Acute Myeloid Leukemia Bone Marrow Research Articles

Coexpression profile of leukemic stem cell markers for combinatorial targeted therapy in AML

Targeted immunotherapy in acute myeloid leukemia (AML) is challenged by the lack of AML-specific target antigens and clonal heterogeneity, leading to unwanted on-target off-leukemia toxicity and risk of relapse from minor clones. We hypothesize that combinatorial targeting of AML cells can enhance therapeutic efficacy without increasing toxicity. To identify target antigen combinations specific for AML and leukemic stem cells, we generated a detailed protein expression profile based on flow cytometry of primary AML (n = 356) and normal bone marrow samples (n = 34), and a recently reported integrated normal tissue proteomic data set. We analyzed antigen expression levels of CD33, CD123, CLL1, TIM3, CD244 and CD7 on AML bulk and leukemic stem cells at initial diagnosis (n = 302) and relapse (n = 54). CD33, CD123, CLL1, TIM3 and CD244 were ubiquitously expressed on AML bulk cells at initial diagnosis and relapse, irrespective of genetic characteristics. For each analyzed target, we found additional expression in different populations of normal hematopoiesis. Analyzing the coexpression of our six targets in all dual combinations (n = 15), we found CD33/TIM3 and CLL1/TIM3 to be highly positive in AML compared with normal hematopoiesis and non-hematopoietic tissues. Our findings indicate that combinatorial targeting of CD33/TIM3 or CLL1/TIM3 may enhance therapeutic efficacy without aggravating toxicity in immunotherapy of AML.

Distinct roles of mesenchymal stem and progenitor cells during the development of acute myeloid leukemia in mice

AbstractDespite increasing evidence for the involvement of bone marrow (BM) hematopoietic stem cell niche in leukemogenesis, how BM mesenchymal stem and progenitor cells (MSPCs) contribute to leukemia niche formation and progression remains unclear. Using an MLL-AF9 acute myeloid leukemia (AML) mouse model, we demonstrate dynamic alterations of BM cellular niche components, including MSPCs and endothelial cells during AML development and its association with AML engraftment. Primary patient AML cells also induced similar niche alterations in xenografted mice. AML cell infiltration in BM causes an expansion of early B-cell factor 2+ (Ebf2+) MSPCs with reduced Cxcl12 expression and enhanced generation of more differentiated mesenchymal progenitor cells. Importantly, in vivo fate-mapping indicates that Ebf2+ MSPCs participated in AML niche formation. Ebf2+ cell deletion accelerated the AML development. These data suggest that native BM MSPCs may suppress AML. However, they can be remodeled by AML cells to form leukemic niche that might contribute to AML progression. AML induced dysregulation of hematopoietic niche factors like Angptl1, Cxcl12, Kitl, Il6, Nov, and Spp1 in AML BM MSPCs, which was associated with AML engraftment and partially appeared before the massive expansion of AML cells, indicating the possible involvement of the niche factors in AML progression. Our study demonstrates distinct dynamic features and roles of BM MSPCs during AML development.

Mebendazole exhibits potent anti-leukemia activity on acute myeloid leukemia

Acute myeloid leukemia (AML) is one of the most common types of acute leukemia in adults with the lowest survival rate of all leukemia. Resistance to cytarabine and anthracycline-based chemotherapy is a major cause of treatment failure. Thus, finding new drugs with anti-leukemia activities and minimal side effect is urgently needed. Here through screening more than 1000 drugs approved by the Food and Drug Administration (FDA) of United States, the anthelmintic drug mebendazole (MBZ) was found to inhibit the growth of AML cell lines (THP-1, U937, NB4 and K562) and bone marrow mononuclear cells (BM-MNCs) from AML patients at pharmacologically achievable concentrations. In contrast, similar concentration of MBZ had little inhibitory effect on the growth of normal peripheral blood mononuclear cells (PBMC) or human umbilical vein endothelial cells (HUVEC). In addition, MBZ induced mitotic arrest and mitotic catastrophe in AML cells based on nuclear morphology, cell cycle distribution, mitotic marker analyses and the number of multinucleated cells and apoptotic cells. Furthermore, MBZ treatment inhibited activation of Akt and Erk in AML leukemic cells. Finally, MBZ repressed the progression of leukemic cells in vivo and prolonged survival in AML xenograft mouse model. Taken together, our results suggest that MBZ could be a potential new therapeutic agent for the treatment of AML patients.

Repolarizing heterogeneous leukemia-associated macrophages with more M1 characteristics eliminates their pro-leukemic effects

ABSTRACT Macrophages exhibit phenotypic heterogeneity under both physiological and pathological conditions. Applications targeting M2-like tumor-associated macrophages (TAMs) improve outcome in solid tumors. Considerable differences are detected between leukemia-associated macrophages (LAMs) and TAMs. However, application to induce M1 characteristics in heterogeneous LAMs has not been established. Here we analyzed clinical relevance of macrophage phenotypes in human acute myeloid leukemia (AML), studied phenotypic evolution of bone marrow (BM) and spleen (SP) LAMs in mouse AML and T cell acute lymphoblastic leukemia (T-ALL) models, explored mechanism leading to different LAM phenotypes and tried to eliminate pro-leukemic effects by inducing M1 characteristics. The results showed that more M2-like LAMs but not total LAMs correlated with worse prognosis in AML patients. Heterogeneity of LAM activation in tissue-specific leukemic microenvironments was observed in both AML and ALL models, i.e. SP LAMs evolved with more M2 characteristics while BM LAMs with more M1 characteristics. Furthermore, IRF7 contributed to M1 characteristics through the activation of SAPK/JNK pathway. Moreover, targeting IRF7-SAPK/JNK pathway to induce M1 characteristics in LAMs contributed to prolonged survival in leukemia mice. Our study provides the potential target for macrophage based immuno-therapy strategy against leukemia.

