IntroductionAcute Myeloid Leukemia (AML) is characterized by genetic and epigenetic dysregulation in myeloid progenitor cells. Histone H3 mutations disrupt chromatin and transcription, contributing to disease, but identifying mutations in histone genes is challenging due to sequence duplication. In particular, histone H2A genes H2AC18 and H2AC19, which share identical sequences, have been largely overlooked. MethodsCancer genomic datasets were interrogated to identify novel variants, and in vivo functional assays were conducted using Drosophila melanogaster tumor models.Resultsa recurrent Alanine to Valine substitution at position 127 (H2A-A127V) was identified in AML and also found in solid tumors, suggesting a broader cancer association. Using a Drosophila melanogaster model, H2A-A127V was shown to promote eye tumor phenotypes and, in the eyeful tumor model, intensified tissue overgrowth. The variant also interacts with the Enhancer of zeste (E(z)), a PRC2 complex member. E(z) downregulation in the presence of H2A-A127V enhanced ectopic growth, suggesting PRC2’s role in disease progression. Analysis of the 1000 Genomes Project revealed that H2A-A127V is an uncommon polymorphism (1.44%), potentially predisposing carriers to cancer.DiscussionThese findings uncover a novel cancer-associated histone variant and emphasize the need to consider duplicated histone genes in mutation analyses to better understand cancer development.

Targeted therapeutics for high-risk cancers remain an unmet medical need. Here we report the results of a large-scale screen of over 11,000 molecules for their ability to inhibit the survival and growth in vitro of human leukemic cells from multiple sources including patient samples, de novo generated human leukemia models, and established human leukemic cell lines. The responses of cells from de novo models were most similar to those of patient samples, both of which showed striking differences from the cell-line responses. Analysis of differences in subtype-specific therapeutic vulnerabilities made possible by the scale of this screen enabled the identification of new specific modulators of apoptosis, while also highlighting the complex polypharmacology of anti-leukemic small molecules such as shikonin. These findings introduce a new platform for uncovering new therapeutic options for high-risk human leukemia, in addition to reinforcing the importance of the test sample choice for effective drug discovery.

Over the years, the overall survival of older patients diagnosed with acute myeloid leukemia (AML) has not significantly increased. Although standard cytotoxic therapies that rapidly eliminate dividing myeloblasts are used to induce remission, relapse can occur due to surviving therapy-resistant leukemic stem cells (LSCs). Hence, anti-LSC strategies have become a key target to cure AML. We have recently shown that previously approved cardiac glycosides and glucocorticoids target LSC-enriched CD34+ cells in the primary human AML 8227 model with more efficacy than normal hematopoietic stem cells (HSCs). To translate these in vitro findings into humans, we developed a mathematical model of stem cell dynamics that describes the stochastic evolution of LSCs in AML post-standard-of-care. To this, we integrated population pharmacokinetic-pharmacodynamic (PKPD) models to investigate the clonal reduction potential of several promising candidate drugs in comparison to cytarabine, which is commonly used in high doses for consolidation therapy in AML patients. Our results suggest that cardiac glycosides (proscillaridin A, digoxin and ouabain) and glucocorticoids (budesonide and mometasone) reduce the expansion of LSCs through a decrease in their viability. While our model predicts that effective doses of cardiac glycosides are potentially too toxic to use in patients, simulations show the possibility of mometasone to prevent relapse through the glucocorticoid’s ability to drastically reduce LSC population size. This work therefore highlights the prospect of these treatments for anti-LSC strategies and underlines the use of quantitative approaches to preclinical drug translation in AML.

