bluuebunny/crossref_metadata_2025 huggingface dataset:
Preclinical Studies Using Polyethylenimine (PEI) Nanoparticles Complexed with Eukaryotic Translation Initiation Factor 5A (eIF5A) siRNA and eIF5AK50R Plasmid DNA Demonstrates Significant Anti-Myeloma Activity in Vitro and in Vivo
Authors: Lust John A. , Thompson John E. , Taylor Catherine A. , Ye Bin , Liu Zhongda , Sun Zhong , Dondero Richard S. , Moon-Tasson Laurie L. , Kline Michael P. , Donovan Kathleen A.
Published: April 2020

Background: Hypusine is a unique amino acid present in all eukaryotes but found in only one highly conserved protein, eukaryotic translation initiation factor 5A. It is formed during a two-step post-translational modification of eIF5A. eIF5A has been identified as a critical factor for cell proliferation in yeast, as a modulator of p53, and recent studies indicate that the unhypusinated form of eIF5A is a pro-apoptotic protein. Because myeloma remains incurable, we hypothesized that induction of apoptosis by modulation of eIF5A may be a useful therapeutic approach. In this study we utilized PEI nanoparticles to deliver plasmid DNA and siRNA to myeloma tumor cells in vitro and in vivo.

Methods: An siRNA was used to suppress levels of endogenous hypusinated eIF5A in tumors while an RNAi-resistant plasmid expressing a mutant of eIF5A (eIF5AK50R), that cannot be hypusinated, was used to increase levels of unhypusinated eIF5A which is proapoptotic. In vitro, KAS-6/1 myeloma cells were incubated with control siRNA/control plasmid, eIF5A siRNA alone, or eIF5A siRNA/eIF5AK50R for 72 hours in the presence or absence of IL-6. Apoptosis was quantitated using flow cytometry to detect annexin V/propidium iodide stained cells; results were corrected for percent apoptosis of the mock control to enable comparison between experiments. In vivo, SCID mice (3/group) were injected with 10 million KAS-6/1 cells SQ in the right flank and approximately 6 weeks later injected intratumorally twice weekly as follows: Group 1 control mice received 20 μg empty vector and 10 μg control siRNA. Group 2 received 20 μg eIF5AK50R plasmid and 10 μg control siRNA. Group 3 received 20 μg eIF5AK50R plasmid and 10 μg eIF5A siRNA. Tumor volumes were quantitated in mm3 using a2b/2 where a is the smaller dimension.

Results: The in vitro results showed the following % apoptosis: Absence of IL-6 - Control: 6.5%, eIF5A siRNA/eIF5AK50R 30.3%. Presence of IL-6–Control:8.2%; eIF5A siRNA alone 36.5%; eIF5A siRNA/eIF5AK50R 45.2%. Western analysis demonstrated that the siRNA successfully inhibited the production of endogenous eIF5A. In vivo, after 24 days of monitoring tumor volume: group 1 mice showed that the KAS-6/1 cells grew from a mean tumor volume of 7±1 up to 102±16 mm3; tumors in group 2 mice remained relatively stable in size from 21±9 to 26±10 mm3. Group 3 mice showed a mean 92% tumor regression from 99±21 down to 7±1 mm3.

Conclusions: Delivery of eIF5A siRNA and eIF5AK50R plasmid by PEI nanoparticles results in significant anti-tumoral reponses in vitro and in vivo. These studies may provide a novel therapy for myeloma patients with end stage disease that have failed standard treatments.
Role of Hypoxia Induced Factor (HIF-1 alpha) in Pulmonary Hypertension in Sickle Cell Disease
Authors: Kumar Mukta , Zhu Xuhui , Buch Shilpa
Published: April 2020

Background. Pulmonary hypertension is a leading cause of mortality in sickle cell disease (SCD), however, the mechanisms leading to the development of pulmonary hypertension remain poorly understood. The pathogenesis of SCD revolves around chronic hypoxia and hypoxic events that are mediated through the upregulation of hypoxia-inducible factor-1alpha (HIF-1alpha), a transcriptional factor, that is involved in regulation of several redox sensitive genes including vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) and ApoE. The objective of this study was to examine the role of HIF-1 and its target genes in SCD-associated pulmonary lung disease.

Methods. In this study, 3 groups of age-matched homo and hemizygous sickle and control C57/Bl6 mice (Jackson Labs) were sacrificed and organs harvested for RNA isolation in Trizol. Using quantitative RT-PCR we assessed the RNA expression of HIF-1 and its target genes, VEGF and PDGF-B from the lungs of homozygous sickle mice and compared these with the C57/BL6 and the hemizygous sickle controls. HPRT was used as the housekeeping gene control.

Results. Homozygous sickle mice demonstrated an upregulation of HIF-1 alpha RNA (almost 2.3 fold) compared with the hemizygous sickle and the C57/Bl6 controls. VEGF RNA expression was elevated ~8.3 to 10.9 fold in sickle mice compared to the two control groups. Interestingly there was a down regulation of PDGF-B expression (0.3 fold) in the sickle mice group compared to the control groups. In contrast, expression of all the HIF-related genes tested was down-regulated in the spleens and brains of sickle mice compared with the respective control tissues.

