bluuebunny/crossref_metadata_2025 huggingface dataset:
ZAP-70 Expression in Chronic Lymphocytic Leukemia: Correlation with Clinical and Biological Features.
Authors: Del Giudice Ilaria , Osuji Nnenna , Matutes Estella , Morilla Alison , Morilla Ricardo , Burford Anna , Maravelaki Sonia , Owusu-Ankomah Kwasi , Swansbury John , Brito-Babapulle Vasantha , Catovsky Daniel
Published: October 2019

One of the most intriguing features of chronic lymphocytic leukemia (CLL) is its clinical heterogeneity. The mutational status of immunoglobulin heavy chain variable region (IgVH) genes is one of the most powerful predictors of overall survival (OS) and progression-free survival (PFS). The expression of the tyrosine kinase ZAP-70 in CLL cells has been proposed as a surrogate marker for the mutational status of IgVH genes. Recent reports suggest that ZAP-70 over-expression is an independent prognostic factor for shorter OS and PFS. There is little information about correlation between ZAP-70 expression and other clinical and biological features in CLL. The aims of this study are 1) to evaluate prospectively ZAP-70 expression in a series of CLL patients; 2) to correlate this with clinical stage, lymphocyte morphology, CD38 expression and chromosomal abnormalities detected by FISH and 3) to analyze the independent prognostic value of ZAP-70 in predicting time to first treatment (treatment free interval, TFI). ZAP-70 expression was analyzed using four-colour flow cytometry (as described by Crespo et al, NEJM2003; 348:1764–75). 201 previously untreated CLL patients were evaluated at diagnosis (32%) or during the disease course. Amongst them, 143 patients were entered in the LRF CLL4 trial. Clinical stage at the time of the study was A in 92 patients (A progressive in 46), B in 63 and C in 46. 28% were ZAP-70 positive (+) (cut-off ≥ 20% of CD5/CD19+ cells). ZAP-70 positivity was associated with prevalence of stage B/C at diagnosis (p=0.004), CLL with >10% prolymphocytes (CLL/PL) or atypical morphology (p<0.001), CD38 positivity (regardless of the cut-off point) (p<0.001), trisomy12 (p=0.001), del(6)(q21) (p=0.05) or no detectable abnormalities (0.002). ZAP-70 negative (−) cases presented more often with del(13)(q14) (p<0.001). There were no significant differences in the frequency of del(11)(q23) or delp53 between ZAP-70(+) and ZAP-70(−) cases. Median time from diagnosis to first treatment (treatment free interval, TFI) was 17.7 months for ZAP-70(+) and 44.6 months for ZAP-70(−) cases (p<0.001). The presence of the following parameters was associated with short TFI in univariate analysis: male sex (p=0.02), atypical morphology (p=0.02), clinical stage B/C (p<0.001), ZAP-70(+) (p<0.001), CD38+ (p<0.001), absence of del(13)(q14) (p<0.001), presence of delp53 (p=0.05). Multivariate analysis is being performed. In conclusion, we document a significant correlation between ZAP-70 over-expression in CLL and other adverse prognostic factors (such as CD38 and advanced stage), provide new data concerning its association with cytogenetic abnormalities detected by FISH and demonstrate the prognostic value of ZAP-70 expression.
Embryonic Stem Cell Transplant (ESCT)-Derived Mixed Hematopoietic Chimerism Prevents Diabetes in NOD Mice.
Authors: Burt Richard K. , Verda Larissa , Kim Duck A. , Statkute Laisvyde , Oyama Yu , Krosnjar Nela , Ikehara Susumu
Published: October 2019

NOD mice are a widely used animal model for studying type I diabetes mellitus characterized by lymphocytic infiltration of pancreatic islets following by development of diabetes by age 3 to 4 months. It has been shown that allogeneic bone marrow transplantation can prevent insulitis and overt diabetes. We have recently demonstrated that embryonic stem cell-derived hematopoietic stem cells (ESC-derived HSC) can reconstitute bone marrow in lethally irradiated mice across MHC barriers without graft versus host disease (GVHD). Herein we report a new ESC-derived HSCT approach for the prevention of autoimmune diabetes in NOD mice. 6-weeks old female mice were sublethally irradiated (8.0 Gy) and transplanted with ESC-derived HSC. To induce differentiation toward HSC in vitro, ESC (R1) were cultured in methylcellulose-based medium supplemented with SCF, IL-3 and IL-6. An enriched c-kit+CD45+ ESC-derived suspension was delivered intra bone marrow (IBM) (mean 0.5 x 106/mouse) (N=8) or intravenously (mean 1 x 106/mouse) (N=6). Six NOD mice were held as control. Mice were followed by blood glucose measurements and chimerism analysis until onset of diabetes or until 32 weeks after transplantation. 7 out of 8 mice from IBM group and 4 out of 6 from IV group did not become hyperglycemic in contrast to control group where 5 out of 6 mice were euthanized because of diabetes. The level of chimerism achieved after transplantation was 8.2%±5.76 in IBM group and 3.2%±2.75 in IV group. Histological examination showed that most of islets were replaced by lymphocytic infiltration or fibrous tissue in controls (even in case of a mouse without clinical evidence of diabetes). In 78% (11/14) of animals from ESC-derived hematopoiesis, remission was confirmed by histology revealing absence of insulinitis and normal immunohistochemical staining of islet cells for insulin. Prevention of diabetes/insulinitis was predicted by the percentage ESC-derived hematopoietic chimerism. All mice with > 5% ESC-derived chimerism remained free of diabetes and insulinitis. These results suggest that allogeneic chimerism achieved with embryonic stem cells can prevent diabetes in NOD mice without evidence of GVHD or use of post transplant immune suppression.
Thrombopoietin-Mediated Activation of the mTOR/S6K/4E-BP Signaling Pathway Is Required for Proliferation in Megakaryocyte Progenitors.
Authors: Drayer Lyndsay , Olthof Sandra , Vellenga Edo
Published: October 2019

