bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phase II Study of Dasatinib in Patients with Relapsed CLL
Authors: Garg Ravin Jain , Wierda William , Fayad Luis , Estrov Zeev , Bickel Susan M , O’Brien Susan 
Published: April 2020

Abstract
Preclinical studies have demonstrated that the Src-related tyrosine kinase Lyn is upregulated and constitutively activated in CLL cells. Lyn inhibitors have been shown to reduce survival of CLL cells in vitro and the Src inhibitor PPW results in the death of murine B cell leukemia. This has spurred interest in the role of Src inhibition as a treatment option for patients with CLL. Dasatinib is a dual Src- and Abl-kinase inhibitor that was recently approved by the FDA for the treatment of patients with CML failing imatinib. It has been shown to induce apoptosis in CLL cells in vitro.
The aim of this study was to investigate the activity of dasatinib in patients with relapsed CLL.
Patients with relapsed CLL, SLL, T-PLL and normal organ function were eligible. The dose of dasatinib was 50 mg PO bid continuously with possible escalation up to 70 mg bid. Each cycle was 4 weeks. Thirteen patients have been treated. The median age was 67 years (range, 42 to 83). The median number of prior treatments was 4 (range, 1 to 8). Five patients (38%) had Rai stage 0–1 disease, the other 8 (62%) had stage II–IV disease. Two patients were inevaluable for response; one changed his mind after only 2 days of treatment and the other patient was hospitalized with Aspergillus 2 weeks into treatment. Of the remaining 11 patients, 6 were treated for 3 cycles, 1 for 6 cycles, 1 is still on treatment, and 3 had therapy stopped after 2, 15, and 34 days for lip numbness, persistent headache, and thrombocytopenia/fatigue, respectively. Five patients had dose escalation after cycle 1, and 1 patient had escalation after cycle 3. The Grade 1 and 2 toxicities included 38% headache, 38% flushing, 46% fatigue, 46% anorexia/nausea, and 23% diarrhea. One patient (8%) had both Grade 3–4 headache and fatigue. One patient (8%) had Grade 3–4 diarrhea and another (8%) had a Grade 3 pleural effusion. Hematologic toxicities included neutropenia in 76%, (grade 3-4 63%), thrombocytopenia in 44% (grade 3–4 22%), and 80% with Grade 1–2 anemia. Eleven patients were evaluable for response. No patient achieved CR or PR based on NCI-WG Criteria. Several patients showed evidence of biologic activity of dasatinib including 1 PR in nodes but worsening anemia, 1 stable disease for 3 cycles, and 2 patients with significant reductions in ALC; one patient had a drop from 76,000/ml to 32,000/ml after only 2 days of dasatinib but stopped therapy because of headache, one had a reduction in WBC from 40,000 to 6,000. One patient continues on therapy. In this trial dasatinib produced no responses by NGI-WG criteria but showed evidence of biologic activity in a very heavily pretreated group of pts with CLL. Accrual continues.
Changes in Immunogenicity of Chronic Lymphocytic Leukemia Cells Mediated by Epigenetic Modifiers
Authors: Dubovsky Jason A , Powers John J. , Sotomayor Eduardo M. , Pinilla Javier 
Published: April 2020

Abstract
The success of immunotherapeutic treatments for B-cell chronic lymphocytic leukemia (B-CLL) hinges upon the circumvention of tolerogenic mechanisms which inhibit the formation of a robust anti-cancer immune response. Recent evidence suggests that DNA methylation inhibitors and histone deacetylase inhibitors can have lasting cancer-specific effects on the expression of highly immunogenic cancer-testis antigens (CTAs) as well as modulating costimulatory and major histocompatability molecule (MHC) surface expression. To investigate the potential efficacy of a combined therapy of methylation (5-aza-2′ytidine) and histone deacetylase (LAQ) inhibitors in human B-CLL we evaluated the expression of CTAs, costimulatory molecules (CD40, CD80, CD86), and MHC molecules (HLA-A, B, C, and HLA-DR) in both cell lines and primary cells from patients with CLL. In addition, we investigated the overall functional immunogenicity of treated B-CLL cells using mixed leukocyte reactions. Results showed that many highly immunogenic CLL-specific CTAs (SSX family, MAGE family, NY-ESO-1, GAGE-2, and NXF2) are significantly upregulated after treatment with both inhibitors. Our data also indicates that the combined effect of both inhibitors was capable of inducing a more immunogenic phenotype (increased CD86, CD80, CD40, HLA-A,B,C, and HLA-DR) in CLL cells consistently, while either drug alone was incapable of producing this effect. In addition treatment with both inhibitors was capable of increasing IFNγ release mediated by allogenic T-cells. Taken together these data indicate that a combined therapy with histone deacetylase and methylation inhibitors, possibly in combination with antigen-specific vaccine immunotherapy, may effectively induce cancer-specific antigen expression and increase costimulatory and antigen presenting capacity of the B-CLL cell.
The Stem Cell Factor-cKIT Pathway Must Be Inhibited to Enable Apoptosis Induced by Bcr-Abl Inhibitors in Chronic Myelogenous Leukaemia Cells
Authors: Belloc Francis , Airiau Kelly , Augis Vanessa , Jeanneteau Marie , Garcia Melissa , Guerin Estelle , Lippert Eric , Moreau-Gaudry François , Mahon Francois-Xavier 
Published: April 2020

