bluuebunny/crossref_metadata_2025 huggingface dataset:
A Predictive Model for Limited Stage Diffuse Large B-Cell Lymphoma (DLBCL): A Retrospective Analysis of 1,252 Cases Performed by the Intergruppo Italiano Linfomi (IIL).
Authors: Cortelazzo Sergio , Luminari Stefano , Bellei Monica , Martelli Maurizio , Vitolo Umberto , Rigacci Luigi , Ambrosetti Achille , Pogliani Enrico , Chisesi Teodoro , Rossi Giorgio , Rossi Andrea , Rambaldi Alessandro , Barbui Tiziano , Brugiatelli Maura , Federico Massimo
Published: October 2019

Introduction: few studies have explored the usefulness of a prognostic index specifically devised for patients with localized DLBCL. The IIL has performed a retrospective analysis of a large group of patients with limited stage DLBCL and developed a new prognostic model.

Results: 1,252 patients with localized (Ann Arbor stage I-II) aggressive B-cell lymphoma (IWF: G or H, WHO:DLBCL) diagnosed from 1988 to 2002 and without CNS involvement, treated by 4 cooperative groups and 2 single institutions, are the subject of this analysis. Patient’s median age was 57 years (range, 17–91) and M/F ratio was 1.26. Clinical stage was I in 239 (19%), IE in 303 (24%), II in 356 (28%) and IIE in 354 (28%) patients, respectively. Supradiaphragmatic disease was present in 56% of patients, 13% had > 3 nodal sites, 53% had extranodal involvement, and 7% had >1 extranodal site. Bulky disease (≥10 cm) was present in 26% of patients, ECOG-PS >1 in 12% and B symptoms in 14%. Abnormal biochemical data included: elevated LDH (28%), β2-microglobulin (B2M;19%) and ESR (38%) and reduced albumin (< 3.5 g/dL) in 21% of the cases. Patients were treated with anthracyclin-containing regimens ± IF-RT. After a median follow-up of 62 months for alive patients (range 1–183 months), 3 and 5-year OS rates were 73% and 71%, respectively. By univariate analysis the following 11 variables were found to be predictive of a short survival: age ≥65 yrs (P=0,0001), stage II nodal (P=0,0001), number of nodal sites (P<0.0001), poor ECOG-PS (P<0.0001), B symptoms (P=0.0009), bulky disease (P=0.0001), elevated ESR (P=0.0001), LDH (P<0.0001), Radiotherapy (P<0.001), B2M (P=0.007) and reduced serum albumin (<0.0001). By Cox multivariate analysis, age ≥65 years (p<0.001), stage II nodal (P<0.001), high LDH (P<0.001) and bulky disease (P<0.01) were indipendent risk factors (RF) for a short survival. The prognostic model was calculated with the sum of scores assigned to each variable; a score of 2 was assigned to advanced age, high LDH, and Bulky; for Stage a score of 1 was considered for stage Ie-IIe and 2 for stage II nodal. Three groups of patients with a different probability of survival (P<0.000001) were identified. Patients at low (Score 0–1; 387 pts), intermediate (Score 2–3; 484 pts) or high risk (Score 4–8; 381 pts) had a 5 years OS of 87%, 77%, 51% respectively. The predictive performance of the model was internally validated through a non parametric Bootstrap method and through residues’ analysis.

Conclusions: This prognostic model, developed and validated on a very large series of patients with localized DLBCL, will allow us to select appropriate therapeutic approaches on the basis of different risk categories.
c-Myc Amplification in Non Hodgkin’s-Lymphomas with Burkitt-Like Features Is Associated with a Poor Prognosis.
Authors: Mossafa Hossein , Damotte Diane , Vincenneau Anne , Amouroux Isabelle , Athken Nareth , Djnabian Arache , Jeandel Roland , Khoury Eli , Martelli Jean-Michel , Samson Thierry , Tapia Sylvie , Flandrin Georges , Troussard Xavier
Published: October 2019

We retrospectively studied 15 newly diagnosed patients presenting with NHL and Burkitt-like cells (BLCs) after morphological examination and histology review (lymph-nodes: 7 cases, peripheral blood: 5 cases, bone marrow: 3 cases and spleen: 1 patient). Conventional cytogenetic analyses were performed at diagnosis on lymph nodes biopsies (n=6), peripheral blood lymphocytes (n=4), bone marrow (n=4) or spleen (n=1). FISH studies used commercially available probes: IGH/c-MYC fusion signals probes, IGH/Bcl-1 fusion signals probes, IGH/Bcl-2 fusion signals probes and c-Myc 8q24 probe to detect t(8;14)(q24;q32), t(11;14)(q13;q32), t(14;18)((q32;q21) and c-Myc amplification, respectively. Morphological examination and/or histology showed BLCs in all patients. Burkitt-like lymphoma (BLL) is a highly proliferative lymphoma that morphologically resembles Burkitt’s lymphoma (BL) but has more polymorph and pleiomorph cells or large lymphoid cells than BL. The mean percentage of Ki-67 positive cells was 80% (range, 70–100%). A normal karyotype was present in 3 cases and a complex karyotype was observed in 12 cases (80%). When combining conventional cytogenetic studies and FISH studies, t(8;14) or the variants t(2;8) or t(8;22) were never detected. In contrast t(11;14)(q13;q32) was found in 4 cases and t(14;18) in 6 cases. Interestingly, c-Myc amplification was observed in all cases with 3 to more than 9 copies in 10–77% metaphase or interphase cells. The diagnosis of follicular lymphoma (FL) was confirmed by a CD5− and CD10− immunologic profile, typical t(14;18) in 4/6 cases and IgH/Bcl-2 fusion gene in all cases. Four cases were classified as mantle cell lymphoma (MCL) with a blastoid variant: MCL diagnosis was established by lymph-node biopsy in 1 case, CD5+ and CD23+ expression in 3/4 cases and 2/4 cases respectively, typical t(11;14)(q13;q32) in 3 cases, complex caryotype including 11 and 14 chromosomal abnormalities in 1 case and IgH/Bcl-1 fusion gene in all cases. Two patients had marginal MZL with a CD5− and CD10− profile and a complex caryotype including +3 and +18. Two patients presented a DLBCL (CD19+, CD20+) with BLCs and one case was classified as T-NHL (CD2+, CD4+, T-cell receptor gene rearrangements) in leukemic phase with BLCs. All these 15 patients have a poor prognosis with a death occurring in 6 patients during the first month after diagnosis. The presence of BLCs was observed independently of the type of lymphomas, FL, MCL or MZL. c-MYC amplification was associated with BLCs and progressive disease. In conclusion, we identified a new subgroup of patients with NHL (14 B-NHL, 1 T-NHL) and a profile including a poor prognosis, Burkitt-like features at presentation without t(8;14)(q24;q32) or its variants and Myc amplification in all cases.
Oxidative Stress Induces Binding of FANCD2 to STAT5 and Facilitates STAT5-Dependent Survival Signals.
Authors: Bagby Grover , Keeble Winifred , Koretsky Tara , Zodrow Dylan , Jove Richard , Hays Laura , Carlson Hanqian
Published: October 2019

