The identification of an anti-thrombin molecule via the screening of semi-random DNA libraries

Thrombosis remains one of the leading causes of mortality and morbidity in the world. Thrombin is a key enzyme involved in the blood clotting processes, which can be intervened by low concentrations of Hirudin. The C-terminal dodecapeptide of Hirudin was capable of inhibiting thrombosis. This peptide has been partially randomized in this report, and the coding sequences have been expressed in yeast as chimerical peptides for secretion into the culture media. Two other semi-random modules have been processed likewise. The supernatant was subsequently tested for anti-thrombin activities. DNA sequencing indicated that the putative positive clone encoded a single serine residue followed by a stop codon. The Ninhydrin assay of the culture supernatant of the positive clone indicated a high content of amino acid. Electrospray Mass Spectrometry showed a distinct peak at 430.5 when the expression products from Pichia pastoris were examined, suggesting that the compound may be a dimannosylated serine, as yeast possesses glycosylation at serine residues. The observed effects of α-Mannosidase treatments on the function of yeast induction products are consistent with this assumption. Partial randomization of peptides and proteins may accelerate directed evolution, yielding unprecedented number of variants for functional interrogation and drug development.

Impact of Additional Chromosomal Abnormalities in Philadelphia-Positive Clone on Prognosis of CML Patients Treated with Tyrosine Kinase Inhibitors

Background: To investigate the impact of additional chromosomal abnormalities(ACA) in Philadelphia-positive clone on response to TKIs and long-term survival of CML patients treated with tyrosine kinase inhibitors (TKIs) in the real world. Methods:In this retrospective study, patients with chronic myelogenous leukemia (CML) in chronic phase treated with TKIs were recruited. The chromosome banding analysis was performed on bone marrow cells at baseline and during the TKIs therapy.The clinic response to TKIs was evaluated according to the recommendation of ELN2013. The overall survival (OS), events free survival (EFS) and progression free survival (PFS) were analyzed. Events were defined as treatment failure, progression to accelerate phase or blast crisis and death. The progression was defined according to criteria of ELN2013 CML recommendation. Results: The characters and cytogenetic analysis of the patients:A total of 451 patients (281 males and 170 females) were enrolled from Jun 2004 to March 2015.The median age was 42 (range 18-79) years old. 414 subjects hadevaluable information of karyotypeat baseline.351 patients (84.4%) had the standard Philadelphia chromosome (Ph), 8(1.9%) showed variant translocation of Ph, 19 had ACA in Ph+ cells (Ph+ACA), only 2(0.5%) showed ACA in Ph- cells (Ph-ACA)£¬11(2.7%) had normal karyotype. 29 patients (6.4%) developed Ph+ACA during the TKIs treatment. Ph-ACA occurred in 8 subjects (6.4%).The response to TKIs: Comparing to the patients without Ph+ACA at baseline, the patients with Ph+ACA had lower cumulative rate of complete cytogenetic response (CCyR) (92.4% vs 68.4%, P=0.038) and prolonger median time to achieving CCyR (19 vs 7 months, P=0.016).The Ph+ACA at diagnosis had no effect on achieving major molecular response (MMR). The OS, EFS and PFS at 7 years for the whole cohort were 91%¡¢54% and 83% respectively. The patients with baseline Ph+ACA had significant inferior survival comparing to the subjects without Ph+ACA (Figure 1). The median time of OS, EFS, PFS for Ph+ACA patients were 108, 50 and 91 months, whereas the patients without Ph+ACA were not reached ( P=0.003 for OS, P<0.001 for EFS, and P=0.004 for PFS). The patients developed Ph+ACA during treatment had similar OS comparing to the subjects with Ph-ACA or without ACA (P=0.115), but they had significant shorter EFS (31 vs 90 months vs not reached£¬P<0.001)and PFS(59 vs 120 months vs not reached£¬P<0.001). Conclusion: The Ph+ACA emergence at diagnosis and during treatment had negative impact on prognosis of CML patients treated with TKIs. Disclosures No relevant conflicts of interest to declare.

