bluuebunny/crossref_metadata_2025 huggingface dataset:
A 48-Week Open-Label Study of Senicapoc (ICA-17043), a Gardos Channel Blocker, in Patients with Sickle Cell Disease.
Authors: Swerdlow Paul , Ataga Kenneth , Smith Wally , Saunthararajah Yogen , Stocker Jonathan W.
Published: April 2020

The Gardos channel, a calcium-activated potassium channel that spans the membrane of red blood cells (RBCs), is a key pathway in RBC dehydration affecting the intracellular concentration of hemoglobin S (Hb S). Inhibition of the Gardos channel in patients with sickle cell disease (SCD) may prevent the formation of dehydrated sickle RBCs, decrease Hb S polymerization, and reduce symptoms. In a Phase II 12-week, randomized, double-blind, placebo-controlled, dose-finding study in 90 patients with SCD, senicapoc (ICA-17043), a novel Gardos channel blocker, demonstrated statistically significant and beneficial hematologic effects including increased hemoglobin levels and decreased indicators of hemolysis. Patients from this study were eligible to enroll in a 48-week, open-label extension study in which all patients received an oral daily dose (10 mg) of senicapoc. Safety assessments included clinical laboratory data, physical exams, vital signs, ECGs, and ophthalmologic exams. Of 56 eligible patients, 44 enrolled in the open-label study. Because all patients received active drug, no formal efficacy comparisons to placebo were available. Within-subject comparison versus baseline levels from the double-blind phase indicated that the beneficial effects of senicapoc were maintained during the open-label study. Patients demonstrated increases in hemoglobin (+6%), hematocrit (+6%), and RBC count (+6%), and decreases in dense cells (−24%), reticulocytes (−19%), indirect bilirubin (−30%), and lactate dehydrogenase (−18%). Senicapoc was generally well tolerated during the open-label extension study. No deaths occurred, and there were no serious adverse events attributable to senicapoc. Twelve of 44 patients discontinued from the 48-week treatment period. Two patients discontinued due to adverse events considered possibly or probably related to study medication (elevation in gamma glutamyl transferase [GGT] level and interstitial nephritis, respectively). Two additional patients discontinued due to adverse events (sickle cell crisis and pain) not considered related to study medication. Of the 8 remaining patients, 1 was lost to follow-up, and 7 discontinued for administrative reasons. The most common adverse events (≥10% of patients) during treatment included sickle cell pain crisis, arthralgia, back pain, headache, upper respiratory tract infection, limb pain, increased GGT, pyrexia, and rash. The only adverse events that occurred in 2 or more patients and considered possibly related to study medication were GGT elevation, rash, and headache. In conclusion, senicapoc 10 mg once daily for 48 weeks appears to be safe and well tolerated in patients with SCD. Hematologic and clinical laboratory data collected in this safety extension study are consistent with the beneficial hematological effects observed during the Phase II double-blind study.
Blocking Adhesion of Sickle Erythrocytes to Endothelial αVβ3 Using RNA Aptamer.
Authors: Nishimura Jun-ichi , Burnette Angela D. , Batchvarova Milena , Nimjee Shahid M. , Zennadi Rahima , Sullenger Bruce A. , Telen Marilyn J.
Published: April 2020

Vaso-occlusive crises are a major clinical feature of sickle cell disease (SCD), and the adhesion of sickle erythrocytes (SS-RBC) to vascular endothelium is crucial to the generation of vaso-occlusion. The αvβ3 integrin is a major endothelial ligand for SS-RBC. Soluble thrombospondin has been thought to serve as a bridging molecule between erythrocyte CD36 and endothelial αvβ3, and high-molecular-weight multimers of von Willebrand factor also promote SS-RBC adhesion to endothelial αvβ3. Our group recently identified SS-RBC adhesion to endothelium via ICAM-4 (LW, CD242)-αvβ3 interactions (Blood, 104, 3774, 2004), suggesting that αvβ3 is a critical target against which to develop reagents to prevent or treat vaso-occlusive crises in SCD. Aptamers are oligonucleotides that bind to molecular targets in a manner conceptually similar to antibodies and have been identified against a wide range of therapeutic targets. RNA aptamers that bind to human integrin αIIbβ3/αvβ3 and inhibit integrin binding to the ligands vitronectin and fibrinogen have previously been identified (Biochem Biophys Res Commun, 338, 956, 2005). We have now tested one of these high-affinity αvβ3 aptamers (clone 17.16: UUCAACGCUGUGAAGGGCUUAUACGAGCGGAUUACCC) for its ability to prevent adhesion of SS-RBC to endothelial cells. Aptamer clone 17.16 bound αIIbβ3 with a Kd of 7–10nM, and the binding was blocked by both Abciximab, a monoclonal antibody against human integrin αIIbβ3/αvβ3 via a common binding motif RGD (arginine-glycine-aspardic acid), and Eptifibatide, a small molecule that binds to the RGD moiety, suggesting the sharing of the binding site at the RGD moiety. To measure its anti-adhesion activity, an in vitro flow chamber assay was adopted. We first induced enhanced expression of αvβ3 on the immortalized HUVEC cell line EC-RF24, using each of the following: TNF-α (10ng/mL for 18.5 hrs at 37°C), thrombin (1nM for 5 min at 37°C), or histamine (25mM for 12 min at RT), and αvβ3 expression by treated and nontreated cells was measured by flow cytometry. The anti-adhesion activity of aptamer clone 17.16 was then confirmed using HUVECs treated with thrombin (1nM for 5mins at 37°C) using a flow chamber assay. Aptamer clone 17.16 (30nM) had anti-adhesion activity similar to LM609, an inhibitory antibody to αvβ3, whereas human complement 8 aptamer (Blood, 106, 57a, 2005, negative control, 30nM) did not have anti-adhesion activity. The anti-adhesion activity of aptamer clone 17.16 (30nM) was enhanced by a dose escalation to 60nM and 120nM. At 2 dynes/cm2, maximal inhibition of adhesion was 57%. In conclusion, we observed significant anti-adhesion activity of αvβ3 aptamer clone 17.16, and it has currently reached a stage suitable for modification to enhance stability and bioavailability. In addition, in vivo experiments in mice will employ intravital microscopy to measure anti-adhesion activity in vivo.

