bluuebunny/crossref_metadata_2025 huggingface dataset:
Syk Is a New Target for AML Differentiation.
Authors: Hahn Cynthia K. , Ross Kenneth N. , Kakoza Rose M. , Carr Steven A. , Du Jinyan , Ong Shao-En , Golub Todd R. , Stegmaier Kimberly
Published: April 2020

One of the challenges of phenotype-based small molecule screening has been the difficulty of protein target identification for newly discovered compounds. For example, we previously performed a gene expression-based small molecule library screen, which identified gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, as an inducer of acute myeloid leukemia (AML) differentiation in cell lines and primary patient cells. Neither EGFR transcript nor protein is expressed in the tested AML cell lines, thus precluding inhibition of this kinase as the mechanism of AML differentiation. However, because multiple EGFR inhibitors induce this phenotype, we hypothesize that a shared off-target kinase is the target in AML differentiation. In order to identify candidate gefitinib targets of AML differentiation, we utilized a proteomics method: peptide immunoprecipitation-HPLC-mass spectrometry. This approach enriches for phospho-tyrosine peptides with immunoprecipitation (IP) after enzyme digestion. In contrast to IP at the protein level, the final mixture following peptide-IP contains primarily phospho-tyrosine-containing peptides. We treated the AML cell line HL-60 with gefitinib versus vehicle and then identified peptide sites with loss of phosphorylation with gefitinib treatment. Spleen tyrosine kinase (Syk) was identified as one of the only kinases with loss of phosphorylation post treatment. Syk is a nonreceptor tyrosine kinase, important in normal B-cell differentiation, and implicated in malignancies such as myelodysplastic syndrome and lymphoma. We first confirmed with IP western immunoblotting the inhibition of Syk phosphorylation with gefitinib treatment in HL-60 cells. We next confirmed with both pharmacological inhibition and genetic loss of Syk the induction of differentiation in the AML cell lines HL-60 and U937. Numerous determinants of myeloid maturation were tested: a complex differentiation gene expression signature, cellular morphology, cell surface proteins CD11b and CD14 expression, and nitro-blue tetrazolium (NBT) reduction, a functional assay for myeloid differentiation. Two reported pharmacological inhibitors of Syk scored positive on all measurements of differentiation. Furthermore, the shRNA construct inducing the most complete loss of Syk also scored the highest on all measurements of differentiation. The most convincing data that Syk is the true target of these inhibitors would be the identification of an inhibitor resistant Syk mutant, analogous to BCR-ABL or c-Kit mutants that confer imatinib resistance. To this end, a Syk random mutagenesis screen is ongoing. In summary, these data identify Syk as a strong candidate target of gefitinib, demonstrate that inhibition of Syk can induce AML differentiation, and identify Syk as a potential target for AML differentiation therapy.
Low Erythrocytic Glutathione Peroxidase-1 Activity Correlates with Hemolytic Rate in Patients with Sickle Cell Disease and Is Elevated on Hydroxyurea.
Authors: Kato Gregory J. , Cho Chun Seok , Yang Seung Ha , Bae Sung Won , Lee Jong Seo , Gladwin Mark T. , Rhee Sue Goo
Published: April 2020

Background: Glutathione peroxidase-1 (GPX1) is the most significant catalytic antioxidant in the red cell. Congenital deficiency of GPX1 has been reported in association with hemolytic anemia due to oxidant stress. Hemolytic rate has been implicated in the development of sickle vasculopathy.

Methods: Red cell pellets were collected from 32 patients with sickle cell disease and 17 healthy African American control subjects, and were stored frozen until assayed. A novel microplate immunoaffinity capture assay was used that measures both immunoreactive GPX1 protein and enzyme activity.

Results: Patients with SCD had higher levels of red cell GPX1 activity than controls (2.42 ± 0.12 vs. 1.86 ± 0.15 units/mg red cell protein (mean ± SEM), p=0.006). GPX1 activity correlated with low reticulocyte count (r=−0.50, p=0.004)(Fig. 1A), low indirect bilirubin (r=–0.45, p=0.01) and high fetal hemoglobin expression (r=0.39, p=0.03). GPX1 activity in SCD patients on hydroxyurea was much higher than those not on hydroxyurea (2.75 ± 0.12, n=21 vs. 1.79 ± 0.13, n=11, p<0.001)(Fig. 1B), suggesting that hydroxyurea might induce GPX1 expression. In the patients off hydroxyurea, low GPX1 activity closely correlated with high serum lactate dehydrogenase level, a marker of high hemolytic rate (r=–0.85, p=0.002), suggesting that low GPX1 levels might contribute to rapid hemolytic rate. In a multivariate model, GPX1 was independently associated with reticulocyte count even when adjusted for fetal hemoglobin level.

Conclusions: In addition to fetal hemoglobin, hydroxyurea may also induce expression of GPX1, a potent antioxidant that might have a role in decreasing hemolysis associated with the robust oxidant stress of SCD. This finding is consistent with previously published evidence of GPX1 induction by hydroxyurea in cancer cell lines, with increased antioxidant function. Additional studies are needed to confirm this phenomenon in a larger cohort of patients with SCD and to further evaluate the combined effect of fetal hemoglobin co-induction, but this finding suggests a potential mechanism by which hydroxyurea may produce clinical benefit in SCD even in the absence of significant fetal hemoglobin induction.

