Correlates of Lenalidomide Induced Immune Stimulation and Response in CLL: Analysis in Patients on Treatment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 979-979 ◽  
Author(s):  
Georg Aue ◽  
Stefania Pittaluga ◽  
Delong Liu ◽  
Larry Stennett ◽  
Susan Soto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
Expression Profiling ◽  
Research Funding ◽  
Immune Stimulation ◽  
Intramural Research Program ◽  
Pre Treatment ◽  
Program Research

Abstract Abstract 979 Lenalidomide's mechanism of action in chronic lymphocytic leukemia (CLL) is not well understood. In vitro data suggest that anti-leukemic immune responses are important. Tumor flare reactions during treatment have been associated with response in some but not other studies. In vivo data that mechanistically link immune stimulation to clinical responses are lacking. We designed an independent, single center, phase II trial of lenalidomide in relapsed/refractory CLL (clinicaltrials.gov: NCT00465127). Here we report final clinical data and results of multiple translational analyses that indicate that an IFNy centered immune response is critical for response. A 3 week on, 3 weeks off treatment scheme (42 day cycles) was chosen to pulse immune stimulation while trying to minimize myelosuppression. The starting dose was 20 mg daily for the first 10 patients and 10 mg for the subsequent 23. Response was measured at 24 weeks. 5 patients, 4 with del 17p, achieved a PR by IWCLL criteria (16%) and were eligible to continue drug for 4 more cycles; the PFS in these patients was 16 months compared to 7 months for all other (p<0.001). Myelosupression remained the limiting side effect. A cytokine release syndrome often accompanied by tumor flare reactions was seen in 78% of patients in cycle 1 and often recurred in subsequent cycles. Compared to other studies it appears that the long treatment free period increased the inflammatory reaction upon restarting of L. All correlative analyses reported here were performed on PBMCs, lymph node (LN) core biopsies and serum obtained from patients during cycle 1 and 2 and included flow cytometry, gene expression profiling (Affymetrix arrays), and cytokine measurements. Nine patients with decreased lymphadenopathy ≥10% (10–85%) on CT after 4 cycles were considered responders (R) for correlative studies. There was a significant decrease in CLL count (median 14% on day 8 and 49% on day 22, p<0.01) and in the number of circulating T (CD3, CD4, CD8) and NK-cells (n=22, p<0.05) with no difference between R and non-responders (NR). In contrast, the CD3 count in LN core biopsies increased 1.4 fold in R compared to matched pre-treatment biopsies (p<0.05) with no change in NR (0.95 fold). In the L free interval CLL cells rebounded to pre-treatment levels. A rapid rebound of CLL counts during treatment interruptions has been previously described but its mechanism is not well understood. In migration assays we observed a 3-fold increased migration towards SDF-1 for L compared to control cells (p=0.03), indicating that increased homing of lymphocytes to tissue sites may be responsible for the rapid decrease in peripheral counts. The cell surface molecules CD40, 54, 86, 95, DR5 were upregulated (p<0.05) while CD5 and 20 were downregulated (p<0.001) on circulating CLL cells. Effects on CD54 and CD5 were stronger in R than NR (p<0.05). Next we performed gene expression profiling on purified PB-CLL cells and LN core biopsies obtained on day 8. L induced upregulation of 95 genes, many of which are known to be regulated by interferon gamma (IFNγ). The comparison with a gene expression signature induced by recombinant IFNγ in CLL cells cultured in vitro confirmed the significant induction of a typical IFNγ response by L in vivo (n=24, p<0.0001). The IFNγ response in PB-CLL cells was no different in R vs NR (n=12, p=0.78), but in LN biopsies it was more prominent in R (n=7) than NR (n=5) (p<0.05). Consistently the IFNG gene was upregulated in LN biopsies of R but actually decreased in NR (p=0.001). Serum IFNγ levels were elevated on L (n=14 at all time points, day 4 p=0.03, day 8 p=0.01, day 22 p=0.02, day 49 p<0.01), but off drug returned to pretreatment levels. Next we sought to determine the source of IFNγ. The tumor cells are ruled out as IFNG was not expressed in purified CLL cells. By flow cytometry the number of IFNγ secreting CD4 T-cells increased on day 8 from 0.8% to 1.5%, p=0.006), an effect that was stronger in R had than NR (p<0.05). IFNγ positive NK cells did not increase on L. These data provide a first mechanistic link between the degree of Lenalidomide induced immune activation to clinical response in CLL. Based on our experience we suggest that continued dosing of L may be superior to dose interruptions. Disclosures: Aue: NHLBI, Intramural Research Program: Research Funding. Off Label Use: Lenalidomide is not FDA approved for CLL. Wiestner:NHLBI, Intramural Research Program: Research Funding.