Abstract 22: A new precision medicine test to guide personalized treatments decision for acute myeloid leukemia patients

Abstract Background and aim: Treatment of acute myeloid leukemia (AML) remains a considerable therapeutic challenge. Complete response (CR) after induction therapy is the first treatment goal in these leukemic patients. A few combinations, based mostly on empirical observations with drugs already known to be more or less effective, are frequently used in the treatment of AML. Because of this, many patients who do not respond to standard chemotherapy need to enter in clinical trials and the use of predictive assays prior to patient treatment represents the ideal scenario to guide or help in treatment decisions. We have now developed an ex vivo test where a initial clinical validation has been achieved in an observational clinical trial in 123 AML patients on single-treatment, 1st-line cytarabine (CYT) plus idarubicin (IDA), achieving 85% clinical correlation. The aim of this study is to provide actionable data to improve disease management in the context of a clinical trial, and provide key information for Precision Medicine (PM) to guide the hematologist among the more sensitive treatments to achieve a CR. Methods: AML bone marrow (BM) samples from adult patients are received at the laboratory within 24 hours from extraction and incubated for 48 hours in 96-well plates containing the single drugs or combinations. The analysis is performed in the automated flow cytometry PharmaFlow platform and 72 hours after the extraction of the sample, an encrypted report is sent to the hematologist before the patient begins treatment. Pharmacologic responses were calculated using pharmacokinetic population models. Induction response was assessed according to the Cheson criteria (2003). Patients attaining a CR/CRi were classified as responders and the remaining as resistant, excluding early deaths. Final scores and treatments ranking are based on a therapeutic algorithm that integrates ex vivo activity; monotherapy dose responses are quantified by the area under the curve (AUC) with limits such as Cmax values, and synergism calculated measuring 8 concentration ratios, requiring consistency in their results in a 3D surface (so-called alpha factor synergism). The PM test attempts to identify the best treatment for predicting sensitivity for each patient. Results: The scoring method was tested using ex vivo results from samples obtained in an observational clinical trial with Spain's PETHEMA group from a cohort of 123 samples from de novo diagnosed AML patients, treated with the standard PETHEMA 1st-line guideline 3+7 with CYT+IDA. The score predicts sensitive patients with 90% accuracy. This accuracy can be compared with an independently derived 92% accuracy in identifying sensitive patients in a statistically significant clinical correlation study. The score is a simplified version of such correlation algorithm. Both methods identify a similar % of all clinically sensitive patients (67% vs 71%). However, the correlation is only valid for CYT-IDA while the PM test can be applied to any treatment. Moreover, for CYT+IDA treatment, the PM test predicts a 3-year overall survival with 75% accuracy. Conclusion: We have developed a novel ex vivo PM test for induction treatment in AML patients to guide hematologists selecting the right treatment to achieve CR in individual patients. Assuming a similar response rate for all these treatments, our test could estimate a net prediction for sensibility to AML treatment higher than 80% in 1st line. This PM test can be used in an Investigator Sponsored Trial as a Companion Diagnostic selecting sensitive patients with higher response rates and survival. Citation Format: Joan Ballesteros, Pau Montesinos, David Martinez-Cuadron, Joaquin Martinez-Lopez, Julian Gorrochategui, Jose Luis Rojas, Cristina Gomez, Pilar Hernandez, Alicia Robles. A new precision medicine test to guide personalized treatments decision for acute myeloid leukemia patients [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 22.

The Cold-Shock Protein Ybx1 Is Required for Development and Maintenance of Acute Myeloid Leukemia (AML) in Vitro and In Vivo

