clinical, cytogenetic and laboratory data were documented. Supervised and unsupervised analyses revealed that aberrant DNAme in MDS is not distributed randomly but rather is highly correlated with disease phenotypes, capturing clinically relevant heterogeneity, beyond what is identified by methodologies used in the past that focused solely on promoter regions. Moreover, this epigenetic information can be harnessed for the development of robust biomarkers predictive of AZA response and integrative approaches combining GE and DNAme data can further improve the predictive performance of these biomarkers, with an AUC score=0.92.\u0000 Citation Format: Maria E. Figueroa. Epigenomic landscapes of MDS [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 IA09.","PeriodicalId":29944,"journal":{"name":"Blood Cancer Discovery","volume":" ","pages":""},"PeriodicalIF":11.2,"publicationDate":"2023-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45018240","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-05-01DOI: 10.1158/2643-3230.BCD-22-0086
Ana Vujovic, Laura de Rooij, Ava Keyvani Chahi, He Tian Chen, Brian A Yee, Sampath K Loganathan, Lina Liu, Derek C H Chan, Amanda Tajik, Emily Tsao, Steven Moreira, Pratik Joshi, Joshua Xu, Nicholas Wong, Zaldy Balde, Soheil Jahangiri, Sasan Zandi, Stefan Aigner, John E Dick, Mark D Minden, Daniel Schramek, Gene W Yeo, Kristin J Hope
Acute myeloid leukemia (AML) is fueled by leukemic stem cells (LSC) whose determinants are challenging to discern from hematopoietic stem cells (HSC) or uncover by approaches focused on general cell properties. We have identified a set of RNA-binding proteins (RBP) selectively enriched in human AML LSCs. Using an in vivo two-step CRISPR-Cas9 screen to assay stem cell functionality, we found 32 RBPs essential for LSCs in MLL-AF9;NrasG12D AML. Loss-of-function approaches targeting key hit RBP ELAVL1 compromised LSC-driven in vivo leukemic reconstitution, and selectively depleted primitive malignant versus healthy cells. Integrative multiomics revealed differentiation, splicing, and mitochondrial metabolism as key features defining the leukemic ELAVL1-mRNA interactome with mitochondrial import protein, TOMM34, being a direct ELAVL1-stabilized target whose repression impairs AML propagation. Altogether, using a stem cell-adapted in vivo CRISPR screen, this work demonstrates pervasive reliance on RBPs as regulators of LSCs and highlights their potential as therapeutic targets in AML.
Significance: LSC-targeted therapies remain a significant unmet need in AML. We developed a stem-cell-adapted in vivo CRISPR screen to identify key LSC drivers. We uncover widespread RNA-binding protein dependencies in LSCs, including ELAVL1, which we identify as a novel therapeutic vulnerability through its regulation of mitochondrial metabolism. This article is highlighted in the In This Issue feature, p. 171.
{"title":"In Vivo Screening Unveils Pervasive RNA-Binding Protein Dependencies in Leukemic Stem Cells and Identifies ELAVL1 as a Therapeutic Target.","authors":"Ana Vujovic, Laura de Rooij, Ava Keyvani Chahi, He Tian Chen, Brian A Yee, Sampath K Loganathan, Lina Liu, Derek C H Chan, Amanda Tajik, Emily Tsao, Steven Moreira, Pratik Joshi, Joshua Xu, Nicholas Wong, Zaldy Balde, Soheil Jahangiri, Sasan Zandi, Stefan Aigner, John E Dick, Mark D Minden, Daniel Schramek, Gene W Yeo, Kristin J Hope","doi":"10.1158/2643-3230.BCD-22-0086","DOIUrl":"10.1158/2643-3230.BCD-22-0086","url":null,"abstract":"<p><p>Acute myeloid leukemia (AML) is fueled by leukemic stem cells (LSC) whose determinants are challenging to discern from hematopoietic stem cells (HSC) or uncover by approaches focused on general cell properties. We have identified a set of RNA-binding proteins (RBP) selectively enriched in human AML LSCs. Using an in vivo two-step CRISPR-Cas9 screen to assay stem cell functionality, we found 32 RBPs essential for LSCs in MLL-AF9;NrasG12D AML. Loss-of-function approaches targeting key hit RBP ELAVL1 compromised LSC-driven in vivo leukemic reconstitution, and selectively depleted primitive malignant versus healthy cells. Integrative multiomics revealed differentiation, splicing, and mitochondrial metabolism as key features defining the leukemic ELAVL1-mRNA interactome with mitochondrial import protein, TOMM34, being a direct ELAVL1-stabilized target whose repression impairs AML propagation. Altogether, using a stem cell-adapted in vivo CRISPR screen, this work demonstrates pervasive reliance on RBPs as regulators of LSCs and highlights their potential as therapeutic targets in AML.</p><p><strong>Significance: </strong>LSC-targeted therapies remain a significant unmet need in AML. We developed a stem-cell-adapted in vivo CRISPR screen to identify key LSC drivers. We uncover widespread RNA-binding protein dependencies in LSCs, including ELAVL1, which we identify as a novel therapeutic vulnerability through its regulation of mitochondrial metabolism. This article is highlighted in the In This Issue feature, p. 171.</p>","PeriodicalId":29944,"journal":{"name":"Blood Cancer Discovery","volume":"4 3","pages":"180-207"},"PeriodicalIF":11.2,"publicationDate":"2023-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10150294/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9682577","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-05-01DOI: 10.1158/2643-3249.aml23-a51
S. Sahoo, C. Goodings, S. Pruett-Miller, M. Lillo, Lei Han, Baranda S Hansen, T. Chang, T. Lammens, M. Hofmans, Marta Derecka, B. De Moerloose, M. Wlodarski
