scholarly journals Development of Anti-CD99scFvs for the Treatment of Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4345-4345
Author(s):  
Yang Zhao ◽  
Jiawei Li ◽  
Vijaya Pooja Vaikari ◽  
Albert Lam ◽  
Atham Ali ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Mouse Model ◽  
Binding Affinity ◽  
Myeloid Leukemia ◽  
Single Chain ◽  
Xenograft Mouse Model ◽  
Acute Myeloid

Abstract CD99 has gained much attention in recent years as a novel therapeutic target in hematological malignancies, due to its upregulation in acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). We have recently shown that targeting CD99 with a knockdown approach or with commercial antibodies results in antileukemia activity in AML cells. We have also developed anti-CD99 nanoparticles and demonstrated excellent in vitro and in vivo antileukemia activity. Here we report the development of human single chain variable fragment targeting CD99 (anti-CD99 scFv) and the preclinical activity in AML cells and in AML xenograft mouse model. The anti-CD99 scFv was developed by inserting CD99scFv DNA sequence into the pFUSE vector, encoding pATL103-CD99 scFvs. To produce anti-CD99 scFv, the plasmids were transfected in Expi293 (HEK293T) cells. Then the medium was harvested and underwent two cycles of dialysis. To determine product purity, proteins were analyzed on SDS-PAGE gel stained with Coomassie blue. For each 240ml cell culture media with recombinant CD99 plasmids, we obtained about 1-2mg anti-CD99 scFv. The binding affinity of CD99 scFv to CD99 surface protein was assessed in 293T cells (CD99 null cells) which exhibited no binding and in MOLM-13 cells and MV4-11 (CD99 positive AML cells) which demonstrated strong binding. Treatment with 5uM of anti-CD99 scFv significantly reduced cell viability in both leukemic cell line MOLM-13 and MV4-11 (MOLM-13: 35.14%, P= 0.008; MV4-11: 30.17%, P=0.002) and primary AML patient blasts (29.37%, P=0.048) compared with control cells. Colony forming assay showed that anti-CD99 scFv treated AML blasts exhibited less number of colonies compared with control cells (plating efficiency (PE): 0.035% vs 0.12%). We also established the in vivo antileukemia activity of anti-CD99 scFv using MOLM-13 cells (FLT3-ITD positive AML cells) NOD scid gamma (NSG) xenograft mouse model. MOLM-13 (2.5x10^6) cells were engrafted into NSG mice via IV tail injection (N=4 mice per group). Mice were treated with PBS (group 1) or 4mg/kg of anti-CD99 scFV on days 10, 14, 18 and 22 post cell engraftment. Mice were euthanized on day 24 and levels of leukemia engraftment were assessed by flow cytometry measurements of huCD45 staining of cells collected from the bone marrow and the peripheral blood and compared between the two groups. Mice treated with four doses of 4mg/kg CD99scFv demonstrated significant reduction in leukemia engraftment in the bone marrow assessed by flow cytometry measurements of huCD45 staining compared with the PBS mice group (huCD45%: 39.7 vs 56, P = 0.0017). In conclusion, we report the development of anti-CD99 single chain variable fragments for the treatment of AML. Our study demonstrates good binding affinity and specificity and a promising preclinical antileukemia activity both in AML cells and in xenograft mouse model. Disclosures Yaghmour: Jazz: Consultancy, Honoraria; Astellas: Consultancy; Takeda: Consultancy; Incyte: Consultancy.

scholarly journals Investigating the Dual Targeting of BCL-2 and CD38 in Models of Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 10-10
Author(s):  
Jayna J Mistry ◽  
Charlotte Hellmich ◽  
Amelia Lambert ◽  
Jamie A Moore ◽  
Aisha Jibril ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Mouse Model ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Xenograft Mouse Model ◽  
Cd38 Expression ◽  
Cytotoxic Treatment ◽  
Bcl2 Expression ◽  
Acute Myeloid

