Targeting IL3 Receptor in Chronic Myeloid Leukemia.

Abstract Abstract 2172 Poster Board II-149 Despite the great success of imatinib therapy in chronic myeloid leukemia (CML), the presence of a residual leukemic clone is detectable in a proportion of patients with CML. Further, patients with accelerated and blast phase of the disease respond poorly to imatinib. Imatinib and other potent tyrosine kinase inhibitors (TKIs) have limited activity against CD34+38- leukemic stem cells, necessitating the need for novel agents capable of eradicating highly resistant CML stem cells. Expression of IL3 receptor, CD123, was demonstrated on CD34+CD38- leukemic stem cells in AML (Jordan et al., Leukemia, 14: 1777, 2000) and CML (Neering et al., Blood, 110: 2578, 2007; Florian et al., Leuk Lymphoma, 47: 207, 2006) and has been shown to be an effective therapeutic target in pre-clinical AML models (Jin et al,,Cell Stem Cell, 5:31, 2009; Feuring-Buske et al., Cancer Res, 62: 1730, 2002). However, its role in CML stem cells has not been investigated. In this study, we examined expression of CD123 on CML progenitor cells and the therapeutic potential of the CD123 targeting agents, DT388IL3 and DT388K116W, both recombinant IL3-diphtheria toxin (DT) conjugates in in vitro and in vivo CML models. DT388IL3 has been shown to eradicate NOD/Scid-initiating AML stem cells and is currently undergoing Phase I/II clinical trials in AML and MDS. DT388K116W is a new DT fusion protein with high binding affinity to the IL3 receptor that demonstrated high potency anti-leukemic activity. These novel agents are directed to the leukemia stem cell surface, trigger receptor-mediated endocytosis, inhibit protein synthesis, and cause programmed cell death. In a series of nine primary CML samples (five from patients with chronic phase CML and four from patients in blast crisis), CD123 was expressed in 86%±5.7% of CD34+CD38- progenitor cells as determined by flow cytometry. Notably, 86%±3.4% of FACS-sorted CD34+38-123+ cells from 7 primary CML samples were Bcr-Abl(+) by fluorescent in situ hybridization analysis, confirming the leukemic origin of this cell population. We next examined the cytotoxic activity of DT-IL3 agents in KBM5 cells and in primary leukemic blasts. DT388K116W induced a dose-dependent decrease in viability and induction of apoptosis in KBM5 (44.6±4.3% apoptotic cells at 10μg/mL, p≤0.001) and in primary CML cells (69.5±15 % apoptosis, n=4, p=0.04) as determined by viable cell counts and annexin V flow cytometry at 72 hours. DT388K116W induced a greater degree of cell death compared to DT388IL3 in KBM5 cells (44.6% vs 21.3%, p=0.009). In two primary CML samples DT-IL3 agents reduced the absolute numbers of CD34+CD38-CD123+ cells by induction of apoptosis (DT388IL3, by 69% (sample#1) and 21% (sample#2); DT388K116W, by 71% and 62%, respectively). Importantly, combination of imatinib with DT-IL3 further enhanced the apoptotic rate in KBM5 (p=0.0001) and primary leukemic cells (n=3, p=0.035). To examine anti-leukemic activity of these agents in vivo, NOD/Scid/IL2Rγ-KO mice were transplanted with leukemic cells from primary myeloid blast crisis CML. After engraftment of the leukemic cells documented by CD45 flow cytometry in murine blood 20 days post transplantation, mice were left untreated or received 5-day intraperitoneal administration of DT388IL3 or DT388K116W at 0.2mg/kg. These IL3 receptor-targeted agents significantly prolonged survival of treated mice compared to vehicle control (median survival: vehicle= 37, DT388IL3 = 48, DT388K116W = 57 days; p= 0.0005) and reduced leukemia burden as detected by CD45 flow cytometry. These data indicate that the IL3 receptor is highly expressed on CD34+38- Bcr-Abl(+) CML stem cells and represents an exciting new and feasible target for therapeutic intervention. Moreover, DT-IL3 conjugates represent a novel therapeutic modality for selective targeting of highly resistant CML stem cells. DT-IL3 agents, alone or in combination with TKIs, might benefit CML patients by reducing/eliminating leukemic stem cells, a concept to be tested in the future clinical trials. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v120.21.413.413 ◽