We previously showed that germline SAMD9 and SAMD9L (SAMD9/9L) disorders are the most common predisposition to childhood myelodysplastic syndromes with bone marrow failure (BMF). Heterozygous mutations in both genes are growth inhibitory and undergo negative selection by acquiring compensatory rescue events in blood. Here, we describe the first report of SAMD9L genetic rescue occurring during embryonal development and resulting in germline triple-allelic mosaicism (1 wildtype (WT) and 2 mutant alleles), where only one mutant allele was transmitted to each of the two diseased children. In a family of 2 affected siblings with hypocellular BMF, the Index case carried germline heterozygous SAMD9L V1512M mutation and her brother was heterozygous for V1512L mutation. The clinical presentation for Index with V1512M was more severe compared to her brother with V1512L mutation. Genetic assessment of the parents showed the asymptomatic mother to be a triple-allelic mosaic, carrying WT SAMD9L allele at ~50% frequency in all tissues, while both V1512M and V1512L mutant alleles “competed” in their allelic distribution depending on the tissue origin. Single-cell DNA sequencing on her peripheral blood (PB) revealed 3 independent diploid clones: V1512M in 14%, V1512L in 68%, and WT (due to revertant UPD7q) in 18% of cells. Because her parents were SAMD9L WT, one of the mutations likely arose de novo and underwent failed embryonic rescue attempt leading to a second mutation. Towards studying the effect on hematopoiesis in vitro, we knocked in V1512M and V1512L mutations in inducible pluripotent stem cells (iPSC). For in vivo functional validation, we created constitutive mouse models with ortholog mutations (V1507M and V1507L). Mutant hematopoietic progenitor cells from iPSC had severely compromised proliferative capacity and yielded fewer erythroid and myeloid cells. This effect was more severe in V1512M vs. V1512L mutants. The divergent mutational phenotypes were also replicated in our mouse models: two-thirds (13/19) of the heterozygous V1507M (V1507Mhet) pups died before 4 weeks of age. In contrast, V1507Lhet mice had overall survival equal to WT mice, while one-third of homozygous V1507L (V1507Lhom) mice showed decreased survival post 19 weeks of age. At baseline, V1507Mhet mice showed severe growth retardation with multisystemic issues, which were absent in the V1507L mutant models. PB cytopenia (anemia, B-cell lymphopenia) though observed in both V1507Mhet and V1507Lhom mice, the degree of severity was high in the V1507Mhet than in the V1507Lhom model. Meanwhile, V1507Lhet mice had blood counts similar to WT. In summary, V1512M compared to V1512L mutation resulted in a more severe phenotype in all analyzed model systems. This observation showed how different mutational permutations of the same amino acid can exert divergent phenotypic effects in SAMD9L BMF disorder. Therefore, together with our genetic analysis, we could postulate that the V1512L mutatio
我们先前表明，种系SAMD9和SAMD9L（SAMD9/9L）疾病是儿童骨髓增生异常综合征伴骨髓衰竭（BMF）的最常见诱因。这两个基因中的杂合突变都具有生长抑制作用，并通过在血液中获得补偿性救援事件而进行负选择。在这里，我们描述了SAMD9L基因拯救在胚胎发育过程中发生并导致种系三等位基因嵌合体（1个野生型（WT）和2个突变等位基因）的第一份报告，其中只有一个突变等基因传递给两个患病儿童中的每一个。在一个由2个患有低细胞BMF的兄弟姐妹组成的家族中，Index病例携带种系杂合子SAMD9L V1512M突变，而她的兄弟是V1512L突变杂合子。与她患有V1512L突变的兄弟相比，患有V1512M的Index的临床表现更为严重。对父母的遗传评估显示，无症状的母亲是一个三等位基因镶嵌体，在所有组织中携带约50%频率的WT SAMD9L等位基因，而V1512M和V1512L突变等位基因根据组织来源在其等位基因分布中“竞争”。在她的外周血（PB）上进行的单细胞DNA测序显示了3个独立的二倍体克隆：14%的细胞为V1512M，68%的细胞为V1 512L，18%的细胞为WT（由于回复子UPD7q）。由于她的父母是SAMD9L WT，其中一个突变可能是新出现的，并经历了失败的胚胎挽救尝试，导致了第二个突变。为了研究体外对造血的影响，我们在诱导型多能干细胞（iPSC）中敲除了V1512M和V1512L突变。为了进行体内功能验证，我们创建了具有直向同源突变（V1507M和V1507L）的组成型小鼠模型。来自iPSC的突变造血祖细胞的增殖能力严重受损，产生较少的红系和髓系细胞。这种作用在V1512M突变体中比在V1512L突变体中更严重。不同的突变表型也在我们的小鼠模型中复制：三分之二（13/19）的杂合V1507M（V1507Mhet）幼崽在4周龄前死亡。相反，V1507Lhet小鼠的总生存率与WT小鼠相当，而三分之一的纯合V1507L（V1507Lhom）小鼠在19周龄后表现出生存率下降。在基线时，V1507Mhet小鼠表现出严重的生长迟缓和多系统问题，这在V1507L突变模型中是不存在的。PB细胞减少症（贫血、B细胞淋巴细胞减少症）尽管在V1507Mhet和V1507Lhom小鼠中都观察到，但V1507Mhe的严重程度高于V1507Lhum模型。同时，V1507Lhet小鼠的血细胞计数与WT相似。总之，与V1512L突变相比，V1512M突变在所有分析的模型系统中都导致了更严重的表型。这一观察结果表明，同一氨基酸的不同突变排列如何在SAMD9L BMF障碍中发挥不同的表型效应。因此，结合我们的基因分析，我们可以推测V1512L突变是在胚胎发生过程中对V1512M的一次失败的拯救尝试。引文格式：Sushree S Sahoo、Charnise Goodings、Shondra M.Pruett Miller、Maria A Lillo、Lei Han、Baranda Hansen、Ti Cheng Chang、Tim Lammens、Mattias Hofmans、Marta Derecka、Barbara De Moerloose、Marcin W Wlodarski。MDS易感SAMD9L点突变在人类、小鼠和细胞模型中的不同作用[摘要]。载：AACR特别会议论文集：急性髓细胞白血病和骨髓增生异常综合征；2023年1月23日至25日；德克萨斯州奥斯汀。费城（PA）：AACR；血液癌症Discov 2023；4（3_Suppl）：摘要编号A51。
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Pub Date : 2023-05-01DOI: 10.1158/2643-3249.aml23-a45
Juan M. Barajas, Masayuki Umeda, Ryan Hiltenbrand, Reethu Krishnan, Tamara Westover, Michael P. Walsh, Jing Ma, Sherif Abdelhamed, J. Klco
Pediatric acute myeloid leukemia (AML) is often refractory to chemotherapy and remains a devastating disease with poor outcomes. Consequently, there is an urgent need to characterize molecular drivers of pediatric AML with the goal of developing more effective molecular therapies. Our group recently identified somatic tandem duplications (TD) in exon 13 of upstream binding transcription factor (UBTF) as a subtype-defining genomic alteration in pediatric AML that is associated with poor outcomes, high relapse rate, and measurable residual disease (MRD) positivity. However, how these alterations drive leukemogenesis is yet to be investigated. Wild-type UBTF regulates rRNA transcription, ribosome biogenesis, and nucleolar formation. Our recent work suggests that UBTF-TD retains these functions. However, we found that expression of UBTF-TD in cord blood CD34+ cells, but not wild-type UBTF, leads to their increased proliferation, self-renewal, and CD117/CD33 surface marker expression, suggesting a potential gain of function as well. Furthermore, we also observed increased expression of HOXB locus genes, PRDM16, MEIS1, and NKX2-3 in the CD34+ UBTF-TD overexpression model relative to our WT control. These expression patterns are similar to what we see in patients, collectively implicating UBTF-TD driving leukemic phenotypes. Based on our localization studies of UBTF-TD to DNA, we hypothesized that UBTF-TD protein may gain the ability to interact with genomic loci that characterize UBTF-TD AML, like the HOXB locus. We therefore wanted to test if UBTF-TD localized to the HOXB cluster and if this lead direct transcriptional upregulation. To test this, we expressed HA-tagged UBTF-TD in cord-blood CD34+ cells and mapped UBTF-TD genomic occupancy using CUT&RUN. We found that UBTF-TD localized to the promoters and gene bodies of key genes, including PRDM16, MEIS1, NKX2-3, and the HOXB cluster. These sites were accompanied by H3K4me3 and H3K27ac active marks without the H3K27me3 repressive mark. To validate these as bone-fide UBTF-TD-occupied regions, we utilized a expressed FKBP12F36V-HA-UBTF-TD fusion in CD34+ cells. This degradation tag (dTAG) system allows for rapid degradation of UBTF-TD protein upon dTAG-13 treatment. We treated our FKBP12F36V-HA-UBTF-TD expressing cord blood CD34+ cells with 1uM of dTAG-13 or DMSO. We found that a 3-day treatment with dTAG-13 led to downregulation of HOXB gene locus expression, but loss of CD117 expression or cell growth required prolonged treatment. Furthermore, our CUT&RUN data showed that dTAG-13 treatment resulted in loss of UBTF-TD protein at the HOXB gene cluster. Collectively, these data show that UBTF-TD interacts with genomic loci at the HOXB gene cluster to directly upregulate their expression. This loss of HOX gene expression leads then leads to loss of a leukemic phenotype. Our data highlights a potential mechanism for UBTF-TD leukemogenesis and sets a foundation where inhibiting the UBTF-TD molecular signa
