Gene Expression In Leukemia Cells Research Articles

MLL-AF4 upregulates 5-lipoxygenase expression in t(4;11) leukemia cells via the ALOX5 core promoter

5-Lipoxygenase (5-LO), encoded by the gene ALOX5, is implicated in several pathologies. As key enzyme in leukotriene biosynthesis, 5-LO plays a central role in inflammatory diseases, but the 5-LO pathway has also been linked to development of certain hematological and solid tumor malignancies. Of note, previous studies have shown that the leukemogenic fusion protein MLL-AF4 strongly increases ALOX5 gene promoter activity. Here, we investigate the upregulation of ALOX5 gene expression by MLL-AF4. Using reporter assays, we first identified the tandem GC box within the ALOX5 promotor sequence as the main target of MLL-AF4. Subsequently, we narrowed down the domains within the MLL-AF4 protein responsible for ALOX5 promoter activation. Our findings indicate that MLL-AF4 binds to the ALOX5 promoter via its CXXC domain and that the AF9ID, pSER and CHD domains redundantly activate transcriptional elongation. Knockdown of the MLL-AF4 gene in the human B cell line SEM revealed that MLL-AF4 is an inducer of ALOX5 gene expression in leukemic cells with lymphoid properties. Finally, we found that the MLL-AF4-related protein MLL-AF9, a driver of acute myeloid leukemia, similarly acts on the ALOX5 promoter. Taken together, we show that two prominent MLL fusion proteins are ALOX5 gene inducers in cells with lymphoid features.

Leveraging Custom CRISPR/Cas9 Screens to Identify AraC-Daunorubicin-Etoposide (ADE), Gemtuzumab Ozogamicin (GO), and Bortezomib Response Modulators Association with Clinical Outcomes in Pediatric AML

Cytarabine, daunorubicin, and etoposide (ADE) remain the standard backbone chemotherapy for pediatric AML. In addition to ADE, gemtuzumab ozogamicin (GO) and bortezomib have been evaluated in the COG's AAML0531 and AAML1031 clinical trials, respectively. Nonetheless, when these experimental agents were combined with ADE, ADE+GO had shown to improve EFS but not OS, while ADE+bortezomib displayed no additional benefit over ADE alone. Thus, treatment options for pediatric AML are still limited, and development of resistance is a major concern, resulting in poor clinical outcomes. To overcome treatment resistance, it is crucial to understand the underlying molecular mechanisms of ADE±GO or bortezomib. Herein, we conducted custom CRISPR/Cas9 synthetic-lethal screens targeting 2440 genes (including AML-relevant genes, pharmacologically relevant genes, and druggable genes), in six AML cell lines with exposure to ara-C, daunorubicin, etoposide, GO and bortezomib followed by integration of the results with outcome data from multiple clinical trials where induction treatment was either standard ADE chemotherapy, ADE+GO or ADE+bortezomib. Six AML cell lines were transduced with a custom library and puromycin selected. Then pool transduced cell populations were then treated with DMSO, the IC30-IC50 levels of cytarabine, daunorubicin, etoposide, GO, or bortezomib for 7-doubling-times at 500x coverage. All samples were processed with genomic DNA extraction, sgRNAs were amplified by PCR, and were subjected to Illumina NovaSeq 6000 SP 100SR sequencing. The abundance of sgRNA was analyzed using MAGeCK-RRA to estimate the RRA drug response score (drug vs. control conditions at 7-doubling times). Genes with an average RRA score of < -1 across six AML cell lines were defined as drug-resistant genes (negative selection), whereas genes with an average RRA score of > 1 were categorized as sensitive genes (positive selection). Next, we investigated leukemic cell gene expression levels of the drug-resistant and drug-sensitive genes with clinical outcomes. At the average RRA score cutoff from CRISPR screens, 459 genes met the significance as resistant or sensitive genes to at least one drug from ADE's screens as ADE genes, 244 genes were significant from ADE genes and GO's screen as ADE+GO genes, and 166 genes were significant from ADE genes and bortezomib'screen as ADE+bortezomib genes ( Fig 1a). ADE genes were evaluated with clinical outcomes with patients treated with ADE AML02 (n=163), ADE arm of AAML0531 (n=201) and AAML1031 (n=411); while ADE+GO genes were evaluated in ADE+GO arm of AAML0531 (n=205) and ADE+bortezomib genes were evaluated in ADE+bortezomib arm of AAML1031 (n=436). Genes showing drug resistance in CRISPR screens and associated with detrimental outcomes (EFS, OS, or MRD1) at high expression were deemed clinically and biologically important drug genes and vice versa. Our preliminary results showed 21 met the criteria as clinically and biologically important drug-resistant or drug-sensitive genes, where genes were significant in multiple CRISPR screens and multiple pediatric AML cohorts ( Fig 1b). Six genes were determined to resist multiple drugs, with higher expression associated with worse outcomes across multiple cohorts. Particularly, ABCC1 encodes the ATP Binding Cassette C1-mediated drug efflux transporter; therefore, knocking out the efflux transporter causing drug resistance, would increase the lethality of AML cells, resulting in poor outcomes in multiple cohorts in patients treated with ADE alone and ADE with GO or bortezomib. Five genesshowed significant as sensitive genes for multiple drugs in our CRISPR screens with high expression association with favorable outcomes in multiple cohorts. Interestingly, two genes were identified as bortezomib-sensitive genes with high expression in patients treated with ADE+bortezemib in AAML1031 cohort had better outcomes. Other genes were revealed to be sensitive to one drug but resistant to another, indicating that antagonistic pleiotropy effects between genes and drugs may affect clinical outcomes. Current research focuses on developing a pipeline to predict different drug combination outcomes for prioritizing drug treatments and identifying novel drug targets to overcome drug resistance to ADE+GO or bortezomib.

Integrated analysis of single-cell RNA-seq and bulk RNA-seq reveals RNA N6-methyladenosine modification associated with prognosis and drug resistance in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a type of blood cancer that is identified by the unrestricted growth of immature myeloid cells within the bone marrow. Despite therapeutic advances, AML prognosis remains highly variable, and there is a lack of biomarkers for customizing treatment. RNA N6-methyladenosine (m6A) modification is a reversible and dynamic process that plays a critical role in cancer progression and drug resistance. To investigate the m6A modification patterns in AML and their potential clinical significance, we used the AUCell method to describe the m6A modification activity of cells in AML patients based on 23 m6A modification enzymes and further integrated with bulk RNA-seq data. We found that m6A modification was more effective in leukemic cells than in immune cells and induced significant changes in gene expression in leukemic cells rather than immune cells. Furthermore, network analysis revealed a correlation between transcription factor activation and the m6A modification status in leukemia cells, while active m6A-modified immune cells exhibited a higher interaction density in their gene regulatory networks. Hierarchical clustering based on m6A-related genes identified three distinct AML subtypes. The immune dysregulation subtype, characterized by RUNX1 mutation and KMT2A copy number variation, was associated with a worse prognosis and exhibited a specific gene expression pattern with high expression level of IGF2BP3 and FMR1, and low expression level of ELAVL1 and YTHDF2. Notably, patients with the immune dysregulation subtype were sensitive to immunotherapy and chemotherapy. Collectively, our findings suggest that m6A modification could be a potential therapeutic target for AML, and the identified subtypes could guide personalized therapy.

