bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Association Between STAT5a Activation, LEF-1 Down Regulation and Leukemic Transformation in Patients with Severe Congenital Neutropenia
Authors: Gupta Kshama , Skokowa Julia , Welte Karl 
Published: April 2020

Abstract
Congenital hematological disorders are excellent models for investigating the regulation of hematopoiesis in humans. For instance, Severe Congenital Neutropenia (CN) is a heterogeneous syndrome characterized by a maturation arrest of granulopoiesis at the level of promyelocytes with no mature neutrophils in the peripheral blood. Even though G-CSF treatment results in increased neutrophil numbers in more than 90 % of CN patients, G-CSF-dependent granulocytic differentiation is severely affected in these patients. CN patients are found to be at increased risk of developing acute myeloid leukemia or myelodysplastic syndrome (AML/MDS) (cumulative incidence ~ 20 %). Since AML/MDS are not observed in cyclic or idiopathic neutropenia patients treated with G-CSF, an underlying defect down stream of G-CSF signaling rather than G-CSF therapy per se predisposes to malignant transformation in CN. STAT5 is activated by G-CSF signaling pathway and has also been found to be activated in AML. Recently we found that downregulation of LEF-1 transcription factor and its target gene C/EBPa are a common pathologic mechanism for CN. Therefore, we investigated the effects of G-CSF on the phosphorylation status of STAT5a in CN and whether it leads to the down modulation of LEF-1 expression and functions. Indeed, we detected elevated phosphoSTAT5 expression in CD34+ cells from CN patients before G-CSF stimulation in vitro, as compared to these cells from healthy individuals. Moreover, treatment with G-CSF resulted in a significantly higher phosphorylation of STAT5a in CN. Intriguingly, levels of phosphoSTAT5 in myeloid blast cells from one CN patient who developed AML was even higher and was in line with undetectable LEF-1 protein expression. Transduction of constitutive active STAT5a (STAT5a 1*6) in CD34+ cells from healthy individuals resulted in significant downregulation of LEF1 levels in a dose dependent manner. A screen of 10 kb upstream region of LEF1 gene revealed two putative STAT5 binding sites (−3891bp to −3909bp and −3714bp to −3732bp) and the specificity of this binding was confirmed in the nuclear extracts of CD34+ cells by chromatin immunoprecipitation assay. We found enhanced and prolonged STAT5a binding to the LEF-1 promoter in G-CSF treated CD34+ cells from CN patients, as compared to healthy individuals. Additionally, transfection of CD34+ cells with LEF-1 cDNA resulted in elevation of LEF-1 promoter activity, which suggests a strong LEF-1 autoregulation. Co-transfection with STAT5a 1*6 significantly disrupted LEF-1- dependent activation of LEF-1 promoter. Moreover STAT5a 1*6 severely abrogated the LEF-1 dependent regulation of C/EBPα gene promoter. Taken together phosphorylation of STAT5 is upregulated in hematopoietic progenitors from CN patients which lead to subsequent down regulation of LEF-1. These downstream effects of activated STAT5a may contribute to the malignant transformation of myelopoiesis in CN.