A Two-Fold Rise of BCR-ABL Transcript Levels Advises BCR-ABL Mutation Analysis in Imatinib-Treated Chronic Myeloid Leukemia (CML) - an Analysis of the Randomized CML-Study IV

Introduction: The clonal selection of a mutant BCR-ABL positive clone can be observed in about one of two patients with imatinib-resistant chronic myeloid leukemia (CML). The early detection of BCR-ABL kinase domain mutations is crucial, since it allows to change the tyrosine kinase inhibitor (TKI) regimen in a timely manner and may therefore prevent disease progression and the accumulation of further genetic lesions. European LeukemiaNet (ELN) recommendations suggest a mutation analysis if optimal response criteria are not achieved at 3, 6, 12 or 18 months, or whenever a loss of optimal response occurs (Soverini et al., Blood 2011). Several attempts have been made to derive this indication from a specific increase of BCR-ABL levels. Here we report on the correlation of a rise in BCR-ABL transcript levels and the prevalence of BCR-ABL kinase domain mutations in imatinib-treated patients of the CML-Study IV. Methods: A total of 1,173 patients were enrolled until 2009 and randomized to one of four imatinib-based treatment arms. BCR-ABLIS of 988 patients was determined in 7,876 samples by quantitative RT-PCR in the central laboratory (median sample number per patient: 8.4, range 1-37; median follow up: 34 months, range 0-86), representing the eligible patients for the study. Thereby, the estimated intra-laboratory variance is assumed to be about 20%. A first rise of BCR-ABLIS to at least two-fold and >0.1% between two samples of a patient's molecular course defined a sample suspected of bearing a mutant BCR-ABL positive clone. A mutation analysis was performed on this critical sample by direct sequencing of ABL exons 4 to 10. Results: A critical rise in BCR-ABLIS was observed in 231 of 988 patients (23%) after a median of 15.2 months on treatment (range 2.8-59.4). In the corresponding sample 33 mutant clones could be detected in 31 patients (13%). Thereby a steeper rise of BCR-ABLIS was correlated with a higher incidence of BCR-ABL mutations in the respective group (table). A total of 18 different mutations could be detected, the most frequent were: M244V, n=7 (21%); E255K, n=4 (12%); T315I, n=3 (9%); L248V, G250E, L387M and F486S, n=2 (6%), respectively. Mutations occur in a substantial proportion (8%) of patients with an only 2 to 3-fold rise of BCR-ABLIS transcript levels (table). Therefore, the most sensitive cut-off should be applied and mutation analysis may be triggered by a doubling of BCR-ABL transcripts at levels >0.1% IS. Conclusion: BCR-ABL kinase domain mutations occur already in a substantial proportion of patients with a doubling of BCR-ABL transcript levels, which should determine mutation analysis. Table 1. Rise of BCR-ABL expression Patients (n) Patients with BCR-ABL mutations (n) Patients with BCR-ABL mutations (%) Inter-sample interval(median, days) 2 to 3-fold 72 6 8.3 98 3 to 5-fold 50 3 6.0 100 5 to 10-fold 39 4 10.3 99 10 to 100-fold 49 10 20.4 98 > 100-fold 21 8 38.1 125 > 2-fold (total) 231 31 13.4 101 Disclosures Hanfstein: Novartis: Research Funding; Bristol-Myers Squibb: Honoraria. Hehlmann:Novartis: Research Funding; Bristol-Myers Squibb: Research Funding. Saussele:Novartis: Honoraria, Research Funding, Travel Other; Bristol-Myers Squibb: Honoraria, Research Funding, Travel, Travel Other; Pfizer: Honoraria, Travel, Travel Other. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Neubauer:MedUpdate: Honoraria, Speakers Bureau. Kneba:Novartis: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Pfirrmann:Novartis: Consultancy; Bristol-Myers Squibb: Honoraria. Hochhaus:Pfizer: Consultancy, Research Funding; ARIAD: Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Müller:Novartis: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.