Translational regulation plays a central role in cell proliferation, survival and cell differentiation through activation of the target of rapamycin (mTOR) signaling pathway. mTOR controls the phosphorylation status of proteins involved in initiating translational control, including ribosomal S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4E-BP). Recently, the mTOR and phosphoinositide 3-kinase (PI3-K) pathways have been linked through the tumor suppressor complex TSC1/2. The PI3-K target PKB inactivates the TSC1/2 complex which acts as a GAP for the Ras homologue Rheb to suppress mTOR signaling. As the regulation of cell number and cell size are important factors during megakaryopoiesis, we investigated the role of mTOR signaling in thrombopoietin (TPO) induced proliferation and differentiation in primary human megakaryocyte progenitors and in megakaryoblastic MO7e cells. Preincubation of isolated CD34+ cells, primary cultured CD61+ cells and MO7e cells with the mTOR inhibitor rapamycin (10 nM) resulted in highly specific inhibition of TPO-mediated 4E-BP1 (S65) phosphorylation, S6K (T389) phosphorylation and its substrate S6 (S235/236), without affecting PKB (S473) phosphorylation. Activation of the mTOR signaling pathway by TPO was dependent on the PI3-K pathway as LY294002 (10 μM) inhibited phosphorylation of 4E-BP1, S6K and S6. Treatment of MO7e cells with rapamycin inhibited TPO-induced proliferation and cell cycling by reducing cells in S-phase and blocking cells in G1. Rapamycin did not induce apoptosis as measured by cells in sub-G0 phase and by Annexin V expression. Suspension cultures of CD34+ cells treated with rapamycin resulted in a 2.3-fold reduction in overall cell proliferation (p=0.01) and a reduction in the percentage of CD61+ megakaryocytic cells (55.2±7.93 vs 35.0±5.42, p=0.08) generated after 7 days. The mean fluorescence intensity of CD61 and CD42 expressing cells was however not decreased. In addition, polyploidisation levels of the CD61 expressing cells cultured in the presence of rapamycin were not decreased, indicating rapamycin inhibited megakaryocyte proliferation, not differentiation. Further analysis revealed that transforming growth factor β1 (TGF-β1), which inhibits proliferation of megakaryocyte progenitors, downregulated TPO-induced S6K/S6 phosphorylation. Concluding, we have shown that the mTOR pathway is activated by TPO and plays a role in regulating proliferation in megakaryocyte progenitors. Part of the effect of the PI3-K/PKB and TGF-β pathways in regulating proliferation may be mediated by the mTOR/S6K/4E-BP1 signaling pathway.
Same Day Dosing of Pegfilgrastim in Elderly Patients Receiving Fractionated Dose Chemotherapy.
Authors: Watt Larry Y. , Redei Istvan , Levin Robert D. , Granick Joel , Neelam Rakhshanda , Musick Robert E.
Published: October 2019

Background: We have previously reported that same day dosing (SDD) of pegfilgrastim (PG) did not result in an increased incidence of negative outcomes as compared to filgrastim (G) administered the next day. (Watt et al. Pharmacotherapy. 2003;23:406. #105). Age (≥ 65 years) is known to be a risk factor for febrile neutropenia in patients undergoing myelosuppressive chemotherapy.

Purpose/Methods: This study aims to assess the efficacy and safety of SDD of PG among older cancer patients for severity of neutropenia (grade 4), infection/neutropenia- related hospitalization, incidence of delayed administration (or dose reduction) of chemotherapy or any unplanned MD visits due to leukopenia (table). G administered subsequent to chemotherapy served as the control group. Patients ≥ 65 years with solid tumors who received fractionated dose chemotherapy at our institution between January 2001 and March 2004 were reviewed retrospectively. Patients who received chemotherapy initially at substandard doses were excluded.

Results: Patient demographics indicated that the two groups were evenly matched, with similar mean baseline absolute neutropenic counts (ANC). Distribution of cancer diagnoses is as follows: PG: breast (7), lung (8), GI (18) and others (2); G: breast (5), lung (9), GI (7) and others (4). Twenty-six (87%) and 21 (84%) patients had stage IV cancer in the PG and G groups, respectively. The mean ANC nadirs for PG and G were 2790±2190 cells/mm³ and 1820±1930 cells/mm³, respectively (p=0.01). For the outcome endpoints, although certain trends seemed to favor SDD of PG, none of the odd ratios (OR) performed reached statistical significance. Doses of PG and G were generally well-tolerated by all patients.

Conclusion: Concurrent administration of PG showed safety and efficacy at least comparable to G in elderly patients receiving fractionated dose chemotherapy. Such administration schedule has the potential to further simplify the outpatient management of chemotherapy-induced neutropenia, providing the needed convenience for elderly patients as well as caregivers. However, further prospective study is warranted.

PG G p-value; OR (95% CI) data were analyzed based on cycles No. patients 30 25 No. cycles 59 54 Age (range) 68.8 (65–79) 68.3 (65–72) Gender (female) 18 (60%) 15 (60%) ANC before chemo±SD(cells/mm³) 4530±1850 4790±2600 p=0.539 ANC nadir±SD (cells/mm³) 2790±2190 1820±1930 p=0.01 Severe Neutropenia (grade IV) 9 (15%) 15 (28%) OR=0.47; (0.19, 1.18) Unplanned MD visit 7 (12%) 3 (5.6%) OR=2.28; (0.56, 9.34) Chemo delayed/dose reduced 9 (15%) 11 (20%) OR=0.70; (0.27, 1.86) Hospitalization 7 (12%) 5 (9.3%) OR=1.32; (0.39, 4.44)
Loss of Runx1 Perturbs Adult Hematopoiesis and Is Associated with a Myeloproliferative Phenotype.
Authors: Growney Joseph D. , Shigematsu Hirokazu , Li Zhe , Lee Benjamin H. , Adelsperger Jennifer , Rowan Rebecca , Curley David P. , Akashi Koichi , Williams Ifor R. , Speck Nancy A. , Gilliland D. Gary
Published: October 2019