Acute myeloid leukemia (AML) is correlated with poor prognosis and a high mortality. Current AML treatment often fails to achieve complete remission and relapse is common, highlighting the need for more targeted treatments. Overexpression of BCL2 is a hallmark of AML progression and is often associated with a poor response to cytotoxic treatment. BCL2 inhibition by the BH3 mimetic Venetoclax has been shown to be effective in promoting AML cell death and recently Venetoclax has received FDA approval for the treatment of AML. However, some patients do not respond or can develop resistance, therefore multiple studies for combinational therapies for Venetoclax have been researched. We have previously demonstrated CD38 inhibition by daratumumab treatment inhibits mitochondrial transfer from mesenchymal stromal cell (MSC) to AML blasts in the bone marrow (BM) microenvironment, which results in a reduction in AML derived oxidative phosphorylation and subsequent reduced leukemia growth and improved animal survival. We therefore investigated the consequences of inhibiting CD38 using daratumumab and BCL2 using Venetoclax on the AML survival. Primary AML blasts were isolated from patient's BM. CD38 expression and BCL2 expression was assessed by flow cytometry analysis of AML. AML had significantly higher BCL2 expression and a slight increase in CD38 expression compared to CD34+ cells. Venetoclax alone caused a significant decrease in cell viability, however daratumumab or in combination with Venetoclax had no additive effect on AML survival. Since AML is highly reliant on the BM microevironment we cultured AML on MSC with either Venetoclax alone, daratumumab alone, or Venetoclax and daratumumab for 24 hours. Cells were then stained with Annexin V-FITC/PI and analysed using flow cytometry. Cells underwent significantly more apoptosis in the combination Venetoclax and daratumumab treatment when compared to control AML cells. To determine the effect of Venetoclax and daratumumab treatment in preclinical models we used an NSG xenograft mouse model of AML, we transplanted MV411-luc or patient derived AML and treated the animals with either vehicle control (PBS) daratumumab (5mg/kg) on day 7 and 14 alone, Venetoclax (100mg/kg/day) alone, or both daratumumab and Venetoclax followed by bioluminescence imaging. In vivo, treatment with combination daratumumab and Venetoclax significantly reduced tumor burden and improved survival compared to control and either drug alone in the patient derived AML xenograft mouse model and in MV411. These data support the further clinical investigation of Venetoclax and Daratumumab combination as a therapeutic approach for the treatment AML. Disclosures Bowles: AbbVie: Research Funding; Janssen: Research Funding. Rushworth:Janssen: Research Funding; AbbVie: Research Funding.

scholarly journals Inhibition of Mnk kinase activity by cercosporamide and suppressive effects on acute myeloid leukemia precursors

Blood ◽  
2013 ◽  
Vol 121 (18) ◽  
pp. 3675-3681 ◽  
Author(s):  
Jessica K. Altman ◽  
Amy Szilard ◽  
Bruce W. Konicek ◽  
Philip W. Iversen ◽  
Barbara Kroczynska ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Mouse Model ◽  
Myeloid Leukemia ◽  
Kinase Activity ◽  
Xenograft Mouse Model ◽  
Key Points ◽  
Acute Myeloid

Key Points The Mnk inhibitor cercosporamide suppresses human leukemic progenitors and exhibits antileukemic effects in a xenograft mouse model. Cercosporamide enhances the antileukemic effects of cytarabine in vitro and in vivo.

scholarly journals A Novel CD49d Targeting Antisense, ATL1102, Effectively Mobilizes Acute Myeloid Leukemia Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3807-3807
Author(s):  
Yann Duchartre ◽  
EunJi Gang ◽  
Hye Na Kim ◽  
Stephanie Nicole Shishido ◽  
Muller Fabbri ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Cell Line ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Surface Expression ◽  
Leukemia Cells ◽  
Acute Myeloid