The family of cold shock proteins (CSPs) is highly conserved and consists of 8 members, including Ybx1-3, Csde1 and Lin28. Ybx1 is a multifunctional DNA/RNA binding protein that modulates gene transcription and translation during inflammation and malignant transformation. Recently, our group identified Ybx1 as a mediator of Jak2 signaling in MPN that protects Jak2-mutated cells from Jak-inhibitor induced apoptosis.In a recently published genome wide CRISPR-Cas9 dropout screen in AML cell lines, depletion of Ybx1 resulted in the highest dropout indices compared to other CSP members, with strongest dependencies in cell lines harboring MLL-rearrangements. Protein expression of Ybx1 in healthy individuals (n=10), primary MDS (n=54) and AML (n=58) bone marrow (BM) biopsies, revealed high protein expression in the majority of AML and MDS cases. Consistently, gene expression data revealed high mRNA expression of Ybx1 in AML samples compared to normal controls.Genetic inactivation of Ybx1 in human AML cell lines by RNAi resulted in reduced proliferative capacity. Therefore, we sought to investigate the requirement for Ybx1 in malignant transformation. We used BM cells from a previously published conventional knockout (ko) mouse model (Lu et al., 2005) in which homozygous deletion is embryonically lethal due to brain malformation. We sorted Lineage-Sca1+Kit+ (LSK-) cells from the BM of heterozygous (Ybx1+/-) and wildtype (Ybx1+/+) mice. Cells were retrovirally infected with either MLL-AF9 (MA9) or HoxA9 and Meis1a (HA9M1) to assess for disease development by serial plating in methylcellulose. Haploinsufficiency for Ybx1 in MA9- or HA9M1 transformed cells limited re-plating capacity to 2-4 rounds. When we injected 2,5x 104 MA9-infected LSK cells into sublethally irradiated recipient mice, recipients of MA9-Ybx1+/- cells (n=8) and MA9-Ybx1+/+ (n=10) showed development of AML. However, recipients of MA9-Ybx1+/- cells had a significant delay in AML development (median survival 67.5 days for Ybx1+/+ versus 101.5 days for Ybx1+/- animals, p=0.0078**). This effect appeared even more pronounced when 1x 106 whole BM cells were transplanted into sublethally irradiated secondary recipients. Besides a significant delay in AML development (median survival 37.5 days for recipients of MA9-Ybx1+/+ versus 79 days for MA9-Ybx1+/- BM, p=0.0042**), disease penetrance was reduced by 40%, indicating that haploinsufficiency for Ybx1 impairs development of MA9 driven AML. In contrast, immunophenotypic abundance of stem- and progenitor cells in Ybx1+/+ versus Ybx1+/- animals revealed comparable numbers in all relevant subpopulations. Serial competitive transplantation of Ybx1+/+ and Ybx1+/- BM into primary and secondary recipient animals showed no competitive disadvantage or lack of self-renewal capacity of Ybx1+/- cells.To address the question whether Ybx1 may also be essential for maintenance of AML, we used RNAi to deplete Ybx1 in already established MA9 driven AML. LSK cells from BL/6 mice transformed with MA9 were injected into primary recipient mice. After AML onset, MA9-LSK cells were sorted and infected with either one of 3 shRNAs against Ybx1 or non-targeting (NT-) control. Lentiviral knockdown of 40% reduced colony formation by more than 50% but did not limit the re-plating capacity in vitro. When injected into sub-lethally irradiated recipient mice, lentiviral knockdown (kd) of Ybx1 resulted in a significant delay in AML development (median survival 39.5 days for NT-control versus 53 days for Ybx1 kd, p=0.0446*).To validate our findings, we used a newly generated conditional ko mouse model for Ybx1, in which exon 3 coding for the cold-shock domain is deleted by activation of an Mx1-Cre-recombinase following pIpC administration. Preliminary results provide first evidence that genetic deletion of Ybx1 after onset of MA9 driven leukemia resulted in improved survival of primary recipient (median survival 73 versus 83 days) and a reduced penetrance in secondary recipient mice.Taken together our results may provide first evidence for a functional role of Ybx1 in MLL-AF9 driven AML. As Ybx1 seems to be dispensable for normal hematopoietic cells, these findings may offer a potential therapeutic index. Experiments to assess for the requirement for Ybx1 in maintenance of murine and human AML as well as analysis on proteomic and transcriptional changes following Ybx1 deletion are currently under way. DisclosuresNo relevant conflicts of interest to declare.

CEBPA mutational analysis in acute myeloid leukaemia by a laboratory-developed next-generation sequencing assay

AimThe presence of biallelic CEBPA mutations is a favourable prognostic feature in acute myeloid leukaemia (AML). CEBPA mutations are currently identified through conventional capillary sequencing (CCS). With the increasing adoption...

Effects of NPM1 gene expression on acute myeloid leukemia cell lines and its mechanism

目的探讨NPM1基因表达对急性髓系白血病（AML）细胞系的影响及其机制。方法选取AML细胞系U937和HL-60细胞，转染NPM1质粒至细胞系构建稳定克隆，采用Western blot法鉴定高表达NPM1蛋白的单克隆细胞。MTT法检测细胞增殖活性，流式细胞术检测细胞周期分布和细胞凋亡率，显微镜下计数检测集落形成能力，Western blot法检测细胞周期相关信号通路蛋白表达，实时荧光定量PCR（RQ-PCR）法检测初诊AML患者骨髓单个核细胞NPM1基因表达水平。结果①U937和HL-60细胞中NPM1高表达组相对细胞增殖率与对照组相比，差异无统计学意义（4.68±1.28对3.89±0.81，3.34±0.37对2.68±0.29，P值均>0.05）。②U937和HL-60细胞中NPM1高表达组S期细胞比例均明显高于对照组[（50.22±3.42）%对（39.78±3.80）%，（59.01±3.27）%对（43.94±2.08）%，P值均<0.05]。③U937细胞NPM1高表达组和对照组相比具有更强的抗凋亡能力[（48.67±3.22）%和（68.77±10.21）%，P<0.05]和集落形成能力（772.7±24.0和652.3±16.5，P<0.05），而HL-60细胞相应的两组细胞上述能力均相似。④NPM1高表达组细胞中CDK4、Cyclin D1、Cyclin D2及Cyclin E表达明显高于对照组，而Cyclin D3表达明显低于对照组。⑤细胞遗传学预后良好组AML患者NPM1定量水平低于预后中等组。结论NPM1蛋白能够促进更多的细胞进入S期，增强抗凋亡和细胞集落形成能力。NPM1定量水平可能预示细胞遗传学的危险度。

The study of expression and prognostic value of CD123 in acute myeloid leukemia bone marrow blasts