Discussion. These findings support the possibility that complex molecular pathways, including dysregulation of HIF pathway, may contribute to the development of pulmonary hypertension in sickle cell disease and underscore role of oxidative stress pathways in the onset of disease pathogenesis.
Integration of DNA Copy Number and Gene Expression Alterations Reveal Novel Insights into the Molecular Pathogenesis and Prognosis of Multiple Myeloma
Authors: Zhou Yiming , Barlogie Bart , Herman Damir , Stephens Owen , Tian Erming , Williams David , Cartron Marie-astrid , van Rhee Frits , Pineda-Roman Mauricio , Alsayed Yazan , Anaissie Elias , Sawyer Jeffrey , Shaughnessy John D.
Published: April 2020

Multiple myeloma (MM) is characterized by a profound genomic instability and a highly variable clinical course. To comprehensively analyze the effects of copy number alterations on gene activity and prognosis, we applied novel bioinformatics and computational approaches to integrate high-resolution aCGH data (Agilent 244K array) and gene expression profiling data (Affymetrix U133Plus2.0 array) from CD138-selected plasma cells from 92 newly diagnosed MM treated with high-dose therapy and stem cell transplantation. We identified what we have termed the “atom region” (AR). ARs are found throughout the genome and define recurrent breakpoint boundaries of sub-chromosomal loss or gain. Genes coding for miRNAs were significantly enriched in regions of the genome defined by ARs. Although many AR-related breakpoints lie in intergenic regions, and thus capture whole genes, a substantial number were identified within genes, making these genes strong candidate disease genes. We found that gain of 1q and loss of 1p represent the most significant copy number alterations linked to early disease-related death. Importantly, 1q gains and 1p loss were significantly linked to a recently described 70-gene expression model of high-risk disease predominated by increased expression of genes mapping to 1q and reduced expression of genes mapping to 1p (1). Gain of 1q and loss of 1p were also highly correlated with a gene-expression-based proliferation index widely used across many tumor types. We provide evidence that gain of 8q24 was also highly correlated with the 70-gene high-risk score, but not the proliferation index. 8q24 gains and survival were linked to elevated expression of EIF2C2/AGO2, a master regulator of microRNA expression and maturation and an overexpressed gene previously identified in our 70-gene high-risk model (1). Interestingly, although the expression of MYC, which also maps to 8q24, was related to copy number changes, its expression was not related to survival. Given the importance of AGO2 in B-cell development (2), and miRNAs in cancer, detailed functional studies of the potential role of AGO2 in MM are underway.
The CD34+CD38LowCD19+ Candidate Leukemic Stem Cell Phenotype Revisited: Useful for Flow MRD Monitoring?
Authors: Wilson Kerrie , Case Marian , Minto Lynne , Bailey Simon , Vormoor Josef , Irving Julie
Published: April 2020

The assessment of minimal residual disease during therapy is a powerful prognostic marker in childhood acute lymphoblastic leukemia but current techniques, whether molecular analysis of antigen receptor gene rearrangements or flow cytometric analysis of aberrant immunophenotypes, track the blast population(s) that predominate(s) at diagnosis. Recent evidence from diagnostic TEL/AML1 positive cases (Hong et al, 2008) suggests that the candidate leukemic stem cell population may be characterised by the immunophenotype CD34+CD38LowCD19+ and thus the identification and quantitation of this cell population at diagnosis and during therapy may have greater clinical significance.

Cytogenetic subgroup Number with candidate population Number without candidate population TEL/AML1 8 1 High Hyperdiploid 6 5 Other 15 16

Therefore, we have sought this proposed leukemic stem cell population in 51 consecutive diagnostic precursor-B acute lymphoblastic leukaemia samples using multi-parametric flow cytometry. Patient blasts were stained with the antibodies CD34PerCP, CD38FITC and CD19APC and 50,000 events acquired on a FACSCalibur. The postulated cancer stem cell population, CD34+CD38LowCD19+, was found in 57% of patients (29 of 51). Correlation with major cytogenetic subgroups showed that 89% (8 of 9) of TEL/AML1 positive cases had evidence of the population, compared to 55% (6 of 11) of high hyperdiploid and 48% (15 of 31) of the remainder (see Table). There was no evidence of the population in ‘normal marrow’ samples which were from children in the latter stages of therapy for ALL i.e. end of treatment bone marrow samples (n=4). We also examined bone marrow samples taken 28 days after the start of treatment in a cohort of patients scoring positive for the candidate population at diagnosis (n=11) and found evidence for persistence of the cells in 4 of them.

CD38 under-expression, in relation to normal B cell precursors, is a common feature of ALL blasts and classification of CD38 expression levels revealed that 92% (24/26) of these under-expressers unsurprisingly housed the population because their blasts were also CD34+ and CD19+. Most importantly, since 43% of the diagnostic samples analysed show no evidence of this population (or it exists below the limits of detection of the assay i.e.<0.1% ), it seems unlikely that this represents a definitive cancer stem cell population in childhood ALL per se and would, therefore, not be appropriate as a generic marker of minimal residual leukemic stem cells.
Integrin Alpha M Chain Expression at Diagnosis Is Inversely Correlated with Cytoreduction Rate and Is Consistently Up-Regulated during Therapy in Acute Lymphoblastic Leukemia (ALL)
Authors: Rhein Peter , Ratei Richard , Mitlohner Rita , Basso Giuseppe , Gaipa Giuseppe , Dworzak Michael N , Stanulla Martin , Schrappe Martin , Ludwig Wolf-Dieter , Karawajew Leonid
Published: April 2020