2012 ◽

Vol 120 (21) ◽

pp. 413-413

Author(s):

Alissa R. Kahn ◽

Kimberly A. Hartwell ◽

Peter G. Miller ◽

Benjamin L. Ebert ◽

Todd R. Golub ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Cord Blood ◽

Leukemic Cells ◽

Leukemic Stem Cells ◽

Minimal Toxicity ◽

Nsg Mice ◽

Cd34 Cells

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

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Cytosine Arabinoside Chemotherapy Does Not Enrich For Leukemic Stem Cells In Xenotransplantation Model Of Human Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.1651.1651 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1651-1651 ◽

Cited By ~ 2

Author(s):

Fabienne de Toni ◽

Robin L. Perry ◽

Estelle Saland ◽

Mayumi Sugita ◽

Marion David ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Leukemic Cells ◽

Leukemic Stem Cells ◽

Frequent Relapses ◽

Cell Cycle Status ◽

Acute Myeloid

Abstract Despite a high rate of complete remission after treatment with conventional genotoxic agents, the overall survival of patients with acute myeloid leukemia (AML) is poor due to frequent relapses caused by the chemoresistance of rare leukemic stem cells (LSCs, also called Scid-Leukemia Initiating Cells). This unfavorable situation leads to a strong need to characterize those cells in order to target them with new specific therapies. Using a robust immunodeficient mouse model (NOD/LtSz-scid IL2Rγchainnull or NSG), we have previously shown that these LSCs were rare and not restricted to the CD34+CD38- immature compartment. This phenotypical heterogeneity of LSCs suggests that pharmacological targeting of LSC will not work if solely based on their cell surface markers. A better understanding of the mechanisms underlying the in vivo chemoresistance is required for the development of innovative targeted therapies. Aracytine (Ara-C, a pyrimidine analog), the most clinically used chemotherapeutic agents for AML patients, inhibits DNA synthesis and, therefore, targets and kills cycling AML cells in S phase of the cell cycle. Based on this mechanism of action, we hypothesized that Ara-C treatment will spare and enrich quiescent LSCs in vivo. We analyzed the response to Ara-C and residual disease in NSG mice engrafted with primary AML cells from 13 patients in two clinical centers (University of Pennsylvania, Philadelphia, USA and Purpan Hospital, Toulouse, France). A sub-lethal treatment of 60 mg/kg Ara-C given daily for five days induced a 5- to 50- fold reduction of peripheral blood blasts and total tumor burden in spleen and bone marrow in all patients tested. For 5 patients, we observed relapse within 4 to 6 weeks post-chemotherapy. Surprisingly, residual viable cells after Ara-C treatment showed no significant enrichment in quiescent cells and CD34+CD38- cells for the majority of primary samples tested (12 and 10 out of 13 total tested, respectively). Of note, the largest fraction (70-90%) of leukemic cells is in G0/G1 phase (including 0.5-20% in G0) in untreated engrafted mice. Moreover, we observed no significant changes in cell cycle profile of residual leukemic cells during the time course of the disease progression for 3 out of 4 patients. Finally, we assessed the frequency of LSCs in Ara-C-treated and control mice using transplantation and limiting dilution analysis in secondary recipients. Interestingly, we observed that Ara-C treatment did not increase the frequency of SL-ICs in residual cells, suggesting that blasts and LSC were equally sensitive to Ara-C in vivo. Our results show that sub-lethal regimen of Ara-C does not lead to enrichment of LSCs and induces cell death of both leukemic bulk and stem/progenitor cells independently of their cell cycle status probably through another in vivo mechanism such as apoptosis, autophagy or necroptosis. This study also suggests that further characterization of chemoresistant leukemic cells beyond phenotype and cell cycle status must rely on more functional properties in order to better elucidate the molecular basis of resistance in AML. Disclosures: Perry: MERCK: Employment. Carroll:Leukemia and Lymphoma Society: Research Funding. Sarry:AFFICHEM SA: Membership on an entity’s Board of Directors or advisory committees.

