Integrated RNAi screening identifies the NEDDylation pathway as a synergistic partner of azacytidine in acute myeloid leukemia

AbstractTreatment of acute myeloid leukemia (AML) remains challenging and novel targets and synergistic therapies still need to be discovered. We performed a high-throughput RNAi screen in three different AML cell lines and primary human leukemic blasts to identify genes that synergize with common antileukemic therapies. We used a pooled shRNA library that covered 5043 different genes and combined transfection with exposure to either azacytidine or cytarabine analog to the concept of synthetic lethality. Suppression of the chemokine CXCL12 ranked highly among the candidates of the cytarabine group. Azacytidine in combination with suppression of genes within the neddylation pathway led to synergistic results. NEDD8 and RBX1 inhibition by the small molecule inhibitor pevonedistat inhibited leukemia cell growth. These findings establish an in vitro synergism between NEDD8 inhibition and azacytidine in AML. Taken together, neddylation constitutes a suitable target pathway for azacytidine combination strategies.

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Integrated RNAi Screening Identifies The NEDDylation Pathway As A Synergistic Partner of Azacytidine In Acute Myeloid Leukemia

10.21203/rs.3.rs-647282/v1 ◽

2021 ◽

Author(s):

Justine Klosner ◽

Konstantin Agelopoulos ◽

Christian Rohde ◽

Stefanie Göllner ◽

Christoph Schliemann ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Synthetic Lethality ◽

Leukemia Cell ◽

Combination Strategies ◽

Suitable Target ◽

Chemokine Cxcl12 ◽

Shrna Library ◽

Acute Myeloid

Abstract Treatment of Acute Myeloid Leukemia (AML) remains challenging and novel targets and synergistic therapies still need to be discovered. We performed a high-throughput RNAi screen in three different AML cell lines and primary human leukemic blasts to identify genes that synergize with common antileukemic therapies. We used a pooled shRNA library that covered 5043 different genes and combined transfection with exposure to either azacytidine or cytarabine analog to the concept of synthetic lethality. Suppression of the chemokine CXCL12 ranked highly among the candidates of the cytarabine group. azacytidine in combination with suppression of genes within the neddylation pathway led to synergistic results. NEDD8 and RBX1 inhibition by the small molecule inhibitor pevonedistat inhibited leukemia cell growth. These findings establish an in vitro synergism between NEDD8 inhibition and azacytidine in AML. Azacytidine and NEDD8 inhibitors are currently undergoing clinical trials in combination with azacytidine. Taken together, neddylation constitutes a suitable target pathway for azacytidine combination strategies.

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

Archives of Medical Science ◽

10.5114/aoms/142465 ◽

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.

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The Vitamin E Derivative Gamma Tocotrienol Promotes Anti-Tumor Effects in Acute Myeloid Leukemia Cell Lines

Nutrients ◽

10.3390/nu11112808 ◽

2019 ◽

Vol 11 (11) ◽

pp. 2808 ◽

Cited By ~ 6

Author(s):

Ghanem ◽

Zouein ◽

Mohamad ◽

Hodroj ◽

Haykal ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Vitamin E ◽

Cell Lines ◽

Myeloid Leukemia ◽

Apoptotic Cell ◽

Leukemia Cell ◽

Therapeutic Effects ◽

Apoptotic Pathway ◽

Acute Myeloid

Acute myeloid leukemia (AML) is a blood cancer characterized by the formation of faulty defective myelogenous cells with morphological heterogeneity and cytogenic aberrations leading to a loss of their function. In an attempt to find an effective and safe AML treatment, vitamin E derivatives, including tocopherols were considered as potential anti-tumor compounds. Recently, other isoforms of vitamin E, namely tocotrienols have been proposed as potential potent anti-cancerous agents, displaying promising therapeutic effects in different cancer types. In this study we evaluated the anti-cancerous effects of γ-tocotrienol, on AML cell lines in vitro. For this purpose, AML cell lines incubated with γ-tocotrienol were examined for their viability, cell cycle status, apoptotic cell death, DNA fragmentation, production of reactive oxygen species and expression of proapoptotic proteins. Our results showed that γ-tocotrienol exhibits time and dose-dependent anti-proliferative, pro-apoptotic and antioxidant effects on U937 and KG-1 cell lines, through the upregulation of proteins involved in the intrinsic apoptotic pathway.