In childhood ALL, detection and quantification of minimal residual disease (MRD) is of crucial prognostic significance. Gene expression pattern of MRD cells undergoes treatment-related changes which provide insights into the mechanisms of therapy response and resistance. Our recent genome-wide analysis of blasts isolated from peripheral blood (PB) of pediatric patients with B-cell precursor (BCP-) ALL at early stages of induction therapy (day 8) revealed an up-regulation of the gene encoding for integrin alpha M chain (ITGAM, CD11b), and pointed to the involvement of adhesion receptors in the therapy response. In the present study we investigate expression of CD11b at protein level and its clinical relevance in BCP-ALL. At diagnosis, ALL cells heterogeneously express CD11b both in PB (mean 1839+/−515 MESF, 160 pts) and in bone marrow (BM) (655+/−114 MESF, 79 pts). At day 8, increased expression of CD11b has been observed in the majority of PB samples (77 of 100 pts, up-regulation range 1.5- to 74-fold, mean 8.2-fold). In BM, the CD11b expression has been consistently up-regulated at the clinically significant MRD timepoints of induction therapy (days 15, 33 and 78; ALL-BFM 2000 protocol). Moreover, since normal CD19+/CD10+ precursor cells are CD11b negative, the detection of CD11b improved discrimination of normal and leukemic blasts in the MRD positive cases with regenerating BM at day 78. Of particular interest, evaluation of the CD11b expression at diagnosis in context of the early therapy response disclosed its potential clinical relevance. In the bivariate data analysis, expression of CD11b significantly correlated with the blast reduction rate (BRR) and blast count (BC) in PB at day 8 (p=0.007 and 0.001, 103 pts). Similarly, the CD11b expression in BM significantly correlated with the BRR and BC in BM at day 15 (p=0.024 and 0.012, 58 pts). In the multivariate regression analysis, excluding BCR-ABL and MLL-AF4 positive ALL cases and using the additional clinical parameters at diagnosis (age, white blood cell count in PB, DNA index), CD11b expression at diagnosis was the most significant variable contributing to BRR at day 8 (p<0.001, standardized beta coefficient 0.61; 98 pts) and to BRR at day 15 (p<0.001, standardized beta coefficient 0.68; 47 pts). Taken together, our data indicate that the expression of CD11b contributes to the early therapy response and has a promising potential as an MRDspecific marker in BCP-ALL.
The Role of Interleukin-15 on Natural Killer Cell Homeostasis and Activity in Patients with Acute Myeloid Leukemia
Authors: Szczepanski Mi roslaw J , Czystowska Malgorzata , Szajnik Marta E , Mandapathil Magis , Hilldorfer Benedict , Welsh Ann , Foon Kenneth , Whiteside Theresa L , Boyiadzis Michael
Published: April 2020

Interleukin-15 (IL-15) has been demonstrated to play a critical role in the regulation of natural killer (NK) cells. IL-15 induces the differentiation of NK cells from hematopoietic progenitors, stimulates the expansion of peripheral NK cells, and supports their survival. We investigated the role of IL-15 as a homeostatic regulator of NK cells in 29 patients diagnosed with acute myeloid leukemia (AML) and the potential role of IL-15 in enhancing the anti-tumor activity of NK cells in AML patients. The percentage of circulating NK cells was lower (p<0.0001) in the AML patients (6%± 0.7, range 1–17%) compared to the NK cells of healthy donors (12%± 1, range 9–17 %). At diagnosis the mean level of IL-15 in patient plasma was 1.9 pg/ml (range 0.03–8.9) and increased (p <0.02) to 5.2 pg/ml (range 0.06–13.4) after the completion of induction chemotherapy, when the NK levels had been reduced to zero cells/microliter. The mean level of IL-15 subsequently decreased to pre-treatment levels in the AML patients who achieved complete remission (mean 1.6 pg/ml, range 0.4–2.3). To assess effects of IL-15 on the NK cytotoxicity, we sorted NK cells from PBMC obtained from AML patients prior to treatment (at diagnosis) and cultured them in the presence of IL-15. Following IL-15 stimulation, a significant increase in NK-cell cytotoxicity against K562 targets and the patients’ autologous leukemic blasts was observed (p<0.05) as was up-regulation in expression of the activating natural cytotoxicity receptors, NKp30 and NKp46 and the C-type lectin receptors NKG2D and NKG2C (p<0.02–0.001). Addition of blocking antibodies to the activating receptors reduced NK-cell cytotoxicity. We determined that IL-15, a homeostatic NK-cell cytokine, increases after severe depletion of NK cells following intensive chemotherapy and this leads to increased NK-cell lytic activity in AML patients. These data suggest that modulation of IL-15 levels in AML could be therapeutically beneficial as IL-15 enhances NK-cell recovery following intensive chemotherapy and increases NK-cell anti-tumor activity.
Characteristics of Refractory and Relapsing Patients with Diffuse Large B-Cell Lymphoma
Authors: Coiffier Bertrand , Salles Gilles , Bosly Andre , Gaulard Philippe , Haioun Corinne , Casasnovas Olivier , Mounier Nicolas , Delfau Marie Helene , Sebban Catherine , Feugier Pierre , Recher Christian , Delarue Richard , Ketterer Nicolas , Tilly Herve , Gisselbrecht Christian
Published: April 2020

Even with the introduction of rituximab, some patients with lymphoma continue to relapse or progress during treatment. To better define these patients, we looked at all patients with aggressive lymphoma included in the GELA trials during the last 20 years or 4 generations of studies: LNH-87, -93, -98, and -03. Each study generation comprise several randomized studies according to different groups of patients, i.e., young or old, low or high risk. ACVBP, the high-dose regimen used by GELA since 1984 was usually one of the 2 arms, except in elderly patients. Rituximab was first introduced in the LNH-98.5 study and was part of nearly all arms in the LNH-03 study. A total of 7806 patients were included in this retrospective analysis, 3116 being treated with ACVBP and 4880 with other regimens. Two analyses were done: one for the 7198 patients treated without rituximab and one for the 608 patients treated with rituximab. Only patients included in a published study were included explaining the lower number for rituximab-treated patients, most of the LNH-03 studies being not yet published.

