bluuebunny/crossref_metadata_2025 huggingface dataset:
Oncoprotein 18 (Op18) : A Differentially Expressed Gene Is a Novel Therapeutic Target in Multiple Myeloma
Authors: Pelluru Dheeraj , Wang Jingyi , Prabhala Rao , Fulciniti Mariateresa , Shammas Masood A. , Amin Samirkumar B , Anderson Kenneth C. , Munshi Nikhil C.
Published: April 2020

Stathmin(Op18) is a ubiquitous cytosolic 18Kda protein regulating microtubule (MT) dynamics via binding and stabilizing activities. The activity of Op18 is down regulated during cell cycle by phosphorylation at four Ser residues. The phospho-Op18 is unable to bind to tubulin allowing the progression of the cell cycle. Two additional Op18-binding proteins, KIS prevents Op18 dephosphorylation and activation while iASPP (inhibitor of apoptosis stimulatory protein phosphatase) binds to WT-p53 and inhibits p53-related apoptosis. We have identidfied Op18 as a differentially expressed gene by suppression subtractive hybridization in myeloma. We have also confirmed differential expression of KIS in myeloma cells compared to normal plasma cells. Overexpression of Op18 was confirmed at both RNA and at protein level by RT –PCR and Western blotting in human myeloma cell lines as well as primary samples compared to normal plasma cells and normal human fibroblasts. In contrast, none of the tonsillar CD138+ plasma cells and normal bone marrow mononuclear cells showed Op18 overexpression. To establish the role of Op18 overexpression in MM cell transformation, the human MM cell lines were treated with antisense Op18 oligodeoxynucleotides (ODN). The growth rate of the ODN treated human myeloma cells was significantly reduced compared to the control cells along with cell cycle arrest in G2/M phase and increase in apoptosis as measured by immunohistochemical staining. Additionally, we have silenced Op18 by transfecting Op18-specific siRNA in MM cells and observed that KIS is translocated to cytoplasm, from nucleus as well as MM cells are arrested in G2/M. Importantly, Op18 silencing increased sensitivity of MM cells to microtubule drugs suggesting possible combination approach for therapeutic application. Its role in networking cellular signal transduction pathways in myeloma is under investigation. Recent publication identifying Op18 as one of the 15 most relevant genes determining outcome in myeloma (Avet-loiseau et al, JCO 2008) adds to the validity of Op18 as a molecule playing important role in myeloma cell growth and survival and warrant investigation as a novel therapeutic target.
C-Myc- Dependent Stabilization of Hif-1alpha in MM: Therapeutic Implications
Authors: Podar Klaus , Zhang Jing , Tonon Giovanni , Grabher Clemens , Lababidi Samir , Raab Marc S , Vallet Sonia , Zhou Yiming , Cartron Marie-Astrid , Sattler Martin , Chauhan Dharminder , Anderson Kenneth C
Published: April 2020

Bone marrow (BM) angiogenesis has been associated with multiple myeloma (MM) progression. Here we demonstrate that oncogenic c-Myc, also prominently associated with adverse prognosis in MM, regulates Hif-1α promoter activity, Hif-1alpha protein levels, and consequently the expression of pro-angiogenic target genes such as VEGF in MM cells. As observed with c-Myc knockdown, anti-MM agents with known anti-angiogenic activity including bortezomib, lenalidomide, and enzastaurin also downregulate Hif-1alpha and VEGF. Moreover, tyrophostin adaphostin induces similar effects, associated with anti-angiogenic activity, in a zebrafish model. The correlation between c-Myc, Hif-1alpha and tumor angiogenesis was further confirmed in a xenograft mouse model of human MM. Mechanistically, our in vitro data show that oncogenic c-Myc stabilizes caspase-8, and thereby protects Hif-1alpha against caspase-8- dependent degradation and attenuation of VEGF secretion. These sequelae also contribute to enhanced VEGF secretion triggered by MM cell-bone marrow stromal cell binding. Indeed, conditioned media from adaphostin- treated co-cultures inhibit endothelial cell growth and tubule formation. In addition, HIF1alpha gene demonstrated significant increased expression (p=0.02, Mann Whitney test) in tumor cells versus plasma cells derived from healthy donors. Importantly, when tested on a dataset of 559 patients, a significant link between HIF1alpha overexpression and poor prognosis was found (p=0,0069). These data further point to the possible relevance of HIF1alpha in the progression of a subset of MM patients. In summary, our data provide strong evidence of a key role of Hif-1alpha in MM angiogenesis and the therapeutic potential of specific Hif-1alpha and c-Myc inhibitors. Moreover they demonstrate additional biologic sequelae of novel MM therapies.
Mcl-1 Fragment Mcl-1(128–350) Induces Inhibition of Multiple Myeloma Cell Proliferation and Apoptosis Via Both Upregulation of C-Jun as Well as Modulation of Its Transcriptional Activity
Authors: Podar Klaus , Zhang Jing , Zimmerhackl Alexander , Barila Daniela , Vallet Sonia , Sattler Martin , Tai Yu-Tzu , Hideshima Teru , Chauhan Dharminder , Anderson Kenneth C.
Published: April 2020

