Survival Of Leukemic Stem Cells Research Articles

Peripheral Blood Flow-Cytometry Chronic Myeloid Leukemia Stem Cells Detection and Quantification during Tyrosine Kinase Inhibitors Therapy

Introduction: In chronic myeloid leukemia (CML), tyrosine Kinase Inhibitors (TKIs) treatment is a potentially life-time therapy for the majority of patients (pts), as few of them, only after achieving a deep and stable molecular response, may discontinue TKIs without recurrence of disease. Available data suggest that relapse after TKIs discontinuation is due to the persistence of leukemic stem cells (LSCs) intrinsically resistant to TKIs. Survival of CML LSCs may be the consequence of activation of several pathways BCR-ABL1 independent. qRT-PCR, the most sensitive assay to monitor disease status in CML pts, may be inappropriate to quantify residual quiescent CML LSCs that are transcriptionally silent. Therefore, the possibility to easily quantify LSCs during TKIs treatment is a great opportunity to better understand the behavior of residual LSCs and potentially to identify those pts candidates to safely discontinue TKIs. Recently, Valent et al described that CD34+/CD38-/Lin- CML LSCs specifically co-express dipeptidylpeptidase IV (CD26) and that CD26 is a potential biomarker for the quantification and isolation of CML LSCs, in bone marrow samples of CML patients. Furthermore, Culen et al. quantified CD26+ LSCs bone marrow compartment in 31 CML patients at diagnosis and their number appears to correlate with response to TKIs treatment. In the present study we wanted to explore the feasibility, rate and potential implication of detecting CD26+ LSCs in peripheral blood (PB) from CML pts during TKI treatment.Methods: CML pts during first line treatment with any approved TKIs, referring to several Italian Hematology Centers, entered this non interventional cross sectional study after signing a proper informed consent. During a routine follow up visit, in which pts were checked for molecular response by standard PB qRT-PCR BCR-ABL1 analysis, additional 3 mls of PB were collected in EDTA and sent within 24 hours to Siena Hematology Lab to detect CD34+/CD38-/CD26+ LSCs by multicolor flow cytometry. After red blood cells lysis, cells were incubated with anti CD45 (BD Biosciences), CD34 (581), CD38 (HIT2), CD26 (M-A261) (BD Pharmigen). After washing, acquisition and analysis were performed by FACSCanto II (BD Biosciences, NR Nannini) using DIVA 8 software (BD, Biosciences). CD45+ cells acquired for each sample ranged from 500,000 to 1,000,000. Isotype controls were included in each staining. In 5 pts a FISH analysis of PB sorted CML LSCs population was also performed.Results: to validate our assay we first performed a FISH analysis of both PB sorted CD34+/CD38-/CD26+ and CD34+/CD38-/CD26- in 5 CML patients at 3-6 months after starting treatment, confirming Ph+ cells only in the CD26+ fraction. Afterward, we checked for circulating CML LSCs a total of 202 CML pts in first line treatment with TKIs for a median of 39 months (range 1-175). Type of TKI, length of treatment, molecular response and quantification of LSCs are summarized in Table 1. PB CML LSCs were detectable in 146/202 (72.3%) pts with a median number of CD26+ of 0,0165 cells/µL (range 0,0018-0,66). Kendall rank correlation coefficient used to analyze the relation between the measurable variables showed no correlation between BCR-ABL/ABLIS ratio (median 0,004 range 0-61) and number of residual LSCs (r 0.118 p=0.097). In 56/202 (27.7%) pts CD26+ LSCs were undetectable, yet we found no correlation with the concomitant degree of molecular response.Conclusions: this study represents the first attempt to measure in a large cohort of CML patients residual circulating LSCs during TKIs treatment. In our hands PB LSCs flow-cytometry assay appeared feasible, specific and sensitive and thus suitable for routine monitoring. As expected, the majority of CML patients, even in deep molecular response, still harbor residual LSCs and the number of PB CD26+ did not correlate with the number of BCR-ABL1 copies. This evidence suggests that the molecular response refers to transcriptionally active CML progenitor cells and not to quiescent, TKIs resistant, CML LSCs. Prospective studies evaluating the behavior of PB CML LSCs during different TKIs treatment, as well as studies monitoring PB CD26+ in CML pts that discontinued TKIs treatment are ongoing. Our goal is to rule out the impact, if any, of a "stem cell response" in addition to the standard molecular response in the management of CML patients mainly to identify those pts candidates for a safe TKI discontinuation. [Display omitted] DisclosuresBocchia:Janssen: Honoraria; Novartis: Honoraria; Bristol-Myers Squibb: Honoraria. Aprile:Novartis: Honoraria. Castagnetti:Pfizer: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; ARIAD Pharmaceuticals: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Tiribelli:Ariad Pharmaceuticals: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau. Breccia:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Ariad: Honoraria; Pfizer: Honoraria. Rosti:Roche: Honoraria, Research Funding, Speakers Bureau; Incyte: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau.