Fanconi anemia (FA) cells are hypersensitive to oxidative stress and exhibit aberrant STAT activation responses to defined extracellular proteins but whether these abnormalities are linked is unclear. Because oxidative stress is known to induce STAT activation, we hypothesized that proper STAT signaling responses in normal cells exposed to H2O2 require intact FA proteins. In fact, we found that FA-C, FA-G, and FA-D2 cells (fibroblasts) showed a significant increase in apoptosis after H2O2-exposure compared to retrovirally-complemented cells. H2O2 induced higher phospho-STAT5 (P-STAT5) expression in complemented cells than in mutant cells. Conversely, mutant cells expressed higher levels of P-STAT3 in both the ground state and after H2O2-induction than complemented cells. Aberrant STAT activation in FA mutant cells was shown to be both nucleus- and JAK2 kinase-dependent. Only low levels of STAT3 and STAT5 were induced in both mutant and complemented cytoplasts and AG490 (a Jak2 inhibitor) significantly suppressed H2O2-induced STAT5 responses. Seeking a direct role of FANCD2 in regulating proper STAT activation responses to H2O2, we carried out immunoprecipitation experiments (with an antibody to the N-terminal fragment of FANCD2) using PD20, a FA-D2 mutant cell line, and FANCD2 complemented PD20. In FANCD2-complemented and normal cells, anti-FANCD2 antibody immunoprecipitated STAT5. However, in mutant cells the same antibody immunoprecipitated STAT3, not STAT5. Thus, mutant (truncated) FANCD2 preferentially binds to and may activate STAT3 in the ground state. In fact, wild type FANCD2 also binds aberrantly to STAT3 in HSC536 (FA-C lymphoblasts) indicating that FANCC may influence the function of wild type FANCD2 and that binding of wild type FANCD2 to STAT3 does not require FANCD2 ubiquitinylation (FANCD2 is not ubiquitinylated in FA-C). Suspecting that in H2O2-exposed cells STAT5 signaling pathways lead to survival while STAT3 pathways lead to apoptosis, we transduced constitutively active mutants (*) of STATs 3 and 5 in mutant D2 and complemented cells. STAT3* increased apoptotic responses to H2O2 in complemented FA-D2 cells and STAT5* decreased apoptotic responses in H2O2-induced FA-D2 cells. In addition, the STAT5 inducible anti-apoptotic gene Bcl-XL was induced in H2O2-exposed complemented FA-D2 cells but not in FA-D2 cells. We conclude that FANCD2 functions to promote survival by ordering proper STAT signaling responses to oxidative stress and that this function of FANCD2 depends in part upon FA-C. We propose that FA cells are hypersensitive to oxidative stress in part because of imbalanced STAT signal transduction responses.
A Comparison of Acute and Delayed Infection Rates among Patients with Indolent Lymphoid Malignancies Receiving Fludarabine(F) and Cyclophosphamide(C) with or without Rituximab(R).
Authors: Tam Constantine S. , Seymour John Francis , Brown Michael , Campbell Philip , Scarlett John D. , Underhill Craig , Ritchie David , Bond Rodney , Grigg Andrew
Published: October 2019

The monoclonal anti-CD20 antibody R improves response rates and survival when added to FC in patients with CLL (Keating Proc ASCO 2004, abstr #6565), but the associated B-cell depletion may increase the infectious risk associated with FC therapy. We therefore compared infectious episodes both during and in the 12 months following therapy in two cohorts of patients treated with FC (F 25mg/m2x3, C 250mg/m2x3 q28 days; n=63) alone or with R (FCR; FC+R 375mg/m2 day 1; n = 91) for indolent lymphoid malignancies. Patients received FC from 10/96 to 12/00 and FCR from 12/00 to 8/03. Both cohorts had similar baseline characteristics including history of infections, neutrophil counts, and Ig levels and were mainly elderly (median age 60 years, range 30–89) with heavily pretreated disease (median number of previous therapies 2, range 1–10) and both received a median of 4 cycles of therapy without routine antibiotic prophylaxis or growth-factors; CLL or follicular lymphomas constituted over two-thirds of group. In comparison to FC, infectious episodes were not significantly increased for FCR either during therapy (FC 15%/cycle vs FCR 20%/cycle, p=0.17) or in the first 12 months of remission (1 infection per 13 patient-months [PM] for both cohorts). There was a trend to increased late neutropenia in the FCR cohort (14% vs 31% at 3 months; P = 0.15). Grade 3+ infections were similar both during therapy (FC 8%/cycle vs FCR 9%/cycle) and in the first 12 months of remission (FC 1 per 84 PM vs FCR 1 per 35PM, p=0.16), as were rates of herpes virus infections (FC vs FCR: during therapy 1% vs 2%/cycle; during follow-up 1 per 48 vs 1 per 94 PM). We conclude that the addition of R to FC does not result in increased early or late infectious risk above that of FC alone.
Allogeneic Peripheral Blood Stem Cell vs. Bone Marrow Transplantation for Patients with Chronic Myeloid Leukemia: Results of an Individual Patient Data Meta-Analysis.
Authors: Schmitz Norbert
Published: October 2019

