scholarly journals Preclinical activity of a novel CRM1 inhibitor in acute myeloid leukemia

Blood ◽  
2012 ◽  
Vol 120 (9) ◽  
pp. 1765-1773 ◽  
Author(s):  
Parvathi Ranganathan ◽  
Xueyan Yu ◽  
Caroline Na ◽  
Ramasamy Santhanam ◽  
Sharon Shacham ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Nuclear Export ◽  
Myeloid Leukemia ◽  
Fold Increase ◽  
In Vivo Studies ◽  
Ic50 Values ◽  
Acute Myeloid

AbstractChromosome maintenance protein 1 (CRM1) is a nuclear export receptor involved in the active transport of tumor suppressors (eg, p53 and nucleophosmin) whose function is altered in cancer because of increased expression and overactive transport. Blocking CRM1-mediated nuclear export of such proteins is a novel therapeutic strategy to restore tumor suppressor function. Orally bioavailable selective inhibitors of nuclear export (SINE) that irreversibly bind to CRM1 and block the function of this protein have been recently developed. Here we investigated the antileukemic activity of KPT-SINE (KPT-185 and KPT-276) in vitro and in vivo in acute myeloid leukemia (AML). KPT-185 displayed potent antiproliferative properties at submicromolar concentrations (IC50 values; 100-500nM), induced apoptosis (average 5-fold increase), cell-cycle arrest, and myeloid differentiation in AML cell lines and patient blasts. A strong down-regulation of the oncogene FLT3 after KPT treatment in both FLT3-ITD and wild-type cell lines was observed. Finally, using the FLT3-ITD–positive MV4-11 xenograft murine model, we show that treatment of mice with oral KPT-276 (analog of KPT-185 for in vivo studies) significantly prolongs survival of leukemic mice (P < .01). In summary, KPT-SINE are highly potent in vitro and in vivo in AML. The preclinical results reported here support clinical trials of KPT-SINE in AML.

The Novel Fluoropyrimidine FdUMP[10] Is Highly Active Against Acute Myeloid Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3302-3302
Author(s):  
Timothy Pardee ◽  
Evan Gomes ◽  
Jamie Jennings-Gee ◽  
David L. Caudell ◽  
William Gmeiner
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Single Agent ◽  
The Novel ◽  
Highly Active ◽  
Ic50 Values ◽  
Acute Myeloid