The Role of the Microenvironment for CLL Proliferation and Survival: Gene Expression Profiling of Leukemic Cells Derived from Blood, Bone Marrow and Lymph Nodes Reveals the B-Cell Receptor and NF κB as Dominant Signaling Pathways

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 356-356 ◽  
Author(s):  
Yair Herishanu ◽  
Berengere Vire ◽  
Delong Liu ◽  
Federica Gibellini ◽  
Gerald E Marti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tumor Cells ◽  
Expression Profiling ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Leukemic Cells ◽  
Activation Markers ◽  
Bcr Signaling

Abstract The host microenvironment is important for proliferation and survival of leukemic cells in chronic lymphocytic leukemia (CLL). Numerous molecules, signaling pathways and cell types have been reported to enhance CLL cell survival. To date, most reports on such interactions are derived from in-vitro studies, where each study focused on a specific ligand/receptor interaction or candidate pathway. Here, we adopted a more global approach to evaluate in-vivo effects of the microenvironment on leukemic cell biology. CLL cells from 15 patients were obtained on the same day from 3 different compartments: peripheral blood (PB), bone marrow (BM) and lymph node (LN), from which a single cell suspension was prepared. Tumor cells from all three compartments were purified by CD19 selection to purity &gt;98%. Patients were assigned to prognostic subtypes based on immunoglobulin sequencing (Ig) and ZAP70 expression: 10 patients had the more progressive subtype (Ig-unmutated, ZAP70+) and 5 patients belonged to the more indolent subtype. Cells were analyzed for surface markers by flow cytometry and by gene expression profiling on Affymetrix HG U133 Plus 2.0 arrays. By flow cytometry, CLL cells in LN expressed higher levels of activation markers including CD69 and CD38 compared to CLL cells in PB (% CD19+/69+; 71 ±27 vs. 35 ±28, p&lt;0.001 and % CD19+/CD38+; 33 ±28 vs. 20±19, p&lt;0.001, respectively). The expression of activation markers in BM derived cells was less consistent and did not reach statistically significant differences. We therefore focused our analysis on a comparison between LN and PB derived cells. First, we confirmed that the expression of a diagnostic CLL gene expression signature established previously for PB derived cells (Klein et al, 2001) was equally present in leukemic cells derived from all three compartments. We then identified a set of about 275 genes that were differentially expressed between LN resident and circulating tumor cells, most of which were up-regulated (fold change &gt;2, FDR &lt;0.2). A large number of these genes encode proteins important for cell cycle control and proliferation: different cyclins, PCNA, Ki67, TOP2A and MYC. We also detected a significant increase in the expression of NF-κB target genes in LN resident tumor cells, including CD83, CD69, JunB, Cyclin D2, GADD45B, CCL3, CCL4 and others. Consistent with activation of the NF-κB pathway in LN, IκB-beta protein levels in tumor cells from LN were lower than levels in matching PB cells. Next we identified genes differentially expressed between CLL subtypes based on Ig-mutation status separately for each of the 3 compartments. Interestingly, these subtype identifying gene sets were only partially overlapping. In Ig-unmutated, ZAP70+ cells several genes were more strongly regulated by the microenvironment then in Ig-mutated, ZAP70 negative cells. Among these genes is LPL, which has been reported to distinguish the CLL subtypes, and other genes induced by B-cell receptor (BCR) signaling. Using in-vitro IgM activation, we show that these genes are indeed induced by BCR stimulation but not by CD40 ligation and that their induction is confined to ZAP70+ CLL cells. In conclusion: interactions between CLL cells and elements of the microenvironment in LN induce cell proliferation and NF-κB activation. The preferential upregulation of BCR regulated genes in ZAP70+ CLL demonstrates a more efficient in-vivo response of ZAP-70+ cells to BCR stimulation. Our results highlight the importance of NFκ κB and BCR signaling in CLL and provide a rationale to focus treatment approaches on these central pathways.

