Development Of Novel Alkylating Chemotherapeutics, Phenyl-Chloroethyl Urea Family, Targeting Mitochondrial Prohibitin For Selective Killing Of Leukemic Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1325-1325
Author(s):  
Hye-Ran Kim ◽  
Hwan-Young Kim ◽  
Trang Nguyen Thi Dai ◽  
Il-Kwon Lee ◽  
Hyeoung-Joon Kim ◽  
...  
Keyword(s):  
Cell Lines ◽  
Conflicts Of Interest ◽  
Mitochondrial Protein ◽  
Leukemic Cells ◽  
Mitochondrial Proteins ◽  
Acute Myelocytic Leukemia ◽  
Dependent Manner ◽  
Mice Model ◽  
Morphological Transformation

Abstract Background Mitochondrial aberrations have been associated with chronic degenerative diseases, aging and cancer. Mitochondrial proteins are interesting targets for the development of selective anticancer drugs in leukemia and other malignancies. We analyzed mitochondrial proteins to develop novel anti-cancer agents targeting selective leukemic cell, especially acute myelocytic leukemia (AML) cells. Materials and Methods Mitochondria were isolated from primary AML cells and AML cell lines (THP-1 and KG-1) by density-gradient ultracentrifugation using swelling buffer and sucrose buffer. Dysregulated mitochondrial protein were identified using 2-DE and mass spectrometry (MALDI-TOF/TOF technology). For in vivo experiments, AML cell grafts were established in 6-week-old Balb/c mice by subcutaneous injection of 1x107 THP-1 AML cells in the right flank at day 0. Results Out of these deregulated proteins, totally 12 and 20 proteins were observed in up- or down-regulated spots, respectively. Interestingly, prohibitin (gi4505773) was highly expressed in all type of leukemic cells, especially primary AML cells (Fig. 1), which is confirmed by immunoblot and immunofluorecenct methods. Aberrant expressed prohibitin induced growth suppression and repressed E2F-mediated transcription. We synthesized potent chemical substances that can alkylate PHB, two molecules of phenyl-chloroethyl urea family such as cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU) from the reaction with 2-ethylisocyanate and 4-cyclohexylaniline and 4-iodoaniline, respectively. They were characterized by 1H NMR and 13C NMR. Time and dose dependent manner of proliferation suppression when treated with CCEU and ICEU was observed in primary AML and AML cell lines. Notably morphological transformation of AML cells was observed when treated with 10 - 100 umol of CCEU and ICEU for 24 hours. The half maximal inhibitory concentration (IC50) was 25umol of most AML cell lines. Cell cycle analysis of CCEU-and-ICEU-treated- AML cells showed a remarkable increase of the sub-G1 phase. Immunoblotting experiment revealed the change of cytoplasmic and nucleoplasmic PHB. The increment expression of cleaved caspase3 and poly ADP-ribose polymerases were also observed in AML cell lines. Moreover, CCEU and ICEU selectively killed AML cells in mouse model (Fig. 2). Conclusion This study showed the development of novel alkylating chemotherapeutics, phenyl-chloroethyl urea family (CCEU and ICEU), targeting mitochondrial prohibitin for selective killing of leukemic cells. Selective eradication effects of these novel chemotherapeutics were further confirmed using in vivo mice model. Disclosures: No relevant conflicts of interest to declare.

Prohibitin Serves as a Novel Target Mitochondrial Protein of Phenyl-Chloroethyl Ureas Involved In Their Antiproliferative Effect on Multiple Myeloma Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3002-3002
Author(s):  
Hye-Ran Kim ◽  
Ha-Young Eom ◽  
Dong Min Kim ◽  
Il-Chul Kim ◽  
Manian Rajesh Kumar ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Mitochondrial Protein ◽  
Mitochondrial Proteins ◽  
Leukemia Cells ◽  
Dependent Manner ◽  
Morphological Transformation ◽  
Novel Target

