bluuebunny/crossref_metadata_2025 huggingface dataset:
The Epigenetic Regulators CBP and p300 Facilitate Leukemogenesis and Represent Therapeutic Targets In Acute Myeloid Leukemia (AML)
Authors: Giotopoulos George , Chan Wai-In , Ruau David , Gallipoli Paolo , Fowler Alexis , Göttgens Berthold , Van Deursen Jan , Cole Philip , Huntly Brian
Published: October 2019

Although molecularly and clinically heterogeneous, AML is characterised by aberrant transcription and abnormal epigenetic regulation. The transcriptional co-activators CBP and p300 have well characterised roles in hematopoiesis and hematopoietic stem cell (HSC) function. These are mutated in AML and also bind a number of AML-associated oncogenes. However, despite this, their roles in the induction and maintenance of AML are poorly understood. To address this question, we have combined genetic and pharmacological inhibition of CBP and p300 in AML and normal hematopoiesis.

Using a murine model where Cbp was conditionally deleted from murine hematopoietic stem and progenitor cells (HSPC), either prior to, or following expression of a number of AML-associated oncogenes, we assessed the role of Cbp in the induction and maintenance of AML. We demonstrated that although not an absolute requirement, Cbp confers a selective advantage for robust immortalisation in vitro, and that Cbp is also an important requirement for the generation and maintenance of AML in vivo. Furthermore, redundancy between Cbp and p300 in myeloid transformation was demonstrated, as p300 knockdown further decreased proliferation in Cbp-/- AML cells.

We next validated CBP/p300 as potential therapeutic targets, using a pharmacological strategy. Using a selective small molecule inhibitor (C646) of the lysine acteyltranferase (KAT) activity of CBP/p300, we demonstrated a significant decrease in growth and clonogenic potential across multiple AML subtypes in vitro. This was mediated through induction of apoptosis and cell cycle arrest. Importantly, no alteration in the growth of normal murine and human hematopoietic progenitors was detected at similar doses. We further demonstrated that inhibition of CBP/p300 KAT activity in human leukemia cells alters a transcriptional programme associated with genomic integrity, linking transcriptional changes to the cellular phenotype. Finally, we demonstrated the efficacy of the HAT inhibitors to decrease clonogenic growth across a panel of primary AML patient samples, representing multiple genetic subtypes. Taken together, these data suggest that CBP/p300 are involved in leukemogenesis across multiple subtypes in AML and that targeting these proteins may be possible with an acceptable side-effect profile.

Disclosures:

No relevant conflicts of interest to declare.
NKL Homeobox Gene MSX1 Reactivates An Oncogenic Network In Lymphoid and Myeloid Malignancies
Authors: Nagel Stefan , Ehrentraut Stefan , Meyer Corinna , Kaufmann Maren , Drexler Hans G. , MacLeod Roderick AF
Published: October 2019

Homeobox genes encode conserved transcription factors (TFs) which regulate fundamental cellular processes during development. Many members of the NKL homeobox gene subfamily are aberrantly expressed in T-cell leukemia and compromise cell differentiation. NKL homeobox gene MSX1 is expressed during embryonic hematopoiesis and its deregulation in Hodgkin lymphoma suggests an oncogenic role of this gene in hematopoietic malignancies. After screening 114 leukemia/lymphoma cell lines by microarray profiling, we detected MSX1 overexpression in three examples each from T-cell acute lymphoblastic leukemia (T-ALL) and mantle cell lymphoma (MCL), and one from acute myeloid leukemia (AML). In silico analysis by R-based statistical tools identified conspicuous expression of MSX1 in 11% of pediatric T-ALL patients, and in 3% each of MCL and AML patients. Thus, we found aberrant MSX1 expression in subsets of both lymphoid and myeloid malignancies. Focusing on MCL and AML we excluded chromosomal rearrangements by classical and molecular cytogenetics at the MSX1 locus underlying overexpression in affected cell lines. However, comparative expression profiling data indicted aberrant histone acetylation involving PHF16 and RTN1, together with TFs FOXC1, HLXB9 and TAL1, as activators of MSX1 transcription. Their involvement was confirmed by siRNA-mediated knockdown and overexpression studies. Reciprocal regulation of MSX1 involved CCND1 and NOTCH signalling. Reporter gene analyses demonstrated that CCND1 and CDKN2D are direct transcriptional targets of MSX1 and its repressive cofactor histone H1C. Fluorescence in situ hybridization showed that t(11;14)(q13;q32) in MCL results in detachment of CCND1 from its corresponding repressive MSX1 binding site. In conclusion, we uncovered a regulatory network around MSX1 in leukemia/lymphoma cells, involving factors and pathways implicated in embryonic hematopoiesis. The reciprocal regulation of MSX1 and the NOTCH pathway in B-cells parallels that of MSX2 in T-cells. These data support the view of a recurrent genetic network involved in hematopoietic development which is reactivated in malignant transformation.

Disclosures:

No relevant conflicts of interest to declare.
An Epigenetic Mechanism Of Imatinib Via Demethylation Of MiR-203
Authors: Umemura Tsukuru , Shibuta Tatsuki , Honda Emi , Shiotsu Hiromichi , Tanaka Yuka , Vellasamy Shalini , Shiratsuchi Motoaki
Published: October 2019

Background

MicroRNAs (miRNAs) are short noncoding RNAs regulating a variety of biological processes by post-transcriptionally silencing via targeting mRNA. Recently there are many reports demonstrating that epigenetic alterations correlate to the characteristics of tumor cells, and that miRNAs were also reported to be regulated by methylation of CpG islands within the promoter region. MiR-203 is epigenetically silenced in human BCR-ABL1-positive leukemic cell lines and primary chronic myelogenous leukemia (CML) cells by the methylation of promoter region. In this study, we analyzed the effect of imatinib, a tyrosine-kinase inhibitor specific to BCR-ABL1 protein, on the expression of miRNA in BCR-ABL1-positive cells.