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871 Construction and evaluation of interleukin 3 (IL3)-zetakine redirected cytolytic T Cells for the treatment of CD123 expressing acute myeloid leukemia

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.871 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A912-A912

Author(s):

Rebecca Moeller ◽

Julian Scherer ◽

Sadik Kassim

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Myeloid Leukemia ◽

Cell Activation ◽

Jurkat Cells ◽

Leukemic Stem Cells ◽

Cd69 Expression ◽

Acute Myeloid

BackgroundAcute Myeloid Leukemia (AML) is an aggressive bone marrow malignancy, characterized by the presence of leukemic blasts in the peripheral blood of patients. Poor AML prognoses1 are largely attributable to high rates of disease relapse, of which CD123+ leukemic stem cells (LSCs) are the primary cause.2 3 CD123, the alpha-chain of the IL3 cytokine receptor,6 has been identified as a favorable therapeutic AML target, overexpressed in both LSCs and blasts.4 5 We sought to direct T cells to CD123+ AML cells via cell surface tethered IL3 (termed ”IL3-zetakine”).7 The use of a zetakine instead of a chimeric antigen receptor (CAR) construct enables structure-guided site-directed mutagenesis to increase binding affinity and alter target cell signaling without detrimental T cell hyperactivation.MethodsZetakine constructs were designed using IL3 sequences bound to a transmembrane domain and intracellular costimulatory and CD3z signaling domains. The constructs were transduced into Jurkat cells with lentiviral vectors (LVV). T cell activation via CD69 expression was assessed via flow cytometry of sorted IL3 zetakine-positive Jurkat cells after co-culture with MOLM13 AML cells. Lead constructs were selected based on initial transduction percentage and activation response. In vitro functionality of each IL3 zetakine was tested with LVV transduced primary T cells by flow cytometry.ResultsZetakine constructs yielded a wide range of transduction percentages in Jurkat cells (0 – 98%) prior to sorting. In co-cultures with CD123+ MOLM13 AML cells, Jurkat cells expressing wildtype IL3 constructs lacking a costimulatory domain induced the highest level of CD69 expression (18.7% CD69+ T cells) in an antigen-specific manner (5.3-fold increase of CD69+ T cells over those cultured with MOLM13 CD123KO cells). The K110E mutant IL3 was reported to exhibit a 40-fold increased affinity over wildtype,8 but it showed no detectable zetakine function. However, additional mutant IL3 zetakines increased Jurkat cell activation up to 5.8-fold. Antigen-specific increases in CD69, as well as CD25, surface expression were also observed with zetakine-transduced primary T cells co-cultured with MOLM13 cells, in addition to target cell killing comparable to antibody-based CD123CAR T-cells.ConclusionsThis work establishes IL3 zetakines as a viable alternative to traditional CD123-targeted CAR constructs. Structure-guided IL3 zetakine mutants with altered affinity and activation profiles will further our understanding of CD123-specific cytotoxicity modulation without inducing acute T cell hyperactivation and exhaustion. These results indicate the ability of IL3 zetakine-expressing T cells to kill CD123-expressing AML cells and illustrate the potential of this novel class of therapeutics.ReferencesGanzel C, et al. Very poor long-term survival in past and more recent studies for relapsed AML patients: the ECOG-ACRIN experience. American journal of hematology 2018:10.1002/ajh.25162.Shlush LI, et al. Tracing the origins of relapse in acute myeloid leukaemia to stem cells. Nature 2017;547(7661):104–108.Hanekamp D, Cloos J, Schuurhuis GJ. Leukemic stem cells: identification and clinical application. International Journal of Hematology 2017;105(5):549–557.Bras AE, et al. CD123 expression levels in 846 acute leukemia patients based on standardized immunophenotyping. Cytometry part B: Clinical Cytometry 2019;96(2):134–142.Sugita M, Guzman ML. CD123 as a therapeutic target against malignant stem cells. Hematology/Oncology clinics of North America 2020;34(3):553–564.Mingyue S, et al. CD123: a novel biomarker for diagnosis and treatment of leukemia. Cardiovascular & Hematological Disorders-Drug Targets 2019;19(3):195–204.Kahlon KS, et al. Specific recognition and killing of glioblastoma multiforme by interleukin 13-zetakine redirected cytolytic T cells. Cancer Res 2004;64(24):9160–6.Bagley CJ, et al. A discontinuous eight-amino acid epitope in human interleukin-3 binds the alpha-chain of its receptor. J Biol Chem 1996;271(50):31922–8.

