AbstractBackgroundAcute Myeloid Leukemia Patients Research Articles

Identification of TIM-3 Expression in Acute Myeloid Leukemia: Clinical and Laboratory Correlation

Abstract Background Acute myeloid leukemia (AML) is an aggressive highly heterogenous hematological malignancy associated with poor prognosis and high mortality rate. Both cancer cells and immune cells participate in tumor initiation, growth and progression and might affect clinical outcomes. Indeed, leukemic cells exploit a variety of mechanisms to evade immune response, leading to disease progression and relapse. T-cell immunoglobulin and mucin domain 3 (TIM-3), an immune checkpoint molecule, is expressed not only on immune cells but also on leukemic stem cells (LSCs) in AML. Studies relating to TIM-3 function in AML are limited, and the role of TIM3 in maintaining leukemic stem cells and contributing to the suppression of the anti-tumor immune response is still poorly understood. Aim of the Work To investigate TIM-3 expression in patients with AML and explore the impact of its expression on the clinico-laboratory characteristics and prognostic behavior of denovo-AML patients. Methods A total of 45 patients newly diagnosed AML patients were tested for TIM-3 expression by flowcytometry at diagnosis. Results TIM-3 was found to be over expressed in patients with AML with significant association with the different FAB subtypes (p = 0.010). TIM-3 positivity was negatively correlated with CD34 expression (p = 0.019.). However, no significant association between TIM-3 expression and extramedullary disease (EMD) as well as cytogenetic risk groups. Conclusion TIM-3 is overexpressed in AML patients. The prognostic value of TIM3 in AML is not clear and remains to be further explored.

Expression Pattern and Prognostic Implication of SALL4 Gene in Acute Myeloid Leukemia

Abstract Background Acute myeloid leukemia (AML) is a clonal disorder arises from the differentiation arrest of myeloid precursor and malignant proliferation of a bone marrow derived, self-renewing stem or progenitor cells in the bone marrow (BM) and blood. Aim of the Work To characterize the expression pattern of SALL4 gene in patients with acute myeloid leukemia and to determine its prognostic impact. Patients and Methods This study was conducted on 20 Egyptian AML patients (recruited from hematology units of Internal Medicine department, Ain shams University Hospitals) and 10 patients as controls. All patients were subjected to full history taking, thorough clinical examination, complete blood count, BM aspiration cytochemical analysis, flow-cytometric immunophenotyping of BM blasts using acute leukemia panel and cytogenetic analysis using FISH technique and RT-PCR to measure SALL4 gene expression in AML cases and controls. Results Our results showed highly significant difference between expression of SALL 4 gene in AML patients than control group with RQ median in AML cases 15.46 and in control group 0.4 (p value 0.000). Non significant results about prognosios was reached Conclusion SALL4 gene expression is constitutive in De-novo AML cases.

How to adjust therapy for elderly AML patients in developing countries?

Abstract Background Acute myeloid leukemia (AML) with advancing age represents a therapeutic challenge. The tools used for comprehensive geriatric assessment (CGA) in elderly AML are not uniform. Methods This is a retrospective study that included 226 elderly AML patients (≥60 years), identified from the electronic database and followed till the end of 2021. Comorbidities were assessed using Charlson Comorbidity Index (CCI), Cumulative Illness Rating Scale-Geriatric (CIRS-G), Hematopoietic cell transplantation-specific comorbidity index (HCT-CI) scores, and Chia-Jen Liu and colleagues simplified proposed models. The primary outcome was 30 days mortality. The secondary outcomes were the achievement of complete remission. Results 226 patients were included, the median age was 67 years, and 86.2% of patients had ECOG greater than or equal to 2 performance status at diagnosis. 82 patients received intensive induction treatment, 85 patients received reduced-intensity treatment, and 59 patients were unfit for therapy. 30-Day mortality was reported in 49.1% of included patients. With multivariate analysis; ECOG-PS (3-4), high CIRS-G or HCT-CI scoring system (≥3), and intermediate-high Chia-Jen Liu score were considered as prognostic factors for early mortality in elderly AML patients. Complete remission (CR) was achieved in 38.3% and 4.7% of the patients who received intensive induction and low-intensity therapy, respectively. Conclusion Geriatric and comorbidity assessment tools can be used to predict early mortality in elderly AML. Higher scores and poor PS are predictors for early death rather than a prediction of response to treatment.

EIF4A inhibition targets bioenergetic homeostasis in AML MOLM-14 cells in vitro and in vivo and synergizes with cytarabine and venetoclax.