AbstractPurpose: Glioblastoma (GBM) is a fatal primary malignant brain tumor. GBM stem cells (GSC) contribute to resistance to the DNA-damaging chemotherapy, temozolomide. The epidermal growth factor receptor (EGFR) displays genomic alterations enabling DNA repair mechanisms in half of GBMs. We aimed to investigate EGFR/DNA combi-targeting in GBM. Experimental Design: ZR2002 is a “combi-molecule” designed to inflict DNA damage through its chlorethyl moiety and induce irreversible EGFR tyrosine kinase inhibition. We assessed its in vitro efficacy in temozolomide-resistant patient-derived GSCs, mesenchymal temozolomide-sensitive and resistant in vivo–derived GSC sublines, and U87/EGFR isogenic cell lines stably expressing EGFR/wild-type or variant III (EGFRvIII). We evaluated its antitumor activity in mice harboring orthotopic EGFRvIII or mesenchymal TMZ-resistant GSC tumors. Results: ZR2002 induced submicromolar antiproliferative effects and inhibited neurosphere formation of all GSCs with marginal effects on normal human astrocytes. ZR2002 inhibited EGF-induced autophosphorylation of EGFR, downstream Erk1/2 phosphorylation, increased DNA strand breaks, and induced activation of wild-type p53; the latter was required for its cytotoxicity through p53-dependent mechanism. ZR2002 induced similar effects on U87/EGFR cell lines and its oral administration significantly increased survival in an orthotopic EGFRvIII mouse model. ZR2002 improved survival of mice harboring intracranial mesenchymal temozolomide-resistant GSC line, decreased EGFR, Erk1/2, and AKT phosphorylation and was detected in tumor brain tissue by MALDI imaging mass spectrometry. Conclusions: These findings provide the molecular basis of binary EGFR/DNA targeting and uncover the oral bioavailability, blood–brain barrier permeability, and antitumor activity of ZR2002 supporting potential evaluation of this first-in-class drug in recurrent GBM.

Acute myeloid leukemia (AML) is an aggressive form of blood cancer. Despite the use of cytotoxic standard-of-care drugs, patients often succumb to the disease partially due to the inability of medically unfit patients to withstand the cytotoxic treatments, regrowth from minimal residual disease and the chemo-resistant nature of leukemic stem cells (LSCs). Hence, novel therapies should focus on targeting the unique biology of LSCs to eliminate and avoid reoccurrence. To overcome challenges inherent of screening rare LSCs in vitro such as culturing and expanding, we optimized the conditions for a 4-week in vitro large-scale expansion (>600 million bulk) and enrichment of the CD34+ LSC-containing fraction (>90% purity) for a primary human AML sample (OCI-AML-8227), functionally validated to be enriched for LSCs in long-term xenotransplant assays (Eppert et al., 2011). Next, we performed a high-throughput screen of 11,140 chemical molecules in 3 stages. First, the viability of AML CD34+ cells and healthy cord blood (CB) CD34+ cells was read out at 1-2 doses using a CellTiter-Glo® Luminescent assay. 61 compounds had >70% inhibition of 8227 CD34+ cells and <30% inhibition on CB CD34+ cells. Next, we determined the dose response and refined the hits to 33 potent compounds with LC50 < 1 μM, including novel compounds and classes previously shown to target bulk and leukemic stem cells in AML. We are now presenting the follow up hits identified from the third stage of validation where we determined the LC 50 specifically in the CD34- (blast) and CD34+CD38- (LSC-enriched) OCI-AML-8227 populations using flow cytometry. We identified 25 novel anti-LSC compounds with high efficacy against CD34+CD38- AML cells (LC 50 < 500 nM). Venetoclax was among the top hits, a compound that revolutionized treatment for poor prognosis AML patients due to its anti-LSC activities, providing internal validation of the screen. We then tested these candidates in a second LSC-enriched model with poor prognosis, OCI-AML-20, to ensure the response of compounds on LSCs is not exclusive to OCI-AML-8227 and to investigate the influence of the microenvironment on drug efficiency. A total of 5 hits have significant responses (>50% reduction in LSC enriched populations) and nontoxic to stroma. Furthermore, we refined our top candidates to 7 compounds based on their efficiencies in the two LSC-enriched models (OCI-AML-20 and 8227) and low toxicity on stroma. To gain insights on their mechanisms of action in LSCs, the two models were treated with the 7 candidates and cytarabine as a control for 3 days and apoptosis was measured by flow cytometry. Out of 7 candidates, 5 induced apoptosis in the LSC-enriched fractions, suggesting elimination of LSCs through apoptosis. The remaining compounds may eliminate LSCs through different mechanisms. From these results, we focused on the three leading compounds and validated them for toxicity on CD34+ hematopoetic stem and progenitor cord blood cells (HSPCs) vs CD34+CD38- (LSC-enriched) OCI-AML-8227 by flow cytometry. Although slight toxicity was observed in HSPCs at higher doses, the LC 50 for the stem cell vs LSC-enriched populations differ by ~6-40-fold, indicating that a significantly lower concentration can be used to eradicate LSCs while sparing HSPCs (LC 50 for CD34+ population CB vs 8227; Compound A: 1558 nM vs 83 nM, Compound B: >2000 nM vs 305 nM and Compound C: 612 nM vs 16 nM, respectively). To functionally validate if HSPCs proliferation and differentiation was impaired and if leukemic progenitors were eradicated, a colony forming unit assay was performed for 12 days after 6 days of treatment. Candidates were found to eliminate leukemic progenitors by 50% or more, reducing leukemia initiating potential and having minimal or no impact on CB progenitor functionality. Overall, compound A is a potential candidate to move forward due to its ability to target and eliminate LSCs through apoptosis in two LSC-enriched models, its low toxicity on stroma and normal cord blood cells. This candidate is classified as an indole, a class shown by Pabst et al., 2014 to reduce CD34+CD15- AML cells in a drug screen for aryl hydrocarbon modulators, suggesting a potential mechanism for LSC elimination through apoptosis. We now aim to examine LSC eradication in a panel of genetically defined primary AMLs to be able to determine the broad applicability of this compound and translate the preliminary results for clinical use.

