Acute Myeloid Leukemia Progenitor Cells Research Articles

Abstract 3003: Inhibiting the mitochondrial enzyme phosphatidylserine decarboxylase (PISD) reduces stemness and increases differentiation in acute myeloid leukemia (AML)

Abstract Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by the accumulation of malignant myeloid cells that have arrested maturation. Most therapeutic regimens approved or under development are cytotoxics. An alternate, but less explored therapeutic approach, is to induce terminal differentiation of AML cells. Upon differentiation, AML cells cease to proliferate or die. Phosphatidylserine decarboxylase (PISD) is a mitochondrial enzyme that converts phosphatidylserine (PS) to phosphatidylethanolamine (PE). Here, we explored the effects of inhibiting PISD on AML growth, stemness and differentiation. Knockout of PISD by CRISPR reduced the growth and clonogenic growth of OCI-AML2 cells. The reported chemical PISD inhibitor, 7-chloro-N-(4-ethoxyphenyl)-4-quinolinamine (aka: MMV007285), reduced growth and viability of OCI-AML2 cells (IC50 = 4.741 μM) and TEX cells (IC50 = 4.868 μM). Using the 8227 primary AML cell culture model, we showed that inhibiting PISD induced cell death in the functionally defined stem cell fraction (CD34+CD38-). MMV007285 also preferentially inhibited the clonogenic growth of primary AML cells (n = 7) over normal hematopoietic cells (n= 3). Moreover, MMV007285 induced AML cell differentiation as evidenced by increased CD11b expression and staining for non-specific esterase. Using high-performance thin layer chromatography (HPTLC), we found that inhibition of PISD with MMV007285 increased intracellular PS. To determine whether increased PS was functionally important, OCI-AML2 cells were treated with PS, resulting in reduced growth and clonogenic growth. Furthermore, PS supplementation targeted AML progenitor cells as it decreased engraftment of TEX cells in mice. Mechanistically, inhibiting PISD induced differentiation and decreased stemness in AML by activating Toll-like receptor (TLR) signaling. Specifically, inhibiting PISD upregulated TLR4 and 8 expression and increased expression of cytokines downstream of TLR activation. We also showed that TLR activation was functionally important to induce AML differentiation. Finally, we evaluated the effects of PISD inhibition in AML mouse models. MMV007285 (300 mg/kg/5 of 7 days orally for 10 days) decreased the growth of OCI-AML2 cells in SCID mice. Moreover, MMV007285 (150 mg/kg/5 of 7 days orally for 5 weeks) impeded the leukemic engraftment of primary AML cell in NOD/SCID mice without toxicity. Using secondary transplants, we showed that MMV007285 also targeted the leukemic stem cells. Taken together, inhibition of PISD altered phospholipid metabolism, inhibited growth and stemness, and increased differentiation in AML cells. Our findings reveal a previously undescribed link between mitochondrial phospholipid metabolism and AML stemness and differentiation, highlighting a potential new therapeutic strategy for AML. Citation Format: Mingjing Xu, Ayesh Seneviratne, Val A. Fajardo, Geethu E. Thomas, G. Wei Xu, Rose Hurren, S. Kim, Neil MacLean, Xiaoming Wang, Marcela Gronda, Danny Jeyaraju, Yulia Jitkova, David Sharon, Ahmed Aman, Rima Al-awar, Steven Chan, Mark D. Minden, Paul LeBlanc, Aaron D. Schimmer. Inhibiting the mitochondrial enzyme phosphatidylserine decarboxylase (PISD) reduces stemness and increases differentiation in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3003.

Venetoclax Synergistically Enhances the Anti-leukemic Activity of Vosaroxin Against Acute Myeloid Leukemia Cells Ex Vivo.

The survival rate for acute myeloid leukemia remains unacceptably low, in large part owing to resistance to chemotherapy and high rates of relapse. There is an urgent need to develop new therapeutic modalities, in particular such that are tolerated by patients over the age of 60years, who form the bulk of new acute myeloid leukemia diagnoses. Vosaroxin (SNS-595), a second-generation topoisomerase II inhibitor and DNA intercalating agent, shows promising preclinical and clinical activity against acute myeloid leukemia. Venetoclax (ABT-199), a selective Bcl-2 inhibitor, was recently approved for the treatment of acute myeloid leukemia. The objective of this study was to determine the anti-leukemic activity and the underlying molecular mechanisms for the combination of venetoclax and vosaroxin in acute myeloid leukemia cell lines and primary patient samples ex vivo. Using both acute myeloid leukemia cell lines and primary patient samples, annexin V/propidium iodide staining and flow cytometry analyses were used to quantify apoptosis induced by venetoclax or vosaroxin, alone or in combination, with subsequent western blotting analyses to assess levels of Bcl-2 family proteins. Alkaline comet assays were performed to quantify DNA damage induced by the two agents and to determine the effect of venetoclax on DNA repair. Finally, colony-forming assays were conducted on normal human CD34+ cord blood cells and primary acute myeloid leukemia patient samples to determine the effect of venetoclax and vosaroxin on normal hematopoietic and leukemic progenitor cells. We found that venetoclax and vosaroxin synergistically induced apoptosis in multiple acute myeloid leukemia cell lines. Although vosaroxin could partially abrogate the increase of Mcl-1 protein induced by venetoclax, it could not abrogate the increased binding of Bim to Mcl-1 induced by venetoclax. Cooperative induction of DNA damage occurred within 8h of treatment with venetoclax plus vosaroxin. Moreover, repair of DNA damage induced by vosaroxin was significantly attenuated by venetoclax. The combination also synergistically induced apoptosis in primary acute myeloid leukemia patient samples and significantly reduced the colony formation capacity of acute myeloid leukemia progenitor cells, while sparing normal hematopoietic progenitor cells. Vosaroxin and venetoclax synergistically induce apoptosis in acute myeloid leukemia cells and cooperatively target acute myeloid leukemia progenitor cells while sparing normal hematopoietic progenitor cells. Our results support the clinical testing of vosaroxin in combination with venetoclax for treating patients with acute myeloid leukemia, especially in the elderly population.