Figure Figure
New Approaches to the Regulation of Erythropoiesis and Hematoglobin Synthesis in β-Hemoglobinopathies: Studies with the Immunomodulatory Drug CC-4047.
Authors: de Parseval Laure A. Moutouh , Verhelle Dominique , Glezer Emilia , Brady Helen , Corral Laura G. , Morris Christopher L. , Muller George , Chan Kyle
Published: April 2020

Sickle cell disease (SCA) and β-thalassemia constitute worldwide public health problems and new therapies are needed. Inhibition of hemoglobin S (HbS) polymerization is a major target for therapeutic approaches in SCA. New experimental therapies including hydroxyurea (HU) have attempted to augment the synthesis of fetal hemoglobin (HbF) and improve upon current treatment. Clinical trial results have demonstrated that lenalidomide (Revlimid®), recently approved by the FDA, reduces or even eliminates the need for red blood cell transfusions in some anemic myelodysplastic patients. We have examined whether CC-4047, another IMiDs® immunomodulatory drug currently under evaluation for the treatment of hematological cancers could regulate erythropoiesis and hemoglobin synthesis. For this purpose, we used an in vitro culture model to differentiate human erythroid progenitors from bone marrow or peripheral blood CD34+ cells. We demonstrate that CC-4047 is a potent inducer of HbF that synergizes with HU, the sole drug approved for SCA, during erythroid differentiation of CD34 progenitors isolated from healthy donors and patients with SCA. In addition CC-4047 modulates erythropoiesis, slowing erythroid maturation and increased proliferation of immature erythroid cells. Unlike other inducers of HbF such as HU, 5-aza-cytidine and butyrate, CC-4047 is not cytotoxic. The percentage of F-cells are increased by 2 to 3 fold with pan-cellular expression of HbF and an increase of Fetal/Adult hemoglobin ratio that are relevant for SCA. Gene expression profiling of erythroid differentiated cells shows that CC-4047 regulates specific erythroid transcription factors and genes that participate in hemoglobin synthesis, and genes involved in cell cycle and cellular differentiation. CC-4047 controls globin gene expression during erythroid differentiation by increasing the rate of γ-globin and ε-globin transcription and by inducing sustained expression of fetal and embryonic hemoglobin synthesis.

Our results support the hypothesis that CC-4047, alone or in combination with certain current approved therapies comprising HU, can restore effective erythropoiesis and increase the ratio of fetal to adult hemoglobin. Moreover, CC-4047 has demonstrated anti-inflammatory and immunomodulatory effect in vivo that could help to limit the inflammatory state in sickle cell patients. In conclusion, administration of CC-4047 may represent an innovative new therapy for β-hemoglobinopathies.
Phase 2 Study of Bortezomib Weekly or Twice Weekly Plus Rituximab in Patients with Follicular (FL) or Marginal Zone (MZL) Lymphoma: Final Results.
Authors: De Vos Sven , Dakhil Shaker R. , McLaughlin Peter , Saleh Mansoor N. , Belt Robert , Flowers Christopher , Holladay Charles , Knapp Mark , Boral Anthony , Zhang Tracy , Goy Andre
Published: April 2020

Background: Bortezomib (btz, VELCADE®) has single-agent activity in relapsed/refractory non-Hodgkin’s lymphoma (NHL), including FL and MZL, on a twice-weekly schedule. Rituximab (R) is approved for relapsed/refractory CD20+ B-cell NHL. Preclinical studies show additive activity in lymphoma cell lines and lymphoma-bearing SCID mice. Btz and R have no overlapping toxicity. Weekly btz is active and safe in multiple myeloma trials.

Methods: Patients (pts) with measurable CD20+ FL or MZL, KPS ≥50%, and a response with TTP ≥4 months to any prior R-containing regimen, were randomized to btz 1.3mg/m2 twice weekly (d 1, 4, 8, 11 of 21-d cycle, 5 cycles, Arm A) or btz 1.6mg/m2 weekly (d 1, 8, 15, 22 of 35-d cycle, 3 cycles, Arm B). R 375mg/m2 was given weekly for 4 weeks from d 1, cycle 1, in both arms. Response was evaluated by independent radiology review (IRR) and by investigators using the IWRC.

Results: 81 pts (41 Arm A, 40 Arm B) were enrolled; median age: 64 yrs (63.0, 64.5); FL: 86% (80%, 93%); Stage IV: 47% (44%, 50%); KPS <90%: 20% (17%, 23%); LDH >ULN: 27% (32%, 23%); ≥1 extranodal site: 43% (41%, 45%); FLIPI score ≥2: 69% (71%, 68%). In total, 56% (54%, 58%) had ≥2 prior lines of therapy, 81% (80%, 82%) had prior CHOP/CVP ±R, 84% (78%, 90%) had prior R (alone or combination), and 8% (7%, 8%) had prior SCT. Median % of planned cumulative btz dose received: 75% (Arm A) and 99% (Arm B); median total btz: 19.5 and 19.0mg/m2; median dosing intensities per week: 1.6 and 1.3mg/m2. Response rates and TTP in evaluable pts are shown in Table 1. TTP curves by IRR are broadly overlapping between the arms (Figure). Most common AEs were fatigue, GI events, and peripheral neuropathy. Grade ≥3 AEs were more common in Arm A (54%) vs Arm B (35%), notably thrombocytopenia (10% vs 0%) and neutropenia (10% vs 3%), as were grade ≥4 AEs and serious AEs.

Conclusion: Weekly and twice weekly btz + R are active and well tolerated in relapsed/refractory CD20+ indolent NHL. The more convenient weekly regimen appears to offer similar efficacy with less toxicity. Weekly btz 1.6mg/m2 + R is being compared with R in a phase 3 study.

