bluuebunny/crossref_metadata_2025 huggingface dataset:
Exploiting ER Stress to Eliminate Inhibitor Antibodies in Hemophilia
Authors: Callaghan Michael U , Kaufman Randal J
Published: April 2020

Hemophilia is the most common severe bleeding disorder, affecting up to 1 in 5,000 males. The most feared complication for patients with hemophilia A is the development of inhibitor antibodies to infused FVIII, occurring in 30% of patients with hemophilia A. Inhibitor antibodies are produced in large numbers by the professional secretory cells of the immune system, the plasma cells, in response to exposures to FVIII. The development of these professional secretory cells has been shown to cause stress in the endoplasmic reticulum (ER) and mechanisms of the unfolded protein response (UPR) are integral to the adaptation of these cells. Proteasome inhibitors, such as bortezomib (Velcade), have been shown to increase ER stress and kill plasma cells by inhibiting the destruction of misfolded proteins and inducing proteotoxicity. Current therapeutic strategies for hemophilia patients with inhibitors revolve around inducing tolerance to FVIII by treating patients with large doses of FVIII daily. We believe this therapy induces antibody production, stressing the plasma cells that manufacture the inhibitor antibodies.

We have tested the hypothesis that the one-two punch of ER stress directed at the anti-FVIII plasma cells and proteasome inhibiting or CHOP inducing agents will induce apoptosis. To this end we have tested bortezomib in vitro and determined that it does potently induce ER stress and the UPR. Further, we have shown that this induces apoptosis and cell death and that cells lacking ATF6α, an upstream mediator of the UPR, are more sensitive to bortezomib while those lacking CHOP, a pro-apoptotic downstream mediator of UPR, are slightly less sensitive. Additionally we have found that the super-oxide scavenger, tiron, protects cells from bortezomib, suggesting a role for reactive oxygen species upstream of UPR activation by bortezomib. We have studied bortezomib in a mouse model of hemophilia A inhibitors and have found that it can effectively eradicate inhibitor antibody producing cells in this model. In contrast when bortezomib was injected into CHOP knock-out inhibitor mice the inhibitor antibodies were not eradicated, in contrast to the wild type controls in which the inhibitor antibodies were eliminated. This is strong evidence in support of CHOP as a primary mediator of plasma cell death induced by bortezomib and suggests that other CHOP inducing agents may also be promising therapeutics for hemophilia inhibitors. To that end we are investigating agents identified through a cell based, luciferase reporter, high throughput screen of 300,000 compounds for CHOP inhibitors. We hope that this work will identify agents that can be used to eradicate inhibitor antibodies in patients with Hemophilia.
Copper Deficiency Remains An Underappreciated Cause of Ineffective Hematopoiesis
Authors: Langston Amelia , Neely Jessica , Heffner Leonard , Griffith D. , Arellano Martha , Esper Gregory , McLemore Morgan L. , Ziegler Thomas , Kempton Christine , Sinha Rajni , Khoury H. Jean , Winton Elliott
Published: April 2020

Copper deficiency has long been recognized as cause of hematopoietic dysfunction, and diagnosis is straightforward if it is pursued. Over a three year period, we diagnosed copper deficiency in seven pts referred to our university-based hematology and BMT outpatient clinics for evaluation and treatment of myelodysplastic syndrome (MDS) or neutropenia/anemia. This represents approximately 3% of new outpatient referrals carrying an ICD-9 code corresponding to MDS or neutropenia. Patient characteristics are shown in Table 1. Three of the seven pts carried a presumptive diagnosis of MDS, and one had received several months of decitabine therapy. Two patients were actually referred for consideration of stem cell transplantation; the others were referred for evaluation of cytopenias. Six of the seven patients had been evaluated by a hematologist prior to referral. Only 3 of 7 pts in our series had recognized risk factors for copper deficiency such as prior gastric bypass or other GI dysfunction; postulated risk factors in the other pts are included in Table1. All pts in our series were anemic and leukopenic, with absolute neutropenia at presentation. In contrast, platelet counts were normal or elevated in all pts, consistent with most previous reports. Five of the seven pts in our series had neuropathy or myelopathy of variable severity. The hematologic abnormalities responded rapidly and completely to oral and/or IV copper replacement in all pts, although neurologic recovery was slow and incomplete.

Conclusions: Our experience indicates that copper deficiency is a relatively common cause of neutropenia and anemia, and the consequences of missing the diagnosis may be substantial, including performance of unnecessary transfusion, chemotherapy, and even stem cell transplantation. Normal or elevated platelet counts and concomitant neuropathy or myelopathy are important clues to the diagnosis. Furthermore, copper deficiency must be considered even in pts without obvious GI absorption abnormalities. Serum copper and ceruloplasmin assays should be incorporated into the routine evaluation of most pts with cytopenias and suspected MDS.

Age/gender Ref. diagnosis Risk factor Associated symptoms Serum copper (80–155 mcg/dl) Ceruloplasmin (21–53 mg/dl 53F MDS–RAEB unknown Severe sensory-motor neuropathy 29 7 43F MDS–RA Excess carbonated soda intake? Severe sensory-motor neuropathy 2 <2 55F Anemia, neutropenia Gastricbypass sensory-motor neuropathy 4 3 45F Possible MDS Gastricbypass None 4 3 66F Anemia, neutropenia Excess tea intake? Bone pain 2 Link 56F Anemia, neutropenia Partial gastrectomy Severe sensory-motor neuropathy 2 <2 36F Anemia, neutropenia Tetrathiomolybdate exposure (lubricants) Moderate sensory-motor neuropathy 1 2
BMI1 Interacts with FANCD2 at DNA Lesions and Prevents Chromosome Breaks
Authors: Chagraoui Jalila , Hebert Josee , Girard Simon , Sauvageau Guy
Published: April 2020

