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

Blood ◽  
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.

High-level ectopic HOXB4 expression confers a profound in vivo competitive growth advantage on human cord blood CD34+ cells, but impairs lymphomyeloid differentiation

Blood ◽  
2003 ◽  
Vol 101 (5) ◽  
pp. 1759-1768 ◽  
Author(s):  
Bernhard Schiedlmeier ◽  
Hannes Klump ◽  
Elke Will ◽  
Gökhan Arman-Kalcek ◽  
Zhixiong Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Therapeutic Window ◽  
Hematopoietic Stem ◽  
Expression Levels ◽  
Cd34 Cells ◽  
Cord Blood Cd34 ◽  
The Impact

Ectopic retroviral expression of homeobox B4 (HOXB4) causes an accelerated and enhanced regeneration of murine hematopoietic stem cells (HSCs) and is not known to compromise any program of lineage differentiation. However, HOXB4 expression levels for expansion of human stem cells have still to be established. To test the proposed hypothesis that HOXB4 could become a prime tool for in vivo expansion of genetically modified human HSCs, we retrovirally overexpressed HOXB4 in purified cord blood (CB) CD34+ cells together with green fluorescent protein (GFP) as a reporter protein, and evaluated the impact of ectopic HOXB4 expression on proliferation and differentiation in vitro and in vivo. When injected separately into nonobese diabetic–severe combined immunodeficient (NOD/SCID) mice or in competition with control vector–transduced cells, HOXB4-overexpressing cord blood CD34+ cells had a selective growth advantage in vivo, which resulted in a marked enhancement of the primitive CD34+ subpopulation (P = .01). However, high HOXB4 expression substantially impaired the myeloerythroid differentiation program, and this was reflected in a severe reduction of erythroid and myeloid progenitors in vitro (P < .03) and in vivo (P = .01). Furthermore, HOXB4 overexpression also significantly reduced B-cell output (P < .01). These results show for the first time unwanted side effects of ectopic HOXB4 expression and therefore underscore the need to carefully determine the therapeutic window of HOXB4 expression levels before initializing clinical trials.

Co-Stimulatory Blockade With CTLA4-Ig Permits Transplantation Of Human Hematopoietic Stem Cells and HLA Incompatible T Cells In NOD/SCID γ Null (NSG) Mouse Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1999-1999
Author(s):  
Annie L. Oh ◽  
Dolores Mahmud ◽  
Benedetta Nicolini ◽  
Nadim Mahmud ◽  
Elisa Bonetti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without T cells as a control. No xenogeneic GVHD was detected at 1:1 CD34:T cell ratio. Engraftment of human CD45+ (huCD45+) cells in mice marrow and spleen was analyzed by flow cytometry. Marrow engraftment of huCD45+ cells at 4 or 8 weeks was significantly decreased in mice transplanted with T cells compared to control mice that did not receive T cells. More importantly, transplantation of T cells at CD34:T cell ratios from 1:50 to 1:0.5 resulted in stem cell rejection since >98% huCD45+ cells detected were CD3+. In mice with stem cell rejection, human T cells had a normal CD4:CD8 ratio and CD4+ cells were mostly CD45RA+. The kinetics of human cell engraftment in the bone marrow and spleen was then analyzed in mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio and sacrificed at 1, 2, or 4 weeks. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb, New York, NY) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. Treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13± 7% engraftment of huCD45+ marrow cells (n=5) which included: 53±9% CD33+ cells, 22±3% CD14+ monocytes, 7±2% CD1c myeloid dendritic cells, and 4±1% CD34+ cells, while CD19+ B cells were only 3±1% and CD3+ T cells were 0.5±1%. We hypothesize that CTLA4-Ig may induce the apoptotic deletion of alloreactive T cells early in the post transplant period although we could not detect T cells in the spleen as early as 7 or 10 days after transplant. Here we demonstrate that costimulatory blockade with CTLA4-Ig at the time of transplant of human CD34+ cells and incompatible allogeneic T cells can prevent T cell mediated rejection. We also show that the NSG model can be utilized to test immunotherapy strategies aimed at engrafting human stem cells across HLA barriers in-vivo. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia. Disclosures: No relevant conflicts of interest to declare.

