bluuebunny/crossref_metadata_2025 huggingface dataset:
Tumor Cell-Associated Tissue Factor Supports Metastatic Potential through Both NK Cell-Dependent and -Independent Mechanisms.
Authors: Palumbo Joseph , Barney Kelley A. , Flick Matthew J. , Talmage Kathryn E. , Kombrinck Keith W. , Hu Zhiwei , La Jeunesse Christine M. , Degen Jay L.
Published: April 2020

Multiple lines of evidence indicate that the hemostatic system contributes to cancer dissemination. Previous studies have shown that tumor cell-associated tissue factor (TF) expression is a crucial determinant of metastatic potential. Furthermore, we have shown that tumor cell-associated TF supports metastatic potential through mechanism(s) dependent on multiple circulating hemostatic system components, including prothrombin, platelets, fibrinogen and, more recently, fXIII. At least two of these circulating hemostatic factors (platelets and fibrinogen) have been shown to support metastatic potential by impeding the clearance of recently embolized tumor cells by natural killer (NK) cells. It is reasonable to hypothesize that tumor cell-associated TF expression also supports metastatic potential by a mechanism coupled to NK cell function. Here, we used C57BL/6-derived, Ras-transformed tumor cell lines expressing wildtype murine tissue factor (TFWT), a mutant TF lacking the intracytoplasmic portion (TFΔTail), or no tissue factor (TFO) to directly examine the interplay between tumor cell-associated TF and NK cell function in determining metastatic potential. Each of these cell lines was capable of robust, comparable tumor growth in wildtype C57BL/6 mice. Loss of either platelet function or fibrinogen significantly diminished the metastatic potential of TFWT cells, but this effect was entirely abrogated by the concomitant loss of NK cells. Similar results were obtained with TFΔTail cells, indicating that the cytoplasmic portion of TF is not critical to these interactions. To determine if the increase in metastatic potential conferred by tumor cell-associated TF expression is entirely linked to NK cell function, we compared the metastatic potential and early survival of TFWT and TFO cells in mice with and without NK cells. TFOcells rarely formed any visible metastatic foci in mice with intact NK cell function, while TFWT cells were aggressively metastatic. Importantly, TF expression remained a significant determinant of metastatic potential even in mice lacking NK cells. Comparisons of the early fate of TFWT and TFO cells revealed that TF expression was not a determinant of initial tumor cell localization within the lungs. Rather, TF expression supported the sustained adherence and/or survival of tumor cells. Taken together, these data indicate that one mechanism linking tumor cell-associated TF expression to metastatic potential is coupled to circulating hemostatic factors and results in impaired NK cell-mediated clearance of recently established micrometastatic foci. However, TF expression also supports metastasis by at least one additional mechanism that is independent of natural killer cell function.
Clinical Significance of JAK2V617F Homozygosity in the Chronic Myeloproliferative Disorders. A Study on 1306 Patients.
Authors: Vannucchi Alessandro M. , Barosi Giovanni , Rambaldi Alessandro , Marchioli Roberto , Barbui Tiziano ,   for the GIMEMA MPD, WP
Published: April 2020

The JAK2V617F mutation is variably found in the Ph’-negative chronic myeloproliferative disorders (MPD); in about 25% of polycythemia vera (PV) or idiopathic myelofibrosis (IM) and in 1–3% of essential thrombocythemia (ET) patients (pts), mitotic recombination causes the mutation to be harboured in the homozygous status. While it has been suggested that homozygosity influences some cellular functions, its clinical significance has not been elucidated yet due to the low number of homozygotes in published series. To this end, a retrospective, multicentric study, that enrolled 1306 pts, was designed within the Myeloproliferative Working Group of the Italian GIMEMA association; all pts who had been typed for JAK2 mutation using the BsaXI digestion assay (Baxter et al, Lancet, 2005) were included. There were 397 PV (30.4%), 639 ET (48.9%), 270 IM (20.7%) pts. JAK2V617F was found in 878 (67.3%), accounting for 82%, 60%, and 64%, respectively, of PV, ET, or IM pts. The homozygotes (Ho) were 183 (20.8%), with a frequency of 26.3% in PV, 2.2% in ET, and 24.1% in IM. Ho pts, irrespectively of the underlying diagnosis, presented higher leukocyte count and hematocrit, while platelet count was lower, as compared to heterozygous (He) or wild-type (WT). The frequency of splenomegaly rose from 26% to 41% to 52% in WT, He and Ho PV pts; from 19% to 22% to 62% in ET; and from 61% to 67% to 89% in IM. Acquagenic pruritus was referred by 12%, 28% and 42% of WT, He, and Ho PV pts, while the corresponding figures were 4%, 12%, and 26% in IM; no difference was noted among ET pts. Also the frequency of systemic symptoms was significantly higher among Ho pts in all three disease groups. 195 pts (14.9% of total) presented major thromboses at diagnosis (71 PV, 114 ET, 10 IM, accounting for 18%, 18%, and 4% in each group, respectively), while 144 events were recorded in the follow-up (15%, 12%, 4% in PV, ET, or IM). The incidence of thrombosis at diagnosis was 12%, 21% and 15% in WT, He or Ho PV pts, while the corresponding figures were 11%, 22% and 50% in ET, and 1%, 7% and 3% in IM. The relative risk (RR) for major thrombosis after diagnosis (adjusted for gender, age, and previous thrombosis) was significantly higher in Ho than in He pts both in ET (RR 4.6 and 1.5, respectively, by taking as 1 the WT pts) and IM (RR 3.2 and 1.6) pts, unlike in PV (RR 0.8 and 0.9, respectively). Ho pts evolved more frequently into myeloid metaplasia than He or WT in both PV (12% vs 0% and 1%, respectively) and ET (14% vs 5%, and 2%). These data support the contention that the JAK2V617F homozygous mutational status associates with a more symptomatic disease across the MPD clinical entities, and identify the unfrequent Ho ET patients as a specific subgroup with a particularly aggressive disease.
Highly Sensitive Real-Time PCR of V617F-JAK2-Mutation To Monitor Minimal Residual Disease and Guide Donor Lymphocte Infusion after Allogeneic Stem Cell Transplantation in Patients with Myelofibrosis.
Authors: Kroeger Nicolaus , Badbaran Anita , Holler Ernst , Hahn Joachim , Kobbe Guido , Bornhauser Martin , Reiter Andreas , Zabelina Tatjana , Zander Axel , Fehse Boris
Published: April 2020