Peperomin E and its orally bioavailable analog induce oxidative stress-mediated apoptosis of acute myeloid leukemia progenitor cells by targeting thioredoxin reductase

The early immature CD34+ acute myeloid leukemia (AML) cell subpopulation-acute myeloid leukemia progenitor cells (APCs), is often resistant to conventional chemotherapy, making them largely responsible for the relapse of AML. However, to date, the eradication of APCs remains a major challenge. We previously reported a naturally occurring secolignan- Peperomin E (PepE) and its analog 6-methyl (hydroxyethyl) amino-2, 6-dihydropeperomin E (DMAPE) that selectively target and induce oxidative stress-mediated apoptosis in KG-1a CD34+ cells (an APCs-like cell line) in vitro. We therefore further evaluated the efficacy and the mechanism of action of these compounds in this study. We found that PepE and DMAPE have similar potential to eliminate primary APCs, with no substantial toxicities to the normal cells in vitro and in vivo. Mechanistically, these agents selectively inhibit TrxR1, an antioxidant enzyme aberrantly expressed in APCs, by covalently binding to its selenocysteine residue at the C-terminal redox center. TrxR1 inhibition mediated by PepE (DMAPE) leads to the formation of cellular selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP), oxidation of Trx, induction of oxidative stress and finally activation of apoptosis of APCs. Our results demonstrate a potential anti-APCs molecular target – TrxR1 and provide valuable insights into the mechanism underlying PepE (DMAPE)-induced cytotoxicity of APCs, and support the further preclinical investigations on PepE (DMAPE)-related therapies for the treatment of relapsed AML.

Deferasirox selectively induces cell death in the clinically relevant population of leukemic CD34+CD38– cells through iron chelation, induction of ROS, and inhibition of HIF1α expression

Despite a high remission rate after therapy, only 40-50% of acute myeloid leukemia (AML) patients survive 5 years after diagnosis. The main cause of treatment failure is thought to be insufficient eradication of CD34+CD38- AML cells. In order to induce preferential cell death in CD34+CD38- AML cells, two separate events may be necessary: (1) inhibition of survival signals such as nuclear factor kappa-beta (NF-κB) and (2) induction of stress responses such as the oxidative stress response. Therefore, regimens that mediate both effects may be favorable. Deferasirox is a rationally designed oral iron chelator mainly used to reduce chronic iron overload in patients who receive long-term blood transfusions. Our study revealed that clinically relevant concentrations of deferasirox are cytotoxic in vitro to AML progenitor cells, but even more potent against the more primitive CD34+CD38- cell population. In addition, we found that deferasirox exerts its effect, at least in part, by inhibiting the NF-κB/hypoxia-induced factor 1-alpha (HIF1α) pathway and by elevating reactive oxygen species levels. We believe that, pending further characterization, deferasirox can be considered as a potential therapeutic agent for eradicating CD34+CD38- AML cells.

Antibody-Targeted Cyclodextrin-Based Nanoparticles for siRNA Delivery in the Treatment of Acute Myeloid Leukemia: Physicochemical Characteristics, in Vitro Mechanistic Studies, and ex Vivo Patient Derived Therapeutic Efficacy.

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults and is associated with high relapse rates. It is known that leukemia stem cells (LSCs), a very small subpopulation of the total number of leukemic cells, maintain the leukemia phenotype (∼80-90% of AML remain the same as at first diagnosis), display chemotherapy resistance, and contribute to disease regeneration. Therefore, targeting LSCs could control the relapse of AML. Small interfering RNA (siRNA), an effector of the RNA interference (RNAi) pathway, can selectively downregulate any gene implicated in the pathology of disease, presenting great potential for treatment of AML. In this study an antibody targeted cyclodextrin-based nanoparticle (NP) (CD.DSPE-PEG-Fab) was developed for siRNA delivery specifically to AML LSCs. The targeted CD.siRNA.DSPE-PEG-Fab formulation, where Fab specifically targets the IL-3 receptor α-chain (IL-3Rα, also known as CD123, a cell surface antigen for human AML LSCs), achieved antigen-mediated cellular uptake in KG1 cells (an AML leukemia stem and progenitor cell line). Efficient delivery of bromodomain-containing protein 4 (BRD4) siRNA using the targeted formulation resulted in downregulation of the corresponding mRNA and protein in KG1 cells and in ex vivo primary AML patient derived samples. The resulting silencing of BRD4 induced myeloid differentiation and triggered leukemia apoptosis. In addition, a synergistic therapeutic effect was detected when administered in combination with the chemotherapeutic, cytarabine (Ara-C). These results indicate the clinical potential of the antibody-tagged cyclodextrin NP for targeted delivery of therapeutic siRNA in the treatment of AML.

The novel BMI-1 inhibitor PTC596 downregulates MCL-1 and induces p53-independent mitochondrial apoptosis in acute myeloid leukemia progenitor cells

Disease recurrence is the major problem in the treatment of acute myeloid leukemia (AML). Relapse is driven by leukemia stem cells, a chemoresistant subpopulation capable of re-establishing disease. Patients with p53 mutant AML are at an extremely high risk of relapse. B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is required for the self-renewal and maintenance of AML stem cells. Here we studied the effects of a novel small molecule inhibitor of BMI-1, PTC596, in AML cells. Treatment with PTC596 reduced MCL-1 expression and triggered several molecular events consistent with induction of mitochondrial apoptosis: loss of mitochondrial membrane potential, BAX conformational change, caspase-3 cleavage and phosphatidylserine externalization. PTC596 induced apoptosis in a p53-independent manner. PTC596 induced apoptosis along with the reduction of MCL-1 and phosphorylated AKT in patient-derived CD34+CD38low/− stem/progenitor cells. Mouse xenograft models demonstrated in vivo anti-leukemia activity of PTC596, which inhibited leukemia cell growth in vivo while sparing normal hematopoietic cells. Our results indicate that PTC596 deserves further evaluation in clinical trials for refractory or relapsed AML patients, especially for those with unfavorable complex karyotype or therapy-related AML that are frequently associated with p53 mutations.

