bluuebunny/crossref_metadata_2025 huggingface dataset:
Posttransplant Lymphoproliferative Disorder in Liver Transplant Recipients: A Report of Seventeen Cases.
Authors: Patel Himisha , Vogl Dan , Aqui Nicole , Stadtmauer Edward A. , Olthoff Kim , Tsai Donald E.
Published: April 2020

Posttransplant lymphoproliferative disorder (PTLD) is a major complication of liver transplantation. The optimal approach to these patients remains to be determined. We report a retrospective analysis of seventeen consecutive cases of PTLD seen at the University of Pennsylvania Medical Center. A total of 1179 liver transplants have been performed, of which 15 (1.3%) patients developed PTLD. There were two additional patients in this series that received their transplant elsewhere. The most common indications for liver transplantation were viral hepatitis (35%) and/or alcoholic cirrhosis (29%). Of the seventeen PTLD patients identified, most were Caucasian (76%) and/or male (76%). The median age at PTLD diagnosis was 50 yrs (range 20–65 yrs) with a median time from liver transplantation to PTLD diagnosis of 25 months (range 3–75 months). The most common presenting symptoms included fevers (47%), weight loss (47%), anorexia (47%), and pain (70%). PTLD location was frequently extranodal (71%) and/or involved the transplanted liver (41%). At diagnosis, immunosuppressive medications included OKT3 (12%), steroids (71%), azathioprine (41%) or mycophenolate mofetil (12%), and cyclosporine (65%) or tacrolimus (35%). PTLD histology consisted of 9 (53%) monomorphic and 8 (47%) polymorphic. EBV was detected by in situ hybridization in 11 (79%) of 14 cases evaluated. Stage at diagnosis was I (29%), II (35%), III (12%), and IV (12%). Initial therapy included reduction in immunosuppression (RI) alone in 13 (76%) of 17 patients. The remaining 4 patients were treated with retransplantation, rituximab + RI, alpha interferon, or chemotherapy. RI alone resulted in 6 (46%) CR and 7 (54%) PD. The addition of rituximab to RI in 1 patient resulted in an additional CR. R±CHOP was used as initial therapy in one patient and in 2 patients as salvage after relapse with all attaining a CR. Current median follow-up is 6 months (range 7 days to 9 years) with PFS of 6 months. Median overall survival following PTLD diagnosis was 6 months (range 7 days to 9 years). Of the 12 expired patients, 6 (50%) died from progressive PTLD, 3 (25%) from liver failure, 2 (17%) from infection, and 1 (8%) from cardiomyopathy. Currently, 5 patients are alive in CR (follow-up times of 6 months, 3.5 yrs, 4.5 yrs, 6 yrs and 8.5 yrs). Although the detection and treatment of PTLD in liver transplant recipients remains problematic and the upfront mortality is still high, long-term survival is possible. Further studies are necessary to better define treatment strategies.
The Anti-FLT3 Monoclonal Antibody EB10 Is Cytotoxic to FLT3 Inhibitor Resistant Cells In Vivo.
Authors: Piloto Obdulio , Griesemer Melissa , Nguyen Bao , Li Li , Li Yiwen , Witte Larry , Hicklin Daniel J. , Small Donald
Published: April 2020

The class III receptor tyrosine kinase, FLT3, has been shown to play a role in leukemogenesis, particularly in acute myeloid leukemias (AML) and acute lymphoblastic leukemias (ALL). This finding has spurred the development of tyrosine kinase inhibitors (TKI) targeting wild-type and mutant FLT3, and these drugs have shown activity in clinical trials. However, as has been observed with Gleevac for treatment of chronic myeloid leukemia, continuous treatment with TKIs may select for resistant clones. Immunotherapy targeting FLT3 may prove efficacious against FLT3-expressing resistant cells since their mode of action is not dependent on FLT3 inhibition. To study resistance to TKIs against FLT3 we developed resistant human cell lines by co-culturing MOLM14 and SEM-K2 cells, expressing FLT3/ITD mutant and wild type FLT3, respectively, with increasing concentrations of the FLT3 inhibitor CEP-701. The resulting cell lines, MOLM14(R) and SEM-K2(R), are resistant to CEP-701 induced cytotoxicity as well as to other selective FLT3 TKIs, CEP-5214 and PKC412. Cross resistance to CEP-701, CEP-5214 and PKC412 was also observed when MOLM14 and SEM-K2 cells were co-cultured with CEP-5214 or PKC412, instead of CEP-701. Western blot analysis reveals that CEP-701, CEP-5214 and/or PKC412 still inhibit FLT3 and downstream STAT5, Akt and MAPK pathways in MOLM14(R) and SEM-K2(R) cells. Sequencing of FLT3 from the resistant clones confirmed that the resistance was not the result of drug resistance mutations in FLT3. This indicates that the observed resistance to CEP-701, CEP-5214 and PKC412 is due to selection of FLT3-independent clones during co-culture with anti-FLT3 TKIs. To see if anti-FLT3 immunotherapy would still be active against resistant cells, MOLM14(R) and SEM-K2(R) cells were injected into NOD/SCID mice and treated with IMC-EB10, an unconjugated monoclonal antibody against FLT3. These in vivo studies demonstrate that IMC-EB10 still reduces leukemic engraftment and prolongs survival of mice injected with the resistant cell lines, to comparable levels observed with the parent cell lines. In conclusion, our data indicates that culturing leukemic cells with increasing concentrations of selective FLT3 TKIs can result in clones that are FLT3 independent and that anti-FLT3 immunotherapy is still cytotoxic to these resistant cells in vivo. An approach combining FLT3 TKIs with anti-FLT3 antibodies may prove superior and result in reduced chance of developing resistance.
Sensitization of Rituximab-Sensitive and Rituximab-Resistant B-NHL Cell Lines/Clones to TRAIL-Induced Apoptosis by Bortezomib and NF-κB Inhibitors.
Authors: Jazirehi Ali R. , Bonavida Benjamin
Published: April 2020

