Sparing Normal Hematopoietic Stem Cells Research Articles

Abstract 5228: Engineering CD123-targeting vδ1-enriched γδT cells with enhanced preclinical efficacy and safety profile

Abstract Acute myeloid leukemia (AML) is an aggressive hematological malignancy that, despite advances in treatment, has an overall 5-year survival rate of less than 30%. There is an urgent unmet need for new and effective therapies for AML. Chimeric antigen receptor (CAR)-modified cell therapies are emerging as promising options. However, the selection of suitable antigens is challenging, due to the heterogeneous expression of potential antigen targets in AML. Most antigens, such as CD123, are expressed on both leukemic cells and normal hematopoietic stem and progenitor cells. In addition, conventional autologous or allogeneic T cell therapies present many limitations, such as cytokine release syndrome (CRS) and graft-vs-host disease (GvHD). We therefore evaluated CD123-targeting CAR designs in vδ1-enriched γδT cells, an innate immune cell platform that is inherently non-MHC restricted, has low risks of CRS and GvHD, and is capable of discriminating between malignant and healthy cells in preclinical models. We engineered vδ1-enriched γδT cells from peripheral blood of healthy individuals to express various CD123-targeting CAR constructs. The CD123-targeting CAR-engineered vδ1-enriched γδT cells expressed high levels of natural cytotoxicity receptors, suggesting capability to target innate receptor ligands on AML cell surface. CD123-targeting CAR-expressing vδ1-enriched γδT cells demonstrated enhanced cytotoxicity in vitro against multiple AML cell lines, in both short-term co-culture and repeated antigen stimulation assays. CD123-targeting CAR-expressing vδ1-enriched γδT cells also showed enhanced efficacy and survival in vivo against mouse models of human AML. Interestingly, attenuation of CAR activity via novel designs preserved the inherent selectivity of vδ1-enriched γδT cells against AML cells relative to normal hematopoietic stem or progenitor cells, which also express CD123. Together, our results demonstrated through rational CAR designing and engineering in vδ1-enriched γδT cells, an allogeneic cell therapy targeting CD123 can be generated with selective cytotoxicity against AML cells while sparing normal hematopoietic stem cells. Such product designs have the potential to be a transformative therapeutic option for a broad population of AML patients. Citation Format: Mei Rosa Ng, Sarah L. Hayward, Di Zhang, Gary Gu, Bri Paulhus, Donjeta Gjuka, Collin Crowley, Sophie McLaughlin, Anya Lublinsky, Angel Mathew, Evans Berreondo Giron, Doanh Mai, Madisen Lee, Megha Wal, Rebecca Alade, Andre Simoes, Robin Lytle, James Gavin, Shira Cramer, Zhong-hua Yan, Taylor Hickman, Rashmi Choudhary, Cori Abikoff, Yana Wang, Istvan Kovacs, Nandhu M. Sobhana, Lan Cao. Engineering CD123-targeting vδ1-enriched γδT cells with enhanced preclinical efficacy and safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5228.

Improved efficacy of quizartinib in combination therapy with PI3K inhibition in primary FLT3-ITD AML cells.

Acute myeloid leukemia is a heterogeneous hematopoietic malignancy, characterized by uncontrolled clonal proliferation of abnormal myeloid progenitor cells, with poor outcomes. The internal tandem duplication (ITD) mutation of the Fms-like receptor tyrosine kinase 3 (FLT3) (FLT3-ITD) represents the most common genetic alteration in AML, detected in approximately 30% of AML patients, and is associated with high leukemic burden and poor prognosis. Therefore, this kinase has been regarded as an attractive druggable target for the treatment of FLT3-ITD AML, and selective small molecule inhibitors, such as quizartinib, have been identified and trialled. However, clinical outcomes have been disappointing so far due to poor remission rates, also because of acquired resistance. A strategy to overcome resistance is to combine FLT3 inhibitors with other targeted therapies. In this study, we investigated the preclinical efficacy of the combination of quizartinib with the pan PI3K inhibitor BAY-806946 in FLT3-ITD cell lines and primary cells from AML patients. We show here that BAY-806946 enhanced quizartinib cytotoxicity and, most importantly, that this combination increases the ability of quizartinib to kill CD34+ CD38-leukemia stem cells, whilst sparing normal hematopoietic stem cells. Because constitutively active FLT3 receptor tyrosine kinase is known to boost aberrant PI3K signaling, the increased sensitivity of primary cells to the above combination can be the mechanistic results of the disruption of signaling by vertical inhibition.

A Phase 1, Open-Label, Safety, Tolerability, and Efficacy Study of NC525 in Subjects with Advanced Myeloid Neoplasms

Background With the exception of certain select subtypes, Acute Myeloid Leukemia (AML) remains incurable for a majority of patients with current therapies. Despite recent drug approvals targeting actionable molecular mutations and BCL-2, relapse remains inevitable for most patients with AML due to leukemia-initiating cells and progenitors {i.e., leukemic stem cells (LSCs)} which are resistant to conventional cytotoxic drugs. Novel agents that preferentially target LSCs to lessen or eliminate the risk for relapse while avoiding significant myelosuppression by sparing normal hematopoietic stem cells (HSCs), are highly sought-after anti-leukemia therapeutics. Leukocyte Associated Immunoglobulin-like Receptor 1 (LAIR-1) is an immune inhibitory receptor that is present on most immune cell subsets, including T cells, NK cells, monocytes, macrophages, dendritic cells, and granulocytes. LAIR-1, a surface leukocyte receptor containing two immune receptor tyrosine-based inhibitory motifs (ITIM), binds to endogenous collagen ligands and plays an important role in inducing an immunosuppressive microenvironment. In AML, the engagement of LAIR-1 inhibits intracellular downstream survival signals, leading to decreased GM-CSF-mediated survival and proliferation of AML blasts. LAIR-1 is expressed on AML blasts and LSCs regardless of FAB subtypes, cytogenetics or molecular mutations. Recent work demonstrates that myeloid leukemia cell lines, primary AML blasts, monoblasts and monocytes, and MDS-EB-2 blasts all express LAIR-1. Critically, while high expression of LAIR-1 is characteristic of LSCs, this receptor is minimally detectable on normal HSCs, making it an ideal anti-leukemic target. NC525 is a humanized immunoglobulin gamma 1, kappa (IgG1κ) monoclonal antibody that targets LAIR-1-expressing AML blasts and LSCs. Extensive IND-enabling preclinical studies have shown the following features of NC525: (i) Inhibition of colony formation of AML CD34+ bone marrow cells; (ii) sparing of colony formation and differentiation ability of healthy CD34+ HSCs; (iii) direct ex vivo killing of AML blasts without killing healthy leukocytes; (iv) suppression of AML in both MV4-11 and THP-1 CDX models; (v) restriction of MV4-11 in a humanized murine model, without depletion or suppression of human T cells; (vi) restriction of MV4-11 tumor growth in murine blood, spleen, and bone marrow; and (vii) reduction of AML disease burden in primary patient-derived AML xenograft (PDX) models. Finally, NC525 completely inhibits leukemia propagation in secondary transplant models, suggesting that NC525 is capable of eradicating LSCs. Furthermore, NC525 destroys leukemic cells by ligation-mediated apoptosis of leukemic blasts and stem cells without evidence of an immunomodulatory effect on other immune subsets. Methods This is an open-label, non-randomized, Phase 1 dose escalation study to determine the safety and tolerability of NC525 in adult patients with advanced myeloid neoplasms. This study will also assess the clinical benefit of NC525. Eligible patients must have relapsed or refractory AML, myelodysplastic syndrome (MDS), or chronic myelomonocytic leukemia (CMML) that has progressed after standard therapy. Participants will receive NC525 by intravenous infusion on Days 1 and 15 of each 28-day cycle. During Phase 1, the safety and tolerability of NC525 will be evaluated by exploring escalating dose levels of NC525. In addition, the pharmacologically active dose (PAD), defined as the dose that provides a maximal biologic effect, such as achieving a complete remission (CR), CR with partial hematological recovery (CRh) or CR with incomplete hematological recovery (CRi), and the maximum tolerated dose (MTD) of NC525 will be determined. Safety and tolerability will be further assessed through the expansion of a few dose levels to determine an optimal recommended Phase 2 Dose (RP2D) and administration schedule of NC525, based on the PK/PD profiles. Taken together, preclinical data suggest that targeting LAIR-1 in AML and other myeloid malignancies may be an effective therapy for these diseases with the advantage of specifically eradicating leukemic stem cells and sparing normal HSCs.