Leukemia Stem Cell Potential of Different Progenitor Subpopulations in Myeloid Blast Phase CML

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3489-3489
Author(s):  
Ross Kinstrie ◽  
Dimitris Karamitros ◽  
Nicolas Goardon ◽  
Heather Morrison ◽  
Richard E Clark ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Research Funding ◽  
Cell Populations ◽  
Novel Therapies ◽  
Blast Phase ◽  
Self Renewal ◽  
Stem Cell Populations

Abstract Blast phase (BP)-CML remains the most critical area of unmet clinical need in the management of CML and novel, targeted therapeutic strategies are urgently needed. In the tyrosine kinase inhibitor (TKI) era, the rate of progression to BP is 1 to 1.5% per annum in the first few years after diagnosis, falling sharply when major molecular response is obtained. Around 10% of patients present with de novo BP-CML and despite the use of TKIs, median survival after the diagnosis of BP-CML is between 6.5 and 11 months.Therefore, improved understanding of the biology of BP-CML and novel therapies to prolong therapeutic responses are urgently sought. Studies of myeloid malignancies show that acquisition of tumor-associated mutations occurs principally in a step-wise manner. Initiating mutations usually originate in an hematopoietic stem cell (HSC) to give rise to preleukemic stem cell populations that expand through clonal advantage. Further mutation acquisition and/or epigenetic changes then lead to blast transformation and disruption of the normal immunophenotypic and functional hematopoietic hierarchy. At this stage, multiple leukemic stem cell (LSC) populations (also termed leukemia initiating cell populations) can be identified. We previously showed, in AML, that the CD34+ LSC populations were most closely related to normal progenitor populations, rather than stem cell populations, but had co-opted elements of a normal stem cell expression signature to acquire abnormal self-renewal potential (Goardon et al, Cancer Cell, 2011). CD34+CD38- LSCs were most commonly similar to an early multi-potent progenitor population with lympho-myeloid potential (the lymphoid-primed multi-potential progenitor [LMPP]). In contrast, the CD34+CD38+ LSCs were most closely related to the more restricted granulocyte-macrophage progenitor (GMP). In chronic phase CML, the leukemia-propagating population is the HSC, and the progenitor subpopulations do not have stem cell characteristics. To date, studies to isolate LSC populations in BP-CML have been limited, identifying the GMP subpopulation only as a possible LSC source (Jamieson et al, NEJM, 2004). Furthermore, in vivo LSC activity has not been assessed. We therefore set out to assess the LSC characteristics of different primitive progenitor subpopulations in myeloid BP-CML both in vitro and in vivo. We isolated different stem and progenitor cell subpopulations using FACS; HSC (Lin-CD34+CD38-CD90+ CD45RA-), multipotent progenitor (MPP; Lin-CD34+CD38-CD90-CD45RA-), LMPP (Lin-CD34+CD38-CD90-CD45RA+), common myeloid progenitor (CMP; Lin-CD34+CD38+CD45RA-CD123+), GMP (Lin-CD34+CD38+CD45RA+CD123+) and megakaryocyte erythroid progenitor (MEP; Lin-CD34+CD38+CD45RA-CD123-). The functional potential of these purified populations was examined in 13 patients by: (i) serial CFC replating assays to study progenitor self-renewal (n=10); (ii) In vivo xenograft studies using NSG mice with serial transplantation to identify populations with LSC potential (n=6). Our data conclusively demonstrate that functional LSCs are present in multiple immunophenotypic stem/progenitor subpopulations in myeloid BP-CML, including HSC, MPP, LMPP, CMP and GMP subpopulations. There was inter-patient variability in terms of both in vitro and in vivo functional properties. Fluorescence in situ hybridisation (FISH) was used to assess clonality in the different progenitor subpopulations and identify which populations contained cells with additional cytogenetic abnormalities (ACAs) with a view to improving our understanding of the clonal hierarchy. Interestingly, there were no significant differences in ACAs in the different progenitor subpopulations in the majority of samples studied, suggesting that clonal evolution tends to occur in the HSC compartment in myeloid BP-CML. Preliminary gene expression profiling studies of the different progenitor subpopulations, using Affymetrix Human Gene 1.0 ST Arrays, demonstrated highly variable gene expression, supporting the functional heterogeneity seen. Taken together, our results demonstrate that myeloid BP-CML is a very heterogeneous disorder with variable LSC populations. Further interrogation of these populations will likely identify novel therapies which will specifically target the LSC. Disclosures Copland: Bristol-Myers Squibb: Consultancy, Honoraria, Other, Research Funding; Novartis: Consultancy, Honoraria, Other; Ariad: Consultancy, Honoraria, Research Funding.

scholarly journals ASXL2 Is Recurrently Mutated in t(8;21) AML and Regulates Hematopoietic Development

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1051-1051
Author(s):  
Vikas Madan ◽  
Lin Han ◽  
Norimichi Hattori ◽  
Anand Mayakonda ◽  
Qiao-Yang Sun ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Research Funding ◽  
Hematopoietic Differentiation ◽  
Wild Type ◽  
Flow Cytometric ◽  
Deficient Mice