目的观察CD123在急性髓系白血病（AML）患者中的表达，探讨其与AML疗效和预后的关系。方法回顾性分析第二军医大学长海医院137例初诊AML（M3除外）患者骨髓异常细胞群中CD123的表达情况，初诊时多参数流式细胞术检测CD123表达占骨髓异常细胞群≥20%定义为阳性，对CD123+与CD123−患者疗效和预后进行比较分析。结果①137例AML患者中CD123+组84例（61.3%），CD123−组53例（38.7%）。所有病例根据细胞遗传学及分子生物学异常进行预后等级分层后，预后良好、预后中等、预后不良患者CD123表达差异无统计学意义（χ2=0.861，P=0.650）。而与CD123−组相比，CD123+组AML患者初诊时WBC更高[47.7（1.0~264.0）对22.4（0.7~211.0），z=−2.592，P=0.010]。②CD123+组首次诱导完全缓解（CR1）率为54.8%（46/84），CD123−组为73.6%（39/53），差异有统计学意义（χ2=5.121，P=0.027），最优疗效达完全缓解的111例患者共有52例复发，总复发率为46.8%，CD123+组与CD123−组复发率分别为50.8%（32/63）对41.7%（20/48），差异无统计学意义（χ2=0.911，P=0.340）。③CD123+组与CD123−组患者的中位总生存（OS）时间分别为20.000（95%CI 13.1~26.9）个月和44.0（95%CI 23.6~47.3）个月（χ2=5.874，P=0.015）；中位无病生存（DFS）时间分别为7.8（95%CI 1.4~14.1）个月和18.6（95%CI 0~39.7）个月，差异无统计学意义（χ2=2.939，P=0.086）。④在预后中等组及年龄≤ 50岁组中，CD123+患者OS及DFS均较CD123−患者差。结论CD123在AML患者中广泛表达，是影响患者CR1及OS的独立危险因素，对评估AML患者诱导化疗反应及生存预后具有重要意义。

Methylation Status and mRNA Expression of DKK-3 and WIF-1 in Acute Myeloid Leukemia Patients

To analyze the promoter methylation status, mRNA expression and clinical significance of DKK-3 and WIF-1 genes in patients with acute myeloid leukemia(AML). Methylation specific polymerase chain reaction (MS-PCR) mothod was carried out to detect DKK-3 and WIF-1 gene promoter methylation status in bone marrow specimen from 56 patients with AML and 20 patients with iron deficiency anaemia(IDA) as control; then the real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to detect mRNA expression of DKK-3, WIF-1 gene and β -catenin in the above-mentioned specinens, and their relationship with the clinical features and survival time was analyzed. The promoter methylation rate of DKK-3 and WIF-1 gene in AML patients were significantly higher than that in control group(χ2=15.330,P<0.001; χ2=17.371,P<0.001). There was no relationship between DKK-3 and WIF-1 gene promoter methylation rate and AML patient's sex, age, clinical typing. The relative expression of DKK-3 and WIF-1 gene mRNA in AML group were 0.840±0.320 and 0.792±0.313, which were lower than those in control group (1.134±0.392 and 1.047±0.334) respectively, the difference was statistically significant (t=3.415,P=0.000; t=3.070, P=0.003). The relative expression of β-catenin mRNA in AML bone marrow specimens in AML group was 0.756±0.304, which was higher than that in control group(0.342±0.105), the difference was statistically significant (t=5.943, P=0.001). The expression of DKK-3 and WIF-1 gene mRNA negatively correlated with β-catenin mRNA(r=-0.543; r=-0.562). Kaplan Meier survival curve analysis showed that overall survival time in AML patients with DKK-3 gene methylation was shorter than that in the AML patients with DKK-3 gene unmethylation(χ2=3.957, P=0.042). Futhermore, the orerall survival time in AML patients with WIF-1 gene methylation was also shorter than that in AML patients with WIF-1 gene unmethylation (χ2=4.520, P=0.029). Wnt/β-catenin signaling pathway is abnormally activated in AML patients, the DKK-3 and WIF-1 gene promoter methylation may be involved in Wnt pathways activation and the pathogenesis of AML.

Abstract 5118: A novel polyamine sulfonamide with anti-leukemic activity

Abstract The greatest challenge in treating Acute Myeloid Leukemia (AML) is refractory disease. Although 60-80% of AML patients achieve complete remission after induction chemotherapy, majority of these patients relapse and die of progressive disease. New treatment options targeting the vulnerabilities of AML biology are highly critical for disease regression, in particular the chemo-resistant leukemia-initiating population, to ensure relapse-free survival in patients. Identifying novel druggable targets that are selective to AML despite their location in the vascular niche is thus highly warranted. We developed an in vitro co-culture model consisting of AML cells and Bone Marrow- derived Endothelial Cells (BMECs), mimicking the protective effect of the vascular niche in the bone marrow microenvironment. Using this unique model, and taking advantage of combinatorial chemistry, we performed high-throughput chemical-phenotypic screening of approximately 30 million novel compounds and identified a novel polyamine sulfonamide 2470-51 that can selectively kill AML cells by overcoming the protective effect of the BMECs. In addition to AML blasts, 2470-51 also exhibited highly selective activity against the leukemic stem and progenitor cell population while sparing normal hematopoietic stem and progenitor cells. In vivo studies using patient-derived xenograft models indicated significant regression in AML engraftment post 2470-51 treatment. Target identification experiments involving unbiased label-free shotgun proteomic analysis in combination with targeted Selected Ion Monitoring (SIM) revealed covalent drug binding targets of 2470-51. By performing differential protein expression analysis using ITRAQ analysis, we identified downstream mechanisms that led to mitotic cell cycle arrest and cell death. Collectively, our findings display the in vitro as well as in vivo efficacy of a novel polyamine sulfonamide in eliminating AML, including the leukemia initiating compartment. We also uncover a novel mechanism in AML that can be taken advantage of for selective toxicity. Furthermore, we establish the role of 2470-51 as a potential therapeutic agent in treating AML. Citation Format: Vindhya Vijay, Amy Meacham, Leylah Drusbosky, Elizabeth Wise, Christopher Cogle. A novel polyamine sulfonamide with anti-leukemic activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5118. doi:10.1158/1538-7445.AM2017-5118