The V617F-JAK2-mutation occurs in about 50 % of patients with myelofibrosis and is a reliable marker to monitor residual disease after allogeneic stem cell transplantation. The establishment of valid complete remission criteria for myelofibrosis especially after allogeneic stem cell transplantation remains a major issue. We developed a new, highly sensitive real-time PCR to monitor and quantify V617F-JAK2-positive cells after dose-reduced allogeneic stem cell transplantation. The mutated differs from the wild-type JAK2 allele by just one nucleotide exchange (G à T) leading to the valine to phenylalanine (V à F) transition. Using PrimerExpress® we designed a TaqMan® PCR where the reverse primer (RP) terminates at the (3′) nucleotide corresponding to this point mutation. Thus, this reverse primer should bind with higher affinity to the mutated than to the wild-type allele. To increase the specificity while conserving optimal sensitivity of the MRD-specific PCR we generated a set of primers shortened each time by one nucleotide at their 5’ end. In parallel, all those shortened primers were designed to contain an additional mutation at the third to last 3’ position. This allowed us to identify the reverse primer combining high specificity with so far not reported sensitivity(0.01 %). After 22 allogeneic stem cell transplantation procedures in 21 JAK2-positive patients with myelofibrosis, 78 % became PCR-negative. In 15 out of 17 patients (88 %), JAK2 remained negative after a median follow-up of 20 months. JAK2-negativity was achieved after a median of 89 days post allograft (range, 19 – 750 days). A significant inverse correlation was seen for JAK2 positivity and donor cell chimerism (r: −0.91, p<0.001). Four of five patients who never achieved JAK2-negativity fulfilled during the entire follow-up all criteria for complete remission recently proposed by the International Working Group, suggesting a major role for JAK2 measurement to determine depths of remission. In one case, residual JAK2 positive cells could be eliminated by donor lymphocyte infusion, supporting the graft versus myelofibrosis effect. In conclusion, allogeneic stem cell transplantation after dose-reduced conditioning induces high rates of molecular remission in JAK2-positive myelofibrosis-patients and quantification. V617F-JAK2 mutation by real-time PCR allows detecting minimal residual disease to guide adoptive immunotherapy.
Injury to the Thymic Niche for DN2/DN3 Developing T Cells Impairs Reconstitution.
Authors: Prockop Susan , Nikolopoulos Sotiris , Myers Aaron , Giancotti Filipo , Petrie Howard T. , O’Reilly Richard J.
Published: April 2020

Despite recovery of most hematopoietic functions, prolonged defects in generating functional T lymphocytes is a common occurrence after T cell depleted bone marrow transplant. While the mechanisms of these defects have not all been elucidated, contributing factors include age, T cell depletion of the graft, therapy with radiation and cytotoxic agents, and graft versus host disease (GvHD). We have designed an in vivo functional assessment of the ability of thymic stroma to support de novo T lymphocyte development. Mice deficient for the alpha chain of the IL-7 receptor (IL7Rα−/−) support robust thymic reconstitution after transplant of limited numbers of congenic precursors. We demonstrated that this capacity for reconstitution of immunodeficient strains depends on the paucity of specific endogenous precursors (DN3) cells in the IL7Rα−/− thymus and reflects the presence of functionally normal but empty stromal niches in this immunodeficient strain. We have proceeded to demonstrate that a variety of chemotherapeutic agents as well as aging, impair the ability of IL7Rα−/− thymic stroma to support de novo T cell development. Some agents allow donor chimerism in the thymus, but not rescue of the hypocellularity (eg cyclophosphamide) while others do not affect reconstitution (eg fludarabine). Multi-agent regimens have been administered and in some instances demonstrate an additive detrimental impact on thymic reconstitution. For several agents, damage has been localized to specific stromal niches by isolating changes in lymphoid subsets and stromal keratin expression. Decreased availability of the stromal niche for DN3 progenitors is associated with a decreased frequency of DN3s and an absolute block in recipient IL7Rα−/− T cell development. In addition, RNA from treated and untreated thymic stromal cells has been used to evaluate the gene expression pattern in thymic stroma of IL7Rα−/− mice treated with cytotoxic agents. In one example to be presented we find decreased thymic reconstitution in mice treated with Busulfan with sustained changes in stromal elements. These findings are consistent with sustained damage to thymic stromal cells. Among other changes, busulfan leads to decreased expression of laminin 5 by cortical thymic epithelial cells. The integrin heterodimerα6β4 is a binding partner for laminin 5 and is expressed uniformly by DN2 thymocytes. The significance of laminin 5/α6β4 signaling during T cell development was assessed using mice with a targeted mutation in the integrin β4 signaling domain. In these experiments mutant fetal liver was used to create hematopoietic chimeras and evaluate T cell development. Our studies provide insight into the nature of damage to thymic stroma by cytotoxic regimens and an understanding of the effect of this damage on subsequent immune reconstitution.
Phase III Study of Denileukin Diftitox (ONTAK®) To Evaluate Efficacy and Safety in CD25+ and CD25- Cutaneous T-Cell Lymphoma (CTCL) Patients.
Authors: Negro-Vilar Andres , Dziewanowska Zofia , Groves Eric , Lombardy Elyane , Stevens Victor
Published: April 2020