Materials & Methods

Two CML cell lines (K562 and KU812) and one AML cell line (HL60) were treated with imatinib for 72 hours. Microarray analysis of miRNAs was conducted by 3D-Gene (TORAY) in K562 cells with/without imatinib. Methylation specific PCR and bisulfite direct sequencing was performed to evaluate methylation status of promoter region of miR-203. Validation of expressions of miRNAs, including miR-203, and mRNAs was analyzed by RT-qPCR. The expression of BCR-ABL protein was confirmed by Western blotting. The function of miR-203 for cell survival was evaluated by the transfection of anti-miRNA.

Results

The microarray analysis showed that 48 miRNAs of CpG-rich 212 miRNAs were upregulated over 2-fold after imatinib treatment, in K562 cells. The demethylated state of the promoter region of miR-203, one of 48 miRNAs, was confirmed by bisulfite direct sequencing. The expression of BCR-ABL mRNA, which is one of the target of miR-203, was inhibited with imatinib to 52% and 26% of the level in control cultures in K562 cells and KU812 cells, respectively. The expression of BCR-ABL protein was also inhibited. The addition of anti-miR-203 increased the expression level of BCR-ABL protein to 68.1% in the K562 cell culture with imatinib treatment. The expression of DNA methyltransferase (DNMT) mRNA was analyzed, and the expressions of DNMT1 and DNMT3B were significantly decreased after imatinib treatments in CML cell lines, whereas the expression of DNMT3A was not changed.

Discussion & Conclusion

We report, for the first time, that imatinib up-regulated miR-203 by inducing demethylation of the promoter region of miR-203 in CML cells. MiR-203 is the important miRNA to inhibit ABL1 and BCR-ABL1 mRNA, and imatinib-induced demethylation of miR-203 is the possible mechanism to suppress growth of BCR-ABL1-positive leukemic cells. It was suggested that the demethylation was partially caused by down regulation of DNMT1 and DNMT3B after imatinib treatments in CML cell lines. In conclusion, imatinib not only inhibits the activity of tyrosine kinase but also induces DNA demethylation of miR-203 in CML cells.

Disclosures:

No relevant conflicts of interest to declare.
A Novel Role Of Tescalcin-Sodium/Hydrogen Exchange Axis Underlying Sorafenib Resistance In FLT3-ITD+ AML
Authors: Man Cheuk Him , Leung Anskar Y.H. , Yuen Lam Stephen Sze , Hei Sun Murphy Ka , Hung Chow Howard Chun , Gill Harinder , Kwong Yok Lam
Published: October 2019

Internal tandem duplication (ITD) of FLT3 (fms-like tyrosine kinase 3) in acute myeloid leukemia (AML) is associated with inferior clinical outcome. Sorafenib is effective in targeting chemo-refractory FLT3-ITD+ AML. However, leukemia progresses invariably. Mechanisms of drug resistance are not completely understood. We hypothesized that a gene encoding tescalcin (TESC), which activate a sodium/hydrogen exchanger 1 (NHE1) and known to be up-regulated at leukemia progression during continuous sorafenib treatment, may underlie TKI resistance. TESC was over-expressed in FLT3-ITD+ AML lines MOLM-13 and MV4-11. Knocking down TESC by siRNA lowered intracellular pH and induced apoptosis. The results were recapitulated by treatment with a NHE1 inhibitor, 5-(N,N-Hexamethylene) amiloride (HMA). Sorafenib resistant MOLM-13 cell line (M13-RE) was generated by long term culture of sorafenib. M13-RE significantly increased its sensitivity to HMA. HMA also enhanced suppression of FLT3 signaling by sorafenib in otherwise resistant cell lines. Treating MOLM-13, MV4-11 and primary FLT3-ITD+ AML cells with HMA significantly reduced leukemia initiation in NOD/SCID mouse xenotransplantation. These observations provided novel information about the pathogenetic role of the TESC-NHE1-pHi axis in mediating sorafenib resistance in AML.

Disclosures:

No relevant conflicts of interest to declare.
IDH1 Mutant Inhibitor Induces Cellular Differentiation and Offers a Combination Benefit With Ara-C In a Primary Human Idh1 Mutant AML Xenograft Model
Authors: Yen Katharine , Lemieux Rene , Popovici-Muller Janeta , Chen Yue , Yang Hua , Straley Kim , Choe Sung , Dorsch Marion , Agresta Samuel , Schenkein David P. , Biller Scott , Su Michael , Wang Fang
Published: October 2019

Somatic point mutations in isocitrate dehydrogenase 1/2 have a gain-of-function neomorphic activity that converts alpha-ketoglutarate to the oncometabolite, R (-)-2-hydroxyglutarate (2HG). Prospective studies of AML patients carrying IDH mutations have shown that intracellular concentrations of 2HG can range from 3-10 mM. This abnormal level of 2HG results in dysregulation of alpha-ketoglutarate dependent enzymes leading to alterations in the epigenetic state of hematopoietic progenitor/stem cells and functionally blocks their ability to fully differentiate.

We have developed a potent and selective, orally available IDH1 mutant inhibitor AGI-14100, that is able to reduce intracellular 2HG concentrations to baseline levels found in wildtype cells. Ex vivo treatment of IDH1 mutant-containing primary human AML patient samples with AGI-14100 induced a proliferative burst followed by cellular differentiation as shown by flow cytometry and cytology.

We next treated a primary human IDH1 (R132H)/FLT3-ITD mutant xenograft model with AGI-14100 either alone or in combination with Ara-c. In these studies, AGI-14100 alone significantly decreased tumor burden in the peripheral blood after 1 month of continuous BID treatment. In combination with a short-term, low-dose course of Ara-C, we also observed a decrease in the bone marrow tumor burden that was better than either treatment alone. Furthermore, this response was sustainable for >3 weeks even after dosing of both drugs had been terminated. Taken together, these data suggest that inhibition of mutant IDH1with AGI-14100 and low dose Ara-c could provide a combination benefit for patients with AML.