Patients with B-NHL respond initially to conventional chemotherapy and/or to immunotherapy with rituximab (alone or in combination with chemotherapy). However, patients develop resistance to these modalities and novel approaches are needed. TRAIL is a cytotoxic molecule that exerts selective anti-tumor cytotoxic activity with minimal toxicity to normal tissues. Further, TRAIL or agonist monoclonal antibody (mAb) to TRAIL receptors, DR4 and DR5, are currently being tested clinically. The present study investigated the sensitivity of B-NHL cell lines to TRAIL-mediated apoptosis using the AIDS-related NHL (ARL) B-cell line, 2F7, and the B-NHL cell lines, Ramos and Daudi. Also, to recapitulate various aspects of acquired rituximab-resistance, we have generated rituximab-resistant (RR) clones from the parental wild type (wt) cells. Rituximab failed to chemo-sensitize the RR clones and the clones exhibited higher resistance to various drugs (e.g., CDDP, VP-16, ADR, Vincristine, Taxol) and to TRAIL (1–250 ng/ml-18 h) compared to the wt cells as analyzed by DNA fragment on assay. The findings demonstrate that the wild type and RR1 cells were resistant to TRAIL-mediated apoptosis at a wide range of TRAIL concentrations. We then examined means to reverse TRAIL resistance. We and others have reported that inhibition of NF-κB activity can sensitize TRAIL-resistant tumor cells to TRAIL-induced apoptosis. Hence, we examined the effect of the proteasome and NF-κB inhibitor, Bortezomib (Velcade), Bay 11–7085 and the specific NF-κB inhibitor DHMEQ (Kikuchi et. al, Cancer Research2003; 63:107). Pretreatment of the NHL tumor cells with Bortezomib, Bay 11–7085 or DHMEQ for 6 h followed by treatment with TRAIL for 18h resulted in significant augmentation of apoptosis and synergy was achieved. Both the rituximab-sensitive and rituximab-resistant tumor cells were sensitized by these inhibitors, though higher concentrations were required for sensitization of the RR clones. Interestingly, detailed analysis of the signaling pathways in the RR clones revealed constitutive hyper-activation of the NF-κB survival pathway leading to over-expression of anti-apoptotic gene products Bcl-2, Bcl-xL and Mcl-1. Based on the findings, we postulate that patients with resistant B-NHL can be treated with combination of TRAIL/anti-DR4 or DR5 mAb and NF-κB inhibitors. Alternatively, these patients can be treated with agents that up-regulate TRAIL expression on host effectors (e.g., T cells, NK cells) in combination with NF-κB inhibitors.
A Dual Bcr-Abl/Lyn Tyrosine Kinase Inhibitor, NS-187, Is a Novel Agent for Imatinib-Resistant Leukemia.
Authors: Kimura Shinya , Naito Haruna , Yokota Asumi , Kamitsuji Yuri , Kawata Eri , Ashihara Eishi , Nakaya Yohei , Naruoka Haruna , Wakayama Tatsushi , Asaki Tetsuo , Niwa Tomoko , Hirabayashi Kazuko , Maekawa Taira
Published: April 2020

Chemical modifications of imatinib mesylate made with the guidance of molecular modeling yielded several promising compounds. Among them, we selected a compound denoted NS-187 (elsewhere described as CNS-9) on the basis of its affinity to Abl, and also to Lyn, which may be involved in imatinib-resistance (Figure). The most striking structural characteristic of NS-187 is its trifluoromethyl (CF3) group at position 3 of the benzamide ring. The presence of the CF3 group strengthened the hydrophobic interactionss of the molecule with the hydrophobic pocket of Abl. Another possible merit of the CF3 group is that it may fix the conformation of the drug by hindering its rotation at the 4-position of the benzamide ring; as a result, a CF3-bearing molecule may be more potent than more flexible compounds such as imatinib. In fact, NS-187 was 25–55 times more potent than imatinib in vitro and and at least 10 times more potent than in vivo. NS-187 also inhibited the phosphorylation and growth of all Bcr-Abl mutants tested except T315I at physiological concentrations. Another special feature of NS-187, in addition to its increased affinity to Abl is its unique spectrum of inhibitory activity against protein kinases. At a concentration of 0.1 μM, NS-187 inhibited only four of 79 tyrosine kinases, that is, Abl, Arg, Fyn, and Lyn. Notably, at 0.1 μM NS-187 did not inhibit PDGFR, Blk, Src or Yes. The IC50 values of NS-187 for Abl, Src and Lyn were 5.8 nM, 1700 nM and 19 nM, respectively, and those of imatinib were 106 nM, >10,000 nM and 352 nM, respectively. These findings indicate that NS-187 acts as a Bcr-Abl/Lyn inhibitor. In this respect, NS-187 may stand out among other novel Abl tyrosine kinase inhibitors, because BMS-354825 inhibits all members of the Src family, while AMN-107 inhibits none of the Src-family kinases. Our proposed docking models of the NS-187/Abl complex support the notion that NS-187 is more specific for Lyn than for Src. The amino acid at position 252 is either Gln or Cys in Src-family proteins. NS-187 inhibited the Gln252-bearing proteins Abl, Fyn and Lyn but had lower activity against the Cys252-bearing Src and Yes. This is probably because Gln, unlike Cys, readily forms hydrogen bonds. The distinguishing characteristic of NS-187, its high affinity for and specific inhibition of Abl and Lyn, may be useful in the treatment of Bcr-Abl-positive leukemia patients.