Denileukin diftitox (ONTAK®) is a genetically engineered fusion protein composed of the enzymatically active domain of diphtheria toxin and the full length sequence of interleukin-2 (IL-2), designed to target malignancies expressing the IL-2 receptor. The drug is approved for the treatment of CTCL in patients expressing the CD-25 component of the IL-2 receptor. L4389-14 is a phase III study designed to further evaluate the efficacy and safety of denileukin diftitox (DD) in distinct subgroups of CTCL patients. The study met its accrual goal of 90 patients and included three subgroups of patients: 1) CD25 (+) patients that crossover from a placebo course of treatment in a companion study (L4389-11) and had progressed or failed to respond during an 8-course placebo treatment (N=34); 2) CD25 (−) patients (N=36) and 3) CD25 (+) patients that had previously been treated with DD, responded, and subsequently relapsed (retreatment group, N=22). Patients entered the study at stages Ia to III and received DD at a dose schedule of 18mcg/Kg/day by IV infusion once daily for 5 days every 3 weeks for up to 8 cycles. Efficacy of treatment was assessed based on tumor burden, lymph nodes, lymphocyte count, and patient status (PGA) at every cycle, beginning on cycle 2. A response assessment required confirmation in two subsequent cycles. Responses were evaluated by an independent Drug Evaluation Review Committee. Assessment of activity of DD across the 3 groups based on an analysis of the data is shown in the table below. Patient demographics were consistent across subgroups and representative of a general CTCL population. Disease status at baseline was Stage IIa or earlier 66% of patients and stage IIb or higher 34% of patients. About 46% of patients presented with mild to severe erythroderma. Adverse events were similar to those previously observed with ONTAK. Serious adverse events of ≥5% incidence were nausea (9%), vomiting (5%), capillary leak syndrome (5%), pyrexia (9%) and infections (10%). The results of this large phase III trial showed very consistent efficacy of denileukin diftitox across treatment-naïve CD25 (+) and CD25 (−) patients, as well as in patients undergoing retreatment with DD. Duration of response and progression free survival were also quite favorable across the different CTCL subgroups.

Response Assessment CD25 (+) Placebo Crossover CD25 (−) CD25 (+) Retreatment N = 34 36 22 ORR (CR/CCR/PR) 47.1% 30.6% 36.4% CR/CCR 17.6% 8.3% 9.1% PR 29.4% 22.2% 27.3% SD 29.4% 44.4% 31.8% PD 20.6% 25.0% 31.8% Duration of Response (days) 820 340 274 Progression Free Survival (days) 870 Not reached 429
The Bcl-2 Protein Family Is Essential for Platelet Survival In Vivo.
Authors: Mason Kylie D. , Carpinelli Marina R. , Collinge Janelle E. , Hilton Adrienne A. , Fletcher Jamie I. , Roberts Andrew W. , Metcalf Donald , Huang David C.S. , Kile Benjamin T.
Published: April 2020

Platelets are small, anuclear cytoplasmic fragments that play an essential role in blood clotting and hemostasis. They are produced by megakaryocytes: large, polyploid cells that reside primarily in the bone marrow. Once shed into the blood stream, platelets are consumed either by their participation in hemostatic processes, or their eventual destruction by the reticulo-endothelial system. The latter occurs when platelets have reached the end of their lifespan, which is 9–10 days in human, 4–5 days in mice. Like the other lineages of blood cells, the steady state level of platelets reflects the balance between their production and destruction. Any imbalance can result in bleeding or clotting tendencies, both of which may be life threatening.

We have found that a pro-survival member of the Bcl-2 protein family is essential for the maintainence of platelet survival in vivo. Mutations in the gene encoding this protein dramatically reduce platelet lifespan in a dose-dependent manner, resulting in thrombocytopenia. Strikingly, no effect on platelet production was observed, allowing us to distinguish the molecular control of platelet survival from that of platelet production. In a complementary approach, we employed a specific small molecule antagonist of the pro-survival Bcl-2-like protein. Treatment with this BH3 mimetic agent triggers features of apoptosis in platelets, dramatically reducing their survival both in vivo and in vitro. Platelet death can be prevented if they are pre-treated with a broad-spectrum caspase inhibitor or if platelets are derived from mice lacking an essential downstream mediator of apoptosis. Furthermore, the heritable thrombocytopenia in mice lacking the specific Bcl-2-like protein is corrected when this downstream target is genetically ablated. Thus, the balance between a Bcl-2-like pro-survival protein and its direct downstream pro-apoptotic target critically determines platelet survival, and hence circulating platelet numbers, in vivo.

