bluuebunny/crossref_metadata_2025 huggingface dataset:
Improvement of Thrombolysis by Rivaroxaban, An Anti Xa Inhibitor. Potential Therapeutic Importance in Patients with Thrombosis
Authors: Varin Remi , Mirshahi Shasultan , Mirshahi Pehzman , Lu-Hong Li , Kierzek Gerald , Vigneau Jean-Francois , Marie Jean-Pierre , Perzborn Elisabeth , Mirshahi Massoud , Soria Claudine , Soria Jeannette
Published: April 2020

Background and objective Rivaroxaban – an oral, direct Factor Xa inhibitor – inhibits thrombus formation and growth in animal models. We have investigated the effects of rivaroxaban on thrombolysis because impaired fibrinolysis is a risk factor for venous thrombosis and it occurs more often in patients who had a myocardial infarction. As the propensity of a clot to be degraded depends on its structure, we tested the effects of rivaroxaban on clot structure and degradability by tissue plasminogen activator (t-PA). This was done in the absence and presence of thrombomodulin because the thrombin - thrombomodulin complex is the activator of TAFI (thrombin-activatable fibrinolysis inhibitor), a potent inhibitor of fibrinolysis.

Methods Clots were formed in a microchamber in the presence or absence of rivaroxaban at pharmacological concentrations (0.15 and 0.25 μg/ml). Clot structure was analyzed by confocal microscopy, and permeability calculated by measuring flow rates. Degradation was evaluated by the amount of D-dimers in the eluate of clot perfused with t-PA, in the presence or absence of thrombomodulin.

Results Microscopy showed that clots formed in the presence of rivaroxaban had thicker fibers and a looser fibrin structure with larger pores than controls, leading to increased permeation rate (Darcy constant 2.16-fold and 2.45-fold higher than controls with rivaroxaban at 0.15 μg/ml and 0.25 μg/ml, respectively). This clot structure modification renders the clots more susceptible to fibrinolytic enzymes. The degradation of clots containing 0.15 μg/ml of rivaroxaban was 3.6-fold higher than that of control clots, after 90 minutes perfusion with t-PA. In addition, when clots are formed in the presence of thrombomodulin, the degradability is decreased in control, while In the presence of rivaroxaban, fibrin degradation remains enhanced.

Conclusion Rivaroxaban increased thrombolysis by t-PA. This was due to a decrease in thrombin generation. Two mechanisms are involved:

modification of clot structure, which makes it more accessible to thrombolytic enzymes; and decrease in TAFI activation by the thrombomodulin–thrombin complex. This property of rivaroxaban may contribute to its antithrombotic effect.
Fatal Disseminated Intravascular Coagulation and Autoimmune Hemolytic Anemia Due to Anti-Ena
Authors: Klapper Ellen B , Xie Joanna J , Garratty George , Pepkowitz Samuel , Arndt Patricia
Published: April 2020

Background: Autoimmune hemolytic anemia (AIHA) caused by warm IgG antibodies is usually characterized by IgG-mediated extravascular hemolysis and anemia that develops slowly. We report a case of AIHA presenting with sudden massive intravascular hemolysis and disseminated intravascular coagulation (DIC), similar to that seen following an ABO incompatible transfusion reaction.

Case Report: A 71-year-old female had a 4-year history of immune thrombocytopenic purpura requiring splenectomy 1 ½ years ago. Six weeks prior to admission, in association with a viral infection, her platelet (plt) count fell from 350,000/mm3 to 150,000/mm3 over 2 weeks. She was treated with oral prednisone, and one week prior to admission her platelet count was 200,000/mm3. Two days before admission she noted reddish urine. Laboratory studies at her doctor’s office showed hemoglobin (Hb) 9 g/dL (had been 13 g/dL 1 week earlier) and bilirubin (bili) 9 mg/dL. On the day of admission, in the emergency department, she complained of generalized weakness, jaundice, and near-syncope. She became increasingly agitated and dyspneic and was intubated. At that time laboratory studies showed Hb 4.5 g/dL, LDH >2,500 U/L, haptoglobin 7 mg/dL, total bili 13.0 mg/dL, indirect bili 11.8 mg/dL, D-dimers >10,000 ng/dL, plts 139,000/mm3, prothrombin time 18.3 seconds, fibrinogen 166 mg/dL, fibrin monomers were positive. She was admitted to the medical intensive care unit with a diagnosis of DIC and hemolysis. The patient’s RBCs were coated with IgG (4+) and C3 (3+); IgM and IgA were not detected. Her plasma and an eluate contained an antiglobulin test reactive autoantibody with anti-Ena specificity (anti-Pr and -Wrb were excluded), an antibody directed at epitopes on glycophorin A. No alloantibodies were detected in the patient’s plasma. The patient was treated with IV steroids and received multiple RBC transfusions over the first few hospital days. Her Hb stabilized at 10 g/dL. The day after admission, she became oliguric and required dialysis throughout her hospital course. Over the next several days, additional signs and symptoms of DIC were noted: ischemic digits and toes, elevated Troponin I, partial thromboplastin time, D-dimers and decreased plt count. She was placed on intravenous heparin. Gastrointestinal bleeding developed and heparin was discontinued on hospital day 9. One week later, the onset of abdominal pain necessitated exploratory laparotomy which revealed ischemic bowel with perforation; vessels showed organized thrombi with recanalization. She remained critically ill and died on hospital day 25.

Conclusion: Complement-mediated intravascular hemolysis in a patient with AIHA due to anti-Ena was associated with renal failure and DIC leading to death of the patient. All of these, even separately, are unusual in warm AIHA. We know of no reports where all 5 events (intravascular lysis, anti-Ena, renal failure, DIC, and death) occurred in one patient.
A Phase I Study of Bortezomib in Combination with ICE (BICE) in Patients with Relapsed/Refractory Classical Hodgkin Lymphoma
Authors: Fanale Michelle A. , Fayad Luis E. , Pro Barbara , Samaniego Felipe , Zachariah Gracy , Nunez Cesar A. , Ji Yuan , Younes Anas
Published: April 2020

Background: The proteasome inhibitor bortezomib induces cell cycle arrest and apoptosis of Hodgkin lymphoma (HL) cells. We have demonstrated that bortezomib’s single agent efficacy was limited in heavily pretreated HL patients. However, preclinical work has shown that bortezomib may synergize with chemotherapy. Thus, we evaluated the combination of bortezomib plus ICE chemotherapy in patients with relapsed/refractory HL.