Abstract Abstract 3002 Background: Prohibitin (PHB) is localized to the mitochondria where it might have a role in the maintenance of mitochondrial function. The diverse function of PHB, together with the emerging evidence that its function can be modulated specifically in certain diseases, implies that PHB would be a potential target for new therapeutics. Materials and Methods: We analyzed mitochondrial proteins and develop new anti-proliferative agents targeting multiple myeloma (MM) cells. Mitochondria were isolated from primary leukemia and MM cell lines (RPMI8226, ARH77, U266 and IM9) by density-gradient ultracentrifugation using swelling buffer and sucrose buffer. Dysregulated mitochondrial protein was identified using 2-DE and mass spectrometry (MALDI-TOF/TOF technology). Results: Out of 38 remarkable deregulated mitochondrial proteins in MM cell lines, prohibitin (PHB) (gi4505773) was highly expressed in primary MM and leukemia cells, which was confirmed by Western blot, immunohistochemistry and immunofluorecenct study in the primary bone marrow cells and sections. Potent chemical substances that can alkylate PHB, two molecules of phenyl-chloroethyl urea family such as cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU), were synthesized independently from the reaction with 2-ethylisocyanate and 4-cyclohexylaniline and 4-iodoaniline, respectively. They were characterized by 1H NMR and 13C NMR. Time and dose dependent manner of proliferation suppression when treated with CCEU and ICEU was observed in MM and leukemia cells. Moreover, notable morphological transformation of MM cells was observed when treated with 10 – 100 umol of CCEU and ICEU for 24 hours. The half of maximal inhibitory concentration (IC50) was 25umol of most MM cell lines. Cell cycle analysis of CCEU-and-ICEU-treated- MM cells showed a remarkable increase of the sub-G1 phase. Immunoblotting experiment revealed the change of cytoplasmic and nucleoplasmic PHB. Expression of cleaved caspase3 and poly ADP-ribose polymerases were also observed to have increased in MM cell lines. Conclusions: By analyzing mitochondrial protein in leukemia and MM cell, we discovered a new molecular marker, PHB, characteristically overexpressed in leukemia and MM cells and developed new anti-cancer agents such as CCEU and ICEU that target against PHB in MM cells. Disclosures: No relevant conflicts of interest to declare.

Discovery of a New Molecular Marker, Prohibitin, and Development of Candidates for New Chemotherapeutic Agents Targeting Prohibitin in Primary AML Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3342-3342
Author(s):  
Hye-Ran Kim ◽  
Myung-Geun Shin ◽  
Sun-Woo Lee ◽  
Hee-Nam Kim ◽  
Il-Kwon Lee ◽  
...  
Keyword(s):  
Molecular Marker ◽  
Cell Lines ◽  
Bone Marrow Cells ◽  
Mitochondrial Protein ◽  
Genetic Mutation ◽  
Chemotherapeutic Agents ◽  
Leukemic Cells ◽  
Mitochondrial Proteins ◽  
Dependent Manner ◽  
Morphological Transformation