Interim Phase I Results of Recombinant Immunotoxin HA22 in Patients with Hairy Cell and Chronic Lymphocytic Leukemias
Authors: Kreitman Robert J. , Wilson Wyndham H. , Stetler-Stevenson Maryalice , Noel Pierre , FitzGerald David J , Pastan Ira 
Published: April 2020

Abstract
For the treatment of B cell malignancies, we have developed a high affinity recombinant immunotoxin (HA22), which is composed of the Fv portion of an anti-CD22 antibody fused to a 38 kDa fragment of Pseudomonas exotoxin A. HA22 is an improved form of immunotoxin BL22, which produced a 50–60% complete remission (CR) rate in cladribine-resistant hairy-cell leukemia (HCL). A partial response (PR) rate of 17% was observed with BL22 in chemoresistant chronic lymphocytic leukemia (CLL). The higher response rate of BL22 in HCL compared to CLL is related to its higher cytotoxicity for HCL than CLL cells ex vivo (median IC50 4.3 ng/ml in 33 HCL patients vs 430 ng/ml in 70 CLL patients, p &lt; 0.0001), and the higher CD22 expression on HCL than on CLL cells (median 44,000 vs 1250 sites/cell).To increase cytotoxic activity, the affinity of the Fv of BL22 was increased about 15-fold by phage display by mutating 3 amino acids in HCDR3 from SSY to THW. Patients with HCL and CLL were treated on 2 separate multicenter phase I protocols. To be eligible, patient required at least 2 prior standard therapies and a medical indication for treatment. HA22 was administered by 30 min infusion every other day for 3 doses (QOD ×3) and patients could be retreated at the same dose level every 4 weeks unless progressive disease or immunogenicity occurred. A standard 3–6 dose escalation scheme was used, allowing new patients to be enrolled at a higher dose level if 0 of 3 or 1 of 6 patients at the current level had dose limiting toxicity (DLT). A total of 16 HCL patients were treated with HA22 at 5, 10, 20, 30, and 40 ug/Kg QOD ×3. Three patients were treated at each of the first 4 dose levels, and a 4th patient received 40 ug/Kg QOD ×3 because one patient at that dose level received only 1 cycle. Of 14 evaluable patients who received a total of 45 cycles, 4 (29%) developed high levels of neutralizing antibodies. No DLT was observed on any cycle, although 1 patient developed a grade II hemolytic uremic syndrome (HUS) during cycle 3 of 30 ug/Kg QOD ×3. Major responses included 6 (38%) CRs and 6 (38%) PRs out of 16 evaluable HCL patients (unmonitored). Responses were observed at all dose levels including 3 of 3 PRs at the lowest dose level, and 1–2 CRs at each of the next dose levels. Like BL22, peak plasma levels of HA22 were inversely related to tumor burden as assessed by circulating malignant cells or spleen size, and increased with repeated doses as patients responded. Phase I testing of HA22 in CLL is at an earlier stage with 11 patients enrolled at dose levels of 5, 10, and 20 ug/Kg QOD ×3, and decreases in circulating CLL cells have been observed. In conclusion, HA22 has not yet shown DLT in phase I testing and is highly active in HCL. Compared to BL22, HA22 shows no evidence of increased toxicity due to enhanced binding affinity to CD22.
Dasatinib Has Activity in Relapsed/Refractory Chronic Lymphocytic Leukemia (CLL/SLL), a Phase II Trial
Authors: Amrein Philip C. , Attar Eyal , Takvorian Tak , Hochberg Ephraim , Ballen Karen K. , Leahy Kathleen M. , Neuberg Donna , Brown Jennifer R. 
Published: April 2020

Abstract
Background: Preclinical studies have shown that CLL cells overexpress lyn kinase protein, and in vitro inhibition of lyn kinase leads to apoptosis of the CLL cells (Contri, J Clin Invest 2005). Because dasatinib has been shown to inhibit lyn kinase in CML cells at concentrations easily achievable in patients, we undertook this phase II study in patients with previously treated CLL/SLL.
Methods: Patients were required to be over 18 years of age, have a diagnosis of CLL/SLL by flow cytometry or immunohistochemistry, and have failed either 1 course of treatment with a fludarabine-containing regimen or 2 non-fludarabine containing regimens. The starting dose of dasatinib was 140 mg daily by mouth. This dose could be reduced to 100 mg or 80 mg daily for toxicity. At baseline all patients had bone marrow biopsies and CT scans, and these were repeated at 2 months. Sequential blood and bone marrow samples were tested for lyn kinase activity.