Degradation of GSPT1 causes TP53-independent cell death in leukemia while sparing normal hematopoietic stem cells.

Targeted protein degradation is a rapidly advancing and expanding therapeutic approach. Drugs that degrade GSPT1 via the CRL4CRBN ubiquitin ligase are a new class of cancer therapy in active clinical development with evidence of activity against acute myeloid leukemia in early-phase trials. However, other than activation of the integrated stress response, the downstream effects of GSPT1 degradation leading to cell death are largely undefined, and no murine models are available to study these agents. We identified the domains of GSPT1 essential for cell survival and show that GSPT1 degradation leads to impaired translation termination, activation of the integrated stress response pathway, and TP53-independent cell death. CRISPR/Cas9 screens implicated decreased translation initiation as protective following GSPT1 degradation, suggesting that cells with higher levels of translation are more susceptible to the effects of GSPT1 degradation. We defined 2 Crbn amino acids that prevent Gspt1 degradation in mice, generated a knockin mouse with alteration of these residues, and demonstrated the efficacy of GSPT1-degrading drugs in vivo with relative sparing of numbers and function of long-term hematopoietic stem cells. Our results provide a mechanistic basis for the use of GSPT1 degraders for the treatment of cancer, including TP53-mutant acute myeloid leukemia.

In Vivo crispr Screening Identifies GLUT1 As a Key Metabolic Regulator of MLL-AF9-Driven Acute Myeloid Leukemia

Disease relapse in patients with acute myeloid leukemia (AML) is associated with a failure of current treatments to eradicate leukemia stem cells (LSCs), a self-renewing population of cells responsible for disease progression and maintenance. Thus, novel therapeutic strategies designed to specifically target LSCs while sparing normal hematopoietic stem cells are needed. To identify dependencies in LSCs that may reveal new treatment opportunities, we performed an in vivo CRISPR/Cas9 dropout screen in the widely used MLL-AF9-driven AML murine model. The pooled lentiviral CRISPR library was designed to target 960 genes encoding cell surface proteins expressed on MLL-AF9 AML cells as these are accessible for therapeutic targeting.The facilitated glucose transporter member 1(GLUT1), a major mediator of cellular glucose uptake, emerged as the highest ranked dependency in the screen, with all 6 sgRNAs depleted more than 10-fold in vivo. Consistent with the results from the screen, validation experiments confirmed that sgRNA-mediated GLUT1 disruption in c-Kit +Cas9 +dsRed +MLL-AF9 cells led to a 5-fold reduction in the establishment of leukemia in both the bone marrow and spleen of recipient mice. In line with these in vivo observations, leukemia cells expressing GLUT1 sgRNAs were rapidly depleted over time in an ex vivo competition assay (p<0.0001). GLUT1 disruption also led to a marked increase in mean survival from 28 to 73 days in mice transplanted with sorted GLUT1 sgRNA-expressing leukemia cells relative to controls. Notably, while GLUT1 loss did not affect apoptosis or cell-cycle state, it led to a more than two-fold increase in the surface expression of the myeloid differentiation marker Gr-1 (p=0.0002). Interestingly, knockdown of GLUT1 lead to reduced mRNA expression levels of key downstream genes of MLL-driven leukemia Meis1 (p<0.0001) and Hoxa9 (p=0.0013) , both of which are commonly downregulated upon differentiation. These findings suggest that GLUT1 ablation arrests AML cell growth at least in part via accelerated differentiation and attenuated cell proliferation.Given GLUT1-mediated glucose transfer constitutes the first rate-limiting step for glucose metabolism, we assessed the metabolic profile of MLL-AF9 AML cells following loss of GLUT1. Bioenergetic profiling revealed that the rate of glycolysis was significantly decreased upon GLUT1 knockdown, as measured by a decrease in extracellular acidification rate (ECAR), glucose uptake, hexokinase activity and extracellular lactate production.To further assess the feasibility of GLUT1 inhibition as a therapy for AML patients, we treated murine cKit +MLL-AF9 leukemia cells with BAY-876, a potent and highly selective GLUT1 inhibitor. BAY-876 impaired tumor growth following 24hr (IC 50 60.3 nM) and 48hr (IC 50 68.8 nM) treatment ex vivo in a dose-dependent manner. Interestingly, the inhibitory effect on the counterpart healthy bone marrow c-Kit + cells was significantly weaker (24hr IC 50 347.7 nM; 48hr IC 50 258.4nM), indicating selective targeting of LSCs. To test the efficacy of BAY-876 as an anti-leukemic agent in vivo, sublethally irradiated mice were transplanted with c-Kit +MLL-AF9 AML cells and 3 days post-injection, were randomised into two groups (Veh n=4; BAY-876 n=6) and orally treated with either vehicle or 4mg/kg of BAY-876 daily. Following 10 days of treatment, mice were sacrificed and leukemia burden was assessed. Notably, substantially lower levels of leukemia cells in the bone marrow (p=0.0095), spleen (p=0.0095), and peripheral blood (p=0.036) were observed in the BAY-876 treatment group with no significant loss of body weight. Consistent with these findings, the average spleen weight was reduced by 66% upon BAY-876 treatment (p=0.0136).Collectively, we demonstrate that MLL-AF9-driven AML cells are dependent on GLUT1 for continued growth and survival. Targeting of GLUT1 downregulates glycolysis and induces cellular differentiation. We report that genetic or pharmacological inhibition of GLUT1 is sufficient to impair leukemic growth in vitro and in vivo, highlighting a potential therapeutic opportunity for disarming intrinsic metabolic dependencies of LSCs. Ongoing studies are aimed at translating these findings to the human disease and exploring combinatorial therapies that may act synergistically to overcome mechanisms of therapy resistance and metabolic plasticity. DisclosuresNo relevant conflicts of interest to declare.