4 groups of patients were defined: refractory or non-responding pts (NR, progression during treatment); patients in PR at the end of treatment (persisting lymphoma cells or PET fixing tumor); relapsing pts with early relapses (ER, during the first year) and late relapses (LR, after one year). 7-year OS was 56% and 7-year PFS was 47.5%, meaning that only 8.5% of the patients were rescued by any treatment at time of progression. Identical results were found in all study generations with 2 groups of patients: those with PR or LR with 7-year OS at 38%, and those with NR or ER, 7-year OS at 12%. Therefore, all generations were grouped (see figure 1). Patients treated with rituximab had a gain of 9% in OS at 6 years (69% vs. 60%). However, they had the same pattern for progression: 6-year OS around 40% for PR and LR patients and around 20% for NR and ER patients. The difference between patients treated with or without rituximab being the percentage of patients in each group: 61% vs. 50% for those without progression and 16% vs. 29% for those with NR or ER. The IPI score does not allow the identification of these poor risk patients. A study is ongoing to characterize the NR+ER patients at diagnosis.

This analysis allowed recognizing 2 patterns of failing therapy. Patients with PR and LR have a disease sensible to chemotherapy while NR and ER pts have a disease not responding to treatment. These patients probably must be treated differently with the introduction of new therapeutic agents into the first line regimen.

Figure Figure
ADAMTS13 Gene Deletion Aggravates Ischemic Brain Damage
Authors: Nishio Kenji , Fujioka Masayuki , Hayakawa Kazuhide , Mishima Kenichi , Fujiwara Michihiro , Banno Fumiaki , Kokame Koichi , Miyata Toshiyuki , Shida Yasuaki , Sugimoto Mitsuhiko , Fukushima Hidetada , Okuchi Kazuo
Published: April 2020

Background: The proteolytic activity of human ADAMTS13 regulates the size of von Willebrand factor (VWF) multimers, controlling excessive platelet aggregation and preventing microvascular thrombus formation. Deficiency of ADAMTS13 causes thrombotic thrombocytopenic purpura (TTP) and patients with TTP often have neurological deficits such as stupor or coma. Therefore, ADAMTS13 appears necessary for vascular homeostasis in the brain and may also influence the response to brain injury during ischemic stroke.

Method and Result: We investigated the role of ADAMTS13 in a mouse middle cerebral arterial occlusion (MCAO) model of ischemia-reperfusion injury in the brain. We compared 24 wild type mice (WT) and 24 ADAMTS13 gene deleted mice (KO), which are healthy and fertile. All mice were males 6–8 weeks of age. Investigators were blinded to the genotype until all analyses were finished. We applied MCAO for 30 minutes followed by 23.5 hours of reperfusion. The cerebral blood flow (CBF) around the cortex of the ischemic region was measured by laser Doppler flowmetry for 60 minutes after MCAO. In both WT and KO mice, the CBF decreased to less than 20% of baseline during MCAO and returned to normal immediately after reperfusion. However, during the subsequent 30 min the CBF decreased to 34.6±5.8% of baseline for KO mice compared to 83.2±6.8% of baseline for WT mice (P < 0.01) and remained significantly decreased at 24 hours, suggesting that ADAMTS13 deficiency promotes thrombosis after reperfusion injury. The infarction volumes of the brain were determined from the area not stained by 2,3,5,-triphenyltetrazolium chloride (TTC) 24 hours after MCAO. KO mice had significantly increased volume of brain infarction compared with WT (31.0±6.5mm3 vs 11.4±1.9 mm3, P < 0.01). The degree of post-ischemic inflammation was estimated by Western blotting of plasma HMGB1 (high-mobility group box1) 24 hours after MCAO. Plasma HMGB1 was increased to 34.4 ± 5.3 ng/ml in KO mice, compared to 19.6 ± 3.5 ng/ml in WT mice (P < 0.05). The KO and WT mice did not differ during the MCAO procedure in body temperature, survival (80% vs 85%) at 24 hours, prothrombin time, arterial pH, pO2 or pCO2.

Conclusion: ADAMTS13 deficiency aggravates the extent of persistent brain ischemia, infarct volume and inflammatory response after brief MCAO. Therefore, ADAMTS13 plays a neuroprotective role against ischemia-reperfusion injury.
Long Term Follow-up Analysis of HD9601 Trial Comparing ABVD Vs Stanford V Vs MOPPABVCAD in Patients with Newly Diagnosed Advanced Stage Hodgkin Lymphoma. A Study from the Intergruppo Italiano Linfomi (IIL)
Authors: Chisesi Teodoro , Bellei Monica , Luminari Stefano , Marcheselli Luigi , Levis Alessandro , Vitolo Umberto , Stelitano Caterina , Pavone Vincenzo , Merli Francesco , Liberati Marina , Baldini Luca , Bordonaro Roberto , Pesce Emanuela , Gobbi Paolo , Federico Massimo
Published: April 2020

PURPOSE: To provide long term follow-up results of HD9601 trial that compared ABVD vs MOPP-EBV-CAD (MEC) vs Stanford V (StV) regimens for the initial treatment of patients with advanced stage Hodgkin Lymphoma (HL)

PATIENTS AND Methods: Patients with stage IIB–III or IV were eligible for randomization among 6 cycles of ABVD, 6 cycles of MEC and 12 weeks of StV; treatment had to be consolidated with optional Involved Field radiotherapy on Bulky or slow responding sites. For the long term, follow-up analysis study database was updated with most recent follow-up data and with information on long-term toxicity.