Inhibition of CML Stem Cells with an Alkaloid That Reduces β-Catenin

Although the majority of CML patients initially respond positively to BCR-ABL tyrosine kinase inhibitors (TKIs), they fail to eradicate the leukemia stem cells (LSCs) from which the disease arises. Only a minority of patients is able to discontinue TKI therapy, presumably due to the survival of LSCs. Therefore, the development of new therapeutics which ablate CML stem cells through a non-TKI, BCR-ABL independent pathway is needed.The Wnt/β-catenin pathway has been identified as an LSC survival pathway which provides proliferative signals and controls the stability of BCR-ABL1 through the increased expression of β-catenin. While previous research has demonstrated that Wnt/β-catenin is necessary for the survival and self-renewal of all CML cells and LSCs, it is not essential for maintenance of normal hematopoietic stem cells (HSCs).Tetrandrine (ES-3000, TET) is a natural product alkaloid used clinically in China as an analgesic and an anti-inflammatory. Its known mechanism of action is the inhibition of voltage-gated calcium channels and calcium activated big potassium (BK) channels which are commonly overexpressed in malignancies. However, TET has recently been demonstrated to inhibit the Wnt/β-catenin pathway resulting in a reduced expression of β-catenin, putatively through the inhibition of CaMKII-γ activation. This study investigated the efficacy of TET in models of CML stem cells.To demonstrate that TET can reduce β-catenin in leukemic cells, an in vitro assay with leukemic K562 cells was performed. Cells were exposed to TET for 24 hours at concentrations between 0-40 μM. Cell lysates were assayed by Western blot for β-catenin and actin. The results demonstrated that TET reduces β-catenin expression in a dose dependent manner.The effectiveness of TET was tested on CML stem cells using an in vivo mouse CML model. After priming donor C57BL/6 (B6) mice with intravenous injections of 5-fluorouracil for four days, bone marrow cells were harvested from femurs and tibia, then transfected twice with retrovirus containing MSCV-BCR-ABL-IRES-GFP. Recipient mice were lethally irradiated by two doses of 550 cGy before bone marrow transplantation by intravenous injection with 5x105 cells/mouse. Blood from recipient mice was tested for disease induction one week after transduction by FACS analysis for GFP. All mice tested positive.Treatments started on day 8 after bone marrow transplantation. Mice were randomized into four groups and treated orally with vehicle [3x/day, 2x], imatinib [100 mg/kg, 2x/day], TET [150 mg/kg, 1x/day] or imatinib + TET [3x/day: 2x with imatinib, 1x with TET]. The results of this study demonstrated that TET given orally once a day is superior to imatinib given twice a day in inhibiting the development of both circulating leukemic cells and leukemic stem cells while the combination of TET with imatinib further improves efficacy (See Figure).To determine whether TET has efficacy on human CML stem-like cells, a colony forming cell (CFC) assay with bone marrow cells from a de-novo CML patient was performed. The bone marrow cells were treated with 10 µM (IC50) TET for 14 days. After treatment, primary and secondary colonies were grown and analyzed by qPCR to determine BCR-ABL or ABL only cells. Replating efficiency of TET treated cells was 54% compared to 67% of solvent controls. In primary colonies, 95% of colonies from solvent control cultures were BCR-ABL positive compared to 70% of colonies treated with ES-3000. In secondary colonies (representing stem-ness), the TET treatment group was negative for BCR-ABL colonies while 79% of solvent control colonies still tested positive for BCR-ABL, indicating efficacy of TET in CML stem-like cells (See Table).We conclude that TET reduces leukemic stem cells in both a murine model of CML and a CFC assay which demonstrates its potential for development as an adjuvant therapy for CML patients demonstrating a lack of optimal response to TKIs, alone. [Display omitted] [Display omitted] DisclosuresMichaels:Escend Pharmaceuticals, Inc.: Equity Ownership. Bates:Escend Pharmaceuticals, Inc.: Equity Ownership.

Potential role of Wnt/β-catenin signaling in blastic transformation of chronic myeloid leukemia: cross talk between β-catenin and BCR-ABL.

Chronic myeloid leukemia (CML) results from malignant transformation of hematopoietic stem cells induced by the BCR-ABL oncogene. Transformation from chronic to blastic phase is the lethal step in CML. Leukemic stem cells (LSCs) are the basic reason for blastic transformation. It has been shown that Wnt/β-catenin signaling contributes to the self-renewal capacity and proliferation of LSCs in CML. However, the role of Wnt/β-catenin signaling in blastic transformation of CML is still obscure. Here, we explored the relationship between BCR-ABL and β-catenin signaling in vitro and in vivo. We found that BCR-ABL stimulated β-catenin via activation of PI3K/AKT signaling in blastic phase CML cells. Inhibition of the kinase activity of BCR-ABL, PI3K, or AKT decreased the level of β-catenin in both K562 cells and a CML mouse model and suppressed the transcription of downstream target genes (c-myc and cyclin D1). In addition, inhibition of the BCR-ABL/PI3K/AKT pathway delayed the disease progression in the CML mouse model. To further explore the role of β-catenin in the self-renewal and survival of CML LSCs, we established a secondary transplantation CML mouse model. Our data revealed that inhibition of the BCR-ABL/PI3K/AKT pathway reduced the tumor-initiating ability of K562 cells, decreased leukemia cell infiltration into peripheral blood and bone marrow, and prolonged the survival of mice. In conclusion, our data indicate a close relationship between β-catenin and BCR-ABL/PI3K/AKT in blastic phase CML. β-Catenin inhibition may be of therapeutic value by targeting LSCs in combination with a tyrosine kinase inhibitor, which may delay blastic transformation of CML.

Epigenetic Reprogramming Sensitizes CML Stem Cells to Combined EZH2 and Tyrosine Kinase Inhibition.

In CML, TKI-persistent LSCs remain an obstacle to cure, and approaches to eradicate them remain a significant unmet clinical need. We demonstrate that EZH2 and H3K27me3 reprogramming is important for LSC survival, but renders LSCs sensitive to the combined effects of EZH2i and TKI. This represents a novel approach to more effectively target LSCs in patients receiving TKI treatment. Cancer Discov; 6(11); 1248-57. ©2016 AACR.See related article by Xie et al., p. 1237This article is highlighted in the In This Issue feature, p. 1197.

Abstract 3338: Pacritinib reduces human myeloid leukemia stem cell maintance in a defined niche

Abstract Introduction Pacritinib, a potent clinical small molecule inhibitor of JAK2, it also suppresses signaling through wild-type and mutant FLT3, IRAK1, and CSF1R. Pacritinib does not cause marrow suppression and has demonstrated single agent activity preclinically in myelofibrosis and other myeloid neoplasms including AML and CMML. Stromal protection was not observed. However, the capacity of pacritinib to eradicate therapy resistant leukemia stem cells (LSC), residing in the bone marrow niche, had not been examined. Thus, we investigated the impact of pacritinib alone or in combination with standard of care therapy on primary blast crisis chronic myeloid leukemia (BC CML), myelofibrosis (MF) and AML LSC survival and self-renewal in a stem cell supportive niche. Methods Genetically engineered mouse bone marrow fibroblasts producing human SCF, IL3 and G-CSF were used as stromal monolayers to support LSC survival and self-renewal. Human primary CD34+ cells were selected from BC CML (n = 5), MF (n = 5) and relapsed AML (n = 4) before and after clinical treatment with azacitidine. As a control, CD34+ cells from age matched normal bone marrow (a-NBM, n = 4) were used for the co-culture. Survival and self-renewal of the cells were investigated by colony forming and replating assays. Pacritinib was used at concentrations ranging from 10 to 50 nM alone and in combination with 1 nM dasatinib. Results Pacritinib alone induced dose-dependent inhibition of self-renewal in a-NBM, AML, MF and CML-BC, with the optimal concentration of 20nM leading to IC50 diversity in the response between normal and leukemia progenitors. AML and MF responded uniformly and inhibition reached 50% at 10nM concentration. BC CML cells were more divergent: 40% demonstrated >50% inhibition, in another 40% it was 20-50% and in 20% inhibition was <20%. Combined treatment with the low dose of dasatinib and pacritinib doses of 10 or 20 nM resulted in a significant (p<0.001, Anova) difference in self-renewal of all BC CML cells, raising a possibility of an additive/synergistic mechanisms. AML cells collected before and after clinical treatment with azacitidine uniformly showed a significant decrease in self-renewal starting with 10 nM pacritinib alone and combined treatment with dasatinib did not enhance the inhibition, suggesting a prospect of using pacritinib as a single agent in the treatment of relapsed AML. Conclusions Together these data indicate that possibly through inhibition of CSF1 and IRAK1 signaling in addition to suppression of JAK2, even in the presence of a LSC supportive niche, readily clinically achievable low nM concentrations of pacritinib alone are effective in reducing self-renewal of MF and relapsed AML. However, a combination of dasatinib and pacritinib is required to eliminate self-renewing LSC in BC CML with minimal toxicity toward normal progenitors. Targeting niche-dependent signaling could represent a robust avenue for treatment of refractory myeloid leukemia. Citation Format: Larisa Balaian, Anna Kulijian, Edward D. Ball, Catriona H.M Jamieson. Pacritinib reduces human myeloid leukemia stem cell maintance in a defined niche. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3338.