Isoform Selective HDAC8 Inhibitor (OCH3) Shows Potency in Selectively Targeting Primary AML Cells

The treatment outcomes for patients diagnosed with acute myeloid leukemia (AML) are still dismal. Recent advances in understanding AML indicate that the lack of efficacy is primarily due to non-specificity of currently used chemotherapeutics targeting both leukemic stem/progenitor cells (LSC) and normal hematopoietic stem cells (HSC). Thus, a critical barrier is the identification of innovative therapies that selectively target LSC. Histone deacetylase 8 (HDAC8) has been shown to enhance p53 protein deacetylation, which results in inactivation of p53, promoting LSC survival. We hypothesize that enzymatic/non-enzymatic role of HDAC8 is critical for LSC survival but not for HSCs. Then, we characterized our two tetrahydroisoquinoline (TIQ)-based selective HDAC8 inhibitors (HDAC8i) BIP and OCH3 for growth inhibition, apoptosis, activation of caspase 3, integrity of mitochondrial membrane potential (MMP), and acetylation of histone H4 in human leukemia cell lines. The growth inhibitory effects observed in cell lines were validated using bone marrow (BM) or peripheral blood (PB) cells from AML patients. Colony forming cell (CFC) assays were performed using AML BM/PB cells treated with OCH3 or BIP. OCH3 and BIP were also tested for hematotoxicity using normal CB CD34+ cells. Furthermore, we compared class I HDAC isoform engagement in human normal cord blood (CB) CD34+ cells and in SET-2 leukemia cells using our novel photoreactive probe TH1143. In CD34+ cells, TH1143 had higher level of engagement for HDAC1 and 2, whereas engagement of HDAC3 and 8 was minimal. In SET-2 cells, HDAC3 and HDAC8 displayed relatively higher engagement with TH1143 indicating HDAC engagement is likely cell type specific.The biological efficacies of OCH3 at 50uM and BIP at 25uM were noted to exert >50% growth inhibition in KG1 and in K562 leukemia cells. Both OCH3 and BIP significantly increased the number of apoptotic cells and there was an enhanced active caspase-3 activity. Furthermore, OCH3 and BIP treated cells displayed lower red/green ratio in comparison to control, indicative of poor MMP and depolarization to induce apoptosis (Table 1.a). OCH3 and BIP were further validated by using BM/PB cells from AML patients showing growth inhibition. This was also accompanied by increase in apoptotic cells by OCH3 and BIP. In contrast to BIP, OCH3 spared CB CD34+ cells as demonstrated by notably lower growth inhibition, apoptotic cells vs control when compared with primary AML cells from patients. Both OCH3 and BIP displayed minimal inhibition of CFU growth in CD34+ cells. However, HDAC8i induced significant CFU growth inhibition in primary AML samples suggesting that HDAC8i spares normal CFU progenitors but not leukemia progenitors (Table 1.b). Notably, both BIP and OCH3 lack ability to exert acetylation of histone H4, unlike broad spectrum HDAC inhibitor TSA (MFI with OCH3=0.96±0.03, BIP=0.77±0, TSA =1.63±0.15) which is consistent with isoform selectivity of OCH3 and BIP.The leukemia growth inhibitory effects at LSC level was demonstrated using ex vivo OCH3 treated AML patient derived BM/PB cells transplantation in humanized immunodeficient NSGS mice. After 10 to 12 weeks of transplantation mice receiving untreated AML cells had 7.73±2.18% while with OCH3 treatment mice had 4.84±1.37% human CD34+ leukemia cells, a 38% reduction in CD34+ leukemia cells, despite only a single ex vivo exposure to OCH3. Furthermore, in a second model, NSGS humanized mice were transplanted (IV) with primary leukemia cells from AML patients and after 4 weeks injected (IP) with OCH3 or vehicle control. After 12 weeks of transplantation in this second model human primary AML cell burden was 5.74±1.31% (OCH3) and 18.13±12.76% (vehicle control), while mice transplanted with normal CD34+ cells treated similarly with OCH3 or vehicle control displayed no detectable inhibition of human myeloid cell chimerism (OCH3:12.28 ± 3.31% vs vehicle control: 17.92±11.96%).Taken together, our data indicate that HDAC8 isoform inhibitor, OCH3 displayed significant inhibition of primary AML patient derived leukemia cells growth in vitro and in vivo in contrast to normal CD34+ cells. Selective inhibition of HDAC8 is sufficient to cause growth inhibition in primary AML progenitors including LSCs in vivo while sparing normal HSCs thus offer opportunities for further development of HDAC8i as new experimental therapeutics in AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Anti-IL1RAP/CD3 Bispecific Antibody (BsAb) Is a Promising Novel and Effective Therapy for Acute Myeloid Leukemia (AML)

