bluuebunny/crossref_metadata_2025 huggingface dataset:
Homocysteine Inhibits Cyclin A Promoter Methylation Via DNMT3 Inactivation in Human Endothelial Cells.
Authors: Jamaluddin S. , Chen Irene , Yang Fan , Liu Xiaoming , Schafer Andrew I. , Durante William , Yang Xiao-feng , Wang Hong
Published: October 2019

Hyperhomocysteinemia is a significant and independent risk factor for cardiovascular disease. We reported previously that homocysteine (Hcy) inhibits endothelial cell (EC) growth by transcriptional inhibition of the cyclin A gene. This is associated with an increase in S-adenosylhomocysteine, a potent inhibitor of methyltransferase. We hypothesized that Hcy inhibits EC growth and cyclin A transcription via hypomethylation and studied the effect of Hcy on epigenetic regulation of the cyclin A gene in EC. We found that the levels of cyclin A mRNA were significantly suppressed by azacytidne (AZC), a potent DNA methyl transferase (DNMT) inhibitor, in human umbilical vein EC (HUVEC). The cyclin A promoter (−518/256) is rich in GC content (59.1%), and has a CpG island spanning a 477 bp region (−277/200). Bisulphite sequencing followed by PCR amplification of the cyclin A core (−267/37) promoter, which contains 23 CpG sites serving as potential methylation sites, showed that Hcy (50 mM) eliminated methylation at two CpG sites, position 1 and 35, in the cyclin A promoter. Hcy selectively inhibited the activity of DNA methyl transferase 1 (DNMT1) by 40%, and had no effect on DNA methyl transferase 3 (DNMT3) activity in HUVEC. Furthermore, chromatin immunoprecipitation (ChIP) assays demonstrated that Hcy reduced the binding of methyl CpG binding protein 2 (MeCP2) and increased the binding of acetylated histone H3 and H4 in the cyclin A promoter in HUVEC but not in human aortic smooth muscle cells. The binding of MeCP2 to the cyclin A promoter was completely suppressed by AZC and trichostatin A, a histone deacetylase (HDAC) inhibitor, indicating that HDAC and DNA methylation mediate MeCP2 binding to cyclin A promoter. Finally, adenovirus tranduced DNMT1 overexpression, but not DNMT3, reversed Hcy-induced growth inhibition. In conclusion, we found that Hcy inhibits EC growth by suppressing cyclin A transcription, and that Hcy exerts this action by inhibiting DNA methylation through the suppression of DNMT1 and HDAC activity.
Kinase Domain Point Mutations in Ph+ Acute Lymphoblastic Leukemia (ALL) and Lymphoid Blast Crisis of Chronic Myeloid Leukemia (CML) and Their Emergence Following Therapy with Bcr-Abl Kinase Inhibitors.
Authors: Jones Dan , Luthra Rayjalakshmi , Kantarjian Hagop M. , Breeden Megan , O’Brien Susan , Faderl Stefan , Cortes Jorge , Thomas Deborah
Published: October 2019

Bcr-Abl kinase domain (KD) point mutations are detected in the dominant clone(s) in approximately 45% of CML at the time of disease resistance, developing after an average of 20–35 months of imatinib therapy. However, low numbers of Philadelphia chromosome (Ph)+ tumor cells with KD mutations could be present at earlier timepoints providing a pool of potential resistant subclones. Since current therapy of Ph+ ALL relies on imatinib maintenance therapy, the pattern of Bcr-Abl KD mutations in this tumor is an important and understudied phenomenon. We assessed the frequency and levels of Bcr-Abl KD mutations at different points in ALL, including at diagnosis, upon relapse and following salvage therapy with kinase inhibitors. We performed Bcr-Abl KD mutational analysis by direct sequencing in 25 cases of Ph+ ALL at the time of diagnosis and 25 cases upon disease persistence/relapse. For comparison, we analyzed 22 cases of lymphoid blast crisis of CML (LyBC), most of which transformed following long-term imatinib monotherapy. To track the emergence of mutated clones, we also performed more sensitive analysis for the T315I mutation by pyrosequencing (5% sensitivity) and allele-specific oligonucleotide probe (ASO) PCR (1:500 sensitivity). KD mutations were not seen by direct sequencing in ALL cases at diagnosis. The T315I mutation was also not detected by pyrosequencing (n =25) or ASO-PCR (n = 10) in newly diagnosed ALL. In contrast, Bcr-Abl KD mutations (Y253H in 3, Q252H, T315I, F317L, E355Q, H396R in 1 each) were seen in 8 of 25 (32%) relapsed/persistent ALL, occurring in patients who had been receiving imatinib for a median of 14 months (range 2–26). An additional 3 patients treated with dasatinib or nilotinib for relapse subsequently developed KD mutations (T315I and Y253H, and F317L) after 1, 4 and 9 months of second therapy. KD mutations were seen in 16 of 22 (73%) patients with lymphoid blast crisis, including T315I in 7, E255K and M244V in 2 each, and Y253H, V299L, F311I, E355G, F359V in 1 each. All KD mutations in LyBC developed following imatinib or nilotinib therapy. As with CML, kinase inhibitor therapy particularly in the relapse/salvage setting is the primary risk factor for emergence of Bcr-Abl KD mutations in Ph+ ALL. There is a high frequency of Bcr-Abl KD mutations associated the lymphoid transformation of CML. However, Bcr-Abl KD mutations develop more rapidly in persistent or relapsed Ph+ ALL than in CML and there is a higher frequency of Y253H mutations noted. These findings will likely have consequences for the timing and dosages of imatinib and other kinase inhibitors in maintenance and relapsed ALL regimens.
ShRNA Targeting p190BCR-ABL Successfully Eliminates Ph-ALL Cells with or without ABL Kinase Domain Mutation.
Authors: Futami Muneyoshi , Hatano Toshiyuki , Soda Yasushi , Kobayashi Seiichiro , Miyagishi Makoto , Tojo Arinobu
Published: October 2019