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E4F1 deficiency results in oxidative stress–mediated cell death of leukemic cells

Journal of Experimental Medicine ◽

10.1084/jem.20101995 ◽

2011 ◽

Vol 208 (7) ◽

pp. 1403-1417 ◽

Cited By ~ 15

Author(s):

Elodie Hatchi ◽

Genevieve Rodier ◽

Matthieu Lacroix ◽

Julie Caramel ◽

Olivier Kirsh ◽

...

Keyword(s):

Cell Death ◽

Cell Lines ◽

Myeloid Leukemia ◽

Tumor Regression ◽

Fetal Liver ◽

Leukemia Cell ◽

Leukemic Cells ◽

Viral Oncoprotein ◽

Mitochondrial Defects

The multifunctional E4F1 protein was originally discovered as a target of the E1A viral oncoprotein. Growing evidence indicates that E4F1 is involved in key signaling pathways commonly deregulated during cell transformation. In this study, we investigate the influence of E4F1 on tumorigenesis. Wild-type mice injected with fetal liver cells from mice lacking CDKN2A, the gene encoding Ink4a/Arf, developed histiocytic sarcomas (HSs), a tumor originating from the monocytic/macrophagic lineage. Cre-mediated deletion of E4F1 resulted in the death of HS cells and tumor regression in vivo and extended the lifespan of recipient animals. In murine and human HS cell lines, E4F1 inactivation resulted in mitochondrial defects and increased production of reactive oxygen species (ROS) that triggered massive cell death. Notably, these defects of E4F1 depletion were observed in HS cells but not healthy primary macrophages. Short hairpin RNA–mediated depletion of E4F1 induced mitochondrial defects and ROS-mediated death in several human myeloid leukemia cell lines. E4F1 protein is overexpressed in a large subset of human acute myeloid leukemia samples. Together, these data reveal a role for E4F1 in the survival of myeloid leukemic cells and support the notion that targeting E4F1 activities might have therapeutic interest.

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The Target Receptor Siglec-3 (CD33) Is Expressed on AML Stem Cells in a Majority of All Patients with AML.

Blood ◽

10.1182/blood.v106.11.4324.4324 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4324-4324

Author(s):

Alexander W. Hauswirth ◽

Stefan FLorian ◽

Maria-Theresa Krauth ◽

Gerit-Holger Schernthaner ◽

Edgar Selzer ◽

...

Keyword(s):

Stem Cells ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Staining Technique ◽

