bluuebunny/crossref_metadata_2025 huggingface dataset:
An Acidic Peptide Inhibits the Interleukin-6-Induced Activities of the Src Family Kinases Hck, Lyn and Fyn in Myeloma Cells.
Authors: Hausherr Amparo , Tavares Rosario , Hallek Michael , Krause Guenter F.
Published: April 2020

Background: Src Family Kinases (SFKs) play pivotal roles in normal B-cell development and in B-cell neoplasias. Our aim is to elucidate the roles of SFKs in signaling cascades resulting in cell proliferation or anti-apoptosis in multiple myeloma (MM). The pleiotropic cytokine interleukin-6 (IL6) is one of the major growth factors for MM cells. We have previously shown that IL6 induces the activation of the SFKs Hck, Lyn and Fyn and that Hck is associated with the IL6R beta chain (gp130) via an acid domain (AD) in gp130.

Aim: We observed that an 18mer peptide (18AD), which is derived from the AD, inhibited the IL-6-dependent growth of myeloma cells. In order to develop a lead structure for small molecule inhibitors we wish to elucidate the cellular and molecular effects of peptide 18AD.

Results: On the cellular level, 50–100μM of a membrane-permeable myristoylated peptide 18AD inhibited factor-dependent proliferation in 7TD-1 (mouse) and INA-6 (human) cells by ~75%. Taking the percentage of annexinV-stained cells after IL6-withdrawal as the reference value for the level of maximal apoptosis, treatment of 7TD1 and INA-6 cells with peptide 18AD resulted in 100 and 60 % of the respective percentages of apoptotic cells. A control peptide (18sc) with an identical amino acid composition but an arbitrarily scrambled sequence had no effect on proliferation and apoptosis. Similar effects of peptides 18AD and 18sc were observed in Baf-EH cells, which overexpress a chimeric erythropoietin receptor-gp130 and human Hck and grew in an erythropoietin dependent manner. Peptide 18AD had no significant growth inhibiting effects on BaF-HE cells cultured in the presence of IL3 or on factor-independent OKT1 hybridoma cells. In 7TD1 cells the expression of a kinase-inactive Hck mutant lead to a reduced proliferative response to IL6. On the molecular level, immunoprecipitation experiments in 7TD1 cells showed that peptide 18AD decreased the complex formation between Hck and gp130 in a concentration dependent way. Immunocomplex kinase assays in INA-6 cells showed that the IL6 induced activation of Hck, Lyn and Fyn was blocked by peptide 18AD, but not by peptide 18sc.

Conclusion: We characterized peptide 18AD, which inhibits the association of Hck and gp130 as well as IL6-induced SFK activity. Thus, the observed anti-proliferative and pro-apoptotic effects of peptide 18AD may be due to inhibition of SFK-mediated pathways.
Histone Deacetylase Inhibitors LBH589 and LAQ824 Deplete Ezh2 and Associated Polycomb Repressive Complex 2/3 Proteins Resulting in Downregulation of HOXA9 and MEIS1 Expression in Human Acute Leukemia Cells.
Authors: Fiskus Warren , Pranpat Michael , Bali Purva , Kumaraswamy Sandhya , Boyapalle Sandhya , Rocha Kathy , Balasis Maria , Atadja Peter , Bhalla Kapil
Published: April 2020

Human enhancer of Zeste homolog (Ezh2) protein belongs to Polycomb Repressive Complex (PRC) 2, which also includes Eed and Suz12. Ezh2 has been shown to promote cellular transformation, and increased Ezh2 expression has been strongly correlated with the invasiveness of prostate and breast cancers. The enzymatically-active Ezh2-containing, PRC2 complex possesses histone methyl transferase (HMTase) activity mediated by the SET domain of Ezh2, which is involved in the methylation of histone (H) 3, lysine (K)-27 and −9. Through this, the PRC2 complex regulates the expression of homeobox domain containing HOX family of transcription factors including HOXA9 and MEIS1, which have been shown to be involved in human leukemogenesis. Co-expression of HOXA9 and MEIS1 is common in acute myeloid leukemia and collaborates in the leukemogenesis. In the present studies, we determined the effect of hydroxamate histone deacetylase inhibitors LBH589 and LAQ824 on Ezh2 and PRC2 complex proteins and their activity in the cultured (K562, LAMA-84, U937 and HL-60) and primary human AML cells. Exposure to 10 to 100 nM of LBH589 or LAQ824 for 24 hours, in a dose dependent manner depleted the protein levels of Ezh2, as well as reduced Suz12 and Eed levels in the cultured and primary leukemia cells. This was associated with decreased levels of the tri- and dimethylated K27 and increased acetylation of K27, both on H3. Correspondingly, these H3 modifications were associated with a significant decline in the levels of HOXA9 and MEIS1. As has been previously reported, treatment with LBH589 and LAQ824 induced p21, as well as caused cell cycle growth arrest in the G1 phase and apoptosis of the cultured and primary AML cells. We next determined whether knockdown of Ezh2 by siRNA to Ezh2 also induces growth inhibitory and cytotoxic effects against the leukemia cells. In the cultured and primary acute leukemia cells, knockdown of Ezh2 expression (by ~80%) by Ezh2 siRNA, but not by the control siRNA, was associated with depletion of Suz12 levels and increased H3K27 acetylation. This was associated with increase in the % of cells in the G1 phase of the cell cycle and significant inhibition of their clonogenic survival in colony growth assays. Co-treatment with LBH589 and siRNA to Ezh2 caused further decline in the Ezh2 levels and increased loss of clonogenic survival of the leukemia cells. These findings suggest that down modulation of Ezh2 and PRC2 complex and its HMTase activity may inhibit clonogenic survival of human acute myeloid leukemia cells. Additionally, combined effect of the knockdown of Ezh2 and its activity along with treatment with LBH589 or LAQ824 may have an improved anti-leukemia efficacy, especially against human AML where HOXA9 and MEIS1 genes are deregulated.
Proteasome Inhibition Sensitizes Leukemic Cells to Anthracyclin-Induced Apoptosis by Increasing the Expression of Bim and Bax Pro-Apoptotic Proteins.
Authors: Pigneux Arnaud , Mahon François X. , Moreau-Gaudry François , Néré Marie L. , Lacombe Francis , Reiffers Josy , Praloran Vincent , Belloc Francis
Published: April 2020