Abstract
Imatinib is an effective first line therapy for early chronic phase chronic myelogenous leukaemia (CML) that acts by targeting the tyrosine kinase activity of BCR-ABL. However, mutations occurred that procure imatinib resistance and, to overcome this, second generation inhibitors of BCR-ABL have been developed. Among these, nilotinib is significantly more potent against BCR-ABL than imatinib but is used in the same order of concentration as imatinib in vivo. Moreover, nilotinib is more specific towards BCR-ABL than imatinib which, at efficient concentration, also inhibits c-KIT. In the current study an in vitro flow cytometry assay to analyze imatinib- and nilotinib-induced apoptosis in CML cells has been developed. Both drugs induced significant apoptosis in CD34 positive cells from 36 CML bone marrow samples collected at diagnosis while CD34 positive cells from 12 other myeloproliferative disorders samples were unaffected, confirming the high specificity of both inhibitors on BCR-ABL positive cells (p&lt;10−9). Nilotinib was confirmed to be 100 times more potent than imatinib to induce apoptosis of CD34 positive cells. When the experiments were performed in the presence of a cocktail of cytokines, inhibitions of 26% for spontaneous apoptosis, 22% for imatinib-induced apoptosis (n=20, p&lt;0.005) and 71% for nilotinib-induced apoptosis (p&lt;10−6) were observed. The inhibition essentially occurred in CD117+/CD34+ cells. This differential inhibition by cytokines was confirmed on K562 cells (34% for imatinib and 71% for nilotinib). A blocking anti-CD117 antibody alleviated the anti-apoptotic effect of cytokines against nilotinib. Moreover, using shRNA against BCR-ABL, we demonstrated that K562 cells were not dependant of BCR-ABL expression as long as the SCF receptor pathway was activated. We conclude that the c-KIT pathway may substitute for BCR-ABL tyrosine kinase to activate survival signals and that c-KIT must be inhibited beside BCR-ABL to allow apoptosis of CML cells.
Imatinib Mesylate Induces BCR-ABL Independent Inhibition of Proteasomal Activity in CML Cells
Authors: Held Stefanie A.E. , Brossart Peter 
Published: April 2020