Acute myeloid leukemia (AML) is an aggressive hematological cancer resulting from uncontrolled proliferation of differentiation-blocked myeloid cells. Seventy percent of AML patients are currently not cured with available treatments, highlighting the need of novel therapeutic strategies. A promising target in AML is the mammalian target of rapamycin complex 1 (mTORC1). Clinical inhibition of mTORC1 is limited by its reactivation through compensatory and regulatory feedback loops. Here, we explored a strategy to curtail these drawbacks through inhibition of an importanteffector of themTORC1signaling pathway, the eukaryotic initiation factor 4A (eIF4A). We tested the anti-leukemic effect of a potent and specific eIF4A inhibitor (eIF4Ai), CR-1-31-B, in combination with cytosine arabinoside (araC) or the BCL2 inhibitor venetoclax. We utilized the MOLM-14 human AML cell line to model chemoresistant disease both in vitro and in vivo. In eIF4Ai-treated cells, we assessed for changes in survival, apoptotic priming, de novo protein synthesis, targeted intracellular metabolite content, bioenergetic profile, mitochondrial reactive oxygen species (mtROS) and mitochondrial membrane potential (MMP). eIF4Ai exhibits anti-leukemia activity in vivo while sparing non-malignant myeloid cells. In vitro, eIF4Ai synergizes with two therapeutic agents in AML, araC and venetoclax. EIF4Ai reduces mitochondrial membrane potential (MMP) and the rate of ATP synthesis from mitochondrial respiration and glycolysis. Furthermore, eIF4i enhanced apoptotic priming while reducing the expression levels of the antiapoptotic factors BCL2, BCL-XL and MCL1. Concomitantly, eIF4Ai decreases intracellular levels of specific metabolic intermediates of the tricarboxylic acid cycle (TCA cycle) and glucose metabolism, while enhancing mtROS. In vitro redox stress contributes to eIF4Ai cytotoxicity, as treatment with a ROS scavenger partially rescued the viability of eIF4A inhibition. We discovered that chemoresistant MOLM-14 cells rely on eIF4A-dependent cap translation for survival in vitro and in vivo. EIF4A drives an intrinsic metabolic program sustaining bioenergetic and redox homeostasis and regulates the expression of anti-apoptotic proteins. Overall, our work suggests that eIF4A-dependent cap translation contributes to adaptive processes involved in resistance to relevant therapeutic agents in AML.

Incidence of Invasive Fungal Infections in Patients With Previously Untreated Acute Myeloid Leukemia Receiving Venetoclax and Azacitidine.

BackgroundAcute myeloid leukemia (AML) is associated with poor prognosis, particularly in elderly patients with comorbidities. Combining azacitidine (AZA) with BCL-2 inhibitor venetoclax (VEN) demonstrated significant improvement in outcomes for newly-diagnosed AML patients compared to AZA alone. However, this regimen is myelosuppressive, and the incidence of invasive fungal infections (IFIs) and impact of antifungal prophylaxis are not well defined.MethodsThis retrospective cohort study evaluated newly-diagnosed AML patients treated with VEN/AZA at the University of Colorado Hospital from January 2014 to August 2020. Patients with history of prior IFI were excluded. Primary outcome was IFI incidence during VEN/AZA therapy. χ2 and Fisher exact tests assessed the impact of patient demographics, AML-specific risk factors, and receipt of antifungal prophylaxis on IFI incidence.Results144 VEN/AZA-treated AML patients were included in the study. 25 (17%) patients developed IFI: 8% (n = 2) “proven,” 24% (n = 6) “probable,” and 68% (n = 17) “possible” per European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium criteria. There was no statistically significant association between IFI incidence with age, sex, or European LeukemiaNet classification. 10 patients received antifungal prophylaxis; none developed IFI. IFI incidence rate per 1000 patient-days was greatest 0–9 days after starting VEN/AZA, at 8.39.ConclusionsIncidence of “proven” and “probable” IFI in our VEN/AZA-treated AML cohort was 5.6%, in-line with incidence rates reported by recent similar studies. Furthermore, IFI incidence decreased as days from starting VEN/AZA therapy increased.

SEMA4D/PlexinB1 promotes AML progression via activation of PI3K/Akt signaling

BackgroundAcute myeloid leukemia (AML) is the most common type of acute leukemia in adults. SEMA4D is a 150 kDa transmembrane protein that belongs to the IV class of the subfamily of semaphorin family. Previous studies have reported that SEMA4D is a multifunctional target in many solid tumors, involving multiple physiological systems, and there are emerging therapies to target these pathways. The role of SEMA4D in AML has not yet been explored.MethodsThe SEMA4D expression prolile, clinical data and potential prognostic analysis were acquired via the cBioPortal and GEPIA databases. SEMA4D expression was measured using real-time quantitative PCR and western blot. Cell counting kit-8 (CCK8) and flow cytometry were used to evaluate the malignant biological characteristics.ResultsWe observed that SEMA4D was increased in AML patients and correlated with risk stratification and prognosis. Moreover, SEMA4D promotes the proliferation and inhibits apoptosis of AML cells by binding to its receptor, PlexinB1, and reduces the sensitivity of AML cells to daunorubicin. In addition, SEMA4D/PlexinB1 promotes the proliferation and survival of AML cells by activating the PI3K/Akt signaling pathway. VX15/2503, an anti-SEMA4D antibody, can inhibit the proliferation of AML cells in xenograft mouse models, thereby inhibiting the development of AML.ConclusionSEMA4D will serve as a unique predictive biomarker and a possible therapeutic target in AML.

