Survival In Acute Myeloid Leukemia Research Articles

Immunomodulatory Drugs Exert Anti-Leukemia Effects in Acute Myeloid Leukemia by Direct and Immunostimulatory Activities.

Immunomodulatory drugs (IMiDs) are anticancer drugs with immunomodulatory, anti-angiogenesis, anti-proliferative, and pro-apoptotic properties. IMiDs are currently used for the treatment of multiple myeloma, myelodysplastic syndrome, and B-cell lymphoma; however, little is known about efficacy in acute myeloid leukemia (AML). We proposed in this study to investigate the relevance of IMiDs therapy for AML treatment. We evaluated the effect of IMiDs on primary AML blasts (n = 24), and the impact in natural killer (NK) cell-mediated immunosurveillance of AML. Using primary AML cells and an immunodeficient mouse leukemia xenograft model, we showed that IMiDs induce AML cell death in vitro and impair leukemia progression in vivo. In addition, treatment of AML blasts with IMiDs resulted in enhanced allogeneic NK cell anti-leukemia reactivity. Treatment by pomalidomide of AML blasts enhanced lysis, degranulation, and cytokine production by primary allogeneic NK cells. Furthermore, the treatment with lenalidomide of patients with myeloid malignancies resulted in NK cell phenotypic changes similar to those observed in vitro. IMiDs increased CD56 and decreased NKp30, NKp46, and KIR2D expression on NK cells. Finally, AML blasts treatment with IMiDs induced phenotypic alterations including downregulation of HLA-class I. The effect of pomalidomide was not correlated with cereblon expression and A/G polymorphism in AML cells. Our data revealed, a yet unobserved, dual effects on AML affecting both AML survival and their sensitivity to NK immunotherapy using IMiDs. Our study encourages continuing investigation for the use of IMiDs in AML, especially in combination with conventional therapy or immunotherapy strategies.

Methylation-associated DOK1 and DOK2 down-regulation: Potential biomarkers for predicting adverse prognosis in acute myeloid leukemia.

DOK-1 and DOK-2 (DOK1/2) are closely related members of downstream of tyrosine kinase (DOK) family genes, which are found to be frequently rearranged in several hematopoietic cancers. However, the clinical implications of DOK1/2 in acute myeloid leukemia (AML) remain largely unknown. To investigate the clinical significance, real-time quantitative PCR (RQ-PCR) was carried out to detect DOK1/2 expressions in 125 de novo AML patients and 28 healthy controls. Real-time quantitative methylation-specific PCR (RQ-MSP) and bisulfite sequencing PCR (BSP) were applied to detect DOK1/2 methylation level and density. DOK1/2 expressions were significantly down-regulated in AML patients. The promoters of DOK1/2 were highly hypermethylated and negatively correlated with DOK1/2 expressions in AML patients. In addition, we also confirmed that DOK1/2 expressions could be restored by DOK1/2 demethylation using 5-aza-2'-deoxycytidine in leukemia cell line THP-1. Survival analyses showed that low-expressed DOK1/2 were associated with markedly shorter overall survival and leukemia free survival in both whole-cohort AML and non-M3 AML patients. Multivariate analyses further revealed that DOK1/2 were act as independent prognostic factors in AML patients. These findings indicate that decreased DOK1/2 expressions associated with their promoter hypermethylations predict adverse prognosis in AML.

HIF1α drives chemokine factor pro-tumoral signaling pathways in acute myeloid leukemia

Approximately 80% of patients diagnosed with acute myeloid leukemia (AML) die as a consequence of failure to eradicate the tumor from the bone marrow microenvironment. We have recently shown that stroma-derived interleukin-8 (IL-8) promotes AML growth and survival in the bone marrow in response to AML-derived macrophage migration inhibitory factor (MIF). In the present study we show that high constitutive expression of MIF in AML blasts in the bone marrow is hypoxia-driven and, through knockdown of MIF, HIF1α and HIF2α, establish that hypoxia supports AML tumor proliferation through HIF1α signaling. In vivo targeting of leukemic cell HIF1α inhibits AML proliferation in the tumor microenvironment through transcriptional regulation of MIF, but inhibition of HIF2α had no measurable effect on AML blast survival. Functionally, targeted inhibition of MIF in vivo improves survival in models of AML. Here we present a mechanism linking HIF1α to a pro-tumoral chemokine factor signaling pathway and in doing so, we establish a potential strategy to target AML.