Response rates and time to events data By investigator By IRR Arm A, N=37 Arm B, N=40* Arm A, N=37* Arm B, N=40* *1 pt with no post-baseline assessment Response rate (CR+CRu+PR), n (%) 21 (57) 21 (53) 17 (46) 15 (38) CR+CRu 7 (19) 6 (15) 4 (11) 1 (3) PR 14 (38) 15 (38) 13 (35) 14 (35) SD 9 (24) 13 (33) 8 (22) 17 (43) PD 7 (19) 5 (13) 11 (30) 7 (18) Median TTP, mo 9.9 9.0 5.2 9.7 Figure Figure
Gene Expression Profile of Idiopathic Thrombocytopenic Purpura (ITP) Reveals Elevated Expression of Interferon Regulated Genes.
Authors: Sood Ruchira , Wong Wendy , Gotlib Jason , Jeng Michael , Zehnder James L.
Published: April 2020

Idiopathic thrombocytopenic purpura (ITP) is an acquired disorder of unknown etiology characterized by reduction in platelet counts. To search for mechanisms underlying the pathophysiology of ITP, we used DNA microarrays to determine the ITP whole blood expression profile. We hypothesized that transcriptional changes associated with the low platelet counts will provide us with new markers for improved diagnosis in addition to novel mechanistic information to understand and treat ITP. We analyzed 29 samples from ITP patients with ages ranging from 1–70 years, of which 15 samples were from either acute or chronic ITP patients with platelet counts ranging from 2,000–56,000 X109/L and additional 14 samples were collected from patients at different stages of recovery from ITP. We used Significance Analysis of Microarrays (SAM) to select genes that best distinguished the 15 ITP samples from 5 controls. We identified 490 differentially expressed genes constituting an ITP expression profile (Figure 1). One of the most significant findings was the relatively high expression of several genes known to be induced by Interferon (IFN) in the ITP samples. Elevated expression of interferon induced genes has also been reported in patients with several autoimmune diseases (Baechler, 2006) including SLE (Baechler, 2003; Bennet, 2003 & Crow, 2003) and systemic sclerosis (Tan, 2005) suggesting that this may a common theme underlying autoimmune diseases, including ITP. In addition, we previously found changes in expression of the interferon simulated genes during a cycle of changing platelet counts in two patients with cyclic thrombocytopenia strengthening a connection between interferon signaling and platelet counts. The greatest differential expression at high significance was observed in the interferon induced genes IFIT1 and IRF2 which showed 12 and 8 fold higher expression in ITP samples relative to normal samples. Additional genes found to be expressed at higher levels in ITP included EPSTI1, PARP 14 and INPP1, while genes with reduced expression in ITP were GNG10, PMX1 and G0S2. The ITP gene signature determined by us indicates a significant role for IFN signaling events in both pediatric and adult ITP, and further identifies additional differentially expressed genes. This approach points to a number of new targets to study in the pathogenesis and prognosis of ITP.

Figure Figure
Peripheral T Cell Apoptosis Is Associated with Homeostatic T Cell Proliferation and Plays a Significant Role in Post-Transplant Immune Deficiency.
Authors: Alpdogan Onder , Patel Neel , Suh David , Grubin Jeremy , McGoldrick Suzanne , Smith Odette M. , Lu Sydney , King Christopher , Kochman Adam , Hubbard Vanessa , van den Brink Marcel R.M.
Published: April 2020

Post-transplant peripheral T cell apoptosis is an important (but ill-defined) contributor to the delay in T cell recovery after an allogeneic bone marrow transplantation (BMT). Using murine bone marrow transplantation (BMT) models to analyze post-transplant peripheral T cell apoptosis, we found that donor BM-derived T cells had a higher percentage of apoptotic cells, with decreased BCL-2 levels in young (3-month old) and old (20-month old) recipients of T cell-depleted (TCD) and T cell-repleted allogeneic BMT. Allogeneic recipients of donor BM deficient of Fas, BAX and TRAIL, or with overexpression of BCL-2 and AKT showed no decrease in donor-derived apoptotic T cells, suggesting that these molecules are not directly responsible for post-transplant peripheral T cell apoptosis. However, we noted that both CD4+ and CD8+ Memory T cells (CD44high) exhibited increased apoptosis when compared to naive T cells post-BMT and therefore used RAG-2-eGFP transgenic mice to study whether T cell maturation affects peripheral T cell apoptosis. We found increased numbers of apoptotic cells in mature (eGFPlow) T cells compared to non-divided (eGFPhigh) recent thymic emigrants in RAG-2-eGFP transgenic mice and in recipients of RAG-2-eGFP transgenic (Tg) BM. Interestingly, most of the apoptotic T cells were of eGFPlowCD44high memory phenotype in both CD4+ and CD8+ T cells, while eGFPlowCD44low naive T cells showed no sign of enhanced apoptosis. Moreover, we found similar increased levels of apoptosis of homeostatically proliferating T cells (CD44high-memory-like cells) in recipients of lineage-depleted allogeneic RAG2-eGFP Tg BM or B6 BM. We then tested the effects of homeostatic T cell proliferation on peripheral T cell apoptosis in recipients of syngeneic CFSE-labeled T cells and found again increased levels of apoptosis in the fast proliferating populations of both CD4+ and CD8+ T cells. These cells expressed high levels of CD44 but down-regulated CD62L on their surface and in contrast to allogeneic proliferation exhibited low levels of CD25 expression and high level of IL-7Rα expression (75%). In addition, BCL-2 expression was down-regulated in the fast-proliferating cell population in both CD4+ and CD8+ T cells.