Abstract In light of the recent studies that showed significant causal relationship between mitochondrial genetic mutation and apoptosis in pathology of various tumors and degenerative diseases, mitochondrial proteins have been interesting targets for the study of apoptosis in leukemia and other malignancies. This observation prompted us to analyze mitochondrial proteins and develop new anti-proliferative agents targeting them. Mitochondria were isolated from AML cells by density-gradient ultracentrifugation using swelling buffer and sucrose buffer. We identified 48 spots corresponding to 38 proteins in primary AML cells using 2-DE and mass spectrometry (MALDI-TOF/TOF technology), the expression of which were altered significantly compared to that of normal hematopoietic cells. Out of these deregulated proteins, 12 and 20 proteins were observed in up- or down-regulated spots, respectively. Interestingly, prohibitin (PHB) (gi4505773) was highly expressed in primary AML cells, which was confirmed by Western blot, immunohistochemistry and immunofluorecenct study in the primary AML bone marrow cells and sections. To assess the functional significance of aberrant prohibitin expression, we applied siRNA delivery for silencing of prohibitin. Transduction with a siRNA 11867 construct resulted in 75% decrease of AML cells as compared to the nonsilencing control construct after two days. Potent chemical substances that can alkylate PHB in AML cells, cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU), were synthesized in our laboratory. Time and dose dependent manner of proliferation suppression when treated with CCEU and ICEU was observed in leukemic cell lines including THP-1, K-562 and Kasumi-1. Moreover, notable morphological transformation of leukemic cells was observed when treated with 50 – 200 umol of CCEU and ICEU for 24 hours. Cell cycle analysis of CCEU-and-ICEU-treated- THP-1 and Kasumi-1 cell lines showed a remarkable increase of the sub-G1 phase. Immunoblotting experiment revealed the change of cytoplasmic and nucleoplasmic PHB in K-562 cell line. Expression of cleaved caspase3 and poly ADP-ribose polymerases were also observed to have increased in primary AML cells and cell lines. By analyzing AML cell mitochondrial protein we discovered a new molecular marker, PHB, characteristically overexpressed in AML cells and developed new anti-cancer agents such as CCEU and ICEU that target against PHB in AML cells.

scholarly journals IGFBP7 As a Potential Therapeutic Target for AML

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5134-5134
Author(s):  
Yina Niu ◽  
Shuiyan Wu ◽  
Shaoyan Hu
Keyword(s):  
Cell Lines ◽  
Conflicts Of Interest ◽  
Fold Increase ◽  
Dependent Manner ◽  
Mice Model ◽  
Cell Counts ◽  
And Migration ◽  
Proliferation And Migration

Abstract Insulin-like growth factor binding proteins (IGFBPs) are secretory factors that play essential roles in regulation of insulin-like growth factors (IGFs) in tissue as well as in modulating IGF binding to its receptors. IGFBP7, known as IGFBP-related protein 1 (IGFBP-rP1), mac25/angiomodulin, function as a potential tumor suppressor in various human solid cancers, including breast, prostate, gastric and liver cancer. We have reported the overexpression of IGFBP7 in the context of acute myeloid leukemia (AML), showing that IGFBP7 expression level in AML patients is significantly increased compared with controls (P<0.001). IGFBP7 expression was obviously decreased in AML patients achieving complete remission (P<0.01), and was significantly increased in relapsed AML patients (P<0.01). In addition, AML patients with high expression of IGFBP7 had shorter overall survival. Here, we investigate the role and mechanism of IGFBP7 in the development and progression of AML. In order to study the role of IGFBP7 in AML, stable cell lines expressing IGFBP7 and control in AML cells were constructed using lentiviral packaging system. Expression microarray assay was carried out to analyze the global gene level changes driven by IGFBP7. MTT and transwell assays were performed to evaluate the effect of IGFBP7 on cell proliferation and migration. Bioinformatics results found that IGFBP7 appeared to utilize multiple cellular processes for its oncogenic roles, including adhesion, migration, and proliferation. Experimental data showed overexpression of IGFBP7 in K562 cells resulted in a 2-3 fold increase in migration in contrast to control cells. Moreover, enforced expression of IGFBP7 also led to phosphorylation of Akt and Erk, whose activities inactivation by pharmacologically inhibitors resulted in the loss of ability to migrating. Finally, knockdown of IGFBP7 in cells with high IGFBP7 level, their migration abilities were significantly decreased. To assess the role of IGFBP7 in leukemogenesis in vivo, the same numbers of K562/IGFBP7 and K562-Vector cells, U937-shIGFBP7 and U937-shNEG cells were injected into NOD-SCID mice by tail vein injection, respectively. About two weeks later, it was showed that mice of K562/IGFBP7 and U937 groups displayed higher white blood cell counts compared with mice of K562-Vector and U937-shIGFBP7 groups, respectively. Mice of K562/IGFBP7 and U937 groups had more severe splenomegaly and hepatomagaly compared with its corresponding control groups. We further characterized the molecular mechanism underlying leukemogenesis driven by IGFBP7 in AML cell lines. The global expression profiling and molecular biological experiments showed PI3K/AKT signaling was activated by overexpression of IGFBP7, and knockdown of IGFBP7 in AML cells led to a decrease of PI3K/AKT activity in PTEN-dependent manner. IGFBP7 promotes proliferation and migration of AML cells, the promotion could be suppressed by both RNA interference and pharmacological inhibition of PI3K/AKT pathway. Immuno-precipitation assay showed that IGFBP7 associated with AXAN2 and induced PTEN degradation. The expression of ANXA2 was significantly positive correlated with the expression ANXA2 in AML patients. The expression of IGFBP7 in AML, overexpression as well as knockdown of IGFBP7 in leukemia cells and in mice model, all suggest that IGFBP7 is a potential proto-oncogene. Collectively this work suggests that targeting IGFBP7 activity may be an effective therapeutic strategy for AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Treatment with the Recombinant SLIT2 Protein Delays AML Leukemogenesis In Vivo