Abstract
Imatinib mesylate (IM; Gleevec) is currently used in the treatment of CML and GIST. IM has been described to influence the function and differentiation of antigen presenting cells, to inhibit the effector function of T lymphocytes and to decrease the immunogenicity of CML cells by downregulation of tumor associated antigens. In the present study, we analyzed the possible effects of IM on antigen processing and presentation. To accomplish this we employed active site labelling of proteasomal subunits to measure proteasomal activities in cells treated with IM and other tyrosine kinase inhibitors (TKIs). Proteasomal activity was determined in cell lysates from established CML cell lines and primary cells from CML patients using a biotinylated active site-directed probe, which, covalently binds and labels proteasomal subunits beta-1, beta-2 and beta-5 depending on their activity. In addition, in order to analyze a possible direct effect of IM, isolated 20S and 26S proteasomes were examined. We found that treatment with IM led to a concentration-dependent decrease of proteasomal activity in BCR-ABL positive cells. In line with these results IM reduced the expression of HLA-class I molecules in HLA-A2 transfected K-562 cells. This inhibitory effect was independent of protein expression of proteasome subunits as analyzed by western blotting and it was not due to the induction of apoptosis as only samples with nuclei fragmentation below 40% were utilized. Furthermore, these effects were not inhibited by addition of zVAD, a pan-caspase inhibitor of apoptosis. The inhibition of proteasomal activity was independent of BCR-ABL as analyzed using IM resistant CML cell lines including BA/F-T315I cell line which is transfected with the multiresistent T315I mutation. Application of BCR-ABL specific siRNA to knock down its expression had no significant influence on proteasomal activity. Accordingly, incubation of isolated 20S and 26S proteasomes with IM resulted in a profound inhibition of their activity as determined by reduced proteolytic cleavage of fluorogenic substrates. Interestingly, this inhibition of the proteasomal activity was not IM specific as nilotinib, a TKI with a higher BCR-ABL affinity had similar effects. Furthermore, the multikinase inhibitor sorafenib but not sunitinib reduced the proteasomal activity in cell lysates and isolated proteasomes. Incubation of cells with the mTOR inhibitor rapamycin inhibited the activity of proteasomal subunits while the PI3 kinase inhibitor LY-294002 that acts upstream of rapamycin had no effect. Our results demonstrate that IM and several other TKIs currently applied in the treatment of patients can affect the immunogenicity of malignant cells by a direct inhibition of the proteasomal activity.
CCN3, a Novel Growth Inhibitory Factor for Chronic Myeloid Leukemia
Authors: Lu Wanhua , McCallum Lynn , Perbal Bernard , Lazar Nourreddine , Irvine Alexandra 
Published: April 2020

Abstract
Chronic Myeloid Leukaemia (CML) is characterized by expression of the constitutively active BCR-ABL tyrosine kinase. Previously, we identified down-regulation of the negative growth regulator, CCN3, as a result of BCR-ABL kinase activity. Reduced CCN3 expression is a prominent feature in both primary human CML cells and cell lines. We now show that CCN3 is growth inhibitory and enhances imatinib induced growth inhibition.
To evaluate the biological consequence of CCN3 expression in CML, K562 cells were stably transfected with a construct containing CCN3 (pCMV82-23) and growth characteristics and activation of signaling pathways were compared to cells transfected with empty vector (control). CCN3 expression was undetected by Real-time PCR in control cells whilst pCMV82-23 cells expressed 2.25 × 106 copies per 50ng of cDNA. pCMV82-23 cells showed reduced colony formation capacity (p=0.003) and reduced cell growth over a period of five days (p=0.005). Investigation of cellular signaling showed CCN3 expression resulted in significant down-regulation of three major signaling pathways and demonstrated reduced phosphorylation of ERK (p=0.002), pAKT (p=0.017) and pSTAT5 (p=0.005) compared to control cells. Protein levels for total ERK, AKT and STAT5 were unaffected by CCN3 expression. Flow cytometry showed that sustained CCN3 expression resulted in an accumulation of cells within the subG0 stage of the cell cycle (11.4% ± 3 (p=0.040)).
To determine if CCN3 expression could influence sensitivity to the BCR-ABL kinase inhibitor, imatinib, pCMV82-23 cells and control cells were treated with imatinib (5uM) for 48h. Control cells treated with imatinib showed moderate growth inihibition (19.6% ± 2.5) compared to untreated control. pCMV82-23 cells showed a significant increase in the magnitude of imatinib induced growth inhibition (63.3% ± 10.5 (p=0.043)). This was associated with an increased accumulation of cells in the subG0 area of the cell cycle, 34.6% ± 5 for pCMV82-23 cells compared to control cells (21.7% ± 8) in response to imatinib treatment (p=0.006). To then determine if these effects could be reproduced using recombinant CCN3 (rCCN3), K562 cells were treated with imatinib (5uM) alone or in combination with rCCN3 (10nM) for 48h. K562 cells treated with the combination of rCCN3 and imatinib showed enhanced growth inhibition (71.8% ± 7.9) compared to cells treated with imatinib alone (81.1% ± 9.2 (p=0.008)).
Loss of CCN3 is consistent with properties associated with the CML phenotype. Sustained expression of CCN3 in K562 cells restores growth control and re-establishes induction of apoptosis. Both increased expression of CCN3 or addition of the recombinant protein provide additional benefit for imatinib induced growth inhibition thus providing a novel avenue for therapeutic intervention.
Synergistically Cooperation of Bortezomib and Arsenic Trioxide on Chronic Myelogenous Leukemia
Authors: Yan Hua , Li Dao , Chen Li , Wu Ying-Li , Li Jun-Ming , Chen Guo-Qiang 
Published: April 2020