Co-Occurrence of Separate BCR-ABL1-Positve and JAK2V617F-Positive Clones in 23 Patients Reveals Biologic and Clinical Importance

Background: The simultaneous detection of a BCR-ABL1 rearrangement and a JAK2V617F mutation in the same patient is a very rare event and has previously been described in case reports or very small series of cases only. Aim: 1) To establish the incidence of cases with concurrent BCR-ABL1 rearrangement and JAK2V617F mutation. 2) Evaluate whether one clone harbours both mutations or whether there are two independent clones. 3) Establish whether these patients have additional concurrent gene mutations and how they influence the evolution of both diseases. Patients and Methods: A total of 27,907 patients with suspected myeloproliferative neoplasms (MPN) where studied in parallel for BCR-ABL1 and JAK2V617F mutation from May 2005 to June 2014 at our institution. BCR-ABL1 analysis was performed by multiplex RT-PCR and JAK2V617F mutation analysis by melting curve based LightCycler assay. A total of 23 patients (0.08%) were positive for both mutations. Eleven patients were male and 12 were female with a median age at diagnosis of 72 years (range 46-80 years). Of fifteen patients 2 or more sample time points were available for follow-up analyses (median follow-up: 4 years, range: 5 months - 9 years). Both BCR-ABL1 and JAK2V617F mutation loads were assessed by quantitative real time PCR. In addition, 22/23 cases were analyzed upon detection of co-occurrence of both clones with a pan-myeloid gene panel consisting of 25 genes: TET2, RUNX1, PHF6, ASXL1, CBL, DNMT3A, SF3B1, TP53, BCOR, BRAF, ETV6, EZH2, FLT3 (TKD), GATA1, GATA2, IDH1, IDH2, KIT, KRAS, MPL, NPM1, NRAS, SRSF2, U2AF1, and WT1. Either complete coding genes or hotspots were first amplified by a microdroplet-based assay (RainDance, Lexington, MA) and subsequently sequenced with a MiSeq instrument (Illumina, San Diego, CA). RUNX1 was sequenced on the 454 Life Sequence NGS platform (Roche 454, Branford, CT). The median coverage per amplicon was 2,215 reads (range 100-24,716). The lower limit of detection was set at a cut-off of 1.5%. Results: At the time point of detection of both mutations morphological assessment was available in 12 patients. The remaining 5 showed features typical for CML. Bone marrow blast count was <5% in all cases. Cytogenetics was available in 18/23 cases (78.3%). The classical t(9;22)(q34;q11) was identifiable in 16/18 patients. Two patients had a normal karyotype as they were in complete cytogenetic remission of their CML (due to TKI treatment) at diagnosis of the JAK2 V617F positive clone. In the majority of patients (n=16) the JAK2V617F mutation predated the BCR-ABL1 clone, in 4 patients CML was known before the detection of the JAK2V617 positive clone, in 1 patient both were diagnosed simultaneously and in another 2 patients information in this regard was lacking. BCR-ABL1 transcript types distributed as follows: b2a2 and/or b3a2 (n=18), and e1a2 (n=5). The continuous quantitative assessment of BCR-ABL1 and JAK2V617F mutational loads in 15 patients showed asynchronous patterns of courses in all cases giving proof of these aberrations representing two different clones in these cases. When treatment with TKI was initiated, the BCR-ABL1 clone decreased while the JAK2V617F clone either remained stable or increased in all 15 cases. Next generation sequencing revealed further mutations in 13/22 analyzed patients (56.5%). One mutation was detected in 8 patients, 4 patients revealed 2, and one patient even 3 different additional mutations. In detail, mutations in the following genes were detected: TET2 (n=8), ASXL1 (n=4), RUNX1 (n=2), CBL (n=1), DNMT3A (n=1), PHF6 (n=1) SF3B1 (n=1) and TP53 (n=1). These mutations were traced and quantified retrospectively. Data suggests that they were most probably present in the JAK2V617F positive clone. This again supports the theory of both clones being independent of each other. Conclusions: 1) Co-occurrence of BCR-ABL1 and JAK2V617F is a very rare event (0.08%). 2) BCR-ABL1 and JAK2V617F represent two different clones. 3) Additional gene mutations are detected in 56% of these cases and all seem to be within the JAK2V617F positive clone. 4) Clinically, the BCR-ABL1 clone is easily controlled with TKI, however, the combined management of the JAK2V617F clone is more challenging especially when a fibrotic phase of the disease takes over. The long-term effect of JAK2-inhibitors in the management of these patients is yet to be established. Disclosures Martin-Cabrera: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