Eliminating Cancer Stem Cells in CML with Combination Transcriptional Therapy

Leukemia stem cells (LSCs) are resistant to current therapies used to treat chronic myeloid leukemia (CML). Abraham etal. (2016) have identified a molecular network critical for CML LSC survival and propose that simultaneously targeting two of their major transcriptional regulators, p53 and c-Myc, may facilitate their eradication.

Acquired expression of osteopontin selectively promotes enrichment of leukemia stem cells through AKT/mTOR/PTEN/β-catenin pathways in AML cells

AimsAcute myeloid leukemia (AML) initiation and progression have been attributed to subpopulations of self-renewing leukemia stem cells (LSCs), which contribute to progression, recurrence and therapeutic resistance in leukemia. Osteopontin (OPN) plays an important role in promoting survival and drug resistance in LSCs. The aim of this study was to explore OPN roles in modulating curcumin-mediated LSC enrichment and survival in AML cell lines and primary CD34+/CD38− bone-marrow-derived AML cells. Materials and methodsThe growth inhibitory effects of curcumin (CUR) were evaluated by MTT assay in U937 and CD34+ KG-1 AML cell lines as well as primary CD34+/CD38− bone-marrow derived AML cells isolated by MACS technique. The proportion of LSC markers (CD34, CD38 and CD123) were evaluated by flow cytometry. The expression levels of OPN, AKT, mTOR, PTEN, β-catenin and NF-κB were investigated by qRT-PCR. Short interfering RNA (siRNA) against OPN was used in AML cells incubated with or without CUR. Key findingsProportions of CD34+/CD38−/CD123+ and CD34+/CD38+/CD123+ LSCs compartment co-expressing an increased level of OPN could be enriched in AML cell lines and in patient's primary cells by CUR treatment. The expression levels of AKT, mTOR, PTEN, and β-catenin and NF-κB1, were also significantly up-regulated concurrently with OPN in the enriched CD34+ AML cells. SignificanceThe increased in CUR-mediated OPN level is involved in a complex interplay of various signaling pathways resulting in cytoprotection and enrichment of CD34+ LSC compartment in CUR-treated AML cells. AKT/mTOR/PTEN/β-catenin/NF-kB signaling pathways may play roles in modulating OPN-mediated LSC cell survival and enrichment.

Regain control of p53: Targeting leukemia stem cells by isoform-specific HDAC inhibition.

Leukemia stem cells (LSCs) are self-renewable, leukemia-initiating populations that are often resistant to traditional chemotherapy and tyrosine kinase inhibitors currently used for treatment of acute or chronic myeloid leukemia. The persistence and continued acquisition of mutations in resistant LSCs represent a major cause of refractory disease and/or relapse after remission. Understanding the mechanisms regulating LSC growth and survival is critical in devising effective therapies that will improve treatment response and outcome. Several recent studies indicate that the p53 tumor suppressor pathway is often inactivated in de novo myeloid leukemia through oncogenic-specific mechanisms, which converge on aberrant p53 protein deacetylation. Here, we summarize our current understanding of the various mechanisms underlying deregulation of histone deacetylases (HDACs), which could be exploited to restore p53 activity and enhance targeting of LSCs in molecularly defined patient subsets.

Abstract P2-03-06: Dysregulation of miR-34a-SIRT1 axis reduced breast cancer stemness

Abstract Recent studies show that enforced expression of miR-34a results in elimination of cancer stem cells (CSCs) in many malignant tumors. Sirtuin-1 (SIRT1) being confirmed as a direct target of miR-34a was reported to be involved in regulation of growth and survival of leukemia stem cells (LSCs). In this study, we aim to understand regulatory mechanism of miR-34a -SIRT1 axis in breast cancer stem cells (BCSCs). Lower endogenous level of miR-34a and higher level of Sirtuin1 (SIRT1) gene were identified in CD44+/CD24- BCSCs than breast cancer cells. Either ectopic expression of miR-34a or silenced SIRT1 in MCF-7 cells inhibited cellular proliferation, and led to cell apoptosis. Overexpression of miR-34a also suppressed expression of ALDH1, BMI1 and Nanog, and decreased capacity of mammosphere formation significantly.. Studies in vivo showed that stable expression of miR-34a reduced tumor burden significantly in nude mice xenografts. Taken together, our results showed that miR-34a inhibit the proliferative potential of BCSCs in vitro and in vivo, at least partially through downregulating SIRT1. miR-34a-SIRT1 axis may play an important role in self-renewal and stemness maintenance of BCSCs. This study may provide a novel BCSCs specific therapeutic strategy to improve breast cancer treatments. Citation Format: Xiao GG, Ma W, Mao J, Li L. Dysregulation of miR-34a-SIRT1 axis reduced breast cancer stemness. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-03-06.

Depletion of γ-catenin by Histone Deacetylase Inhibition Confers Elimination of CML Stem Cells in Combination with Imatinib.

Quiescent leukemia stem cells (LSCs) that are insensitive to BCR-ABL tyrosine kinase inhibitors confer resistance to imatinib in chronic myelogenous leukemia (CML). Identifying proteins to regulate survival and stemness of LSCs is urgently needed. Although histone deacetylase inhibitors (HDACis) can eliminate quiescent LSCs in CML, little is known about the underlying mechanism that HDACis kill LSCs. By fishing with a biotin-labeled probe, we identified that HDACi JSL-1 bound to the protein γ-catenin. γ-Catenin expression was higher in LSCs from CML patients than normal hematopoietic stem cells. Silencing γ-catenin in human CML CD34+ bone-marrow (BM) cells sufficiently eliminated LSCs, which suggests that γ-catenin is required for survival of CML LSCs. Pharmacological inhibition of γ-catenin thwarted survival and self-renewal of human CML CD34+ cells in vitro, and of murine LSCs in BCR-ABL-driven CML mice. γ-Catenin inhibition reduced long-term engraftment of human CML CD34+ cells in NOD.Cg-Prkdcscid II2rgtm1Sug/JicCrl (NOG) mice. Silencing γ-catenin by shRNA in human primary CD34+ cells did not alter β-catenin, implying a β-catenin-independent role of γ-catenin in survival and self-renewal of CML LSCs. Taken together, our findings validate that γ-catenin may be a novel therapeutic target of LSCs, and suppression of γ-catenin by HDACi may explain elimination of CML LSCs.