We conclude that newly generated donor BM-derived T cells have increased peripheral T cell apoptosis, which is associated with homeostatic proliferation and does not involve Fas, BAX, TRAIL, and AKT. This mechanism of peripheral T cell apoptosis, which has not been recognized previously, plays a significant role in post-transplant T cell deficiency and represents a promising target for novel strategies to overcome post-transplant immune deficiency in BMT recipients.
Rapid and Massive Expansion of Cord Blood Derived Cytokine Induced Killer (CIK) Cells: An Innovative Proposal for the Treatment of Leukemia Relapse after Cord Blood Transplantation.
Authors: Introna Martino , Franceschetti Marta , Ciocca Alice , Borleri Gianmaria , Conti Elena , Golay Josee , Rambaldi Alessandro
Published: April 2020

Cytokine induced killer cells (CIK) are CD3+/CD56+ T/NK cells with cytotoxic potential against leukemic and other tumor cells but not normal bone marrow in vitro and in vivo. They are expanded in vitro with rhIL-2 after stimulation of peripheral blood mononuclear cells with OKT3 and IFN-γ. We have shown in a recent phase I study that 107/kg allogeneic CIK cells can be safely given to patients relapsing after allogeneic bone marrow transplantation and show evidence of anti-leukemic activity in vivo with very little GVHD. Cord blood (CB) transplantation is progressively becoming an extensively used treatment for patients with malignant disorders. One major limitation of this procedure is the lack of donor derived cells to perform donor lymphocyte infusions in case of relapse. In order to be able therefore to extend the use of CIK cells to the CB transplantation setting, we have standardised a 21 days expansion protocol to produce CIK cells starting from very small amounts of nucleated cells isolated from cord blood. Using this protocol, 15x106 mononuclear cells (MNC) from CB containing a mean 0.3x106 CD3+/CD56+ yielded on average 805 x 106 MNC (50 fold expansion) containing 630 x106 CD3+/CD56+ cells (corresponding to a fold expansion of 1860 for CIK). In order to transfer the method to a clinical setting, we explored the possibility of expanding the residual cells recovered from the empty bags after CB transplantion. Three used CB bags were returned to the laboratory after transplantation and repeatedly washed. An average of 22 x106 nucleated cells could be recovered, yielding a mean 473 x106 CD3/CD56+ cells at the end of the culture period (1485 fold expansion of CIK cells). CIK cells generated from CB showed strong cytotoxic activity against a variety of tumor target cell lines including B and T lymphomas and myeloid leukemias (42–72% killing at a 30:1 E:T ratio). More importantly, they were cytotoxic against AML blasts isolated from 2 patients (41% lysis). During expansion CB derived CIK cells upregulated the NKG2D marker from a mean fluorescence intensity of 49 to 209. Furthermore they expressed perforin and granzyme molecules in >90% of cells. These observations open up the possibility of a future clinical application of this protocol, performed in GMP conditions. Patients relapsing following cord blood transplantation may be treated with CIK cells expanded from the same cord blood unit, where donors would not be anymore available for cell mediated immunotherapy.
Dynamics of BCR-ABL Kinase Domain Mutations in Patients with Chronic Myeloid Leukemia (CML) after Treatment with One, Two or Three Tyrosine Kinase Inhibitors (TKI).
Authors: Jabbour Elias , Jones Dan , Kantarjian Hagop , O’Brien Susan , Garcia-Manero Guillermo , Giles Francis , Wierda William , Cortes Jorge
Published: April 2020

Dasatinib (D) and nilotinib (N) are potent tyrosine kinase inhibitors (TKIs) with activity against many imatinib (IM) resistant BCR-ABL kinase domain mutants, except T315I. In vitro mutant models have selected specific mutations occurring after incubation with IM, D and N. Therapy with these new TKI may select for patients with T315I or other mutations relatively insensitive to them. We assessed the change in mutation status of the bcr-abl kinase domain (codons 220 to 500) in 113 patients (pts) with CML who received therapy with D and/or N after imatinib failure. Median age was 60 years (range, 21 to 82 years). Seventy-one (63%) pts received prior interferon (IFN). Median time on imatinib was 28 months (range, 2 to 78 months). At the time of imatinib failure, mutations were detected in 46 of 85 (54%) pts who had DNA sequencing. The evolution of mutations after a second TKI was as follows (Table 1).

Twenty pts received a third TKI after failing IM and a second TKI. The evolution of mutations in this cohort was as follows (Table 2).

Overall, 19 of 101 evaluable pts (19%), cases had new mutations emerge following TKI switch 17 after a 2nd TKI (12 nilotinib, 5 dasatinib), and 2 after a 3rd TKI (2 dasatinib). We analyzed whether these N- and D-associated new mutations were at sites that have been detected following D and N treatment in vitro (Burgess et al, PNAS 2005; Bradeen et al, Blood 2006; Von Bubnoff et al, Blood 2006). Only 14/46 (30%) kinase domain mutations that developed after D (7) or N (7) corresponded with an in vitro-identified site. Only 5 of 134 (4%) mutations identified were T315I (3 after dasatinib, 2 after nilotinib), but the mutation status of these patients was unknown after IM. We conclude that the spectrum of mutations that develops in vivo after TKI switch is broader and includes common imatinib-resistance sites as well. There appears to no marked increase in the incidence of T315I mutation after TKI switch.

Table 1. Dynamics of mutations after 2nd TKI Post IM mutation No. Post-2nd TKI Mutation (New + Same + Lost) *1 pt acquired new mutation with persistence of pre-existing mutation, 1 lost 3 mutations and acquired 1, and 1 pt lost 2 mutations. Nilotinib Dasatinib Absent 39 8+NA+NA/21 3+NA+NA/18 Present 46 3+20+3/26 2+16+2*/20 Unknown 28 8/9 13/19

Table 2. Dynamics of mutations after 3rd TKI Post IM mutation No. Post-3nd TKI Mutation (compared to status after 2nd TKI) (New + Same + Lost) Nilotinib Dasatinib Absent 5 0/1 1+NA+NA/4 Present 12 0+1+0/1 2+6+3/11 Unknown 3 1/3 NA
The Racial Disparity Gap in Pediatric Leukemia and Lymphoma Survival Has Been Eliminated in Children but Not in Older Adolescents and Young Adults.
Authors: Viny Aaron , Hilden Joanne M. , Stallion Anthony , Bleyer W. Archie
Published: April 2020