Immunoprofiling of Normal Human Donor Blood Identified Potential Pharmacodynamic Markers for JNJ-63709178 (CD123xCD3) Duobody® Antibody Treatment

BackgroundJNJ-63709178 (CD123xCD3) is a promising new bispecific antibody currently being investigated for the treatment for acute myeloid leukemia (AML), a disorder of early hematopoietic progenitor cells characterized by the proliferation and accumulation of immature, clonal, myeloid cells. Constitutive overexpression of the α-subunit of the interleukin-3 receptor (ie, IL-3αR/CD123) is a hallmark of the early AML progenitor cell population (ie, leukemic stem cells [LSC]), as well as leukemic blasts. CD123 is unique to the IL-3 receptor and is responsible for the high specificity and low affinity binding of IL-3. Under physiological conditions, activation of the IL-3 receptor induces proliferative, anti-apoptotic, and differentiating signals. JNJ-63709178, a humanized IgG4 bispecific DuoBody® antibody with high affinity anti-CD123 and -CD3 arms, recognizes CD123 on myeloid cells and CD3 on T cells, leading to T-cell activation, with subsequent lysis of CD123+ AML blasts. JNJ-63709178 induces potent tumor cell killing in vitro, ex vivo, and in murine AML models, and is currently being evaluated in first-in-human clinical trials. Although CD123 is distinctively expressed on leukemic cells, some populations of normal myeloid cells have been reported to express CD123. The goals of this study were to determine CD123 receptor density on various leukocyte populations in normal human peripheral blood and evaluate the effects of JNJ-63709178 on those cells to identify potential pharmacodynamic markers.MethodsPeripheral blood samples from 6 normal human donors were analyzed ex vivo for CD123 expression on various leukocyte populations to determine receptor density. Receptor density on cells was quantified using Quantibrite PE enumeration kit. Subsequently, blood samples were treated with escalating concentrations of JNJ-63709178 (CD123xCD3) or the control compound CNTO 7008 (nullxCD3) DuoBody® antibodies and incubated at 37°C for 48 hours. After incubation, blood samples were stained and processed for analysis of T-cell activation (CD25 expression) and population persistence or depletion (frequencies within leukocytes). In the myeloid compartment, basophils, myeloid dendritic cells (mDCs), monocytes, neutrophils, and eosinophils were analyzed. In the lymphoid compartment, plasmacytoid dendritic cells (pDCs), B cells (naïve and memory), T cells (helper and cytotoxic), and natural killer (NK) cells (CD56bright and CD56dim) were analyzed.ResultsIn normal human peripheral blood samples, CD123 expression was highest on pDCs and basophils (above 10,000 receptors per cell). Intermediate expression (300-3,000 receptors per cell) was observed on mDCs, monocytes, eosinophils, and naïve B cells. Low expression (10-200 receptors per cell) was observed on neutrophils, memory B cells, T cells, and NK cells. These data are in agreement with previous studies (Busfield et al. ASH 2013 abs3598). Following incubation with JNJ-63709178, potent activation of CD4+ and CD8+ T cells was observed at sub-nanomolar levels, with EC50values of 0.07 nM and 0.1 nM, respectively. Higher CD123 expression and T-cell activation correlated strongly with profound depletion of basophils and pDCs, as well as partial depletion of eosinophils and monocytes. Consistent with low CD123 expression, T cells and NK cells did not show substantial dose-dependent depletion. In contrast to CD123 expression levels, B cells and neutrophils showed partial depletion, despite low CD123 expression, while mDCs did not show dose-dependent depletion, despite higher CD123 levels. The activation of T cells and cytotoxic effect on CD123-positive cells was observed only with JNJ-63709178. The control compound CNTO 7008 (nullxCD3) had no effect on T-cell activation or any of the investigated cell populations.ConclusionsThese findings highlight the potential pharmacodynamic markers of JNJ-63709178 (CD123xCD3) bispecific treatment and provide a better understanding of its mechanism of action and a rationale for receptor expression monitoring during ongoing clinical trials. DisclosuresBabich:Janssen: Employment. Li:Janssen: Employment. Gaudet:Janssen Pharmaceuticals R&D: Employment, Other: Stock options, Patents & Royalties: pending, not yet issued. Huang:Janssen Research & Development, LLC: Employment, Other: I am an employee of Janssen and a stock owner . Sasser:Johnson & Johnson: Equity Ownership; Janssen Pharmaceuticals R&D: Employment. Salvati:Janssen Pharmaceuticals R&D: Employment, Other: stock options, Patents & Royalties: patent. Kalota:Janssen Pharmaceuticals R&D: Employment, Other: stock.

Selective Death of Leukemia Initiating Cells Induced By Deferasirox

Despite high remission rate after therapy, only 30-40% of acute myeloid leukemia (AML) patients survive 5 years after diagnosis. The main cause of treatment failure is thought to be insufficient eradication of leukemia initiating cells (LIC). Identifying drugs that can efficiently eradicate LICs is therefore imperative. HIF1α is essential for LIC maintenance and targeting HIF1α selectively eliminates LIC. Deferasirox is an iron chelator used to reduce chronic iron overload in patients receiving long-term blood transfusions. Our aim is to study the ability ofdeferasirox to induce apoptosis and to target HIF1α in AML LICs.CD34+CD38- LIC-like cells, sorted from the KG1a cell line, exhibited increased sensitivity to deferasirox with an IC50 of 1.3μM compared to 8.9μM calculated for the more mature CD34+CD38+ cells. The LIC-like cells, which were more sensitive to deferasirox, were less sensitive to ARA-C compared to the CD34+CD38+ cells. Deferasirox was >2-fold more efficient in inducing apoptosis in the CD34+CD38− cells, compared to the CD34+CD38+ cells (74±7.2% & 32±6.2% apoptosis, respectively). Deferasirox demonstrated a synergistic cytotoxic effect with ARA-C on both the CD34+CD38− and CD34+CD38+ KG1a cell fractions. Similar results were observed with CD34+CD38-CD123+ LICs and CD34+CD38+ CD123+ progenitor cells isolated from AML patients. As shown with the cell line, AML patient LICs were 2-fold more sensitive to deferasirox treatment showing a 62±15% induction of cell death compared to only a 34±9% induction in leukemic progenitor cell death (p<0.01). The increase in cell death was accompanied by an increment of reactive oxygen species (ROS) levels which was more prominent in the LICs compared to the progenitor cells. Furthermore, deferasirox enhanced the cytotoxic effects of ARA-C on both the LSCs and the leukemic progenitor cells. These data indicate that deferasirox is highly cytotoxic to AML cells; however, it is significantly more specific to AML initiating cells. In addition, deferasirox exhibited significant inhibitory effect on the proliferation of both the CD34+CD38− and CD34+CD38+ KG1a cell fractions accompanying by an S-phase arrest. The inhibition of proliferation was also demonstrated using proliferation dye and colony assay on both the LIC-like cells and the LICs isolated from AML patient (p<0.001). Since deferasirox is an NFkB inhibitor which regulates HIF1α levels, and since HIF1α is selectively activated in AML stem cells and is essential for maintenance of these cells, we studied the effect of deferasirox on this pathway. We found that NFkB translocation to the nucleus was reduced by over 80% and as a result the binding of NFkB to the HIF1α promoter was decreased in the LIC-like cells treated with deferasirox. HIF1α was downregulated transcriptionally (~45% reduction) and translationally (~55% reduction) in the LIC-like cells while it was upregulated in the CD34+CD38+KG1a cells following deferasirox treatment. In order to understand which of deferasirox’s properties is crucial for its apoptotic effect, we used the iron chelator, deferipron, which does not effect on NFkB or HIF1α and siHIF1α. Each one of these treatments alone did not lead to an apoptotic effect in LIC-like cells or in the CD34+CD38+ kg1a cells. Conversely, the combination of the two lead to an increase in apoptosis in the LIC-like cells suggesting that only a combination of iron chelation and HIF1α reduction is sufficient to result in an apoptotic response. The apoptotic effect wasn’t observed in the CD34+CD38+ fraction. These data hint that AML-LICs are selectively targeted by deferasirox.We describe a novel and unique anti-LIC property of deferasirox, which was originally developed as an iron chelator. We found clinically relevant concentrations of deferasirox to be cytotoxic in vitro to AML progenitor cells but even more potent against LICs. We believe that deferasirox exerts its cytotoxic effect, at least in part, by downregulating HIF1α levels in the LIC population. Pending further characterization, deferasirox can be considered as a potential therapeutic agent for eradicating LICs. DisclosuresNo relevant conflicts of interest to declare.