High PARP-1 Expression Predicts Poor Survival in Acute Myeloid Leukemia and PARP-1 Inhibitor and Saha-Bendamustine Hybrid Inhibitor Combination Treatment Synergistically Enhances Anti-Tumor Effects

PARP-1 plays a critical role in DNA damage repair and contributes to progression of cancer. To address the role of PARP-1 in AML, we analyzed the expression of PARP-1 in acute myeloid leukemia (AML) using RT-PCR. We found high expressers had higher levels of blast cells in bone marrow (P=0.003) and WBC in peripheral blood (P=0.008) and were associated with a more frequent FLT3-ITD mutation (28.2% vs 17.3%, P=0.031). The overall survival (OS) and event free survival (EFS) of the high expression group were significantly shorter than those of the low expression group (P=0.005 and P=0.004, respectively). High PARP-1 expression predicts poor survival in AML patients. Then, we investigated the efficacy and mechanism of PARP inhibitor BMN673 (Talazoparib) combined with, NL101, a novel SAHA-bendamustine hybrid in vitro and in vivo. The combination significantly enhanced apoptosis of AML cells and induced G2/M cell cycle arrest. Mechanistically, BMN673 and NL101 combinatorial treatment promoted DNA damage. In vivo, the combination effectively delayed the development of AML and prolonged survival. The synergistic effects of PARP inhibitor BMN673 in combination with SAHA-bendamustine hybrid, NL101, provide a new therapeutic strategy against AML. Funding: This work was supported by the National Natural Science Foundation of China (Grant number: 81370643, 81470305, and 81670124) and Zhejiang Provincial Key Innovation Team (Grant number: 2011R50015). Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: This study was approved by the Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University (Hangzhou, China). Informed consent was obtained from all patients according to institutional guidelines.

An unexpected protein interaction promotes drug resistance in leukemia

The overall survival of patients with acute myeloid leukemia (AML) is poor and identification of new disease-related therapeutic targets remains a major goal for this disease. Here we show that expression of MPP1, a PDZ-domain-containing protein, highly correlated with ABCC4 in AML, is associated with worse overall survival in AML. Murine hematopoietic progenitor cells overexpressing MPP1 acquired the ability to serially replate in methylcellulose culture, a property crucially dependent upon ABCC4. The highly conserved PDZ-binding motif of ABCC4 is required for ABCC4 and MPP1 to form a protein complex, which increased ABCC4 membrane localization and retention, to enhance drug resistance. Specific disruption of this protein complex, either genetically or chemically, removed ABCC4 from the plasma membrane, increased drug sensitivity, and abrogated MPP1-dependent hematopoietic progenitor cell replating in methylcellulose. High-throughput screening identified Antimycin A as a small molecule that disrupted the ABCC4–MPP1 protein complex and reversed drug resistance in AML cell lines and in primary patient AML cells. In all, targeting the ABCC4–MPP1 protein complex can lead to new therapies to improve treatment outcome of AML, a disease where the long-term prognosis is poor.

Identification of miRNA-mRNA Network Associated with Acute Myeloid Leukemia Survival.

BackgroundAcute myeloid leukemia (AML) is a common hematologic malignancy of adults. The pathophysiological mechanism of AML is not well understood. The purpose of this study was to examine the crucial miRNAs and mRNAs associated with AML survival.Material/MethodsThe full clinical dataset of miRNA and mRNA expression profiling of AML patients was downloaded from The Cancer Genome Atlas database. Univariate Cox regression analysis was performed to obtain those miRNAs and mRNAs associated with AML survival. A miRNA-mRNA interaction network was constructed. The underlying functions of mRNAs were predicted through Kyoto Encyclopedia of Genes and Genomes (KEEG) pathway enrichment. The expression levels of miRNAs and mRNAs were detected by quantitative real-time polymerase chain reaction (qRT-PCR).ResultsFourteen miRNAs and 830 mRNAs associated with AML survival were identified. Of the 14 miRNAs, hsa-mir-425, hsa-mir-1201, and hsa-mir-1978 were identified as risk factors and the other 11 miRNAs were identified as protective factors of AML survival. For target-genes of miRNAs, GTSF1, RTN4R, and CD44 were the top risk factor target-genes associated with AML survival. An interaction network was constructed that including 607 miRNA-target gene pairs associated with AML survival. Target-genes associated with AML survival were significantly enriched in several pathways including pancreatic secretion, calcium signaling pathway, natural killer cell mediated cytotoxicity, and Alzheimer’s disease. The qRT-PCR results were consistent with our bioinformatics analyses.ConclusionsThe miRNA hsa-mir-425 was identified as the top risk factor miRNA of AML survival and CD44 was identified as one of the top three risk factor target-genes associated with AML survival. Both hsa-mir-425 and CD44 may play key roles in progression and development of AML through calcium signaling pathway and natural killer cell mediated cytotoxicity.