Abstract Chromosomal translocation t(8;21) (q22;q22) leading to generation of oncogenic RUNX1-RUNX1T1 fusion is a cytogenetic abnormality observed in about 10% of acute myelogenous leukemia (AML). Studies in animal models and recent next generation sequencing approaches have suggested cooperativity of secondary genetic lesions with t(8;21) in inducing leukemogenesis. In this study, we used targeted and whole exome sequencing of 93 cases (including 30 with matched relapse samples) to profile the mutational landscape of t(8;21) AML at initial diagnosis and post-therapy relapse. We identified recurrent mutations of KIT, TET2, MGA, FLT3, NRAS, DHX15, ASXL1 and KMT2Dgenes in this subtype of AML. In addition, high frequency of truncating alterations in ASXL2 gene (19%) also occurred in our cohort. ASXL2 is a member of mammalian ASXL family involved in epigenetic regulation through recruitment of polycomb or trithorax complexes. Unlike its closely related homolog ASXL1, which is mutated in several hematological malignancies including AML, MDS, MPN and others; mutations of ASXL2 occur specifically in t(8;21) AML. We observed that lentiviral shRNA-mediated silencing of ASXL2 impaired in vitro differentiation of t(8;21) AML cell line, Kasumi-1, and enhanced its colony forming ability. Gene expression analysis uncovered dysregulated expression of several key hematopoiesis genes such as IKZF2, JAG1, TAL1 and ARID5B in ASXL2 knockdown Kasumi-1 cells. Further, to investigate implications of loss of ASXL2 in vivo, we examined hematopoiesis in Asxl2 deficient mice. We observed an age-dependent increase in white blood cell count in the peripheral blood of Asxl2 KO mice. Myeloid progenitors from Asxl2 deficient mice possessed higher re-plating ability and displayed altered differentiation potential in vitro. Flow cytometric analysis of >1 year old mice revealed increased proportion of Lin-Sca1+Kit+ (LSK) cells in the bone marrow of Asxl2 deficient mice, while the overall bone marrow cellularity was significantly reduced. In vivo 5-bromo-2'-deoxyuridine incorporation assay showed increased cycling of LSK cells in mice lacking Asxl2. Asxl2 deficiency also led to perturbed maturation of myeloid and erythroid precursors in the bone marrow, which resulted in altered proportions of mature myeloid populations in spleen and peripheral blood. Further, splenomegaly was observed in old ASXL2 KO mice and histological and flow cytometric examination of ASXL2 deficient spleens demonstrated increased extramedullary hematopoiesis and myeloproliferation compared with the wild-type controls. Surprisingly, loss of ASXL2 also led to impaired T cell development as indicated by severe block in maturation of CD4-CD8- double negative (DN) population in mice >1 year old. These findings established a critical role of Asxl2 in maintaining steady state hematopoiesis. To gain mechanistic insights into its role during hematopoietic differentiation, we investigated changes in histone marks and gene expression affected by loss of Asxl2. Whole transcriptome sequencing of LSK population revealed dysregulated expression of key myeloid-specific genes including Mpo, Ltf, Ngp Ctsg, Camp and Csf1rin cells lacking Asxl2 compared to wild-type control. Asxl2 deficiency also caused changes in histone modifications, specifically H3K27 trimethylation levels were decreased and H2AK119 ubiquitination levels were increased in Asxl2 KO bone marrow cells. Global changes in histone marks in control and Asxl2 deficient mice are being investigated using ChIP-Sequencing. Finally, to examine cooperativity between the loss of Asxl2 and RUNX1-RUNX1T1 in leukemogenesis, KO and wild-type fetal liver cells were transduced with retrovirus expressing AML1-ETO 9a oncogene and transplanted into irradiated recipient mice, the results of this ongoing study will be discussed. Overall, our sequencing studies have identified ASXL2 as a gene frequently altered in t(8;21) AML. Functional studies in mouse model reveal that loss of ASXL2 causes defects in hematopoietic differentiation and leads to myeloproliferation, suggesting an essential role of ASXL2 in normal and malignant hematopoiesis. *LH and NH contributed equally Disclosures Ogawa: Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

scholarly journals Tirapazamine As a Strategy to Overcome Hypoxia-Induced Drug Resistance in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4436-4436
Author(s):  
Barbara Muz ◽  
Pilar De La Puente ◽  
Micah John Luderer ◽  
Farideh Ordikhani ◽  
Abdel Kareem Azab
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Drug Resistance ◽  
Cell Survival ◽  
Research Funding ◽  
High Dose ◽  
Drug Resistant ◽  
Hypoxic Conditions