Abstract LB-290: Oncometabolites induce a BRCAness state that can be exploited by PARP inhibitors

Abstract 2-Hydroxyglutarate (2HG) exists as two enantiomers, R-2HG and S-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase-1 and -2 (IDH1/2) mutations, while the latter is produced under pathologic process such as hypoxia. Recurring IDH1/2 mutations were first identified gliomas and acute myeloid leukemia (AML), and subsequently they were found in multiple other tumor types. Many IDH1/2-mutant tumors are known to be chemo- and radiosensitive, although the mechanisms underlying this enhanced sensitivity have been elusive. Here, we report that IDH1/2 mutations induce a homologous recombination (HR) defect which renders tumor cells exquisitely sensitive to Poly (ADP-Ribose) polymerase (PARP) inhibitors. Remarkably, this “BRCAness” phenotype can be completely reversed by small molecule mutant IDH1/2 inhibitors, and it can be entirely recapitulated by treatment with either 2HG enantiomer in cells with intact IDH1/2. We performed a comprehensive series of studies directly implicate two αKG-dependent dioxygenases, KDM4A and KDM4B, as key mediators of the observed phenotype. In addition, we demonstrate that 2HG-induced HR suppression cannot be explained by mutant IDH1/2-associated alterations in NAD+ levels. We have demonstrated IDH1/2-dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells and AML bone marrow cultures in vitro, as well as genetically-matched tumor xenografts in vivo. Finally, we have extended these findings to several structurally related and clinically relevant oncometabolites. We demonstrate profound synthetic lethality with PARP inhibitors in tumors which produce these other oncometabolites, and our data suggest a similar mechanism of action via which HR is suppressed. Small molecule inhibition of oncogenic kinases is a pillar of precision medicine in modern oncology, and this approach has been extrapolated to treat IDH1/2-mutant and other oncometabolite-producing cancers with small molecule inhibitors which block the neomorphic activity of the mutant proteins. The findings present here directly challenge this therapeutic strategy, and they instead provide a novel approach to treat these tumors oncometabolite-producing tumors with DNA repair inhibitors. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair and genetic instability. We previously reported that hypoxia suppresses HR, driving genetic instability and conferring a BRCAness phenotype in hypoxic tumor cells. It is tempting to speculate that the findings reported here provide a novel commonality between hypoxia and IDH1/2 mutations as mediating a “hit-and-run” mechanism for genetic instability and tumor progression through 2HG, but at the same time bestowing a vulnerability to PARP inhibition that can be therapeutically exploited. Based on these findings, we are planning a multi-center Phase II trial testing the efficacy of olaparib for the treatment of recurrent IDH1/2-mutant tumors, and we anticipate this trial will be open for enrollment later this year. Citation Format: Parker Sulkowski, Christopher Corso, Nathaniel Robinson, Susan Scanlon, Karin Purshouse, Hanwen Bai, Yanfeng Liu, Ranjini Sundaram, Denise Hegan, Nathan Fons, Gregory Breuer, Yuanbin Song, Ketu Mishra, Henk De Feyter, Robin de Graaf, Yulia Surovtseva, Maureen Kachman, Stephanie Halene, Murat Gunel, Peter Glazer, Ranjit S. Bindra. Oncometabolites induce a BRCAness state that can be exploited by PARP inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-290. doi:10.1158/1538-7445.AM2017-LB-290

An atlas of bloodstream-accessible bone marrow proteins for site-directed therapy of acute myeloid leukemia.

The concept of arming antibodies with bioactive payloads for a site-specific therapy of cancer has gained considerable interest in recent years. However, a successful antibody-based targeting approach critically relies on the availability of a tumor-associated target that is not only preferentially expressed in the tumor tissue but is also easily accessible for antibody therapeutics coming from the bloodstream. Here, we perfused the vasculature of healthy and acute myeloid leukemia (AML)-bearing rats with a reactive ester derivative of biotin and subsequently quantified the biotinylated proteins to identify AML-associated bone marrow (BM) antigens accessible from the bloodstream. In total, >1400 proteins were identified. Overall, 181 proteins were >100-fold overexpressed in AML as compared with normal BM. Eleven of the most differentially expressed proteins were further validated by immunohistochemistry and confocal microscopic analyses, including novel antigens highly expressed in AML cells (for example, adaptor-related protein complex 3 β2) and in the leukemia-modified extracellular matrix (ECM) (for example, collagen-VI-α-1). The presented atlas of targetable AML-associated BM proteins provides a valuable basis for the development of monoclonal antibodies that could be used as carriers for a site-specific pharmacodelivery of cytotoxic drugs, cytokines or radionuclides to the BM in AML.

Specific Depletion of Leukemic Stem Cells: Can MicroRNAs Make the Difference?