Results: Between 1996 and 2000, 355 patients were registered into the trial and were randomly assigned to one of the three arms (ABVD 122 pts, MEC 106 pts, and StV 107 pts). Radiotherapy was administered to 62%, 47%, and 66% of cases in the 3 arms respectively. As previously described response rates at the end of treatment program were 89%, 94%, and 76% in the 3 arms, respectively. Initial results were published in 2005 with a median f-up of 61 months. Median follow-up of current analysis is 86 months. Compared with previous data we observed 2 additional relapses, both in StV arm. Moreover, we observed 7 additional deaths in patients who had achieved a CR after initial treatment. Overall, 20 patients died after achievement of CR with 7 additional events compared with our previous report (Gobbi et al. JCO 2007). Overall analyzing data by study arm we observed 4 (+3), 11 (+3), and 5 (+1) deaths in CR patients randomized toABVD, MEC, and StV, respectively. Additional deaths in CR from previous report were further analyzed and were caused by pulmonary embolism (3 cases: 2 ABVD, 1 MEC), sepsis (2 cases: 1 MEC, 1 StV), LMA (1 MEC), and second cancer NOS (1 ABVD). No additional significant longterm toxic event was recorded. Long-term analysis resulted in 8-yr OS of 88%, 84%, and 77% for ABVD, MEC, and StV, respectively without differences among study arms (P=0.337).

Conclusions: The long-term analysis of HD9601 trial confirmed the results of the initial analysis. Few additional events observed were mostly represented by deaths in CR patients but these events did not substantially modify survival rates of the three arms.
Stimulation of T Cells by Lenalidomide Involves Putative Lenalidomide Binding Proteins CD3-Epsilon-Associated Protein and GDP-Mannose Pyrophosphorylase a
Authors: Gandhi Ani ta K , Rogovitz Audrey , Lopez-Girona Antonia , Mendy Derek , Morrison Lisa , Xie Weilin , Schafer Peter H
Published: April 2020

Introduction: Lenalidomide is approved in the US for the treatment of transfusion-dependent patients with anemia due to Low- or Intermediate-1-risk myelodysplastic syndromes associated with a del (5q) cytogenetic abnormality, with or without additional cytogenetic abnormalities. Lenalidomide is also approved for use in the US and Europe in combination with dexamethasone in previously treated multiple myeloma patients. In vitro and ex vivo studies have shown that lenalidomide has a direct antiproliferative effect against tumor cells and induces antiangiogenesis. It has also been shown to have immunomodulatory activity, including co-stimulatory effects on T and NK cells. Using the yeast 3-hybrid system, based on a leukemia cDNA library and biotinylated lenalidomide analog, we identified 16 putative lenalidomide-binding proteins from 700,000 clones screened. These included CD3-epsilon-associated protein (CAST) and GDP-mannose pyrophosphorylase A (GMPPA). CAST binds to the T cell receptor and is also part of the RNA polymerase 1 complex. GMPPA is a nucleotidyl transferase that converts mannose-1-phosphate and GTP to GDP-mannose, which is involved in the production of N-linked oligosaccharides. To determine whether these proteins are required for lenalidomide-induced immunomodulatory activity, we evaluated the lenalidomide-induced upregulation of interleukin-2 (IL-2) production in primary T cells transfected with siRNA against CAST and GMPPA.

Methods: Primary human peripheral blood T cells were transfected with CAST or GMPPA siRNA, or mock siRNA as a control. Reduction of CAST and GMPPA expression was confirmed by qRT-PCR. After 24 hours, transfected cells were stimulated with anti-CD3 mAb in the presence or absence of 0.1 and 1 μM lenalidomide 48 hours. Cells were lysed after 48 hours, and IL-2 mRNA and mature protein levels were quantified using qPCR and ELISA, respectively.

Results: CAST gene expression in T cells was reduced by 60% (p<0.05) in CAST siRNA-transfected cells. GMPPA gene expression was reduced by 66% (p<0.05) in GMPPA siRNA-transfected T cells. Lenalidomide-induced IL-2 mRNA production was reduced from 9-fold in controls to 6-fold in CAST siRNA transfectants (p<0.05) and from 15-fold in controls to 6-fold in GMPPA siRNA transfectants (p<0.05). Lenalidomide-induced IL-2 protein production was reduced from 22-fold in controls down to 8-fold in CAST siRNA transfectants (p<0.05) and from 5.6-fold in the controls down to 1.6-fold in GMPPA siRNA transfectants (p<0.05).

Conclusion: These findings support the hypothesis that CAST and GMPPA play an important role in the lenalidomide IL-2 induction mechanism in primary T cells.
A Stapled p53 Helix Targets HDMX to Overcome Nutlin-3 Resistance and Reactivate the p53 Tumor Suppressor Pathway in Cancer
Authors: Bernal Federico , Wade Mark , Silverstein Amy M. , Verdine Gregory L. , Wahl Geoffrey M. , Walensky Loren D.
Published: April 2020

p53 is a transcription factor that induces cell cycle arrest or apoptosis in response to DNA damage and cellular stress, and thereby plays a critical role in protecting cells from malignant transformation. The E3 ubiquitin ligase HDM2 controls p53 levels through a direct binding interaction that neutralizes the transactivation activity of p53 and targets it for degradation via the ubiquitylation-proteasomal pathway. Whereas the HDM2-homologue HDMX lacks ubiquitin ligase function, it participates in regulating the p53 axis by heterodimerizing with HDM2 and sequestering p53 through protein interaction. Loss of p53 activity, either by deletion, mutation, or HDM2/HDMX overexpression, is the most common defect in human cancer. Tumors expressing wild type p53 are rendered vulnerable by pharmacologic approaches that stabilize and upregulate p53. In this context, HDM2 and HDMX have emerged as independent therapeutic targets for restoring p53 activity and resensitizing cancer cells to apoptosis in vitro and in vivo. The small molecule nutlin-3 is an effective antagonist of the p53-HDM2 interaction. However, several studies have demonstrated the inability of nutlin-3 to disrupt the p53-HDMX complex, rendering tumor cells that overexpress HDMX nutlin-3-resistant. We have previously described the synthesis and characterization of a hydrocarbon-stapled alpha-helical p53 peptide (SAH-p53-8) that binds HDM2 with low nanomolar affinity, targets HDM2 in situ, and reactivates the p53 tumor suppressor pathway in HDM2-overexpressing osteosarcoma cells. We now report that SAH-p53-8 binds HDMX with even higher affinity, co-immunoprecipitates with endogenous HDMX, and induces apoptosis and cell cycle arrest in nutlin-3-resistant cancer cells that overexpress HDMX. Thus, by inserting a chemical staple into a peptide fragment of the p53 transactivation domain, we have generated the first bifunctional inhibitor of HDM2 and HDMX, enabling the investigation and pharmacologic modulation of both targets in human cancer.
Development of a Novel Polyamide-Based Agent to Inhibit EVI1 Function
Authors: Sicot Géraldine , Vogel Marion , Zhang Yi , Harki Daniel A , Lezon-Geyda Kimberly , Dervan Peter , Perkins Archibald S.
Published: April 2020