These findings have notable implications for our understanding of platelet biology, strategies to promote platelet survival, and approaches to remove excess platelets that contribute to pro-thrombotic states.
IVIg-Primed Leukocytes Offer Longer Protection in the Amelioration of ITP Than IVIg.
Authors: Crow Andrew R. , Brinc Davor , Siragam Vinayakumar , Freedman John , Lazarus Alan H.
Published: April 2020

Using a murine model of ITP, we have previously demonstrated that both IVIg and a monoclonal antibody which reacts with red cells (TER-119), ameliorates thrombocytopenia. We have also recently demonstrated that passive transfer of IVIg-primed splenic leukocytes can completely recapitulate the effects of standard IVIg therapy in the amelioration of murine ITP, and further showed that CD11c+ dendritic cells were the primary cell mediating IVIg functions (Nat Med2006;12:688–692). However, the duration of action of IVIg, TER-119 and IVIg-primed leukocytes in the inhibition of murine ITP has not been evaluated. Herein, we have evaluated the efficacy and duration of action of IVIg-primed leukocytes compared with standard therapeutic delivery of IVIg and TER-119. On day 1, mice were pre-treated with 50 ug TER-119 or 50 mg IVIg, or with only 106 leukocytes that had been primed for 30 minutes with IVIg, followed by extensive washing to remove all extraneous IVIg. Alternatively, leukocytes were also primed as above with BSA as a negative control. On days 2, 5, 8 and 11, mice from each group were injected with an antibody directed to integrin αIIβ to induce immune thrombocytopenia and bled the following day for platelet enumeration. On days 3 and 6, platelet counts revealed that mice receiving IVIg-primed leukocytes, IVIg or TER-119 were all protected from thrombocytopenia to the same extent. Mice that were pre-treated with leukocytes primed with BSA were not protected, as expected. However, at day 9, mice that received IVIg-primed leukocytes were still completely protected from thrombocytopenia, while mice receiving IVIg or TER-119 were not protected. By day 12, none of the treatment regimens conferred protection from thrombocytopenia. We are currently repeating these experiments with IVIg-primed bone marrow-derived dendritic cells and these results will also be presented. These data confirm that in the treatment of murine immune thrombocytopenia, pre-treatment of mice with IVIg-primed leukocytes offers up to 50% longer duration of protection from thrombocytopenia than standard therapies with IVIg or TER-119.
A SERPINE1 (PAI-1) Single Nucleotide Polymorphism Predicts Osteonecrosis in Children Treated for Acute Lymphoblastic Leukemia: Results of the Children’s Oncology Group (COG) Study AALL03B2.
Authors: Hamilton Leo H. , Mattano Leonard A. , Sather Harland N. , Relling Mary V.
Published: April 2020

As intensity of glucocorticoid use has increased, osteonecrosis has become an increasingly frequent complication of modern intensive chemotherapy for pediatric patients with acute lymphoblastic leukemia (ALL). Although patient age > 10 years is a well-known risk factor, other host risk factors that may predispose to this complication are not well described. We tested whether 12 polymorphisms in 11 candidate genes were associated with osteonecrosis among up to 344 patients ≥ 10 years old treated on the Children’s Cancer Group protocol CCG 1882 for newly diagnosed high-risk ALL, adjusting for gender, age, and treatment arm (augmented versus standard treatment). Genes tested (TYMS, ER, MTHFR, ABCB1, BGLAP (osteocalcin), ACP5, LRP5, ESR, SERPINE1, VDR, PTH, and PTHR) were chosen because they are candidates based on the putative mechanisms underlying osteonecrosis risk, including involvement in antifolate pharmacodynamics, drug pharmacokinetics, thrombosis risk, and possible hormonal influence on glucocorticoid effects. All children received dexamethasone during the delayed intensification phase of therapy, with doses varying by treatment arm (one dexamethasone pulse in the standard chemotherapy arm vs. two dexamethasone pulses in the augmented chemotherapy arm). Of the 11 candidate polymorphisms, a single nucleotide polymorphism (dbSNP rs6092) in the SERPINE1 gene (plasminogen activator inhibitor type 1 or PAI-1) was the only polymorphism associated with an increased risk of osteonecrosis in both univariate (p=0.0039) and multivariate (p=0.0019) analyses (adjusting for gender, age, and treatment arm), with an odds ratio of 2.51 (95% CI 1.32 to 4.74). Overall, 23.9% of the 88 children heterozygous or homozygous for the SERPINE1 T allele, versus 11.1% of the 234 children homozygous for the C allele, developed osteonecrosis. The combined CT or TT genotypes were present in a greater percentage (27%) of whites than in blacks (12%, p = 0.016), consistent with a higher risk (p = 0.003) of osteonecrosis in whites than blacks that was originally reported for CCG-1882 (J Clin Oncol18:3262–72, 2000). In a multivariate analysis, only the SERPINE1 polymorphism and sex predicted AVN, with the incidence of osteonecrosis lower in boys (odds ratio 0.49, 95% confidence interval 0.25 to 0.96, p = 0.034). No statistically significant gene-gene interactions between SERPINE1 and any other tested genotype were noted. SERPINE1 polymorphisms and PAI-1 serum levels have previously been associated with a clinical pro-thrombotic state. We conclude that polymorphisms in SERPINE1 may contribute to the risk of osteonecrosis in patients treated with glucocorticoids.
The Effect of Low Molecular Weight Heparin (LMWH) on Cancer Survival. A Systematic Review and Meta-Analysis (MA) of Randomized Trials.
Authors: Lazo-Langner Alejandro , Goss Glenwood D. , Spaans Johanna N. , Rodger Marc A.
Published: April 2020

Several studies have supported the existence of an antitumor effect of anticoagulants and several MA of studies comparing unfractionated heparin and LMWH for treatment of venous thromboembolism have suggested that the risk of mortality in the subgroup of cancer patients receiving LMWH might be reduced; however other authors have reported contrary findings. We conducted a systematic review and MA of randomized trials comparing LMWH with placebo/no intervention studying as the main outcome the impact on survival in cancer patients. Data sources were Medline, EMBASE, HEALTHstar, Cochrane Library, and grey literature. Data extraction was done by one reviewer and verified by a second reviewer. Discrepancies were resolved by consensus. Main outcomes were death at 1 year and major bleeding episodes. Secondary outcome was death at 2 years. A MA was done using a DerSimonian and Laird random-effects model with odds ratio (OR) and relative risk (RR) as summary statistics. We conducted a MA of survival rates using censored end-points and a MA of hazard ratios (HR). If not reported, HR were extracted from the survival curves and pooled using the generic inverse variance method (random effects model). 283 potential references were identified and 7 were fully evaluated. Four studies which enrolled 898 patients with solid malignancies (the majority with extensive disease) were included; 3 of the 4 studies included previously treated patients. OR and RR of death are shown in the table.