Disclosures:

Yen: Agios Pharmaceuticals: Employment, Equity Ownership. Lemieux:agios Pharmaceuticals: Employment, Equity Ownership. Popovici-Muller:agios Pharmaceuticals: Employment, Equity Ownership. Chen:agios Pharmaceuticals: Employment, Equity Ownership. Yang:Agios Pharmaceuticals: Employment, Equity Ownership. Straley:Agios Pharmaceuticals: Employment, Equity Ownership. Choe:agios Pharmaceuticals: Employment, Equity Ownership. Dorsch:agios Pharmaceuticals: Employment, Equity Ownership. Agresta:agios Pharmaceuticals: Employment, Equity Ownership. Schenkein:agios Pharmaceuticals: Employment, Equity Ownership. Biller:agios Pharmaceuticals: Employment, Equity Ownership. Su:agios Pharmaceuticals: Employment, Equity Ownership. Wang:Agios Pharmaceuticals: Employment, Equity Ownership.
Strong Therapeutic Effect Of Anti-IL1RAP Immunotherapy In a Xenograft Model Of Acute Myeloid Leukemia
Authors: Agerstam Helena , Askmyr Maria , Hansen Nils , Rissler Marianne , Juliusson Gunnar , Richter Johan , Wunderlich Mark , Järås Marcus , Mulloy James C. , Fioretos Thoas
Published: October 2019

Acute myeloid leukemia is a genetically heterogeneous disease characterized by expansion of immature hematopoietic cells in the bone marrow. The disease is associated with a general poor survival and thus the need for novel treatment strategies is urgent. We have previously shown that the IL1 receptor accessory protein (IL1RAP) is highly expressed on primitive chronic myeloid leukemia (CML) cells and that a polyclonal antibody against IL1RAP can direct natural killer cells to specifically target and destroy CML CD34+CD38- cells in an in vitro-based antibody dependant cell-mediated cytotoxicity (ADCC) assay (Järås et al, PNAS, 2010). We recently also demonstrated that IL1RAP is upregulated in about 80% of AML patients and that primitive AML cells can be targeted by a monoclonal antibody against IL1RAP and selectively killed by ADCC (Askmyr et al, Blood, 2013).

In this study, we applied these findings to a human AML xenograft model. NOD/SCID mice were engrafted with lethal doses of MA9Ras cells, previously generated by transformation of human umbilical cord blood CD34+ cells by retroviral integration of cDNAs directing the expression of an MLL/AF9 fusion and an activated NRAS gene. Leukemic mice were treated twice weekly with Mab81.2, a monoclonal antibody targeting IL1RAP, or a corresponding isotype control antibody. Notably, the seven mice treated with Mab81.2 displayed a significant reduction of leukemic cells in the peripheral blood 35 days after transplantation compared to the seven mice in the isotype control treated group (0.10±0.04% vs. 0.69±0.45% CD45+ cells, p=0.0043). This reduction was even more significant at 62 days (3.75±2.50% vs. 46.46±24.92% CD45+ cells, p=0.0007). Treatment with the IL1RAP-targeting antibody also resulted in an increased overall survival compared to the isotype control treated group (median survival 75 days vs 64 days, p=0.0089). Furthermore, at time of sacrifice, the Mab81.2-treated mice were shown to have significantly lower frequency of leukemic cells in the bone marrow (4.99±3.34% vs. 59.51±25.18% CD45+ cells, p=0,0001) and spleen (17.19±10.21% vs. 49.71±23.09% CD45+ cells, p=0,0052).

In conclusion, we show that in vivo delivery of the Mab81.2 monoclonal antibody results in significant suppression of human MA9Ras cells and prolongs survival in a xenograft setting, demonstrating that anti-IL1RAP immunotherapy provides a new promising treatment approach for AML.

Disclosures:

Richter: Cantargia: Consultancy, Equity Ownership. Järås:Cantargia AB: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Fioretos:Cantargia AB: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Research Funding.
ODE-Adefovir As Potential Therapeutic Agent In AML
Authors: Khoury Haytham , Ruijuan Hu , Schimmer Aaron , Hostetler Karl , Minden Mark D
Published: October 2019

Adefovir is an antiviral adenosine monophosphate analogue from the class of acyclic nucleoside phosphonate (ANP). During 0n-patent drug library screen, we discovered adefovir-dipivoxil (adefovir-DP) as potentially active against AML. Second-generation formulations Of adefovir, including ODE-adefovir and HDP-adefovir, have been designed to overcome adefovir-DP pharmacodynamic disadvantages. To characterize the activity of the above-mentioned agents in AML, we undertook this study. In AML cell lines (OCI/AML-1, -2, -3, -4 and -5) adefovir-DP appeared to be highly potent with IC50 ranging from 45.8 to 252.5 nM. ODE-adefovir was equally potent as adefovir-DP (IC50 from 51.7 nM to 205.3 nM), whereas HDP-adefovir was significantly less potent (224.8±19.6 to 956.3±14.4 nM). Similar results were obtained when we tested the activity of these agents in primary AML patient samples. Interestingly, ODE-adefovir was remarkably less toxic than adefovir-DP in normal hematopoietic cells, with IC50 at 40 and 1µM, respectively. In addition, ODE-adefovir at dose 15mg/kg/day showed potent activity against AML in NOD/SCID-mouse human-leukemia model with reduction in human leukemia in mouse bone marrow > 50%in all mice tesed within 14 days. Based on its chemical structure, we hypothesized that the plausible mechanisms of ODE-adefovir cytotoxicity in AML are cell-cycle arrest and DNA damage. Indeed, treatment of OCI/AML-2 cells with ODE-adefovir induced increase of the cells in the S phase from 9% to 30%. Moreover, ODE-adefovir increased the expression of pH2Ax, a hallmark of DNA damage, within 2h of treatment. Plus, there was increase in phosphorylation of P53; transactivation of proapoptotic genes, Puma, Noxa and BAX; and induction of the intrinsic apoptotic pathway, as shown by the cleavage of caspases 7 and 9. Further investigation unveiled strong synergism between ODE-adefovir and cytarabine, a mainstay drug in the treatment of AML, without any cross-resistance between both agents, thus making their co-administration potentially beneficial. MRP4 expression appeared to influence AML cells response to ODE-adefovir and its inhibition potentiates this response. Taken together, our findings emphasize the potential cytotoxic activity of adefovir in AML and the importance of choice of appropriate formulation for such purpose. Further, they indicate that the development of ODE-adefovir for clinical testing in AML is warranted.