The EVI1 gene at chromosome 3q26 is associated with acute myeloid leukemogenesis, due to both chromosomal rearrangement and to overexpression in the absence of rearrangement. Some rearrangements such as t(3;3) and inv(3) result in overexpression of EVI1 protein, while translocation t(3;21) yields an AML1-MDS1-EVI1 (AME) fusion protein. EVI1 possesses two zinc finger domains, an N-terminal domain with fingers 1–7, which binds to GACAAGATA, and a C-terminal domain (fingers 8–10) which binds GAAGATGAG. Inhibition of EVI1 function with a small molecule compound may provide a targeted therapy for EVI1-expressing leukemias. As a first step towards inhibiting the leukemogenic function of EVI1, we performed structure-function studies on both EVI1 and AME protein to determine what domains are critical for malignant transformation activity. Assays were

Rat1 fibroblasts in a soft agar colony forming assay for EVI1; primary bone marrow cells in a serial replating assay for AME.

Both assays revealed that mutation of arginine 205 in zinc finger 6 of EVI1, which completely abrogates sequencespecific DNA binding via the N-terminal zinc finger domain, resulted in complete loss of transforming activity; mutations in other domains, such as the C-terminal zinc finger domain, CtBP binding domain, and the domains of AML1 had less of an effect or no effect on transforming activity. In an effort to inhibit EVI1 leukemogenic function, we developed a polyamide, DH-IV-298, designed to block zinc fingers 1–7 binding to the GACAAGATA motif. DNAseI footprinting revealed a specific interaction between DH-IV-298 and the GACAAGATA motif; no significant interaction was observed elsewhere; a mismatch polyamide failed to footprint at equivalent concentrations; and DH-IV-298 failed to bind to a control DNA lacking the GACAAGATA motif. Electromobility shift assay showed that, at a 1:1 polyamide:DNA ratio, DH-IV-298 lowered EVI1:DNA affinity by over 98%, while mismatch was significantly less effective (74% reduction). To assess the effect of DH-IV-298 on EVI1 binding to DNA in vivo, we performed CAT reporter assays in a NIH-3T3-derived cell line with a chromosome-embedded tet-inducible EVI1-VP16 as well as a EVI1-responsive CAT reporter. Removal of tetracycline resulted in a four-fold increase in CAT activity that was completely blocked by DH-IV-298. The mismatch polyamide was significantly less effective than DH-IV-298. Further studies are being performed to assess the effect on endogenous gene expression, and on growth of leukemic cells that express EVI1. These studies provide evidence that a cell permeable small molecule compound may effectively block the activity of a leukemogenic transcription factor.
Development of mPF4/Heparin Antibodies in a Murine Platelet Activation Model
Authors: Suvarna Shayela , Qi Rui , Arepally Gowthami M
Published: April 2020

Heparin-Induced Thrombocytopenia (HIT) is an immune-mediated thrombotic disorder caused by antibodies that recognize complexes of Platelet Factor 4 (PF4) and heparin. We have previously shown that exogenous administration of murine PF4 (mPF4) and heparin (mP+H) daily for five days into mice leads to development of high titer PF4/heparin autoantibodies (anti-mP+H; Suvarna Blood, 2007). While this experimental model has been extremely useful for understanding various biological aspects of heparin sensitization, this murine model requires extremely high-doses of mP+H to elicit antibody production. To circumvent this non-physiologic approach, we studied effects of in vivo platelet activation in mice with or without concomitant heparin administration. After receiving approval from our Institutional Animal Care and Use Committee, we initially performed dose-finding studies of various platelet agonists {arachidonic acid (AA), ADP, collagen and PAR-4} to identify the maximal tolerated dose associated with in vivo platelet activation (assays of circulating PF4 levels) and associated with increased morbidity. Once optimal doses of platelet agonists were identified, we injected agonists in the presence or absence of heparin daily for five days and monitored for development of PF4/heparin antibodies as previously described (Suvarna, Blood, 2005). Of the various agonists tested, we noted development of anti-mP+H in mice injected with collagen (coll), but not in mice injected with AA, ADP and PAR-4. Moreover, anti-mP+H responses in mice injected with collagen showed heparin dependence, as shown in the Table below.

Mean A450nm Collagen (coll) alone (n=5) 0.04 ± 0.01 coll + 0.25 U/mL hep (n=5) 0.1 ± 0.04 coll + 0.5 U/mL hep (n=5) 0.1 ± 0.02 coll + 1 U/mL hep (n=5) 0.32 ± 0.27* (P<0.05 v collagen alone)

Additional cohorts injected with coll + 1U/mL heparin showed significantly higher rates of seroconversion (mean A450nm= 0.25 ± 0.06, n=15) as compared to mice injected with coll alone (mean A450nm= 0.07 ± 0.02, n=15; p=0.007 for coll+hep v. coll alone, See Figure). In mice treated with coll+ 1U/mL Hep, anti-mP+H were specific for mP+H (mean A450nm= 0.358 ± 0.133) as compared to mPF4 alone (mean A450nm= 0.06±0.007). Anti-mP+H did not recognize rat albumin (mean A450nm= 0.015 ±0.006) or ELISA wells coated with PBS (mean A450nm=0.019 ±0.003). In summary, these studies indicate that anti-mP+H can be elicited in mice after platelet activation and heparin exposure. Further refinements of this model will be important in advancing our understanding the physiologic basis of heparin sensitization.