Abstract BACKGROUND: Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Acute myeloid leukemia comprises approximately one-fifth of pediatric leukemias and is the seventh most common pediatric malignancy. In children, relapse following primary therapy approaches 40%, and the 5-year event-free survival (EFS) rate is only approximately 50%. Treatment is dominated by generic chemotherapeutic agents. Novel therapeutic strategies are highly warranted. The bone marrow microenvironment has been shown to promote cell adhesion-mediated drug resistance in leukemia cells. Breaking adhesive bonds of AML cells with their protective niche to mobilize them from the bone marrow to the peripheral blood may make drug treatment more efficient. Our studies have suggested the adhesion molecule CD49d as an anchor molecule for ALL and AML cells in the bone marrow. However, as of today, no drug targeting CD49d is approved for use in leukemia. Here, we evaluate a novel human specific CD49d targeting antisense, ATL1102, in clinical development for Multiple Sclerosis, in human AML cells. METHODS: We determined CD49d expression in the human AML cell line HL-60 treated with a CD49d targeting antisense ATL1102 and antisense control by qPCR and flow cytometry. Annexin V/DAPI and BrdU stainings were used for viability determination and cell cycle assay respectively by flow cytometry. A NOD/SCID IL2Rγ-/- (NSG)xenograft model of human HL-60 cell line was used for an in vivo mobilization assay. RESULTS: To assess the on-target effect of ATL1102 on CD49d, HL-60 cells were nucleoporated with either ATL1102 or control antisense.mRNA expression of CD49dwas significantly decreased by ATL1102 treatment cells (85.2%±15.4 expression inhibition using ATL1102 1µM after 24h compared to control, p<0.001) as assessed by RT-PCR. The FACS analysis 72 hours after treatment revealed a significant decrease of surface expression of CD49d in a dose-dependent manner (99%±0.4 (1µM, *), 87.9%±8.7 (3µM) and 57.8%±7.2 ATL1102 (10µM, ***), 55.9±13.5 (30µM, **) vs 99.7%±0.1 for control antisense (30 µM), P<0.001, n=3). No significant effect on apoptosis or cell cycle was observed after ATL1102 treatment. We also evaluated the in vivo effect of ATL-1102 on mobilization of leukemia cells in a pilot experiment. For this purpose, HL-60 cells (5x106/per mouse) were injected via the tail vein in sublethally irradiated NSG mice. Presence of human ALL cells (hCD45) was determined weekly by flow cytometry of white blood cells isolated from peripheral blood (PB). 23 Days post-leukemia injection, mice were treated with either antisense control (CTRL) (n=3), ATL1102 (50mg/kg, n=2). Peripheral blood was drawn before and 24 hours after ATL1102-treatment. ATL1102 induced a strong mobilization of AML cells to the PB of leukemia-recipient mice compared to control antisense treated-mice (69.1% and 87.7% vs 1.1%, 0.2% and 28.1% for ATL1102 (50mg/ml) and CTRL treated-mice respectively. The mobilized cells show a decrease of surface expression of CD49d (16.8%±9.2% vs 32.8%±16.7%), although this was not of statistical significance in this pilot experiment. Experiments to repeat this assay with large numbers of mice are in progress as well as experiments to determine the initial location of the mobilized AML cells and synergy of ATL1102 with chemotherapy are ongoing. CONCLUSION: We demonstrate that ATL1102 can efficiently decrease CD49d expression in AML cell line in vitro and in vivo, and that ATL1102 leads to mobilization of AML cells to the peripheral blood. Disclosures Wayne: NIH: Patents & Royalties; Medimmune: Honoraria, Other: travel support, Research Funding; Kite Pharma: Honoraria, Other: travel support; Pfizer: Honoraria; Spectrum Pharmaceuticals: Honoraria, Other: travel support, Research Funding. Tachas:Antisense Therapeutics Ltd: Employment, Equity Ownership, Patents & Royalties.

scholarly journals An Advanced Preclinical Mouse Model for Acute Myeloid Leukemia Using Patients' Cells of Various Genetic Subgroups and In Vivo Bioluminescence Imaging

PLoS ONE ◽  
2015 ◽  
Vol 10 (3) ◽  
pp. e0120925 ◽  
Author(s):  
Binje Vick ◽  
Maja Rothenberg ◽  
Nadine Sandhöfer ◽  
Michela Carlet ◽  
Cornelia Finkenzeller ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Mouse Model ◽  
Myeloid Leukemia ◽  
Bioluminescence Imaging ◽  
In Vivo Bioluminescence Imaging ◽  
Acute Myeloid

scholarly journals Acute myeloid leukemia induces protumoral p16INK4a-driven senescence in the bone marrow microenvironment

Blood ◽  
2019 ◽  
Vol 133 (5) ◽  
pp. 446-456 ◽  
Author(s):  
Amina M. Abdul-Aziz ◽  
Yu Sun ◽  
Charlotte Hellmich ◽  
Christopher R. Marlein ◽  
Jayna Mistry ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Stromal Cell ◽  
Cell Senescence ◽  
Senescent Phenotype ◽  
Age Related ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is an age-related disease that is highly dependent on the bone marrow (BM) microenvironment. With increasing age, tissues accumulate senescent cells, characterized by an irreversible arrest of cell proliferation and the secretion of a set of proinflammatory cytokines, chemokines, and growth factors, collectively known as the senescence-associated secretory phenotype (SASP). Here, we report that AML blasts induce a senescent phenotype in the stromal cells within the BM microenvironment and that the BM stromal cell senescence is driven by p16INK4a expression. The p16INK4a-expressing senescent stromal cells then feed back to promote AML blast survival and proliferation via the SASP. Importantly, selective elimination of p16INK4a+ senescent BM stromal cells in vivo improved the survival of mice with leukemia. Next, we find that the leukemia-driven senescent tumor microenvironment is caused by AML-induced NOX2-derived superoxide. Finally, using the p16-3MR mouse model, we show that by targeting NOX2 we reduced BM stromal cell senescence and consequently reduced AML proliferation. Together, these data identify leukemia-generated NOX2-derived superoxide as a driver of protumoral p16INK4a-dependent senescence in BM stromal cells. Our findings reveal the importance of a senescent microenvironment for the pathophysiology of leukemia. These data now open the door to investigate drugs that specifically target the “benign” senescent cells that surround and support AML.