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Combination of BCL-2 Inhibition and Triptolide Causes Synthetic Lethality in Acute Myeloid Leukemia through Preventing Reciprocal Resistance

Blood ◽

10.1182/blood-2019-123722 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2083-2083

Author(s):

Bing Xu ◽

Yuanfei Shi ◽

Long Liu ◽

Bing Z Carter

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Research Funding ◽

Synthetic Lethality ◽

Leukemic Cells ◽

Apoptotic Pathway ◽

P53 Status ◽

Acute Myeloid

BCL-2 inhibition exerts effective pro-apoptotic activities in acute myeloid leukemia (AML) but clinical efficacy as a monotherapy was limited in part due to the treatment-induced MCL-1 increase. Triptolide (TPL) exhibits anti-tumor activities in part by upregulating pro-apoptotic BCL-2 proteins and decreasing MCL-1 expression in various malignant cells. We hypothesized that combined BCL-2 inhibition and TPL exert synergistic anti-leukemia activities and prevent the resistance to BCL-2 inhibition in AML. We here report that TPL combined with BCL-2 inhibitor ABT-199 synergistically induced apoptosis in leukemic cells regardless of p53 status through activating the intrinsic mitochondrial apoptotic pathway in vitro. Although ABT-199 or TPL alone inhibited AML growth in vivo, the combination therapy demonstrated a significantly stronger anti-leukemic effect. Mechanistically, TPL significantly upregulated BH3 only proteins including PUMA, NOXA, BID and BIM and decreased MCL-1 but upregulated BCL-2 expression in both p53 wild type and p53 mutant AML cell lines, while the combination decreased both BCL-2 and MCL-1 and further increased BH3 only BCL-2 proteins. MCL-1 and BCL-2 increases associated with respective ABT-199 and TPL treatment and resistance were also observed in vivo. Significantly downregulating MCL-1 and elevating BH3 only proteins by TPL could not only potentially block MCL-1-mediated resistance but also enhance anti-leukemic efficacy of ABT-199. Conversely, BCL-2 inhibition counteracted the potential resistance of TPL mediated by upregulation of BCL-2. The combination further amplified the effect, which likely contributed to the synthetic lethality. This mutual blockade of potential resistance provides a rational basis for the promising clinical application of TPL and BCL-2 inhibition in AML independent of p53 status. Disclosures Carter: Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding.

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Phase I Dose Escalating Trial of Bortezomib (Velcade®) in Combination with Idarubicin and Cytarabine in Patients with Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v104.11.1799.1799 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1799-1799 ◽

Cited By ~ 8

Author(s):

Eyal C. Attar ◽

Daniel J. DeAngelo ◽

Karen K. Ballen ◽

Emily Learner ◽

Elizabeth G. Trehu ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Phase I ◽

Dose Level ◽

Myeloid Leukemia ◽

Science Studies ◽

Leukemia Cell ◽

Maximum Tolerated Dose ◽

Synergistic Cytotoxicity ◽

Acute Myeloid

Abstract Conventional therapy for acute myeloid leukemia (AML) consists of a combination of cytarabine and an anthracycline such as idarubicin. Currently, most patients ultimately fail treatment due to leukemia cell resistance to drug therapy. In vitro experiments have shown that the addition of a proteasome inhibitor to an anthracycline results in synergistic cytotoxicity to leukemia cells. Hence, we initiated this phase I trial in patients to see if bortezomib could be safely added to conventional treatment. Patients over age 60 with AML or any patient 18 or older with relapsed disease after a remission of at least 3 months (not refractory) were eligible. All patients received idarubicin 12 mg/m2 on days 1–3 and cytarabine 100 mg/m2 by continuous infusion days 1–7. In addition, patients received bortezomib by IV bolus on days 1, 4, 8, and 11. Cohorts of 3 to 6 patients were entered using increasing doses of bortezomib in order to determine the maximum tolerated dose (MTD). The first cohort received 0.7 mg/m2 of bortezomib with each bolus. If dose limiting toxicity (DLT) was encountered, then cohort advancement was restricted. DLTs included prolonged myelosuppression, neuropathy, and other grade 3 or 4 toxicities. Dose escalation would proceed to 1.0 mg/m2 and then to 1.3 mg/m2 if tolerated. No escalation was planned beyond 1.3 mg/m2. To date 14 patients have been entered on this study. In the first cohort of 3 patients with bortezomib at 0.7 mg/m2, a DLT due to prolonged neutropenia was encountered, so an additional 3 patients were entered at this dose level. No DLTs were encountered among these additional patients, so 3 more patients were entered with bortezomib at 1.0 mg/m2. One of these patients experienced prolonged thrombocytopenia and thus 3 additional patients were enrolled at 1.0 mg/m2. No DLTs were encountered among these additional patients, and thus the next cohort of patients with bortezomib at 1.3 mg/m2 was opened. To date, two patients have been enrolled at this dose level. The plan is to enroll a third patient at this level and to assess for possible DLTs. Among the 12 patients evaluable for response, there have been 4 patients achieving complete remission, 3 patients achieving remission without complete recovery of platelet count (CRp defined as having met criteria for CR but with 25,000–99,000 platelets/μl), 2 patients achieving a partial remission (CR but with 5–24% bone marrow blasts), and 3 patients failing to respond. In conclusion, bortezomib at 0.7 mg/m2 and 1.0 mg/m2 in the day 1, 4, 8, and 11 schedule can be added to idarubicin and cytarabine with acceptable toxicity. This study continues in an attempt to determine whether bortezomib can be escalated safely to 1.3 mg/m2 in this combination. Additional patients will be enrolled at the candidate MTD to gain confidence in the safety and activity at this level. Correlative science studies are planned.