Acute myeloid leukemia (AML) is an aggressive form of blood cancer defined by the uncontrolled proliferation and clonal expansion of immature myeloblast cells in the blood and bone marrow, leading to hematopoietic failure. Despite the use of aggressive and cytotoxic standard-of-care drugs, patients often relapse and succumb to the disease partially due to the inability of medically unfit patients to withstand the cytotoxic treatments, regrowth from minimal residual disease and the chemo-resistant nature of leukemic stem cells (LSCs) which can remain in a quiescent state and reside in a protective bone marrow niche. Hence, novel therapies targeting unique leukemic stem cell biology are highly needed to eliminate and avoid reoccurrence.

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.

The leukemia stem cell (LSC) populations of acute myeloid leukemia (AML) exhibit phenotypic, genetic, and functional heterogeneity that contribute to therapy failure and relapse. Progress toward understanding the mechanistic basis for therapy resistance in LSCs has been hampered by difficulties in isolating cell fractions that enrich for the entire heterogeneous population of LSCs within individual AML samples. We previously reported that CD200 gene expression is upregulated in LSC-containing AML fractions. Here, we show that CD200 is present on a greater proportion of CD45dim blasts compared with more differentiated CD45high cells in AML patient samples. In 75% (49 of 65) of AML cases we examined, CD200 was expressed on ≥10% of CD45dim blasts; of these, CD200 identified LSCs within the blast population in 9 of 10 (90%) samples tested in xenotransplantation assays. CD200+ LSCs could be isolated from CD200+ normal HSCs with the use of additional markers. Notably, CD200 expression captured both CD34- and CD34+ LSCs within individual AML samples. Analysis of highly purified CD200+ LSC-containing fractions from NPM1-mutated AMLs, which are commonly CD34-, exhibited an enrichment of primitive gene expression signatures compared with unfractionated cells. Overall, our findings support CD200 as a novel LSC marker that is able to capture the entire LSC compartment from AML patient samples, including those with NPM1 mutation.

BackgroundCardiac glycosides are approved for the treatment of heart failure as Na+/K+ pump inhibitors. Their repurposing in oncology is currently investigated in preclinical and clinical studies. However, the identification of a specific cancer type defined by a molecular signature to design targeted clinical trials with cardiac glycosides remains to be characterized. Here, we demonstrate that cardiac glycoside proscillaridin A specifically targets MYC overexpressing leukemia cells and leukemia stem cells by causing MYC degradation, epigenetic reprogramming and leukemia differentiation through loss of lysine acetylation.MethodsProscillaridin A anticancer activity was investigated against a panel of human leukemia and solid tumor cell lines with different MYC expression levels, overexpression in vitro systems and leukemia stem cells. RNA-sequencing and differentiation studies were used to characterize transcriptional and phenotypic changes. Drug-induced epigenetic changes were studied by chromatin post-translational modification analysis, expression of chromatin regulators, chromatin immunoprecipitation, and mass-spectrometry.ResultsAt a clinically relevant dose, proscillaridin A rapidly altered MYC protein half-life causing MYC degradation and growth inhibition. Transcriptomic profile of leukemic cells after treatment showed a downregulation of genes involved in MYC pathways, cell replication and an upregulation of hematopoietic differentiation genes. Functional studies confirmed cell cycle inhibition and the onset of leukemia differentiation even after drug removal. Proscillaridin A induced a significant loss of lysine acetylation in histone H3 (at lysine 9, 14, 18 and 27) and in non-histone proteins such as MYC itself, MYC target proteins, and a series of histone acetylation regulators. Global loss of acetylation correlated with the rapid downregulation of histone acetyltransferases. Importantly, proscillaridin A demonstrated anticancer activity against lymphoid and myeloid stem cell populations characterized by MYC overexpression.ConclusionOverall, these results strongly support the repurposing of proscillaridin A in MYC overexpressing leukemia.

