bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutational Analysis of Thrombopietin, c-MPL and Jak2 Genes in Essential Thrombothethymia Patients - Identification of Concurrent Mutations of c-MPL and Jak2.
Authors: Ding Jianmin , Komatsu Hirokazu , Iida Shinsuke , Ito Asahi , Yano Hiroki , Ri Masaki , Inagaki Atsushi , Kusumoto Shigeru , Ishida Takashi , Nitta Masakazu , Ueda Ryuzo
Published: October 2019

The molecular pathogenesis in chronic myeloproliferative disorder including essential thrombocythemia (ET) has been recently clarified. We previously identified a novel activating mutation of c-MPL gene (S505N) as a cause of familial ET. The Jak2 mutation (Jak2V617F) is also reported most frequently as a constitutive activating mutation in ET. Here we screened the mutations in the open reading frames of thrombopoietin (TPO), c-MPL and Jak2 in the 26 Japanese (sporadic) ET patients. None had any somatic mutations in TPO. We found Jak2V617F mutation in 7 patients. Interestingly, one of the patients had the c-MPL somatic mutation (W515L). The c-MPL W515L transfectants presented the autonomous STAT5 and MEK1/2 phosphorylation in the absence of the ligand, which show the c-MPL W515L is a constitutive activating mutation for signalling. The concurrent mutations of Jak2V617F and c-MPL W515L are likely to contribute to the pathogenesis of the thrombocythemia in this patient. Now we are focusing on the issue whether the single clone with both of two mutations occupy or the two clones with either Jak2 or c-MPL mutation coexist in the bone marrow of this ET patient (Single cell PCR is on going with the megakaryocytes of this patient).
A Molecular Cytogenetics Profile of Chromosome 9p in CMPD.
Authors: Vaughan Beverley R. , Brazma Diana , Grace Colin , Howard Julie , Nacheva Elisabeth
Published: October 2019

Chronic Myeloproiferative Disorders (CMPDs) form a continuum of haematological malignancies of which the molecular pathogenesis remains obscure. Chronic Myeloid Leukaemia (CML) being the only disorder with a recognisable pathogenomic abnormality, the Ph chromosome. Tyrosine kinases are clearly implicated in the disease pathogenesis, in particular the role of JAK2. It has been shown that over 80% of Polycythaemia Vera (PV) patients and 30% of Essential Thrombocythaemia (ET) patients demonstrate a clonal and recurrent mutation V617F in the JH2 pseudo-kinase domain of JAK2 located on 9p24.1. Array CGH investigations of cell lines, revealed a complex pattern of imbalances affecting the short arm of chromosome 9: on the background of 2–3-fold amplification, a loss of the 9p24.1 region, which harbours the JAK 2 along with a number of other kinases, was repeatedly observed. The aim of this study was to clarify the aCGH observations using a series of BAC DNA probes mapping from the telomere of 9p toward the centromere. Results indicate the presence of imbalances (amplifications & deletions) and rearrangements, involving the region encompassing JAK2. In all CML-BC cell lines the FISH data supports the observation of a relative loss of the 9p24.1 region on the background of amplification of chromosome 9. An on-going study of CMPD and MDS patients with both normal and abnormal karyotypes has revealed imbalances affecting the short arm of chromosome 9. In 23% (7/30) of patients there were imbalances; the majority of these (56%) demonstrated a relative loss of information in this region. The pathegenomic significance of imbalances involving 9p remains unclear. It may however represent an acquired homozygousity as a consequence of mitotic recombination, with a ‘second hit’ removing a wild type allele, leading on to further, as yet un-clarified changes.
Expression of Nuclear Factor-kappa B Pathway Genes and Their Correlation with Clinical Features in Multiple Myeloma.
Authors: Almeida Manuella S.S. , Vettore Andre L. , Felix Roberta S. , Andrade Valeria C.C. , Carvalho Fabricio , Yamamoto Mihoko , Chauffaille Maria de Lourdes L.F. , Zago Marco Antonio , Colleoni Gisele W.B.
Published: October 2019

NF-k B is a transcription factor that controls the expression of a variety of genes involved in different cellular processes, including apoptosis regulation, cell proliferation and expression of adhesion molecules. In multiple myeloma (MM) patients, NF-k B confers longer survival and drug resistance to tumor cells. Therefore, a better understanding of molecular mechanisms involved in NF-k B pathway can contribute to identify new therapeutic targets for this incurable disease.

Purpose: To evaluate the expression of NF-k B pathway genes in MM patients by real-time quantitative PCR and its possible correlation with disease clinical features and survival.

Patients and methods: Expression of eight genes related to NF-k B pathway (NFKB, IKB, RANK, RANKL, OPG, IL6, VCAM and ICAM) were studied in 53 bone marrow samples from newly diagnosed MM patients and in seven normal controls, using Taqman system. Genes were considered overexpressed when tumor expression level was at least four times higher than the observed in normal samples. These results were compared to clinical variables using Pearson’s Qui-square and T-Test. Overall survival (OS) was estimated using Kaplan-Meier method.

Results: 18.9% of MM cases were classified as ISS 1, 34% ISS 2 and 39.6% ISS 3. The percentages of overexpression of the eight genes were: NFKB 0%, IKB 22.6%, RANK 15.1%, RANKL 31.3%, OPG 7.5%, IL6 39.6%, VCAM 10% and ICAM 26%. We found association between IL6 expression level and the International Staging System (ISS) (p = 0.01), meaning that MM patients with high ISS scores have more chance of overexpression of IL6. We also found association between IL6 overexpression and presence of chromosome 13 deletion (p= 0.035), a well-known marker of worse prognosis in MM. Mean value of ICAM relative expression was also associated with ISS score (p = 0.02). Regarding OS, cases with IL6 overexpression present worse evolution than cases with IL6 normoexpression (p=0.04).

