bluuebunny/crossref_metadata_2025_split huggingface dataset:
Everolimus Is Comparably Effective to Rapamycin Acting Synergistically with Imatinib in the Treatment of Constitutively Active Mutations of the Juxtamembrane Domain of c-Kit.
Authors: Corbacioglu Selim , Prill Jan-Michael , Wagner Tobias , Debatin Klaus-Michael 
Published: October 2019

Abstract
Kit cITD is a complex internal tandem duplication involving the juxtamembrane (JMD) and kinase domain of stem cell factor receptor c-kit which was identified recently in 7% of childhood AML. kit cITD induces constitutive proliferation and apoptosis resistance that can be synergistically blocked with imatinib (im) and rapamycin (ra).
Due to a long half-life ra has disadvantageous pharmacological characteristics making drug handling especially difficult in children with serum concentrations quickly raising to toxic levels. We therefore compared ra with its ester analogue everolimus (RAD001, ev) which has a better bioavailability and a shorter half-life in order to assess equivalence and potential functional superiority. Ba/F3 cells, stably transfected with c-kit WT and kit cITD, were treated with im, ra and ev alone and in combination. In proliferation assays of WT and cITD cell lines ev was a significantly more potent inhibitor than ra. When ra or ev were combined with low im concentrations proliferation was synergistically inhibited with both drugs but again significantly better with ev. In cITD expressing cells ra and ev alone were comparably poor inducers of apoptosis. In assays combining im with ra or ev both combinations showed a significant and comparable synergism with an additional dose effect of the mTOR inhibitors between 3nM and 0.3 nM. In a NOD/SCID animal model all animals injected with kit cITD succumbed due to multi-organ infiltration of c-kit positive cells within 20 days. Survival was prolonged in animals treated with im/ra and ev. We therefore conclude that ev is at least equivalent to ra and can replace ra exploring a molecular based therapeutic attempt in susceptible children with identified mutations of the JMD of c-kit.
Long-Term Safety and Efficacy of Stem Cell Gene Therapy for ADA-SCID.
Authors: Aiuti Alessandro , Benninghoff Ulrike , Cassani Barbara , Cattaneo Federica , Callegaro Luciano , Andolfi Grazia , Mirolo Massimiliano , Scaramuzza Samantha , Marktel Sarah , Tabucchi Antonella , Carlucci Filippo , Eibl Martha , Aker Memet , Slavin Shimon , Ciceri Fabio , Miniero Roberto , Bordignon Claudio , Roncarolo Maria Grazia 
Published: October 2019

Abstract
Severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA) deficiency is a fatal congenital disorder of the immune system associated with systemic toxicity due to accumulation of purine metabolites. We previously showed that retroviral-mediated ADA gene transfer into autologous hematopoietic stem/progenitor cells (HSC) allowed restoration of immune and metabolic functions. We have now enrolled eight ADA-SCID children (age: 7–67 months) in our phase I/II gene therapy trial in which HSC are combined with low intensity conditioning with busulfan (total dose 4 mg/Kg i.v.). Previous treatment included haploidentical bone marrow transplant (n=3) or long-term (>1 year) enzyme replacement therapy (PEG-ADA) (n=4) associated with insufficient immune reconstitution or severe autoimmunity. In the latter case, PEG-ADA was discontinued to favour the growth advantage for gene corrected cells. The patients received a median dose of 8.8x106/Kg bone marrow CD34+ cells (range 0.9–10.8), containing on average 26.2±9.6% transduced CFU-C. Five patients experienced ANC <0.5x109/L, which was extended beyond day +30 in two patients. With a median follow up of 3.1 years (range 0.4–5.9), no adverse events related to gene transfer have been observed. Long-term engraftment of transduced HSC was demonstrated by stable multilineage marking, persisting more than 5 years from gene therapy. The average proportion of transduced cells in the peripheral blood at one year post-gene therapy (n=6) was 5% for granulocytes, 95% for T cells, 56% for B cells and 62% for NK cells. Comparison of the insertion sites retrieved ex vivo from patients with those identified in pre-transplant transduced CD34+ cells showed no skewing in the profile of genome distributions or in the gene families hit by the vector, and no clonal expansion. In the six children with a follow-up >1 year after gene therapy, we observed a progressive increase in lymphocyte counts which was sustained over time (median at 1.5 years 1.6x109/L), polyclonal thymopoiesis and normalization of T-cell functions in vitro. Serum Ig levels improved and evidence of antigen-specific antibodies was obtained, leading to IVIG discontinuation in five patients. All the children are currently healthy and thriving, and none of them showed severe infections. Sustained ADA activity in lymphocytes and RBC resulted in a dramatic reduction of RBC purine toxic metabolites (dAXP<30 nmoles/ml in 5 patients) and amelioration of children’s growth and development. In summary, these data confirm that gene therapy is safe and efficacious in correcting both the immune and metabolic defect in ADA-SCID, with proven clinical benefit.
Prevalence and Prognostic Significance of FLT3 Mutations in Acute Myeloid Leukemia: Association of ITDs with Poor Outcome in Patients with Normal Cytogenetics.
Authors: Palmisano Michela , Ottaviani Emanuela , Grafone Tiziana , Testoni Nicoletta , Paolini Stefania , Rondoni Michela , Baccarani Michele , Martinelli Giovanni 
Published: October 2019