Methods: Eligibility:

Relapsed/refractory classical HL, Only 1 prior chemotherapy regimen, Prior treatment with anthracycline-containing regimen, No prior ASCT.

Regimen: ICE (Moskowitz, C et al 2001). Bortezomib on D1 and D4 at dose levels of 0 = 1 mg/m2, 1 = 1.3 mg/m2, 2 = 1.5 mg/m2. Neulasta given on D5. Cycles were repeated on D14 if platelet count is ≥ 100,000/mm3 and ANC is ≥ 1,000/mm3. After 3 cycles CTs and PET/CT were performed to evaluate response. Hematologic DLT was defined as grade 4 thrombocytopenia or neutropenia lasting greater than 2 weeks.

Results: 13 patients enrolled (6 primary refractory and 7 relapsed HL). Median age 32 (range 21–39). 3 patients were treated at dose levels 0 and 1 and 7 patients were treated at dose level 2. 12 patients were evaluable for toxicity and response. The ORR was 75% with 25% CR (1 primary refractory, 2 relapsed) and 50% PR (1 primary refractory, 5 relapsed). PET/CT scans showed negativity in all patients who responded to treatment. 8/9 patients who responded to treatment underwent ASCT (1 patient declined ASCT). All patients who underwent ASCT had successful pheresis and engraftment. At median follow-up of 12 months all 8 patients treated with ASCT are free of relapse. Treatment was well tolerated with no DLTs noted. Reversible grade 4 neutropenia and thrombocytopenia occurred respectively in 33% and 50% of the patients. Median day for retreatment for cycle 2 was D20 and for cycle 3 was D21. There have been no toxicities of peripheral neuropathy, febrile neutropenia, or infection.

Conclusions: Our data suggests favorable tolerability and efficacy of bortezomib plus ICE in patients with relapsed/refractory Hodgkin lymphoma. We are planning to confirm these promising results in a randomized phase II trial.
Phase I Trial of Bortezomib in Combination with Rituximab-HyperCVAD/Methotrexate and Cytarabine for Untreated Mantle Cell Lymphoma
Authors: Romaguera Jorge , Fayad Luis , McLaughlin Peter , Pro Barbara , Rodriguez Maria Alma , Wang Michael , Weaver Pamela , Hartig Kimberly , Kwak Larry , Feldman Tatyana , Smith Judy , Ford Peggy , Goldberg Stuart , Pecora Andrew , Goy Andre
Published: April 2020

Background: Though mantle cell lymphoma (MCL) outcome has improved over the last two decades - especially with dose-intense or high-dose therapy - MCL patients still relapse particularly in >65yo. Bortezomib has shown single agent activity of 33% in relapsed MCL and has been safely combined with conventional as well as intensified doxorubicin-containing regimens, but not with high doses of the methotrexate/cytarabine combination, where there is concern about lung toxicity.

Materials and methods: MCL pts from ages 18 through 79, with adequate organ function unless due to lymphoma, and without HIV-1 infection or any significant medical/mental condition were treated under IRB-approved study using R-HCVAD alternating with R-M/A (JCO October 1, 2005) with doxorubicin given by bolus. Bortezomib was added to R-HCVAD as a fixed dose of 1.3 mg/m2 IV given at the end of the first infusion of cyclophosphamide on day 2 and after doxorubicin bolus on day 5. Bortezomib was added to R-M/A after rituximab on day 1 and on day 6, in increasing doses of 0.7, 1, and 1.3 mg/m2 in cohorts of 3 patients. As an additional precaution, the first cohort of patients received a −1 dose level of the cytarabine adjusted by age. Dose limiting toxicity was defined initially as a drop of 10% from baseline in diffusing lung capacity (DLCO) and later increased to 25% (grade 1 toxicity according to National Cancer Institute criteria) after the first cycle of R-MA with bortezomib.

Results: Since April 2007, sixteen patients have been entered on the trial, ages 40 to 70 years (median 58). Currently 2/3 patients have been entered in the last cohort. There has been no DLT. After 59 cycles, toxicities according to NCI criteria were as follows:

BR-HCVAD Phase I Toxicity in 59 cycles

Variable Grade1 (%) Grade2 (%) Grade3 (%) Grade4 (%) *First R-M/A cycle of each patient. 15/16 patients evaluable. Thrombocytopenia 7 (12) 3 (5) 8 (14) 26 (44) Neutropenia 3 (5) 5 (8) 4 (7) 28 (47) Anemia 11 (19) 23 (39) 11 (19) 2 (3) Neutropenic fever 0 0 5 (8) 1 (2) Dyspnea 0 0 0 0 DLCO* 2 (3) 0 0 0 Neurological 5 (8) Sensory 1 (2) 0 0

As expected, responses are excellent with 11 complete responders and 4 partial responders out of 15 evaluable patients, of whom 3 are still on treatment.