The Pan-Bcl-2 Family Inhibitor 97C1 Targets Blast Crisis Chronic Myeloid Leukemia Stem Cells but Spares Normal Cord Blood Progenitor Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 516-516 ◽  
Author(s):  
Daniel Goff ◽  
Alice Shih ◽  
Angela Court Recart ◽  
Larisa Balaian ◽  
Ryan Chuang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Blast Crisis ◽  
Chemotherapy Resistance ◽  
Chronic Phase ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 516 Introduction: Several studies have demonstrated the role of leukemia stem cells (LSC) in the development and maintenance of human chronic myeloid leukemia (CML). These cells, which first develop in chronic phase CML (CP CML) with acquisition of the BCR-ABL fusion protein, are often quiescent and can be highly resistant to apoptosis induced by drugs and radiotherapy that target rapidly dividing cells. Data has also shown that CML LSC become increasingly resistant to BCR-ABL inhibition with progression to blast crisis CML (BC CML). Bcl-2 family proteins are key regulators of apoptosis and have been shown by numerous studies to regulate cancer resistance to chemotherapy. This family of proteins has also been implicated in the development of BC CML, however most studies have focused on CML cell lines and their expression of Bcl-2 family proteins in vitro. Thus, there is relatively little data on expression of Bcl-2 family proteins in primary CML LSC and on the role of these proteins in regulating chemotherapy resistance in CML LSC in vivo. As Bcl-2 family proteins are known regulators of chemotherapy resistance we hypothesized that human BC CML LSC may overexpress these proteins compared to normal hematopoietic stem cells. We analyzed Bcl-2 family mRNA and protein expression in CP CML and BC CML LSC and compared this expression to normal cord blood stem and progenitor cells. We also analyzed whether these cells were sensitive to chemotherapy treatment in vitro. Finally, we tested whether a high potency pan-Bcl-2 inhibitor, 97C1, could effectively kill CML LSC in vitro and in vivo. Methods: Bcl-2 and Mcl-1 protein expression was measured in primary CP CML, BC CML, and normal cord blood cells using intracellular FACS. We also measured Bcl-2, Mcl-1, Bcl-X, and Bfl-1 mRNA expression in FACS sorted CD34+CD38+lin− cells (LSC) from these samples. For all drug studies we used either serially transplanted CD34+ cells derived from primary BC CML patient samples or primary CD34+ normal cord blood cells. In vitro drug responses were tested by culturing CD34+ cells either alone or in co-culture with a mouse bone marrow stromal cell line (SL/M2). Effects on colony formation and replating were also tested by culturing sorted CD34+CD38+lin− cells in methylcellulose in the presence and absence of drug. For in vivo testing of 97C1 we transplanted neonatal RAG2-/-yc-/- mice with CD34+ cells from 3 different BC CML and cord blood samples. Transplanted mice were screened for peripheral blood engraftment at 6–8 weeks post-transplant and engrafted mice were then treated for 2 weeks with 97C1 by IP injection. Following the treatment period the mice were sacrificed and hemotapoietic organs were analyzed for human engraftment by FACS. Results: BC CML progenitors expressed higher levels of Bcl-2 and Mcl-1 protein compared to normal cord blood and chronic phase CML cells. mRNA expression of Mcl-1, Bcl-X, and Bfl-1 was also increased in BC CML progenitors compared to CP CML progenitors. While BC CML LSC cultured in vitro were resistant to etoposide and dasatinib-induced cell death, 97C1 treatment led to a dose-dependent increase in cell death along with a dose-dependent decrease in the frequency of CD34+CD38+lin− cells compared to vehicle treated controls. While cord blood progenitor cells were also sensitive to 97C1 treatment they had an IC50 around 10 times higher than that for the BC CML cells (100nM versus 10nM). Importantly, 97C1 treatment did not inhibit cord blood colony formation or colony replating in vitro. Mice transplanted with BC CML LSC developed CML in 6–8 weeks post-transplant with diffuse myeloid sarcomas and engraftment of human CD34+CD38+lin− cells in the peripheral blood, liver, spleen, and bone marrow. In vivo treatment with 97C1 led to a significant reduction in both total human engraftment and engraftment of CD34+CD38+lin− cells in all hematopoietic organs analyzed. Conclusion: Our results demonstrate that BC CML LSC are resistant to conventional chemotherapy but are sensitive to 97C1 in vitro and in vivo. Broad-spectrum inhibition of Bcl-2 family proteins may help to eliminate CML LSC while sparing normal hematopoietic stem and progenitor cells. Disclosures: Jamieson: CoronadoBiosciences: Research Funding; CIRM: Research Funding.

