scholarly journals Inhibiting the Mitochondrial Sulfhydryl Oxidase Alr Reduces Cox17 and Alters Mitochondrial Cristae Structure Leading to the Differentiation of AML and Stem Cells

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 881-881
Author(s):  
Danny V. Jeyaraju ◽  
Veronique Voisin ◽  
Changjiang Xu ◽  
Samir H. Barghout ◽  
Dilshad H. Khan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Respiratory Chain ◽  
Research Funding ◽  
Annexin V ◽  
Leukemic Cells ◽  
Copper Chaperone ◽  
Leukemic Stem Cells ◽  
Sulfhydryl Oxidase

Abstract The vast majority of mitochondrial proteins are encoded in the nucleus, translated in the cytoplasm and then imported into the mitochondria. A subset of these imported proteins are folded into their mature and functional forms in the mitochondrial inter-membrane space (IMS) by the Mitochondrial IMS Assembly (MIA) pathway. We found that genes encoding substrates of the MIA pathway are over-expressed in leukemic stem cells compared to bulk AML cells. Therefore, we assessed the effects of inhibiting the MIA pathway in AML. We knocked down the mitochondrial sulfhydryl oxidase ALR, a key regulator of the MIA pathway. Knockdown of ALR with shRNA reduced the growth and viability of OCI-AML2, TEX and NB4 leukemia cells. In addition, knockdown of ALR reduced the engraftment of TEX cells into mouse marrow, demonstrating an effect on the leukemia initiating cells. The small molecule selective ALR inhibitor, MitoBloCK-6, mimicked the effects of ALR knockdown and killed AML cells with an IC50 of 5-10 μM. MitoBloCK-6 preferentially reduced the clonogenic growth of primary AML cells (n=4/5) over normal hematopoietic cells (n=4). However, only 3/10 bulk AML cells were sensitive to MitoBloCK-6 induced cell death by Annexin V/PI staining. Next, we evaluated the efficacy and toxicity of ALR inhibition in vivo . We injected primary AML cells or normal cord blood into the femurs of mice and then treated mice with MitoBloCK-6 (80 mg/kg i.p. 5 of 7 days x 2 weeks). MitoBloCK-6 strongly reduced the engraftment of primary AML samples but did not affect engraftment of cord blood. In secondary transplants, MitoBloCK-6 also targeted leukemic stem cells. No change in mouse body weight, serum chemistries, or organ histology was seen. As expression levels of ALR substrates are increased in AML stem cells, we assessed the effects of ALR inhibition on differentiation in AML. Genetic or chemical inhibition of ALR induced the differentiation of AML cells as evidenced by increased CD surface marker expression and increased non-specific esterase. In addition, ALR inhibition was preferentially cytotoxic towards undifferentiated cells and stem cells over differentiated bulk AML cells. Interrogation of the effects of ALR inhibition on its substrates identified the mitochondrial copper chaperone, Cox17 as the primary downstream target in leukemic cells. Inhibition of ALR selectively reduced levels of Cox17 protein and altered mitochondrial cristae structure. Validating the functional importance of these findings, knockdown of Cox17 phenocopied ALR inhibition and reduced AML proliferation, induced differentiation of AML cells, and altered mitochondrial cristae structure, without changing respiratory chain activity or oxygen consumption. Of note, cristae remodelling independent of respiratory chain function has been recently implicated in cellular differentiation and in yeast, Cox17 regulates the cristae organizing machinery. Thus, we have identified novel mechanisms by which mitochondrial pathways regulate the fate and differentiation of AML cells and stem cells Moreover, inhibition of ALR may be a novel therapeutic strategy to promote the differentiation of AML cells and stem cells. Disclosures Schimmer: Takeda Pharmaceuticals: Research Funding; Medivir: Research Funding; Novartis Pharmaceuticals: Honoraria.