Figure Figure
Radiosensitization of Human Cancers by Smac Agonists.
Authors: Giagkousiklidis Stavros , Vogler Meike , Westhoff Andrew , Kasperczyk Hubert , Debatin Klaus-Michael , Fulda Simone
Published: April 2020

Resistance to current treatment regimens such as radiation therapy remains a major concern in oncology and may be caused by defects in apoptosis programs. Since “Inhibitor of apoptosis proteins” (IAPs), which are expressed at high levels in many tumors, block apoptosis at the core of the apoptotic machinery by inhibiting caspases, therapeutic modulation of IAPs could target a key control point in resistance. Here, we report for the first time that full length or mature Smac, an inhibitor of IAPs, significantly enhanced γ-irradiation-induced apoptosis and reduced clonogenic survival in neuroblastoma, glioblastoma or pancreatic carcinoma cells. Notably, Smac had no impact on DNA damage/DNA repair, activation of NF-κB, upregulation of p53 and p21 proteins or cell cycle arrest following γ-irradiation indicating that Smac did not alter the initial damage and/or cellular stress response. Smac enhanced activation of caspase-2, -3, -8 and -9, loss of mitochondrial membrane potential and cytochrome c release upon γ-irradiation. Inhibition of caspases also blocked γ-irradiation-induced mitochondrial perturbations, indicating that Smac facilitated caspase activation, which in turn triggered a mitochondrial amplification loop. Interestingly, mitochondrial perturbations were completely blocked by the broad range caspase inhibitor zVAD.fmk or the relatively selective caspase-2 inhibitor zVDVAD.fmk, whereas caspase-8 or caspase-3 inhibitors only inhibited the increased drop of mitochondrial membrane potential provided by Smac, suggesting that caspase-2 was acting upstream of mitochondria upon γ-irradiation. In conclusion, our findings provide evidence that targeting IAPs, e.g. by Smac agonists, is a promising strategy to enhance radiosensitivity in human cancers.
Overcoming Resistance to Imatinib Mesylate in a CML Cell Line by Downregulation of P-gp with siRNAs.
Authors: Lima Raquel T. , Guimaraes Jose E. , Vasconcelos M. Helena
Published: April 2020

Imatinib Mesylate (Gleevec, formely STI571 from NOVARTIS) is a highly selective drug, capable of inhibiting the BCR-ABL tyrosine kinase which is responsible for CML. However, resistance to this drug has been documented, mainly due to an overexpression of the BCR-ABL protein, to mutations in the tyrosine-kinase domain of BCR-ABL itself or to an overexpression of the multidrug resistance protein 1 (P-gp). In the current study we investigated if downregulation of P-gp expression with siRNAs, in a cell line resistant to Imatinib which overexpressed P-gp (K562Dox), would allow to overcome resistance to this drug. In order to optimise transfection of the siRNAs, we used a fluorochrome labelled control-siRNA and fluorescence microscopy to evaluate the % of uptake of the siRNA under different transfection conditions. The optimised conditions (200 nM siRNA using the jetSi™ transfection reagent) allowed us to obtain uptake of siRNAs in 53% of cells. In order to confirm if the siRNAs were capable of downregulating the expression of P-gp we carried out, 24h after transfection, Real-Time Quantitative PCR studies and Western Blots of cells: i) treated with medium only (Blank), ii) transfected with the siRNAs suspension buffer, iii) transfected with a control siRNA (CRNAi), iv) transfected with two different siRNAs for P-gp (MDR-FE or MDR-CR). We verified that in cells treated with the MDR-CR siRNA there was a reduction in the P-gp mRNA expression of 26%, when compared to the CRNAi. Furthermore, when cells were treated with the MDR-FE or MDR-CR siRNAs, protein levels were also reduced by 33% and 57% respectively, when compared to the CRNAi. It was then verified if this downregulation of MDR expression caused sensitization of cells to Imatinib, in comparison to control cells, using both viability (Trypan Blue) and apoptosis (TUNEL) assays. For this purpose, Imatinib Mesylate was added to the cells previously transfected with siRNAs (for P-gp or control siRNAs) and to cells in exponential growth (Blank cells). Results showed that there was an enhancement of the effect of Imatinib in cells previously transfected with both P-gp siRNAs (MDR-FE or MDR-CR). This increase in the effect of Imatinib was due to an increase in cellular apoptosis. Indeed, the % of apoptotic cells increased from 3 % in cells treated with solvent to 7 % in cells treated with 1 μM Imatinib. It was concluded that downregulation of P-gp protein expression by RNAi technology permitted sensitisation of CML cells to Imatinib, suggesting that P-gp inhibition may be considered as an adjuvant therapy in cases of Imatinib resistance mediated by this mechanism.
Gene Expression Profiling (GEP) in Multiple Myeloma (MM): Comparison of Purified MM Cells (PMM), Random Bone Marrow Biopsies (RBX) and Fine Needle Biopsies from Focal Lesions (FNBX).
Authors: Huang Yongsheng , Zhan Fenghuang , Barlogie Bart , Shaughnessy John
Published: April 2020

Background: GEP has become a useful tool for the distinction of genetic subgroups of patients with malignant disease of a common pathologic diagnosis, including MM. The aim of this study was to determine whether whole bone marrow biopsies could replace the laborious magnetic bead purification of CD 138-positive plasma cells, with the potential added advantages of: 1. less sample manipulation and thus facilitating shipping to a central processing and analysis facility; 2. encompassing the bone marrow micro-environment as an important contributor to MM survival and drug resistance.

Patients and Methods: Samples included paired PMM and RBX from 190 newly diagnosed patients treated on Total Therapy protocols TT2 (n = 85) and TT3 (n = 105); 29 of these patients also had CT-guided fine needle biopsies (FNBX); 5 subjects with MGUS and 5 normal donors were used for comparison. Total RNA was applied to Affymetrix U133 Plus2 microarray chips. Unsupervised hierarchical clustering (UHC) was performed on Log2 transformed GEP intensity values of probe sets with >95% present detection calls and >0.5 standard deviation.