Abstract Introduction: Multiple myeloma (MM) is a lymphoplasmacytic malignancy characterized by the continuous spread of MM cells in and out of the bone marrow (BM). Despite the introduction of novel therapies, cancer patients relapse due to the development of drug resistant cells, which are, at least in part, promoted by hypoxia. Therefore, in this study we aimed to overcome drug resistance in MM by inhibition of the hypoxic responses in these cells. Tirapazamine (TPZ) is a hypoxia-activated pro-drug causing cell apoptosis, which has been shown to improve the outcome of patients with solid tumors when combined with radiotherapy; however, it has not been tested in MM. We used TPZ for the first time in MM to target the drug resistant cancer cells and sensitize them to therapy. Methods: To test the effect of TPZ on tumor survival in vitro, MM cell lines (MM1.s, H929, OPM1, RPMI8226) were exposed to normoxia (21% O2) or hypoxia (1% O2) for 24 hours with different concentrations of TPZ in order to obtain an IC50, and cell survival was assessed using MTT assay. Also, a combination of bortezomib and carfilzomib with or without TPZ was tested on cell survival. For in vivo study, 5 x 106 MM1s-Luc-GFP cells were injected intravenously (IV) into SCID mice and tumor progression was monitored for 3 weeks by bioluminescent imaging. First, we tested the hypoxic status of mice treated with and without a high-dose bortezomib (1.5mg/kg). Pimonidazole (PIM) was injected intraperitoneally (IP) into mice and 4 hours later BM was harvested, stained with anti-PIM-APC antibody and followed by measuring PIM signal in MM1s-GFP+ cells using flow cytometry. Second, we developed drug resistant cells by treating mice with a high-dose bortezomib (1.5mg/kg), and then treated with (1) bortezomib only (0.5mg/kg; n=3), or (2) bortezomib and TPZ (40mg/kg; n=3), all administered IP sequentially twice a week. The number of residual MM1s-GFP+ cells was calculated by flow cytometry. Results: We found that TPZ was active in a dose-dependent manner only in hypoxic conditions in MM cell lines. We showed that residual MM cells in the BM after high-dose bortezomib are hypoxic, as demonstrated by PIM staining. The combination of TPZ with bortezomib and carfilzomib resensitized cancer cells to death in hypoxia, overcoming hypoxia-induced drug resistance in vitro. Moreover, TPZ-treatment in combination with bortezomib further decreased residual MM cells in vivo. Conclusions: We reported that MRD was hypoxic and that TPZ, which was cytotoxic for MM cells only in hypoxic conditions, overcame hypoxia-induced drug resistance in vitro and killed bortezomib-resistant residual MM cells in vivo. This is the first study to show the efficacy of TPZ in MM. This data provides a preclinical basis for future clinical trials testing efficacy of TPZ in MM. Disclosures Azab: Selexys: Research Funding; Karyopharm: Research Funding; Cell Works: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner ; Verastem: Research Funding.

scholarly journals Cryopreserved Platelets: From in Vitro Thrombin Generation Potential to in Vivo Safety

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2339-2339 ◽  
Author(s):  
Mariasanta Napolitano ◽  
Lucio Lo Coco ◽  
Giorgia Saccullo ◽  
Piera Stefania Arfò ◽  
Giuseppe Tarantino ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Leukemia ◽  
Thrombin Generation ◽  
Buffy Coat ◽  
Severe Thrombocytopenia ◽  
Activation Markers ◽  
Hematological Patients ◽  
Pre Treatment

