bluuebunny/crossref_metadata_2025 huggingface dataset:
Antileukemia Activity of JNJ-26481585, a Potent Second-Generation Histone Deacetylase Inhibitor
Authors: Tong Weigang , Wei Yue , Stevenson William , Kuang Shao-Qing , Arts Janine , Garcia-Manero Guillermo
Published: April 2020

Background: Histone Deacetylases (HDACs) play key roles in tumor cell proliferation, survival and angiogenesis. HDAC inhibitors have been shown to induce cell cycle arrest, terminal differentiation and/or apoptosis in broad spectrum of human tumors and xenograft animal models. JNJ-26481585 is a hydroxamic acid derivative second-generation pan-HDAC inhibitor with nM potency in pre-clinical studies. In animal models, no evidence of cardiac toxicity has been observed, which suggests a better therapeutic index than other HDAC inhibitors.

Aim: We evaluated the anti-leukemia and molecular activity of JNJ-26481585 in human leukemia cell lines (HL60, MOLT4, THP1 and U937) and primary leukemia cells.

Results: JNJ-26481585 potently inhibited proliferation of leukemia cells, with IC50 between 25 nM and 250 nM after 24-hour of treatment. In contrast, analysis of other HDAC inhibitors, including vorinostat and MGCD0103, had IC50 in the μM range. JNJ-26481585 also induced dose-dependent apoptosis (aprox. 25–35% at concentration of 25 nM) of leukemia cell lines by Annexin V staining after 24-hour treatment. A synergistic apoptotic effect was observed by co-treating with hypomethylating agent 5-aza-2′-deoxycytidine (25% with JNJ-26481585 25 nM, 5% with decitabine 1 μM, and 70% for the combination). Acetylation of histones H3, H4 and phosphorylation of H2AX was also observed after exposure to JNJ-26481585 at concentration of 12.5 nM or higher. We also studied fresh primary leukemia cells from 9 patients (ALL N=1, MDS N=1, AML N=7). Six of the 9 patients (67%) had relapsed/refractory disease. Their median white cell counts were 32×103/L (range 6–407×103/L), with 25% to 95% blasts in the peripheral blood. Treatment with JNJ-26481585 for 24-hours induced significant growth arrest and apoptosis (20–50% apoptosis at 25 nM) in a dose-dependent manner (between 12.5–500 nM), but not in mononuclear cells from healthy donors (N=2). It also induced acetylation of histones H3, H4, phosphorylation of H2AX, induction of p21 and HSP70, activation of caspase-3, cleavage of PARP in these primary leukemia cells after 24-hour treatment at concentrations of 12.5–500 nM.

Conclusion: JNJ-26481585 is a potent second generation pan-HDAC inhibitor with activity in human leukemias. It is a promising new agent for future clinical studies either as single agent or combination epigenetic therapy with DNA hypomethylating agents and should be tested in clinical trials.
Small Molecule Inhibition of Cdc7, a Key Cell Cycle Regulator and Novel Therapeutic Target, Successfully Inhibits Leukemia Cell Growth in Vitro and in Vivo
Authors: Frattini Mark G. , Shum David , O’Dwyer Kristen M , Brentjens Renier J. , Yeh Ray , Maslak Peter , Heaney Mark Lawrence , Jurcic Joseph , Djaballah Hakim , Kelly Thomas J
Published: April 2020

Cdc7 is a heterodimeric serine/threonine protein kinase that is a key regulator in the process of initiation of DNA replication and the G1 to S phase transition. Both the kinase and its known substrates are over-expressed in the majority of human cancers. As a result of the recent progress in the areas of pharmacogenetics and high throughput screening technology, identifying specific small molecule inhibitors of cell cycle regulated protein kinases has provided a means not only to study these signal transduction pathways but also to identify potential novel therapeutic agents. To this end, we have developed an assay for Cdc7 kinase inhibitory activity using a highthroughput screening (HTS) approach, screening over 250,000 natural and synthetic small molecules. As a result, we have identified and confirmed seventeen compounds, representing nine different chemical scaffolds, with Cdc7 kinase inhibitory activity. Based on potency, we selected the lead compound (CKI-7) which was further characterized using kinase profiling, microarray experiments, and standard cell based cytotoxicity assays. These latter studies demonstrated that CKI-7 induced cytotoxicity of established leukemia and lymphoma cell lines in culture with inhibitory concentrations (IC50s) in the low nanomolar range. Significantly, CKI-7 likewise induced cytotoxicity of MDR1 overexpressing cell lines with similar IC50s, demonstrating that this novel compound can overcome a major mechanism of chemotherapy resistence in human tumor cells. We additonally demonstrate that CKI-7 induces cytotoxicity of patient-derived primary acute leukemia tumor cells (both chemotherapy naïve and relapsed/refractory samples) in vitro at similarly low nanomolar concentrations. In vivo dose-dependent anti-tumor activity of CKI-7 was subsequently demonstrated in a SCID-Beige mouse systemic tumor model utilzing a recently isolated Philadelphia chromosome positive acute lymphoblastic leukemia cell line (PhALL3.1). Standard cell cycle synchronization studies established that exposure to CKI-7 results in cell cycle dependent caspase 3 activation and apoptotic cell death. This cell death is the direct result of Cdc7 kinase inhibition by CKI-7 as demonstrated using a substrate biomarker assay. In conclusion, our data confirm that Cdc7 is a new promising target for cancer therapy, and that CKI-7, a selective small molecule inhibitor of this enzyme, is an equally promising novel cancer therapeutic agent.
Diagnosis and Classification of Amyloidosis in Abdominal Subcutaneous Fat Aspiration Specimens Using Mass Spectrometry Based Proteomics
Authors: Vrana Julie A. , Zeldenrust Steven R. , Theis Jason D. , Gamez Jeffrey D. , Kurtin Paul J. , Dogan Ahmet
Published: April 2020

Abdominal subcutaneous fat aspiration is one of the most practical, sensitive and specific methods for the diagnosis of systemic amyloidosis. One limitation of this method, compared to more invasive tissue biopsy based approaches, remains the technical difficulties in further classification of the amyloidosis as commonly used methods, such as immunohistochemistry, are not readily applicable to fat aspiration specimens. To overcome these difficulties we developed a method using nano-flow liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS) that could identify amyloid subtypes in freshly obtained Congo Red positive fat aspirate specimens with great accuracy.