Disclosures:

Off Label Use: Adefovir dipivoxil is a drug used for the treament of hepatitis B. WE found it is cytotoxic for AML.
Replacing Gemtuzumab Ozogamicin With Idarubicin In Frontline Fludarabine, Cytarabine and G-CSF Based Regimen Does Not Compromise Outcome In Core Binding Factor Acute Myelogenous Leukemia
Authors: Borthakur Gautam , Cortes Jorge E. , Ravandi Farhad , Gonzalez Graciela Nogueras , Luthra Rajyalakshmi , Jabbour Elias , Garcia-Manero Guillermo , Kadia Tapan M. , Bass Joyce , Brandt Mark , Kantarjian Hagop M.
Published: October 2019

A regimen comprising of fludarabine, cytarabine, G-CSF and gemtuzumab ozogamicin (FLAG-GO) has been our frontline treatment regimen for patients with core binding factor acute myelogenous leukemia (CBF-AML) and among 50 patients with median follow up of over 3 years, this regimen resulted in overall survival (OS) and relapse free survival (RFS) of 78% and 85% respectively. This is clearly better than our historical data with idarubicin and cytarabine based regimens (Borthakur et al. JCO. Vol 28, No 15 suppl; 2010:6552). After withdrawal of GO from the market, it has been substituted by low dose idarubicin at 6 mg/m2 on days 3 and 4 in induction and in one post remission cycle during cycles 3-6 (FLAG-Ida). So far 38 patients have been treated with FLAG-Ida (median follow up 1 year) with all patients achieving complete remission. The current report is part of the planned analysis to ensure that patient outcomes have not been compromised by the change in regimen.

Univariate and multivariate (MVA) Cox proportional hazards regression was used to identify association of the clinical variables with overall survival (OS), event free survival (EFS) and time to relapse (TTR). Event is defined as death from any cause or relapse. Treatment regimen (FLAG-GO or FLAG-Ida) were included as variables in the analysis. Apart from relevant clinical variables, reduction in fusion transcript ratio (in reference to ABL gene transcript compared to that at diagnosis) at time points 1 month (≥3 log reduction yes/no) and 3 month (≥3 log reduction yes/no) and presence of any mutation (RAS, KIT, FLT3 yes/no) were also added as variables. Stepwise backwards selection method was used to remove variables that did not remain significant in the multivariate model (p ≥ 0.15).

T(8;21) is the cytogenetic abnormality in 52% of all 88 patients. Median age of all patients is 51 (range, 19-78 years) and 48% are female. Median time to 3 log reduction in transcript ratio was 1 month (range, 1-22 months). Complete remission rate with or without platelet recovery has been 98% with 2 induction deaths.

Kaplan-Meier analysis showed OS (Fig.1), EFS and TTR were not significantly different FLAG-GO and FLAG-Ida regimens. By MVA, OS, EFS and TTR are not different among these regimens (p > 0.5). Three log or more reduction in transcript ration at 1 month was associated with better OS (p=.03) and EFS (p=.008) (not TTR) in MVA. Presence of KIT, RAS or FLT3 gene mutation did not impact outcomes studied.

In conclusion, replacing GO with low dose idarubicin in a front-line FLAG regimen does not seem to compromise the excellent outcomes with such a regimen in CBF AML.

Disclosures:

No relevant conflicts of interest to declare.
The Effects Of Hydrogen Sulphide On Adipocyte Viability In Human Adipocyte and Adipocyte Derived Mesenchymal Stem Cells Culture Under Ischemic Conditions
Authors: Aykan Andac , Ozturk Serdar , Avcu Ferit , Sahin Ismail , Ural Ali Ugur , Ilikci Rahsan , Isik Selcuk
Published: October 2019

Hydrogen Sulfide (H2S) is accepted as a gasotransmitter, which has a gaseous structure like the other two gasotransmitters; carbon monoxide and nitric oxide. It is known that H2S increases the resistance to ischemia in some tissues and decreases apoptosis, however the exact mechanism is still unclear. Morover there is no research about its effects on adipose tissue in the literature.

The aim of this study was to evaluate the in vitro effects of H2S on Adipocyte survival under ischemic conditions and to explain the possible mechanisms of its apoptotic process in details.

For this purpose mesenchymal stem cell (MSCs) culture was prepared from subcutaneous adipose tissue sample of a human donor. Adipose derived MSCs were differentiated into adipogenic direction and mature adipocyte culture was obtained. Adipose derived MSCs culture and mature adipocyte culture were divided into six groups separately. Sodium Hydrogen Sulfide (NaHS) was used as a H2S donor. After applying NaHS to all groups with a concentration of 0, 0.1, 1, 10, 100 and 1000μM, cell cultures were incubated in special gas incubator with %1 oxygen at 37°C for 24 hours. Following the ischemia period, cell culture groups were evaluated by the rates of proliferation/cytotoxicity within MTT test, apoptosis and necrosis rates with flow cytometry, apoptotic (bax, caspase3) and anti-apoptotic genes (bcl-2) expression levels within reverse transcriptase polymerase chain reaction.

Statistically significant increase in proliferation rates were found in MSCs groups which was applied low dose (0,1 and 1 μM) NaHS (p<0,05). For every different dose applications, statistically significant decrease was found in apoptosis levels on the mature adipocyte cultures (p<0,05). In both cell culture groups bcl-2 gene expression was increased and caspase-3 gene expression was decreased.

As a conclusion, H2S has protective effect on both MSCs and mature adipocytes and this effect is mediated by elevation of anti-apoptotic gene expression. Furthermore, this study suggests that, administration of H2S to fat graft before fat graft injection may be therapeutic benefit in the fat graft viability.