Despite increasing insight of the mechanisms of leukemogenesis, implementation of risk-stratification treatment strategies and use of molecular targeting therapeutics, only a minority of AML patients are cured with chemotherapy-based regimens. Most patients are either primary refractory to induction therapy or subsequently relapse following a brief remission likely due to persistence of chemo-resistant leukemia stem cell (LSC)-enriched subpopulations. Therefore there are urgent unmet therapeutic needs. The principle of CD3 based BsAb therapy is to recruit and activate T cell effector function upon engaging target cells expressing a tumor associated antigen (TAA). Several TAA (e.g.CD33, CD123) have been found to be differentially overexpressed on the surface of AML blasts. Recently, the expression of IL-1 receptor accessory protein (IL1RAP) was reported to be higher on LSCs and more differentiated leukemic progenitor cells (LPCs) than on normal hematopoietic stem cells (HSC), making it an attractive TAA target for immunotherapy. Thus, we hypothesize that an anti-IL1RAP/CD3 BsAb could preferentially target LSCs and LPCs while sparing normal HSCs, thereby offering a window of therapeutic opportunity for AML patients.To test this hypothesis we first determined the expression pattern of IL-1RAP. IL1RAP was differentially expressed on AML cell lines: HL60 and MV4-11 (high), THP-1 (intermediate), and KG-1a (low). Its expression levels were variable on primary blasts from AML patients (n=10). CD34+ peripheral blood mononuclear cells (PBMC) from healthy donors showed little or no IL1RAP expression. Next, we screened 12 anti-IL1RAP monoclonal antibodies from our human phage display antibody library. Based on the results of the BIAcore and antibody dependent cellular cytotoxity (ADCC) assays performed on MV4-11 cells, we selected one lead candidate with binding affinity of ~20nM and EC50 4.9nM for further development. Using knobs-into-holes strategy, we paired this IL1RAP antibody with an anti-CD3 antibody. The dual binding ability of this BsAb was confirmed by flow cytometric analysis. We subsequently proved T cell dependent cytotoxicity (TDCC) of this BsAb using both AML cell lines and AML primary blasts as targets. The anti-IL1RAP/CD3 BsAb induced over 95% of specific killing on all tested AML samples at E: T (Effector : Target) ratio of 1:1 compared with no cytotoxicity effect on the IL-1RAP-negative Raji cells in the 48-hour long term killing (LTK) assay. In line with the cytotoxic data, there was also substantial release of cytokines including IFN-γ, TNF-α and Granzyme B from the AML cells but not Raji cells. TDCC reported over 90% of IL1-RAP specific killing in all but one (only 60%) patient samples (n=10), which had the lowest IL1RAP expression level. The killing of leukemia cells was also observed by time-lapse live cell microscope imaging using luciferase-expressing MV4-11luci+ cells with addition of 100ng/ml BsAb and T cells at E:T ratio of 5:1, which documented effective T cell recruitment and cytolytic activities against MV4-11luci+ cells. We also tested the anti-leukemic activity of this BsAb in vivo using a NSG xenograft mouse model. NSG mice transplanted with 1x106 MV4-11luci+ cells were treated with 200µg anti-IL1RAP/CD3 BsAb plus 3x106 unstimulated T cells, vehicle, T cells alone or anti-CD19/CD3 BsAb (control Ab) plus T cells twice a week for 30 days. In vivo imaging demonstrated effective elimination of the leukemia signal (P<0.001) in MV4-11luci+-engrafted NSG mice treated with T cells and anti-IL1RAP/CD3 BsAb (photon flux (PF) =5.5x105) compared with intense leukemia infiltration in controls treated with either vehicle (PF=3.5x109), T cells alone (PF=4x109), or control anti-CD19/CD3 BsAb plus T cells (PF=2x109). In a separate experiment, treatment with anti-IL1RAP/CD3 BsAb plus T cells of NSG mice engrafted with primary AML blasts led to significant decrease in disease burden than treatment with isotype antibody plusT cells (p=0.004). Further characterization and determination of disease response in these animals is ongoing.In conclusion, we have successfully engineered and characterized an anti-IL1RAP/CD3 BsAb derived from our human naïve phage display library. Our data demonstrated that this BsAb can effectively eradicate AML cells in vitro and in vivo, supporting the clinical translation of this novel immunotherapeutic agent for treatment of AML patients. DisclosuresKwak:InnoLifes: Consultancy, Equity Ownership; Pepromene Bio: Consultancy, Equity Ownership; Celltrion, Inc: Consultancy. Stein:Stemline: Consultancy; Amgen: Consultancy, Speakers Bureau.

Cancer Stem Cells in the Development of Novel Therapeutics for Refractory Pediatric Leukemia.

Although treatment strategies for pediatric leukemia have improved overall survival rates in the recent past, relapse rates in certain subgroups such as infant leukemia remain unacceptably high. Despite undergoing extensive chemotherapy designed to target the rapidly proliferating leukemia cells, many of these children experience relapse. In refractory leukemia, the existence of cell populations with stemness characteristics, termed leukemia stem cells (LSCs), which remain quiescent and subsequently replenish the blast population, has been described. A significant body of evidence exists, derived largely from xenograft models of adult acute myeloid leukemia, to support the idea that LSCs may play a fundamental role in refractory disease. In addition, clinical studies have also linked LSCs with increased minimal residual disease, higher relapse rate, and decreased survival rates in these patients. Recently, a number of reports have addressed effective ways to utilize new-generation genomic sequencing and transcriptomic analyses to identify targeted therapeutic agents aimed at LSCs, while sparing normal hematopoietic stem cells. These data underscore the value of timely translation of knowledge from adult studies to the unique molecular and physiological characteristics seen in pediatric leukemia. We aim to summarize this article in the rapidly expanding field of stem cell biology in hematopoietic malignancies, focusing particularly on relevant preclinical models and novel targeted therapeutics, and their applicability to childhood leukemia.

Targeting miRNA-551b, a “Stemness”-like microRNA, to Eradicate AML (Stem) Cells

Despite high complete remission (CR) rates achieved after chemotherapy, only 30-40% of patients with Acute Myeloid Leukemia (AML) survive five years after diagnosis. The main cause of this treatment failure is insufficient eradication of a subpopulation of chemotherapy-resistant leukemia cells with stem cell properties, named “leukemic stem cells” (LSCs). LSCs use a variety of mechanisms to resist chemotherapy and targeting them is one of the major challenges in AML treatment. Since miRNAs can target multiple genes/pathways simultaneously, their modulation (downregulation or upregulation) may have great potential for the successful elimination of therapy-resistant leukemic (stem) cells (Martiañez Canales et al. Cancers 2017). Here, we show that miRNA-551b, previously identified by us as a stem cell-like miRNA, can be a potential novel target to specifically eradicate AML stem-like cells.Aiming at identification of miRNA-based therapy to specifically eradicate LSCs, while sparing normal Hematopoietic Stem Cells (HSCs), we determined expression of miRNAs in normal HSCs, Leukemic Stem Cells (LSCs) and leukemic progenitors (LP) all derived from the same AML patient's bone marrow. Using this approach, we identified miRNA-551b as being highly expressed in normal HSCs residing both in healthy and AML bone marrows. In AML, high expression of miR551b demonstrated to be associated with an adverse prognosis. Moreover, miRNA-551b was highly expressed in immature AML cases and its expression in a cohort of patients coincided with the expression of stem cell genes (De Leeuw et al. Leukemia 2016).To further elucidate the link between miRNA-551b and AML “stemness” and to test whether downregulation of miRNA-551b affects the survival of AML (stem/progenitor) cells, proliferation and the balance between differentiation and “stemness”, we reduced miRNA-551b expression, either by lentiviral transduction of antagomirs or by adding locked nucleotide acid (LNA)-oligonucleotides to AML cell lines and primary AML cells. Downregulation of miRNA-551b in the stem cell-like AML cell line KG1a led to inhibition of cell growth in vitro, which was due to inhibition of proliferation rather than induction of apoptosis. KG1a tumor growth in an in vivo mouse model was also reduced when miRNA-551b was downregulated. In primary AML, miRNA-551b knockdown resulted in a significant decrease in the survival of leukemic progenitors and LSCs, while hematopoietic stem cells (HSCs) and normal progenitors from healthy bone marrows were not affected. These results suggest that a therapeutic approach inhibiting miRNA-551b expression might specifically eradicate leukemic progenitors and LSCs from primary AML, while sparing HSCs. We are currently studying miRNA-551b targets which can be responsible for this specific LSCs elimination.In conclusion, our results suggest that inhibition of miRNA-551b could be a promising approach to eliminate stem cell-like AML cells, thereby decreasing relapse rates and improving AML treatment outcome. DisclosuresOssenkoppele:Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Research Funding; Roche: Consultancy, Honoraria; Celgene: Honoraria, Research Funding; Johnson & Johnson: Consultancy, Honoraria, Research Funding; Genmab: Research Funding.