Abstract Abstract 3302 Acute Myeloid Leukemia (AML) is an aggressive myeloid malignancy that leads to marrow failure and death. This disease affects approximately 12,000 people per year in the United States, causing 9,000 deaths. Despite decades of research, therapy remains essentially unchanged and outcomes are poor. In patients over the age of 60 less then 10% of patients survive 5 years from diagnosis. There is a desperate need for the identification of new active agents with favorable toxicity profiles. The novel polymeric fluoropyrimidine (FP) FdUMP[10] is an oligodeoxynucleotide pro-drug of the thymidylate synthase (TS)-inhibitory FP metabolite 5-fluoro-2'-deoxyuridine-5`-O-monophosphate (FdUMP). The observation that this compound was highly active against several leukemia lines in the NCI 60 cell line screen prompted us to evaluate its activity in several preclinical models of AML. In vitro, FdUMP[10] exhibited remarkable activity against 3 human acute leukemia cell lines, HL60, Jurkat and THP-1, with IC50 values of 3.378 nM (95% CI 2.984 to 3.825), 5.438 nM (4.609 to 6.417) and 4.093 nM (3.413 to 4.907) respectively. We next tested its efficacy against a more genetically defined murine model of AML driven by expression of MLL-ENL. FdUMP[10] exhibited even greater activity against all murine lines tested. The IC50 values of FdUMP[10] against two MLL-ENL driven murine AML cell lines were 214 pM (95%CI 178.9 to 255.9) and 292.3 pM (251.8 to 339.4). The IC50 values observed for FdUMP[10] for all the murine lines tested were lower than both Ara-C (30-40 nM) and doxorubicin (2-4 nM). We then determined the cytotoxic mechanism for FdUMP[10] in vitro. Upon treatment with FdUMP[10] both the human and murine cell lines undergo extensive apoptosis as indicated by Annexin V and propidium iodide staining. Treated cells developed γH2AX foci, rapid and complete TS inhibition and display trapped Topoisomerase I (Topo I) cleavage complexes. FdUMP[10]-mediated induction of apoptosis was p53 independent as murine AML cells that had p53 knocked down by RNAi demonstrated resistance to both Ara-C and doxorubicin, but not to FdUMP[10]. We next tested the efficacy of FdUMP[10] in vivo. The MLL-ENL driven murine AML model results in blasts that can be transplanted into sublethally irradiated, immunocompetent, syngeneic recipients. The recipients develop a fatal and therapy-resistant AML. Lines were generated that expressed a luciferase reporter. Animals were imaged 6–7 days after injection of the leukemias to ensure engraftment and then began treatment with either the combination of Ara-C plus doxorubicin, single-agent FdUMP[10], or observation. Studies were performed using 2 doses of FdUMP[10] at 150 or 300 mg/kg injected on days 1 and 3 and compared to animals treated with 100 mg/kg Ara-C and 3mg/kg doxorubicin injected on days 1 through 5. Both treatments resulted in a statistically significant survival advantage over observation. A preliminary toxicology study compared FdUMP[10], 150 mg/kg daily, to 5-fluorouracil (5 FU), 150 mg/kg daily, or the combination of Ara-C at 100 mg/kg plus doxorubicin at 3 mg/kg daily. All groups were treated for 3, 4 or 5 days. On day 6 animals were sacrificed and organs harvested, sectioned, and stained. Slides were then reviewed by a veterinary pathologist. Tissues most affected were the small intestine, colon, and the bone marrow. The 5FU-treated animals had severe villous blunting and fusion with crypt necrosis in both large and small intestine. In contrast, FdUMP[10]-treated animals had only mild crypt epithelial apoptosis with mitoses. The 5 FU and Ara-C plus doxorubicin groups had a severe pan-cytopenia in the marrow compared to FdUMP[10] treated animals that showed only minimal to mild apoptosis. These data support the assertion that FdUMP[10] has lower toxicity then either Ara-C plus doxorubicin or identically dosed 5 FU. In summary FdUMP[10] exhibited remarkable activity against AML cells in vitro and in vivo. Additionally, FdUMP[10] had decreased toxicity compared to treatment with either single agent 5 FU or combination treatment with Ara-C plus doxorubicin. Disclosures: Gmeiner: Salzburg Therapeutics: Equity Ownership.

scholarly journals Anti-leukemia properties of cadmium nanoparticles in the in vitro and in vivo condition: A chemobiological study

2021 ◽  
Author(s):  
Yudi Miao ◽  
Behnam Mahdavi ◽  
Mohammad Zangeneh
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Aqueous Extract ◽  
Myeloid Leukemia ◽  
Antioxidant Properties ◽  
Leukemia Cell ◽  
Sem Images ◽  
Acute Myeloid