Acute myeloid leukemia (AML) develops in a step-wise manner from pre-leukemic clonal expansion to full-blown disease driven by aberrant epigenetic changes. Indeed, regulators of the epigenome such as DNMT3A, TET2, IDH1/2, EZH2 and ASXL1 are often mutated in pre-leukemia and myeloid malignancies. We and others identified K27M/I mutations in histone H3 in AML (Boileau et al. Nat Commun, 2019; Lehnertz et al. Blood, 2017). We demonstrated that K27 mutations are found in pre-leukemic hematopoietic stem cells (HSCs), are enriched in secondary AML, expand the functional human HSC pool and increase leukemic aggressiveness. Transcriptomic and epigenomic analysis determined that K27 mutations alter gene expression through a global decrease in promoter H3K27 tri-methylation and a gene-specific increase in H3K27 acetylation in leukemic cells (Boileau et al. Nat Commun, 2019). Here, we have analyzed the effects of the K27M mutation on HSCs at the single-cell level to understand its role in pre-leukemic clonal expansion. Healthy CD34+CD38- human cord blood cells were transduced with HIST1H3H WT or K27M and injected intrafemorally into sub-lethally irradiated NSG mice. After 14 weeks, bone marrow cells from the femur were collected and sorted for CD34+ transduced (GFP+) cells. Single-cell transcriptomics were performed by generating gene expression libraries from ~8,000 CD34+ cells using the 10X Genomics technology and sequenced using HiSeq4000. We have performed initial clustering and dimensionality reduction (t-SNE and UMAP) and identified 10 and 11 distinct clusters in the WT and K27M samples, respectively. Gene sets distinguishing the individual clusters have been determined. Using published gene lists for primitive hematopoietic cell types, the clusters have been assigned to specific cell types such as HSC, granulocyte-monocyte progenitors (GMP), common myeloid progenitors (CMP), multi-lymphoid progenitors (MLP) and megakaryocyte-erythroid progenitors (MEP) (Laurenti et al. Nat Immunol, 2013). Preliminary joint clustering analysis indicates the presence of two distinct clusters for the WT and K27M samples that were both assigned as "HSCs" in individual clustering. Further analysis to identify the differences in the clusters and cell populations between WT and K27M samples is being performed and will be presented at this meeting. Overall, this single-cell transcriptomic analysis will aid in determining the mechanism of action of the K27M mutant histone in pre-leukemic HSC clonal expansion. In addition, we will be performing similar single-cell analysis on HSCs expressing mutant ASXL1 as a comparison. Further understanding of the role of mutations in epigenetic regulators, such as histone H3 and ASXL1, in pre-leukemic clonal hematopoiesis will provide valuable insight on how to better prevent and treat AML and other myeloid malignancies. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is an aggressive form of blood cancer defined by the uncontrolled proliferation of immature myeloblast cells in the blood and bone marrow, leading to hematopoietic failure. The 5-year survival rate is 28% in patients aged 20 years and older and 64% in patients aged 19 years and younger (SEER 2019). A large portion of these patients succumb to the disease partially due to the chemo-resistant nature of leukemic stem cells (LSCs). Hence, novel therapies targeting unique LSC biology that spare hematopoietic stem cells (HSCs) are needed to eliminate and avoid reoccurrence of this disease. We had previously identified FDA-approved anti-inflammatory glucocorticoids mometasone, halcinonide, and budesonide as compounds that induce terminal differentiation of the LSC (CD34+CD38-) and progenitor cell (CD34+CD38+) populations to leukemic blast cells (CD15+CD34-) in refractory human AML (Laverdière & Boileau et al., Blood Can. J. 2018). Following the paradigm of successful differentiation treatment in AML (acute promyelocytic leukemia with all-trans retinoic acid), the effects and mechanism of action of the glucocorticoids on LSCs need to be further investigated for other AML subtypes. Furthermore, dexamethasone, a glucocorticoid currently used to successfully treat acute lymphoblastic leukemia (ALL), is being studied in a Phase II clinical trial for induction and post-remission chemotherapy in older patients with de novo or therapy-related AML (clinicalTrials.gov, NCT03609060). To identify the subtypes of AML that are sensitive to steroid-induced LSC differentiation, we began by screening a panel of cell lines (F36P, MOLM-13, Kasumi-6, Kasumi-1 and K562) and observed that only Kasumi-1, a pediatric leukemia carrying the t(8;21) mutation leading to the fused RUNX1-RUNX1T1 gene, was responsive to glucocorticoid treatment, although without differentiation. This is consistent with the finding of Simon et al. who observed a loss of bulk AML cells in RUNX1 AML samples following dexamethasone treatment (Simon et al., Clin Cancer Res. 2017). However, we observed expansion of bulk cells following differentiation of LSCs in primary AML, indicating different mechanisms of steroid response in different samples: differentiation of LSCs or overall loss of AML cells. We will further investigate these compounds in a panel of 10 genetically defined primary AML samples to classify which oncogenetic drivers or subtypes of AML are linked to sensitivity to the three glucocorticoids, including which drive cell death vs LSC differentiation. We will perform ex vivo and in vivo studies of the glucocorticoids to assess the extent of engraftment in treated versus DMSO treated samples. This additional data will be presented at the annual meeting. In addition, to explore the mechanism of action of these steroids in AML, we investigated the roles of the cytokines interleukin-3 (IL-3), interleukin-6 (IL-6), stem cell factor (SCF), granulocyte colony stimulating factor (GCSF), thrombopoietin (TPO) and FMS-like tyrosine kinase 3 ligand (FLT3L), used to culture AML, on the differentiation effects induced by the glucocorticoids. We observed that only FLT3L was required for the complete differentiation of LSCs. In summary, we have observed that the three glucocorticoid steroids (mometasone, halcinonide, and budesonide), as well as dexamethasone to a lesser extent, can induce two different responses in a sample-dependent manner: terminal differentiation of LSCs or overall cell loss. We have also observed that the differentiation response requires FLT3L for maturation of the AML cells. Our current studies involve in vivo and genomic assays to determine the effect on functional LSCs and the genetic markers of sensitivity and we will present these results. Disclosures Minden: Trillium Therapetuics: Other: licensing agreement.