IL1RAP Antibodies Block IL1-Induced Expansion of Primitive CML Cells and Display Therapeutic Effects in Xenograft Models

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1118-1118
Author(s):  
Helena Ågerstam ◽  
Nils Hansen ◽  
Sofia Von Palffy ◽  
Carl Sandén ◽  
Kristian Reckzeh ◽  
...  
Keyword(s):  
Stem Cells ◽  
Research Funding ◽  
Effector Cell ◽  
Leukemic Cells ◽  
Nsg Mice ◽  
Xenograft Models ◽  
Equity Ownership ◽  
In Vivo Effects

Abstract Chronic myeloid leukemia (CML) is currently treated with tyrosine kinase inhibitors (TKIs) but these do not effectively eliminate the CML stem cells. As a consequence, CML stem cells persist and cause relapse in most patients upon drug discontinuation. Furthermore, no effective therapy exists for the advanced stages of the disease. Thus, there is still a need for novel treatment strategies in CML. We have previously shown that Interleukin 1 receptor accessory protein (IL1RAP), a co-receptor of IL1R1, is highly expressed on primitive CML cells and that a polyclonal IL1RAP antibody can direct natural killer (NK) cells to specifically target and destroy CD34+CD38- CML cells in an in vitro-based antibody dependent cell-mediated cytotoxicity (ADCC) assay (Järås et al, PNAS, 2010). The aim of the present study was to investigate the consequences of IL1RAP expression on primitive CML cells and the in vivo therapeutic efficacy of monoclonal IL1RAP antibodies against CML cells. Primary chronic phase (CP) CD34+ CML cells were cultured in medium supplemented with cytokines known to signal through receptor complexes involving IL1RAP. The addition of IL1 to the cultures resulted in a marked cellular expansion specifically for the primitive CD34+CD38- CML cells. Moreover, the CD34+CD38- cells showed phosphorylation of the downstream mediator of IL1-signaling NFKB. RNA-sequencing confirmed the activation of NFKB and of genes involved in cell cycling, indicating that IL1 stimulation of CD34+CD38- CML cells induced proliferation. Upon addition of an IL1RAP antibody capable of blocking IL1-signaling to the suspension cultures, the IL1-induced expansion and NFKB phosphorylation of CD34+CD38- CML cells was suppressed. Interestingly, both the IL1RAP expression and the response to IL1 as measured by NFKB phosphorylation was retained during TKI treatment of the cells. To assess the in vivo effects of IL1RAP antibodies in CML models, we first engrafted NOD/SCID mice with BCR/ABL1 expressing BV173 cells and treated the mice with the monoclonal IL1RAP antibody mAb81.2. Mice receiving treatment with mAb81.2 displayed a prolonged survival compared to controls, accompanied by reduced levels of leukemic cells in the BM. In vitro studies showed that mAb81.2 lacked a direct effect on cellular expansion or apoptosis. Instead, the IL1RAP antibody could direct NK cells to elicit killing of the leukemic cells, thereby suggesting effector cell mediated mechanisms to be an important in vivo mode-of-action. To validate the in vivo effects on primary CML cells, we next engrafted CP or blast phase (BP) CML cells into immunodeficient mice. Following engraftment of CP CD34+ CML cells into NSG mice and subsequent treatment with mAb81.2, a reduction of human myeloid cells was observed, suggesting that the treatment targeted the leukemic graft. Importantly, mAb81.2 treatment also reduced the levels of candidate CD34+CD38-IL1RAP+ CML stem cells. Finally, BP CML cells were engrafted into NOD/SCID mice that have a more intact effector cell function compared to NSG mice. Following treatment with mAb81.2 a significant reduction of leukemic cells in the BM as well as in the periphery was observed compared to control mice. Importantly, secondary transplantations revealed a therapeutic effect also on the BP CML stem cells. In vitro ADCC assays confirmed that CML BP cells, including a sample with the highly TKI-resistant T315I mutation, could be targeted and killed using mAb81.2. We conclude that IL1RAP antibodies can suppress IL1-induced expansion of primitive CML cells and that in vivo administration of IL1RAP antibodies in CML xenograft models has anti-leukemic effects that extend to the CML stem cells. These results show that an antibody-based therapy against IL1RAP can be used to efficiently target CML stem cells. Disclosures Richter: BMS: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Järås:Cantargia AB: Equity Ownership. Fioretos:Cantargia AB: Equity Ownership.