scholarly journals Silencing long non-coding RNA XIST suppresses drug resistance in acute myeloid leukemia through down-regulation of MYC by elevating microRNA-29a expression

2020 ◽  
Vol 26 (1) ◽  
Author(s):  
Chong Wang ◽  
Lingling Li ◽  
Mengya Li ◽  
Weiqiong Wang ◽  
Yanfang Liu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Cellular Viability ◽  
Down Regulation ◽  
Acute Myeloid

Abstract Background Long non-coding RNAs (lncRNAs) are biomarkers participating in multiple disease development including acute myeloid leukemia (AML). Here, we investigated molecular mechanism of X Inactive-Specific Transcript (XIST) in regulating cellular viability, apoptosis and drug resistance in AML. Methods XIST, miR-29a and myelocytomatosis oncogene (MYC) expression in AML bone marrow cells collected from 62 patients was evaluated by RT-qPCR and Western blot analysis. Besides, the relationship among XIST, miR-29a and MYC was analyzed by dual luciferase reporter assay, RIP, and RNA pull down assays. AML KG-1 cells were treated with anti-tumor drug Adriamycin. The role of XIST/miR-29a/MYC in cellular viability, apoptosis and drug resistance in AML was accessed via gain- and loss-of-function approaches. At last, we evaluated role of XIST/miR-29a/MYC on tumorigenesis in vivo. Results XIST and MYC were up-regulated, and miR-29a was down-regulated in AML bone marrow cells. Silencing XIST inhibited cellular activity and drug resistance but promoted cellular apoptosis of KG-1 cells by down-regulating MYC. XIST inhibited miR-29a expression to up-regulate MYC. Moreover, silencing XIST inhibited tumorigenesis of AML cells in vivo. Conclusions Overall, down-regulation of XIST decreased MYC expression through releasing the inhibition on miR-29a, thereby reducing drug resistance, inhibiting viability and promoting apoptosis of AML cells.

Cellular pharmacokinetics of daunorubicin: relationships with the response to treatment in patients with acute myeloid leukemia.

1988 ◽  
Vol 6 (5) ◽  
pp. 802-812 ◽  
Author(s):  
E Kokenberg ◽  
P Sonneveld ◽  
W Sizoo ◽  
A Hagenbeek ◽  
B Löwenberg
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Response To Treatment ◽  
Remission Induction ◽  
Pharmacokinetic Parameters ◽  
Induction Treatment ◽  
Blast Cells ◽  
Acute Myeloid

In an attempt to identify pharmacokinetic factors that determine the response of acute myeloid leukemia (AML) patients to induction chemotherapy, we determined the concentrations of daunorubicin (DNR) and the main metabolite daunorubicinol (DOL) in vivo and particularly evaluated the concentrations in blood and bone marrow nucleated cells. Cell measurements were obtained in 37 evaluable patients during their first remission induction treatment with DNR and cytarabine (ara-C) and directly compared with the plasma distribution kinetics of DNR. We show that (1) plasma DNR concentrations do not correlate with DNR concentrations in bone marrow nucleated cells; but (2) plasma area under the curve (AUC) values of DNR correlate inversely (P less than .01) with AUC values of DNR in WBCs; (3) concentrations of DNR in WBCs correlate positively (P less than .01) with DNR concentrations in bone marrow nucleated cells; and (4) the concentrations of DNR in WBCs show a negative correlation (P less than .01) with the numbers of peripheral blast cells at diagnosis. We then tested whether the pharmacokinetic parameters had predictive value for the clinical outcome of therapy, but none of the plasma levels or WBC and bone marrow concentrations of DNR predicted treatment outcome. The inverse correlation between the concentrations of DNR in WBC and the numbers of peripheral blast cells suggests that the effective DNR concentrations achieved intracellularly are mainly a function of the tumor load so that lesser amounts of DNR accumulate intracellularly when the AML cell numbers in blood are higher.

