High-Throughput Chemical Screen on Acute Myeloid Leukemia Stem Cells Identifies Novel Anti-LSC Compounds

Abstract Acute myeloid leukemia (AML) is an aggressive form of blood cancer defined by the uncontrolled proliferation and clonal expansion of immature myeloblast cells in the blood and bone marrow, leading to hematopoietic failure. Despite the use of aggressive and cytotoxic standard-of-care drugs, patients often relapse and succumb to the disease partially due to the inability of medically unfit patients to withstand the cytotoxic treatments, regrowth from minimal residual disease and the chemo-resistant nature of leukemic stem cells (LSCs) which can remain in a quiescent state and reside in a protective bone marrow niche. Hence, novel therapies targeting unique leukemic stem cell biology are highly needed to eliminate and avoid reoccurrence. High-throughput screens of human AML LSCs are not performed due to technical issues such as low LSC frequency within primary samples, an inability to purify LSCs, and the difficulty maintaining and expanding primary patient samples and LSCs in vitro. We were able to optimize conditions for a 4-week in vitro large-scale expansion (>600 million bulk) of the primary human AML sample 8227 (OCI-AML-8227), functionally validated to be enriched for LSCs in long-term xenotransplant assays (Eppert et al., 2011). These optimized conditions enabled the isolation and maintenance of the LSC-containing fraction for a chemical screen. We isolated the CD34+ LSC-containing fraction (>90% purity) and performed a high-throughput screen of 11,166 chemical molecules using a CellTiter Glo assay followed by a counter screen against normal CD34+ cord blood (CB) hematopoietic stem and progenitor cells. From this HT screen, a total of 61 hits had >70% inhibition on CD34+ 8227 cells and <30% inhibition on CD34+ CB cells. We also identified glucocorticoids, which were also identified in our prior small-scale anti-LSC screen where they were found to specifically drive human LSCs to terminally differentiate (Laverdière & Boileau, et al., 2018). We then performed dose response assays for each candidate compounds and confirmed 35 potent anti-LSC compounds with IC 50 < 1 μM. This refined the types of compounds to including anti-apoptotic inhibitors, GSK inhibitors, protease inhibitors, metabolism inhibitors, HDAC inhibitors, BET inhibitors, nucleic acid synthesis inhibitors, cell cycle inhibitors and Wnt/β-catenin inhibitors. This is interesting as some of the classes of these compounds (inhibitors of GSK, BET, nucleic acid synthesis, Wnt/β-catenin and metabolism) have been shown to target bulk and leukemic stem cells in AML in vitro and in vivo. We now aim to examine LSC eradication in a panel of genetically defined primary AMLs confirmed through in vitro and in vivo assays. Our goal is to be able to understand and establish the molecular mechanisms and biomarkers on primary functional LSCs. Disclosures No relevant conflicts of interest to declare.

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Novel Therapeutics That Can Target and Eradicate Leukemic Stem Cells in Acute Myeloid Leukemia: Investigating FDA-Approved Anti-Inflammatory Compounds

Blood ◽

10.1182/blood-2019-125338 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5052-5052

Author(s):

Isabella Iasenza ◽

Meaghan Boileau ◽

Andrea Neumann ◽

Héloïse Frison ◽

Mark D. Minden ◽

...

Keyword(s):

Stem Cells ◽

Acute Myeloid Leukemia ◽

Mechanism Of Action ◽

Myeloid Leukemia ◽

Terminal Differentiation ◽

Dependent Manner ◽

Leukemic Stem Cells ◽

Anti Inflammatory ◽

Acute Myeloid

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

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Specific Targeting of Acute Myeloid Leukemia By the Use of Non-Virally Engineered CIK (Cytokine-Induced Killer) Cells Expressing the Anti-CD33 Chimeric Antigen Receptor (CAR)

Blood ◽

10.1182/blood-2018-99-114572 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2201-2201 ◽

Cited By ~ 1

Author(s):

Maria Caterina Rotiroti ◽

Silvia Arcangeli ◽

Chiara Buracchi ◽

Chiara Francesca Magnani ◽

Claudia Cappuzzello ◽

...