The Stem Cell Trialist’s Collaborative Group. An individual-patient data meta-analysis using data from 9 randomized trials enrolling 1,111 patients (pts.) to compare allogeneic peripheral blood stem cell transplantation (PBSCT) with bone marrow transplantation (BMT) has recently been completed (Hematology J2004;5:s89). Here, we report the results of this analysis for patients suffering from CML in 1st chronic phase (CP1) (n=422) or accelerated phase/blast crisis (AP/BC) (n=50). Overall survival at five years (70%) was not significantly different in patients who were transplanted with peripheral blood (PB) as compared to bone marrow (BM) cells if all pts. [absolute difference (AD) 3.3 %, p=0.16] or pts. in CP1 [AD 1.1 %, p=0.72] were considered. However, pts. transplanted for AP or BC survived significantly longer following PBSCT than BMT [AP 40 %, p=0.015]. The difference between two subgroups was significant (test of interaction chi²=4.3;p=0.04). Disease-free survival (DFS) was significantly better after PBSCT for all pts. [AD 15.2 %, p=0.004], for pts. in CP1 [AD 11.5 %, p=0.069], and AP/BC [AD 30.3 %, p=0.009]. Test of interaction between two subgroups was chi²=4.3;p=0.07. Relapse rates for all pts. [AD 18.6 %, p=0.00005] and pts. in CP1 [AP 17.0 %, p=0.0002] were significantly lower after PBSCT; no statistical difference was found for pts. in AP/BC most likely due to small patient numbers. Extensive chronic GVHD occurred more often after PBSCT [AD 20%, p<0.00001 ) than after BMT.Treatment-related mortality (TRM) at 5 yrs was 27% for all pts and 26% for patients in CP phase with no significant differences between pts. transplanted with PB instead of BM.TRM was higher in the BMT arm (62% vs. 22%, p=0.02) for pts. in AP/BC. The difference between two subgroups was statistically significant (chi²=5.5;p=0.02). Pts. with CML in AP/BC should be offered a transplant with G-CSF mobilized PB. For pts. in CP1 the decision to use PB or BM needs to value the higher risk of attracting extensive cGVHD against the lower risk of relapse after PBSCT. Further follow up will be necessary to see if the difference in relapse rates after BMT as compared to PBSCT continues to increase over time.
Second Autologous or Allogeneic Transplantation after the Failure of First Autograft in Multiple Myeloma.
Authors: Qazilbash Muzaffar H. , Saliba Rima , de Lima Marcos , Couriel Daniel , Hosing Chitra , Aleman Ana , Lei X , Champlin Richard E. , Giralt Sergio A.
Published: October 2019

Background: Most patients undergoing high-dose therapy and autologous transplant for multiple myeloma eventually relapse. The optimal salvage treatment for these patients is not very well defined. Both autologous and allogeneic hematopoietic stem cell transplantations have been used for salvage. We analyzed the outcomes of second autologous or allogeneic transplants, performed as salvage in patients relapsing after an autograft.

Methods: Fourteen patients received a second autograft for salvage, while thirty-four patients underwent allogeneic transplantation (related 24, unrelated 10). The median age at transplant was 52 years in the autologous group, and 51 years in the allogeneic group. The median interval between the first and the second transplant was 25 months in the autologous group and 17 months in the allogeneic group. The disease characteristics were similar in both autologous and allogeneic groups.

Results: With a median follow-up of 10 months among survivors in each group, both autologous and allogeneic transplant groups had a response rate (complete + partial) of 64%. One hundred day nonrelapse mortality was 7% in the autologous group and 12% in the allogeneic group. Median disease-free survival (DFS) was 11 months in the autologous and 6 months in the allogeneic group. Median overall survival (OS) was 29 moths in the autologous and 14 months in the allogeneic group. 1-year DFS was 40% in the autologous group and 22% in the allogeneic group (p = 0.2). 1-year overall survival was 70% in the autologous and 53% in the allogeneic group (p = 0.3). The most common causes of non-relapse mortality were graft vs. host disease (62%) in the allogeneic group, and infections (100%) in the autologous group. On univariate analysis for DFS in allogeneic group, an interval of >1 year between the first and the salvage transplant was the only factor associated with a significantly better outcome (p = 0.01). Disease status at transplant, type of donor, tumor mass, β2 microglobulin level, and serum albumin level did not show any impact on the outcome.

Conclusions: Both autografting and allografting are feasible as salvage for myeloma patients relapsing after the first autograft. A slightly better outcome with salvage autografting may be due to decreased toxicity.
Allogeneic Hematopoietic Stem Cell Transplantation as a Promising Treatment for Natural Killer-Cell Neoplasms.
Authors: Murashige Naoko , Kami Masahiro , Kishi Yukiko , Kim Sung-Won , Takeuchi Masami , Matsue Kosei , Kanda Yoshinobu , Hirokawa Makoto , Kawabata Yoshinari , Matsumura Tomoko , Kusumi Eiji , Hirabayashi Noriyuki , Nagafuji Koji , Suzuki Ritsuro , Takeuchi Kengo , Oshimi Kazuo
Published: October 2019

The efficacy of allogeneic hematopoietic stem-cell transplantation (allo-HSCT) for NK-cell neoplasms is unknown. We investigated the results of allo-HSCT for NK-cell neoplasms between 1990 and 2003 through questionnaires. A hematopathologist reclassified the pathological diagnoses according to the WHO classification. Of 345 patients from 76 hospitals who underwent allo-HSCT for malignant lymphoma, 32 had NK-cell neoplasms: extranodal NK/T-cell lymphoma (n=27), blastic NK-cell lymphoma (n=3), and aggressive NK-cell leukemia (n=2). Fifteen were chemosensitive and 17 chemorefractory. Nine were in CR at the time of allo-HSCT. Twenty-five had matched related donors, 3 mismatched related, 2 matched unrelated, and 1 mismatched unrelated donors. Stem-cell source was bone marrow in 11 and mobilized peripheral blood in 21. Conditioning regimens were myeloablative (n=26) and non-myeloablative (n=6). Total body irradiation was given to 23 patients.