Myeloid cell leukaemia-1 (Mcl-1) is an anti- apoptotic member of the Bcl-2 family commonly expressed in multiple myeloma (MM). Drugs (e.g. bortezomib) can induce a 28kD Mcl-1 fragment (Mcl-1Δ128–350) in a caspase- dependent manner, which induces inhibition of MM cell proliferation and apoptosis. Here we sought to delineate molecular sequelae downstream of Mcl-1Δ128–350 which mediate its anti- proliferative and pro-apoptotic effects in MM and other malignant cells. Our results demonstrate that exogenous Mcl-1Δ128–350 induces upregulation and nuclear accumulation of c-Jun, as well as generation of a pro-apoptotic 60kD c-Abl fragment (c-Abl Δ). Bortezomib treatment triggered c-Jun upregulation in Mcl-1wt/wt, but not Mcl-1 null, murine embryonic fibroblasts (MEFs), and neither transfection with exogenous c-Jun nor with exogenous 60kD c-Abl Δ triggered the generation of Mcl-1Δ128–350. Moreover, drug-induced generation of Mcl-1Δ128–350 was not abrogated by specific transient knockdown of c-Jun or c-Abl by small interfering RNA, further supporting the requirement of Mcl-1Δ128–350 for c-Jun upregulation. Our studies also identified mechanisms downstream of upregulated c-Jun which trigger inhibition of MM cell proliferation and apoptosis. Interestingly and similar to c-Jun and c-Abl Δ, Mcl-1Δ128–350 accumulates within the nuclear fraction. Indeed, interaction of Mcl-1Δ128–350 with c-Jun, as well as subsequent enhanced AP-1 reporter activity, demonstrate a direct regulatory role of Mcl-1Δ128–350 in c-Jun- dependent gene transcription. Finally, gene profiles in MM cells transfected with either Mcl-1wt or Mcl-1Δ128–350 identify differentially expressed genes associated with MM cell proliferation, survival and drug resistance. Taken together, these data both delineate the role of Mcl-1 in MM pathogenesis and further support targeting Mcl-1 in novel MM treatment strategies.
The Novel, Orally Available Multi-Kinase Inhibitor BAY 73-4506 in Multiple Myeloma
Authors: Breitkreutz Iris , Raab Marc S , Christensen Olaf , Zimmerhackl Alexander , Zhang Jing , Blotta Simona , Hideshima Teru , Chauhan Dharminder , Wilhelm Scott , Anderson Kenneth C , Podar Klaus
Published: April 2020

Agents targeting not only myeloma cells directly but also bone marrow stromal cells (BMSCs), endothelial cells, and osteoclasts (OCLs) that cause enhancement of tumor cell growth, angiogenesis, and MM bone disease, respectively, are promising new treatment modalities for multiple myeloma. Here we investigated the novel, orally available multi-kinase inhibitor BAY 73-4506 (BAY), currently in phase II clinical trials, for its therapeutic effect in MM. BAY is a potent inhibitor of angiogenic (VEGFR 1–3, PDGFR-β), as well as oncogenic, (cKIT, RET, FGFR, Raf) kinases We first tested the ability of BAY to suppress proliferation and survival in a wide array of MM cell lines, including those resistant to conventional chemotherapeutics. Our data show that BAY, in a low micromolar range that is well below concentrations achieved in patient plasma during the first clinical trial in solid tumors, induces apoptosis by caspase-9 and caspase-3 activation in all cell lines tested. Importantly, BAY also overcomes the growth advantage conferred in a BMSC-MM, as well as an endothelial cell-MM, co-culture system. BAY treatment abrogates growth factor-stimulated MEK, ERK and AKT phosphorylation at sub-micromolar concentrations. Since the VEGF signaling pathway is a potent inducer of angiogenesis and BAY targets VEGFR 1–3, we examined its anti-angiogenic properties. BAY inhibits endothelial cell growth and endothelial cell tubule formation in vitro at concentrations less than 1μM; moreover, it also markedly inhibited VEGF-induced cell migration on fibronectin. Activation of MAP kinase is a critical event during OCL differentiation, activation, and survival; and importantly, BAY also inhibits osteoclastogenesis, evidenced by blockade of M-CSF/RANKL-triggered differentiation of mononuclear cells to TRAP-positive osteoclasts. Finally, BAY significantly delays tumor growth and abrogates blood vessel formation in vivo in a xenograft mouse model of human MM. These in vitro and in vivo results provide the basis for further clinical evaluation of BAY to improve patient outcome in MM.
A Myeloproliferative Disorder in the Tc1 Mouse Model of Down Syndrome
Authors: Alford Kate A. , Vanes Lesley , Li Zhe , Orkin Stuart H. , Fisher Elizabeth M. C. , Tybulewicz Victor L. J.
Published: April 2020

Down syndrome (DS) children have a one in ten chance of being diagnosed with leukemia within the first ten years of life. Acute megakaryoblastic leukemia (AMKL) is a subtype of acute myeloid leukemia (AML) that accounts for nearly 50% of these leukemias. AMKL is associated with a self-regressing neoplasia found almost exclusively in DS newborns called Transient Myeloproliferative Disorder (TMD). In all cases of TMD and DS-AMKL, leukemic blast cells show mutations in the gene encoding the hematopoietic transcription factor GATA1, resulting in production of a truncated form of the protein called GATA1s. Mutations in GATA1 are not seen in non-DS-AMKL or other DS leukemias and it is clear both trisomy of human chromosome 21 (HSA21) and a mutation in GATA1 are required for the development of both TMD and AMKL. However, it is unknown which genes on HSA21 need to be trisomic in order to predispose an individual with DS to AMKL. Our group has generated mice (termed the Tc1 mice) that contain an almost complete, freely segregating copy of HSA21. These mice display phenotypic features of DS. We have examined adult hematopoiesis in these mice. Blood samples taken from a cohort of Tc1 mice were examined from 4 weeks until 60 weeks of age. Complete blood cell counts show that whilst the mice do not develop leukemia they displayed persistent macrocytosis and had reduced erythrocyte numbers. Crossing the Tc1 mice with mice that express GATA1s protein did not perturb or exacerbate this phenotype. Over the age of 15 months more than 50% of Tc1 mice examined were found to have developed splenomegaly. These mice displayed megakaryocyte hyperplasia and had increased numbers of cells of the erythroid lineage. In vitro colony forming assays demonstrated an increase in the frequency of megakaryocytic and granulocyte-macrophage progenitors in the spleen, consistent with extramedullary hematopoiesis. In the bone marrow, no abnormalities were seen in the lineage-, c-Kit+, Sca1+ (LSK) compartment, however there was a significant increase in the percentage of common myeloid progenitors (CMP) and a corresponding decrease in megakaryocyte-erythrocyte progenitors (MEP). This suggests a possible block in development from CMP to MEP. These data demonstrate defects in hematopoietic development in a proportion of adult Tc1 mice. However, preliminary data suggest that these mice do not develop a neonatal myeloproliferative disorder that is comparable with human TMD. It may be that the phenotype seen in the adult Tc1 mice is due to defects in hematopoietic progenitors that are different to those responsible for development of TMD and DS-AMKL. This mouse model may therefore provide a useful tool to examine the role of HSA21 genes in adult hematopoietic disorders.
Outcome of Patients with Myeloproliferative Disorders Treated with Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Donato Michele L. , Prann Angela , Ullrich Anna , Feldman Tatyana , Goldberg Stuart L. , Goy Andre , Masood Aisha , Pecora Andrew L. , Siegel David S. , Rowley Scott D.
Published: April 2020