IntroductionThe present study investigated the anti-acute myeloid leukemia effects of Ziziphora clinopodides Lam leaf aqueous extract conjugated cadmium nanoparticles.Material and methodsTo synthesize CdNPs, Z. clinopodides aqueous extract was mixed with Cd(NO3)2 .4H2O. The characterization of the biosynthesized cadmium nanoparticles was carried out using many various techniques such as UV-Vis. and FT-IR spectroscopy, XRD, FE-SEM, and EDS.ResultsThe uniform spherical morphology of NPs was proved by FE-SEM images with NPs the average size of 26.78cnm. For investigating the antioxidant properties of Cd(NO3)2, Z. clinopodides, CdNPs, and Daunorubicin, the DPPH test was used. The cadmium nanoparticles inhibited half of the DPPH molecules in a concentration of 196 µg/mL. To survey the cytotoxicity and anti-acute myeloid leukemia effects of Cd(NO3)2, Z. clinopodides, CdNPs, and Daunorubicin, MTT assay was used on the human acute myeloid leukemia cell lines i.e., Murine C1498, 32D-FLT3-ITD, and Human HL-60/vcr. The IC50 of the cadmium nanoparticles was 168, 205, and 210 µg/mL against Murine C1498, 32D-FLT3-ITD, and Human HL-60/vcr cell lines, respectively. In the part of in vivo study, DMBA was used for inducing acute myeloid leukemia in mice. CdNPs similar to daunorubicin ameliorated significantly (p≤0.01) the biochemical, inflammatory, RBC, WBC, platelet, stereological, histopathological, and cellular-molecular parameters compared to the other groups.ConclusionsAs mentioned, the cadmium nanoparticles had significant anti-acute myeloid leukemia effects. After approving the above results in the clinical trial studies, these cadmium nanoparticles can be used as a chemotherapeutic drug to treat acute myeloid leukemia in humans.

scholarly journals The Salt-Inducible Kinase inhibitor YKL-05-099 suppresses MEF2C function and acute myeloid leukemia progressionin vivo

2019 ◽  
Author(s):  
Yusuke Tarumoto ◽  
Shan Lin ◽  
Jinhua Wang ◽  
Joseph P. Milazzo ◽  
Yali Xu ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Disease Progression ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Clinical Investigation ◽  
Genetic Targeting ◽  
Acute Myeloid

AbstractLineage-defining transcription factors (TFs) are compelling targets for leukemia therapy, yet they are among the most challenging proteins to modulate directly with small molecules. We previously used CRISPR screening to identify a Salt-Inducible Kinase 3 (SIK3) requirement for the growth of acute myeloid leukemia (AML) cell lines that overexpress the lineage TF MEF2C. In this context, SIK3 maintains MEF2C function by directly phosphorylating histone deacetylase 4 (HDAC4), a repressive cofactor of MEF2C. Here, we evaluated whether inhibition of SIK3 with the tool compound YKL-05-099 can suppress MEF2C function and attenuate disease progression in animal models of AML. Genetic targeting of SIK3 or MEF2C selectively suppressed the growth of transformed hematopoietic cells underin vitroandin vivoconditions. Similar phenotypes were obtained when exposing cells to YKL-05-099, which caused cell cycle arrest and apoptosis in MEF2C-expressing AML cell lines. An epigenomic analysis revealed that YKL-05-099 rapidly suppressed MEF2C function by altering the phosphorylation state and nuclear localization of HDAC4. Using a gatekeeper allele ofSIK3, we found that the anti-proliferative effects of YKL-05-099 occurred through on-target inhibition of SIK3 kinase activity. Based on these findings, we treated two different mouse models of MLL-AF9 AML with YKL-05-099, which attenuated disease progressionin vivoand extended animal survival at well-tolerated doses. These findings validate SIK3 as a therapeutic target in MEF2C-positive AML and provide a rationale for developing drug-like inhibitors of SIK3 for definitive pre-clinical investigation and for studies in human patients with leukemia.Key PointsAML cells are uniquely sensitive to genetic or chemical inhibition of Salt-Inducible Kinase 3in vitroandin vivo.A SIK inhibitor YKL-05-099 suppresses MEF2C function and AMLin vivo.

The BTK Inhibitor Ibrutinib Blocks SDF1/CXCR4 Mediated Migration of Acute Myeloid Leukemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 915-915
Author(s):  
Stuart A Rushworth ◽  
Lyubov Zaitseva ◽  
Megan Y Murray ◽  
Matthew J Lawes ◽  
David J MacEwan ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Cell Trafficking ◽  
Acute Myeloid