In the majority of Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (Ph-ALL) cases, the resulting BCR-ABL gene generates 190 kD active tyrosine kinase (p190) which is responsible for leukemogenesis and can be a molecular target for therapy. Although a series of ABL kinase inhibitors including imatinib, nilotinib and dasatinib reveal potent activities against Ph-ALL, acquired resistance caused by point mutations in the kinase domain such as T315I still remains to be overcome. That is why a novel strategy is desired in the treatment of Ph-ALL. We previously reported that lentiviral delivery of maxizyme targeting p190 specifically induced apoptosis of Ph-ALL cells (Blood 104:356, 2004). Since RNA interference proved to be a more powerful tool in selective gene silencing, we applied this technology to test whether specific and efficient killing of Ph-ALL cells could be achieved by down-regulation of p190. We designed a series of 21-mer and 27-mer small hairpin RNA (shRNA) targeting p190 mRNA and constructed plasmid vectors expressing these shRNA, which were screened by transfection of 293T/p190 cells to determine optimal target sites. As a result, three candidate sequences were identified; junctional 27-mer, junctional 21-mer and ABL 21-mer. Then, we inserted each of the shRNA expression cassettes into the lentiviral vector (HIV-U6/shRNA) and prepared high titer virus stock for infection of leukemia cells. shBCR-ABL/21, but not shBCR-ABL/27, induced significant and specific cell death of p190+ Ph-ALL cells in a time-dependent manner. shABL was more potent than shBCR-ABL/21 and also active against p210+ CML cells as well as 293 cells, but did not substantially affect Ph-negative leukemia cells. Both shABL and shBCR-ABL/21 completely inhibited growth of Ba/F3 cells harboring either wild-type or mutant p190 which renders those resistant to imatinib. Furthermore, both shRNA at low multiplicity of infection additively cooperated with imatinib in growth inhibition of Ba/F3-p190 cells. These data suggest that shRNA targeting p190 may become a therapeutic option in Ph-ALL by improvement of its delivery system like liposome.

Growth of BA/F3-p190BCR-ABL Cells transduced with shRNA Targeting p190BCR-ABL Growth of BA/F3-p190BCR-ABL Cells transduced with shRNA Targeting p190BCR-ABL
Inhibition of p38 MAPK Signalling Impairs the Homing of B Lineage ALL Cells to the Bone Marrow of NOD/SCID Mice.
Authors: Juarez Julius , Pena Aileen Dela , Baraz Rana , Bradstock Kenneth Francis , Bendall Linda J.
Published: October 2019

The mechanisms regulating the migration of B lineage acute lymphoblastic leukemia (ALL) cells between the blood and bone marrow compartments remain obscure. The chemokine SDF-1 regulates the motility and proliferation of ALL cells in vitro. We have previously demonstrated that the chemotaxic response of these cells to SDF-1 requires signalling through p38 MAPK and that signalling through PI-3K and MEK/ERK pathways plays only a minor role in a subset of cases. This contrasts with normal CD34+ hematopoietic progenitors and CD34+CD19+ B cell progenitors where signalling through PI-3K is the predominant pathway required for chemotaxis. In this study we examined the requirement for SDF-1 and p38 MAPK signalling in the homing of the B lineage ALL cells to the bone marrow of NOD/SCID mice. CFSE-labelled ALL cells were pre-treated with either the specific CXCR4 antagonists AMD3100 (100μM) or TC14012 (100μM), the p38 MAPK inhibitor SB203580 (10μM), the AKT inhibitor LY294002 (6.65μM) or the MEK inhibitor PD98059 (20μM) for 0.5–1 h at 37°C. Control or inhibitor treated cells were then injected intravenously into irradiated NOD/SCID mice which were either pre-treated with control (vehicle) or the inhibitors AMD3100 (200μg/mouse), TC14012 (200μg/mouse), SB203580 (0.1 mg/mouse), the AKT inhibitor LY294002 (2 mg/mouse) or the ERK inhibitor PD98059 (0.2 mg/mouse). Homing was assessed 4 hours after the injection of cells before significant proliferation of homed cells could occur. AMD3100 and TC14012 significantly inhibited the homing of ALL cells to the bone marrow by up to 80% and 67% respectively. Inhibition of p38 MAPK also decreased the homing of ALL cells to the bone marrow of NOD/SCID mice by 40% compared with the DMSO controls (p<0.05). No inhibition of homing was observed with the inhibitors of PI-3K or MEK in ALL cells. These results support our previous in vitro findings with regards to the importance of the p38 MAPK pathway in SDF-1 induced motility of ALL cells and demonstrate that the same pathway plays an essential role in the homing of these cells to the bone marrow of NOD/SCID mice.
Pediatric T-ALL but Not cALL Blasts Express Ligands for Siglec-7 Inhibiting NK Cell Mediated Lysis.
Authors: Viebahn Susanne , Gieseke Friederike , Pfeiffer Matthias , Handgretinger Rupert , Müller Ingo
Published: October 2019

Changes in the glycosylation pattern of cell surface proteins occur during early stages of malignant transformation. These alterations in the glycan repertoire may contribute to numerous processes of tumor cell survival such as adhesion, migration and immune evasion. Sialic acid is the most common terminal carbohydrate moiety of glycan structures in humans binding to a family of eleven receptors termed sialic acid binding Ig-like lectins (siglecs). Siglecs are expressed on virtually all effector cells of the immune system and several members function as inhibitory receptors. Siglec-7 is known as an inhibitory receptor expressed by NK cells and T cells. Therefore, siglec-7 ligands may contribute to immune evasion of malignant cells.

In order to analyze the expression of siglec-7 ligands by malignant cells, we cloned the extracellular domain of human siglec-7. The recombinant protein was expressed in 293 cells to allow glycosylation, which is supposed to be important for specificity. As the interaction of a single glycan and lectin is of low affinity, we tetramerized siglec-7 in analogy to MHC-tetramers to increase avidity. This strategy allowed for reliable use of siglec-7 fusion protein in flow cytometry. Analysis of PBMC from healthy donors revealed that siglec-7 ligands are expressed on all subpopulations of PBMC: expression was detected on CD4+ and CD8+ T-cells as well as on monocytes and NK cells. B cells could be subdivided into a positive and negative population after staining with siglec-7 tetramers.