Overcoming disparities in the successful treatment of minority cancer patients has been a major challenge. In particular, blacks/African Americans face a higher likelihood of developing and dying from each of the four most common malignancies in the United States (breast, prostate, colon, and lung cancer) as well as from other cancers. Among children, leukemia and lymphoma have also shown a worse outcome in black patients versus whites. Given the success of the Children’s Oncology Group (COG) in reducing the racial deficit in leukemia and lymphoma, we examined national survival data from the U.S. Surveillance, Epidemiology and End Results (SEER) program for trends in survival difference between white and blacks with leukemia and lymphoma, as a function of age at diagnosis. The annual 5-year relative survival rate trends during 1975 to 2000 for all leukemia and all lymphoma (including Hodgkin lymphoma) showed convergence in white and black children (age <10), comparable trends in 10- to 19-year-olds, and divergence in those 20 to 29 years of age with blacks doing progressively worse than whites. The age dependent patterns were qualitatively similar for acute lymphoblastic leukemia, acute myelogenous leukemia, and non-Hodgkin lymphoma, with convergence in <10 year-olds and divergence in 20- to 29-year-olds apparent in each type of leukemia and lymphoma. For the year 2000, the trends in 20- to 29-year-olds project a 20 to 25% survival deficit for blacks versus whites for acute myelogenous leukemia and a 5 to 10% deficit for other leukemia and lymphomas. The fact that the deficits between white and black patients with leukemia and lymphoma have been overcome for pediatric patients in the United States indicates that this can also be achieved in young adults and older adolescents. A major solution would be access to clinical trials through COG or other clinical trials for the next older age group. The current National Cancer Institute Progress Review Group offers recommendations that are relevant to these disparate outcomes observed in this study; these will be reviewed.

Figure Figure
RUNX1/AML1 DNA Binding Domain and ETO/MTG8 NHR2 Dimerization Domain Are Critical to AML1−ETO9a Leukemogenesis.
Authors: Yan Ming , Hiebert Scott , Zhang Dong-Er
Published: April 2020

The 8;21 translocation, which involves the gene encoding the RUNX family DNA binding transcription factor AML1 (RUNX1) on chromosome 21 and the ETO (MTG8) gene on chromosome 8, generates AML1−ETO fusion proteins. Previous analyses have demonstrated that full length AML1−ETO blocks AML1 function and requires additional mutagenic events to promote leukemia in mice. More recently, we have identified an alternatively spliced form of AML1−ETO, AML1−ETO9a, from t(8;21) AML patient samples (Yan et al. Nat. Med.12:945–949, 2006). AML1−ETO9a lacks the C−terminal NHR3 and NHR4 domains of AML1−ETO and is highly leukemogenic in mice. Here, we report that the AML1 DNA binding domain and the ETO NHR2 dimerization domain, but not the ETO NHR1 domain are critical for the induction of acute myeloid leukemia by AML1−ETO9a. Using retroviral mediated gene expression and hematopoietic cell transplantation in recipient mice, we examined AML1−ETO9a, AML1−ETO9a without the NHR1 domain [AML1−ETO9a (dNHR1)] or the NHR2 domain [AML1−ETO9a(dNHR2)], without a histone deacetylase/Sin3A interacting domain between NHR1 and NHR2 [AML1−ETO9a(d350–428)], and mutant AML1−ETO9a proteins that have lost the capacity to bind DNA [AML1−ETO9a(L148D)] and [AML1−ETO9a(R173Q)] in leukemogenesis. All of the mice transplanted with AML1−ETO9a (n =11) and AML1−ETO9a(dNHR1) (n = 12) expressing cells developed acute myeloid leukemia with a similar phenotype (Lin−/c−kit+) within 21 weeks. The median survival times of mice with AML1−ETO9a and AML1−ETO9a(dNHR1) are 9.4 weeks and 10.5 weeks, respectively. Furthermore, all of the mice expressing AML1−ETO9a(d350–428) (n = 11) also developed leukemia with a median survival time of 17.2 weeks. Significant numbers of AML1−ETO9a(d350–428) expressing cells are positive for myeloid markers CD11b and Gr1 in these leukemic mice. In contrast, none of the mice with AML1−ETO9a(dNHR2) (n = 14), AML1−ETO9a(L148D) (n = 8), and AML1−ETO9a(R173Q) (n = 8) expressing hematopoietic cells developed leukemia. Taken together, these data suggest that the AML1 DNA binding domain and the ETO NHR2 domain are required for AML1−ETO9a induced leukemia development and the region between amino acids 350 and 428 of AML1−ETO9a also affects the differentiation stage and latency of leukemogenesis.
Inactivation of the Myc−Induced DNA Damage Response Promotes Lymphomagenesis and Treatment Resistance In Vivo.
Authors: Reimann Maurice , Loddenkemper Christoph , Rudolph Cornelia , Stein Harald , Schlegelberger Brigitte , Dorken Bernd , Schmitt Clemens A.
Published: April 2020

Activation of the DNA damage response (DDR) was recently claimed to serve as a critical anti−cancer barrier in early human tumorigenesis (Bartkova−J et al., and Gorgoulis−VG et al., Nature, 2005). As a consequence, deregulation of the DDR machinery may be required for tumor manifestation.

Myc activation is an oncogenic event frequently detected in malignant lymphomas. It is known that constitutive Myc expression provokes apoptosis via the ARF/p53 pathway as a cellular failsafe mechanism to counteract malignant transformation, but the potential role of Myc signaling to the DDR machinery, mediated via the Atm (ataxia telangiectasia mutated) kinase, as another putative tumor suppressive restraint remains still elusive.

Utilizing a tamoxifen−regulatable Myc system as well as RNA interference in vitro, we found in cell−based models that acute induction of Myc, associated with an increased production of reactive oxygen species (ROS) and marks of DNA strand breaks, leads to p53 activation which is mainly mediated by Atm and its upstream regulator PP5.