Figure Figure
High-Dose Therapy with Peripheral Blood Stem Cell Transplantation for Patients with Multiple Myeloma: Prognostic Impact of Chromosomal Aberrations and Correlation with the ISS-Score
Authors: Neben Kai , Jauch Anna , Hose Dirk , Cremer Friedrich , Heiss Christiane , Hielscher Thomas , Bertsch Uta , Klein Ulrike , Hillengass Jens , Ho Anthony D. , Goldschmidt Hartmut
Published: April 2020

Multiple Myeloma (MM) is a malignant lymphoproliferative B-cell disease characterized by the accumulation of monoclonal plasma cells in the bone marrow. Acquired genomic aberrations have been shown to significantly impact response to chemotherapy and survival in MM. The aim of our study was to assess the clinical relevance of genomic abnormalities in 306 MM patients treated with high-dose chemotherapy (HDCT) and peripheral stem cell transplantation (PBSCT) in our center.

We analyzed 171 males and 135 females with a median age of 60 years (range 25 – 73 years). According to the international staging system (ISS), MM patients were classified as stage I (46.6%), stage II (36.1%) and stage III (17.4%) at the onset of chemotherapy. All patients underwent frontline HDCT with 200 melphalan mg/m2 and PBSCT according or in analogy to the GMMG-HD3- or GMMG-HD4-trials. Interphase-FISH-analysis was performed on CD138-purified plasma cells using probes for chromosomes 1q21, 6q21, 8p21, 9q34, 11q23, 13q14.3, 15q22, 17p13, 19q13, and 22q11, as well as IgH-translocations t(4;14)(p16.3;q32.3) and t(11;14)(q13;q32.3). For the entire group, the median overall survival (OS) and progression-free survival (PFS) after HDCT was 6.4 and 2.2 years, respectively.

Table 1. Chromosomal abnormalities with significant results (a-level=0.05) on PFS or OS (univariate analysis, unadjusted p-values)

Aberration yes vs. no Frequency % 3-year PFS % P value 3-year OS % P value del(8p21) 19 26 vs. 37 0.01 58 vs. 78 0.02 del(13q14.3) 46 23 vs. 53 <0.001 65 vs. 83 0.03 del(17p13) 10 22 vs. 40 0.02 44 vs. 82 <0.001 t(4;14) 14 10 vs. 42 <0.001 54 vs. 81 0.04 +1q21 35 20 vs. 46 <0.001 67 vs. 85 0.002 +19q13 54 49 vs. 22 0.03 76 vs. 73 0.92

In a first step, we analyzed the prognostic impact of each individual chromosomal aberration on PFS and OS (Table 1). After adjustment for the ISS-score, del(8p21), del(13q14.3), del(17p13), t(4;14) as well as gains of 1q21 and 19q13 preserved significant impact on PFS, while del(17p13), t(4;14) and gain of 1q21 were of statistical significance for OS, indicating that these chromosomal aberrations give prognostic information in addition to the ISS-score. Subsequently, we performed a multivariate analysis including all the chromosomal aberrations analyzed. While del(17p13) and gain of 1q21 showed significant results on OS, del(13q14.3), del(22q11) and gain of 15q22 were significant for PFS.

In conclusion, our results show that the heterogeneity seen in the clinical course of MM patients after HDCT can be correlated with distinct chromosomal aberrations. This analysis may have implications for the risk-adopted management of patients with MM.
Analytical Approaches for the BOAC SNP Panel Association with Progression Free Survival in Myeloma
Authors: Van Ness Brian G. , Ramos Christine , Kumar Vipin , Steinbach Michael , Durie Brian GM , Crowley John , Barlogie Bart , Hoering Antje , Haessler Jeff , Johnson David C , Morgan Gareth J
Published: April 2020

The Bank On A Cure (BOAC) has established DNA banks from multiple cooperative and institutional clinical trials, and platforms for examining the association of genetic variations (SNPs) with disease risk and outcomes in myeloma. We have previously described the development and content of a novel custom SNP panel that contains 3,404 SNPs in 983 genes, representing cellular functions and pathways that may influence disease response, toxicities, complications, and survival. Although survival certainly varies according to tumor heterogeneity (ie. chromosomal abnormalities, gene expression variations) germline variations that influence the microenvironment, drug distribution, drug transport and metabolism, may also have an association with event free survival outcomes. To explore SNP associations with progression free survival (PFS) we compared the BOAC SNP profiles of short term PFS (less than 1 year, n=70) versus long term PFS (greater than 3 years, n=73) in two phase III clinical trials (ECOG E9487 and SWOG S9321). A variety of analytical approaches was undertaken including univariate rank ordering, recursive partitioning, and support vector machine learning tools (SVM). Each of these approaches has advantages and limitations in dealing with type I false positive errors as well as sensitivity and specificity. We included subset validation approaches and randomization of classes to address how robust and predictive different approaches were. From our analysis we conclude germline genomic variations do have an impact on progression free survival, with a subset of SNPs from the panel reaching 76% predictive association and hazard ratios of PFS of 9.6 (CI 4.5, 20.5), p<0.001, using SVM analysis. Based on univariate approaches, we find the most significant variations associated with PFS differences were genes that could be functionally categorized as pharmacologic. The presentation will focus on the analytical approaches, and refinements necessary to assure predictive value compared to random associations. Notwithstanding the clear importance of tumor cell variations in genetic deregulation, we conclude that various functions within the bone marrow and drug response likely interplay as a complex influence on disease progression, response, and survival. This suggests combining gene expression profiles of the tumors with germline SNP profiles may provide more accurate prognosis. These combined analytical approaches are currently being developed with BOAC data bases, and examples will be discussed.
Regulation of Multiple Myeloma Survival and Progression by CD1d
Authors: Spanoudakis Emmanouil , Hu Ming , Naresh Kikkeri , Terpos Evangelos , Melo Valeria , Reid Alistair G , Kotsianidis Ioannis , Rahemtulla Amin , Karadimitris Anastasios
Published: April 2020