Keyword(s):

Bone Marrow ◽

Myeloid Leukemia ◽

Cytokine Production ◽

Cik Cells ◽

Cell Immunotherapy ◽

Cik Cell ◽

Acute Myeloid

Abstract Background Acute Myeloid Leukemia (AML) is still associated with high relapse rates when treated with conventional chemotherapeutic and hematopoietic transplantation regimens. Thus, new treatment options are urgently needed. Immunotherapy adopting T cells engineered to express tumor-directed Chimeric Antigen Receptors (CARs) has shown striking results particularly in the context of B-cell malignancies, sparking a keen interest in extending this approach also to other hematological malignancies such as AML. Among the surface molecules identified, the CD33 molecule represents so far one of the main validated target in AML and, being broadly expressed on AML blasts, represents a suitable antigen to be targeted with CAR-T cells. Objectives The aim of the present study is to preclinically evaluate the efficacy and safety profiles of CD33.CAR redirected Cytokine Induced Killer (CIK) cells alone and in combination with standard chemotherapeutic agents. Methods Donor derived- and autologous-CIK cells were stably or transiently transduced with a third generation anti-CD33.CAR by Sleeping Beauty transposon- or mRNA-mediated engineering. In vitro anti-AML activity has been assessed by means of Flow cytometry-based cytotoxicity (AnnV-7AAD staining), proliferation (Ki67 staining and CFSE dilution) and cytokine production (intracellular IFNg and IL2 detection) assays, upon challenge with AML samples. In vivo efficacy has been evaluated in NSG mice transplanted with MA9-NRas AML cell line or primary AML samples. Moreover, an already established xenograft chemotherapy model has been exploited to examine the potential benefit of combining CD33.CAR-CIK cells with standard AML induction therapy (Ara-C and doxorubicin). Results CD33.CAR stably expressing CIK cells were able to induce a potent anti-leukemic activity in vitro, in terms of specific killing either in short term (>70% at 4h, E:T ratio 5:1) and long term cytotoxic assays (>90% at 1 week, E:T ratio 1:10), with statistically significant differences as compared to the unmanipulated condition. Moreover, CD33.CAR-CIK cells were able to retain a significant cytotoxic activity when re-challenged with the CD33+ target following a previous stimulation (up to 65%). The proliferative response to AML target cells was also considerable and CAR-specific (up to 60% of Ki67+CAR-CIK cells and up to 70% of CFSE diluted CAR-CIK cells), as well as the cytokine production (up to 35% of IFN-γ producing CAR-CIK cells and up to 25% of IL-2 producing CAR-CIK cells). CIK cells transiently expressing the CD33.CAR were also effective towards the AML target. In vivo results showed that CD33.CAR-CIK cells were able to control the disease in MA9 grafted mice in all the districts analyzed (peripheral blood, bone marrow, spleen, liver and kidney), as compared to untreated mice. To evaluate the effect of CD33.CAR-CIK cell immunotherapy particularly on Leukemia Initiating Cells (LICs), CD33.CAR-CIK cells were administered as an early treatment approach, treating mice 5 days after i.v. injection of a secondary transplanted PDX sample. We observed a clear engraftment reduction in the treated cohort, nearly undetectable in 2 out 5 mice, while a high leukemic burden has been detected in untreated mice (up to 70% of engraftment in bone marrow). Furthermore, by exploiting CD33.CAR-CIK cell treatment in mice experiencing disease recurrence after the "5+3" chemotherapy-induction protocol, preliminary data showed that CD33.CAR-CIK cells were also capable to target chemotherapy resistant/residual AML cells. Conclusions Considering our in vivo preliminary results, we aim to further evaluate CD33.CAR-CIK cell immunotherapy efficacy, particularly against chemotherapy resistant/residual AML cells. Concerning the safety aspect, since the CD33 targeting raises concerns for a potential myelotoxicity, we will assess the potential long-term off-target effects of CD33.CAR-CIK cells (comparing stably with transiently expressed CD33.CARs) on normal hematopoietic stem/myeloid progenitor cells. Disclosures No relevant conflicts of interest to declare.

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The Actin Binding Protein Plastin-3 Is Involved in the Pathogenesis of Acute Myeloid Leukemia

Cancers ◽

10.3390/cancers11111663 ◽

2019 ◽

Vol 11 (11) ◽

pp. 1663 ◽

Cited By ~ 1

Author(s):

Arne Velthaus ◽

Kerstin Cornils ◽

Jan K. Hennigs ◽

Saskia Grüb ◽

Hauke Stamm ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Binding Protein ◽

Actin Binding ◽

Bone Marrow Niche ◽

Actin Binding Protein ◽

Acute Myeloid

Leukemia-initiating cells reside within the bone marrow in specialized niches where they undergo complex interactions with their surrounding stromal cells. We have identified the actin-binding protein Plastin-3 (PLS3) as potential player within the leukemic bone marrow niche and investigated its functional role in acute myeloid leukemia. High expression of PLS3 was associated with a poor overall and event-free survival for AML patients. These findings were supported by functional in vitro and in vivo experiments. AML cells with a PLS3 knockdown showed significantly reduced colony numbers in vitro while the PLS3 overexpression variants resulted in significantly enhanced colony numbers compared to their respective controls. Furthermore, the survival of NSG mice transplanted with the PLS3 knockdown cells showed a significantly prolonged survival in comparison to mice transplanted with the control AML cells. Further studies should focus on the underlying leukemia-promoting mechanisms and investigate PLS3 as therapeutic target.