Abdominal subcutaneous fat aspirate specimens were obtained from 73 patients with clinical suspicion for systemic amyloidosis. One half of the specimen was stained with Congo red and used for diagnosis of amyloidosis and the other half was processed and enzyme digested for LC-MS/MS analysis. The resulting LC-MS/MS data was correlated to theoretical fragmentation patterns of tryptic peptide sequences from the Swissprot database using Scaffold (Mascot, Sequest, and X!Tandem search algorithms). Peptide identifications were accepted if they could be established at greater than 90.0% probability and protein identifications were accepted if they could be established at greater than 90.0% probability and contain at least 2 identified spectra. The identified proteins were subsequently examined for the presence or absence of amyloid related peptides. Of the 73 cases studied, 41 were positive for Congo red consistent with systemic amyloidosis. In Congo red positive cases, LC-MS/MS peptide profiles consistent with AL-lambda (28/31), AL-kappa (6/7), and ATTR (2/3) were observed. Only one case in the Congo red negative control group (31/32) gave a kappa light chain profile which was attributed to a high level of kappa in the serum (285 mg/dL). Of the 35 out of 41 cases of systemic amyloidosis successfully classified by LC-MS/MS, additional clinical and pathology data validating the amyloid type was available. In each of these cases the MS/MS results accurately predicted the amyloid type.

In conclusion, LC-MS/MS proteomic analysis of abdominal subcutaneous fat aspiration specimens involved by amyloidosis provides a highly specific (97% specificity) and sensitive (>85% sensitivity) method for diagnosis and classification of amyloidosis. The method is rapid and readily applicable in a clinical setting and will greatly improve the clinical management of amyloidosis patients.
A Novel Targeted Therapeutic Approach to Multiple Myeloma Using Tumor Specific Anti-Sense RNA Sequences
Authors: Li Zhi-Wei , Shalitin Dror , Shen Jing , Share Michael , Li Mingjie , Wang Cathy S , Sanchez Eric , Steinberg Jeffrey A , Chen Haiming , Bonavida Benjamin , Berenson James R.
Published: April 2020

Despite recent advances in the therapy of multiple myeloma (MM), the disease remains incurable. Current drugs for MM target not only MM but also normal cells. This limits the efficacy of these agents and may lead to significant morbidities for patients. Therefore, novel strategies are needed that allow targeting of tumor cells in these patients without affecting nonmalignant cells. MM is a malignancy of clonal antibody-secreting plasma cells with specific rearrangement of DNA that is transcribed into a unique mRNA sequence that is translated into the tumor specific monoclonal antibody. These tumor specific transcripts are abundant in all of the malignant cells of these patients. We have explored the possibility of using a unique sequence from this transcript, the complementarity determining region (CDR) gene sequence, to direct drug release into MM cells. Using a quenched fluorescein-labeled oligonucleotide sequence complementary to the CDR3 gene sequence from the MM cell line RPMI8226 as a probe, our results have demonstrated that quenched fluorescent signals of this RPMI8226-specific oligonucleotide were released upon binding to the CDR3 gene sequence of RPMI8226, but not to oligo(dT), random sequences or the CDR3 gene sequence from another MM cell line, U266. Similarly, using a quenched fluorescein-labeled oligonucleotide sequence complementary to the CDR3 gene sequence from U266 as a probe, our results have also shown that quenched fluorescent signals of the U266-specific oligonucleotide were released upon binding to the CDR3 gene sequence of U266, but not to oligo(dT), random sequences or the CDR3 gene sequence from RPMI8226. In addition, transfection of 8226- or U266-specific oligonucleotides into 8226 and U266 cells indicated that the oligonucleotides specifically discriminated the two cell lines. Furthermore, these oligonucleotides are stable in PBS or in transfected cells for at least 48hrs at 37°C, long enough for killing of MM cells with most currently available anti-MM drugs. Currently, we are evaluating the efficacy of this targeted approach to specifically identify tumor cells, and developing therapeutic modification of the oligonucleotide to allow drug delivery specifically to the tumor cells. Our studies validate a new strategy for MM treatment that may introduce a new targeted therapeutic approach in MM as well as other B cell malignancy, without impacting nonmalignant cells, which should allow much higher concentrations of drugs to be delivered into the tumor cells. Updated results using this novel targeted treatment approach will be reported at the meeting.
Identification of a Novel Mode of Kinase Inhibitor Resistance: An F604S Exchange in FIP1L1-PDGFRA Modulates FIP1L1-PDGFRA Protein Stability in a SRC-Dependent Manner
Authors: Gorantla Sivahari P. , von Bubnoff Nikolas , Peschel Christian , Duyster Justus
Published: April 2020