NF-k B is constitutively activated in the majority of acute myelogenous leukemia (AML) specimens. Anthracyclines used in AML treatment can activate NF-k B and inhibit apoptosis through synthesis of IAPs upregulation. Because proteasome activity is necessary for NF-k B activation and p53 degradation, inhibitors of the proteasome have been proposed as pro-apoptotic agents to sensitize leukemic blast cells to anthracycline chemotherapy. Apoptosis was quantified in THP-1 cells, incubated with increasing concentrations of (IDA) alone or in association with a low concentration (0.25 μM) of MG132 as a proteasome inhibitor. A low apoptotic rate was found with either MG132 or IDA alone with a range of 5 to 20 ng/ml. The combination of both drugs induced a percentage of apoptotic cells which was significantly higher than the addition of the effects of both drugs separately (p<0.01). A low concentration of IDA (5ng/ml) induced, in the presence of MG132 as much apoptosis as 20 ng/ml of IDA alone. Above 40 ng/ml of IDA, the sensitizing effect of MG132 was no more significant. This shows that proteasome inhibition lowers the threshold of sensititvity of THP-1 cells to IDA-induced apoptosis. Such a sensitization was not found in a MG132-resistant THP-1 derived cell line. This difference was not related to the uptake of the drug since no difference in IDA incorporation by THP-1 cells was found in the presence or the absence of MG132. Western blot analysis of I-k B phosphorylation and degradation failed to show any difference between MG132 and control-treated cells. Conversely increased expression of Bax and Bim pro-apoptotic proteins was evidenced. The increase in Bim precedes the induction of apoptosis and participates in Idarubicin-induced apoptosis. Bortezomib gave the same results for apoptosis induction and synergistic effect with IDA, increased expression of Bax and Bim pro-apoptotic proteins was also evidenced. MG132 also potentiated IDA-induced apoptosis in primary blast cells from 22 AML patients while no effect of MG132 was found on the IDA-induced apoptosis normal lymphocytes, PHA-stimulated lymphocytes, normal cord blood CD34+ cells and bone marrow normal myeloid cells. These data confirm that proteasome inhibition induces a specific sensitization of leukemic cells to IDA and suggest that an increase in the pro-apoptotic potential (Bim, Bax) are involved in the process.
In Vivo Anti-Tumor Activity of Atiprimod on SCID Models of Multiple Myeloma.
Authors: Neri Paola , Tassone Pierfrancesco , Shammas Masood A. , Batchu Ramesh B. , Blotta Simona , Samala Sushma , Catley Laurence , Hamasaki Makoto , Hideshima Teru , Jacob Gary S. , Picker Donald H. , Venuta Salvatore , Anderson Kenneth C. , Munshi Nikhil C.
Published: April 2020

Atiprimod (N-N-diethl-8, 8-dipropyl-2-azaspiro [4.5] decane-2-propanamine) is an orally bio-available cationic amphiphilic agent previously studied for its in vivo anti-inflammatory activity. We previously demonstrated that Atiprimod, in a time- and dose-dependent manner, inhibits myeloma cell growth and survival in IL-6 independent (OPM1) as well as dependent (INA-6) MM cell-lines with an IC50 in the range of 0.5–2 μM. We have now further characterized the molecular changes in MM cells induced by Atiprimod, evaluating the gene expression profile of INA-6 and OPM-1 cells exposed to this drug. Following 24 h treatment, Atiprimod induced significant down-regulation of genes involved in growth and cell-cycle control (CDC14, CDC2-like 5, Cyclin-E binding protein 1 and MDM2), adhesion (ITGA-6, ITGA-X, ADAM-17, CDH-3, CDH-6, CTNND-1, PECAM-1 and MADCAM-1) cell-signalling pathways (PRKAB-1, Mapk-7, GPR-125 and GPR-32), up-regulation of genes implicated in apoptotic cascades (TNFAIP-3, TNFSF-10, TNFRSF-10c, CDKN2A and CDKN1A) and in negative regulation of signal transduction (RGS-4 and IGFBP-5). We next evaluated the in vivo activity of Atiprimod using three SCID mouse models of human MM: 1) To evaluate effects of Atiprimod directly on MM cells, we used SCID mice bearing subcutaneous OPM1 tumors and treated i.p. with Atiprimod or vehicle alone (PBS) on alternate days for 7 days. In this model 31%, 48% and 55% inhibition of tumor growth were observed in mice treated with Atiprimod at 20, 30 and 50 mg/kg, respectively, compared to control group. 2) To evaluate effects of Atiprimod on MM cells in the context of a human BM microenvironment, we used SCID mice implanted with a human fetal bone chip (SCID-hu) engrafted with IL-6-dependant INA-6 cells and treated i.p. with Atiprimod (40 mg/kg) or vehicle alone (PBS) for two weeks. The response was evaluated by detection of serum soluble human IL-6 receptor (shuIL-6R) released by MM cells in murine serum. We observed a 60% reduction in shuIL-6R level in mice treated with Atiprimod versus control group. 3) Finally, to evaluate effects of Atiprimod on primary patient cells in a human microenvironment, we used SCID-hu mice engrafted with patient MM cells (IgG λ) and treated i.p. with Atiprimod (50 mg/kg) or vehicle alone (PBS) for 4 weeks. Treatment with Atiprimod induced a reduction in both human IgG and λ light chain levels in mouse sera, whereas levels of both proteins continued to rise in mice treated with vehicle alone. Taken together these data demonstrate the in vivo anti-tumor activity of Atiprimod and provide a rational for its clinical evaluation in MM. Based on these data the drug is presently in a multi-site Phase I/IIa clinical trial in patients with relapsed or refractory multiple myeloma (MM).
Dkk1 Transgenic Mice for the Study of Bone Lesions in Human Multiple Myeloma.
Authors: Fujita Ken-ichi , Kim Joong Su , Eckhardt Laurel A. , Shaughnessy John D. , Janz Siegfried
Published: April 2020