Bone marrow niche-mediated survival of leukemia stem cells in acute myeloid leukemia: Yin and Yang.

Acute myeloid leukemia (AML) is characterized by the accumulation of circulating immature blasts that exhibit uncontrolled growth, lack the ability to undergo normal differentiation, and have decreased sensitivity to apoptosis. Accumulating evidence shows the bone marrow (BM) niche is critical to the maintenance and retention of hematopoietic stem cells (HSC), including leukemia stem cells (LSC), and an increasing number of studies have demonstrated that crosstalk between LSC and the stromal cells associated with this niche greatly influences leukemia initiation, progression, and response to therapy. Undeniably, stromal cells in the BM niche provide a sanctuary in which LSC can acquire a drug-resistant phenotype and thereby evade chemotherapy-induced death. Yin and Yang, the ancient Chinese philosophical concept, vividly portrays the intricate and dynamic interactions between LSC and the BM niche. In fact, LSC-induced microenvironmental reprogramming contributes significantly to leukemogenesis. Thus, identifying the critical signaling pathways involved in these interactions will contribute to target optimization and combinatorial drug treatment strategies to overcome acquired drug resistance and prevent relapse following therapy. In this review, we describe some of the critical signaling pathways mediating BM niche-LSC interaction, including SDF1/CXCL12, Wnt/β-catenin, VCAM/VLA-4/NF-κB, CD44, and hypoxia as a newly-recognized physical determinant of resistance, and outline therapeutic strategies for overcoming these resistance factors.

Survival regulation of leukemia stem cells.

Leukemia stem cells (LSCs) are a subpopulation cells at the apex of hierarchies in leukemia cells and responsible for disease continuous propagation. In this article, we discuss some cellular and molecular components, which are critical for LSC survival. These components include intrinsic signaling pathways and extrinsic microenvironments. The intrinsic signaling pathways to be discussed include Wnt/β-catenin signaling, Hox genes, Hh pathway, Alox5, and some miRNAs, which have been shown to play important roles in regulating LSC survival and proliferation. The extrinsic components to be discussed include selectins, CXCL12/CXCR4, and CD44, which involve in LSC homing, survival, and proliferation by affecting bone marrow microenvironment. Potential strategies for eradicating LSCs will also discuss.

Identification of a Small Molecule Inhibitor of RAD52 to Induce Synthetic Lethality in BRCA-Deficient Leukemias

Altered DNA repair mechanisms are responsible for survival of leukemia stem cells (LSCs) and/or leukemia progenitor cells (LPCs) accumulating numerous lethal DNA double-strand breaks (DSBs). DSBs resulting from stalled/broken replication forks in proliferating cells are primarily repaired by RAD51-mediated homologous recombination repair (HR), which depends on BRCA1-PALB2-BRCA2-RAD51 paralogs (BRCA pathway), while RAD52 pathway serves as redundant back-up mechanism. Enhanced self-renewal of LSCs and high proliferation rate of LPCs commit them to HR.It has been reported that inhibition of RAD52 either by the knockout, specific shRNA, or a small peptide aptamer induced synthetic lethality in BRCA pathway-deficient tumor cell lines and primary leukemia cells. Yet pharmacological inhibition of RAD52, which binds single-stranded DNA (ssDNA) and lacks enzymatic activity, has not been demonstrated.Here, we applied high-throughput screening and structure-based selection followed by biochemical assays and computer modeling to identify three leading compounds: (1) 20264, (2) RU-0084339, and (3) D-I03. Compound 20264 appeared to interact with the hotspot in RAD52 DNA binding domain 1 to interfere with ssDNA binding. RU-0084339 is a major allosteric inhibitor of RAD52 ssDNA binding domain which disassembles undecamer ring structure of RAD52. D-I03 abrogated RAD52-mediated ssDNA annealing and ssDNA pairing. RAD52 small molecule inhibitor (RAD52smi) reduced recruitment of RAD52 to DNA damage-induced nuclear foci and suppressed RAD52-mediated DNA double-strand break (DSB) repair activity in cells with negligible effects on other DSB repair pathways. Importantly, RAD52smi selectively eliminated cancer cell lines carrying BRCA1/2 inactivating mutations.Since inactivating mutations in BRCA pathway are rare in leukemias, individual BRCA pathway-deficient leukemias were identified by Gene Expression and Mutation Analysis (GEMA). Gene Expression approach applied microarrays, qRT-PCR, and/or flow cytometry to identify individual leukemias displaying downregulation of at least one gene in BRCA pathway. On the other hand, Gene Mutation strategy detected individual leukemias expressing an oncogene causing downregulation of BRCA pathway gene(s) (e.g., BCR-ABL1, MLL-AF9, AML1-ETO - mediated downregulation of BRCA1 and/or BRCA2) and harboring inactivating mutations in BRCA pathway (e.g., BRCA2 = FANCD1, and other FA genes). BRCA-deficient cells from individual patients indentified by GEMA were selectively sensitive to RAD52smi alone or in combination with already approved cytotoxic drugs.RAD52 is a promising new target because it is "druggable" by small molecule inhibitors. Moreover, inhibition of RAD52 by genetic knockout and small peptide aptamer did not exert any major negative effects in normal cells and tissues. Altogether, this work provided foundation for precision medicine guided synthetic lethality in BRCA-deficient leukemias exerted by small molecule inhibitors targeting novel mechanism - RAD52 dependent DSB repair. DisclosuresNo relevant conflicts of interest to declare.

The Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) Regulates the Function and Migration of Leukemic Blasts through CD63/PI3K/AKT/P21 Axis