scholarly journals In Vivo Analysis of PML-RARA in a Humanized Mouse Model

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1020-1020
Author(s):  
Hiromichi Matsushita ◽  
Takashi Yahata ◽  
Yin Sheng ◽  
Yoshihiko Nakamura ◽  
Yukari Muguruma ◽  
...  
Keyword(s):  
Cord Blood ◽  
Conflicts Of Interest ◽  
Nuclear Bodies ◽  
Leukemic Cells ◽  
Human Cord Blood ◽  
Immunodeficient Mice ◽  
Cd34 Cells ◽  
Nog Mice

Abstract Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML) characterized by the formation of a PML-RARa fusion protein, which leads to the accumulation of abnormal promyelocytes. Xenograft mouse models with human leukemic cells have advantages for analyzing the human leukemias in vivo, especially for genetic analyses. However, human primary APL cells are difficult to engraft even in very severely immunodeficient mice, such as NOD/shi-SCID IL2Rg-/- (NOG) mice. In order to understand the mechanisms involved in human APL leukemogenesis, we established a humanized in vivo APL model using the transplantation of PML-RARA-transduced CD34+ cells from human cord blood into NOG mice. The expression of PML-RARa in the CD34+ cells disrupted the nuclear bodies in vitro. The clonogenic assay showed that PML-RARa inhibited the total colony formation, but favored the growth of myeloid colonies. When CD34+ cells with PML-RARA were transplanted, they proliferated in the NOG mice for more than three to four months after transplantation (in 24 out of the 34 mice). All 16 mice with more than 3,000 PML-RARA-transduced CD34+ cells were engrafted, while the engraftment was only detected in eight out of 18 mice when the cell density used for transplantation was less than 3,000 cells. These cells possessed abundant azurophilic abnormal granules in the cytoplasm, and some of them had bundles of Auer rods. They expressed CD13, CD33 and CD117, but not HLA-DR or CD34. In addition, the gene expression analysis revealed that these cells and human primary APL were clustered together among various types of AML, suggesting that these induced APL cells well recapitulated human primary APL. Similar to human primary APL, the induced APL cells possessed the ability for myeloid differentiation after treatment with all-trans retinoic acid in vitro and in vivo, and a very low potential for re-transplantation, which was similarly observed in both unsorted induced APL cells and the CD34- fraction. When human cord blood was fractionated before the PML-RARA transduction, the CD34+/CD38+ cells and common myeloid progenitors (CMP) in the CD34+/CD38+ cells led to the efficient development of APL in vivo. These findings demonstrate that CMP is a target for PML-RARA in APL, whereas the resultant CD34- APL cells may share the ability to maintain the leukemia. Disclosures No relevant conflicts of interest to declare.

scholarly journals Leukemic Stem Cells of Monocytic AMLs Are Not-Resistant to BCL-2 Inhibition

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3469-3469
Author(s):  
Simon Renders ◽  
Aino-Maija Leppä ◽  
Alexander Waclawiczek ◽  
Maike Janssen ◽  
Elisa Donato ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Research Funding ◽  
Independent Study ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Monocytic Differentiation ◽  
Color Flow ◽  
Travel Costs ◽  
Nsg Mice