Figure Figure
Hypermethylation of DNA and Increased Expression of DNMT1 May Represent a Protective Mechanism from Oxidative Stress and DNA Damage in MDS.
Authors: Jankowska Anna M. , Gondek Lukasz P. , Szpurka Hadrian , Nearman Zachary P. , Tiu Ramon , Maciejewski Jaroslaw P.
Published: April 2020

Epigenetic changes including altered cytosine methylation have been hypothesized to play a pathogenic role in myelodysplastic syndrome (MDS) and other myeloid malignancies. While the specific functional consequences of CpG island methylation depend on the pattern of silenced promoters, the triggers for changes in the methylation program are unknown. DNA damage is likely to interfere with faithful heritability of cytosine methylation patterns during cell division. For example, formation of 8-oxoguanine (8-OG) in CpG islands may result in hypomethylation through inhibition of methylation-sensitive human maintenance methyltransferase DNMT1. Alternatively, hypermethylation may be a part of cellular response to oxidative damage e.g., through stimulation of DNMT1 activity. When we investigated expression of various DNA repair genes in highly purified CD34+ progenitor and stem cells of patients with MDS (19 RARS/RA, 16RAEB1/2/sAML, 2 CMML) using expression arrays, we found a significant and concordant up-regulation of N-methylpurine DNA glycosylase MPG; this finding was confirmed by TaqMan PCR in 21/37 MDS patients tested. Over-expression of MPG may be in response to increased DNA alkylation, but MPG has been described to co-localize in methylated CpG islands and could reflect their increased methylation. We have stipulated that up-regulation of MPG is a consequence of ongoing DNA damage and chose to study other parameters that could support this theory. For example, incorporation of 8-oxoguanine can be measured by flow cytometry and when we used this method, we determined that an increased oxidative DNA load/stress was found in 67% of all MDS cases studied (n=24) and correlated with advanced stage of MDS (p<.006), cytopenia (e.g., neutropenia p<.002) and a greater propensity for AML transformation (p<.02). We also studied expression of DNA glycosylase/AP lyase OGG1 with the capacity to excise oxidized guanine from DNA and found up-regulation of OGG1 mRNA levels in 11/37 patients. To investigate whether oxidative/alkylating DNA damage is indeed associated with alteration of methylation we have measured 2 parameters:

global methylation using an ELISA-based colorimetric assay specific for methylcytosine and expression of DNMT1 using TaqMan PCR.

We determined that patients with over-expression of OGG1 were characterized by lower expression of DNMT1 (>2x control levels) and inversely correlated with global methylation levels. In contrast, those patients with over-expression of DNMT1, a corresponding increase in global methylation was observed. In sum, our results are consistent with the inverse relationship between hypermethylation and oxidative stress or alternatively that hypermethylation may be a protective response to an ongoing DNA damage in a subset of MDS patients.
CliniMACS® Cell Enrichment Using NKp46 — A Large Scale, Single-Step NK Cell Isolation Method.
Authors: Bondzio Ilka , Schmitz Jurgen , Huppert Volker
Published: April 2020

Natural Killer (NK) cells show potent anti-leukemic activity in vitro and thus NK cells isolated using the CliniMACS® Cell Separation System are currently under clinical evaluation as part of a treatment for acute myeloid leukemia (AML). (Uharek L et al., 2003, Gentilini C et al., 2005; Koehl U et al., 2004, 2005; Passweg, JR et al., 2005, 2006; Miller J et al., 2005). Currently, the processing of highly purified, clinical-grade NK cells requires a two-step method using the CliniMACS Instrument: Depletion of CD3+ cells followed by enrichment of CD56+ cells (Iyengar R et al., 2003). This procedure shows excellent performance (reviewed in Passweg et al., 2005) with yields of 30–60%, purities of >85%, > 99.97%/3.5 log T cell depletion but is time consuming. Simpler one-step procedures have thus been tried to reduce the processing time (CD3 depletion, CD3/CD19 depletion, CD6 depletion, CD56 enrichment) but result in a more heterogeneous final cell product that usually contains less than 60% NK cells. NKp46 (CD335) has been demonstrated to be primarily expressed by NK cells and may thus be a suitable selection marker for one-step NK cell enrichment. We evaluated an NKp46 enrichment strategy using the autoMACS™ Pro Separator, NKp46-Biotin, and Anti-Biotin MicroBeads. We could enrich CD56+CD3- cells from peripheral blood mononuclear cells (PBMCs) to a purity of 90% with a yield of 40–60% and a >99.92%/3.1 log depletion of T cells (n = 2). These results encouraged the evaluation of this procedure on a clinical scale by using the CliniMACS Cell Separation System. Using the CliniMACS Plus Instrument, NK cells could be enriched to 73–87% purity (n = 3) from Buffy Coats derived from whole blood. T cells were efficiently depleted (>99.92%/3.0 log) to such a degree that NKp46-selected NK cell products may fulfil the release criteria for NK cell donor lymphocyte infusions (NK-DLI) in the context of haploidentical stem cell transplantation. While CD56+ CD3- NK cells are not activated by antibody binding during cell separation, magnetic labelling using anti-NKp46 antibodies may trigger NK cell activation. NKp46-mediated NK cell activation may be synergistically enhanced by the use of IL-2. Functional studies of NK cells isolated by different methods will be required to further evaluate their feasibility for use in NK-DLIs.
Phase II Trial of the mTOR Inhibitor RAD001 in Relapsed and/or Refractory Waldenstrom Macroglobulinemia: The Dana Farber Cancer Institute Experience.
Authors: Ghobrial Irene M. , Chuma Stacey , Sam Amy , Leduc Renee , Nelson Marybeth , O’Connor Kelly , Harris Brianna , Soumerai Jacob , Warren Diane , Jacobsen Eric , DeAngelo Daniel , Anderson Kenneth C. , Richardson Paul G. , Witzig Thomas E. , Treon Steven P.
Published: April 2020

INTRODUCTION: Previous studies have demonstrated the clinical activity of the mTOR inhibitor RAD001 in low- grade lymphomas. Our preclinical studies demonstrated activity of mTOR inhibitors in Waldenstrom Macroglobulinemia (WM) cell lines and patient samples. This phase II study aimed to determine safety and activity of the oral mTOR inhibitor RAD001 (Novartis Pharmaceutical, MA) in patients with relapsed or refractory WM.