Disclosures:

No relevant conflicts of interest to declare.
Disease-Associated Chromatin Conformation and Therapeutic Implications In Leukemia
Authors: Cheng Jason X , Anastasi John , Vardiman James W.
Published: October 2019

Disease- or treatment-associated chromatin conformation has yet to be illustrated. Here we first demonstrate that monocytic and erythroid leukemia cell lines have distinctly different chromatin conformation at the PU.1 locus: a looped, connected, RNA-polymerase-II-bound, active conformation in the former and a disconnected, inactive conformation in the latter. These conformations undergo opposite transformations, becoming more active in the erythroid leukemia line and less active in the monocytic leukemia line, in response to both DNA and histone hypomethylating drugs.To explore the underlying mechanisms we developed a novel method to analyze DNA modifications. We demonstrate that the erythroid leukemia line has a marked drug-responsive change in both hydroxymethyl-CpG and 5-meythyl-CpG at the promoter, whereas the monocytic leukemia line has a higher level bivalent histone/chromatin with co-localized H3K4me3 and H3K27me3 at the enhancer. Consequently, the erythroid leukemia line is more sensitive to DNA hypomethylation while the monocytic leukemia line is more sensitive to histone hypomethylation. Further studies on clinical leukemia samples confirm the findings, and demonstrate that the leukemic cells from the clinical samples have different chromatin conformations with much more bivalent chromatin and denser DNA/histone modifiers and are more sensitive to hypomethylating drugs, compared to normal controls. Different chromatin conformations dictate preferential drug responses and open new diagnostic and therapeutic avenues.

Disclosures:

No relevant conflicts of interest to declare.
Inhibition Of PI3K/mTOR Overcomes Nilotinib Resistance In BCR-ABL1 Positive Leukemia Cells
Authors: Ding Jie , Romani Julia , Zaborski Margarete , MacLeod Roderick AF , Nagel Stefan , Drexler Hans G. , Quentmeier Hilmar
Published: October 2019

Expression of the Philadelphia chromosome (Ph), i.e., the t(9;22) chromosomal translocation and the formation of the BCR-ABL1 fusion protein, is the hallmark of chronic myeloid leukemia (CML). Tyrosine kinase inhibitors (TKI), such as imatinib and nilotinib, have emerged as leading compounds to treat CML. Translocation t(9;22) does not only occur in CML, 20-30% of acute lymphoblastic leukemia (ALL) are also found to carry the Ph. However, TKIs are not as effective in the treatment of Ph+ ALL as in CML. In this study, the Ph+ cell lines JURL-MK2 and SUP-B15 were used to investigate TKI resistance mechanisms and sensitization of Ph+ tumor cells to TKI treatment. The annexin V/PI (propidium iodide) assay revealed that nilotinib induced apoptosis in JURL-MK2 cells, but not in SUP-B15 cells. There was no mutation in the tyrosine kinase domain of BCR-ABL1 in both cell lines. SUP-B15 cells were not generally irresponsive to TKI, evidenced by dephosphorylation of the BCR-ABL1 downstream target GAB2 (Grb-associated binder-2). The resistance to apoptosis after nilotinib treatment was accompanied by the constitutive and nilotinib irresponsive activation of the phosphoinositide 3-kinase (PI3K) pathway. Treatment of SUP-B15 with the dual PI3K/mammalian target of rapamycin (mTOR) inhibitor BEZ235 alone induced apoptosis in a low percentage of cells, combination of nilotinib and BEZ235 led to a synergistic effect. Caspase 3 and PARP (poly ADP ribose polymerase) cleavage confirmed that apoptosis was induced after combined treatment. The main role of PI3K/mTOR inhibitor BEZ235 and the reason for apoptosis in the nilotinib-resistant cells was the block of the translational machinery, leading to the rapid down-regulation of anti-apoptotic protein MDM2 (human homolog of the murine double minute 2, hMDM2). The current findings suggest that MDM2 may be a therapeutic target to increase TKI-mediated apoptosis and that the combination of PI3K/mTOR dual inhibitor and TKI inhibitor might turn out to be a novel strategy for TKI-resistant BCR-ABL1 positive leukemia.

Disclosures:

No relevant conflicts of interest to declare.
The Preparation Of PLGA-PLL-Peg Nanoparticles Simultaneously Loaded With Daunorubicin and Tetrandrine By Modified Double-Emulsion Method
Authors: Liu Ran , Wang Yonglu , Wang Liyao , Cheng Jian , Xia Guohua , Bao Wen , Zhang Xiaoping , Guo Liting , Chen Baoan , Xu Yuanlong , Chen Wei , Yin Haixiang
Published: October 2019

A functional polymer composed of PLGA, PLL and PEG was synthesized, which was used as carrier material for fabricating drug delivery system of nanoparticles. PLGA-PLL-PEG nanoparticles simultaneously loaded with daunorubicin (DNR) and tetrandrine (Tet) were prepared in order to inhibit MDR activity and enhance the antitumor activity of DNR. A modified double-emulsion solvent evaporation/diffusion method was used to increase the incorporation of DNR (hydrophilic) and Tet (hydrophobic) into PLGA-PLL-PEG nanoparticles (NPs). The influence of various processing parameters on particle size and drug loadings were investigated systematically, such as the molecular weight, such as the molecular weight and concentration of PLGA-PLL-PEG, volume ratio of acetone to dichloromethane, PVA concentration in the external aqueous phase, the volume ratio of internal aqueous phase to external aqueous phase and the surfactants of internal aqueous phase. The particle size of the nanoparticles produced by optimized formulation and preparation was 213.0±12 nm (n = 3) with low polydispersity index (0.075 ± 0.023, n = 3). Transmission electron microscopy (TEM) examination showed that the morphology of the prepared nanoparticles was spherical in shape with a smooth surface. The drug loadings were 3.63±0.15% for DNR and 4.27±0.13% for Tet (n = 3). The entrapment efficiencies were 70.23±1.91% for DNR and 86.5±0.7% for Tet (n = 3). The release of DNR and Tet were sustained over one week. The PLGA-PLL-PEG-NPs formulation was potentially useful for hydrophilic and hydrophobic drugs that require efficient delivery to cancer cells.