Conclusion: Bortezomib can be safely combined with R-M/A. Accrual is ongoing and more patients and dose levels will be reported on at the meeting.
Factor V as Anticoagulant Cofactor for Activated Protein C in Factor Va Inactivation
Authors: Cramer Thomas J , Griffin John H. , Gale Andrew J.
Published: April 2020

Factor V (FV) is a cofactor that promotes inactivation of activated factor VIII (FVIIIa) by the activated protein C and protein S complex (APC/protein S). Cleavage in FV at Arg506 is required for proteolytic inactivation of FVa, but also for the anticoagulant function of FV as cofactor for APC in the inactivation of FVIIIa. This is demonstrated by the well known FVLeiden mutant with Arg506 mutated to glutamine (Q506), causing APC resistance due to both impaired sensitivity of Q506FVa to APC and reduced cofactor activity of Q506FV for APC inactivation of FVIIIa. However, FVIIIa loses activity rapidly due to dissociation of the A2 domain, and this may be the primary mechanism of FVIIIa inactivation. Thus, we question whether the APC-mediated inactivation of FVIIIa is relevant to the FVLeiden thrombophilic phenotype. Rather, we hypothesized that FV can function as an anticoagulant cofactor for the APC/protein S complex in the inactivation of activated FV (FVa). To test this hypothesis, we designed a coagulation assay initiated by tissue factor that was sensitive to FV but was insensitive to FVIII. FV was titrated into FV deficient plasma and clotting times were measured in absence and presence of APC to determine an APC sensitivity ratio (APCsr). An increase in the APCsr was observed as the level of FV was increased, suggesting an anticoagulant function of FV. Similar titrations were done with Q506FV, showing no increase in clotting time when APC was present and an APCsr of 1.0 in the presence of Q506 FV. Control experiments confirmed that this clotting assay was insensitive to the presence or absence of FVIII; thus, these assays were reflecting FVa inactivation. The potential anticoagulant effect of FV as cofactor for APC in FVa inactivation was further investigated by monitoring proteolysis of purified FVa by APC over time using SDS PAGE. Recombinant purified FVa was labeled with a fluorescent dye, and then subjected to proteolysis by APC/protein S in the absence or presence of FV in a time course. The resulting FVa fragments seen on SDS gels reflected the known cleavages at Arg306 and Arg506, and the FVa cleavage products were quantified by digital fluorescent scanning of the gel. FV stimulated a small but statistically insignificant increase in the rate of FVa cleavage by APC/protein S. Thus, in our experimental conditions, we found a significant anticoagulant effect of FV in clotting assays that were sensitive to FV but not sensitive to FVIII whereas in purified reaction mixtures there was not a significant enhancement by FV of APC proteolysis of FVa. These data contrasting FV’s apparent APC-cofactor activities between plasma and purified reaction mixtures lead us to speculate that other factors or mechanisms present in plasma also contribute to the anticoagulant function of APC in a FV dependent manner.
Relapse of Children with Aplastic Anemia after Immunosuppressive Therapy
Authors: Kamio Takuya , Ito Etsuro , Ohara Akira , Kosaka Yoshiyuki , Tsuchida Masahiro , Mugishima Hideo , Yabe Hiromasa , Morimoto Akira , Ohga Shouichi , Yagasaki Hiroshi , Bessho Fumio , Nakahata Tatsutoshi , Kojima Seiji
Published: April 2020

Although the therapeutic outcome for acquired aplastic anemia (AA) has improved markedly with the introduction of immunosuppressive therapy (IST) with antithymocyte globulin (ATG) and cyclosporine A (CyA), a significant portion of patients treated with IST subsequently relapse and require second-line therapy. However, detailed analysis of the relapse cases has not been reported. In this study, we assessed the relapse rate, response to second-line treatment after relapse and prognosis. From November 1992 to July 2007, 473 newly diagnosed AA children (213 female, 260 male) entered two consecutive prospective studies (AA-92 and AA-97). The median age was 8 years, ranging from 1 to 18 years. Patients with very severe (n = 210), severe (n = 149) and non-severe disease (n = 114) received initial IST consisting of ATG and/or CyA. At six months after the initial IST, 280 patients (59.2%) achieved CR (n = 94) or PR (n=186). Relapse is defined by conversion to no response (NR) from partial or complete response (PR/CR). Among the 280 patients who responded to IST, 49 (17.5%) relapsed. The cumulative incidence of relapse was 19.6% at 10 years and the median time to relapse was 18 months, ranging 7 to 138 months. Among the 49 patients who relapsed, 22 received a second round of IST with ATG and CyA. Fourteen patients (63.6%) responded to the second round. However, three relapsed, and two developed paroxysmal nocturnal hemoglobinuria. Eight patients received CyA after relapse. Four patients responded to CyA, but two relapsed subsequently. Hematopoietic stem cell transplantation (HSCT) was attempted in 26 patients who relapsed after initial responses. Before HSCT, nine received a second course of IST with ATG and CyA and two received CyA. Fifteen patients progressed directly to HSCT. Bone marrow transplantation (BMT) from an HLA-matched sibling (n =7), HLA-matched family member (n = 1), or HLA-mismatched family member (n = 2) was performed in 10 patients, and all are presently alive. BMT from an unrelated donor was attempted in 13 patients, and four died of complications related to BMT. Cord blood transplantation from an unrelated donor was attempted in two patients and two patients are alive. Of the 26 patients who received HSCT, 21 are alive and well, 22 to 137 months (median 72 months) following transplantation. There were seven deaths out of total 49 who initially relapsed. The causes of death were HSCT-related complications (n = 5), MDS/acute myelogenous leukemia (n = 1), bacteremia (n = 1). Eight of 9 patients who received HSCT following a second round of IST are alive and well. The present study suggests that a second round of IST should be offered to the patients who relapse, and HSCT should be considered for the patients who fail to respond to the second round of IST.
Comprehensive Ultra High Resolution Analysis of Copy Number and Allele Status Including Novel Microdeletions Spanning NF1 in Acute Myelogenous Leukemia
Authors: Malek Sami , Ouillette Peter , Wright Whitney , Dressel Amanda , Karp Judy , Bockenstedt Paula , Al-Zoubi Ammar , Talpaz Moshe , Kujawski Lisa , Shedden Kerby , Shakhan Sajid , Li Cheng , Erba Harry , Parkin Brian
Published: April 2020

Karyotypic abnormalities are of dominant importance in AML risk prognostication and therapy selection. A comprehensive description of subchromosomal genomic copy number changes and allele status together with gene mutation analysis and identification of chromosomal translocations is needed to fully harvest the prognostic and biological power of genomic changes in AML.