Myeloproliferative disorders (MPD) are characterized by marrow clonal proliferation leading to ineffective hematopoiesis. Between 1998 and 2007, 27 patients (pts) with myeloproliferative disorders were treated with hematopoietic stem cell transplantation. Patients included 9 males (33%) and 18 females (67%) with a median age of 52 years (range 30–68). Diseases included Philadelphia chromosome-negative chronic myelogenous leukemia (CML) (n=2, 7%), myelofibrosis (n=21, 78%), polycythemia vera (n=2, 7%) and essential thrombocythemia (n=2, 7%). Twelve patients (44%) had abnormal cytogenetics, 24 pts (89%) had splenomegaly and 9 pts (33%) had a prior splenectomy. Preparative regimens included busulfan and cyclophosphamide (n=11, 41%), cyclophosphamide and total body irradiation (n=3, 11%), fludarabine and busulfan (n=3, 11%), fludarabine and cyclophosphamide (n=1, 4%), fludarabine and melphalan (n=7, 26%), and fludarabine with low dose total body irradiation (n=2, 7%). Patients with unrelated donors also received ATG. Twelve patients received an unrelated donor product, 7 pts received bone marrow and 20 pts received peripheral blood stem cells.

The 5-year overall Kaplan-Meier product-limit survival estimates (figure 1.) was 60.9%. The day 100 transplant-related mortality was 18.5%, all in patients receiving either cyclophosphamide and total body irradiation or cyclophosphamide and busulfan. Overall, there were twice as many deaths in patients receiving a non-fludarabine-based regimen (n=7, 25.9%) compared with patients receiving a fludarabine-based regimen (n=3, 11.1%) though the difference was not statistically significant (p=0.15). The overall incidence of grade II–IV acute GVHD was 44% and chronic GVHD was 64%. The overall relapse rate was 16%. No significant differences in relapse rates by acute (p=0.40) and chronic GVHD (p=0.53) or by fludarabive vs non-fludarabine based regimens (p=0.24) were observed. In univariate Cox proportional hazard models, chronic GVHD was the only significant factor associated with worse survival (p=0.035).

In conclusion, myeloproliferative disorders can be successfully treated with allogeneic hematopoietic stem cell transplantation. Chronic GVHD significantly reduces overall survival. The optimal preparative regimen still remains to be determined.

Figure 1. Kaplan-Meier product-limit survival estimates of MPD treated with allogeneic hepatopoietic stem cell transplantation (n=27). Figure 1. Kaplan-Meier product-limit survival estimates of MPD treated with allogeneic hepatopoietic stem cell transplantation (n=27).
Previously Unknown Interactions Between AML1 and MLL Provide Epigenetic Regulation of Gene Expression in Normal Hematopoiesis and in Leukemia
Authors: Huang Gang , Elf Shannon , Yan Xiaomei , Wang Lan , Liu Yan , Sashida Goro , Gural Alex , Menendez Silvia , Lee Jennifer , Yang Youyang , Zhao Xinyang , Nimer Stephen D.
Published: April 2020

In all organisms, the fundamental process of transcriptional regulation requires transcription factors, which bind to DNA in response to extra-cellular signals and regulate transcription of target genes. In eukaryotes, this process also involves epigenetic regulation, which includes DNA and histone modifications. Hematopoiesis and leukemia are excellent model systems for studying the higher eukaryotic regulations of gene expression and for identifying important molecules involved in genetic and epigenetic transcriptional regulation. The Mixed-Lineage Leukemia (MLL) protein, a Set1-like H3K4 methyltransferase, and the heterodimeric transcriptional factor AML1/CBFβ are critical for definitive and adult hematopoiesis. They are required for the generation of all hematopoietic lineages and act as tumor suppressors in human leukemia. We have previously shown that the regulation of PU.1 by AML1 is mediated by 3 AML1 binding sites in the PU.1 upstream regulatory element (URE), located −14 kb relative to the transcription start site in mice (Huang et al. Nat Genet. 2008). To understand whether AML1 plays a critical role in regulating the PU.1 locus at the chromatin level, we have utilized this PU.1 regulation system as a model to study the potential interplay between AML1/CBFβ and MLL. We found that MLL binds to the evolutionarily conserved Runt-domain of AML1, enhances the formation of the AML1/CBFβ heterodimer, and blocks the ubiquitin-proteasome mediated degradation of AML1. We also found that AML1/CBFβ is required for maintenance of the H3K4-me3 histone mark at the PU.1 upstream regulatory element (URE) and promoter region, and that MLL is required for AML1 to regulate PU.1 expression. In contrast, we found that several leukemia-associated MLL fusion proteins, including MLL-AF4, MLL-AF9, MLL-ENL, and MLL-AF10, no longer stabilize AML1 but rather induce AML1 degradation and downregulate PU.1 expression. Taken together, our data indicate that MLL and some of its leukemia-associated fusion proteins physically and functionally interact with AML1. Their effects on target genes, particularly PU.1, may be critical for the normal regulation of hematopoiesis and for the aberrant regulation that underlies acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL).
Aberrant Splicing of the E-Cadherin Gene Is a Novel Mechanism of Gene Silencing in Chronic Lymphocytic Leukemia Cells
Authors: Sharma Sanjai , Lichtenstein Alan
Published: April 2020