Our ability to manage acute myeloid leukemia (AML) is limited by our incomplete understanding of the epigenetic disruption central to leukemogenesis, including improper histone methylation. Here we examine 16 histone H3 genes in 434 primary AML samples and identify Q69H, A26P, R2Q, R8H and K27M/I mutations (1.6%), with higher incidence in secondary AML (9%). These mutations occur in pre-leukemic hematopoietic stem cells (HSCs) and exist in the major leukemic clones in patients. They increase the frequency of functional HSCs, alter differentiation, and amplify leukemic aggressiveness. These effects are dependent on the specific mutation. H3K27 mutation increases the expression of genes involved in erythrocyte and myeloid differentiation with altered H3K27 tri-methylation and K27 acetylation. The functional impact of histone mutations is independent of RUNX1 mutation, although they at times co-occur. This study establishes that H3 mutations are drivers of human pre-cancerous stem cell expansion and important early events in leukemogenesis.

ABSTRACTTargeting MYC oncogene remains a major therapeutic goal in cancer chemotherapy. Here, we demonstrate that proscillaridin, a cardiac glycoside approved for heart failure treatment exhibit anticancer selectivity towards high MYC expressing leukemic cell lines and leukemia stem cells. At a clinically relevant concentration, proscillaridin induced a rapid downregulation of MYC protein level, due to a significant decrease in MYC protein half-life. Proscillaridin treatment induced a downregulation of gene sets involved in MYC pathway, and a concomitant upregulation of genes involved in hematopoietic differentiation. Proscillaridin induced a significant loss of lysine acetylation in histone H3 (K9, K14, K18 and K27) and in non-histone proteins such as MYC, MYC target proteins, and a series of histone acetylation regulators. Loss of lysine acetylation correlated with a rapid downregulation of histone acetyltransferase protein levels, involved in histone and MYC acetylation (CBP, P300, GCN5, TIP60, and MOZ), preferentially in MYC overexpressing leukemia as compared to other cancer cells. These results support the repurposing of proscillaridin in MYC overexpressing leukemia and propose a novel strategy to target MYC in cancer.

