Acute Myelogenous Leukemia Model Research Articles

Control of acute myeloid leukemia and generation of immune memory in vivo using AMV564, a bivalent bispecific CD33 x CD3 T cell engager.

Off-the-shelf immunotherapeutics that suppress tumor growth and provide durable protection against relapse could enhance cancer treatment. We report preclinical studies on a CD33 x CD3 bivalent bispecific diabody, AMV564, that not only suppresses tumor growth, but also facilitates memory responses in a mouse model of acute myelogenous leukemia (AML). Mechanistically, a single 5-day treatment with AMV564 seems to reduce tumor burden by redirection of T cells, providing a time window for allogeneic or other T cells that innately recognize tumor antigens to become activated and proliferate. When the concentration of bispecific becomes negligible, the effector: target ratio has also shifted, and these activated T cells mediate long-term tumor control. To test the efficacy of AMV564 in vivo, we generated a CD33+ MOLM13CG bioluminescent human cell line and optimized conditions needed to control these cells for 62 days in vivo in NSG mice. Of note, not only did MOLM13CG become undetectable by bioluminescence imaging in response to infusion of human T cells plus AMV564, but also NSG mice that had cleared the tumor also resisted rechallenge with MOLM13CG in spite of no additional AMV564 treatment. In these mice, we identified effector and effector memory human CD4+ and CD8+ T cells in the peripheral blood immediately prior to rechallenge that expanded significantly during the subsequent 18 days. In addition to the anti-tumor effects of AMV564 on the clearance of MOLM13CG cells in vivo, similar effects were seen when primary CD33+ human AML cells were engrafted in NSG mice even when the human T cells made up only 2% of the peripheral blood cells and AML cells made up 98%. These studies suggest that AMV564 is a novel and effective bispecific diabody for the targeting of CD33+ AML that may provide long-term survival advantages in the clinic.

Therapeutic Targeting of TIM-4-L with Engineered T Cells for Acute Myeloid Leukemia.

Disruption of lipid bilayer asymmetry is a common feature observed in cancer cells and offers novel routes for therapeutic targeting. We used the natural immune receptor TIM-4 to interrogate for loss of plasma membrane phospholipid polarity in primary acute myelogenous leukemia (AML) samples and evaluated the anti-leukemic activity of TIM-4-L-directed T-cell therapy in preclinical AML models. We performed FACS analysis on 33 primary AML bone marrow specimens and correlated TIM-4-L expression frequency and intensity with molecular disease characteristics. Using Kasumi-1 and MV-4-11 AML cell lines, we further tested the anti-leukemic effects of TIM-4-L-directed engineered T cells in vitro and in vivo. We found that 86% of untreated AML blasts displayed upregulation of cell surface TIM-4-L. These observations were agnostic to AML genetic classification, as samples with mutations in TP53, ASXL1, and RUNX1 displayed TIM-4-L upregulation similar to that seen in favorable and intermediate subtypes. TIM-4-L dysregulation was also stably present in AML cell lines. To evaluate the potential of targeting upregulated TIM-4-L with adoptive T-cell therapy, we constructed TIM-4-L-directed engineered T cells, which demonstrated potent anti-leukemic effects, effectively eliminating AML cell lines with a range of endogenous TIM-4-L expression levels both in vitro and in vivo. These results highlight TIM-4-L as a highly prevalent target on AML across a range of genetic classifications and novel target for T-cell-based therapy in AML. Further investigations into the role of TIM-4-L in AML pathogenesis and its potential as an anti-leukemic target for clinical development are warranted.

Platelet Factor 4 Binds to Low-Density Lipoprotein Receptor to Block Leukemic Stem Cell Expansion and Recurrence

Healthy hematopoietic stem cells (HSCs) reside in highly specialized microenvironments within the bone marrow, commonly referred to as niches. HSC activity is tightly regulated by various cellular and molecular niche constituents that ensure healthy hematopoiesis. However, through the accumulation of mutations, hematopoietic stem and progenitor cells can give rise to leukemia-initiating cells or leukemic stem cells (LSCs), which play a vital role in the onset, progression, and treatment outcome of acute myelogenous leukemia (AML). In AML, the expansion of the leukemic clone is associated with impaired healthy hematopoiesis, resulting in severe anemia, thrombocytopenia, and immunodeficiency, which can lead to severe morbidity. Current therapeutic options mainly eliminate the bulk of leukemic cells but do not efficiently target LSCs, leading to the high rates of relapse seen in AML patients. Recent studies suggest that LSCs hijack and alter the healthy bone marrow niche to better support their maintenance and proliferation. Understanding how LSCs interact with the bone marrow microenvironment is crucial for eradicating LSCs and preventing AML relapse. Our previous work showed that platelet factor 4 (PF4), a chemokine stored in platelet alpha-granules and secreted by bone marrow megakaryocytes, can induce quiescence in healthy myeloid-biased HSCs ( Nature Medicine 2014; Developmental Cell 2018). Here, we investigate the regulation of malignant LSCs by PF4 using mouse and human AML models. In an aggressive mouse AML model, MLL-AF9, our results show that recombinant PF4 blocks the proliferation of phenotypic LSCs (Lineage − IL7Rα − Sca1 − MLL-AF9-GFP + c-Kit high CD34 + FcγRII/III high) in vitro (70% reduction; P=0.0279). Strikingly, cell cycle analyses revealed that in vivo, both LSCs and bulk AML proliferation are significantly inhibited 24 hours post-PF4 (80 mg/kg) injection, resulting in a significantly lower leukemic burden. By contrast, Pf4 -/- mice exhibited rapid AML disease progression via increased LSC proliferation. Furthermore, using a genetic mouse model ( Pf4-Cre; iDTR) where PF4-secreting megakaryocyte cells can be depleted in vivo in leukemic mice, our results revealed that megakaryocyte depletion led to poor AML survival due to accelerated disease progression (Log-rank P<0.0017). These results demonstrate that megakaryocyte-derived PF4 can directly regulate the proliferation of leukemia cells. Interestingly, our results indicate that AML drives striking alterations in bone marrow megakaryocytes, including a lower frequency, reduced cell maturation, and reduced secretion of PF4 (~80% reduction, P<0.0001) compared to healthy controls. Accordingly, PF4 levels are reduced in the serum of myelodysplastic syndrome patients where hyperproliferative malignant cells are found alongside dysplastic megakaryocytes. To assess whether recombinant PF4 can alter the course of AML relapse in a preclinical experimental setting, we treated moribund leukemic mice with the chemotherapeutic drug Cytarabine followed by 3 daily I.V. injections of PF4 (80 mg/kg) or saline. Strikingly, this short treatment with PF4 preferentially reduced the frequency of LSCs in vivo during the relapse phase of AML (75% reduction; P=0.05). Furthermore, mice transplanted with MLL-AF9 AML cells that were pre-treated with Cytarabine and PF4 in vitro had prolonged overall survival (Log-rank P<0.0132) compared to Cytarabine alone. Importantly, two of six mice had no detectable AML up to 150 days post-transplantation. Finally, single-cell RNA sequencing experiments revealed that PF4 signals through Low-Density Lipoprotein Receptor (LDLR) on LSCs, whose expression is significantly upregulated upon Cytarabine treatment, during the relapse phase. Functional studies with fluorescently labeled low-density lipoprotein (LDL) confirmed that PF4 limits LSC uptake of LDL via LDLR to block LSC proliferation ( P=0.0044), suggesting an important role for PF4 in AML metabolism. Altogether, our studies reveal an unknown function for PF4 in AML and highlight PF4's potential as an effective adjuvant therapy in preventing AML relapse by inhibiting the recurrence of chemotherapeutic-resistant LSCs.