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MOZ-TIF2, but Not BCR-ABL, Confers Properties of Leukemic Stem Cells to Committed Murine Hematopoietic Progenitors.

Blood ◽

10.1182/blood.v104.11.62.62 ◽

2004 ◽

Vol 104 (11) ◽

pp. 62-62

Author(s):

Brian J.P. Huntly ◽

Hirokazu Shigematzu ◽

Kenji Deguchi ◽

Benjamin Lee ◽

Shinichi Mizuno ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mononuclear Cells ◽

Hierarchical Organization ◽

Leukemic Stem Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Acute Myeloid ◽

Myeloid Progenitors

Abstract The existence of leukemia stem cells has been demonstrated in acute myeloid and lymphoblastic leukemias (AML and ALL). The origins of these cells are unknown, but it has been suggested that they result from the transformation of adult hematopoietic stem cells (HSC). To challenge this hypothesis we tested the ability of representative leukemia oncogenes to transform committed myeloid progenitor cells that lack the capacity for self-renewal. Flow-sorted populations of common myeloid progenitors (CMP), and granulocyte-monocyte progenitors (GMP) were transduced with the fusion oncogenes MOZ-TIF2 and BCR-ABL, respectively and their self-renewal and leukemogenic potential were tested in in vitro and in vivo assays. Utilizing the same experimental design we were also able to address the poorly understood question of the contribution of the cell of transformation to the eventual leukaemia phenotype. In contrast to CMP or GMP transduced with BCR-ABL or non-transduced control cells, CMP or GMP that were retrovirally transduced with MOZ-TIF2 could be serially replated in methylcellulose cultures, and continuously propagated in liquid culture media containing IL-3. In further contrast, transplantation of CMP or GMP transduced with MOZ-TIF2 into recipient mice also resulted in an acute myeloid leukemia (AML). This leukaemia could be transplanted to secondary recipients, documenting the long-term self-renewal properties of the leukemic stem cells, yet in limiting dilution experiments did not cause disease below a transplanted cell dose of 1 x104 cells, suggesting that the probability of transferring leukemia to secondary recipients relates to the frequency of self-renewing leukemic stem cells within the total leukemic population. This in turn suggests that our retroviral bone marrow transduction and transplantation models have the same hierarchical organization of self-renewal as has been shown for human AML. The phenotype of the leukemias were virtually indistinguishable regardless of whether the initially transduced cell population was CMP, GMP or the control populations of whole bone marrow mononuclear cells or HSC, suggesting that MOZ-TIF2 may also have a dominant effect upon the eventual leukaemia phenotype. These observations indicate that MOZ-TIF2, but not BCR-ABL, can confer properties of leukemic stem cells to committed myeloid progenitors. Control experiments conducted with with MOZ-TIF2 point mutants that do not cause leukemia in the murine BMT system and with BCR-ABL, a fully active leukemogenic tyrosine kinase, were insufficient to cause in-vitro changes in self-renewal or leukaemia. Together, these data argue strongly that retroviral insertional mutagenesis alone cannot explain these results. However, we cannot exclude the possibility that an active MOZ-TIF2, but not BCR-ABL, can collaborate with mutations induced by retroviral mutagenesis to confer properties of leukemic stem cells to committed progenitors. These findings have important implications regarding the origin of leukemic stem cells, and provide tools for understanding the transcriptional programs that confer properties of self-renewal in malignant and non-malignant cells.

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Personalized Approach To Treatment of Acute Myeloid Leukemia Using a High-Throughput Chemosensitivity Assay

Blood ◽

10.1182/blood.v122.21.483.483 ◽

2013 ◽

Vol 122 (21) ◽

pp. 483-483 ◽

Cited By ~ 2

Author(s):

Ka Yee Yeung ◽

C. Anthony Blau ◽

Vivian G. Oehler ◽

Su-In Lee ◽

Christopher P. Miller ◽

...

Keyword(s):