Up-Regulation of TRIM32 Associated With the Poor Prognosis of Acute Myeloid Leukemia by Integrated Bioinformatics Analysis With External Validation.

BackgroundAcute myeloid leukemia (AML) is a malignant and molecularly heterogeneous disease. It is essential to clarify the molecular mechanisms of AML and develop targeted treatment strategies to improve patient prognosis.MethodsAML mRNA expression data and survival status were extracted from TCGA and GEO databases (GSE37642, GSE76009, GSE16432, GSE12417, GSE71014). Weighted gene co-expression network analysis (WGCNA) and differential gene expression analysis were performed. Functional enrichment analysis and protein-protein interaction (PPI) network were used to screen out hub genes. In addition, we validated the expression levels of hub genes as well as the prognostic value and externally validated TRIM32 with clinical data from our center. AML cell lines transfected with TRIM32 shRNA were also established to detect the proliferation in vitro.ResultsA total of 2192 AML patients from TCGA and GEO datasets were included in this study and 20 differentially co-expressed genes were screened by WGCNA and differential gene expression analysis methods. These genes were mainly enriched in phospholipid metabolic processes (biological processes, BP), secretory granule membranes (cellular components, CC), and protein serine/threonine kinase activity (molecular functions, MF). In addition, the protein-protein interaction (PPI) network contains 15 nodes and 15 edges and 10 hub genes (TLE1, GLI2, HDAC9, MICALL2, DOCK1, PDPN, RAB27B, SIX3, TRIM32 and TBX1) were identified. The expression of 10 central genes, except TLE1, was associated with survival status in AML patients (p<0.05). High expression of TRIM32 was tightly associated with poor relapse-free survival (RFS) and overall survival (OS) in AML patients, which was verified in the bone marrow samples from our center. In vitro, knockdown of TRIM32 can inhibit the proliferation of AML cell lines.ConclusionTRIM32 was associated with the progression and prognosis of AML patients and could be a potential therapeutic target and biomarker for AML in the future.

986. Incidence of Invasive Fungal Infections in Previously Untreated Patients with Acute Myeloid Leukemia Receiving Venetoclax and Azacitadine

Abstract Background Acute myeloid leukemia (AML) is associated with poor prognosis, particularly in elderly patients with co-morbidities. Low-intensity therapies like azacitidine (aza) were the standard of care and were associated with low response rates and limited survival. Combining venetoclax (ven) with aza demonstrated significant improvements in responses and survival compared to aza alone, and represents the new standard of care for this population. However, as a myelosuppressive regimen, infectious complications, especially invasive fungal infections (IFI), are a potential concern. The incidence of IFI and the role for antifungal prophylaxis have not been well defined for newly-diagnosed AML patients receiving ven/aza. Methods We conducted a retrospective cohort review of AML patients treated with ven/aza at the University of Colorado Hospital from January 2014 to August 2020. Duration of therapy was defined as the time from initiation of treatment through one of the following endpoints (1) patient discontinuation, (2) progression of disease, (3) bone marrow transplantation, or (4) death. Four patients with a history of prior IFI were excluded. We assessed the impact of patient age, sex, duration of neutropenia, antifungal prophylaxis, and AML specific risk factors on the incidence of IFI as defined by the European Mycoses Study Group. Results One hundred forty-four AML patients were included in the study. Ten patients received antifungal prophylaxis and none developed IFI (p=0.21). Twenty-five (17%) patients developed IFI: 2 (8%) had proven IFI, 6 (24%) probable IFI, and 17 (68%) possible IFI. Invasive pulmonary aspergillosis represented all 25 cases of proven, probable, and possible IFI. There was a statistically significant association between prolonged neutropenia ( &amp;gt;60 days) and IFI (p=0.007), whereas age, sex, and SWOG classification were not significantly associated with IFI. Conclusion The incidence of IFI in our AML cohorts treated with ven/aza was 17%, lower than that reported at other institutions. Neutropenia &amp;gt; 60 days was significantly associated with IFI in our AML cohort treated with ven/aza. Although we were not powered to determine whether antifungal prophylaxis impacted IFI, there was no significant difference in IFI for patients who received prophylaxis. Disclosures All Authors: No reported disclosures