Bmi1 is a major determinant of hematopoietic stem cell (HSC) self-renewal. To date, much of our understanding of Bmi1 function comes from studies linking it to p16ink4a-Rb and p19arf-p53 pathways. However, several observations have raised the possibility that Bmi1 requirement for the maintenance of stem cell self-renewal ability extends far beyond its role in repressing the INK4a/ARF locus. The phenotypical similarities shared between Bmi1 deficiency in mouse and Fanconi Anemia (FA) patients (e.g., congenital aplastic anemia) together with the reported mono-ubiquitin ligase activities of proteins in both complexes and the emerging concept connecting DNA repair to stem cell properties, prompted us to evaluate Bmi1 implication in DNA damage repair. We now report that, like FA cells, Bmi1-deficient HSCs accumulate at G2/M phase of cell cycle. Moreover, Bmi1 is rapidly recruited to DNA damage foci in a PI3 kinase like kinase (PIKK)-dependant manner, in several cell types including hematopoietic stem cells. Upon induction of DNA lesions, Bmi1 interacts with several proteins involved in DNA damage response including γ-H2AX, MDC1, RAP80 and the monoubiquitinated form of FANCD2. We found that Bmi1 depletion did not hinder DNA damage-induced γ-H2AX foci formation and that RNF8-dependent signaling cascade is functional in absence of Bmi1. Furthermore, as observed in FA, loss of Bmi1 is associated with occurrence of spontaneous chromosome breaks and increased sensitivity to clastogenic agents. These chromosome breaks occur although FANCD2 monoubiquitination and loading onto chromatin is preserved. Together this work reveals an unsuspected role for Bmi1 in securing chromosome integrity and connects Polycomb genes to FA repair pathway.
Defective T Cell Migration in Chronic Lymphocytic Leukemia Is Repaired by Lenalidomide
Authors: Ramsay Alan G. , Svensson Lena , Hogg Nancy , Gribben John G.
Published: April 2020

We have previously demonstrated that multiple gene expression abnormalities are induced in T cells from chronic lymphocytic leukemia (CLL) patients including defects within the actin cytoskeleton signaling pathways that control immune recognition and motility (Gullu et al. JCI, 2005). T cell immune surveillance requires rapid migratory responses and LFA-1 (CD11a/CD18; αLβ2) is a promigratory receptor that engages the cytoskeleton to control migration. We hypothesized that CLL T cells may exhibit dysfunctional migration in response to ICAM-1, the principal ligand for LFA-1. Using time lapse microscopy, we observed significantly reduced chemokine SDF-1 (CXCL12) induced migration on ICAM-1 of CLL CD4 and CD8 T cells compared to age-matched healthy donor T cells. Healthy T cells tracked for 45 min displayed a random course of migration with an average speed of ~ 8 μm/min, whereas CLL T cells were slower ~ 5 μm/min (n=14, ~ 30% reduction, p<0.01). We further postulated that direct contact of CLL tumor cells with healthy T cells would induce this migratory defect. Healthy CD4 or CD8 T cells were cocultured with either allogeneic CLL B cells or allogeneic healthy B cells and subsequently used in migration assays. Co-culture with CLL cells resulted in significantly reduced T cell migration compared with co-culture with healthy B cells (~ 44% reduction in migration, n=6, p<0.01). Evidence that direct contact was required to induce this migratory defect was shown when no effect was observed when cell-cell adhesion was prevented by pretreatment of CLL cells with anti-ICAM-1 blocking antibody prior to primary co-culture with healthy T cells. This cancer-induced migratory defect was repaired when CLL T cells were pretreated with the immunomodulatory drug Lenalidomide (1μM for 1hr). Treatment with this agent enhanced the migratory potential of CLL T cells to a speed comparable to untreated and treated healthy T cells. The finding that lenalidomide can restore rapid migration in patient T cells provides evidence that this agent may increase immune surveillance in CLL patients.
Treatment with Lenalidomide (Revlimid®) Upregulates CD40 Expression on CLL Cells and Increases Their Sensitivity to CD40-Mediated Cytotoxicity in Vitro
Authors: Glezer Emilia , Ferrajoli Alessandra , Reuben James M. , Corral Laura G
Published: April 2020

Lenalidomide, an approved drug for the treatment of multiple myeloma and myelodysplastic syndromes with 5q deletion is currently undergoing clinical investigation in chronic lymphocytic leukemia (CLL). Although the exact mechanism of action of lenalidomide in CLL is not known, it was shown to augment anti-CD40 induced cytotoxicity in human multiple myeloma cells (Tai et al. Cancer Res. 2005) and is currently being evaluated in combination with an anti-CD40 agent in a multiple myeloma clinical trial. While signaling of the costimulatory receptor CD40 promotes survival, proliferation, and differentiation of normal B cells, it can cause activation-induced cell death in malignant B cells. In addition, ligation of CD40 by CD40L (CD154) was shown to sensitize B-CLL cells to apoptosis (Dicker et al. Blood 2006). In a phase II clinical study (Ferrajoli et al. Blood 2008), blood samples from 12 CLL patients were collected at baseline and after 1 week, 4 weeks, 3 months and 6 months of treatment with lenalidomide. Samples were shipped overnight to (San Diego) for the phenotypic monitoring of CD40 expression on CD19+CD5+ B-CLL cells by flow cytometry. Changes in CD40 expression were also measured in B-CLL cells treated in vitro with lenalidomide. In addition, samples were used for in vitro cytotoxicity assays that included treatment with fludarabine or valproic acid in combination with lenalidomide; and a CD40-induced cytotoxicity assay using CD40 Ligand (CD154)-transfected murine fibroblasts and lenalidomide. In 6 out of 12 patients, lenalidomide therapy significantly upregulated the expression of CD40 on B-CLL cells above pretreatment levels (4.5±4.5% vs. 49.5±32.7 %, p=0.020). Moreover, B-CLL cells obtained at baseline from 6 patients and treated in vitro with 1 μM of lenalidomide (a concentration corresponding to clinically achievable plasma levels) for 24h and/or 48h exhibited significantly higher expression of CD40 than B-CLL cells exposed to DMSO vehicle (1.95±0.23 fold increase; p=0.038). Exposure to lenalidomide in vitro neither caused apoptosis of B-CLL cells nor increased their sensitivity to fludarabine or valproic acid cytotoxicity (n=9). However, when B-CLL cells obtained at baseline from 6 patients were treated in vitro with lenalidomide, they became more sensitive to apoptosis induced by CD40 Ligand (CD154)-transfected murine fibroblasts than DMSO treated cells (62.6±16.8% lenalidomide vs. 43.2±16.1% DMSO; p=0.032). Lenalidomide had no significant effect on the viability of B-CLL cells co-cultured with control non-transfected murine fibroblasts. Our data suggest a potential therapeutic use of lenalidomide in combination with CD40 ligation in CLL and warrant further investigation.
Richter Transformation of Chronic Lymphocytic Leukemia: Incidence, Risk Factors and Outcome
Authors: Alipour Sina , Leitch Heather , Vickars Linda M , Foltz Lynda M , Galbraith Paul F , Leger Chantal S , Ramadan Khaled M
Published: April 2020