Abstract
Acute Myeloid Leukemia (AML) is a difficult disease to treat, and better treatments are needed. Molecular targeted therapy represents a novel therapeutic approach.
Activating mutations of FMS-like tyrosine kinase 3 (FLT3) are present in approximately one third of patients with de novo AML and have been implicated in its pathogenesis. The leukemic blasts of most AML patients have the internal tandem duplications (ITDs) in the juxtamembrane region or point mutations in Asp835 and Iso836 codons in the activation loop of the kinase domain (TKD) of the FLT3 receptor. Both mutations result in constitutive FLT3 receptor activity and may play a significant role in leukemogenesis.
In this study we have analyzed the incidence and type of FLT3 mutations in a large series of newly diagnosed AML patients. Furthermore, we have evaluated the prognostic impact of FLT3 mutations.
The FLT3/ITD was determined by polymerase chain reaction (PCR). The mutations of D835 and I836 codons were determined by PCR followed by restriction enzyme digestion (PCR-RFLP). For the estimation of the statistic significance of the differences in the clinical-biological characteristics, between the mutated patients and wild-type patients, it has been used the Student’s test t for independent data. The probabilities of overall survival (OS) and disease free survival (DFS) were analysed by Kaplan-Meier method; the differences of OS and DFS, between the mutated patients and wild-type patients, were assessed using the log-rank test.
Both FLT3/ITD and FLT3/TKD mutations were found in 15%. Dual mutations were found in 2% of 126 patients. Among the FAB subtypes of AML, the rate of FLT3 aberrations was higher in M4 (27%) and M5 (26%). FLT3/ITD was associated to leukocytosis (106.8x10(e)9/l vs 30x10(e)9/l in FLT3-wt, p=0.015) and high percentage of circulating blast cells (82% vs 42% in FLT3-wt, p<0.0001). Differently, FLT3/TKD mutations were not associated with high white blood cells count and blast cells percentage.
FLT3 mutations were more prevalent in patients with normal karyotype (51%). In this group, DFS and OS were significantly inferior for patients with FLT3/ITD than patients withouth mutations (0 vs 5, p=0.0032; 5 vs 9, p=0.049, respectively). We have identified the FLT3/ITD as an independent poor prognostic factor in AML patients with normal cytogenetics. Therefore, targeting FLT3 mutations represents a potential therapeutic target for AML. These results suggest that new treatment modalities, such as therapy with a FLT3 tyrosine kinase inhibitor, are clearly needed for this group of patients with “standard risk” profile.
Involvement of the NOTCH1 and NOTCH2 Genes in B-Cell Lymphomagenesis.
Authors: Wlodarska Iwona , De Keersmaecker Kim , Cools Jan , De Wolf-Peeters Chris , Bosly Andre , Delos Monique , Michaux Lucienne , Vandenberghe Peter , Hagemeijer Anne , Marynen Peter 
Published: October 2019