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.

scholarly journals Imetelstat Significantly Reduces Leukemia Stem Cells in Patient-Derived Xenograft Models of Pediatric AML

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3352-3352
Author(s):  
Sonali P. Barwe ◽  
Fei Huang ◽  
E. Anders Kolb ◽  
Anilkumar Gopalakrishnapillai
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Research Funding ◽  
Annexin V ◽  
Telomerase Activity ◽  
Leukemia Stem Cells ◽  
Patient Derived Xenograft ◽  
Pediatric Aml ◽  
Pdx Models

Abstract Introduction Acute myeloid leukemia (AML) is the deadliest malignancy in children. Despite the use of maximally intensive therapy, 20% of patients experience recurrent disease. These patients are also burdened with significant treatment-related toxicities. To improve survival in pediatric AML, novel targeted therapies that are more effective and less toxic are needed. Telomerase inhibition has been shown to be effective in reducing leukemic burden and eradicating leukemia stem cells (LSCs) in syngeneic mouse models of AML and in patient-derived xenograft (PDX) models of adult AML (Bruedigam et al., 2014). Recent transcriptome analyses demonstrate that the genomic landscape of pediatric AML is distinct from adult AML (Bolouri et al., 2018). In fact, mutations in the telomerase complex components are infrequent in pediatric AML unlike adult AML patients (Aalbers et al., 2013). However, similar to what is seen in adult patients, Aalbers et al. identified that telomere lengths in pediatric AML cells were shortened compared to normal leukocytes, and pediatric AML patients with the shortest telomere length tend to have shorter overall survival. Furthermore, the 5-year survival rate was 88% for pediatric AML patients who had lower telomerase activity, and 43% for those patients with higher telomerase activity, suggesting telomerase activity could be an important prognostic factor in pediatric AML patients (Verstovsek et al., 2003). Imetelstat is an oligonucleotide that specifically binds with high affinity to the RNA template of telomerase and is a potent, competitive inhibitor of telomerase enzymatic activity (Asai et al., 2003; Herbert et al., 2005). In this study, we evaluated if imetelstat has anti-leukemia activity in pediatric AML PDX models. Results The PDX lines tested in this study were derived using samples from pediatric AML patients who were 1-14 years old, representing different FAB subtypes. Mouse passaged pediatric AML PDX lines (n=6) were treated ex vivo with imetelstat or mismatch oligo control and the viability of LSC (CD34+CD38low population) was determined at 48 or 96 h by staining with BV785-human CD45, APC-human CD34, Pacific blue-human CD38, FITC conjugated annexin V and propidium iodide (PI). Imetelstat treatment significantly increased apoptosis/death (PI+/annexin V+) of the LSC population in a dose-dependent manner in all PDX lines evaluated (Fig. 1A, B), while it had limited activity on LSCs in normal pediatric bone marrow samples (n=4). The efficacy of imetelstat either alone or in combination with chemotherapy or azacitidine was evaluated in two distinct PDX models of pediatric AML in vivo. Mice engrafted with both NTPL-377 and DF-2 lived longer when treated with imetelstat than the untreated mice (Fig. 1C, D, n=5 each, P<0.05). Mice receiving standard chemotherapy consisting of cytarabine and daunorubicin or azacitidine showed prolonged survival compared to the untreated mice. Interestingly, sequential administration of imetelstat following chemotherapy treatment provided additional benefit over chemotherapy alone (P<0.01). Concurrent treatment of azacitidine and imetelstat further extended survival of these mice compared to azacitidine alone (P<0.05). At the end of the in vivo studies, the percentage of LSC population was evaluated in the bone marrow of mice post euthanasia. There was a significant reduction of LSC population in mice treated with imetelstat compared to those treated with the mismatch oligo (Fig. 1E, F, P<0.05). Neither chemotherapy nor azacitidine alone affected LSC population compared to untreated mice. However, imetelstat significantly reduced the LSC population when combined with chemotherapy or azacitidine compared to single agent (P<0.05). These results were confirmed by secondary transplantation in mice, which showed delayed engraftment of cells isolated from imetelstat treated mice (Fig. 1G, H). Conclusions Imetelstat treatment of pediatric AML PDX samples showed significant dose- and time-dependent effects on the viability of the LSCs to induce cell apoptosis/death. These results were corroborated in vivo in two distinct PDX models which showed reduced LSC population and increased median survival in mice with imetelstat treatment. Combining imetelstat with chemotherapy or azacitidine further enhanced activity against LSCs, suggesting imetelstat could represent an effective therapeutic strategy for pediatric AML. Figure 1 Figure 1. Disclosures Barwe: Prelude Therapeutics: Research Funding. Huang: Geron Corp: Current Employment, Current equity holder in publicly-traded company. Gopalakrishnapillai: Geron: Research Funding.