Tumor SHB gene expression affects disease characteristics in human acute myeloid leukemia

The mouse Shb gene coding for the Src Homology 2-domain containing adapter protein B has recently been placed in context of BCRABL1-induced myeloid leukemia in mice and the current study was performed in order to relate SHB to human acute myeloid leukemia (AML). Publicly available AML databases were mined for SHB gene expression and patient survival. SHB gene expression was determined in the Uppsala cohort of AML patients by qPCR. Cell proliferation was determined after SHB gene knockdown in leukemic cell lines. Despite a low frequency of SHB gene mutations, many tumors overexpressed SHB mRNA compared with normal myeloid blood cells. AML patients with tumors expressing low SHB mRNA displayed longer survival times. A subgroup of AML exhibiting a favorable prognosis, acute promyelocytic leukemia (APL) with a PMLRARA translocation, expressed less SHB mRNA than AML tumors in general. When examining genes co-expressed with SHB in AML tumors, four other genes ( PAX5, HDAC7, BCORL1, TET1) related to leukemia were identified. A network consisting of these genes plus SHB was identified that relates to certain phenotypic characteristics, such as immune cell, vascular and apoptotic features. SHB knockdown in the APL PMLRARA cell line NB4 and the monocyte/macrophage cell line MM6 adversely affected proliferation, linking SHB gene expression to tumor cell expansion and consequently to patient survival. It is concluded that tumor SHB gene expression relates to AML survival and its subgroup APL. Moreover, this gene is included in a network of genes that plays a role for an AML phenotype exhibiting certain immune cell, vascular and apoptotic characteristics.

Receipt of Initial Chemotherapy for Newly Diagnosed Acute Myeloid Leukemia (AML)

The use of chemotherapy can improve quality of life and survival in AML; however, many newly diagnosed patients do not receive any chemotherapy. Non-biological factors may influence the utilization of chemotherapy and can impact quality of care.

Abstract 3123: Identification of TIFA as a novel therapeutic target in acute myeloid leukemia

Abstract Aurora A-dependent NF-κB signaling portends poor prognosis of cancers including acute myeloid leukemia (AML). Our previous study demonstrated that phosphorylation-dependent oligomerization of TRAF-interacting protein with FHA domain (TIFA) triggers the activation of NF-κB. The present study identifies that Aurora A is an essential kinase for the Thr9 phosphorylation of TIFA, and that TIFA functionally mediates the Aurora A-driven NF-κB survival pathway in AML. Overexpression of TIFA occurred concurrently with Aurora A and NF-κB signaling factors in de novo AML patients but not healthy individuals, and also correlated with poor prognosis. Silencing of TIFA specifically attenuated leukemic cell growth and enhanced chemosensitivity of AML cells via down-regulation of pro-survival factors Bcl-2 and Bcl-XL that support NF-κB-dependent anti-apoptotic events. In addition, molecular targeting of TIFA perturbed leukemic cytokine secretion and significantly lowered the IC50 of chemotherapeutic drugs to treat AML cells. Furthermore, in vivo delivery of TIFA-inhibitory fragments potentiates the clearance of leukemic myeloblasts in the bone marrow of xenograft-recipient mice via enhanced chemotoxicity, similar to the effect of anti-inflammatory drug treatments. Collectively, we proposed that TIFA functionally supports the positive feedback between TNF-α, Aurora A, and NF-κB to facilitate AML survival signaling, and impairment in this pathway can enhance the efficacy of AML treatments. Citation Format: Pei-Yu Wu, Tong-You Wade Wei, Ting-Jung Wu, Ming-Daw Tsai. Identification of TIFA as a novel therapeutic target in acute myeloid leukemia [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 3123. doi:10.1158/1538-7445.AM2017-3123

Azelaic Acid Exerts Antileukemic Activity in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an acute leukemia common in most adults; its prevalence intensifies with age. The overall survival of AML is very poor because of therapeutic resistance. Azelaic acid (AZA) is non-toxic, non-teratogenic, and non-mutagenic and its antitumor effect on various tumor cells is well established; Nonetheless, its therapeutic effects in AML cells are largely unknown. In this study, it was shown that AZA significantly inhibits the cell viability and induces apoptosis in AML cells in a dose-dependent manner. Additionally, AZA suppressed the expression of phosphorylated Akt, Jab1 and Trx, and this suppression was enhanced by treatment with Jab1 siRNA. Furthermore, AZA sensitized AML cells to Ara-c chemotherapy. The suppressive effect of AZA on tumor growth was examined in vivo by subcutaneously inoculated AML cells in a tumor model using nude mice. These findings indicate that AZA is useful as an effective ingredient in antineoplastic activity.