Homozygous loss of function of Runx1 during murine development results in an embryonic lethal phenotype characterized by a complete lack of definitive hematopoiesis. In light of recent reports of disparate requirements for hematopoietic transcription factors during development as opposed to adult hematopoiesis, we employed a conditional gene targeting strategy to effect loss of Runx1 function in adult mice. In contrast with the critical role of Runx1 during development, Runx1 was not essential for hematopoiesis in the adult hematopoietic compartment, although there were a number of significant hematopoietic abnormalities observed. Runx1 excision had significant lineage specific effects on B- and T-cell maturation, as well as pronounced inhibition of common lymphocyte progenitor production. Runx1 excision also resulted in inefficient platelet production. Of note, Runx1 deficient mice developed a mild myeloproliferative phenotype characterized by an increase in peripheral blood neutrophils, an increase in myeloid progenitor populations, and extramedullary hematopoiesis comprised of maturing myeloid and erythroid elements. These findings indicate that Runx1 deficiency has markedly different consequences during development compared with adult hematopoiesis, and provides insights into the phenotypic manifestations of Runx1 deficiency in hematopoietic malignancies.
Prognostic Significance of CD20 in Children with B-Precursor Acute Lymphoblastic Leukemia.
Authors: Jeha Sima , Cheng Cheng , Sandlund John , Ribeiro Raul , Razzouk Bassem , Rubnitz Jeffrey , Hijiya Nobuko , Howard Scott , Behm Fred , Pui Ching-Hon
Published: October 2019

CD20 expression is associated with inferior survival in adults with ALL. Preliminary reports indicate that regimens incorporating rituximab improve outcome in this subgroup. We analyzed the prognostic impact of CD20 in 353 children with B-cell precursor ALL treated between 12/20/91 and 5/17/99 on three consecutive St. Jude Total Therapy studies. CD20 expression (>20%) was found in 169 patients (48%). Expression was higher in patients between 1 and 10 years (51% of 252 patients), as compared to those less than 1 year of age (7% of 15 patients) or more than 10 year old (43% of 86 patients), p=0.0008. African American patients were more likely to express CD20 (66%), as compared to Caucasians (45%) or other ethnic groups (45%), p=0.047. None of 14 patients with MLL-AF4 expressed CD20. There was no association between CD20 expression and E2A-PBX, TEL-AML1, ploidy, WBC at diagnosis, or gender. In contrast to the experience in adult ALL, our patients with CD20 expression tended to have a better treatment outcome than those without the expression: 5-year EFS 84% ± 2.9% versus 78% ± 3.1% (p=0.08); 5-year overall survival 88% ± 2.5% versus 83% ± 2.8% (p=0.13). This data suggest that CD20 expression is not associated with inferior outcome in pediatric patients treated with contemporary regimens.
Generation and Analysis of Target Genes Libraries of the Erythropoietic Transcription Factor GATA-1.
Authors: Katsantoni Eleni Z. , Horsman Sebastiaan , Moorhouse Michael J. , de Jager Victor C.L. , van der Spek Peter , Grosveld Frank , Strouboulis John
Published: October 2019

GATA-1 is essential for the generation of the erythroid, megakaryocytic, eosinophilic and mast cell lineages. It acts as an activator and repressor of different target genes. In erythroid cells it represses cell proliferation and early hematopoietic genes while activating erythroid genes. In order to elucidate further the role of GATA-1 we applied an in vivo tagging methodology for the specific, quantitative biotinylation of this factor in mammalian cells (de Boer et al., 2003). We applied this method for identification of novel target genes of GATA-1 by performing pull-downs of crosslinked chromatin using streptavidin. We have also performed chromatin immunoprecipitations, where crosslinked chromatin was immunoprecipitated with antibodies against GATA-1, thus enriching for sequences bound in vivo by this factor in the immunoprecipitated DNA. Libraries of in vivo bound DNA targets were generated and a number of clones were sequenced. In order to facilitate the bioinformatics analysis of these libraries we generated TF Target Mapper (Transcription Factors Target Mapper). This is a BLAST search tool that allows rapid extraction of annotated information on genes around each hit and combines sequence cleaning/filtering, pattern searching and comparisons of the output list of genes or gene ontology IDs with user implemented lists. This tool was successfully applied to analyze sequences bound in vivo by the transcription factor GATA-1 and efficiently extracted information on genes around ChIPed sequences, thus identifying known and potentially novel GATA-1 gene targets. Using TF Target Mapper, 95 sequences were processed and annotated information on 372 genes 50kb upstream and downstream of each hit was extracted in 27 minutes. Among these genes, known targets of GATA-1, such as α-globin and ζ-globin, were readily identified by comparing to a list of known GATA-1 targets. This work is anticipated to provide a genome wide map of GATA-1 target genes in vivo. The identification of target genes and elucidation of their functions in hematopoiesis will allow the construction of complex transcriptional pathways that control lineage commitment and differentiation decisions.
Ideal (IBW) Rather Than Actual (ABW) Body Weight Should Be Used To Calculate Cell Dose in Allogeneic HSCT.
Authors: Singhal Seema , Evens A. , Frankfurt O. , Gordon L. , Meagher R. , Tallman M. , Williams S. , Winter J. , Grinblatt D. , Kaminer L. , Mehta J.
Published: October 2019