Abstract
The NOTCH pathway is a key regulator of developmental choices, differentiation and function throughout the hematopoietic system. An aberrant activation of this pathway may underlie leukemogenesis, as shown in T-ALL cases with the rare t(7;9)(q35;q34) targeting NOTCH1 or with mutation(s) of NOTCH1 found in approximately 50% of T-ALL cases. We report here evidence for NOTCH1 and NOTCH2 rearrangements in B-cell lymphoma. Involvement of NOTCH1 was identified by the presence of dic(9;14)(q34;q32) and trisomy 12 in a case of chronic lymphocytic leukemia (CLL) at the time of transformation, and by st(9;22)(q34;q11) accompanied by t(14;18)(q32;q21)/IGH-BCL2 and t(8;14)(q24;q32)/IGH-CMYC in a follicular lymphoma (FL). FISH analysis of dic(9;14) and t(9;22) demonstrated the respective involvement of the IGH and IGL loci, and mapped both 9q34 breakpoints to the 5′ end of NOTCH1. Western blot analysis with cleaved Notch1 (Val1744) antibody was performed in both cases and hyperactive NOTCH1 signaling was found in the first case only. This particular case displayed in addition a mutation in the PEST domain of NOTCH1. No mutations, neither in the PEST, nor in HD domain of NOTCH1 (2 domains frequently mutated in T-ALL) were found in the FL case. FISH analysis of 30 additional lymphoma cases documented by structural aberrations of 9q33q34 revealed a normal status of NOTCH1.
NOTCH2 rearrangement was identified by FISH in 1 out of the 3 leukemia/lymphoma cases we could collect with t(1;14)(p13;q32). This particular CLL case showed a 3-way translocation, t(1;14;18)(p13;q32;q21), targeting also BCL2. So far, this patient did not show any clinical features of CLL transformation.
We hypothesize that IG-translocations affecting NOTCH genes in B-cell malignancies contribute to the lymphomagenesis by deregulating the transcription of these genes resulting in an aberrant ligand-independent NOTCH signaling. Mutation in the PEST domain of NOTCH1 as found in one case with dic(9;14) may further contribute to the process by stabilization of the aberrant product. Further molecular and immunohistochemical investigations of these cases are in progress.
Inhibition of Polo-Like Kinase 3 by CIB1 Facilitates Outside-In Signaling through αIIbβ3 in Platelets.
Authors: Naik Ulhas P. , Naik Meghna U. 
Published: October 2019

Abstract
Integrin αIIbβ3, the platelet fibrinogen receptor, is involved in bi-directional signaling during platelet activation. It has been shown that the β3 subunit of this receptor is phosphorylated on both threonine and tyrosine. Although, the role of tyrosine phosphorylation is well studied, the role of threonine phosphorylation is not well understood. Here we identify the kinase responsible for threonine phosphoruylation of β3 and provide a mechanistic significance of this phosphorylation in outside-in signaling. We found that calcium- and integrin-binding protein 1 (CIB1), which specifically interacts with the cytoplasmic domain of αIIb, is not involved in inside-out signaling, but regulates outside-in signaling through αIIbβ3. CIB1 is known to interact with and regulate several serine/threonine protein kinases. Among these, p21 activated kinase 1 (PAK-1) is highly expressed in platelets. We found that CIB1 does not colocalize with PAK-1 during outside in signaling induced by adhesion of platelets to immobilized fibrinogen. When searched for the expression of other kinases that are known to interact with CIB1, we found that polo-like kinase 3 (Plk3) is expressed in platelets and is responsible for the constitutive phosphorylation of β3. Plk3 is constitutively active in resting platelets but is rendered inactive during outside in signaling as a result of CIB1 binding in a calcium dependent manner. Plk3 becomes colocalized with CIB1 at the filopodia when platelets change shape in response to an agonist and remain associated with both CIB1 and αIIb as platelets spread on immobilized fibrinogen, as indicated by immunoprecipitation experiments. These results suggest that in resting platelets, constitutively active Plk3 maintains β3 in a threonine phosphorylated state which is nonpermissive for activation. Upon platelet activation by agonist CIB1 associates with Plk3 and renders it inactive in a calcium-dependent manner. This allows serine/threonine phosphatases, such as PP1-c to dephosphorylate β3 on threonine and facilitate outside-in signaling. Thus we have identified the kinase responsible for threonine phosphorylation of β3 and have provided a possible mechanism of the regulation of outside-in signaling.
Congestive Heart Failure Is a Rare Event in Patients (pts) Receiving Imatinib Therapy.
Authors: Atallah Ehab , Durand Jean-Bernard , Kantarjian Hagop , Jabbour Elias , O’Brien Susan , Rios Mary , Cortes Jorge 
Published: October 2019