Subsequently, we analyzed primary lymphatic blasts from childhood leukemias. Interestingly, T-ALL blasts expressed ligands for siglec-7, whereas these ligands were absent from cALL blasts. As cALL blasts are known to be better targets for NK cell-mediated cytotoxicity than T-ALL blasts, we hypothesized that siglec-7 ligands on the surface of leukemic blasts inhibit NK cells. To test the functional relevance of siglec-7 ligand expression for NK cell-mediated cytotoxicity, we used soluble monomeric siglec-7 as competitive inhibitor in cytotoxicity assays. In these competition assays, NK cell-mediated specific lysis of leukemic cells increased roughly twofold in the presence of soluble siglec-7.

These results show that engagement of inhibitory siglecs on the surface of effector cells might contribute to immune escape mechanisms of malignant cells. It underlines the function of siglec-7 as a non-MHC-specific inhibitory receptor on NK cells and the important role of altered glycans expression on malignant cells in tumor immunology.
Blasts Express Phospho-Akt in the Majority of Adult Patients with Acute Lymphocytic Leukemia (ALL).
Authors: Advani Anjali S. , Gibson Sarah , Jin Tao , Tiu Ramon , Ramsingh Giridharan , Kalaycio Matt , Bolwell Brian , Sobecks Ronald , Sekeres Mikkael , Sungren Shawnda , Hsi Eric D.
Published: October 2019

Background: Akt, a serine/threonine protein kinase, is a critical regulator of cellular apoptosis, proliferation, and differentiation. Constitutive activation (phosphorylation) of Akt is frequently observed in various types of cancer, and may represent a therapeutic target. Akt inhibitors, such as perifosine, are in clinical development. We evaluated the expression of phosphorylated Akt (pAkt) in patients with newly diagnosed ALL.

Methods: Between 1998 and 2005, patients with newly diagnosed ALL and an available diagnostic bone marrow biopsy performed at the Cleveland Clinic Foundation were evaluated. B5-fixed bone marrow core biopsies were reviewed for areas with the highest concentration of blasts. A tissue microarray was constructed using 1 mm tissue cores. The cores were arrayed in duplicate in the majority of samples, with a few samples having only 1 core. Immunohistochemistry was performed for pAkt with a monoclonal antibody to pAkt (Rockland Immunochemicals), using automated stainers and heat induced epitope retrieval. pAkt staining of blasts was estimated in 10% increments. The pattern of staining for pAkt was classified as positive (activated) if nuclear or cytoplasmic staining occurred in more than 10% of the blasts.

Results: Eighty-eight patients with newly diagnosed ALL were treated at our institution during this time. Forty-five patients were evaluable. Thirty-three cases were not evaluable for the following reasons: bone marrow cores not available (9), bone marrow aspirate performed at an outside hospital (20), bone marrow aspirate sample not sufficient (1), and diagnostic bone marrow aspirate not performed (3). The median age was 37 [range 18–81]. Sixteen patients (35%) had poor risk cytogenetics, 12 (27%) normal karyotype, 9(20%) miscellaneous abnormalities, and 8 (18%) unknown cytogenetics as defined by CALGB criteria. Forty-one patients (91%) had precursor B-cell ALL, and 4 patients (9%) precursor T cell ALL. The median white count was 6.6 × 109/L [range 0.93–278], and median LDH 772 U/L [range 155–4608]. The expression of pAkt was characterized predominately by cytoplasmic staining with 8 cases exhibiting nuclear staining. Akt was activated (phosphorylated) in 32 out of 45 patients (71%) with newly diagnosed ALL. The median percentage of blasts with pAkt in the cytoplasm was 30%, with 19 patients (42%) having ≥50% blasts with cytoplasmic pAkt. There was a trend towards more patients with Philadelphia chromosome (Ph) positive ALL having ≥50% blasts with pAkt in the cytoplasm (7/10 patients) versus patients with other cytogenetic abnormalities (12/33) (p=0.079).

Conclusions: Akt was activated in the majority of patients with newly diagnosed ALL. Akt may be a therapeutic target in ALL, and especially for patients with Ph+ ALL. Larger studies will be needed to define the prognostic significance of Akt activation in ALL.
Factors Influencing Survival after Relapse from Childhood ALL: A Children’s Oncology Group Study.
Authors: Nguyen Kim , Cheng Su-Chun , Raetz Elizabeth , Sather Harland , Carroll William L. , Winick Naomi , Hunger Stephen P. , Gaynon Paul , Loh Mignon L.
Published: October 2019

Despite improvements in front-line therapy for acute lymphoblastic leukemia (ALL), treatment after relapse remains a critical challenge. Examination of outcome after relapse in a large, recently treated, and unselected population provides a useful benchmark for evaluating new approaches.

Survival after relapse was tracked on Children’s Cancer Group (CCG) ALL trials. Relapses were categorized by site (isolated or combined bone marrow relapse (MR); isolated central nervous system relapse (CNSR); isolated testicular relapse (TR)), and time from diagnosis to relapse (early < 18 months; intermediate between 18 and 36 months; late > 36 months). We examined the impact of age, sex, ethnicity, initial risk assignment (standard risk (SR) or higher risk (HR)), immunophenotype, length of first remission (CR1), and site of relapse on outcome in multivariate analyses.

A total of 9,585 infants, children, and adolescents with newly diagnosed acute lymphoblastic leukemia were enrolled on CCG trials conducted between 1988 and 2002. We examined survival after relapse for 1,874 children with MR, CNSR, or TR.