Abstract Treatment with Hypomethylating agents (HMA) such as 5-Azazytidine (AZA) in combination with the BCL-2 inhibitor Venetoclax (VEN) has recently become the standard of care for AML patients unsuitable for intensive induction chemotherapy and shows results superior to treatment with AZA alone (DiNardo et al., 2020, NEJM). However upfront resistance and relapse following initial response remain major obstacles. It has recently been proposed that monocytic differentiation predicts resistance to AZA/VEN treatment in AML (Pei et al., 2020 Cancer Discovery). This appears to be due to increased expression of other anti-apoptotic proteins such as MCL-1 in monocytic AMLs, which conveys resistance to AZA/VEN therapy, as survival of leukemic cells in these patients is no longer dependent on BCL-2. However, an independent study found no impaired outcome in patients with monocytic AMLs treated with HMA/VEN (Maiti et al., 2020, Blood, ASH abstract). Here, we show that monocytic AML cell lines and bulk cells of monocytic primary AML cells are indeed intrinsically resistant to AZA/VEN treatment. However, in a collective of 30 patients treated with HMA/VEN at Heidelberg University Medical Center between 2018 and 2020, monocytic differentiation assessed by flow cytometry was not an independent risk factor for refractory disease. We hypothesized that the conflicting data may be caused by intra-patient heterogeneity of AZA/VEN sensivitity and assessed killing efficiency in various immunophenotypic subpopulations of 12 primary AML patient samples in vitro. The CD64 +CD11b +, differentiated blast population made up &gt;50% of leukemic cells in monocytic and &lt;20% in primitive samples and showed high levels of resistance to AZA/VEN therapy in both primitive and monocytic leukemias but did not engraft when transplanted into NSG mice, arguing they do not contain leukemic stem cells (LSC). In contrast, we found immature CD64 -CD11b - GPR56 + LSC to be sensitive to AZA/VEN treatment irrespective whether they were derived from monocytic or primitive types of primary AMLs. As expected, LSCs from either monocytic or primitive AMLs initiated disease in NSG mice, highlighting that targeting LSCs is essential for the effect of AML therapy. Next, we investigated expression of BCL-2, MCL-1 and BCL-xL in the same primary patient samples and observed high MCL-1 expression in monocytic AML samples. However, MCL-1 expression was restricted to the CD64 +CD11b + population whereas in the LSC sub-populations robust expression of BCL-2 but low levels of MCL-1 and BCL-xL were detected, independent of whether monocytic or primitive AMLs were analyzed. To further validate the sensitivity of LSCs of monocytic AML to BCL-2-I, we established a platform combining BH-3 profiling with multi-color flow cytometry, allowing for single cell assessment of cellular dependencies on independent apoptotic pathways. We found that LSCs of both AML types show high VEN/BAD but low MS-1 induced apoptosis, functionally confirming the expression patterns of BCL-2 and MCL-1. As LSCs are rare in monocytic samples, investigation of samples in bulk are dominated by MCL-1 expressing and resistant non-LSCs, explaining the overall higher MCL-1 expression/survival of monocytic compared to immature AML cells. However, our data uncovers sensitivity of LSCs to AZA/VEN independent of overall monocytic or primitive sample classification and provide a mechanistic explanation for the clinical data of Maiti et al. and our Heidelberg AML collective, which found no increased resistance of monocytic AMLs to AZA/VEN treatment. Disclosures Unglaub: JazzPharma: Consultancy, Other: travel costs/ conference fee; Novartis: Consultancy, Other: travel costs/ conference fee. Schlenk: Abbvie: Honoraria; Agios: Honoraria; Astellas: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria; Daiichi Sankyo: Honoraria, Research Funding; Hexal: Honoraria; Neovio Biotech: Honoraria; Novartis: Honoraria; Pfizer: Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria, Research Funding; AstraZeneca: Research Funding; Boehringer Ingelheim: Research Funding. Müller-Tidow: Janssen: Consultancy, Research Funding; Bioline: Research Funding; Pfizer: Research Funding.