Bone Marrow ◽

Complete Remission ◽

High Throughput ◽

Myeloid Leukemia ◽

In Vitro Cytotoxicity ◽

Personalized Approach ◽

New Diagnosis ◽

Acute Myeloid ◽

Refractory Aml

Most patients with acute myeloid leukemia either relapse or fail to respond to initial therapy. Moreover, each patient’s AML contains multiple mutations that although varying from one population to another, are unique to that individual, prompting development of individualized approaches to AML therapy. We have devised a high-throughput sensitivity assay for 160 drugs; 45 are FDA approved and 115 are investigational, representing multiple mechanisms of action and signaling pathways. 30 primary patient blood marrow samples and 15 acute leukemia cell lines have been analyzed. Peripheral blood blasts from individual patients were thawed, viable cells isolated and purified to >80% using magnetic bead separations. In vitro chemotherapy cytotoxicity testing is performed in a 384 well high throughput format, with eight concentrations of each drug. The output is cell survival assessed via a luminescent detection method after a 4-day incubation with various drugs. Fitted curves (idbs XLfit) were derived from plots of survival versus drug concentrations used in the study. Leukemia cells are tested adherent to coated plates to mimic adhesion in the bone marrow microenvironment, a property that confers drug resistance. The assay exhibits excellent reproducibility from independently thawed samples from the same patients, with a Spearman correlation coefficient of 0.9 (p=1.6 X 10-141). Gene expression microarrays were also performed for the same 30 patients. Our assay of the 30 patients revealed that there were over 50 drugs that exhibited cytotoxicity in at least some patients. There was wide variation in the drug sensitivity patterns exhibited by the patient blasts samples, and each was unique. Notably, all patient samples were susceptible to several drugs with IC50s in the range that might predict clinical response. For patients who achieved complete remission, we showed statistically significant association with in vitro cytotoxicity in response to 7 drugs that are commonly employed to treat AML, including mitoxantrone (p=0.002 for 0.1 µM, p=0.01 for 0.3 µM), clofarabine (p=0.0009 for 0.1 µM, p=0.003 for 0.3 µM, p=0.007 for 1µM), daunorubicin (p=003 for 0.1 µM, p=0.005 for 0.3 µM), etoposide (p=0.02 at for 0.1 µM, p=0.01 for 0.3 µM, p=0.01 for 1 µM), and fludarabine (p=0.05 for 0.3 µM, p=0.002 for 1 µM). In addition, we used a multivariate statistical method to identify drug combinations that are effective in predicting the complete remission of AML patients. In particular, we found that the in vitro cytotoxicity data of a combination of three drugs (BAY 11-7085, TPCA-1, ON 01910.Na) are more predictive of the complete remission of patients compared to using each of these drugs individually (i.e. 91.2% accuracy compared to 84.8%, 84.2%, 79.1% respectively). We also performed linear regression analyses to study the relationship between in vitro cytotoxicity in the high throughput screen of each drug versus the patients’ clinical features (including complete remission, having received the tested drug, new diagnosis vs. relapse status, age, gender, bilirubin, albumin, lactate dehydrogenase, white blood count, platelet count, blast, absolute neutrophil count, % bone marrow blasts, hemoglobin, fibrinogen, cytogenetics risk category). The following patients’ clinical features showed significant correlations with the in vitro cytotoxicity of our assays in selected drugs: complete remission for mitoxantrone (p= 0.04 for 0.3 µM), flavoperidol (p= 0.02 0.1 µM), and fludarabine (p= 0.01 0.3 µM, p=0.02 for 1 µM); drug the patient received for clofarabine (p= 0.03 for 0.3 µM, p=0.007 for 1 µM); new diagnosis vs. relapse for clofarabine (p= p= 0.02 for 0.3 µM, p=0.006 for 1 µM). This assay serves as the basis for a new clinical trial (ClinicalTrials.gov Identifier: NCT01872819) now open to enrollment for “personalized” leukemia therapy, for which patients with refractory AML are assigned drugs based on the results of this test. This new personalized approach to individualized therapy for refractory AML may provide novel drugs to patients and new insights into leukemia drug resistance. Disclosures: No relevant conflicts of interest to declare.

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Proteomic, Gene Expression, and Micro-RNA Analysis Of Bone Marrow Mesenchymal Stromal Cells In Acute Myeloid Leukemia Identifies Pro-Inflammatory, Pro-Survival Signatures In Vitro and In Vivo

Blood ◽

10.1182/blood.v122.21.3685.3685 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3685-3685

Author(s):

Michael Andreeff ◽

Rui-yu Wang ◽

Richard E. Davis ◽

Rodrigo Jacamo ◽

Peter P. Ruvolo ◽

...