Results: UHC analysis of RBX (MM, MGUS, normal) identified 2 major subgroups: one containing both normal and MGUS samples (“normal-like”, N-L) and the other comprising only MM samples (“myeloma-like”, M-L) (Figure 1). In the context of previously defined 7 GEP subgroups (Zhan et al, Blood 2004), the N-L group contained higher proportions of hyperdiploid and normal karyotype cases, whereas the M-L group comprised the majority of MAF/MAFB, FGFR3/MMSET and proliferation cases. Good-risk standard laboratory features (non-IgA isotype, lower B2M and CRP levels, higher Hb concentrations) were over-represented in the N-L group. When applied to TT2 patients with a median follow-up of 3 years, N-L patients had superior 3-yr EFS (85% vs 55% for M-L group; p=.02). A further UHC analysis addressed similarities/differences among PMM, RBX and FNBX samples available in 29 patients. The dendrogram had 2 major branches; one comprising all PMM samples and the other all BX with sub-branches separating RBX and FNBX subgroups. Finally, 90% of all MAF, MAFB, FGFR3, MMSET and CCND1 “spikes” identified in PMM (with well established prognostic implications) were also detectable in RBX and FNBX samples.

Conclusion: GEP analysis of RBX distinguished 2 subgroups, M-L and N-L, in the context of comparison with normal subjects and MGUS. Compared to M-L, N-L patients (comprising more favorable genetic and standard laboratory features) had superior EFS. Both RBX and FNBX were adequate tissue sources for the detection of prognosis- and possibly therapy-relevant translocation-associated “spiked” genes. Genes differentiating PMM, RBX and FNBX will be reported at the meeting. BX as tissue source should make GEP studies of MM feasible in the context of multi-institutional trials.
Risk of Monoclonal Gammopathy of Undetermined Significance (MGUS) and Subsequent Multiple Myeloma among African-American and White Veterans in the U.S.
Authors: Landgren Ola , Gridley Gloria , Turesson Ingemar , Caporaso Neil E. , Goldin Lynn R. , Baris Dalsu , Fears Thomas R. , Hoover Robert N. , Linet Martha S.
Published: April 2020

Background: The age-adjusted incidence of multiple myeloma (MM) is 2-fold higher in African-Americans than in Whites, which is paralleled by reports of a higher prevalence of monoclonal gammopathy of undetermined significance (MGUS) in African Americans. Etiological factors for MGUS and determinants for transformation of MGUS to MM are unknown, but data on the race-specific prevalence of MGUS and progression of MGUS to MM may provide clues to etiology.

Objective: To determine the prevalence and the probability of progression from MGUS to MM in African-Americans compared to Whites.

Patients and Methods: The cohort was identified from review of discharge diagnoses in hospital medical records for inpatient hospitalizations at 142 nationwide U.S. Veterans Affairs (VA) hospitals between October 1, 1980 and September 30, 1996. The target population for calculation of MGUS prevalence included all African-American (N=749,020) and White (N=3,248,795) veterans hospitalized at least once at age 18 or older. For estimating the risk of malignancy, all subjects without a prior discharge diagnosis of malignancy were followed from one year after index hospital discharge (MGUS diagnosis for MGUS cases, and first discharge for any reason for all others) until the diagnosis of a first malignancy, death, or the end of the observation period (September 30, 1996), whichever came first. Using the Kaplan-Meier procedure, we calculated the cumulative probability of developing MM among MGUS cases according to race, testing for statistical significance using the Wilcoxon-test appropriate for censored data. The Cox proportional hazards model was applied. Poisson regression was used for analyses comparing risk for MM among MGUS versus non-MGUS cohorts.

Results: We identified 734 cases of MGUS in African-Americans and 1,312 in Whites. The age-adjusted prevalence-rate for MGUS was 3.0-fold (2.7–3.3 95% CI) higher in African-Americans than in Whites. Among MGUS cases, the estimated cumulative risk of developing MM during the first 10 years of follow-up was similar (Wilcoxon-test P=0.37) for African-Americans (17%) and for Whites (15%). The risk of developing MM among all MGUS (versus non-MGUS) cases was very high (RR=89.1; 74.7–106.3, 95%CI).

Conclusions: In the largest study to date, we suggest that the excess risk of MM in African-Americans results from an increase in risk of MGUS rather than an increased risk of progression from MGUS to MM.
Molecular Classification of Multiple Myeloma: A Distinct Transcriptional Profile Characterizes Patients Expressing CCND1 and Negative for 14q32 Translocations.
Authors: Agnelli Luca , Bicciato Silvio , Mattioli Michela , Fabris Sonia , Intini Daniela , Verdelli Donata , Baldini Luca , Morabito Fortunato , Callea Vincenzo , Zanella Alberto , Deliliers Giorgio Lambertenghi , Lombardi Luigia , Neri Antonino
Published: April 2020

The deregulation of CCND1, CCND2 and CCND3 genes represents a common event in multiple myeloma (MM), being at least one of them deregulated in almost all MM tumors. A recently proposed TC classification1 grouped MM patients into five classes on the basis of their cyclins D expression profiles and the presence of the main translocations involving the immunoglobulin heavy-chain (IGH) locus at 14q32. The aim of our study was to identify the putative transcriptional fingerprints associated with the deregulation of the different D-type cyclins and the presence of IGH translocations. The cyclin D expression levels obtained by high-density oligonucleotide microarray analysis of purified plasma cells from 50 MM cases were used to stratify the samples into the five TC classes, along with the molecular characteristics. The cyclin D expression data were validated by means of real-time quantitative PCR analysis; fluorescence in-situ hybridization was used to investigate the cyclin D loci arrangements, and to detect the main IGH translocations and the chromosome 13q deletion. A multi-class classification analysis was performed on the gene expression data and used to identify the transcriptional fingerprints of the 5 TC groups. 112 probe sets were selected as characterizing the TC1, TC2, TC4 and TC5 groups, whereas the TC3 samples showed heterogeneous phenotypes and no marker genes. In particular, TC1, TC4 and TC5 groups were characterized by the molecular signatures associated with the primary IGH translocations target genes. The TC2 group, showing significantly extra copies of the CCND1 locus (P=5.9×10−3) and neither IGH translocations nor the chromosome 13q deletion (P=1.7×10−3), was characterized by the overexpression of 30 genes, mainly involved in protein biosynthesis at translational level. Among the most specifically modulated transcripts within the group we identified a novel gene containing a BTB/POZ domain, typical of many zinc finger transcription factors and associated with transcriptional repression activity. A meta-analysis performed on two publicly available MM datasets, containing almost 250 cases, validated the identified gene expression signatures with a global classification rate (indicating the correct prediction of the TC class for the independent set) of 86% and 90%, respectively. Our data contribute to the understanding of the molecular and biological features of distinct MM subtypes; the identification of a distinctive gene expression pattern in TC2 patients may improve risk stratification and indicate novel therapeutic targets.
Aberrant Autocrine and Paracrine CD40L Signaling Promotes Primary Myeloma Cell Survival and Attenuates Bortezomib-Induced Apoptosis.
Authors: Chen-Kiang Selina , Ely Scott , Di Liberto Maurizio , Wu Kaida , Shaughnessy John , Moore Malcolm , Niesvizky Ruben , Hatada Eunice
Published: April 2020