Establishing an AML Ex Vivo Platform to Generate Physiologically Relevant Models

Acute myelogenous leukemia (AML) comprises a highly polyclonal and heterogeneous disease containing leukemic stem cells, progenitors, and differentiated blasts. Despite advances in treatment, the long-term relapse-free survival for AML remains poor. Due to complex mutational profiles and highly divergent subtypes in AML, the use of physiologically relevant, patient-derived models is needed. Traditional cell line models of AML almost exclusively represent differentiated blasts, are homogenous in clonality, and often do not encompass major genetic subtypes. To address these gaps, we have established a culture system that supports the growth of higher-order AML samples ex vivo. By optimizing culture conditions, we successfully cultivated 70% (10/14) of AML disseminated PDX models ex vivo and demonstrated they continue to proliferate after long term cultures. We established a 16-color flow panel to characterize their cell maturity state and show they maintain cell surface heterogeneity. Importantly, these models have not ‘drifted’ transcriptomically after multiple passages. Our platform includes 2 NPM1c mutant and 3 IDH1/2 mutant ex vivo lines, in addition to other subtypes underrepresented in traditional culture methods. Furthermore, we treated AML PDXs in vivo with a clinically-relevant dose of venetoclax + azacytidine (Ven/AZA) for 2 cycles (14 days) and then evaluated post-VEN/AZA cells in our ex vivo assay. Growing these models ex vivo, we show using our flow panel that Ven/AZA treatment shifts the differentiation status from a stem-like state to a more mature-like cell state which is consistent with literature evidence of treatment resistance. Additionally, a second PDX model treated with three cycles of Ven/AZA in vivo and harvested in ex vivo culture conditions maintained both Ven and AZA resistance. Interestingly, compared with the vehicle treatment, Ven/AZA treatment caused resistance to other cell death agents including selective MCL1 and dual Bcl2/xL inhibitors. Lastly, we performed a 7-day cell compound panel screen using common standard of care agents for the treatment of AML. Many of the targeted agent activity correlated with expected genetic subtypes, such as FLT3 inhibitors in FLT3-ITD mutants and venetoclax in WT TP53 models. This established ex vivo platform provides physiologically relevant models to explore novel target biology, better understand compound mechanism of action (MoA), and generate drug resistant models through in vivo driven clinically relevant dose scheduling using standard of care agents.

Targeting Unc51-like Autophagy Activating Kinase 1 (ULK1) Overcomes Adaptive Drug Resistance in Acute Myelogenous Leukemia.

ULK1 drives a cell-intrinsic adaptive resistance in AML and targeting ULK1-mediated autophagy can synergize with existing and emerging AML therapies to overcome drug-resistance and induce apoptosis.

Dual mTORC1/2 Inhibition Synergistically Enhances AML Cell Death in Combination with the BCL2 Antagonist Venetoclax.

Acute myelogenous leukemia (AML) is an aggressive disease with a poor outcome. We investigated mechanisms by which the anti-AML activity of ABT-199 (venetoclax) could be potentiated by dual mTORC1/TORC2 inhibition. Venetoclax/INK128 synergism was assessed in various AML cell lines and primary patient AML samples in vitro. AML cells overexpressing MCL-1, constitutively active AKT, BAK, and/or BAX knockout, and acquired venetoclax resistance were investigated to define mechanisms underlying interactions. The antileukemic efficacy of this regimen was also examined in xenograft and patient-derived xenograft (PDX) models. Combination treatment with venetoclax and INK128 (but not the mTORC1 inhibitor rapamycin) dramatically enhanced cell death in AML cell lines. Synergism was associated with p-AKT and p-4EBP1 downregulation and dependent upon MCL-1 downregulation and BAK/BAX upregulation as MCL-1 overexpression and BAX/BAK knockout abrogated cell death. Constitutive AKT activation opposed synergism between venetoclax and PI3K or AKT inhibitors, but not INK128. Combination treatment also synergistically induced cell death in venetoclax-resistant AML cells. Similar events occurred in primary patient-derived leukemia samples but not normal CD34+ cells. Finally, venetoclax and INK128 co-treatment displayed increased antileukemia effects in in vivo xenograft and PDX models. The venetoclax/INK128 regimen exerts significant antileukemic activity in various preclinical models through mechanisms involving MCL-1 downregulation and BAK/BAX activation, and offers potential advantages over PI3K or AKT inhibitors in cells with constitutive AKT activation. This regimen is active against primary and venetoclax-resistant AML cells, and in in vivo AML models. Further investigation of this strategy appears warranted.