To further examine the role of Atm during Myc−driven tumorigenesis in vivo, we intercrossed Atm knockout mice to Eμ−myc transgenic mice that develop B−cell lymphomas. Lymphomas formed significantly faster in the absence of Atm, mostly due to a significant apoptotic defect in Atm−/− lymphomas compared to Atm+/+ derived (referred to as ‘control’) lymphomas.

In accordance with our in vitro findings, we detected significantly elevated levels of ROS−induced oxidative DNA damage and γ−H2AX foci in Myc−driven lymphomas. Importantly, constitutive expression of Myc selected against components of the Atm−governed DDR in a subset of control lymphomas. Like Atm deficiency, these lesions predetermined a poor response in subsequent cytotoxicity assays in vitro and anticancer therapy in vivo, since DNA damaging treatments also produce apoptosis via PP5/Atm.

The role for ROS as inducers of DNA damage during the execution of Myc−induced apoptosis was further substantiated by the finding that Eμ−myc transgenic mice continuously exposed to the ROS scavenger N−acetylcysteine (starting prenatally) developed lymphomas with significantly reduced levels of spontaneous apoptosis and with less marks of DNA damage (i.e. γ−H2AX foci), but a retained ability to respond to anticancer therapy in vitro and in vivo.

Hence, Atm eliminates pre−neoplastic lesions by converting oncogenic signaling into apoptosis, while selection for an attenuated Atm response permits lymphoma formation, and, in turn, has profound implications for reduced efficacy of DNA damaging therapies.
Evi-1 Is Essential for Expansion and Maintenance of Hematopoietic Stem Cells.
Authors: Goyama Susumu , Yamamoto Go , Sato Tomohiko , Ogawa Seishi , Chiba Shigeru , Kurokawa Mineo
Published: April 2020

The ecotropic viral integration site-1 (Evi-1) was first identified as a gene located at the integration site of an ecotropic retrovirus leading to murine myeloid leukemia. Since its identification, Evi-1 has been recognized as one of the dominant oncogenes associated with human myeloid leukemia and myelodysplastic syndrome. Evi-1 is a member of the SET/PR domain family of transcription factors and it contains two separated zinc-finger DNA-binding domains, which recognize cognate DNA sequences respectively. Recently, it was shown that Evi-1 is predominantly expressed in hematopoietic stem cells (HSCs), and HSC activity is significantly impaired in Evi-1-deficient embryos, which succumb to the multiple organ defects before birth. In order to investigate a role of Evi-1 in adult hematopoiesis, we generated the mutant mice harboring Cre recombinase recognition sites (loxP sites) flanking exon 4 of the Evi-1 gene (Evi1flox/flox), together with the mice in which the same region was completely deleted (Evi-1−/−). Evi-1−/− mice died around embryonic day 13–16. In Evi-1−/− embryos, Lineage−Sca1+cKit+ cells (LSK cells) and colony forming cells were severely reduced. Furthermore, Evi-1−/− fetal liver cells could not reconstitute hematopoiesis of sublethally irradiated mice in the transplantation experiments, indicating an apparent defect in hematopietic stem/progenitor cells. In contrast, more committed cells was unaffected in Evi-1−/− fetal liver. Then, we ablated Evi-1 by breeding Evi-1flox/flox mice with mice expressing Cre-recombinase under the control of the Tie2 promoter and enhancer, a marker of endothelial and hematopoietic stem cells. These mice virtually phenocopied Evi-1−/− mice, demonstrating that Evi-1 function is distinctly required in Tie2+ cells to establish HSCs. Next, we assessed the requirement of Evi-1 in adult hematopoiesis using an interferon (IFN) -inducible Mx-Cre gene-targeting method, in which floxed alleles can be effectively deleted in hematopoietic cells upon injection of IFN-inducer pIpC. Immediately upon injection of pIpC, platelet counts modestly declined in Evi-1flox/flox/Mx-Cre mice, while white blood cell counts or hemoglobin levels were not affected. Platelet recovery after 5FU-treatment is also delayed in Evi-1-deficient mice compared to the control mice. Furthermore, two weeks after pIpC infection, LSK cells in Evi-1-deficient mice decreased to about half the number of those in the control mice. By 10 weeks, however, LSK cells in these mice began to recover. At this time point, a majority of hematopoietic cells in the bone marrow retained the intact Evi-1 alleles, suggesting that HSCs can not maintain hematopoiesis in the absence of Evi-1 and are outcompeted by a fraction of HSCs that escaped Cre-mediated Evi-1 exicision. Together, these results show that Evi-1 plays an indispensable role not only in the establishment of HSCs during embryogenesis but also in the maintenance of HSCs in adults.
Ott1(Rbm15) Is Essential for Pre-B Differentiation and Has an Inhibitory Role in Myeloid and Megakaryocytic Lineages.
Authors: Raffel Glen D. , Mercher Thomas , Shigematsu Hirokazu , Williams Ifor R. , Cullen Dana E. , Akashi Koichi , Bernard Olivier A. , Gilliland D. Gary
Published: April 2020