Abstract Introduction Despite recent significant progress in the understanding of the biology of acute myeloid leukemia (AML) the clinical outcomes for the majority of patients diagnosed with AML presently remain poor. Consequently, there is an urgent need to identify pharmacological strategies in AML, which are not only effective but can be tolerated by the older, less well patient. Recently our group and others have shown that there is high Bruton’s Tyrosine Kinase (BTK) phosphorylation and RNA expression in AML. Moreover, our recent study described for the first time that ibrutinib and BTK-targeted RNA interference reduced factor-induced proliferation of both AML cell lines and primary AML blasts, as well as reducing AML blast adhesion to bone marrow stromal cells. Inhibition of BTK has been shown to regulate chronic lymphocytic leukemia, mantle cell lymphoma and multiple myeloma cell migration by inhibiting SDF1 (stromal derived factor 1) induced CXCR4 regulated cell trafficking. Here we report that in human AML ibrutinib in addition functions in a similar way to inhibit SDF1/CXCR4-mediated AML migration at concentrations achievable in vivo. Methods To investigate the role of BTK in regulating AML migration we used both pharmacological inhibitor ibrutinib and genetic knockdown using a lentivirus mediated BTK targeted miRNA in primary AML blasts and AML cell lines. We examined migration of AML blasts and AML cells to SDF-1 using Transwell permeable plates with 8.0µM pores. Western blotting was used to examine the role of SDF-1 in regulating BTK, AKT and MAPK activation in primary AML blasts. Results We initially examined the expression of CXCR4 in human AML cell lines and found that 4/4 cell lines were positive for CXCR4 expression. Next we examined the effects of ibrutinib on the migration of the AML cell lines U937, MV4-11, HL60 and THP-1 in response to SDF1. We found that ibrutinib can inhibit the migration of all AML cell lines tested. We tested the in-vitro activity of ibrutinib on SDF-1 induced migration in a spectrum of primary AML blasts from a wide age spectrum of adult patients and across a range of WHO AML subclasses and found that ibrutinib significantly inhibits primary AML blast migration (n=12). Next we found that ibrutinib can inhibit SDF-1 induced BTK phosphorylation and downstream MAPK and AKT signalling in primary AML blast. Finally to eliminate the problems associated with off target ibrutinib activity we evaluated migration of AML cells lines using genetic inhibition of BTK. The introduction of BTK-specific miRNA dramatically inhibited the expression of BTK in THP-1 and HL60 and reduced SDF1 mediated migration confirming that BTK is involved in regulating AML migration in response to SDF1. Conclusions These results reported here provide a molecular mechanistic rationale for clinically evaluating BTK inhibition in AML patients and suggests that in some AML patients the blasts count may initially rise in response to ibrutinib therapy, analgous to similar clinical observations in CLL. Disclosures No relevant conflicts of interest to declare.

Vgamma9Vdelta2 T Cells-Based Immunotherapy Targeting BTN3 Molecules in Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3726-3726
Author(s):  
Daniel Olive ◽  
Audrey Benyamine ◽  
Aude Le Roy ◽  
Rémy Castellano ◽  
Julie Gertner-Dardenne ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Mevalonate Pathway ◽  
Conflicts Of Interest ◽  
Tumor Burden ◽  
Acute Myeloid