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Myeloid Leukemia ◽

Micro Rna ◽

Leukemia Cells ◽

Rna Analysis ◽

Nsg Mice ◽

Acute Myeloid

Abstract The bone marrow microenvironment (BME) in acute myeloid leukemia (AML) generates resistance signals that protect AML cells/stem cells from chemotherapy. The mechanisms how the BME might support leukemia cell survival are unclear but elucidation of this process could prove useful for therapy design. Here we report new insights specific to stroma functionality in AML. A series of novel experimental approaches were developed including : 1) nanostring micro-RNA and proteomic analysis using reverse-phase protein arrays (RPPAs) of MSC derived from AML patients and normal donors; 2) genome-wide RNA analysis of FACS-sorted MSCs using Illumina arrays of genetically-defined human and murine AML cell lines/primary AML samples after co-culture with normal MSC in vitro; 3) in vivo interaction between genetically-defined murine AML and stromal cells in syngenic C57BL/6J mice, followed by harvesting and FACS-isolation of specific MSC after leukemia engraftment; 4) use of genetically-modified human MSC in vivo in our ectopic humanized bone marrow model in NSG mice (Blood 2012 : 119,4971), followed by bioluminescence growth and homing analysis of human leukemia cells. This model allows the study of in vivo effects of altered MSC on human AML development. 1) Proteomic and transcriptomic analysis of primary MSC from AML patients (n = 106) and normal MSC (n = 71) by RPPA using validated mAbs to 150 proteins and phospho-proteins demonstrated major differences by hierarchical clustering analysis: GSKA, STAT1, PDK1, PP2A, CDKN1A, CDK4, and STAT5AB were significantly over-expressed in AML- vs. normal MSC, while STMN1, SIRT1, SMAD1, SMAD4, HSP90 and EIF2S1 were under-expressed. Differences were observed between MSC from newly diagnosed vs. relapsed AML-MSC. Nanostring analysis of 38 AML-MSC and 24 normal MSC identified differential expression of numerous miRs, a select group of which has been validated so far by qRT-PCR. AML MSC express reduced levels of let-7g, let-7c, miR 21 and miR93, and elevated levels of miR410 compared to normal MSC. Pathways were identified in MSC that might contribute to leukemia survival. 2) Analysis OCI-AML3 cells co-cultured with normal -MSC revealed upregulation of a variety of genes in MSC encoding cytokines and chemokines and gene set enrichment analysis (GSEA) identified activation of NFkB in MSC as a potential cause of these changes. When the ectopic humanized bone marrow model system in NSG mice was used, suppression of NFkB in MSC resulted in a ∼ 50% reduction of AML burden. When murine MSC cultured with wt p53 MLL/ENL-Luc-FLT3-ITD cells were compared to isogenic cells with deleted p53, striking differences were seen in the MSC transcriptome: 429 differentially expressed genes were identified that distinguished co-cultures with p53wt and p53-/- cells, suggesting that AML cells may communicate signals to their microenvironment in a p53-dependent manner. GSEA identified NFkB and HIF-1a as targets, data were confirmed independently, and HIF-1a knockout MSC were found to be inhospitable for AML in the ectopic in vivo model. 3) These syngenic cells were introduced into B57BL/6J mice and MSC were isolated after leukemia engraftment: 147 genes were consistently upregulated and 236 genes downregulated in MSC by their interactions with AML in vivo. Upregulated genes included CTGF, CXCL12, genes related to complement (C4A, C4B, Serpin G1), and IGFBP5, an inhibitor of osteoblast differentiation. Identification of CXCL12 was intriguing as Link's group recently reported the critical role of CXCL12 produced by early MSC in normal hematopoiesis (Nature 2013 : 495,227). Both AML-ETO and MLL-ENL leukemias caused upregulation of CTGF, metalloproteinases, adhesion molecules, and NFkB-related genes in vivo. IPA analysis showed responses in BM-MSC associated with inflammation, cellular movement, cell-cell signaling, cellular growth and proliferation and immune cell trafficking. Conclusion AML cells induce changes in MSC, in short term co-cultures in vitro, or in syngenic systems in vivo, that are consistent with pro-survival, anti-apoptotic, and growth-stimulatory signals that mimic inflammatory responses. Large-scale analysis of primary AML-derived MSC confirms and extends this data. Results facilitate the development of therapeutic strategies to render the BM microenvironment inhospitable to leukemia cells but supportive of normal hematopoiesis. Disclosures: Lowe: Blueprint Medicines: Consultancy; Constellation Pharmaceuticals: Consultancy; Mirimus Inc.: Consultancy.

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Disulfiram, an clinically used anti-alcoholism drug has the potential to target acute myeloid leukemia cells in a copper-dependent manner in vivo

Blood ◽

10.1182/blood.v122.21.5040.5040 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5040-5040

Author(s):

Bing Xu ◽

Rongwei Li ◽

Huijuan Dong ◽

Feili Chen ◽

Yuejian Liu ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Specific Antigen ◽