Richter transformation (RT) is a rare complication of chronic lymphocytic leukemia (CLL). There is little information in the literature about its risk and outcome. In this study we assessed the incidence, presenting characteristics and outcomes of patients (pts) with CLL who developed RT. An electronic database search of pts with CLL who presented at St Paul’s Hospital between 1969 and 2007 was performed. Among 465 pts with CLL, 24 pts (5%) developed RT. Presenting features included B-symptoms (17%), lymph node enlargement (58%), progressive cytopenia (29%), hypercalcemia (4%), and spleno/hematomegaly (13%). The median age at diagnosis of CLL and RT were 64 y (range 33–80 y) and 67 y (range 48–81 y) respectively. The median time to transformation from CLL diagnosis was 61 months (range 1–257 m). Twenty one patients (88%) had been previously treated for CLL. Seventeen patients (71%) had received >1 prior therapy. The median lymphocyte count at diagnosis was 12 ×109/L (range 4–120 ×109/L). Six patients (25%) are still alive with a median follow up of 38 m (range: 3–66 m). The only predictive factor for better survival post-transformation on univariate analysis was age of less than 60 y at CLL diagnosis (p=0.01). Other factors such as CLL Rai stage, lymphocyte count at diagnosis were not predictive for survival. This group of patients was compared with randomly selected group of patients with CLL but did not have RT. The baseline characteristics of the groups are presented in the table. No significant differences were found between the two groups in terms of gender, age at diagnosis, Rai stage or median lymphocyte count at diagnosis. The 5 and 10 year OS for the RT group were 76% and 39% compared to 93% and 84% for the CLL group (p= 0.002), respectively. In summary, RT significantly shortens the survival of CLL patients. There were no obvious predictive factors for RT in CLL pts at diagnosis.

Table: Baseline characteristics of Richter and CLL groups.

Parameter Richter group (%) CLL group (%) P value Number 24 37 Sex: M/F (ratio) 16/8 (2:1) 22/15 (1.5:1) 0.052 Age at Diagnosis: Median (range) 64 (33–80) 60 (37–85) 0.6 Rai stage at diagnosis: 0, 1+2, 3+4 7, 16,1 (29, 67, 4) 23, 14, 0 (62, 38, 0) 0.3 Median lymphocyte count at diagnosis (range) 12 ×109/1 (4–120) 8 × 109/1 (5–394) 0.056

Fig: OS of pts with RT compared with CLL pts and no RT Fig:. OS of pts with RT compared with CLL pts and no RT
The RALGEF Pathway Contributes to the Pathogenesis of Chronic Myelogenous Leukemia by BCR/ABL
Authors: Fredericks Jessica , Parikh Chaitali , Subrahmanyam Ramesh , Ren Ruibao
Published: April 2020

Selected inhibitors of the BCR/ABL tyrosine kinase have shown a remarkable clinical activity in patients with chronic myelogenous leukemia (CML). However, these drugs do not completely eradicate leukemic cells and drug resistance emerges. Identification of additional contributors to the pathogenesis of CML remains to be important for developing strategies for overcoming resistance to BCR/ABL kinase inhibitors and for eradicating leukemic cells. We and others have previously shown that expression of BCR/ABL in mouse bone marrow cells by retroviral transduction and transplantation (BMT) efficiently induces a CML-like myeloproliferative disorder (MPD). Using this BMT model for CML, we have found that the Grb2 SH2 binding site Y177 of BCR/ABL, which is critical for BCR/ABL to activate the RAS pathway, is required for the induction of CML-like disease by BCR/ABL. We have further shown that oncogenic NRAS (NRASG12D) can rescue the defect of the Y177F mutant BCR/ABL in the induction of CML-like disease. To further elucidate the pathways downstream of NRAS responsible for this “rescue” effect, we utilized three well-characterized NRAS effector mutants, G12D/T35S (activating RAF but is impaired in binding PI3K and RALGEF), G12D/E37G (activating RALGEF but not RAF or PI3K), and G12D/Y40C (activating PI3K but not RAF or RALGEF). None of the three NRAS effector mutants could induce hematological malignancies by themselves, but all were capable of “rescuing” the CML defect of BCR/ABLY177F to some extent. Interestingly, the NRAS G12D/E37G effector mutant was the most efficient. We further examined the role of RALGEF pathway by introducing an activated RALGDSCAAX into our BCR/ABLY177F mouse model. RALGDS-CAAX was able to cooperate with BCR/ABLY177F to induce a CML-like MPD in nearly half of mice whereas RALGDSCAAX alone did not induce diseases in any mice. As a complement to this study, we also introduced dominant negative RAL A or RAL B in the background of BCR/ABL. This resulted in a significant delay in disease onset. Taken together, this data sheds light on the importance of the RALGEF pathway in BCR/ABL-mediated CML induction and supports the notion of inhibition of this pathway as a potential therapeutic strategy.
Combination Therapy of Small Molecule Inhibitor PHA-739358 and Tyrosine Kinase Inhibitor Imatinib Yields Synergistic Antiproliferative Effects and Suppresses Emergence of Resistance of Chronic Myeloid Leukemia in Vitro
Authors: Balabanov Stefan , Gontarewicz Artur , Keller Gunhild , Moll Jürgen , Bokemeyer Carsten , Holyoake Tessa L , Brummendorf Tim H.
Published: April 2020