儿童急性髓细胞白血病（AML）通常对化疗难以治愈，并且仍然是一种预后不佳的毁灭性疾病。因此，迫切需要表征儿童AML的分子驱动因素，以开发更有效的分子疗法。我们的研究小组最近确定，上游结合转录因子（UBTF）外显子13中的体细胞串联重复（TD）是定义儿童AML基因组改变的一种亚型，与不良预后、高复发率和可测量的残留疾病（MRD）阳性相关。然而，这些改变如何驱动白血病的发生尚待研究。野生型UBTF调节rRNA转录、核糖体生物发生和核仁形成。我们最近的工作表明，UBTF-TD保留了这些功能。然而，我们发现脐血CD34+细胞中UBTF-TD的表达，而不是野生型UBTF的表达，会导致其增殖、自我更新和CD117/CD33表面标记物表达增加，这也表明功能的潜在获得。此外，我们还观察到，与我们的WT对照相比，在CD34+UBTF-TD过表达模型中，HOXB基因座基因、PRDM16、MEIS1和NKX2-3的表达增加。这些表达模式与我们在患者中看到的相似，共同暗示UBTF-TD驱动白血病表型。基于我们对UBTF-TD与DNA的定位研究，我们假设UBTF-TD蛋白可能获得与表征UBTF-TD-AML的基因组基因座（如HOXB基因座）相互作用的能力。因此，我们想测试UBTF-TD是否定位于HOXB簇，以及这是否导致直接的转录上调。为了测试这一点，我们在脐血CD34+细胞中表达了HA标记的UBTF-TD，并使用CUT&RUN绘制了UBTF-TD的基因组占有率。我们发现UBTF-TD定位于关键基因的启动子和基因体，包括PRDM16、MEIS1、NKX2-3和HOXB簇。这些位点伴有H3K4me3和H3K27ac活性标记，而没有H3K27me3抑制标记。为了验证这些是真正的UBTF-TD占据区，我们在CD34+细胞中使用了表达的FKBP12F36V-HA-UBTF-TD融合物。该降解标签（dTAG）系统允许在dTAG-13处理时快速降解UBTF-TD蛋白。我们用1uM的dTAG-13或DMSO处理表达FKBP12F36V-HA-UBTF-TD的脐血CD34+细胞。我们发现，dTAG-13治疗3天会导致HOXB基因座表达下调，但CD117表达或细胞生长的丧失需要延长治疗时间。此外，我们的CUT&RUN数据显示，dTAG-13处理导致HOXB基因簇处的UBTF-TD蛋白损失。总之，这些数据表明，UBTF-TD与HOXB基因簇的基因组基因座相互作用，直接上调其表达。HOX基因表达的这种损失随后导致白血病表型的损失。我们的数据强调了UBTF-TD白血病发生的潜在机制，并为抑制UBTF-TD分子特征可能是一种可行的治疗选择奠定了基础。引文格式：Juan M Barajas、Masayuki Umeda、Ryan Hiltenbrand、Reethu Krishnan、Tamara Westover、Michael P Walsh、Jing Ma、Sherif Abdelhamed、Jeffery M Klco。UBTF-TD分子改变通过直接相互作用和调节HOXB基因座来驱动儿童AML中的白血病发生[摘要]。载：AACR特别会议论文集：急性髓细胞白血病和骨髓增生异常综合征；2023年1月23日至25日；德克萨斯州奥斯汀。费城（PA）：AACR；血液癌症Discov 2023；4（3_Suppl）：摘要编号A45。
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Pub Date : 2023-05-01DOI: 10.1158/2643-3249.aml23-a20
Audrey Lasry, B. Nadorp, M. Fornerod, D. Nicolet, Huiyun Wu, C. Walker, Zhengxi Sun, Matthew T. Witkowski, Anastasia N. Tikhonova, Maria Guillamot, Geraldine Cayanan, Anna H. Yeaton, T. Gruber, Ann-Kathrin Eisfeld, I. Aifantis
Acute myeloid leukemia (AML) is a hematopoietic malignancy with poor prognosis and limited treatment options. Here we provide a comprehensive single cell RNA-Sequencing census of the bone marrow immune microenvironment in adult and pediatric AML patients. We characterize unique inflammation signatures in a subset of AML patients, associated with inferior outcomes. We identify atypical B cells, a dysfunctional B cell subtype enriched in high-inflammation AML patients, as well as an increase in CD8+ GZMK+ and regulatory T cells, accompanied by a reduction in T cell clonal expansion. We derive an inflammation-associated gene score (iScore) that associates with poor survival outcomes in AML patients. Addition of the iScore refines current risk stratifications for AML patients and may enable identification of patients in need of more aggressive treatment. This work provides a first framework for classifying AML patients based on their immune microenvironment and a rationale for consideration of the inflammatory state in clinical settings. Citation Format: Audrey Lasry, Bettina Nadorp, Maarten Fornerod, Deedra Nicolet, Huiyun Wu, Christopher J Walker, Zhengxi Sun, Matthew T Witkowski, Anastasia N Tikhonova, Maria Guillamot, Geraldine Cayanan, Anna Yeaton, Tanja A Gruber, Ann-Kathrin Eisfeld, Iannis Aifantis. Inflammation remodels the immune microenvironment in acute myeloid leukemia [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 A20.
急性髓性白血病(AML)是一种预后不良且治疗方案有限的造血恶性肿瘤。在这里，我们提供了成人和儿童AML患者骨髓免疫微环境的全面单细胞rna测序普查。我们在AML患者的一个亚群中描述了独特的炎症特征，与不良预后相关。我们鉴定了非典型B细胞，一种在高炎症性AML患者中富集的功能失调B细胞亚型，以及CD8+ GZMK+和调节性T细胞的增加，伴随着T细胞克隆扩增的减少。我们得出了与AML患者不良生存结果相关的炎症相关基因评分(iScore)。iScore的加入细化了AML患者目前的风险分层，并可能使识别需要更积极治疗的患者成为可能。这项工作提供了基于免疫微环境对AML患者进行分类的第一个框架，并为临床环境中考虑炎症状态提供了依据。引文格式:Audrey Lasry, Bettina Nadorp, Maarten Fornerod, Deedra Nicolet，吴慧云，Christopher J Walker，孙正曦，Matthew T Witkowski, Anastasia N Tikhonova, Maria Guillamot, Geraldine Cayanan, Anna Yeaton, Tanja A Gruber, Ann-Kathrin Eisfeld, Iannis Aifantis。炎症重塑急性髓性白血病的免疫微环境[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;血癌杂志[j]; 2009;4(3 -增刊):摘要/ Abstract
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Pub Date : 2023-05-01DOI: 10.1158/2643-3249.aml23-a30
Feven Tameire, P. Wojnarowicz, S. Fujisawa, Sharon Huang, O. Reilly, C. Dudgeon, Nick Collette, J. Drees, Kathryn T. Bieging-Rolett, Takashi O. Kangas, Weiyu Zhang, M. Fumagalli, Iman Dewji, Yunfang Li, Anissa S. H. Chan, Xiaohong X. Qiu, B. Harrison, Ashley LaCayo, K. Staschke, A. C. Rigby, S. Ramurthy, E. Lightcap, D. Surguladze, N. Bose
The integrated stress response (ISR) is an adaptive signaling pathway that cells utilize to respond to a wide range of extrinsic and intrinsic stresses, many of which are important for tumorigenesis. Activation of ISR plays a dual role in cell fate decisions; while the ISR promotes survival, prolonged activation of ISR induces apoptosis. Activation of General Control Nonderepressible 2 (GCN2), an ISR kinase that senses and responds to nutrient stress conditions results in anti-tumor activity. We are developing HC-7366, a first-in-class, first-in-human GCN2 activator, and are currently evaluating it in a phase 1 clinical trial in solid tumors (NCT05121948). Here we present a series of studies characterizing the antitumor effects of HC-7366 in acute myeloid leukemia (AML). Higher expression of GCN2 and ISR markers such as ATF4 has been observed in primitive or minimally differentiated AML cells, suggesting that AML may be particularly sensitive to HC-7366. Encouragingly, in vivo efficacy studies in MOLM-16 and KG-1 tumor models showed 100% complete response and 100% tumor growth inhibition, respectively. Analysis of tumors from treated mice by IHC demonstrated activation of ISR as evidenced by increased expression of the ATF4 targets, ASNS and PSAT1, confirming that HC-7366 is functioning as a GCN2 activator in vivo. In the MV4-11 model, a differentiated subtype of AML that shows limited response to venetoclax, the combination of HC-7366 and venetoclax produced strong benefit resulting in 26% tumor