Abstract
Background: A recent report suggested that imatinib is cardiotoxic and can lead to severe left ventricular dysfunction and heart failure.
Study Aim: To evaluate the incidence of congestive heart failure (CHF) in pts receiving imatinib.
Methods: We reviewed all Grade III and IV cardiac or what could be considered cardiac-related adverse events (eg, shortness of breath, dyspnea on exertion, edema, etc) occurring in pts on clinical trials at MDACC involving imatinib.
Results: 1276 pts were enrolled on clinical trials with imatinib from 7/28/1998 to 7/27/2006. After reviewing all reported adverse events, particularly those that could be considered of at least possible cardiac origin, 22 pts (1.8%) were identified as having symptoms that could be attributed to CHF. The median age for these 22 pts was 70 yrs (range, 49 to 83 yrs); 12 were male. Their diagnosis at the time of imatinib was started was chronic myeloid leukemia in chronic phase (11 of 561 pts, 1.96%), accelerated phase (4 of 384 pts, 1.04%), or blast phase (2 of 123 pt, 1.62%), myeloproliferative disorder (4 of 124 pts, 3.2%), acute lymphoblastic leukemia (1 of 74 pts, 1.35%) and none in 10 pts with c-kit positive AML. Twelve pts (55%) had received prior interferon therapy and 3 pts had received anthracyclins. The median time from start of imatinib therapy to a cardiac adverse event was 162 days (2–2045 days). Imatinib dose at the time of AE was 300mg/d in 1 pt, 400mg/d in 8 pts, 600mg/d in 9 pts, and 800mg/d in 5 pts. At the time these events were reported, 8 were considered possibly or probably related to imatinib. Eighteen pts had previous medical conditions predisposing to cardiac disease: CHF (6 pts, 27%), DM (6 pts, 27%), hypertension (10 pts, 45%), coronary artery disease (CAD) (8 pts, 36%), arrhythmia (3 pts, 14%) and cardiomyopathy (1 pt 5%). Fifteen pts had an echocardiogram or MUGA scan at the time of the event, and 9 of them had documented low ejection fractions (<50%); in only 2 of these 9 pts was there an echocardiogram prior to imatinib (LVEF >50% in both). Of the 9 pts with low ejection fractions at the time of the event , 6 had prior cardiac conditions (3 CAD, 2 CHF and 1 cardiomyopathy), one patient’s low EF etiology could not be determined as no further cardiac evaluation was performed because of progressive disease, and one pt was on anagrelide prior to the event. Eleven of the 22 pts continued imatinib therapy with dose adjustments and management for the CHF symptoms with no further complications.
Conclusions: Imatinib therapy as a causal factor of CHF is uncommon and is mainly seen in elderly patients with pre-existing cardiac conditions. Patients with a previous cardiac history should be monitored closely and treated aggressively with diuretics if they develop fluid retention.
In Vitro and In Vivo Activity of SKI-606, a Novel Abl/Src Inhibitor, Against Imatinib Resistant BCR-ABL+ Neoplastic Cells.
Authors: Puttini Miriam , Coluccia Addolorata M.L. , Boschelli Frank , Franceschino Anna , Tornaghi Lucia , Ahmed Shaheen , Redaelli Sara , Piazza Rocco , Magistroni Vera , Andreoni Federica , Scapozza Leonardo , Formelli Franca , Gambacorti Carlo 
Published: October 2019

Abstract
Resistance to imatinib represents an important scientific and clinical issue in CML and Ph+ ALL and is usually due to the selection of cells harbouring kinase point mutations of the BCR-ABL gene or presenting BCR-ABL gene amplification. In order to improve Bcr-Abl inhibition, several inhibitors with higher potency compared to imatinib have been identified including dasatinib and nilotinib, which show clinical activity in patients resistant to imatinib. In the present study the effects of the novel inhibitor SKI-606 on various models of resistance to imatinib were studied. SKI-606 is a selective dual Src/Abl inhibitor potently active against several CML cell lines and transfectants with IC50 values in the low nanomolar range, 2 logs lower than those obtained with imatinib. Cells expressing activated forms of c-KIT (HMC1560 and GIST882) or PDGFR (Ba/F3 Tel-PDGFRβ) were unaffected by SKI-606, in contrast to imatinib and dasatinib. SKI-606 reduced proliferation rate of cells where resistance to imatinib was caused by BCR-ABL gene amplification, such as Lama84R and of imatinib-resistant K562R and KCL22R in which no known mechanism have been identified. D276G, Y253F and E255K kinase point mutants were affected by SKI-606, resulting in inhibition of cell proliferation with IC50 values of 25, 40 and 394 nM (imatinib:1147, 1888 and 3174 nM). No activity was observed against T315I mutant. These results were confirmed in in vivo experiments, in models where resistance was not caused by mutations, as well as in cells carrying the Y253F, E255K and D276G mutations. In the first one SKI-606 was able to eradicate tumors, while significant delay in tumor growth was observed using Ba/F3 transfectants. Modelling considerations attribute the superior activity of SKI-606 to its ability to bind the intermediate (1M52) conformation of Bcr-Abl, in contrast to imatinib.
Excluding the T315 residue involved in direct hydrophilic interactions with both SKI-606 and imatinib, Y253 and E255 make electrostatic interactions stabilizing the inactive conformation of Abl nucleotide binding loop, thus affecting in a more pronounced way the binding of imatinib. Similar considerations have been made for the D276 residue affecting a-Helix C inactive conformation. The extended profile of activity performed here allows us to conclude that SKI-606 is a fairly active and specific inhibitor, with a very limited number of targets outside the Abl and Src families of TK.
Orally Active Inhibitors of the Imatinib Resistant Bcr-Abl Mutant T315I.
Authors: Shakespeare William C. , Wang Frank , Xu Qihong , Zhu Xiaotian , Narasimham Narayana , Metcalf Chester A. , Wang Yihan , Sundaramoorthi Raji , Huang Wei-Sheng , Zou Dong , Thomas Mathew , Romero Jan , Wen David , Chen Ingrid , Cai Lisi , Liu Shuangying , Wardwell Scott , Ning Yaoyu , Keats Jeff , Snodgrass Joseph , Russian Karin , Broudy Marc , O’Hare Thomas , Smith Caitlyn , Corbin Amie , Druker Brian , Iuliucci John , Dalgarno David , Sawyer Tomi K. , Clackson Tim 
Published: October 2019