Survival after relapse by site and time to relapse Site MR CNSR TR Time to Relapse n *survival(%) n *survival(%) n *survival(%) *3-year survival Early 498 10 175 49 15 50 Intermediate 378 23 179 73 35 57 Late 488 53 52 77 54 70

Overall survival for any bone marrow relapse was best for late MR and worst for early MR. Early MR was linked to HR presenting features and T-cell disease. Survival after intermediate or late MR was superior for SR patients (p <.05). Survival was better for CNSR or TR than for MR. Older age at diagnosis, male sex, T cell immunopheno-type, and CNS disease at presentation were significant adverse factors for survival after adjustment for site and time to relapse in multivariate analyses (p<.05).

Importantly, in the same multivariate analysis, patients treated on more contemporary trials (CCG1952CCG1953CCG1961, and 1962) had inferior survival rates after relapse when compared to patients treated on earlier trials (CCG 1881CCG 1882CCG 1883CCG 1891CCG 1901, and 1922) (p<.001). Importantly, these data from the analysis of a large, unselected group of patients indicates that features at initial presentation and prior therapy influences outcome following relapse. The inferior survival rates of those relapsing on more intensive protocols likely reflects particularly aggressive and chemoresistant disease leading to poor salvage. Increasing the intensity of retrieval therapy is unlikely to improve after relapse and novel therapeutic approaches that target these resistant leukemias are urgently needed for successful treatment of relapsed ALL.
The Role of Cranial Irradiation (CRT) or Extended Triple Intrathecal Therapy (T.I.T.) in Childhood T-Cell ALL; the ALL Israel National Studies (INS) 89 and 98 (Modified ALL-BFM 86 and 95) Report.
Authors: Stark Batia , Attias Dina , Avrahami Gali , Ballin Ami , Bielorai Bela , Burstein Yoav , Elhasid Ronit , Gavriel Herzel , Kapelushnik Josef , Miskin Hagit , Sthoeger Dalia , Weintraub Michael , Steinberg David , Izraeli Shai
Published: October 2019

Patients with T-cell ALL are at increased risk of CNS relapse and require more intensive CNS directed therapy. CRT is considered to be the most effective therapy, but is associated with serious adverse late effects and secondary brain tumors. We evaluated retrospectively, in the context of the ALL-BFM-based protocols, the impact of CRT or extended T.I.T. on outcome within High Risk (HR: Prednisone poor responders) and non-HR (Prednisone good responders) subgroups.

In INS 89, modified ALL-BFM 86/90, all Non-HR patients received extended T.I.T (×18) as CNS preventive treatment, and VP-16 was added systemically in between the HD-MTX (5g/m2). In INS 98, based on ALL-BFM 95, VP-16 was omitted and pulses of VCR+DEXA were added in maintenance. Patients with T-ALL Non-HR and WBC >100000, were assigned to receive CRT 12 Gy, following the results of the AIEOP-ALL 91 (Conter J.C.O. 15:2786–2790, 1997) reporting inferior outcome in this group when comparing T.I.T only to BFM with irradiation. T-ALL HR patients were treated without modification and were all assigned to CRT 18 or 12Gy.

Results: Between 1989 and 2002, 145 T-ALL children aged <18 years were enrolled in the INS 89 (84 patients, 1989–97) and INS 98 (61 patients, 1998–2002). With a median follow-up of 12 and 5 years in INS 89 and INS 98 respectively, the 5 year event-free-survival (EFS) was 61.9±5.3% and 75.4±5.5%, respectively (p=0.14), and CNS relapse (isolated and combined) rates were 4.1% and 1.8%, respectively (p=0.4). Overall 5 yrs EFS, DFS, and CNS relapse rate of all the T-ALL patients were 67.4%, 78.8%, and 3.1% respectively. Outcome of Non-HR patients with WBC <100000 (all were non irradiated 72 pts) was; EFS 74.7%, and no CNS relapse. In the Non-HR with WBC >100000 (without CNS involvement) 9 patients were irradiated and 18 patients received the Extended T.I.T. These groups had 5 yr EFS of 78%±14% and 76%±10% respectively, and CNS relapse rate 11.1% and 5.6% respectively (p=0.59). In the HR group (34 pts) the 8 patients who refused CRT fared significantly worse than the irradiated patients, with EFS of 38%±17% Vs 73%±11% (p=0.08), and CNS relapse rate of 17% Vs 0 (p=0.14). T-cell ALL Non-HR children (prednisone good responders), in the context of the ALL-BFM intensive protocols and extended T.I.T may not require preventive CRT regardless of their WBC at presentation.
ABCA3 Expression in AML Blast Cells: Association with Poor Disease Outcome and Intracellular Drug Sequestration.
Authors: Chapuy Bjoern , Radunski Ulf , Corsham Sabrina , Koch Rafael , Cheong Naeun , Inagaki Nobuya , Griesinger Frank , Reinhardt Dirk , Truemper Lorenz , Wulf Gerald G.
Published: October 2019

Analysing the intrinsic drug efflux capacity of leukemic stem cell with the side population (SP) phenotype, we recently found the intracellular ABC transporter ABCA3 expressed in blast cells from patients with acute myeloid leukaemia (AML). Here we extend our observations to a larger cohort of AML samples and addressed the role of ABCA3 for cytostatic drug resistance. Applying quantitative RT/PCR (Taqman), we detected significant levels of ABCA3 in 59 of 92 blast cell samples from 58 pediatric and 34 adult patients with AML. The expression of ABCA3 correlated positively (coefficient 0.83) with the expression of CD34, while there was no association with the acquisition of markers for myeloid and monocytic maturation. As for disease outcome analysed by univariate analysis, we found a significantly worse outcome in both event free survival (p=0.048) and overall survival (p=0.034) for the patients with high-level ABCA3 expression. Functionally, the expression of ABCA3 was restricted to the cytoplasm in the AML blast cells, and co-localisation studies revealed an association with endosomal markers. Ectopic expression of ABCA3 augmented the lysosomal compartment in HEK 293 cells, and enhanced intracellular anthracyclin sequestration into vesicles of the endosomal system. Correspondingly, we observed a significant reduction of cytostatic efficacy from anthracyclins in stably ABCA3 expressing HEK293 cells compared to the parental cell line. In conclusion, we found the intracellular transporter ABCA3 expressed in the blast cells of most patients with AML, and elevated ABCA3 levels associated with poor disease outcome. Functional data revealed intracellular anthracyclin sequestration by ABCA3, suggesting a role of ABCA3 for drug resistance in patients with AML.
Differential Expression of SLAM Family Receptor Markers in Normal Human Hematopoietic Stem Cells and Their Malignant Counterpart in MDS and AML.
Authors: Tiu Ramon V. , Powers Jennifer J. , Haddad Abdo , Jiang Ying , Maciejewski Jaroslaw P.
Published: October 2019