For over 40 years the standard treatment for acute myeloid leukemia (AML) patients has been a combination of chemotherapy consisting of cytarabine and an anthracycline such as daunorubicin. This standard treatment results in complete remission (CR) in the majority of AML patients. However, despite these high CR rates, only 30–40% (<60 years) and 10–20% (>60 years) of patients survive five years after diagnosis. The main cause of this treatment failure is insufficient eradication of a subpopulation of chemotherapy resistant leukemic cells with stem cell-like properties, often referred to as “leukemic stem cells” (LSCs). LSCs co-exist in the bone marrow of the AML patient with residual healthy hematopoietic stem cells (HSCs), which are needed to reconstitute the blood after therapy. To prevent relapse, development of additional therapies targeting LSCs, while sparing HSCs, is essential. As LSCs are rare, heterogeneous and dynamic, these cells are extremely difficult to target by single gene therapies. Modulation of miRNAs and consequently the regulation of hundreds of their targets may be the key to successful elimination of resistant LSCs, either by inducing apoptosis or by sensitizing them for chemotherapy. To address the need for specific targeting of LSCs, miRNA expression patterns in highly enriched HSCs, LSCs, and leukemic progenitors, all derived from the same patients’ bone marrow, were determined and differentially expressed miRNAs between LSCs and HSCs and between LSCs and leukemic progenitors were identified. Several of these miRNAs are specifically expressed in LSCs and/or HSCs and associated with AML prognosis and treatment outcome. In this review, we will focus on the expression and function of miRNAs expressed in normal and leukemic stem cells that are residing within the AML bone marrow. Moreover, we will review their possible prospective as specific targets for anti-LSC therapy.

High CD123 levels enhance proliferation in response to IL-3, but reduce chemotaxis by downregulating CXCR4 expression.

High expression of the α chain of the interleukin-3 receptor (IL-3Rα; CD123) is a hallmark of acute myeloid leukemia (AML) leukemic stem cells (LSCs). Elevated CD123 expression is part of the diagnostic immunophenotyping of myeloid leukemia, and higher expression is associated with poor prognosis. However, the biological basis of the poorer prognosis is unclear, and may include heightened IL-3 signaling and non-cell autonomous interactions with the bone marrow (BM) microenvironment. We used TF-1 cells expressing different levels of CD123 and found elevated CD123 levels amplified the proliferative response to exogenous IL-3 and maintained viability in reducing IL-3 concentrations. This was associated with stronger activation of STAT5, Akt, and extracellular signal-regulated kinase 1/2 in vitro. Surprisingly, in vivo e14.5 fetal liver cells transduced with retroviral constructs to express high CD123 failed to engraft in syngeneic recipients. In exploring the underlying mechanism for this, we found that CXCR4, a key molecule involved in LSC/BM interactions, was specifically downregulated in CD123 overexpressing cells in a manner dependent on IL-3 signaling. CXCR4 downregulation was sufficient to alter the chemotactic response of hematopoietic cells to stromal derived factor-1 (SDF-1). Thus, we propose that the overexpression of CD123 in AML LSC dictates their location by altering CXCR4/SDF-1 interaction in the BM, raising the possibility that this mechanism underpins the egress of BM AML LSC and more mature cells into the circulation.

Prognostic value of FLT3-ITD detection on acute myelogenous leukemia by DNA extracted from stored bone marrow slides by chemical method

Objective To investigate the prognostic value of Fms-like tyrosine kinase3, intenal tandem duplication (FLT3-ITD) detection by DNA extracted from stored bone marrow slides in chemical method. Methods Trace DNA was extracted from 58 bone marrow slides which were stored for 1-5 years below 20 ℃, including 48 patients with de novo acute myeloid leukemia (AML) and 10 controls without hematologic malignancies. Polymerase chain reaction (PCR) was used to detect the FLT3-ITD of these bone marrow slides samples. Results There were 6 patients of FLT3-ITD+ detected in these 48 AML patients(12.5%, 6/48). No FLT3-ITD was found in 10 healthy controls. AML patients with FLT3-ITD+ had low complete time compared with FLT3-ITD- patients (χ2= 7.274, P= 0.007). Splenohepatomegalia and FLT3 mutation were the risk factors affecting AML patients with CR after the first chemotherapy (OR= 7.2, P= 0.12; OR= 36.3, P= 0.10). FLT3-ITD was a risk factor of poor prognosis in patients with newly diagnosed AML (RR= 9.088, P= 0.029). Conclusion Extraction of AML bone marrow slides trace DNA by using chemical method can be widely applied in clinic and is a key experimental way to study the molecular biology retrospectively. Furthermore, the detection of FLT3-ITD by trace DNA extracted from stored bone marrow slides can be used to predict the prognosis of AML. Key words: Leukemia, myeloid, acute; Fms-like tyrosine kinase 3; Internal tandem duplication; Prognosis

Single-cell genomic profiling of acute myeloid leukemia for clinical use: A pilot study.

Although bulk high-throughput genomic profiling studies have led to a significant increase in the understanding of cancer biology, there is increasing awareness that bulk profiling approaches do not completely elucidate tumor heterogeneity. Single-cell genomic profiling enables the distinction of tumor heterogeneity, and may improve clinical diagnosis through the identification and characterization of putative subclonal populations. In the present study, the challenges associated with a single-cell genomics profiling workflow for clinical diagnostics were investigated. Single-cell RNA-sequencing (RNA-seq) was performed on 20 cells from an acute myeloid leukemia bone marrow sample. Putative blasts were identified based on their gene expression profiles and principal component analysis was performed to identify outlier cells. Variant calling was performed on the single-cell RNA-seq data. The present pilot study demonstrates a proof of concept for clinical single-cell genomic profiling. The recognized limitations include significant stochastic RNA loss and the relatively low throughput of the current proposed platform. Although the results of the present study are promising, further technological advances and protocol optimization are necessary for single-cell genomic profiling to be clinically viable.