We have analyzed DNA from 96 AML-derived pure blast populations (purified using column-based multi parameter negative selection followed by multi-gated FACS sorting) compared with paired buccal DNA using the Affymetrix 6.0 SNP platform. To support data analysis and display, we have developed the software tools PLUT and LOH tool version 2 and have refined dChipSNP. Data were supplemented with blast karyotypes and mutation status of Flt3, NPM1, p53, N-ras and K-ras.

Results: AML cases carried between zero and thirty-four subchromosomal losses and gains. Approximately 23% of all cases had ≥3 subchromosomal lesions and approximately 50% of all cases had no such lesions. Of the 22 cases with complex and hypercomplex genomic changes by SNP profiling, ~50% had mutations in p53 exons 5–9 and ~50% were p53 wild-type by sequence analysis. Monoallelic deletion of p53 as part of various deletions at 17p was found in 7 of 96 (7%) cases. Importantly, 7/96=7% additional cases demonstrated UPD at 17p which spanned the p53 locus. Of all 14/96=15% of cases with LOH at 17p (with and without copy loss) 9/14=64% carried p53 mutations and 11/14=79% had complex karyotypes. Of the AML cases with NPM1 mutations (19 of 96 cases or 20%), 75% carried no detectable subchromosomal lesions, while 25% carried between 1 and 4 such lesions.

Previously unidentified microdeletions resulting in monoallelic NF1 loss were identified on 17p in a total of 10 of 96 (10%) cases, thus suggesting activation of the Ras pathway independent of Ras mutations (all affected cases had wild-type N-ras and K-ras) in a substantial subset of AML cases. Mutational analysis of all coding exons of NF1 on the retained allele in the affected cases is ongoing.

Additional recurrent microdeletions were identified on chromosomes 3p and 3q as well as 12p, the latter encompassing the genes ETV6 and p27, as previously reported.

Finally, analysis of the prognostically and therapeutically important deletions 5q and 7q identified large deletions without recurrent microdeletions. Regions of minimal loss on 5q have been delineated.
The T(4;11) Fusion Protein MLL/AF4 Regulates TERT Expression
Authors: Gessner Andreas , Thomas Maria , Castro Patricia Garrido , Heidenreich Olaf , Greil Johann
Published: April 2020

About 50% of all infants suffering from acute lymphoblastic leukaemia (ALL) show the translocation t(4;11)(q21;q23) which creates the fusion genes MLL/AF4 and AF4/MLL. This reciprocal translocation identifies a therapy resistant form of leukaemia with a poor prognosis. In order to gain a better insight into the molecular mechanisms of t(4;11) leukaemias we used the two ALL cell lines SEM and RS4;11, both harbouring the t(4;11) translocation, however with different fusion sites. Specific small interfering RNAs (siRNAs) against the two fusion site variants of the MLL/AF4 fusion transcript were designed and transfected into the respective cells via electroporation, using very mild conditions. Serial electroporations at two-day intervals resulted in sustained depletion of the MLL/AF4 transcript up to 70–80%, and depending on the experimental setup, cells were analysed after two or three electroporations. Knock-down of MLL/AF4 resulted in strong inhibition of proliferation and clonogenicity in the two t(4;11)-positive cell lines SEM and RS4;11, along with induction of apoptosis. MLL/AF4 depletion resulted in a 65% decrease in telomerase activity in both SEM and RS4;11 cells, with telomerase reverse transcriptase (TERT) expression being reduced twofold on both transcript and protein levels. Notably, TERT reduction was even stronger (90% depletion) when apoptosis caused by MLL/AF4 knock-down was suppressed with the caspase inhibitor zVAD. In contrast, levels of the RNA component of the telomerase, TERC, were not affected by MLL/AF4 knock-down. Additionally, MLL/AF4 knock-down was associated with reduced expression of several members of the HOXA gene cluster, HOXA6 (65% reduction), HOXA7 (85% reduction), HOXA9 (60% reduction) and HOXA10 (75% reduction). Interestingly, siRNA-mediated knock-down of the MLL/AF4 target gene HOXA7 also induced apoptosis and resulted in a 70% decrease of TERT levels in two t(4;11) positive cell lines without affecting MLL/AF4. Chromatin immunoprecipitation assays revealed HOXA7 binding to the promoter of TERT. Therefore, MLL/AF4 regulates TERT expression, at least in part, via HOXA7. These data suggest that t(4;11) positive cells with substantially lower TERT expression undergo apoptosis, and that TERT may play an antiapoptotic role in t(4;11) positive ALL. Furthermore, these studies identify TERT as a putative new therapeutic target in this therapy-resistant infant leukaemia.
The in Vivo Response of Patients Suffering from Severe Congenital Neutropenia (CN) to G-CSF Is Due to “Emergency” Granulopoiesis Induced by C/EBPβ
Authors: Dan Lan , Thakur Basant Kumar , Skokowa Julia , Welte Karl
Published: April 2020