Members of the signaling lymphocytic activation molecule (SLAM) family, including CD150, CD48 and CD244, were shown to precisely distinguish more committed lineage restricted progenitor cells from pluripotent and multipotent murine hematopoietic stem cells (HSC; Kiel et al; 2005 Cell). Similar SLAM profiles may also be present on HSC subsets in humans. We hypothesized that these SLAM markers may be indicators not only of stem cell potential in normal hematopoiesis but also distinguish a subset of the most immature malignant precursors of leukemia. In agreement with the concept of a “cancer stem cell,” the presence of leukemic stem cell population may be an indicator of important clinical and biological properties. We first tested the distribution of CD150, CD48 and CD244 antigens on human CD34+ cells derived from 7 control individuals using 4-color flow cytometry. CD34+ cells were measured in the blast gate based on side scatter and CD45 expression. Within CD34+ blasts, expression of CD48, CD150, and CD244 was detected on 16.71%±9.69, 6.53%±2.93, and 26.92%±6.95 of cells respectively. Subsequently, we investigated SLAM expression in 9 immature leukemic cell lines, including KG-1, K562, U937, HEL, HL60, MKN-95, NB-4, Kasumi and UT7, and found increased expression of SLAM markers in KG-1 (CD48+, CD150+, CD244+) and Kasumi (CD48−, CD150−, CD244+). Consequently, none of the leukemic cells showed pluripotent/multipotent SLAM profiles. We then compared the SLAM marker expression on blasts from patients with AML and MDS with that of CD34+ cells from normal controls. We studied a total of 28 patients: 11 MDS (2 low grade, 5 advanced MDS, 3 MDS/MPD overlap) and 10 AML (FAB: 3 M1, 2 M2, 1 M3, 2 M4/M4E0 and 2 M6). In our cohort, 8/10 AML patients expressed one of the three SLAM markers; 6/10 were CD150−CD48−CD244+ (63.57%±6.96) and 2/10 were CD150+CD48−CD244−(46%±10.96) suggestive of the presence of either pluripotent or multipotent leukemic stem cell phenotype. In the MDS cohort, 8/11 patients expressed one of three SLAM markers, 7/11 were CD150−CD48−CD244+ (41.21% ± 8.9) and 1/11 were CD150+CD48−CD244− (1.26%±0.59) again consistent with a profile derived from either pluripotent or multipotent stem cells. None of the MDS and AML patients had either co-expression of CD244 and CD48 or increased expression of CD48 alone. Two of the M1 type AML patients with CD150−CD48−CD244+ phenotype received prior chemotherapy and achieved complete remission on bone marrow biopsy and flow cytometry using traditional blast markers. In some, we conclude that the SLAM receptor markers may be associated with certain types of leukemic blasts and may be useful in the identification of leukemic stem cell population in both MDS and AML.
TAT-Apoptin Mediated Induction of Apoptosis in Leukaemic Cells.
Authors: Gaken Joop , Pericleous Louisa , Farzaneh Farzin , Mufti Ghulam J. , Tavassoli Mahvash
Published: October 2019

We have studied the specific targeting of leukaemic cells using the Chicken Anaemia Virus (CAV)-derived protein VP3 (Apoptin) linked to the protein transduction domain (PTD) from HIV TAT with the aim of using this strategy for in vitro purging. Apoptin is a 13.6 kDa protein which induces apoptosis specifically in cancer cells whilst leaving normal cells unaffected. Expression of Apoptin in normal cells results in its cytoplasmic localisation. In tumour cells Apoptin resides initially in the cytoplasm and subsequently translocates to the nucleus and induces apoptosis. Apoptin is phosphorylated both in vitro and in vivo in tumour cells but negligibly in normal cells at threonine 108. A gain-of-function point mutation (T108E) results in accumulation of Apoptin in the nucleus and the killing of normal cells, implying that phosphorylation is a key factor of the tumour-specific properties of Apoptin.

We have demonstrated that Apoptin induces apoptosis in a variety of human solid tumour cell lines, but not in normal fibroblast and epithelial cells. Apoptin induced apoptosis in HSC3 head and neck cancer cells acts through the mitochondrial pathway and was blocked (>75%) by shRNA against PUMA, a BH3 Only protein which induces Bax and BAK resulting in loss of mitochondrial membrane potential and release of cytochrome C. Furthermore, activation of the p53 family member, p73, substantially increased (5–10 fold for p73 β and γ) sensitivity of Saos2 tumour cells to Apoptin-induced killing.

For efficient protein delivery, Apoptin was fused to a TAT PTD and addition of this protein to normal and tumour cells resulted in the selective killing of tumour cells. To increase the stability and solubility of TAT-Apoptin we have fused it to the maltose binding protein (MBP), this modification significantly increases both yield and the solubility of Apoptin while retaining its biological function. Apoptin tumour specific toxicity was assessed in a range of leukaemic and solid tumour cell lines. Addition of MBP-TAT-Apoptin protein to HL60, K562 and Jurkat cells resulted in 50%, 55% and 75% cell death by apoptosis as judged by PARP cleavage, respectively, at day 4 as compared to MBP-TAT control whilst normal B cells, fibroblasts and epithelial cells are unaffected. Fluorescent microscopy demonstrated that MBP-TAT-Apoptin was rapidly internalised in almost 100% of cells within 24hrs in all cell types tested. Direct injection of Apoptin expressing Ad vectors also showed clear regression of established tumours in mice.