Therapy for acute myeloid leukemia (AML) involves intense cytotoxic treatment and yet approximately 70% of AML are refractory to initial therapy or eventually relapse. This is at least partially driven by the chemo-resistant nature of the leukemic stem cells (LSCs) that sustain the disease, and therefore novel anti-LSC therapies could decrease relapses and improve survival. We performed in silico analysis of highly prognostic human AML LSC gene expression signatures using existing datasets of drug-gene interactions to identify compounds predicted to target LSC gene programs. Filtering against compounds that would inhibit a hematopoietic stem cell (HSC) gene signature resulted in a list of 151 anti-LSC candidates. Using a novel in vitro LSC assay, we screened 84 candidate compounds at multiple doses and confirmed 14 drugs that effectively eliminate human AML LSCs. Three drug families presenting with multiple hits, namely antihistamines (astemizole and terfenadine), cardiac glycosides (strophanthidin, digoxin and ouabain) and glucocorticoids (budesonide, halcinonide and mometasone), were validated for their activity against human primary AML samples. Our study demonstrates the efficacy of combining computational analysis of stem cell gene expression signatures with in vitro screening to identify novel compounds that target the therapy-resistant LSC at the root of relapse in AML.

Therapy resistance and relapse in acute myeloid leukemia (AML) are driven by leukemia stem cells (LSCs). Recent evidence highlighting functional and genetic heterogeneity among LSC subclones underscores the importance of capturing the entire LSC compartment in studies of LSC biology. Although LSCs are often enriched in the CD34+CD38- cell fraction, they are frequently detected in other phenotypic fractions, and in some cases are restricted to the CD34- population. In order to discover novel LSC markers, we examined genes differentially expressed between functionally-validated LSC+ and LSC- cell fractions obtained from primary AML samples, and identified CD200 as a candidate cell surface marker for LSCs.

Quiescent and proliferating leukemia cells accumulate highly lethal DNA double-strand breaks that are repaired by 2 major mechanisms: BRCA-dependent homologous recombination and DNA-dependent protein kinase-mediated (DNA-PK-mediated) nonhomologous end-joining, whereas DNA repair pathways mediated by poly(ADP)ribose polymerase 1 (PARP1) serve as backups. Here we have designed a personalized medicine approach called gene expression and mutation analysis (GEMA) to identify BRCA- and DNA-PK-deficient leukemias either directly, using reverse transcription-quantitative PCR, microarrays, and flow cytometry, or indirectly, by the presence of oncogenes such as BCR-ABL1. DNA-PK-deficient quiescent leukemia cells and BRCA/DNA-PK-deficient proliferating leukemia cells were sensitive to PARP1 inhibitors that were administered alone or in combination with current antileukemic drugs. In conclusion, GEMA-guided targeting of PARP1 resulted in dual cellular synthetic lethality in quiescent and proliferating immature leukemia cells, and is thus a potential approach to eradicate leukemia stem and progenitor cells that are responsible for initiation and manifestation of the disease. Further, an analysis of The Cancer Genome Atlas database indicated that this personalized medicine approach could also be applied to treat numerous solid tumors from individual patients.

Acute myeloid leukemia (AML) has a relatively poor 5-year survival rate of 30% in adults, largely due to high rates of relapse, which is thought to be driven by chemoresistant leukemic stem cells (LSCs). A therapy would need to eradicate all LSCs to obtain durable responses in AML patients. Unlike typical cancerous cells, LSCs are often quiescent, allowing them to evade standard therapies and serve as a reservoir for relapse. Thus, the identification of novel anti-LSC therapies could result in the improvement of the clinical outcome of patients suffering from AML.

Acute myeloid leukemia (AML) is a hierarchical disease in which the bulk blast population is sustained by a minority population of leukemia stem cells (LSC). Evidence of functional heterogeneity in the LSC compartment, including variable responses to chemotherapy, underscores the importance of examining the entire stem cell compartment in studies of LSC biology. However, there are currently no phenotypic markers that can consistently segregate LSCs within the leukemic blast population. Although LSC activity is most often enriched in the CD34+CD38- cell fraction, LSCs are also frequently detected in other phenotypic fractions, and in some cases are absent from CD34+ fractions. Thus, LSC studies that focus only on CD34+CD38- blasts may miss biologically important clones that are present in other phenotypic cell fractions. To identify novel markers that will enable better enrichment of LSC activity, we examined gene expression data obtained from functionally validated LSC+ and LSC- cell fractions sorted from primary AML samples, and identified CD200 as a candidate cell surface marker for LSCs.