Mortality in cancer patients receiving LMWH versus placebo/no treatment All Patients Patients with advanced disease OR 95% CI P OR 95% CI P OR odds ratio; CI confidence interval; RR relative risk 1-year mortality 0.70 0.49, 1.00 0.05 0.75 0.57, 0.99 0.04 2-year mortality 0.57 0.34, 0.96 0.03 0.59 0.42, 0.84 0.004 RR 95% CI P RR 95% CI P 1-year mortality 0.87 0.77, 0.99 0.04 0.89 0.80, 0.99 0.03 2-year mortality 0.90 0.84, 0.97 0.007 0.92 0.86, 0.98 0.02

The results of the survival meta-analysis showed that the pooled survival proportions at 1 and 2 years were 0.43 and 0.19 for the LMWH and 0.35 and 0.11 for the control group, respectively; differences were statistically significant (1 year p=0.018, 2 year p=0.001). The MA of HR is shown in the figure.

Figure Figure

There was no increase in major bleeding episodes. Sensitivity analyses including only patients with advanced disease did not modify the findings; an analysis according to tumor type was not conducted due to a lack of information. LMWH improves overall survival in cancer patients independent of the stage. Additional trials are required to define the tumor types, disease stages, and dosing schedules most likely to derive the greatest survival benefit. Since the majority of patients included were previously treated, exploring the benefit of LMWH in newly diagnosed patients is necessary.
Immunomagnetic Depletion of CD25-Expressing T Cells from Donor Blood Removes a Mixed Population of Regulatory and Effector T Cells - Implications for Graft Engineering.
Authors: Melenhorst J. Joseph , Lu Jun , Sosa Edgardo , Hensel Nancy F. , Barrett A. John
Published: April 2020

CD4+CD25+FOXP3+ regulatory T cells (TR) control proliferative CD4 and CD8 T cell responses to self and foreign antigens such as cytomegalovirus (CMV) and tumor-specific antigens. Thus, depletion of CD25-expressing cells from a resting population of T cells prior to antigen stimulation could boost the generation of antigen-specific T cells for adoptive transfer to treat viral infection or tumors. We depleted peripheral blood mononuclear cells (PBMC) from nine CMV seropositive donors using Miltenyi CD25 microbeads (20 μl/107 cells). CD25-depleted or unmanipulated PBMC were stimulated with CMV pp65-expressing antigen presenting cells for 10–14 days with low dose IL-2, and intracellular interferon-gamma (IFNγ) production by flow cytometry was compared between CD25-depleted and -undepleted cultures. An absolute increase in antigen-specific CD4+ and CD8+ T cells was seen after CD25 depletion in 4/8 and 5/8 cultures respectively. However, in other cultures there was a decrease or no change in IFNγ+ CD4+ T cells in CD25-depleted cultures, suggesting that the pp65-specific precursor cells had also been removed. We then used 4 μl beads per 107 PBMC to selectively remove only the CD25bright (predominantly Treg) population in nine donors and confirmed by flow cytometry that only CD25bright cells had been removed from the starting population. However, real-time quantitative PCR (Q-PCR) showed that even though the CD25+ fraction was enriched in FOXP3-expressing cells, a substantial proportion of the CD25-depleted PBMC still expressed FOXP3. Flow cytometric analysis of FOXP3 expression by CD25+ and CD25-negative CD4+ T cells showed that a substantial proportion of CD25- cells expressed FOXP3, confirming that CD25 is not a suitable single marker for depletion of Tregs. Again no reproducible augmentation of antigen-specific T cell responses was observed: one and five donors showing an increase in CD4 and CD8+ antigen-specific T cells, respectively, while the remainder showed a decrease or no change in CD4+ and CD8+ IFNγ-producing cells. These results suggest that removal of CD25+ cells from PBMC using CD25 microbeads removes both Treg and pp65-specific effector CD4+ and CD8+ T cells. Further, since FOXP3 is induced in responder cells as confirmed by FOXP3 Q-PCR, depletion of Treg at the start of the cultures may only transiently alleviate the negative regulation of the antigen response. Thus, CD25 depletion using microbeads is not a reliable method to boost antigen specific T cell expansions because of the inadvertent removal of a portion of the memory response to the antigen. Since it recently has been demonstrated that Treg act as an IL-2 sink, the addition of this cytokine should functionally silence the Tregs while preserving the inflammatory response.
Modulating AML1-ETO with Signature-Based Small Molecule Library Screening.
Authors: Stegmaier Kimberly , Corsello Steven M. , Ross Kenneth N. , Golub Todd R.
Published: April 2020