Abstract As they can kill Acute Myeloid Leukemia (AML) blasts in vitro and in vivo, Vg9Vd2T cells are key players in the design of new strategies of immunotherapy. AminoBisphonates (NBP) can enhance their activation in vitro and in vivo. Their combination with low-dose IL2 has shown promising results in 2 patients with AML who underwent partial remission. NBP treatment of blasts inhibits the Mevalonate pathway. The subsequent accumulation of Isopentenyl Diphosphate sensitize AML blasts to Vg9Vd2T cells killing but some AML cell lines blasts are resistant to this TCR mediated-lysis. Butyrophilin 3 A1 (BTN3A1) has been shown to be involved in IPP recognition and Vg9Vd2 T cells activation. Agonist monoclonal antibodies (mAb) recognizing the 3 isoforms of BTN3, can trigger BTN3 on tumor cell lines and sensitize them to Vg9Vd2 T cells lysis. We show that primary AML blasts from patient at diagnosis are heterogeneously killed by allogenic-IL-2-NBP-expanded Vg9Vd2 T. Some are resistant to this lysis and/or poorly sensitized by NBP. BTN3 molecules are highly expressed by blasts of AML cell lines and primary AML samples. We show that treatment of primary AML blasts with agonist anti-BTN3 mAb can overcome the resistance to Vg9Vd2 cells lysis in vitro. We assess this effect in vivo, showing that the addition of agonist anti-BTN3 mAb to Vg9Vd2 cells infusion decreased the tumor burden and increased the survival of NOG mice xenografted with luciferase-transduced U937 cell line. We confirm this effect in a model of mice xenografted with primary AML blasts, showing that treatment with anti-BTN3 mAb added to Vg9Vd2 cells infusion can decrease the number of blastic cells in the spleen, bone marrow and the blood, without requiring additional cytokine infusion. This drastic effect on sensitization of primary AML blasts to Vg9Vd2T cells killing could be of great interest especially in cases of refractory or relapsing AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals T cell engaging bispecific antibodies targeting CD33 IgV and IgC domains for the treatment of acute myeloid leukemia

2021 ◽  
Vol 9 (5) ◽  
pp. e002509
Author(s):  
Sayed Shahabuddin Hoseini ◽  
Mallika Vadlamudi ◽  
Madelyn Espinosa-Cotton ◽  
Hoa Tran ◽  
Yi Feng ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Bispecific Antibodies ◽  
Immunodeficient Mice ◽  
Cytotoxicity Assays ◽  
Acute Myeloid

BackgroundAcute myeloid leukemia (AML) remains one of the most challenging hematological malignancies. Despite progress in therapeutics, majority of patients succumb to this neoplasm. CD33 is a proven therapeutic target, given its expression on most AML cells. Almost all anti-CD33 antibodies target the membrane distal immunoglobulin V (IgV) domain of the CD33 extracellular domain.MethodsIn this manuscript, we present data on three bispecific antibodies (BsAbs) against the CD33 IgV and membrane proximal immunoglobulin C (IgC) domains. We use in vitro binding and cytotoxicity assays to show the effect of these BsAbs on AML cell lines. We also use immunodeficient mice-bearing leukemias from cell lines and patient-derived xenografts to show the effect of these BsAbs in vivo.ResultsIn vitro, the IgV-targeting BsAb had higher binding to AML cell lines using flow cytometry and delivered more potent cytotoxicity in T-cell-dependent cytotoxicity assays; importantly, the IgC domain-targeting outperformed the IgV domain-targeting BsAb in medullary and extramedullary leukemia animal models.ConclusionsThese data support further clinical development of this BsAb for first-in-human phase I clinical trial.

The PI3K Inhibitor GDC-0941 Induces Growth Arrest and Apoptosis of Acute Myeloid Leukemia Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3787-3787
Author(s):  
Laura Sun ◽  
Leanne Berry ◽  
Changchun Du ◽  
Xiaoju Max Ma ◽  
Lily Shi ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Pi3k Inhibitor ◽  
Employment Equity ◽  
Disease Heterogeneity ◽  
Equity Ownership ◽  
Acute Myeloid