Leukemia Cell ◽

Leukemia Cells ◽

Control Group ◽

Acute Myeloid

Abstract Background Disulfiram(DS), an old drug clinically used for alcoholism, was reported to have antitumor effects, recent studies have found that Copper(Cu) can significantly enhance the DS-induced cell death in vitro in a variety of tumor cells. Our previous studies also demonstrated that disulfiram/copper (DS/Cu) couldtarget human leukemia cell lines(like KG1α,Molt4) through the activation of JNK, in vitro. However, there is few report about the ability of DS/Cu in killing cancer cells in vivo. Aims This study aims to explore the effect of DS/Cu on acute myeloid leukemia cell line KG1αin vivo and clarify the underlining mechanism. Methods 6-8 week old female NOD/SCID mice were sublethally irradiated with 2Gy X-ray the day before transplantation, followed by intravenous injection of KG1α cells (1×107 cells) suspended in 0.2 mL of PBS. 5 weeks after transplantation mice were randomly divided into three treatment groups: vehicle (0.9% saline), a combination of DS and Cu daily for 2 weeks, Ara-C alone twice before killing. Mice were sacrificed after 2 weeks treatment with tissues of spleen, liver, bone marrow being observed using histopathology method to detect the invasion of leukemia. The DS/Cu-induced p-c-jun activation was also examined by western blot using tissues of spleen, liver, bone marrow. Statistical analysis was carried out with one-way ANOVA to assess statistical significance (*p < 0.05). Results 4 weeks after transplantation, mice were dispirited with low appetite, down-bent gait, wrinkled fur, slow move, just like suffered from leukemia. What’s more, immature blasts like morphology similar to KG1α were found in the peripheral blood of the mice(11%±3.41). All the mice were sacrificed after 2 weeks treatment, mice in control group were observed with slightly larger spleen and liver with the morphology of invasion of leukemia such as a granular appearance than the other two groups. Histopathology examination showed that leukemia cells infiltrate liver, spleen and bone marrow, and the immunohistochemistry examination found that the leukemia cells in spleen, liver and bone marrow expressed human specific antigen CD45 with the highest expression level in the control group. Moreover, solid tumor could be observed in the peritoneal cavity of two mice in the control group with expression of human specific antigen CD45detected by immunohistochemistry examination. Western blot in this study showed DS/Cu complex induced phosphorylation of c-Jun expression in the spleen, liver and bone marrow. Conclusion DS/Cu complex could effectively target the acute myeloid leukemia cells in the acute leukemia NOD/SCID mice while inhibiting the invasion of leukemia to some extent, and the activation of JNK might play a functional role in DS/Cu mediated antileukemic effects. Disclosures: No relevant conflicts of interest to declare.

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Therapeutic Targeting of Mesothelin in Acute Myeloid Leukemia with Chimeric Antigen Receptor T Cell Therapy

Blood ◽

10.1182/blood-2020-143140 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 11-11 ◽

Cited By ~ 1

Author(s):

Quy Le ◽

Sommer Castro ◽

Thao T. Tang ◽

Anisha Loeb ◽

Amanda R. Leonti ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Cell Surface ◽