FIP1L1-PDGFR alpha is a constitutively activated protein kinase which was reported in chronic eosinophilic leukemia (CEL) and in cases of hypereosinophilic syndrome and mastocytosis with eosinophilia. Imatinib is clinically active against FIP1L1-PDGFRA positive disease. However, clinical resistance to imatinib has been observed in FIP1L1-PDGFRA positive leukemia and was shown to occur due to a secondary mutation (T674I) in the PDGFR alpha kinase domain. Using a screening strategy to identify imatinib resistant mutations, we generated numerous imatinib resistant cell clones. Analysis of the PDGFRA kinase domain in these cell clones revealed a broad spectrum of resistance mutations including the clinically reported exchange T674I. Interestingly, one of the abundant mutations was a Phe to Ser exchange at position 604 (F604S), which occurred alone or in combination with other exchanges. Surprisingly, FIP1L1-PDGFRA/F604S in contrast to D842H and F604+D842H did not increase the biochemical or cellular IC50 value to imatinib when compared to wild-type (wt). However, F604S and F604S+D842H transformed Ba/F3, NIH3T3 and mouse bone marrow more efficiently compared to wt and D842H, respectively. Also, F604S and F604S+D842H showed strong activation of Stat5, ERK and Akt compared to wt and D842H. Immunoprecipitation and immunoblotting indicated increased amounts of FIP1L1-PDGFRA protein in F604S versus wt cells. Moreover, SRC coimmunoprecipitated with FIP1L1-PDGFRA in wt, but not F604S cells. We hypothesized that F604S might interfere with FIP1L1-PDGFRA protein stability, and that SRC might be involved in this process. GST pull down experiments using SRC-SH2 domain showed lesser binding of FIP1L1-PDGFRA/F604S compared to wt. Similarly, using a GST-PDGFRA fragment, more SRC was precipitated with wt compared to F604S. Importantly both, the SRC inhibitor PD166326 and SRC siRNA mimicked the F604S phenotype and resulted in stabilization of the wt protein. Also, co-expression of SRC in 293T cells augmented degradation of wt, but not F604S FIP1L1-PDGFRA, indicating that SRC is a negative regulator of FIP1L1-PDGFRA protein stability. Similar results were obtained with an exchange in near proximity to F604. Kinase-defective SRC had no effect on FIP1L1-PDGFRA stability, indicating that kinase activity of SRC is necessary for its effect on FIP1L1-PDGFRA stability. Moreover, kinase defective FIP1L1-PDGFRA (G610R) was not degraded indicating that kinase activity of FIP1l1-PDGFRA is necessary for its own degradation. Taken together, imatinib resistance screening in FIP1L1-PDGFRA identified a novel class of resistance mutations, that do not act by impeding drug binding to the target, but rather increase target protein levels by interfering with its SRC mediated degradation.
Lyl1 and Scl Dosage Is Critical for Adult Hematopoietic Stem Cell Function and Survival
Authors: Souroullas George , Salmon Jessica , Curtis David J. , Goodell Margaret
Published: April 2020

The Stem Cell Leukemia gene (Scl) and the Lymphoblastic Leukemia gene 1 (Lyl1) genes encode two related basic-helix-loop-helix transcription factors that are involved in T-cell acute lymphoblastic leukemia via chromosomal translocations or aberrant expression due to other genetic or epigenetic abnormalities. Previous studies have shown that Scl is necessary for embryonic hematopoiesis and erythropoiesis, but dispensable for adult hematopoietic stem cell (HSC) function. Lyl1 on the other hand is important for adult HSC function and B-cell development. In order to elucidate in more detail the functions of Lyl1 and Scl in adult HSCs, we generated double knock-out mice and studied the function of their HSCs. Double knock-out mice demonstrate a striking genetic interaction between the two genes, with a clear dose-dependence for the presence of Scl or Lyl1 alleles for HSC function.

Specifically, we carried out competitive fetal liver transplantation assays using mice with different combinations of Lyl1 and Scl alleles and analyzed their potential to repopulate the bone marrow of recipient mice. Peripheral blood chimerism of recipient mice shows that HSC function is lost with progressive loss of Lyl1 or Scl dosage, suggesting an overlap of function of the two genes in the HSCs (Figure)

We have further investigated the function of Lyl1 and Scl in adult hematopoiesis by generating Lyl1/Scl-conditional double knock-out mice and studied adult HSC function measured by bone marrow repopulation assays. Donor engraftment dramatically decreased after deletion of the floxed Scl allele, demonstrating rapid loss of adult HSC function. Furthermore, Scl deletion in a Lyl1−/− background causes hematopoietic progenitors to rapidly undergo apoptosis in a cell-intrinsic fashion. These results demonstrate that at least one allele of either Lyl1 or Scl must be present for adult HSC survival, and show that their functions partly overlap in HSCs, suggesting that Lyl1 and Scl are crucial regulators of adult, as well as embryonic, hematopoiesis.

Figure Figure
Human T-Cell Leukemia Virus Type 1 Tax Activates Caveolin-1 Gene Expression
Authors: Sawada Shigeki , Ishikawa Chie , Tanji Hiroe , Mori Naoki
Published: April 2020

Caveolae are plasma membrane invaginations that function as regulators of signal transduction. Caveolins are a class of oligomeric structural proteins that are both necessary and sufficient for caveolae formation. Caveolin-1 has been shown to regulate multiple cancer-associated processes including cellular transformation, tumor growth, cell migration and metastasis, cell death and survival, multidrug resistance, and angiogenesis. Adult T-cell leukemia (ATL) is a mature CD4+ T-cell malignancy etiologically associated with human T-cell leukemia virus type 1 (HTLV-1). We examined expression of caveolin-1 and caveolin-2 in HTLV-1-infected T-cell lines and primary ATL cells. These cells expressed high levels of caveolin-1 mRNA compared with uninfected T-cell lines and normal PBMCs. However, caveolin-2 mRNA expression was not restricted to HTLV-1-infected T cells. Immunohistochemical staining of caveolin-1 in ATL lymph nodes and skin lesions showed that primary ATL cells were indeed stained strongly positive for caveolin-1. In vitro infection of a human T-cell line with HTLV-1 induced caveolin-1 mRNA expression. The viral protein Tax transcriptionally activated caveolin-1 gene. Using Tax mutants, we demonstrated that Tax-induced caveolin-1 expression required the NF-kappaB and CREB signaling pathways. TGF-beta is an inhibitor of T-cell proliferation and cytotoxicity. HTLV-1-infected T-cell lines and ATL cells are known to be resistant to TGF-beta-induced growth inhibition. We demonstrated that caveolin-1 is able to suppress TGF-beta signaling at the transcriptional level in T cells. Caveolin-1 colocalized with TGF-beta type I receptor in an HTLV-1-infected T-cell line. Thus, we describe a new function of Tax, as a repressor of TGF-beta signaling through caveolin-1 expression, which may play a critical role in ATL leukemogenesis.
FLIPI and F2 Index – Prognostic Indices in Advanced Stage Follicular Lymphoma
Authors: Unterhalt Michael , Hoster Eva , Dreyling Martin , Buske Christian , Hiddemann Wolfgang ,   on behalf of the German Low-Grade Lymphoma Study Group (GLSG)
Published: April 2020