Abstract Background: Cryopreservation of platelets (PLTs) at -80°C with dimethyl sulfoxide (DMSO) can extend their shelf life up to 2 years. Cryopreserved PLTs (CRY-PLTs) are reported to have a greater in vivo hemostatic effect than liquid-stored PLTs. Aims of this study were: i. to evaluate the thrombin generation potential of buffy coat derived cryopreserved PLTs (CRY- BC PLT) in comparison with fresh buffy coat derived platelets concentrates; ii. to determine the efficacy and safety of CRY-PLTs transfusion in hematological patients with severe thrombocytopenia. Materials and methods: BC PLTs were obtained from 5 buffy coats and pooled. The final PLTs concentrates were leukoreduced by filtration and transferred to a 650 mL patented cryopreservation kit (Promedical ®) which allowed mixing with DMSO 25% in a closed system and following removal of supernatant without further manipulations. BC-PLTs were washed prior freezing, suspended in homologous plasma from 1 of the 5 donors to a final concentration of 200 mL and frozen at - 80°. CRY- BC PLTs were preserved at -80°C with 6% DMSO. A system of 3 accessory bags directly connected to the mother bag was adopted for the in vitro study, to avoid repeated freezing/thawing of samples. In vitro assays were performed before freezing and at 3,6 and 9 months after thawing. Before assay, CRY-BC PLT were thawed at 37°C and diluted in plasma to adjust to 300× 109/L PLTs. Fresh BC PLTs underwent the same dilution to adjust to 300 ×109/L PLTs. Thrombin generation (TGA) was tested in CRY BC-PLTs and compared to TG potential of fresh BC PLTs. TGA was triggered by the addition of 0.5 pmol/L of recombinant human tissue factor. Endogenous thrombin potential (ETP) and peak height (PH) were determined. Flow Cytometry assays for PLTs activation markers and thromboelastography were also determined on each sample. CRY-BC PLTs, separately prepared according to the above described method for in vivo study, were infused in five hematological patients with acute leukemia (AL) and severe thrombocytopenia (PLTs <10 ×109/L) participating to the trial NCT02032134.CRY-BC PLTs were transfused to control epistaxis (n=2) and for prophylaxis (n=3). Patients were observed up to 7 days after infusion and the occurrence of any side effect was registered. An increase in PLTs count was observed only in one case, under prophylaxis, but bleeding was successfully controlled or prevented in all cases. Plasma from patients transfused with CRY-BC PLTs was tested for TGA pre-treatment and 24 hours after treatment Results Fourty nine BC-PLTs from 245 healthy volunteer donors (145 males and 100 females, mean age: 48.16.±18.91) were prepared, cryopreserved and analyzed up to 9 months after storage. Cryopreserved PLTs show a good thrombin generation potential that is stably maintained up to 9 months after cryopreservation [ETP (nM min): 529.25±98.64 at T0, 558.82±114.67 at T3, at 548.57±93.38 T6 and 533.04±103.15 at T9 months, respectively; PH(nM): 132.77±44.9 at T0, 103.4±44.9 at T3, 108.0±36.7 at T6 and 132.0±44.6 at T9 months, respectively]. At TGA, fresh BC-PLTs (n=35) had a mean ETP of 760.13±130.11, PH was 138.9±40.2. Thrombin generation of CRY-BC PLTs is comparable to fresh BC-PLTs, even if slightly decreased. Infusion of CRY-BC PLT (1U) was effective in controlling mucosal bleeding (epistaxis) in two patients with AL and severe thrombocytopenia. CRY-PLT were also effective when administered for prophylaxis in 3 patients with very low platelets count secondary to chemotherapy. In vivo, thrombin generation is stably maintained up to 24 hours after infusion of 1 Unit of CRY-BC PLTs, without any adverse effect (mean ETP pre-treatment was: 414.13±160.60, 24 hours after transfusion: 326.95±152.54). CRY-BC PLTs were safe and they did not determine any thrombotic event. At flow-cytometry, CRY-BC PLTs expressed higher activation markers (CD62P,CD63) than fresh BC PLTs. CRY-BC PLTs are able to significantly decrease the time to clot formation and clot strength, as measured also by thromboelastography. CRY-BCPLTs activation/deterioration is accompanied by an effective hemostatic in vivo function. Conclusions: Cryopreserved PLTs have an enhanced hemostatic activity and a good thrombin generation potential. They are effective and safe in preventing and controlling bleeding, being available in emergency/urgency settings also for patients with acute leukemia and severe thrombocytopenia. Disclosures Reina: Promedical: Consultancy.

scholarly journals Sox6 as a new modulator of renin expression in the kidney

2020 ◽  
Vol 318 (2) ◽  
pp. F285-F297 ◽  
Author(s):  
Mohammad Saleem ◽  
Conrad P. Hodgkinson ◽  
Liang Xiao ◽  
Juan A. Gimenez-Bastida ◽  
Megan L. Rasmussen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Stromal Cells ◽  
Expression Profiling ◽  
Muscle Cells ◽  
Afferent Arteriole ◽  
Adult Kidney

Juxtaglomerular (JG) cells, major sources of renin, differentiate from metanephric mesenchymal cells that give rise to JG cells or a subset of smooth muscle cells of the renal afferent arteriole. During periods of dehydration and salt deprivation, renal mesenchymal stromal cells (MSCs) differentiate from JG cells. JG cells undergo expansion and smooth muscle cells redifferentiate to express renin along the afferent arteriole. Gene expression profiling comparing resident renal MSCs with JG cells indicates that the transcription factor Sox6 is highly expressed in JG cells in the adult kidney. In vitro, loss of Sox6 expression reduces differentiation of renal MSCs to renin-producing cells. In vivo, Sox6 expression is upregulated after a low-Na+ diet and furosemide. Importantly, knockout of Sox6 in Ren1d+ cells halts the increase in renin-expressing cells normally seen during a low-Na+ diet and furosemide as well as the typical increase in renin. Furthermore, Sox6 ablation in renin-expressing cells halts the recruitment of smooth muscle cells along the afferent arteriole, which normally express renin under these conditions. These results support a previously undefined role for Sox6 in renin expression.