Lovastatin inhibits erythroleukemia progression through KLF2-mediated suppression of MAPK/ERK signaling

BackgroundLovastatin, an HMG-CoA inhibitor and an effective cholesterol lowering drug, exhibits anti-neoplastic activity towards several types of cancer, although the underlying mechanism is still not fully understood. Herein, we investigated mechanism of growth inhibition of leukemic cells by lovastatin.MethodsRNAseq analysis was used to explore the effect of lovastatin on gene expression in leukemic cells. An animal model of leukemia was used to test the effect of this statin in vivo. FAM83A and DDIT4 expression was knocked-downed in leukemia cells via lentivirus-shRNA. Western blotting, RT-qPCR, cell cycle analysis and apoptosis assays were used to determine the effect of lovastatin-induced growth suppression in leukemic cells in vitro.ResultsLovastatin treatment strongly inhibited cancer progression in a mouse model of erythroleukemia induced by Friend virus. In tissue culture, lovastatin inhibited cell proliferation through induction of G1 phase cell cycle arrest and apoptosis. Interestingly, lovastatin induced most known genes associated with cholesterol biosynthesis in leukemic cells. Moreover, it suppressed ERK1/2 phosphorylation by downregulating FAM83A and DDIT4, two mediators of MAP-Kinase signaling. RNAseq analysis of lovastatin treated leukemic cells revealed a strong induction of the tumor suppressor gene KLF2. Accordingly, lentivirus-mediated knockdown of KLF2 antagonized leukemia cell suppression induced by lovastatin, associated with higher ERK1/2 phosphorylation compared to control. We further show that KLF2 induction by lovastatin is responsible for lower expression of the FAM83A and DDIT4 oncogenes, involved in the activation of ERK1/2. KLF2 activation by lovastatin also activated a subset of cholesterol biosynthesis genes that may further contribute to leukemia suppression.ConclusionsThese results implicate KLF2-mediated FAM83A/DDIT4/MAPK suppression and activation of cholesterol biosynthesis as the mechanism of leukemia cell growth inhibition by lovastatin.

Genome-Wide CRISPR/Cas9 Screen Identifies AraC-Daunorubicin-Etoposide (ADE) Response Modulators Associated with Clinical Outcomes in Pediatric AML

Cytarabine, daunorubicin, and etoposide (ADE) remain the standard backbone chemotherapy in pediatric AML despite newly approved drugs. However, many pediatric AML develop treatment resistance and relapse, resulting in poor survival. To overcome treatment resistance, it is crucial to understand the molecular mechanisms behind ADE treatment outcomes. In this study, we performed a genome-wide CRISPR/Cas9 synthetic-lethal screening to identify ADE response modulators and integrated CRISPR-screen results with gene expression and clinical outcomes in pediatric AML treated with ADE. K-562 cells were transduced with the Brunello CRISPR/Cas9 library and puromycin selected before the transduced cell population was treated with DMSO, IC30 of cytarabine, daunorubicin, or etoposide (37uM, 0.1uM, and 0.85uM, respectively) for four days at 400x coverage. Genomic DNA was collected for all samples, sgRNAs were PCR amplified, and then sequenced on Illumina Hiseq 4000. The abundance of sgRNA was analyzed using MAGeCK-MLE to obtain normalized beta score per gene to identify treatment-related (treatment minus control beta values [diff-beta]) genes. Genes with diff-beta <2 standard deviations (SD) were associated with drug resistance (negative selection), and genes with diff-beta >2 SD were associated with increased drug sensitivity (positive selection). For translational impacts of CRISPR screen results with outcomes in ADE-treated AML patients, leukemic cell gene expression was evaluated for association with clinical endpoints such as event-free survival (EFS), overall survival (OS), minimal residual disease after induction 1 (MRD1) and relapsed/refractory disease (RR) in pediatric AML from two clinical cohorts, which multisite AML02 trial (n=163) and publicly available TARGET database (only patients treated with the ADE arm of AAML0531, n=213) were included. At the SD cut-off, 888, 114, and 878 are identified as drug sensitive genes, while 306, 1301, and 412 are resistant genes to cytarabine, daunorubicin, or etoposide, respectively (Figure 1). These significant genes are further tested for associations between expression levels and clinical endpoints in ADE-treated pediatric AML in two independent cohorts. Venn diagram in Figure 2 shows the overlapped genes between CRISPR hits and genes with significant clinical outcome associations in AML02 and TARGET-AAML0531. Of particular interest, five genes are significant CRISPR hits for all three drugs (all are sensitive to cytarabine and etoposide but resistant to daunorubicin), as well as for the clinical outcome associations. Among these, GABARAP, GYPA, and SUCLG2 are associated with better outcomes, while C11orf58 and MATR3 are associated with detrimental outcomes. Furthermore, Figure 2 also shows a heat map that illustrates the association between expression levels and clinical outcomes of these five genes with the available EFS, OS, MRD1, and RR (with and without risk adjustment) in two cohorts. Notably, GYPA (CD235a) is an erythroid cell membrane protein, and high leukemic cell GYPA expression is shown to be associated with favorable EFS and OS in AML02 (EFS HR=0.48, p=0.004 with risk-adjusted HR=0.45 p=0.002; OS HR=0.46, p=0.012 with risk-adjusted HR=0.43, p=0.006) and EFS in TARGET-AAML0531 (HR= 0.67, p=0.0038 with risk-adjusted HR=0.72 p=0.08). Another notable gene with detrimental outcome associations is MATR3, which has been shown to have a deleterious effect on cell fate transitions, chromatin structure, and cell differentiation (PMID: 34716321). Overall, our study coupled the results of a genome-wide CRISPR/Cas9 screen with leukemic cell gene expression and clinical outcomes identified clinically and functionally significant ADE genes. C11orf58, MATR3, GABARAP, GYPA, and SUCLG2 are potential novel biomarkers associated with prognosis in the context of pediatric AML treated with ADE. Our future work is focused on identifying the most optimal combination of induction regimens based on the transcriptomic signature of patients’ leukemic cells. Acknowledgments: NIH-R01-CA132946 (Lamba and Pounds), NIH-T32-HG008958 (UF PARADIGM Training Program), University of Florida Opportunity Seed Grant (Lamba), American Lebanese Syrian Associated Charities (ALSAC). We thank Dr. Roya Rafiee for CRISPR/Cas9 drug screen contribution; Drs. Campana, Coustan-Smith, and Susana Raimondi for MRD and cytogenetic data. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A ten-gene DNA-damage response pathway gene expression signature predicts gemtuzumab ozogamicin response in pediatric AML patients treated on COGAAML0531 and AAML03P1 trials.