Myeloid Leukemia ◽

Car T Cells ◽

Car T ◽

Acute Myeloid

Background: Acute myeloid leukemia (AML) is one of the most highly refractory hematologic malignancies despite intensive combination chemotherapy and bone marrow stem cell transplantation. Lack of curative treatments is in large part due to our poor understanding of the disease biology and paucity of therapeutic targets. In an effort to identify actionable targets, we recently completed the largest genome, epigenome and transcriptome profiling of AML in nearly 3000 children and young adults. This discovery effort has led to the identification of a library of novel AML-restricted targets (high expression in AML, minimal-to-no expression in normal hematopoiesis) for therapeutic development. One such target was MSLN which encodes for mesothelin, a cell surface adhesion molecule that is highly expressed in 30-50% of AML cases in pediatric (Children Oncology Group) and adult (MD Anderson) cohorts and is entirely absent in normal bone marrow and peripheral blood CD34+ cells. MSLN expression in normal tissues is confined to mesothelial cells lining the pleura, pericardium, and peritoneum. Previous studies targeting MSLN in solid tumors have demonstrated clinical efficacy with minimal toxicities. Given that T cells genetically modified to express chimeric antigen receptors (CARs) are extremely effective at eradicating relapsed and refractory malignancy, we developed MSLN-directed CAR T cells for pre-clinical evaluation in AML. Methods: From primary patient samples, we verified MSLN expression by RT-PCR and confirmed mesothelin surface protein expression on leukemic blasts by flow-cytometry as well as detected soluble mesothelin in the plasma by ELISA. The VH and VL sequences from Amatuximab were used to create the scFv domain of the standard CAR (41-BB and CD3Zeta). For in vivo CAR T study, Nomo-1 cells, which express endogenous level of MSLN, and Kasumi-1 cells engineered to express MSLN with a lentivirus construct (Kasumi-1 MSLN+) were transplanted into NSG mice. Mock transduced MSLN-directed CAR T cells were infused 1 week (Nomo-1) and 2 weeks (Kasumi-1 MSLN+) following leukemic cell injection. Leukemic burden was measured by bioluminescence IVIS imaging weekly. For in vitro study, Nomo-1 cells were treated with GM6001 (50uM), a metalloprotease inhibitor, or DMSO control for 48 hr prior to evaluation of surface mesothelin by flow cytometry and soluble mesothelin in the culture supernatant by ELISA. Results: In vivo cytotoxicity of CAR T cells against Nomo-1 and Kasumi-1MSLN+ AML models demonstrated potent, target-dependent tumor killing. After 1- and 2-weeks post CAR T infusion, leukemic cells were eradicated in both Nomo-1 (p<0.0005, week 2, Figure 1A) and Kasumi-1 MSLN+ xenografts (p<0.005 at week 2, Figure 1B). Mesothelin undergoes shedding at the cell membrane as a result of ADAM17-mediated cleavage. Blocking ADAM17 activity with GM6001 in Nomo-1 cells led to increased cell surface mesothelin (Figure 1C) with a corresponding reduction in the shed form (Figure 1D), suggesting that GM6001 treatment stabilizes mesothelin on the cell surface. Furthermore, GM6001 treatment during co-culture of Nomo-1 and CAR T cells enhanced cytolytic activity of CAR T cells (Figure 1E). GM6001 treatment did not significantly impact cell viability of Nomo-1 cells in the absence of CAR T cells (data not shown). Conclusion: In this study, we demonstrate that mesothelin is a viable therapeutic target and a potential diagnostic biomarker in AML. We show that MSLN CAR T cells were highly effective in eliminating MSLN-positive AML cells in vitro and in vivo. Shedding contributes to the loss of mesothelin antigen and provides a source of soluble mesothelin that may interfere with antibody-based therapies, including CAR T cells. Modulating MSLN shedding by inhibiting ADAM17-mediated cleavage resulted in stabilized mesothelin and improved CAR T cell functionality. This work warrants further evaluation of MSLN CAR T cells to be tested in clinical trials for AML and demonstrates that inhibiting MSLN shedding is a promising approach to improve CAR T efficacy. Disclosures No relevant conflicts of interest to declare.

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Accurate Detection of Residual Leukemic Stem Cells In Remission Bone Marrow Predicts Relapse In Acute Myeloid Leukemia Patients

Blood ◽

10.1182/blood.v116.21.759.759 ◽

2010 ◽

Vol 116 (21) ◽

pp. 759-759 ◽

Cited By ~ 3

Author(s):

Monique Terwijn ◽

Arjo P Rutten ◽

Angèle Kelder ◽

Alexander N Snel ◽

Willemijn J Scholten ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Consolidation Therapy ◽