Concurrent targeting Akt and sphingosine kinase 1 by A-674563 in acute myeloid leukemia cells

Akt signaling plays a pivotal role in acute myeloid leukemia (AML) development and progression. In the present study, we evaluated the potential anti-AML activity by a novel Akt kinase inhibitor A-674563. Our results showed that A-674563 dose-dependently inhibited survival and proliferation of U937 AML cells and six lines of human AML progenitor cells, yet sparing human peripheral blood mononuclear leukocytes (PBMCs). A-674563 activated caspase-3/9 and apoptosis in the AML cells. Reversely, the pan-caspase inhibitor z-VAD-CHO dramatically alleviated A-674563-induced AML cell apoptosis and cytotoxicity. For the molecular study, we showed that A-674563 blocked Akt activation in U937 cells and human AML progenitor cells. Further, A-674563 decreased sphingosine kinase 1 (SphK1) activity in above AML cells to deplete pro-survival sphingosine-1-phosphate (S1P) and boost pro-apoptotic ceramide production. Such an effect on SphK1 signaling by A-674563 appeared independent of Akt blockage. Significantly, K6PC-5, a novel SphK1 activator, or supplement with S1P attenuated A-674563-induced ceramide production, and subsequent U937 cell death and apoptosis. Importantly, intraperitoneal injection of A-674563 at well-tolerated doses suppressed U937 leukemic xenograft tumor growth in nude mice, whiling significantly improving the animal survival. The results of the current study demonstrate that A-674563 exerts potent anti-leukemic activity in vitro and in vivo, possibly via concurrent targeting Akt and SphK1 signalings.

Preclinical evaluation of WYE-687, a mTOR kinase inhibitor, as a potential anti-acute myeloid leukemia agent

Mammalian target of rapamycin (mTOR) as a potential drug target for treatment of acute myeloid leukemia (AML). Here, we investigated the potential anti-leukemic activity by WYE-687, a potent mTOR kinase inhibitor. We demonstrated that WYE-687 potently inhibited survival and proliferation of established (HL-60, U937, AML-193 and THP-1 lines) and human AML progenitor cells. Yet, same WYE-687 treatment was non-cytotoxic to the primary peripheral blood mononuclear leukocytes (PBMCs) isolated from healthy donors. WYE-687 induced caspase-dependent apoptotic death in above AML cells/progenitor cells. On the other hand, the pan-caspase inhibitor (Z-VAD-FMK), the caspase-3 specific inhibitor (Z-DEVD-FMK) or the caspase-9 specific inhibitor (z-LEHD-fmk) attenuated WYE-687-induced cytotoxicity. At the molecular level, WYE-687 concurrently inhibited activation of mTORC1 (p70S6K1 and S6 phosphorylations) and mTORC2 (AKT Ser-473 and FoxO1/3a phosphorylations), whiling downregulating mTORC1/2-regulated genes (Bcl-xL and hypoxia-inducible factor 1/2α) in both HL-60/U937 cells and human AML progenitor cells. In vivo, oral administration of WYE-687 potently inhibited U937 leukemic xenograft tumor growth in severe combined immunodeficient (SCID) mice, without causing significant toxicities. In summary, our results demonstrate that targeting mTORC1/2 by WYE-687 leads to potent antitumor activity in preclinical models of AML.

Targeting GLI1 Suppresses Cell Growth and Enhances Chemosensitivity in CD34+ Enriched Acute Myeloid Leukemia Progenitor Cells

Background/Aims: Resistance of leukemia stem cells (LSCs) to chemotherapy in patients with acute myeloid leukemia (AML) causes relapse of disease. Hedgehog (Hh) signaling plays a critical role in the maintenance and differentiation of cancer stem cells. Yet its role in AML remains controversial. The purpose of the present study is to investigate the role of GLI1, the transcriptional activator of Hh signaling, in AML progenitor cells and to explore the anti-AML effects of GLI small-molecule inhibitor GANT61. Methods: The expression of GLI1 mRNA and protein were examined in AML progenitor cells and normal cells. The proliferation, colony formation, apoptosis and differentiation of AML progenitor cells were also analyzed in the presence of GANT61. Results: Kasumi-1 and KG1a cells, containing more CD34⁺ cells, expressed higher level of GLI1 compared to U937 and NB4 cells with fewer CD34⁺ cells. Consistently, a positive correlation between the protein levels of GLI1 and CD34 was validated in the bone marrow mononuclear cells (BMMC) of AML patients tested. GANT61 inhibited the proliferation and colony formation in AML cell lines. Importantly, GANT61 induced apoptosis in CD34⁺ enriched Kasumi-1 and KG1a cells, whereas it induced differentiation in U937 and NB4 cells. Furthermore, GANT61 enhanced the cytotoxicity of cytarabine (Ara-c) in primary CD34⁺ AML cells, indicating that inhibition of GLI1 could be a promising strategy to enhance chemosensitivity. Conclusions: The present findings suggested that Hh signaling was activated in AML progenitor cells. GLI1 acted as a potential target for AML therapy.