OTT1(RBM15) is a putative transcriptional repressor related to the spen/Mint/SHARP family of proteins which was originally identified as the 5′ translocation partner of the MAL(MKL1) gene in t(1;22)(p13;q13) infant acute megakaryocytic leukemia (AMKL). OTT1 contains RNA recognition motifs (RRMs) and a transactivation/repression spen paralog and ortholog (SPOC) homology domain however no physiologic function has yet been attributed to it. To define the role of OTT1 in hematopoiesis and help gain an understanding of potential dysregulated pathways in t(1;22) AMKL, a conditional allele of Ott1 was generated in mice. Germline deletion of Ott1 was early embryonic lethal, however, deletion in adult mice utilizing the Mx1-Cre transgene affected multiple lineages within the hematopoietic compartment. Deletion of Ott1 caused a loss of peripheral B cells as a result of block in pro/pre-B differentiation. An expansion myeloid cells was seen in the spleen and bone marrow and increased numbers of myeloid CFU were detected in the bone marrow. Progenitor analysis demonstrated in increase in Lin−Sca-1+c-Kit+ (LSK) cells with an altered clonogenicity shifting towards granulocytic cell fate. Finally, increased numbers of megakaryocytes were found in the bone marrow and spleen suggesting a negative role for Ott1 in megakaryocytic proliferation/survival. These data indicate a requirement for Ott1 in B lymphopoiesis and inhibitory roles within the myeloid, megakaryocytic and progenitor compartments. Accordingly, the ability of Ott1 to affect hematopoietic cell fate and expansion within multiple lineages including megakaryocytes may underlie the mechanism of OTT1-MAL-mediated leukemogenesis.
Ubiquitin Ligase Component Fbw7 Regulates the Quiescence of Hematopoietic Stem Cells.
Authors: Matsuoka Sahoko , Oike Yuichi , Onoyama Ichiro , Takubo Keiyo , Ito Keisuke , Arai Fumio , Ikeda Yasuo , Nakayama Keiko , Nakayama Keiichi , Suda Toshio
Published: April 2020

Fbw7 is a SCF ubiquitin ligase component that catalyzes the ubiquitination of c-Myc, Cyclin E, and Notch. In several human cancer cell lines and primary cancer cells, Fbw7 is mutated and functions as a tumor suppressor gene. Previously we have reported that Fbw7-deficient mice died at embryonic day 10.5–11.5 with deficiencies in hematopoietic and vascular development, indicating that Fbw7 has a pivotal role in hematopoiesis (Tsunematsu R et al. J Biol Chem. 2004). Fbw7 is widely expressed in various hematopoietic lineages in BM of adult mice, but little has been known about the function of Fbw7 in hematopoiesis. To assess the requirement of Fbw7 in adult hematopoietic cells, we generated Fbw7-deficient mice by conditional gene targeting. Fbw7 was conditionally deleted from Mx-1-Cre;Fbw7fl/− adult mice by injection of pIpC over 1 week to induce Cre expression. We examined Fbw7fl/+ littermates as a control. We found progressive pancytopenia in Fbw7-deficient mice. Furthermore, most Fbw7-deficient mice developed leukemia (mainly ALL) within 3 months after pIpC treatment, suggesting that Fbw7 is essential to maintain normal hematopoiesis and loss of Fbw7 accelerates leukemogenesis. The portion of Fbw7-deficient Lin−Sca-1+c-Kit+CD34− hematopoietic stem cells (HSCs) in the G0 phase was 2.5-fold decreased and the frequency of cell division of Fbw7-deficient HSCs markedly increased in culture. These data suggest that Fbw7 promotes quiescence of HSCs. To examine the function of Fbw7-deficient HSCs, we transplanted 1500 Lin−Sca-1+cKit+ BM cells from Fbw7-dificient mice or littermate controls into lethally irradiated recipient mice with 4×105 normal BM mononuclear cells. In the result, Fbw7-deficient HSCs are impaired in long-term repopulating activity and multipotency. It has been reported that c-Myc controls the self-renewal activity of HSCs through the cell adhesion to the osteoblastic niche (Wilson A et al. Genes Dev. 2004). We found that c-Myc is significantly accumulated in Fbw7-deficient Lin−Sca-1+cKit+ BM cells, suggesting that HSCs leave the niche and show the active cell cycling. We propose that a ubiquitin ligase, Fbw7 is a key mediator of HSC quiescence and self renewal capacity.
A New Prognostic Index (MIPI) for Patients with Advanced Stage Mantle Cell Lymphoma.
Authors: Hoster Eva , Dreyling Martin , Unterhalt Michael , Hasford Joerg , Hiddemann Wolfgang
Published: April 2020

On behalf of the German Low Grade Lymphoma Study Group (GLSG) and the European MCL Network.

Background: There is no generally established prognostic classification system for patients with mantle cell lymphoma (MCL), as the International Prognostic Index (IPI) and the Follicular Lymphoma International Prognostic Index (FLIPI) have been developed based on data of patients with diffuse large cell and follicular lymphoma, respectively.

Methods: The data of 455 patients with advanced stage MCL treated first-line within three clinical trials of GLSG and European MCL Network have been analyzed to clarify the prognostic relevance of IPI and FLIPI and to derive a new prognostic index of overall survival (OS). Age, sex, ECOG performance status, Ann Arbor stage, B-symptoms, number of extranodal sites, number of involved nodal areas, tumor size, serum LDH activity, WBC count, platelet count, hemoglobin, albumin, β2-microglobulin and cell proliferation (Ki-67) were considered as candidate prognostic factors. Statistical methods included Kaplan-Meier estimates and logrank test for the validation of IPI and FLIPI and multiple Cox regression with backward variable selection for the derivation of the new prognostic index.

Results: IPI showed a significant impact on OS, but low-intermediate and high-intermediate risk groups comprised more than two thirds of the patients and were not well separated. According to the FLIPI, only 6% of the patients were classified as low risk and almost two thirds of the patients as high risk, and low and intermediate risk groups were not separated. Four of the candidate prognostic factors were independently associated with OS, namely age, ECOG performance status, LDH and WBC count. The relative risk was 1.42 (95% confidence interval 1.18 to 1.72, p = 0.0002) for an increased age by ten years, 2.01 (1.19 to 3.39, p = 0.0088) for an ECOG greater than one, 1.51 (1.13 to 2.02, p = 0.0059) for a 2 fold elevation of LDH and 2.56 (1.66 to 3.95, p < 0.0001) for a 10 fold increase of WBC count. According to these four parameters, patients could be classified into a low risk (44% of the patients, median OS not reached), an intermediate risk (35%, median OS 51 months), and a high risk group (21%, median OS 29 months).