Scid Mice ◽

Leukemic Cells ◽

Cytostatic Drug ◽

Leukemic Stem Cells ◽

Targeting Therapy ◽

Acute Myeloid

Abstract The cell surface antigen Siglec-3 = CD33 is becoming increasingly important as target of therapy in acute myeloid leukemia (AML). In particular, a conjugate consisting of the humanized CD33 antibody P67.6 (gemtuzumab) and the cytostatic drug calicheamicin has been developed for clinical use and was found to work as an effective antileukemic agent (Mylotarg®) in patients with CD33+ AML. In normal myelopoiesis, expression of CD33 is restricted to advanced stages of differentiation, whereas primitive stem cells do not express CD33 (Siglec-3). In line with this notion, CD33-targeting therapy is a non-myeloablative approach. In the present study, we asked whether leukemic stem cells in patients with AML express CD33. For this purpose, a multicolor-staining technique was applied in eleven patients with AML. Leukemic stem cells were defined as CD34+/CD38−/CD123+ cells. In all patients in whom the majority of myeloblasts expressed CD33 (=CD33+ AML, n=8), the AML progenitor cells reacted with the CD33 antibody P67.6. Repopulation experiments utilizing NOD/SCID mice confirmed that the AML stem cells in these patients reside within the CD33+ subpopulation of leukemic cells. Moreover, AML stem cells (CD34+/CD38−/CD123+ cells) highly purified (>98% purity) from patients with (CD33+) AML by cell sorting, were found to express CD33 mRNA in RT-PCR analyses. To demonstrate that AML stem cells can also reside within the CD33-negative fraction of the AML clone, we purified CD33-negative cells in a patient with AML in whom a majority of leukemic stem cells were found to lack CD33. In this particular patient, the CD33-negative cells were found to repopulate NOD/SCID mice with leukemias in the same way as the entire leukemic clone did. The CD33 antigen was neither detectable on CD34+/CD38− cells in the normal bone marrow nor on leukemic stem cells in patients with CD33-negative AML. In summary, our data show that leukemic stem cells in patients with CD33+ AML frequently express the target receptor CD33. This observation is in favor of novel treatment concepts employing CD33-targeting antibodies (Mylotarg®) in acute myeloid leukemia.

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The Transcription Factor ELF4 Promotes Survival of Myeloid Leukemic Stem Cells.

Blood ◽

10.1182/blood.v116.21.1209.1209 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1209-1209

Author(s):

Chun Shik Park ◽

Koramit Suppipat ◽

H. Daniel Lacorazza

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Blast Crisis ◽

Philadelphia Chromosome ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Leukemic Stem Cells ◽

Hematopoietic Stem

Abstract Abstract 1209 Chronic myeloid leukemia (CML) is a myeloproliferative disease that originate in hematopoietic stem cells (HSCs) as a result of the t(9;22) translocation, giving rise to the Ph (Philadelphia chromosome) and BCR-ABL oncoprotein. Although treatment of CML patients with tyrosine kinase inhibitor can efficiently eliminate most leukemic cells, chemoresistant leukemic stem cells (LSCs) can survive and drive recurrence of CML in these patients. A number of genes have been described to promote or inhibit proliferation of LSCs. Some of them have similar roles in normal HSCs. The transcription factor ELF4 promotes cell cycle entry of quiescent HSCs during homeostasis (Lacorazza et al., 2006). Thus, to investigate the function of ELF4 in CML initiation and maintenance, we developed a BCR-ABL-induced CML-like disease using retroviral transfer of BCR-ABL in Elf4-null bone marrow (BM) cells. We first investigated whether ELF4 is required for the induction of CML. Recipient mice of BCR-ABL-transduced WT BM cells developed CML and died with a latency 16–23 days, whereas recipient mice of BCR-ABL-transduced Elf4-/- BM cells showed longer latency of 45–47 days (n=20; p<0.0005). Progression of leukemia was monitored in peripheral blood, BM and spleen by flow cytometry. In mice transplanted with BCR-ABL-transduced Elf4-null BM cells, Gr-1+ leukemic cells expanded the first two weeks after BM transplantation followed by a decline at expense of a secondary expansion of B220+ cells. In contrast, Gr-1+ leukemic cells continuously expanded in mice receiving BCR-ABL-transduced WT BM cells. These results suggest that loss of ELF4 causes a profound abrogation in BCR-ABL-induced CML, while allowing progression of B-cell acute lymphocytic leukemia. Since loss of Elf4 led to impaired maintenance of myeloid leukemic cells, we postulated that ELF4 may affect survival of LSCs. Thus, we analyzed the frequency of Lin-c-Kit+Sca-1+ (LSK) cells that are BCR-ABL positive in BM and spleen. We found that BCR-ABL+ LSK cells were significantly reduced in recipients of BCR-ABL-transduced Elf4-/- BM cells. These studies indicate that ELF4 is essential to maintain the LSC pool in CML acting as a molecular switch between myeloid and lymphoid blast crisis. Disclosures: No relevant conflicts of interest to declare.