In multiple myeloma (MM), impaired apoptosis is critical for clonal expansion of malignant MM cells in the bone marrow (BM), although the precise underlying mechanism has not been defined. During physiologic plasma cell differentiation, clonal expansion of antigen-activated B cells in the germinal center requires CD40 signalling, which mediates CD40L signals from activated T cells to promote B cell activation and survival. Terminal differentiation of plasma cells from activated B cells is then accompanied by a progressive reduction of CD40 expression, suggesting that the life span of normal plasma cells may be limited by the loss of CD40L signalling. Here we show that CD40L and CD40 are both aberrantly expressed in primary BM MM cells, but not in normal BM plasma cells, their likely normal counterparts. CD40L is also copiously expressed in multiple cell types in myeloma and normal bone marrows, indicating that autocrine and paracrine CD40L signalling may prolong the survival of MM cells in the bone marrow microenvironment. Supporting this hypothesis, disruption of CD40L signalling by an antagonizing CD40L antibody markedly accelerates apoptosis of BM MM cells ex vivo. In the majority of MM cases (N=50), CD40 is expressed at either greater than 90% of BM MM cells or lower than 30% of BM MM cells in vivo, but rarely in between. However, there is no apparent correlation between the frequency of CD40 expression in BM MM cells in vivo and immunoglobulin isotype or treatment history, including bortezomib therapy. Ex vivo, the level of CD40 expression in BM MM cells is enhanced in response to bortezomib, which rapidly activates CD40 transcription. Moreover, killing of primary BM MM cells by bortezomib ex vivo is attenuated by CD40L signalling, at least in part through sustaining NF-?B activation that is required for MM cell survival. Together, our findings suggest that autocrine and paracrine CD40L signalling is critical for the survival of MM cells in the bone marrow and may contribute to the development of drug resistance.
A Multicenter, Single-Arm, Open-Label Study To Evaluate the Efficacy and Safety of Single-Agent Lenalidomide in Patients with Relapsed and Refractory Multiple Myeloma; Prelininary Results.
Authors: Richardson Paul , Jagannath Sundar , Hussein Mohamad , Berenson James , Singhal Seema , Irwin David , Williams Stephanie F. , Bensinger William , Badros Ashraf Z. , Vescio Robert , Kenvin Laurie , Yu Zhinuan , Olesnyckyj Marta , Faleck Herbert , Zeldis Jerome , Knight Robert , Anderson Kenneth C.
Published: April 2020

INTRODUCTION: Lenalidomide (REVLIMID®; CC-5013) is a novel, orally active immunomodulatory drug under investigation for the treatment of multiple myeloma (MM). Phase 1 dose-escalation studies in patients (pts) with relapsed and refractory MM determined that the maximum tolerated dose (MTD) of lenalidomide was 25 mg/day, based upon myelosuppression encountered beyond 28 days, which was manageable with growth factor support and dose reduction. In a multicenter phase 2 study to determine optimal dose and schedule, 102 pts with relapsed or refractory MM were randomized to receive lenalidomide at either 15 mg bid (n=34) or 30 mg qd (n=68), for 21 days every 4 wks. Both treatment arms showed significant activity with manageable toxicity. An increased incidence of cytopenia was noted in the 15-mg bid group and thus the 30 mg qd schedule was taken forward.

METHODS: The objective of this multicenter, phase 2, open-label study (CC-5013-MM-014) was to further evaluate the effectiveness and safety of single-agent lenalidomide administered at a dose of 30 mg qd for 21 days every 28 days (28-day cycle) in pts with relapsed and refractory MM. Eligible patients included those who had received prior thalidomide, bortezomib, or SCT.

RESULTS: 222 pts were enrolled into the study. All patients had received at least 2 prior anti-myeloma treatments, including bortezomib (41%), thalidomide (80%), and SCT (44%). Table 1 shows Best Response data, excluding patients in whom responses were not evaluable (n=10). Partial response or better occurred in 25% of patients and SD or better in 71%. Time to Progression was a median of 22.4 wks (range 1.8– 66 wks). The median survival has not been reached (the lower bound of the 95% CI exceeds 15 months). The most common treatment-related AEs (those reported in ≥10% of patients overall) included upper respiratory tract infection, neutropenia and thrombocytopenia. AEs that most frequently led to dose reduction or interruption by percentage of cases were neutropenia (40%), thrombocytopenia (23%), fatigue (5%), and anemia (5%).

CONCLUSION: Oral lenalidomide in relapsed and refractory MM patients achieved PR+CR in 25%, stable disease or better in 71%, a median TTP of approximately 6 months and a median survival that has not been reached. Toxicity has been manageable with a very low incidence of DVT and minimal treatment-emergent neuropathy.