Effects of LSD1 Inhibition with Img-7289 on the Leukemia Initiating Cell Population with Induction of Differentiation and Cell Death in Pediatric Relapsed/Refractory AML

Background: Outcomes for pediatric patients with relapsed or refractory acute myelogenous leukemia (AML) are poor, with <30% overall survival in recurrent settings. Additionally, patients harboring high-risk molecular features, including KMT2A/MLL-rearrangements or GLIS2 fusion genes, experience poor outcomes. LSD1 inhibition has been evaluated as a potential therapeutic approach in adult patients with AML, but its role in pediatric AML remains limited. In this study, we characterize the anti-leukemic effect of an LSD1 inhibitor, IMG-7289, across a panel of pediatric AML models in vitro and in vivo and evaluate its effect on leukemia initiating cells (LICs). Methods: Dose-response effect of IMG-7289 on cell viability was assessed across 11 pediatric leukemia cell lines. Induction of apoptosis and cell cycle arrest was assessed by flow cytometric analysis of Annexin V and 7-AAD respectively. Flow cytometry was also used to analyze a panel of cell surface markers to assess for differentiation effect. Pediatric AML patient-derived xenograft (PDX) mouse models, including an acute megakaryoblastic leukemia (AMKL) model and a KMT2A-rearranged (KMT2A-r) AML model, established by implanting AML cells into NSG-S mice, were used for in vivo validation studies and limiting dilution secondary transplantation studies. PDX models were treated with IMG-7289 (25 mg/kg PO) or Vehicle daily for 21 days (n=6/arm). Engraftment was determined using flow cytometry to assess expression of human CD45 (hCD45) in the bone marrow. Leukemia initiating cell (LIC) frequency was evaluated by transplanting serial dilutions (2x105, 105, 104, 103, and 102 cells) of bone marrow cells derived from Vehicle- or IMG-7289-treated PDX models into untreated donor mice. The LIC frequency was estimated using the Extreme Limiting Dilution Analysis (ELDA) software (Walter and Eliza Hall Bioinformatics Institute of Medical Research), and log-rank analysis was used to compare event-free survival (EFS), defined as time from secondary transplant to engraftment. Results: Seven pediatric AML cell lines demonstrated sensitivity to IMG-7289, including 2 AMKL cell lines, M07e (IC50: 0.03 μM ± 0.01) and CMK (IC50: 0.065 μM ± 0.003), and 2 KMT2A-r AML cell lines, MV4;11 (IC50: 0.007 μM ± 0.001) and MOLM14. Treatment with IMG-7289 leads to induction of apoptosis (DMSO 8.8% vs IMG-7289 30.3%, P=0.0005) and cell-cycle arrest (G0/G1 phase populations: DMSO 65.5% vs IMG-7289 74.7%, P<0.0001) in MV4;11 cells. To investigate the effect of IMG-7289 on induction of cell differentiation, cell surface marker changes were evaluated over time in IMG-7289-treated AML. MV4;11 and M07e cells treated over 25 days showed increased expression of the monocytic cell surface markers CD14 and CD86 compared to DMSO control. Additionally, decreased expression of megakaryocytic markers CD41 and CD42b were observed in IMG-7289-treated M07e cells. To evaluate the in vivo activity of IMG-7289, a KMT2A-r AML PDX model was treated with IMG-7289 for 21 days. We observed a significant reduction in hCD45+ cells in IMG-7289-treated mice (27.5% hCD45+) vs Vehicle-treated mice (76% hCD45+, P=0.0012). To evaluate effects of IMG-7289 on the LIC population, serial transplantation of cells derived from Vehicle-treated or IMG-7289-treated AMKL PDX model was performed. Assessment of leukemia engraftment (flow cytometric analysis for hCD45+ cells in mouse bone marrow) at 20 weeks post-secondary transplant demonstrated an LIC frequency of 1 in 4.9x104 in IMG-7289-treated mice representing a ~500-fold reduction in LIC number compared to Vehicle control. Furthermore, a significant improvement in EFS was observed in IMG-7289-treated mice (median survival = 33 days) compared to Vehicle control (median survival = 76 days, P=0.0009, log-rank). Conclusions: In conclusion, treatment with IMG-7289 results in decreased viability, as demonstrated by induction of apoptosis and cell cycle arrest, as well as differentiation induction in pediatric AML cell lines. The in vitro and in vivo activity observed in pediatric AML is also observed in models with high-risk phenotypes (KMT2A-r AML and AMKL). Activity of LSD1 inhibition on the LIC population may represent a promising strategy to mitigate relapse or refractory disease.

N-Heterocyclic 3-Pyridyl Carboxamide Inhibitors of DHODH for the Treatment of Acute Myelogenous Leukemia.

Acute myelogenous leukemia (AML), a disease of the blood and bone marrow, is characterized by the inability of myeloblasts to differentiate into mature cell types. Dihydroorotate dehydrogenase (DHODH) is an enzyme well-known in the pyrimidine biosynthesis pathway; however, small molecule DHODH inhibitors were recently shown to induce differentiation in multiple AML subtypes. Using virtual screening and structure-based drug design approaches, a new series of N-heterocyclic 3-pyridyl carboxamide DHODH inhibitors were discovered. Two lead compounds, 19 and 29, have potent biochemical and cellular DHODH activity, favorable physicochemical properties, and efficacy in a preclinical model of AML.

TAS1553, a small molecule subunit interaction inhibitor of ribonucleotide reductase, exhibits antitumor activity by causing DNA replication stress

Ribonucleotide reductase (RNR) is composed of two non-identical subunits, R1 and R2, and plays a crucial role in balancing the cellular dNTP pool, establishing it as an attractive cancer target. Herein, we report the discovery of a highly potent and selective small-molecule inhibitor, TAS1553, targeting protein-protein interaction between R1 and R2. TAS1553 is also expected to demonstrate superior selectivity because it does not directly target free radical or a substrate binding site. TAS1553 has shown antiproliferative activity in human cancer cell lines, dramatically reducing the intracellular dATP pool and causing DNA replication stress. Furthermore, we identified SLFN11 as a biomarker that predicts the cytotoxic effect of TAS1553. Oral administration of TAS1553 demonstrated robust antitumor efficacy against both hematological and solid cancer xenograft tumors and also provided a significant survival benefit in an acute myelogenous leukemia model. Our findings strongly support the evaluation of TAS1553 in clinical trials.