Conclusions: IL6 overexpression was found in 39.6% of MM cases and confirms its participation in myeloma progression, regulating growth and survival of tumor cells. Besides, IL6 overexpression was significantly associated with advanced stage disease and del(13) and had impact on MM patient’s survival. ICAM was overexpressed in 26% of cases and its level was associated with ISS scores. Therefore, ICAM seems to have some potential as therapeutic target in MM.
Imatinib Induces Apoptosis of Myeloma Cells by Inhibiting c-Kit Signaling Transduction.
Authors: Li Juan , Huang Beihui , Zhao Ying , Luo Shaokai
Published: October 2019

Objective To evaluate the mechanism and influence of Imatinib on myeloma cells which express c-Kit receptor.

Methods KM3 cells were treated with Imatinib for indicated time and indicated concentration and cell growth index were evaluated by XTT assay. Cell cycle were examined by flow cytometry. Apoptosis were assessed by Annexin V/PI. c-Kit and phosphate c-Kit were examined by Western blot.

Results Imatinib inhibited proliferation of KM3 cells in a concentration more than 0.25μmol/L, induced a progressive decline in S phase cell fraction and an increase in G0/G1 cells. Annexin V/PI staining and DNA ladder indicated that Imatinib had a substantial effect on apoptosis of KM3 cells in a dose-dependent manner and induced pro-caspase-3 and PARP to cleave. Imatinib treatment inhibited expression of c-Kit and provoked a decreased of IL-6 induced c-Kit phosphorylation.

Conclusion Imatinib inhibites KM3 myeloma cells proliferation and induces apoptosis of KM3 myeloma cells by inhibiting c-Kit signaling transduction.
Selective Inhibition of Glycogen Synthase Kinase-3 by Facilitators of Insulin Response Promotes Proliferation and Survival in Multiple Myeloma Cells.
Authors: Jernberg-Wiklund Helena , Stromberg Thomas , Hammarberg Anna , Georgii-Hemming Patrik , Osterborg Anders , Nilsson Kenneth
Published: October 2019

There is ample evidence for a central role of IGF-1R in multiple myeloma (MM) cell survival and resistance to apoptosis. Recently, we have demonstrated the use of inhibitors of the IGF-1R tyrosine kinase (RTK) activity as a promising therapeutic strategy in MM in vitro and in vivo (Stromberg et al Blood 2006,107:669–78, Menu et al Int J Cancer 2007,121:1857–61). In the present investigation, we have examined the role of glycogen synthase kinase-3 (GSK3), a target of the IGF-1R/PI 3-K/AKT pathway, in proliferation and apoptosis of MM. Unlike most kinases GSK3 is constitutively active, but may be inhibited by e.g. insulin, EGF and FGF. GSK3 is also a converging point of the Wnt and the IGF-1R/PI 3-K/AKT survival pathway. Reduction of GSK3 activity resulted from its phosphorylation at the regulatory residues Ser21 (α isoform) and Ser9 (β isoform) catalysed by AKT. GSK3 inhibitors LiCl, AR-A014418, the maleimides SB216763 and SB41528, and the highly selective aminopyrimidine derivatives CT98014 and CT99021 (Novartis, Emeryville, CA), were investigated for their effects on MM cells when growth and survival were challenged by cytotoxic stress and/or interference with different signaling proteins. Apoptosis induced by serum starvation was reduced by CT99021, an effect comparable to the survival-promoting effect of IGF-1. The selective GSK3 inhibitor also restored survival in MM cells induced by glucocorticoids and combinations increasing the susceptibility to apoptosis using PI 3-K inhibitors LY294002, the IGF-1R antagonist αIR3, the mTOR inhibitor rapamycin and the MEK inhibitor PD98059. In addition to the anti-apoptotic effect, CT99021 was also demonstrated to counteract G0/G1 accumulation induced by serum starvation, LY294002, αIR3 and rapamycin. However, accumulation of cells in the G2/M phase induced by doxorubicin and melphalan, were not counteracted by GSK3 inhibitors. Substrates of GSK3 most prominently include beta-catenin and the IκB-analog Bcl-3, both targeted for degradation upon GSK3 activation. Selective GSK inhibition restituted protein levels of beta-catenin, Bcl-3, p21 cip1/WAF1, p27kip1 and cyclin B1, D2 and E corroborating with the effects on survival and proliferation. These data demonstrate that targeting of the GSK3 using highly selective inhibitors is associated with potent anti-apoptotic effects and propagation through the cell cycle in MM.
IgG-Derived Tregitope Peptides Suppress T Cell Responses in Vitro and in Vivo
Authors: De Groot Anne S. , Moise Leonard , Su Yan , McMurry Julie A , Martin William D , Scott David W.
Published: October 2019

We have identified a set of putative natural T regulatory epitopes (Tregitopes) which, when co-administered with an antigen, cause the expansion of antigen-specific adaptive Tregs in vitro and in vivo. They have the following characteristics:

they bind, in most cases, with high affinity to multiple MHC class II molecules and, when co-administered with antigen, they suppress effector T cell immune responses to the antigen and up-regulate Treg associated cytokines and chemokines.

T cells responding to Tregitopes exhibit a T regulatory phenotype (CD4+/CD25hiFoxP3+). To test whether Tregitopes derived from immunoglobulin (Ig) suppress immune responses to antigen co-administered in vivo, we performed two types of experiments. In the first, we dosed three groups of HLA DR4 mice every other week for six weeks with either

a peptide antigen (pAg) alone, pAg with murine Fc, or pAg with mTregitope289, the murine homolog of the human Tregitope289.

Mice were sacrificed and spleens harvested for assay. While the mice dosed with murine Fc demonstrated a reduced IL-4 ELIspot response to pAg, remarkably, the reduction was even greater in the mice treated with Tregitope. In a second model, C57Bl/6 mice were injected with LPS-stimulated B cells that were pulsed with either

ovalbumin (OVA) alone, mTregitopes 167 and 289 or with OVA together with the two mTregitopes.