The success story of Imatinib (IM) in chronic myeloid leukemia (CML) is comprised by primary or acquired resistance to IM, insufficient therapeutic options for advanced stages of disease and limited effects on immature hematopoietic stem cells emphasizing the need for novel therapeutic strategies. We recently reported on PHA-739358, which shares activity of a pan-Aurora and an ABL kinase inhibitor. In this study, the individual contribution of both pathways to the effect of PHA-739358 was further investigated and a dose dependent reduction of BCR-ABL activity starting at very low concentrations was observed. In contrast, inhibition of the phosphorylation of Aurora kinase down stream target phospho-histone H3-Ser10 required higher amount of the inhibitor suggesting a predominant BCR-ABL mediated effect of PHA-739358 at low concentration. Simultaneous short-term treatment with PHA-739358 and IM resulted in pronounced apoptosis of wild type or low-grade IM-resistant BCR-ABL expressing cells while no such effects were observed in BCR-ABL negative or highly IM-resistant T315I mutants. In primary CD34+ cells of CML patients including non-dividing quiescent leukemic stem cells combination therapy with IM and PHA-739358 revealed a synergistic antiproliferativ activity which also affected immature CD34+38-cells. However, neither mono- nor combination therapy led to a significant induction of apoptosis. In addition, PHA-739358 did not influence quiescent stem cells analysed in the CSFE assays. Another focus of interest was the emergence of resistance secondary to treatment with PHA-739358, where resistant clones were found considerably less frequent than in IM-treated cells. Interestingly, PHA-739358 resistant cells did not reveal mutations in Aurora kinases A and B or in BCR-ABL kinase domain and remained IM-sensitive. Furthermore, simultaneous administration of PHA-739358 and IM considerably reduced the number of resistant clones and virtually abolished emergence of resistance at 2-times IC50 concentration of PHA- 739358 (plus IM at IC50). In conclusion, PHA-739358 potently inhibits proliferation of CD34+ stem und progenitor cells including immature CD38- subpopulation. However, in line with other tyrosine kinase inhibitors no induction of apoptosis in quiescent stem cells can be achieved. Remarkably, combination of PHA-739358 and IM prevents emergence of resistance rising the hope for a durable control of CML.
Patients with Chronic Myeloid Leukemia with Variant Philadelphia Chromosome (Ph) Translocations Have a Similar Outcome as Those with Classic Ph When Treated with Imatinib or 2nd Generation TKI
Authors: Batty Nicolas , Kantarjian Hagop , Borthakur Gautam , Ravandi Farhad , O’Brien Susan , Estrov Zeev , Shan Jianqin , Verstovsek Srdan , Cortes Jorge
Published: April 2020

Background: Variant Philadelphia chromosome (Ph) translocations frequently involving 1-2 additional chromosomes besides 9 and 22 and represent 5–10% of patients (pts) with chronic myeloid leukemia (CML). The European LeukemiaNet recommendations provide a warning for patients with variant translocations, although there is limited information about their outcome after therapy with tyrosine kinase inhibitors (TKI). Our prior analysis mostly among pts who had failed prior interferon suggested that these pts had similar outcome to those with classic Ph translocations when treated with imatinib (El-Zimaity et al; Br. J Haematol 2004).

Aims: To explore the characteristics and outcome of patients with variant translocations treated with frontline imatinib or 2nd generation TKI (dasatinib or nilotinib) after imatinib failure.

Methods: We reviewed the outcome of all pts with CML treated at our institution in 3 groups:

early chronic phase (CP) receiving imatinib as initial therapy, and CP treated with 2nd generation TKI after Imatinib failure, accelerated phase (AP) treated with 2nd TKI after imatinib failure.

Results of pts with variant Ph were compared to those with classic Ph.

Results: Among 554 pts (278 CP frontline imatinib, 190 CP post imatinib failure, 86 AP post Imatinib failure) 33 (6%) had variant Ph (21[8%], 6[3%], 6[7%], in each of the 3 groups, respectively). Median follow up is 55 months (mo) (2 – 90), 24 (1 – 53) mo and 29 (5 – 46) mo, respectively, for the 3 groups. Results are summarized in the following tables:

Frontline Imatinib Therapy Percentage Variant Ph Classic Ph P value N=21 N=255 MCyR 95 95 1 CCyR 86 89 0.49 2-yr EFS 83 93 0.93 2-yr TFS 94 96 0.7 2-yr OS 100 99 0.48 Second generation TKI Variant Ph Chromosome Variant Ph Classic Ph P value Chronic Phase N = 6 N = 78 MCyR 100 75 0.34 CCyR 100 72 0.34 2-yr EFS 100 80 0.27 2-yr OS 100 98 0.71 Accelerated Phase N=6 N=80 MCyR 33 38 1 CCyR 33 32 1 2-yr EFS 25 41 0.41 2-yr OS 100 89 0.44

Conclusion: Pts with variant Ph have a similar prognosis to those with classic Ph translocations when treated with imatinib as initial therapy or with 2nd generation TKI after imatinib failure. The warning category for these patients may no longer be needed in the era of TKI.
Imatinib Induced Molecular Response in CD34+ Hematopoietic Stem and Progenitor Cells of Patients with Chronic Myeloid Leukemia Following the First Week of Therapy
Authors: Bruennert Daniela , Czibere Akos , Bruns Ingmar , Pechtel Sabrina , Gattermann Norbert , Kronenwett Ralf , Haas Rainer , Frank Neumann
Published: April 2020