Abstract Abstract 3787 Poster Board III-723 Acute myeloid leukemia (AML) is a biologically and molecularly heterogeneous malignancy characterized by an accumulation of myeloid-lineage cells in the blood and bone marrow. Phosphatidylinositol 3' kinase (PI3K) -mediated signaling is frequently dysregulated in cancer and controls fundamental cellular functions such as cell migration, growth, survival and development of drug resistance in many cancers, including AML, and therefore represents an attractive therapeutic target, even against the backdrop of AML disease heterogeneity. In AML, for example, activating mutations in Ras and Flt3 are common and a number of autocrine and paracrine signaling mechanisms have been proposed as important disease mediators; all converge on PI3K as an important signal transduction node to provide growth- and survival-promoting effects. Here, we demonstrate that PI3K p110-a,b,d catalytic subunits are all prevalent across a panel of ∼30 cell lines and that the majority of cell lines as well as primary patient samples show evidence of PI3K pathway activation. We demonstrate in vitro with both cell lines and patient cell isolates, that a potent and selective pan-isoform PI3K inhibitor, GDC-0941, modulates pharmacodynamic markers such as p-Akt, p-4E-BP1, and p-FOXO-3a, and that treated cells show a G0/G1 cell-cycle arrest. A strong induction of apoptosis is observed by annexin/PI staining with upregulation of cleaved caspases and PARP. Preliminary observations suggest significant induction of p27 and Bim as direct transcriptional targets of FOXO-3a, as well as downregulation of c-Myc levels; these targets may represent key mediators of observed cellular effects and are the subject of ongoing work. Additional studies are currently underway to identify clinical standard-of-care agents and new molecular entities (pipeline NMEs) that combine favorably with PI3k-inhibition in vitro, and the results of several in-vivo xenograft studies will also be presented. Disclosures: Sun: Genentech Inc.: Employment. Berry:Genentech: Employment, Patents & Royalties. Du:Genentech, Inc.: Employment, Equity Ownership. Ma:Genentech: Employment, Patents & Royalties. Shi:Genentech: Employment, Patents & Royalties. Medeiros:Genentech: Research Funding. Ebens:Genentech, Inc.: Employment, Equity Ownership, Patents & Royalties.

Dendrogenin_A : A Natural Liver X Receptor Modulator for the Treatment of Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3767-3767
Author(s):  
Christian Recher ◽  
Marion David ◽  
Philippe de Medina ◽  
Cécile Bize ◽  
Nizar Serhan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Equity Ownership ◽  
Kg1 Cells ◽  
Acute Myeloid

Abstract Acute Myeloid Leukemia (AML) is the most common type of leukemia in adults. Despite intensive research, current treatments remain unsatisfactory with only 40% of younger (<60 years) and less than 10% of older (>60 years) AML patients achieving long-term complete remission. Consequently, drugs with novel mechanism of action are urgently needed to improve the outcome of these patients. We have recently identified Dendrogenin A (DDA) as a cholesterol metabolite present in normal cells but undetectable in various cancer cell lines including AML (de Medina et al, Nat Commun, 2013). DDA, the first steroidal alkaloid identified in mammals, exhibited strong anticancer effects against different tumor models in vitro and in vivo. In this study, we investigated the antileukemic potency of DDA in AML. We demonstrated that DDA exerts potent cytotoxic effect in a large panel of AML cell lines and cytogenetically and molecularly diverse primary AML patient samples (n=50) with a median IC50 of 3.3 µM (range 1.2-10 µM). We determined that DDA triggers both apoptosis and cytotoxic autophagy on AML cells. Macroautophagy was characterized by the accumulation of autophagic vacuoles and the stimulation of autophagic flux. As opposed to conventional chemotherapies, the antileukemic effect of DDA was similarly efficient in both immature stem/progenitor CD34+CD38-CD123+ subpopulation and leukemic bulk. Interestingly, the antileukemic activity of DDA on AML patient samples was not correlated to usual prognostic factors such as adverse cytogenetic risk karyotype, clonogenic ability, white blood cells count and FLT3-ITD or NPM status. Pharmacokinetic studies revealed that both per os (PO) and intraperitoneal (IP) administration led to a good absorption with calculated bioavailability of 74% (PO) and 48% (IP), showing that these modes of administration are relevant to in vivo preclinical studies. We then examined the in vivo anti-leukemic efficacy of DDA in NOD/SCID mice injected subcutaneously with HL60 and KG1 cells. We demonstrated that daily administration of DDA (20 mg/kg IP or 40 mg/kg PO) significantly reduced KG1 and HL60 tumor growth. Immunohistochemical analysis revealed that AML xenografts from mice exposed to DDA display a 3.5 fold increase of LC3 punctated cells and a decreased P62 level highlighting that DDA induces autophagy in vivo. Furthermore, DDA significantly kills AML cells in bone marrow and brain (55±5.6% reduction of viable CD45+ cells), and strongly reduces (57±7.8%) the total cell tumor burden in bone marrow and spleen in established disease models (eg. orthotopically engraftment of HL60 cells and three primary AML patient cells via tail vein injection in NOD/SCID/IL2Rγc-deficient mice). In addition, we showed that DDA is well tolerated in mice at effective dose and spares normal hematopoietic stem/progenitor cells from healthy donor. Mechanistic studies revealed that DDA is a natural modulator of the Liver X Receptor (LXR), a nuclear receptor involved in cholesterol homeostasis, immunity and proliferation. We found that the silencing of LXRβ gene prevents the capacity of DDA to trigger both cell death and autophagy on AML cells in vitro. In addition, DDA failed to block tumor development and to trigger autophagy on LXRβ-invalidated KG1 cells xenografted on NOD/SCID mice. Moreover, DDA strongly stimulates the expression of the myeloid leukemogenesis tumor suppressors Nur77 and Nor1 through an LXRβ-dependent mechanism. Interestingly, DDA triggers the relocation of Nur77 to the mitochondria, a process associated with both apoptosis and autophagic cell death. This study provides a strong rationale to bring DDA in clinical trials for patients with AML. Disclosures de Medina: Affichem: Employment. Bize:Affichem: Employment. Paillasse:Affichem: Employment. Noguer:Affichem: Employment. Sarry:Affichem: Equity Ownership. Silvente-Poirot:Affichem: Equity Ownership. Poirot:Affichem: Equity Ownership.