The cancer specific toxicity of Apoptin has potential value for a range of therapeutic applications such as purging of autologous bone marrow as used for the treatment of multiple myeloma and possibly direct treatment of leukaemias either alone or linked to antibodies for targeting of specific types of leukaemias.
Heme Oxygenase-1 (HO-1)/Heat Shock Protein 32 (Hsp32) as a Novel Survival Factor and Target in AML.
Authors: Kneidinger Michael , Gleixner Karoline V. , Kondo Rudin , Samorapoompichit Puchit , Vales Anja , Krauth Maria T. , Mayerhofer Matthias , Gruze Alexander , Pickl Winfried F. , Greish Khaled , Boehm Alexandra , Sperr Wolfgang R. , Maeda Hiroshi , Valent Peter
Published: October 2019

Heme oxygenase 1 (HO-1), also known as heat shock protein 32 (Hsp32), has recently been identified as a stress-related survival molecule that acts anti-apoptotic and cytoprotective in inflammatory reactions. Recent data suggest that HO-1/Hsp32 is also expressed in neoplastic cells in various malignancies. In the present study, we provide evidence that HO-1 is constitutively expressed in primary leukemic cells in patients with acute myeloid leukemia (AML, n=17) and in various AML cell lines such as HL60, KG1, KG1a, and U937. Expression of HO-1 mRNA was demonstrable by RT-PCR, and the HO-1 protein by immunocytochemistry and Western blotting. In addition, we were able to demonstrate expression of HO-1 mRNA and of HO-1 protein in the CD34+/CD38− progenitor/stem cell fraction in the leukemic clone in patients with AML. The HO-1 inductor hemin (10 μM) was found to promote expression of HO-1 in AML cells. Incubation with the HO-1-targeting drugs pegylated zink protoporphyrin (PEG-ZnPP) or styrene maleic acid-conjugated ZnPP (SMA-ZnPP), resulted in a dose-dependent inhibition of growth of leukemic cells at pharmacologic concentrations (IC50: 5–20 μM for cell lines and primary AML cells). The SMA-ZnPP-induced growth-inhibition of AML cells were found to be associated with induction of apoptosis as evidenced by light microscopy, electron microscopy, and by a Tunel assay. In consecutive experiments, combination experiments were performed using SMA-ZnPP and AML cell lines. In these experiments, SMA-ZnPP was found to synergize with cytarabine in producing growth inhibition in all AML cell lines tested. In summary, these data show that HO-1/Hsp32 is a novel survival factor and interesting target in AML. The clinical significance of this observation remains to be determined in forthcoming trials.
Triptolide Cooperates with Cytarabine To Induce Apoptosis in Acute Myeloid Leukemia.
Authors: Pigneux Arnaud , Uhalde Maïalen , Mahon François X. , Lacombe Francis , Praloran Vincent , Milpied Noel-Jean , Belloc Francis
Published: October 2019

Triptolide, a diterpene triepoxide isolated from the Chinese herb Tripterygium wilfordii Hook.f, has shown antitumor activity in a broad range of solid tumors and on leukemic cells in vitro. Here, we examined its pro-apoptotic effects on THP1 myeloid cell line and primay AML blasts in association with cytarabine. THP1 cells were treated with increasing concentrations of Ara-C in the presence or not of Triptolide (5 ng/ml). Apoptosis was measured by flow cytometry at the mitochondrial level by using the ΔΨm sensitive probe DiOC6(3). This low dose of Triptolide did not induce any apoptosis in 24 hrs. However, it significantly increased the apoptosis induced by Ara-C in the range 0.1–2 μg/ml (p<0.001) (figure 1). At 48 hrs, this cooperative effect was observed for lower doses of Ara-C (0.05–0.1 μg/ml)(figure 2).

Fig. 1 Fig. 1. Fig. 2 Fig. 2.

When measuring the IκB content by western blot, although triptolide alone was found able to decrease IκB (p<0.05), there was no effect on the IκB content of Ara-C-treated cells. Similarly, no effect of the Ara-C/Triptolide association was found in the DNA binding activity of NFκB. The DNA synthesis activity was measured by flow cytometry using the BrdUrd method and a dose dependent DNA synthesis inhibition was found with Triptolide from 2 to 20 ng/ml. Moreover, the association of 5 ng/ml Triptolide with 0.05 or 0.1 μg/ml of Ara-C showed an increase in the DNA synthesis inhibition as compared with both drugs separately. However, while Ara-C induced an accumulation of cells in the S compartment of the cell cycle, the cell cycle distribution was not significantly altered by Triptolide alone, suggesting that this drug induced also a slowing down during all the cell cycle. The association Triptolide/Ara-C was also investigated in vitro on primary blast cells from 26 AML patients. The blood or bone marrow mononuclear cells were cultured for 48 to 72 hrs in the presence of a cytokine cocktail (Stem α.4B) to recruit the cells into the cell cycle. The samples were then treated with 7.5 ng/ml Triptolide, 0.05 or 0.1 ng/ml Ara-C or both drugs in combination for 24 and 48 hrs. Apoptosis was measured by flow cytometry, using FITC-Annexin V. The blast cells were discriminated on the basis of their low CD45 expression. The combination of both drugs yielded a percentage of apoptotic cells that was significantly higher than each drug administered separately (p<0.05). These results show that low concentrations of Triptolide which were not shown to induce apoptosis by themselves were able to potentiate the pro-apoptotic and anti-proliferative effects of Ara-C. The potentiation by triptolide of the antileukemic activity of cytarabine in vitro warrants further clinical investigations for the treatment of AML patients, specially in elderly patients in which low-dose cytarabine treatment could be improved by an association with Triptolide.
Rational for Specific Inhibition of Both PI3K/AKT and mTORC1 Activities in Acute Myelogenous Leukaemia.
Authors: Tamburini Jerome , Chapuis Nicolas , Bardet Valerie , Park Sophie , Verdier Frederique , Dreyfus Francois , Lacombe Catherine , Bouscary Didier
Published: October 2019