C/EBP transcription factors are crucial for the regulation of granulopoiesis in vitro and in vivo. C/EBPa is considered to be the master regulator of “steady state” granulopoiesis via upregulation of several myeloid genes (e.g. ELA2, CSFR3, etc.). The absence of C/ EBPa results in a complete loss of neutrophils. We were able to show that in patients with severe congenital neutropenia (CN) harbouring HAX-1 or ELA2 mutations C/EBPa is severely abrogated secondary to defective expression of LEF-1 (Skokowa J, et al. Nat Med.12, 1191–7 (2006)). Therefore, we were interested, whether other transcription factors are capable of substituting C/EBPa, since these patients respond to G-CSF with slight increase in neutrophils from less than 200/ul to above 1500/ul depending on the dose of G-CSF. C/EBPβ has recently been shown to be required for cytokine induced “emergency” granulopoiesis (Hirai H, et al. Nat Immunol.7, 732-9 (2006)). Therefore, we investigated the expression pattern of C/EBPβ during G-CSF treatment of CN patients. Indeed, C/EBPβ mRNA was upregulated 2.8-fold in CD33+ myeloid cells from CN patients by G-CSF treatment, as compared to healthy individuals. It was associated with upregulation of G-CSFR mRNA and –protein expression as well as ligand binding to G-CSFR in myeloid cells, and elevated levels of biologically active G-CSF in serum from CN patients. To confirm C/EBPβ-dependent activation of G-CSFR and G-CSF gene expression, we performed reporter gene assays in CD34+ bone marrow hematopoietic progenitor cells from two CN patients co-transfected with C/EBPβ and reporter constructs containing upstream regulatory regions of G-CSF or G-CSFR genes, −1470bp and −670bp, respectively. Indeed, C/EBPβ activated G-CSFR promoter 3.2-fold and G-CSF promoter 5.4-fold. These data demonstrate that in CN patients, G-CSF induces C/EBPa independent granulopoiesis and that C/EBPβ is required for the response to G-CSF treatment in these patients. C/EBPβ leads in response to G-CSF to induction of differentiation of neutrophil precursors to mature neutrophils in vivo. Our hypothesis is that in CN the steady state granulopoiesis is abrogated whereas the emergency granulopoiesis still leads to sufficient numbers of neutrophils.
Loss of Id4 Accelerates CLL Progression in TCL1 Mice
Authors: Chen Shih-Shih , Johnson Amy J. , Claus Rainer , Sablitzky Fred , Plass Christoph , Byrd John C.
Published: April 2020

The inhibitor of DNA binding protein 4 (ID4) is a member of dominant negative basic helix loop helix (bHLH) transcription factor family that has a HLH domain but lacking DNA-binding domain. Methylation of ID4 in B-cell lineage malignancies including CLL and germinal center non-Hodgkin lymphomas (GC-NHLs) has been described. To date, the impact of Id4 loss in CLL has not been demonstrated. We utilized the TCL1 transgenic mouse model of human CLL to study the importance of loss of ID4 in disease progression. We demonstrated that ID4 was silenced by methylation only at the time mice had symptomatic leukemia. We therefore sought to investigate if earlier loss of Id4 would accelerate CLL progression. We generated mice with haploid loss of Id4 in TCL1 transgenic background by breeding homozygous TCL1 transgenic mice and mice with heterozygous Id4 mutant gene. By comparing to the control littermates with heterozygous TCL1 oncogene, mice with the loss of Id4 occur to have significantly accelerated CLL disease progression with shorter overall survival (12 versus 16 months, p-value<0.0001). The pathological analysis on Id4 mutant mice demonstrated that the CLL symptoms such as the elevated white blood cell counts, enlarged spleen and lymphoid tissues. We then performed the microarray studies on 1 month old TCL1 mice with control or mutant Id4 to identify ID4-dependent transcriptional changes in primary B-cells. Among the identified targets, genes involved in cell proliferation and anti-apoptosis were then verified. Supportively, B-cells from Id4 mutant TCL1 mice have significantly (p-value<0.001) diminished apoptosis in response to dexamethasone treatment. Additionally, both significant in vitro (p-value<0.001) and in vivo (p-value=0.025) increased proliferation of Id4 mutant TCL1 B-cells after the stimulation by CpG685NO168 was demonstrated. These data provide support that Id4 acts as tumor suppressor in transformed B-lymphocytes and is silenced through the process of methylation. The Effort to identify binding partners of Id4 in CLL cells and targeting its re-expression is warranted.
The Association Between STAT5a Activation, LEF-1 Down Regulation and Leukemic Transformation in Patients with Severe Congenital Neutropenia
Authors: Gupta Kshama , Skokowa Julia , Welte Karl
Published: April 2020

Congenital hematological disorders are excellent models for investigating the regulation of hematopoiesis in humans. For instance, Severe Congenital Neutropenia (CN) is a heterogeneous syndrome characterized by a maturation arrest of granulopoiesis at the level of promyelocytes with no mature neutrophils in the peripheral blood. Even though G-CSF treatment results in increased neutrophil numbers in more than 90 % of CN patients, G-CSF-dependent granulocytic differentiation is severely affected in these patients. CN patients are found to be at increased risk of developing acute myeloid leukemia or myelodysplastic syndrome (AML/MDS) (cumulative incidence ~ 20 %). Since AML/MDS are not observed in cyclic or idiopathic neutropenia patients treated with G-CSF, an underlying defect down stream of G-CSF signaling rather than G-CSF therapy per se predisposes to malignant transformation in CN. STAT5 is activated by G-CSF signaling pathway and has also been found to be activated in AML. Recently we found that downregulation of LEF-1 transcription factor and its target gene C/EBPa are a common pathologic mechanism for CN. Therefore, we investigated the effects of G-CSF on the phosphorylation status of STAT5a in CN and whether it leads to the down modulation of LEF-1 expression and functions. Indeed, we detected elevated phosphoSTAT5 expression in CD34+ cells from CN patients before G-CSF stimulation in vitro, as compared to these cells from healthy individuals. Moreover, treatment with G-CSF resulted in a significantly higher phosphorylation of STAT5a in CN. Intriguingly, levels of phosphoSTAT5 in myeloid blast cells from one CN patient who developed AML was even higher and was in line with undetectable LEF-1 protein expression. Transduction of constitutive active STAT5a (STAT5a 1*6) in CD34+ cells from healthy individuals resulted in significant downregulation of LEF1 levels in a dose dependent manner. A screen of 10 kb upstream region of LEF1 gene revealed two putative STAT5 binding sites (−3891bp to −3909bp and −3714bp to −3732bp) and the specificity of this binding was confirmed in the nuclear extracts of CD34+ cells by chromatin immunoprecipitation assay. We found enhanced and prolonged STAT5a binding to the LEF-1 promoter in G-CSF treated CD34+ cells from CN patients, as compared to healthy individuals. Additionally, transfection of CD34+ cells with LEF-1 cDNA resulted in elevation of LEF-1 promoter activity, which suggests a strong LEF-1 autoregulation. Co-transfection with STAT5a 1*6 significantly disrupted LEF-1- dependent activation of LEF-1 promoter. Moreover STAT5a 1*6 severely abrogated the LEF-1 dependent regulation of C/EBPα gene promoter. Taken together phosphorylation of STAT5 is upregulated in hematopoietic progenitors from CN patients which lead to subsequent down regulation of LEF-1. These downstream effects of activated STAT5a may contribute to the malignant transformation of myelopoiesis in CN.
Interim Phase I Results of Recombinant Immunotoxin HA22 in Patients with Hairy Cell and Chronic Lymphocytic Leukemias
Authors: Kreitman Robert J. , Wilson Wyndham H. , Stetler-Stevenson Maryalice , Noel Pierre , FitzGerald David J , Pastan Ira
Published: April 2020