Premature termination codons (PTCs) in tumor suppressor genes can contribute to oncogenesis. PTCs prematurely terminate transcription, leading to expression of a truncated, mRNA. The nonsense-mediated degradation (NMD) pathway degrades the aberrant transcripts resulting in loss of expression. To identify RNA transcripts with PTCs in chronic lymphocytic leukemia specimens (CLL), we employed a microarray technique to screen for genes which upregulate their RNA signal upon NMD blockade. In the list of genes with highest upregulation, we identified the E-cadherin gene, a known tumor suppressor gene. On further investigation, we found that the PTC was due to stabilization of an alternatively spliced E-cadherin transcript. This transcript completely lacks exon 11 (exon skipping), resulting in a frameshift and a PTC which is recognized by the NMD pathway and degraded. RT-PCR analysis demonstrated that the exon 11 skipping occurs in normal B cells as well but at a much lower frequency. A real time PCR based strategy to quantify the relative amount of spliced transcript confirmed a 10–30 fold increase in exon 11 skipping in CLL cells (n=29) as compared to normal B cells (n=4) and the degree of skipping increased with increased Rai stage. In addition, real time PCR also demonstrated a significant decrease in total wild type E-cadherin RNA expression in 58% of CLL specimens compared to normal B cells. As prior work demonstrates an upregulated wnt/β-catenin pathway in CLL and, and that E-cadherin can be a physiologic regulator of this pathway, we tested wnt pathway reporter expression in CLL specimens. High expression was identified and ectopic expression of E-cadherin in some samples with E-cadherin loss was sufficient to inhibit the wnt-reporter activity. This suggests that E-cadherin loss, possibly due to exon 11 alternative splicing and exon skipping plays a role in upregulated wnt signaling. As there are no mutations in exon 11 or its flanking intronic regions in CLL cells, our results suggest a novel mechanism of E-cadherin gene inactivation in which the trans-factors/splicing factors in malignant B lymphocytes induce an increased nonproductive splicing of a tumor suppressor gene.
MicroRNA-181b Inhibits ATRA Induced Differentiation in the APL Cell Line NB-4 Via Negative Regulation of the Tumorsupressor RASSF1A
Authors: Braeuer Daniela , Hartmann Jens-Uwe , Christopeit Maximilian , Behre Gerhard
Published: April 2020

MicroRNAs (miRNAs) are small non-coding RNAs of 19–25 nucleotides that are involved in the regulation of critical cell processes such as apoptosis, cell proliferation and differentiation. However, little is known about the role of miRNAs in AML and granulopoiesis. In this study, we analysed the expression of miRNAs in the acute promyelocytic leukemia cell line NB-4 during all-trans-retinoic acid (ATRA) treatment by using a miRNA microarray platform (Exiqon) and quantitative PCR analysis. We identified several ATRA regulated microRNAs. Among them we found microRNA-181b as negatively regulated by ATRA. MicroRNA-181b is known to be overexpressed in several cancers such as thyroid papillary carcinoma, colorectal cancer, acute lymphocytic leukemia (ALL) and also acute promyelocytic leukemia (APL). To determine the role of microRNA-181b in the formation and maintenance of the APL-blast phenotype, we performed LNA knock down and transient overexpression experiments. Here we could show in quantitative PCR analysis an inhibitory effect of microRNA-181b on the transcription of the ATRA induced granulocytic differentiation markers C/EBPα and C/EBPβ. Furthermore in FACS analysis we could observe a reduction of the ATRA stimulated formation of the granulocytic surface marker CD11b under microRNA-181b overexpression conditions. To identify the mechanism how microRNA-181b affects ATRA induced differentiation we performed data base analysis to identify putative targets of microRNA-181b. One promising candidate is the tumorsuppressor RASSF1A. RASSF1A was already shown to be involved in proliferation and differentiation by regulation of cell cycle processes. As first group we were able to show in this study the ATRA induced upregulation of RASSF1A in NB-4 cells by quantitative PCR analysis. To demonstrate the negative effect of microRNA-181b on RASSF1A we analysed RASSF1A protein level under LNA knock down and overexpression conditions of microRNA-181b. Here we could observe an upregulation of RASSF1A protein level when microRNA-181b was downregulated. And in addition, a higher amount of microRNA-181b elicits the reduction of RASSF1A protein level. The regulatory effect of RASSF1A in the ATRA induced differentiation of NB-4 cells we confirmed by using siRNA against RASSF1A. In this experiment the FACS analysis showed a reduction of ATRA induced CD11b expression under siRNA mediated knock down of RASS1A. In conclusion, these findings support our hypothesis that microRNA-181b can act as oncogenic microRNA and thereby promotes the leukemic phenotype of APL-blasts by negative regulation of the tumorsuppressor RASSF1A.
NK Cell Anti-Tumor Reactivity Is Impaired by Platelet-Derived TGF-B through NKG2D Downregulation
Authors: Kopp Hans-Georg , Placke Theresa , Schmiedel Benjamin J , Kanz Lothar , Krusch Matthias , Salih Helmut R
Published: April 2020