Introduction. The Tissue Inhibitor of MetalloProteinases-1 (TIMP-1) is a member of the inflammatory network that dramatically increases in response to inflammation. First described as an inhibitor of MMPs, TIMP-1 exerts pleiotropic effects in the hematopoietic microenviroment. Along with the finding that hematopoietic stem progenitor cells (HSPCs) directly respond to inflammation, a persistent inflammatory stimulation may hinder HSPC functions and eventually lead to hematological malignancies. Specifically, the inflammatory pathways activated by the bone marrow (BM) microenvironment influences the transformation and progression of leukemia. In hematological malignancies, TIMP-1 promotes proliferation in lymphoma cells and increased TIMP-1 serum levels are associated with poor prognosis in a variety of cancers. However, recent studies have revealed the dichotomy of TIMP-1 in cancer progression.Here, in the attempt to provide further evidence for the critical role of inflammation in leukemic microenvironment, we investigated the role of TIMP-1 in leukemic blasts from patients with Acute Myeloid Leukemia (AML) and elucidated the downstream pathway ignited by rhTIMP-1.Methods. Human CD34+ HSPCs were isolated from cord blood (CB) units as control samples, while leukemic cells were collected from AML patients at diagnosis (percentage of blasts >90%; n=36). Cell proliferation was assessed by cell cycle analysis, and CFSE staining. In addition, we performed colony-forming unit assays. TIMP-1's contribution to cell survival was evaluated by AnnexinV/PI staining. The expression of the tetraspannin receptor CD63 (TIMP-1's putative receptor) was assessed by flow cytometry. Leukemic blasts were assayed towards a CXCL12 gradient after exposure to rhTIMP-1 or pre-treated with LY294002 (PI3K inhibitor); migrated cells were counted and characterized for CD63 expression. Leukemic blasts were sorted in two separate fractions, CD63- and CD63+, and their capability to respond to rhTIMP-1 was assessed. Downstream molecular targets of TIMP-1 (such as PI3K, pAkt, p21) were also confirmed by flow cytometry. RT-PCR was employed to detect the expression of hypoxia-inducible factor-1 (HIF-1α). Finally, co-culture system was performed with CFSE-positive leukemic blasts and mesenchymal stromal cells (MSCs) from normal or AML patients in the presence of rhTIMP-1.Results. We firstly found that TIMP-1 levels are increased in the plasma of BM of AML patients at diagnosis. Similarly to CD34+ HSPCs from CB, the clonogenic potential of primary human AML blasts is increased upon exposure to rhTIMP-1. Moreover, rhTIMP-1 promotes in vitro AML blast survival, possibly due to anti-apoptotic effects and a slight but significant increase in the proportion of leukemic cells entering S-phase of the cell cycle. Such effect is related to the downregulation of cyclin-dependent kinase inhibitor p21. As for the dissection of TIMP-1 signalling pathway, our data revealed that the tetraspannin CD63 receptor is required for TIMP-1's cytokine functions. Indeed, about half of leukemic blasts expresses CD63, whose activation leads to PI3K recruitment and Akt phopshorylation, key modulators of survival/proliferation pathways. Exposure of AML blasts to rhTIMP-1 resulted in the induction of HIF-1α, which is known to play an important role in survival of leukemic stem cells. Moreover, rhTIMP-1 increased the SDF1-driven migration of leukemic blasts through the activation of PI3K. Interestingly, migrating AML blasts are highly enriched in CD63+ cells, whereas the CD63-negative pre-sorted fraction of leukemic cells showed reduced response to migration and pAkt expression after exposure to rhTIMP-1.Finally, in the co-culture system between AML cells and normal versus leukemic MSCs, we defined the particular role of TIMP-1 in normal and leukemic microenviroment. In particular, our results suggest that a defective capacity of AML-MSCs in sustaining the proliferation and migration of AML blasts may be restored by rhTIMP-1.Conclusions. In our study TIMP-1 emerges as an important factor in the leukemic BM microenvironment, modulating leukemic blasts survival, migration and function. Specifically, our work suggests that TIMP-1 promotes survival and migration via CD63/PI3K/AKT/p21 signalling. Taken together, our findings indicates TIMP-1 pathway as a potential novel therapeutic target in AML. DisclosuresCavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria.

CITED2 Cooperates with Low PU.1 and DNMT3A to Maintain Self-Renewal in Hematopoietic Stem Cells

CITED2 has a conserved role in the maintenance of normal hematopoiesis. We have recently shown that ~70% of acute myeloid leukemia (AML) patients display enhanced CITED2 expression levels. Interfering with CITED2 expression is detrimental for leukemia maintenance in vitro and in vivo, demonstrating that CITED2 is critically important for the survival of leukemic stem cells (LSCs). Ectopic expression of CITED2 in normal CD34+ stem and progenitor cells (HSPCs) led to significantly better human engraftment in transplanted NSG mice, consistent with the maintenance of very primitive lin- CD34+ CD38- CD90+ CD45RA- HSCs within the bone marrow 28 weeks after transplantation. Although the CITED2-engrafted mice displayed enlarged spleens, blood development appeared normal, as measured through myeloid, B and T cell staining. This indicates that CITED2 as a single hit is not sufficient to transform human CD34+ cells.CITED2 expression frequently coincides with low expression of the myeloid transcription factor PU.1, suggesting that combined effects, rather than single events are important during AML development. To investigate this, we combined lentiviral downregulation of PU.1 with overexpression of CITED2 (PU.1Low-CITED2High) and studied hematopoietic development. CITED2 increased the percentage of immature CD34+ CD38- cells 5-fold, which was not further increased by the additional downregulation of PU.1. However, functional analysis through limiting dilution LTC-iC assays indicated that combining PU.1 down-, with CITED2 upregulation led to a synergistic 8.5-fold increase in LTC-iC frequency, whereas only changing PU.1 or CITED2 induced a respective 1.4 to 3-fold change in HSC frequency. To more stringently assess self-renewal, we cultured transduced cells for 4 weeks on MS5 cells under myeloid differentiating conditions (G-CSF, IL3 and TPO) and subsequently performed CFC assays. Whereas after 4 weeks all groups displayed similar colony numbers, secondary and tertiary replatings demonstrated that self-renewal could only be maintained for more than 10 weeks when CITED2 upregulation was combined with PU.1 downregulation. This replating capacity of PU.1Low-CITED2High cells was limited to CD34+ CD38- HSCs, as replating of CD34+ CD38+ progenitor-derived colonies did not yield new CFCs.In order to investigate the underlying mechanisms, we performed transcriptome analysis on human HSCPs after knockdown of PU.1, overexpression of CITED2 or the combination of both. PU.1Low-CITED2High cells displayed a gene expression pattern different from the PU.1Low or CITED2High only cells, suggesting that the two events have synergistic effects. Some genes, like HLX and SF3B1 have been shown to cause or are mutated in AML, demonstrating that the synergistic changes are related to AML. When comparing the differentially regulated genes in the PU.1Low -CITED2High cells to the gene expression in the Hemaexplorer database, a similar pattern was observed, when compared between AML and normal cells.In order to investigate the effects of the PU.1low CITED2high combination on AML development, we resorted to a PU.1-dependent mouse model of AML development. CITED2 expression in BM cells from PU.1KD/KD mice (in which deletion of an Upstream Regulatory Element leads to an 80% downregulation of PU.1), led to a steady increase of GFP+ cells over time as compared to control cells and demonstrated a dramatic expansion of Gr-1+ Mac-1+ cells, a hallmark of AML in these mice. This suggests that CITED2 contributes to a faster progression towards AML upon lowering of PU.1.To identify if our model corresponds to AMLs with a specific subset of mutations, we clustered publically available AML data (TCGA), based on the gene expression changes in the PU.1Low -CITED2High cells. The majority of AMLs clustered together in 2 groups, in which FLT3, p53 and DNMT3A mutations were most prevalent. FLT3 mutations, through its activation of STAT5, are consistent with high CITED2 expression, whereas p53 mutations are consistent with our data indicating that CITED2 loss regulates HSCs in a p53-dependent manner. The presence of DNMT3A mutations suggests that DNA methylation changes collaborate with high CITED2 and low PU.1 during leukemogenesis. This is currently under investigation.In summary, our data imply that CITED2, low PU.1 and potentially changes in DNA methylation all contribute to maintenance of self-renewal and leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Anti-Leukemic Activation of Immune Cells in a Leukemic-Induced Microenvironment By CXCR4 Inhibition