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2368-2368
Author(s):  
Luise A de Albuquerque Simoes ◽  
Isabel Weinhäuser ◽  
Diego A Pereira-Martins ◽  
César Alexander Ortiz Rojas ◽  
Thiago Mantello Bianco ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Cancer Progression ◽  
Functional Role ◽  
Conflicts Of Interest ◽  
Therapeutic Potential ◽  
Leukemic Cells ◽  
Control Group

Abstract Accumulating evidence suggest that the axon guidance molecules SLIT and ROBO are not only implicated in physiological process but also in cancer progression. Depending on the type of cancer the SLIT-ROBO axis can either act as a tumor suppressor gene, in which case the SLIT2 promoter site is frequently hypermethylated or as an oncogene, whereby high expression is often associated with poor prognosis. In the context of acute myeloid leukemia (AML), low expression of SLIT2 has been associated with low overall survival (OS) (Golos et al., 2019), while the functional role of SLIT2 remains largely unknown. Recently, we showed that the knockdown of SLIT2 increased cell proliferation of acute promyelocytic leukemia (APL) cells resulting in a more aggressive course of disease progression in vivo using the murine transgenic APL model (Weinhäuser et al., 2020). Here, we aimed to study the functional role of SLIT2 in a more heterogeneous disease, such as AML. Using different publicly available datasets. (GSE58477, normal karyotype blasts: 62, healthy CD34 +: 10; GSE63409, LSC: 14, HSC: 5) we detected increased methylation at the SLIT2 promoter site of AML leukemic cells compared to healthy CD34 + cells suggesting SLIT2 tumor suppressive functions. In addition, we measured decreased levels of SLIT2 in the bone marrow (BM) plasma of AML patients compared to healthy donors. To assess the biological role of SLIT2, we treated AML cell lines (KASUMI1, MV411, and MOLM13) with recombinant SLIT2 (50 ng/mL) in vitro. Administration of SLIT2 reduced AML cell growth, colony formation and induced cell cycle arrest in the G1 phase for all AML cell lines. Conversely, the knockdown of SLIT2 promoted increased THP-1 and OCI-AML3 cell proliferation. Next, we determined whether the treatment with SLIT2 could delay leukemogenesis in vivo using the AML cell line MV4-11. Engraftment was monitored by luciferase bioluminescent signal and NSGS mice were either treated with recombinant SLIT2 using a dose of 25 ng/g of body weight or vehicle (control group). SLIT2 therapy resulted in a lower disease burden, decreased leukemic infiltration in the BM and spleen, reduced spleen size, and increased OS compared to the control group (p&lt;0.05). In conclusion, we showed that SLIT2 methylation is recurrent in AML patients and that the level of SLIT2 in the plasma of AML patients is reduced. Moreover, SLIT2 treatment appears to have a cytostatic effect on different AML cell lines delaying leukemogenesis in vivo. Overall, our study reveals the therapeutic potential of SLIT2 in hematological malignancies, which could be used as an adjuvant in the clinic. Disclosures No relevant conflicts of interest to declare.