Combination of Dasatinib with Conventional Chemotherapy Is Associated with a High Response Rate in High Risk Acute Myeloid Leukemia (AML)

Background: High risk AML patients (pts) have poor outcome with low complete remission (CR) rate and long term survival despite intensive chemotherapy.Tyrosine kinases play an important role in AML pathogenesis. Pre-clinical studies performed in our center (Blood. 2013 122:1900) have shown that Src family tyrosine kinases (SFK) including Lyn, Hck and Fgr are abnormally activated in AML compared to normal hematopoietic stem cell and progenitor cells. Other studies have shown that the c-Kit receptor is over-activated in a subset of AML pts and contributes to abnormal leukemia cell growth. We further show that the small molecule SFK and c-Kit inhibitor dasatinib reduces proliferation and survival of AML stem and progenitor cells. Importantly, dasatinib enhances the sensitivity of AML stem and progenitor cells to chemotherapeutic agents by inhibiting Akt signaling, increasing mdm2 phosphorylation and enhancing p53 activity in AML cells. Based on this data we conducted a phase I/II clinical study of the combination of dasatinib with conventional cytarabine-idarubicin ("7+3")-based induction chemotherapy in high-risk AML.Methods: Between September 20013 and July 2015, 18 adult AML pts were enrolled in the study. Eligibility criteria were high-risk AML, age >18 years, and suitability for intensive therapy. High risk AML was defined by one of the following criteria: older age (> 60 yrs), poor-risk cytogenetic and molecular abnormalities (ELN criteria), secondary disease (AML evolving from myelodysplasia or myeloproliferative neoplasm), therapy related (t-AML), or relapsed/refractory.This Phase I study used a 3 + 3 dose escalation design for dasatinib while keeping a fixed dose of cytarabine and idarubicin (cytarabine 200 mg/m2 CIV days 1-7, idarubicin 12 mg/m2 IV days 1-3). Dasatinib was started at a dose of 70 mg (dose level 1; DL-1) days 1-7, and escalated to 100 mg orally days 1-7 (DL 1). Pts who failed to achieve CR received second re-induction with the same regimen. Pts who achieved CR received consolidation high dose cytarabine and/or allogeneic stem cell transplantation (SCT) based on donor availability.Results: Of the 18 pts enrolled on the study, 7 pts (39%) had secondary AML, 5 pts (28%) relapsed AML, 2 pts (11%) t-AML, and 4 (22%) were newly diagnosed older AML pts (one with complex karyotype, one with trisomy 8 and one cytogenetically normal with FTL3-ITD). The median age of all pts was 62 yrs (range 27-73). Of the 18 pts, 13 pts are currently evaluable for response; of which 10 (77%) achieved CR/Cri. Of the 5 non-evaluable pts, one pt withdrew from study after 2 days of therapy, one pt was taken off study after 3 days due to cytarabine neurotoxicity, one pt died of intracranial hemorrhage before the day 14 bone marrow biopsy, one pt had therapy interrupted secondary to pneumonia and sepsis, and one is still receiving therapy. None of the pts required more than one induction to achieve CR. Of the 10 pts who achieved CR/Cri, 8 patients went on to receive allogeneic SCT. As expected, the most commonly reported grade 3 and 4 adverse events (AE) were anemia 50%, thrombocytopenia 44%, neutropenia 38%, and fatigue 27%. The most common grade 1-2 AE were GI toxicities 61% and rash 33%.Correlative studies performed on blood samples obtained from pts on day 3 after initiation of treatment (n=9) demonstrated significant decrease in of SRC activity (as indicated by reduction in phospho-SRC levels on immunoblotting, 0.52±0.11 of control, p=0.01), and increased expression of p53-target genes as evaluated by Q-RT-PCT [including Puma (16.2±6.9 fold, p=0.015), P21 (4.9±1.1 fold, p=0.004), DR5 (3.4±0.9, p=0.004), Bax (3.7±0.9 fold, p=0.001) and HDM2 (2.1±0.5, p=0.02)].Conclusion: Combination of dasatinib with conventional "7+3" induction chemotherapy is feasible in high-risk AML and leads to higher CR rate compared to historical data without increase in toxicity rate, allowing more pts to receive allogeneic transplantation. Correlative laboratory studies are consistent with pre-clinical studies suggesting that this combination is associated with significant inhibition of SRC activity and enhanced activation of p53-target genes. DisclosuresDos Santos:Amgen: Employment. Stein:Amgen: Speakers Bureau. Chen:Seattle Genetics: Consultancy, Research Funding, Speakers Bureau; Millennium: Consultancy, Research Funding, Speakers Bureau; Merck: Consultancy, Research Funding; genentech: Consultancy, Speakers Bureau. Khaled:Sequenom: Research Funding.

BH3 mimetic ABT-737 induces apoptosis in CD34+ acute myeloid leukemia cells and shows synergistic effect with conventional chemotherapeutic drugs.