Gemtuzumab ozogamicin (GO) is an anti-CD33 monoclonal antibody linked to calicheamicin, a DNA damaging agent, and is a well-established therapeutic for treating acute myeloid leukemia (AML). In this study, we used LASSO regression modeling to develop a 10-gene DNA damage response gene expression score (CalDDR-GEx10) predictive of clinical outcome in pediatric AML patients treated with treatment regimen containing GO from the AAML03P1 and AAML0531 trials (ADE + GO arm, N = 301). When treated with ADE + GO, patients with a high CalDDR-GEx10 score had lower complete remission rates (62.8% vs. 85.5%, P = 1.7 7 * 10-5) and worse event-free survival (28.7% vs. 56.5% P = 4.08 * 10-8) compared to those with a low CalDDR-GEx10 score. However, the CalDDR-GEx10 score was not associated with clinical outcome in patients treated with standard chemotherapy alone (ADE, N = 242), implying the specificity of the CalDDR-GEx10 score to calicheamicin-induced DNA damage response. In multivariable models adjusted for risk group, FLT3-status, white blood cell count, and age, the CalDDR-GEx10 score remained a significant predictor of outcome in patients treated with ADE + GO. Our findings present a potential tool that can specifically assess response to calicheamicin-induced DNA damage preemptively via assessing diagnostic leukemic cell gene expression and guide clinical decisions related to treatment using GO.

Genome-Wide Association Study (GWAS) Identifies SNPs in GPR56 and IGF1R Predictive of Leukemic Cell In Vitro cytarabine Sensitivity in Pediatric AML

Introduction: Acute myeloid leukemia (AML) is the second most common pediatric leukemia with the worst prognosis of all major cancers in pediatrics. Resistance is the most common reason for treatment failure and adverse side effects also contribute to morbidity and mortality. Since cytarabine is the mainstay of AML treatment, the wide interpatient variability in response to cytarabine complicates chemotherapy further. This necessitates the identification of additional genetic variants important in the susceptibility to cytarabine cytotoxicity in order to allow for personalized treatment that maximizes efficacy and minimizes toxicity. Previous studies have utilized genome-wide gene expression signatures to identify markers associated with cytarabine response. Few studies have also utilized lymphoblastoid cell lines from normal individuals available through HapMap project to perform genome-wide association analysis (GWAS) to identify genetic-variants and gene-expression associated with in vitro cellular cytotoxicity to cytarabine. However, to best of our knowledge no GWAS study has so far been performed on in vitro cytarabine chemosensitivity of leukemic cells obtained from pediatric AML patients.Hypothesis: To identify SNPs predictive of cytarabine in vitro chemosensitivityof leukemic cells obtained from pediatric AML patients (St Jude AML02 clinical trial) at diagnosis.Methods: Leukemic cells from diagnosis obtained from N=65 patients were treated with varying concentrations of cytarabine and IC50 values were obtained.Samples were divided into resistant (n= 15; IC50 > 5ng/μl) and sensitive groups (n=50; IC50 < 5ng/μl), Figure 1A. Genome-wide SNPdata were generated using Illumina Omni 2.5M and Exome Beadchip SNP array at University of Miami, Hussman Institute for Human Genomics. We performed the standard GWAS QC procedure in order to remove SNPs with call rate < 95%, monomorphic SNPs, SNPs with MAF < 5% and samples with call rate < 95%. Following QC, GWAS was performed on the remaining high quality SNPs (n=1,317,146) to identify if any of these are significantly associated with resistant or sensitive groups. Second, genes to which SNPs passing the suggestive threshold (p < 1x10-4) map and genes within +/-500kb of these SNPs were tested for differential expression between sensitive and resistant groups using T-test. Third, SNPs mapping to genes or SNPs within 500kb from genes that were differentially expressed (nominal p<0.05) were then tested for their eQTL effect using T-test or ANOVA.Results: Although none of the SNPs reached genome-wide significance (p<5.0 × 10-8), 112 SNPs located within or near 68 unique genes reached the suggestive level (p<1x10-4,Figure 1B). Among the top candidate SNPs were GPR56 rs1376041 G>A and rs75400242 G>ASNP found within 500kb of IGF1R gene. For both these SNPs the minor allele A was significantly associated with cytarabine resistance (p=1.42x10-5 and p=2.19x10-5, respectively). Furthermore, the minor allele of rs1376041 and rs75400242 was associated with a significantly higher level of GPR56 and IGF1R gene expression level (p=0.019 and p=0.044). Results for GPR56 for association of rs1376041 SNP with cytarabine resistance and GPR56 gene expression are shown in Figure 2A and 2C. Consistent with above observation for both GPR56 (Figure 2B) and IGF1R higher leukemic cell gene expression was associated with resistant group as compared to sensitive cases (p=0.0313 and p=0.0270).Conclusions: These results suggest that these SNPs might contribute to cytarabine in vitro sensitivity through regulation of gene expression. This is biologically relevant since it has been documented that GPR56 expression is associated with cytarabine resistance and poor outcomes in AML. In fact, recent data suggests high expression of GPR56 to contribute to AML development identified it as a potential target for antibody targeted therapy. For IGF1R, consistent with our results it has been associated with promoting growth of AML cells and cytarabine resistance as well. Future functional pharmacogenomic studies will help in better understanding the mechanisms behind the SNP associations reported here.Acknowledgments: We are thankful for funding from NIH R01CA139246 and Dr. McDonough for helpful discussions regarding GWAS analysis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

GATA1, GATA2, and TAL1 Regulate the Expression of Neurotrophic Receptor Tyrosine Kinase in Leukemia Cells

Introduction Acute myeloid leukemia (AML) is a heterogeneous disease with poor overall survival and a high relapse rate. Transcription factors GATA1, GATA2, and TAL1 are key regulators of hematopoietic gene expression during normal embryonic and adult blood cell differentiation. Still, their roles in the regulation of cancer-specific genes during AML progression and therapy response remain unclear. Aims: Here, we aimed at the description of transcriptomic signatures of GATA1, GATA2, and TAL1 and identification of novel therapeutic targets in AML. Methods By the lentiviral transduction we have obtained leukemia (HL-60, Kasumi-1, THP-1) and neuroblastoma (SH-SY5Y) cell lines with overexpression of GATA1 and GATA2. Confocal microscopy was used to detect TrkA protein localization in cells. By qRT-PCR, we measured the expression of genes coding GATA1, GATA2, TAL1, and TrkA at mRNA levels. We used 5-azacytidine (5-aza) to induce changes in gene expression in leukemia cells. Cell viability was measured by trypan blue exclusion in the Neubauer chamber. Results First, we performed extensive data mining: analysis of publicly available transcriptome analysis data of 422 AML patients revealed genes differentially expressed (DEGs) with GATA1, GATA2, and TAL1. We found 125 genes common for GATA1, GATA2, and TAL1 including NTRK1 (encoding TrkA protein). Notably, by k-means clustering, we showed that patients with the induced level of GATA/TAL DEGs have poor overall survival. Previously we showed that leukemia cells have specific intracellular localization of TrkA protein. Ectopic expression of GATA1 and GATA2 in AML cells induced NTRK1 expression at mRNA and protein levels. 5-aza treatment of HL-60 cells resulted in the upregulation of GATA1, TAL1, and NTRK1 at mRNA and protein levels. Also, recombinant NGF (TrkA ligand) was able to rescue AML cells from 5-aza induced death. Conclusions Our data provide a novel mechanism of the GATA1, GATA2, and TAL1 involvement in AML progression. We show that NTRK1 expression is regulated by GATA1, GATA2, and TAL1 in leukemia but not in neuroblastoma cells. Our findings suggest the existence of GATA1, GATA2, and TAL1-driven control of non-hematopoietic genes in AML cells.