scholarly journals Targeting mTORC1/2 by a mTOR Kinase Inhibitor (PP242) induces Apoptosis In AML Cells Under Conditions Mimicking Bone Marrow Microenvironment

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 778-778
Author(s):  
Zhihong Zeng ◽  
Yuexi Shi ◽  
Twee Tsao ◽  
Yihua Qiu ◽  
Steven M. Kornblau ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Signaling Pathways ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Annexin V ◽  
Mtor Signaling ◽  
Leukemic Cells ◽  
Survival Signaling

Abstract Abstract 778 The prognosis of patients with acute myeloid leukemia (AML) remains poor. Our studies have demonstrated that chemoresistance of AML is not solely due to increased survival signaling in AML cells, but is also enhanced by microenvironment/leukemia interactions. Bone marrow-derived mesenchymal cells (MSC) comprise an essential component of the leukemia bone marrow microenvironment. MSC have the capacity to support normal and malignant hematopoiesis and protect leukemic cells from chemotherapy. We have previously reported that co-culture of AML cells with MSC results in activation of multiple pro-survival signaling pathways in leukemic cells, from which phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling is the key upstream regulator of survival and chemoresistance (Tabe et al., 2007 Cancer Res. 2007). In this study, we investigated the role of mTOR signaling in primary AML cells co-cultured with stroma and in the in vivo leukemia mouse model utilizing a novel TOR kinase inhibitor PP242 (Intellikine, La Jolla, CA). Unlike rapamycin and its analogs, which suppress TORC1 only partially and do not acutely inhibit TORC2, PP242 has been reported to achieve greater inhibition of both TOR complexes, resulting in broader suppression of the PI3K/AKT/TOR signaling in Ph+ B-ALL and T-cell lymphoma (Feldman, et al., PLoS Biol 2009; Janes, et al., Nat Med. 2010). We first employed reverse phase protein array (RPPA) technique profiling of 53 proteins to determine the changes in activation of signaling pathways in leukemic cells from 20 primary AML samples co-cultured with murine stromal line MS-5. Co-culture with stroma resulted in activation of multiple signaling pathways in primary AML cells, inducing upregulation of pAKT(Thr308) in 18, mTOR in 17, pERK(Thr202/204) in 14, and pSTAT3(Ser727) in 12 of the 20 pt samples. This resulted in significant decrease of spontaneous apoptosis in primary AML samples (average 33.7 ± 3.8% annexin V(+) cells in primary AML without co-culture vs. 19.6 ± 3.1% in primary AML co-cultured with MS5, p = 0.027, n = 20). In a next set of experiments, blockade of mTOR signaling with PP242, in a dose dependent fashion, effectively induced apoptosis in primary AML samples (n = 9) cultured with or without stroma: at 60nM, 6.4 ± 1.8% and 8.8 ± 2.4% specific apoptosis (annexin V+), respectively; at 190nM, 10.5% ± 52.8% and 14.9% ± 3.9%; at 560nM, 17.6.9 ± 5.7%; and 21.9 ± 4.9% at 1.67uM, 27.2 ± 6.1% and 27.3 ± 5.8%; at 5uM, 38.8 ± 6.5% and 37.1 ± 7.2%. Importantly, at low nanomolar concentrations, PP242 attenuates the activities of both TORC1 and TORC2, resulting in inhibition of phosphorylation of AKT at S473, S6K at S240/244 and 4EBP1 at T37/46 in both, primary AML cells and most importantly in MSC cultured alone or co-cultured with AML. In the in vivo leukemia mouse model utilizing GFP/luc-labeled Baf3-FLT3/ITD cells, PP242 (60mg/kg/QD gavage) exerted significantly greater anti-leukemia activity compared with TORC1 inhibitor rapamycin (0.1mg/kg/QD IP, p = 0.03). PP242 suppressed leukemia progression as determined by bioluminescence imaging (average luminescence intensity 5.65 ± 1.75 in control vs. average 2.75 ± 0.65 in PP242 group) and significantly extended survival (p = 0.005). In summary, our findings indicate a novel therapeutic strategy to target leukemia within the BM microenvironment through efficient blockade of mTOR/AKT signaling with novel selective TORC kinase inhibitor. This research is funded by Intellikine. Disclosures: Liu: Intellikine: Employment. Rommel:Intellikine: Employment. Fruman:Intellikine: Research Funding. Konopleva:Intellikine: Research Funding.