Graft-versus-host disease (GVHD) prophylaxis was cyclosporin and short-term methotrexate in 27 patients. Grade 2–4 acute GVHD and chronic GVHD developed in 13 and 9, respectively. Nine died of disease progression, 4 of infection, 2 of veno-occlusive disease, 2 of acute GVHD, 1 of interstitial pneumonitis, and 1 of thrombotic microangiopathy. Two-year progression-free and overall survivals were 33% and 38%, respectively (median follow-up, 32 months). All patients who did not relapse/progress within 10 months achieved progression-free survival during the follow-up. In multivariate analysis, stem cell source (BM vs. PB; relative risk 3.33) and acute GVHD (grade 2–4 vs. grade 0–1; relative risk 2.56) significantly affected progression-free survival. Allo-HSCT is a promising treatment for NK-cell neoplasms.

Figure Figure
The Polycomb Group Transcriptional Repressor Bmi-1 Is Constitutively Expressed in Multiple Myeloma (MM) Cells and Modulates Proliferation through a Mechanism Independent of the Ink4a/ARF Locus.
Authors: Desarnaud Frank J. , Shammas Masood , Neri Paola , Koley Hemanta , Prabhala Rao , Cheng Li , Ryung Suk Kim , Chauhan Dharminder , Tassone Pierfrancesco , Bae Jooeun , Anderson Kenneth C. , Munshi Nikhil C.
Published: October 2019

The polycomb group protein Bmi-1 is required for self-renewal of adult hematopoietic stem cells by inhibiting cell senescence and apoptosis through repression of p16INK4a and p14/p19ARF expression. Based on gene expression profile studies showing absence of Bmi-1 expression in normal plasma cells versus upregulation in all MM cell lines and primary patient MM cells, we evaluated its role in MM pathobiology. Bmi-1 expression in MM cell lines is constitutive, and is not modulated by IL-6 and IGF-1. Immunoprecipitation and immunoblotting studies confirmed both Bmi-1 overexpression and nuclear localization. Bmi-1 was found to be tyrosine- and serine-phosphorylated. We next inhibited Bmi-1 expression in MM cell lines (ARP and MM.1S) by stably transfecting a PU6 vector expressing siRNA targeting Bmi-1. Following G418 selection, cell growth, as measured by H3 thymidine incorporation and MTT assay, was stimulated 1.4 fold in MM.1S and 2 fold in ARP cells compared to control cells transfected with a PU6 vector. Gene expression profiling showed: up-regulation of IL-6 and IGF-1 receptors, as well as Gab-1, which plays a critical role in MM cell proliferation and survival induced by IL-6; Phospholipase D1 and proteinase 3 which potentiate cell proliferation by repression and cleavage of p21cip1/waf1; stem cell growth factor, HOXA9, which modulate bone marrow stem cell survival and proliferation. In contrast, p21cip1/waf1 and GADD45a, GADD45b which moderate cell growth arrest were down regulated. However, no change in expression of either p16INK4a or p14/p19ARF was detected by microarray and immunoblotting. We therefore conclude that Bmi-1 is abnormally expressed in MM cells and contrary to its classic action, modulates MM cell growth and survival by modulating cytokine signaling rather than INK4a/ARF. Our studies further suggest that Bmi-1 represents a promising target for novel MM therapeutics.
HSP70 Induces IL-6 in Stromal Cells and Stat-3 Activation in Myeloma Cells.
Authors: Nimmanapalli Ramadevi , Gerbino Elvira , Dalton William S. , Alsina Melissa
Published: October 2019

Multiple myeloma (MM) is characterized by the clonal proliferation of malignant plasma cells that accumulate preferentially in the bone marrow. In spite of high dose chemotherapy and novel targeted therapies this disease remains incurable with a median survival of 3–6 years mainly because of the emergence of drug resistance. Improved survival requires new strategies to prevent relapse. Heat shock proteins (HSPs) are a super family of highly conserved proteins, which are induced in plant, yeast, bacterial and mammalian cells in response to an array of physiological and environmental stress cues. Among heat shock protein families, HSP70 is one of the most highly conserved and is the only protein expressed in response to cellular stress. Exogenous HSP70 has been demonstrated to act as a cytokine to human monocytes by stimulating rapid calcium influx, activating nuclear factor (NF)-kB and up-regulating the expression of IL-1b, IL-6 and tumor necrosis factor alpha (TNF-a) (Asea A et al., 2000). Adhesion of myeloma cells to bone marrow stromal cells mediates IL-6 secretion and tumor cell proliferation in part mediated by STAT-3 activation (Cheung WC et al., 2001). We have shown that adhesion of myeloma cells to bone marrow stromal cells enhances IL-6 secretion by stromal cells and HSP70 secretion by myeloma cells. When we inhibited the HSP70 expression using either KNK437 (HSF-1 inhibitor) or RNAi to HSP70, IL-6 secretion by stromal cells as well as activation of STAT-3 in myeloma cells was inhibited in dose-dependent manner. These results suggest that HSP70 released from myeloma cells is enhancing IL-6 secretion from stromal cells. Incubation of stromal cells with recombinant HSP70 did not enhance IL-6 secretion in stromal cells suggesting that some other soluble factor released from myeloma cells cooperates with HSP70 to enhance IL-6 secretion by stromal cells, We examined whether HSP70 can modulate IL-6 mediated STAT-3 activation by stimulating 8226 cells with IL-6 in the presence or absence of KNK437 and RNAi to HSP70 and measuring phospho-STAT-3 by western analysis. HSP70 inhibition attenuated IL-6 induced STAT-3 activity, but not ERK1/2 activity, indicating that HSP70 mediated IL-6 signaling is very specific to STAT-3. The signal transduction cascade by which HSP70 induces IL-6 secretion and the mechanism by which HSP70 mediates IL-6 induced STAT-3 activity are currently under investigation.
Inhibition of Multiple Myeloma Cell Proliferation and Increase of Apoptosis through Regulation of the NF-κB and JNK Pathways by Silencing TRAF6 C-Domain mRNA.
Authors: Chen Haiming , Campbell Richard A. , Zhu Daocheng , Li Sonia Guangxu , James Christine Pan , Santos Janice , Wang Cathy S. , Bonavida Benjamin , Berenson James R.
Published: October 2019