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

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1884-1884
Author(s):  
Alissa R. Kahn ◽  
Kimberly A. Hartwell ◽  
Peter G. Miller ◽  
Benjamin L. Ebert ◽  
Todd R. Golub ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Lines ◽  
Serotonin Receptor ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Nsg Mice ◽  
Cell Kill ◽  
Serotonin Receptor Antagonist

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

Drug Screening of Primary Human Pediatric Pre-B Cell Acute Lymphoblastic Leukemia (pre-B ALL) Cells in Their Stromal Niche

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4295-4295
Author(s):  
Jae-Hung Shieh ◽  
Tsann-Long Su ◽  
Jason Shieh ◽  
Malcolm A.S. Moore
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Culture System ◽  
Lymphoblastic Leukemia ◽  
Nsg Mice ◽  
Cd34 Cells ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Abstract 4295 Pre-B cell acute lymphoblastic leukemia (pre-B ALL) is the most common leukemia in children and is treatable. However, no in vitro nor in vivo models are available to investigate their pathophysiology other than a number of established cell lines that grow in the absence of any cytokine dependence or stromal interaction. We developed a serum-free MS-5 cell (a murine bone marrow stromal cell line) co-culture system that is capable of expanding human primary pre-B ALL CD34+CD19+ cells in vitro. To define a population of pre-B ALL initiating cells, our study reveals that a sorted CD34bright population displays a slow proliferation and maintains a high % of CD34+ cells. In contrast, CD34dim cells/CD34− cells fraction shows a higher proliferation but expanded cells lost CD34 antigens. A group of alkylating molecules (BO-1055, -1090, 1099, -1393 and -1509) was evaluated for proliferation of the pre-B ALL CD34+ cells, the pre-B ALL CD34− cells, human mesenchymal stem cells (hMSC), murine MSC (MS-5 cells and Op9 cells), human bone marrow derived endothelial cells (BMEC), and human cord blood (CB) CD34+ cells, as well as for a week 5 cobblestones area forming (CAFC) assay with CB CD34+ cells. BO-1055 molecule efficiently suppressed the growth of pre-B ALL CD34+ cells (IC50 = 0.29 μM) and CD34− cells (IC50 = 0.31 μM). In contrast, IC50 of BMEC, MSC, CB CD34+ cells and CAFC are >10, >25, 8, and >5 μM, respectively. Pre-B ALL cells expressing green fluorescent protein (GFP) and luciferase (GFP-Lu-pre-B ALL) were created, and a xenograft of the GFP-Lu-pre-B ALL cells to NOD/SCID IL2R gamma null (NSG) mice was established. The in vivo effect of BO-1055 to the GFP-Lu-pre-B ALL cells in NSG mice is under investigation. Our stromal culture system supports primary pre-B ALL cells and closely recapitulates the growth of primary human pre-B ALL cells in their niche in vivo. Based on this co-culture system, we identified BO-1055 as a potential therapeutic agent with an excellent toxicity window between pre-B ALL cells and normal tissues including BMEC, MSC and hematopoietic progenitor/stem cells. The in vitro stromal co-culture system combined with the xenograft model of GFP-Lu-pre-B ALL cells provides an efficient and powerful method to screen new drugs for pre-B ALL therapy. Disclosures: No relevant conflicts of interest to declare.

Fingolimod Expands Human Umbilical Cord Blood Cells (UCB) in Vitro and Improves Engraftment Rate of Human UCB in Sublethally Irradiated NOD SCID Gamma (NSG) Mice

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2413-2413
Author(s):  
Basem M. William ◽  
Stephen Moreton ◽  
Jane Reese ◽  
Pingfu Fu ◽  
Julia V. Busik ◽  
...  
Keyword(s):  
Statistical Model ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Diabetic Patients ◽  
Nsg Mice ◽  
Cd34 Cells ◽  
In Vivo Treatment