regression. Enhanced activation of ISR pathway was again observed when HC-7366 was combined with venetoclax. HC-7366 treatment also impacted possible venetoclax resistance mechanisms by increasing the levels of PUMA while reducing levels of S100A8/A9 proteins. To investigate the effects of the compound on primary AML, we performed an ex vivo screen using cells from AML patients and treatment with HC-7366 showed a remarkable decrease in cell proliferation. Furthermore, in a xenotransplantable model of patient-derived AML, we found that HC-7366 significantly reduced mature myeloid (CD33+) AML cells in the bone marrow as compared to standard of care (SOC) agents, including venetoclax. We confirmed that activation of ISR, reduction in cell viability, and inhibition of protein synthesis following treatment with HC-7366 was dependent on GCN2 using CRISPR knockout cells. In addition, HC-7366 reduced mitochondrial respiration in MOLM-16 cells, suggesting effects on cellular bioenergetics. Metabolomics analyses of AML xenograft tumors showed that HC-7366 significantly altered metabolites associated with amino acid metabolism, urea cycle, and oxidative stress. Together, our in vitro and in vivo results demonstrate that HC-7366 is a potent GCN2 activator with strong antitumor activity in AML as a single agent and in combination with venetoclax, supporting its investigation in clinical trials in patients with AML. Citation Format: Feven Tameire, Paulina M. Wojnar
综合应激反应(integrated stress response, ISR)是一种适应性信号通路，细胞利用它来响应广泛的外在和内在应激，其中许多应激对肿瘤的发生很重要。ISR的激活在细胞命运决定中起双重作用;虽然ISR促进存活，但ISR的长时间激活会诱导细胞凋亡。GCN2是一种ISR激酶，可感知并响应营养胁迫条件，其激活可导致抗肿瘤活性。我们正在开发HC-7366，这是一种同类首创、首次用于人体的GCN2激活剂，目前正在实体肿瘤的1期临床试验中进行评估(NCT05121948)。在这里，我们提出了一系列研究表征HC-7366在急性髓性白血病(AML)中的抗肿瘤作用。在原始或最低分化的AML细胞中观察到GCN2和ISR标记物如ATF4的高表达，表明AML可能对HC-7366特别敏感。令人鼓舞的是，MOLM-16和KG-1肿瘤模型的体内疗效研究分别显示100%完全缓解和100%肿瘤生长抑制。经免疫组化处理的小鼠肿瘤分析显示，ATF4靶点、ASNS和PSAT1的表达增加，证明了ISR的激活，证实HC-7366在体内发挥GCN2激活剂的作用。在MV4-11模型(一种对venetoclax反应有限的分化亚型AML)中，HC-7366与venetoclax联合使用产生了强大的益处，肿瘤消退率为26%。当HC-7366与venetoclax联合使用时，ISR通路的激活再次增强。HC-7366处理还通过增加PUMA水平同时降低S100A8/A9蛋白水平影响了可能的venetoclax耐药机制。为了研究该化合物对原发性AML的作用，我们使用AML患者的细胞进行了离体筛选，用HC-7366治疗显示细胞增殖显著减少。此外，在患者源性AML的异种移植模型中，我们发现与标准护理(SOC)药物(包括venetoclax)相比，HC-7366显著减少骨髓中成熟髓系(CD33+) AML细胞。我们证实，使用CRISPR敲除细胞，HC-7366处理后，ISR的激活、细胞活力的降低和蛋白质合成的抑制依赖于GCN2。此外，HC-7366降低了MOLM-16细胞的线粒体呼吸，提示对细胞生物能量学有影响。AML异种移植肿瘤的代谢组学分析显示，HC-7366显著改变了与氨基酸代谢、尿素循环和氧化应激相关的代谢物。总之，我们的体外和体内实验结果表明，HC-7366是一种有效的GCN2激活剂，在AML中具有很强的抗肿瘤活性，无论是单独使用还是与venetoclax联合使用，都支持其在AML患者的临床试验中的研究。引用格式:7 . Tameire, Paulina M. Wojnarowicz, Sho Fujisawa, Sharon Huang, Owen Reilly, Crissy Dudgeon, Nicholas Collette, Jeremy Drees, Kathryn Bieging-Rolett, Takashi O Kangas，张卫宇，Maria Fumagalli, Iman Dewji，李云芳，Anissa SH Chan，邱晓红，Ben Harrison, Ashley LaCayo, Kirk A. Staschke, Alan C. Rigby, savthri Ramurthy, Eric S. Lightcap, David Surguladze, Nandita Bose。HC-7366激活GCN2，在AML模型中无论是单独治疗还是与Venetoclax联合治疗，均具有显著的抗肿瘤疗效[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/ Abstract
{"title":"Abstract A30: Activation of GCN2 by HC-7366 results in significant anti-tumor efficacy as monotherapy and in combination with Venetoclax in AML models","authors":"Feven Tameire, P. Wojnarowicz, S. Fujisawa, Sharon Huang, O. Reilly, C. Dudgeon, Nick Collette, J. Drees, Kathryn T. Bieging-Rolett, Takashi O. Kangas, Weiyu Zhang, M. Fumagalli, Iman Dewji, Yunfang Li, Anissa S. H. Chan, Xiaohong X. Qiu, B. Harrison, Ashley LaCayo, K. Staschke, A. C. Rigby, S. Ramurthy, E. Lightcap, D. Surguladze, N. Bose","doi":"10.1158/2643-3249.aml23-a30","DOIUrl":"https://doi.org/10.1158/2643-3249.aml23-a30","url":null,"abstract":"\u0000 The integrated stress response (ISR) is an adaptive signaling pathway that cells utilize to respond to a wide range of extrinsic and intrinsic stresses, many of which are important for tumorigenesis. Activation of ISR plays a dual role in cell fate decisions; while the ISR promotes survival, prolonged activation of ISR induces apoptosis. Activation of General Control Nonderepressible 2 (GCN2), an ISR kinase that senses and responds to nutrient stress conditions results in anti-tumor activity. We are developing HC-7366, a first-in-class, first-in-human GCN2 activator, and are currently evaluating it in a phase 1 clinical trial in solid tumors (NCT05121948). Here we present a series of studies characterizing the antitumor effects of HC-7366 in acute myeloid leukemia (AML). Higher expression of GCN2 and ISR markers such as ATF4 has been observed in primitive or minimally differentiated AML cells, suggesting that AML may be particularly sensitive to HC-7366. Encouragingly, in vivo efficacy studies in MOLM-16 and KG-1 tumor models showed 100% complete response and 100% tumor growth inhibition, respectively. Analysis of tumors from treated mice by IHC demonstrated activation of ISR as evidenced by increased expression of the ATF4 targets, ASNS and PSAT1, confirming that HC-7366 is functioning as a GCN2 activator in vivo. In the MV4-11 model, a differentiated subtype of AML that shows limited response to venetoclax, the combination of HC-7366 and venetoclax produced strong benefit resulting in 26% tumor regression. Enhanced activation of ISR pathway was again observed when HC-7366 was combined with venetoclax. HC-7366 treatment also impacted possible venetoclax resistance mechanisms by increasing the levels of PUMA while reducing levels of S100A8/A9 proteins. To investigate the effects of the compound on primary AML, we performed an ex vivo screen using cells from AML patients and treatment with HC-7366 showed a remarkable decrease in cell proliferation. Furthermore, in a xenotransplantable model of patient-derived AML, we found that HC-7366 significantly reduced mature myeloid (CD33+) AML cells in the bone marrow as compared to standard of care (SOC) agents, including venetoclax. We confirmed that activation of ISR, reduction in cell viability, and inhibition of protein synthesis following treatment with HC-7366 was dependent on GCN2 using CRISPR knockout cells. In addition, HC-7366 reduced mitochondrial respiration in MOLM-16 cells, suggesting effects on cellular bioenergetics. Metabolomics analyses of AML xenograft tumors showed that HC-7366 significantly altered metabolites associated with amino acid metabolism, urea cycle, and oxidative stress. Together, our in vitro and in vivo results demonstrate that HC-7366 is a potent GCN2 activator with strong antitumor activity in AML as a single agent and in combination with venetoclax, supporting its investigation in clinical trials in patients with AML.\u0000 Citation Format: Feven Tameire, Paulina M. Wojnar","PeriodicalId":29944,"journal":{"name":"Blood Cancer Discovery","volume":" ","pages":""},"PeriodicalIF":11.2,"publicationDate":"2023-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47780604","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-05-01DOI: 10.1158/2643-3249.aml23-a08