Gene expression profiling of in vitro-derived and in vivo-derived bovine blastocysts using microarray analysis

2011 ◽  
Vol 22 ◽  
pp. S53-S54
Author(s):  
Digdem Aktoprakligil Aksu ◽  
Cansu Agca ◽  
Soner Aksu ◽  
Haydar Bagis ◽  
Tolga Akkoc ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Microarray Analysis ◽  
Expression Profiling

Terminal Differentiation of FLT3/ITD AML Blasts In Patients Treated with the FLT3 Inhibitor AC220

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 660-660
Author(s):  
Mark J. Levis ◽  
Amy Sexauer ◽  
Trivikram Rajkhowa ◽  
Donald Small ◽  
Michael J. Borowitz
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Peripheral Blood ◽  
Terminal Differentiation ◽  
Flt3 Inhibitor ◽  
Microscopic Evaluation ◽  
Pre Treatment

Abstract Abstract 660 AML is characterized by abnormal proliferation of myeloid cells that have a block in differentiation. FLT3/ITD mutations are relatively common in AML, and previous in vitro studies have demonstrated that signaling from ITD-mutated FLT3 blocks myeloid differentiation through repression of CEBP/a. As part of an ongoing phase 2 trial, we treated 6 patients with FLT3/ITD AML who were refractory to either primary induction therapy or salvage therapy after relapse with the highly potent and selective FLT3 inhibitor AC220. At the start of therapy, all 6 patients had circulating blasts (mean 9864 blasts/mm3; median 2970) and the median blast percentage in the bone marrow was 71.5%. Western blotting revealed a high level of sustained in vivo FLT3 inhibition in all patients. By Day 8, no patient had detectable blasts in the peripheral blood. After 14 days of treatment with AC220, all 6 patients displayed striking differentiation to the myelocyte stage within the bone marrow. By light microscopic evaluation of bone marrow aspirates, myelocytes (promyelocytes, myelocytes, and metamyelocytes) increased from a median of 10.5% pre-treatment to 52% after 2 weeks. Most patients were neutropenic on Day 1 of treatment (mean 574, median 560 neutrophils/mm3), but rose to a mean of 3275 neutrophils/mm3 after 4–8 weeks of treatment (median time to peak 34 days). By Day 28 of treatment, marrows were most often still hypercellular, but consisted primarily of fully differentiated neutrophils. Marrow blasts were markedly reduced or absent by Day 28 in all 6 cases (mean 2.3%, median 1.5%). In all 6 patients the FLT3/ITD mutation originally detected at the beginning of treatment was present in the marrow and peripheral blood despite the absence of circulating blasts after the first week of therapy. The FLT3 mutant allelic ratio did not change between pre-therapy and Day 28. Neutrophils were isolated to homogeneity (confirmed by cytospin) from peripheral blood by double ficoll density centrifugation. Using genomic DNA obtained from these purified neutrophils, we confirmed by PCR that the FLT3/ITD mutation was present, at a similar ratio as compared with the pre-treatment blasts. However, there was no detectable expression of FLT3 either by RNA (quantitative PCR) or protein (western blotting and flow cytometry) in these neutrophils. The isolated neutrophils morphologically resemble normal neutrophils by light microscopy, and by flow cytometry they express the differentiation antigen CD15 and CD11b, and have lost expression of immature markers such as cKIT and CD34. Stimulation of these neutrophils by endotoxin results in normal respiratory burst activity, as measured by reduction of nitroblue tetrazolium. They also express lactoferrin and MMP-9, proteins typically expressed in mature neutrophils. Clinically, lung nodules and fever occurred in 3 of the 6 patients within 14 days of the peak neutrophil count. They were not treated with steroids, but rather with antibiotics, and in all cases resolved. Other patients on this trial have developed Sweet's syndrome during the neutrophil surge. CEBPa transcript levels in Molm14 cells (an AML cell line with a FLT3/ITD mutation) rose 3–5-fold over baseline following treatment with AC220. This is consistent with our previously published findings, and suggests at least one mechanism for the observed release of the differentiation block observed in the AC220-treated patients. These clinical and correlative laboratory results suggest that effective, sustained in vivo FLT3 inhibition in AML patients with FLT3/ITD mutations induces terminal differentiation in blasts in many ways similar to that seen with all trans retinoic acid in acute promyelocytic leukemia. Furthermore, these findings demonstrate the direct link between the growth factor receptor pathway and control of differentiation, and provide new insight into mechanisms of leukemogenesis. Disclosures: Levis: Ambit Biosciences, Inc: Consultancy.