Purpose: Extracellular antagonists of the cellular WNT signalling pathway can be divided into two classes depending on the mechanism by which ligand-receptor interactions are inhibited. Members of the first class including SFRP (secreted frizzled-related proteins), WIF1 (WNT inhibitory factor 1) and CER1 (cerberus homolog 1) bind to WNT proteins in the extracellular milieu. Members of the second class including Dickkopf (DKK) proteins bind to the WNT receptor subunits LRP5/6. Reduced WNT signalling may play a role in the natural history of the bone disease that is a hallmark of multiple myeloma (MM). Gene expression profiling of MM plasma cells has shown that over-expression of the Dickkopf family member DKK1 is linked with abundance and size of osteolytic lesions (N Engl J Med 2003, 349:2483–94). The biological mechanism of DKK1-mediated bone destruction is not known.

Experimental procedure: To elucidate the mechanism by which DKK1 facilitates osteolysis, we generated transgenic mice in which mouse Dkk1 is expressed under control of the mouse immunoglobulin Eα enhancer. This enhancer is located 3′ of the immunoglobulin heavy-chain locus and comprised of four individual elements that span ~30 kb of genomic DNA. These elements were subcloned into a contiguous stretch of DNA, which we refer to as “reconstituted” Eα enhancer or HS1234 (Int Immunol 2001, 13:1003–12). Here we appended HS1234 to a Dkk1 cDNA whose expression is controlled by a mouse immunoglobulin VH promoter inserted 5′ of the transcriptional start site of Dkk1. Because of an internal ribosome entry site (IRES) 3′ of Dkk1, the gene encoding enhanced green fluorescence protein (EGFP) is co-expressed with Dkk1; i.e., it is a reporter of Dkk1 expression. A scheme of the structure of the Eα-Dkk1 transgene is depicted in Figure 1.

Figure 1 Figure 1.

Results: We generated 14 founder lines of Eα-Dkk1 transgenic mice that expressed Dkk1 and the EGFP-encoding gene by RT-PCR (Figure 2).

Figure 2 Figure 2.

The mice contained a variable number of Eα-Dkk1 copies, ranging from 2–3 to more than 100. There was but a loose association of transgene expression (qPCR of Dkk1 mRNA levels) and copy number (qPCR of genomic Ea-Dkk1 fragments); i.e., the high-copy number mice exhibited higher average mRNA levels than the low-copy number mice, although there were outliers in each group. Consistent with Eα-dependent restriction of Dkk1 expression to B/plasma cells, Dkk1 mRNA was readily detectable in lymphoid tissues, such as spleen, mesenteric lymph node and bone marrow, but not in liver.

Conclusion: The newly developed Eα-Dkk1 transgenics may provide a valuable model system to elucidate DKK1-dependent bone disease in human MM.
Macrophage Inflammatory Protein (MIP)-1alpha (CCL3) Is a Downstream Target of FGFR3 and RAS Signaling in Multiple Myeloma.
Authors: Masih-Khan Esther , Trudel Suzanne , Li ZhiHua , Nadeem Vincent , Wei Ellen , Paterson Joshua , Claudio Jaime O. , Stewart A. Keith
Published: April 2020

The identification of the t(4;14) translocation in Multiple Myeloma (MM) provided the first indication that fibroblast growth factor 3 (FGFR3) could act as an oncogene. Several studies have assessed the potential of FGFR3 as a drug target and have shown that inhibition of FGFRs using tyrosine kinase inhibitors induces cell death of FGFR3 expressing MM cells. To clarify the signaling pathways implicated in FGFR3 dependent myeloma, we assessed gene expression changes associated with treatment of MM cell lines with three known small molecule FGFR inhibitors, PD173074, SU5402 and a third novel compound using Human U133_Plus2 arrays. In addition to describing the unique pharmacogenomic signals of each drug in myeloma, 845 differentially expressed genes, common to all three FGFR3 inhibitors, were identified. Cell cycle, DNA replication and ATP binding proteins were the most common (47%) functional classes of genes effected. Further validation and refinement of this gene list was conducted using FGFR3 siRNA inhibition and FGF ligand induction. 120 genes were altered between FGFR3 RNAi and scrambled control and 892 were induced by FGF ligand. Ten genes were commonly identified as significantly regulated by all 4 FGFR3 inhibitors and induced in the opposing direction by ligand. Of these macrophage inflammatory protein (MIP)-1alpha, and dual specific phosphatase 6 (DUSP6) were positively regulative while ANXA9, CR2, AL531683, ZNF589, AW274468, FRMD3, LTB and WDR42A were negatively regulated by FGFR3 signaling. Validating our findings, a feedback loop between FGFR/ERK/MAP kinase activation and DUSP6 has previously been described in the literature. MIP-1alpha however was the most significantly altered (12 fold) on array, and MIP-1alpha regulation in response to FGFR3 pathway stimulation was confirmed by flow cytometry and Western blot. Mining of publicly available array datasets on 174 MM patients significantly associated MIP1-alpha with FGFR3 expression (p=0.01). Of note down-regulation of MIP-1alpha was not observed following FGFR3 inhibition in MM cells with RAS mutations. Based on these, and previous findings, we hypothesized that MIP-1alpha was regulated by the FGFR3/ERK/MAP kinase pathway. Indeed, inhibition of ERK in FGFR inhibitor resistant cells with RAS mutations also led to down regulation of MIP-1alpha. Because of the recognized role of MIP1-alpha (CCL3) in survival and proliferation of MM cells and in MM bone disease, our observations raise the possibility that pharmacological inhibition of MIP-1 alpha may hold therapeutic promise in t(4;14) MM and may serve as a biomarker for successful FGFR3 or RAS signaling inhibition.
Decitabine, a Potential Targeted Therapeutic for Juvenile Myelomonocytic Leukemia.
Authors: Liu Y. Lucy , Emanuel Peter Dean , Castleberry Robert P.
Published: April 2020