Single-Cell Transcriptomic Profiling of De Novo and Relapsed Acute Myeloid Leukemia Identifies a Leukemic Stemness Program Shared across Diverse Phenotypes

Introduction: Relapse remains the major cause of mortality in acute myeloid leukemia (AML). Prior work indicates that a rare subset of leukemic stem cells (LSCs) self-renew and propagate AML. However, characterizing LSCs is complicated by their scarcity, the lack of universal markers and the heterogeneity across patients. Here, we aim to define a transcriptional program associated with LSCs in patient samples. Methods: We performed single-cell RNA sequencing (scRNA-seq) scRNA-seq of bone marrow from 19 AML samples (14 patients) using the 10X Chromium 3' v2 platform. These samples span multiple morphologies, genetic alterations, and disease stages. Leukemic and normal cells were distinguished based on agreement of three methods: (i) canonical marker expression, (ii) clustering analysis in a multi-sample dataset, and (iii) inferred chromosomal alterations. Leukemic cells were mapped to a panel of signatures from the Human Cell Atlas to infer the most similar normal cell-type, using single-cell gene-set enrichment analysis. Transcription factor activity was inferred at the single-cell level using the SCENIC workflow. Cell state trajectories were constructed using Monocle v2. Common driver mutations were detected at the bulk level using targeted gDNA sequencing and in single cells with targeted amplification of cDNA libraries. A validation cohort of samples was processed with the CITE-seq protocol to capture single-cell gene expression and surface protein levels for CD34, CD38, CD123, CLL1, and TIM3. Results: We captured a total of 55,355 cells meeting quality thresholds, with a median of ~2,800 cells/sample. We observed a large inter-patient heterogeneity with cells segregating largely by sample (Fig. 1A), which was not explained by morphological subtype, treatment received, or driver mutations. As previously described, similarity in gene expression of longitudinal samples did not depend on time before relapse. However, we found transcriptional similarity in a group of samples with relatively silent CNV profiles, suggesting that large chromosomal alterations are a main driver of inter-patient variability. We also observed variation in terms of nearest normal cell assignment: while some samples contained cells resembling diverse mature cell types, others had an abundance of stem-like cells, confirmed by high activity of transcription factors involved in self-renewal (e.g. HOXA9, GATA2). To analyze intrasample variation, we performed Principal Component Analysis and found that, in over half of the samples, LSC and maturation genes were the main source of transcriptional variation. A gradient of activation of known LSC signatures was detected in these samples (Fig 1B). Cell state trajectory reconstruction indicated a continuum of LSC gene expression in leukemic cells. Interestingly, expression of known LSC genes was mostly diffuse is a small subset of samples, a finding that suggests that LSC activity may be widespread in these cases but remains to be validated functionally. Finally, we derived a stemness signature correlated with LSC in our cohort, by extracting concordant genes in a ranked correlation analysis and reconstruction of gene regulatory networks. This yielded a recurrent stemness signature that included previously described LSC-associated genes that were not part of our input, as well as novel factors with expression highly specific to the most LSC-like cells (Fig 1C). To validate this novel stemness signature, we experimentally determined LSC frequencies in a separate cohort (N=5) by xenotransplantation according to expression of CD34 and CD38, and confirmed higher expression of our signature in the LSC fraction. Conclusions: Within a genetically and phenotypically diverse cohort of patients, we could identify, at single-cell resolution, recurrent programs of stemness and myeloid maturation. Altogether, we provide novel candidates for a transcriptional program of putative LSC drivers with therapeutic relevance in AML. Figure Disclosures Johnson: AbbVie: Research Funding; Roche/Genentech, Merck, Bristol-Myers Squibb, AbbVie: Consultancy; Roche/Genentech, Merck: Honoraria. Assouline:Takeda: Research Funding; AbbVie: Consultancy, Honoraria, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding. Mercier:Sanofi-Genzyme: Consultancy.

Cirmtuzumab Consolidation for Treatment of Patients with Detectable CLL on Venetoclax