B. Carter, Poo Yee Mak, W. Tao, Lauren B Ostermann, Yuki Nishida, S. Boettcher, M. Andreeff
The Bcl-2 inhibitor venetoclax (VEN)/hypomethylation agent (HMA) combination achieves high response rates, has improved outcomes for many patients with AML and is now considered standard of care for patients who are older or unfit to receive intensive chemotherapy. However, the median overall survival is only 14.7 months on this regimen and only 2.5 months post relapse. Molecular analysis demonstrates that mutations in TP53 and oncogenic kinases are key determinants of lower response rates and early relapse. Preclinical studies also show that increased kinase signaling in AML stem/progenitor cells in TP53 mutant AML. The heat shock protein 90 (HSP90) chaperone, a key regulator of proteostasis, is responsible for the correct folding of kinases and transcription factors. HSP90-associated-epichaperomes, formed in malignant cells, are complexes consisting of HSP90, co-chaperones, and associated proteins that support the maturation, activity, and stability of many cancer-associated kinases and transcription factors including mutated TP53. Hence, HSP90 epichaperome inhibition has the potential of targeting TP53 mutant AML. In contrast to other HSP90 ATP inhibitors, PU-H71 (zelavespib) is a competitive inhibitor specific for the ATP binding site of HSP90 epichaperomes. We here investigate the therapeutic potentials of targeting HSP90 epitherachorme with PU-H71 in TP53 mutated AML. Western blot analysis found increased HSP90 and several signaling proteins in TP53 knockout and mutant Molm13 cells generated by CRISPR/cas-9 or by exposing to idasanutlin, compared to the isogeneic wild-type controls. Using a fluorochrome-labelled PU-H71 and flow cytometry, we demonstrate the presence of HSP90 epichaperomes in AML cells and AML stem/progenitor cells with TP53 mutations, but not in normal bone marrow and bone marrow stem/progenitor cells. PU-H71 effectively kills AML cells and AML stem/progenitor cells with various TP53 mutations, and prolongs survival in TP53-mutant AML xenograft mice with minimal effects on normal CD34+ bone marrow cells and hematopoiesis. PU-H71 increased Bim expression and enhanced VEN activity in AML cells and AML stem/progenitor cells with TP53 mutations. Importantly, in a mixture of TP53 wild-type/R248W Molm13 cells (1000:1), nutlin3a selectively killed TP53 wild-type but enriched TP53 mutant Molm13 cells; VEN treatment favored the outgrowth of TP53-mutant cells, while PU-H71 effectively killed TP53 wild-type and mutant cells. Furthermore, PU-H71 exhibited anti-leukemia activity against both TP53 WT and mutant AML cells, which was further enhanced by VEN in vivo in a xenograft model of mixed TP53 WT and mutated Molm13 cells (10:1). Our data support that the HSP90 epichaperome is essential for the growth and survival of AML and AML stem/progenitor cells harboring mutant TP53. Inhibition of HSP90 by PU-H71 targets AML cells/stem/progenitor cells enhances VEN activity and prevents outgrowth of VEN-resistant TP53 mutant AML cells. This co
Bcl-2抑制剂venetoclax (VEN)/低甲基化剂(HMA)联合治疗获得了高缓解率，改善了许多AML患者的预后，现在被认为是老年或不适合接受强化化疗的患者的标准治疗。然而，该方案的中位总生存期仅为14.7个月，复发后仅2.5个月。分子分析表明，TP53和致癌激酶的突变是低反应率和早期复发的关键决定因素。临床前研究还表明，在TP53突变型AML中，AML干细胞/祖细胞中的激酶信号传导增加。热休克蛋白90 (HSP90)伴侣是蛋白质稳态的关键调节因子，负责激酶和转录因子的正确折叠。恶性细胞中形成的HSP90相关表粒是由HSP90、共伴侣和相关蛋白组成的复合物，支持许多癌症相关激酶和转录因子(包括突变的TP53)的成熟、活性和稳定性。因此，抑制HSP90表染色质具有靶向TP53突变型AML的潜力。与其他HSP90 ATP抑制剂相比，PU-H71 (zelavespib)是HSP90表体ATP结合位点特异性的竞争性抑制剂。我们在此研究了用PU-H71靶向HSP90表色素在TP53突变的AML中的治疗潜力。Western blot分析发现，与等基因野生型对照相比，通过CRISPR/cas-9或暴露于idasanutlin产生的TP53敲除和突变的Molm13细胞中HSP90和一些信号蛋白增加。利用荧光标记的PU-H71和流式细胞术，我们证实了HSP90表粒存在于TP53突变的AML细胞和AML干细胞/祖细胞中，而不存在于正常骨髓和骨髓干细胞/祖细胞中。PU-H71能有效杀死各种TP53突变的AML细胞和AML干细胞/祖细胞，延长TP53突变的AML异种移植小鼠的生存期，对正常CD34+骨髓细胞和造血功能的影响最小。在TP53突变的AML细胞和AML干细胞/祖细胞中，PU-H71增加了Bim表达，增强了VEN活性。重要的是，在TP53野生型/R248W Molm13细胞的混合物中(1000:1)，nutlin3a选择性地杀死TP53野生型但富集TP53突变型Molm13细胞;VEN处理有利于TP53突变细胞的生长，而PU-H71有效地杀死了TP53野生型和突变型细胞。此外，PU-H71对TP53 WT和突变AML细胞均表现出抗白血病活性，在TP53 WT和突变Molm13细胞混合的异种移植模型中，VEN在体内进一步增强了这种活性(10:1)。我们的数据支持HSP90表染色体对于携带突变TP53的AML和AML干细胞/祖细胞的生长和存活至关重要。PU-H71抑制HSP90靶向AML细胞/干细胞/祖细胞，可增强VEN活性并阻止VEN抗性TP53突变型AML细胞的生长。这一概念值得临床评估。引文格式:Bing Carter, Po Yee Mak, Wenjing Tao, Lauren B Ostermann, Yuki Nishida, Steffen Boettcher, Michael Andreeff。靶向HSP90表表观体治疗TP53突变型AML[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 A08。
{"title":"Abstract A08: Targeting HSP90 epichaperome in TP53 mutant AML","authors":"B. Carter, Poo Yee Mak, W. Tao, Lauren B Ostermann, Yuki Nishida, S. Boettcher, M. Andreeff","doi":"10.1158/2643-3249.aml23-a08","DOIUrl":"https://doi.org/10.1158/2643-3249.aml23-a08","url":null,"abstract":"\u0000 The Bcl-2 inhibitor venetoclax (VEN)/hypomethylation agent (HMA) combination achieves high response rates, has improved outcomes for many patients with AML and is now considered standard of care for patients who are older or unfit to receive intensive chemotherapy. However, the median overall survival is only 14.7 months on this regimen and only 2.5 months post relapse. Molecular analysis demonstrates that mutations in TP53 and oncogenic kinases are key determinants of lower response rates and early relapse. Preclinical studies also show that increased kinase signaling in AML stem/progenitor cells in TP53 mutant AML. The heat shock protein 90 (HSP90) chaperone, a key regulator of proteostasis, is responsible for the correct folding of kinases and transcription factors. HSP90-associated-epichaperomes, formed in malignant cells, are complexes consisting of HSP90, co-chaperones, and associated proteins that support the maturation, activity, and stability of many cancer-associated kinases and transcription factors including mutated TP53. Hence, HSP90 epichaperome inhibition has the potential of targeting TP53 mutant AML. In