One week later, mice were challenged with OVA 323–339 peptide in adjuvant. Two weeks after challenge, draining lymph nodes were harvested and LN cells stimulated with OVA 323–339 for measurement of T-cell proliferation by thymidine incorporation and by IFN-γ secretion by ELIspot. The mice receiving B cells previously pulsed with OVA alone demonstrated a robust IFN-gamma response to OVA re-stimulation. In contrast, the mice receiving B cells previously pulsed with OVA + Tregitopes demonstrated a comparatively reduced response. When sera were assayed for anti-OVA antibodies by ELISA, antibody response to OVA also declined following treatment with B cells co-administered with Tregitope. The mechanism of suppression appears to be due to the induction of antigen-specific adaptive tolerance induction (De Groot AS et al. Activation of natural regulatory T cells by IgG Fc-derived Peptide “Tregitopes” Blood112: in press, 2008).
Identification of the Involvement of the Tyrosine Kinase C-Ros in the Pathogenesis of Chronic Myelomonocytic Leukemia (CMML)
Authors: Cilloni Daniela , Carturan Sonia , Bracco Enrico , Defilippi Ilaria , Maffè Chiara , Iacobucci Ilaria , Pautasso Marisa , Rotolo Antonia , Giugliano Emilia , Messa Emanuela , Martinelli Giovanni , Saglio Giuseppe
Published: October 2019

The abnormal activation of tyrosine kinases are a common finding in chronic myeloproliferative disorders. Perturbation of RTK signalling by genetic alterations results into an abnormal proliferation advantage and finally into a malignant transformation. c-Ros is an orphan RTK displaying transforming activity whose role has been established in the development of neuronal neoplasia. The aim of this study was to evaluate the involvement of c-Ros in the pathogenesis of chronic myelomonocytic leukemia (CMML) and to establish the effects of c-Ros activation. c-Ros expression was evaluated by RQ-PCR in 133 samples collected from 96 CMML patients at diagnosis (96 BM and 37 PB) and 60 healthy donors (30 PB and 30 BM). The protein amount and localization was analyzed by westen blot and immunofluorescence assay. In order to establish the effects of c-Ros activation we generated a chimeric receptor containing the extracellular domain derived from epidermal growth factor receptor (EGFR) and the transmembrane and cytoplasmic domains from c-ros (ER). The chimeric receptor was then transfected in NIH3T3 and HEK293T cells. Transfected and control cells were then stimulated with 100 nM EGF ligand and proliferation and apoptosis evaluated by incorporation of 3H timidine and MTT assay and by FACS for the detection of annexin V, respectively. We found that Ros is undetectable in healthy subjects but it is overexpressed in CMML (p<0,0001) in both BM and PB cells with a median value of 2−ΔΔCt in BM of 380 (range 10–63303) and 212 in PB (range 6–30012). WB confirmed the presence of c-Ros protein in CMML cells but not in normal controls. Immunofluorescence staining localized the protein within the cytoplasm. We found that ROS is highly expressed in CD34+ cells and monocytes from CMML patients but not in their normal counterparts. Sequence analysis revealed the absence of mutations of c-Ros promoter. SNPs analysis exclude the presence of duplications or deletions of the gene. Moreover we found that the EGF induced activation of c-Ros affects proliferation by increasing of 3.5 folds the proliferation rate as compared to cells transfected with the empty vector and stimulated with EGF under the same conditions. Furthermore cell adhesion was 4 folds decreased as compared to control. By contrast apoptosis is not affected by c-ROS activity (p=0,2). This study demonstrates that c-Ros is abnormally expressed in patients with CMML. The abnormal activation of c-Ros is responsible for loss of adhesion and increased proliferation. In conclusion, we identified a new tyrosine kinase which may be responsible for the proliferation defect typical of CMML cells and could represent a target for molecular therapies.
Protein Phosphatase 2B Inhibition Promotes the Secretion of Von Willebrand Factor from Endothelial Cells
Authors: Nolasco Leticia , Gushiken Francisca C. , Patel Vimal , Turner Nancy , Pradhan Subhashree , Dong Jing-fei , Moake Joel L. , Vijayan K. Vinod
Published: October 2019

Ultra-large von Willebrand factor (ULVWF) multimers secreted from the Weibel-Palade bodies (WPBs) of endothelial cells plays a crucial role in the development of thrombotic microangiopathies. Secretion of WPB contents is regulated, in part, by the phosphorylation of vesicle trafficking proteins that constitutes the endothelial exocytotic machinery. In comparison to agonist-induced, protein kinase-dependent signaling pathways that regulate the exocytosis of WPB contents, a role for protein phosphatases in regulating endothelial exocytosis is currently undefined. In this study, we show that inhibition of serine/threonine protein phosphatase 2B (PP2B) activity by cyclosporine A (CsA), tacrolimus (FK506) or a cell-permeable PP2B autoinhibitory (AI) peptide promotes the secretion of hyper-adhesive ULVWF from human umbilical vein endothelial cells (HUVECs) in the absence of any other endothelial cell-stimulating agent. PP2B inhibitor-induced secretion and anchorage of ULVWF strings from HUVECs induce the tethering of adhesive platelets. In support of a role for PP2B in VWF secretion, we demonstrated that the catalytic subunit of PP2B expressed as a GST fusion protein interacts with the vesicle trafficking protein, Munc18c. Serine phosphorylation of Munc18c, which promotes granule exocytosis in other secretory cells, was significantly increased in CsA-treated HUVECs, suggesting that this process may be involved in CsA-mediated WPB exocytosis and secretion of VWF. Furthermore, plasma VWF antigen levels were also enhanced in CsA-treated mice, and siRNA-mediated knockdown of PP2Bb in HUVECs significantly enhanced VWF secretion. These observations suggest that CsA promotes VWF release, at least in part by inhibition of PP2B activity. These observations are compatible with the clinically observed association of CsA treatment and increased plasma VWF levels in humans, as well as the association between prolonged exposure to CsA and thrombotic microangiopathy in a subset of exposed patients.
Hematopoietic Differentiation of Human Induced Pluripotent Stem Cells
Authors: Choi Kyung-Dal , Yu Junying , Smuga-Otto Kimberly , Dias Jessica , Salvagiotto Giorgia , Vodyanik Maxim , Thomson James , Slukvin Igor
Published: October 2019

Induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity for modeling of human diseases in vitro as well as for developing novel approaches for regenerative therapy based on immunologically compatible cells. In the present study, we employed an OP9 differentiation system to characterize the hematopoietic differentiation potential of seven human iPSC lines obtained from human fetal, neonatal, and adult fibroblasts through reprogramming with POU5F1, SOX2, NANOG, and LIN28 and compared it with the differentiation potential of five human embryonic stem cell lines (hESC; H1, H7, H9, H13, and H14). Similar to hESCs, all iPSCs in coculture with OP9 generated all types of colony forming cells (CFCs) as well as CD34+ cells that can be separated into distinct subsets based on differential expression of CD43 and CD31. CD34+CD31+CD43− cells obtained from all iPSCs expressed molecules present on endothelial cells and readily formed a monolayer when placed in endothelial conditions, while hematopoietic CFC potential was restricted to CD43+ cells. iPSC-derived CD43+ cells could be separated into three major subsets based on differential expression of CD235a/CD41a and CD45: CD235a+CD41a+/− (erythro-megakaryocytic progenitors), and lin-CD34+CD43+CD45− (multipotent), and lin-CD34+CD43+CD45+ (myeloid-skewed) primitive hematopoietic cells. Both subsets of primitive hematopoietic cells expressed genes associated with myeloid and lymphoid development, although myeloid genes were upregulated in CD45+ cells, which are skewed toward myeloid differentiation. Cytogenetic analysis demonstrated that iPSCs and derived from them CD43+ cells maintained normal karyotype. In addition short tandem repeat analysis of CFCs generated from IMR90-1 cells has been performed to confirm that blood cells are in fact derived from reprogrammed IMR90 cells, and not from contaminating hESCs. While we observed some variations in the efficiency of hematopoietic differentiation between different iPSCs, the pattern of differentiation was very similar in all seven tested iPSC and five hESC lines. Using different cytokine combinations and culture conditions we were able to expand iPSC-derived myeloid progenitors and induce their differentiation toward red blood cells, neutrophils, eosinophils, macrophages, ostoeclasts, dendritic and Langerhans cells. Although several issues remain to be resolved before iPSC-derived blood cells can be administered to humans for therapeutic purposes, patient-specific iPSCs can already be used for characterization of mechanisms of blood diseases and to identify molecules that can correct affected genetic networks.
Improved Outcome by Addition of Lomustine (CCNU) to Idarubicin and Cytarabine in Elderly Patients with De Novo Acute Myeloid Leukemia. A Report from the GOELAMS Group
Authors: Pigneux Arnaud , Witz Francis , Sauvezie Mathieu , Bene Marie-Christine , Luquet Isabelle , hunault-Berger Mathilde , Recher Christian , Vey Norbert , Lioure Bruno , Himberlin Chantal , Escoffre-Barbe Martine , Berthou Christian , Lissandre Severine , Fegueux Nathalie , Cahn Jean-Yves , Jourdan Eric , Bouscary Didier , Dreyfus Francois , Reiffers Josy M. , Milpied Noel , Harousseau Jean-Luc , Ifrah Norbert
Published: October 2019

In elderly patients with acute myeloid leukemia (AML) treated intensively, no improvement has been shown in the last 20 years. We performed a retrospective study in 847 patients over 60 years old, prospectively enrolled in 3 trials conducted in France between 1995 and 2005, with the aim to investigate prognostic factors for complete remission (CR) achievement and survival. Induction therapy consisted in the association of Idarubicin 8mg/m2 d1-5 and Cytarabine 100mg/m2 d1-7 (Group I, 339 patients) or the same drugs with the addition of lomustine (10mg\m2 orally at day 1)(Group II, 508 patients). Consolidation therapy consisted of anthracycline and cytarabine courses at lower doses, preceded or not by a first course with intermediate dose cytarabine. The patients’ characteristics were similar between the two groups concerning sex, WBC count, ECOG, and cytogenetics, yet patients were older in Group II versus Group I (55% versus 45% over 69 years of age, p<0.0001).The CR rate was significantly higher for patients in Group II compared to Group I (67 % vs 57%, p= 0.002). The toxic death rate was not different between groups. In multivariate analysis, three good prognostic factors emerged for achieving complete remission: good or intermediate cytogenetics (p<0.0001), ECOG < 2 (p<0.0001), and adjunction of lomustine to induction chemotherapy (p=0.002). The median overall-survival was significantly improved for patients treated with lomustine (12.7± 2.2 months vs 8.7± 2.7 months, p=0.004). In multivariate analysis, five prognostic factors affected positively overall survival: adjunction of lomustine to induction chemotherapy (p < 0.0001), age < 69 years (p =0.001), ECOG < 2 (p =0.001), FAB other than AML0,6 or 7 (p = 0.004) and good or intermediate cytogenetics(p = 0.007). The median event-freesurvival was also improved for patients treated with lomustine (10.7± 2.2 months vs 7± 2.7 months, p=0.002). Event-free-survival was affected by the same prognostic factors as overall survival. We conclude that lomustine might be added in standard induction therapy as it allowed to obtain both better CR rate and survival in this retrospective study.
Myc Drives Chromosomal Gain in Acute Myeloid Leukemia
Authors: Jones Letetia , Sevcikova Sabina , Phan Vernon , Jain Sachi , Shieh Angell , Dubansky Joshua , Li Min , Maunakea Mei Lin , Zhu George , Tennant Thelma R , Lowe Scott , Le Michelle M. , Kogan Scott C.
Published: October 2019