AML Cell Vaccines Co-Expressing CD80 and IL-15/IL-15 Receptor Alpha Induce Activation and Cytolytic Activity in Post Remission Autologous Patient PBMC Ex Vivo

There is a critical need for more effective therapy for acute myelogenous leukemia (AML). Although many patients achieve remission, most relapse with poor outcomes. Even after allogeneic Stem Cell Transplantation (SCT), 30-50% of patients relapse due to the persistence of residual disease.To address the poor immunogenicity of AML cells and the diminished immune responsiveness of patients, our candidate autologous AML vaccine is lentivirally engineered, in each patient's leukemic cells, to express CD80, IL-15, and IL-15 Receptor alpha (IL-15Rα). In prior studies in a syngeneic 32Dp210 murine AML model, CD80-mediated co-stimulation of T-cells combined with immune activation by the IL-15/IL-15Rα heterodimer showed unprecedented synergy in induction of anti-leukemic cytolytic activity (Shi, Y. et al, 2018). This was observed in both ex vivo co-culture and in vivo where vaccinated leukemic mice had >80% cure rates. No local skin, organ, or systemic toxicity was observed, nor was there evidence of systemic cytokine release of IL-6 or TNFα after SC or IV injection of up to 10 8 transduced irradiated AML cells.We confirmed the feasibility of producing patient-derived AML vaccines by transduction of 16 independent AML samples with a tri-cistronic lentiviral vector (TLV) that contains human CD80, IL-15 and IL-15Rα. Transduction levels were 11-71% of cells (median 38.6%).To define the minimum transduction level required for PBMC activation and to assess synergy of co-expressed human CD80, IL-15, and IL-15Rα, allogenic U937 leukemia cells were initially used as stimulators. Transduced U937 (U937-TLV) had high-level surface expression of CD80 and IL-15, secreted IL-15 (7 ng/ml/24 hours from 2 x 10 6 cells/ml) and activated CD3+ T-cells from an AML patient (Fig.1). Mixtures of irradiated U937-TLV with non-transduced U937 were created at fixed ratios (100%, 80%, 40%, 20%, 10%, 5%, 0%) for overnight co-culture with patient PBMC. At 24 hours, the T-cells were analyzed for activation by measurement of the frequency of CD69+ CD4 or CD8 T cells (Fig. 1), normalized to expression of unstimulated PBMC (0%) and the percentage of maximal CD69 expression with 100% U937-TLV (100%). Background levels of activation due to the presence of allogenic U937 were negligible. Co-culture with as little as 10% transduced U937-TLV reliably activated patient T-cells. To assess the synergy of CD80, IL-15 and IL-15Rα expression, parallel experiments were performed with PBMC co-cultured in IL-15 containing supernatants from U937-TLV cells (Fig. 1). The frequencies of activated T-cells were significantly higher after co-culture with U937/U937-TLV cells than after stimulation with IL-15-containing supernatants from similar ratios of U937/U937-TLV, confirming the synergy of CD80 and IL-15/IL-15Rα in the transduced cells.To better, model the clinical setting, we assessed induction of immune responses of patient T cells to autologous transduced AML. PBMC were stimulated with transduced or non-transduced autologous AML cells vs stimulation with allogeneic U937-TLV, or with anti-CD3/CD28 beads to define maximal stimulation. Negative controls included culture of PBMC alone. All patients had T-cell activation, as measured by induction of CD69, HLA-DR and CD95 (Fas) expression, although there was heterogeneity in the nature of responses, e.g., disparate induction of the markers in individual patients (Fig. 2A and B). Induction of cytotoxic effector pathways was confirmed by detection of CD178 (FasL) and perforin expression (Figure 2C and D). Overall, all patients' PBMC had the capacity to mount T-cell responses of similar magnitude to both allogeneic U937-TLV and autologous vaccine.These studies establish that autologous AML cells transduced with CD80, IL-15 and IL-15Rα can elicit specific anti-leukemic T-cell responses, even in the face of prior lymphodepleting chemotherapy. A strength of this autologous vaccine strategy is that it is agnostic to which AML proteins are immunogenic for each patient. Although uniformly detected, there was heterogeneity in the induction of activation markers and effector pathways, which may reflect host and/or disease-related differences. The mechanisms underlying differences in the nature of responses in patients will be important to understand and will provide the basis for future immune correlative studies for our Phase 1 vaccine trial in transplant ineligible AML patients. [Display omitted] DisclosuresKohn: Lyrik Therapeutics: Membership on an entity's Board of Directors or advisory committees; MyoGene Bio: Membership on an entity's Board of Directors or advisory committees; ImmunoVec: Membership on an entity's Board of Directors or advisory committees; Pluto Immunotherapeutics: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees; UC Regents: Patents & Royalties; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Sangamo Biosciences: Membership on an entity's Board of Directors or advisory committees.

BCL-2 Inhibitor ABT-737 Effectively Targets Leukemia-Initiating Cells with Differential Regulation of Relevant Genes Leading to Extended Survival in a NRAS/BCL-2 Mouse Model of High Risk-Myelodysplastic Syndrome.

AbastractDuring transformation, myelodysplastic syndromes (MDS) are characterized by reducing apoptosis of bone marrow (BM) precursors. Mouse models of high risk (HR)-MDS and acute myelogenous leukemia (AML) post-MDS using mutant NRAS and overexpression of human BCL-2, known to be poor prognostic indicators of the human diseases, were created. We have reported the efficacy of the BCL-2 inhibitor, ABT-737, on the AML post-MDS model; here, we report that this BCL-2 inhibitor also significantly extended survival of the HR-MDS mouse model, with reductions of BM blasts and lineage negative/Sca1+/KIT+ (LSK) cells. Secondary transplants showed increased survival in treated compared to untreated mice. Unlike the AML model, BCL-2 expression and RAS activity decreased following treatment and the RAS:BCL-2 complex remained in the plasma membrane. Exon-specific gene expression profiling (GEP) of HR-MDS mice showed 1952 differentially regulated genes upon treatment, including genes important for the regulation of stem cells, differentiation, proliferation, oxidative phosphorylation, mitochondrial function, and apoptosis; relevant in human disease. Spliceosome genes, found to be abnormal in MDS patients and downregulated in our HR-MDS model, such as Rsrc1 and Wbp4, were upregulated by the treatment, as were genes involved in epigenetic regulation, such as DNMT3A and B, upregulated upon disease progression and downregulated upon treatment.