Several members of the tumor necrosis factor receptor-associated factor (TRAF) family, including TRAF2, TRAF5, and TRAF6 have been implicated in regulating signal transduction from various TRAF family members. However, the unique biological function of TRAF6 is largely determined by its TRAF-C domain, which does not interact with peptide motifs that are recognized by TRAF1, -2, -3 or -5.

Based on TRAF6, CD40, and RANKL sequences and crystal structures, we targeted the TRAF6 C-domain residues from 420 to 440 because the TRAF6 interaction domain with CD40 or RANKL resides in residues 333 to 508. We found that silencing TRAF6 mRNA with the C-terminal domain siRNA significantly inhibited MM cell proliferation maximally at 72 hours whereas effects on inducing MM cell apoptosis were most prominent at 48 hours. The decrease in cell proliferation and increase in cell apoptosis occurred in a concentration (of siRNA)-dependent fashion. Furthermore, NF-κB mRNA expression and protein levels were also reduced using siRNA of the TRAF6 C-domain. We also examined the effect of TRAF6 siRNA on the JNK pathway since this signaling pathway is also associated with cell cycle effects in myeloma. We measured JUN kinase kinase (JNKK), which activates the MAP kinase homologues SAPK and JNK in response to IL-1 receptor stimulation. The results showed that the phosphorylation of JNKK is clearly reduced after knockdown of TRAF6 gene expression by siRNA. Furthermore, we examined c-Jun, a component of the transcription factor complex AP-1, which binds and activates transcription at TRE/AP-1 elements. The transcription activity of c-Jun is regulated by SAPK/JNK binding to c-Jun and phosphorylation of c-Jun at Ser63/73. We found that total endogenous c-Jun is reduced after silencing TRAF6 mRNA in the RPMI8226 MM cell line. In contrast, our data demonstrated that TRAF6 C-domain does not affect TRAF5 or TRAF2 gene expression. In addition, introduction of siRNA derived from the Zn-finger sequence into RPMI8226 MM cells failed to inhibit TRAF6 production. These studies suggest that the TRAF6 C-Domain may be an excellent target to block myeloma cell signaling important for the survival and proliferation of MM cells.
Bortezomib-Induced Reversal of CAM-DR Phenotype in Myeloma Is Associated with AKT and ERK1/2 Signaling.
Authors: Nimmanapalli Ramadevi , Hazlehurst Lori , Enkemann Steven , Alsina Melissa , Dalton William S.
Published: October 2019

An increasing body of evidence suggests that interactions between myeloma cells and the microenvironment are critical for tumor cell survival, proliferation and drug resistance. These pro-survival interactions include binding of soluble factors, and direct contact with stromal cells, or extra cellular matrix (ECM) components such as fibronectin (FN). More specifically, adhesion of multiple myeloma (MM) to FN via ν1 integrins has been demonstrated to confer resistance to a host of chemotherapeutic agents (Shain et al., 2000). This anti-apoptotic phenomenon, termed cell adhesion-mediated drug resistance (CAM-DR), is an intrinsic pathway for evading the therapy-induced apoptosis (Damiano et al., 2000). Bortezomib (PS-341), a specific inhibitor of proteosome, has been shown to cause apoptosis of several tumor cell lines including multiple myeloma. In contrast to conventional cytotoxics, Bortezomib, showed more apoptosis in 24 hours for 8226 myeloma cells adhered to FN (25 % 10 nM) compared to cells in suspension (10 % 10 nM) in a dose -dependent manner. To investigate the signal transduction mechanism responsible for the reversal of CAM-DR phenotype by Bortezomib, we conducted Affymetrix GeneChipÒ Expression of 8226 cells cultured either in suspension, or adhered to FN in the presence and absence of Bortezomib (50 nM) for 4 and 24 hours using the Affymetrix U133A chips. A total of 197 genes were upregulated on FN compared to suspension, among which only 12 genes were increased with Bortezomib and 105 genes were decreased with PS-341. A total of 870 genes were downregulated on FN adhesion, among which the majority of them were increased with Bortezomib. Similar patterns of gene expression profiles were observed after 24 hrs of treatment with Bortezomib. To examine how Bortezomib is reversing CAMDR phenotype, we chose to examine the genes that were changed with FN adhesion and reversed when treated with Bortezomib. The adhesion of 8226 cells to FN enhanced both ERK1/2 and AKT signaling pathways, and both pathways were down regulated when treated with Bortezomib. The main indicators of the AKT signaling on FN are upregulation of PYK2, AKT2, MDM2 and downregulation of p21 and BAD. In conformation with our micro-array data, PYK2 phosphorylation and ERK1/2 phosphorylation were enhanced when 8226 cells were adhered to FN. The mechanisms to understand role of PYK2 phosphorylation and downstream AKT signaling in Bortezomib-induced reversal of CAMDR are under investigation.
B-Lymphocyte Stimulator Protein (BLYS) Is Expressed by Bone Marrow Mast and Lymphoplasmacytic Cells in Waldenstrom’s Macroglobulinemia, and Provides Signaling for Growth, Survival and IgM Secretion.
Authors: Santos Daniel Ditzel , Tournilhac Olivier , Xu Lian , Evdoxia Hatjiharrisi , Hunter Zachary , Branagan Andrew , Manning Robert , Anderson Kenneth C. , Grewal Iqbal , Treon Steven P.
Published: October 2019