In vivo administration of vascular endothelial growth factor (VEGF) and its antagonist, soluble neuropilin-1, predicts a role of VEGF in the progression of acute myeloid leukemia in vivo

Blood ◽  
2002 ◽  
Vol 100 (13) ◽  
pp. 4622-4628 ◽  
Author(s):  
Gunter Schuch ◽  
Marcelle Machluf ◽  
Georg Bartsch ◽  
Masashi Nomi ◽  
Henri Richard ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Vascular Endothelial Growth Factor ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Vascular Endothelial ◽  
Neuropilin 1 ◽  
Endothelial Growth Factor ◽  
Acute Myeloid

Recent findings implied that the progression of hematologic malignancies, like that of solid tumors, is dependent on neovascularization. Recent studies on patients with acute myeloid leukemia (AML) showed increased levels of leukocyte-associated vascular endothelial growth factor (VEGF) and neovascularization of the bone marrow. Murine (32D, M1) and human (HEL, U937, and UKE-1) leukemic cell lines and freshly isolated leukemic cells were analyzed for the expression of VEGF and VEGF receptor mRNA. The expression of VEGF and VEGF receptors KDR and neuropilin-1 (NRP-1) was detected in these cells. In a murine chloroma model, delivery of VEGF165using microencapsulation technology resulted in enhanced tumor growth and vascularization, whereas treatment with a VEGF antagonist soluble NRP-1 (sNRP-1) inhibited tumor angiogenesis and growth. In a systemic leukemia model, survival of mice injected with adenovirus (Ad) encoding for Fc-sNRP-1 (sNRP-1 dimer) was significantly prolonged as compared with mice injected with Ad-LacZ. Further analyses showed a reduction in circulating leukemic cells and infiltration of liver and spleen as well as bone marrow neovascularization and cellularity. Taken together, these results demonstrate that angiogenic factors such as VEGF promote AML progression in vivo. The use of VEGF antagonists as an antiangiogenesis approach offers a potential treatment for AML. Finally, our novel in vivo drug delivery model may be useful for testing the activities of other peptide antiangiogenic factors.

The Growth Inhibition and Apoptosis Induction of Acute Myeloid Leukemia Blast Population by the Blocking IGF-IR Pathway.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4515-4515
Author(s):  
Si Sun ◽  
Yanli He ◽  
Xingbing Wang ◽  
Wei Liu ◽  
Jun Liu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Pi3k Pathway ◽  
Leukemia Cell Line ◽  
Free System ◽  
Acute Myeloid ◽  
Marrow Cells

Abstract The insulin-like growth factor-1receptor (IGF-1R) is overexpressed in a variety of tumors and has been associated with cancer development. Here, we analysis the IGF-IR expression on the bone marrow cells from 45 newly diagnosed patients with acute myeloid leukemia (AML) by flow cytometry. IGF-1R universally expressed on AML blasts and the leukemia cell line HL-60, did not show significant correlation with FAB subtypes. However, the bone marrow cells from AML patients with high myeloblast counts (&gt;80%) generally showed brighter IGF-IR expressions, which indicated the IGF-IR pathway might play an important role for AML blast proliferation and survival. Indeed, blocking the IGF-1R pathway by neutralizing monoclonal antibodies could reduce the proliferation of HL-60 cells by 38.28% at 48 hr. This inhibitory effect on blast growth was observed in 4 of 5 AML samples. In the same IGF-1R blocking treatment, the apoptosis of HL-60 cells was significantly induced, resulting in apoptosis of 57% of the cell population with the measurement of Annexin V vs PI staining by flow cytometry. The control contained only 20% apoptotic cells. We also demonstrated that the blockade of the IGF-1R pathway inhibited the phophorylation of the PI3K pathway component Akt in HL-60 cells when cultured in a serum free system with a supplement of 50ng/ml exogenous IGF. Since PI3K pathway activation greatly contributes to the proliferation, survival and drug resistance of AML, it is of interest to study whether blockading IGF-IR could also inhibit the PI3K pathway in primary AML blasts and synergize other anti-leukemia agents to improve the therapeutic effectiveness. Conclusions: IGF-IR may play an important role in the proliferation and survival of the AML blast population; Blocking the IGF-IR pathway could significantly inhibit the growth of AML blasts and considerably induce the apoptosis of AML blasts; IGF-IR could become a critical molecular target in anti-leukemia drug discovery.

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