Juvenile myelomonocytic leukemia (JMML) is a mixed myelodysplastic /myeloproliferative disorder (MDS/MPD). It occurs in infancy and young children with a progressive course leading to death within one year after diagnosis. It is characterized by monocytosis, leukocytosis, elevated fetal hemoglobin, hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF), a low percentage of myeloblasts in the bone marrow, and absence of the Philadelphia chromosome or the BCR/ABL fusion gene. Mutations or other abnormalities in RAS, NF1, and PTPN11, have been linked to be responsible for the pathogenesis of JMML in up to 75% of cases. Treatment has been very difficult for JMML. Only allogeneic stem cell transplantation (SCT) can extend survival. However, the relapse rate from allogeneic SCT is inordinately high in JMML (28–55%), with 5-year disease-free survival rates of 25–40%. Decitabine, as one of the second generation of hypomethylating agents, has been demonstrated to produce encouraging responses in adult patients with chronic myelogenous leukemia or other hematopoietic disorders. Our recent studies have demonstrated that PTEN deficiency is detected in 67% of JMML patients. We hypothesize that hypermethylation of the PTEN promoter is one of the causes that lead to PTEN deficiency in JMML, and that a hypomethylating agent may improve PTEN expression in JMML cells, and thus inhibit hypersensitivity to GM-CSF. In order to test our hypothesis, we conducted an in vitro pilot study with JMML cells. Hypermethylation of the PTEN promoter was detected in 23/30 (77%) of JMML patients using Methylation-specific PCR. Sequencing confirmed that the CpG islands of the PTEN promoter were hypermethylated. A CFU-GM formation assay was used to evaluate the therapeutic sensitivity of Decitabine to JMML cells. Frozen mononuclear cells from peripheral blood samples of 5 JMML patients were plated in 0.3% agar medium with Decitabine ranging in concentration from 1nM to 1000nM. Significant inhibition of spontaneous CFU-GM growth was observed in all cultures in a dose-dependent fashion. The effective Decitabine concentrations in the cultures were lower or equivalent to the safe and tolerable plasma concentrations achievable in adult patients in clinical settings (30 mg/m2/day). Our data suggests that hypermethylation of the PTEN promoter is a common event in JMML, and Decitabine may be a potentially safe and effective reagent to treat JMML. Further pharmacokinetic studies should be conducted in the clinic to clarify the plasma concentration in pediatric patients, and the mechanism of Decitabine in inhibiting hypersensitivity of JMML cells to GM-CSF should be further explored since multiple genes are hypermethylated in cancers.
Sequential Cytogenetic Analyses of 577 Patients with Myelodysplastic Syndromes: Correlations between Initial Karyotype, Cytogenetic Dynamics, and Clinical Course.
Authors: Steidl Christian , Schabla Rainer , Germing Ulrich , Hildebrandt Barbara , Noesslinger Thomas , Pfeilstoecker Michael , Giagounidis Aristoteles , Kunzmann Regina , Haas Peter , Luebbert Michael , Truemper Lorenz , Haase Detlef
Published: April 2020

Myelodysplastic syndromes are dynamic diseases presenting with different clinical courses ranging from almost stable courses to rapid progression to acute myeloid leukemias. Karyotype is one of the most important prognostic factors and defines subgroups of favorable, intermediate and adverse prognosis. So far, comparably low attention has been payed on karyotypic changes occuring in sequential cytogentic examinations during the course of the disease. We retrospectively examined karyotypes of 577 patients with MDS or AML with previous history of MDS and at least two successfully performed metaphase analyses. The cytogenetic and clinical data was collected from 5 different centres of the “Kompetenznetz Akute und Chronische Leukaemien” (Duesseldorf, Duisburg, Freiburg, Goettingen, Vienna). Compared to the inital karyotype, karyotype evolution was defined as acquisition of additional aberrations, expansion of an aberrant clone by more than 20% or development of a completely aberrant karyotype after a former mosaic karyotype. According to these criteria, we found karyotype evolution in 155 cases (27%). 2–8 cytogenetic examinations have been performed per case. In 121 cases additional aberrations occured and in 34 cases the aberrant clone expanded in a subsequent analysis. In the group of patients with expansion of the aberrant clone the most frequent karyotypes were 5q- (9x) and +8 (7x). The most frequent aberrant karyotypes later acquiring additional aberrations were complex (22x) and karyotypes with two aberrations (11x), but in the vast majority of cases additional aberrations occurred on basis of a normal karyotype (70x). The most frequent additional aberrations were −7/7q- (23x), 5q- (11x) and +8 (11x). In the group of initially normal karyotypes patients with karyotype evolution had a shorter survivial (p<0.05). In summary, partial or complete momosomy 7 is the most frequent additional aberration in sequential cytogenetic analyses, indicating progression of disease. Due to their genetic instability complex karyotypes or karyotypes with 2 aberrations typically acquire additional anomalies in the course, whereas karyotypes with 5q- and +8 tend to have comparably stable courses. Furthermore, we show that also cases with a normal karyotype can harbour genetic instability as in 12% of all cases a normal karyotype evolved into an aberrant karyotype which was associated with a worse prognosis compared to stable normal karyotypes. Subgroup analyses are necessary to address correlations with therapy, time to AML progression, and the dependency on examination intervals.
Incidence and Clinical Profile of JAK2 V617F Mutation in Myelofibrosis with Myeloid Metaplasia.
Authors: Barosi Giovanni , Marchetti Monia , Massa Margherita , Rosti Vittorio , Vannucchi Alessandro , Invernizzi Rosangela , Campanelli Rita , Viarengo Gianluca , Magrini Umberto , Bergamaschi Gaetano
Published: April 2020