We previously reported that constitutive activation of PI3K/AKT activity in primary blasts from patients with AML is related to the expression of the p110d isoform of class IA PI3K which is activated by an upstream, actually unknown kinase (Sujobert P, Blood, 2005, 106, 1063). Activation of PI3K activity might make AML cells susceptible to mTORC1 inhibition, which was recently shown to be activated in AML cells. mTORC1 inhibition by Rapamycin or RAD001 may control both proliferation and survival of leukemic cells. The mechanisms leading to mTORC1 activation in AML are actually unknown

In this report, we studied the interactions between PI3K and mTORC1 pathways in primary blast cells from the bone marrow of patients with primary AML at diagnosis. We observed that specific inhibition of mTORC1 activity with RAD001 upregulates PI3K activity, as evidenced by an increased phosphorylation of AKT on Ser 473. In 8 patients, the mean increase of AKT phosphorylation in the presence of RAD001 was 200% (130%–500%). Such results were observed in most patients with constitutive activation of PI3K, but very rarely in patients without constitutive PI3K activity.

We then studied the activation of the mTORC1 complex when a full inhibition of PI3K activity with IC87114, a specific inhibitor of p110d isoform, was performed. mTORC1 activity, as assessed by phosphorylation of downstream targets, p70S6K, S6 ribosomal protein and 4EBP1 was not inhibited by IC87114 (10μM) in PI3K+ patients, whereas RAD001 (10nM) completely or near completely inhibited all phosphorylation events. Similar results were detected in the MV4-11 cell line. Moreover, we detected constitutive activation of mTORC1 in several PI3K- patients. These results clearly indicate that mTORC1 activation is no related to PI3K activity. In accordance with that, we showed that simultaneous inhibition of PI3K and mTORC1, with IC87114 and RAD001 respectively, additively inhibited the proliferation of blast cells and the clonogenicity of leukemic progenitor cells. Our results provide evidence that RAD001 relieve mTORC1 mediated feedback inhibition on AKT activation, as recently described in multiple myeloma and some cancer cell lines. We also demonstrate that mTORC1 activation occurs independently of PI3K activity. We are currently studying the upstream mechanisms resulting in mTORC1 activation. Finally, functional studies support a clear strategy for combining an mTORC1 inhibitor with an inhibitor of PI3K.
SHP-1 (PTPN6) Gene Supression by FLT3/ITD Defines an Aggressive Subgroup of Myeloid Malignancies.
Authors: Mena-Duran Armando V. , Pajuelo Juan C. , Cervera-Zamora Jose , Garcia-Casado Zaida , Valencia Ana , Barragan Eva , Benlloch Luis , Mustelin Tomas , Sanz Guillermo , Sanz Miguel A.
Published: October 2019

Recently, Chen et al (J Biol Chem.2005;280:5361–9) showed SHP-1 (PTPN6) gene supression by FLT3/ITD in TF-1 cell lines and in primary cells from AML patients. To address the prognostic impact that both together FLT3/ITD and SHP-1 gene suppression may have in myeloid malignancies patients, we have studied 85 patients diagnosed with de novo AML and MDS. SHP-1 gene expression was quantified by real time PCR and compared with normal CD34+ cells from healthy donors. FLT3/ITD was detected by conventional agarose gel electrophoresis after PCR. FLT3/D835 was detected by melting curve methodology. FLT3 mutations were correlated with higher WBC and blasts count at diagnosis (p=0.001). FLT3/ITD had a negative impact on DFS compared with wt or D835 mutations (log-rank p=0.054). Median levels for SHP-1 were lower in leukemic blasts than in normal peripheral blood CD34+ cells (p=0.001). SHP-1 lowest expression was found in those patients where FLT3 mutations were present (p=0.001). SHP-1 lowest expression was associated with higher blasts count in BM at diagnosis (p=0.017).

Having both Flt3 mutations and lowest expression for SHP-1 were strongly associated with higher WBC (> 20 x109/L) (p=0.005). OS univariate analysis yielded a worse prognosis for patients having both lower SHP-1 expression and harboring an Flt3 mutation (p=0.021).

Figure Figure

In summary, SHP-1 (PTPN6) gene supression by FLT3 constitutively activating mutations confers a bad prognosis for myeloid malignancies patients and is associated with hyperleukocytosis at diagnosis.
Differences in the Localization of Receptor Tyrosine Kinase FLT3 in Human Hematopoietic Stem and Progenitor Cells and Leukemic Blasts.
Authors: Koch Sina , Ryser Martin , Bornhaeuser Martin , Ehninger Gerhard , Thiede Christian
Published: October 2019

Activation of type III receptor tyrosine kinase FLT3 by its ligand controls cell proliferation and differentiation in normal hematopoiesis. Constitutively activating mutations of FLT3 are common abnormalities in acute myeloid leukemia. Internal tandem duplication (ITD) mutations of the FLT3 receptor are found in approximately one third of patients with AML and normal karyotype and have been shown to be associated with inferior response towards chemotherapy. One possible way how mutant FLT3 receptors may be mediating its pathological effects is by altering the normal localization of the receptor protein. To determine the intracellular localization of wild type and mutant FLT3 receptors, confocal laser scanning microscopy was performed in cultured leukemic cell lines (THP-1, EOL-1, MV4-11) and primary hematopoietic stem cells (HSC) from healthy donors. HSC (CD34+) cells were prepared from human bone marrow and leukapheresis products of healthy donors by MACS technique using CD34 and CD135 (FLT3) coupled microbeads. Additionally, different leukemic cell lines were transduced with retroviral vectors encoding FLT3wt or FLT3-ITD receptors.