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Treatment with 8F4, An Anti-PR1/HLA-A2 T Cell Receptor-Like Antibody, Reduces Established Acute Myeloid Leukemia and Eliminates Leukemia Stem Cells in Mice

Blood ◽

10.1182/blood.v118.21.766.766 ◽

2011 ◽

Vol 118 (21) ◽

pp. 766-766

Author(s):

Anna Sergeeva ◽

Hong He ◽

Kathryn Ruisaard ◽

Karen Clise-Dwyer ◽

Lisa S St. John ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Cell Receptor ◽

Leukemia Stem Cells ◽

Nsg Mice ◽

Secondary Transfer

Abstract Abstract 766 PR1 (VLQELNVTV) is an HLA-A2-restricted leukemia-associated peptide from proteinase 3 and neutrophil elastase that is recognized by PR1-specific cytotoxic T lymphocytes that contribute to cytogenetic remission of myeloid leukemia. We developed a high affinity T cell receptor (TCR)-like mouse monoclonal antibody (8F4) that binds to a conformational epitope of the PR1/HLA-A2 complex. Flow cytometry and confocal microscopy of 8F4-labeled cells showed significantly higher PR1/HLA-A2 expression on AML blasts compared with normal leukocytes. Moreover, 8F4 mediated complement dependent cytolysis of AML blasts and Lin−CD34+CD38− leukemia stem cells (LSC), but not normal leukocytes. To investigate in vivo biological effects 8F4 on established leukemia, we established xenografts of primary human HLA-A2-positive AML in sublethally irradiated NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Leukemia engraftment was monitored in peripheral blood by flow cytometry. Mice with established PR1/HLA-A2-expressing leukemia were treated with twice-weekly intravenous injections of 200 μg 8F4 or isotype control antibody. Flow cytometry and histology analysis of tissues was used to assess leukemia burden and level of engraftment. After 5 weeks of treatment AML was reduced 300-fold in bone marrow of 8F4-treated mice compared to isotype-treated control animals (0.07 ± 0.06% hCD45+cells versus 20.4 ± 4.1%, n=5 mice per group). Moreover, leukemia stem cells (LSC, CD34+CD38−Lin-) were no longer detected in bone marrow of 8F4-treated mice, compared to 0.88 ± 0.24% in isotype-treated mice. Equally, AML was evident in the liver and spleen of isotype-treated mice (1.1 ± 0.16% and 0.32 ± 0.17%, respectively), but was undetectable in 8F4-treated mice (p<0.001). Similar results were obtained with AML from two additional patients, one with secondary AML (CMML) and one with AML-M7. Bone marrow contained 6.2 ± 3.0% (n=3) AML versus 41 ± 15% (n=2 mice; p=0.06) in the first case and 0.16 (n=1) versus 7.0 ± 4.1 (n=2) in the second case after 2–3 weeks of twice-weekly injection. To confirm 8F4-mediated elimination of LSC, we performed secondary transfer experiment with 1×106 bone marrow cells from 8F4- and isotype-treated mice, transplanted into recipient NSG mice, irradiated with 250 cGy. AML was undetectable in mice that received bone marrow from 8F4-treated animals versus 4.1 ± 2.4% (n=4) in bone marrow of mice that received cells from isotype- treated mice, determined at 16 weeks after secondary transfer. Because PR1/HLA-A2 expression on normal hematopoietic cells (HSC) is similar to LSC in AML patients, we sought to determine whether 8F4 treatment of NSG mice xenografted with CD34-selected umbilical cord blood resulted in elimination of xenograft. Fourteen weeks after transplant stable chimerism (4.1 - 7.7% hCD45+ cells) was established, mice were treated with 50 μg 8F4 intravenously and peripheral blood was monitored weekly for chimerism. Human CD45+ cells decreased to 0.35 – 0.95% by week 1, but increased to 1.9 – 2.1 % hCD45+ cells at week 3. Bone marrow at week three contained myeloid (CD13+CD33+) and lymphoid (CD19+) cells showing that while 8F4 has off- target effects against normal hematopoietic cells, HSC are preserved. This is consistent with our previous studies that showed no 8F4-mediated effect on colony formation of normal bone marrow cells. In conclusion, these results show that anti-PR1/HLA-A2 monoclonal antibody 8F4 is biologically active in vivo and selectively eliminates LSC, but not normal HSC. This justifies continued study of 8F4 as a novel therapy for AML. Disclosures: No relevant conflicts of interest to declare.