Imatinib is a selective bcr-abl tyrosine-kinase inhibitor, successfully used as standard therapy for patients with chronic myelogenous leukemia (CML) in chronic phase. We examined the effect of Imatinib on the gene expression transcriptome pattern of CD34 positive hematopoietic stem and progenitor cells of 6 patients with newly diagnosed CML following the first week of therapy. Affymetrix U133A 2.0 microarrays covering 21.722 probe sets were used to analyze and compare the gene expression profiles of CD34+ cells from peripheral blood of 6 patients with newly diagnosed CML in chronic phase before and 7 days after the initiation of Imatinib first line therapy. One patient was female and 5 male. Their median age was 61 years (range 32–79 years). In addition, quantitative molecular monitoring of bcr-abl was performed using Light Cycler technology. Using a supervised hierarchical cluster analysis of the gene expression data we found that 303 genes were differentially expressed with a lower bound of at least 1.2 fold (−2.58 to −1.2 and 1.2 to 2.09). Of these, 183 genes were down-regulated and 120 genes were up-regulated comparing day 1 with day 7. A considerable number of the down-regulated genes are involved in the regulation of cell cycle (CDC2, CDC20, CCNB1, CCNB2) and DNA replication (TOP2A, POLE2, MCM-complex). In contrast, genes coding for proteins governing adhesion and cell migration were up-regulated (CD44, L-Selectin). The observations of the gene expression data of the aforementioned genes could be confirmed by quantitative real-time PCR. After 10 months of Imatinib therapy (range 6–13 months) all 6 patients reached in median a 3 log reduction (range 1.5–3 log reduction) of their bcr-abl mRNA. Bcr-abl FISH analysis confirmed that the majority of CD34+ cells examined were Ph-Chromosome positive. These data indicate that in the early phase of treatment Imatinib mainly acts as an inhibitor of cell cycle and replication which results in a decreased proliferation. The silencing of the Ph-Chromosome positive cells is associated with an up-regulation of genes coding for adhesion during the first week of therapy.
Role of the Urokinase Receptor (uPAR) in the Cross-Talk of Hematopoietic Stem Cells with the Bone Marrow Microenvironment
Authors: Montuori Nunzia , Ricci Patrizia , Serio Bianca , Visconte Valeria , La Penna Claudio , Pesapane Ada , Risitano Antonio M. , Rotoli Bruno , Rossi Guido , Ragno Pia , Selleri Carmine
Published: April 2020

The urokinase-type plasminogen activator receptor (uPAR) is a cell-surface receptor involved in cell adhesion and migration. uPAR binds urokinase (uPA) and vitronectin (VN) and interacts with integrins and chemotaxis receptors. Soluble forms of uPAR (suPAR) have been detected in human plasma and urine. A cleaved form of suPAR (c-suPAR), lacking the N-terminal domain and exposing the sequence SRSRY (aa 88–92), stimulates cell migration by activating fMLP receptors. We recently demonstrated uPAR involvement in G-CSF-induced CD34+ hematopoietic stem cell (HSC) mobilization. We also demonstrated that c-suPAR could induce mobilization of hematopoietic stem/progenitor cells in mice. Since HSC mobilization and homing to bone marrow (BM) are mirror image processes which utilize the same mediators and similar signaling pathways, we investigated whether uPAR and its ligands could play a role in regulating CD34+ HSC interactions with the BM stroma, thus also contributing to HSC homing and engraftment to the BM. We found expression of uPA and VN in cultures of human BM stroma cells. Interestingly, stroma cells also produced suPAR and high amounts of c-suPAR, exposing the chemotactic SRSRY sequence. The role of the different soluble forms of uPAR produced by stroma cells in regulating HSC interactions with the BM microenvironment was analyzed by long term cultures (LTC) of BM and G-CSF mobilized CD34+ HSCs, in the presence of suPAR or the uPAR-derived uPAR84–95 peptide, corresponding to the active site of c-suPAR. Both suPAR and the uPAR84–95 peptide increased the number of adherent and released clonogenic progenitors from LTC of BM and G-CSF mobilized HSCs. To elucidate the mechanism of suPAR and c-suPAR effects on CD34+ HSC interactions with the stromal microenvironment, in vitro adhesion and proliferation assays were performed on CD34+ KG1 cells. suPAR treatment determined a significant increase in CD34+ KG1 cell adhesion whereas c-suPAR increased cell proliferation. Taken together, our results indicate that BM stroma produces soluble forms of uPAR that regulate CD34+ HSC interactions with BM microenvironment, their local proliferation and trafficking from and to BM.
Monitoring of Donor Chimerism in CD34+ Peripheral Blood Progenitors Allows to Detect Minimal Residual Disease after Allogeneic Stem Cell Transplantation -Results of a Randomized Trial
Authors: Bornhaeuser Martin , Oelschlaegel Uta , Bug Gesine , Platzbecker Uwe , Lutterbeck Karin , Kiehl Michael G , Schetelig Johannes , Kiani Alexander , Illmer Thomas , Schaich Markus , Theuser Catrin , Mohr Brigitte , Brendel Cornelia , Fauser Axel A , Klein Stefan , Martin Hans , Ehninger Gerhard , Thiede Christian
Published: April 2020