Leukemic Stem Cells ◽

Prognostic Information ◽

Induction Cycle ◽

Cd34 Expression ◽

Acute Myeloid

Abstract Abstract 759 Detecting leukemia initiating cells or leukemic stem cells (LSCs) in acute myeloid leukemia (AML) is a challenge. AML is a heterogeneous disease and since the first studies on LSCs, different immunophenotypic cell compartments have been reported to contain LSCs. For CD34 positive (CD34+) patients, the most important LSC compartment is most likely CD34+CD38-. However, both LSCs and normal hematopoietic stem cells (HSCs) reside in this compartment. Therefore, LSC detection requires the ability to accurately discriminate between LSCs and HSCs. Previous research showed that LSCs, in contrast to HSCs, expressed aberrant markers (AM, including lymphoid lineage markers and the LSC marker CLL-1). Although AM- cells are often presumed to be normal HSCs, AM expression can be absent or very weak on LSCs and AM- cells can therefore be malignant too. We previously described that in 30% of the patients, besides the AM expression, additional FACS parameter (forward scatter (FSC) and sideward scatter (SSC) and/or CD34 expression levels) could also be used to detect LSCs (Terwijn et al., ASH 2009 abstract 399). To prove that the CD34+CD38- fraction contains both normal and malignant SCs, and in addition, to prove that AM- cells can be malignant too, we studied molecular aberrancies (3 cases of FLT3-ITD positive AML) in the relevant cell fractions. 1: CD34+CD38-AM+ cells proved to be FLT3-ITD positive in all three cases. In addition, these cells initiated leukemic engraftment in NOD-SCID IL-2Rγ -/- mice in 2/2 cases. 2: The CD34+CD38-AM- cells with a FSC/SSC low or CD34 high character were FLT3-ITD negative in 3/3 cases and initiated multilineage engraftment in mice in 2/2 cases, indicating that these are HSCs. 3: The CD34+CD38-AM- cells with a FSC/SSC high or CD34 low phenotype were FLT3-ITD positive in 2/2 cases and therefore malignant, indicating that AM expression alone is not always sufficient in discriminating HSCs from LSCs. Since therapy surviving LSCs are thought to be responsible for relapses, we hypothesized that LSC frequency, as a fraction of total WBC, detected in remission bone marrow (BM) would provide prognostic information. Using AM as a primary gating strategy and, when applicable, FSC/SSC and/or CD34 expression, as a secondary gating step, LSC analysis was performed in 112 CD34+ AML patients (<60 years). In 76/112 patients, LSCs were detected using the most specific AM (CLL-1 (n=45), CD7 (n=19), CD11b (n=4), CD56 (n=3), CD22 (n=2), CD19 (n=2) and CD36 (n=1)) as a primary gating strategy. In 20 of these patients, a secondary gating step in FSC/SCC (n=16) and CD34 expression (n=4) refined the malignant population. In 36/112 patients, no AM was expressed on the CD34+CD38- cells, but in 25 cases, FSC/SSC could be used to gate the malignant fraction, in 6 cases combined with CD34 expression. Thus, in total, 101/112 patients were monitored for LSC frequency. After first cycle of chemotherapy, using a cut-off 5 × 10−6, patients with LSC frequency above cut-off (LSC+) showed shorter overall survival (OS, p=0.06) and especially relapse-free survival (RFS, p=0.001) as compared to LSC- patients. Median RFS was 11 months vs >36 months, respectively (fig A). Also after second induction cycle, high LSC frequency (>5 × 10−6) predicted shorter OS and RFS (both p<0.001). Median RFS of LSC+ patients was 8 months, that of LSC- patients >36 months (fig B). After consolidation therapy, 2 × 10−6 was the most optimal cut-off to define LSC+ and LSC- patients. OS and RFS were significantly shorter in LSC+ patients (p=0.037 and p=0.001, respectively), with a median RFS of 13 months vs. > 36 months (figure C). The relative risk of relapse was 5.0 (95% C.I 1.8–14.0) after first induction cycle, 4.7 (95% C.I.2.2-10.1) after second cycle and 8.5 (95% C.I 1.8– 41.4) after consolidation therapy. Multivariate analysis including WBC count, cytogenetic risk profile and the number of cycles needed to reach CR showed that LSC frequency is an independent factor for RFS (1st cycle p=0.01, 2nd cycle p<0.001, consolidation p=0.038). With this gating strategy, 90% of CD34+ AML patients could be monitored for residual LSCs, resulting in accurate prognostic information which is valuable for risk stratification-based treatment. In addition, the prospective isolation of LSCs and HSCs from diagnosis AML BM might identify more selective therapies that eradicate LSCs, while leaving HSCs intact. This work was supported by Netherlands Cancer Foundation KWF. Disclosures: No relevant conflicts of interest to declare.

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HOXB6 overexpression in murine bone marrow immortalizes a myelomonocytic precursor in vitro and causes hematopoietic stem cell expansion and acute myeloid leukemia in vivo

Blood ◽

10.1182/blood-2004-04-1583 ◽

2005 ◽

Vol 105 (4) ◽

pp. 1456-1466 ◽

Cited By ~ 82

Author(s):

Neal A. Fischbach ◽

Sofia Rozenfeld ◽

Weifang Shen ◽

Stephen Fong ◽

Daniel Chrobak ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Hox Genes ◽

Cooperative Interaction ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Acute Myeloid

AbstractThe HOX family of homeobox genes plays an important role in normal and malignant hematopoiesis. Dysregulated HOX gene expression profoundly effects the proliferation and differentiation of hematopoietic stem cells (HSCs) and committed progenitors, and aberrant activation of HOX genes is a common event in human myeloid leukemia. HOXB6 is frequently overexpressed in human acute myeloid leukemia (AML). To gain further insight into the role of HOXB6 in hematopoiesis, we overexpressed HOXB6 in murine bone marrow using retrovirus-mediated gene transfer. We also explored structure-function relationships using mutant HOXB6 proteins unable to bind to DNA or a key HOX-binding partner, pre–B-cell leukemia transcription factor-1 (PBX1). Additionally, we investigated the potential cooperative interaction with myeloid ecotropic viral integration site 1 homolog (MEIS1). In vivo, HOXB6 expanded HSCs and myeloid precursors while inhibiting erythropoiesis and lymphopoiesis. Overexpression of HOXB6 resulted in AML with a median latency of 223 days. Coexpression of MEIS1 dramatically shortened the onset of AML. Cytogenetic analysis of a subset of HOXB6-induced AMLs revealed recurrent deletions of chromosome bands 2D-E4, a region frequently deleted in HOXA9-induced AMLs. In vitro, HOXB6 immortalized a factor-dependent myelomonocytic precursor capable of granulocytic and monocytic differentiation. These biologic effects of HOXB6 were largely dependent on DNA binding but independent of direct interaction with PBX1.

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