Bayesian variable selection for high-dimensional data with an ordinal response: identifying genes associated with prognostic risk group in acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) is a heterogeneous cancer of the blood, though specific recurring cytogenetic abnormalities in AML are strongly associated with attaining complete response after induction chemotherapy, remission duration, and survival. Therefore recurring cytogenetic abnormalities have been used to segregate patients into favorable, intermediate, and adverse prognostic risk groups. However, it is unclear how expression of genes is associated with these prognostic risk groups. We postulate that expression of genes monotonically associated with these prognostic risk groups may yield important insights into leukemogenesis. Therefore, in this paper we propose penalized Bayesian ordinal response models to predict prognostic risk group using gene expression data. We consider a double exponential prior, a spike-and-slab normal prior, a spike-and-slab double exponential prior, and a regression-based approach with variable inclusion indicators for modeling our high-dimensional ordinal response, prognostic risk group, and identify genes through hypothesis tests using Bayes factor.ResultsGene expression was ascertained using Affymetrix HG-U133Plus2.0 GeneChips for 97 favorable, 259 intermediate, and 97 adverse risk AML patients. When applying our penalized Bayesian ordinal response models, genes identified for model inclusion were consistent among the four different models. Additionally, the genes included in the models were biologically plausible, as most have been previously associated with either AML or other types of cancer.ConclusionThese findings demonstrate that our proposed penalized Bayesian ordinal response models are useful for performing variable selection for high-dimensional genomic data and have the potential to identify genes relevantly associated with an ordinal phenotype.

Platelet transcriptome profiles provide potential therapeutic targets for elderly acute myelocytic leukemia patients

BackgroundAcute myeloid leukemia (AML) is the most common acute leukemia in adults, with a median age of 68 in clinical diagnosis. About 60% patients are over 60 years old. There are various treatment options for AML patients. But for elderly patients, the complete remission rates are disappointing due to genetic, molecular, and age-related factors. Development of next-generation sequencing technologies makes it possible to seek individual strategies for patients in different ages. This study analyzed transcriptome profiles in platelets of AML patients in different ages for the first time.MethodsPlatelet RNA sequencing in AML of ten elderly and seven young patients were performed with Illumina TruSeq Stranded mRNA library Prep Kit and Illumina HiSeq4000 sequencing instrument. With the FASTQ sequencing data obtained, statistical analyses between elderly with young AML patients were analyzed by R program. GO and KEGG enrichment analyses were performed via R package clusterProfiler. TOP 10 down-regulated/up-regulated genes in elderly patients compared to young patients were selected with the threshold of |L2FC| > 2 and padj ≤ 0.0001. The down-regulated gene ATF4 was chosen by GSEA analysis and ROC analysis with AUC > 0.95.ResultsWe found 3059 genes with differential transcript levels (GDTLs) in AML patients of different age. Among them, 2048 genes are down-regulated and 651 genes are up-regulated in elderly patients. We found that gene transcript profiles in elderly patients is obviously different from those in young patients, including a collection of down-regulated genes related to proteins processing in endoplasmic reticulum and immunity. We further identified that genes of pathway in cancer and mitogen activated protein kinase (MAPK) pathway, involved in natural immunity and metabolism, are significantly down-regulated in elderly patients. Among all screened genes with decreased transcript levels, we believe that activating transcription factor 4 (ATF4) is a biomarker indicating different chemotherapy strategies for elderly patients.ConclusionsIn summary, gene transcript profiles are different in platelets of elderly and young AML patients. And ATF4 can be a useful biomarker indicating different chemotherapy strategies for AML patients with different ages.

Abstract 1018: SEL120, a CDK8/CDK19 inhibitor, possesses strong multilineage differentiation potential in AML

Abstract Background Acute myeloid leukemia (AML) is a heterogeneous disease characterized by rapid proliferation of leukemic blasts and high rate of acquired resistance to drugs. Differentiating agent ATRA has been established as a backbone of APL treatment, however its activity in other leukemias is limited. Knowing that Cyclin Dependent Kinase 8 (CDK8) can maintain tumor dedifferentiation and embryonic stem cell pluripotency, we investigated whether CDK8 inhibitor SEL120 could effectively target leukemia by induction of lineage commitment. SEL120 is a specific, selective inhibitor of CDK8 and its paralog CDK19. A first-in-human phase Ib clinical trial with SEL120 in AML or HR-MDS patients is currently ongoing. To better understand the mechanism of action of SEL120 in AML, we studied effects of SEL120 on differentiation as one of the important anti-leukemic activities of the compound. Methods Global transcriptional changes were analyzed by RNAseq at different time points to capture early and long-term effects of SEL120. Genome-wide profiling of DNA-binding was performed by CHIPseq. Cell cycle, proliferation and lineage specific markers were studied by flow cytometry. Differentiation potential of AML cells was studied in semi-solid methylcellulose-based media to asses colony formation of SEL120 treated blasts. Results SEL120 treatment leads to decreased CDK8 occupancy and increased RNA Pol II occupancy as well as changes in the peak distribution among promoter and enhancer regions. SEL120 could repress many “stemness” genes and induce the expression of genes involved in lineage commitment, including regulators of erythroid/megakaryocytic fate, such as RGS18, KLF1, FLI1 and GATA1/2. Moreover, for the first time, we showed that prolonged exposure of AML CD34+ cells to SEL120 could lead to colony formation in semi-solid media. Detailed analysis by flow cytometry at early and late time points reflected sequential changes in the expression of lineage-specific surface markers, characterizing differentiation and the presence of cells of myeloid or erythroid/megakaryocytic origin. Conclusion In addition to established role of CDK8 in regulation of tumor suppressor genes we present evidence for an essential role of CDK8 in lineage controlling-functions. Previously we reported specific cytotoxicity of SEL120 on cells positive for stemness markers such as CD34+. We further expanded on these studies, showing profound morphological changes in AML blasts during prolonged exposure to SEL120, correlating with increased expression of myeloid and erythroid/megakaryocytic markers. Based on these findings further studies are warranted to investigate the efficacy of SEL120 in anemia associated with bone marrow failures in AML and MDS. Combination of direct effects of SEL120 on viability of cells, with a strong differentiation potential, represents a promising profile for a drug in successful leukemia treatment. Citation Format: Urszula Pakulska, Elzbieta Adamczyk, Katarzyna Dziedzic, Katarzyna Wiklik, Michal Combik, Michal Mikula, Aniela Golas, Marta Obacz, Magdalena Masiejczyk, Przemyslaw Juszczynski, Magdalena Cybulska, Milena Mazan, Krzysztof Brzozka, Tomasz Rzymski. SEL120, a CDK8/CDK19 inhibitor, possesses strong multilineage differentiation potential in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1018.