Chemopreventive Potential of Major Flavonoid Compound of Methanolic Bark Extract of Saraca asoca (Roxb.) in Benzene-induced Toxicity of Acute Myeloid Leukemia Mice.

Background:Saraca asoca (SA) (Roxb.) is one of the folk medicinal plants found in India, Bangladesh, and Sri Lanka. Its major biological activity appears due to the presence of flavonoid group of compounds in its bark extract.Objective:In this study, our research aims to analyze the chemopreventive effect of flavonoids, especially a natural phenol catechin present in the bark methanolic extract of SA on acute myeloid leukemia (AML) mice.Materials and Methods:The total bark extract was partitioned and analyzed on thin-layer chromatography (TLC) plate. The yellow-brown material of spot 4 was analyzed and identified as catechin. The yellowish brown material (YBM) was tested for their chemopreventive potential. An in vivo AML mice model was used to test the efficacy. Hematological parameters (Hb %, red blood cell, and white blood cell count), expression of cell cycle regulatory proteins, and DNA fragmentation analysis were performed.Results:After treatment of benzene-exposed mice with the major flavonoid compound, namely catechin, the above parameters increase significantly (P < 0.05). There was an upregulation of p53 and p21, caspase 11 myeloperoxidase, bcl2, and CYP2EI in catechin-treated group. DNA was less fragmented in flavonoid-treated group compared to that of control (P ≤ 0.05). The present study indicates that the secondary metabolites of SA methanolic bark extract, comprising flavonoid catechin as major constituents, have modulatory effect in cell cycle deregulation and hematological abnormalities induced by benzene in mice.Conclusions:Our data suggest that catechin from methanolic bark extract of SA effectively attenuates benzene-induced secondary AML in bone marrow, which is likely associated with the anticell cycle deregulation properties of this flavan-3-ol. This study was supported by the observation that catechin (YBM), like doxorubicin, can act as the neutralizer and protector of mortality in cancer cases.SUMMARY The catechin from methanolic bark extract of Saraca asoca has chemoprotective activity in benzene-induced secondary acute myeloid leukemia.(AML) in bone marrowHematological parameters, structural analysis of DNA showed that the purified catechin attenuates the conditions responsible for the development of AMLThe purified flavonol, catechin has a modulatory effect on different cell cycle deregulations induced by benzene in AML model.

Ex Vivo Activity Profile of the CD123xCD3 Duobody® Antibody JNJ-63709178 Against Primary Acute Myeloid Leukemia Bone Marrow Samples

Background/Aims:Acute myeloid leukemia (AML) is a hematologic malignancy that affects the normal production of neutrophils, red blood cells, and platelets. CD123 (IL-3 receptor alpha) is over-expressed on AML stem cells and blasts, compared with normal hematopoietic progenitor and stem cells, and represents a promising target for the development of antibody-based AML therapies (Jordan et al., Leukemia 2000;14:1777-1784).The outcome for patients with AML remains poor, and effective therapeutics are needed in this patient population. JNJ-63709178 is a promising new bispecific antibody that recruits T cells to tumor cells through a tumor-specific antigen-binding arm, CD123, and a T-cell-specific CD3 arm. JNJ-63709178 is a humanized antibody on an IgG4 scaffold with potent in vitro and in vivo T-cell-mediated killing of CD123-positive leukemic cells. The Fc domain of JNJ-63709178 was also engineered to diminish binding to Fcγ receptors (to reduce the potential for nonselective T-cell activation), yet preserve binding to human FcRn to maintain a long serum half-life. JNJ-63709178 is currently being explored in a Phase 1 study in patients with relapsed or refractory AML.Methods: To evaluate the ex vivo effects of this class of bispecific antibodies on primary samples from patients with AML (15 newly diagnosed, 1 previously treated and 1 unknown), patient bone marrow samples were collected and received within 24-48 hours at Vivia Biotech and analyzed by Vivia's proprietary ExviTech flow cytometry system. This platform preserves the whole bone marrow native environment by retaining all cell types, matrix, and proteins important for long-term growth of primary human bone marrow and blood. CNTO 9958, a close analog of JNJ-63709178 with similar affinities for CD123 and CD3 and functionally similar to JNJ-63709178, was utilized for these studies. Patient samples were evaluated for blast count, CD123 quantification on blast cells (QuantiBRITE PE beads, Beckton Dickinson), T-cell count and activation markers (CD25, ICOS, and PD1), and effector-to-target (E:T) ratios were determined. Following 4 to 120 hours of culturing with CNTO 9958, the bone marrow aspirate was extensively profiled by an automated multi-parameter flow cytometry system at Vivia Biotech. Growth and depletion of leukemic blasts and T-cell subsets were monitored upon CNTO 9958 antibody treatment and expansion and activation of CD4+ and CD8+T-cells were measured at multiple time points.Results: CD123 expression was detected in 16 of 17 primary AML bone marrow samples examined, and expression ranged from ~1300 to ~24000 receptors/cell on AML blasts. Blast cell depletion was measured in the same 17 primary AML bone marrow samples across different time points and after incubating with varied concentrations of CNTO 9958. CNTO 9958 was able to effectively reduce blasts levels with EC50 <0.5 µg/mL (median EC50: 0.11 µg/mL, range: 0.001-500 µg/mL) at 72 hours’ incubation in 14 of 16 samples. Measurement of T-cell subtypes showed proliferation of both CD4+ and CD8+T cells as well as increased activation (CD25+ and ICOS+) on effector T cells. There was a correlation between proliferation of cytotoxic T cells and blast depletion. There was no correlation between effect and CD123 level. E:T ratios as low as 1:54.3 (range 1:2.6-1:54.3) resulted in efficient depletion of leukemic blasts with CNTO 9958. Baseline activation of T cells (CD8+CD25+) was observed to correlate with the efficacy of CNTO 9958 on blast depletion.Conclusions: Vivia Biotech's ExviTech flow cytometry system provides a biologically relevant platform to evaluate patient bone marrow samples ex vivo, preserving the tumor microenvironment of the bone marrow sample, including blasts and white blood cells. Using this system to evaluate AML patient samples, CD123 was expressed in 16 of 17 primary AML bone marrow samples, with expression densities ranging from ~1,300 to ~24,000 receptors/cell. The CD123xCD3 bispecific antibody CNTO 9958 was able to recruit and activate endogenous patient T cells to efficiently kill CD123-positive AML cells in a time- and dose-dependent manner, even at the very low E:T ratios observed and irrespective of CD123 receptor density in AML patient samples. These results support clinical evaluation of JNJ-63709178 in patients with AML. DisclosuresForslund:Janssen Pharmaceuticals Research and Development: Employment. Syed:Janssen Pharmaceuticals Research and Development: Employment. Axel:Janssen Pharmaceuticals Research and Development: Employment. McDaid:Janssen Pharmaceuticals Research and Development: Employment. Li:Janssen: Employment. Ballesteros:Vivia Biotech: Employment, Equity Ownership. Gaudet:Janssen Pharmaceuticals R&D: Employment, Other: Stock options, Patents & Royalties: pending, not yet issued. Attar:Janssen: Employment. Salvati:Janssen Pharmaceuticals R&D: Employment, Other: stock options, Patents & Royalties: patent. Huang:Janssen Research & Development, LLC: Employment, Other: I am an employee of Janssen and a stock owner . Sasser:Johnson & Johnson: Equity Ownership; Janssen Pharmaceuticals R&D: Employment.