Acute Myeloid Leukemia (AML) is a disease characterized by diverse genetic pathogenesis, including both balanced and unbalanced chromosomal aberrations. Much is known regarding the pathogenic effects of balanced rearrangements in AML, whereas our understanding of how unbalanced aberrations contribute to leukemia is more limited. The balanced t(15;17) chromosomal rearrangement is a nearly constant feature of acute promyeloctyic leukemia (APL), a subtype AML. The translocation fuses the promyelocytic leukemia gene (PML) to the retinoic acid receptor α gene (RARA). Trisomy 8 is the most common secondary karyotypic lesion observed in APL, and it has been speculated but not proven that the MYC gene contributes to this chromosomal gain. We previously reported that mouse chromosome 15, which contains the mouse Myc gene in a region syntenic to human chromosome 8q24, is commonly gained in the MRP8 PML-RARA mouse model of APL. We now report our work to assess the hypothesis that increased MYC cooperates with PML-RARα to accelerate disease and that gain of MYC/Myc drives +8 in humans and +15 in mice. Expressing MYC with a retroviral vector in PML-RARA bone marrow led to the rapid development of APL-like leukemias (3 months vs. 8.5 months with PML-RARA alone). Chromosome 15 was not gained in any of the leukemias, although 70% had other clonal karyotypic abnormalities. This finding suggests that when MYC is overexpressed, there is no selective pressure to gain chromosome 15, supporting our hypothesis that Myc is driving this gain. We also generated PML-RARA mice haploinsufficient for Myc to examine the effect of decreasing MYC levels. The median latency among leukemic animals was 258 days for mice with PML-RARA and two wild-type Myc alleles, whereas the latency was increased to 339 days for PML-RARA Myc haploinsufficient mice. Hence, lower MYC expression served as a check on leukemic transformation. Furthermore, the majority of the leukemias that arose in Myc haploinsufficient mice had gained wild-type Myc. These data demonstrate a selective pressure for Myc gain. Additional experiments showed that

as MYC expression increases there is a decrease in both latency and genetic complexity of leukemias that arise, that MYC and PML-RARα interact to disrupt myeloid differentiation in vivo and that although MYC cooperates with PML-RARα to cause leukemia, additional events are required for completing transformation even at high levels of MYC.

Altogether our studies of increased and decreased MYC expression in PML-RARA mice show a strong correlation between MYC dosage and leukemic transformation. Our results suggest that agents that target MYC might be useful for the treatment of AML.
Functional Comparison of Freshly Isolated and Ex-Vivo Expanded CD4+CD25+Foxp3+ Regulatory T Cells in Suppressing Murine Acute GvHD
Authors: Sega Emanuela I , Leveson-Gower Dennis , Nguyen Vu H. , Negrin Robert
Published: October 2019

Graft versus host disease (GVHD) is a major complication of hematopoietic stem cell transplantation resulting from donor T cell reactivity against host tissue antigens. CD4+CD25+Foxp3+ regulatory T cells (Treg) are known to be important in maintaining self tolerance and preventing autoimmunity. Using murine models of acute GVHD in which allogeneic bone marrow cells are transplanted into lethally irradiated hosts, we and others have shown that donor Treg are able to suppress GVHD induced by donor allogeneic T cells and dramatically improve survival. Treg are rare and suppression of GVHD requires adequate numbers of Treg in relation to the number of conventional T cells (Tcon). To overcome this problem, expansion of Treg has been performed, however there has not been a head to head comparison of the function of expanded vs fresh Treg. Highly purified CD4+CD25+Foxp3+ T cells (>98% purity) were expanded using anti-CD3/anti-CD28 dynabeads and 1000 U/ml IL-2. Under these conditions, after five days Treg expanded up to 13 fold while maintaining high Foxp3 expression levels (85–90%). Longer expansion periods result in more T cell expansion but an overgrowth of Foxp3 negative T cells. In a mixed lymphocyte reaction assay, the ex-vivo expanded Treg efficiently suppressed the proliferation of alloreactive T cells. The expanded Treg were evaluated in an in vivo acute GVHD mouse model in direct comparison with freshly isolated Treg using a novel bioluminescent imaging assay that allowed for assessment of Tcon proliferation in addition to traditional metrics of GVHD severity including weight gain, survival and GVHD score. Initial experiments show that, similar to freshly isolated Treg, the ex-vivo expanded Treg suppress GVHD symptoms and improve survival, although a greater number of expanded Treg were required comparable to freshly isolated Treg. The mean GVHD score for the Tcon alone group was 5.8±1.02. Fresh Treg added at 1:1 ratio decreased the GVHD score to 0.75±0.25 (p=0.0036). Ex-vivo expanded Treg demonstrated a dose-dependent decrease in GVHD score, although four times more expanded Treg were needed to obtain a similar reduction in GVHD score (0.50±0.5, p=0.0036). This observed difference in potency was not due to the ex-vivo expanded Treg being short-lived when infused in mice. Bioluminescence imaging of luciferase positive (luc+) cultured Treg showed the same in vivo persistence as freshly isolated Treg. The ability to expand ex-vivo generated Treg is greater than the difference in potency, making ex-vivo expanded Treg potentially a viable option for treatment of GVHD, however, increased ratios of Treg:Tcon are likely to be required.
Engineering CD19-Redirected T Lymphocytes to Enhance Their Safety and Efficacy
Authors: Hoyos Valentina , Savoldo Barbara , Vera Juan F , Quintarelli Concetta , Rooney Cliona M , Heslop Helen E. , Brenner Malcolm K , Dotti Gianpietro
Published: October 2019