Background: Venetoclax (Ven)-based regimens are highly effective in treating patients with chronic lymphocytic leukemia (CLL), effecting clearance of detectable minimal residual disease (MRD) in many patients after just 1 year of therapy. Approximately, 30% of patients fail to clear detectable MRD within this time period and risk developing, progressive disease (PD) despite continued Ven therapy. Moreover, these patients have a relatively short progression free survival (PFS) after stopping fixed-duration therapy. Therefore, development of consolidation strategies to eradicate detectable MRD and/or to mitigate the risk of PD in patients who fail to clear MRD after 1 year of Ven-based therapy is of vital clinical importance. In particular, patients who fail to clear MRD after 1 year of Ven-based therapy harbor CLL cells with high-level expression of ROR1, an oncoembryonic protein expressed by a broad array of incurable malignancies. Patients who are refractory to MRD-clearance on Ven-based therapies have gene-expression signatures reflective of high-level ROR1-signaling, which can be inhibited by the anti-ROR1 mAb, Cirmtuzumab (Choi et al., Cell Stem Cell. 2018; 22: 951-959). Prior studies demonstrated that Cirmtuzumab and Ven have at least additive activity in promoting cytotoxicity of CLL cells in vitro (Rassenti et al. PNAS. 2017;114(40):10731-10736). As such, cirmtuzumab may be an effective consolidation strategy for patients who fail to clear MRD following completing 1 year of Ven-based therapy. Study Design and Methods: Study Design: We initiated a single center, phase 2 Simon's two-stage-designed study to determine the efficacy of Cirmtuzumab plus Ven combination therapy in patients who fail to clear detectable MRD (defined as &amp;gt; 0.01% of the blood mononuclear cells, as assessed by flow cytometry) after ≥1 year of Ven-based therapy (NCT04501939). Enrolled patients will receive cirmtuzumab at 600 mg IV, q14 days for 2 doses and then monthly thereafter. Patients will continue oral Ven at 400mg daily and be monitored for safety and PD. Following 6 months of Cirmtuzumab + Ven, patients will be assessed for MRD in the blood. At the discretion of the treating physician, patients may continue combination treatment for up to one year (Figure 1). Major Eligibility Criteria: Patients must have a diagnosis of CLL with detectable MRD after having received ≥ 12 months of Ven-based treatment . Patients will be excluded from the study if they have an uncontrolled medical co-morbidity, which compromises their capacity to complete the study, or if they are receiving treatment for CLL with agents other than Ven. End Points: The primary end-point of the proposed study is clearance of detectable MRD in the blood after 6 months of cirmtuzumab + Ven therapy. Secondary end-points include safety and time to next CLL treatment. Exploratory end-points include changes in gene expression in leukemia cells, including analysis of archival pre-Ven treatment samples, when available. Statistical Methods: We used a Simon's two-stage minimax design at 5% significance level to test the scientific hypothesis that 6 cycles of cirmtuzumab + Ven will achieve undetectable MRD (a 'response') in more than 5% of patients, against the null hypothesis of less than a 5% response rate. In the first stage, we plan to accrue 12 patients. If none of these patients respond at 6 months, the study will close. Otherwise, we will accrue an additional 4 patients for a total of 16 patients. If three or more patients respond, the primary endpoint of the trial will have been met, demonstrating an MRD clearance rate greater than 5%. The trial is designed to have 80% power to provide such evidence at the 5% significance level if the true rate of clearance of MRD is 25% or greater. Disclosures Heyman: Oncternal Therapeutics, INC: Research Funding. Choi:Pharmacyclics/Abbvie: Research Funding; Genentech: Consultancy. Jamieson:Forty Seven Inc: Patents &amp; Royalties; Bristol-Myers Squibb: Other. Kipps:Genentech/Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; VelosBio: Research Funding; Oncternal Therapeutics, Inc.: Other: Cirmtuzumab was developed by Thomas J. Kipps in the Thomas J. Kipps laboratory and licensed by the University of California to Oncternal Therapeutics, Inc., which provided stock options and research funding to the Thomas J. Kipps laboratory, Research Funding; Celgene: Honoraria, Research Funding; Ascerta/AstraZeneca, Celgene, Genentech/F. Hoffmann-La Roche, Gilead, Janssen, Loxo Oncology, Octernal Therapeutics, Pharmacyclics/AbbVie, TG Therapeutics, VelosBio, and Verastem: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pharmacyclics/ AbbVie, Breast Cancer Research Foundation, MD Anderson Cancer Center, Oncternal Therapeutics, Inc., Specialized Center of Research (SCOR) - The Leukemia and Lymphoma Society (LLS), California Institute for Regenerative Medicine (CIRM): Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Paired Analyses of AML at Diagnosis and Relapse By Single-Cell RNA Sequencing Identifies Two Distinct Relapse Patterns

Introduction: Despite achievement of complete remission (CR) following chemotherapy, Acute Myelogenous Leukemia (AML) relapses in the majority of adult patients. While relapsed AML is almost always clonally related to the disease at diagnosis, the actual molecular and cellular contributors to chemotherapy resistance and to AML relapse remain incompletely understood. Some molecular determinants of relapse have been identified in genomic, epigenetic and proteomic aberrations, while cellular relapse reservoirs have been identified in leukemia stem cells as well as in more mature leukemic cell compartments. Here, we set out to determine the cellular composition, gene mutation status and gene expression of paired AML specimens procured at diagnosis and at relapse aiming at a better understanding of the AML relapse process. Methods: We employed the drop-seq 3' single cell RNA sequencing (scRNA-seq) method (Macosko 2015) with minor modifications to analyze the mRNA expression in single cells derived from 12 paired AML specimens procured at diagnosis and at relapse from prior CR. We obtained scRNA-seq data on 1000-2000 single cells per sample detecting approximately 2000-3000 unique molecular identifiers (UMIs) and 800-1500 genes per cell. Using WES or panel-based sequencing we determined mutations in known driver genes. Subsequently, we optimized novel methods for detection and mapping of mutated driver genes to individual cells using mutation specific PCR conditions and novel bioinformatics approaches. We annotated scRNA-seq expression profiles of the diagnosis and relapsed AML specimens individually using publicly available data for cell type-specific RNA markers derived from sorted normal cell populations and further compared the scRNA-seq data to scRNA-seq data of 5 pooled normal human bone marrows generated for this study. Results: Through analyses of scRNA-seq data of paired diagnosis and relapse AML specimens via principle components analyses (PCA) or t-distributed stochastic neighbor embedding (t-SNE) we detected varying degrees of separation of cell clusters in all cases analyzed indicative of substantial changes in single cell gene expression between AML diagnosis and relapse. A few of these observed cluster shifts were paralleled by gain or loss of mutated genes (e.g. FLT3-ITD) at relapse while most others lacked obvious clonal genomic markers. Through subsequent comparison of the expression similarities of single AML cells to sorted normal human bone marrow cells we detected two distinct AML relapse patterns: i) a pattern of relapse suggesting simple leukemia regrowth as evidenced by similar proportions of leukemia cells mapping onto discrete normal bone marrow cells (e.g. monocyte-like or GMPs or CMPs), and, ii) a pattern of relapse whereby the gene expression of relapsed cells (but not diagnosis cells) had similarity to normal hematopoietic cells that are conventionally placed more apical in the classical hematopoiesis differentiation cascade (HSCs, MPPs, CMPs; a phenotypic shift to immaturity). In addition, no leukemia sample mapped to just one classically defined bone marrow cell type but instead to multiple cell types, suggesting that most AML leukemia cells harbor aberrant hybrid cell gene expression patterns. Finally, we detected quantitative shifts in T cells and NK cells in some samples at relapse, which will be analyzed in greater detail. Conclusions: The comparative analysis of scRNA-seq data of paired AML specimens procured at diagnosis and relapse, identifies frequent and previously unrecognized changes in gene expression in leukemia cells at relapse. Through a comparison of gene mutation and gene expression at single cell resolution we identify two distinct AML relapse patterns in adult AML. Disclosures No relevant conflicts of interest to declare.