The bone marrow microenvironment (BMM) provides a protective niche that supports growth and survival of leukemic stem cells. Stromal-derived factor-1 (SDF-1)/CXC receptor 4 (CXCR4) plays pivotal roles in the cross-interactions between blasts and the BMM to prevent retention and mobilization of leukemic cells, as well as in normal hematopoiesis including the development of immune cells. Here, we show that the CXCR4 antagonist, Plerixafor, decreased the level of CXCR4 expression and inhibited SDF-1-induced migration of leukemic cells. Further, the inhibition of the interaction between leukemic cells and the BMM by the CXCR4 antagonist enhanced cytotoxic activity of immune cells as a result of increased susceptibility of leukemic cells to chemotherapeutic agents such as cytosine arabinoside (Ara-C) in a mouse model of acute myeloid leukemia (AML), suggesting biological effects of the BMM through immune cell activation. To examine the level of CXCR4 expressed by primary AML blasts and murine leukemic cells and the role of CXCR4 in migration at various concentration, 19 AML samples and murine C1498 and human Jurkat cells were subjected to FACS analysis and migration assay. CXCR4 expression in C1498 cells was about 31.29%, compared to 99.7% in the CXCR4+ Jurkat cell line and RNA expression was also confirmed. C57Bl/6J mice were used to construct a syngeneic AML animal model by infusion of 2x106 C1498 cells, and peripheral blood from a facial vein and samples from multiple organs from the sacrificed mice were obtained until day 30 post-injection. Total RNA was extracted from BM cells, liver, and spleen of mice at day 15 and 1ug RNA was reverse transcribed into cDNA using a Transcriptor First Strand cDNA Synthesis Kit. All data were normalized to the amount of GAPDH expression, with samples run in triplicate. The quantitative real-time PCR assay for IFN-γ, perforin, and granzyme B, main cytokines produced by cytotoxic T and NK cells, was performed to examine the functional capacity of subsets. Data revealed that both normal human samples and AML mononuclear cells highly expressed CXCR4. CXCR4 expression in both groups was significantly decreased by CXCR4 inhibition, when treated with 5 µM Plerixafor. Our migration assay clearly showed the inhibitory effect of Plerixafor on SDF-1α-induced migration of primary AML and C1498 cells. Under SDF-1α conditions (100 ng/ml), C1498 cells were co-cultured with the CXCR4 antagonist at various concentrations. Migrations of both C1498 and primary AML cells were similarly inhibited. To test the direct role of the CXCR4 antagonist in apoptosis, C1498 cells were cultured with or without Ara-C. The Ara-C and Plerixafor dual-treated group (termed P+A group) displayed no significant difference in apoptosis when compared to the Ara-C only group, suggesting that apoptosis is exclusively controlled by Ara-C, but not Plerixafor, in vitro. However, leukemic blasts synergistically and significantly decreased in the P+A group, compared to those of the other groups, in a syngeneic leukemic mouse model experiments, suggesting an unexpected role for Plerixafor in leukemic blast suppression specifically in the AML niche. The frequency of CD4 and CD8 cells was maintained in the P+A group, compared to the Ara-C-only, Plerixafor treated only, and C1498 injected groups, implying that Plerixafor cannot independently alter the frequency of immune cells. Most of all, the expression levels of IFN-γ, perforin, and granzyme B in the spleens of the P+A group were significantly increased, compared to those of the control groups including the Plerixafor only group. This study demonstrates that the effects of CXCR4 inhibition on blast suppression and immune cell function in the tumor microenvironment and chemotherapy with Plerixafor represents an advanced therapeutic strategy of targeting the leukemic niche. DisclosuresNo relevant conflicts of interest to declare.

RNA Splicing Modulation Impairs Acute Myeloid Leukemia Stem Cell Maintenance

IntroductionDisease relapse is the leading cause of death in secondary AML (sAML), which evolves from antecedent hematologic disorders like myelodysplastic syndrome (MDS) or myeloproliferative neoplasms (MPNs) or following exposure to chemotherapy. Persistence of therapy-resistant leukemia stem cells (LSC) harboring enhanced survival and self-renewal capacity has been linked to high relapse rates in sAML. Previously, we showed that missplicing of a stem cell regulatory gene, GSK3 b, and splice isoform switching favoring pro-survival BCL2 family isoform expression promoted generation of therapy-resistant LSC (Abrahamsson et al PNAS 2009; Goff et al Cell Stem Cell 2013). However, whether aberrant pre-mRNA splicing promotes sAML LSC generation, in the absence of mutation, and if pharmacological splicing modulation impairs LSC maintenance, in a mutation-independent manner, has not been elucidated.Methods and ResultsComparative RNA-sequencing and gene set enrichment analyses revealed significant alterations in splicing factor gene expression in purified progenitors from untreated sAML compared with normal samples. In addition, using an isoform-specific alignment algorithm, we established a sAML LSC splice isoform expression signature that identified increased expression of select transcripts, e.g. CD82 and PTK2B. Thus, we investigated the LSC inhibitory efficacy of a stable, potent splicing modulatory agent, 17S -FD-895, in humanized AML LSC stromal co-culture and primagraft assays. Notably, there was a dose-dependent reduction in AML LSC (n=4) survival and self-renewal after in vitro 17S -FD-895 treatment, with a favorable therapeutic index compared to normal controls (n=3, p<0.01). Splicing reporter activity and PCR analyses revealed rapid and potent 17S -FD-895-induced alterations in splicing, promoting pro-apoptotic isoform expression and intron inclusion in the stem cell regulatory gene MCL1. Also, 17S -FD-895 restored normal expression patterns of PTK2B, and MCL1-L/S and BLC2-L/S expression ratios.Flow cytometric analyses in AML LSC primagraft models treated with 17S -FD-895 (5-10 mg/kg delivered intravenously in 3 doses over 2 weeks) revealed a decrease in human stem (CD45+ CD34+ CD38- Lin-, 68% reduction in the spleens of the 10 mg/kg group versus vehicle controls, n=5 mice per group, p<0.05) and progenitor (CD45+ CD34+ CD38+ Lin-, 80% reduction to nearly zero in the spleens of the 10 mg/kg group versus vehicle controls, p=0.08) cell frequencies. Furthermore, MCL1-L/S and BCL2-L/S expression ratios were significantly reduced in LSC-enriched fractions from 17S -FD-895-treated mice compared to vehicle controls. Consistent with a reduction in functional LSC burden after 17S -FD-895 treatment, subsequent serial transplantation studies showed a 47-65% reduction in leukemic burden in the hematopoietic tissues of recipients of CD34+ cells from mice in the 10 mg/kg treatment group versus vehicle controls (n=5 mice per group, p<0.05).ConclusionsHere we demonstrate that a potent and stable splicing modulatory agent, 17S -FD-895, normalized sAML-specific splice isoform expression patterns as well as MCL1-L/S and BLC2-L/S ratios. Moreover, pharmacologic splicing modulation reduced AML LSC survival and self-renewal in a dose-dependent manner in both in vitro and in vivo models with a favorable therapeutic index. Further evaluation of this compound as a splicing-targeted single agent or combined with standard of care therapy may reduce or eradicate LSC burden in therapy-resistant sAML. In addition, LSC-specific splice isoforms may represent important biomarkers that could be developed as companion diagnostics for splicing-targeted therapies in sAML and other recalcitrant malignancies. DisclosuresJamieson:Johnson & Johnson: Research Funding; GlaxoSmithKline: Research Funding.