For the treatment of B cell malignancies, we have developed a high affinity recombinant immunotoxin (HA22), which is composed of the Fv portion of an anti-CD22 antibody fused to a 38 kDa fragment of Pseudomonas exotoxin A. HA22 is an improved form of immunotoxin BL22, which produced a 50–60% complete remission (CR) rate in cladribine-resistant hairy-cell leukemia (HCL). A partial response (PR) rate of 17% was observed with BL22 in chemoresistant chronic lymphocytic leukemia (CLL). The higher response rate of BL22 in HCL compared to CLL is related to its higher cytotoxicity for HCL than CLL cells ex vivo (median IC50 4.3 ng/ml in 33 HCL patients vs 430 ng/ml in 70 CLL patients, p < 0.0001), and the higher CD22 expression on HCL than on CLL cells (median 44,000 vs 1250 sites/cell).To increase cytotoxic activity, the affinity of the Fv of BL22 was increased about 15-fold by phage display by mutating 3 amino acids in HCDR3 from SSY to THW. Patients with HCL and CLL were treated on 2 separate multicenter phase I protocols. To be eligible, patient required at least 2 prior standard therapies and a medical indication for treatment. HA22 was administered by 30 min infusion every other day for 3 doses (QOD ×3) and patients could be retreated at the same dose level every 4 weeks unless progressive disease or immunogenicity occurred. A standard 3–6 dose escalation scheme was used, allowing new patients to be enrolled at a higher dose level if 0 of 3 or 1 of 6 patients at the current level had dose limiting toxicity (DLT). A total of 16 HCL patients were treated with HA22 at 5, 10, 20, 30, and 40 ug/Kg QOD ×3. Three patients were treated at each of the first 4 dose levels, and a 4th patient received 40 ug/Kg QOD ×3 because one patient at that dose level received only 1 cycle. Of 14 evaluable patients who received a total of 45 cycles, 4 (29%) developed high levels of neutralizing antibodies. No DLT was observed on any cycle, although 1 patient developed a grade II hemolytic uremic syndrome (HUS) during cycle 3 of 30 ug/Kg QOD ×3. Major responses included 6 (38%) CRs and 6 (38%) PRs out of 16 evaluable HCL patients (unmonitored). Responses were observed at all dose levels including 3 of 3 PRs at the lowest dose level, and 1–2 CRs at each of the next dose levels. Like BL22, peak plasma levels of HA22 were inversely related to tumor burden as assessed by circulating malignant cells or spleen size, and increased with repeated doses as patients responded. Phase I testing of HA22 in CLL is at an earlier stage with 11 patients enrolled at dose levels of 5, 10, and 20 ug/Kg QOD ×3, and decreases in circulating CLL cells have been observed. In conclusion, HA22 has not yet shown DLT in phase I testing and is highly active in HCL. Compared to BL22, HA22 shows no evidence of increased toxicity due to enhanced binding affinity to CD22.
Dasatinib Has Activity in Relapsed/Refractory Chronic Lymphocytic Leukemia (CLL/SLL), a Phase II Trial
Authors: Amrein Philip C. , Attar Eyal , Takvorian Tak , Hochberg Ephraim , Ballen Karen K. , Leahy Kathleen M. , Neuberg Donna , Brown Jennifer R.
Published: April 2020

Background: Preclinical studies have shown that CLL cells overexpress lyn kinase protein, and in vitro inhibition of lyn kinase leads to apoptosis of the CLL cells (Contri, J Clin Invest 2005). Because dasatinib has been shown to inhibit lyn kinase in CML cells at concentrations easily achievable in patients, we undertook this phase II study in patients with previously treated CLL/SLL.

Methods: Patients were required to be over 18 years of age, have a diagnosis of CLL/SLL by flow cytometry or immunohistochemistry, and have failed either 1 course of treatment with a fludarabine-containing regimen or 2 non-fludarabine containing regimens. The starting dose of dasatinib was 140 mg daily by mouth. This dose could be reduced to 100 mg or 80 mg daily for toxicity. At baseline all patients had bone marrow biopsies and CT scans, and these were repeated at 2 months. Sequential blood and bone marrow samples were tested for lyn kinase activity.

Results: Among the 15 patients enrolled there were 10 male and 5 female subjects with a median age of 59 years (40–78 years). ECOG performance status was 0 in 9 subjects, 1 in 3, and 2 in 3 subjects. All patients had previously received fludarabine, and 5 patients required treatment within 6 months of their last regimen. The median number of prior treatments was 3 (range: 1 to 7). By cytogenetic/FISH analysis there were 5 patients with del(17p) and 6 patients with del(11q). All patients required treatment by NCI–WG criteria. The major toxicity encountered was myelosuppression: grade 3 + 4 neutropenia in 10 subjects, grade 3 + 4 thrombocytopenia in 4 subjects. Gastrointestinal toxicity was minor with only 1 subject experiencing grade 3 diarrhea. Other toxicities: 1 patient had a grade 2 pleural effusion, 1 patient had a transient serum K=9.9 (likely an artifact of high white count and without clinical sequelae), and 1 patient had a transiently prolonged QTc of 516 ms. There were no fatal events, and all toxicities were reversible. The median duration on study was 10 weeks, but 5 responding or stable patients have remained on treatment for over 9 months. Partial responses (PR) by NCI-WG criteria were achieved in 2 of the 15 patients (13% with 90% CI 2%–36%). An additional 2 patients would have qualified for PR (lasting >2 months) except for myelosuppression. Among the remaining 11 patients, 6 had nodal responses (2 CR and 4 PR) by physical exam (PE) without a 50% reduction in lymphocytosis. CT scans confirmed nodal responses in 3 of the 10 patients with nodal responses by PE. The relationship between clinical response and lyn kinase, bcl-2, and mcl-1 expression will be presented at the meeting.