Abstract 4777: Cancer stemness and resistance: Napabucasin (BBI-608) sensitizes stemness-high cancer cells to Paclitaxel by inhibiting the STAT3-MUC1 pathway

Abstract Stemness-high cancer cells, or cancer stem cells (CSC) represent a subpopulation of cancer cells with enhanced tumorigenic capacity, metastasis-forming potential, and resistance to conventional chemotherapy and radiation. One such key CSC pathway is regulated by STAT3, a transcription factor that is downstream of several cytokines and growth factor receptors which controls the expression of a broad range of target genes and plays essential roles in CSC biology in many tumor types. Cancer stemness inhibitor Napabucasin (BBI-608), a small molecule that inhibits gene transcription driven by STAT3, can inhibit stemness gene expression, block spherogenesis, and kill CSCs. In vivo, BBI-608 effectively blocks cancer relapse and metastasis in xenograft models while sparing normal hematopoietic stem cells, suggesting that targeting stemness-high cancer cells is a feasible approach for developing next-generation cancer therapeutics to combat cancer recurrence. SOX2 (sex-determining region Y-box protein 2), a transcription factor that is essential for self-renewal and pluripotency, is amplified in various cancer types and has been shown to be required for CSC self-renewal and maintenance. Mucin 1 (MUC1), a glycoprotein normally expressed at the apical surface of epithelial cells, is overexpressed in most human epithelial cancers. MUC1 has been implicated in regulating tumor proliferation, metabolism, invasion, angiogenesis, chemoresistance, and inflammation. Here, we generated a stemness-high culture system based on the reporter activity of a SOX2-regulatory region construct in MKN28 gastric cancer cells (MKN28 SOX2-reporter GFP+ cells). BBI-608 treatment inhibited the STAT3-MUC1 pathway in stemness-high cells. High MUC1 status in stemness-high cells was associated with Paclitaxel resistance. Down-regulation of MUC1 sensitized stemness-high cells to Paclitaxel. Moreover, BBI-608 synergized with Paclitaxel in inhibiting spherogenesis of stemness-high cells. This study provides a new mechanism for the association of cancer stemness with drug resistance. Our findings support this combination therapy, which pairs a conventional chemotherapy (Paclitaxel) with a cancer stemness inhibitor (BBI-608), as a promising strategy to combat cancer. Citation Format: Harry A. Rogoff, Juying Li, Chiang Li. Cancer stemness and resistance: Napabucasin (BBI-608) sensitizes stemness-high cancer cells to Paclitaxel by inhibiting the STAT3-MUC1 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4777. doi:10.1158/1538-7445.AM2017-4777

The Relevance of Stem Cell Load at Diagnosis for the Development of Relapse in Pediatric Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy which has an incidence of 40 children on average per year in the Netherlands and Belgium combined (population of 28 million). Despite the fact that almost all children achieve complete remission (CR), 30-40% of patients eventually relapse. Since survival after relapse is poor (35-40%), the overall survival (OS) of pediatric AML remains relatively low (~70%) compared to the increasing remission rates. The most effective strategy to improve the dismal outcome of AML patients is thus to prevent relapse.Over the last decades accumulating evidence is suggesting that AML develops in a hierarchical structure; originating from hematopoietic stem cells which are transformed to leukemia initiating cells, also referred to as leukemic stem cells (LSC). LSC possess self-renewal capacity and are more resistant to therapy. Therefore, LSC are supposed to be responsible for outgrowth of both the initial leukemia and the relapse. This holds true for adult AML, where the frequency of LSC at diagnosis has shown to be of importance for clinical outcome. However, while it has been shown that a high number of immature cells (CD34+/CD38-/CD45-/low) associates with an increased risk of relapse in pediatric AML, relatively little is known about the prognostic impact of LSC within this compartment.RAEB-t. Flow-cytometric LSC characterization was performed on either bone marrow (BM) or peripheral blood (PB) from AML patients collected at diagnosis. Purified white blood cells (WBC) were obtained after lysing the red blood cells using lysing solution (Pharm Lyse or FACSLysing, BD Biosciences) and were subsequently incubated with monoclonal antibody combinations and analyzed using an 8-color flow cytometer approach. LSC were defined as CD34+ CD38- cells with aberrant expression of CD123, CD7, CD56 or CD2.Results: Data were available from 103 patients and patient characteristics are listed in Table 1. In this cohort relapse-free survival (RFS) was 59.4%. Fortunately 56.4% of relapsed patients achieved a second CR and OS was 85.4%. LSC-load is defined as % of aberrant CD34+ CD38- cells within the CD34+ compartment. In our pediatric cohort, 17 patients (16.5%) had no expression of CD34 on blast cells at all (CD34null) and consequently no aberrant CD34+ CD38- LSC could be detected. Absence of CD34 has often been associated with good prognosis in literature. In our cohort relapse-rate seemed to follow this trend (4 out of the 17 CD34null patients (23.5%) vs. 34 out of the 83 CD34positive patients (41%)) but did not differ significantly (Plogrank = 0.196).ROC curve analysis showed that a cut-off of 17.2% LSC at diagnosis was associated with the occurrence of developing relapse with a specificity of 92%. Kaplan-Meier survival analysis, as depicted in Figure 1, showed a significant association between a high LSC-load and impaired RFS (34% relapses in LSClow vs. 64% relapses in LSChigh) (Plogrank= 0.027). Univariate analysis showed that next to LSC percentage, FLT3 mutation status and WBC count were significantly associated with RFS. After multivariate adjustment (taking into account cytogenetic risk groups and mutational status) LSC frequency was the only independent predictor of relapse or RFS (HR 2.3, 95% CI 1.1-4.8, p=0.032).Conclusion: Our results confirm data from adults and reveal that the frequency of LSC at diagnosis in pediatric AML patients can distinguish patients more likely to fail current treatment regimens as these patients develop significantly more relapses). Identifying this patient group creates opportunities for more personalized medicine and the development of therapies directed against LSC, sparing normal hematopoietic stem cells. [Display omitted] [Display omitted] DisclosuresKaspers:Janssen-Cilag: Research Funding. Cloos:Takeda: Honoraria.