Relapse of hematological malignancy remains a major complication after allogeneic stem cell transplantation. This is especially true for patients receiving minimal or reduced-intensity conditioning therapy. Analysis of donor chimerism (DC) is an important diagnostic tool to assess the risk of relapse after allogeneic stem cell transplantation, especially in patients lacking a specific marker suitable for monitoring of minimal residual disease. The sensitivity of a standard PCR assay amplifying short-tandem-repeat sequences can be improved significantly by investigating sorted CD34+ peripheral blood cells. We prospectively compared the serial analysis of DC in selected CD34+ cells and unmanipulated whole blood (WB) within a randomised study in 131 patients with CD34+ hematological malignancies (AML, ALL and MDS) surviving more than 100 days after transplantation. The primary end-point was the association between decreasing CD34+ DC and haematological relapse. Whenever a decreasing CD34+ or WB DC was confirmed and no signs of active GvHD were present, a rapid taper of CsA or tracolimus (50% every 5–7 days) was suggested. If no GvHD occurred within 14 days after the stop of CsA or tacolimus, patients were scheduled to receive donor lymphocyte infusions (DLI) in incremental doses. The cumulative incidence of relapse was significantly increased in patients with decreasing or incomplete CD34+ DC (62% vs. 38%, p=0.01). This was associated with a lower probability of overall survival (20% vs. 39%, p=0.03). The interval between the decrease in CD34+ DC and hematological relapse was 35 days (range 0–567) in the study group compared to only 8 days (range 0–63) in the control group monitored by WB DC analysis (p=0.05). The median time between a decrease in CD34+ DC and WB DC was 14 days (range, 0 to 445). Patients receiving preemptive therapy triggered by decreasing CD34+ DC had a significantly higher probability of disease-free survival compared to cases monitored and treated according to WB DC (19% vs. 0%, p=0.009). Multivariate analysis revealed age, disease-risk and decreasing CD34+ DC as independent risk-factors for overall survival in the study group. In summary, we could demonstrate that the quantification of DC in CD34+ selected cells is a sensitive method to predict relapse and survival after allogeneic SCT. Although this technology opens a window for preemptive therapy, new treatment approaches have to be employed to improve the overall outcome of patients with recurrence of residual disease after allogeneic stem cell transplantation.
Similar Outcomes Using Myeloablative Versus Reduced Intensity and Non-Myeloablative Allogeneic Transplant Preparative Regimens for AML or MDS: From the Center for International Blood and Marrow Transplant Research
Authors: Luger Selina , Ringden Olle , Peréz Waleska S. , Zhang Mei-Jie , DiPersio John F. , Sierra Jorge , Tallman Martin , Weisdorf Daniel J. , Pulsipher Michael
Published: April 2020

Use of reduced intensity conditioning (RIC) and nonmyeloablative (NMA) regimens in allogeneic transplantation (allo HCT) for AML and MDS has rapidly increased over the past decade. Efficacy of these approaches compared to myeloablative (MA) conditioning has not been clearly established. We compared disease status, donor, graft and recipient characteristics and outcomes of 3731 MA HCT with 1500 RIC/NMA procedures performed at 217 centers between 1997 and 2004 (median f/u 58 vs. 40m, MA vs. RIC/NMA). Patients in the MA group were younger (median age 42 (18–68) vs. 55 (18–69) years), more frequently in CR1, and more often were transplanted from matched sibling grafts, compared to recipients of RIC/NMA. NMA recipients were less often transplanted with >10% blasts and had a lower performance scores. RIC recipients received fully matched unrelated donor (URD) grafts and PBSC as hematopoietic source more often than MApatients. Proportion with adverse cytogenetics, IPSS categories for MDS patients, median time to first relapse in AML CR2 patients, and marrow blasts at HCT were similar in the conditioning groups. 5-years univariate probabilities and multivariate relative risk (RR) outcomes of relapse, transplant-related mortality (TRM), leukemia-free (LFS) and overall survival (OS) are shown below:

Outcome: MA RIC PBSC RIC BM NMA * overall p<0.001 Univariate: Relapse, % 32 (31–34) 39 (35–43) 42 (36–48) 43 (38–48) TRM,% 34 (32–36) 34 (30–38) 37 (31–43) 36 (31–41) Adjusted LFS, % 33 (31–34) 30 (26–35) 28 (23–34) 24 (20–29) Adjusted OS, % 34 (33–36) 33 (29–37) 32 (27–38) 26 (21–30) Multivariate: Relapse* 1.00 1.05 (0.92–1.21) 1.51 (1.23–1.85) 1.64 (1.38–1.94) TRM* 1.00 1.26 (1.03–1.54) 1.36 (1.01–1.83) 1.55 (1.20–1.99) Treatment 1.00 1.03 (0.90–1.17) 1.17 (1.01–1.36) 1.28 (1.12–1.45) failure* Mortality* 1.00 1.03 (0.91–1.16) 1.12 (0.96–1.30) 1.20 (1.05–1.36)

MA regimens were associated with significantly less relapse (RR with RIC/NST 1.32 (0.19–1.46), p <0.001). While early TRM was substantially less with RIC/NMA approaches, 5 year TRM is equivalent (p=0.48). This leads to marginally better 5 year LFS (RR 1.08, p=0.05) but similar OS (RR 1.05, p=0.25) with MA vs. RIC/NST. These data indicate that late TRM negates any early advantage offered by RIC and NMA regimens, resulting in similar but slightly better 5 year LFS in recipients of MA conditioning. Similar outcomes with these RIC/NMA vs. MA regimens suggest the need for prospective trials comparing these transplant intensities in patients eligible for either approach.
Agricultural Pesticide Use Ã‚Â and the T(14;18) Connection to Lymphomagenesis
Authors: Roulland Sandrine , Agopian Julie , Lecluse Yannick , Navarro Jean-Marc , Gac Anne-claire , Briand Mélanie , Grenot Pierre , Gauduchon Pascal , Ruminy Philippe , Lebailly Pierre , Nadel Bertrand
Published: April 2020