METHODS: Patients who had at least one previous therapy for WM, and who had symptomatic relapsed or refractory disease were eligible. NCI CTCAE v3.0 was used for toxicity assessment. All patients received daily RAD001 at 10 mg. A cycle was considered 28 days. Patients were allowed to stay on therapy until progression of disease or excessive toxicity. This study was conducted in a collaborative effort between Dana Farber Cancer Institute (DFCI) and Mayo Clinic College of Medicine. Here, we report the data on the patients accrued at DFCI.

RESULTS: 19 pts (15 men and 4 women) have been treated to date. All patients had symptomatic disease and required therapy. The median number of lines of prior treatment was 3 (range 1 – 5) including included rituximab, nucleoside analogues (fludarabine or 2-CDA), combination chemotherapy (e.g. CHOP, CVP), chloramucil, and bortezomib. The median IgM at baseline was 3330 mg/dL (range 1010– 7410). The median follow on RAD001 was 8 months (range 3 – 22 months). Eighteen pts are currently evaluable for response. Best responses to RAD001 after 2 cycles using IgM monoclonal protein were as follows: partial remission in 8 (44%), minimal response in 5 (28%). Progressive disease occurred in 4 (22%) and stable disease occurred in 1 (6%). The overall response rate (PR+MR) was 72%. The median duration of response has not been reached (3–22+ months). Patients tolerated therapy well without significant toxicities. Grade 3 and 4 toxicities included grade 4 thrombocytopenia in 1 patient, grade 3 pneumonia in 1 patient, grade 3 hyperglycemia in 1 patient and grade 3 mucositis in 1 patient. Other adverse events of grade 2 or lower included nail cracking, mucositis, diarrhea, and fatigue. Attributable toxicities otherwise proved manageable with appropriate supportive care, and RAD001 was generally well tolerated. One patient enrolled on the study withdrew consent and changed to hospice care within 3 weeks of therapy, and passed away due to disease progression.

CONCLUSIONS: The use of the oral RAD001 single agent RAD001 in patients with relapsed or refractory WM was welltolerated and demonstrated significant activity achieving an overall response rate in 72% of patients. Future studies of combination of this agent with rituximab and bortezomib are currently being planned.
Activity of the Multi-Targeted Kinase Inhibitor, AT9283 on Imatinib-Resistant CML Models.
Authors: Tanaka Ruriko , Squires Matthew S , Kimura Shinya , Yokota Asumi , Mallett Kirsty , Smyth Tomoko , Thompson Neil T , Nagao Rina , Yamauchi Takahiro , Ushiki Takashi , Lyons John F , Maekawa Taira
Published: April 2020

CML is caused by a consistent genetic abnormality, termed the Philadelphia chromosome, that results from a reciprocal (9;22) translocation leading to the expression of the BCR-ABL fusion protein. Although treatment has been revolutionized by the introduction of tyrosine kinase inhibitors which target Abl activity, reactivation of Abl signaling via several different point mutations remains problematic. In particular the mutation of Threonine 315 to Isoleucine (T315I) confers resistance to all existing therapies with tyrosine kinase inhibitors in the clinical settings. We describe the in vitro and in vivo effects of AT9283, a potent inhibitor of several protein kinases, including Abl kinase (wild type BCR-ABL and several of the drug resistant mutant variants that have arisen in clinical practice e.g. T315I), JAK2, JAK3 and Aurora kinases A and B, on imatinib-resistant CML cells including those harboring BCR-ABL (T315I). AT9283 has potent anti-proliferative activity in a panel of BaF3 and human cell lines expressing the BCR-ABL or its mutant forms. In BaF3 BCR-ABL wild-type and T315I mutant cells and K562 CML cells we observed inhibition of substrates of both BCR-ABL (STAT5) and Aurora B (Histone H3) at concentrations >300nM and <100nM, respectively, suggesting that AT9283 is capable of inhibiting Aurora and BCR-ABL simultaneously in these cell lines. The in vivo effects of AT9283 were examined in several mouse models engrafted either subcutaneously or intravenously with BaF3, human CML cell lines or primary CML patient samples expressing the BCR-ABL or its mutant forms. Specifically AT9283 prolonged the survival of mice engrafted intravenously with either BaF3 BCR-ABL T315I, or E255K cells when administered intraperitoneally twice daily at doses of either 6.25 or 10mg/kg or once daily at 15mg/kg when administered 5 days in every week repeated twice. Maximal survival advantage was conferred at either 10mg/kg twice daily or 15mg/kg once a day. Similar data were obtained in an intravenous model using primary CML cells taken from a patient harbouring the BCR-ABL E255K mutation. We also present data from ongoing studies showing increased survival rates in these in vivo model systems following multiple cycles of AT9283 administered on the 15mg/kg once daily schedule. These data together support further clinical investigation of AT9283 in patients with treatment resistant CML.
Pre- vs. Post-National Coverage Determination (NCD) Blood Utilization and Hemoglobin Values among Medicare Patients Treated with Erythropoietic-Stimulating Agents (ESAs) for Chemotherapy-Induced Anemia (CIA)
Authors: Burton Tanya , Larholt Kay , Apgar Elizabeth , Pashos Chris , Bookhart Brahim , Corral Mitra , Piech Catherine Tak , Mckenzie R. Scott
Published: April 2020

Background: In July 2007, the Centers for Medicare and Medicaid Services issued ESA coverage limitations for cancer patients with CIA through a national coverage determination (NCD). Clinical outcomes based on ESA dosing described in the NCD have not been reported in prospective observational or clinical trial data. To understand hematologic outcomes in the Medicare population treated in pre- and post-NCD time period, an analysis of hematologic outcomes from the DOSE (Dosing and Outcomes Study of Erythropoiesis-Stimulating Therapies) registry, an ongoing prospective observational study, was conducted.