Background: The follicular lymphoma international prognostic index (FLIPI) has become a widely accepted tool for stratification of patients with follicular lymphoma according to their risk profiles (Solal-Celigny et al., Blood 2004). Its prognostic relevance has been proven in patients treated with standard chemotherapy as well as with immunochemotherapy (Buske et al., Blood 2006). Recently, a new prognostic index, the F2 index, has been proposed as possible alternative to the FLIPI (Federico et al., ICML-10, 2008). Methods: We used the collective of patients with advanced stage follicular lymphoma treated first-line within the recent trials of the GLSG to explore the prognostic relevance of the F2 prognostic index in comparison to the FLIPI. Outcome parameters were time to treatment failure (TTF) and overall survival (OS). Statistical methods included Kaplan-Meier plots to validate the prognostic indices and multiple Cox regression to explore the relevance of the prognostic factors.

Results: Of the 1562 patients, the F2 index could only be assessed in 758 patients (50%), of those 355 had had a treatment failure and 124 had died. The low number of evaluable patients was mainly due to the use of β2-microglobulin as prognostic factor, which was missing in 45% of the patients. The FLIPI could be assessed in 1424 patients (93%), of those 697 had had a treatment failure and 255 had died. The F2 index classified 7% of the patients as low risk, 58% as intermediate risk, and 35% as high risk, in comparison to 14%, 41% and 45% according to the FLIPI. Both F2 and FLIPI separated three risk groups with regard to TTF (p<0.0001). However, the F2 index did not separate risk groups in the three FLIPI risk subgroups. In addition, in multiple regression analysis of the F2 and FLIPI prognostic factors, two of the three new F2 prognostic factors, namely bulk > 6 cm and elevated β2-microglobulin showed no prognostic relevance for TTF. Furthermore, after backward elimination, all the FLIPI risk factors (age > 60 years, LDH > ULN, hemoglobin > 120 g/l and more than 4 involved nodal regions) remained independently significant together with bone marrow involvement, whereas bulk > 6cm and b2-microglobulin were of no relevance. With regard to OS, the FLIPI separated three groups, whereas according to the F2 index low and intermediate risk groups were overlapping.

Conclusion: The F2 index did not show superior relevance to the FLIPI in our evaluation and should not replace the FLIPI as prognostic index in follicular lymphoma in general. However, a simplification of the FLIPI with regard to the assessment of the number of involved nodal areas and the incorporation of the performance status would be challenging.
Phase 1 Study of Weekly Vincristine Sulfate (VCR) Liposomes Injection (VSLI, Marqibo™) Plus Dexamethasone in Adults with Relapsed or Refractory Acute Lymphoblastic Leukemia (ALL)
Authors: Thomas Deborah A. , Kantarjian Hagop M , Stock Wendy , Heffner Leonard , Hirabayashi Sue , Lu Biao , Deitcher Steven R. , O’Brien Susan
Published: April 2020

Novel formulations of standard chemotherapy allowing increased drug delivery without additional toxicity may improve outcomes for patients with relapsed or refractory acute lymphoblastic leukemia (ALL). Vincristine sulfate liposomes injection (VSLI, Marqibo®) is VCR encapsulated in sphingomyelin (55%)/cholesterol (45%) nanoparticle liposomes called Optisomes™. Pharmacokinetic studies have shown that the altered distribution and elimination phases of VSLI may lead to increased VCR exposure compared to traditional VCR, and may account for the increased efficacy observed in preclinical models. Activity of VCR is dose and time-dependent, but neurotoxicity limits dosing to 1.4 mg/m2 (capped at 2.0 mg). VSLI, however, may be given without dose capping. We conducted a phase 1, open-label, dose-escalation study of VSLI in adults with relapsed or refractory ALL to determine the safety, maximum tolerated dose (MTD), and activity of this formulation. Subjects received VSLI intravenously weekly at doses of 1.5, 1.825, 2.0, 2.25 or 2.4 mg/m2. Dexamethasone 40 mg was given days 1–4 and 11–14 of each 4 week cycle. Thirtysix eligible subjects, all of whom had been previously treated with VCR, received at least 1 dose of VSLI. All were Philadelphia chromosome negative except for one. Median number of doses received for all subjects was 4; total medium cumulative dose was 9.09 mg/m2 (19.20 mg). MTD of VSLI was 2.25 mg/m2 based on dose-limiting toxicities of grade 3 motor neuropathy, grade 4 seizure and grade 4 hepatotoxicity observed in 1 subject each at the 2.4 mg/m2 dose level. The most common toxicities (constipation [67%], fatigue [61%], pyrexia [50%], anemia [50%], peripheral neuropathy [50%; mostly grade 1–2]) were as expected. Complete response (CR) was achieved in 7 of 36 (19%) subjects based on intent to treat analysis (Table). Overall response rate (including 1 PR) was 22%. Four subjects (11%) achieved hematologic improvement, 13 (36%) had stable disease, and 9 (25%) progressed. CR rate was 29% for the 7 subjects treated with VSLI as second salvage. Five of 7 subjects who achieved CR were able to undergo allogeneic stem cell transplant (SCT). In conclusion, VSLI appears to be an effective therapeutic option which may permit potentially curative SCT. A phase 2 international multi-center trial of single agent VSLI in subjects with relapsed ALL is currently accruing.