Results: Among the 15 patients enrolled there were 10 male and 5 female subjects with a median age of 59 years (40–78 years). ECOG performance status was 0 in 9 subjects, 1 in 3, and 2 in 3 subjects. All patients had previously received fludarabine, and 5 patients required treatment within 6 months of their last regimen. The median number of prior treatments was 3 (range: 1 to 7). By cytogenetic/FISH analysis there were 5 patients with del(17p) and 6 patients with del(11q). All patients required treatment by NCI–WG criteria. The major toxicity encountered was myelosuppression: grade 3 + 4 neutropenia in 10 subjects, grade 3 + 4 thrombocytopenia in 4 subjects. Gastrointestinal toxicity was minor with only 1 subject experiencing grade 3 diarrhea. Other toxicities: 1 patient had a grade 2 pleural effusion, 1 patient had a transient serum K=9.9 (likely an artifact of high white count and without clinical sequelae), and 1 patient had a transiently prolonged QTc of 516 ms. There were no fatal events, and all toxicities were reversible. The median duration on study was 10 weeks, but 5 responding or stable patients have remained on treatment for over 9 months. Partial responses (PR) by NCI-WG criteria were achieved in 2 of the 15 patients (13% with 90% CI 2%–36%). An additional 2 patients would have qualified for PR (lasting &gt;2 months) except for myelosuppression. Among the remaining 11 patients, 6 had nodal responses (2 CR and 4 PR) by physical exam (PE) without a 50% reduction in lymphocytosis. CT scans confirmed nodal responses in 3 of the 10 patients with nodal responses by PE. The relationship between clinical response and lyn kinase, bcl-2, and mcl-1 expression will be presented at the meeting.
Conclusions: Dasatinib has modest activity in CLL, and combinations with standard agents, perhaps in an intermittent schedule, should be considered in subsequent trials.
Defensine Alpha1 as a New Marker of Imatinib Resistance in Chronic Myelogenous Leukemia
Authors: Etienne Gabriel , Dupouy Maryse , Chollet Claudine , Pasquet Jean-Max , Lippert Eric , Reiffers Josy , Mahon François- Xavier , Turcq Beatrice 
Published: April 2020

Abstract
Imatinib (IM) is the first therapeutic option for patients with newly diagnosed CML chronic phase (CML-CP). As alternative therapeutic options are available, identification of predictive factors of IM resistance may be of interest.
To identify specific early predictive biomarkers of IM resistance, we performed pangenomic microarray analysis in four patients with IM secondary resistance. All the four patients were in complete hematologic response at the time of analysis and RNA were extracted from peripheral mononuclear cells. Comparison of gene profile expression -was done between a sample corresponding to -BCR-ABL transcript decrease preceding the best response to IM and a sample corresponding at the time of IM resistance. BCR-ABL transcript levels were equivalent between the two points. Each patient was analyzed independently in order to limit genetic variability. We identified 22 genes differentially expressed at the time of resistance (4 up regulated, 18 down regulated). Among them, the defensin alpha 1 (DEFA1) and alpha 4 (DEFA4) genes were strongly over expressed at the time of IM resistance, 63-fold and 30-fold respectively.
Using Q-RT/PCR we next performed a prospective analysis of DEFA1 expression in 15 patients treated with standard dose (400mg/day) of IM. Nine of them were IM responders while 6 exhibited IM secondary resistance. IM responders were characterized by a strong and durable decrease in DEFA1 transcript level with a DEFA1-β actin ratio above 103. Patients resistant to IM were characterized by a lower DEFA1 expression before IM start and a dramatic increase of DEFA1 expression. Moreover the increasing of DEFA1 expression in IM secondary resistant patients occured while BCR-ABL transcript level continue to decrease. The median time between the increase of DEFA1 and the increase of BCR-ABL trancript level was 6 months (range from 0 to 23 months).
Finally we investigated DEFA1 expression in 39 CML-CP (19 early chronic phase, 20 late chronic phase)-. before IM started-. Twenty eight patients were IM responders and 11 were IM resistants (primary resistance in 2, secondary resistance in 9). DEFA1 expression before IM start was significantly lower in the IM resistant group (p=0.03) than IM responder group.