Table 1. Best Response Best Response* n (%) *Excluding patients not evaluable (n=10); CR=complete response and PR=partial response (EBMT criteria) ≥PR (CR + PR) 53 (25) Stable disease 152 (71)
BCL6 Attenuates DNA Damage Sensing in Normal and Malignant B-Cells by Directly Repressing ATR.
Authors: Ranuncolo Stella M. , Polo Jose M. , Dierov Jamil , Shaknovich Rita , Carroll Martin , Melnick Ari M.
Published: April 2020

The BCL6 (B-Cell-Lymphoma-6) transcriptional repressor is a critical oncogene in B-cell lymphomas and is required for establishment of germinal centers by normal B-cells. However, the mechanisms by which BCL6 licenses germinal center formation and lymphomagenesis are unknown. To characterize this mechanism we identified BCL6 target genes by expression arrays and high throughput chromatin immunoprecipitations. Remarkably, a number of these target genes were critical mediators of DNA damage sensing checkpoints including ATR and p53. Therefore, we hypothesized that BCL6 could attenuate DNA damage sensing by silencing these genes, which is likely a critical attribute for survival and proliferation of germinal center B-cells cells undergoing somatic hypermutation (SHM) and class switch recombination (CSR). Accordingly, we found that expression of BCL6 in normal diploid fibroblasts could block cellular sensing of DNA damage as demonstrated by loss of histone 2AX (H2AX) phosphorylation and delayed repair of double strand breaks. Repression of ATR (but not p53 or other targets) was required for this phenotype. This is a physiological effect since the same result was observed when BCL6 was expressed in purified primary human tonsilar mature B-cells. Reciprocally, shRNA knockdown of BCL6 in B-cell lymphomas rescued repression of ATR, enhanced H2AX phosphorylation and accelerated repair of double strand breaks, independent of the status of p53. shRNA knockdown of BCL6 caused a marked increase of apoptosis in lymphoma cells in response to DNA damage, due to restored DNA damage checkpoint functions. Importantly, BCL6 knockdown had an identical effect on ATR levels, H2AX phosphorylation, DNA damage, and survival in purified primary human germinal center centroblasts. These results suggest that a major role of BCL6 in germinal center formation is to attenuate cellular response to DNA damage occurring as a byproduct of CSR and SHM. The same mechanism also seems to be required for lymphomagenesis, since we observed that sustained BCL6 expression in human primary mature B-cells leads to aberrant survival properties and genomic instability. Moreover, BCL6 blockade using a specific inhibitor molecule designed by our lab induces apoptosis in lymphoma cells and synergizes with DNA damaging agents. Therefore, we have identified a critical mechanism of action of the BCL6 oncoprotein in normal and pathogenic states and show that specific targeting of BCL6 could synergize with chemotherapy drugs for the therapy of B-cell lymphomas.
The Proteasome Inhibitor PR-171 Inhibits Cell Growth, Induces Apoptosis, and Overcomes De Novo and Acquired Drug Resistance in Human Multiple Myeloma Cells.
Authors: Ivancsits Douglas , Nimmanapali Rama , Sun Megan , Shenk Kevin , Demo Susan D. , Bennett Mark K. , Dalton William S. , Alsina Melissa
Published: April 2020

Proteasome inhibitors are a new class of drugs that have shown to be cytotoxic and induce apoptosis in tumor cells, particularly, multiple myeloma (MM). This led to the clinical evaluation and subsequent FDA approval of the proteasome inhibitor, bortezomib, for the treatment of refractory and relapsed multiple myeloma. However, continued occurrence of refractoriness, development of resistance, and the toxicity of bortezomib in treated patients has led to the recent development of novel agents with increased binding efficiency and specificity to the proteasome. PR-171 is a member of the epoxomicin-class of compounds that act as potent and selective irreversible inhibitors of the chymotrypsin-like protease activity of the 20S proteasome resulting in accumulation of ubiquinated proteins and apoptosis of tumor cells (Ming et al., PNAS96:10403). In this study, we evaluated the effects of PR-171 on human MM cells’ survival, proteasome activity and drug resistance. PR-171 induced growth inhibition and apoptosis in the drug sensitive human MM cell lines 8226/S, MM1, H929, and U266, with IC50 values ranging from 9 to 21nM. PR-171 retains activity on cells resistant to melphalan, mitoxantrone, and zarnestra, but has reduced activity on cells resistant to doxorubicin. Pretreatment with verapamil restored sensitivity of the doxorubicin resistant cells (8226/Dox40) to PR-171 back to levels seen in the parental, drug sensitive cell line (8226/S). Proteasome activity was inhibited by PR-171 in the drug sensitive and drug resistant MM cell lines, although much less inhibition was observed in the doxorubicin resistant cells, consistent with the reduced effects of PR-171 on cell viability. In freshly isolated MM cells from patients, PR-171 at doses of 10–250nM induced apoptosis in 9/9 samples tested from both newly diagnosed and relapsed refractory patients. To test the effects of PR-171 on Cell Adhesion Mediated Drug Resistance (CAM-DR), MM cell lines were treated with drug after adhesion to fibronectin. PR-171 at 1–100nM overcomes CAM-DR in 8226/S and H929 cell lines, but not in MM1. The ability of PR-171 to overcome CAM-DR was also seen in primary MM cells from patients in 6 out of 9 samples tested. In summary, we have shown that PR-171 directly inhibits proteasome activity, proliferation, and induces apoptosis in human MM sensitive and resistant cell lines, induces apoptosis in isolated MM cells from newly diagnosed and relapsed refractory patients, and overcomes de novo drug resistance. These studies provide the foundation for clinical investigation of PR-171 to improve the outcome of patients with MM and other hematological malignancies.
R-Etodolac and a Novel Indole-Pyran Structural Analog, SDX-308, Induce Cytotoxicity and Overcome Drug Resistance in Multiple Myeloma.
Authors: Yasui Hiroshi , Hideshima Teru , Neri Paola , Jin Janice , Kiziltepe Tanyel , Shiraishi Norihiko , Ishitsuka Kenji , Laurence Catley P. , Raje Noopur , Podar Klaus , Tassone Pierfrancesco , Chauhan Dharminder , Richardson Paul G. , Leoni Lorenzo M. , Kanekal Sarath , Elliott Gary T. , Munshi Nikhil C. , Anderson Kenneth C.
Published: April 2020