Identification and targeting leukemia stem cells: The path to the cure for acute myeloid leukemia.

Accumulating evidence support the notion that acute myeloid leukemia (AML) is organized in a hierarchical system, originating from a special proportion of leukemia stem cells (LSC). Similar to their normal counterpart, hematopoietic stem cells (HSC), LSC possess self-renewal capacity and are responsible for the continued growth and proliferation of the bulk of leukemia cells in the blood and bone marrow. It is believed that LSC are also the root cause for the treatment failure and relapse of AML because LSC are often resistant to chemotherapy. In the past decade, we have made significant advancement in identification and understanding the molecular biology of LSC, but it remains a daunting task to specifically targeting LSC, while sparing normal HSC. In this review, we will first provide a historical overview of the discovery of LSC, followed by a summary of identification and separation of LSC by either cell surface markers or functional assays. Next, the review will focus on the current, various strategies for eradicating LSC. Finally, we will highlight future directions and challenges ahead of our ultimate goal for the cure of AML by targeting LSC.

CD44v6-targeted T cells mediate potent antitumor effects against acute myeloid leukemia and multiple myeloma

AbstractGenetically targeted T cells promise to solve the feasibility and efficacy hurdles of adoptive T-cell therapy for cancer. Selecting a target expressed in multiple-tumor types and that is required for tumor growth would widen disease indications and prevent immune escape caused by the emergence of antigen-loss variants. The adhesive receptor CD44 is broadly expressed in hematologic and epithelial tumors, where it contributes to the cancer stem/initiating phenotype. In this study, silencing of its isoform variant 6 (CD44v6) prevented engraftment of human acute myeloid leukemia (AML) and multiple myeloma (MM) cells in immunocompromised mice. Accordingly, T cells targeted to CD44v6 by means of a chimeric antigen receptor containing a CD28 signaling domain mediated potent antitumor effects against primary AML and MM while sparing normal hematopoietic stem cells and CD44v6-expressing keratinocytes. Importantly, in vitro activation with CD3/CD28 beads and interleukin (IL)-7/IL-15 was required for antitumor efficacy in vivo. Finally, coexpressing a suicide gene enabled fast and efficient pharmacologic ablation of CD44v6-targeted T cells and complete rescue from hyperacute xenogeneic graft-versus-host disease modeling early and generalized toxicity. These results warrant the clinical investigation of suicidal CD44v6-targeted T cells in AML and MM.

Identification of Small Molecules Selectively Targeting Leukemic Stem Cells within Their Stromal Niche

Abstract 413Current therapies for acute myeloid leukemia (AML) are highly toxic, yet the relapse rate remains high. New therapies are needed to improve cure rates while decreasing toxicity. Because therapies may be affected by the tumor niche, we aimed to test new compounds on leukemic stem cells (LSCs) within their stromal microenvironment. A niche-based high throughput screen identified candidate small molecules potentially toxic to MLL-AF9 murine leukemic stem cells (LSCs) while sparing normal hematopoietic stem cells (HSCs) and bone marrow stroma (Hartwell et al, Blood 118, Abs 760, 2011.) Three such compounds, including a selective serotonin receptor antagonist highly specific for the 5-HT1B receptor, SB-216641, and two antihelminthics, parbendazole and methiazole, were found to be effective and selected for studies on human leukemias.We first examined SB-216641, studying the effects of this compound on 7 human primary AML samples. We began by assessing the compound’s effect on LSCs using the week 5 cobblestone area forming cell (CAFC) assay, a standard in vitro stem cell assay. CD34+ cells were isolated with immunomagnetic beads. The leukemic cells were pulse treated for 18 hours and washed prior to placement on MS-5 murine stroma. We performed serial drug dilutions using the CAFC assay with the human primary samples as well as with HSCs derived from cord blood. All human leukemic samples formed cobblestone areas in the control setting (46-200 CAFCs/106 cells plated). IC50 for the human primary leukemia CAFCs was 630 nm, and at 10 μM all LSCs were killed while normal human HSCs had 100% survival. A combination of the AML cell line HL60 transduced with GFP-luciferase and normal cord blood CD34+ cells (1:200) were then pre-incubated overnight with SB-216641 at 5 and 10 μM and injected into Nod Scid IL2R-gamma null (NSG) mice. The control mice had leukemic engraftment by luciferase imaging and flow cytometry and the mice that received treated cells had no leukemic engraftment but normal multilineage engraftment of cord blood. Primary patient AML samples were also pre-incubated overnight with SB-216641 at 10 μM and injected into NSG mice. As shown by flow cytometry, control mice engrafted with leukemia and mice that received pre-treated cells had no engraftment following exposure to SB-216641. Finally, an in vivo study was completed on NSG mice injected intraperitoneally with 20 mg/kg/day beginning on day 1 or day 8 after inoculation with HL60 (500 cells). The mice were imaged at 2 and 3 week time points and both treatment groups had significantly less leukemia on imaging than the control group with minimal toxicity noted. Another specific 5-HT1B receptor antagonist, SB-224289, was found to have similar activity to SB-216641 against leukemic cells and to spare HSCs in preliminary studies.Similar CAFC studies with serial dilutions on primary AML samples were performed on the two anti-helminthic agents. IC50 for parbendazole was 1.25 μM and for methiazole 5 μM. As shown by luciferase imaging and flow cytometry, when injected with combined HL60 and cord blood pre-incubated overnight at 5 and 10 μM with each compound as described above, the control mice engrafted with leukemia and the mice that received treated cells had no leukemic engraftment but normal multilineage engraftment of cord blood. NSG mice were then injected with primary AML pretreated overnight with parbendazole at 10 μM. As shown by flow cytometry, control mice engrafted with leukemia and mice that received pre-treated cells had significantly lower engraftment following exposure to parbendazole (p = 0.01).Two new avenues of leukemia therapy were discovered warranting further investigation. SB-216641, an agent with a completely novel receptor target in leukemia therapy, has shown both in vitro success in human leukemia as well as preliminary success in vivo with minimal toxicity. We aim to move forward with this agent while also testing parbendazole in vivo, as this compound is already known to have good pharmacokinetics and minimal toxicity in animals. The high toxicity to LSCs and sparing of normal HSCs give both these agents an attractive profile for future clinical trials. Disclosures:Ebert:Genoptix: Consultancy; Celgene: Consultancy.