Table. VSLI Clinical Activity (CR) by Dose Level and Salvage Status

1.5 mg/m2 n = 5 1.825 mg/m2 n = 3 2 mg/m2 n = 3 2.25 mg/m2 n = 18 2.4 mg/m2 n = 7 Total n = 36 1st salvage 1/1 0/1 -- 2/7 1/4 4/13 (31) 2nd or later salvage 1/4 1/2 0/3 1/11 0/3 3/23 (13)
Direct Transcriptional Repression of BCL2 by BCL6 in Germinal Centre B Cells and Its Disruption in B Cell Lymphomas with BCL2 Locus Alterations
Authors: Piovan Erich , Saito Masumichi , Basso Katia , Novak Urban , Shen Qiong , Pasqualucci Laura , Dalla-Favera Riccardo
Published: April 2020

The human proto-oncogene BCL6 encodes a BTB/POZ-zinc finger transcriptional repressor that is required for germinal centre (GC) development and is expressed in the majority of normal GC B cells and in the majority of B cell lymphoma (B-NHL), including follicular lymphoma (FL) and a subset of diffuse large B cell lymphomas (DLBCLs). Deregulation of BCL6, by chromosomal translocation or somatic hypermutation, is implicated in the pathogenesis of B-NHL. The precise function of BCL6 in GC development and lymphomagenesis is still unclear also due to the very few direct BCL6 target genes that have been identified. In order to identify physiologically relevant BCL6 direct target genes, we used a novel approach combining high-throughput biochemical (ChIP-on-chip), bioinformatics (ARACNe) (Basso et al., Nature Genetics, 2005) and gene expression profile analysis (Klein et al., Proc. Natl. Acad. Sci., 2004) of normal GC B cells. The results (see abstract by Saito et al.) have identified a core of bona fide target genes whose promoter is bound by BCL6, whose transcription is dynamically linked to BCL6, and whose expression is down-regulated in GC B cells. Among the genes meeting these stringent criteria, we found BCL2, encoding the anti-apoptotic molecule whose expression is deregulated by chromosomal translocations or gene amplification in the majority of FL and in a subset of DLBCL. Further investigations showed that BCL6 represses BCL2 transcription by binding specific DNA sites within the BCL2 promoter region. Suppression of BCL6 expression via specific siRNAs leads to increased levels of BCL2, and, conversely, constitutive BCL6 expression prevents B cell receptor (BCR)-induced upregulation of BCL2 in B cells. Consistent with a physiological role for BCL6- mediated BCL2 suppression, immunohistochemical analysis shows that BCL2 expression is absent in GC B cells where BCL6 is highly expressed. Notably, the comparative analysis of BCL6 and BCL2 expression in B-NHL cell lines showed a conserved inverse correlation between BCL6 and BCL2 levels in cell lines carrying a normal BCL2 locus. However, cell lines carrying chromosomal translocations or amplifications affecting the BCL2 gene displayed the pathologic co-expression of both proteins, suggesting that the alterations affecting the BCL2 locus prevent BCL6-mediated suppression. These results indicate that one critical role of BCL6 is the modulation of antiapoptotic function in GC B cells via BCL2 transcriptional repression, suggesting a mechanism by which B cells may die in the GC if not rescued by BCR and CD40 engagement, both of which downregulate BCL6 while inducing BCL2 expression. Alterations of the BCL2 locus may contribute to lymphomagenesis by making the gene resistant to suppression by BCL6, and therefore allowing B cells to avoid apoptosis in the absence of antigen- and T-cell-mediated rescue.
Secondary Acute Myeloid Leukemia (sAML) Treated with Amonafide (AS1413) + Cytarabine: Durable Responses in Poor-Risk AML
Authors: Allen Steven L , Erba Harry P , Rizzieri David A. , O’Donnell Margaret , Powell Bayard , Lundberg Ante S , Bennett John M , Capizzi Robert L
Published: April 2020

Background: Secondary acute myeloid leukemia (sAML), evolving from prior MDS or leukemogenic therapy (tAML), is associated with a poor prognosis. Many sAML patients are elderly with significant comorbidities. The incidence of unfavorable cytogenetics and expression of multidrug resistance (MDR) phenotype is higher in sAML blasts. Amonafide (AS1413) is a DNA intercalating agent and unique topoisomerase II (Topo II) inhibitor that, unlike the classical Topo II inhibitors (DNR, IDA, mitoxantrone, etoposide), does not affect the DNA/Topo II cleavable complex that results in DNA fragmentation. It does not act as either a substrate or an inhibitor of the MDR efflux pump, P-glycoprotein (Pgp), nor is it cross-resistant with classical Topo II inhibitors. A complete remission (CR) rate of 38.6%, with an additional 3.4% of patients achieving CRp, has previously been reported for this phase II trial. Updated findings from this trial including duration of remission data are reported here.

Methods: Patients received a 4 hr infusion of amonafide 600 mg/m2/day × 5 and continuous IV infusion cytarabine 200 mg/m2/day × 7. 14 patients received a second course on day 14 for persistent leukemia. Patients received consolidation with stem cell transplant or intermediate/high dose cytarabine, depending on age. Central bone marrow review was performed for patients in CR. The primary endpoint was CR + CRp. Secondary endpoints included duration of CR/CRp. The last patient was treated in December 2006.