Targeting the Akt, GSK-3, Bcl-2 axis in acute myeloid leukemia.

Over the last few decades, there has been significant progress in the understanding of the pathogenetic mechanisms of the Acute Myeloid Leukemia (AML). However, despite important advances in elucidating molecular mechanisms, the treatment of AML has not improved significantly, remaining anchored at the standard chemotherapy regimen "3+7", with the prognosis of patients remaining severe, especially for the elderly and for those not eligible for transplant procedures. The biological and clinical heterogeneity of AML represents the major obstacle that hinders the improvement of prognosis and the identification of new effective therapeutic approaches. To date, abundant information has been collected on the genetic and molecular alterations of AML carrying prognostic significance. However, not enough is known on how AML progenitors regulate proliferation and survival by redundant and cross-talking signal transduction pathways (STP). Furthermore, it remains unclear how such complicated network affects prognosis and therapeutic treatment options, although many of these molecular determinants are potentially attractive for their druggable characteristics. In this review, some of the key STP frequently deregulated in AML, such as PI3k/Akt/mTOR pathway, GSK3 and components of Bcl-2 family of proteins, are summarized, highlighting in addition their interplay. Based on this information, we reviewed new targeted therapeutic approaches, focusing on the aberrant networks that sustain the AML blast proliferation, survival and drug resistance, aiming to improve disease treatment. Finally, we reported the approaches aimed at disrupting key signaling cross-talk overcoming resistances based on the combination of different targeting therapeutic strategies.

Leukemic blasts program bone marrow adipocytes to generate a protumoral microenvironment

AbstractDespite currently available therapies, most patients diagnosed with acute myeloid leukemia (AML) die of their disease. Tumor-host interactions are critical for the survival and proliferation of cancer cells; accordingly, we hypothesize that specific targeting of the tumor microenvironment may constitute an alternative or additional strategy to conventional tumor-directed chemotherapy. Because adipocytes have been shown to promote breast and prostate cancer proliferation, and because the bone marrow adipose tissue accounts for up to 70% of bone marrow volume in adult humans, we examined the adipocyte-leukemia cell interactions to determine if they are essential for the growth and survival of AML. Using in vivo and in vitro models of AML, we show that bone marrow adipocytes from the tumor microenvironment support the survival and proliferation of malignant cells from patients with AML. We show that AML blasts alter metabolic processes in adipocytes to induce phosphorylation of hormone-sensitive lipase and consequently activate lipolysis, which then enables the transfer of fatty acids from adipocytes to AML blasts. In addition, we report that fatty acid binding protein-4 (FABP4) messenger RNA is upregulated in adipocytes and AML when in coculture. FABP4 inhibition using FABP4 short hairpin RNA knockdown or a small molecule inhibitor prevents AML proliferation on adipocytes. Moreover, knockdown of FABP4 increases survival in Hoxa9/Meis1-driven AML model. Finally, knockdown of carnitine palmitoyltransferase IA in an AML patient-derived xenograft model improves survival. Here, we report the first description of AML programming bone marrow adipocytes to generate a protumoral microenvironment.

Prognostic Significance of the Percentage of Blasts with CD34+/CD38low/-/CD123+ Phenotype in Acute Myeloid Leukemias

To investigate the percentage of blasts with the CD34+/CD38low/-/CD123+ phenotype in de novo acute myeloid leukemia (AML) patients and analyse its correlation with prognosis. The percentage of CD34+/CD38low/-/CD123+ cells in the blast population of 148 newly diagnosed patients with AML was determined by using flow cytometry and its correlation with complete response, disease-free survival and overall survival were evaluated. The median percentage of CD34+/CD38low/-/CD123+ cells in newly diagnosed patients was 2.8% (ranged from 0.01 to 67%). The high expression of CD34+/CD38low/-/CD123+ in AML patients positively correlated with the NPM1 wild-type (χ2=5.194,P<0.05), but did not relate with the positive FLT3-ITD mutations (χ2=0.418,P>0.05). Further multivariable analysis showed that the higher expression of the CD34+/CD38low/-/CD123+ was associated with lower complete remission (P<0.05), worse disease-free survival(P<0.01) and shorter overall survival(P<0.01) in AML patients. The percentage of CD34+/CD38low/-/CD123+ cells at diagnosis significantly correlates with the response to treatment and survival. This prognostic marker may be used to rapidly identify the risk of treatment failure in clinical practice.