The Expansion Of CML Clones Initiates At The CMP Stage, and Is Associated With The Down-Regulation Of IRF8 and GFI1

In the chronic phase of chronic myeloid leukemia (CML-CP), leukemic stem cells do not necessarily depend on the BCR-ABL tyrosine kinase activity for their growth and survival and thus resistant to tyrosine kinase inhibitors (TKIs). In this study, we aimed to identify the initial progenitor population that is getting switched on BCR-ABL growth signaling and tried to elucidate the underlying molecular mechanisms of BCR-ABL dependent cell growth. We thus intensively analyzed the involvement status of CML clones in each developmental stage at diagnosis. To identify the hematopoietic stem or progenitor cell stage that is responsible for CML clone expansion, bone marrow cells from 13 newly-diagnosed CML-CP patients were analyzed by FACS, and purified stem/progenitor populations were tested for the t(9;22) involvement by FISH. Gene expression signature of each purified population was also evaluated by cDNA microarray. CD34+CD38- HSC fraction was markedly diminished in all CML-CP patients compared to healthy volunteers (<1% of CD34+ cells in patients vs. ∼10% in volunteers), whereas CD34+CD38+ myeloid progenitors expanded. Interestingly, the t(9;22) positive ratios in the HSC fraction were greatly diversified among patients (Figure 1). Of note, in 4 patients, the involvement of t(9;22) positive clone was less than 10%, suggesting that their leukemic stem cells have not outgrown normal HSCs. Among CD34+CD38+ myeloid progenitors, common myeloid progenitors (CMPs) robustly expanded and were composed more than 90% of t(9;22) positive clone in all cases. Downstream of CMPs, megakaryocyte/erythrocyte progenitors (MEPs) but not granulocyte/macrophage progenitors (GMPs) were dominantly involved in leukemia (t(9;22) positive ratio; 96.0+/-5.5% vs. 56.3+/-37%). The expression level of BCR-ABL is not different among these progenitor populations. These observations collectively suggest that in CMP-CP, BCR-ABL signaling becomes effective on cell proliferation especially at the CMP stage. Gene expression analysis of stem/progenitor populations in CML patients revealed that IRF8 and GFI1, transcription factors playing critical roles in myeloid differentiation and cell proliferation, were down-regulated specifically in CMPs as compared with that in normal controls (Figure 2). In order to substantiate the role of IRF8 and GFI1 in CML pathogenesis, we used a CML mouse model established by enforced retroviral expression of BCR-ABL. As in analysis of CML patients, BCR-ABL expressing CMPs but not stem/multipotent progenitor cells acquired growth advantage over normal counterparts. Importantly, the expression of IRF8 and GFI1 became undetectable after BCR-ABL transduction in the expanding CMPs. Our observations revealed that, in CML-CP hematopoiesis, BCR-ABL dependent cell proliferation initiates at the CMP stage, and is accompanied with the down–regulation of IRF8 and GFI1. Because IRF8 knockout mice develop myeloproliferative disorders, and because CMPs expand in GFI1 null mice, the attenuation of these molecules could be downstream effector of BCR-ABL dependent myeloid cell growth. Taken together, the reactivation of these molecules might be useful to develop alternative therapeutic strategies for CML-CP, for example, with TKI-resistant BCR-ABL mutants. Disclosures: Miyamoto: Kyushu University Hospital: Employment.