Conclusions: Dasatinib has modest activity in CLL, and combinations with standard agents, perhaps in an intermittent schedule, should be considered in subsequent trials.
Defensine Alpha1 as a New Marker of Imatinib Resistance in Chronic Myelogenous Leukemia
Authors: Etienne Gabriel , Dupouy Maryse , Chollet Claudine , Pasquet Jean-Max , Lippert Eric , Reiffers Josy , Mahon François- Xavier , Turcq Beatrice
Published: April 2020

Imatinib (IM) is the first therapeutic option for patients with newly diagnosed CML chronic phase (CML-CP). As alternative therapeutic options are available, identification of predictive factors of IM resistance may be of interest.

To identify specific early predictive biomarkers of IM resistance, we performed pangenomic microarray analysis in four patients with IM secondary resistance. All the four patients were in complete hematologic response at the time of analysis and RNA were extracted from peripheral mononuclear cells. Comparison of gene profile expression -was done between a sample corresponding to -BCR-ABL transcript decrease preceding the best response to IM and a sample corresponding at the time of IM resistance. BCR-ABL transcript levels were equivalent between the two points. Each patient was analyzed independently in order to limit genetic variability. We identified 22 genes differentially expressed at the time of resistance (4 up regulated, 18 down regulated). Among them, the defensin alpha 1 (DEFA1) and alpha 4 (DEFA4) genes were strongly over expressed at the time of IM resistance, 63-fold and 30-fold respectively.

Using Q-RT/PCR we next performed a prospective analysis of DEFA1 expression in 15 patients treated with standard dose (400mg/day) of IM. Nine of them were IM responders while 6 exhibited IM secondary resistance. IM responders were characterized by a strong and durable decrease in DEFA1 transcript level with a DEFA1-β actin ratio above 103. Patients resistant to IM were characterized by a lower DEFA1 expression before IM start and a dramatic increase of DEFA1 expression. Moreover the increasing of DEFA1 expression in IM secondary resistant patients occured while BCR-ABL transcript level continue to decrease. The median time between the increase of DEFA1 and the increase of BCR-ABL trancript level was 6 months (range from 0 to 23 months).

Finally we investigated DEFA1 expression in 39 CML-CP (19 early chronic phase, 20 late chronic phase)-. before IM started-. Twenty eight patients were IM responders and 11 were IM resistants (primary resistance in 2, secondary resistance in 9). DEFA1 expression before IM start was significantly lower in the IM resistant group (p=0.03) than IM responder group.

In conclusion our data suggest that variation of DEFA1 expression is early associated with IM resistance while low expression of DEFA1 at the beginning of IM treatment may be predictive of IM resistance independently of BCR-ABL level.
The Aurora Kinase Inhibitor MLN8237 Has Potent Anticancer Activity in CML and Ph+ ALL Models and Significantly Increases the Efficacy of Nilotinib
Authors: Nawrocki Steffan T. , Medina Ernest , Smith Sabrina , Swords Ronan , Kelly Kevin , Padmanabhan Swaminathan , Ecsedy Jeffrey , Giles Francis J , Carew Jennifer
Published: April 2020

The serine/threonine kinase Aurora A is a key regulator of mitosis. Its expression and activation are necessary for mitotic entry, centrosome maturation, and mitotic spindle assembly and function. Aurora A gene amplification and/or overexpression of its encoded protein has been observed in many types of human tumors and is hypothesized to promote genetic instability, a hallmark feature of malignant cells that contributes to disease progression and drug resistance. The central and obligatory role of Aurora A in mitosis and its dysregulation in cancer makes it an attractive target for therapeutic inhibition. MLN8237 is a novel orally available Aurora A inhibitor developed for cancer therapy by Millennium Pharmaceuticals that has entered Phase I trials in humans. We investigated the preclinical efficacy and mechanism of action of MLN8237 in models of chronic myeloid leukemia (CML) and Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL). Sub-micromolar concentrations of MLN8237 potently inhibited the in vitro growth and survival of human CML and Ph+ ALL cell lines and primary cells derived from CML and Ph+ ALL patients. In vitro kinase assays demonstrated that MLN8237 effectively inhibits the activity of wildtype and drug-resistant mutant forms of BCR-ABL. Notably, MLN8237 possessed equipotent anticancer activity against Ba/F3 cells expressing wildtype (p210) BCR-ABL and the imatinib, nilotinib, dasatinib, and bosutinib-resistant mutant T315I form of BCR-ABL. Loss of function of the p53 tumor suppressor contributes to resistance to imatinib and many other anticancer agents. We assessed the potential impact of loss of p53 function on cellular sensitivity to MLN8237 by achieving stable shRNA-mediated p53 knockdown in Ba/F3 cells expressing p210 BCR-ABL. Impairment of p53 function did not significantly affect the anticancer activity of MLN8237, indicating that it may be an effective agents for patients with p53 defects. Daily oral administration of MLN8237 (20 mg/kg BID) to immunodeficient mice bearing xenografts of the K562 human CML blast crisis cell line was very well tolerated and significantly reduced tumor growth in vivo. Moreover, co-administration of MLN8237 and nilotinib resulted in significantly greater tumor growth inhibition than what was achieved by either single agent alone. These data suggest that MLN8237 is a promising new agent for the treatment of CML and Ph+ ALL patients that fail to respond to conventional therapy.
Erythropoietin Stimulates Bone Formation In Vivo By Targeting Both HSCs and MSCs
Authors: Jung Younghun , Ziegler Anne , Shiozawa Yusuke , Wang Zhuo , Song Junhui , Wang Jianhua , Wang Jincheng , Pienta Michael J. , Lee Clara H. , Havens Aaron M. , Pedersen Elisabeth A. , Krebsbach Paul H. , Taichman Russell S.
Published: April 2020