Residual HSPC in the Leukemia Microenvironment Are Reprogrammed Via Extracellular Vesicle Trafficking

Several groups have shown that leukemic cells create a self-reinforcing bone marrow (BM) niche that functionally impairs normal hematopoietic stem and progenitor cells (HSPC) indirectly through stroma-secreted factors. We recently demonstrated an alternative mechanism whereby extracellular vesicles (EVs) from acute myeloid leukemia (AML) patients and cell lines, but not BM CD34 controls, suppress their clonogenicity through EV trafficking of microRNA that directly downregulate critical transcription factors (c-Myb and HoxA9). Here, we aimed to clarify the fate of residual HSPC in in vivo AML xenografts, as well as ex vivo intrafemural (IF) injection and in vitro exposure of EVs experiments. Among KSL cells we observed a significant increase in the frequency of the long-term hematopoietic stem cell (SLAM, CD150+CD48−) subpopulation, but not the multipotent progenitors even at low levels of AML infiltration or direct IF injection of EVs. The HSPC pool redistribution was accompanied by cell cycle alterations in residual HSPC that showed AML EVs consistently induced quiescence (G0) in KSL (cKit+Sca1+Lin−) HSPC populations. When we assessed their DNA damage, residual HSPC showed a distinct increase in the gH2AX foci relative to control non-engrafted mice as well as the transcriptional upregulation of Rad51 and P21 genes along with gains in phosphorylation of the tumor suppressor p53. Yet, the reprogrammed KSL showed no evidence of apoptosis indicated by the lack of upregulation of the p53 target, Puma, and Annexin V staining, nor evidence of senescence (P16 and Sparc transcripts). To gain additional insight, we performed a tandem mass tag (TMT) proteomic profiling of AML-EV exposed HSPC with or without exposure to EVs derived from AML cells. The results showed significant enrichment of DNA methylation regulatory pathway such as DNMT1, HELLS and UHRF1 as well as inflammatory pathways including IL1b, NOS, CEBPB and NFkB pathway-targets, confirmed by transcriptional profiling of KSL from xeno-transplanted mice. Based on our recent report that miR-1246 is one of the most highly enriched miRNA in AML derived EVs and proceeded to determine its target transcripts using an attenuated RISC complex (RISC-Trap), followed by high-throughput sequencing. Bioinformatics analysis identified a set of 27 miR-1246-specific targets relative to control microRNAs. Strikingly, the target set was selectively enriched for a panel of negative cell-cycle regulator genes (CDK1, CDK7, CDK11, CCNF, HDAC2 and GATA3) as well as the DNA methylation regulators (DNMT1 and HELLS).Collectively, our results demonstrated that residual HSPC in the AML BM are phenotypically reprogrammed and suppressed in their proliferation along with DNA damage accumulation via paracrine EV microRNA trafficking. Our study provides insight into HSPC fates in the AML niche and echoes observations of cell competition, as a mode of non-cell autonomous regulation where p53 activation in the reprogrammed cells leads to a progressive decline in proliferation and fitness. We propose that AML EV trafficking of miR-1246 specifically may contribute to the altered fate of residual HSPC via transcriptional regulation of proliferation-related genes. Disclosures No relevant conflicts of interest to declare.