TIM-3 Is a Promising Target to Eradicate Acute Myeloid Leukemia Stem Cells Sparing Normal Hematopoietic Stem Cells

Abstract 559Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for permanent cure. To selectively kill AML LSCs sparing normal hematopoietic stem cells (HSCs), one of the most practical approaches is to target the AML LSCs-specific surface or functionally indispensable molecules. Based on differential transcriptome analysis of prospectively-purified CD34+CD38− LSCs from AML patient samples and normal HSCs, we found that T-cell immunoglobulin mucin-3 (TIM-3) was highly expressed in AML LSCs but not in normal HSCs (Kikushige et al., Cell Stem Cell, 2010). In normal hematopoiesis, TIM-3 is mainly expressed in mature monocytes and a fraction of NK cells, but not in granulocytes, T cells or B cells. In the bone marrow, TIM-3 is expressed only in a fraction of granulocyte/macrophage progenitors (GMPs) at a low level, but not in HSCs, common myeloid progenitors, or megakaryocyte/erythrocyte progenitors. In contrast, in human AML, TIM-3 was expressed on cell surface of the vast majority of CD34+CD38− LSCs and CD34+CD38+ leukemic progenitors in AML of most FAB types, except for acute promyelocytic leukemia (M3). FACS-sorted TIM-3+ but not TIM-3− AML cells reconstituted human AML in the immunodeficient mice, indicating that the TIM-3+ population contains most of functional LSCs. To selectively eradicate TIM-3-expressing AML LSCs, we established an anti-human TIM-3 mouse IgG2a antibody, ATIK2a, possessing antibody-dependent cellular cytotoxic and complement-dependent cytotoxic activities in leukemia cell lines transfected with TIM-3. We first tested the effect of ATIK2a treatment on reconstitution of normal HSCs in a xenograft model. ATIK2a was intraperitoneally injected to the mice once a week after 12 hours of transplantation of human CD34+ cells. Injection of ATIK2a did not affect reconstitution of normal human hematopoiesis except removing TIM-3-expressing mature monocytes. In contrast, injection of TIM-3 to the mice transplanted with human AML samples markedly reduced leukemic repopulation. In some mice transplanted with AML bone marrow, only normal hematopoiesis was reconstituted after anti-TIM-3 antibody treatment, suggesting that the antibody selectively killed AML cells, sparing residual normal HSCs. To further test the inhibitory effect of ATIK2a on established human AML, eight weeks after transplantation of human AML cells, engraftment of human AML cells was confirmed by blood sampling and thereafter ATIK2a was injected to these mice. In all cases tested, ATIK2a treatment significantly reduced human TIM-3+ AML fraction as well as the CD34+CD38− LSCs fraction. In addition, to verify the anti-AML LSCs effect of ATIK2a treatment, human CD45+AML cells from the primary recipients were re-transplanted into secondary recipients. All mice transplanted from primary recipients treated with control IgG developed AML, whereas none of mice transplanted with cells from ATIK2a-treated primary recipients developed AML, suggesting that functional LSCs were effectively eliminated by ATIK2a treatment in primary recipients.Thus, TIM-3 is a promising surface molecule to target AML LSCs. Our experiments strongly suggest that targeting this molecule by monoclonal antibody treatment is a practical approach to eradicate human AML. Disclosures:No relevant conflicts of interest to declare.

Selective Targeting of Leukemic Over Normal Stem Cells by the Serotonin Receptor Antagonist SB-216641

Abstract 1884Acute myeloid leukemia (AML) is a common and aggressive hematologic malignancy affecting both children and adults which continues to have high mortality rates as well as high morbidity from toxic therapies. New treatments are needed to improve cure rates and decrease morbidity. A niche-based high throughput screen done in a murine system identified candidate small molecules potentially toxic to leukemic stem cells (LSCs) while sparing normal hematopoietic stem cells (HSCs) and bone marrow stroma (Hartwell KA, Miller, PG et al., in preparation). One such compound, SB-216641, demonstrated dose-dependent activity against leukemia in both a cell autonomous and non-autonomous manner, by modifying niche–based support. SB-216641 is a selective serotonin receptor antagonist specific for the 5-HT1B receptor, highlighting a pathway not previously investigated in the context of AML or leukemia stem cell biology.We examined the effects of this candidate small molecule on 7 human primary AML samples. CD34+ cells were isolated from these samples with immunomagnetic beads. Using the colony forming assay to assess kill of progenitor cells, all samples had ≥99% cell kill at 25 μM (10 times the IC-50 found in the murine system). We then assessed the compound’s effect on LSCs using the cobblestone area forming cell (CAFC) assay, a standard in vitro stem cell assay. The leukemic cells were pulse treated for 18 hours and washed to remove residual SB-216641 prior to placement on MS-5 murine stroma and therefore only the direct effect on the leukemic cells was measured in this assay. CAFCs were read out at week 5, or week 2 when the sample was FLT3-ITD+ (Chung KY et al, Blood 2005, Vol 105, 77–84). We first tested five samples at 25 μM. All samples formed cobblestone areas in the control setting (46–200 CAFCs/106 cells plated). Four samples had no CAFC formation with SB-216641 and the remaining sample had >95% decrease in CAFC formation. We then performed serial dilutions using the CAFC assay in the human primary samples as well as in HSCs derived from cord blood to obtain the IC-50 for human AML and to ensure that our differential cell kill of LSCs versus normal HSCs held true in the human samples. IC-50 for the human primary leukemias was found to be 630 nanomolar and at 10 μM all leukemic samples were fully killed with 100% survival of normal human HSCs [see figure 1]. As a confirmatory study, using HL60 and U937 human AML cell lines transduced with GFP-luciferase, 500 cells were preincubated with SB-216641 at 25 μM or DMSO control and then injected IV into Nod Scid IL2R-gamma null (NSG) mice and imaged at 5 weeks. In both cell lines, the control mice had engraftment and the mice that received treated cells had no engraftment. HL60 cells were then preincubated with SB-216641 at lower doses (10 and 5 μM) and injected into NSG mice and imaged at 3 weeks. Again, the control mice had engraftment and the mice that received treated cells had no engraftment. [Display omitted] 5-HT1B receptor antagonists have not previously been known to be active against AML or leukemic stem cells. Some hematopoietic cells including platelets express serotonin receptors and T-cells specifically have been found to express the 5-HT1b receptor. Selective 5-HT1B receptor antagonists have found to have apoptotic effects in vitro against cell lines of other cancers and may be involved in MAP kinase and P13K/Akt signaling pathways. SB-216641 is a highly promising compound which warrants further investigation. Its high toxicity to LSCs and sparing of normal HSCs make it appealing for possible clinical use in the future. Disclosures:No relevant conflicts of interest to declare.