In Vitro and In Vivo Anti-Leukemic Effects of KPT-9274, a Reported PAK4 Allosteric Modulator, in Acute Myeloid Leukemia: Promising Results Justifying Further Development in This Disease

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2471-2471
Author(s):  
Shaneice Mitchell ◽  
Shelley Orwick ◽  
Matthew Cannon ◽  
Virginia M. Goettl ◽  
Taylor D. LaFollette ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Flow Cytometric Analysis ◽  
Time Dependent ◽  
Inhibition Of Proliferation ◽  
Acute Myeloid

Abstract Acute Myeloid Leukemia (AML) is the most common adult acute leukemia and is characterized by numerous driver mutations and/or cytogenetic rearrangements that promote disruption of stem cell/early myeloid progenitor differentiation, apoptosis, and proliferation. Identification of both personalized targets specific to a mutation or genomic abnormality and also global ubiquitous tumor-related targets relevant to AML represents a high priority to improve therapy. p21 protein (Cdc42/Rac)-activated kinase 4 (PAK4) is involved in disease progression for several solid tumors but its expression and contribution to disease pathogenesis in AML has not been examined. Since multiple cellular pathways important in AML (e.g., RAS and Wnt/β-catenin) are regulated by PAKs, we hypothesized PAK4 (and other family members) could represent an attractive pharmacologic target. We first evaluated the expression of PAK4 in AML cell lines and patient blasts (55 patients) using RNA sequencing and Western blot confirmation. This demonstrated PAK4 to be abundantly expressed at the mRNA and protein level in virtually all the analyzed samples. We then tested the in vitro effects of the PAK4 allosteric modulators (PAMs) KPT-9274 (clinical candidate) and KPT-9331 (tool compound) on AML cell lines. These included MV4-11, HL-60, THP-1 and Kasumi-1. Cell lines were treated for 24, 48 and 72 with PAMs KPT-9274 and KPT-9331 at dosages ranging from 1nM to 10uM. Proliferation was measured by MTS assay. All cell lines showed a dose-and time- dependent decrease in cell proliferation with IC50 ranging from 0.14 to 0.28 µM for both compounds. Cell lines with low protein and mRNA expression of PAK4, such as HL-60, were sensitive to PAM treatment suggesting possible alternative targets of these agents. To determine the effect of PAMs on apoptosis, MV4-11 and THP-1 cell lines were treated with KPT-9331 at IC50 concentration (~0.25 µM) and cell death was measured via Annexin-V/PI flow cytometric analysis after 24, 48h and 72h treatment. KPT-9331 induced a time dependent increase in apoptosis in both cell lines. In MV4-11 cells, KPT-9331 caused cell cycle arrest and inhibition of proliferation after 24hrs. We also tested the effect of PAMs in primary AML cells. Patient samples cocultured with a human stromal cell lines were treated with PAMs for 96 hours. IC50 values ranged from 0.14 - 0.19 µM. A dose dependent decrease in proliferation following PAM treatment was observed in all the five analyzed samples irrespective of genetic subtype. PAMs treatment for 48hrs using a whole blood viability assay from normal donors showed no significant cytotoxic effect on T and NK cells, but modest toxicity to normal B cells. Normal hematopoietic colony forming cell assays are being initiated and will be presented. We next utilized a human AML leukemia xenograft model with MV4-11 cells to assess the in vivo activity of KPT-9274. Mice were dosed once daily via oral gavage with KPT-9274 (150 mg/kg) or vehicle control. KPT-9274 dramatically inhibited tumor growth, prevented invasion of MV4-11 cells, and improved overall survival with all mice (n=7) being alive (median not reached) at day 49 of experiment as compared to 1 out of 7 vehicle-treated mice (median survival 36 days) being alive at this time. In summary, KPT-9274 demonstrates promising activity in pre-clinical AML models and warrants further investigation in this disease. Ongoing efforts include validating the specificity of the reported target in AML (versus alternative targets), in vivo exploration in primary human AML xenograft models, and understanding the effects of this compound on normal hematopoiesis and function. Disclosures Baloglu: Karyopharm Therapeutics Inc.: Employment, Equity Ownership. Senapedis:Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Blum:Gilead Sciences: Research Funding. Byrd:Acerta Pharma BV: Research Funding.