Downregulation of conventional HLA molecules from the surface of tumour cells is an important mechanism for tumour immune evasion, survival and progression. Whether CD1d, a non-conventional, glycolipid-presenting HLA class I-like molecule can affect tumour cell survival is not known. To test this we studied expression of surface CD1d on plasma cells from different stages of multiple myeloma (MM) using flow-cytometry. Expressing results as the ratio of the Geo MFI CD1d/isotype IgG1 we found that while CD1d expression was comparable between MGUS (n=8) and newly diagnosed MM patients (n=14; Geo MFI MGUS: 8.61±4.3 vs new MM: 7.1±4.72, p>0.05), in relapsed/advanced disease CD1d was significantly lower (Geo MFI:1.92±0.9, p<0.003 vs MGUS and new MM) and completely lost in 4 out of 5 myeloma cell lines at protein and RNA level. Further, 4 out of 8 paired, same-patient trephine biopsies stained with anti- CD1d showed drastic loss of CD1d expression in advanced compared to early disease. These results confirmed loss of CD1d expression during disease progression and suggested that CD1d impacts negatively on myeloma cell survival. Consistent with this, we found that engagement of CD1d by 2 different anti-CD1d mAbs and as compared to isotypic IgG or media control, induces cell death (i.e., Annexin+) of the CD1d-expressing B lymphoblastoid cell line C1R-CD1d, of myeloma cell lines with retrovirally restored expression of CD1d and purified, CD1d-expressing primary myeloma cells in a dose- and time-dependent manner, coincident with loss of mitochondrial membrane potential (MMP) as assessed by DioC3 staining. Biochemical analysis of relevant cell death pathways showed that MMP loss is associated with overexpression of the pro-apoptotic protein Bax but as demonstrated by immunoblotting and pharmacological inhibition it is caspase- independent. By introducing appropriate CD1d retroviral constructs into CD1d- myeloma cell lines we showed that anti-CD1d-induced cell death requires the cytoplasmic tail but not a Tyr residue critical for lysosomal sorting of CD1d. Finally, we found that anti-CD1d co-operates with anti-myeloma agents in the killing of myeloma cells. Thus, these findings provide evidence linking a novel function of CD1d in the regulation of cell death with tumour survival and progression and might have pathogenetic and therapeutic implications for other CD1d-expressing hematopoietic malignancies as well as myeloma.
Homozygous Deletions Can Be Used to Define a Cell Death Specific Gene Expression Signature Able to Predict Outcome in Myeloma
Authors: Dickens Nicholas J , Walker Brian , Leone Paola E , Jenner Matthew W , Davies Faith , Morgan Gareth J
Published: April 2020

Defining biologically relevant prognostic indices in myeloma is a challenge. The International Staging System (ISS) is important but based on simple clinical data. Cytogenetics, in particular switch translocations and 17p-, are more important biologically, being based on underlying genetic changes. However, these changes do not capture all of the important biological data and so signatures based on global expression analysis have been developed to address these shortcomings. Most signatures to date have been based on expression features correlated with outcome rather than using structural genetic change to define biologically relevant expression features and, as such, although predictive, they may be acting as “bystander” markers rather than being pathologically important themselves. We have used homozygous deletions (HD) to define relevant genes and gene signatures predictive of outcome in a large homogeneously treated set of myeloma patients.
Depletion of Host CD122+ Cells Facilitates Widespread Skeletal Multiple Myeloma Engraftment in NOD/SCID Recipients
Authors: Freeman Lisa M , Petcu Eugene , Smith Robert , Salajegheh Ali , Diamond Peter , Zannettino Andrew Christopher William , Evdokiou Andreas , Luff John , Khalil Dalia , Vari Frank , Catley Lawrence , Hart Derek , Vuckovic Slavica
Published: April 2020

To facilitate human multiple myeloma (MM) engraftment into NOD/SCID recipients, mice were depleted of CD122+ cells (NK and myeloid cells) by antibody-mediated ablation prior to transplantation with the MM cell lines (RPMI8226, RPMI8226-TGL or U226). The MM engraftment, skeletal MM distribution, osteolysis, lambda chain paraprotein and associating disease symptoms in CD122+ cell-depleted and CD122+ cellreplete mice were compared. The CD122+ cell-depleted mice engrafted at a significantly higher frequency with human CD38+, CD56+, PCA-1+ and CD138+ cells. In the CD122+ cell-depleted mice, bioluminescence MM signal involved the whole mouse compared to limited imaging signal in the CD122+ cell-replete mice. The majority 88%–100% of CD122+ cell-depleted mice developed MM engraftment throughout the appendicular and axial skeletons with osteolysis and rare subcutaneous plasmacytomas (11% of mice). Serum paraprotein appeared earlier at 4–5 weeks post-transplant in CD122+ cell-depleted mice and continued to increase during the 12–13 weeks of analysis. The majority, 92% of CD122+ cell-depleted mice developed hind-limb paralysis and had a significantly shortened 45 day survival. Thus, depletion of CD122+ cells reduced resistance to the human MM and produced a new MM xenograft-NOD/SCID model that recapitulates the clinical manifestations of MM and eliminates the major limitations associated with the published MM xenograft-mouse model.