Graft CD34+ cell content based on ABW affects outcome after conventional allografts (Singhal et al. BMT2000;26:489–96). Engraftment after autologous (Ali et al. BMT2003;31:861–4) and allogeneic (Cilley et al. BMT2004;33:161–4) HSCT correlates better with CD34+ cell dose based on IBW than ABW. 63 patients (27–66 y, median 52) underwent submyeloablative HSCT from HLA-matched sibling (n=37), 10/10 allele-matched unrelated (n=19), or 1-locus/allele mismatched (n=7) donors for hematologic malignancies after 100 mg/m2 melphalan with (n=42; no prior autograft) or without (n=21; prior autograft) 50 mg/kg cyclophosphamide. GVHD prophylaxis comprised cyclosporine-mycophenolate with HLA-matched sibs and tacrolimus-mycophenolate with the rest. G/GM-CSF were not used. Supportive care was uniform. ABW and IBW values were 45–147 kg (median 79) and 52–85 kg (median 67) respectively. The ABW-IBW difference was −24% to +133% (median +16%); 9 patients were >5% underweight and 41 were >5% overweight. Chemotherapy doses were based on ABW for underweight patients, and adjusted weight for overweight (IBW + 25% of the ABW-IBW difference). The target CD34+ cell dose was 2–8 x106/kg IBW; preferably >6 and ≤ 8 based on prior findings (Singhal et al. ASH 2004). The CD34+ cell dose infused was 1.4–11.8 (median 5) by IBW and 1.2–9.3 (median 4.5) by ABW. The CD3+ cell dose (108/kg) was 0.9–14.9 (median 3) by IBW and 0.7–19.7 (median 2.7) by ABW. The 2-y cumulative incidence of relapse and transplant-related mortality (TRM) was 55% and 24% respectively. The 2-y probabilities of overall (OS) and disease-free (DFS) survival were 35% and 21% respectively.

CD34+ cell dose by IBW CD34+ cell dose by ABW >6 and ≤ 8 (n=11) 6 or >8 (n=52) ≤ P >6 and ≤ 8 (n=8) 6 or >8 (n=55) ≤ P 3-y OS 61% 27% 0.040 31% 25% 0.22 3-y DFS 41% 13% 0.043 0% 14% 0.53 1-y TRM 0% 27% 0.029 0% 25% 0.090 1-y Relapse 45% 48% 0.34 63% 47% 0.70 CD3+ cell dose by IBW CD3+ cell dose by ABW <3 (n=31) 3 (n=32) ≥ P <3 (n=36) 3 (n=27) ≥ P 3-y OS 34% 18% 0.032 27% 24% 0.54 3-y DFS 24% 4% 0.043 18% 7% 0.58 1-y TRM 10% 35% 0.008 17% 30% 0.12 1-y Relapse 52% 44% 0.66 53% 41% 0.67

As the tables show, the CD34+ and CD3+ cell doses by IBW affected OS, DFS and TRM significantly. When ABW was used with the same numeric thresholds, the differences were less marked and not significant. The figures show the effect of the CD34+ cell dose on DFS and the CD3+ cell dose on OS.

Figure Figure Figure Figure

Other cell dose thresholds based on ABW were also not found to be significant (data not shown). Based on these findings and our prior work on engraftment, we suggest using IBW rather than ABW to calculate cell doses for HSCT; for statistical analyses and for clinical practice if a specific cell dose is being targeted.
Culture-Expanded Human Bone Marrow Stromal Stem Cells Organize Functional Bone Marrow Niches In Vivo.
Authors: Miura Yasuo , Gao Zhigang , Miura Masako , Seo Byoung-Moo , Sonoyama Wataru , Chen WanJun , Gronthos Stan , Zhang Li , Shi Songtao
Published: October 2019

Bone marrow stromal stem cells (BMSSCs) are mesenchymal stem cells that are capable of differentiating into osteoblasts, chondrocytes, adipocytes, muscle cells and neural cells. Upon in vivo transplantation, BMSSCs form bone and associated hematopoietic marrow elements. However, the functional role of BMSSC-associated bone marrow is still unknown. In this study, we demonstrated that human BMSSCs organized ectopic bone marrow niche microarchitecture that contained hematopoietic progenitors and multiple lineages of cells including myeloid, lymphoid, erythroid and megakaryocytic cells originated from recipients. Significantly, transplantation of the ectopically generated bone marrow cells can rescue lethally irradiated mice with successful hematopoietic engraftment, suggesting a therapeutic potentiality of human BMSSC-organized hematopoietic progenitor cells. In addition, systemically administrated bone marrow cells derived from long bones of donor mice homed to BMSSC-associated ectopic bone marrow niche microenvironments. These data demonstrate that BMSSC-organized ectopic bone marrow has functional analogy to physiological bone marrow. Mechanistically, platelet-derived growth factor (PDGF)-BB was found to promote hematopoiesis in the BMSSC transplants through an up-regulated expression of β-catenin. Conversely, inhibition of PDGF signaling in BMSSCs by PDGF receptor β siRNA blocked hematopoiesis and osteogenesis in the BMSSC transplants. Moreover, the BMSSC-organized ectopic bone/marrow structure could be utilized as an organ to be re-transplanted into secondary recipient mice to deliver hematopoietic cells in the recipient’s circulation system, implying a therapeutic potential of transplanting bone/marrow organ system containing hematopoietic stem cell niches. In summary, these evidences demonstrate that regeneration of BMSSC-organized functional bone marrow niches contain hematopoietic progenitor/stem cells and therefore provide promise for clinical therapy.
Enhancing the Therapeutic Potential of an Anti-Leukemic Peptide.
Authors: Palermo Christine M. , Wimley William C. , Hemenway Charles S.
Published: October 2019