B-lymphocyte stimulator protein (BLYS) is a member of the tumor necrosis family of ligands which is expressed by monocytes and neutrophils and regulates B-cell homeostasis and immunoglobulin production through its receptors BCMA, TACI and BAFF-R. In recent studies, we have shown that bone marrow (BM) mast cells (MC) are increased in WM patients and support tumor cell growth. We therefore evaluated sorted WM BM MC (CD117+FceRI+) and lymphoplasmacytic cells (LPC) for BLYS, BCMA, TACI AND BAFF-R by multicolor flow cytometry and RT-PCR. Results were as follows:

Flow Cytometry RT-PCR WM MC WM MC BLYS 5/7 (71%) 8/15 (53%) 25/26 (96%) 20/23 (61%) BCMA 4/6 (66%) 7/8 (87%) 20/20 (100%) 8/13 (61%) TACI 15/16 (93%) 7/7 (100%) 20/20 (100%) 12/15 (80%) BAFF-R 11/16 (68%) 5/8 (62%) 20/20 (100%) 11/11 (100%)

By RT-PCR, BLYS was detected in MC from 5/7 normal donors. However, in contrast to MC from WM patients, BLYS was not expressed on the cell surface of MC from 7 normal donors using multicolor flow cytometry (p=0.02). Importantly, recombinant human BLYS stimulated proliferation, enhanced survival and/or induced IgM secretion of WM LPC at concentrations of up to 1 ug/mL. Paradoxically, at higher concentrations (5, 10 ug/mL) BLYS induced apoptosis of WM LPC. These studies therefore demonstrate that MC and LPC in WM patients express BLYS and BLYS receptors, and provide the framework for specific targeting of MC-LPC interactions in WM.
Clinical and Biological Correlates of CD45 Expression on Bone Marrow Plasma Cells from Patients with Multiple Myeloma.
Authors: Kumar Shaji , Rajkumar S. Vincent , Kimlinger Teresa , Greipp Philip R. , Witzig Thomas E.
Published: October 2019

Background: Multiple myeloma (MM) is characterized by accumulation of clonal malignant plasma cells in the bone marrow (BM). CD45 was initially characterized as a leucocyte common antigen and can be found on all hematopoietic cells and is a key regulator of antigen mediated signaling and activation in B and T-lymphocytes. The expression of CD45 appears to be heterogeneous among the plasma cells in MM and this characteristic has been suggested to have biological and prognostic significance.

Goals: To study CD45 expression by flow cytometry (FC) on BM plasma cells from patients with different stages of MM, and compared CD45 expression to key clinical and biological characteristics including clinical outcome, labeling index (%S-phase), bone marrow angiogenesis, and bone disease. In addition, the expression profile of various adhesion molecules on CD45+ vs CD45− plasma cells was studied.

Results: BM from seventy-five patients seen at the Mayo Clinic during 1995 and diagnosed with newly diagnosed MM (29), relapsed MM (17), smoldering MM (12), and MGUS (17) were studied for CD45 expression.. CD45+ was defined as >20% of gated plasma cells expressing CD45. A mean of 30% of the plasma cells expressed CD45 by flow cytometry (median, 14%; range, 0.1 to 100%). Among the entire cohort of patients, 33 (44%) of the patients were CD45+. Patients with early disease (MGUS and SMM) had a higher percentage of plasma cells expressing CD45 compared with patients with advanced disease (new or relapsed MM) (43% vs. 22%; P = 0.005). Among those with early disease 69% were CD45 positive compared to 28% among those with advanced disease (P = 0.0008). Among the 46 patients with advanced disease, the mean percentage of CD45+ plasma cells was 14% (range, 0.1 – 85) for those with bone lesions compared to 34% (2 – 99) for those with none; P = 0.02. Also, those with high labeling index (>=1%) had a higher percentage of CD45 expressing plasma cells than those with a low labeling index (30% for high LI vs. 15% for low LI; (P = 0.08). Among those with advanced disease, patients with low-grade angiogenesis had a higher percentage of CD45+ plasma cells; 31% vs. 13% (P = 0.03). The median overall survival for the CD45 positive group was 39 mos vs. 18 mos for the CD45 negative group, but the difference was not statistically significant (P=0.1). The expression of CD138 (syndecan-1), CD56 (NCAM) and CD54 (ICAM-1) were higher among the CD45 neg plasma cells. Other adhesion molecules, especially LFA-1, Mac-1, VLA-5, and CD44 were more often expressed on the CD45 + plasma cells.