Results: 88 patients were enrolled; median age was 62.5 (range 23–87); prior MDS, 48.9%, (43/88); tAML, 51.1% (45/88); unfavourable cytogenetics, 46.6% (41/88). The most common non-hematologic grade ≥ 3 AEs seen were febrile neutropenia (35.2%), hypotension (15.9%), pneumonia (14.8%), hypokalemia (12.5%), and bacteremia (11.4%). Death rate within 28 days was 20.5%. Overall CR + CRp rate: 42.0% (37/88), including 38.6% (34/88) CR; 3.4% (3/88) CRp. CR by age < 60: 39.4% (13/33); ≥ 60: 43.6% (24/55). 30/37 patients received post-remission therapy with BMT (7), high/intermediate dose cytarabine (21) or both (2).

Kaplan-Meier estimate of the proportion of responders in continuous CR/CRp at 18 months is 41% for all sAML patients, and is similar for all poor-risk subgroups including age ≥ 60, 43%; tAML, 57%; and unfavorable cytogenetics, 38%. Median overall survival is 209 days for all patients and 400 days for patients with CR/CRp.

Conclusions: Durable CRs have been achieved in patients with sAML/tAML treated with amonafide plus cytarabine. This efficacy was similar across subgroups independent of age or the presence of various poor risk characteristics. Phase III evaluation of amonafide plus cytarabine vs. daunorubicin plus cytarabine (3+7) is currently underway.

Duration of Remission Duration of Remission
Clinical Predictors of Prophylaxis Use Prior to the Onset of Acute Venous Thromboembolism in Hospitalized Patients: Swiss Venous ThromboEmbolism Registry (SWIVTER)
Authors: Kucher Nils , Spirk David , Kalka Christoph , Mazzolai Lucia , Nobel Daniel , Banyai Martin , Frauchiger Beat , Bounameaux Henri
Published: April 2020

Background: Venous thromboembolism (VTE) prophylaxis among hospitalized patients remains underutilized. We investigated the use of appropriate prophylaxis and predictors for using prophylaxis prior to the onset of acute VTE.

Methods: In 14 academic and non-academic Swiss hospitals, 567 consecutive patients (306 medical and 261 surgical patients) with objectively confirmed acute VTE and hospitalization within 30 days prior to the VTE event were enrolled. Data on acute and chronic comorbidities, VTE risk factors, and prophylaxis use prior to VTE were prospectively recorded.

Results: Among the 567 patients (62±16 years), deep vein thrombosis (DVT) alone was diagnosed in 222 (39%), acute pulmonary embolism (PE) in 218 (38%), and PE plus DVT in 127 (22%). Overall, prophylaxis was used in 329 (58%) patients prior to the VTE event. Among the medical patients, 146 (48%) received prophylaxis, and among the surgical patients, 183 (70%) received prophylaxis (p <0.001). The indication for prophylaxis was present in 262 (86%) medical patients and in 217 (83%) surgical patients. Among the patients with an indication for prophylaxis, 135 (52%) of the medical patients and 165 (76%) of the surgical patients received prophylaxis (p <0.001). Admission to the intensive care unit (OR 3.28, 95% CI 1.94–5.57), recent surgery (OR 2.28, 95% CI 1.51–3.44), bed rest >3 days (OR 2.12, 95% CI 1.45–3.09), obesity (OR 2.01, 95% CI 1.03–3.90), prior DVT (OR 1.71, 95% CI 1.31–2.24), and prior PE (OR 1.54, 95% CI 1.05–2.26) were independent predictors of prophylaxis. In contrast, cancer (OR 1.06, 95% CI 0.89–1.25), age (OR 0.99, 95% CI 0.98–1.01), acute heart failure (OR 1.13, 95% CI 0.79–1.63), and acute respiratory failure (OR 1.19, 95% CI 0.89–1.59) were not predictive of prophylaxis.

Conclusions: Although an indication for prophylaxis was present in most patients who suffered acute VTE as a complication during or shortly after hospitalization, almost half did not receive any form of prophylaxis. Future effort should focus on the improvement of prophylaxis in hospitalized patients, particularly in patients with cancer, acute heart or respiratory failure, and in the elderly.
Natural History of Mesenteric Venous Thrombosis: A Large Multicentre Study
Authors: Dentali Francesco , Ageno Walter , Witt Dan , Malato Alessandra , Clark Nathan , Garcia David , Siragusa Sergio , McCool Kathleen , Dyke Shannon , Crowther Mark A.
Published: April 2020

Introduction: Mesenteric venous thrombosis (MVT) is an uncommon but potentially life-threatening disease, accounting for 5–15% of mesenteric ischemic events. Most cases of MVT are either identified at laparotomy or at autopsy, with a mortality rate of approximately 50% in old series. Advances in imaging techniques have facilitated the early diagnosis of MVT and, thus, have contributed to a decrease in mortality in the more recent series. The treatment of MVT involves anticoagulation alone or in combination with surgery. Experts suggest that MVT can safely be managed without surgery if there is no evidence of bowel infarction. However, information on the natural history of MVT and on efficacy and safety of long term treatment with oral anticoagulant (OAT) is based on small uncontrolled series of patients with a limited follow up. Therefore, the aim of our study is to clarify the natural history of this disease in a large cohort of patients recently diagnosed with MVT.

Methods: The charts of all patients with splanchnic vein thrombosis who are currently attending or who have attended four anticoagulation clinics (Denver, Albuquerque, Varese and Palermo) were reviewed. At these centres, patients are regularly followed up for the monitoring of OAT and information on clinical events is documented and registered in a computerized database. All patients with objectively diagnosed MVT, were selected. The charts of eligible patients were reviewed for baseline clinical characteristics including sex, age, prior history of cardiovascular disease and use and type of anticoagulant therapy. Information on mortality and all objectively confirmed recurrent venous thromboembolic events were noted.