Despite ongoing success in the treatment of childhood acute lymphoblastic leukemia (ALL), patients harboring translocations involving the genetic locus 11q23 continue to have a poor prognosis. The majority of 11q23 translocations result in the formation of a functional fusion protein consisting of the N-terminus of MLL fused to the C-terminal portion of one of more than 30 fusion partners. This translocation results in a functional chimeric protein critical for leukemogenesis. Recently, our lab has identified an interaction between two common MLL fusion partners, AF4 and AF9. Through a series of mapping experiments we identified a small region of human AF4 to be sufficient for its interaction with AF9. Based on these studies, a synthetic peptide (PFWT) that mimics the AF9 binding site on AF4 was developed. Treatment of leukemia cells that express the MLL-AF4 protein with PFWT results in apoptosis with no observable affects on CD34+ hematopoietic stem cells, suggesting the AF9-AF4 interaction is a promising chemotherapeutic target. To improve upon the therapeutic potential of PFWT, we developed a high-throughput enzyme-linked colorimetric assay to identify peptidomimetics that block the AF9-AF4 interaction. A combinatorial peptide library was synthesized in which each position of the 10-mer sequence was substituted with either the α- or corresponding β-amino acid. β-Amino acids which are similar to α-amino acids but contain an additional carbon in their backbone were chosen because of their resistance to proteases (210= 1024 possible unique sequences). To date, 30 of the peptides screened compare favorably with PFWT for disrupting the AF4-AF9 interaction. Sequencing of the peptides by MS/MS revealed substitutions at the N- and C-terminal ends are well tolerated. In addition, peptides can incorporate as many as three β-amino acids and still retain biological activity. These data are important for establishing a sequence with improved pharmacokinetic properties as compared to PFWT and serve as the first step in our design towards an optimal peptide sequence for drug development. They also validate the utility of a high-throughput assay system for drug screening. Future studies for the identified peptides include determining their biological half-lives, AF9 binding affinities, and ability to induce apoptosis in leukemia cells.
Dexmethasone Down-Regulates Stromal Cell-Derived Factor-1 Production in Bone Marrow Stromal Cells: Implications in Myeloma Cell Biology.
Authors: Kim Seong-Woo , Hwang Jin-Hee , Yun Hwan-Jung , Kim Samyong , Jo Deog-Yeon
Published: October 2019

Stromal cell-derived factor-1 (SDF-1) plays a role in the homing of myeloma cells to bone marrow. In addition, SDF-1 modestly enhances the proliferation of myeloma cells and inhibits Dexmethasone (Dex)-induced apoptosis of the cells. Dex is currently used to treat multiple myeloma, based on its apoptic effects. In this study, we investigated the regulatory effects of Dex on SDF-1 production in bone marrow stromal cells (BMSCs) and on CXCR4 expression in myeloma cells. As previously reported, it was evident that primary myeloma cells (CD138+ cells obtained from patients with multiple myeloma) and Dex-resistant myeloma cell line RPMI8226 expressed CXCR4 and responded to SDF-1, resulting in chemotaxis. SDF-1 modestly stimulated the proliferation of primary myeloma cells and RPMI8226 cells and protected the cells from Dex-induced apoptosis. Human umbilical vein endothelial cells transduced with the SDF-1 gene using adenoviral vectors better supported the formation of cobblestone areas of primary myeloma cells and RPMI8226 cells in co-culture, similar to hematopoietic progenitor cells; this was blocked by pretreating the myeloma cells with pertussis toxin, indicating that SDF-1 plays a critical role not only in migration of the cells underneath the SDF-1-producing stromal cells but also in proliferation of the cells in contact. Dex up-regulated CXCR4 expression in RPMI8226 cells; however, its regulatory effects on CXCR4 in primary myeloma cells differed among patients. RT-PCR and Northern blot analyses revealed that Dex down-regulated SDF-1 mRNA expression in both primary BMSCs and murine stromal MS-5 cells in a dose-dependent manner. Western blot analysis and ELISA assay confirmed that Dex inhibited SDF-1 production in BMSCs. Furthermore, Dex inhibited cobblestone area formation of RPMI8226 cells in co-culture with MS-5. Interestingly, Dex up-regulated CXCR4 mRNA expression and cytoplasmic CXCR4 in BMSCs. These results indicate that Dexamethasone induces the down-regulation of SDF-1 production in BMSCs, which might mediate, at least in part, its anti-myeloma effects in vivo.
Pegylated Liposomal Doxorubicin (PLD), Vincristine and Reduced-Dose Dexamethasone (DVd) in the Treatment of a Predominantly African American Population with Multiple Myeloma.
Authors: Jajeh Ahmad , Catchatourian Rosalind , Osafo David , Tamkus Deimante , Zalzaleh Ghassan , Bamrolia Anshul
Published: October 2019

Recently there has been an important recognition of potentially different responses to pharmaceuticals based on genetic predisposition, with the first FDA advisory panel recommending approval of a heart failure drug for African Americans alone. Liposomal doxorubicin (DOXIL; PLD) is a microscopic pegylated phospholipid vesicle with a core containing conventional doxorubicin. The pegylated coat protects the liposomes from detection by mononuclear phagocytes increasing blood circulation time (t½ = 55 hours). Due to its prolonged half-life, PLD provides a similar effect to using continuous infusion doxorubicin, but administered over 1-hour, transforming the regimen into an outpatient treatment. PLD has also been shown to have a significantly better cardiac safety profile than conventional doxorubicin. A phase II trial using DVd was started in October 2000 and is still enrolling (PLD 40 mg/m2, vincristine 2 mg IVP, and dexamethasone 40 mg PO 1–4 d every 4-weeks). Twenty-seven patients have been enrolled (11 males/16 females; mean age 56 years [range 41–75]). The majority of patients enrolled in this study are African American (74%), a patient population not commonly studied. Patients presented with relatively advanced disease (stages II – III). Baseline mean serum albumin level was 3.5 mg/dL (range 1.4 to 4.4), beta-2 microglobulin 3.38 (range 1.0 – 8.97), fourteen patients had IgG Kappa, three patients has IgG Lambda, six patients with IgA, and four patients with light chain disease. Eighteen patients completed six cycles of therapy, with two patients completing five cycles. Six patients underwent autologous bone marrow transplant following their response to DVd.