Arsenic disulfide synergizes with the phosphoinositide 3-kinase inhibitor PI-103 to eradicate acute myeloid leukemia stem cells by inducing differentiation

Although dramatic clinical success has been achieved in acute promyelocytic leukemia (APL), the success of differentiating agents has not been reproduced in non-APL leukemia. A key barrier to the clinical success of arsenic is that it is not potent enough to achieve a clinical benefit at physiologically tolerable concentrations by targeting the leukemia cell differentiation pathway alone. We explored a novel combination approach to enhance the eradication of leukemia stem cells (LSCs) by arsenic in non-APL leukemia. In the present study, phosphatidylinositol 3-kinase /AKT/mammalian target of rapamycin (mTOR) phosphorylation was strengthened after As(2)S(2) exposure in leukemia cell lines and stem/progenitor cells, but not in cord blood mononuclear cells (CBMCs). propidium iodide-103, the dual PI3K/mTOR inhibitor, effectively inhibited the transient activation of the PI3K/AKT/mTOR pathway by As(2)S(2). The synergistic killing and differentiation induction effects on non-APL leukemia cells were examined both in vitro and in vivo. Eradication of non-APL LSCs was determined using the nonobese diabetic/severe combined immunodeficiency mouse model. We found that a combined As(2)S(2)/PI-103 treatment synergized strongly to kill non-APL leukemia cells and promote their differentiation in vitro. Furthermore, the combined As(2)S(2)/PI-103 treatment effectively reduced leukemia cell repopulation and eradicated non-APL LSCs partially via induction of differentiation while sparing normal hematopoietic stem cells. Taken together, these findings suggest that induction of the PI3K/AKT/mTOR pathway could provide a protective response to offset the antitumor efficacy of As(2)S(2). Targeting the PI3K/AKT/mTOR pathway in combination with As(2)S(2) could be exploited as a novel strategy to enhance the differentiation and killing of non-APL LSCs.

Defining Leukemia Stem Cells in MLL-Translocated Leukemias: Implications for Novel Therapeutic Strategies

Hematological malignancies and probably many other tumors are dependent on highly proliferating and self-renewing cancer stem cells. An important question in the development of novel, less toxic antileukemic strategies specifically targeting leukemia stem cells is how closely leukemia stem cells are related to normal hematopoietic stem cells. It has been recently demonstrated that leukemia stem cells can be derived from different stages in normal hematopoiesis and have unique phenotypic and genetic features. Introduction of Mixed-lineage leukemia ( MLL)-fusion oncoproteins, frequently found in infant leukemias and therapy-related leukemias, into differentiated hematopoietic progenitor cells results in the generation of leukemias with a high frequency of leukemia stem cells. The progenitor-derived leukemia stem cells ectopically express a limited stem cell program while maintaining the global identity of differentiated myeloid cells. Development of therapeutic strategies that specifically target the leukemia stem cell program while sparing normal hematopoietic stem cells may represent a novel therapeutic approach in human leukemias with high efficacy yet less side effects.

Pioglitazone inhibits the growth of human leukemia cell lines and primary leukemia cells while sparing normal hematopoietic stem cells

Peroxisome proliferator-activated receptors (PPARs) compose a subfamily of nuclear hormone receptors functioning as transcriptional regulators. Originally, the PPARgamma ligand known as thiazolidinedione (TZD) was used for the treatment of diabetic patients. However, recent studies have shown that TZD also has an antitumor effect that inhibits cell growth in several types of human malignant neoplasms, including leukemia cell lines. Since pioglitazone is the only TZD currently available in clinics in Japan and the role of TZD in normal human hematopoietic cells or primary leukemia cells has not been previously reported, we investigated the effect of pioglitazone on human normal hematopoietic progenitor cells, primary leukemia cells, and leukemia cell lines (HL60, K562, U937, HEL, CEM, Jurkat, and NALM1). Pioglitazone inhibited the proliferation of leukemia cells in a dose-dependent manner. The viable cell numbers of HL60, K562, and Jurkat leukemia cell lines were profoundly reduced when the cells were cocultured with pioglitazone. Colony formation in the leukemia cell lines as well as the primary leukemia cells was significantly inhibited to 20-71% and 1-25% of that in control cultures by the addition of 100 and 300 microM of pioglitazone, respectively. However, the CFU-E and CFU-GM colonies of cells obtained from healthy volunteers were not altered in the presence of 100 microM of pioglitazone. Pioglitazone (300 microM) induced slight decrease of CFU-E and CFU-GM. BFU-E was more sensitive to pioglitazone than CFU-E and CFU-GM. Pioglitazone-induced growth inhibition in HL60 cells was associated with cell cycle arrest at the G1 phase, as has been reported for another TZD, troglitazone. Similar levels of PPARgamma protein were observed in both leukemia and normal bone marrow cells by Western blotting, suggesting that the expression of PPARgamma protein was not associated with the inhibitory potency of pioglitazone. In conclusion, our results suggest that pioglitazone may offer a new therapeutic approach to aid in the treatment of leukemia.

Mechanisms Controlling Selective Death of Leukemia Stem Cells in Response to Parthenolide.

Studies have shown that human acute myelogenous leukemia (AML) originates from a rare population of leukemic stem cells (LSCs). LSCs are found in nearly all AML subtypes and are sufficient to initiate and maintain leukemic growth in both long-term cultures and NOD/SCID mice. Since conventional chemotherapy drugs typically target actively cycling leukemic blast cells, the quiescent LSC population is likely to be drug resistant. Recently, we have described agents that can selectively kill LSC while sparing normal hematopoietic stem cells (HSC), thus demonstrating that quiescent LSC can be effectively destroyed. In particular, the sesquiterpene lactone parthenolide (PTL) is active as a single agent and can induce robust LSC-specific apoptosis. Therefore, we sought to further elucidate the mechanisms that result in leukemia-specific death. To this end, we have shown that PTL inhibits NF- κB and activates p53 in AML cells. Moreover, we have found that AML cells are highly-oxidized relative to their normal less-oxidized counterparts as determined by dihydrofluorescein diacetate (H2DCF-DA) fluorescence. Although PTL treatment was found to increase the basal oxidative load of both normal and leukemic cells, these increases were ultimately associated with only leukemia-specific death. High levels of reactive oxygen species are associated with genotoxic stress. Therefore, we hypothesized that PTL-mediated increases of the oxidative load in AML cells would be sufficient to produce genotoxic stress in AML cells, but not normal cells. We thus examined pathways involved in sensing and responding to oxidative and genotoxic stress. These studies demonstrate that upon PTL treatment, AML cells phosphorylate p53 on serine 15, and increase transcription of several downstream p53 pro-apoptotic genes, including GADD45, Bax, and p21. In addition, we detected phosphorylation of ATM on serine 1981, γ-H2AX, and phosphorylation of Chk2 on threonine 68. Importantly, these events are not observed in normal hematopoietic cells treated with PTL. These findings suggest a DNA damage/repair response to PTL in AML cells, and that normal cells appear to be resistant to this mechanism. Collectively, these data indicate that oxidative stress and DNA damage pathways may be a central component of PTL-induced apoptosis. We propose a model whereby AML cells exist in a physiological state in which PTL is able to preferentially invoke genotoxic stress in AML. Based on empirical evidence, we suggest that AML LSC also exist in such a state, and that PTL mediated inhibition of NF- κB and induction of stress leads to selective apoptosis of malignant cells, sparing normal cell populations.