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Disulfiram, an clinically used anti-alcoholism drug has the potential to target acute myeloid leukemia cells in a copper-dependent manner in vivo

Blood ◽

10.1182/blood.v122.21.5040.5040 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5040-5040

Author(s):

Bing Xu ◽

Rongwei Li ◽

Huijuan Dong ◽

Feili Chen ◽

Yuejian Liu ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Specific Antigen ◽

Leukemia Cell ◽

Leukemia Cells ◽

Control Group ◽

Acute Myeloid

Abstract Background Disulfiram(DS), an old drug clinically used for alcoholism, was reported to have antitumor effects, recent studies have found that Copper(Cu) can significantly enhance the DS-induced cell death in vitro in a variety of tumor cells. Our previous studies also demonstrated that disulfiram/copper (DS/Cu) couldtarget human leukemia cell lines(like KG1α,Molt4) through the activation of JNK, in vitro. However, there is few report about the ability of DS/Cu in killing cancer cells in vivo. Aims This study aims to explore the effect of DS/Cu on acute myeloid leukemia cell line KG1αin vivo and clarify the underlining mechanism. Methods 6-8 week old female NOD/SCID mice were sublethally irradiated with 2Gy X-ray the day before transplantation, followed by intravenous injection of KG1α cells (1×107 cells) suspended in 0.2 mL of PBS. 5 weeks after transplantation mice were randomly divided into three treatment groups: vehicle (0.9% saline), a combination of DS and Cu daily for 2 weeks, Ara-C alone twice before killing. Mice were sacrificed after 2 weeks treatment with tissues of spleen, liver, bone marrow being observed using histopathology method to detect the invasion of leukemia. The DS/Cu-induced p-c-jun activation was also examined by western blot using tissues of spleen, liver, bone marrow. Statistical analysis was carried out with one-way ANOVA to assess statistical significance (*p < 0.05). Results 4 weeks after transplantation, mice were dispirited with low appetite, down-bent gait, wrinkled fur, slow move, just like suffered from leukemia. What’s more, immature blasts like morphology similar to KG1α were found in the peripheral blood of the mice(11%±3.41). All the mice were sacrificed after 2 weeks treatment, mice in control group were observed with slightly larger spleen and liver with the morphology of invasion of leukemia such as a granular appearance than the other two groups. Histopathology examination showed that leukemia cells infiltrate liver, spleen and bone marrow, and the immunohistochemistry examination found that the leukemia cells in spleen, liver and bone marrow expressed human specific antigen CD45 with the highest expression level in the control group. Moreover, solid tumor could be observed in the peritoneal cavity of two mice in the control group with expression of human specific antigen CD45detected by immunohistochemistry examination. Western blot in this study showed DS/Cu complex induced phosphorylation of c-Jun expression in the spleen, liver and bone marrow. Conclusion DS/Cu complex could effectively target the acute myeloid leukemia cells in the acute leukemia NOD/SCID mice while inhibiting the invasion of leukemia to some extent, and the activation of JNK might play a functional role in DS/Cu mediated antileukemic effects. Disclosures: No relevant conflicts of interest to declare.

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Therapeutic inhibition of GAS6-AS1/YBX1/MYC axis suppresses cell propagation and disease progression of acute myeloid leukemia

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-02145-9 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Hao Zhou ◽

Wei Liu ◽

Yongming Zhou ◽

Zhenya Hong ◽

Jian Ni ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Disease Progression ◽