Metabolic Capability to Induce the Pasteur Effect Mediates Sensitivity of Human Leukemic Cells to Metformin

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2601-2601
Author(s):  
Sarah Scotland ◽  
Estelle Saland ◽  
Lindsay Peyriga ◽  
Rémi Peyraud ◽  
Elizabeth Micklow ◽  
...  
Keyword(s):  
Cell Lines ◽  
Research Funding ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Pasteur Effect ◽  
Cytotoxic Response ◽  
Leukemic Cell Lines

Abstract Abstract 2601 An emerging hallmark of cancer cells is the reprogramming of intermediary and energy metabolism these cells undergo. Several epidemiological studies have shown that metformin, widely used to treat patients with type 2 diabetes, may reduce their risk of cancer. Despite several reports of anti-neoplastic activity of metformin, the mechanisms responsible for this activity have not been fully elucidated in cancer or leukemic cells. We hypothesized that metformin elicits a metabolic reprogramming driven by alterations in mitochondrial function and signaling, which induces apoptosis in leukemic cells, and that metabolic flexibility determines the variation(s) of the cytotoxic response to metformin among different leukemic cell lines. We first demonstrated that metformin markedly decreased oxygen consumption of six leukemic cell lines in a concentration-dependent manner. We also observed that the cytotoxic effect of metformin varies between cell lines reflecting their energetic capacity to compensate for the mitochondrial inhibition induced by metformin (eg. to induce the Pasteur effect). Importantly, metformin-insensitive leukemic cells did not exhibit a Pasteur effect in response to metformin. All leukemic cells exhibited high basal conversion of glucose to lactate (eg. aerobic glycolysis) and specific expression of key metabolic genes as compared to normal mononuclear cells. Despite dependence on glucose catabolism, metformin sensitivity was associated with relative resistance to glucose starvation. Metformin effects in drug-resistant cells were potentiated by the addition of a glycolytic inhibitor, but not by inhibitors of the pentose phosphate pathway or glutaminolysis. Leukemic cells with broad metabolic capacities to utilize other energetic substrates in response to diverse nutrient starvation showed insensitivity to metformin. Metformin induced a significant decrease in metabolites of the upper segment of glycolysis and the oxidative branch of the pentose phosphate pathway as well as a clear increase of PRPP and IMP biosynthesis. Energy charge, the nucleotide phosphate pool and lactate/glucose ratio remained stable after metformin treatment. Furthermore, our results showed that basal glucose uptake/consumption and the activity of the lower segment of the glycolytic pathway are key determinants of a cytotoxic response to metformin. In addition, high glutathione, malate, IMP and orotate content were observed in metformin-insensitive leukemic cells. Moreover, the cytotoxic effect of metformin was independent of AMPK/LKB1 status of the leukemic cells while p53 expression abrogated this effect. The presence of wild-type p53 appears to partially protect tumor cells from glucose starvation and metformin cytotoxicity and prevents the induction of the Pasteur effect. Finally, we demonstrated that metformin increased the cytotoxicity of chemotherapy agent, cytarabine, on all leukemic cell lines in vitro and significantly reduced leukemic colony-forming units (CFU-L) from six primary AML patient samples in a concentration-dependent manner. Additional experiments on metabolic and signaling pathways as well as in vivo studies are in progress to better understand the cytotoxic response of metformin in both AML cell lines and primary AML patient specimens that impact the therapeutic potential of metformin in vivo. Disclosures: Carroll: Agios Pharmaceuticals: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Sanofi Aventis Corporation: Research Funding; Glaxo Smith Kline, Inc.: 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.

scholarly journals A NATURAL PRODUCT DECURSIN ENHANCES THE RADIOSENSITIZATION OF IONIZING RADIATION AGAINST DMBA-INDUCED TUMOR

