Aryl Hydrocarbon Receptor (AhR) Antagonist Stemregenin 1 (SR1) Enhances in Vitro- and in Vivo-Derived Platelets (PLTs) From Human Megakaryocytes (MKs)

Abstract Abstract 3450 Our goal is to generate sufficient PLTs from ex vivo-generated MKs for clinical utility in PLT transfusions. A critical step in this process begins with ex vivo-generated hMKs and deriving clinically relevant PLTs. We reported that infused mature, high-ploidy murine (m) MKs derived from fetal liver (FL) cells increased PLT counts in recipient mice in a clinically relevant fashion, thus avoiding the need to generate ex vivo functional PLTs. We examined whether this strategy applies to hMKs derived from FL cells (gestational age, 17–21 weeks) and bone marrow (BM) cells in a xeno-transfusion model using immunodeficient NOD/SCID/IL-2Rγcnull (NSG) mice. Infused hPLTs isolated from blood had a half-life (T1/2) of 10 hours (hrs), compared to 24 hrs for infused murine PLTs. The hPLTs were functional in NSG mice as demonstrated by their incorporation into growing thrombi in situ. Both hFL hematopoietic mononuclear cells and hBM-CD34+ cells were cultured in serum-free media supplemented with optimized cytokine cocktails to generate hMKs. In contrast to the murine studies where the FL cell-derived mMKs were the most efficient source of derived mPLTs, FL cell-derived hMKs had low ploidy (0% ≥ 8N ploidy), gave rise to ∼16 PLTs/infused hMK, and had a short T1/2 (6 hrs). In contrast, 17% of hBM cell-derived MKs had a ploidy of ≥ 8N, and after infusion into NSG mice, resulted in a wave of MKs transiently entrapped in the pulmonary microvasculature and then over ∼0.5–3 hrs released PLTs with a T1/2 of 10 hrs, comparable to infused hPLTs. Maximally, we achieved a level of 5% of circulating total PLTs being derived from human cells with ∼32 PLTs/infused hMK. These hPLTs were normal in size, displayed normal levels of surface markers, were functional, and incorporated into growing thrombi. One strategy to increase hPLT yield is to expose developing MKs to drugs reported to increase MK maturation, thrombopoiesis, and/or facilitate hematopoietic progenitor cell expansion. Such drugs include dimethylfasudil (diMF) (an inhibitor of several kinases involved in polyploidization), UNC0638 (a G9a histone methyltransferase inhibitor), SR1 (an AhR antagonist), and nicotinamide (a sirtuin histone/protein deacetylases inhibitor). Although diMF promoted size and polyploidization of hMKs, diMF markedly worsened yield of PLTs/infused hMK and decreased PLTs T1/2 in vivo. UNC0638 led to significant cell expansion, but lowered hMKs ploidy and PLTs/infused hMK yield. Nicotinamide increased maturation, size and polyploidization of hMKs, but PLT release following MK infusion needs further study. Of note, SR1 that has been reported to promote the expansion of human HSC, not only increased size and ploidy of hMKs, but also hPLT release in vitro and in vivo. SR1-treated hMKs resulted in a 3-fold increased yield of normal size, T1/2 and functional PLTs/infused hMK compared to a DMSO-treated control. In summary, like mMKs, infused hMKs into mice release PLTs in the pulmonary vasculature though at a lower efficiency. Released hPLTs were functional and T1/2 was as expected. diMF enhanced MK ploidy, but worsened PLT yield and T1/2, while an AhR antagonist SR1 that also improved MK ploidy appears to markedly enhance yield of PLT/infused hMK, while maintaining T1/2. The ability of SR1 to enhance PLT release from induced pluripotent stem cells (iPSCs)-derived MKs remains to be tested, but this drug appears to be a strong candidate for a therapeutic strategy to take ex vivo-grown hMKs and generate PLTs in clinical relevant numbers. Disclosures: No relevant conflicts of interest to declare.

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Screen for Small Molecules Capable of Expanding Human Hematopoietic Stem Cell Ex Vivo

Blood ◽

10.1182/blood.v118.21.1919.1919 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1919-1919

Author(s):