contrast to other HSP90 ATP inhibitors, PU-H71 (zelavespib) is a competitive inhibitor specific for the ATP binding site of HSP90 epichaperomes. We here investigate the therapeutic potentials of targeting HSP90 epitherachorme with PU-H71 in TP53 mutated AML. Western blot analysis found increased HSP90 and several signaling proteins in TP53 knockout and mutant Molm13 cells generated by CRISPR/cas-9 or by exposing to idasanutlin, compared to the isogeneic wild-type controls. Using a fluorochrome-labelled PU-H71 and flow cytometry, we demonstrate the presence of HSP90 epichaperomes in AML cells and AML stem/progenitor cells with TP53 mutations, but not in normal bone marrow and bone marrow stem/progenitor cells. PU-H71 effectively kills AML cells and AML stem/progenitor cells with various TP53 mutations, and prolongs survival in TP53-mutant AML xenograft mice with minimal effects on normal CD34+ bone marrow cells and hematopoiesis. PU-H71 increased Bim expression and enhanced VEN activity in AML cells and AML stem/progenitor cells with TP53 mutations. Importantly, in a mixture of TP53 wild-type/R248W Molm13 cells (1000:1), nutlin3a selectively killed TP53 wild-type but enriched TP53 mutant Molm13 cells; VEN treatment favored the outgrowth of TP53-mutant cells, while PU-H71 effectively killed TP53 wild-type and mutant cells. Furthermore, PU-H71 exhibited anti-leukemia activity against both TP53 WT and mutant AML cells, which was further enhanced by VEN in vivo in a xenograft model of mixed TP53 WT and mutated Molm13 cells (10:1). Our data support that the HSP90 epichaperome is essential for the growth and survival of AML and AML stem/progenitor cells harboring mutant TP53. Inhibition of HSP90 by PU-H71 targets AML cells/stem/progenitor cells enhances VEN activity and prevents outgrowth of VEN-resistant TP53 mutant AML cells. This co","PeriodicalId":29944,"journal":{"name":"Blood Cancer Discovery","volume":" ","pages":""},"PeriodicalIF":11.2,"publicationDate":"2023-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43076389","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-05-01DOI: 10.1158/2643-3249.aml23-a05
M. Muftuoglu, M. Yilmaz, Mahesh Basyal, Li Li, N. Daver, M. Andreeff
Targeted therapies(TT) combining FLT3 inhibitors with hypomethylating agents (HMA) and Venetoclax (Ven) are highly effective against FLT3-ITD acute myeloid leukemia (AML) with high response rates. Signaling mutations along with emergence of rarer mutations are the key contributors to primary and secondary resistance to TT. We hypothesize that resistant leukemia clones have unique proteomic profiles that facilitate survival under therapy pressure(TP) and deciphering proteomic profiles at the single-cell level will delineate resistance mechanisms and adaptive responses driven by TP. We performed multiplexed single-cell proteomic analysis of serial PB and BM samples (n:162) from patients treated with FLT3i+HMA+/-VEN using CyTOF. Unsupervised analysis identified leukemia cells and non-malignant cellular elements of the leukemia compartment in an unbiased manner. Next, we interrogated the leukemia proteomic landscape to identify leukemia associated proteomic features. Since Ven targets BCL2 and FLT3i modulates the expression of anti-apoptotic molecules we assessed the expression of apoptosis regulators and found that leukemia cells with immature phenotype, including those with leukemia stem cell(LSC) phenotype, almost always expressed moderate-high levels of BCL2. Monocytic cells(MC) lacked BCL2 expression and had the highest levels of MCL1. Despite expressing moderate levels of MCL1 and BCL-XL, TT was effective in substantially eliminating CD34+ immature leukemia cells. We observed a relative enrichment of MCs, either benign or malignant, after TT. Since cells with MC phenotype were inherently resistant to TT we hypothesized that leukemia cell subsets having similar proteomic profiles to MCs will persist after TT. Phenotypic interrogation of leukemia cells (UMAP) revealed that LSCs generally clustered on the opposite pole distant from MCs and leukemia cells on poles facing MCs were more differentiated. Remarkably, FLT3-ITD mutation partners differentially altered the proteomic landscape. NPM1 mutant FLT3-ITD AML cells displayed a less diverse leukemia architectural organization with differentiation block. Contrarily, signaling mutations diversified the leukemia landscape into a diverse continuum of differentiation states. We also observed that RAS/MAPK mutations could overcome differentiation block and gave rise to a differentiation continuum encompassing less-differentiated, transitional and differentiated leukemia cells. The transitional CD34+ leukemia cells, mapped in close vicinity to MCs, and differentiated MCs that preferentially persisted at D28, had active signaling pathways and expressed CD36. The presence of CD34+ leukemia cells with persistent signaling at D28 was indicative of poor clinical outcomes. Conclusion: Multiplexed single-cell proteomic analysis identified a unique mode of resistance and proteomic features of surviving cells in with FLT3-ITD AML patients treated with TT, and elucidated how different mutation partners in FLT3-IT
靶向治疗(TT)联合FLT3抑制剂与低甲基化药物(HMA)和Venetoclax (Ven)治疗FLT3- itd急性髓性白血病(AML)非常有效，反应率高。信号突变以及罕见突变的出现是导致TT原发性和继发性耐药的关键因素。我们假设耐药白血病克隆具有独特的蛋白质组学特征，有助于在治疗压力(TP)下生存，而在单细胞水平上破译蛋白质组学特征将描述TP驱动的耐药机制和适应性反应。我们使用CyTOF对接受FLT3i+HMA+/-VEN治疗的患者的一系列PB和BM样本(n:162)进行了多重单细胞蛋白质组学分析。无监督分析确定白血病细胞和白血病室的非恶性细胞成分在一个公正的方式。接下来，我们询问白血病蛋白质组学景观，以确定白血病相关的蛋白质组学特征。由于Ven靶向BCL2和FLT3i调节抗凋亡分子的表达，我们评估了凋亡调节因子的表达，发现未成熟表型的白血病细胞，包括白血病干细胞(LSC)表型的白血病细胞，几乎总是表达中高水平的BCL2。单核细胞(MC)缺乏BCL2表达，MCL1水平最高。尽管表达了中等水平的MCL1和BCL-XL，但TT有效地消除了CD34+未成熟白血病细胞。我们观察到TT后良性或恶性MCs的相对富集。由于具有MC表型的细胞固有地抵抗TT，我们假设具有与MCs相似的蛋白质组学特征的白血病细胞亚群在TT后会持续存在。白血病细胞表型分析(phenotype interrogation of leukemia cells, UMAP)显示，LSCs一般聚集在远离MCs的另一端，而面向MCs的另一端的白血病细胞分化程度更高。值得注意的是，FLT3-ITD突变伙伴不同程度地改变了蛋白质组学格局。NPM1突变体FLT3-ITD AML细胞表现出分化阻滞的白血病结构组织多样性较低。相反，信号突变使白血病景观多样化，进入分化状态的多样化连续体。我们还观察到RAS/MAPK突变可以克服分化障碍，并产生包括低分化、过渡性和分化白血病细胞的分化连续体。移行性CD34+白血病细胞，定位于MCs附近，分化的MCs优先持续在D28，具有活跃的信号通路并表达CD36。CD34+白血病细胞在D28处具有持续的信号传导，表明临床结果较差。结论:多重单细胞蛋白质组学分析确定了接受TT治疗的FLT3-ITD AML患者存活细胞的独特耐药模式和蛋白质组学特征，并阐明了FLT3-ITD AML中不同突变伙伴如何改变蛋白质组学格局以驱动TT耐药。引用格式:Muharrem Muftuoglu, Musa Yilmaz, Mahesh Basyal, Li Li, Naval Daver, Michael Andreeff。FLT3-ITD AML景观的多重CyTOF分析确定了与靶向治疗耐药相关的蛋白质组学特征[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 - A05。