CR, and nCR was achieved in nine patients, partial responses were achieved in seven patients, minor response in two patients, and progressive disease in five patients, based on Blade Response Assessment. Median follow up is twenty-four months (range 3 months – 5 years). Overall medium time to progression is approximately 1 year. Twenty patients are still alive, one patient has been lost to follow up, and six deaths have occurred. Four early deaths were due to disease progression and sepsis. Two died after one year of therapy due to progressive refractory disease. One died after the second cycle because of sudden cardiac death with sepsis. Three of the early deaths had amyloidosis. No episodes of cardiac dysfunction were observed.

Conclusion: African Americans have a 3-fold higher risk of cardiac toxicity with conventional doxorubicin. The use of DVd in this predominantly African American patient population was cardiac safe and provided an easy administered outpatient option, with an overall response rate of ~66% in stage II–III patients.
Myc Targets Cks1 To Provoke Proliferation and Lymphomagenesis.
Authors: Keller Ulrich , Old Jennifer B. , Nilsson Jonas , Nilsson Lisa , Maclean Kirsteen , Yang Chunying , Spruck Charles , Boyd Kelli , Reed Steven I. , Cleveland John L.
Published: October 2019

Reduced levels of the cyclin dependent kinase inhibitor p27Kip1 connote poor prognosis in cancer. In human Burkitt lymphoma, and in pre-cancerous B cells and lymphomas arising in Eμ-Myc transgenic mice, p27Kip1 expression is markedly reduced. Furthermore, the Cks1 component of the SCFSkp2 complex that is necessary for p27Kip1 ubiquitylation and degradation, and to a lesser extent Skp2, are induced by Myc ex vivo and in Eμ-Myc B-cells and lymphomas, and up-regulation of CKS1 and SKP2 are hallmarks of Burkitt lymphoma. While loss of Skp2 has rather modest effects, the deletion of Cks1 in Eμ-Myc B-cells elevates p27Kip1 levels, reduces proliferation and delays lymphoma development. In contrast, Myc-induced apoptosis and transcriptional activity are not affected by Cks1 (or Skp2) loss. Therefore, Myc accelerates cell proliferation and promotes tumorigenesis through its ability to selectively induce Cks1.
Heparins Inhibit Tumor Angiogenesis by Sequestering Fibroblast Growth Factor (FGF) from Tumor Vascular Endothelial Cells.
Authors: Smiley Shannon L. , Henry Dale O. , Chen Shang-Chiung , Wong Michael K.K.
Published: October 2019

The association between cancer and thromboembolic disease is a well-known phenomenon and contributes to the morbidity and mortality of cancer patients. Clinical studies of thrombosis in these patients show that heparins may have beneficial effects on survival. Antithrombotic agents have been shown to exert an anti-tumor effect in various experimental models however the underlying mechanism remains unknown. We show that heparins inhibit in vivo tumor angiogenesis and offer molecular evidence that heparins exert an anti-angiogenic effect by directly sequestering fibroblast growth factor (FGF) from its receptor on tumor derived endothelial cells (TDECs). NIH-3T3 fibroblasts were stably transfected with an expression construct that results in the constitutive excretion of FGF-1 (Clone C). Clone C gives rise to aggressive and highly angiogenic xenograft tumors. Clone C was inoculated into nude mice and therapeutic doses of Low Molecular Weight (LMW) heparins were injected daily beginning on Day 2. Tumors in the control group were grossly angiogenic and highly vascularized. In contrast, the heparin treated tumors were pale and possessed only scant peri-tumoral vessels. In order to assess the biologic mechanism of this, murine TDECs were isolated and cultured as previously published. Unfractionated and LMW heparins inhibit FGF-induced TDEC mitogenesis in a concentration- and time-dependent manner. FGF overcame and rescued heparin-induced inhibition suggesting that an FGF-heparin interaction is responsible. In order to test the hypothesis that heparin strips and sequesters FGF off its receptor on TDECs, we used a FGF protein fused to a hemagglutinin peptide tag at the carboxyl-terminus end (FGF-HA). FGF-HA is biologically identical to wild type FGF, but its detection limit is 10X more sensitive. FGF-HA was allowed to bind to FGFR on TDECs. These cells were subsequently incubated with Heparin covalently linked to Sepharose beads (Heparin-Sepharose) or to Sepharose alone. These beads were removed, and TDEC growth analyzed prospectively. Heparin-Sepharose treatment results in significant TDEC growth inhibition as compared to incubation with Sepharose alone. Western blot analysis shows that FGF was sequestered only on the Heparin-Sepharose beads.

Conclusion: The anti-angiogenic mechanism of heparins resides, in part, in its ability to sequester angiogenic cytokines such as FGF from its receptor on tumor endothelium.
Secondary Malignancies after Hematopoietic Stem Cell Transplantation in Patients Treated with Total Body Irradiation.
Authors: de la Fuente Cristina , Berrocal Maria I. , Cabrera J. Rafael , Regueiro Carlos A. , Fores Rafael , Regidor Carmen , Millan Isabel , Nuñez Luis , Valcarcel Francisco , Fernandez Manuel N. , Sanjuan Isabel , de la Torre Alejandro
Published: October 2019