We sought to develop a strategy to pharmacologically modulate AML1-ETO, the most common gene rearrangement associated with acute myeloblastic leukemia (AML). Like many other oncogenic transcription factors associated with the acute leukemias, AML1-ETO has been considered undruggable. In principle, these well-characterized somatic mutations identified in the acute leukemias represent unique, tumor-specific therapeutic targets. In practice, acute leukemia therapy continues to focus on nonspecific cytotoxic agents. In order to address this challenge, we developed a genomic, signature-based small molecule library screening approach, Gene Expression-based High-throughput Screening (GE-HTS). This approach uses gene expression signatures as surrogates for different biological states in a small molecule library screen. We focused our initial efforts on identifying modulators of AML1-ETO. First, a 25-gene signature for AML1-ETO abrogation was defined by transcriptional profiling of t(8;21) Kasumi-1 cells with and without AML1-ETO-directed RNA interference and with a U937 inducible model of AML1-ETO. The signature was confirmed in microarray data from t(8;21)-containing primary patient leukemias (p < 0.001). Next, the ability of 2,600 FDA-approved drugs and bioactive agents to induce this abrogation signature was evaluated by ligation mediated amplification (LMA) and bead-based fluorescence detection. The screen identified 16 hits confirmed on repeat testing, including seven corticosteroids. Five hits were selected for further study based on their chemical diversity, ability to induce a robust expression signature, and potential for clinical translation: 5-aza-deoxycytidine, floxuridine, methotrexate, methylprednisolone, and pyrimethamine. Next, whole genome effects of these compounds with microarray-based expression profiling were evaluated. Using Gene Set Enrichment Analysis (GSEA), we determined that all five compounds induce both whole genome effects consistent with AML1-ETO knockdown and a whole genome program consistent with neutrophil maturation. In a subset of these compounds, we also see changes in cell surface markers and morphological features consistent with myeloid maturation. Intriguingly, two of the hits are well-characterized DNA methyltransferase inhibitors, and two of the hits are dihydrofolate reductase inhibitors that increase S-adenosylhomocysteine, an inhibitor of methyltransferases. Thus, reversal of AML1-ETO-mediated gene silencing by demethylation may overcome its repressive effects. This application of knockdown-derived expression signatures to small molecule library screening should enable the targeting of nearly any oncogenic transcription factor.
The Polycistronic miRNA Cluster miR-17-92 Is Over-Expressed in Early Phase Chronic Myeloid Leukemia (CML) CD34+ Cells.
Authors: Venturini Letizia , Battmer Karin , Castoldi Mirco , Schultheis Beate , Hochhaus Andreas , Muckenthaler Martina , Ganser Arnold , Eder Matthias , Scherr Michaela
Published: April 2020

Micro RNAs (miRNAs) are small non-coding, single-stranded RNAs regulating gene expression by hybridization to complementary sequences in target mRNAs. Aberrant miRNA expression has been found in a variety of human malignancies including B-cell leukemias and lymphomas. The polycistronic miR-17–92 cluster, which includes seven miRNAs (miR-17-5p, miR-17-3p, miR-18a, miR-19a, miR-20a, miR-19b-1, miR-92-1), resides in intron 3 of the C13orf25 gene located on human chromosome 13 and has recently been described to harbor oncogenic potential in a murine transplantation model. In addition, expression of the polycistron can be regulated by c-myc, which is also activated by the Bcr-Abl oncogene product. To study miRNA expression in CML, K562 cells were analyzed by microarray analysis (miCHIP) for normalized expression of 244 miRNAs following treatment either with

imatinib to inhibit Bcr-Abl kinase activity or with specific anti bcr-abl shRNA to knock-down gene expression.

Both of these treatment strategies led to a 2–4-fold reduction of six mature miRNAs (miR-17-5p, miR-17-3p, miR-18a, miR-20a miR-19b-1, miR-92–1) as compared to untreated control cells. The reduction of the miR-17–92 miRNA cluster expression was confirmed for each individual mature miRNA species encoded in the cluster by real-time RT-PCR. Treatment with either imatinib or anti-bcr-abl shRNA also down-regulates expression of mature miRNA miR-19a (>3-fold) in K562 cells as quantified by real-time RT-PCR. We next used PCR based quantification to analyze miR-17–92 expression in purified normal (n=4) and CML CD34+ cells harvested at diagnosis of chronic phase disease (n=16). Individual mature miRNAs of the cluster were up-regulated (2–6-fold) in CML as compared to normal CD34+ cells. Additionally, the pri-miRNA for the miR-17–92 cluster was up-regulated in 70% (7/10) of CML samples. Interestingly, retrovirus-mediated over-expression of the miR-17-19b-1 cluster in K562 cells mediates enhanced colony-formation of transduced cells. Furthermore, K562 cells expressing high levels of the miR-17-19b-1 cluster are more resistant to proliferation inhibition induced by specific anti-myc RNAi, indicating some functional role of the miR-17-92 cluster on proliferation and survival in this model. In summary, the polycistronic miR-17–92 cluster is up-regulated in early chronic phase CML CD34+ cells and may contribute to cellular transformation by the Bcr-Abl oncogene product.
CD44 Is Selectively Required for the Homing and Engraftment of BCR-ABL-Expressing Leukemic Stem Cells.
Authors: Krause Daniela S. , Lazarides Katherine , von Andrian Ulrich H. , Van Etten Richard A.
Published: April 2020