A 5-Gene Ara-C, Daunorubicin and Etoposide (ADE) Drug Response Score As a Prognostic Tool to Predict AML Treatment Outcome

Introduction: Cytarabine, daunorubicin and etoposide (ADE) are commonly used for remission and intensification of pediatric acute myeloid leukemia (AML). However, development of drug resistance is a major cause of treatment failure. In this study, we performed a comprehensive evaluation of expression levels of genes of pharmacological significance (pharmacokinetic /pharmacodynamic) to ADE and derived a drug response score predictive of treatment outcomes in pediatric AML patients. Methods: This study included 163 cases (median age=8.79 year, range= (0.013-21.1)) with AML enrolled in the multicenter AML02 clinical trial (ClinicalTrials.gov Identifier: NCT00136084) with Affymetrix U133A microarray gene expression and clinical data available. We used a penalized LASSO regression algorithm (glmnet R-package) to fit a cox regression model on diagnostic leukemic cell gene expression levels of 66 genes of pharmacological significance to ADE. We performed 1000 bootstraps of LASSO regression with event free survival (EFS) as the outcome variable and the five genes represented in at least 95% of the models were included to build an ADE-Response Score (ADE-RS) equation. Patients were classified into low or high score groups using recursive portioning implemented in Rpart-R package and evaluated for association with minimal residual disease after induction I (MRD1), EFS and overall survival (OS). ADE response score equation was further validated using RNA-Seq gene-expression data obtained from diagnostic samples of 432 pediatric AML patients enrolled in Children's Oncology Group (COG) AAML0531 and AAML03P1 treatment protocols. Results: After applying LASSO regression, we defined the equation: ADE-RS = (0.128 x DCTD) - (0.0993 x TOP2A) + (0.212 x ABCC1) - (0.113 x MPO) - (0.126 x CBR1) to develop ADE-response score (ADE-RS), followed by classifying patients into low (60%; 98 patients) or high (40%; 65 patients) score groups. Patients in the high ADE-RS group had significantly worse EFS (HR=4.07(2.43-6.84), P &amp;lt; 0.0001; Figure 1A) and OS (HR= 4.54(2.42-8.49), P&amp;lt;0.0001) and higher proportion of MRD1 positive patients (P=0.014; Figure 1B) compared to patients in the low ADE-RS group. These results were validated in an independent COG cohort, where patients in the high score group demonstrated higher MRD1 positivity (P=0.0005; Figure 1D) and inferior EFS (HR=1.32(1.01-1.73), P=0.044; Figure 1C). We recently developed a six-gene leukemic stem cell score (pLSC6 score) that was associated with risk-groups (P&amp;lt;0.00001) and outcome (oral presentation ASH2018, Under-review, Leukemia). We further integrated pLSC6 and ADE-RS and observed significantly better prediction of treatment outcomes in AML02, COG and TCGA cohorts. Based on pLSC6 and ADE-response scores, patients were classified into three groups; 1) Low/Low:pLSC6/ADE-RS; for patients with low pLSC6 and low ADE-RS. 2) Low/High:pLSC6/ADE-RS: for patients in low pLSC6 and high ADE-RS or vice versa.3) High/High:pLSC6/ADE-RS: for patients in high pLSC6 and high ADE-RS. In all study cohorts, patients in low/low pLSC6-ADE-RS group demonstrated better outcomes compared to the low-high and the high/high score groups (EFS in AML02 cohort; Figure 1E and OS; Figure 1F). In a multivariable cox-regression models that included pLSC6-ADE response score groups, MRD1 status, risk groups, WBC at diagnosis and age in AML02 cohort, high pLSC6-ADE score group was found significantly associated with poor EFS (HR=6.02(2.71-13.2), P&amp;lt;0.00001; Figure 1G) and was the only significant predictor of poor OS (HR=8.3(2.9-23.99), P&amp;lt;0.00001; Figure 1H) Discussion: In this study, we defined a pharmacological response score focused on key genes of PK/PD significance to ADE. We further integrated LSC score with the ADE response score to improve our ability to predict treatment outcome in AML patients across different clinical trials. ADE-RS was composed of five genes: DCTD, which is a deaminase, involved in ara-C inactivation; CBR1, a carbonyl reductase involved in inactivation of daunorubicin (DNR); MPO, myeloperoxidase, an etoposide activator; ABCC1, an efflux transporter of DNR and etoposide; and TOP2A, DNA topoisomerase II alpha, which is a target for DNR and etoposide. Integrated pLSC6 and ADE-RS has a potential to predict treatment outcomes using diagnostic gene expression levels and accordingly develop treatment strategies to improve treatment outcome. Figure 1 Disclosures Gruber: Bristol-Myers Squibb: Consultancy. Rubnitz:AbbVie: Research Funding.

Inactivation of Prostaglandin E2 as a Mechanism for UGT2B17-Mediated Adverse Effects in Chronic Lymphocytic Leukemia

High expression of the metabolic enzyme UDP-glucuronosyltransferase UGT2B17 in chronic lymphocytic leukemia (CLL) cells was associated with poor prognosis in two independent studies. However, the underlying mechanism remains unknown. We hypothesized that UGT2B17 impacts intracellular levels of hormone-like signaling molecules involved in the regulation of gene expression in leukemic cells. We initially confirmed in a third cohort of 291 CLL patients that those with high UGT2B17 displayed poor prognosis (hazard ratio of 2.31, P = 0.015). Consistent with the unfavorable prognostic significance of elevated UGT2B17 expression in CLL patients, high UGT2B17 expression was associated with enhanced proliferation of MEC1 and JVM2 malignant B-cell models. Transcriptomic analyses revealed that high UGT2B17 was linked to a significant alteration of genes related to prostaglandin E2 (PGE2) and to its precursor arachidonic acid, both in cell models and a cohort of 448 CLL patients. In functional assays, PGE2 emerged as a negative regulator of apoptosis in CLL patients and proliferation in cells models, whereas its effect was partially abrogated by high UGT2B17 expression in MEC1 and JVM2 cells. Enzymatic assays and mass-spectrometry analyses established that the UGT2B17 enzyme inactivates PGE2 by its conjugation to glucuronic acid (GlcA) leading to the formation of two glucuronide (G) derivatives. High UGT2B17 expression was further associated with a proficient inactivation of PGE2 to PGE2-G in CLL patient cells and cell models. We conclude that UGT2B17-dependent PGE2 glucuronidation impairs anti-oncogenic PGE2 effects in leukemic cells, thereby partially contributing to disease progression in high UGT2B17 CLL patients.

Tecfidera Modulates Activation of Malignant B-Cells: Correlative Analysis from a Phase 1 Clinical Trial in Patients with Chronic Lymphocytic Leukemia