Alox-5 As a Potent Therapeutic Target on Overcoming TKI-Resistance in Chronic Myeloid Leukemia with T315I Mutation in Bcr-Abl

Background&Significance: Chronic myeloid leukemia(CML) is a malignant disease of a primitive haematological cell, characterised by inappropriate expansion of myeloid cells. Although the disease is readily controlled by Tyrosine kinase inhibitors, approximately one third of patients will eventually fail treatment. And we believed it corresponds to insensitive leukemia stem cells(LSCs) with unresponsive genes to the kinase inhibitors in CML and mutation of Bcr-Abl. 5-lipoxygenase gene(Alox5) was proved as a novel therapeutic target in cancer stem cells of CML. It encodes a member of the lipoxygenase gene family and plays a role in the synthesis of leukotrienes from arachidonic acid. Without Alox5, Bcr-Abl fails to induce CML in mice due to the impairments of the functions of LSCs. However, recent report suggest that Alox5 regulation may not be important for the development of CML in human. Interestingly, we found specific upregulation of Alox5 in CML patients with strongly positive expression of p210 in mRNA level, including the patients primary diagnosed as CML and who suffered in relapse for TKI resistance. Therefore, we characterized the function and regulation of Alox5 in TKI-resistant CML.Results: Firstly, we verified the upregulation of Alox5 by real-time PCR on sorted human CML progenitor populations with strongly positive expression of Bcr-Abl(p210), but not on CML patients obtained remission after treatment of TKI. To evaluate the function and regulation of Alox5, we silenced Alox5 by siRNA and chemical inhibitior in human CML cell lines K562, its TKI-resistant cell lines K562R, murine CML cell lines BaF3wild, and its TKI-resistant cell line BaF3T315I. As a result, the apoptotic rate induced by Alox5 inhibition alone in K562 and BaF3wild cells was lower than by TKI. Conversely, blockage of Alox5 in BaF3T315I cells caused increasingly apoptotic rate, which was higher than imatinib treatment. To further study if Alox5 could play an important role in impairing leukemia stem cells in CML, we cultured LSCs(Lin-c-Kit+Sca-1+) derived from CML patients with Bcr-Abl-T315I mutation in the presence of Alox5 inhibitor or imatinib alone. For 24h treatment, obvious apoptosis was observed in cells cultured with Zileuton, a kind of Alox5 inhibitor, but not in cells cultured with imatinib. Therefore, apoptosis related genes were detected, significant downregulation of Bcl-2 was found compared to in LSCs without Alox5 knockouted. In addition, downregulation of Alox5 followed Bcr-Abl inhibition in CML, we hypothesised that Alox5 was a downstream of Bcr-Abl, genomic array was used to uncover the signaling pathway connected Alox5 with Bcr-Abl. The results shown us that PKC-β was significantly downregulated when Bcr-Abl was inactivated by TKI. Selectivly inhibited PKC-β could decreased Alox5 expression in mRNA level. As for this mechanism, we found p38/MAPK signaling pathway mediated regulation of Alox5 by PKC-β. Next, we evaluated the in vivo anti-CML effect of Alox5 inhibition with a xenograft mice model. Two weeks after the transplantation of human CML cells with mutant Bcr-Abl-T315I(n=12). NOD/SCID/IL2Rg-KO(KOG) mice were treated with normal saline, 10mg/kg Zileuton or imatinib intraperitoneally on everyday schedule. At first, we confirmed that Zileuton doesn't affect normal hematopoiesis(n=3), but eventually inhibite LSCs growth(n=3). Eight to ten weeks after the transplantation, the frequencies of human CD45+ CML cells and LSCs were significantly reduced by Zileuton treatment in bone marrow((BM) of the recipient mice compared with normal saline-treated control mice and imatinib treated mice, indicating that Alox5 inhibition can inhibit the survival of CML-T315I mutant cells and LSCs in vivo.Conclusion: Together, these results suggest that Alox5 would be a potent therapeutic target on overcoming TKI-resistance involved in Bcr-Abl-T315I mutation. DisclosuresNo relevant conflicts of interest to declare.

A Role for IL1RAP in Acute Myelogenous Leukemia Stem Cells Following Treatment and Progression