Conclusions: This study demonstrates that CD45 expression is more common in early disease and is lost with disease progression. There are important immunophenotypic and biologic differences in the CD45+ vs CD45− plasma cells that may have implications for the design of immunotherapy for MM.
Characterization of a R115777 Resistant Human Multiple Myeloma Cell Line with Cross-Resistance to PS-341.
Authors: Buzzeo Robert W. , Enkemann Steven A. , Nimmanapalli Ramadevi , Alsina Melissa , Lichtenheld Mathias G. , Dalton William S. , Beaupre Darrin M.
Published: October 2019

Multiple myeloma is an incurable disease where new treatment strategies are required to improve on current treatment standards. Based on this knowledge we have performed a phase II clinical trial testing the farnesyltransferase inhibitor R115777 in patients with relapsed myeloma (Alsina M. et al, Blood 2004). R115777 was found to have a favorable toxicity profile and 64% of patients achieved disease stabilization. RAS mutation and inhibition of farnesyltransferase did not correlate with clinical efficacy; findings consistent with our prior observation that R115777 induces apoptosis via a Ras independent mechanism (Beaupre D.M. et al, Mol. Cancer Ther. 2004). In order to further characterize the mechanisms by which R115777 induces apoptosis in myeloma cells and to investigate drug resistance, we have identified and characterized a R115777 resistant human myeloma cell line. 8226/S cells were cultured continuously in increasing concentrations of R115777 for over 6 months. 8226/R5 cells were found to be nearly 50 times more resistant to R115777 compared to the parent cell line. K-Ras remained prenylated in both resistant and sensitive cells after R115777 treatment; however, HDJ-2 farnesylation was inhibited in both lines implying that farnesyltranseferase (the drug target) is not modified. 8226/R5 cells were also resistant to a diverse group of anti-tumor agents including PS-341. Many 8226 lines that acquire drug resistance have elevated expression of P-glycoprotein. We found that P-glycoprotein expression is not increased in the 8226/R5 line and furthermore influx and efflux of R115777 was similar in both parent and resistant cells. Expression of the drug resistance proteins hsp27, 70, and 90 were also not increased. Comparison of 8226/S and 8226/R5 gene expression profiles revealed increased expression of JAK2 in resistant cells. Interestingly, STAT3 phosphorylation was significantly increased in the 8226/R5 line consistent with its reported role in myeloma drug resistance. Experiments are underway to delineate the contribution of JAK2 to the multidrug resistant phenotype. Characterization of aberrant JAK2 activation is relevant since constitutive STAT3 activity is frequently observed in primary myeloma isolates.
In Vivo Regulatable Mouse Models of NRAS(V12)-Driven Acute Systemic Mastocytosis and Acute Myeloid Leukemia.
Authors: Wiesner Stephen M. , Hasz Diane E. , Jones Jamie M. , Largaespada David A.
Published: October 2019

NRAS is commonly mutated in acute myeloid leukemia (AML), chronic myeloproliferative disease (MPD), and myelodysplastic syndrome (MDS). Despite intense scrutiny, the specific contribution Ras activation makes in the development of these diseases has not been determined. In an effort to create a model in which to study the effects of Ras dysregulation in hematopoietic disease, we have developed separate founder lines of transgenic mice with the tetracycline transactivator (tTA) driven by the Vav hematopoietic promoter in one line, and NRAS(V12) driven by the tetracycline responsive element (TRE) in the other. Multiple lines were established for each transgene. The TRE-NRAS(V12) lines also have a linked internal ribosome entry site (IRES) - human truncated CD2 cDNA so that transgene expression can be monitored easily by flow cytometry. When the Vav-tTA and TRE-NRAS(V12) lines are crossed, doubly transgenic animals uniformly develop Mast Cell Leukemia (MCL) or Aggressive Systemic Mastocytosis (ASM) between two and four months of age. Disease is characterized by tissue infiltrates of large, well-differentiated C-kit, Gr-1+ mast cells in the spleen, liver, skin, lungs and lymph nodes. Skin lesions are especially prominent in affected mice. Analysis of bone sections show small to large foci of similarly well differentiated mast cells. Expression of the TRE-NRAS(V12) transgene and mast cell disease is repressible by administration of doxycycline in the drinking water of affected animals showing that NRAS(V12) is required to initiate and maintain ASM/MCL in this model. One of the TRE-NRAS(V12) lines, when crossed to Vav-tTA transgenic mice, results in AML (characterized by peripheral white blood cell counts greater that 30X10^3/ul and a differential count of greater that 20% blasts in some animals) with underlying mild mast cell disease. In conclusion, these results demonstrate that expression of oncogenic NRAS is sufficient to initiate myeloid leukemia and the leukemic cells require NRASV12 expression for continued survival. These models will be useful for studying the role of RAS dysregulation in hematopoietic disease in general, as well as in mast cell MPD’s and AML. Mice develop disease with 100 per cent penetrance in a period of time amenable to pharmacologic intervention and cooperating mutagenesis studies. In addition, the model offers the opportunity to evaluate changes in the hematopoietic system in response to transgene repression and derepression by virtue of the tetracycline transactivator system. This work was sponsored by the Leukemia and Lymphoma Society of America (LLS 7019-04, Specialized Center of Research) and the Leukemia Research Fund.
Cooperative Pathways to Acute Myeloid Leukemia Include the Combining of Transcription Factor Alterations: PML-RARα Cooperates with SOX4.
Authors: Kogan Scott C. , Maunakea Mei Lin , Himmel Karen L. , Yin Bin , Le Beau Michelle M. , Largaespada David A.
Published: October 2019

One paradigm for transformation of normal myeloid cells into acute myeloid leukemia (AML) is the combining of transcription factor alteration with activation of signaling pathways, including cytokine receptor activation. To identify possible alternative pathways to leukemogenesis we crossed MRP8-PML/RARA transgenic mice with BXH-2 mice, which harbor an endogenous retrovirus that causes AML. Approximately half of the leukemias that arose in this cross showed features of acute promyelocytic leukemia (APL). We identified 22 insertions sites in 8 APL-like leukemias. Of these, 7 represented common insertion sites in the Mouse Retroviral Tagged Cancer Gene Database (RTCGD, http://rtcgd.ncifcrf.gov/mm4/index.html). We introduced into a retroviral vector cDNAs encoding 6 genes located at retroviral insertion sites identified in PML/RARA leukemias (Sox4, Meis1, Lck, Sfpi1, Nfil3, Cblb). PML/RARA transgenic bone marrow was transduced with these retroviruses and the marrow was used to reconstitute lethally irradiated recipient animals. Recipients of PML/RARα transgenic marrow transduced with a SOX4 retrovirus developed APL-like leukemias in 3 months. SOX4 is an HMG box transcription factor that exhibits sequence specific DNA binding and transactivation. It is the most common insertion site in RTCGD, and has been found to be overexpressed in small cell lung cancer and medulloblastoma, as well as other tumors. In light of other data indicating that combining transcription factor abnormalities can rapidly induce leukemia (e.g. Hoxa9 + Meis1), our finding that SOX4 + PML/RARα is potently leukemogenic supports the hypothesis that such cooperativity represents another important paradigm in myeloid leukemogenesis.
Targeting RAS Signaling in Multiple Myeloma (MM): Synergistic Inhibition by Co-Treatment with the MEK Inhibitor U0126 and Prenylation Inhibitors.
Authors: Reuter Christoph W.M. , Morgan Michael A. , Sebil Tarkan , Ganser Arnold
Published: October 2019