Methods: ESA-treated cancer patients with CIA were selected based on Medicare primary coverage, available baseline hemoglobin (Hb) value, and receipt of at least two ESA administrations. Data were categorized into two timeframes based on date of initial ESA administration: pre-NCD (4/06-4/07) and post-NCD (10/07-5/08). Baseline demographics, Hb values during ESA treatment, and blood utilization patterns were analyzed.

Results: 288 patients were identified (Pre-NCD – 230; Post-NCD – 58) from 41 sites. Patient groups were similar with respect to age, gender, weight, and tumor type. ESA treatment duration was significantly greater in the pre-NCD group (70 days vs. 54 days, p = 0.0011). As shown in the table, differences with regard to blood utilization and Hb values were observed between the Pre-NCD and Post-NCD populations. A significantly greater proportion of patients required a blood transfusion and the number of units administered per study patient was significantly higher in the Post-NCD group. Hb levels were significantly lower at all time points of observation in the Post-NCD group.

Pre-NCD Post-NCD p-value Transfusion Outcomes Proportion of Patients Transfused 18.3% 32.8% p = 0.0157 Mean No. of Units per Study Patient 0.5 1.1 p = 0.0089 Hematologic Outcomes: Mean Hb (SD) Baseline 10.6 (0.8) 9.6 (0.5) < 0.0001 Week 4 11.1 (1.3) 9.9 (1.1) < 0.0001 Week 8 11.2 (1.3) 10.4 (1.3) 0.013 Week 12 11.1 (1.3) 9.8 (1.2) 0.0002 Week 16 11.0 (1.1) 9.7 (0.2) 0.018

Conclusion: Greater blood utilization and lower Hb values were observed in Medicare CIA patients treated with ESAs during the Post-NCD time period compared to the Pre- NCD time period. The impact of the NCD on patient outcomes is important to providers and hospital systems and warrants further research.
Wnt/β-catenin and Pten/Akt Signaling Interaction Drives Hematopoietic Stem Cell Self-Renewal and Leukemogenesis.
Authors: Perry John M. , Grindley Justin C. , He Xi , Li Linheng
Published: April 2020

Self-renewal is the central property of stem cells but remains inadequately defined. Separately, the Pten/Akt and Wnt/β-catenin signaling pathways have been implicated in regulating hematopoietic stem cells (HSCs) and in leukemia development. Using an HSC-specific conditional mutant model, we studied the effects of Pten deletion combined with constitutively active β-catenin. HSC-specific deletion of Pten leads to a relatively moderate HSC expansion in spleen with increased myeloid differentiation. In contrast, HSC-specific activation of β-catenin results in functional failure by differentiation blockage. However, unlike single mutants, double mutant mice exhibit a novel phenotype including dramatic expansion in HSCs without extensive differentiation both in vitro and in vivo. Furthermore, β-catenin deficiency prevents the relatively moderate expansion of HSCs in Pten mutants. Our data reveal that these two pathways interact to coordinately drive HSC proliferation while inhibiting both differentiation and apoptosis. Together, the Pten/Akt and Wnt/β-catenin pathways cooperate to drive self-renewal, with uncontrolled self-renewal leading to leukemia development.
Specific Induction of Granzyme B in Human B Cells by Viral Antigens.
Authors: Hagn Magdalena , Maier Julia , Syrovets Tatjana , Simmet Thomas , Jahrsdoerfer Bernd
Published: April 2020

B cells are not currently known to produce granzyme B (GrB) in (patho-) physiological settings. We recently reported that B-chronic lymphocytic leukemia cells and normal B cells treated with interleukin-21 (IL-21) and anti-B cell receptor antibodies (anti-BCR) produce functional GrB. Here we demonstrate for the first time that viral antigens can also induce specific peripheral B lymphocytes to produce and secrete substantial amounts of active GrB. Using FACS, ELISpot, immunofluoresence and western blot we show that B cells from subjects recently vaccinated against tick-borne encephalitis virus (TBEV) but not unvaccinated subjects respond to viral TBEV antigens with GrB secretion in a dosedependent manner. This response is direct and occurs only in the presence of co-activation with certain IL-2 family cytokines such as IL-21. Similar results were found with other viruses including hepatitis B and rabies. GrB production in B cells required activation of JAK1 and STAT3 and inhibition of JAK1 by pyridone 6 completely abrogated GrB induction by viral antigens or anti-BCR. Our findings suggest GrB secretion by B cells may be part of a novel, anti-viral immune response mechanism. Further studies will elucidate whether or not granzyme B-secreting B cells can act as cytotoxic cells towards virus-infected cells.
Proteomic Analysis of Chronic Malignant B-Cell Derived Microparticles Reveals CD148 as a Potential Antigenic Marker for Mantle Cell Lymphoma Diagnosis.
Authors: Miguet Laurent , Fornecker Luc-Matthieu , Felden Claire , Gervais Carine , Herbrecht Raoul , Sanglier Sarah , van Dorsselaer Alain , Mauvieux Laurent
Published: April 2020