Results: Seventy seven patients (mean age 49.2 years; 45 males) were included. Thirteen patients had a previous thromboembolic event. Thirty two were idiopathic. Forty six patients were on long term OAT. Median follow up was 36 months (Range 2–204 months). Seven patients had a recurrent thromboembolic event (5 splancnic vein thromboses and two PEs) for an incidence rate of 23.4 events/1000 year patient. Two patients (2.6%) had a major bleeding event (one subdural haematoma and one gastroenteric bleeding). Five patients had a VTE recurrence when suspended OAT for an incidence rate of 45.9 events/1000 year patient. Seven patients (9.1%) died during follow up (4 were related to cancer).

Conclusion: patients with a previous episode of MVT have a low risk of recurrent thromboembolic events while on oral anticoagulant treatment. This risk is clearly increased after treatment is stopped. In these patients, long term anticoagulant therapy appears as a safe and effective option.
Substitution of Four Residues Revealed by High-Throughput Screening of Human Factor VIII A2 Domain Generated a Recombinant Molecule That Escapes to Anti-Factor VIII Antibodies
Authors: Plantier Jean-Luc , Saboulard Didier , Delcourt Marc , Enjolras Nathalie , Negrier Claude
Published: April 2020

Using Massive Mutagenesis technique® we performed a high-throughput alanine substitution of 206 residues between aminoacids 376 to 649 from factor VIII (FVIII) A2 domain. The pattern of activity and the levels of production of FVIII mutants were assessed following transient expression in COS-1 cells. FVIII mutants that kept at least 50% of wild-type activity were then screened in an inhibitor assay against total immunoglobulin G (IgG) fractions from patients with severe hemophilia A who had developed inhibitory antibodies (n=4; range 6–15 BU/mL) or a non immune IgG as control. In this assay, the cell culture supernatants containing FVIII were incubated in a volume of FVIII-depleted plasma for 1h30 in the presence of IgG. The residual activity was then measured in a chronometric assay. No single mutations were able to significantly allow FVIII to escape inhibitors. Four mutations (S409A, L462A, E507A, L629A) having a tendency to resist to inhibitors were selected and recombined two by two leading to a significant but insufficient resistance to anti-FVIII antibodies. The effect of the mutations was additive since a molecule (FVIII-4A2) combining the 4 substitutions significantly resisted to the inhibitory antibodies. Residual activity of FVIII-4A2 ranged from 8% to up to 82% of the initial activity depending on the inhibitor plasma whereas this residual activity never exceeded 30% for control wild-type FVIII. Following production by CHO cells, purified FVIII-4A2 demonstrated a similar pattern of resistance to the four IgG fractions already assayed. FVIII-4A2 was then assayed against 11 additional unrelated inhibitors (range 3–2662 BU/mL) and displayed also a resistance against 10 out of the 11 IgG fractions. The resistance was in all case only partial in relation with the likely presence of anti-C2 and/or anti-A3-C1 inhibitors within the IgG fractions. As detected in a solid-phase assay, the decrease in inhibitory effect was for some of the IgG fractions partly related to a decrease in their binding capacity. As a control experiment, FVIII-4A2 was poorly recognized by the monoclonal antibody GMA012 directed against the A2 domain. In contrast, the binding to ESH4, an anti-C2 monoclonal antibody was not affected. Such combination of mutations opened the perspective for the generation of a recombinant FVIII molecule that can be used as an effective substitutive FVIII therapy in patients with inhibitors.
Embryoid Body Defect in Mouse Rps19-Haploinsufficient Embryonic Stem Cell Model of Diamond Blackfan Anemia
Authors: Singh Sharon , Dsilva Sehba , Lipton Jeffrey Michael , Ellis Steven , Liu Johnson M.
Published: April 2020

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome that is characterized by erythroid hypoplasia, risk of other cytopenias, congenital anomalies and a cancer predisposition. Thus far, all the genes identified as mutated in DBA encode ribosomal proteins (RPS19, RPS17, RPS24, RPL5, RPL11, and RPL35a). In the 25% of DBA patients with RPS19 mutations, haploinsufficiency of RPS19 has been linked to faulty ribosome biogenesis, which ultimately predisposes erythroid precursors to apoptosis through as yet unknown mechanisms. Previous attempts by others to apply targeted mutagenesis to Rps19 were unsuccessful because of compensatory Rps19 expression from the non-targeted allele. We have concentrated our efforts on characterizing the murine Rps19-mutated embryonic stem (ES) cell, S17-10H1, which was generated using a genetrap strategy. The gene-trap vector contains a strong splice acceptor-β-geo cassette-poly A termination, and following insertion, it should cause splicing with the exon upstream and termination at the poly A signal, effectively cutting Rps19 in half. S17-10H1 was sequenced using 3′ RACE (rapid amplification of cDNA ends) to confirm insertion of the vector between exons 2 and 3 of Rps19. PCR with primers against the β-geo sequence was also used to confirm insertion of the gene trap vector into the mutant ES cells. Western blot analysis of two different ES cell samples confirmed at least 50% less Rps19 protein than found in the wild-type parental ES cell line, AK7. The ES cells were subsequently induced to undergo primary differentiation into embryoid bodies (EBs). Although there was no significant difference in the EB size or shape at day 5 of culture, the number of EBs that formed in the mutant cultures was decreased by at least three-fold. Preliminary experiments indicated no obvious morphological differences in day 13 EBs derived from parental or mutant ES cells. We attempted to create chimeric mice by microinjection of the S17-10H1 cell line into 36 blastocysts. Six chimeric mice were set up in mating pairs with C57BL/6J partners. Analysis of more than 60 pups from the 60% chimeric male revealed a lack of germline transmission, possibly indicating that this mutation leads to embryonic lethality or inability to complete gametogenesis. We conclude that this ES cell differentiation model mimics the human disease in leading to Rps19 haploinsufficiency and provides a new and potentially powerful tool that can be used to elucidate molecular mechanisms and test potential therapies in DBA.
Raf1 Is Required for Induction of a Bcr-Abl Positive CML Like Myeloproliferative Disease in Mice
Authors: Albers Corinna , Illert Anna Lena , Miething Cornelius , Peschel Christian , Duyster Justus
Published: April 2020