CCL3 Signaling Is Essential for Leukemia Progression but Dispensable for Hematopoietic Stem Cell Maintenance

Background: Hematologic malignancies remodel the bone marrow microenvironment (BMME), reducing support for normal hematopoiesis while increasing support for the malignancy. The chemokine CCL3 has been demonstrated to play a role in BMME dysfunction in multiple hematologic malignancies including myeloma, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), and myelodysplastic syndrome. Therefore we used murine models to analyze the requirement for CCL3 expression in leukemia progression and the viability of CCL3 signaling as a target in myelogenous leukemias.Methods/Results: We utilized genetically altered mice with a global loss of CCL3 (CCL3KO) on a C57bl/6 background. We used sorted hematopoietic stem and progenitor cells (HSPC) (Lineage-sca1+ckit+) from CCL3KO mice or wild type controls to generate 2 models of AML, the BCR/ABL+Nup98/HoxA9 model of blast crisis CML (bcCML), and the MLL/AF9 model of AML. In the bcCML model, CCL3KO leukemic cells do not generate a leukemic disease when transplanted into naïve WT recipient mice in contrast to WT leukemic cells that will generate an acute myeloid leukemia with 100% penetrance over the course of 15-20 days. In the MLL/AF9 AML model, the disease progression of CCL3KO leukemic cells is significantly delayed as compared to WT controls (WT vs. CCL3KO p<0.05 n=15 mice/group). Therefore CCL3 modulates leukemogenesis in two genetically divergent AML models. The small molecule maraviroc is FDA approved and inhibits ligand binding to the chemokine receptor CCR5, one of the receptors for CCL3. Due to the poor pharmacokinetics of maraviroc in mice we utilized a novel micelle nanoparticle (NP) delivery platform that is targeted to the BMME through the use of a tartrate-resistant acid phosphatase (TRAP) binding peptide (TBP). Using this platform we can delay the release of maraviroc over 48 hours, and target that release to the bone marrow. To verify that CCL3 signaling is targetable in hematologic malignancies, and that the BMME targeted NP platform is a viable method of drug delivery we loaded maraviroc into TBP-NPs and treated the murine model of bcCML every 2 days over the course of 10 days. The bcCML model was used for these studies due to its exquisite sensitivity to CCL3 expression. In bcCML mice the BMME is altered including an increase in phenotypic MSC populations and HSPC populations. Following treatment with maraviroc TBP-NPs the increase in MSC and HSPC populations is reversed and leukemic burden was decreased >2 fold in the bone marrow (VEH vs Maraviroc TBP-NP treated p<0.01 n=10 mice/group). These data suggest that CCL3 can play a critical role in the progression of myelogenous leukemias and is a potential target for therapy.If CCL3 signaling is to be a viable therapeutic target in AML it must be specific to leukemic cells while sparing the normal hematopoietic system. Therefore we sorted Lineage-Sca1+Ckit+Flt3- (Flt3-LSK) bone marrow cells enriched for long term repopulating HSPCs (LT-HSCs) in order to establish stem cell activity on a per cell basis through competitive transplantation. Upon secondary transplantation, the true test of LT-HSC function, CCL3KO Flt3-LSK donor cells engrafted in recipient mice at a higher rate (2-way ANOVA, p<0.05 over 16 weeks, n=8 mice/group). These results confirm that CCL3KO mice maintain a functional LT-HSC population.Conclusions: These results demonstrate that in multiple mouse models of myelogenous leukemia CCL3 plays an important, and in some cases, indispensable role in leukemogenesis, likely involving dysruption of the normal BMME. Importantly however, a long-term engrafting normal HSC population is clearly maintained even in the complete absence of CCL3 suggesting that anti-CCL3 therapy would be well-tolerated by the normal hematopoietic system. DisclosuresNo relevant conflicts of interest to declare.

Mining for METTL3 inhibitors to suppress cancer.

The RNA methyltransferase METTL3 catalyzes N6-methyladenosine (m6A) modification of messenger RNAs (mRNAs). It is overexpressed in many types of cancer, including acute myelogenous leukemia (AML), and promotes cancer cell growth and tumorigenicity. Now, a selective small molecule inhibitor of METTL3 shows significant antileukemic effects in preclinical AML models, highlighting the promise of pharmacological METTL3 inhibition as a new cancer therapy.

Dual CXCR4 and E-Selectin Inhibition (GMI-1359) Rapidly Increases AML Cellular Motility Prior to Intravasation and Vascular Niche Depletion Observed By Intravital Bone Marrow Two-Photon Microscopy