2019 ◽  
pp. 14-16
Author(s):  
ADEEB SHEHZAD ◽  
HIRA ZAHID ◽  
WAZIR MUHAMMAD ◽  
RIZWAN AHMAD ◽  
EBTESAM A AL-SUHAIMI
Keyword(s):  
Radiation Therapy ◽  
Ionizing Radiation ◽  
Cell Lines ◽  
Cancer Cell ◽  
Underlying Mechanism ◽  
Cancer Cell Lines ◽  
Skin Lesions ◽  
Dependent Manner ◽  
Mice Model

Objective: Radiation therapy has gained significant attention for the treatment and prevention of solid and malignant human tumors. However, after periodical exposures, radiation therapy losses its efficacy against cancer cells displaying radio-resistant phenotypes. Therefore, decursin might improve the efficiency of radiotherapy against a variety of human cancers. Methods: The chemopreventive efficacy of decursin was evaluated against B16F10 cancer cell lines and DMBA/croton oil-induced skin carcinogenesis in BALB/c mice. Decursin was administered intraperitoneal at the dose of 20 mg/kg/day for 8 weeks following exposure to 5 Gy of ionizing radiation (IR) after 1 month of DMBA application. Western blot was performed for underlying mechanism of radioresistance. Results: Decursin suppressed the proliferation and viability of melanoma cancer cell lines in a concentration- and time-dependent manner. The in vivo data collected from mice model revealed that decursin reduced the precancerous skin lesions and the incidence of tumor bearing in radiation-exposed mice. Decursin also enhanced the effect of IR by downregulation of Akt/NFκB pathway through activation of IκBα. Conclusion: Our results suggest that the activation of Akt/NFκB establishes a pro-survival response to radiation that may account for the development of radioresistance. Decursin blocks the abnormal expression of these proteins and potentiates the radiotherapeutic effect.

scholarly journals Inhibiting the Core Autophagy Enzyme ATG4B with Novel Drugs Sensitizes Resistant Leukemic Stem/Progenitor Cells to Standard Targeted Therapy

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 933-933 ◽  
Author(s):  
Katharina Rothe ◽  
Akie Watanabe ◽  
Donna L. Forrest ◽  
Sharon Gorski ◽  
Robert Young ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Interaction Analysis ◽  
Leukemic Cells ◽  
In Vivo Model ◽  
Autophagic Flux ◽  
Proof Of Concept