Azacitidine (AZA) Prolongs Overall Survival in Older Patients with Acute Myeloid Leukemia (AML) with Poor Prognostic Karyotypes Compared with Conventional Care Regimens (CCR)

Azacitidine (AZA) Prolongs Overall Survival in Older Patients with Acute Myeloid Leukemia (AML) with Poor Prognostic Karyotypes Compared with Conventional Care Regimens (CCR)

CSF1R Is Associated with Poor Overall Survival in AML and Mediates Supportive Interactions Between AML and Stromal Cells in the AML Microenvironment

Bone marrow (BM) stromal cells produce factors important for the growth and maintenance of normal hematopoietic, as well as leukemic cells. Interactions between hematopoietic and stromal cells as mediated by classic ligand-receptor, cytokine and metabolic elements help to create a supportive microenvironmental niche. Colony stimulating factor 1 (CSF1) is a cytokine produced by BM stromal cells. CSF1 exists as both secreted and membrane-bound isoforms, with the latter being predominant in the BM. CSF1 acts on the CSF1 receptor (CSF1R), which is expressed on phagocytic cells to induce their growth and differentiation into monocytes, macrophages and dendritic cells. Acute myeloid leukemia (AML) arises from mutations that lead to a block in differentiation of myeloid cell precursors (myeloblast or blast cells), the accumulation of which interferes with normal hematopoiesis. In this study, we examined the role of the CSF1-CSF1R interaction in AML as related to patient outcome and its influence on AML-stromal cell interactions.Analysis of cell surface protein levels of CSF1R by flow cytometry revealed that a subset of AML patient samples express high levels of CSF1R (CSF1Rhigh). Clinically, we found that CSF1Rhigh patients have shorter overall survival compared to patients with low CSF1R expression. To examine the CSF1-CSF1R interaction experimentally, CSF1Rhigh AML cells (patient samples and cell lines) were grown in in-vitro co-cultures with murine MS5 stromal cells engineered to overexpress the membrane-bound form of human CSF1 (MS5-hCSF1). We found that MS5-hCSF1 stromal cells display an increased growth rate, altered cytokine profile and signaling changes (namely mTOR pathway activation) compared to cells that do not express CSF1. In co-culture assays, we found that MS5-hCSF1 stromal cells enhanced the long-term growth (2 weeks) and survival of CSF1Rhigh AML cells by several fold. This growth advantage was not observed for CSF1RlowAML cells. Pre-culture of AML cells with MS5-hCSF1 stromal cells for 72 hours led to increased colony numbers in colony forming unit (CFU) assays compared to pre-culture on non-CSF1-expressing stroma. This suggests that exposure to CSF1 may enhance the clonogenic potential of AML cells. Co-culture of AML cells with MS5-hCSF1 stromal cells led to increased surface expression of the stem cell markers CD34 and CD117 (c-kit), suggestive of an immature, progenitor-like AML phenotype driven by CSF1. Furthermore, co-culture of AML and MS5-hCSF1 stromal cells led to distinct changes in the cytokine profiles of both AML and stromal cells, with increased levels of IL-8, IL-10 and IL-1 RA and RANTES in AML cells, and increased levels of IL-3, IL-6 and VEGF in stromal cells.Taken together, these results suggest that the CSF1-CSF1R interaction may be important in promoting a pro-leukemic phenotype. In particular, stromal support of AML is enhanced in a CSF1-dependent manner, with effects on AML growth, differentiation and production of signaling molecules. These effects in turn, have implications for modulating stromal cells themselves. Studying and understanding the complex interplays between AML and stromal cells as driven by the CSF1-CSF1R interaction may aid in the development of therapeutic approaches to target AML and its niche environment. DisclosuresJain:Roche Canada: Research Funding.

Hispanic Ethnicity Is Associated with Younger Age at Presentation and Worse Survival in AML