In conclusion our data suggest that variation of DEFA1 expression is early associated with IM resistance while low expression of DEFA1 at the beginning of IM treatment may be predictive of IM resistance independently of BCR-ABL level.
The Aurora Kinase Inhibitor MLN8237 Has Potent Anticancer Activity in CML and Ph+ ALL Models and Significantly Increases the Efficacy of Nilotinib
Authors: Nawrocki Steffan T. , Medina Ernest , Smith Sabrina , Swords Ronan , Kelly Kevin , Padmanabhan Swaminathan , Ecsedy Jeffrey , Giles Francis J , Carew Jennifer 
Published: April 2020

Abstract
The serine/threonine kinase Aurora A is a key regulator of mitosis. Its expression and activation are necessary for mitotic entry, centrosome maturation, and mitotic spindle assembly and function. Aurora A gene amplification and/or overexpression of its encoded protein has been observed in many types of human tumors and is hypothesized to promote genetic instability, a hallmark feature of malignant cells that contributes to disease progression and drug resistance. The central and obligatory role of Aurora A in mitosis and its dysregulation in cancer makes it an attractive target for therapeutic inhibition. MLN8237 is a novel orally available Aurora A inhibitor developed for cancer therapy by Millennium Pharmaceuticals that has entered Phase I trials in humans. We investigated the preclinical efficacy and mechanism of action of MLN8237 in models of chronic myeloid leukemia (CML) and Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL). Sub-micromolar concentrations of MLN8237 potently inhibited the in vitro growth and survival of human CML and Ph+ ALL cell lines and primary cells derived from CML and Ph+ ALL patients. In vitro kinase assays demonstrated that MLN8237 effectively inhibits the activity of wildtype and drug-resistant mutant forms of BCR-ABL. Notably, MLN8237 possessed equipotent anticancer activity against Ba/F3 cells expressing wildtype (p210) BCR-ABL and the imatinib, nilotinib, dasatinib, and bosutinib-resistant mutant T315I form of BCR-ABL. Loss of function of the p53 tumor suppressor contributes to resistance to imatinib and many other anticancer agents. We assessed the potential impact of loss of p53 function on cellular sensitivity to MLN8237 by achieving stable shRNA-mediated p53 knockdown in Ba/F3 cells expressing p210 BCR-ABL. Impairment of p53 function did not significantly affect the anticancer activity of MLN8237, indicating that it may be an effective agents for patients with p53 defects. Daily oral administration of MLN8237 (20 mg/kg BID) to immunodeficient mice bearing xenografts of the K562 human CML blast crisis cell line was very well tolerated and significantly reduced tumor growth in vivo. Moreover, co-administration of MLN8237 and nilotinib resulted in significantly greater tumor growth inhibition than what was achieved by either single agent alone. These data suggest that MLN8237 is a promising new agent for the treatment of CML and Ph+ ALL patients that fail to respond to conventional therapy.
Erythropoietin Stimulates Bone Formation In Vivo By Targeting Both HSCs and MSCs
Authors: Jung Younghun , Ziegler Anne , Shiozawa Yusuke , Wang Zhuo , Song Junhui , Wang Jianhua , Wang Jincheng , Pienta Michael J. , Lee Clara H. , Havens Aaron M. , Pedersen Elisabeth A. , Krebsbach Paul H. , Taichman Russell S. 
Published: April 2020

Abstract
Previously we showed that HSCs isolated from stressed animals (an acute bleed) regulates osteoblast (OBs) differentiation from bone marrow stromal cells (BMSCs). This activity suggests that HSCs regulate the development of their own niche. The molecular basis for this activity is the production of BMP-2 and BMP-6 by HSCs. Yet what stimulates HSCs to produce BMPs is unknown. We hypothesized that erythropoietin (Epo) may activate HSCs to produce BMPs, or Epo directly activates BMSCs to form OBs. To test these possibilities, Epo serum levels in the bled vs. non-bleed mice were determined and found to match the time frame of HSC activation. Surprisingly, EpoR expression was identified on HSCs isolated using the SLAM-family of receptors, and its expression on HSCs increased in bled animals. In vitro stimulation with Epo activated the JAK-STAT signaling pathways, and importantly, BMP production by HSCs.