HOTAIR expression and prognostic impact in acute myeloid leukemia patients

BackgroundAcute myeloid leukemia (AML) is a disorder characterized by a rapid onset of symptoms attributable to bone marrow failure due to clonal proliferation of primitive hematopoietic stem cells or progenitor cells. Epigenetic abnormalities play an important role in the development and progression of acute leukemia. Long non-coding ribonucleic acid (lncRNA) plays an important role in epigenetic regulation. Homeobox (Hox) transcript antisense intergenic RNA (HOTAIR) is a lncRNA which has been determined to be a negative prognostic indicator in various solid-tumor patients. However, its role in hematopoietic tumors as AML is to be assessed. This study aimed at measuring lncRNA HOTAIR expression level on bone marrow (BM) mononuclear cells in newly diagnosed AML patients and correlating its expression with their outcome and different prognostic variables. This provides new prospective for a novel marker involved in development and progression of AML which can be used as a diagnostic marker and a target of therapy. The current study included 65 subjects divided into 35 newly diagnosed AML adult patients (before initiation of chemotherapy) and 30 non-leukemic adult patients who are candidates for BM aspiration for causes other than hematological malignancies as immune thrombocytopenic purpura and hypersplenism as controls. HOTAIR expression was measured on BM mononuclear cells by quantitative reverse transcription polymerase chain reaction (qRT-PCR).ResultsHOTAIR expression was found to be significantly upregulated in AML patients (probability (p) value = 0.000) and it can be used as a diagnostic biomarker of AML as confirmed by a significant difference between cases and controls using receiver operating characteristic curve (ROC) analysis. However, it was not significantly correlated with event free survival (EFS) or prognostic variables.ConclusionThis study showed that the expression of HOTAIR is upregulated in de novo AML patients and can be used as a diagnostic marker. However, highly expressed HOTAIR is not associated with poor prognosis.

Radotinib enhances cytarabine (Ara-C)-induced acute myeloid leukemia cell death

BackgroundAcute myeloid leukemia (AML) is a heterogeneous disease that frequently relapses after standard chemotherapy. Therefore, there is a need for the development of novel chemotherapeutic agents that could treat AML effectively. Radotinib, an oral BCR-ABL tyrosine kinase inhibitor, was developed as a drug for the treatment of chronic myeloid leukemia. Previously, we reported that radotinib exerts increased cytotoxic effects towards AML cells. However, little is known about the effects of combining radotinib with Ara-C, a conventional chemotherapeutic agent for AML, with respect to cell death in AML cells. Therefore, we investigated combination effects of radotinib and Ara-C on AML in this study.MethodsSynergistic anti-cancer effects of radotinib and Ara-C in AML cells including HL60, HEL92.1.7, THP-1 and bone marrow cells from AML patients have been examined. Diverse cell biological assays such as cell viability assay, Annexin V-positive cells, caspase-3 activity, cell cycle distribution, and related signaling pathway have been performed.ResultsThe combination of radotinib and Ara-C was found to induce AML cell apoptosis, which involved the mitochondrial pathway. In brief, combined radotinib and Ara-C significantly induced Annexin V-positive cells, cytosolic cytochrome C, and the pro-apoptotic protein Bax in AML cells including HL60, HEL92.1.7, and THP-1. In addition, mitochondrial membrane potential and Bcl-xl protein were markedly decreased by radotinib and Ara-C. Moreover, this combination induced caspase-3 activity. Cleaved caspase-3, 7, and 9 levels were also increased by combined radotinib and Ara-C. Additionally, radotinib and Ara-C co-treatment induced G0/G1 arrest via the induction of CDKIs such as p21 and p27 and the inhibition of CDK2 and cyclin E. Thus, radotinib/Ara-C induces mitochondrial-dependent apoptosis and G0/G1 arrest via the regulation of the CDKI–CDK–cyclin cascade in AML cells. In addition, our results showed that combined treatment with radotinib and Ara-C inhibits AML cell growth, including tumor volumes and weights in vivo. Also, the combination of radotinib and Ara-C can sensitize cells to chemotherapeutic agents such as daunorubicin or idarubicin in AML cells.ConclusionsTherefore, our results can be concluded that radotinib in combination with Ara-C possesses a strong anti-AML activity.