The diagnosis of mature B-cell neoplasms remains difficult in a number of cases, especially leukemic phases of non Hodgkin lymphomas, for which discriminating criteria or marker are often lacking. In order to identify new surface markers, we developed an original proteomic approach based on mass spectrometry analysis of plasma membrane microparticles derived from chronic B-cell lymphoproliferations: chronic lymphocytic leukemia/ small cell lymphoma (CLL/SLL) and mantle cell lymphoma (MCL). The approach consisted of protein extraction from plasma membrane microparticles (MPs), generated following actinomycin D stimulation and recovered using differential centrifugation. The complex protein mixture was further separated using 1D-gel electrophoresis separation and gel bands were systematically cutted at 2 mm intervals, trypsin digested and the resulting peptide mixtures was subsequently analysed by tandem mass spectrometry prior to protein identification. The lists of the proteins obtained for each pathology were then examined with respect to the membrane localization of the proteins in order to fulfill biological requirements needed for its further clinical use. Comparison of the lists of the proteins obtained for each pathology identified CD148, a membrane receptor with phosphatase activity, as a candidate for a discriminating marker detected in MCL but not in CLL in this approach. Flow cytometry studies, performed on 55 patients and 10 controls, showed that an anti-CD148 antibody stained significantly higher MCL (n=22) than CLL/SLL (n=33) circulating cells (p<0.0001). Even if the reason of such a regulated expression of CD148 remains unknown, our results indicate that a medium or high CD148 expression level may exclude the diagnosis of CLL/SLL and high CD148 expression levels may be strongly in favor of MCL diagnosis.

Figure Figure
B-Cell Receptor Profiling Suggests a Limited Number of Antigens Involved in the Pathogenesis of Chronic Lymphocytic Leukemia.
Authors: Binder Mascha , Müller Fabian , Frick Mareike , Wehr Claudia , Pantic Milena , Pfeifer Dietmar , Pasqualini Renata , Arap Wadih , Veelken Hendrik , Mertelsmann Roland H. , Trepel Martin
Published: April 2020

B-cell receptors (BCR) on chronic lymphocytic leukemia (CLL) cells have stereotyped hypervariable regions, categorizing approximately 20% of all patients into one of almost 50 BCR subsets with nearly identical receptors. This suggests common antigen recognition in some CLL patients. We explored the binding pattern of recombinant CLL BCRs Fab fragments by selecting epitope mimicking peptides from phage display libraries. We performed selections with multiple libraries using different peptide lengths and constraints. Selection was successful on seven of nine Fab fragments with at least one library. The selected peptides specifically bound the Fab fragment they were selected on as well as the native BCR expressed on parental CLL cells. Subsequently, we validated in one example that the selected peptide truly mimics an epitope recognized by CLL BCRs. As CLL BCRs have long been suspected of interacting with a set of different antigens, we screened a random panel of CLL cases for binding six representatives of the selected epitope mimics described above. Surprisingly, all CLL samples recognized one or several of the epitope mimics. The majority of CLL samples could be assigned to one of 14 categories with specific epitope binding patterns. These categories were not predictable from the amino acid sequence of the complementarity-determining region 3 although samples expressing homologous BCRs preferentially clustered in the same category. In addition, we showed that this ligand-binding categorization is not only functionally but also clinically relevant. Patients belonging to the largest binding category had a poorer prognosis with a shorter time to first treatment than control patients. Our data suggest that CLL BCRs have a surprisingly confined structural diversity and that, if antigenic drive exists in CLL pathogenesis, the number of potential antigens may be much smaller than previously anticipated. Moreover, the peptides identified in this study may have potential as a targeting moiety for CLL-directed drug therapy.
Outcomes of Allogeneic Stem Cell Transplant in Patients with Myeloid Leukemias Over 65 Years of Age. Rationale for Aggressive Therapy with Curative Intent.
Authors: Giralt Sergio , Saliba Rima M , Rondon Gabriela , de Padua Silva Leandro , Alousi Amin M. , Popat Uday , De Lima Marco , Andersson Borje , Kebriae Partow , Anderlini Paolo , Hosing Chitra , Jones Roy , Shpall Elizabeth J. , Champlin Richard E. , Khouri Issa F.
Published: April 2020

Background: Improvements in supportive care and the use of reduced intensity conditioning regimens (RIC) have expanded the use of allogeneic stem cell transplantation (SCT) to older patients with myeloid leukemia (AML, MDS, CML or other myeloproliferative disorders). No large single or multi-institutional study looking at outcomes of patients over the age of 65 has been performed, and the results of allografting in septuagenarians (>70 years of age) have not been reported.

Rationale: To explore the long-term outcomes of allogeneic transplant in patients with hematologic malignancies over the age of 65 and determine the impact of age and other prognostic factors in this subset of patients.

Patients and Methods: From 1/96 to 12/07 a total of 96 patients over the age of 65 underwent an allogeneic SCT for a myeloid leukemia. Patient, treatment characteristics and outcomes are summarized in the accompanying table.

Outcomes: For AML/MDS patients in remission between 65 and 70 years of age the OS at 2 years is 54% (95%CI:27–75) and 21% (95%CI:1–59) for patients 70 years or older (p=0.3); for AML/MDS patients not in remission OS @ 2 years was 21% (95%CI:9–36) for patients between 65 and 70 years of age and 0% for patients 70 yrs or older (p=0.06).

Conclusion: Allogeneic SCT can result in long term disease control in selected patients over the age of 65. Disease status at the time of transplant remains the most important prognostic factor for outcomes in this patient population. Patients over the age of 70 with AML/MDS in remission or with MPD can undergo allografting safely and achieve long term benefits, patients over 70 with refractory disease should be treated primarily on clinical trials aimed at improving outcomes in this patient population.