Disclosures:

No relevant conflicts of interest to declare.
Drug-sensitivity of resistant acute myeloid leukemia cell line to Doxorubicin is enhanced by Triptolide through down-regulation of HIF-1α and Nrf2
Authors: Xu Bing , Chen Feili , Liu Yuejian , Wang Shiyun , Li Rongwei , Dong Huijuan
Published: October 2019

Background

Relapse and treatment-related complication have been the hurdle for the cure of acute myeloid leukemia patients. Finding a new drug-sensitizer could significantly prolong the survival time of patients. Triptolide (TPL) has been shown to enhance the drug-sensitivity of a variety of cancer cells when used in a low-concentration. HL60/DOX is a kind of acute myeloid leukemia cell line which is resistant to a commonly used drug Doxorubicin. This kind of cell line is widely used in the study of drug-sensitization.

Aim

This study aims to investigate whether low-dose TPL could enhance the drug-sensitivity of resistant acute myeloid leukemia cell line HL60/DOX to Doxorubicin and related mechanism.

Method

HL60/DOX cells were subjected to different treatments and thereafter MTT assay, flow cytometry and Western blot or RT-PCR were used to determine IC50, apoptotic status and expression of Nrf2, HIF-1α and their target genes.

Results

HL60/DOX cells were exposed to increasing concentrations of Doxorubicin with TPL at a constant IC20 concentration (14nM) for 48h. In comparison with anticancer agent alone, TPL enhanced the cytotoxicity of Doxorubicin (IC50:14.36±2.23 vs. 7.9±0.33μM, 1.82 fold; P=0.008) to HL60/DOX. Results of combination index showed that TPL and anti-cancer agents had synergistic effects when fraction affected was below 60%. Flow cytometry analysis also showed that apoptoticratio of cells treated by Doxorubicin together with TPL was significantly increased compared to cell streated by Doxorubicin alone (19.55±1.70%vs. 72.62±4.83%, P<0.01). Next, we explored the underlying mechanism. Hypoxia-inducible factor- 1α (HIF-1α) is the major mediator of hypoxic responses in malignant cells. High expression of it is correlated with bad clinical outcome in patients. It is also important for the survival of leukemia stem cells which are responsible for relapse. Nrf2 is an important protective factor which is correlated with chemo-resistance in cancer cells. When combined with TPL, Doxorubicin down-regulates Nrf2 and HIF-1α expression at protein and mRNA levels. Down-stream genes of Nrf2 e.g NQO1, GSR and HO-1 as well as target genes of HIF-1α e.g BNIP3, VEGF and CAIX are also down-regulated at mRNA level.

Conclusion

Inconclusion, our study demonstrates that TPL could enhance drug-sensitivity of HL60/DOX in vitro through down-regulation of Nrf2 and HIF-1α, providing the experimental base for further study.

Disclosures:

No relevant conflicts of interest to declare.
Gamma-Tocotrienol and Simvastatin Synergistically Induce Cytotoxicity In Leukemia Cell Lines, K-562 and HL-60
Authors: Maung Ko , Palau Victoria , Lightner Janet , Brannon Marianne , Krishnan Koyamangalath
Published: October 2019

Introduction

Statins and tocotrienols modulate the cholesterol biosynthesis pathway by inhibiting the 3-hydroxy-3- methylglutaryl coenzyme A (HMG-CoA) reductase. Tocotrienols modulate HMG-CoA reductase by post-transcriptional downregulation. In addition, tocotrienols contain a farnesol moiety in its side-chain that triggers degradation of HMG-CoA reductase. These effects lead to suppression of cell proliferation, cell cycle arrest, and apoptosis. Several studies have shown that statins have suppressive effects in in vitro experiments on acute myelocytic leukemia cell lines. Since both statins and gamma-tocotrienol are associated with decreased cholesterol biosynthesis, we hypothesized that if the cytotoxicity of these drugs on cancer cells is related to impaired biosynthesis of cholesterol, combination of them could synergize in cytotoxicity on leukemic cells.

Materials and Methods

K-562 and HL-60 leukemia cells were grown in Iscove's Modified Dulbecco's medium with penicillin/streptomycin, 10% fetal bovine serum and 20% fetal bovine serum added respectively. K-562 and HL-60 leukemia cells were seeded in 96 well plates, grown overnight, and treated for 24, 48 and 72 hours with simvastatin in concentrations of 1,2,4, and5 µM; gamma-tocotrienol in concentrations of 20, 40, and 80 µM; and a combination of the two drugs in the same concentrations. For 24 hour dose, cells were seeded at a density of 5000/well and for 48 and 72 hour doses, at  3500/well. Following the treatment, MTS/PMS reagent (Promega, Madison, WI), [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] mixed with and an electron coupling reagent (phenazine methosulfate), was added at 40 µg/well and incubated at 37°C for 2 hours. We measured the solubilized formazan crystals at 450 nm as an indicator of cytotoxicity. The presence of ATP  as an indicator of cell viability was measured with the CellTiter Glo®  assay (Promega). Cells were again seeded, grown overnight, and dosed as indicated above.  Following treatment, the assay was conducted as specified by the manufacturer.

Results

Both simvastatin and gamma-tocotrienol induce cytotoxicity in K-562 and HL-60 cell lines by the MTS and Cell Titer Glo Assays.  The IC50 at 72 hour incubation are as follows 1) HL-60: simvastatin - 16.543 uM, gamma tocotrienol - 36.297 uM;  2) K-562: simvastatin - 5.235 uM, gamma tocotrienol - 34.947 uM.  We used CompuSyn to calculate the IC50.  When combined, simvastatin and gamma-tocotrienol exhibit synergy at lower concentrations when examined by isobologram analysis.