BACKGROUND: Acute myelogenous leukemia (AML) cells that reside in bone marrow (BM) receive a great deal of protection from the cytotoxic effects of therapeutic agents. In contrast, the circulating leukemia cells are typically more chemosensitive compared to those embedded in BM niches. The BM homing of AML cells is mediated by multiple adhesive and chemokinetic interactions including, respectively, by sialylated glycoproteins on the cancer cells binding to E-selectin on the endothelium and by CXCR4-mediated sensing of SDF-1/CXCL12 gradients. Consequently, both E-selectin and CXCR4 inhibitors have been pursued as targets for add-on therapeutic strategies aimed to "mobilize" AML cells out of the BM. A next generation "mobilizing" agent GMI-1359 combines, in one molecular structure, the E-selectin and CXCR4 inhibitory moieties. In a previous study, GMI-1359 markedly reduced leukemia cell adhesion to endothelial cells, leukemia cellularity in BM (Zhang et al., 2016) and it enhanced the survival of cytarabine/daunorubicin therapy in a FLT3-mutated AML model (Zhang et al., 2015). APPROACH: Here, we used intravital 2-photon microscopy of calvarial BM to study the behavioral response of AML cells to the dual CXCR4 and E-selectin inhibition (GMI-1359) in vivo. We used a mTurquoise2 fluorescence-tagged transplantable mouse AML model (characterized by MLL, ENL-FLT3, ITD, p53-/-). To delineate the bone and vascular niches, the syngeneic immune-competent recipient mice harbored cell lineage fluorescence reporter genes such as Col2.3-GFP, hCD2-DsRed and CD11c-EYFP with the bone collagen and blood highlighted by, respectively, second harmonic generation (SHG) and fluorescent dextran. Intravenously infused AML cells (5x10E4) homed to BM where they gradually displaced most endogenous cells. In this experimental system, GMI-1359 or vehicle were infused into the tail vein while recording AML cell motilities in calvarial BM stroma in 3-D over 4 hours. RESULTS: In untreated or control vehicle treated mice, AML cells were slowly motile, migrating with the average velocity of ~2 µm/min. Cellular trajectories were random within the average 400 µm confinement radius corresponding to the size of BM cavities. Most AML cells localized in the vascular niche, defined as within a 50 µm distance to the nearest blood vessel. A minority of AML cells were also present in the proximity to osteoblasts and the bone (i.e. in the bone niche). In contrast, GMI-1359 infusion resulted in a 66% increase of the average speed of cellular motility of AML cells within 20 min and up to 100% increase within 3.5 hours. The motility pattern remained largely random within the bone cavity confinement volume and was concentrated in the vicinity of vasculature. Thereafter, we observed multiple instances of AML cell intravasation into the capillary lumens followed by several minute-long vessel wall attachment and sudden outflow. This cellular dynamics resulted in a substantial, yet structurally biased decrease of BM AML cellularity whereby the vascular niche was emptied preferentially whilst the bone niche remained populated by AML cells. In no case we observed a complete BM depletion of AML cells. Interestingly, as we reported before, a single, CXCR4-only inhibitor caused a protracted (24-48 h) depletion of AML cells in BM without significant cellular motility enhancement. CONCLUSIONS: Our observations reveal an unexpected mechanism of action by a dual E-selectin/CXCR4 inhibitor involving cellular migratory motility enhancement in BM stroma prior to AML cell intravasation. We envisage that, besides the designed blocking of E-selectin and CXCR4 molecules from binding their corresponding ligands, the dual moiety GMI-1359 agent has a capacity to generate motility-enhancing signals in AML cells that single inhibitors do not seem to trigger. Among several mechanisms possible, this action could involve E-selectin and CXCR4 crosslinking by a dual moiety molecular structure or binding avidity enhancement, requiring further investigation. In addition, our results highlight a likely mechanism of AML resistance to therapeutic "mobilization", one that involves cancer cell persistence in the bone/osteoblastic niches. Disclosures Zal: Daiichi-Sankyo: Research Funding; Moleculin Biotech, Inc.: Research Funding. Fogler:GlycoMimetics: Current Employment, Current equity holder in publicly-traded company, Patents &amp; Royalties. Magnani:GlycoMimetics, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties. Andreeff:Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Amgen: Research Funding; Centre for Drug Research &amp; Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees.

LSD1 Inhibitor CPI-482 Shows Efficacy and Prolongs Survival in Mouse Models of AML and Post-MPN AML in the Context of Constitutive JAK-STAT Pathway Activation

LSD1 Inhibitor CPI-482 Shows Efficacy and Prolongs Survival in Mouse Models of AML and Post-MPN AML in the Context of Constitutive JAK-STAT Pathway Activation

Combined Blockage of E-Selectin and CXCR4 (GMI-1359) Enhances Anti-Leukemia Effect of FLT3 Inhibition (Sorafenib) and Protects Hematopoiesis in Pre-Clinical AML Models

Acute myelogenous leukemia (AML) is characterized by an accumulation of abnormal white blood cells. Internal tandem duplications in the fms-like tyrosine kinase 3 (FLT3-ITD) account for 30% of adult AML cases and confer poor prognosis (Nakao et al., Leukemia 1996). FLT3 inhibitors like sorafenib efficiently eliminate circulating leukemia blasts, but frequently not in the bone marrow (BM), which suggests a protective effect of the BM niche for leukemic stem cell survival (Zhang et al., JNCI 2008). The homing of AML cells in BM is mediated chiefly by the adhesion to E-selectin on endothelial cells (ECs) and by CXCR4-directed cellular migration to stromal CXCL12 (SDF1) sources (Chien et al., Blood 2013; Peled and Tavor, Theranostics 2013). In many respects, BM homing signals are shared between leukemia and hematopoietic stem cells (HSCs). Our previous study demonstrated that targeting E-selectin/CXCR4 with the dual E-selectin/CXCR4 antagonist GMI-1359 markedly reduced leukemia cell adhesion to ECs and mesenchymal stem cells, reduced the BM-mediated protection of leukemic cells during FLT3-targeted therapy in vitro, and effectively reduced leukemia cellularity in the BM in vivo (Zhang et al., Can Res suppl 2016). Further, GMI-1359 combined with cytarabine/daunorubicin provided a profound survival benefit in mice with FLT3-mutated leukemia (Zhang et al., Blood suppl 2015). In the present study, we sought to evaluate dual E-selectin/CXCR4 blockage in the context of FLT3 inhibition by sorafenib in vivo, and to better understand the underlying mechanism. We compared expression levels of E-selectin ligands and CXCR4 in FLT3 inhibitor-sensitive Ba/F3-FLT3-ITD cells and their inhibitor-resistant counterparts Ba/F3-FLT3-ITD+D835Y and Ba/F3-FLT3-ITD+F691L. Resistant cells expressed 1.7 to ~5.6-fold higher levels of total E-selectin ligand detected by a soluble E-selectin reagent, and 10-fold higher levels of CXCR4. In addition, BM-mimetic hypoxia culture profoundly upregulated the cell surface expression of E-selectin ligands and CXCR4 on leukemia cells. We evaluated anti-leukemia effects of co-targeting E-selectin/CXCR4 and FLT3 with GMI-1359 and sorafenib in a patient-derived AML xenograft (PDX) model harboring FLT3-ITD and WT1 mutations. We observed that addition of GMI-1359 to sorafenib greatly reduced leukemia cellularity compared to sorafenib alone, and as much as by 92%, 82%, 69% and 45% in, respectively, liver, lung, spleen and BM (Fig. 1) as compared with vehicle-treated mice (p &amp;lt; 0.05). As expected, the number of circulating leukemia cells transiently increased. The GMI-1359/sorafenib combination improved mouse survival (median survival 138.5 versus 109, 87 and 126 days for the GMI-1359/sorafenib versus vehicle, GMI-1359 and sorafenib, respectively, p &amp;lt; 0.001). Using intravital 2-photon microscopy, we observed AML cell behavior in calvarial BM and their response to acute GMI-1359 bolus infusion. Remarkably, AML cell mobility began to increase in the BM microenvironment as soon as 20 min after treatment (Fig. 2), followed by intravasation and cellular outflow through the BM capillary vasculature over the next 2-4 hours. Moreover, although BM homing signals are thought to be shared between leukemia and HSCs, the combination therapy improved hematopoiesis parameters compared to sorafenib alone. In particular, this important effect was associated with increased numbers of megakaryocytes (2.1-fold), myelocytes (2.1-fold), and erythrocytes (7.1-fold) in BM (p &amp;lt; 0.01). The underlying mechanism(s) of hematopoiesis protection by GMI-1359 are under investigation. Conclusion: Co-inhibition of E-selectin/CXCR4 enhances the anti-leukemia efficacy of FLT3 inhibition and preserves hematopoiesis in the BM in a PDX model of AML. Disclosures Fogler: GlycoMimetics: Current Employment, Current equity holder in publicly-traded company, Patents &amp; Royalties. Magnani:GlycoMimetics, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties. Zal:Daiichi-Sankyo: Research Funding; Moleculin Biotech, Inc.: Research Funding. Andreeff:Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research &amp; Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