Deletion of Rac2 inhibits Proliferation of Chronic Myelogenous Leukemia (CML) Stems Cells and Progenitors (HSC/P) In Vivo and Promotes Survival of Scl/p210-BCR-ABL Mice.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3253-3253
Author(s):  
Amitava Sengupta ◽  
Jorden Arnett ◽  
Susan Dunn ◽  
Jose Cancelas
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Chronic Myelogenous Leukemia ◽  
Research Funding ◽  
Myelogenous Leukemia ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Rac Gtpases ◽  
And Migration

Abstract Abstract 3253 Poster Board III-1 Chronic myelogenous leukemia (CML) is a hematopoietic stem cell (HSC) malignancy induced by p210-BCR-ABL and is characterized by myeloproliferation in the bone marrow (BM) and egress of leukemic stem cells and progenitors (LSC/P) to extramedullary sites. Persistence of BCR-ABL+ HSCs in patients under imatinib suggests that inhibition of ABL-kinase alone is not sufficient to completely eliminate the LSC/P population. Rac GTPases represent integrative molecular switches for p210-BCR-ABL-induced HSC transformation and combined pharmacological and genetic attenuation of Rac GTPases significantly prolong survival in vivo, as reported in a retroviral transduction/transplantation model (Thomas EK & Cancelas JA et al, Cancer Cell 2008). Here, we analyzed the role of Rac2 GTPase in the leukemic maintenance and in the interaction of LSC/P with the leukemic microenvironment in vivo. We used a stem cell leukemia (Scl) promoter-driven, tet-off, Scl-tTA x TRE-BCR-ABL (Scl/p210-BCR-ABL) binary transgenic mouse model (Koschmieder S et al., Blood 2005), where expression of BCR-ABL is restricted to the HSC/P compartment, allowing the study of the intrinsic molecular changes in LSC/P during leukemogenesis. In these mice, Scl-driven expression of BCR-ABL is active in HSC (Lin-/Sca1+/c-kit+; LSK) and progenitors (Lin-/c-kit+/Sca-1-; LK), and CML development is associated with the activation of downstream signaling effectors CrkL, p38-MAPK and JNK. Additionally, Scl/p210-BCR-ABL mice had increased cycling of LSK cells and expansion of circulating and splenic, but not BM, LSC/P, suggesting egress of LSC/Ps from the marrow. These mice share all the characteristics of HSC/P transformation in CML, including increased HSC/P proliferation and survival, severely reduced adhesion to fibronectin, increased migration towards CXCL12, increased cell surface expression of CD44 and decreased expression of L-selectin. Myeloproliferative disease (MPD) in these mice is transplantable into recipient mice, and CML splenocytes have a 10-fold increase in homing to the spleen than towards BM (P<0.05). Leukemic splenocytes are also enriched in endosteal lodging progenitors, compared to the BM-derived progenitors (1.9-fold, P≤0.05). In order to determine the contribution of Rac2 GTPase in the transformation phenotype of leukemic stem cells and progenitors, Scl/p210 mice were intercrossed with Rac2-/- mice. Interestingly loss of Rac2 GTPase alone significantly prolongs survival of the leukemic mice (P≤0.001). Prolonged survival, as observed in Scl/p210 x Rac2-/-, is associated with significantly reduced proliferation of leukemic LK (3-fold, P<0.05) and LSK (6-fold P<0.005) cells, both in BM as well as in spleen, in vivo. Scl/p210 x Rac2-/- mice are also characterized by increased apoptosis (1.7-fold) and lower frequency of LSK cells (2-fold) compared to the Scl/p210 mice in vivo. However, deletion of Rac2 does not significantly reverse the adhesion and migration transformation phenotype of LSC/P. In summary, Rac2 deficiency induces a significant survival of CML mice in a HSC-initiated model of disease through decrease proliferation and survival but does not reverse the transformation phenotype affecting adhesion and migration. This murine model may represent an adequate in vivo system to dissect out the specific signaling pathways involved in p210-BCR-ABL-induced stem cell transformation. Disclosures: Cancelas: CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.