Conclusion

Gamma-tocotrienol, an isoform of vitamin E, and simvastatin, a cholesterol lowering drug exhibit synergy in induction of cytotoxicity in K-562 and HL-60 leukemia cell lines. Rescue experiments and mechanistic pathway analysis are being explored to confirm these observations.

Disclosures:

No relevant conflicts of interest to declare.
TIM-3 Is Highly Expressed On Blast Cells In Patients With Acute Myeloid Leukemia
Authors: Li Caixia , Yu Xiao , Zhu Yibei , Wu Xiaojin , Ma Xiao , Liu Hong , Ye Lu , Ma Chao , Xia Rui , Sun Aining , Ruan Changgeng , Chen Suning , Depei Wu
Published: October 2019

T cell immunoglobulin-3(TIM-3) is known as a negative regulator in anti-tumor immunity through its reaction with TIM-3 ligand, galectin-9. It has been confirmed that TIM-3 is expressed on Th1 cells, dendritic cells, monocytes, macrophages, malignant stem cells and so on. But the expression of TIM-3 and its clinical implications in patients with acute myeloid leukemia(AML) remains unknown. In this study, we sought to determine the expression and clinical implications of TIM-3 in AML. From August 2012 to June 2013, in total of 32 AML patients with sixteen male and sixteen female were enrolled in this study. We collected their peripheral blood before they received any treatment and then obtained their peripheral blood mononuclear cells(PBMC). Monoclonal antibody was added into PBMC and cell population was analyzed by flow cytometry. Blast cells were identified with SSC CD45±and mature lymphocytes with SSC CD45+. The average expression of TIM-3 on blast cells was 43.46%, while on mature lymphocytes was 13.78% (P<0.001). In univariate analysis, the level of expression was not correlated to the percentage of blast cells and there was no difference between each type of AML. Complete remission was similar between different levels of TIM-3 expression(P>0.05). These results demonstrated that TIM-3 was highly expressed on blast cells than on mature cells in AML, which indicated that TIM-3 could be associated with the differentiation of blast cells and a potential marker to detect the tendency of relapse. TIM-3-targeted antitumor therapy presents a perspective possibility.

Disclosures:

No relevant conflicts of interest to declare.
Immunophenotyping Versus Morphological Evaluation Of Fresh and Stabilized Cerebrospinal Fluid As Diagnostic Tool For CNS Involvement In Childhood Acute Lymphoblastic Leukemia
Authors: Sonneveld Edwin , Zwaan Christian M. , van der Sluijs-Gelling Alita J , de Haas Valerie , Pieters Rob , van der Velden Vincent H.J.
Published: October 2019

Introduction

Presence of malignant cells in cerebrospinal fluid (CSF) is a risk factor in pediatric acute lymphoblastic leukemia (ALL). Consequently, these patients receive extra intrathecal treatment. We evaluated the concordance between morphological and flow-cytometric (FCM) results, both on freshly analyzed and on stabilized, overnight transported samples.

Methods

Diagnostic CSF samples of 61 newly diagnosed pediatric ALL patients were divided in two aliquots. One was sent to the local laboratory and processed within a few hours after sampling (lab1). A second aliquot was 1:1 diluted with sterile medium and sent to the reference laboratory (lab2). For all samples, a MGG (May-Grünwald-Giemsa) stained cytocentrifuged slide was morphologically evaluated and two 6-color FCM stainings were performed. Samples were considered positive (CNS2) by MGG if at least one blast was seen and by immunophenotyping if a cluster (≥ 10 events) of ALL cells was detected.

Results

Comparison of morphological data between both laboratories showed concordance in 33 samples (53%). In 20 of the 28 discordant samples only 1 or 2 blasts were reported by a single laboratory. Comparison of FCM data between laboratories was concordant in 58 samples (95%). Three samples (tumor load 15%-18%) were reported positive by one laboratory only; in two of these <100 cells could be acquired per tube. Comparison between FCM and morphology showed discordant results in 25 samples (41%) for lab1 and 19 (31%) in lab2. One discordant case was positive by FCM but negative by morphology, in all others only morphology was reported positive. The latter cases mostly had 1 or 2 blasts reported by morphology, generally not confirmed by the other laboratory.

Conclusions

This study shows that morphological analysis of CSF samples is non-reproducible if only 1 or 2 blasts are suspected. In contrast, FCM analysis of CSF is highly reproducible between fresh and stabilized samples. Although immunophenotyping still seems less sensitive than morphology, discordant samples generally have only 1 or 2 blasts reported by MGG, which is highly irreproducible. Nevertheless, by using a single 8-color tube higher cell numbers may be acquired and the FCM sensitivity may be increased.

Disclosures:

No relevant conflicts of interest to declare.
High Resolution Melting Analyses For Genetic Variants in ARID5B and IKZF1 with Childhood Acute Lymphoblastic Leukemia Susceptibility Loci In Taiwan
Authors: Peng Ching-Tien
Published: October 2019

Introduction

Childhood acute lymphoblastic leukemia (ALL) is a heterogeneous disease including multiple subtypes, defined on the basis of cell lineage and chromosome anomalies. Previous genome-wide association (GWA) studies report that several ARID5B and IKZF1 single nucleotide polymorphisms (SNPs) are associated with the incidence of ALL. However, high resolution melting analysis (HRM) supplies a faster and more convenient technique to detect SNPs. We applied HRM to detect the SNPs in ARID5B and IKZF1 gene.

Method

Seventy-nine pediatric ALL patients and 80 healthy controls were enrolled. Polymorphic variants of IKZF1 (rs6964823, rs4132601, and rs6944602) and ARID5B (rs7073837, rs10740055, and rs7089424) were detected by HRM method. The SNPs were further analyzed the association with childhood ALL.

Result

We evaluated 6 mutations by PCR amplicons (81 -176 bp) using the 96-well Light Cycler system. The primer detail was listed in Table 1. The melting curve data showed the difference in normalized temperature and we easily and accurately extended application of HRM analysis to genotyping of IKZF1 (Figure 1) and ARID5B (Figure 2) gene variants.