Introduction: Previous studies have suggested differences in presentation, prognostic factors and treatment outcomes between blacks and whites with acute myeloid leukemia (AML). Such differences, however, are not well described in Hispanics, who are otherwise known to have higher rates of B-ALL and APL compared to whites. This study aimed to examine the relationship of the hispanic ethnicity with non-APL AML baseline characteristics and treatment outcomes in comparison to non-Hispanic whites.Methods: All patients diagnosed with AML between the years 2000-2010 were identified within the Montefiore Medical System in the Bronx, NY. Descriptive statistics were used to compare differences in baseline patient and disease characteristics between white and hispanic subjects. Kaplan Meier survival function was utilized to compare the survival experience of patients, stratified by key prognostic factors. Cox PH multivariable models were constructed to look at ethnic differences in overall survival (OS), adjusted for clinically important and statistically significant prognostic variables.Results: 248 evaluable subjects with newly diagnosed AML were identified, among which 153 were whites (n=80) and Hispanics (n=73). Hispanics were significantly younger at presentation compared to whites [median age 59 (45-71) vs 70yrs (57-82), p<0.001]. The cytogenetic profile of AML in Hispanics (favorable 8.8%, intermediate 49.1%, high-risk 42.1%) was not significantly different when compared to Caucasians (favorable 3.8%, intermediate 58.5%, high-risk 37.7%, p=0.51) A subset of patients (n=81) had mutational testing done, that revealed a higher frequency of p53 mutations in hispanic (7/33, 21%) compared to white patients (1/25, 4%). Other mutations were not significantly different between the groups. 66% of Hispanics were treated with "7+3" induction chemotherapy, compared to only 50% of whites, due to the higher proportion of elderly AML patients in the latter group (p=0.09). Only 68% of those Hispanic AML patients induced, however, achieved complete remission compared to 76% of whites (p=0.45). The rates of allogeneic transplantation were similar in both ethnic groups (H: 12.7%, W: 11.3%, p=0.27). In multivariable Cox PH analysis, adjusted for age, cytogenetic risk group and initial treatment modality, Hispanics were noted to have significantly lower OS compared to whites (HR 1.72, 95%CI 1.02 - 2.88, p=0.04).Conclusion: In this multiethnic, multiracial, single-center, inner-city adult AML cohort, we report for the first time that even though AML in Hispanics presents at an earlier age, it is associated with worse OS. The higher frequency of deleterious p53 mutations in hispanic AML patients could explain their poorer outcomes. Ongoing mutational studies are testing larger cohorts of patients and will be reported at ASH. DisclosuresNo relevant conflicts of interest to declare.

CD99 Is Highly Expressed in Acute Myeloid Leukemia (AML) and Presents a Viable Therapeutic Target

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by clonal proliferation and block of differentiation of myeloid precursors. Overall survival for patients with AML remains dismal (<50% for younger patients and <10% for older patients) due to high relapse rate. In search for novel therapeutic targets in AML, we compared gene expression data of normal hematopoietic vs AML cells from 7 datasets (GSE13159, GSE13164, GSE7186, GSE1159, GSE995, GSE31174 and TCGA Leukemia) available on Oncomine. We identified CD99 to be significantly upregulated in AML cells compared with normal cells in all data sets with available measurements of CD99 expression (median ranking 155, p = 0.013); other genes among the top 10 genes identified in this analysis were FLT3 (median ranking 102, p<0.001) and WT1 (median ranking 120, p<0.001); both are known to play a role in AML. CD99 was significantly over-expressed (p<0.001) in 542 AML patients as compared with PBMCs from 74 healthy donors from the GSE13159 dataset. In the GSE13164 dataset, CD99 was significantly over-expressed in 257 AML patients as compared with PBMCs from 58 healthy donors. Consistently, in the GSE7186 dataset, CD99 was significantly over-expressed (p<0.001) in 23 AML patients as compared with 6 normal bone marrow samples and in the GSE1159 dataset, CD99 was significantly over-expressed (p=0.001) in 285 AML patients as compared with 5 normal bone marrow, and 3 normal blood samples. We also analyzed CD99 expression in cells obtained from 23 patients with AML and sorted according to their CD34 and CD38 expression levels (GSE3077 dataset). We found that CD99 expression was significantly higher (p<0.001) in the CD34+CD38+ and CD34+CD38- subpopulation compared with CD34-CD38- and CD34-CD38+; suggesting a possible role of CD99 in AML stem cells.Interestingly, analysis of three datasets (GSE22848, GSE6891, GSE15434) via R2: Genomics Analysis and Visualization Platform showed a correlation between CD99 expression and the presence of FLT3-ITD mutation. In the GSE22848 dataset, CD99 was significantly over-expressed (p=0.007) in 48 patients with FLT3-ITD as compared with 189 patients with FLT3 wildtype. In the GSE6891 dataset, 126 patients with FLT3-ITD had a significant over-expression of CD99 (p=0.006) as compared with 334 patients with FLT3 wildtype and in the GSE15434 dataset 90 FLT3-ITD positive patients had significantly higher levels of CD99 (p<0.001) as compared with 161 patients with the wildtype gene.CD99 (E2, MIC2), a 32-kD cell surface glycoprotein, is known to be involved in the transendothelium migration of neutrophils, T-cell adhesion, and T-cell death by a caspase-independent pathway. In cancer cells, CD99 was found to be highly expressed on the cell surface of Ewing’s sarcoma tumors and in gliomas. Importantly, CD99 expression levels were found to be correlated with tumor invasiveness and with lower survival rates. In order to examine the role of CD99 in AML, we assessed CD99 expression by flow cytometry in nine AML cell lines (KG-1, KG-1A, MOLM13, MV4-11, Kasumi-1, THP-1, NB4, U937, UOC-M1); we found CD99 to be expressed in all cell lines. To determine whether CD99 is a potential therapeutic target in AML, we treated leukemia cells with anti-CD99 mAb (mAb 0662) at 5 µg/mL and analyzed cell viability 48 hours post-treatment. We found significant decrease in cell viability; 15% in MV4-11 cells (p=0.02), 32% in MOLM13 cells (p=0.002) and 18% in THP-1 cells (p<0.001) as compared with untreated controls measured by Alamar blue assay. Furthermore, inhibiting CD99 led to a decrease in migration of MV4-11, MOLM13 and THP-1 cells when analyzed using a trans-well migration assay.In conclusion, CD99 is highly expressed in AML, and this expression is significantly higher in less differentiated leukemia cells and in patients with FLT3-ITD mutation. Functional studies using CD99 antibodies revealed a possible role of this gene in cell survival and cell migration. Further studies are needed to establish CD99 as a potential therapeutic target and further investigations are ongoing to determine the mechanism by which CD99 regulates cell survival in AML. DisclosuresNo relevant conflicts of interest to declare.