Abstract
Chronic Myelogenous Leukemia (CML) is one of myeloproliferative disorders characterized by the chromosome translocation (9; 22), which causes the generation of the Bcr-Abl fusion protein. Latter activates a number of signal transducers and transcription factors, thus leading to multi-resistance to chemotherapeutic agents. Introduction of imatinib mesylate (Gleevec) significantly increased CML patients’ response but still some of patients got disease progression. Therefore, it still remains a high priority to develop new approaches to treat Bcr-Abl+ leukemia. Aim: We aim to investigate the synergy of bortezomib and arsenic trioxide on inducing the apoptosis on Bcr-Abl+ K562-s as well as K562-r cells, which was sensitive or resistant to imatinib treatment respectively. Material and methods: Bcr-Abl+ K562-s and K562-r cells were chosen to evaluate the drugs’ effect. We observed the inhibition of cell growth and cell viability after bortezomib and/or arsenic treatment. Flow cytometric evaluation and western blot analysis were performed to detect the development of apoptosis, the changeable expression of Bcr-Abl protein and the apoptosis-related proteins. Results: K562-s and K562-r cells had different response to imatinib treatment. As expected, 0.25μM of imatinib can inhibit half of K562-s cell growth whereas 10–15 more times of concentration of imatinib were necessary to reach the same inhibitory effect in K562-r cells. Combined bortezomib at 12nM with 1μM of ATO, the concentration inducing 50% of K562-s growth arrest, synergistically induced apoptosis in K562-s cells after 48hrs of cotreatment, confirmed by notable elevation of Annexin V+ cells through flow cytometric analysis, when compared to those in the control or single treatment (p&lt;0.01), and immunoblotting detection of caspase-3 cleavage, degradation of poly-adenosine diphosphate-ribose polymerase (PARP), and decreased expression of Bcr-Abl protein. More surprisingly, 24nM concentration of bortezomib and 2μM of ATO, 2 times of concentration needed for 50% of K562-s cell growth inhibition respectively, inhibited 50% of K562-r cells growth, far less than the increased proportion needed during imatinib treatment. And more of important, although single treatment of bortezomib or ATO had no or little effect on inducing apoptosis, combined treatment significantly induced the apoptosis in K562-r cells (p&lt;0.05), associated with activation of apoptosis-relevant proteins and obvious degradation of Bcr-Abl protein.
Conclusion: Taken together, these findings indicate that bortezomib combined with ATO could be developed into a novel therapeutic strategy for CML. More study will be needed to further explore its potent mechanism involved during cotreatment.
Oligoclonal Vβ21 with Different Vα Partner in T Cells Associated with CML Cell Antigens
Authors: Li Yangqiu , Zha Xianfeng , Chen Shaohua , Yang Lijian , Lu Yuhong , Yu Zhi 
Published: April 2020

Abstract
Chronic myeloid leukemia (CML) is one of most common leukemia, although target therapy with imatinib is significant improved the survival rate, disease relapse and drug resistant with abl gene mutant are still the major clinical problem, improvement strategy to reverse the outcome of CML is the key point for the researchers. T-cell immunodeficiency was suggested in tumor patients for many years, understanding T cell immune status in leukemia patients might suggest treatment strategies to enhance immune competence in all. In order to investigate the clonal expansion and the feature of CDR3 sequence of T cells in patients with CML, the CDR3 of TCR Vα 29 and TCR Vβ 24 subfamily genes were amplified in peripheral blood mononuclear cells (PBMCs) from two case with CML using RT-PCR, the PCR products were further analyzed by genescan technique to evaluate clonality of the detectable TCR Vα and Vβ subfamilies. The oligoclonal PCR products were analyzed by sequencing to define the sequence of CDR3. Oligoclonal Vβ21 T cells could be identified in both CML cases, which might partner with different oligoclonal Vα subfamilies, including Vα7, Vα13 or Vα18. The CDR3 sequences from two Vβ21 and three Vα oligoclonal TCR genes were identified in Vβ21NDβNJβ1.1, Vβ21NDβNJβ2.7, Vα7NJα23, Vα13NJα49, Vα18NJα5 respectively. The length and motifs of CDR3 seem different in all five TCR genes, however, the motifs in both Vβ21 genes (GAV or LRV) share the valine as last residue of CDR3 motif, and all of three Vα gene (Vα7:G; Vα13:GDEAD or Vα18:GG ) share the Glycine as first residue in the CDR3 motif. All of five TCR sequences were already submitted to the Genbank (Accession No: EU395806, EU379940, FJ009444, EU589347 or EU770971 respectively). The results indicated that oligoclonal Vβ21 genes with different Vα partners which share part of CDR3 motifs might recognize the similar CML associated antigen epitope in CML patients. Farther investigation will be constructed the recombination vector containing the antigen specific TCRs for gene transfer to establish TCR modified-T cells, which was expected to employ for specific immunotherapy in CML.
Outcome of Patients with Chronic Myeloid Leukemia with T315I BCR-ABL Mutation
Authors: Pagnano Kati a BB , Silveira Rosana A , Nardinelli Luciana , Conchon Monika , De Souza Carmino , Llacer Pedro ED , Lorand-Metze Irene , Funke Vaneuza , Pasquini Ricardo , Mascarenhas Cintia C , Zanichelli Maria Aparecida , Clementino Nelma CD , Bendit Israel 
Published: April 2020