scholarly journals Extracellular ATP and CD39 Regulates Mitochondrial Function and Cytarabine Resistance through Intrinsic PKA-ATF-PGC1a Pathway in Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2737-2737
Author(s):  
Jean-Emmanuel Sarry ◽  
Christian Recher ◽  
Nesrine Aroua
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Leukemic Cells ◽  
Nucleoside Triphosphate ◽  
Preclinical Model ◽  
Acute Myeloid

Abstract Relapses in acute myeloid leukemia (AML) are caused by chemoresistant leukemic populations and new therapeutic approaches that specifically target these cells are urgently needed. Based on transcriptomic analyses of relevant PDX preclinical model of the resistance to cytarabine (AraC) and of the residual disease in patients, we identified ecto-nucleoside triphosphate diphosphohydrolase-1 CD39 (ENTPD1) overexpressed in the residual leukemic cells in vivo after chemotherapy. By flow cytometry, we confirmed that AraC increased cell surface CD39 expression in AML cell lines in vitro and in vivo as well as in 24 diverse patient-derived xenograft models. We further observed this increase in 100 patients at 35-days post-intensive chemotherapy compared to their respective diagnosis. Interestingly, high CD39 expression on AML patients was associated with a worse response to AraC in vivo. Furthermore, we showed that FACS-sorted CD39high AML cells had increased mitochondrial mass and activity, and were resistant to AraC in vitro and in vivo. We demonstrated that CD39 downstream signaling pathway was dependent on cAMP-PKA-PGC1a-TFAM axis and its inhibition by H89 sensitized AML cells to AraC through the inhibition of mitochondrial OxPHOS biogenesis and function. Finally, pharmacological inhibition of CD39 ATP hydrolase activity or genetic invalidation of CD39 protein using two inhibitors or shRNA markedly enhanced AraC cytotoxicity in AML cell lines and primary patient samples in vitro and in vivo. Together, these results indicate CD39 as a new player of the intrinsic chemoresistance pathway and a new therapeutic target to specifically overcome AraC resistance and eradicate these leukemic cells responsible for relapses in AML. Disclosures No relevant conflicts of interest to declare.

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