Discussion: IPI and FLIPI showed only modest prognostic discrimination in our external validation data set of patients with advanced stage MCL. In contrast, age, ECOG performance status, LDH and WBC were identified as independent prognostic factors of OS, and three reasonably sized and well separated risk groups were defined. With four clinical prognostic factors readily determined in clinical routine, our new prognostic index (MIPI) is superior to the IPI. Bootstrap validation confirmed the separation of three risk groups, but external validation is still needed. The MIPI may prove to be an important tool to facilitate risk-adapted treatment decisions for patients with advanced stage MCL.
A New Target in Multiple Myeloma: Inhibition of Notch Pathway Induces Apoptosis and Enhances Drug Sensitivity of Myeloma Cells In Vitro and In Vivo.
Authors: Nefedova Yulia , Verali Krista , Sullivan Daniel M. , Dalton William S. , Gabrilovich Dmitry I.
Published: April 2020

Bone marrow (BM) microenvironment and particularly BM stromal cells play a critical role in de novo drug resistance of multiple myeloma (MM) cells. BM stromal cells express Notch ligands and activate Notch signaling in MM cells. We have previously demonstrated that Notch signaling is one of the major mechanisms of BM stroma mediated MM cell protection from chemotherapeutic drugs. Here we investigated whether the pharmacological inhibition of Notch signaling with γ-secretase inhibitor (GSI) could affect viability of MM cells and overcome BM stroma mediated resistance of MM cells to chemotherapy. GSI (6–8 μM) induced apoptosis of MM cells cultured in suspension or on monolayer of BM stroma via specific inhibition of Notch signaling. The effect of GSI was evaluated in four different MM cell lines and primary MM cells isolated from BM of four patients with MM. Treatment with GSI alone significantly reduced the viability of MM cells with IC50 almost 5-fold lower than that for peripheral blood mononuclear cells or BM cells from healthy donors. In addition, treatment of MM cells with GSI reversed BM stroma mediated protection of MM cells from drug-induced apoptosis. This effect of GSI was associated by dramatic up-regulation of pro-apoptotic protein NOXA and down-regulation of anti-apoptotic proteins bcl-xL, bcl-2, and Akt. Inhibition of NOXA with siRNA canceled the cytotoxic effect of GSI on MM cells indicating that NOXA could mediate the direct effect of GSI on MM cells. To test the effect of GSI on MM cells in vivo we used SCID/NOD mouse model. Mice were injected s.c. with human MM RPMI-8226 cells. These cells express both Notch ligands and Notch receptors so Notch signaling is activated by interaction between MM cells themselves. Tumor became visible in ~3 weeks after inoculation and grew as a solid tumor which allowed easily monitoring tumor size. Mice were treated with doxorubicin (1.5 mg/kg, i.p., once a 4 days, 3 times), GSI (5mg/kg/day i.p. for 14 days), combinations of doxorubicin and GSI, or vehicle control (PBS). Tumor size was constantly monitored during treatment and 3 weeks after the treatment. Treatment with doxorubicin and GSI alone resulted in moderate decreased in tumor burden as compared with control group. In contrast, combination of GSI and doxorubicin induced dramatic antitumor effect. Thus, our study, for the first time, demonstrates that inhibition of Notch signaling with GSI can be a new promising approach for therapeutic intervention in MM.
Aurora Kinases as Therapeutic Targets in Multiple Myeloma.
Authors: Reiman Tony , Evans Robert P. , Naber Claudia , Steffler Tara , Keats Jonathan J. , Perry Troy , Maxwell Christopher A. , Chau Heidi , Belch Andrew R. , Pilarski Linda M.
Published: April 2020

BACKGROUND: We have previously found that in multiple myeloma there is amplification of the centrosome, the organelle that nucleates the mitotic spindle. We have reported that RHAMM is a component of the centrosome which interacts with TPX2, the protein that targets Aurora A kinase to the mitotic spindle. We have also shown that RHAMM expression and alternative splicing in myeloma correlate with centrosome amplification, aggressive disease and poor survival. These findings led us to speculate that Aurora A and possibly other Aurora kinases are potential therapeutic targets in myeloma.

METHODS: We examined the expression of Aurora A, B and C kinases in 5 myeloma cell lines and autoMACS-purified CD138+ myeloma bone marrow plasma cells from 20 patients. We assessed the anti-proliferative and pro-apoptotic effects of Aurora A knockdown in myeloma cell lines with RNA interference. We investigated the anti-myeloma activity of two potent, selective Aurora kinase inhibitors, VE-465 (Merck/Vertex) and AZD1152 (AstraZeneca), in 5 myeloma cell lines, in CD138+ bone marrow plasma cells from 2 myeloma patients, and in a NOD/SCID murine xenograft model.

RESULTS: Aurora A, B and C kinases are ubiquitously expressed in both myeloma cell lines and myeloma bone marrow plasma cells. Expression levels vary among patients. Aurora A and B are expressed in myeloma plasma cells at levels comparable to that seen in the CD138- cells from the same marrow sample, and comparable to the levels seen in normal marrow from control individuals. Aurora C, while expressed at low levels, is consistently ectopically overexpressed in myeloma plasma cells relative to coexisting CD138- cells and normal marrow. In myeloma cell lines, Aurora A knockdown with RNA interference induces apoptosis and cell killing. In all five myeloma cell lines tested, and in myeloma bone marrow plasma cells from two patients, both VE-465 and AZD1152 induce apoptosis and myeloma cell killing at nanomolar concentrations, to varying degrees (20–80% reduction in cell viability). VE-465 is known to inhibit all three Aurora kinases with comparable specificity, while AZD1152 is known to inhibit Aurora B and C more selectively than Aurora A. Despite these differences in activity, both compounds have comparable pre-clinical efficacy against myeloma. Myeloma cell lines treated with either agent demonstrate a phenotype consistent with target inhibition. Both drugs show additive effects on killing of cell lines and primary myeloma cells when combined with dexamethasone, even in dexamethasone-resistant cells. Anti-myeloma activity was seen with single agent Aurora kinase inhibition in the murine model, at well tolerated doses.

CONCLUSIONS: Aurora kinases are potential therapeutic targets in myeloma. Aurora kinase inhibitors comprise an emerging class of anti-cancer drug therapy that deserves further evaluation for myeloma patients.