Abstract
The most frequent causes of imatinib resistance are BCR-ABL mutations, usually selected during treatment. Most of them destabilize the inactive conformation necessary for imatinib binding. The T315I mutation, located at the ATP binding pocket is resistant to imatinib and other available kinase inhibitors and is associated with a poor outcome. The aim of this study was to evaluate the frequency of T315I mutation in patients with CML resistant to imatinib and to describe their outcome. Mutation analysis was performed in two institutions, through direct automated sequencing. Total RNA was extracted from peripheral blood or bone marrow and amplification of the kinase domain of ABL from BCR/ABL was performed, using a semi-nested RT-PCR, to cover amino acids 244–486. PCR product was submitted to direct automated sequencing and compared with normal sequences of BCR-ABL gene (M14752, GenBank). A total of 173 resistant imatinib resistant patients were analyzed. BCR-ABL mutations were detected in 47 out of 173 (27%) patients. Among these patients, 14 (30%) presented T315I mutation. The mutation was found in ten patients during imatinib treatment, in one before start of imatinib, in three after dasatinib (2) and nilotinib (1) failure. Three patients have more than one mutation analysis, performed during different time-points of treatment: imatinib resistance, dasatinib resistance (3) and bone marrow transplantation relapse (1). Overall survival from diagnosis was 39,5% and 17,5% from mutation detection, in a median time of 69 and 13 months respectively. After imatinib resistance, eight patients were treated with dasatinib, one with nilotinib and had no hematological response; tree were treated with Hydrea and two were submitted to allogeneic bone marrow transplantation. Five patients died due to disease progression in median time of 19 months after mutation detection. One patient died after bone marrow transplantation, due to GVHD and infection. Eight patients are alive, three in CP, two in BC, two in hematological response and one in complete cytogenetic response after bone marrow transplantation. In conclusion, in this study we confirmed the high frequency of T315I mutation in imatinib resistant patients and their poor prognosis. Most of them started imatinib in a late chronic or advanced phase, with exception of two patients which started imatinib 800mg in early chronic phase, suggesting that there was inhibition of Ph positive sensitive cells and selection of resistant clones.
Myelodysplasia and Acute Myeloid Leukemia during Treatment of Chronic Myeloid Leukemia with Dasatinib
Authors: Conchon Monika , Bendit Israel , Ferreira Patricia , Dias Lucia , Kumeda Cristina , Chamone Dalton , Dorlhiac-Llacer Pedro 
Published: April 2020

Abstract
The incidence and significance of Philadelphia negative aberrant clones in patients treated with the second generation of tyrosine kinase inhibitors, including dasatinib, are infrequent. Here we report 2 patients with myelodysplastic syndrome associated with monosomy 7 in Philadelphia negative cells during consecutive imatininb and dasatinb therapy. No previous exposure to chemotherapeutic agents was reported in both patients. In case 1, retrospective assessment of the bone marrow sample collected prior to treatment with dasatinib did not show any abnormal clone, which became evident only after dasatinib was commenced. In case 2, monsomy 7 were found already in 15% Philadelphia negative cells during imatinib treatment and persisted during dasatinib therapy. In the 2 cases described, we assumed that the consecutive imatinib/dasatinib therapy, could abrogate the proliferation of the Philadelphia positive clone and allow pre-existing monosomy 7 clone to emerge.