BackgroundAcute Myelogenous Leukemia (AML) evolves as many patients who are responsive to therapy upfront are resistant to the same agents when applied at relapse. We previously reported the results of our prospective efforts to formally assess the evolution of the leukemia stem cell (LSC) population(s) during patients' clinical courses. We identified a 9-90 fold increase in LSC activity and greatly increased phenotypic diversity of the LSC population. To identify the potential mechanisms underlying these changes we further characterized functionally-defined LSC populations from paired diagnosis and relapse samples.MethodsPrimary bone marrow and peripheral blood samples were collected on IRB approved protocols from patients with newly diagnosed AML undergoing induction therapy as well as normal donors. Twenty-five patients who relapsed after achieving a complete remission were selected for further study. Screening studies identified seven patients whose pre-therapy samples demonstrated sustained engraftment of NSG mice following transplantation. Transcriptional profiling of highly enriched LSC populations from seven patients was performed using ABI TaqMan® Low Density Array (TLDA) qPCR analyses following pre-amplification using a novel 153 gene expression platform. Protein expression levels of interleukin-1 receptor accessory protein (IL1RAP) on bulk leukemia cells and LSC populations from 25 patients were assessed by flow cytometry. The impact of loss of IL1RAP was assessed using lentiviral based shRNA targeting all IL1RAP isoforms followed by assessment of proliferation, apoptosis, colony forming unit (CFU) activity and NSG engraftment capacity in human cell lines as well as in primary patient samples. Downstream signaling events for IL1RAP were probed using a small molecule inhibitor approach.ResultsWhile the majority of the LSC populations' gene expression profile remained stable, twelve genes were differentially expressed between pre-treatment and relapsed LSC populations including IL1RAP. Flow cytometric analyses confirmed that IL1RAP is overexpressed on both bulk leukemia populations as well as LSC populations at diagnosis and relapse in comparison to normal hematopoietic stem cell (HSC) populations. Targeting ILRAP1 using shRNA in both cell lines and primary AML samples resulted in impaired proliferation, increased apoptosis, a marked loss of CFU capacity and impaired NSG engraftment. IL1 signaling is known to involve both the MAPkinase and NFKappB pathways. To determine which pathways are involved in IL1RAP mediated LSC survival, we performed a small molecule inhibitor screen targeting elements in both signaling cascades. Established inhibitors of the NFKappaB pathway resulted in loss in loss of leukemic cell function while MAPK signaling inhibition had minimal to no effect.ConclusionsWe identified IL1RAP as being overexpressed in both bulk leukemia and functionally defined LSC populations from pre-treatment and relapsed AML samples. Loss of IL1RAP was associated with a marked decline in LSC function. Preliminary studies support a primary role for the NF Kappa B pathway in LSC function. Our findings support a critical role for IL1RAP in LSC function and support its development as a target for AML therapy in both the upfront and relapse setting. DisclosuresWang:Immunogen: Research Funding. Calvi:Fate Therapeutics: Patents & Royalties. Becker:Millenium: Research Funding.

Chlorpromazine, an Inhibitor of Intracellular Trafficking of FLT3-ITD and KIT D816V, Shows Prominent Anti-Leukemic Activities Against AML Cells and AML Stem Cells in Vitro and in Vivo.

Activating mutations of receptor tyrosine kinases (RTKs) are involved in the pathophysiology of various types of cancers. Among them, FLT3 internal tandem duplications (ITD) and a KIT point mutation at D816V are frequently found in acute myeloid leukemia (AML) and associated with poor prognosis. Although a number of clinical trials were conducted, an effective RTK inhibitor with acceptable toxicity hasn’t been developed.Recent studies have demonstrated these oncogenic RTKs are mislocalized in the cytoplasm, where they transmit aberrant leukemogenic signals to downstream molecules in a compartment-specific manner. So, we speculated that manipulation of their intracellular trafficking between intracellular compartments might be promising as a new therapeutic approach for AML with mutant RTKs. In this study, we evaluated the in vitro and in vivo anti-leukemic effects of chlorpromazine (CPZ) as an inhibitor of membrane trafficking, which is a well-established antipsychotic drug. All of the protocols were approved by the Institutional Review Board of Kinki University Faculty of Medicine, and we obtained AML samples after the written informed consent was given from all the patients.First, to examine the effect of CPZ on the intracellular trafficking of RTK mutants, we introduced murine FLT3-ITD and KIT-D814V (corresponding to human KIT D816V) into murine embryonic fibroblasts (MEFs) isolated from E14.5 fetuses, respectively. Both FLT3-ITD and KIT-D814V were distributed in cytoplasm with a punctate pattern, both of which were mainly located close to endoplasmic reticulum (ER) and Golgi. In contrast, when treated with CPZ, both RTK mutants were predominantly localized in the compartment apart from ER and Golgi in a diffused pattern, indicating that the intracellular trafficking system that retains mutated RTKs at ER or Golgi is disrupted by CPZ treatment.Next, we evaluated the effect of CPZ on cell growth and survival using several human leukemia cell lines and primary AML cells with or without mutant RTK. As a result, CPZ severely inhibited the growth and survival of leukemia cell lines and primary AML cells with mutant RTK in a dose-dependent manner (up to 95% inhibition; IC50: between 1.5 μM and 10.7 μM). In addition, CPZ inhibited the growth of the cells without mutant RTK, whereas the IC50 levels were 3- to 4-fold higher than those seen in the cells with mutant RTK. We also examined the effects of CPZ on the growth and survival of CD34+38- leukemic stem cells (LSCs) derived from AML patients (n=4), and found that CPZ effectively inhibited the growth and/or survival of LSCs, especially in LSCs with mutant RTK (IC50=7.5 μM). As for this mechanism, we found that CPZ significantly suppressed the activities of mutant RTKs and their downstream molecules such as Stat5 in all tested FLT3-ITD(+) LSCs. These results indicate that CPZ inhibits cell growth and/or survival of AML cells and LSCs harboring mutant RTKs. Next, we evaluated the in vivo anti-leukemic effect of CPZ with a xenograft model. Three days after the transplantation of human AML cells with mutant RTKs (n=8), NOD/Scid/IL2Rg-KO (NOG) mice were treated with normal saline or 10 mg/kg CPZ intraperitoneally on everyday schedule. At first, we confirmed that plasma concentration levels of CPZ on this schedule were within the non-toxic range as previously reported (0.03 to 0.71 μM, n=3) and that CPZ doesn’t affect normal hematopoiesis (n=3). Eight to ten weeks after the transplantation, the frequencies of human CD45+ AML cells and CD34+38- LSCs were significantly reduced by CPZ treatment in the bone marrow (BM) of the recipient mice compared with normal saline-treated control (ctrl) mice (CD45+ 58.2% vs 8.47%, p<.001; LSCs 0.51% vs 0.07%, p<.005), indicating that CPZ can inhibit the growth and survival of both AML cells and LSCs in vivo. We further tested the anti-leukemic activities of CPZ on established human AML in a zenograft model. After confirming the engraftment of human AML cells by BM sampling six weeks after transplantation of FLT3-ITD(+) AML cells (n=3), these mice were treated with CPZ or normal saline. In all cases tested, CPZ treatment for 4 weeks significantly reduced human AML fraction in the BM. (18.4% vs 1.99%, p<.005)Together, these results suggest that CPZ, an inhibitor of the intracellular trafficking of leukemogenic RTKs, would be a promising therapeutic drug to eradicate AML cells with an established safety profile. DisclosuresRai:Shionogi & Co., Ltd. : Research Funding. Tanaka:Shionogi & Co., Ltd. : Research Funding. Tanimura:Shionogi & Co., Ltd. : Research Funding. Matsui:Shionogi & Co., Ltd. : Research Funding. Kanakura:Shionogi & Co., Ltd. : Research Funding. Matsumura:Shionogi & Co., Ltd. : Research Funding.