The genotype distribution and the allele frequencies of IKZF1 (rs6964823, rs4132601, and rs6944602) and ARID5B (rs7073837, rs10740055, and rs7089424) gene polymorphisms among cases and controls and their association with the risk of childhood ALL were shown in Table 2. The distribution of genotype rs7073837 in ARID5B was significantly different between the ALL group and the control group (P = 0.046), while the distributions for IKZF1 (rs6964823, rs4132601, and rs6944602) and ARID5B (rs10740055, and rs7089424) were not significantly different. In addition, we analyzed the association for those SNPs with B lineage ALL and found rs7073837 in ARID5B conferred a higher risk for B lineage ALL (odds ratio, OR = 1.70, 95% confidence interval, CI = 1.01-2.87, P = 0.049).

Conclusion

HRM was a practical method to detect SNPs in ARID5B and IKZF1 gene. We determined the rs7073837 in ARID5B was associated with the risk of childhood B lineage ALL.

Disclosures:

No relevant conflicts of interest to declare.
Diagnostic and Prognostic Significance Of Isolated Trisomy 2 In Hematopoietic Malignancies
Authors: Aypar Umut , Waltman Lindsey , Reichard Kaaren , Van Dyke Daniel L.
Published: October 2019

Isolated trisomy 2 in hematopoietic malignancies is rare, having only been reported in eight cases in the literature. Of these cases, the majority are older male patients (7/8) ranging in age from 64 to 84 years. The underlying hematologic malignancies include: myelodysplastic syndrome (MDS), refractory anemia (RA) subtype; MDS, RA with excess blasts (RAEB-II) subtype; MDS, RAEB in transformation (RAEB-t); chronic myelomonocytic leukemia (CMML-t) in transformation; MDS transformed into AML; acute monoblastic and monocytic leukemia (AMoL; FAB M5); and AML in relapse. The molecular pathogenesis and prognostic significance of isolated trisomy 2 remains unknown due to the small number of reported cases. Herein, we report 11 cases of isolated trisomy 2 in hematologic disorders seen in the Mayo Clinic Cytogenetics laboratory from 1996-2012. The majority of patients were older males (7/11) ranging in age from 63 to 93 years. The underlying bone marrow pathologic diagnoses include: hypercellular bone marrow without diagnostic features of malignancy (cases 1 and 2); MDS, refractory cytopenia with multilineage dysplasia (RCMD) subtype (cases 3 and 4); RAEB-1 (cases 5 and 6); long-standing history of primary myelofibrosis now with 7% bone marrow blasts (case 7); acute myeloid leukemia (AML), not otherwise specified (cases 8 and 9); AML with myelodysplasia-related changes (cases 10 and 11). Trisomy 2 has been suggested to represent an age-related phenomenon as it is seen predominantly in older individuals demonstrating this abnormality. Our data suggests that this could be a possible explanation since all of the eleven cases were ages 63 and over. Based on the limited clinical information in our study, it appears that isolated trisomy 2 harbors little prognostic significance and that, rather, the prognostic significance is driven by the underlying pathologic diagnosis. For example, 3 of the 4 AML cases and the case of PMF with increasing bone marrow blasts survived only 7, 8, 6 weeks and 21 weeks post bone marrow biopsy/cytogenetic evaluation, respectively. Although our study only has two cases that lack diagnostic features of malignancy, one of these cases survived 10 years following the identification of the cytogenetic abnormality. Therefore, trisomy 2 as a sole abnormality should not be considered as definitive evidence for MDS in the absence of diagnostic morphological criteria (similar to trisomy 8 and 20q deletion).

Disclosures:

No relevant conflicts of interest to declare.
Acute Lymphoblastic Leukemia Treated With Modified Hyper C-VAD Protocol In Iran
Authors: Jalaeikho Hassan , deylami Ahmad Ahmadzadeh , Keyhani Manoutchehr
Published: October 2019

Background

Relapse and mortality remains high in Acute Lymphoblastic Leukemia (ALL) with reported 5-year survival less than 39%. Bone marrow transplant (BMT) has been offered to some patients; however, 70% of patients do not have a donor-matched sibling and transplant beds are limited in Iran. We treated a cohort of 251 patients by inducing a remission and then treating with Hyper-CVAD rather than treating with Hyper-CVAD at induction and examined survival compared to reports in the literature.

Methods

We conducted a retrospective review of survival of 251 treated with a modified protocol of Hyper-CVAD between 2005 and 2012. The treatment protocol used 4-week induction regimen with Vincristine, Daunorubicin, and Dexamethasone. BCR-ABL positive patients also received daily Imatinib. Intrathecal methotrexate and cytarabine were given to patients with brain involvement. Remission was evaluated by a bone marrow biopsy and aspiration. If patient had T-Cell ALL then we add cyclophosphamide to the induction regimen. Hyper-CVAD treatment was initiated two weeks after last dose of Vincristine. Any patient with brain or bone involvement received radiotherapy post induction. BMT was offered to all BCR-ABL positive patients or those who had a suitable donor. All patients in complete remission received monthly vincristine prednisone for 5 days ,6- mercaptopurine every night and methotrexate every week up to 30 months .Patient with persistent liver enzyme elevation received cyclophosphamide instead of methotrexate.

Result

The mean age was 26 (range 14 to 70). The cohort included patient with T ALL (11%), Burkitt’s type ALL (15%), Pre-B ALL (67%), Pro-B ALL (4%) and BCR-ABL (3%). Sixteen patients died during the first month. Mortality increased to 45 patients at the end of six months. Relapse was the main cause of death. BMT was conducted after complete second remission and suitable patient with BCR-ABL positive patients on 13 occasions but none survived. At 5 years of follow up 129 patients were alive (50.1% survival).

Conclusion

Our patients experienced an improved survival compared to reports in the literature and many did not have complication of radiotherapy. A modified Hyper-CVAD protocol should be the subject of further investigation for the treatment of ALL.

Disclosures:

No relevant conflicts of interest to declare.