In chronic myeloid leukemia (CML) patients treated by autologous hematopoietic stem cell (HSC) transplantation, malignant progenitors in the graft can contribute to relapse of leukemia (Deisseroth et al., Blood1994; 83:3068), but the mechanisms of homing and engraftment of leukemic CML stem cells are unknown. Although the frequency of autografting in CML has decreased following the introduction of imatinib, most imatinib-responsive patients harbor residual BCR-ABL-expressing stem cells (Graham et al., Blood2002; 99:319) and some will develop progressive leukemia. Autografting with cells harvested at the time of minimal residual disease could be an important salvage therapy, but methods to selectively block the engraftment of leukemic stem cells are needed. In this study, we show that CD44 expression is increased on murine BCR-ABL-expressing stem/progenitor cells and contributes to the ability of these cells to bind to Selectins. In a retroviral transduction/transplantation model of CML, BCR-ABL-transduced progenitors from CD44-deficient donors were defective in homing to recipient marrow, resulting in defective engraftment and impaired induction of CML-like myeloproliferative disease. By contrast, CD44-deficient stem cells transduced with empty retrovirus engrafted as efficiently as wild-type HSC. In addition, CD44 was not required for induction of acute B-lymphoblastic leukemia (B-ALL) by BCR-ABL, indicating that the engraftment requirement for CD44 is specific to leukemic cells initiating CML, not B-ALL. The requirement for donor CD44 was bypassed by direct intrafemoral injection of BCR-ABL-transduced CD44-deficient stem cells, or by co-expression of human CD44 with BCR-ABL. Treatment of BCR-ABL-transduced stem/progenitor cells from wild-type donors with antibody to CD44 attenuated the induction of CML-like leukemia in recipients. These results demonstrate a major role for CD44 in homing and engraftment of BCR-ABL-expressing leukemic stem cells, possibly through adhesive interactions with Selectins and/or hyaluronan in the recipient bone marrow niche. They further argue that BCR-ABL-transduced stem/progenitor cells depend to a greater extent on CD44 for engraftment than do normal HSC, and suggest that CD44 blockade may be beneficial in autologous transplantation in CML.
Psychosocial Factors as Measured by the Transplant Evaluation Rating Scale (TERS) Predict Length of Hospitalization and Transplant Outcomes Following Hematopoietic Stem Cell Transplantation.
Authors: Speckhart Dawn S. , Solomon Scott R.
Published: April 2020

Although psychological and social factors are recognized as being important in the evaluation of patients for hematopoietic stem cell transplantation (HSCT), no standard approach to psychosocial assessment currently exists. In solid organ transplantation, psychosocial assessments have been integrated into the selection of appropriate candidates, and certain psychosocial variables, such as active substance abuse, have been shown to negatively impact outcomes in solid organ transplant patients. To determine whether similar factors impact outcomes in patients undergoing HSCT, we prospectively conducted psychosocial assessments on 221 consecutive patients (155 autologous, 66 allogeneic) undergoing HSCT. The relationship between psychosocial variables, such as those assessed on the Transplant Evaluation Rating Scale (TERS), and objective outcomes, such as length of hospitalization and survival was evaluated. Based on the patient’s TERS score, each patient was stratified into one of two groups (low/moderate risk (n=187) vs. high risk (n=34)) based on their predicted psychosocial risk for problems during transplant. Although the two groups were similar in regards to known pre-transplant prognostic factors such as age, performance status, disease risk, and transplant type, there was a significant difference in the median length of hospitalization between patients who score low/moderate (10 days) and those who scored high (21.5 days) on the TERS. This difference was significant both for patients receiving autologous (9 vs. 15 days, p<.02) and allogeneic transplants (16 vs. 45 days, p<.001). Furthermore, 2-year overall survival was significantly improved in allogeneic transplant patients who score low/moderate vs. those that scored high on the TERS (72% vs. 46%; p<=.02). These findings suggest a strong correlation between pre-transplant psychosocial risk factors, resource utilization and patient outcome in HSCT.

Figure 1. Overall survival following allogeneic stem cell transplantation according to TERS score Figure 1. Overall survival following allogeneic stem cell transplantation according to TERS score
Allelically Mismatched Replacement Therapy Due to Common African−Restricted Haplotypes of the Factor (F)VIII Protein May Underlie the Increased Incidence of FVIII Inhibitors Observed in Hemophilia−A Patients of African−Descent.
Authors: Howard Tom E. , Viel Kevin R. , Fernstrom Karl M. , Deshpande Seema , Ameri Afshin , Ali M. Showkat , Channell Cynthia , Iyer Rathi V. , Watts Raymond G. , Lutcher Charles , Nakaya Shelley , Kasper Carol K. , Thompson Arthur R. , Abshire Thomas C. , Almasy Laura
Published: April 2020

Alloantibodies that inhibit the function of wildtype (wt) FVIII molecules constitute the most serious complication of replacement therapy for hemophilia (H)A patients. Although it is not possible to accurately predict which patients will develop inhibitors, ethnicity is a known risk factor as alloimmunization occurs ~2−fold more often in African Americans (AAs) than in Caucasians (Cs). Analysis of data from a resequencing study revealed that the FVIII gene (F8) contains four nonsynonymous−single nucleotide polymorphisms (ns−SNPs) that encode six distinct wt FVIII proteins designated haplotype (H)1−H6. While AAs express all but H6, only H1 and H2, the two recombinant (r)−FVIII proteins used clinically, are expressed by Cs. About 25% of AAs express H3, H4, or H5, proteins that differ structurally from r−FVIII at three ns−SNPs (R484H, D1241E, M2238V), two of which are located in dominant inhibitor epitopes and have AA−restricted minor−alleles. We designed the Pharmacogenetics of Inhibitor Risk (PIR) study to test the hypothesis that these ns−SNPs, when allelically mismatched in replacement therapy, are pharmacogenetic risk factors that contribute to the greater incidence of inhibitor development in patients of African−descent. We enrolled 94 of 223 total AA HA patients from collaborating treatment centers. Table 1 presents characteristics of the first 47 subjects (whose functional F8 regions have been resequenced entirely) including: FVIII haplotype (exposure) − H3/H4/H5 vs H1/H2; inhibitor status (outcome) − yes/no; F8 mutation − high−risk vs low−risk types; other covariates and potential confounders. A preliminary analysis demonstrated an odds ratio of 3.6 for inhibitors developing in the exposed subjects (P=0.10; 95% CI=0.7–17.6). Our finding suggests that pharmacogenetic factors may indeed contribute to the increased incidence of this alloimmune complication in AAs. To increase the power to detect a true association and allow adjustment for confounders, we are currently completing F8 resequencing in the next 47 patients.