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Targeting CD96 For Antibody Based Elimination Of Leukemic Stem Cells In AML: A New Strategy In Stem Cell Transplantation

Blood ◽

10.1182/blood.v122.21.3972.3972 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3972-3972 ◽

Cited By ~ 2

Author(s):

Matthias Staudinger ◽

Christian Kellner ◽

Matthias Peipp ◽

Natalie Schub ◽

Andreas Humpe ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Autologous Stem Cell Transplantation ◽

Target Cells ◽

Leukemic Stem Cells ◽

Hematopoietic Stem

Abstract Although the mortality of autologous stem cell transplantation in contrast to allogeneic is low, in AML patients the lack of immune surveillance as well as contamination of the transplant with residual leukemic stem cells (LSC) limits its use. Therefore, elimination of LSC by targeted therapy may represent a promising therapeutic approach. Recently, CD96 was identified as marker antigen on AML-LSC (Hosen et al., PNAS 104: 11008, 2007). Here, by addressing CD96 with magnetic cell sorting (MACS) or using antibody dependent cellular cytotoxicity (ADCC), new strategies for engineering autologous stem cell grafts or for in vivo targeting of residual AML stem cells are presented. To evaluate the efficacy of depletion of LSC by MACS technology, grafts containing hematopoietic stem cells were spiked with CD96 positive AML cells. Using biotinylated CD96 antibody TH111 raised in our laboratory in combination with anti-biotin-micro beads (Miltenyi Biotech, Bergisch Gladbach, Germany) up to a 1000-fold depletion of targeted cells was achieved. The viability, cell count and the potential of hematopoietic progenitor cells (HPC) to proliferate and differentiate were not affected by this procedure as documented by flow cytometry and colony forming assays. As residual LSC residing within the patient may also account for AML relapse after high-dose chemotherapy and subsequent SCT, eradication of AML stem cells in vivo is desirable. To target CD96+ AML-LSC by ADCC, chimeric antibodies containing wild type or affinity maturated variable regions in combination with an optimized human IgG1Fc were generated by recombinant DNA technologies. Both recombinant antibodies were expressed in Hek 293 cells enriched to homogeneity by affinity chromatography and analyzed for their functional properties. As shown by flow cytometry, the antigen binding affinity of the maturated antibody was enhanced (EC50 0.6 μg/ml vs. 2 μg/ml). Moreover, as analyzed in standard ADCC assays, NK cell mediated lytic properties against CD96-positive target cells were elevated (maximum lysis: 52%) using the affinity maturated chimeric CD96 antibody (EC50: 0.02 μg/ml vs. 0.15 μg/ml). Thus, this CD96 purging strategy avoids unwanted transplantation of AML-LSC and may help to revitalize autologous stem cell transplantation in this indication. Although, specific side effects by CD96 application will have to be considered, this may allow for an additional therapeutic avenue to eliminate in vivo residual AML-LSC in autologous as well as in allogeneic situations. Disclosures: No relevant conflicts of interest to declare.