SDX-101, the less toxic R-isomer of the commercially available non-steroidal inflammatory drug R,S-etodolac (Lodine®), lacks COX inhibitory activity and is being investigated in Phase II clinical trials in chronic lymphocytic leukemia. Recently, we reported that R-etodolac, at clinically relevant concentrations, induces potent in vitro cytotoxicity in drug-sensitive and conventional drug-resistant multiple myeloma (MM) cell lines, as well as in primary tumor cells from MM patients. R-etodolac triggers caspase/poly-ADP-ribose polymerase (PARP) cleavage and downregulates of cyclin D1 expression (Yasui et al. Blood 2005). Importantly, R-etodolac at sub-cytotoxic doses upregulates Mcl-1s and synergistically enhances dexamethasone (Dex)-induced caspase-dependent apoptosis in Dex-sensitive MM.1S cells. Combination of R-etodolac with Dex enhances cytotoxicity in Dex resistant OPM1 MM cells and in Dex-resistant patient MM cells in vitro. We further studied the in vivo anti-tumor effect of combined R-etodolac and Dex in SCID mice injected subcutaneously with OPM1 human MM cells. While oral treatment of SCID mice with R-etodolac alone (250 mg/kg/d) or Dex alone (1 mg/kg/d) did not induce any significant reduction of tumor volume compared with control (PBS), the combination of R-etodolac and Dex inhibited tumor growth synergistically (synergism quotient = 1.6) and significantly (p = 0.023), suggesting that R-etodolac may reverse Dex resistance in MM. Finally, we demonstrated that racemic SDX-308, a novel indole-pyran structural analog of etodolac, has 10-fold more potent cytotoxicity than R-etodolac in MM cell lines both sensitive and resistant to conventional therapies, as well as in patient’s MM cells. Moreover, SDX-308, like R-etodolac, can overcome the viability and proliferative enhancing effects of exogeneous IL-6, IGF-1, or bone marrow stroma cells. These combined observations indicate that SDX-308 is a promising more potent second generation analog of R-etodolac for MM therapy. Our data suggest that R-etodolac and its novel analog SDX-308 overcome resistance to some conventional therapeutics used for MM, and provide preclinical rationale to conduct clinical trials of R-etodolac and SDX-308 to treat MM.
Targeting Src- Family Kinase Activation and Downstream Molecular Events To Overcome MM Cell Growth, Survival, and Drug- Resistance.
Authors: Podar Klaus , Tai Yu-Tzu , Abtahi Dean , Raab Marc S. , Yasui Hiroshi , Ishitsuka Kenji , Shiraishi Norihiko , Catley Laurence P. , Sattler Martin , Raje Noopur , Hideshima Teru , Chauhan Dharminder
Published: April 2020

IL-6 induces growth and survival factor in multiple myeloma (MM) via ERK and Akt signaling cascades. Our recent studies difined early signaling events mediating phosphorylation of ERK and Akt-1: specifically, a gp130/Hck- containing complex plays a pivotal role in Hck- mediated phosphorylation of Grb2- associated binder (Gab) family adapter proteins thereby mediating downstream signaling (Podar et al, 2004). We have also shown that IL-6 triggers resistance to dexamethasone via a PI3kinase/ Akt-1 signaling pathway by specific activation of SHP2 phosphatase, withsubsequent blockade of related adhesion focal tyrosine kinase (Chauhan et al, 2000). In the present study we first evaluated AP 23464, a potent adenosine 5′-triphosphate (ATP)- based inhibitor of Src kinase (ARIAD Pharmaceuticals Inc., Cambridge, MA), for its effect on proliferation and survival in MM cells. Our results show that AP23464 markedly inhibits both IL-6- triggered Hck activation and the association of Gab adapter protein Gab1 with SH2- domain containing protein- tyrosine phosphatase - 2 (SHP2). Consequently, AP23464 both inhibited MM cell proliferation, as evidenced by 3H[dT] uptake, and induced apoptosis, evidenced by marked increase in sub-G1 phase cells. In contrast, proliferation of normal donor peripheral blood mononuclear cells remained unchanged. Next we investigated the role of Gab-1- SHP2 association mediating IL-6- induced resistance to dexamethasone, and conversely, the effect of inhibition by AP23464. Specifically, we determined whether IL-6- triggered Gab1- SHP2 binding is required for both RAFTK binding to SHP2 and associated IL-6- mediated dexamethasone resistance in MM cells. Utilizing wild- type Gab1 and SHP2- Gab1 binding mutants, our results show marked reduction in IL-6- triggered SHP2- RAFTK association. Moreover, IL-6- triggered resistance to dexamethasone was markedly inhibited in MM cells transfected with SHP2- Gab1 mutants, but not with wild type- Gab1. Importantly, AP23464 downregulated IL-6- mediated resistance to dexamethasone in a dose- dependent manner. Taken together, the present study shows anti-MM effects of AP23464, including abrogation of IL-6- induced dexamethasone resistance via inhibition of SHP2/ RAFTK- binding to Gab1. These studies provide the preclinical basis for the therapeutic evaluation of AP23464, alone or in combination with dexamethsoneto improve patient outcome in MM.
A Green Tea Polyphenol, Epigallocatechin-3-Gallate, Induces Selective Apoptosis in Multiple Myeloma Cells: Mechanism of Action and Therapeutic Potential.
Authors: Shammas Masood A. , Batchu Ramesh B. , Koley Hemanta , Bertheau Robert C. , Neri Paola , Goyal Raj , Anderson Kenneth C. , Munshi Nikhil C.
Published: April 2020