We have analyzed the incidence and risk factors of developing a secondary malignancy after total body irradiation (TBI) and hematopoietic stem cell transplantation (HSCT). From March 1986 to December 2002, 205 patients received TBI as a part of the HSCT conditioning regimen. TBI was administered in 6 fractions, twice a day, up to a total dose of 12 Gy, with a median dose rate of 11.44 cGy/min. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) was performed in 119 patients and the other 86 patients received an autologous hematopoietic stem cell transplantation (AHSCT). Median age was 30 years (5–63). We have calculated the cumulative incidence of solid tumors and secondary hematologic malignancies among these patients. Death due to noncancerous causes and patients lost to follow-up were entered as a competitive risk. With a median follow-up of 32 months (0.2–229)- including patients deceased in the first three months- 13 (6.3%) developed a secondary malignancy, 7 of them (3.4%) developed a solid tumor and 6 (2.9%) developed a secondary hematologic malignancy. The 7 patients who developed a solid tumor-1 glioblastoma, 2 head and neck carcinoma, 2 basocelular carcinoma, 1 osteosarcoma and 1 cervical intraepithelial neoplasia- had received an allo-HSCT. The 6 patients that developed a secondary hematologic malignancy- 5 therapy-related leukemia/myelodisplasia (t-AML/MDS) and 1 B cell non Hodgkin’s lymphoma- had received an AHSCT. The overall probability of developing a secondary malignancy after HSCT is 2.5% at 3 years (95% confidence interval (CI) 1.1– 6); 5% at 10 years (95% CI 2.6–9.3), and 9% at 15 years (95% CI 5–16.5). The probability of developing a solid tumor after HSCT is 0.5% at 3 years (95% CI 0.1–3.6), 1.8 % at 10 years (95% CI 0.6–5.5), and 6 % at 15 years (95% CI 2.6–13.7) and the probability of developing a secondary hematologic malignancy is 2 % at 3 years (95% CI 0.8–5.3), and 3,1 % at 10 and 15 years (95% CI 1.4–6.9). Median time to develop a solid tumor was 134 months (29–229). Median time to develop a secondary hematologic malignancy was 31 (3–60) months. Multivariate analysis proved that allo-HSCT was the only risk factor of developing a solid tumor, and that AHSCT and advanced age were risk factors of developing secondary hematologic malignancy (mean age 30 vs. 50 years ). To conclude, the probability of developing a solid tumor after HSCT is higher if an allo-HSCT has been performed and increases with time. AHSCT and advanced age are risk factors for the development of a secondary hematologic malignancy, a risk that decreases 5 years after AHSCT.
Presence of a Flt-3 Internal Tandem Duplication in AML Blasts Predicts Inhibition of Differentiation towards Leukemic Dendritic Cells.
Authors: Houtenbos Ilse , Westers Theresia M. , Ossenkoppele Gert J. , van de Loosdrecht Arjan A.
Published: October 2019

In the search for new treatment modalities to eradicate minimal residual disease cells (MRD) in acute myeloid leukemia (AML), immunotherapy provides an attractive option. AML blasts show differentiation towards leukemic dendritic cells (DC), providing the unique opportunity to generate DC harbouring the full range of tumour antigens. In a large cohort of AML samples (n=154) AML-DC were generated by two culture methods, i.e. in presence of cytokines GM-CSF, TNF-α, SCF, Flt-3L, IL-3 and IL-4 (n=147) or calcium ionophore (CI) and IL-4 (n=108). Median AML-DC yield, defined by phenotypical DC characteristics, in the cytokine-based cultures was 12% (range:0–70%). Considering cultures yielding ≥10% AML-DC successful, 58% (85/147) of cytokine-based cultures were successful and 61% (66/108) of CI cultures. Overall, functional AML-DC generated with either method was possible in 66% (101/154) of patients. Identification of AML blast populations with DC differentiation capacity is important to select patients eligible for immunisation programs. Interestingly, presence of Flt-3 internal tandem duplication (ITD) was strongly correlated with decreased DC differentiation capacity in both culture methods (cytokine-based culture: p<0.001; CI-culture: p=0.03) suggesting that constitutive activation of tyrosine kinase receptors inhibits differentiation towards DC. In multiparameter regression analysis, powerful predictors for cytokine-based AML-DC culture outcomes were Flt-3 ITD (regression coefficient B:-3.41, p<0.001), CD14 (B:3.28, p<0.001) and TNFα-RI (B:2.82, p<0.001). This regression model predicts 88% of culture outcomes. ROC curves show high sensitivity (95%) and specificity (76%) with an AUC of 0.93 (p<0.001). In 25% of unsuccessful cytokine-based cultures, the CI-based culture method provides an alternative. This percentage increases to 56% if Flt-3 ITD+ AML samples are left out, emphasizing Flt-3 ITD+ blasts’ inability to differentiate towards leukemic DC. In conclusion, AML-DC cultures are successful in most patients. Selection of patients is well possible based upon the presence of Flt-3 ITD and the expression of CD14 and TNFα-RI. Based on these results, we are currently entering patients in a phase I/II clinical vaccination trial.
Studies on GM-CSF Induced Signaling and Cell Growth in JMML: An Experimental Model To Identify Mechanism Directed Targeted Therapeutics.
Authors: Ding Yu , Teja Bijan , Blackmore Chris , Dingwa Sumita , Ujack Eva , Brown Christopher , Narendran Aru
Published: October 2019

Juvenile myelomonocytic leukemia (JMML) is considered to be one of the most difficult to treat and deadliest diseases of early childhood. The pathogenesis of JMML involves deregulated cytokine signal transduction, especially a selective hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). Current treatment modalities do not provide significant long-term disease control. We describe an experimental model system in which the effect of targeted agents can be studied in the context of GM-CSF mediated growth and survival of JMML cells.