An aberrant tyrosine kinase signalling due to non-receptor tyrosine kinase JAK2 V617F mutation has been highlighted in the pathogenesis of polycythemia vera (PV). In myelofibrosis with myeloid metaplasia (MMM) this mutation has been reported to be present in approximately 50% of the patients and its pathogenetic role is not elucidated. Here we report the results of JAK2 V617F mutation in a retrospective analysis of blood samples from 170 patients with MMM. Search for JAK2 mutation was performed by an allele specific PCR from DNA purified from granulocytes. To evaluate whether the mutation was carried in the homozygous or heterozygous state, digestion of PCR products with BsaXI restriction enzyme was performed. The overall frequency of JAK2 V617F mutation was 60% and homozygosity for the mutation was found in 39.2% of mutant samples. Disease duration was similar in JAK2 mutated and wild type patients. Patients who harboured an homozygous mutation had an higher myeloproliferative severity score (that indexed leukocytosis, thrombocytosis and splenomegaly) than patients who had a wild type or heterozygous genotype. In post-PV MMM, the mutant gene was present in 22/22 (100%) of patients, and the frequency of homozygosity was 59% of the mutated cases. In post-ET MMM (n=13), the mutant gene was present in 46% of the patients, and the frequency of homozygosity was 16% of the mutant samples. In idiopathic MMM (n=135), the incidence of the mutational state was 54.8%, with 35.1% of homozygote mutation. Patients who had received a diagnosis of prefibrotic myelofibrosis (WHO classification) had an incidence of the mutation significantly lower than patients who were diagnosed in the fibrotic stage (6/18, 33% vs 68/112, 60.7%; P=0.001). By considering only patients not receiving cytoreductive or disease modifying agents, patients who had an heterozygote mutation, had a mean Hb value higher than wild type patients (9.4 g/dL vs. 11.4 g/dL; P=0.004). Moreover, patients who had an homozygote mutation had the myeloproliferative severity score higher than both heterozygote and wild type patients (2.26 vs 1.59, P=0.008, and 2.26 vs.1.52, P=0.001, respectively). We conclude that JAK2 V617F mutation is significantly represented in MMM patients. It is a necessary event in the transformation from PV to MMM while not in the transformation from ET to MMM. Patients who harboured an heterozygote state maintained higher Hb values than patients with a wild type genotype, while the homozygote mutation was associated with leukocytosis, thrombocytosis and splenomegaly.
Phase I Clinical Trial of IPI-504, a Novel, Water-Soluble Hsp90 Inhibitor, in Patients with Relapsed/Refractory Multiple Myeloma (MM).
Authors: Jagannath Sundar , Siegel David , Richardson Paul , Mazumder Amitabha , Sydor Jens , Goddard Jill , Walker John , Patterson Jon , Normant Emmanuel , Tong Jeffrey , Palombella Vito , Anderson Ken , Adams Julian , Grayzel David
Published: April 2020

IPI-504 is a novel, water-soluble Heat Shock Protein 90 (Hsp90) inhibitor. It is the chemical reduction product of 17-AAG, a well-characterized Hsp90 inhibitor. 17-AAG is currently in clinical trials, but suffers from very poor aqueous solubility and must be administered to patients in either suboptimal DMSO formulations, or newer cremophor or emulsion formulations. IPI-504, however, exists as a hydrochloride salt which is soluble in water in excess of 100 mg/mL. It has previously been shown that IPI-504 inter-converts with 17-AAG and exists in a pH and enzyme-mediated dynamic redox equilibrium. When IPI-504 or 17-AAG is administered to mice, rats, or monkeys a dynamic equilibrium occurs in vivo in which both species are present in plasma as well as tissue. Thus, IPI-504 recapitulates all of the Hsp90 inhibitory properties and biological activity of 17-AAG without its formulation liabilities. In preclinical studies IPI-504 has demonstrated in vitro and in vivo anti-tumor effects in a variety of cancers including xenograft and orthotopic models of multiple myeloma. Therefore an open-label phase I dose-escalation trial of IPI-504 infused on days 1, 4, 8 and 11 of a 21 day cycle is being conducted in pts with relapsed/refractory MM. IPI-504 is infused in 250cc of normal saline over thirty minutes. Pts receive IPI-504 for one cycle of treatment. The primary objectives of the study are to determine the maximum tolerated dose (MTD) and characterize the pharmacokinetic (PK) and pharmacodynamic (PD) marker effects of IPI-504. An accelerated titration design was utilized during the first three dose levels. Dose limiting toxicity (DLT) is defined as grade 4 or greater hematologic toxicity and/or grade 3 or greater non-hematologic toxicity within the first 21 days of treatment. Results demonstrate that the in vivo dynamic equilibrium of IPI-504 and 17-AAG is detectable in human plasma PK samples and the water-based formulation is well-tolerated. Data on PK, dose proportionality, safety, tolerability, PD markers, and anti-tumor activity of IPI-504 will be reported. In conclusion, clinical evaluation of this agent is ongoing to define the safety, tolerability and potential activity of IPI-504 to treat pts with advanced MM. These results may help clarify the effects of Hsp90 inhibition using a water-soluble, ansamycin-based Hsp90 inhibitor without the confounding effects of DMSO-based formulations.
A Multicenter Phase II Study of Combination Treatment with Melphalan, Arsenic Trioxide and Vitamin C (MAC) for Patients with Relapsed or Refractory Multiple Myeloma.
Authors: Berenson James , Boccia Ralph , Siegel David , Bozdech Marek , Bessudo Alberto , Stadtmauer Edward , Pomeroy J. Talisman , Steis Ronald , Flam Marshall , Lutzky Jose , Jilani Syed , Volk Joseph , Lindgren Tiberio , Moss Robert , Patel Ravi , Ferreti Delina , Yeh Howard , Bevivino Sal
Published: April 2020