Abstract
Resistance to the Bcr-Abl kinase inhibitor imatinib in patients with chronic myeloid leukemia (CML) is associated with emergence of Bcr-Abl point mutations that preclude effective drug binding. Although most mutants are effectively inhibited with the second generation inhibitors dasatinib and nilotinib, neither compound inhibits the T315I mutant which represents ~ 25% of all clinically observed mutants. Through our program of structure-guided design, we have identified a series of compounds that inhibit the T315I mutant of Bcr-Abl both in vitro and in vivo. AP24534, a representative member of this new series, inhibited the kinase activity of both the wild-type enzyme and the T315I point mutant with IC50s of 3 and 31 nM respectively, and inhibited the proliferation of their respective BaF3-derived cell lines with IC50s of 2 and 14 nM. Additionally, AP24534 inhibited the proliferation of BaF3 cells expressing the clinically relevant mutants Y253F, E255K, H396P, or M351T with IC50s of 2, 7, 1, and 1 nM respectively. Inhibition of cellular proliferation directly correlated with decreased cellular phosphorylation of Bcr-Abl. Daily oral administration of AP24534 to mice bearing subcutaneous xenografts of Bcr-Abl-T315I-expressing BaF3 cells elicited dose-dependent tumor shrinkage, with complete tumor regression observed at the highest doses. In a separate model, daily oral administration of AP24534 significantly prolonged the survival of mice injected intravenously with these cells, at doses ranging from 5–30 mg/kg. These data indicate that this class of inhibitors has the potential to address CML refractory to current targeted agents.
Epigenetic Dysregulation of Candidate Cis-Regulatory Sequences in Hematological Malignancies.
Authors: Thompson Reid F. , Figueroa Maria E. , Melnick Ari M. , Greally John M. 
Published: October 2019

Abstract
Epigenetic changes (in particular, altered cytosine methylation) have been described in a variety of tumors. The CpG Island Methylator Phenotype (CIMP) is a well-known instance of this phenomenon wherein cytosine methylation is markedly dysregulated (normally hypomethylated loci shift to a methylated state). CIMP has been demonstrated in a number of different cancer types including hematological malignancies like AML. While methylation status has been studied predominantly at CpG islands, we used a novel assay (HELP; Khulan et al., Genome Res. 2006) to look for changes in cytosine methylation in large contiguous regions of the genome. We assessed global patterns of cytosine methylation by HELP analysis in a variety of tumor samples including leukemias and lymphomas. We found significant changes in the global methylation patterns of malignant cells, confirming prior observations of epigenetic dysregulation in these tumor types. We also discovered that the majority of the changes in cytosine methylation are occurring not at CpG islands but at other loci in the genome, including constitutively hypomethylated loci that we are finding to be candidate cis-regulatory sequences. We conclude that cytosine methylation changes in cancer occur much more extensively than analysis of CpG islands alone would indicate, and that the epigenetic dysregulation in cancer may be predominantly targeted to cis-regulatory sequences rather than to promoters.