To determine if Epo directly acts on BMSCs, BMSC co-cultures were established with HSCs derived from bled (B-HSC) or non-bled (NB-HSCs) animals, or treated with rhEpo alone for 14d. Differentiation along the OB lineage was evaluated for CFU-fibroblastic and osteoblastic (CFU-F, CFU-OB) formation, QRT-PCR for Runx2 (OB-specific transcription factor), bone sialoprotein and osteocalcin, or for bone mineral formation (alizarin red staining). BMSCs responded to the presence of NB-HSCs by increasing their generation of CFU-OB and CFU-F compared to no HSCs. Co-culture with B-HSCs or rhEpo treatments were significantly better able to induce CFU-OB and CFU-F differentiation of the BMSCs. QRT-PCR demonstrates that the OB-specific transcripts were increased with rhEpo or co-culture with B-HSC compared to NB-HSC or vehicle. Alizarine red staining also demonstrated an increase in mineral apposition in response to Epo or B-HSC.
To directly determine if Epo stimulates bone formation in vivo, newborn mice were treated with rhEpo (1,500U-6,000U/kg, 3x/week), anabolic PTH (as a positive control) or vehicle for 4 weeks. rhEpo increased the hematocrit and hemoglobin levels in a dose dependant manner. As expected, PTH increased the bone formation. rhEpo treatments enhanced OB numbers in long bone sections, and micro-CT measurements of the vertebra revealed a significant increase in bone formation parameters including bone mineral density (BMD), bone volume fraction (BVF) and trabecular number (Tb.N.).
These data for the first time demonstrate that Epo regulates the formation of the HSC niche by both direct and indirect pathways, and further demonstrates a coupling between hematopoesis and osteopoiesis in the marrow. These results also suggest that targeting the Epo/EpoR pathway may serve as a therapeutic modality to treat skeletal or mesenchymal abnormalities in humans.
Role of BCR in Down-Modulation of BCR-ABL Oncogenicity in CML
Authors: Perazzona Bastianella , Wang Yan , Arlinghaus Ralph B. 
Published: April 2020

Abstract
Bcr-Abl acquires its transforming ability through its up-regulated Abl tyrosine kinase activity. Bcr is a phosphoprotein with a novel serine/threonine kinase activity encoded by its first exon. Over-expression of BCR in K562 cells produces a phosphoserine form of Bcr and interferes with the oncogenic effects of BCR-ABL in mice (Lin et al., Oncogene 2001). We have recently shown the inhibitory effects of Bcr on Bcr-Abl, in a nude mouse solid tumor model. Expression of BCR/GFP in TonB210 cells used for injection delayed tumor formation and tumors were 50% smaller compared to the TonB210/GFP control. In contrast two point mutants in the BCR kinase domain (Y360F and S354A), not only blocked Bcr’s inhibitory effects but enhanced the oncogenic effects of BCRABL (Perazzona et.al. Oncogene, 2008). Similar Bcr effects were observed in a mouse leukemia model. We are investigating the mechanism of the interaction between Bcr and Bcr-Abl proteins. Using TonB210 cells, in which BCR-ABL expression is controlled by a tetracycline-inducible promoter and Bcr is stably transduced by lentivirus infection, we observed that increasing levels of Bcr-Abl expression increased the levels of the Bcr protein. Treatment of TonB210 cells with imatinib mesylate decreased the levels of Bcr- Abl and surprisingly the Bcr protein as well, indicating that the tyrosine kinase function of Bcr-Abl is required to up-regulate Bcr protein expression. In addition, withdrawal of doxycycline also reduced Bcr-Abl and Bcr protein levels, confirming that Bcr-Abl is required for increased expression of the Bcr protein. In order to examine the levels of Bcr in cells lacking Bcr-Abl, we transduced BCR/GFP with lentivirus infection into BaF3 and 32D cells. Surprisingly, these cell lines expressed extremely low levels of Bcr, despite 90% expression of the GFP marker. Expression of Bcr was restored by overnight treatment with the proteasome inhibitor calpain inhibitor I. Forced expression of Bcr-Abl in BCR- transduced cells restored high expression of Bcr protein, confirming that Bcr- Abl is required for preventing degradation of the Bcr protein. Together these findings indicate that Bcr-Abl up-regulated Bcr expression by interfering with proteasomemediated degradation of the Bcr protein. Additional studies indicated that Bcr increases expression of the myeloid membrane surface marker Mac-1 in Bcr-Abl TonB210 cells, which originated from the mouse pro-B cell BaF3. We propose that Bcr may play a role in generating the myeloid phenotype caused by Bcr-Abl in CML patients and may be an important player in the chronic phase of CML by down-modulating Bcr-Abl.