Two rare cases of acute myeloid leukemia with t(8;16)(p11.2;p13.3) and 1q duplication: case presentation and literature review

BackgroundAcute myeloid leukemia (AML) is a complex hematological disease characterized by genetic and clinical heterogeneity. The identification and understanding of chromosomal abnormalities are important for the diagnosis and management of AML patients. Compared with recurrent chromosomal translocations in AML, t(8;16)(p11.2;p13.3) can be found in any age group but is very rare and typically associated with poor prognosis.MethodsConventional cytogenetic studies were performed among 1,824 AML patients recorded in our oncology database over the last 20 years. Fluorescence in situ hybridization (FISH) was carried out to detect the translocation fusion. Array comparative genome hybridization (aCGH) was carried out to further characterize the duplication of chromosomes.ResultsWe identified three AML patients with t(8;16)(p11.2;p13.3) by chromosome analysis. Two of the three patients, who harbored an additional 1q duplication, were detected by FISH and aCGH. aCGH characterized a 46.7 Mb and 49.9 Mb gain in chromosome 1 at band q32.1q44 separately in these two patients. One patient achieved complete remission (CR) but relapsed 3 months later. The other patient never experienced CR and died 2 years after diagnosis.ConclusionA 1q duplication was detected in two of three AML patients with t(8;16)(p11.2;p13.3), suggesting that 1q duplication can be a recurrent event in AML patients with t(8;16). In concert with the findings of previous studies on similar patients, our work suggests that 1q duplication may also be an unfavorable prognostic factor of the disease.

Abstract 6217: Synergistic effect of CDK8 and BCL-2 inhibition in AML through regulation of MCL-1 and BIM balance

Abstract Background Acute myeloid leukemia (AML) is characterized by rapid proliferation of myeloid blood cells. Due to its heterogeneity and the high rate of acquired drug resistance, new treatment modalities are needed. SEL120 is a specific type I selective inhibitor of Cyclin-dependent kinase 8 (CDK8) and Cyclin-dependent kinase 19 (CDK19). A first- in- human phase Ib clinical trial with SEL120 in patients with AML or HR-MDS was initiated in June 2019. Preclinical studies demonstrated high efficacy of SEL120 in experimental AML models via mechanisms involving differentiation and induction of apoptosis. Transcriptomic analysis of hematological cell lines demonstrated that SEL120 treatment upregulated expression of an apoptotic activator BIM from BCL-2 family of proteins. Methods Efficacy of the compound alone or in combination was tested in viability assays in a broad panel of cancer cell lines. Activity and mechanism of action of CDK8 inhibitor - SEL120 alone and in combination was investigated by flow cytometry, western blotting, co-immunoprecipitation, differential gene expression and ChIPseq analysis. In vivo efficacy was tested in mice injected with MV4-11 cell line both subcutaneously and intravenously. Results Here we provide a strong rationale for combination of SEL120 and BCL-2-selective inhibitor Venetoclax (ABT-199). We found that SEL120 synergistically induced apoptosis with Venetoclax in AML cells. Combination of both compounds significantly reduced levels of prosurvival protein MCL-1 and induced hallmarks of apoptosis including Caspase-3 activation and PARP cleavage. Previous studies associated Venetoclax resistance with increased sequestration of proapoptotic BIM by high levels of MCL-1. While a SEL120 treatment alone had no effects on MCL-1 levels, combination of both compounds resulted in sensitization of Venetoclax-resistant AML cells. We demonstrated that mechanism of Venetoclax resistance can be abrogated by the cotreatment with SEL120 leading to changes in proportions of BIM and MCL-1 levels. Synergistic interaction between SEL120 and Venetoclax resulted in apoptotic cell death in established cell lines and patient derived AML cells. Finally, using murine models of subcutaneous or disseminated AML, we found complete remissions of AML and associated recovery of normal cells in bone marrow of animals treated with both SEL120 and Venetoclax. Conclusion Taken together, these data provided rationale for a novel clinical strategy that may lead to durable responses in AML patients. Citation Format: Tomasz Rzymski, Marta Obacz, Milena Mazan, Marion Chappelier, Marcus Järås, Michal Mikula, Elżbieta Adamczyk, Katarzyna Wiklik, Michal Combik, Aniela Golas, Magdalena Masiejczyk, Przemyslaw Juszczynski, Jerzy Ostrowski, Krzysztof Brzózka. Synergistic effect of CDK8 and BCL-2 inhibition in AML through regulation of MCL-1 and BIM balance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6217.