Tecfidera (Dimethylfumarate, DMF) is an immunomodulatory drug currently approved in the United States for the treatment of patients with multiple sclerosis (MS). DMF formulations have also been utilized for the treatment of patients with psoriasis. The ability of DMF to Inhibit autoreactive T-lymphocytes may also be applicable to malignant T- and B- lymphocytes. Indeed, preclinical studies by multiple groups have highlighted its potential efficacy as a treatment for patients with various lymphoid cancers, though inhibition of STAT, NF-KB, Wnt signaling pathways, and glycolysis. This information provided rationale to conduct a phase 1 clinical trial of DMF as a treatment for patients with relapsed chronic lymphocytic leukemia (CLL). In this study we report the correlative assessment of the initial patients who received DMF.Patients with relapsed or refractory CLL received DMF 120 mg PO BID (based on preclinical modeling with primary CLL cells in vitro and in vivo and 50% lower than the standard DMF dose for patients with MS). Treatment was planned for a short duration of 2 months to evaluate tolerability and effects on CLL cell signaling and tolerability of the drug by patients with CLL. Peripheral blood leukemic cell were isolated and analyzed by RNAseq and phosphoprotein immunoblotting at times just prior to, and 6 hours immediately following drug administration.Consistent with preclinical results (Brennan et al, PLOS one 2015; Selman et al, Science Trans Med 2018), STAT1 phosphorylation at Y701 was decreased 6 hours following the initial dose, compared to high baseline levels. Differential expression analyses for RNAseq of total RNA isolated from peripheral blood CLL cells weere performed using limma models, which compared pretreatment versus post-treatment at a specific time point. Functional annotations of these genes revealed a significant enrichment related to B-cell activation, B-cell receptor (BCR) signaling, B-cell differentiation, and immune signaling generally. Specifically, several known CLL therapy targets including SYK, LYN, and BTK were significantly downregulated. Gene-set-enrichment analysis (GSEA) revealed that the “CLL-BCR gene signature” previously described by Herishanu et al. (Blood 2011) was consistently down-regulated after DMF treatment in samples from different time points (C1D1 post, C1D8 and C2D1) compared to pretreatment samples (FDR q of 0.24, 0.01 and 0.04, respectively).In conclusion, this is the first report of the clinical use of DMF for patients with CLL in which inhibition of leukemic cell activation has been demonstrated. The inhibitory effect of DMF on leukemic cell gene expression, in particular its antagonism of BCR signaling, is valuable information for the development of novel combination strategies for CLL or other B-cell malignancies. DisclosuresKipps:F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Research Funding; Genentech Inc: Consultancy, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Choi:Gilead: Speakers Bureau; Genentech: Speakers Bureau; Rigel: Consultancy; AbbVie, Inc: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Research Funding, Speakers Bureau.

Gene Aberrations in Adult Patients with Ph-Negative ALL in the Czech Republic

IntroductionPhiladelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is an identified high-risk B-lineage ALL subtype, accounting for approximately 20% of adult patients with B-ALL. Ph-like ALL is characterized by poor outcome, very high risk of disease relapse and poor overall survival (OS). The leukemic cell gene expression profile of Ph-like ALL is similar to that of Ph-positive ALL. However, instead of BCR-ABL1, such patients harbor a highly diverse range of genetic alterations. The gene aberrations detected in Ph-like ALL patients are associated with JAK/STAT, Ras, Ikaros and ABL signaling pathways. These aberrations can be subdivided into five distinct subgroups based on the type of cytokine receptor or kinase fusion present: 1) rearrangements of CRLF2; 2) ABL-class gene rearrangements; 3) JAK2 and EPOR rearrangements; 4) sequence mutations or deletions activating JAK-STAT or MAPK signaling pathways and 5) other rare kinase alterations. The study aimed to define the frequency of high-risk genetic aberrations associated with Ph-like subtype in adults with Ph-negative ALL from the Czech Republic. The genetic basis in adult patients with Ph-like ALL has not been studied to date.Patients and MethodsThe molecular genetic analysis was performed in 74 Ph-negative ALL adult patients from routine hematologic practice. DNA and RNA were isolated from patient´s peripheral blood or bone marrow. The mutational analysis was focused on the detection of the P2RY8-CRLF2 fusion gene (real-time PCR) and the “hot spot” region of the JAK2 gene (PCR/Sanger sequencing) in patient´s cohort. Minimal residual disease (MRD) value was also assessed in all patients prior to the first consolidation cycle (week 11) by real-time PCR. The detection of copy number variations (MLPA method) and targeted sequencing (NGS) was performed in a selected set of patients.ResultsThe patient cohort of Ph-negative ALL consisted of 46.6% young adults (age 15 to 39 years), 35.6% adults (age 40 to 59 years), and 17.8% older adults (age 60 to 75 years). Among all patients, increasing age was associated with an inferior outcome of the disease. The difference in OS was significant for young adults compared with adults and older adults (median: 126.7, 23.5 and 22.6, respectively; P=0.0087). The above-mentioned gene aberrations were identified in 36.5% of patients with Ph-negative ALL. The 3-year OS for patients with and without gene aberrations was 36% and 49%. Copy number alterations were detected in almost a third of patient cohort in several genes (IKZF1, PAX5, BTG, ETV6, EBF1, CDKN2A/2B, and SHOX). Deletions in IKZF1 were the most frequent aberration (17.8%). Within the patient cohort, the presence of IKZF1 deletions did not influence the OS significantly. The presence of P2RY8-CRLF2 fusion gene was identified in 16.4% patients with markedly inferior OS in comparison to a patient group without detected P2RY8-CRLF2 (median: 33.2 and 126.7). Sequence mutations of TP53, IL7R, and JAK2 genes occurred in 14.3%, 2.9%, and 1.6% patients, respectively. All patients with the detected sequence variant were carriers of at least one other gene aberration. Achieving MRD negativity in patients with Ph-like aberrations tended to more favorable OS in comparison to those MRD positive, but this trend was not statistically significant enough.ConclusionsOur findings demonstrate the representation of selected gene aberrations in Ph-negative ALL adults patients. Also they have led to the introduction of molecular genetic diagnostics of Ph-like ALL to routine hematological practice in the Czech Republic. MRD negativity and younger age are the most important prognostic factors in this subgroup of patients according to our analysis.This study was supported by TA CR (TE02000058), MH CZ - DRO (FNBr, 65269705) and CEITEC 2020 (LQ1601), and MEDGENET 2020 (692298). DisclosuresFolber:Affimed: Research Funding. Doubek:Roche: Consultancy, Honoraria; Affimed: Research Funding; AbbVie: Consultancy, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Novartis: Consultancy.

Genome-wide association analysis identifies SNPs predictive of in vitro leukemic cell sensitivity to cytarabine in pediatric AML.

Cytarabine has been an integral part of acute myeloid leukemia (AML) chemotherapy for over four decades. However, development of resistance and high rates of relapse is a significant impediment in successfully treating AML. We performed a genome-wide association analysis (GWAS) and identified 113 (83 after adjusting for Linkage Disequilibrium) SNPs associated with in vitro cytarabine chemosensitivity of diagnostic leukemic cells from a cohort of 50 pediatric AML patients (p<10-4). Further evaluation of diagnostic leukemic cell gene-expression identified 19 SNP-gene pairs with a concordant triad of associations: i)SNP genotype with cytarabine sensitivity (p<0.0001), ii) gene-expression with cytarabine sensitivity (p<0.05), and iii) genotype with gene-expression (p<0.1). Two genes from SNP-gene pairs, rs1376041-GPR56 and rs75400242-IGF1R, were functionally validated by siRNA knockdown in AML cell lines. Consistent with association of rs1376041 and gene-expression in AML patients siRNA mediated knock-down of GPR56 increased cytarabine sensitivity of AML cell lines. Similarly for IGF1R, knockdown increased the cytarabine sensitivity of AML cell lines consistent with results in AML patients. Given both IGF1R and GPR56 are promising drug-targets in AML, our results on SNPs driving the expression/function of these genes will not only enhance our understanding of cytarabine resistance but also hold promise in personalizing AML for targeted therapies.