Bone marrow and peripheral mononuclear cells from JMML patients were enriched by immunoaffinity chromatography and grown in the presence of GM-CSF. Purity of these cells was confirmed by FACS and their absolute dependence on GM-CSF for growth and survival was assessed. A direct role for GM-CSF was confirmed by the addition of neutralizing antibodies. Molecular techniques showed the lack of excess GM-CSF secretion by these cells that proved receptor hypersensitivity as the principal driving force behind the excessive proliferation. To understand the signaling pathways involved in this process, we constructed a capture antibody array with antibodies to critical signaling molecules. GM-CSF treated and control lysates were incubated with the arrays and the expression and phosphorylation of the signaling molecules were detected by anti p-tyr, p-Ser and p-Thr antibodies. Blots were then quantified by an automated image analysis program. Results showed consistent activation of a group of signaling molecules mediated by the addition of GM-CSF. These include IRS-1, FAK, IkappaB and LIFR. Using this assay system we then looked at the effects of a number of novel therapeutic agents to inhibit the growth and specifically interfere with the GM-CSF mediated signaling cascade. These novel therapeutic agents and targeted kinase inhibitors such as 17-AAG, DMAG, arsenic trioxide, rebeccamycin, AMN-107, and Prima-1. (IC50: 0.1, 0.01. 0.5, 0.1, 0 and 10, uM respectively). These agents were studied in combination to identify agents that exhibit synergistic and additive properties. Significant additive effects were seen with certain combinations, notably in combination with Hsp90 inhibitors.

We describe an effective experimental model to identify the players and pathways involved in the abnormal growth properties of JMML. We discuss in detail the implications of these findings in the development of potential anti JMML therapies in the future.
Platelet-Derived Microparticles Accelerate Engraftment of Peripheral Blood Stem Cells in Human Allogeneic Transplantation.
Authors: de Rop Christiane , Priesack Jan , Tiede Andreas , Trummer Arne
Published: October 2019

While the procoagulant activity of platelet derived microparticles (PMP) has been widely accepted, knowledge regarding their immunological and adhesive qualities is still limited. It has been shown that murine BM cells covered with PMP engrafted lethally irradiated mice significantly faster than those not covered, indicating that PMPs play an important role in the homing of peripheral blood stem cells (PBSC). Here we studied the impact of PMP on engraftment in human allogeneic PBSC transplants for patients with hematological malignancies. PBSC samples were collected in buffered citrate from transplantation bags after infusion of transplants into patients with hematological malignancies (AML = 5, ALL = 1). Conditioning regimens included busulfan/cyclophosphamide (Bu/Cy), anti-CD66b-radioimmunotherapy (RIT)/Bu/Cy, and reduced intensity regimens with fludarabin/busulfan (Flu/Bu) and FLAMSA. Platelet-poor plasma (PPP) was prepared (1500g for 20min), immediately shock-frozen in liquid nitrogen and stored at −80°C. For further analysis PPP’s were carefully thawed at room temperature (RT). 90μl of PPP was stained with 5μl of CD41-PE and CD62P-FITC each for 15min at RT in the dark (IgG1-FITC and -PE served as negative controls, TRAP-6 (10μM) stimulated whole blood processed in same way as samples as positive control). To stop staining 900μl PBS/BSA 2% was added and 500μl of this solution were transferred into BD Trucount tubes by reverse pipetting giving a final concentration of 100 beads/μl. Samples were analyzed immediately using Coulter FC500 flow cytometer with CXP software. As expected the CD34 cell count (mean=5.1x106/kg body weight, SD=2.0x106/kg) showed a significant correlation (p=0.0197, Pearson r=−0.83) with the time to engraftment (mean=15.7days, SD=2.0d). The amount of CD62P positive microparticles (mean=423/μl, SD=119/μl) and the conditioning regimen showed no significant correlation with CD34 cell count or time to engraftment with leucocytes >1000/μl. In contrast, CD41-PMP count (mean=1223/μl, SD=857μl) correlated significantly with the CD34 cell count (p=0.0086, Pearson r=0.92) and the time to engraftment (p=0.0039, Pearson r = −0.95). Therefore, PBSCT contain significant amounts of PMP which are most likely generated during apheresis. Preliminary results show a stronger correlation with time to engraftment than does CD34 cell count. We conclude that PMP may accelerate engraftment of PBSC in humans. However, this function seems unrelated to P-Selectin expression. Therefore, further studies aiming to identify other adhesion molecules involved in PMP-mediated engraftment of PBSCT are warranted.
Gene Correction in Human Cells Is Promoted by the Bacteriophage Protein Redβ.
Authors: Porter Andrew C. , Stewart Francis , Xu Ke
Published: October 2019

Correction of a disease gene mutation by homologous recombination (HR) with a transfected DNA repair template represents an ideal form of gene therapy for many inherited hematological disorders. Therapeutic gene correction via HR in hematopoietic stem cells is unlikely to succeed, however, without major improvements in its efficiency. One promising approach is to use an engineered zinc finger endonuclease to specifically cleave the target gene and so promote HR between a dsDNA template and its chromosomal target (Urnov et al (2005) Nature 435:646-51). We are exploring an alternative approach based on the expression of Redβ, a single stranded (ss) DNA annealing protein from bacteriophage λ. In bacteria, Redβ promotes HR-mediated gene modification using templates with short regions of homology (e.g. 30–100nt). Such HR is independent of RecA-mediated host cell HR. For double stranded (ds) DNA templates, co-expression of λ exonuclease (the red α gene product) is required, but ssDNA templated modifications are particularly efficient and require only Redβ expression. Such properties could be useful for therapeutic gene correction if reproduced in human cells. Preliminary experiments in mouse embryonic stem cells suggested that Redβ can function in mammalian cells (Zhang Y. et al, (2003) BMC Mol. Biol. 4). To explore this further, we have generated a human cell line in which large amounts of Redβ can be expressed inducibly in the nucleus. In contrast to the over-expression of endogenous HR proteins Rad51 (RecA homologue) or Rad52 (ssDNA annealing protein), sustained Redβ over-expression is well tolerated, with no impairment of cell proliferation or viability. We find that Redβ does indeed promote HR in human cells. Surprisingly, dsDNA and ssDNA templated gene modification are stimulated to similar extents by Redβ. Stimulations are modest, but similar to those achieved by human Rad51 over-expression. The effects of co-expressing Red α are under investigation. We have also shown that ssDNA-templated gene correction is stimulated by transient, RNAi-mediated, depletion of the mismatch repair protein MSH2, and that such stimulation is additive with Redβ-stimulated gene correction.