NK cells can recognize and eliminate malignant cells thereby preventing both local tumor progression and metastatic spread. Besides by the direct interaction with their target cells, NK cell activity is influenced by the interplay with other hematopoietic cells influencing the activity of both involved cell types. Recently, we and others demonstrated that thrombocytopenia inhibits metastasis in mice, which is dependent on the presence of NK cells as shown by NK cell depletion experiments (Jin et al., Nat Med2006, 12: 557; Nieswandt et al., Cancer Res1999, 59: 1295). These data suggest that thrombocytopenia indirectly inhibits tumor dissemination by allowing NK cells to exert their anti-tumor effector functions. However, yet nothing is known regarding the molecular mechanisms underlying platelet-NK cell interaction, especially in humans. Here we report that human platelets express high levels of MHC class I, and incubation of MHC class I-negative tumor cells with platelets induced formation of tumor cell-platelet aggregates resulting in tumor MHC class I “pseudoexpression” which can inhibit NK cell reactivity via KIR triggering. Furthermore, tumor cell-induced platelet aggregation, alike stimulation with classical platelet agonists such as thrombin or collagen, resulted in secretion of platelet-derived soluble factors (releasate) containing, among others, high levels of the immunoregulatory cytokine TGF-b. Presence of releasate in cocultures of NK cells and tumor target cells was found to mediate a substantial inhibition of NK cell cytotoxicity and IFN-g production. Of note, platelet releasate caused a marked downregulation of the activating immunoreceptor NKG2D expressed on NK cells, which was partially restored by a neutralizing TGF-b antibody. Confirming the functional relevance of this finding, neutralization of TGF-b in platelet releasate by the same antibody also partially restored both the impaired NK cell cytotoxicity and IFN-g production in cocultures with tumor cells. Taken together, our data demonstrate that platelets may substantially influence NK cell anti-tumor reactivity by various mechanisms including inhibition of NKG2D-mediated NK cell immunosurveillance. Further elucidation of platelet ability to confer expression of immunregulatory surface molecules to tumor cells and the various soluble factors carrying NK cell-modulatory activity may reveal novel targets to prevent tumor dissemination.
Down Regulation of Hepcidin and Interleukin 1-Alpha in Pbmc from Patients with Beta thalassemia
Authors: Lai Maria-Eliana , Vacquer Stefania , Carta Maria Paola , Mulas Claudia , Cocco Pierluigi , Dessi Sandra
Published: April 2020

Thalassemia is an inherited blood disease characterized by low levels or absence of normal globin chains. Severe forms of thalassemia, thalassemia major (TM), require regular blood transfusions, the main cause of secondary hemosyderosis. However, iron overload is a potential complication, even in patients (pts) with thalassemia intermedia (TI), who do not require transfusions. Among them, iron overload is mainly the result of an excessive absorption of dietary iron, caused by a down-regulation of hepcidin, an hepatic hormone that acts as a major regulator of systemic iron homeostasis. Very low mRNA levels of hepcidin have been reported in urine as well as in liver biopsies from patients with TI. It has been also reported that, interleukin-1 alpha (IL-1α) stimulates hepcidin transcription, raising the question as to whether a down-regulation of this cytokine may be responsible, at least partially, for the excessive iron absorption occurring in patients with TI. Here, we assessed whether IL-1α was involved in hepcidin regulation in β-thalassemia. Peripheral blood mononuclear cells (PBMCs) were isolated from 6 TM pts, 8 TI pts and 6 controls. mRNA was obtained from PBMCs by RT-PCR. Hepcidin mRNA levels were reduced in both TM and TI pts when compared to the age matched normal controls. Neverthless, hepcidin mRNA levels were significantly lower in TI pts, as compared to TM pts receiving regular blood transfusions. Among TI pts lower hepcidin mRNA level was associated with lower level of IL-α. Tumor necrosis factor alpha (TNF-α) does not seem to be involved in the regulation of hepcidin transcription. Interestingly, the levels of mRNA expression of acyl-CoA-cholesterol acyltransferase (ACAT), the enzyme responsible for intracellular cholesterol ester accumulation, and thus, for atherosclerotic plaque formation were strongly induced in PBMCs in TI pts. These results suggest that PBMCs hepcidin and IL-1-α measurements could possibly be used in the future as simple, ease and sensible diagnostic tools for the detection of iron overload in patients with thalassemia. ACAT expression may even be used as therapeutic target in preventing atherosclerotic complications such as pulmonary thromboembolism, cerebral thrombosis, and leg ulcers. frequently occurring in such patients.
Long-Term Efficacy of the Terminal Complement Inhibitor Eculizumab in a Patient with Cold Agglutinin Disease (CAD)
Authors: Roeth Alexander , Huettmann Andreas , Duehrsen Ulrich , Philipp Thomas
Published: April 2020

Cold agglutinin disease (CAD) is a rare form of autoimmune hemolysis in which monoclonal or polyclonal IgM autoantibodies can activate the complement system in colder areas of the body (e.g. extremities), resulting in red blood cell destruction. Typically prednisone is ineffective and, although chemotherapy is often used, only on rare occasions does it suppress autoantibody production. Although CAD is often mild and self-limited, some cases require hospitalization and can be life-threatening. Supportive transfusions of red blood cells may also be necessary. Based on what is known about the pathophysiology of CAD, blockade of the terminal complement cascade by eculizumab could be a possible therapeutic approach. Eculizumab, a monoclonal antibody targeting complement factor C5, has shown high efficacy in patients with hemolytic paroxysmal nocturnal hemoglobinuria (PNH) through inhibition of the terminal complement system. We tested the efficacy of eculizumab in a transfusion-dependent patient with CAD refractory to extensive previous treatment. Eculizumab was dosed as follows: 600 mg IV every 7±2 days × 4; 900 mg 7±2 days later; and then 900 mg every 14±2 days. The patient has continued dosing for approximately one year. As eculizumab treatment may increase the risk for meningococcal disease, a standard vaccination scheme was administered prior to treatment. Transfusion requirements as well as clinical and biochemical indicators of hemolysis were monitored. Our results showed that intravascular hemolysis, measured by lactate dehydrogenase (LDH), was reduced by 46% from 780±50.5 U/L (mean±SE) during the 1 year period prior to treatment to 446±35.5 U/L during 1 year of eculizumab treatment. Reduction in hemolysis resulted in improvement of anemia (from a mean hemoglobin level of 10.0 g/dL 1 year prior to treatment to 11.8 g/dL during 1 year of eculizumab treatment) with a significant reduction in the number of PRBC units transfused (from a mean of 18 units during the pre-treatment period to 0 units during eculizumab treatment). Ferritin levels also decreased from 666±70.8 μg/L (mean±SE) before treatment to 313±8.9 μg/L during treatment. The patient reported an improvement of fatigue and overall quality of life. There were no adverse events observed and the ongoing therapy with eculizumab continues to be safe and well tolerated in this patient. In summary, this is the first report of eculizumab efficacy in a patient with CAD. Chronic blockade of terminal complement with eculizumab resulted in reductions in hemolysis and transfusion requirements and improvements in anemia and fatigue. As no alternative effective treatment options are available, these promising results provide a rationale for a clinical trial for the use of eculizumab in patients with CAD.
A Molecular Signature for a Cytokine Receptor Survival Pathway in AML Identifies Unique Prognostic Indicators and Therapeutic Targets
Authors: Thomas Daniel , Powell Jason A , Barry Emma F , To Luen Bik , Lopez Angel , Guthridge Mark A
Published: April 2020