Less than 70 years of age 70 years of age or greater N 71 25 Median Age (range) 66 (65–69) 71 (70–76) AML/MDS 61 22 CML/MPD 10 3 Disease Stage @ SCT CR1/CR2/CP1 11/5/1 2/4/0 Other 54 19 Regimen Type Ablative 10 1 RIC 48 14 Non Ablative 13 10 % Matched Sibling Donor 60 60 % ATG in Conditioning 39 40 %OS @ 2 yrs (95%CI) 35 (24–46) 13 (3–30) p=0.08 %PFS @ 2 yrs (95%CI) 30 (19–42) 13 (3–30) %NRM @ 1 yr (95%CI) 24 (16–37) 44 (28–72) p=0.05 %aGVHD 2 or greater 28 (20–41) 35 (20–61) %cGVHD @ 2 yrs 35 (25–50) 26 (12–56) Figure Figure
Prognostic Significance of SALL4 Expression Levels in Paediatric Acute Myeloid Leukaemia (AML).
Authors: Ballerini Paola , Boelle Pierre-Yves , Deswarte Caroline , Auvrignon Anne , Couchy Gabrielle , Parker Judith Landman- , Robert Alain , Nelken Brigitte , Adam Mircea , Lapillonne Helene , Delabesse Eric , Perot Christine , Lai Jean Luc , Dastugue Nicole , Preudhomme Claude , Zucman-Rossi Jessica , Leverger Guy
Published: April 2020

Pediatric AML still represent an unfavourable disease resulting from the heterogeneous clonal expansion of malignant transformed haematopoietic stem or progenitor cell. The leukemia cell population is continuously replenish by rare, functionally distinct “leukaemia stem cells” (LSC) endowed with the capacity to self renew as well with the ability to generate clonogenic leukemic progenitors The AML-LSCs have been well documented and seem to behave like quiescent or slowly dividing hematopoietic stem cells. Therefore, LSC are considered less sensitive to treatments, which rather target actively dividing cells, and responsible for relapse. Recently, Y. Ma et al. suggested a major role of SALL4 gene both in stemness activity and leukemia transformation of normal hematopoietic stem cells. We sought to evaluate the expression of SALL4 gene in a panel of 88 pediatric AML, 60 Acute Lymphoblastic Leukemia (T and B ALL) and a few hematopoietic normal tissues. SALL4 expression was determined by quantitative RT-PCR in pre-treatment bone marrow samples (BM) (median blasts: 80%) and in normal tissues. SALL4 expression was much higher in AML compared to ALL (p<0.0001) and normal tissue (p < 0.0001). Values varied greatly among AML samples (range: 0 to 144; median 0.3). AML samples were dichotomized at SALL4’s median expression into“low” (n=44) or “high“(n=44) expressers. High expression was correlated to cytogenetic risk, as defined by MRC classes (p= 0.0008) but not to FAB categories, WBC, MLL rearrangements or FLT3, N RAS and NPM1 genotypes. Low expression was significant associated to t(8;21) translocation (P=0.02). SALL expression progressively increased from MRC1 class (low risk) to MRC3 (high risk) (p=0.002). High SALL4 patients had a shorter OS (OS: 46.8%±8% vs 74%±7%, p= 0.035) and a shorter EFS (EFS: 40% ± 8% vs 58% ±8%; P= 0.2) compared to low SALL4 expressers, although the latter association was less strong. As the highest values of SALL4 correlated with intermediate (MRC2) and high (MRC3) cytogenetic risk, we looked at fitting a model based upon the 4 quartiles of SALL4 expression. The upper quartile (values > 16, n= 12) had the worst outcome compared to the three others. Once stratified on MRC groups, MRC2 patients in the upper SALL4 quartile had 3.2 times more risk of relapse (HR= 3.2, CI95%: 1.3–7.8, P=0.02) and 5.4 more risk to die (HR=5.4, CI95%: 1.8–7.6; P= 0.0005) than MRC2 patients in the three others quartiles. In conclusion, SALL4 expression level may define an important risk factor in AML, particularly among patients with cytogenetic intermediate risk.
Study of the Mechanisms by Which Angptl5 and IGFBP2 Support Ex Vivo Expansion of Human Cord Blood Hematopoietic Stem Cells.
Authors: Zheng Junke , Zhang Chengcheng
Published: April 2020

We previously showed that angiopoietin-like protein 5 (Angptl5) and IGF Binding Protein 2 (IGFBP2) support dramatic ex vivo expansion of human hematopoietic stem cells (HSCs). To understand the mechanisms of their action, here we studied the effects of Angptl5 and IGFBP2 on the surface phenotype, signaling activation, self-renewal, apoptosis, differentiation, and homing of human cord blood CD34+ cells. Using immunofluorescence staining, we showed that Angptl5 and IGFBP2 activate certain signaling pathways such as MAPK and Stat5 in human cord blood CD34+ cells. IGFBP2 and Angptl5 increased the expression of transcription factors HoxB4, Bmi-1, EZH2, and survivin, measured by intracellular staining flow cytometry analysis and real-time RT-PCR. IGFBP2 and Angptl5 also inhibit expression of certain transcription factors important for differentiation of myeloid, erythroid, and lymphoid lineages. To test whether IGFBP2 and Angptl5 affect the homing of HSCs, we cultured human cord blood CD34+ cells in serum-free medium supplemented with SCF, TPO, Flt3-L, IGFBP2 or Angptl5, and transplanted them into sublethally irradiated NOD/SCID mice intraveneously or intrafemorally. Both IGFBP2 and Angptl5 support ex vivo expansion of SRCs in intrafemorally injected mice, suggesting the expansion-stimulating effects elicited by both factors are not caused by modulation of HSC homing. Interestingly, when we used intrafemoral injection, we found that Angptl5 treated HSCs have enhanced engraftment in non-injected bone marrow. This suggests Angptl5 treated HSCs further facilitate the mobilization of HSCs in vivo. We conclude that IGFBP2 and Angptl5 support self-renewal and inhibit differentiation of human cord blood HSCs. Our data also suggest that a combination of expression of transcription factors important for self-renewal, survival, and differentiation of HSCs can be used as a “stemness index” that predicts the activity of cultured human HSCs.
Naïve B Cells Differentiate into Highly Activated Granzyme B-Secreting B Cells in Response to Interleukin 21 and B Cell Receptor Stimulation
Authors: Schwesinger Elisabeth , Hagn Magdalena , Simmet Thomas , Jahrsdoerfer Bernd
Published: April 2020