Background: Despite the recent increase in treatment options for patients with multiple myeloma (MM), the disease remains largely incurable. Both arsenic trioxide (ATO) and melphalan have shown clinical activity in MM. Recent in vitro and in vivo studies in our laboratory have shown that arsenic trioxide sensitizes chemoresistant MM cells to melphalan-induced cytotoxicity; the addition of ascorbic acid (AA) further improves this effect. We conducted a multi-center clinical trial to evaluate the safety and efficacy of this steroid-free combination, melphalan, ATO and vitamin C (MAC), for patients with relapsed/refractory MM.

Methods: MM pts who relapsed after responding to 1st-line therapy and/or were refractory to prior treatment were enrolled. During week 1 of each 6-week cycle, pts received ATO, 0.25 mg/kg IV, followed by ascorbic acid (AA), 1 g IV, days 1–4. ATO followed by AA was given twice-weekly for the next 4 weeks of each cycle. Low-dose melphalan (0.10 mg/kg) was administered orally for the first 4 days of each cycle. Pts received a maximum of 6 cycles followed by weekly maintenance treatment with ATO and AA. The primary objectives of this study were to determine response rate and safety and tolerability of MAC therapy.

Results: 65 patients have been enrolled and 51 are currently evaluable for response. 26 (1 CR, 10 PR, 15 MR) of the 51 evaluable patients (51%) had an objective response and an additional 14 patients achieved stable disease, resulting in a total of 40 patients (78%) with disease control. Among patients with elevated serum creatinine levels at baseline, renal function improved for those with responsive or stable disease. 20 of the 26 responding patients had failed ≥ 2 prior therapies: 19 pts had received prior thalidomide or lenalidomide therapy and 8 pts had received prior bortezomib. The regimen was well-tolerated with few significant side effects reported. Mild cytopenias occurred infrequently and were reversible.

Conclusions: The results from this large multi-center phase II trial show that the MAC regimen is active in a group of MM patients who had either relapsed or were refractory to standard and/or investigational MM treatments. The regimen was well-tolerated even in this heavily pre-treated patient population. These findings are consistent with preclinical studies that showed the efficacy of this combination from both in vitro and in vivo studies.
Reduced Expression of CXCR4 on Circulating CD34+ Cells Is Associated with Hematopoietic Progenitor Cells (HPC) Mobilization in Patients with Myelofibrosis with Myeloid Metaplasia (MMM).
Authors: Massa Margherita , Rosti Vittorio , Vannuccchi Alessandro M. , Campanelli Rita , Pecci Alessandro , Viarengo Gianluca , Meli Valentina , Marchetti Monia , Guglielmelli Paola , Bruno Edward , Xu Ming , Hoffman Ronald , Barosi Giovanni
Published: April 2020

Spontaneous mobilization of HPC is a key feature of MMM. We investigated the CXCR4/SDF-1 (stromal derived factor-1) axis in 52 consecutive MMM patients. The percentage of circulating CD34+ cells co-expressing CXCR4 at the cytofluorimetric analysis was significantly lower (P=0.02) in MMM patients (median, 37%, range, 0 to 92%) than in the normal controls (n=12; median, 75%; range, 39 to 91%). CXCR4 mean fluorescence intensity was also highly significantly lower in MMM patients (median 0.73; range, 0.06 to 7.05) than in normal controls (median, 1.84; range, 0.41 to 6.69; P=0.004). By analyzing the association between CXCR4 expression and the mobilization of progenitor cells, an inverse correlation was found between CXCR4 expression and the number of circulating CD45+/CD34+ HPC (R=−0.40; P=0.005). By restricting the relation analysis of CXCR4 expression and clinical features to those patients who were not receiving cytoreductive treatment (n=41), there was no correlation between CXCR4 expression and patients’ sex, hemoglobin concentration, WBC, prefibrotic stage. By contrast, CXCR4 expression was inversely correlated with clinical characteristics that portrayed an advanced disease, such as older age (R=−0.31; P=0.026), longer disease duration (R=−0.37; P=0.008), larger splenomegaly (r=−0.33; P=0.015) and lower platelet count (r=0.42; P=0.007). The levels of CXCR4 mRNA, measured in peripheral blood CD34+ cells of patients with MMM were lower as compared with those of cells purified from normal peripheral blood (ΔCT by real time RT-PCR using GAPDH as housekeeping reference gene: 5.33 ± 0.49 vs. 1.53 ± 0.79, mean ± SD; P=0.003). SDF-1 plasma levels were significantly increased in patients with MMM but no correlation was documented with CXCR4 expression. We conclude that reduced expression of CXCR4 on CD34+ cells is a mechanism for the constitutive mobilization of HPC in MMM.
Detection of JAK2 V617F in the Diagnosis of Erythrocytosis: Feasibility and Diagnostic Value in Clinical Practice.
Authors: James Chloé , Delhommeau François , Marzac Christophe , Teyssandier Irène , Le Couédic Jean-Pierre , Giraudier Stéphane , Roy Lydia , Saulnier Patrick , Tulliez Michel , Vainchenker William , Casadevall Nicole , Ugo Valérie
Published: April 2020