Acute myeloid leukemia (AML) is an immunophenotypically heterogenous malignant disease. The early immature CD34+ AML cell subpopulation is frequently impervious to intensive chemotherapy, making them largely responsible for relapse of AML. CD34+ AML cells have higher level of Bcl-2 protein expression than the CD34- subpopulation. As such, development of drugs that specifically target the Bcl-2 may have the potential to eliminate immature CD34+ AML progenitor cells and provide therapeutic benefit. In this work, we made an attempt to investigate the cytotoxic effect of a novel Bcl-2 family inhibitor, ABT-737, on CD34+ AML cell lines (KG1a and Kasumi-1) as well as CD34+ primary AML cells. Primary human CD34+ cells were isolated from bone marrow mononuclear cells using CD34 MicroBead kit. The growth inhibitory effect was measured by cell counting kit-8. Apoptosis was analyzed by annexin V/PI assays. Protein expression was determined by Western blotting analysis. Inhibition of Bcl-2 by ABT-737 effectively inhibited growth and induced apoptosis in CD34+ AML cell lines and CD34+ primary AML cells without affecting CD34+ normal hematopoietic cells. Furthermore, Western blot analysis showed that ABT-737 induced apoptosis associated with caspase-3 activation and poly ADP-ribose polymerase (PARP) degradation. Finally, ABT-737 synergistically enhanced the cytotoxic effect of cytarabine and daunorubicin in CD34+ AML cells. Taken together, these findings indicate that ABT-737 may offer as a promising molecular targeting agent in CD34+ AML.

Targeting Mitochondria with Avocatin B Induces Selective Leukemia Cell Death.

Treatment regimens for acute myeloid leukemia (AML) continue to offer weak clinical outcomes. Through a high-throughput cell-based screen, we identified avocatin B, a lipid derived from avocado fruit, as a novel compound with cytotoxic activity in AML. Avocatin B reduced human primary AML cell viability without effect on normal peripheral blood stem cells. Functional stem cell assays demonstrated selectivity toward AML progenitor and stem cells without effects on normal hematopoietic stem cells. Mechanistic investigations indicated that cytotoxicity relied on mitochondrial localization, as cells lacking functional mitochondria or CPT1, the enzyme that facilitates mitochondria lipid transport, were insensitive to avocatin B. Furthermore, avocatin B inhibited fatty acid oxidation and decreased NADPH levels, resulting in ROS-dependent leukemia cell death characterized by the release of mitochondrial proteins, apoptosis-inducing factor, and cytochrome c. This study reveals a novel strategy for selective leukemia cell eradication based on a specific difference in mitochondrial function.

Prognostic Impact and Targeting of BMI-1 in Acute Myeloid Leukemia

A minor fraction of leukemia cells, leukemia stem cells, have been shown to be highly resistant to current therapies and thought to be responsible for recurrence. BMI-1, a part of polycomb repressive complex 1 (PRC1) is essential for the self-renewal of normal hematopoietic and leukemia stem cells. PTC-209 is a novel selective transcriptional inhibitor of BMI-1, which has been shown to have antitumor activity against cancer-initiating cells in colorectal cancer. We investigated the prognostic significance of BMI-1 in acute myeloid leukemia (AML) using reversed phase protein array and effects of the BMI-1 inhibitor PTC-209 on primary and leukemia cell lines. BMI-1 protein expression was determined in bulk AML blasts from 511 newly diagnosed patients. BMI-1 expression was higher in unfavorable cytogenetics (n=252, median 0.068) compared to intermediate (n=225, median -0.116, P = 0.017) or favorable cytogenetics (n=34, median -0.338, P = 0.0007 versus unfavorable, 0.05 versus intermediate). Higher BMI-1 levels were associated with shorter median overall survival (42.8 versus 55.3 weeks, P = 0.046 Log Rank test). There was no correlation between BMI-1 levels and percentages of CD34 -positive cells (r = 0.07). A total of 6 AML (MOLM-13, OCI-AML3, MV4-11, NB4, HL60 and U-937) and 5 ALL (Reh, NALM6, Jurkat, Raji and MOLT-4) cell lines were exposed to PTC-209 for 48 hours. PTC-209 exhibited dose- and time-dependent anti-proliferative and cytotoxic activities. The IC50 values (concentration at which cell growth is inhibited by 50% at 48 hours of exposure) were 0.33 ± 0.04 µM (mean ± SEM) for AML and 0.55 ± 0.09 µM for ALL, indicating potent anti-proliferative effects. In contrast, PTC-209 showed differential cytotoxic effects between AML and ALL cells. The ED50 values (effective concentration inducing 50% killing as measured by Annexin V positivity) were no more than 2.5 µM in 5 of 6 AML lines while they were higher than 10 µM in 3 out of 5 ALL cell lines, implicating that BMI-1 is more critical in AML than ALL. Treatment with PTC-209 triggered several molecular events consistent with induction of apoptosis in sensitive lines (e.g. MV4-11 and MOLM-13): conformational change of BAX (i.e., BAX activation), loss of mitochondrial membrane potential (MMP), caspase-3 activation and DNA fragmentation in addition to phosphatidylserine (PS) externalization. Eighteen-hour treatment of MV4-11 cells with 2.5 µM PTC-209 led to compound-specific induction of conformationally active BAX (31%), MMP loss (80%) and caspase-3 cleavage (38%). qRT-PCR showed reduced transcript level of BMI-1 (61% reduction) after 6-hour PTC-209 exposure in MV4-11 cells. PTC-209 induced PS externalization in primary AML cells (82.5 ± 4.3% after 48-hour treatment with 2 µM PTC-209, n = 6) and to a lesser degree, in ALL cells (33.7 ± 13.4%, n = 4, p < 0.05). Importantly, CD34+CD38– AML progenitor cells were as sensitive to PTC-209 as C34– more mature AML cells. Normal lymphocytes were resistant to PTC-209 (9.1 ± 4.6% even at 10 µM). Collectively, BMI-1 inhibition by small molecule inhibitors could be developed into a novel therapeutic strategy. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of Fatty Acid Oxidation with Avocatin B Selectively Targets AML Cells and Leukemia Stem Cells