Agricultural pesticide use has been repeatedly, but not consistently, associated with risk of non-Hodgkin lymphoma (NHL). Over the past years, it has become clear that NHL heterogeneity is setting many hurdles to this connection and that pertinent bio-markers must be identified according to malignant subtypes. Among those, t(14;18) translocation constitutes the early and promoting event of a cascade of causative effects leading to Follicular Lymphoma (FL): ectopic BCL2 expression leads to the rescue, accumulation, and developmental block of germinal center (GC) B-cells; there, protracted AID activity would favor the occurrence of complementary oncogenic hits and malignant progression, which arise from mistakes during somatic hypermutation (SHM) and class-switch recombination (CSR) processes. Although the significance of t(14;18) in blood from healthy individuals is yet unclear, evidence for a higher prevalence has been reported among farmers occupationally exposed to pesticides. Furthermore, recent findings have established that pesticide exposure is significantly associated with risk of t(14;18)+ NHL but not with t(14;18)-negative cases, suggesting that pesticides may act through a t(14;18)-dependant pathway. To get molecular insights into this connection, we followed and compared the status and clonal evolution of t(14;18)+ cells over a median of 9 years in blood samples issued from a cohort of farmers exposed to pesticides (n=112) with those of non-farmer controls (n=25). We report here that exposed individuals exhibit a much higher increase of t(14;18) frequency in blood, and that this increase result far less from a genotoxic effect of pesticides than from an immunogenic effect leading to an extensive clonal expansion of activated t(14;18)+ B cells. Strikingly, we demonstrate that such clones recapitulate the hallmark features of developmentally blocked FL cells as they 1) express high levels of BCL2/JH fusion transcripts; 2) retain the CD10 surface expression normally lost upon GC exit; and 3) sustain constitutive AID activity with the most advanced ones bearing, unexpectedly, early stigmata of AID-mediated genomic instability. Altogether these results show that expanded t(14;18)+ clones in farmers exposed to pesticides constitute bona fide FL precursors standing at various stages of tumor progression and support the notion of a direct connection between t(14;18) frequency in blood and malignant progression that could be could helpful in the identification of individuals at “high” risk for transformation to overt FL.
Signaling Diversity in Human Lymphoma B Cells and in Tumor Infiltrating T Cells Correlates with Follicular Lymphoma Patient Clinical Outcomes
Authors: Irish Jonathan M. , Houot Roch , Myklebust June H. , Czerwinski Debra K. , Nolan Garry P. , Levy Ronald
Published: April 2020

Introduction: Flow cytometry analysis of live cells from cryopreserved tumor samples provides the opportunity to study simultaneously the biology of both cancer cells and the patient’s immune system cells. Furthermore, single cell based approaches can identify and quantify subsets within mixed populations, such as heterogeneous primary tumors, without the need to physically separate cells. We have previously used flow cytometry to identify differences in signaling mechanism between healthy human B cells from two differentiation stages and to compare signaling kinetics of tumor and non-malignant B cells within primary human lymphoma specimens1.

Methods: Here we used barcoded phospho-specific flow cytometry to measure signaling events in live lymphoma B cells and tumor-infiltrating T cells from follicular lymphoma (FL) tumor samples obtained before patients received therapy. Patients were then stratified according to signaling and clinical outcome was examined within the resulting groups. We examined response to initial chemotherapy, which was a combination of cyclophosphamide, vincristine, and prednisone (cvp), and overall survival, measured as the time from diagnosis to last follow up or mortality.

Results: Differences in B cell receptor (BCR) signaling strength and kinetics characterized previously unappreciated diversity within the lymphoma B cell population. In some cases, BCR crosslinking triggered robust phosphorylation of AKT and ERK in one subset of lymphoma cells, while in another lymphoma subset within the same tumor sample, BCR crosslinking did not lead to phosphorylation of either protein. In these cases, both lymphoma B cell subsets expressed BCL2 and surface immunoglobulin heavy and light chain restricted to the tumor isotype. Patients whose lymphoma tumor contained a BCR insensitive cell subset (Group 2) had significantly worse responses to cvp therapy (p = 0.001) and lower overall survival (p = 0.003) than patients whose lymphoma cells displayed more homogeneous BCR signaling (Group 1). We next examined tumor infiltrating T cell signaling in the FL cases from Group 1, where no significant BCR insensitive subset was observed. Within Group 1, differences in the magnitude of IL-2, IL-7, and IL-15 mediated STAT5 phosphorylation in tumor infiltrating T cells further distinguished a set of patients with significantly higher overall survival (p = 0.04).

Conclusions: These results identify BCR signaling in lymphoma B cells and cytokine signaling in tumor infiltrating T cells as clinically relevant biomarkers for tracking and isolating lymphoma cell subsets and for monitoring immune system activity during therapy. By following patients over time, we can now determine whether cell intrinsic signaling diversity enables the emergence of therapy insensitive cancer cell subsets.
17-DMAG Targets the NF-κB Family of Proteins to Induce Apoptosis in CLL: Clinical Implications of Hsp90 Inhibition
Authors: Wagner Amy J , Hertlein Erin K , Raymond Chelsey A , West Derek A , Flynn Joseph M , Lin Thomas , Johnson Amy J , Byrd John C
Published: April 2020