In normal HSC, only 4–13% are expressing FLT3, and unexpectedly FLT3 staining was observed both in the plasma membrane and evenly distributed in the cytoplasm. The intracellular FLT3wt was not localized to the endoplasmic reticulum (ER), but part of it co-localized with the Trans-Golgi-network (TGN) and early endosomes.

In leukemic cell lines endogenously expressing either wt or mutant FLT3, wtFLT3 localized to the plasma membrane and uniformly to the cytoplasm whereas mutant FLT3 strongly accumulated in a perinuclear region but did not localize to the plasma membrane. Stable transfection of leukemic cell lines with wt or mutant FLT3 confirmed this localization pattern. In leukemic cell lines neither FLT3wt nor FLT3-ITD accumulated in the ER or in the Golgi apparatus, but instead FLT3wt and FLT3-ITD seem to reside in the TGN and in early endosomes as shown by immunofluorescence costaining with the organelle markers.

These data indicate that although perinuclear accumulation of mutant FLT3 is observed in all cells analyzed, wtFLT3 is also found intracellularly. FLT3-ITD might reside in a different state concerning glycosylation and/or phosphorylation (Schmidt-Arras et al., Mol Cell Biol, 2005) and therefore it may activate alternative or additional signaling cascades by having access to different or additional substrates. Further studies will clarify, whether intracellularly located wtFLT3 in healthy HSC and FLT3wt-expressing leukemic cells is also involved in FLT3 signaling.
Donor CD8+ T Cells Mediate GVL without GVHD in Recipients Conditioned with Anti-CD3 mAb.
Authors: Zhang Chunyan , Lou Jingwei , Li Naninong , Todorov Ivan , Lin Chia-Lei , Cao Yu-An , Contag Christopher H. , Kandeel Fouad , Forman Stephen , Zeng Defu
Published: October 2019

Donor CD8+ T cells play a critical role in mediating graft versus leukemia (GVL), but also induce graft versus host disease (GVHD) in recipients conditioned with total body irradiation (TBI). Here, we report that injections of donor C57BL/6 (H-2b) or FVB/N (H-2q) CD8+ T with bone marrow cells induced chimerism and eliminated BCL1 leukemia/lymphoma cells without GVHD in anti-CD3-conditioned BALB/c (H-2d) recipients. In contrast, the same dose of donor CD8+ T and marrow cells induced lethal GVHD in TBI-conditioned recipients. In addition, the anti-CD3-conditioned long-term complete chimeras without prior exposure to host-type BCL1 cells also eliminated the tumors when being challenged with BCL1 cells 120 days after HCT. This is in contrast to the report that long-term complete chimeras induced with delayed donor lymphocyte infusion lost GVL activity. Using in vivo and ex vivo bioluminescent imaging, we observed that donor CD8+ T cells expanded rapidly and infiltrated GVHD target tissues in TBI-conditioned recipients, but donor CD8+ T cell expansion in anti-CD3-conditioned recipients was confined to lympho-hematological tissues. This confinement was associated with lack of up-regulated expression of α4β7 integrin and chemokine receptors (i.e. CXCR3) on donor CD8+ T cells. In addition, host-reactive donor CD8+ T cells in anti-CD3-conditioned recipients were only partially deleted, and the residual cells were rendered heterogeneous: some unresponsive/anergic, some Tc2, some Foxp3+ suppressive cells, and some effector cells. The whole population of residual donor CD8+ T cells from anti-CD3-conditioned recipients mediated GVL without GVHD in TBI-conditioned secondary recipients. These results indicate that anti-CD3-conditioning separates GVL from GVHD via confining donor CD8+ T cell expansion to host lympho-hematological tissues as well as tolerization of the residual donor CD8+ T cells, in which the residual host-reactive effector cells mediate persistent GVL, and the regulatory CD8+ T cells prevent them from damaging host tissues.
Ape-1/ref-1 Mediated Redox Regulation of Retinoic Acid Receptor Transcription Function in Myeloid Leukemia Cell Differentiation and Apoptosis.
Authors: Robertson Kent A. , Colvin Edwin S. , Luo Meihua , Reed April , Kelley Mark R.
Published: October 2019

Ape1/ref-1is a multifunctional base excision DNA repair protein that is involved in the repair of abasic sites in DNA. However, it also has a distinct role in the redox regulation of a variety of cellular proteins, such as Fos, Jun, p53, NFkB, PAX, HIF-1a, HLF, and others. Ape-1/ref-1 maintains these proteins in a reduced state thereby facilitating their DNA binding and transcriptional activation capability. HL-60 cells are known to respond to retinoic acid (RA) with terminal granulocytic differentiation and apoptosis, which is mediated through the RA receptors. Previous experiments suggested that Ape1/ref-1 expression is related to apoptosis. To further define this relationship, we used retroviral gene transduction to over-express HA-tagged Ape1/ref-1 in HL-60 myeloid leukemia cells. We observed that the RA-induced growth inhibition, apoptosis, and differentiation of HL-60 cells over-expressing Ape1/ref-1 was significantly enhanced compared to wild type HL-60 cells. To further understand the mechanism of this effect we performedgel shift experiments in vitro with Ape1/ref-1, retinoic acid receptor alpha (RAR-α), and a retinoic acid response element (RARE) under varying redox conditions andco-transfection experiments in CV-1 cells with Ape1/ref-1 and RAR-α using an RARE linked to a luciferase reporter.

Results:gel shift experiments demonstrate a redox dependent binding of RXR-α and RAR-α to their RARE which is mediated by Ape1/ref-1;western blot analysis of transfected CV-1 cells revealed proper expression of each transfected construct including RAR-α, RXR-α and Ape1/ref-1; andexamination of RA-treated CV-1 cells for RARE-linked luciferase expression demonstrated Ape1/ref-1 enhancement of RAR activated transcription of the luciferase reporter.

In conclusion, our data supports the contention that Ape1/ref-1 expression may be important for enhancing RA-induced myeloid differentiation and programmed cell death through a redox based mechanism in transcription of target genes.