Acute myeloid leukemia (AML) is an aggressive malignant disease characterized by poor patient outcome and suboptimal front-line chemotherapy. To identify novel anti-AML compounds, we performed a high-throughput screen of a natural products library (n=800). This screen was performed against the AML cell line (TEX), which has several properties of leukemia stem cells, the cells responsible for disease pathophysiology and patient relapse. Here, avocatin B was identified as a potent and novel anti-leukemia agent. Avocatin B, at concentrations as high as 20µM, had no effect on normal peripheral blood stem cell viability. In contrast, it induced death of primary AML cells with an EC50 of 1.5-5.0 µM. Selective toxicity towards a functionally defined subset of primitive leukemia cells was also demonstrated. Avocatin B (3µM) reduced clonogenic growth of AML progenitor cells with no effect on clonogenic growth of normal hematopoietic stem cells. Further, treatment of primary AML cells with avocatin B (3µM) diminished their ability to engraft into the bone marrow of pre-conditioned, NOD/SCID mice (t18=6.5; p<0.001). Together, these results confirm that avocatin B is a novel anti-AML agent with selective toxicity toward leukemia and leukemia stem cells.Mechanistically, avocatin B-induced reactive oxygen species (ROS)-dependent leukemia cell apoptosis that was characterized by the release of mitochondrial proteins, cytochrome c and apoptosis inducing factor (AIF). Cytochrome c and AIF were detected in the cytosol of avocatin B treated TEX cells by flow cytometry. Avocatin B-induced apoptosis, as measured by the Annexin V/Propidium iodide assay, DNA fragmentation and PARP cleavage, was abolished in the presence of anti-oxidants confirming the functional importance of ROS. Next, we further evaluated the role of mitochondria in avocatin B-induced apoptosis. First, we generated leukemia cells lacking mitochondria by successive culturing in media containing ethidium bromide. The drastic (>80%) reduction in mitochondria were confirmed by nonyl acridine orange staining and flow cytometry and a near absence of the mitochondria specific proteins ANT and ND1, as measured by Western blotting. Avocatin B’s activity was abolished in leukemia cells lacking mitochondria. Next, using lentiviral knockdown, we generated leukemia cells lacking CPT1, the enzyme that facilitates transport of 16-20 carbon lipids into mitochondria. Avocatin B’s activity was abolished in cells with reduced CPT1 expression (>70% as measured by qPCR analysis). To further confirm the importance of CPT1 in avocatin B-induced death, we chemically inhibited CPT1 with etomoxir. Avocatin B’s activity was blocked in the presence of etomoxir, further demonstrating that avocatin B accumulates in mitochondria.Since avocatin B is a lipid that targets mitochondria and that mitochondria can oxidize fatty acids for energy, we next assessed the impact of avocatin B on fatty acid oxidation, using the Seahorse Bioanalyzer. Avocatin B inhibited leukemia cell fatty acid oxidation (>40% reduction in oxygen consumption at 10µM) and this occurred at a 10-fold less concentration than etomoxir, the standard experimental molecule used to probe this pathway. Further, avocatin B resulted in a 50% reduction in levels of NADPH, an important co-factor generated during fatty acid oxidation that participates in catabolic processes during cell proliferation.These results show that avocatin B accumulates in mitochondria to inhibit fatty acid oxidation and reduce NADPH to result in ROS-mediated leukemia cell apoptosis. This highlights a novel AML-therapeutic strategy by which mitochondria are targeted to impair cellular metabolism leading directly to AML cell death. DisclosuresNo relevant conflicts of interest to declare.

The Expression Pattern of CD33 Antigen Can Differentiate Leukemic from Normal Progenitor Cells in Acute Myeloid Leukemia.

Leukemic stem cells (LSC) in acute myeloid leukemia (AML), defined by CD34 and CD38 antigens also express CD33 similar to normal hematopoietic stem cells. Residual LSC are believed to be responsible for relapse in AML after chemotherapy. Leukemic progenitor cell compartments were defined by CD34 and CD38 expression by flow cytometry in 61 new cases of AML. In each of four compartments thus defined, CD34+CD38-, CD34+CD38+, CD34-CD38- and CD34-CD38+, the pattern and intensity of expression of CD33 were studied in comparison to similar progenitor cell compartments in normal bone marrow and peripheral blood stem cell harvests. Post induction bone marrow samples from 10/61 cases were studied for aberrant CD33 expression. The intensity and pattern of expression of CD33 in AML progenitor cells were significantly different compared to normal progenitor cells. In two cases who were in morphological remission post induction, aberrant CD33 expressing progenitor cells were detectable at a frequency of 1.6 and 0.5% respectively in the bone marrow. Aberrant CD33 expression in bone marrow LSC identified as CD34+CD38- cells in the CD45 dim/low side scatter region on flow cytometry may be useful as minimal residual disease marker after AML therapy. The method involves the use of a limited number of reagents and can be applied to all cases of AML.

Selective Targeting Of Inv(16)+ AML Stem Progenitor Cells By Inhibiting HDAC8

Chromosomal inversion inv(16)(p13.1q22) which leads to the fusion of the transcription factor gene CBFb and the MYH11 gene, occurs in over 8% of acute myeloid leukemia (AML) cases. The fusion product CBFβ-SMMHC (CM) inhibits differentiation of hematopoietic stem and progenitor cells (HSPCs) and creates pre-leukemic populations predisposed to acute myeloid leukemia (AML) transformation. The mutations of tumor suppressor p53 occur in approximately half of all cases of human cancer, but TP53 mutations are relatively rare in inv(16) AML. We have previously shown that CM expression leads to reduced acetylation of p53 and impaired p53 target gene activation through formation of aberrant protein complex with p53 and HDAC8 (Blood, 2012,120: A772.). Here, we showed that CM interacts with p53 both in CM transformed mouse primary bone marrow cells as well as in AML stem and progenitor cells from inv(16) patients. When HDAC8 selective pharmacological inhibitor 22d directed against its catalytic sites (ChemMedChem 2012, 7:10, 1815-24;) were used to treat inv(16) mouse primary bone marrow progenitor cells and inv(16)+ CD34+ stem progenitor cells from patients, Ac-p53 levels were remarkably increased as shown by western blot. We further assessed the p53 target genes expression after HDAC8 inhibitor 22d treatment by qRT-PCR assay in inv(16)+ CD34+ stem progenitor cells (n=8), and observed variable levels of activation in p53 targets (Fold activation: p21:2.25-fold, hdm2:1.17-fold, 14-3-3σ: 3.12-fold, puma: 2.39-fold), indicating p53 was re-activated. Similar results were also shown in CM transformed mouse bone marrow progenitor cells. Importantly, we found that 22d treatment significantly inhibit the growth of inv(16)+ AML CD34+ cells (n=9) rather than normal CD34+ cells (n=7) , (AML IC50= 6.509 μM, vs Normal cells IC50=13.83 μM, p=0.0003). Meanwhile, 22d selectively induces apoptosis of inv(16)+ AML stem and progenitor cells while sparing normal HSPCs (AML LD50= 10.24 μM, vs NL LD50= 46.36 μM, p=0.001). To evaluate whether the effect of HDAC8i is mediated by p53, we knocked down p53 with a lentiviral vector expressing shRNA against p53 (or non-silencing shRNA) in AML CD34+ cells, and treated the cells with HDAC8 inhibitor 22d (5-20 µM). We showed that despite the inter-sample variability, knocking down p53 expression in all AML samples tested (n=3) led to reduced HDAC8i-induced apoptosis, suggesting that p53 contributes to the apoptosis effect induced by HDAC8i (22d) in inv(16)+ AML cells. Importantly, by taking advantage of our conditional knock-in mouse model (Cbfb56M/+/Mx1-Cre), which develops AML under induced expression of CBFß-SMMHC (Cancer Cell, 2006, 9:1, 57-68), we were able to perform the ex vivo treatment assay by treating primary leukemic cells (marked with dTomato) with either DMSO (as vehicle control) or with HDAC8 inhibitor 22d (10μM) for 48h, followed by transplantion into congenic mice (control group n=8, treatment group n=7). We observed reduced short-term engraftment of leukemic cells that are treated with 22d (10 μM) at 4 weeks post-transplantation in the peripheral blood (Donor cell%: control group=5.99%, treatment group=0.178%, P=0.0093). Interestingly, engraftment of cord blood CD34+ cells at 16 weeks post-bone marrow transplantation was not reduced after treatment with 22d (10 μM) (human CD45+ %: control=66.2% versus treatment=63.4%, p=0.9), indicating the effect by HDAC8 inhibition is selective for leukemic cells. In conclusion, we have identified a novel mechanism whereby CBFβ-SMMHC inhibits p53 fucntion, and may further implicate inhibition of HDAC8 as a promising approach to selectively target inv(16)+ AML stem and progenitor cells. Disclosures:No relevant conflicts of interest to declare.