{"title":"Abstract A05: Multiplexed CyTOF analysis of FLT3-ITD AML landscape identifies proteomic profiles associated with resistance to targeted therapies","authors":"M. Muftuoglu, M. Yilmaz, Mahesh Basyal, Li Li, N. Daver, M. Andreeff","doi":"10.1158/2643-3249.aml23-a05","DOIUrl":"https://doi.org/10.1158/2643-3249.aml23-a05","url":null,"abstract":"\u0000 Targeted therapies(TT) combining FLT3 inhibitors with hypomethylating agents (HMA) and Venetoclax (Ven) are highly effective against FLT3-ITD acute myeloid leukemia (AML) with high response rates. Signaling mutations along with emergence of rarer mutations are the key contributors to primary and secondary resistance to TT. We hypothesize that resistant leukemia clones have unique proteomic profiles that facilitate survival under therapy pressure(TP) and deciphering proteomic profiles at the single-cell level will delineate resistance mechanisms and adaptive responses driven by TP. We performed multiplexed single-cell proteomic analysis of serial PB and BM samples (n:162) from patients treated with FLT3i+HMA+/-VEN using CyTOF. Unsupervised analysis identified leukemia cells and non-malignant cellular elements of the leukemia compartment in an unbiased manner. Next, we interrogated the leukemia proteomic landscape to identify leukemia associated proteomic features. Since Ven targets BCL2 and FLT3i modulates the expression of anti-apoptotic molecules we assessed the expression of apoptosis regulators and found that leukemia cells with immature phenotype, including those with leukemia stem cell(LSC) phenotype, almost always expressed moderate-high levels of BCL2. Monocytic cells(MC) lacked BCL2 expression and had the highest levels of MCL1. Despite expressing moderate levels of MCL1 and BCL-XL, TT was effective in substantially eliminating CD34+ immature leukemia cells. We observed a relative enrichment of MCs, either benign or malignant, after TT. Since cells with MC phenotype were inherently resistant to TT we hypothesized that leukemia cell subsets having similar proteomic profiles to MCs will persist after TT. Phenotypic interrogation of leukemia cells (UMAP) revealed that LSCs generally clustered on the opposite pole distant from MCs and leukemia cells on poles facing MCs were more differentiated. Remarkably, FLT3-ITD mutation partners differentially altered the proteomic landscape. NPM1 mutant FLT3-ITD AML cells displayed a less diverse leukemia architectural organization with differentiation block. Contrarily, signaling mutations diversified the leukemia landscape into a diverse continuum of differentiation states. We also observed that RAS/MAPK mutations could overcome differentiation block and gave rise to a differentiation continuum encompassing less-differentiated, transitional and differentiated leukemia cells. The transitional CD34+ leukemia cells, mapped in close vicinity to MCs, and differentiated MCs that preferentially persisted at D28, had active signaling pathways and expressed CD36. The presence of CD34+ leukemia cells with persistent signaling at D28 was indicative of poor clinical outcomes. Conclusion: Multiplexed single-cell proteomic analysis identified a unique mode of resistance and proteomic features of surviving cells in with FLT3-ITD AML patients treated with TT, and elucidated how different mutation partners in FLT3-IT","PeriodicalId":29944,"journal":{"name":"Blood Cancer Discovery","volume":" ","pages":""},"PeriodicalIF":11.2,"publicationDate":"2023-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43106890","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-05-01DOI: 10.1158/2643-3249.aml23-a50
S. Loghavi, Yoheved Gerstein, M. Routbort, K. Patel, Koichi Takahashi, M. Daniels, Julie B Moskowitz, D. Hammond, K. Chien, L. Medeiros, F. Ravandi, H. Kantarjian, G. Garcia-Manero, B. Arun, Joseph D. Khoury, C. Dinardo
The incorporation of panel-based germline genetic testing for assessment of pathogenic or likely pathogenic variants associated with increased risk of breast, ovarian, prostatic and pancreatic cancer in NCCN guidelines and wide adoption of cell-free circulating DNA sequencing (liquid biopsy) for patients with advanced solid tumors has increased the identification of pathogenic TP53 variants in blood of patients with solid tumors. The variant allelic frequency (VAF) of the pathogenic variant is often used to help infer the compartment of origin (germline vs somatic) with near heterozygous (~50%) VAF favoring germline; however, when VAF is low (sub heterozygous) potential considerations include mosaic Li Fraumeni syndrome, clonal hematopoiesis, or incidental myeloid neoplasms, and determining the origin of the TP53 mutation can be a challenge with important surveillance and treatment implications. We describe a cohort of patients with solid tumors or hematologic malignancies in whom pathogenic or likely pathogenic TP53 variants of uncertain origin were identified by clinical genetic testing of peripheral blood, next generation sequencing (NGS) using an 81 gene hematologic malignancy-based panel, or cell-free DNA sequencing/liquid biopsy. In a subset of patients for whom solid tumor, adjacent normal tissue and/or bone marrow (BM) was available, immunohistochemistry for p53 protein was performed. Patients (n= 17) included 11 (61%) women and 7 (39%) men with a median age of 54 years (range 8-7) at initial cancer diagnosis, with 17 unique TP53 variants with a median VAF of 16% (range, 2-50%). The most common indication for referral for additional investigation of the TP53 origin was sub-heterozygous VAF on germline testing of blood (8/47%) or saliva (1/6%); TP53 variant identified in plasma when the concurrent solid tumor was TP53 wild-type (3/18%) followed by presence of a pathogenic TP53 variant in remission BM of patients with history of hematologic malignancies (4/24%). Additional germline testing of skin fibroblasts and/or BM examination was performed in in 9 (53%) and 8 (47%) patients, respectively. p53 IHC was performed on 3 BM biopsies and 2 solid tumor tissues. A multimodality/multidisciplinary interpretation of these results allowed appropriate classification of the TP53 variants in 12/18 patients (67%). These included Li Fraumeni syndrome (5/12; 42%); clonal hematopoiesis (5/12; 42%); donor-derived clonal hematopoiesis (1/12;8%) and therapy-related myelodysplastic syndrome (1/12; 8%) patients. Appropriate awareness and distinction of clonal hematopoiesis and potential myeloid neoplasms from mosaic germline TP53 in the setting of subheterozygous VAF identified on peripheral blood or bone marrow analyses can be challenging. Multimodality testing and a multidisciplinary approach for accurate interpretation is required. Immunohistochemical staining for p53 can be useful in making this distinction in a subset of patients. Citation Format: Sana