ASXL1 Mutations Promote Myeloid Transformation Through Inhibition of PRC2-Mediated Gene Repression

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 405-405 ◽  
Author(s):  
Omar Abdel-Wahab ◽  
Mazhar Adli ◽  
Lindsay Saunders ◽  
Jie Gao ◽  
Alan H. Shih ◽  
...  
Keyword(s):  
Gene Expression ◽  
Research Funding ◽  
Hematopoietic Cells ◽  
Loss Of Function ◽  
Genome Wide ◽  
Hoxa Gene Expression ◽  
Primary Leukemia ◽  
Hoxa Gene

Abstract Abstract 405 Somatic mutations in ASXL1 have been identified in patients with myeloid malignancies and are associated with worsened overall survival in AML and MDS patients. However the mechanisms of myeloid transformation of ASXL1 mutations had not been delineated. We therefore performed extensive in vitro and in vivo studies to assess the functional implications of ASXL1 mutations in the hematopoietic compartment. Transcriptional and Western blot analysis demonstrated loss of ASXL1 protein in primary leukemia samples with endogenous ASXL1 mutations indicating that these mutations are loss-of-function disease alleles. Further, ASXL1 depletion by shRNA in normal and malignant hematopoietic cells leads to robust upregulation of a set of genes including the posterior HOXA cluster (HoxA5-HoxA13). Increased HoxA gene expression was confirmed in human hematopoietic stem progenitor cells targeted with ASXL1 siRNA and in mice with conditional deletion of Asxl1 in the hematopoietic compartment. Previous studies in Drosophila had revealed that Asxl forms the polycomb-repressive deubiquitinase (PR-DUB) complex with BAP1, which normally opposes the function of polycomb repressive complex 1 (PRC1) by removing H2AK119 ubiquitination. We verified that wild-type, but not mutant ASXL1 associates with BAP1 in co-immunoprecipitation studies. However, BAP1 depletion in hematopoietic cells did not result in significant changes in HoxA gene expression, suggesting that ASXL1 regulates gene expression in hematopoietic cells independent of its role in the PR-DUB complex. We therefore performed CHIP sequencing for known activating and repressive chromatin marks and histone mass spectrometry to elucidate the genome-wide effects of ASXL1 loss on chromatin state in hematopoietic cells. This allowed us to show that ASXL1 loss resulted in genome-wide loss of the transcriptionally repressive mark H3K27me3 in hematopoietic cells and primary patient samples with ASXL1 mutations. These data were supported by western blot analysis and histone mass spectrometry demonstrating a significant loss of H3K27 trimethylation in ASXL1-mutant cells. Moreover, ASXL1 mutations in primary leukemia samples are characterized by loss of H3K27 trimethylation at the HoxA locus. These data led us to hypothesize that ASXL1 interacts with the PRC2 complex; co-immunoprecipitation studies revealed that ASXL1 associates with members of the PRC2 complex including EZH2 and SUZ12 but not with the PRC1 repressive complex. Importantly, ASXL1 downregulation resulted in loss of EZH2 recruitment to the HOXA locus indicating a role of ASXL1 in recruiting the PRC2 complex to known leukemogenic loci. We next assessed the effects of ASXL1 loss in vivo by generating a conditional knock-out model of ASXL1 and also by employing shRNA to deplete ASXL1 in hematopoietic cells expressing the NRASG12D oncogene. Consonant with the in vitro data, we observed HOXA9 overexpression with ASXL1 loss/depletion in vivo. Preliminary analysis reveals that conditional, hematopoietic specific ASXL1-knockout (ASXL1fl/fl Vav-Cre) mice are characterized by progressive expansion of LSK and myeloid progenitor cells in mice less than 6 months of age. After 6 months of age a significant proportion of ASXL1fl/fl Vav-Cre mice developed leukocytosis, anemia, thrombocytopenia, and splenomegaly; pathologic analysis of tissues revealed a phenotype consistent with myelodysplasia with myeloproliferative features. Moreover, loss of ASXL1 in cooperation with expression of NRasG12D resulted in impaired survival, increased myeloproliferation, and progressive anemia consistent with MPN/MDS in vivo. Taken together, these results reveal that ASXL1 mutations result in a loss-of-function and suggest a specific role for ASXL1 in epigenetic regulation of gene expression by facilitating PRC2-mediated transcriptional repression of known leukemic oncogenes. Moreover, our in vivo data validate the importance of ASXL1 mutations in the pathogenesis of myeloid malignancies and provide insight into how mutations that inhibit PRC2 function contribute to myeloid transformation through epigenetic dysregulation of specific target genes. Disclosures: Carroll: Agios Pharmaceuticals: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Sanofi Aventis Corporation: Research Funding; Glaxo Smith Kline, Inc.: Research Funding.

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