Modification of primary T cells to express chimeric antigen receptors (CARs) is an attractive strategy for the generation of tumor-specific T cells for adoptive therapies. However, their efficacy is limited by poor expansion within the tumor microenvironment. The addition of co-stimulatory endodomains, such as CD28, to the CAR may enhance cell expansion in response to the antigen, but cell growth and survival remain suboptimal since the co-stimulation provided through these endodomains cannot recapitulate the spatiotemporal sequence of signals T cells receive during physiologic activation. To further potentiate the expansion and survival of CAR-modified T lymphocytes, we generated a new vector encoding 3 molecules: CAR.19 incorporating the CD28 endodomain, codon optimized hIL15 to enhance cell survival and growth, and an inducible suicide gene based on the expression of Caspase9 (iCasp9) to increase the margin of safety associated with transgenic expression of an autocrine growth factor. These three sequences were linked using 2A-like peptide sequences. We compared the proliferative capacity, cytotoxic activity and in vivo anti-tumor effects of T lymphocytes expressing either CAR.19-28z alone or CAR.19-28z, IL15 and the suicide gene. T lymphocytes were activated with OKT3/CD28 antibodies and then transduced with retroviral supernatants. Phenotypic analysis showed 70±10% and 75±5% transduction efficiency for iCasp9/CAR19-28z/IL15 and CAR19-28z T cells, respectively. Only the iCasp9/CAR19-28z/IL15 T cells produced IL15 (>100pg/mL) after stimulation with CD19+ tumor cells. In co-culture experiments, T cells expressing CAR.19-28z alone or iCasp9/CAR19-28z/IL15 both completely eliminated CD19+ tumor cells within 72 hours. However, labeling with carboxyfluorescein diacetate succinimidyl ester (CFSE) showed that the proliferation of the iCasp9/CAR19-28z/IL15+ T cells in response to CD19+ tumor cells was greater than that of CAR.19-28z control+ T cells. Finally, the activation of the suicide gene iCasp9 with a small-molecule dimerizer (CID 50ng/mL) rapidly induced >90% apoptosis of T cells expressing iCasp9/CAR19-28z/IL15. Hence all three transgenes were functional. To assess the antitumor effects of the modified cells in vivo, we used a xenograft SCID mouse model and an in vivo bioluminescence system. CD19+ Daudi cells (1×106) expressing firefly Luciferase (FL) were injected i.p., and on day 4, mice received i.p iCasp9/CAR19-28z/IL15+ or CAR19-28z+ or control T cells (10×106). By day 30 the tumor signal was significantly reduced in mice receiving iCasp9/CAR19-28z/IL15+ T cells (ROI<7×107) compared to mice receiving CAR19-28z or control T cells (ROI>2.0×109). In conclusion, our data indicate that a tricistronic vector can effectively be expressed in tumor-redirected human T cells, improving their survival and allowing their destruction should unwanted effects occur.
Pentostatin, Cyclophosphamide, and Rituximab (PCR) Achieve High Response Rates in Indolent B-Cell Lyphoma without Prolonged Myelosuppression
Authors: Samaniego Felipe , Fanale Michelle , Pro Barbara , Hagemeister F.B. , McLaughlin Peter , Romaguera Jorge , Neelapu Sattva , Rodriguez Maria Alma , Fayad Luis , Younes Anas , Kwak Larry W.
Published: October 2019

Background: The addition of rituximab to combination chemotherapy has improved treatment outcomes of indolent lymphomas. Combination therapy with purine analogs, akylating agents, and monoclonal antibodies is a promising approach for treating indolent B-cell lymphoma. Nucleoside analog-based regimens selectively target lymphoid cells, making them attractive drugs for lymphoid cancers. Pentostatin is a nucleoside analog that compared with other nucleoside analogs is reported to have less bone marrow cell toxicity. The combination of pentostatin, cyclophosphamide, and rituximab (PCR) is an effective regimen for relapsed chronic lymphocytic leukemia.

Methods:In this study, we examined the efficacy of pentostatin, cyclophosphamide, and rituximab for the treatment of B-cell lymphoma. Pentostatin (4 mg/m2), cyclophosphamide (600 mg/m2), and rituximab (375 mg/m2) were given on day 1 of a 21-day cycle with planned 6 or 9 cycles and restaging after every 3 cycles. Patients received prophylaxis with acyclovir 400 mg/po bid and trimethoprim-sulfamethoxazole 3 times per week. Small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) tissues were stained for ZAP70 protein.

Results: At the time of abstract submission, 80 patients with a median age was 60 years were treated, 43 patients with follicular lymphoma, 27 with SLL/CLL, and 9 with mucosa-associated lymphoid tissue. We observed an overall response of 77 out of 80 (96%), CR/CRu in 65 out of 80 (81%), PR in 12 out of 80 (15%), and mixed response or progression in 3 out of 80 (4%). Neutropenia (49% < 1000/uL), thrombocytopenia (2% < 100,000/uL), nausea (15% grade 3), fatigue (37% grade 3 and 4), and muscle pain (21% grade 3) was observed. ZAP70 staining and response will be reported. Two patients experienced prolonged pancytopenia that resolved and no cases of myelodysplasia were observed.

Conclusions: PCR therapy is an effective regimen in indolent B cell lymphoma with tolerable toxicity.
Small Molecule XIAP Inhibitors Cooperate with TRAIL to Trigger Apoptosis in Childhood Acute Leukemia Cells and Overcome Bcl-2-Mediated Resistance
Authors: Fakler Melanie , Löder Sandra , Vogler Meike , Schneider Katja , Jeremias Irmela , Debatin Klaus-Michael , Fulda Simone
Published: October 2019