在NCCN指南中纳入基于小组的种系基因检测，用于评估与乳腺癌、卵巢癌、前列腺癌和胰腺癌风险增加相关的致病性或可能致病性变异，以及在晚期实体瘤患者中广泛采用无细胞循环DNA测序(液体活检)，增加了实体瘤患者血液中致病性TP53变异的鉴定。致病变异的变异等位基因频率(VAF)经常被用来帮助推断起源区室(种系vs体细胞)，近杂合(~50%)的VAF倾向于种系;然而，当VAF较低(亚杂合)时，潜在的考虑因素包括马赛克Li Fraumeni综合征、克隆造血或偶发髓系肿瘤，确定TP53突变的起源可能是一个具有重要监测和治疗意义的挑战。我们描述了一组患有实体肿瘤或血液恶性肿瘤的患者，通过外周血的临床基因检测，使用基于81个基因的血液恶性小组的下一代测序(NGS)或无细胞DNA测序/液体活检，确定了起源不确定的致病性或可能致病性TP53变异。在实体瘤、邻近正常组织和/或骨髓(BM)可用的患者亚组中，对p53蛋白进行免疫组化。患者(n= 17)包括11名(61%)女性和7名(39%)男性，初始癌症诊断时的中位年龄为54岁(范围8-7岁)，有17种独特的TP53变异，中位VAF为16%(范围2-50%)。转诊进一步调查TP53起源的最常见适应症是血液(8/47%)或唾液(1/6%)种系检测的亚杂合子VAF;当并发实体瘤为TP53野生型时，血浆中发现TP53变异(3/18%)，随后在有血液系统恶性肿瘤病史的缓解期患者中发现致病性TP53变异(4/24%)。分别有9例(53%)和8例(47%)患者进行了皮肤成纤维细胞的生殖系检测和/或BM检查。3例BM活检和2例实体瘤组织行p53免疫组化。对这些结果的多模式/多学科解释允许对12/18(67%)患者的TP53变异进行适当分类。其中包括Li Fraumeni综合征(5/12;42%);克隆造血(5/12;42%);供体来源克隆造血(1/12,8%)和治疗相关骨髓增生异常综合征(1/12;8%)患者。在外周血或骨髓分析中发现亚杂合性VAF的情况下，适当认识和区分克隆造血和潜在的髓系肿瘤是具有挑战性的。需要多模态测试和多学科方法来进行准确的解释。免疫组织化学染色的p53可用于使这种区分在一个子集的患者。引文格式:Sanam Loghavi, Yoheved Gerstein, Mark J Routbort, Keyur P Patel, Koichi Takahashi, Molly Daniels, Julie Moskowitz, Danielle E Hammond, Kelly Chien, L. Jeffrey Medeiros, Farhad Ravandi, Hagop M Kantarjian, Guillermo Garcia-Manero, Banu Arun, Joseph D Khoury, Courtney D DiNardo。通过种系检测或无细胞循环DNA测序鉴别TP53致病性突变实体瘤患者中嵌合李氏综合征、克隆造血和偶发髓系肿瘤[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/ Abstract
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Pub Date : 2023-05-01DOI: 10.1158/2643-3249.aml23-a46
Timothy M. Chlon, Emily Stepanchick, Analise Sulentic, Andrew F. Wilson, D. Starczynowski
Germline mutations in the RNA Helicase gene DDX41 are the most common cause of inherited susceptibility to adult MDS and AML. These mutations are always heterozygous and are typically frameshifts, causing loss of functional protein. We recently reported that at least one functional copy of DDX41 is essential for hematopoiesis, and that DDX41 is required for ribosome biogenesis (Chlon et al., Cell Stem Cell 2021). While biallelic DDX41 mutations cause dramatic defects in hematopoiesis, the role of heterozygous mutations in MDS pathogenesis is not yet understood. DDX41 mutation carriers frequently experience idiopathic cytopenias of unknown significance (ICUS) prior to MDS onset, suggesting that underlying hematopoietic defects contribute to MDS/AML (Choi et al., Haemotologica 2021). The majority of DDX41-mutant MDS patients have refractory anemia, indicating that the erythroid lineage is particularly affected in these patients (Sebert et al., Blood 2019). Since ribosome defects are a common cause of inherited anemias and also contribute to MDS pathogenesis, we characterized the effect of heterozygous DDX41 mutations on erythropoiesis in murine and human models. Mice that were transplanted with Ddx41+/− bone marrow develop anemia at 12-15 months post-transplant. At younger ages, these mice have normal hematopoietic indices, but when stress erythropoiesis is induced by Pheylhydrazine-treatment, the Ddx41+/− mice have prolonged anemia. These observations indicate that heterozygous loss of DDX41 causes defects in erythropoiesis during stress and aging. We further characterized the effect of Ddx41-hetrozyogisty on erythroid progenitor function in vitro. In colony assays, we found that Ddx41+/− HSPC form fewer BFU-E but comparable numbers of myeloid colonies. In liquid culture erythroid differentiation cultures, we found that Ddx41+/− HSPC produce fewer CD71+ Ter119+ progenitors than controls. Mechanistically, we found that in vitro-derived erythroid progenitors from both mice and cell line models had decreased protein translation, suggesting that ribosome defects underlie the observed inefficiency in erythropoiesis. In congenital ribosomopathy diseases, ribosome defects lead to p53 activation which contributes to defects in erythropoiesis. Interestingly, TP53 mutations are common in DDX41-mutant MDS/AML (Sebert et al., Blood 2019), suggesting that the ribosome deficiency selects for these mutations. To assess the role of p53 mutations in the development of DDX41-mutant MDS, we generated Ddx41+/−;p53+/− mice and transplanted the bone marrow into lethally-irradiated recipients and followed the mice for 1 year. These mice developed a lethal MDS-like phenotype at 7-12 months post-transplant while control mice lived until the end of the study period. The sick mice had anemia and other cytopenias accompanied by enlarged spleens and dysplastic myeloid cells. Collectively, these results indicate that p53 mutations cooperate with Ddx41-heterozygosity to promo
RNA螺旋酶基因DDX41的种系突变是成人MDS和AML遗传易感性的最常见原因。这些突变总是杂合的，通常是移码的，导致功能蛋白的损失。我们最近报道，DDX41的至少一个功能拷贝对造血至关重要，并且DDX41是核糖体生物发生所必需的（Chlon等人，Cell Stem Cell 2021）。虽然双等位基因DDX41突变会导致造血功能的显著缺陷，但杂合突变在MDS发病机制中的作用尚不清楚。DDX41突变携带者在MDS发病前经常经历意义不明的特发性细胞减少症（ICUS），这表明潜在的造血缺陷导致MDS/AML（Choi等人，Haemotologica 2021）。大多数DDX41突变MDS患者患有难治性贫血，这表明这些患者的红系谱系尤其受到影响（Sebert等人，Blood 2019）。由于核糖体缺陷是遗传性贫血的常见原因，也有助于MDS的发病机制，我们在小鼠和人类模型中表征了杂合DDX41突变对红细胞生成的影响。用Ddx41+/−骨髓移植的小鼠在移植后12-15个月出现贫血。在较年轻的时候，这些小鼠的造血指数正常，但当苯肼治疗诱导应激性红细胞生成时，Ddx41+/−小鼠会出现长期贫血。这些观察结果表明，DDX41的杂合缺失会导致应激和衰老过程中红细胞生成的缺陷。我们进一步表征了Ddx41杂合性对体外红系祖细胞功能的影响。在菌落测定中，我们发现Ddx41+/−HSPC形成的BFU-E较少，但髓系菌落数量相当。在液体培养红系分化培养中，我们发现Ddx41+/−HSPC产生的CD71+Ter119+祖细胞比对照组少。从机制上讲，我们发现来自小鼠和细胞系模型的体外衍生红系祖细胞的蛋白质翻译减少，这表明核糖体缺陷是观察到的红细胞生成效率低下的原因。在先天性核糖体疾病中，核糖体缺陷会导致p53活化，从而导致红细胞生成缺陷。有趣的是，TP53突变在DDX41突变型MDS/AML中很常见（Sebert et al.，Blood 2019），这表明核糖体缺乏选择了这些突变。为了评估p53突变在DDX41突变MDS发展中的作用，我们产生了DDX41+/−；p53+/-小鼠，并将骨髓移植到致命辐射的受体中，并对小鼠进行1年的随访。这些小鼠在移植后7-12个月时出现了致命的MDS样表型，而对照小鼠则一直存活到研究期结束。患病小鼠出现贫血和其他细胞减少，伴有脾脏肿大和骨髓细胞发育异常。总之，这些结果表明p53突变与Ddx41杂合性协同作用，促进血液系统恶性肿瘤。引文格式：Timothy M Chlon，Emily Stepanchick，Analise Sulentic，Andrew Wilson，Daniel T Starczynowski。DDX41的杂合突变导致红系祖细胞缺陷，并与p53突变协同导致血液系统恶性肿瘤[摘要]。载：AACR特别会议论文集：急性髓细胞白血病和骨髓增生异常综合征；2023年1月23日至25日；德克萨斯州奥斯汀。费城（PA）：AACR；血液癌症Discov 2023；4（3_Suppl）：摘要编号A46。
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