Role of Daunorubicin Dose Intensification for Induction Therapy in Acute Myeloid Leukemia Patients with FLT3-ITD Mutants

BackgroundPatients with FLT3-ITD mutated acute myeloid leukemia (AML) have generally poor survival. Recent update of ECOG trial comparing standard- vs. high-dose daunorubicin showed that daunorubicin dose intensification improved survival in AML with FLT3-ITD mutants (Blood 2016;127:1551). In subgroup analysis of our previous randomized trial, high-dose daunorubicin seemed to be more effective than idarubicin in AML patients with FLT3-ITD mutants (ASH abstract No. 2535, 2015). In this retrospective investigation, we aimed to evaluate the role of daunorubicin dose intensification for induction therapy in AML patients with FLT3-ITD mutants who were treated at a single institute.MethodsWe analyzed data from 120 patients of newly diagnosed FLT3-ITD mutated AML patients who received induction chemotherapy between January 2002 and March 2016. The regimens consisted of high-dose daunorubicin (HD-DN, 90 mg/m2/d x 3d, n=39), standard-dose daunorubicin (SD-DN, 45 mg/m2/d x 3d, n=48), or idarubicin (IDA, 12 mg/m2/d x 3d, n=33) in combination with cytarabine (200 mg/m2/d x 7d). Patients with acute promyelocytic leukemia were not included.ResultsAfter the first round of induction chemotherapy, 53 patients had persistent leukemia; 50 received the second round of induction chemotherapy consisting of daunorubicin (45 mg/m2/d x 2d) or idarubicin (8 mg/ m2/d x 2d) in addition to cytarabine (200 mg/m2/d x 5d) and 3 received other regimens. A total of 81 patients achieved CR, and the CR rates were 76.9%, 58.3%, and 69.7% in HD-DN, SD-DN, and IDA, respectively (P=0.175). The 4-year cumulative incidence of relapse (CIR) of these 81 patients was 48.8%. With the median follow-up duration of survivors of 59.9 months (range, 4.6-170.7), 4-year overall survival (OS) and event-free survival (EFS) were 57.1%/27.7%/35.7% (P=0.025) and 45.2%/23.9%/36.0% (P=0.042) in HD-DN, SD-DN, and IDA, respectively. HD-DN showed statistically higher OS (hazard ration [HR], 0.424; P=0.005) and EFS (HR, 0.497; P=0.01), and lower CIR (P=0.036) than SD-DN, while OS and EFS differences between HD-DN and IDA were not statistically significant.ConclusionDaunorubicin dose intensification for induction therapy seemed to be effective in AML patients with FLT3-ITD mutants. Further studies are needed to investigate whether HD-DN is superior to IDA in this population. Considering high relapse rate, combination strategies of daunorubicin dose intensification and targeted agents such as FLT3 inhibitors should be developed. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The High Affinity CXCR4 Inhibitor, BL-8040, Induces Apoptosis of AML Blasts and Their Terminal Differentiation By Blocking AKT/ERK Survival Signals and Downregulating BCL-2, MCL-1 and Cyclin-D1 through Regulation of Mir-15a/16-1 Expression