Children with high risk acute lymphoblastic leukemia (ALL) do not respond well to current treatments. This failure is, at least in part, due to defects in apoptosis programs. Therefore, new strategies are required that counter apoptosis resistance in order to improve the poor prognosis of high risk pediatric acute leukemia. Since XIAP, a member of “Inhibitor of Apoptosis Proteins” (IAPs), is expressed at high levels in acute leukemia and blocks apoptosis at a central point of the apoptotic machinery, XIAP may present a suitable molecular target for therapeutic intervention. Here, we report that neutralizing XIAP by small molecule inhibitors is a novel and effective approach to sensitize childhood acute leukemia cells for TRAIL- or chemotherapy-induced apoptosis. XIAP inhibitors at subtoxic concentrations, but not a structurally related control compound, synergize with TRAIL to induce apoptosis in acute lymphoblastic leukemia cells. Also, XIAP inhibitors act in concert with TRAIL to reduce clonogenic growth of ALL cells demonstrating that they suppress long-term survival. Analysis of signaling pathways reveals that XIAP inhibitors enhance TRAIL-induced activation of caspases, loss of mitochondrial membrane potential and cytochrome c release in a caspase-dependent manner, indicating that they promote a caspase-dependent feedback mitochondrial amplification loop. Intriguingly, XIAP inhibitors overcome Bcl-2-mediated resistance to TRAIL by enhancing Bcl-2 cleavage and Bak conformational change. Thus, XIAP inhibitors combined with TRAIL even break Bcl-2-imposed resistance, a defect in the apoptotic pathway that is common in acute leukemia and associated with poor prognosis. Further, XIAP inhibitors prime ALL cells for apoptosis induced by various anti-leukemic drugs, e.g. cytarabine, doxorubicin, etoposide and 6-mercaptopurine, or by agonistic anti-CD95 antibody. Notably, XIAP inhibitors kill leukemic blasts from children with ALL ex vivo and cooperate with TRAIL to induce apoptosis. In contrast to malignant cells, XIAP inhibitors at equimolar concentrations alone or in combination with TRAIL are non-toxic to normal peripheral blood mononuclear cells despite expression of the apoptosis-inducing TRAIL receptors on the cell surface, pointing to a therapeutic window. Most importantly, XIAP inhibitors significantly reduce leukemic burden in vivo in a mouse model of pediatric ALL engrafted in NOD/SCID mice. Thus, small molecule XIAP inhibitors present a promising novel approach for apoptosis-based therapy of childhood acute leukemia.
Combination of Lenalidomide, Melphalan, Prednisone and Thalidomide (RMPT) in Relapsed/Refractory Multiple Myeloma: Results of a Multicenter Phase II Clinical Trial
Authors: Palumbo Antonio , Falco Patrizia , Sanpaolo Grazia , Falcone Antonietta , Ferderico Vincenzo , Canepa Letizia , Crugnola Monica , Baldini Luca , Larocca Alessandra , Magarotto Valeria , Petrucci Maria Teresa , Boccadoro Mario
Published: October 2019

Background: In newly diagnosed multiple myeloma (MM) patients the addition of lenalidomide or thalidomide or bortezomib to the standard oral melphalan and prednisone (MP) combination significantly increased response rate and event-free survival. In advanced MM, the 4 drug combination VMPT further improves response rate. In this multicenter open label phase I/II trial the safety/efficacy profile of the 4 drug combination, lenalidomide, melphalan, prednisone and thalidomide (RMPT) was evaluated in patients with relapsed/refractory myeloma.

Methods: Oral lenalidomide was administered at 10 mg/day on days 1–21, oral melphalan at 0.18 mg/kg on days 1–4, oral prednisone at 2 mg/kg on days 1–4. Thalidomide was administered at 50 mg/day (Arm A) or 100 mg/day (Arm B) on days 1–28. Each course was repeated every 28 days for a total of 6 courses. Aspirin 100 mg/day was given as a prophylaxis for thrombosis. Maintenance therapy included lenalidomide alone at 10 mg/day on days 1–21.

Results: Forthy-four patients, median age 69 years (range 47–80), with relapsed or refractory MM were enrolled. Twenty-six patients received RMPT as second line of therapy, 18 as third line. Twenty patients received prior autologous transplant, 10 thalidomide-based regimen, 9 bortezomib-based regimen and 3 allogeneic stem cell transplant. After a median of 2 courses, 75.8% of patients achieved at least a partial response (PR), including 30% very good partial response (VGPR). Among patients who received RMPT as second line therapy the PR rate was 81.8%, including VGPR 36.4%.Among patients who received thalidomide 100 mg, the PR rate was 93.3% (including VGPR 46.7%) compared to 64.7% of thalidomide 50 mg. The 1-year-progressionfree survival was 48.6% and the 1-year survival from study entry was 90%. Grade 3–4 hematologic adverse events included: neutropenia (66.6%), thrombocytopenia (36.3%) and anemia (30.2%). Grade 3–4 non hematologic adverse events included: infections (21.2%), neurological toxicity (6%) and fatigue (9%). No thromboembolic events were reported.

Conclusion: Initial results showed that RMPT is an effective salvage therapy with a high proportion of responses. Toxicities were manageable. No thromboembolic complications were reported.
The SH2-B Adaptor Protein Negatively Regulates EPO-Dependent Signalling Via Interaction with Erythropoietin Receptor Ptyr-343
Authors: Javed Mojib Javadi , Tschirch Edda , Beattie Bryan K , Stickle Natalie , Huang Kai , Jaster Robert , Carter-Su Christin , Barber Dwayne
Published: October 2019