Hyperfunction of CD4 CD25 regulatory T cells in de novo acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) is a common hematopoietic malignancy that has a high relapse rate, and the number of regulatory T cells (Tregs) in AML patients is significantly increased. The aim of this study was to clarify the role of Tregs in the immune escape of acute myeloid leukemia.MethodsThe frequencies of Tregs and the expression of PD-1, CXCR4 and CXCR7 were examined by flow cytometry. The expression of CTLA-4 and GITR was tested by MFI. Chemotaxis assays were performed to evaluate Treg migration. The concentrations of SDF-1α, IFN-γ and TNF-α were examined by ELISA. Coculture and crisscross coculture experiments were performed to examine Treg proliferation and apoptosis and the effect of regulatory B cells (Breg) conversion.ResultsThe frequencies of Tregs in peripheral blood and bone marrow in AML patients were increased compared with those in healthy participants. AML Tregs had robust migration towards bone marrow due to increased expression of CXCR4. AML Treg-mediated immunosuppression of T cells was achieved through proliferation inhibition, apoptosis promotion and suppression of IFN-γ production in CD4+CD25− T cells. AML Bregs induced the conversion of CD4+CD25−T cells to Tregs.ConclusionIn AML patients, the Breg conversion effect and robust CXCR4-induced migration led to Treg enrichment in bone marrow. AML Tregs downregulated the function of CD4+CD25− T cells, contributing to immune escape.

Expression of G Protein–Coupled Receptor 56 (GPR56) in Acute Myeloid Leukemia

Abstract Background Acute myeloid leukemia (AML) is a heterogeneous marrow-based clonal group of neoplasms that affects hematopoietic cells responsible for the production of myeloid lineages. Prognosis of AML is multifactorial and has been extensively studied, yet it’s highly dependent on the presence of leukemic stem cells (LSCs). G protein–coupled receptor 56 (GPR56) was introduced as a novel human LSC marker in AML patients as acknowledged by the engraftment potential of GPR56+ cells sorted from selected AML specimens, which contributed to leukemia propagation in mice. Aim of the work The aim of this study is to analyze GPR56 expression in de novo AML patients using flow cytometry. The results of GPR56 expression will be correlated with the clinical outcome of the patients as well as other laboratory parameters. Subjects and Methods The study was carried out at Ain Shams University hospitals on a total number of 40 AML patients attending hematology-oncology unit during the period from November 2016 to July 2017, in addition to 20 healthy control subjects. Patients were assessed at day 28 after induction of treatment by bone marrow examination and minimal residual disease analysis, and follow up of the disease course was done. Results GPR56 was highly expressed above mean in 62.5% of AML patients. High values were significantly related to failure to attain complete remission, whereas it lacked prognostic significance to patient outcome and cytogenetic subgroups. Conclusion The poor prognostic impact of GPR56 was partially validated in our study. Recommendations Flow cytometric evaluation of GPR56 should be incorporated into the initial laboratory work-up for all newly diagnosed AML patients.

DNA Methylation-Based Classification Highlights the Role of the JAK-STAT Pathway in Acute Myeloid Leukemia

DNA Methylation-Based Classification Highlights the Role of the JAK-STAT Pathway in Acute Myeloid Leukemia