Background: Acute Myeloid Leukemia (AML) is a heterogeneous group of diseases characterized by uncontrolled proliferation and survival of hematopoietic stem and progenitor cells. The chemokine CXCL12 and its receptor CXCR4 are key players in the survival, bone marrow (BM) retention and the maintenance of AML blasts in their stemness state. CXCR4 overexpression is associated with poor prognosis in AML patients. Signaling activated through CXCR4 was shown to be detrimental by increasing survival of tumor cells and promoting resistance to therapy.Objective: To study the effect of the CXCR4-antagonist, BL-8040, on the survival of human AML blasts and to investigate the molecular mechanism by which inhibition of CXCR4 signaling leads to leukemia cell death.Methods: Human AML cell lines and human primary AML samples were used for in vitro studies. The in-vivo effect of BL-8040 was tested using the MV4-11, U-937, THP-1 cells and human primary AML cells engrafted in NOD scid gamma (NSG) mice.Results: We found that BL-8040 directly induced apoptosis of AML cells both in FLT3-ITD and FLT3-WT AML, in-vitro and in-vivo. BL-8040 treatment triggered mobilization of AML blasts from their protective BM microenvironment and induced their terminal differentiation, in-vitro and in-vivo. The apoptosis of AML cells induced by BL-8040 was attributed to miR-15a/miR-16-1 up-regulation resulting in down-regulation of their target genes BCL-2, MCL-1 and cyclin-D1. The increase in miR-15a/miR-16-1 levels directly induced AML cell death. Moreover, CXCR4 blockade by BL-8040 also inhibited survival signals by the ERK/AKT kinases enhancing the apoptosis effect. Survival of AML cells was found to be dependent on BCL-2 as demonstrated by the ability of the BCL-2 inhibitor, ABT-199, to induce dose dependent apoptosis in vitro. It was reported that the MCL-1 protein plays a key role in acquiring resistance to ABT-199. We found that BL-8040 synergizes with ABT-199 in inducing AML cell death. This could be attributed to the reduction of both, AKT/ERK and MCL-1 levels, by treatment with BL-8040. In addition, BL-8040 synergizes with the FLT3 inhibitor AC220 in the induction of AML cell death both in-vivo and in-vitro. The combined treatment of BL-8040 and AC220 was found to prolong survival and reduce minimal residual disease in-vivo. Interestingly, the combined treatment was also associated with a significant reduction in the expression of BCL-2 and ERK signaling.Conclusions: BL-8040 can be a potential therapeutic option in AML by targeting not only AML anchorage in the BM but also AML survival and differentiation. Our results demonstrate that BL-8040 in AML regulates the expression of miR-15a/16-1 and their target genes BCL-2, MCL-1 and cyclin-D1. Furthermore, these results indicate that the CXCR4 antagonist, BL-8040 may tip the balance toward cell death by down- regulating survival signals through miR-15a/16-1 pathway and inhibition of the ERK/AKT survival signaling cascade in AML cells. Our results provide rational for combination of BL-8040 with ABT-199 to overcome potential acquired resistance to ABT-199 in AML patients. The synergistic effect of BL-8040 with AC220 could provide a rational basis for the combination of BL-8040 with FLT3 inhibitors in FLT3-ITD AML patient population. [Display omitted] [Display omitted] [Display omitted] DisclosuresAbraham:Biokine Therapeutics Ltd: Employment. Bulvik:Biokine Therapeutics Ltd: Employment. Wald:Biokine Therapeutics Ltd: Employment. Eizenberg:Biokine Therapeutics Ltd: Employment. Pereg:BioLineRx Ltd: Employment. Peled:Biokine Therapeutics Ltd: Consultancy, Employment.

Mir-130a Is Aberrantly Overexpressed in Adult Acute Myeloid Leukemia with t(8;21) and Its Suppression Induces AML Cell Death

Emerging evidence has revealed that miRNAs can function as oncogenes or tumor suppressor genes in leukemia. By profiling miRNA expression across a cohort (n=156) with de novo acute myeloid leukemia (AML), we identified miR-130a as significantly overexpressed in AML with t(8;21). Expression of miR-130a decreased significantly once patients with t(8;21) achieved complete remission, but increased sharply at the time of relapse. In patients with t(8;21) AML, KIT mutational status was associated with miR-130a expression - with higher expression associated with KIT activating mutations. Increased miR-130a expression in t(8;21) AML was associated with worse event-free survival, however no impact on overall survival was observed. Knockdown of RUNX1/RUNX1T1 (AML1/ETO) protein in the SKNO-1 cell line resulted in decrease of expression of miR-130a. Direct binding of RUNX1/RUNX1T1 fusion protein with the promoter sequence of miR-130a was detected with luciferase reporter gene assay. Following miR-130a knockdown, SKNO-1 demonstrated increased sensitivity to etoposide. Taken together, our data suggest that miR-130a is directly activated by RUNX1/RUNX1T1, and can be used to predict leukemia burden, event-free survival and chemotherapy sensitivity in AML with t(8;21). DisclosuresNo relevant conflicts of interest to declare.