Erythropoiesis is a developmentally important process, whereby multipotent hematopoietic stem cells differentiate into mature erythrocytes. Erythropoietin (EPO) is a critical regulator in this process and mediates its signal via the erythropoietin receptor (EPO-R) and the primary associated tyrosine kinase, JAK2. EPO, EPO-R and JAK2 play a crucial role in erythropoiesis, as deficiency in any of these proteins results in an embryonic lethal anemia. Structural-functional studies and murine knock-in models have shown EPO-R pTyr-343 to play a critical role in EPO mediated signalling. STAT-5 is activated by EPO-R pTyr-343, but STAT5ΔN mice do not have any profound erythroid abnormalities. Such evidence has led our group to hypothesize that other SH2 containing effectors interact with EPO-R pTyr-343. Cloning of Ligand Target screening was utilized to demonstrate that EPO-R pTyr-343 binds to adaptor protein SH2-Bβ. SH2-B contains multiple protein-protein interaction domains including multiple proline-rich regions, a PH domain and an SH2 domain. Although SH2-B does play a role in a number of signaling pathways, it is not required for embryonic development. Since SH2-B is a potent regulator of JAK2 in context of Growth Hormone and Leptin signaling, and it can directly interact with EPO-R pTyr-343, we hypothesize that SH2-B functions as an important adaptor protein downstream of the EPO-R.H2-B constitutively associates to the inactive EPO-R, an interaction that is independent of JAK2 binding to the EPO-R. Upon EPO stimulation, enhanced SH2-dependent binding of SH2-B to pTyr-343 and pTyr-401 of the EPO-R was confirmed utilizing a panel of EPO-R truncation mutants. The EPO mediated interaction between SH2-B and activated EPO-R is both dose and time dependent. EPO stimulation also results in SH2-B serine and threonine phosphorylation. Importantly, the interaction of SH2-B and EPO-R was observed in erythroid cell lines and primary murine splenocytes. The function of SH2-B in EPO signaling was investigated via knocking down SH2-B in Ba/F3-EPO-R cells. Knock down of SH2-B results in hypersensitive EPO-dependent phosphorylation of multiple targets including the EPO-R, JAK2, STAT5 and Erk1/2. It is evident that SH2-B is a global negative regulator of EPO-dependent signaling via its ability to affect EPO-dependent JAK2 activation.
Lnk Controls Hematopoietic Stem Cell Self-Renewal through Direct Interactions with JAK2 and Contributes to Oncogenic JAK2-Induced Myeloproliferative Diseases in Mice
Authors: Bersenev Alexey , Wu Chao , Balcerek Joanna , Tong Wei
Published: October 2019

Hematopoietic stem cell (HSC) homeostasis and self-renewal are regulated by intrinsic cytokine signaling pathways. One important signaling axis for HSC is the cell surface receptor, Mpl, and its ligand, thrombopoietin (Tpo). Upon Tpo stimulation, Mpl activates Janus Kinase (JAK2) that triggers a cascade of downstream signal transduction pathways that regulates many aspects of cell development. Under steady-state conditions, mice lacking the inhibitory adaptor protein Lnk harbor an expanded HSC pool with enhanced self-renewal. Surprisingly, we found that Lnk−/− HSCs have an increased quiescent fraction, decelerated cell cycle kinetics, and enhanced resistance to repeated 5-Florouracil (5-FU) treatments in vivo compared to wild type HSCs. We further provided genetic evidence demonstrating that Lnk controls HSC quiescence and self-renewal predominantly through Mpl. Consistent with this observation, Lnk deficiency in HSCs potentiates JAK2 activation in response to TPO. Biochemical experiments reveal that Lnk directly binds to phosphorylated tyrosine residues in JAK2 following TPO stimulation. Dysregulation of cytokine receptor signaling pathways leads to hematological malignancies. Abnormal activation of JAK2 by a chromosomal translocation between the transcription factor Tel and JAK2 (Tel/JAK2) was shown to cause atypical Chronic Myelogenous Leukemia (aCML). Recently, the JAK2 V617F mutation has been observed at high frequencies in several myeloproliferative diseases (MPDs). The JAK2V617F mutant retains Lnk binding ability, suggesting Lnk status could modify MPD development. Indeed, we found that loss of Lnk accelerates oncogenic JAK2- induced CML/MPD in the mouse transplant models. Therefore, we identified Lnk as a physiological negative regulator of JAK2 in stem cells that may contribute to leukemic transformation conferred by oncogenic JAK2.
No Influence on the Incidence of Osteonecroses When Dexamethasone Replaces Prednisone during Induction Treatment for Childhood ALL: Results of Trial ALL-BFM 2000.
Authors: Möricke Anja , Zimmermann Martin , Schrauder Andre , Stanulla Martin , Schmid Hansjörg , Fengler Rüdiger , Frühwald Michael , Mann Georg , Schrappe Martin
Published: October 2019

Osteonecroses (ON) are common complications associated with the treatment of acute lymphoblastic leukemia (ALL). For symptomatic ON, incidences of 1.6% to 9.3% have been published in the literature. The range of incidence rates may be related to treatment, data acquisition methods and patients’ age distribution. Between 09/99 and 06/06, 3,048 protocol patients with ALL (age 1–17 years) were enrolled into the multicenter trial ALL-BFM 2000 (Germany, Austria, Switzerland). Data on symptomatic ON were prospectively collected and were reported in 111 patients (3.6%, 95% confidence interval (CI) 3.0–4.4%). The median age of the patients with ON was 12.8 years (range 1.3–17.9 years), the median time from ALL diagnosis until diagnosis of ON was 15 months (range 2–73 months; data available from 92 patients). The incidence in adolescents, in particular adolescent girls, was significantly higher than in younger children (age <10 years: girls 0.8%, boys 0.7%, p=0.68; ≥ 10 years: girls 18.4%, boys 7.6%, p<0.001). The corticosteroid preparation used in the induction therapy (randomization 60 mg/m2/day prednisone vs. 10 mg/m2/day dexamethasone for three weeks plus tapering) did not influence the ON rate (prednisone 3.2%, dexamethasone 3.0%, p=0.77, 95% CI for difference: −1.2 – 1.6%). Various treatment attempts of ON were reported; 10 patients have been documented to be free of symptoms. At least 38 patients underwent surgical interventions, mainly core decompressions. Total endoprostheses of one or both hips were implanted in at least 10 patients. Final evaluation of all patients in particular with regard to long-term sequelae is not yet possible. The existing data demonstrate the far-reaching consequences of ON for the quality of life in a considerable proportion of the patients. Comparing these results with the ON data of study ALL-BFM 95 (Bürger et al., Pediatr Blood Cancer, 2005) which were collected in a predominantly retrospective way, the data suggest an increase in ON incidence in the current trial ALL-BFM 2000 (ALL-BFM 95: 1.6%). As the control treatment arms of the two trials were quite similar and the randomized treatment modifications in ALL-BFM 2000 had no influence on the ON rate, the higher incidence may be attributed to the prospective data collection in the current study and possibly to a higher attention to these complications by the attending doctors.