Abstract Leukemic stem cell (LSC) persistence is a major cause of therapy failure and relapse in patients, thus warranting further investigations into the cells' molecular properties to aid in meaningful clinical decisions. Chronic myeloid leukemia (CML) is a clear example and excellent study model with rare, propagating LSCs that are not eradicated by BCR-ABL1-directed tyrosine kinase inhibitor (TKI) monotherapy such as Imatinib Mesylate (IM), since the LSCs do not exclusively rely on BCR-ABL1 for their survival. We and others have shown that the persistence of primitive leukemic cells is mediated by macroautophagy (autophagy), a catabolic cellular recycling process. In particular, we discovered that transcript and protein expressions of all ATG4 family members, including ATG4A, B, C, and D, are significantly increased in CD34+ CML cells compared to CD34+ normal bone marrow (BM) cells. ATG4B expression is also significantly higher in pre-treatment CD34+ CML cells from IM-nonresponders vs. IM-responders, including LSCs. Moreover, we revealed that stable suppression of ATG4B significantly suppressed autophagy, impaired survival of IM-nonresponder stem/progenitor cells and sensitized leukemic cells to TKI treatment. Thus, we identified ATG4B as a critical therapeutic target in CML (Rothe et al., Blood, 2014). To further explore whether targeting of ATG4B would be a viable strategy in the successful treatment of various leukemia, two lead ATG4B inhibitors were recently developed for pre-clinical proof-of-concept studies. Compound 4-28, a styrylquinoline, was identified by in silico screening and high content cell-based screening. Its structure-activity relationship (SAR)-based optimization led to a more stable and potent compound, LV-320. LV-320 was further evaluated by Microscale Thermophoresis and showed consistent Kd values for binding to the ATG4B enzyme (Kd=16±1 μM). LV-320 can inhibit autophagic flux, shows excellent tolerability and a good PK profile. It is also characterised as a non-competitive inhibitor of ATG4B and displays similar potency against ATG4A. Interestingly, inhibition of ATG4B with compound 4-28 decreased viability by 40-60% and increased apoptosis by 30-40% in different BCR-ABL1+ leukemic cell lines upon serum-deprivation as compared to the same cells without treatment of 4-28 (p<0.05). 4-28 treatment also efficiently inhibited autophagic flux in these cells as shown by Western blot analysis of LC3-II/I and p62 accumulation. In addition, compound 4-28 was able to inhibit the clonal growth of patient-derived CML stem/progenitor cells from IM-nonresponders, in particular when combined with various TKIs compared to single agents (20 vs. 50%, p<0.05). However, this combination approach also showed slight toxic effects on healthy BM cells. We then tested the more stable and potent compound LV-320 with superior results. Treatment of several drug-resistant and mutated CML and aggressive BCR-ABL1+ B-ALL cell lines with LV-320 increased apoptosis up to 90% compared to controls and reached almost 100% when combined with various TKIs (p<0.05). Moreover, LV-320 sensitized IM-nonresponder stem and progenitor cells to TKIs in colony-forming short-term and long-term cell assays as compared to single agents (10 % vs. 40 %, p<0.05), but was not toxic to primitive BM cells from healthy donors (up to 10µM). Mechanistically, we found that LV-320 effectively inhibited autophagic flux in leukemic cells: Confocal microscopy demonstrated an increase in LC3-II-positive punctae (autophagosomes) and the presence of yellow punctae (blocked autophagosome-lysosome fusion) when leukemic cells where transduced with a mRFP-GFP-LC3 construct and treated with LV-320 or a combination of LV-320 and TKIs. Drug interaction analysis further indicated synergy between LV-320 plus IM (CI value ≤ 0.9). A novel in vivo model is currently being investigated to validate our proof-of-concept. Together, our results suggest that targeting of ATG4B with novel autophagy inhibitors in combination with TKIs may be able to circumvent drug resistance in CML and possibly other aggressive leukemia. Disclosures No relevant conflicts of interest to declare.

The BRAF Pseudogene Is a Proto-Oncogenic Competitive Endogenous RNA

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 263-263
Author(s):  
Florian Karreth ◽  
Markus Reschke ◽  
Bjoern Chapuy ◽  
Margaret A. Shipp ◽  
Roberto Chiarle ◽  
...  
Keyword(s):  
Cell Lines ◽  
Conflicts Of Interest ◽  
Human Cancer ◽  
Disease Onset ◽  
B Cell Lymphoma ◽  
Mapk Signaling ◽  
Dependent Manner ◽  
Genomic Locus