The IL-3 receptor regulates myeloid cell survival and proliferation and is overexpressed on AML blasts and CD34+CD38- AML “stem-like” cells suggesting it plays a role in leukemic cell survival that could be exploited by targetted therapy. We have previously shown that Serine 585 in the cytoplasmic domain of the beta subunit of the IL-3 receptor is phosphorylated in response to sub-picomolar concentrations of cytokine, binds 14-3-3 zeta adaptor and is required for cytokine mediated survival (Guthridge et al, 2006, EMBO). We wished to examine the phosphorylation status of this receptor in AML blasts from patients and the expression levels of downstream Ser585 target genes.

Results: We show that Ser585 of the beta common subunit is constitutively phosphorylated in the majority of leukaemic patients regardless of Flt3 status or cytogenetics (18/21 patients). In contrast, tyrosine phosphorylation of the receptor is not deregulated and remains cytokine-dependent (20/21). We have identified a panel of 139 Ser585-regulated genes by microarray (WT receptor vs Ser to Gly mutant) and show that a specific subset of these genes is deregulated in AML blasts confirmed by RT-PCR compared to normal mononuclear cells consistent with constitutive activation of this pathway in AML. We have identified a 4-gene signature that is a molecular correlate of the Ser585-survival pathway in AML. We further identify Ser585-regulated genes that are prognostic indicators of clinical outcome in a cohort of adult patients AML (n=33) independent of WCC or cytogenetic abnormalities. Targetting Ser585 survival genes using siRNA or blocking mAbs induces apoptosis in AML blasts validating Ser585-regulated genes as potential targets for therapy.

Conclusions: Given the emerging problem of resistance to tyrosine kinase inhibitors (TKI) and limited success of Flt3 inhibitors in AML, new strategies for therapy are needed. By interrogating a cytokine survival pathway that functions independently of tyrosine phosphorylation we have uncovered novel Ser585 gene targets with promising clinical utility in leukaemia.
Mechanisms of Suppression Used by Regulatory T Cells in Patients Newly Diagnosed with Acute Myeloid Leukemia
Authors: Szczepanski Miroslaw J , Szajnik Marta , Czystowska Malgorzata , Mandapathil Magis , Welsh Ann , Foon Kenneth , Whiteside Theresa L , Boyiadzis Michael
Published: April 2020

An elevated frequency of CD4+CD25high regulatory T cells (Treg) has been reported in the peripheral blood in patients with various solid tumors and hematologic malignancies. Although the increase in Treg seems to be a characteristic feature of most tumors, the functional role of Treg and the mechanisms of suppression, especially in patients with hematologic malignancies, have been less well defined. We investigated Treg-mediated suppression and the responsible mechanisms in thirty newly diagnosed acute myeloid leukemia (AML) patients prior to any treatment and twenty five healthy donors (NC). The percentage of circulating CD4+ CD25high Treg was higher (p <0.0001) in the AML patients (4.5 ±0.2%, range 1.7–8.2%) compared to NC (1.5 ± 0.08%, range 0.9–3.1 %). To evaluate the suppressive function, CD4+CD25high T cells (S) were co-cultured with sorted, CFSE-labeled autologous CD4+CD25high T cells (R) at different S/R ratios. Suppression mediated by Treg co-incubated with proliferating autologous responders was significantly higher (p<0.001) in AML than that mediated by control Treg. To evaluate the role of cytokines produced by Treg in suppression and a need for cell-to- cell contact, transwell analysis of S/R co-cultures was performed. Co-incubation in the presence of transwell inserts (TRI) resulted in significant reduction of suppression (p<0.05), and the addition of neutralizing antibodies to IL-10 and TGF-β1 in the presence of TRI further decreased suppression mediated by Treg. These data suggest that both immunoinhibitory cytokine production and cell-to-cell contact are necessary for suppression. To explore other potential mechanisms of Treg suppression, we evaluated the expression by Treg of ectonucleotidases CD39 and CD73 and the capability of Treg to produce adenosine. CD4+CD25high T cells of AML patients had higher expression (p<0.01) of CD39 and more efficiently hydrolyzed ATP to adenosine relative to Treg in NC. These data indicate that various mechanisms of suppression may be utilized by Treg in patients with AML. The increase frequency of Treg mediating potent suppression by various mechanisms is likely to play a role in host anti-tumor immune responses. Therefore, modulation of the frequency and functions of Treg might provide new immunotherapeutic approaches in AML.
Activation of p53 Signaling Is Synergistically Enhanced by Bcl-2 Inhibition through Induction of Noxa and Bak/Bax Heterodimers Resulting in Apoptosis of AML Stem Cells
Authors: Konopleva Marina , Tsao Twee , Ling Xiaoyang , Kojima Kensuke , Watt Julie , Mak Duncan H. , Verhaegen Monique , Bornmann William , Soengas Maria , Andreeff Michael
Published: April 2020