Abstract Background: Umbilical cord blood transplantation (UCBT) has emerged as an alternative for allogeneic hematopoietic stem cell transplantation for patients who don't have a matched donor. However, cell dose remains an obstacle to successful UCBT and strategies aimed at improving rate and speed of engraftment are needed. Acid sphingomyelinase (ASM), a glycoprotein enzyme that converts sphingomyelin to ceramide, was recently shown to modulate inflammatory and cell stress responses. In vitro treatment with docosahexaenoic acid (DHA), an ASM inhibitor, restores colony formation ability of CD34+ cells from diabetic patients and that paralleled reduction of ASM in such cells (Tikhonenko et al. PLoS One 2013). Fingolimod (FTY720) is a novel immunosuppressant that is FDA approved for the treatment of relapsing multiple sclerosis. Fingolimod was shown to inhibit ASM activity in vitro through a mechanism that is similar to desipramine (a known inhibitor of ASM) yet at much lower micromolar concentrations (Dawson et al. Biochem Biophys Res Commun 2011). Hypothesis: Fingolimod expands UCB cells and improves engraftment of UCB cells in NSG mice. Methods: CD34+ cells, separated by magnetic-activated cell sorting (MACS) from pooled human UCB units, were cultured for 72 hours at 37 °C in RPMI-1640 + 10% FBS + 100 ng/ml each of SCF, TPO, and FL. Cells were be divided into 3 plates; the third plate had fingolimod added to culture (at 1 uM concentration) and the first 2 plates had no fingolimod added. Sphingomyelinase activity was measured using the Amplex Red Sphingomyelinase Assay Kit according to the manufacture protocol (Molecular Probes, Eugene, OR). Cells were then plated into methylcellulose for 7-10 days before colony forming units (CFU) were counted. Cells from each plate were injected into a corresponding group of sublethally irradiated (225cGy) NSG mice (n=5 per group) as follows: group 1 was injected with control cells, group 2 was injected with control cells, but mice received 5 mcg/kg/day fingolimod by intraperitoneal injection starting on the day of transplant for 6 weeks, and group 3 was injected with cells cultured with fingolimod. Engraftment of human cells was measured by flow cytometry for CD45 surface marker in peripheral blood of recipient mice at 3 weeks and 8 weeks. Engraftment rate of human UCB cells in NSG mice was estimated using a mixed statistical model. Results: Adding fingolimod to cell culture resulted in a robust expansion of UCB cells as evident by doubling in CFU (p<0.0001). Fingolimod also decreased acid, but not neutral sphingomyelinase activity by 25%. In NSG mice, the engraftment rate for control group was 0.106, which was not statistically significant from zero (p = 0.104). The slope for fingolimod treated group was 0.106, which was not significant from zero, either (p = 0.104). However, the slope for fingolimod injected group was 0.197, which was significant from zero (p = 0.0066) as shown in figure below. Conclusions: Fingolimod expands CFU activity of human UCB cells in culture. In addition, in vivo treatment of NSG mice with fingolimod accelerates engraftment of human CD34+ selected UCB cells. Figure: In vivo treatment of NSG mice with fingolimod accelerates engraftment of human UCB cells. Rate of engraftment estimated using a mixed statistical model (p=0.0066). Figure:. In vivo treatment of NSG mice with fingolimod accelerates engraftment of human UCB cells. Rate of engraftment estimated using a mixed statistical model (p=0.0066). Disclosures No relevant conflicts of interest to declare.

Pronounced Anti-Leukemia Activity in Vivo of the Novel Peptidic CXCR4 Antagonist LY2510924 As a Single Agent and in Combination with Chemotherapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3745-3745
Author(s):  
Byung-Sik Cho ◽  
Zhihong Zeng ◽  
Hong Mu ◽  
Zhiqiang Wang ◽  
Teresa McQueen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cultured Cells ◽  
Single Agent ◽  
Leukemic Cells ◽  
Cxcr4 Antagonist ◽  
Nsg Mice ◽  
Xenograft Models