The Feature of SALL4 and BMI-1 Expression in Placenta and Umbilical Cord Blood

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4800-4800
Author(s):  
Shaohua Chen ◽  
Qi Shen ◽  
Lijian Yang ◽  
Bo Li ◽  
Yupo Ma ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Research Funding ◽  
Mononuclear Cells ◽  
Expression Level ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Expression Levels

Abstract Abstract 4800 The SALL4 is a newly discovered pluripotency stem cell factor involved in the self-renewing, hematopoietic stem cells (HSCs) and leukemic stem cells. Its important role in normal HSCs and leukemic stem cells is supported by its interactions with several key players, like BMI-1, a putative oncogene of the Polycomb group family, controlling specific target genes involved in development, cell differentiation, proliferation, and senescence. Little is known the expression feature of SALL4 and BMI-1 mRNA in placenta and umbilical cord blood (UCB). In this study, the mRNA expression level of SALL4 and BMI-1 in 10 cases placenta tissues and the mononuclear cells from the corresponding UCB (CBMCs) were measured by real-time PCR with SYBR Green I technique, β2-microglobulin gene (β2M) was used as an endogenous reference, mononuclear cells from 10 healthy adults peripheral blood (PBMCs) and 2 cases of adult thymic cells served as control. The result showed that the expression level of both SALL4 and BMI-1 in placenta (Median: 34.36 and 17.55 respectively) were significantly higher than those from CBMCs (Median: 11.3 and 2.07 respectively) (p=0.007, p=0.001) and PBMCs (Median: 0.64 and 0.03 respectively) (P<0.001, P<0.001). Moreover, both SALL4 and BMI-1 expression levels in CBMCs were significant higher than those from PBMCs (P=0.002, P=0.001). And the expression of SALL4 could be detected in thymic cells. Correlation analysis was performed among the relative expression levels of SALL4 and BMI-1. The results showed that significant positive correlation between the expression levels of SALL4 and BMI-1 was observed in placenta tissues and CBMCs, as well as in PBMCs (rs=0.648, P=0.043, rs=0.721, P=0.019, rs=-0.697, P=0.025). In conclusions, the expression level of SALL4 and BMI-1 might be related to the numbers of hematopoietic stem cells, placenta tissues may be as an appropriate model for investigating gene regulation of SALL4 and BMI-1, while the significance of expression both genes in PBMCs and thymic cells is needed further investigation. Disclosures: Chen: National Natural Science Foundation of China(no. 30871091): Research Funding; the Fundamental Research Funds for the Central Universities (No. 21610603): 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 Using a lentiviral Tet-regulated miR-E shRNA dual color vector to evaluate gene function in human leukemic stem cells in vivo