Management of Chronic Myelogenous Leukemia Using Therapeutic Drug Monitoring of Imatinib: The French Experience of a Centralized Laboratory
Authors: Molimard Mathieu , Bouchet Stephane , Etienne Gabriel , Legros Laurence , Rea Delphine , Huguet Francoise , Titier Karine , Moore Nicholas , Reiffers Josy , Belanger Coralie , Mahon Francois-Xavier 
Published: April 2020

Abstract
Pharmacokinetic monitoring is widely used in different medical specialities, but it has been rarely applied in clinical oncology practice. The current gold standard treatment of chronic myelogenous leukemia (CML) is imatinib, a tyrosine kinase inhibitor. We have previously shown the necessity to obtain a trough plasma threshold of 1000 ng/mL for efficient treatment with imatinib. We routinely perform centralized quantification for patients in France and this has allowed the assessment of imatinib therapeutic monitoring and its use in a real-life setting.
After 16 months of data collection, we had gathered 1607 samples for 1044 CML patients (mean age 55 years, F/M sex ratio 0.67) treated with imatinib 400 mg (median) range (100–800mg). We received only one sample for 739 patients and more than one sample for 305 patients.
The mean trough plasma concentration of imatinib (Cmin) was 1043 ng/mL (median: 876 ng/mL) and 596 of the 1044 CML patients (57%) had a Cmin &lt;1000ng/ml at first determination. Plasma concentration increased with dose, but there was a large inter-individual variability (64%) and intra-individual variability was twice as small. For plasma concentrations &lt; 1000 ng/mL, mean dose was 420 mg and for those ≥ 1000 ng/mL, this was 510 mg.
For the 189 patients having had at least 2 correct Cmin determination, 70% had initial Cmin&lt; 1000 ng/mL (mean concentration of 1st determination: 583 ng/mL). Among the 62 patients who initially had a Cmin below 1000 ng/mL that subsequently rose above this threshold, 63% had their imatinib dose increased; the rest did not have a dose modification. For the latter, it is probable in view of low intra-individual variability that this was due to enhanced compliance. For the 32 patients with a first Cmin &lt;1000 and no CCyR, none of those with Cmin remaining below 1000 ng/mL achieved CCyR, wheras 5 (28%) achieved CCyR when Cmin rose above 1000 ng/mL. In cases where there was suspicion of a drug–drug interaction, the most frequently combined drugs were proton pump inhibitors (such as omeprazole), diuretics, allopurinol and NSAIDs. The most recurrent adverse effects were digestive, hematological and muscular.
Although the studied population had characteristics generally described for this pathology (age, sex ratio), there was probably selection bias at the beginning of study: we received first and foremost the patients having an insufficient response, and therefore low plasma concentration. Therapeutic drug monitoring of imatinib appears to be helpful for the management of CML patients and the resulting database allows a better understanding and use of this treatment.