Table 1. Relevant characteristics of the first 47 African-American hemophilia-A PIR study patients*
A Variant of Recombinant Factor VIIa with Enhanced Procoagulant and Antifibrinolytic Activities in a Model of Hemophilia.
Authors: Wolberg Alisa S. , Persson Egon , Hedner Ulla , Ezban Mirella , Allen Geoffrey A.
Published: April 2020

Recombinant factor VIIa (rFVIIa, NovoSeven; Novo Nordisk A/S, Copenhagen, Denmark) has proven efficacy in treating bleeding in hemophilia patients with inhibitors. We have shown that high concentrations of rFVIIa improve parameters of thrombin generation and fibrin clot formation and stability in an in vitro model of hemophilia (Wolberg et al. 2005 Brit J Haem 131:645–55). Recently, new rFVIIa analogs with increased procoagulant activity have been described. One of these analogs, rFVIIa with mutations V158D/E296V/M298Q (NN1731), exhibits increased activity in in vitro and in vivo models. In the present study, we compared the abilities of rFVIIa and NN1731 to specifically modulate factor X activation, platelet activation, thrombin generation, and fibrin clot formation and stability in an in vitro model of hemophilia. RFVIIa and NN1731 similarly increased factor X activation on tissue factor−bearing monocytes; however, NN1731 exhibited 30−fold higher factor Xa generation rates on activated platelets than similar concentrations of rFVIIa. We employed a complete, cell−based, reconstituted model of coagulation to compare the activities of rFVIIa and NN1731 in hemophilic conditions. The model system consists of tissue−factor bearing monocytes, purified pro− and anti−coagulant proteins (XI, X, IX, VIII, VIIa, V, II, AT, TFPI), and freshly−isolated, unactivated platelets. Fibrinogen is included in certain wells to examine clot formation. “Hemophilia” is simulated by omitting factors VIII and IX. In this assay, NN1731 produced 4 – 10−fold higher maximal thrombin generation rates than equal concentrations of rFVIIa. In contrast to even very high concentrations of rFVIIa (up to 500 nM), NN1731 was able to normalize the rate of thrombin generation. NN1731 did not directly activate platelets and thrombin generation was not detected prior to platelet activation, suggesting that the presence of NN1731 would not lead to thrombin generation in the absence of injury. Both rFVIIa and NN1731 shortened clotting times in the absence of factors IX and VIII; however, NN1731 did so at lower concentrations than were required of rFVIIa. In a plasmin challenge assay, in which clot formation is initiated in the presence of plasmin, both rFVIIa and NN1731 increased fibrin formation and the length of time that fibrin was present in the absence of factors IX and VIII; however, NN1731 was effective at lower concentrations than were required of rFVIIa. Our results indicate that NN1731 increases factor Xa and thrombin generation and promotes clot formation and stability to a greater degree than equal concentrations of rFVIIa. For these reasons, NN1731 may be especially efficacious in situations in which rapid formation of a firm fibrin clot with increased resistance to fibrinolysis is necessary to achieve hemostasis.
Leukemia Stem Cells in Murine MLL−AF9 AML Are Downstream Myeloid Lineage Cells That Have Acquired Stem−Like Biological and Genetic Properties.
Authors: Somervaille Tim C.P. , Cleary Michael L.
Published: April 2020

An essential prerequisite for the development of more effective targeted therapies in AML is a characterization of the frequency and biological properties of leukemia stem cells (LSCs), which sustain the disease and mediate relapse. Previous studies have shown that hematopoietic stem cells, as well as progenitors, may be targeted by MLL oncoproteins to give rise to AML, however it was unclear whether these lineage negative cells also functioned as LSCs to sustain disease. To address this issue, we have identified and characterized LSCs in a somatic, genetic mouse model of leukemia that faithfully recapitulates many of the pathologic and clinical attributes of human leukemia initiated by the MLL−AF9 oncogene. In this model, CFCs in the bone marrow and spleen of leukemic mice were demonstrated to be LSCs, based on the observation that secondary transplantation of the progeny of individually isolated AML CFCs cultured in vitro for seven days or longer invariably resulted in transfer of short latency disease. These self−renewing cells were remarkably frequent, accounting for 25–30% of myeloid lineage cells at late−stage disease. Unexpectedly, they expressed mature myeloid lineage antigens (91.3 ± 3.8% Mac1+ Gr1+ immunophenotype, versus fewer than 0.2% lineage negative), placing them downstream of the known hematopoietic progenitor compartment. Furthermore, LSCs in this model generated a phenotypic, morphologic and functional leukemia cell hierarchy loosely defined by expression of c−kit. When compared with immortalized progenitors, LSCs exhibited a markedly enhanced ability to engraft secondary recipients, which was not due to differences in bone marrow homing, which was equivalently poor in the compared populations. Rather, LSCs exhibited an acquired ability to proliferate in response to stromal cell derived cytokines, an enhanced SDF1 induced chemotaxis, and increased proliferation in contact with OP9 stromal cells demonstrating that LSCs exhibit altered microenvironmental interactions by comparison with the oncogene immortalized CFCs that initiated the disease. Thus, the LSCs responsible for sustaining, expanding and regenerating MLL−AF9 AML are downstream myeloid lineage cells, outside of the normal stem and progenitor compartment. They have acquired an aberrant Hox−associated self−renewal program as well as other biologic features of hematopoietic stem cells. Our findings support a revision of the prevailing hypothesis that AML LSCs are always rare and solely located within the most immature bone marrow progenitor compartment. Furthermore, LSCs exhibit markedly different microenvironmental interactions, by comparison with cells simply immortalized by MLL−AF9, indicating that acquisition of sensitivity to stromal derived survival and proliferative signals is a critical feature of LSCs, in addition to their extensive self−renewal capabilities.