Abstract Non-coding RNAs have long been viewed as non-functional genomic relicts of evolution, but recetn findings have implicated their importance in physiology and disease. Recently, in vitro experiments demonstrated that the pseudogenes of PTEN and KRAS operate as natural miRNA decoys (competitive endogenous RNAs or ceRNAs) that regulate the expression of their parental genes. However, in vivo evidence for a causal role of pseudogenes in cancer development is lacking. To investigate whether the BRAF pseudogene (BRAFps) possesses oncogenic properties we generated transgenic mice carrying a Tet-inducible BRAF pseudogene allele. Global BRAFps overexpression resulted in the development of aggressive B-cell lymphoma after 6-12 months. These tumors were characterized by a profound expansion of B-lymphocytes in the spleen, as well as splenomegaly, lymphadenopathy and infiltration of the kidneys, lungs, and liver by neoplastic cells. The BRAFps-induced lymphoma was polyclonal, transplantable, dependent on continuous BRAFps expression, and cooperated with heterozygous loss of PTEN to accelerate disease onset. Mechanistically, we propose that BRAFps functions as a ceRNA that sequesters miRNAs from BRAF and possibly other targets. Indeed, overexpression of BRAFps results in elevated levels of BRAF in a Dicer-dependent manner. This, in turn, increased BRAF-dependent MAPK signaling and proliferation. To further validate the ceRNA activity of BRAFps, we engineered mice to express only the 3’UTR or CDS of BRAFps as each portion of the pseudogene may individually engage in miRNA-mediated crosstalk with BRAF. Notably, both BRAFps-CDS and BRAFps-3’UTR increased spleen and lymph node weights 6 months after induction. Interestingly, BRAFps-3’UTR elicited a lymphoma phenotype similar to full length BRAFps, while mice expressing BRAFps-CDS developed a more indolent form of this phenotype, suggesting that lymphomagenesis is primarily mediated by the BRAFps 3’UTR. BRAFps transcript was undetectable in primary human B-cells, but was aberrantly expressed in primary human DLBCL and human DLBCL cell lines. Expression of BRAF and BRAFps was positively correlated in human primary DLBCL and human DLBCL cell lines. In addition, gains or amplifications of the genomic locus containing BRAFps were found in various human cancer types. Overexpression of BRAFps in human lymphoma cells elevated BRAF levels, MAPK activation, proliferation and growth in xenografts. Our results demonstrate for the first time the oncogenic potential of a pseudogene in an engineered mouse model and indicate that ceRNA- mediated regulation is an important regulatory mechanism of gene expression in vivo. Disclosures No relevant conflicts of interest to declare.

Loss of the mitochondrial phosphate carrier SLC25A3 induces remodeling of the cardiac mitochondrial protein acylome

2021 ◽  
Author(s):  
Jessica N. Peoples ◽  
Nasab Ghazal ◽  
Duc M. Duong ◽  
Katherine R. Hardin ◽  
Janet R. Manning ◽  
...  
Keyword(s):  
Energy Production ◽  
Mitochondrial Protein ◽  
Atp Synthesis ◽  
High Energy ◽  
Mitochondrial Proteins ◽  
Isocitrate Dehydrogenase 2 ◽  
Signaling Organelles ◽  
Specific Pathway ◽  
Phosphate Carrier

Mitochondria are recognized as signaling organelles because, under stress, mitochondria can trigger various signaling pathways to coordinate the cell's response. The specific pathway(s) engaged by mitochondria in response to mitochondrial energy defects in vivo and in high-energy tissues like the heart are not fully understood. Here, we investigated cardiac pathways activated in response to mitochondrial energy dysfunction by studying mice with cardiomyocyte-specific loss of the mitochondrial phosphate carrier (SLC25A3), an established model that develops cardiomyopathy as a result of defective mitochondrial ATP synthesis. Mitochondrial energy dysfunction induced a striking pattern of acylome remodeling, with significantly increased post-translational acetylation and malonylation. Mass spectrometry-based proteomics further revealed that energy dysfunction-induced remodeling of the acetylome and malonylome preferentially impacts mitochondrial proteins. Acetylation and malonylation modified a highly interconnected interactome of mitochondrial proteins, and both modifications were present on the enzyme isocitrate dehydrogenase 2 (IDH2). Intriguingly, IDH2 activity was enhanced in SLC25A3-deleted mitochondria, and further study of IDH2 sites targeted by both acetylation and malonylation revealed that these modifications can have site-specific and distinct functional effects. Finally, we uncovered a novel crosstalk between the two modifications, whereby mitochondrial energy dysfunction-induced acetylation of sirtuin 5 (SIRT5), inhibited its function. Because SIRT5 is a mitochondrial deacylase with demalonylase activity, this finding suggests that acetylation can modulate the malonylome. Together, our results position acylations as an arm of the mitochondrial response to energy dysfunction and suggest a mechanism by which focal disruption to the energy production machinery can have an expanded impact on global mitochondrial function.

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