Polycythemia Vera (PV) is a myeloproliferative disorder (MPD), whose diagnosis is currently based on an association of clinical and biological criteria following the WHO or the PVSG classification. It is characterized by a primitive absolute erythrocytosis, and formation of endogenous erythroid colonies (EEC). Recently, we described a point mutation in JAK2 (JAK2 V617F) and showed that this activating mutation was the cause of the disease (James et al, Nature, 2005). This molecular abnormality was therefore likely to be a diagnostic marker of PV, as bcr-abl for chronic myeloid leukemia (CML). Nevertheless, JAK2 V617F is also found in other MPDs, sharing some common features with PV, as essential thrombocythemia, idiopathic myelofibrosis, and other rare MPDs. One major criterion for the diagnosis of PV requires the demonstration of increased red cell mass as measured by isotopic methods. We assessed the value of detection of JAK2 V617F as a first intention diagnostic test in 88 patients with hematocrit values above 51% (=erythrocytosis) and showed that the mutation correlated with the diagnosis of PV according to the WHO (R=0.879) and the PVSG (R=0.717) criteria, with a positive predictive value of 100% in the context of erythrocytosis. Besides, the presence of the mutation strongly correlated with EEC formation in 81/87 patients (R=0.824) and only weakly with the serum erythropoietin level (R=0,416). PCR-based genotyping techniques are less time-consuming, less expensive, than DNA sequencing and are easier to perform in hematology diagnostic laboratories. Therefore, we studied the feasibility of the detection of JAK2 V617F with widespread instruments commonly used in routine. We analyzed 119 samples from patients with a suspicion of myeloproliferative disease and showed that JAK2 V617F was efficiently detected by LightCycler® and TaqMan® genotyping technologies, these latter being a little more sensitive than sequencing. For 50 patients, peripheral blood and bone marrow samples were both available. In all cases (34 mutated, 16 non-mutated) the mutation was identically detected. Based on these results, we propose that the detection of JAK2 V617F in granulocytes has a first place in the diagnostic chart of an erythrocytosis, as bcr-abl in CML, avoiding, if positive, an isotopic red cell mass measurement and bone marrow EEC assays. The presence of JAK2 V617F in a patient with erythrocytosis would then lead to the diagnosis of MPD of PV type. Further prospective studies will be necessary to assess if all the MPDs patients bearing JAK2 V617F can be grouped in a new subset within the MPD entity, especially in term of thrombotic and neoplasic risk.
Clonality Assay (X-CIP) and JAK 2 V617P Mutation: Clustering Patients with Essential Thrombocythemia at High Risk for Thrombosis.
Authors: Fiorini Alessia , Reddiconto Giovanni , Farina Giuliana , Chiusolo Patrizia , Teofili Luciana , Bonomo Pierluigi , Rossi Elena , Za Tommaso , Palladino Mariangela , De Stefano Valerio , Leone Giuseppe , Sica Simona
Published: April 2020

We have previously demonstrated that clonal hemopoiesis in female ET patients was significantly associated to the risk of thrombosis. When data on JAK2 V617P mutation became available we aimed to evaluate the incidence of this mutation according to clonality status. Ninety-two female patients, median age 32 (19–65) with a diagnosis of ET according to PVSG criteria were studied for clonality with X-CIP, 83 patients were evaluable for endogenous erythroid colonies (EECs) growth and 44 patients were evaluable for JAK2 V617P mutation. Twenty episodes of thromboses (21.7%) were detected and they include splanchnic (11), CNS (5), limb (3) thromboses and pulmonary embolism (1). Thromboses were mainly detected at diagnosis. Thirthy-seven patients showed clonal hemopoiesis (40%), 27 had polyclonal hemopoiesis (29.5%) and 28 were considered uninterpretable due to constitutional skewing (30.5%). Thromboses were overrepresented in the monoclonal group in respect to the polyclonal one (15/37 vs 2/27, p=0.003). DNA for analysis of JAK2 V617P by allele-specific PCR was available for 44 of these patients. Thirty-two patients of 44 (73%) showed JAK2 V617P mutation; JAK2 V617P was found in 16/21 patients with monoclonal hemopoiesis (76%), in 9/13 in the polyclonal group (69%) and in 7/10 (70%) in patients with constitutional skewing. Thirteen of the 32 patients with JAK2 V617P mutation (41%) had thromboses while no thrombotic event was recorded in wild type patients (p=0.008); among the 13 patients with thrombosis and JAK2 V617P mutation, 9 (69%) had monoclonal hemopoiesis, 2 (15%) had polyclonal hemopoiesis, and 2 had constitutive skewing. Thus there was a significant increase in thrombotic events in patients with JAK2 V617P mutation and monoclonal hemopoiesis (p=0.04). JAK2 V617P mutation was also significantly associated to the presence of EECs (p=0.02); in fact 22/26 patients with EECs growth showed JAK2 mutation (85%). Finally when patients with splanchnic thromboses were analyzed 9/11 had a monoclonal X-CIP (81.8%) and 7 of them who were evaluable for JAK2 V617P mutation were invariably carriers of the mutation. Thus we confirm that X-CIP in young female ET is correlated to the risk of thrombosis. JAK2 V617P mutation is significantly associated to the development of thrombosis and is present in the vast majority of patients with monoclonal hemopoiesis. The mechanisms underlying monoclonal hemopoiesis in the absence of JAK2 V617P mutation are unclear, notwithstanding these patients are clinically characterized by both absence of EECs and thrombosis.
Analysis of JAK2 Mutation in Essential Thrombocythemia (ET) Patients with Monoclonal and Polyclonal X-Chromosome Inactivation Patterns (XCIPs).
Authors: Kiladjian Jean-Jacques , Elkassar Nahed , Hetet Gilles , Balitrand Nicole , Conejero Carole , Giraudier Stephane , Cassinat Bruno , Gardin Claude , Fenaux Pierre , Chomienne Christine , Grandchamp Bernard
Published: April 2020

Background: ET is considered as a hematopoietic stem cell disorder, closely related to polycythemia vera (PV). Several studies in female ET patients, using XCIPs analysis, have shown the persistence of polyclonal patterns in a subset of patients, the significance of this finding being unclear (Blood: 1997;89:128 and 1999;93:417). Patients with polyclonal patterns were suggested to be at lower risk of thrombosis. The mechanism of sustained megakaryocytic proliferation, in the absence of a reactive cause, is still to be found in this disease. The recent description of an activating V617F JAK2 mutation in PV prompted us to study its relevance in ET, especially with respect to XCIPs analysis results.