Chronic lymphocytic leukemia (CLL) is a disease of accumulating B-cell lymphocytes for which there is currently no curative therapy. One attractive therapeutic target currently being explored in CLL is Hsp90, a chaperone which stabilizes various client proteins (Akt, Raf, ZAP-70) which are important for survival of CLL cells. Interfering with Hsp90 protein binding to these chaperone proteins leads to their rapid degradation. Our group and others have demonstrated that 17-allylamino-17-demethoxy-geldanamycin (17-AAG) depletes only select chaperone proteins and promotes only modest cytotoxicity in CLL. 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG) is a novel Hsp90 inhibitor with improved solubility, bioavailability and cytotoxicity in other cancer cell lines as compared to 17-AAG. We demonstrate that 17-DMAG more potently induces caspase dependent apoptosis of primary CLL cells, as compared to 17-AAG, but does not mediate apoptosis of normal T-cells and NK-cells. In primary CLL cells, 17-DMAG treatment leads to cellular depletion of a broader range of Hsp90 client proteins (Akt, Cdk9, ZAP-70 and IKK). IKK is the activating kinase of the NF-kB family of transcription factors. To validate the down-stream significance of this we used EMSA to confirm that NF-kB is constitutively active in CLL as compared to normal B-cells. We demonstrate that 17-DMAG effectively reduces NF-kB DNA binding and thereby decreases subsequent transcription of known NF-kB target genes (Mcl-1 and XIAP). In addition to the direct role of NF-kB on target genes, NF-kB inhibition indirectly increases Bim transcription through regulation of the FoxO3a transcription factor. Furthermore, the reciprocal regulation transcription of Bcl-2 family members Mcl-1 and Bim are early events that initiate membrane depolarization and activation of the caspase cascade, leading to 17-DMAG induced cytotoxicity. siRNA experiments to elucidate the direct importance of Bim to 17-DMAG treatment are ongoing at this time. In conclusion, our data demonstrate that the Hsp90 inhibitor 17-DMAG represents a novel multi-targeted inhibitor of several critical kinases, transcription factors and anti-apoptosis genes relevant to CLL survival. Given its oral formulation, which allows administration of 17-DMAG by continuous dosing and uninterrupted inhibition of Hsp90, initiation of phase II clinical trials in CLL that include detailed pharmacodynamic studies monitoring NF-kB target genes are indicated.

This work is supported by the Leukemia and Lymphoma Society of America and the D. Warren Brown Foundation.
Epithelial Cells of Donor Origin after Allogeneic HCT: Result of Cell Fusion, Transdifferentiation (plasticity) or Transfer of Epithelial Cell Precursors with the Graft?
Authors: Khan Faisal , Sy Sarah , Louie Polly , Smith Megan , Chernos Judy , Lee Robyn , Berka Noureddine , Sinclair Gary D. , Leigh Richard , Proud David , Russell James A. , Storek Jan
Published: April 2020

Hematopoietic stem cells (HSC) appear to carry a degree of developmental plasticity that allows them to differentiate across boundaries of lineage, tissue, and germ layer, as evidenced by findings of donor-type epithelial cells in recipients of hematopoietic cell transplantation (HCT). However, these claims are often subject to criticism due to either the artifacts associated with determining the recipient versus donor origin of cells using XY-FISH or cross-lineage cell fusion. Alternatively, donor-type epithelial cells post-transplant could originate from epithelial cells or their precursors transferred with the graft. Here we wished to determine whether donor nasal epithelial cells can be detected in post-transplant patients, and if so, then the underlying mechanism. We collected nasal scrapings and whole blood from 23 allo-HCT survivors, either early (2–3 months, n=5) or late (4–22 years, n=18) post-transplant. To avoid the limitations of XY-FISH, chimerism of the epithelial cells was determined as follows: Nasal cells were stained with cytokeratin (CK) and CD45 antibodies. True epithelial cells (CK+CD45−) were laser captured. DNA extracted from the captured epithelial cells and blood leukocytes was PCR amplified for a panel of 15 autosomal short tandem repeat (STR) markers and an XY-differentiating locus (ABI-Identifiler™). In addition, a combination of immunofluorescence microscopy for CK and FISH for two autosomes was used in 8 patients to assess cross-lineage cell fusion. Magnetic separation using CD36 microbeads and staining with CK was utilized to identify epithelial cell precursors in 3 additional peripheral blood stem cell graft specimens. In all 23 HCT survivors, epithelial cells of donor origin were identified accounting for 2.4% to 12.3% of the nasal epithelial cells. The percentage of donor origin nasal epithelial cells was significantly higher (p=0.004) in late (mean=7.45%) compared to early (mean = 3.88%) post-transplant. None of the nasal epithelial cells in 8 HCT survivors exhibited the presence of cell fusion, but CD36+CK+ epithelial cells were identified in all of the three graft specimens. In conclusion, even with a method obviating the artifacts of XY-FISH; donor origin epithelial cells were identified. This appears to be due to trans-differentiation of HSC into epithelial cells or the transfer of epithelial cells (or their precursors) with the graft, but not due to cell fusion.
Incidence and Risk Factors for Hypertension during Induction Chemotherapy for Pediatric Acute Lymphoblastic Leukemia
Authors: Freiberg Andrew S. , Kees-Folts Deborah , Dyer Anne-Marie , Taylor Megan
Published: April 2020

Background: The most common form of cancer in children is acute leukemia, with acute lymphoblastic leukemia (ALL) comprising approximately seventy-five percent of these. The initial month of therapy (induction) for all children with ALL includes high dose steroids. One of the side effects of these steroids is hypertension.

Objective: To determine the incidence and risk factors of hypertension during induction chemotherapy for acute lymphoblastic leukemia in children.

Methods: Retrospective analysis was performed of data collected from all children diagnosed with acute lymphoblastic leukemia (ALL) at Hershey Medical Center (HMC) in 2004 and 2005 (n = 37). Each blood pressure from presentation through the four weeks of induction was recorded. To minimize the effect of a single high blood pressure measurement, median systolic and diastolic pressures were determined for each of the four weeks of induction therapy. These pressures were compared to published age matched norms. For each patient and each week of therapy, the percentile (<50, 50–90, 90–95, >99) was determined.

Results: Thirty-seven children (21 male, 16 female) met study criteria, with a median age of five. Thirteen of these patients received prednisone and twenty-four received dexamethasone. Overall, 95% of children had mean systolic or diastolic pressures >90th percentile, 81 had pressures >95th percentile, and 51% were above the 99th percentile for at least one week of induction. By contrast, only 32%, 19%, and 11% had pressures at these levels at initial presentation. The incidence of significant hypertension increased significantly during the four weeks of induction. Only three children (10%) with pressures above the 95th percentile were clearly noted in the medical record to be hypertensive, and only two children (6.7%) with pressures this high were treated.

Conclusions: Hypertension is common and underrecognized during induction chemotherapy for childhood ALL. The incidence of hypertension increases weekly during induction chemotherapy.