Human B cells have so far not been known to express the cytotoxic molecule granzyme B (GrB). Recently we found malignant B cells from patients with chronic lymphocytic leukemia, activated with interleukin 21 (IL-21), secrete high amounts of GrB. Here we demonstrate healthy human naïve B cells differentiate into GrB-secreting B cells in response to IL-21 and B cell receptor (BCR) cross-linking. After 72 hours up to 90% of viable B cells were differentiating. GrB-secreting B cells were more resistant to apoptosis than unstimulated B cells and non-viable B cells did not contain GrB, suggesting that the primary function of B-cell-derived GrB is not self-regulation. Differentiation was associated with a CD19+CD27-IgD-CD38-phenotype, increased dextran uptake and strong upregulation of molecules involved in cell adhesion (CD54), antigen-presentation (MHC class II) and co-stimulation (CD86). In summary we describe a novel differentiation pathway of naïve B cells into highly activated GrB-positive B cells in response to IL-21 and BCR stimulation. Our data indicate this differentiation serves potential interactions with target cells including antigen uptake and presentation, but not self-regulation. Our findings may have significant implications for understanding the role of B cells in immunity, and may open novel immunotherapeutic approaches.
IL-12 Stimulates Interferon-Gamma Mediated Inhibition of Tumor-Induced Regulatory T Cell Proliferation and Enhances Tumor Clearance
Authors: Cao Xuefang , Collins Lynne I. , Cai Sheng F. , Mayer Joshua C. , Walter Michael J. , Piwnica-Worms David , Schreiber Robert D. , Ley Timothy J.
Published: April 2020

We have recently demonstrated that regulatory T (Treg) cells in the tumor environment (but not in the peripheral lymphoid tissues) utilize granzyme B to suppress anti-tumor immunity (Immunity 2007 Oct; 27(4):635–46), which suggests that Treg cells may be subject to regulation by local factors during anti-tumor immune responses. In order to identify such factors, we have employed an unbiased approach. First, we performed cytokine profiling on the tumor ascites fluid (tumor environment) and plasma samples harvested from naïve and tumor-bearing mice by using rodent Multi-Analyte Profiles (MAPs) provided by Rules-Based Medicine (RBM). Second, we performed gene expression profiling on Treg cells purified from the tumor ascites, spleens of tumor-bearing mice, and spleens of naïve mice by using Affymetrix MOE430v2 arrays. By matching the profiles of local cytokines and their receptor expression on tumor-associated Treg cells, we identified candidate cytokines that may modulate Treg cell function. In addition, we developed an in vitro system to study Treg cell proliferation in the tumor setting. Co-culturing irradiated tumor cells with unfractionated splenocytes (but not purified Treg cells) induces significant expansion of CD4+Foxp3+ Treg cells (2–4 fold in 5 days), suggesting that accessory cells and/or factors produced by them are responsible for Treg expansion. Using this system, we tested candidate cytokines for potential effects on Treg proliferation. Remarkably, IL-12 treatment completely blocked Treg expansion in an IL-12 receptor-dependent fashion. However, absence of IL-12 receptor mRNA in Treg cells suggested an indirect mechanism for this effect. Notably, an Interferon-gamma (IFNg) neutralizing antibody partially abolishes this IL-12 effect, and IFNg treatment exhibits similar (but lesser) inhibition of Treg proliferation. Conversely, Treg cells in IFNg receptor-deficient cultures are partially resistant to IL-12 inhibition, but completely resistant to IFNg inhibition. Mechanistic analyses revealed that IFNg signaling directly causes cell cycle arrest in Treg cells. Furthermore, IL-12 treatment enhances tumor clearance in vivo, and is more efficacious than Treg cell depletion. These findings indicate that IL-12 can stimulate IFNg-mediated inhibition of Treg cell proliferation, which may partially account for the anti-tumor effect of this cytokine. This study provides definitive evidence that an IL-12/IFNg axis can function to inhibit Treg cell proliferation, and that Treg cells, as immune suppressors themselves, are subject to cytokine-mediated suppression during anti-tumor immune responses. Because antigen-based tumor vaccines have been found to activate Treg cells that can considerably dampen antitumor immunity, such Treg-inhibiting cytokines may have the potential to serve as an adjuvant for vaccine-based immunotherapy.
APRIL Provides Springtime for Antibody-Producing Plasma-Cell Lifetime
Authors: Huard Bertrand , Belnoue Elodie , Kee Thomas Mc , Matthes Thomas , Siegrist Claire-Anne , Roosnek Eddy
Published: April 2020

Antibody-producing plasma cells depend on their environment for survival, but the molecules involved in this process are still not well defined. Plasma cells are fully equipped to respond to a proliferation inducing ligand (APRIL) from the tumor necrosis factor (TNF) superfamily, by virtue of their constitutive expression of the B-cell maturation antigen (BCMA), as canonical receptor from the TNF receptor superfamily, and the heparan sulfate proteoglycan (HSPG), CD138, as co-receptor. Here, we report that APRIL promoted the in vitro survival of plasma cells by upregulating expression of several anti-apoptotic molecules, such as bcl-2, bcl-xL and mcl-1. We further observed an in situ localization for APRIL consistent with this pro-survival role, both in mucosa-associated lymphoid tissues (MALT) and the bone marrow. In upper MALT, the tonsillar epithelium produced APRIL. Upon infection, APRIL production increased considerably when APRIL-secreting neutrophils, recruited from the blood, infiltrated the crypt epithelium. HSPG retained secreted APRIL in the sub-epithelium of the infected zone to create APRIL-rich niches, wherein IgG-producing plasma cells accumulated. In lower MALT, neutrophils were the unique source of APRIL giving rise to similar niches for IgA-producing plasmocytes in villi of lamina propria. The requirement on an inflammatory reaction in niche establishment implies that plasma-cell survival in mucosa is associated to pathogen presence, and must be short as a consequence. We observed also APRIL in the bone marrow. In this latter organ, maturating granulocytes produced constitutively APRIL. Such constitutive expression of a plasma cell pro-survival explains, at least in part, why plasma-cell longevity in the bone marrow can be so long lasting. These in situ human observations were confirmed in vivo with APRIL-deficient mice.
Prolonged Inhibition of BCR Signaling and Suppression of B Cell Lymphoma through Irreversible Inhibition of Bruton’s Tyrosine Kinase
Authors: Evans Erica , Sheets Michael , Bernard Hugues , Martin Thia St. , Nacht Mariana , Tester Rich , Jewell Chuck , Mazdiyasni Hormoz , Petter Russell , Westlin William , Singh Juswinder
Published: April 2020