Methods: We looked for V617F JAK2 mutation in whole blood DNA in a series of 43 ET female patients for whom XCIPs had previously been reported by our group (Blood: 1997;89:128). Monoclonal pattern was defined by greater than 75% expression of one allele of an X-chromosome polymorphic gene. ET diagnosis was made according to PVSG criteria, after careful exclusion of PV by red cell mass measurement, CML by karyotype and/or BCR-ABL RT-PCR, and idiopathic myelofibrosis by bone marrow biopsy. V617F JAK2 mutation was screened by direct DNA sequencing (sensitivity of the assay: about 5%).

Results: V617F JAK2 mutation was found in 12 of the 43 samples (28%), in agreement with recent reports. No significant differences were found between JAK2 mutated and non-mutated patients for age (43 vs 57 years), clinical symptoms at presentation (33% vs 35%), platelet count (866 vs 907 G/l), or cytoreductive therapy (41% vs 61%), respectively. Of the 35 patients with a monoclonal pattern in granulocytes (and polyclonal T lymphocytes), 8 (23%) had JAK2 mutation, as compared to 4 of the 8 patients (50%) with polyclonal pattern in granulocytes (p=0.185, Fisher’s exact test).

Conclusion: The 50% incidence of JAK2 mutation we found in ET patients with a polyclonal pattern of hematopoiesis may be due to a better sensitivity of the JAK2 mutation assay by comparison to XCIPs clonality assays. It shows that many ET with a XCIPs polyclonal pattern are in fact clonal disorders. On the other hand, the absence of JAK2 mutation in about 75% of patients with a monoclonal pattern suggests that other molecular events can lead to clonal megakaryocytic proliferation in a large proportion of ET patients.
Activating Notch1 Mutations in Mouse Models of T-ALL.
Authors: O’Neil Jennifer , Calvo Jennifer , McKenna Keith , Krishnamoorthy Veena , Aster Jon C. , Bassing Craig H. , Alt Frederick W. , Kelliher Michelle , Look A. Thomas
Published: April 2020

NOTCH1 was discovered through its involvement in the t(7;9) chromosomal translocation found in less than 1% of T-ALLs. Recent studies have demonstrated a broader involvement of NOTCH1 in human T-ALL. The majority of T-ALL patients have activating mutations that disrupt either the heterodimerization domain or the PEST domain of NOTCH1. We sought to determine whether these mutations are also acquired in mouse models of T-ALL. We have sequenced the heterodimerization domain and PEST domain of notch1 in our mouse model of TAL1-induced leukemia and have found that 74% of the tumors harbor activating mutations in notch1. Cell lines derived from these tumors undergo G0/G1 arrest and often apoptosis when treated with a γ-secretase inhibitor (DAPT). In addition, we found activating notch1 mutations in 38% of thymic lymphomas that occur in mice deficient for various combinations of the H2AX, p53 and Rag2 genes. Thus, notch1 mutations are often acquired as a part of the molecular pathogenesis of T-ALL occurring in mice predisposed to develop the disease because they are transgenic for overexpression of the tal1 oncogene or because they have lost key tumor suppressor genes known to promote genomic instability.
Self-Association Contributes to E2a-Pbx1-Mediated Oncogenesis.
Authors: Wang Zhong , Cleary Michael L.
Published: April 2020

Pbx1 is a proto-oncogene that was originally discovered at the site of t(1;19) chromosomal translocations in pediatric acute B cell precursor leukemia. It codes for a TALE (three amino acid loop extension) class homeodomain transcription factor, which is a component of hetero-oligomeric protein complexes that regulate developmental gene expression. As a result of chromosomal translocations, Pbx1 is oncogenically activated by in-frame fusions with the E2a bHLH protein, which confers strong transcriptional activator properties and constitutive nuclear localization, bypassing the need for dimerization with Meinox homeodomain proteins to stabilize and import Pbx1 into the nucleus. E2a-Pbx1 oncoproteins retain an ability to bind DNA as a complex with Hox transcription factors, and co-expressed HoxA9 accelerates leukemogenesis. Using a biochemical approach, we have recently observed that E2a-Pbx1 self-associates through the Pbx moiety of the chimeric protein to form higher-order oligomers. Structure/function studies suggest that self-association is required for oncogenic activity since mutant E2a-Pbx1 proteins unable to self-associate are transformation defective in an in vitro myeloid progenitor serial replating assay. Interestingly, their oncogenic activity in this assay is rescued using synthetic oligomerization domains of FKBP. The drug AP21998, which disrupts FKBP-mediated oligomerization, blocks the proliferation of transformed myeloid progenitors and facilitates their terminal myeloid differentiation. In addition to self-association, the DNA binding homeodomain of Pbx1 is also required for transformation, but mutagenesis studies indicate that Pbx1 domains involved in cooperative DNA binding with Hox partners are dispensable. These studies suggest an alternative mechanism for leukemogenesis in which E2a-Pbx1 deregulates subordinate gene expression as an oligomeric complex that circumvents interactions with heterologous homeodomain proteins that otherwise modulate Pbx1 nuclear localization, DNA binding, and transcriptional activity.