2019 ◽  
Author(s):  
Henny Maat ◽  
Jennifer Jaques ◽  
Edo Vellenga ◽  
Gerwin Huls ◽  
Vincent van den Boom ◽  
...  
Keyword(s):  
Stem Cells ◽  
Overall Survival ◽  
Gene Function ◽  
Leukemic Cells ◽  
Human Leukemia ◽  
Dependent Manner ◽  
Leukemic Stem Cells ◽  
Dual Color ◽  
Color Vector

AbstractRNA interference is a powerful tool to study loss-of-gene function in leukemic cells. Still, in order to identify effective novel treatment strategies to target and eradicate leukemic stem cells (LSCs), it is critically important to study gene function in a well-controlled and time-dependent manner. We implemented a lentiviral Tet-regulated miR-E shRNA dual color vector in our in vitro and in vivo human leukemia models. Thus, we were able to efficiently introduce doxycycline-inducible and reversible gene repression and trace and isolate transduced miR-E shRNA expressing cells over time. As proof of concept we focused on the non-canonical PRC1.1 Polycomb complex, which we previously identified to be essential for LSCs (1). Here, we show that inducible downmodulation of PCGF1 strongly impaired the growth of primary MLL-AF9 cells. Next, a Tet-regulated miR-E PCGF1 human xenograft MLL-AF9 leukemia mouse model was established, which revealed that early knockdown of PCGF1 at the onset of leukemia development significantly reduced peripheral blood chimerism levels and improved overall survival. In contrast, knockdown of PCGF1 when leukemia was already firmly established in the bone marrow proved insufficient to enhance overall survival. Despite these findings, FACS analysis of MLL-AF9/miR-E PCGF1/CD45+/GFP+ populations suggested that particularly cells with inefficient PCGF1 knockdown contributed to leukemogenesis. In conclusion, by building in vivo xenograft leukemia inducible RNAi models, we show that timing of gene knockdown critically impacts on the efficacy of leukemia treatment and that clonal drift still plays a major role in the escape of LSCs.

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.

A Phase I/II Trial of Plerixafor/G-CSF Combined with IV Bu/Flu Conditioning Regimen In AML/MDS Patients Undergoing Allogenic Stem Cell Transplantation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2358-2358 ◽  
Author(s):  
Marina Konopleva ◽  
Zeng Zhihong ◽  
Rui-Yu Wang ◽  
Peter F. Thall ◽  
Gloria McCormick ◽  
...  
Keyword(s):  
Stem Cells ◽  
Phase I ◽  
Research Funding ◽  
Fold Increase ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Preparative Regimen ◽  
Relapsed Disease ◽  
Fish Cells ◽  
Over Time