B-cell receptor (BCR) signaling is essential for normal B cell development and proliferation. In addition, functional BCR signaling contributes to the proliferation and survival of many B cell lymphomas through constitutive low level or tonic signaling. Disruption of this tonic signaling pathway results in cell death or decreased viability in many B cell lymphoma cells. Bruton’s tyrosine kinase (Btk) is a member of the Tec family of protein tyrosine kinases and plays a crucial role in the development and activation of B cells through association with the BCR signalosome. We have developed a novel, potent, irreversible inhibitor of Btk that selectively disrupts the BCR signaling pathway as measured by inhibition of both Btk Y223 autophosphorylation and phosphorylation of the Btk substrate PLCg2. AVL101 inhibits Btk but leaves upstream BCR kinase activity intact; both Syk phosphorylation at Y525/526 and transphosphorylation of Btk on residue Y551 are unaltered. By mass spectrometry, we have shown that AVL101 covalently modifies Btk specifically at Cys-481, and in cell-based assays, this irreversible binding leads to prolonged inhibition of Btk kinase activity. After removal of the compound, inhibition of Btk activity is maintained in cultured B cell lymphoma cells for > 8 hours. This prolonged inhibition correlates with the half-life of the Btk protein, which we have found to be greater than 8 hours, irrespective of BCR activation state or modification by the inhibitor. We have demonstrated that AVL101 functionally inhibits BCR signaling in vitro and in vivo. In vitro, AVL101 inhibits BCR ligand-induced calcium flux as well as proliferation of the B cell lymphoma cell lines DOHH2 and WSU-DLCL2. In vivo, we have shown AVL101 is orally bioavailable and can inhibit Btk-dependent B cell function. Our data demonstrate that Btk is a valid therapeutic target for B cell lymphomas and other disorders dependent on BCR signaling. Furthermore, irreversible inhibition of Btk enables complete and prolonged inactivation of the target kinase, and suggests that clinically, a rapid and sustained therapeutic response may be possible using an orally available, irreversible Btk inhibitor.
Characteristics of Chronic-Phase CML Patients Having Durable Cytogenetic Response to Low-Dose Imatinib
Authors: Kawaguchi Tatsuya , Hamada Akinobu , Nakashima Reiko , Horikawa Kentaro , Ishihara Sonoko , Saito Hideyuki , Mitsuya Hiroaki
Published: April 2020

Imatinib mesylate (IM), a BCR-ABL tyrosine kinase inhibitor, has been established as first-line treatment of chronic-phase chronic myeloid leukemia (CML). On the basis of the IRIS study, 400mg/day IM has been recommended as an initial dose for adult CML patients. Although most patients usually tolerate the recommended dose, serious adverse effects such as grade 3 or 4 cytopenia may occur and require dose reduction of IM to 200–300mg/day. In fact, 8% of patients were receiving less than 400mg IM during 5-year follow-up in the IRIS study (Druker et al, New Engl J Med, 2006). In clinical settings, low-dose IM is actually effective for some patients; however, clinical and laboratory features of such patients have not been well investigated. Recently, it was demonstrated that the effective plasma threshold for trough IM levels should be maintained above 1,002 ng/ml to obtain maximal effects in CML patients (Picard et al, Blood, 2007). In this regard, determination of trough IM plasma levels may help to predict efficacy of low-dose IM. To evaluate the association of optimal IM doses with trough IM levels and patients’ basic characteristics such as body surface area (BSA), we assessed trough plasma concentrations of IM using high performance liquid chromatography (Hamada et al, J Pharmacol Exp Ther, 2003) in 31 chronic-phase CML patients, who were treated in Kumamoto University Hospital during 2003 to 2007. An optimal dose of IM was determined by a favorable response to IM achieving complete cytogenetic response and tolerable adverse events. Twenty-seven patients tolerated IM therapy during the observation period: optimal doses of IM were 400mg/day in 13 patients, 300mg/day in 9 patients, and 500 or 600 mg/day in 5 patients. Four patients discontinued IM for some reasons: two for toxicity, one for a concomitant unrelated disease and one for inefficacy due to drug-resistance. The trough plasma concentrations of IM were 1.64±0.68 μg/ml (mean±SD) in patients on the standard dose of 400mg/day IM (standard dose group) and 1.38±0.53 μg/ml in patients on the reduced dose of 300mg/day IM as an optimal dose (reduced dose group). Both mean trough levels of two groups showed no significant difference and exceeded the effective plasma threshold. Interestingly, BSA was significantly smaller in patients of the reduced dose group than those of the standard dose group (1.50± 0.16 vs. 1.75 ± 0.15 m2 (mean± SD), p=0.001). An optimal IM dose was found to be significantly associated with age and gender as well as BSA. These results suggest that the reduced dose of 300mg/day IM may be sufficient for the treatment of CML patients with smaller body sizes. Monitoring of trough IM levels should enable proper management of individual patients in combination with regular monitoring of optimal response to IM.