Multiple myeloma (MM) is a fatal hematological malignancy associated with disruption of RAS-to-MAP kinase (MAPK/ERK) signaling. Prenylation inhibitors such as farnesyltransferase inhibitors (FTIs) and the competitive HMG-CoA reductase inhibitor lovastatin have been shown to block RAS post-translational modification and thus transforming ability. Targeting down-stream members of the RAS signaling cascade with compounds such as the MEK inhibitor U0126 is another strategy used to successfully disrupt oncogenic RAS signal transduction. In order to assess the effects of co-treating MM cells with U0126 and prenylation inhibitors (e.g. FTI L-744,832 and lovastatin), six MM cell lines were assayed for viability using the MTT assay, induction of apoptosis by TUNEL assay, and MEK activation by a phospho-MEK-specific FACS assay. Treated cells were also subjected to Western blotting to determine levels of phosphorylated MAP kinase as well as processed and unprocessed RAS and RAS-related proteins. Co-treating MM cells with U0126 and FTI L-744,832 or lovastatin synergistically inhibited MM cell proliferation. Incubation of NCI-H929 cells with U0126 caused a concentration dependent increase of cells in the G0/G1 phase of the cell cycle. Accordingly, 48 h treatment of NCI-H929 cells with 20 μM U0126 caused a reduction in activated MEK in both G0/G1 and G2/M cell cycle phases (41.9 and 39.5%, respectively) while 50 μM reduced activated MEK levels by 64.8% in G0/G1 and 74.5% in G2/M. Lovastatin treatment (48 h, 10 μM) of NCI-H929 led to an accumulation of cells at the G1/S boundary of the cell cycle and >90% loss of activated MEK in both G0/G1 and G2/M. Lovastatin-induced changes in cell cycle distribution and loss of MEK activation could not be rescued by farnesyl pyrophosphate, but were restored to levels similar to those measured in untreated cells by geranylgeranyl pyrophosphate or mevalonate. Co-treating OPM-2 cells 48 h with 10 μM lovastatin and 50 μM U0126 led to >96% decrease in activated MEK in G0/G1 and >76% reduction in G2/M. Lovastatin treatment also caused a loss of prenylated RAS and RAS-family proteins as demonstrated by Western blot analyses. Additionally, Western blotting showed that U0126 treatment (5 μM, 30 min) alone or in combination with lovastatin (5 μM, 30 min) blocked IL-6 stimulated activation of MAP kinase in OPM-2 cells but had no effect on pAKT levels. Our results support that inhibition of RAS processing and down-stream signaling are the major mechanisms of action through which U0126 and lovastatin synergistically inhibit MM cell growth. Furthermore, these data demonstrate the potential therapeutic usefulness of these compounds in the treatment of multiple myeloma.
A Novel Ring-Substituted Diindolylmethane 1,1-bis [3′-(5-methoxyindolyl)]-1-(p-t-butylphenyl) Methane Abrogates ERK Activation and Induces Apoptosis in Acute Myeloid Leukemia (AML).
Authors: Contractor Rooha , Samudio Ismael J. , Estrov Zeev , Harris David , McCubrey James A. , Safe Stephen H. , Andreeff Michael , Konopleva Marina
Published: October 2019

We investigated the antileukemic activity and molecular mechanisms of action of a newly synthesized ring-substituted diindolylmethane (DIM) derivative, named, 1,1-bis [3′-(5-methoxyindolyl)]-1-(p-t-butylphenyl) methane (DIM #34), in myeloid leukemic cells. DIM #34 inhibited leukemic cell growth via induction of apoptosis. DIM #34 inhibited clonogenic growth and induced apoptosis of AML CD34+ progenitor cells but spared normal progenitors. DIM #34 induced loss of mitochondrial membrane potential, which was accompanied by the release of cytochrome c into the cytosol and early cleavage of caspase-9 followed by the cleavage of caspases -8, and -3. Bcl-2 overexpression and caspase-9-deficient cells were partially protected against DIM #34-induced apoptosis, suggesting activation of the intrinsic apoptotic pathway. DIM #34 induced Bax cleavage, and Bax knockout cells were partially resistant to cell death. Furthermore, DIM #34 transiently inhibited the phosphorylation and the activity of the extracellular-regulated kinase (ERK) and abrogated Bcl-2 phosphorylation. Because other methylene substituted DIM analogs transactivate the nuclear receptor PPARγ, we studied the role of PPARγ in apoptosis induction. Although the co-treatment of cells with a selective PPARγ antagonist T007, and a low dose of DIM #34 partially diminished apoptosis, apoptosis was not inhibited at higher concentrations of DIM #34, suggesting the involvement of both, receptor-dependent and independent mechanisms. Co-treatment with RXR- and RAR-ligands enhanced DIM #34-induced cell death. Together, these findings showed that substituted DIMs represent a new class of compounds that selectively induce apoptosis in AML cells through interference with ERK and activation of PPARγ signaling pathways.