CK2 Inhibitor CX4945 Shows Strong In Vivo Anti Leukemia Effect in AML Via Augmented Ikaros-Mediated Regulation of Global Epigenetic Landscape

Over-expression of Casein Kinase II (CK2) pro-oncogenic kinase in Acute Myelogenous Leukemia (AML) is associated with poor prognosis. Inhibition of CK2 by siRNAs or with the specific inhibitor, CX-4945, shows strong cytotoxic effects. CK2 is a ubiquitous, constitutively active serine/ threonine kinase which has been implicated in leukemia progression via multiple mechanisms. One of the well-established mechanism is CK2 mediated phosphorylation and impairment of tumor suppressor function of Ikaros transcription factor. Impaired function of Ikaros has been associated with the development of leukemia including AML. In B-cell lymphoblastic leukemia, CK2 inhibition has been shown to exert a therapeutic effect via restoration and/or enhancement of the tumor suppressor activity of the Ikaros protein. The mechanism of therapeutic action of CK2 inhibition in AML is largely unknown and in vivo efficacy CK2 inhibitors in AML has never been established. Here we report anti-leukemia effect of CX-4945 in preclinical models of AML and demonstrate mechanism of action of CK2 inhibitor via restoration of Ikaros driven global regulation of epigenetic landscape. In order to study the effect of CK2 inhibition on Ikaros function in AML, we used U937 - a human myelomonocytic leukemia cell line with high baseline CK2 expression. U937 cells were treated with specific CK2 inhibitor, CX4945 for 72 hours. CK2 activity and Ikaros phosphorylation was measured using CK2 kinase assay and in vivo labeling - radio immunoblot assay. Results showed significant decrease in CK2 activity and Ikaros phosphorylation with no change in overall expression of the Ikaros protein. Expression analysis using RNA sequencing showed that treatment with CX-4945 caused significant upregulation of genes controlling immunity, and inflammation; and downregulation of genes involved in nucleic acid metabolism, RNA processing, and translation. Analysis of global genome-wide Ikaros occupancy using Chromatin Immunoprecipitation followed by next generation sequencing (ChIP-seq) of CX4945 treated U937 cells demonstrated that CX-4945 treatment significantly increased the number of Ikaros binding sites as well as increased peak strength while minimally re-distributing Ikaros' global genomic occupancy. While increased binding to the Promoter, Gene Body, and Gene Desert elements in CX-4945-treated cells was similar, the increase in Ikaros' DNA binding to enhancers was particularly pronounced. Further analysis showed that enhanced Ikaros DNA binding following treatment with CX-4945, directly induces formation of de novo enhancers. In order to test whether enhanced DNA binding of Ikaros is accompanied by augmented Ikaros function in the global epigenetic regulation of gene expression. We determined and compared chromatin accessibility of U937 cells before and after treatment with CX-4945, using ATAC-seq. Results indicate that Ikaros-induced de novo open chromatin at distal regulatory regions controls genes involved in the negative regulation of biological processes and cellular metabolism. Overall, these data demonstrate that, augmented Ikaros DNA-binding following CK2 inhibition resulted in 1) Ikaros' pioneering activity, 2) Ikaros' ability to induce the de novo formation of enhancers and super-enhancers, and 3) Ikaros' ability to induce the de novo formation of active enhancers and to activate poised enhancers. Together, these data uncover novel Ikaros functions in regulating the epigenetic landscape and identifies CK2 as a critical regulator of Ikaros activity. Next, we treated AML xenograft model of luciferase labelled U937 cells with CX4945 via oral gavage at dose 200mg/kg/day for 3 weeks and demonstrated significantly lower leukemia burden in treated group as measured by decreased bio-luminescence imaging. In summary, these results demonstrate for the first time that CK2 inhibitor, CX-4945 has strong anti-leukemia effect in AML preclinical models. One of the mechanisms by which CX4945 exert a therapeutic effect in AML involves enhancing Ikaros' function as regulator of global epigenomic landscape. These results provide strong mechanistic basis to develop novel targeted combination therapies using CK2 inhibitors for treatment of AML. Further studies evaluating combination therapies using patient derived xenograft (PDX) models of AML are underway. Disclosures Payne: Elf Zone, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Pevonedistat, a Nedd-8 Activating Enzyme Inhibitor, Upregulates NOXA to Increase Effectiveness of Azacitidine and Venetoclax in Preclinical Models of Acute Myelogenous Leukemia