Introduction: Chronic myelogenous leukemia (CML) results from neoplastic transformation of hematopoietic stem cells (HSC), characterized by a chromosomal translocation t(9;22)(q34;q11). This aberration leads to the expression of the oncogenic tyrosine kinase Bcr-Abl, which mediates signals for proliferation, transformation and anti-apoptosis via various different pathways including the Raf/MEK/ERK cascade. The cytoplasmic protein Raf1 is a key molecule within this cascade. Recent studies have revealed an additional function of the Raf-1 kinase that is independent of the activation of the MAPK cascade and whose effect is to increase resistance to apoptosis. Therefore Raf1 is an interesting target for molecular therapies and more effective Raf1 inhibitors have recently been developed by the pharmaceutical industry. Here we report the impact of Raf1 signalling for Bcr-Abl mediated transformation.

Methods: We exerted a siRNA based approach in combination with a murine bone marrow transplantation model. To this end we designed a MSCV based retrovirus encoding both the Raf1 microRNA and the Bcr-Abl oncogene on a single construct. This approach ensured knockdowns of more than 90% of Raf1 in every Bcr-Abl transformed cell.

Results: Methylcellulose assays demonstrated that bone marrow coexpressing Raf1 microRNA and Bcr-Abl had a 2 fold decreased colony forming ability compared to control cells. We then transduced bone marrow (BM) with retrovirus coexpressing Raf1 microRNA and p185 Bcr-Abl and transplanted lethally irradiated recipient Balb/C mice. The onset and progression of leukemia was significantly delayed in mice transplanted with Raf1 microRNA and Bcr-Abl compared with the Bcr- Abl transduced control microRNA group. Raf1 knockdown mice showed only a moderate rise of white blood cell (WBC) counts and prolonged overall survival (median survival 39 ± 7.1 days) in comparison to control mice (23.3 ± 2.4 days). However, we were not able to completely avoid the development of leukemia by Raf1 knockdown.

Conclusion: Taken together our data demonstrate that Raf1 is important for the development of a myeloproliferative disease by Bcr-Abl in mice. Therefore Raf1 inhibition in combination with Bcr-Abl kinase inhibition depicts an interesting approach towards eradication of Bcr- Abl positive leukemia. In addition, this study describes a novel and versatile approach to express an oncogene and a microRNA using a single retroviral construct. Thus this powerful tool can be used to systematically screen drugable signalling targets involved in oncogenesis.
Nilotinib in Elderly Chronic Myeloid Leukemia Patients in Chronic Phase (CML-CP) with Imatinib Resistance or Intolerance: Efficacy and Safety Analysis
Authors: Lipton Jeffrey H. , le Coutre Philipp D. , Wang Jim , Yang Mindy , Szczudlo Tomasz , Giles Francis
Published: April 2020

Background: Nilotinib is a potent and highly selective BCR-ABL kinase inhibitor approved for the treatment of Philadelphia chromosome-positive chronic myeloid leukemia (Ph+ CML) patients in chronic (CML-CP) or accelerated phase (CML-AP) who have failed prior therapy including imatinib.

Methods: This subanalysis of the open-label, single-arm, phase 2 study evaluated the efficacy and safety of nilotinib in elderly (≥65 years) CML-CP patients who were resistant or intolerant to imatinib. Nilotinib was dosed at 400 mg twice daily.

Results: A total of 321 CML-CP patients (71% imatinib-resistant; 29% imatinib-intolerant) were enrolled. Thirty percent (98/321) of patients were ≥65 years; 8% (8/98) of these patients were 380 years. The baseline characteristics among patients ≥65 and <65 years were similar. Discontinuation of nilotinib due to adverse events was uncommon, and did not differ among the two age groups (18% in both groups). Efficacy was maintained in elderly patients, with 48% of patients achieving MCyR and 38% achieving CCyR, compared with 63% and 44% of patients <65 years achieving MCyR and CCyR, respectively. Duration of cytogenetic response was also consistent among age groups with 84% and 85% of responding elderly patients maintaining MCyR and CCyR at 18 months, compared with 85% and 89% of patients <65 years maintaining MCyR and CCyR at 18 months, respectively. Estimated overall survival (OS) rates at 12 months were 97% and 91%, for patients <65 years and ≥65 years, respectively. Overall, the safety profile of nilotinib was similar among the two age groups. Biochemical laboratory abnormalities were transient, clinically asymptomatic, and consistent in both age groups; elevated lipase occurred in 14% and 23%, and elevated total bilirubin occurred in 9% and 3% of patients <65 years and ≥65 years, respectively. The most common grade 3/4 hematological laboratory abnormalities were also comparable; neutropenia was reported in 31% and 30%, thrombocytopenia in 26% and 36%, and anemia in 8% and 15%, of patients <65 years or ≥65 years, respectively. The incidence of grade 3/4 pleural/pericardial effusions were <1% and 1% and grade 3/4 bleeding events were <1% and 1% in patients <65 years or ≥65 years, respectively. The incidence of myocardial infarction (<1% vs 4%), congestive heart failure (2% vs 1%), and QTcF prolongation >500 msec (<1% vs 2%) were also similar among patients <65 years or ≥65 years.

Conclusions: Nilotinib is highly active and induced durable clinical responses in CML-CP patients regardless of age. Importantly, the safety profile of nilotinib is maintained in elderly patients and there was no increase in the incidence of cardiac evens making it an excellent therapeutic option for patients with Ph+ CML, regardless of age.