Myeloid Leukemia ◽

Target Genes ◽

Leukemia Cell ◽

Treatment Strategies ◽

Network Analyses ◽

Acute Myeloid

Abstract Background Acute myeloid leukemia (AML) is the most common type of leukemia in adults. Its therapy has not significantly improved during the past four decades despite intense research efforts. New molecularly targeted therapies are in great need. The proto-oncogene c-Myc (MYC) is an attractive target due to its transactivation role in multiple signaling cascades. Deregulation of the MYC is considered one of a series of oncogenic events required for tumorigenesis. However, limited knowledge is available on which mechanism underlie MYC dysregulation and how long non-coding RNAs (lncRNAs) are involved in MYC dysregulation in AML. Methods AML microarray chips and public datasets were screened to identify novel lncRNA GAS6-AS1 was dysregulated in AML. Gain or loss of functional leukemia cell models were produced, and in vitro and in vivo experiments were applied to demonstrate its leukemogenic phenotypes. Interactive network analyses were performed to define intrinsic mechanism. Results We identified GAS6-AS1 was overexpressed in AML, and its aberrant function lead to more aggressive leukemia phenotypes and poorer survival outcomes. We revealed that GAS6-AS1 directly binds Y-box binding protein 1 (YBX1) to facilitate its interaction with MYC, leading to MYC transactivation and upregulation of IL1R1, RAB27B and other MYC target genes associated with leukemia progression. Further, lentiviral-based GAS6-AS1 silencing inhibited leukemia progression in vivo. Conclusions Our findings revealed a previously unappreciated role of GAS6-AS1 as an oncogenic lncRNA in AML progression and prognostic prediction. Importantly, we demonstrated that therapeutic targeting of the GAS6-AS1/YBX1/MYC axis inhibits AML cellular propagation and disease progression. Our insight in lncRNA associated MYC-driven leukemogenesis may contribute to develop new anti-leukemia treatment strategies.

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Zebularine inhibits human acute myeloid leukemia cell growth in vitro in association with p15INK4B demethylation and reexpression

Experimental Hematology ◽

10.1016/j.exphem.2006.10.005 ◽

2007 ◽

Vol 35 (2) ◽

pp. 263-273 ◽

Cited By ~ 38

Author(s):

Stuart A. Scott ◽

Ashakumary Lakshimikuttysamma ◽

David P. Sheridan ◽

Stephen E. Sanche ◽

C. Ronald Geyer ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Growth ◽

Myeloid Leukemia ◽

Leukemia Cell ◽

Acute Myeloid Leukemia Cell ◽

Human Acute Myeloid Leukemia ◽

Acute Myeloid ◽

Myeloid Leukemia Cell

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Differential Expression of Mirna* Species in Cancer and the Contribution of MiR-223* to the Development of Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v114.22.2960.2960 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2960-2960

Author(s):

Florian Kuchenbauer ◽

Sarah M Mah ◽

Andrew McPherson ◽

Michael Heuser ◽

Bob Argiropolous ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Leukemia Cell ◽

Mirna Sequence ◽

Human Leukemia ◽

Mirna Species ◽

Mirna Duplex ◽

Acute Myeloid

Abstract Abstract 2960 Poster Board II-936 Processing of the pre-miRNA through Dicer1 generates a miRNA duplex, consisting of a miRNA and miRNA* strand. While the functional roles of miRNAs are now well established, the potential roles of miRNA* species remain unclear. However, recent evidence suggests that the star strand of some miRNAs can be abundant and enter the RISC complex. Since the abundance of miRNA*s has not been comprehensively assessed in mammals and we took advantage of 10 deep sequencing libraries from a variety of human and murine cells to determine the most abundant complementary strand for non-annotated miRNA*s. We then calculated the ratio of miRNA/miRNA* for each miRNA duplex. In contrast to previous assumptions that one strand is highly dominant, we found that approximately 50% of the investigated miRNA duplexes exhibit high ratios with a dominating strand (ratio >100), 20% have intermediate ratios (ratio between 100-10) and a remarkable 10% show low ratios (ratio <10), indicating comparable expression of both strands. In addition, we found that ∼10% of all miRNA/miRNA* duplexes display inverse ratios (ratio<1), indicating incorrect annotation in miRBase. Comparing miRNA/miRNA* ratios across the miRNA sequence libraries revealed that most ratios remain constant across tissues and species. This could possibly allow for a novel classification of miRNAs into a-duplexes, miRNAs duplexes with a dominant strand and b-duplexes with both strands being abundant. However, certain ratios were highly variable across the libraries examined as exemplified for the ratio of miR-223/miR-223* which ranged from 0.11 (317:2684 read counts) to 19.6 (13006:660 read counts) in murine and human leukemia cell lines. Bioinformatics analysis on predicted miR-223* targets showed an enrichment for cancer associated genes (p<0.05), suggesting a tumor suppressor-like role for miR-223. Consistent with this, an analysis of samples from 94 AML patients with normal karyotype revealed an inverse correlation of miR-223* with CD34 expression (p=0.018), a negative prognostic marker in AML. In addition, in vitro experiments with mutated miR-223 and miR-223* constructs revealed regulatory potential for miR-223* in myeloid progenitor cells. Taken together, we propose a new classification for miRNA duplexes and provide evidence for a possible role a miRNA* in the development of acute myeloid leukemia. Disclosures: No relevant conflicts of interest to declare.

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