Introduction: Venetoclax (VEN), a selective BCL-2 inhibitor, has yielded exceptional response rates in patients with acute myeloid leukemia (AML). VEN binds BCL-2 to directly inhibit sequestration of pro-apoptotic proteins such as the activator BIM. Free BIM can bind to BAX, enabling its oligomerization with BAK, mitochondrial outer membrane permeabilization (MOMP) and subsequent apoptosis (PMID 24074954, 9687260, 19641500, 20164920). VEN has limited efficacy in relapsed-refractory AML as a single agent, but when used in combination with DNA methyltransferase inhibitors (DNMTi; 5'azacitidine-AZA-or decitabine) or low-dose cytosine arabinoside (LDAC), 50-70% of untreated patients in recent clinical trials achieved complete remission (PMID 27520294, 30361262, 30892988). Despite this progress in AML therapy, the majority of AML patients treated with VEN ultimately relapse, and a large subset of patients never respond (PMID 30361262, 30892988). One postulated route of resistance to VEN is cellular upregulation of myeloid cell leukemia-1 protein (MCL-1), which, similarly to BCL-2, functions as an anti-apoptotic protein. VEN in combination with selective MCL-1 inhibitors has demonstrated added benefit over either agent alone in AML cells in vitro and in xenograft models but efficacy of this combination in the clinic has yet to be reported (PMID 30185627). Both MCL-1-dependent and MCL-1-independent mechanisms of VEN resistance are emerging (PMID 31048321, 30148320, 31262744) and new approaches aimed at addressing these are moving toward the clinic. Pevonedistat (PEV) was developed as a targeted inhibitor of NEDD-8 activating enzyme (NAE), which disrupts protein turnover mediated by Cullin-RING ligases (PMID 19360080) and has been shown to have activity in combination with AZA (PMID 29348128). PEV and AZA both upregulate NOXA, a BCL-2 family member that is known to suppress MCL-1 (PMID 26045051, Jin, Cojocari, Purkal et al.,unpublished data), so we postulated that PEV/AZA in combination synergizes with VEN in a triple combination with efficacy superior to VEN/AZA alone. Methods/Results: AML cell lines were treated with PEV, AZA and VEN alone, and compared with various VEN/PEV/AZA combinations revealing improved combinatorial activity with the triplet in the majority of cell lines and associated NOXA induction. Similar results were seen in primary AML patient samples ex vivo (Figure 1). These combinations were compared in vivo in an OCI-AML2 cell line xenograft model, also illustrating significantly improved response with the PEV/AZA/VEN combination (Figure 2). CRISPR/Cas 9 deletion of PMAIP1 in AML cell lines demonstrated that NOXA deletion abrogates this synergy. Discussion: Together, these results demonstrate that, in several pre-clinical models of AML, the PEV/AZA/VEN triple combination provides stronger anti-tumorigenic activity than either agent alone or the VEN/PEV and VEN/AZA combinations. Importantly, the three-drug combination may be effective in AML cells which do not respond to VEN/AZA alone. Further studies to delineate this mechanism of action, and a clinical trial (NCT03862157) testing the combination in newly diagnosed AML are underway. Figure 1 Disclosures Cojocari: AbbVie Inc: Employment, Other: DC, JP, ERB, JDL, and DCP are employees of AbbVie. JP, ERB, JDL &amp; DCP are stockholders of AbbVie Inc. The design study conduct, and financial support for this research were provided by AbbVie. AbbVie Inc. participated in the interpretation of data, review. Purkal:AbbVie Inc: Employment, Other: DC, JP, ERB, JDL, and DCP are employees of AbbVie. JP, ERB, JDL &amp; DCP are stockholders of AbbVie Inc. The design study conduct, and financial support for this research were provided by AbbVie. AbbVie Inc. participated in the interpretation of data, review. Leverson:AbbVie Inc: Employment, Other: Stock or options. Boghaert:AbbVie Inc: Employment, Other: DC, JP, ERB, JDL, and DCP are employees of AbbVie. JP, ERB, JDL &amp; DCP are stockholders of AbbVie Inc. The design study conduct, and financial support for this research were provided by AbbVie. AbbVie Inc. participated in the interpretation of data, review. Phillips:AbbVie Inc: Employment, Other: DC, JP, ERB, JDL, and DCP are employees of AbbVie. JP, ERB, JDL &amp; DCP are stockholders of AbbVie Inc. The design study conduct, and financial support for this research were provided by AbbVie. AbbVie Inc. participated in the interpretation of data, review. Savona:AbbVie: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; Boehringer Ingelheim: Patents &amp; Royalties; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Selvita: Membership on an entity's Board of Directors or advisory committees.

SEL120 - a First-in-Class CDK8/19 Inhibitor As a Novel Option for the Treatment of Acute Myeloid Leukemia and High-Risk Myelodysplastic Syndrome - Data from Preclinical Studies and Introduction to a Phase Ib Clinical Trial

SEL120 - a First-in-Class CDK8/19 Inhibitor As a Novel Option for the Treatment of Acute Myeloid Leukemia and High-Risk Myelodysplastic Syndrome - Data from Preclinical Studies and Introduction to a Phase Ib Clinical Trial

Central Role of Cell Cycle Regulation in the Antitumoral Action of Ocoxin.

Nutritional supplements which include natural antitumoral compounds could represent safe and efficient additives for cancer patients. One such nutritional supplement, Ocoxin Oral solution (OOS), is a composite formulation that contains several antioxidants and exhibits antitumoral properties in several in vitro and in vivo tumor conditions. Here, we performed a functional genomic analysis to uncover the mechanism of the antitumoral action of OOS. Using in vivo models of acute myelogenous leukemia (AML, HEL cells, representative of a liquid tumor) and small-cell lung cancer (GLC-8, representative of a solid tumor), we showed that OOS treatment altered the transcriptome of xenografted tumors created by subcutaneously implanting these cells. Functional transcriptomic studies pointed to a cell cycle deregulation after OOS treatment. The main pathway responsible for this deregulation was the E2F–TFDP route, which was affected at different points. The alterations ultimately led to a decrease in pathway activation. Moreover, when OOS-deregulated genes in the AML context were analyzed in patient samples, a clear correlation with their levels and prognosis was observed. Together, these data led us to suggest that the antitumoral effect of OOS is due to blockade of cell cycle progression mainly caused by the action of OOS on the E2F–TFDP pathway.