Disruption of Mdm2-p53 interaction by the small molecule Nutlin-3a activates p53 signaling and induces apoptosis in acute myeloid leukemia (AML) (Kojima et al., Blood 2005). ABT-737 binds to the hydrophobic BH3-binding groove of the Bcl-2 family members Bcl-XL, Bcl-2, Bcl-w but not Mcl-1 and exerts its pro-apoptotic function by preventing anti-apoptotic Bcl-2 family members from sequestering activating BH3 proteins (Konopleva et al., Cancer Cell 2006). We have recently reported that Nutlin-3a and ABT-737 induced Bax conformational change and mitochondrial apoptosis in AML cells in a strikingly synergistic fashion (Kojima et al., Cell Cycle 2006). In this study, we investigated molecular mechanisms of this synergism. Nutlin-3a and ABT-737 synergistically induced apoptosis in OCI-AML3 and MOLM13 cells with wt-p53, but not in p53-null HL-60 cells or in OCI-AML3 stably transfected with p53 shRNA. Both, Nutlin-3a and ABT-737 induced Noxa protein levels resulting in Noxa/Mcl-1 heterodimers and therefore inactivation of Mcl-1. Lentiviral transfection of MOLM13 cells with Noxa shRNA protected cells from ABT-737/Nutlin-induced apoptosis. As reported by us, ABT-737 disrupted Bcl-2/Bak leading to formation of pro-apoptotic Bak/Bax heterodimers. Despite increased p53 levels there was no change in p53/Bcl-2 or p53/Bak association; however, combined exposure of cells to Nutlin-3a/ABT-737 induced massive Bak/Bax heterodimers and facilitated direct binding of p53 to Bax. HCT-116 cells deficient in Bax were largely protected from pro-apoptotic effects of the Nutlin-3a/ABT-737 combination. Taken together, these findings indicate that in leukemic cells p53-dependent apoptosis is executed through pro-apoptotic Bcl-2 family proteins, which are also targeted by BH3 mimetics. Bcl-2 is critical for AML leukemogenesis (Padua et. al., ASH 2008) and is overexpressed in AML stem cells. In primary AML samples, the Nutlin-3a/ABT-737 combination synergistically depleted CD34+CD38−CD123+ AML stem cells (combination index 0.5). Since high Bcl-2 and increased Bcl-2/Bax ratios are associated with poor clinical response in AML, the combination strategy proposed here may have considerable therapeutic promise in targeting AML stem cells.
Trib1 and Trib2 but Not Trib3 Degrade C/EBPá and Induce Acute Myelogenous Leukemia
Authors: Dedhia Priya , Keeshan Karen , Vega Maria , Uljon Sacha , Xu Lanwei , Shestova Olga , Romany Candice , Blacklow Stephen C , Pear Warren S.
Published: April 2020

Trib1, Trib2, and Trib3 are mammalian homologues of the Tribbles protein family, an evolutionarily conserved group of proteins that can mediate proteasome-dependent degradation. Evidence suggests that these proteins function as adapters, where they recruit E3 ligases and enhance ubiquination of the target protein in order to promote its degradation. To date, increased Trib1 and Trib2 mRNA expression has been shown to correlate with acute myelogenous leukemia (AML) in humans and induces AML in mice; whereas Trib3 has not been associated with AML. In order to understand the effects of Trib family members in hematopoietic cells, we reconstituted mice with hematopoietic progenitors retrovirally expressing Trib1, Trib2, or Trib3. Trib1 and Trib2 mice developed AML whereas Trib3 mice did not. Our previous data suggested that Trib2-mediated degradation of the transcription factor, C/EBPα, is important for leukemogenesis. We now show that Trib1, like Trib2, strongly binds C/EBPα and induces its degradation. In contrast, Trib3 weakly binds C/EBPα and fails to induce its degradation. Consistent with the ability to strongly bind and degrade C/EBPα, Trib1 and Trib2, but not Trib3, block differentiation of myeloid cells. We are currently mapping the domains that account for the differences between Trib1/Trib2 and Trib3 in leukemogenesis. Together, our results strengthen the correlation between Trib-induced C/EBPα degradation and induction of AML. Furthermore, our data show that different Tribbles family members have distinct targets and understanding this specificity may provide opportunities to therapeutically target Tribbles.
Outcome of First Salvage Therapy in Core Binding Factor Associated Acute Myelogenous Leukemia Is Less Than Optimal
Authors: Fava Carmen , Blamble Deborah , O’Brien Susan , Garcia-Manero Guillermo , Pierce Sherry , Kantarjian Hagop , Borthakur Gautam
Published: April 2020

Background: Core binding factor (CBF) associated acute myelogenous leukemia (AML) is considered to have a better prognosis compared to that of other patients with AML. Reinduction with cytarabine based therapy followed by allogeneic stem cell transplant is a standard salvage approach.

Method: We performed a retrospective analysis of outcome in patients with CBF AML.

Results: Between the years 1992 and 2005, 107 patients with CBF AML were treated at M.D. Anderson Cancer Center. Sixty-six (62%) patients had inv 16 abnormality and 41 (38%) had t(8;21) abnormality. Induction chemotherapy regimens included fludarabine and cytarabine with or without granulocyte colony stimulating factor (G-CSF) (66 patients) or idarubicin and cytarabine with/without G-CSF (41 patients). One hundred and one (94%) patients achieved complete remission (CR). After a median CR duration of 159 weeks, 37 (37%) patients relapsed. Relapse rate was 26/66 (39%) among patients with inv 16 abnormality and 11/41 (27%) among those with t (8;21) abnormality. Higher WBC count predicted for relapse (p=.001) and relapse rate did not differ among induction regimens (p=0.1).