The Choice of Treatment for Elderly AML-Patients: 7+3 or Low Doses ARA-C

Background Acute myeloid leukemia (AML) in older adults is a biologically and clinically distinct entity. These patients often have comorbidities, and their treatment must be chosen with caution. In AML patients over 60y old, cure rates are under 10% even after intensive chemotherapy (CT). Aim To compare the efficacy of different therapeutic approaches in elderly AML-pts treated in NRCH. Methods From 2002 till 2019, NRCH has conducted a prospective non-randomized study which included 80 AML-patients 60-81y (Me - 67y): 60-65 yy (n=53) and &gt;65y (n=27); M/F - 35/45; de novo AML n=61 (76,25%), AML from MDS - n=13 (16,25%), «secondary» AML - n=6 (7,5%); cytogenetic risk: favorable n=1 (1,25%), intermediate n=49 (61,25%), poor n=30 (37,5%). The patients were stratified to different treatment approaches according to age. Patients 60-70y (n=40) mostly received 1-2 induction cycles 7+3 (ARA-C 100 mg/m2 bid; Dauno - 45-60 mg/m2 ), then 2 consolidation cycles 7+3 (Dauno - 45 mg/m2) and 2 years maintenance (5+5 with 6-MP). Patients &gt;70y (n=22) were usually treated with 1-2 induction and 2 consolidation cycles of low dose Ara-C (LDAC) (10 mg/m2 sc bid, 28-days) and 3 years maintenance with 21-28-days LDAC. In some cases, fit patients over the age of 70y have got 7+3 (n=5) and some younger with comorbidities - LDAC (n=13). The analysis was done in May 2019. We evaluated treatment outcome according to age, cytogenetics and type of CT. Results The CR rate in the whole group of elderly AML-pts was 57,5% (46/80) with a median CR-duration - 10 mon (1-138 mon), early death - 16,25% (13/80) and resistance - 26,25% (21/80) with no major differences in the two age cohorts (&lt;&gt;70y). In order to assess of the efficacy of two chemotherapy options we have compared 7+3 and LDAC in patients aged 60-65 and older. In patients aged 60-65 CR-rate was higher -75% (21/28) after 7+3 vs 50% (2/4) after LDAC, with less resistant forms - 7% (2/28) vs 25% (1/4), respectively. In &gt; 65y group CR-rate was identical in pts after 7+3 (47%, 8/17) and after LDAC (55%, 17/31) with similar numbers of resistant forms: 41% vs 29%. Early death rate did not differ among the groups. There was statistically higher CR-rate and lower resistant forms on 7+3 in pts aged 60-65 compared to older pts. - 75% vs 47% (p&lt;0.05) and 7% vs 41% (&lt;0.005) (Tab.1). Long-term results in the whole cohort of elderly patients were as follows: 1-y OS - 46%, DFS - 52,2%, 5-y ОS - 13%, DFS - 9% with high relapse rate (70%) development, mostly with early occurrence (Me - 12.8 mon). The median survival was statistically longer in patients aged 60-65y (n=32) comparing to older ones (n=48) - 15.4 vs 8 mon, respectively (p=0.0542), but DFS was equal (Fig.1). Higher incidence (87% vs 74%) and earlier relapse development (12 vs 16 mon) were registered in the group with poor cytogenetics comparing to favorable/intermediate (p=0,022). We didn't observe significant difference in long-term outcome of AML-pts older or younger 70y. Standard LDAC duration is 10-14 days, twice less than we used in our study. A landmark-analysis (from day 30 of induction) has shown that, if the 1st course-duration was less than 28 days, the median survival was shorter - 5,9 mo in comparison with 14,9 mo in pts with 28-days LDAC (p=0.12). The efficacy of 28-days LDAC was very similar to 7+3, mainly in patients aged 66 and older (Fig.2). Conclusion The outcome in elderly AML-patients is determined by age (more or less 65y) and the group of cytogenetic prognosis (poor vs favorable/intermediate). Intensive induction (7+3) is more preferable in patients aged 60-65 y as it produced the higher CR-rate and less resistant forms, results that are comparable to younger AML-patients. In patients older than 65y - CR-rate after 7+3 or 28-day LDAC was identical, and 28-days LDAC may become the choice of treatment for patients older than 65y. However long-term results are generally poor so new therapeutic strategies for elderly AML-patients are highly needed. Disclosures No relevant conflicts of interest to declare.

Nuclear factor of activated T-cells, NFATC1, governs FLT3ITD-driven hematopoietic stem cell transformation and a poor prognosis in AML

BackgroundAcute myeloid leukemia (AML) patients with a high allelic burden of an internal tandem duplication (ITD)-mutated FMS-like Tyrosine Kinase-3 (FLT3) have a dismal outcome. FLT3ITD triggers the proliferation of the quiescent hematopoietic stem cell (HSC) pool but fails to directly transform HSCs. While the inflammatory transcription factor nuclear factor of activated T-cells 2 (NFAT2, NFATC1) is overexpressed in AML, it is unknown whether it plays a role in FLT3ITD-induced HSC transformation.MethodsWe generated a triple transgenic mouse model, in which tamoxifen-inducible Cre-recombinase targets expression of a constitutively nuclear transcription factor NFATC1 to FLT3ITD positive HSC. Emerging genotypes were phenotypically, biochemically, and also transcriptionally characterized using RNA sequencing. We also retrospectively analyzed the overall survival of AML patients with different NFATC1 expression status.ResultsWe find that NFATC1 governs FLT3ITD-driven precursor cell expansion and transformation, causing a fully penetrant lethal AML. FLT3ITD/NFATC1-AML is re-transplantable in secondary recipients and shows primary resistance to the FLT3ITD-kinase inhibitor quizartinib. Mechanistically, NFATC1 rewires FLT3ITD-dependent signaling output in HSC, involving augmented K-RAS signaling and a selective de novo recruitment of key HSC-transforming signaling pathways such as the Hedgehog- and WNT/B-Catenin signaling pathways. In human AML, NFATC1 overexpression is associated with poor overall survival.ConclusionsNFATC1 expression causes FLT3ITD-induced transcriptome changes, which are associated with HSC transformation, quizartinib resistance, and a poor prognosis in AML.