Previously we showed that HSCs isolated from stressed animals (an acute bleed) regulates osteoblast (OBs) differentiation from bone marrow stromal cells (BMSCs). This activity suggests that HSCs regulate the development of their own niche. The molecular basis for this activity is the production of BMP-2 and BMP-6 by HSCs. Yet what stimulates HSCs to produce BMPs is unknown. We hypothesized that erythropoietin (Epo) may activate HSCs to produce BMPs, or Epo directly activates BMSCs to form OBs. To test these possibilities, Epo serum levels in the bled vs. non-bleed mice were determined and found to match the time frame of HSC activation. Surprisingly, EpoR expression was identified on HSCs isolated using the SLAM-family of receptors, and its expression on HSCs increased in bled animals. In vitro stimulation with Epo activated the JAK-STAT signaling pathways, and importantly, BMP production by HSCs.

To determine if Epo directly acts on BMSCs, BMSC co-cultures were established with HSCs derived from bled (B-HSC) or non-bled (NB-HSCs) animals, or treated with rhEpo alone for 14d. Differentiation along the OB lineage was evaluated for CFU-fibroblastic and osteoblastic (CFU-F, CFU-OB) formation, QRT-PCR for Runx2 (OB-specific transcription factor), bone sialoprotein and osteocalcin, or for bone mineral formation (alizarin red staining). BMSCs responded to the presence of NB-HSCs by increasing their generation of CFU-OB and CFU-F compared to no HSCs. Co-culture with B-HSCs or rhEpo treatments were significantly better able to induce CFU-OB and CFU-F differentiation of the BMSCs. QRT-PCR demonstrates that the OB-specific transcripts were increased with rhEpo or co-culture with B-HSC compared to NB-HSC or vehicle. Alizarine red staining also demonstrated an increase in mineral apposition in response to Epo or B-HSC.

To directly determine if Epo stimulates bone formation in vivo, newborn mice were treated with rhEpo (1,500U-6,000U/kg, 3x/week), anabolic PTH (as a positive control) or vehicle for 4 weeks. rhEpo increased the hematocrit and hemoglobin levels in a dose dependant manner. As expected, PTH increased the bone formation. rhEpo treatments enhanced OB numbers in long bone sections, and micro-CT measurements of the vertebra revealed a significant increase in bone formation parameters including bone mineral density (BMD), bone volume fraction (BVF) and trabecular number (Tb.N.).

These data for the first time demonstrate that Epo regulates the formation of the HSC niche by both direct and indirect pathways, and further demonstrates a coupling between hematopoesis and osteopoiesis in the marrow. These results also suggest that targeting the Epo/EpoR pathway may serve as a therapeutic modality to treat skeletal or mesenchymal abnormalities in humans.
Role of BCR in Down-Modulation of BCR-ABL Oncogenicity in CML
Authors: Perazzona Bastianella , Wang Yan , Arlinghaus Ralph B.
Published: April 2020

Bcr-Abl acquires its transforming ability through its up-regulated Abl tyrosine kinase activity. Bcr is a phosphoprotein with a novel serine/threonine kinase activity encoded by its first exon. Over-expression of BCR in K562 cells produces a phosphoserine form of Bcr and interferes with the oncogenic effects of BCR-ABL in mice (Lin et al., Oncogene 2001). We have recently shown the inhibitory effects of Bcr on Bcr-Abl, in a nude mouse solid tumor model. Expression of BCR/GFP in TonB210 cells used for injection delayed tumor formation and tumors were 50% smaller compared to the TonB210/GFP control. In contrast two point mutants in the BCR kinase domain (Y360F and S354A), not only blocked Bcr’s inhibitory effects but enhanced the oncogenic effects of BCRABL (Perazzona et.al. Oncogene, 2008). Similar Bcr effects were observed in a mouse leukemia model. We are investigating the mechanism of the interaction between Bcr and Bcr-Abl proteins. Using TonB210 cells, in which BCR-ABL expression is controlled by a tetracycline-inducible promoter and Bcr is stably transduced by lentivirus infection, we observed that increasing levels of Bcr-Abl expression increased the levels of the Bcr protein. Treatment of TonB210 cells with imatinib mesylate decreased the levels of Bcr- Abl and surprisingly the Bcr protein as well, indicating that the tyrosine kinase function of Bcr-Abl is required to up-regulate Bcr protein expression. In addition, withdrawal of doxycycline also reduced Bcr-Abl and Bcr protein levels, confirming that Bcr-Abl is required for increased expression of the Bcr protein. In order to examine the levels of Bcr in cells lacking Bcr-Abl, we transduced BCR/GFP with lentivirus infection into BaF3 and 32D cells. Surprisingly, these cell lines expressed extremely low levels of Bcr, despite 90% expression of the GFP marker. Expression of Bcr was restored by overnight treatment with the proteasome inhibitor calpain inhibitor I. Forced expression of Bcr-Abl in BCR- transduced cells restored high expression of Bcr protein, confirming that Bcr- Abl is required for preventing degradation of the Bcr protein. Together these findings indicate that Bcr-Abl up-regulated Bcr expression by interfering with proteasomemediated degradation of the Bcr protein. Additional studies indicated that Bcr increases expression of the myeloid membrane surface marker Mac-1 in Bcr-Abl TonB210 cells, which originated from the mouse pro-B cell BaF3. We propose that Bcr may play a role in generating the myeloid phenotype caused by Bcr-Abl in CML patients and may be an important player in the chronic phase of CML by down-modulating Bcr-Abl.