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Autophagy Promotes the Survival and Therapy Resistance of Human Acute Myeloid Leukemia Cells Under Hypoxia

Blood ◽

10.1182/blood.v124.21.2236.2236 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2236-2236 ◽

Cited By ~ 1

Author(s):

Dirkje W Hanekamp ◽

Megan K Johnson ◽

Scott Portwood ◽

Joshua Acklin ◽

Eunice S. Wang

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Acute Myeloid Leukemia ◽

Tyrosine Kinase Inhibitor ◽

Myeloid Leukemia ◽

Kinase Inhibitor ◽

Single Agent ◽

Xenograft Models ◽

Acute Myeloid

Abstract Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy occurring primarily in older adults. Despite high remission rates following upfront therapy, the disease eventually recurs in most patients, and overall cure rates remain only 20-30%. Preclinical studies have recently demonstrated that the marrow microenvironment in acute leukemic hosts to be intrinsically hypoxic, with AML progression associated with further hypoxia. Moreover, human AML cells and primary AML colonies cultured under hypoxia are markedly less sensitive to cytarabine chemotherapy than normoxic cells. We hypothesized that AML cells may respond to hypoxic stress and mediate chemoresistance in part by invoking autophagy, a highly regulated catabolic process by which cells evade apoptosis by degrading damaged cellular components. To test our hypothesis, we investigated the effects of two known autophagy inhibitors (bafilomycin A1 (Baf) and chloroquine (CQ)) on the sensitivity of human AML cells to various therapeutic agents under differing oxygen levels. Methods: We treated HEL (FLT-3 wildtype) and MV4-11 (FLT-3 ITD mutant) AML cells with autophagy inhibitors (Baf and CQ) alone and in combination with a chemotherapeutic drug (cytarabine (AraC), doxorubicin (Dox), decitabine (Dac)) or a tyrosine kinase inhibitor (sorafenib, SFN) under normoxic (21% O2) or hypoxic (1% O2) conditions. Apoptosis /cell death and proliferation were measured by flow cytometry for Annexin-PI and MTT assays, respectively. Autophagy was assessed by flow cytometry using Cyto-ID Green Dye (Enzo Life Sciences), fluorescent microscropy for acridine orange dye accumulation, and western blot analysis. Results: Autophagy in human ALL and AML cell lines was significantly increased following 24-72 hours of hypoxia (1% O2) as compared with normoxia and was a relatively late response to prolonged low oxygen levels (> 24 hours). Treatment with cytotoxic agents (AraC or Dox) or hypomethylating agent (Dac) resulted in a dose-dependent increases in the number of autophagic vesicles in AML cells consistent with autophagy induction. Low-doses of Baf which selectively inhibits the vacuolar H+ ATPase to prevent lysosomal acidification, and CQ, which blocks lysosome-autophagosome fusion by raising the pH of lysosomes and endosomes, both resulted in buildup of autophagic vesicles by flow cytometry consistent with inhibition of autophagic flux in human AML cells. Combination treatment with an autophagy inhibitor (Baf, CQ) and cytotoxic chemotherapy (AraC, Dox) significantly enhanced apoptosis and cell death over single agent therapy. Treatment with Baf combined with hypomethylating therapy (Dac) synergistically improved the anti-leukemic effects as compared with monotherapy (CI 0.09-0.31)(see Figure). The addition of Baf also improved cell death induced by sorafenib (SFN) on FLT-3 ITD mutant human AML cells (MV4;11) (CI 0.36-0.9) (see Figure). Single agent Baf or CQ treatment resulted in significantly higher levels of apoptosis and cell death in AML cells under hypoxia. The anti-tumor activity of almost all combination regimens was consistently improved under hypoxic versus normoxic culture conditions. In vivo CQ treatment (25-50 mg/kg i.p. daily) in preclinical human AML xenograft models significantly inhibited systemic leukemia progression as a single agent. Further experiments investigating the in vivo effects of CQ combined with other chemotherapeutic agents in preclinical AML xenograft models are ongoing. Conclusions: Our data suggest that human AML cells preferentially induce autophagy to promote survival under chronic hypoxia and following cytotoxic, hypomethylating, and FLT-3 tyrosine kinase inhibitor therapy. Strategies targeting autophagy therefore may have the potential to improve therapeutic responses and overcome chemoresistance of AML cells within the hypoxic bone marrow microenvironment. Figure 1 Figure 1. Figure 2 Figure 2. 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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