Cathecholamines Differently Regulate Human AML and Normal Hematopoietic Progenitor Cell Motility Via miR126 and RGS16

Abstract Introduction Motility, proliferation, bone marrow (BM) retention and egress to the circulation of hematopoietic stem and progenitor cell (HSPCs) are key elements in normal hematopoiesis and also in the pathogenesis of acute myeloid leukemia (AML). HSPCs are tightly regulated in the BM niche by various molecules. Among them, CXCL12 and its major receptor CXCR4, expressed by both HSPCs and BM niche components, govern HSPC retention in the BM. Administration of CXCR4 antagonists lead to mobilization of both normal HSPCs and leukemia-inducing stem cells (LICs) from the BM to the circulation. We have previously found that normal human HSPCs functionally express b2-adrenergic receptors (b2-AR) that are up-regulated by myeloid cytokines such as G-CSF. The catecholaminergic neurotransmitters epinephrine and norepinephrine (NE) activate both Wnt and GSK3β signaling pathways via b2-AR, leading to enhanced HSPC proliferation, motility and BM repopulation (Spiegel et al, Nat Immunol 2007; Lapid et al, JCI 2013). These findings indicate HSPC regulation by dynamic interactions of the sympathetic nervous and hematopoietic systems. However, the role of catecholamines in regulation of AML remains elusive. Results We found that several human AML cell lines from different FAB subtypes express b2-AR. NE, a b2-AR activating ligand, increased b2-AR expression and the CXCL12-induced migration of AML primary patients’ cells and cell lines. In addition, NE treatment significantly enhanced CXCL12induced actin polymerization, which drives most of the cellular movements. Looking for a downstream effector of b2-AR, we focused on RGS16, a G-protein signaling regulator, which negatively regulates CXCL12/CXCR4 axis (Berthebaud et al., Blood 2005). Concurrently with the increase in cell migration, NE decreased RGS16 expression (both in protein and mRNA level) in monocytic AML cells. However, no effect on either cell migration or RGS16 expression was observed in non-monocytic AML cells. These data provided evidence for the involvement of catecholamines in the regulation of AML cell migration and showed a correlation between AML FAB subtypes, cell motility and RGS16 expression. One of the regulators of RGS16 levels is miR126, which is highly expressed in AML and normal HSPCs and plays an important role in mobilization and proliferation of normal HSPCs. Indeed, in search for the mechanisms underlying the above observed differences, we found that the enhancing effect of NE on CXCL12-induced migration of monocytic AML cells was accompanied by up-regulation of miR126 expression concurrently with down-regulation of RGS16 expression, whereas in non-monocytic AML cells we observed the opposite effects, suggesting that NE differently regulates AML cells belonging to different FAB subtypes. Upon studying human normal HSPCs, we found that in steady state, normal cells express low levels of β2-AR and NE did not affect either RGS16 expression or CXCL12-induced migration of both mononuclear and CD34+ cells derived from human cord blood and BM. Conclusions Our results demonstrate that while normal and AML cells share common mechanisms that govern their motility, there are unrevealed yet mechanisms, apparently cell-type dependent, which uniquely lead to opposite effects in normal HSPCs, monocytic and non-monocytic AML cells. Altogether, these findings suggest that targeting of miR126 and RGS16 pathways by specific agonists and antagonists may serve as a new approach for selective eradication of LICs. Disclosures: No relevant conflicts of interest to declare.

The Src and c-Kit kinase inhibitor dasatinib enhances p53-mediated targeting of human acute myeloid leukemia stem cells by chemotherapeutic agents

AbstractThe SRC family kinases (SFKs) and the receptor tyrosine kinase c-Kit are activated in human acute myeloid leukemia (AML) cells. We show here that the SFKs LYN, HCK, or FGR are overexpressed and activated in AML progenitor cells. Treatment with the SFK and c-KIT inhibitor dasatinib selectively inhibits human AML stem/progenitor cell growth in vitro. Importantly, dasatinib markedly increases the elimination of AML stem cells capable of engrafting immunodeficient mice by chemotherapeutic agents. In vivo dasatinib treatment enhances chemotherapy-induced targeting of primary murine AML stem cells capable of regenerating leukemia in secondary recipients. Our studies suggest that enhanced targeting of AML cells by the combination of dasatinib with daunorubicin may be related to inhibition of AKT-mediated human mouse double minute 2 homolog phosphorylation, resulting in enhanced p53 activity in AML cells. Combined treatment using dasatinib and chemotherapy provides a novel approach to increasing p53 activity and enhancing targeting of AML stem cells.