Dichloromethane fraction of Melissa officinalis induces apoptosis by activation of intrinsic and extrinsic pathways in human leukemia cell lines

Various components from medicinal plants are currently used in cancer therapy because of their apoptosis-inducing effects. The present study has aimed to investigate the growth inhibitory and apoptotic effects of Melissa officinalis on tumor cells. We prepared different fractions of this plant to investigate their inhibitory effects on two leukemia cell lines, Jurkat and K562. Fractions with the highest inhibitory effects were examined for induction of apoptosis by the annexin V/propidium iodide assay and cell cycle changes by flow cytometry. Real-time polymerase chain reaction evaluated the changes in expression of apoptosis-related genes. Among different fractions, dichloromethane and n-hexane dose-dependent showed the strongest inhibitory effects on both K562 and Jurkat cells. The dichloromethane fraction significantly induced apoptosis at concentration of 50 µg/ml on Jurkat (85.66 ± 4.9%) and K562 cells (65.04 ± 0.93%) at 24 h after treatment (p < 0.002). According to cell cycle analysis, more than 70% of the cells accumulated in the sub-G1 phase when cultured in the presence of the dichloromethane fraction. This fraction up-regulated Fas and Bax mRNA expression as well as the Bax/Bcl-2 ratio according to cell type, showing its effect on the activation of both extrinsic and intrinsic pathways of apoptosis. The expression of apoptosis-related genes did not significantly change following treatment with the n-hexane fraction. These data indicated that the dichloromethane fraction of M. officinalis had the ability to induce apoptosis and change apoptosis-related gene expression in leukemia cells.

Integration of Different “-omics” Technologies Identifies Inhibition of the IGF1R-Akt-mTOR Signaling Cascade Involved in the Cytotoxic Effect of Shikonin against Leukemia Cells

Hematological malignancies frequently have a poor prognosis and often remain incurable. Drug resistance, severe side effects, and relapse are major problems of currently used drugs, and new candidate compounds are required for improvement of therapy success. The naphthoquinone shikonin derived from the Chinese medicinal herb, Lithospermum erythrorhizon, is a promising candidate for the next generation of chemotherapy. The basal cellular mechanism of shikonin is the direct targeting of mitochondria. Cytotoxicity screenings showed that the compound is particularly effective against leukemia cells suggesting an additional cellular mechanism. mRNA and miRNA microarrays were used to analyze changes in gene expression in leukemia cells after shikonin treatment and combined with stable-isotope dimethyl labeling for quantitative proteomics. The integration of bioinformatics and the three “-omics” assays showed that the PI3K-Akt-mTOR pathway was affected by shikonin. Deregulations of this pathway are frequently associated with cancerogenesis, especially in a wide range of hematological malignancies. The effect on the PI3K-Akt-mTOR axis was validated by demonstrating a decreased phosphorylation of Akt and a direct inhibition of the IGF1R kinase activity after shikonin treatment. Our results indicate that inhibiting the IGF1R-Akt-mTOR signaling cascade is a new cellular mechanism of shikonin strengthening its potential for the treatment of hematological malignancies.

Terpenic fraction of Pterodon pubescens inhibits nuclear factor kappa B and extracellular signal-regulated protein Kinase 1/2 activation and deregulates gene expression in leukemia cells

BackgroundPlant derived compounds have been shown to be important sources of several anti-cancer agents. As cell cycle deregulation and tumor growth are intimately linked, the discovery of new substances targeting events in this biochemical pathway would be of great value. The anti-leukemic effect of an ethanolic extract of Pterodon pubescens seeds (EEPp) has been previously demonstrated and now we show that a terpenic subfraction (SF5) of EEPp containing farnesol, geranylgeraniol and vouacapan derivatives induces apoptosis in the human chronic myelogenous leukemia cell line K562. This work addresses SF5’s antiproliferative mechanisms in these cells since they are still unclear.MethodsDNA synthesis in K562 cells was assessed by [3H]-methyl-thymidine incorporation and cell cycle status by flow cytometry. The expression of cyclins D1 and E2, of the cell cycle inhibitor p21 and of the proto-oncogene c-myc was evaluated by semi-quantitative RT-PCR. Extracellular-signal-regulated kinases (ERK) 1/2 and nuclear factor kappa B (NF-κB) activation was evaluated by western blotting.ResultsIn K562 cells, SF5 treatment induced a higher inhibition of DNA synthesis and cell growth than the original EEPp hexanic fraction from which SF5 originated, and also arrested the cell cycle in G1. Exposure of these cells to SF5 led to a decrease in cyclin E2 and c-myc expression while p21 mRNA levels were increased. Furthermore, SF5 inhibited the activation of mitogen-activated protein kinase (MAPK) ERK 1/2 and NF-κB.ConclusionsThis work suggests that the anti-leukemic action of SF5 is linked to the inhibition of ERKs, NF-κB and c-myc signaling pathways resulting in reduced cyclin E2 mRNA expression and cell cycle arrest in the G1 phase.

Use of qChIP to identify genes targeted by the Ikaros tumor suppressor

The IKAROS gene encodes a DNA binding zinc finger protein that is essential for hematopoietic differentiation and acts as a tumor suppressor in leukemia. The Ikaros protein is known to both activate and repress target gene expression and to participate in chromatin remodeling. The specific mechanisms by which Ikaros exerts it tumor suppressor activities are unknown. We are using Chromatin Immunoprecipitation (ChIP) assay combined with quantitative PCR (qPCR), called qChIP, to identify genes whose expression is directly regulated by Ikaros. Using this technique we calculate the difference in Ikaros binding to the promoter region of the target genes versus nonspecific binding (control). qChIP assays show that Ikaros binds to the promoter regions of the MYC oncogene, a known Ikaros target, and two other novel target genes. The promoter regions of MYC and the two novel target genes were cloned into luciferase reporter constructs and used to transiently transfect HEK cells that were co‐transfected with Ikaros or empty vector. Luciferase reporter assays showed that Ikaros suppresses the promoter activity of MYC and the two novel target genes. Our data confirm that the MYC oncogene is an Ikaros target and identify two novel Ikaros target genes. These data suggest that Ikaros exerts its tumor suppression function by inhibition of target gene expression in leukemia cells.Supported by R01 HL095120, a St. Baldrick's Foundation Career Development Award, the Four Diamonds Fund of Pennsylvania State University, College of Medicine (to SD), the NIDDK Short‐term Education Program for Under‐Represented Persons (to NFO) and a La Sierra University Dept. of Chemistry and Biochemistry stipend (to JLP).