Abstract LY2510924 is a novel selective peptidic CXCR4 antagonist that blocks SDF-1α from binding to its receptor. We have demonstrated that LY2510924 at nanomolar concentrations durably disrupts the SDF-1α/CXCR4 axis in acute myeloid leukemia (AML) cells and exerts anti-leukemia effects as a single agent (AACR 2014: #4768). We further investigated the pronounced anti-leukemia activity of LY2510924 and the mechanisms underlying the anti-leukemia effect. To test the efficacy of LY2510924 in combination with chemotherapy, we injected OCI-AML3/luc/GFP cells into NSG mice. Mice were randomized into 4 groups (10 mice per group) on day 8: control, chemotherapy (cytarabine [50 mg/kg, daily for 5 days, intravenous or intraperitoneal]/doxorubicin [1.5 mg/kg, daily for 3 days, co-delivered intravenously]), LY2510924 (2.5 mg/kg, daily for 3 weeks, subcutaneously), or chemotherapy and LY2510924. Bioluminescence imaging demonstrated that LY2510924 exerted an anti-leukemia effect equal to that achieved with chemotherapy (P=0.249), and the combination therapy group had the lowest luciferase activity. LY2510924-treated mice had prolonged survival (Figure 1) compared to controls (52 days vs. 40 days, p=0.006), and combination therapy extended survival even further (62 days vs. 52 days, p=0.004). Next, we examined anti-leukemia efficacy of LY2510924 in primary human AML xenograft models. NSG mice were injected with primary AML cells and randomized into 2 groups on day 25, after engraftment was documented: control (n=13) and treatment with LY2510924 (n=15; 2.5 mg/kg subcutaneously, daily). First, we examined AML cell mobilization by measuring the proportion of circulating leukemic cells after daily LY2510924 administration. Mice treated with LY2519024 had a significant increase of circulating leukemic cells at 3 hours (2.1-fold, P=0.008), and further increases at 24 hours (2.7-fold, P=0.008) and 48 hours (3.0-fold, P=0.009) compared to controls. Flow cytometry showed a sustained inhibition of CXCR4 12G5 surface expression at 3 and 24 hours after the first LY2510924 injection. Thereafter, weekly examination of circulating leukemic cells in both groups revealed slower progression of leukemia in the LY2510924-treated group (54% vs. 86% circulating AML cells on day 45, P<0.001). Additionally, we sacrificed 3 mice per group on days 35 and 45 and demonstrated that LY2510924-treated mice had significantly lower leukemic cell burden in the spleen (22% vs. 51%, P=0.001) on day 35, and in both spleen (20% vs. 60%, P<0.001) and bone marrow (72% vs. 90%, P=0.012) on day 45 by flow cytometry. CXCR4 blockade with LY2510924 was associated with reduced AKT and/or ERK signaling in leukemic cells of spleen, bone marrow, and blood as measured by multi-parametric phospho-flow cytometry. This anti-leukemia effect translated into a significant prolongation of survival in LY2510924-treated mice (56 days vs. 44 days, p<0.001, Figure 2). Our previous study (AACR 2014:#4768) demonstrated that LY2510924 did not induce AML cell death in vitro on its own but inhibited AML cell growth in co-cultures with human marrow stromal cells (hMSC). To explore how CXCR4-mediated signaling in AML cells elicits anti-leukemia effects, we performed whole gene expression profiling of FACS-sorted OCI-AML3 cells co-cultured with hMSC for 48 hours and co-treated with LY2510924, in duplicates. Among genes modified by CXCR4 antagonist, we found that CTNNB1 (human beta-catenin), JARID1C (lysine-specific demethylase 5C), RARA (retinoic acid receptor alpha), RARRES2 (chemerin), and COQ4 (coenzyme Q) were downregulated in co-cultured OCI-AML3 cells treated with LY2510925, when compared to either mono-cultured cells or co-cultured cells without LY2510924. These findings are currently being validated by using functional in vitro assays. In conclusion, our findings demonstrate that CXCR4 antagonist LY2510924 inhibits AML progression in leukemia xenograft models in vivo and has a synergistic anti-leukemia effect in combination with chemotherapy. LY2510924 efficiently inhibits CXCR4 signaling in primary AML cells in vivo and induces mobilization of leukemic cells into circulation. This results in pronounced anti-leukemia activity as a single agent. LY2510924's potency and durable occupancy of CXCR4 receptors will likely translate into greater anti-leukemia potency in future clinical applications. Disclosures Peng: Eli Lilly & Company: Employment. Thornton:Eli Lilly & Company: Employment, stocks Other.

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