Salvage chemotherapy included cytarabine based regimen in 22 (59%) patients, clofarabine and idarubicin (2 patients), topoisomerase inhibitor (5 patients), histone deacetylase inhibitor (2 patients) and miscellaneous regimens (6 patients). Sixteen (43%) of the patients with relapsed CBF AML achieved CR after first salvage therapy. Eleven of 26 (42%) patients with inv 16 and 7/11 (64%) of patients with t (8;21) were resistant to first salvage therapy. Thirteen patients (10 in CR) underwent allogeneic stem cell transplant and 12 of them remained in CR post-transplant. Overall survival was significantly worse among patients who relapsed compared to the ones who did not (p=.001).

Conclusion: A significant proportion of patients with CBF AML relapse and second remissions can be achieved in less than half the patients. This highlights the need for better induction/consolidation regimens in this group of patients with ‘good-risk’ AML.
Hypomethylating Therapy in Patients with AML and High-Risk MDS and Chromosome 5 and 7 Abnormalities Is Associated with An Improved Outcome Compared to Conventional Chemotherapy
Authors: Ravandi Farhad , Issa Jean-Pierre , Garcia-Manero Guillermo , O’Brien Susan , Pierce Sherry , Shan Jenny , Verstovsek Srdan , Faderl Stefan , Cortes Jorge , Kantarjian Hagop
Published: April 2020

Outcome of patients with AML and high-risk MDS with chromosome 5 and 7 abnormalities [excluding del 5(q)] has been poor with fewer than 10% of patients alive at 2 years. We investigated whether the use of hypomethylating agents, 5-azacytidine and decitabine, is associated with an improved outcome. Between January 2004 and December 2007, a total of 81 pts [including 37 (46%) with AML (≥ 20% blast) and 44 (54%) with high-risk MDS] with chromosome 5 and 7 abnormalities were treated with hypomethylating agents as their initial therapy. This included 68 patients with complex abnormalities and 13 with less than 3 aberrations. During the same period, 151 patients (126 AML, 25 MDS) with chromosome 5 and 7 abnormalities (128 complex, 23 noncomplex) were treated with intensive cytotoxic chemotherapy (including cytarabine based regimens in 72% and other regimens in 28%). The median age for the two groups was 66 and 61 years, respectively [(ranges (37–85) and (19–89)]. Thirty three (41%) pts in the hypomethylating group achieved CR versus 53 (35%) pts in the chemotherapy group (p=0.395). With a median follow up of 51 weeks (range 12–101) and 40 weeks (range, 5–128), 22/33 pts in the hypomethylating group and 33/53 pts in the chemotherapy group have relapsed giving median CR duration of 45 weeks for the hypomethylating group and 23 weeks for the chemotherapy group (p=0.153). The overall survival was superior for the hypomethylating group as compared to the chemotherapy group (Figure). We conclude that treatment with hypomethylating agents may be superior to chemotherapy in patients with chromosome 5 and 7 abnormalities who are traditionally resistant to cytotoxic agents.

Figure Figure
Alternate Donor HCT for Fanconi Anemia (FA): Results of a Total Body Irradiation (TBI) Dose De-Escalation Study
Authors: MacMillan Margaret L. , Blazar Bruce R. , DeFor Todd , Ma Linan , Tolar Jakub , Zierhut Heather , Wagner John E.
Published: April 2020

FA is the most common heritable marrow failure syndrome with a high predisposition to myelodysplastic syndrome and leukemia. Great strides have been made in optimizing the conditioning regimen and GVHD prophylaxis, necessarily tailored to these patients as a result of their underlying defect in DNA repair. Prior to 1995, survival rates after alternate donor hematopoietic cell transplantation (AD-HCT) were exceptionally poor (<20%). After 1999, the addition of fludarabine (FLU) significantly improved engraftment and survival. However, risks of infection and late effects remained important challenges. Seeking to reduce these risks, we conducted a single center, single arm, TBI dose deescalation trial designed to determine the lowest possible dose of TBI required for engraftment in recipients of T cell depleted AD marrow (TCD BM). All patients received cyclophosphamide (CY) 10 mg/kg × 4 days, FLU 35 mg/m2 × 4 days, ATG 30 mg/kg ×5 days and a single fraction of TBI with CT guided thymic shielding (TS). TBI dose deescalation strata were: TBI 300 cGy (cohort 1); TBI 150 cGy (cohort 2); no TBI (cohort 3). All patients received CSA and methylprednisolone as GVHD prophylaxis and G-CSF 5 ug/kg/day until engraftment. The decision to proceed with each stepwise decrease in TBI was based upon achieving primary neutrophil engraftment in 10 of 10 patients at each dose level, or 14 of 15 patients if one graft failure is observed in any of the first 10 patients. More than 1 graft failure in 15 patients was considered unacceptable and the next higher TBI dose level was then considered the optimal dose. Between July 2006-May 2008, 19 FA patients were enrolled with 17 in cohort 1 and 2 in cohort 2. Five patients received 5–6/6 unmanipulated UCB because a marrow donor could not be identified. All patients achieved primary engraftment at a median of 11 days after AD-HCT. However, 2/2 patients who received TBI 150 developed secondary graft failure at 76 and 114 days after HCT. The dose de-escalation was stopped with TBI 300 cGy identified as the lowest possible dose in the context of FLU/CY given concomitantly. Thus far, 17 patients have been treated with TBI 300. As shown, results compare favorably with prior regimens using TBI 450 with and without TS (table).

Outcomes after AD-HCT in Standard Risk FA Patients

N Neutrophil Engraftment Acute GVHD Chronic GVHD 1 Year Survival TS = thymic shielding TBI 450 no TS 21 95% 24% 19% 67% TBI 450 +TS 12 92% 42% 8% 83% TBI 300 +TS 17 94% 25% 0% 92%

Our results demonstrate that TBI 300 cGy is sufficient for consistent engraftment in recipients of CY-FLU-ATG and HLA matched or mismatched TCD AD BM or UCB and represents a new standard of care for this patient population. Longer follow up is needed to quantitate the impact of lower dose radiation on immune recovery, risks of infection, cancer risk and other therapy-related late effects.