Epigallocatechin-3-gallate (EGCG), a polyphenol extracted from green tea, induces dose and time dependent cell death in both IL-6-dependent and independent multiple myeloma cell lines and primary patient cells, with minimal or no effect on the growth of normal cells. The cell death is apoptotic as determined by annexin V staining and is not inhibited by IL-6. Evaluation of molecular mechanism of action by gene expression profiling indicated that EGCG had a profound effect on transcription of major regulatory genes involved in distinct pathways controlling cell growth arrest and apoptosis: Exposure of myeloma cells to EGCG induced the expression of: 1) Fas ligand, Fas, and caspase 4, the initiators and mediators of death receptor dependent apoptosis; 2) death-associated protein kinase 2, a multifunctional pro-apoptotic protein kinase; 3) P53-like proteins, p73, p63; 4) CARD10 and CARD14, positive regulators of apoptosis and NF-kappaB activation; and 5) Cyclin-dependent kinase inhibitors, p16 and p18. In a subset of these selected genes, the expression data is also confirmed with western blot analyses. We have also demonstrated that the transcript and protein levels of a metastasis associated laminin receptor 1 are significantly elevated in myeloma cell lines and patient samples compared to normal cells. RNAi mediated inhibition of laminin receptor 1, abrogated EGCG-induced apoptosis in myeloma cells thus indicating that the profound anti-cancer effect of this compound is probably mediated through this receptor. The selective expression of this receptor explains the selective activity of EGCG in multiple myeloma cells without adversely affecting normal cells.

Taken together these data confirm significant and selective anti-cancer activity of EGCG, a natural product, in MM and provides the basis for its clinical evaluation.
A Humanized Anti-CD70 Monoclonal Antibody Targets CD70-Expressing Multiple Myeloma.
Authors: McEarchern Julie A. , McDonagh Charlotte , Ho Allen , Tai Yu-Tzu , Francisco Leigh , Klussman Kerry , Morris-Tilden Carol , Gordon Kristine , Oflazoglu Ezogelin , Kissler Kim , Munshi Nikhil , Anderson Kenneth C. , Treon Steven P. , Carter Paul , Wahl Alan F. , Grewal Iqbal S. , Law Che-Leung
Published: April 2020

The tumor necrosis factor family member CD70 (CD27L) is an integral membrane protein transiently expressed on antigen-activated T and B lymphocytes. CD70 regulates lymphocyte survival and differentiation of CD27 positive resting T cells and antigen-primed B cells. Aberrant CD70 expression has been detected on hematologic tumors including diffuse large cell, follicular, and mantle cell lymphomas, chronic lymphocytic leukemia and Reed-Sternberg cells in Hodgkin’s disease. Here we report for the first time the expression of CD70 on multiple myeloma (MM). Nine of thirteen MM cell lines examined expressed detectable surface CD70 with receptor copy numbers ranging from 9,000 to 145,000. Furthermore, seven of twelve primary MM isolates and six of six Waldenstrom’s macroglobulinemia (WM) isolates tested were positive for CD70. The presence of CD70 on MM and WM, combined with the antigen’s limited expression on normal non-hematopoietic tissues makes CD70 an attractive target for antibody-directed therapy of plasma cell disorders. We have engineered chimeric and humanized variants (human IgG1 isotype) of the murine anti-CD70 monoclonal antibody, 1F6. Both chimeric and humanized 1F6 variants retain the binding specificity of the murine parent monoclonal antibody (mAb) and acquired Fc-dependent effector functions including antibody-dependent cellular cytotoxicity (ADCC), complement dependent cytoxicity (CDC) and macrophage-mediated antibody-dependent cellular phagocytic (ADCP) activities against CD70+ tumor cells. Specific lysis (ADCC, CDC) or tumor cell uptake (ADCP) was observed when engineered anti-CD70 mAb were used at ≥10 ng/mL against a panel of CD70-expressing hematologic tumors. The significance of these in vitro observations was further established by the ability of anti-CD70 mAb to significantly improve the survival of mice bearing disseminated CD70-expressing tumors. The in vitro function and in vivo efficacy of anti-CD70 were dependent upon Fc-FcγR interactions, as activity and survival benefit was lost with anti-FcγR blockade or when the targeting antibody was engineered to eliminate FcγR binding. Our data suggest that humanized anti-CD70 mAb possesses potent antitumor activity and has potential utility for therapy of hematologic cancers including MM and WM.
Arginine (R) at Position 595 in FLT3 Is Critical for Transforming Potential of FLT3-Length Mutations.
Authors: Vempati Sridhar , Schwab Ruth , Malamoussi Theodora , Dugas Martin , Mellert Gudrun , Schnittger Susanne , Hiddemann Wolfgang , Spiekermann Karsten
Published: April 2020

Activating mutations in the juxtamembrane region of FMS-like tyrosine kinase-3 (FLT3-length mutations, FLT3-LM)-are found in 20–25% of patients with acute myeloid leukemia (AML). FLT3-LM consists of duplications and additional insertions that show a variable length and site of insertion. Although these different length mutations lead to constitutive activation of FLT3 and subsequent downstream signalling pathways, several questions still remain unanswered. Here, we focussed on the question whether a common sequence motif in the duplicated region can be identified in patients carrying FLT3-LM. To address this topic we sequenced the juxtamembrane region of FLT3 from 274 patients with acute leukemias. We found that the length of mutation (tandem duplications) varied from 2–42 amino acids with a median of 17 AA. Furthermore, the duplicated amino acids centered around the motif DFREYEY of FLT3. Since the length of the motif DFREYEY varied from patient to patient, we focussed our study on the frequency of single amino acids within the duplicated region. We found that arginine (R) 595 in the motif DFREYEY is duplicated in 77% of patients. Further studies showed that a single duplication of arginine at position 595 in FLT3 is able to confer factor independent growth to Ba/F3 cells. In vitro, deletion or substitution of duplicated R 595 in two FLT3-LMs with different lengths showed a 50% reduction in the factor independent growth when compared to undeleted/non-substituted FLT3-LMs. The reduced proliferative capacity of Ba/F3 cells expressing FLT3-LM with deletions of R-595 was associated with a reduction of STAT5 activation.

Our data provide important insights into the molecular mechanisms of transformation by FLT3-LM and define duplicated R 595 as a critical mediator of the leukemic potential of these mutants.