Abstract Abstract 2358 Allogeneic stem cell transplantation (alloSCT) is an effective treatment for pts with acute myeloid leukemia (AML) in first remission. However, only 10–20% of pts with relapsed disease achieve a durable remission. Microenvironment/leukemia interactions play a major role in chemoresistance of leukemic stem cells residing in the bone marrow niches. In pre-clinical in vivo leukemia models, inhibition of chemokine receptor CXCR4 results in mobilization of leukemic cells into circulation and sensitization to chemotherapy. We hypothesized that mobilization of leukemic stem cells by CXCR4 inhibition and G-CSF will result in improved anti-leukemia activity of a standard preparative regimen followed by alloSCT. In this Phase I/II study, G-CSF is administered at a standard dose beginning on day -9 daily for 6 days, and the CXCR4 inhibitor plerixafor (Mozobil®) from day -7 at one of the 4 dose levels 0 (control), 0.08, 0.16, or 0.24 mg/kg, 8 hours prior of each four daily doses of a standard preparative regimen (Fludarabine, 40mg/m2 and IV Busulfan, 130mg/m2, days -6 through -3). Twenty seven pts have been enrolled in the study to date with a median age of 48 yrs (range 25–65). Baseline characteristics include 13 pts (48%) with de novo AML, 6 (22%) with secondary AML, 5 with MDS and 3 with CML. Among the 24 AML/MDS pts, 14 (58%) had intermediate and 10 (42%) poor risk cytogenetics. Twelve pts (50%) had primary refractory AML, 5 were in 1st or 2nd relapse, 2 were untreated, 3 were in CR1 and 2 in CR2. The source of stem cells was sibling donor in 16 and unrelated donor in 11. After phase I plerixafor dose escalation in 16 pts, 11 pts received 0.24 mg/kg in Phase II. Common grade ≥ 3 adverse events which consisted primarily of neutropenic fever, infections, or rash were seen in 24/27 (89%) pts. There were no toxicities ascribed to the G-CSF/plerixafor component of the regimen. No evidence of significant delays in neutrophil (ANC >500/mm3, median 12.5d, range 10–19) or platelet recovery (plt >20k/mm3, median 12d, range 9–74d) were observed. Grade I-II GVHD was seen in 10/27 pts (37%), with no occurrences of Grade III-IV GVHD. Of the 19 pts with active disease at study entry, 18 achieved a CR. Treatment failure was due to persistent disease in 1 pt (4%), relapsed disease in 10 pts (37%) and early death due to complications from intracranial hemorrhage in 1 pt (4%). Median progression-free survival (PFS) for all pts was 26.6 wks (95%CI: 18.1–33.9 wks) and 15.7 wks (95% CI: 12.1–26.6 wks) in relapsed pts. Median follow-up for all study pts was 19.14 wks (range: 0.7–54.6 wks). Correlative studies analyzed from 16 pts enrolled in the Phase I portion of the trial demonstrate that G-CSF/plerixafor mobilizes CD34+ cells, with the mean fold increase of 5.9-fold at 0.08 mg/kg plerixafor; at 0.16 mg/kg, 13-fold; and at 0.24 mg/kg, 14.2-fold. Based on fitted longitudinal linear mixed models, G-CSF had a significant effect on cell mobilization over time (WBC and CD34+). In contrast, plerixafor at the doses of 0.16 and 0.24 mg/kg was significantly associated with increased cellular CXCR4 expression levels and with mobilization of CXCR4+ cells over time (p<0.02). To determine relative proportion of mobilization of leukemic and non-leukemic cells, we performed FISH analysis on peripheral blood samples from pts with informative cytogenetic abnormalities (n=12). Both, FISH+ and FISH- cell counts increased from day -8 to day -6 and remained relatively stable or decreased thereafter (between day -6 and day -3), with the initial increase much larger for the plerixafor dose level 0.16 mg/kg (mean fold increase FISH+, 24.3; FISH-, 10.3). Over time, the relative increase of FISH+ cells was significantly higher than that of FISH- cells, indicating preferential mobilization of cytogenetically abnormal leukemic over normal cells (p=0.005). In summary, G-CSF/plerixafor is safe in combination with the established IV busulfan/fludarabine preparative regimen for alloSCT in pts with advanced disease. Our data indicate preferential mobilization of clonal leukemic over normal cells. The objective of the ongoing Phase II study is to determine if the combination of G-CSF/plerixafor with busulfan/fludarabine improves PFS compared to historical controls receiving busulfan/fludarabine alone. We hypothesize that interventions disrupting stroma-leukemia interactions may enhance chemosensitivity and therefore the therapeutic efficacy in hematological malignancies. Disclosures: Konopleva: Genzyme: Research Funding. Off Label Use: Plerixafor for transplant in AML. Andreeff: Genzyme: Consultancy, Research Funding. Champlin: Genzyme: Consultancy, Research Funding.

scholarly journals Targeting miRNA-551b, a "Stemness"-like microRNA, to Eradicate AML (Stem) Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1494-1494
Author(s):  
Tània Martiáñez ◽  
Noortje Van Gils ◽  
David Christian De Leeuw ◽  
Eline Vermue ◽  
Arjo Rutten ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Research Funding ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Inhibition Of Proliferation ◽  
Novel Target

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

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