Iman Hatem Fares ◽

Jalila Chagraoui ◽

Jana Krosl ◽

Denis-Claude Roy ◽

Sandra Cohen ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Fold Increase ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Abstract 1919 Hematopoietic stem cell (HSC) transplantation is a life saving procedure whose applicability is restricted by the lack of suitable donors, by poor responsiveness to mobilization regimens in preparation of autologous transplantations, by insufficient HSC numbers in individual cord blood units, and by the inability to sufficiently amplify HSCs ex vivo. Characterization of Stemregenin (SR1), an aryl hydrocarbon receptor (AHR) antagonist that promotes HSC expansion, provided a proof of principle that low molecular weight (LMW) compounds have the ability to promote HSC expansion. To identify novel putative agonists of HSC self-renewal, we initiated a high throughput screen (HTS) of a library comprising more than 5,000 LMW molecules using the in vitro maintenance of the CD34+CD45RA- phenotype as a model system. Our study was based on the fact that mobilized peripheral blood-derived CD34+CD45RA- cells cultured in media supplemented with: stem cell factor, thrombopoietin, FLT3 ligand and interleukin 6, would promote the expansion of mononuclear cells (MNC) concomitant with a decrease in CD34+CD45RA- population and HSC depletion. LMW compounds preventing this loss could therefore act as agonists of HSC expansion. In a 384-well plate, 2000 CD34+cells were initially cultured/well in 50μl medium comprising 1μM test compounds or 0.1% DMSO (vehicle). The proportions of CD34+CD45RA− cells were determined at the initiation of experiment and after a 7-day incubation. Six of 5,280 LMW compounds (0.11%) promoted CD34+CD45RA− cell expansion, and seventeen (0.32%) enhanced differentiation as determined by the increase in proportions of CD34−CD45RA+ cells compared to control (DMSO). The 6 LMW compounds promoting expansion of the CD34+CD45RA− cell population were re-analyzed in a secondary screen. Four out of these 6 molecules suppressed the transcriptional activity of AHR, suggesting that these compounds share the same molecular pathway as SR1 in stimulating HSC expansion, thus they were not further characterized. The remaining 2 compounds promoted, similar to SR1 or better, a 10-fold and 35-fold expansion of MNC during 7 and 12-day incubations, respectively. The expanded cell populations comprised 65–75% of CD34+ cells compared to 12–30% determined for DMSO controls. During 12-day incubation with these compounds, the numbers of CD34+ cells increased ∼25-fold over their input values, or ∼ 6-fold above the values determined for controls. This expansion of CD34+ cells was associated with a ∼5-fold increase in the numbers of multilineage CFC (granulocyte, erythroid, monocyte, and megakaryocyte, or CFU-GEMM) compared to that found in DMSO control cultures. The ability of the 2 newly identified compounds to expand functional HSCs is currently being evaluated in vivo usingimmunocompromised mice. In conclusion, results of our initial screen suggest that other mechanism, besides inhibition of AhR, are at play for expansion of human HSC. Disclosures: No relevant conflicts of interest to declare.

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Assessment of antitumor function of NK cells expanded with exosomes from K562.mb21 cells.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.7_suppl.132 ◽

2017 ◽

Vol 35 (7_suppl) ◽

pp. 132-132 ◽

Cited By ~ 1

Author(s):

Jeremiah Oyer ◽

Sarah B. Gitto ◽

Sara Khederzadeh ◽

Kari Shaver ◽

Dean A. Lee ◽

...

Keyword(s):

Nk Cells ◽

Cell Expansion ◽

Nk Cell ◽

Ex Vivo ◽

K562 Cells ◽

In Vitro Cytotoxicity ◽

Feeder Cell ◽

Nsg Mice

132 Background: NK cells can kill malignant cells to provide innate immunity against tumors. Due to their low abundance in blood, a focus is to expand NK cells ex vivo having enhanced anti-tumor cytotoxicity to be used as a treatment. Our group has pioneered a cell-free method using plasma membrane (PM) particles derived from K562 cells expressing 41BBL and membrane-bound IL-21 (K562.mb21) which were developed for NK cell expansion. Compared to feeder cell based methods for NK cell expansion, PM21-particles improve safety and allow for potential wide-spread dissemination, and also allows direct in vivo use. Exosomes, vesicles naturally secreted by cells, may yet be another novel feeder cell free way for NK cell expansion and may have further advantageous therapeutic dimensions. Methods: EX21-exosomes and PM21-particles were prepared from K562.mb21 cells and characterized by Nanosight and Western blot analysis. CD3-depleted PBMCs were cultured with EX21 for 14 days, NK cell amounts were monitored and media changed every 2-3 days. In vitro cytotoxicity against K562 cells were comparatively assessed for EX21-NK cells and PM21-NK cells. In vivo anti-tumor efficacy of EX21- and PM21-NK cells was assessed in NSG mice implanted ip with SKOV3_luc ovarian tumor cells (1 x 106 cells seeded for 4 days). SKOV3-bearing mice were treated with vehicle, or two doses of EX21-NK cells or PM21-NK cells (1 x 107, in 5 day intervals), and with or without in vivo administration of EX21 (10 µg, 3x/week) or PM21-particles (600 µg, 3x/week). All groups were injected ip with IL-2 (10 KU, 3x/week). Survival analysis was performed with a Log-rank (Mantel-Cox) test. Results: NK cells cultured with EX21 expanded 530 fold (344-710) over 14 days compared to 735 fold (667-802) in presence of PM21-particles. Treatment of SKOV3 engrafted NSG mice with NK cells, expanded with either EX21 or with PM21, allowed significant ( < 0.0001) increase in survival compared to untreated animals (41-44 vs 29 days post treatment). Ip delivery of EX21 to SKOV3 bearing mice had no effect on survival in either untreated control or EX21-NK cell treated groups. Conclusions: EX21 efficiently expands NK cells and EX21-NK cells have equal anti-tumor effect as PM21-NK cells, both in vitro and in vivo.

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Therapeutic Targeting of CCR1 to Prevent Dissemination of Multiple Myeloma Plasma Cells

Blood ◽

10.1182/blood-2019-121974 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3099-3099

Author(s):

Mara N Zeissig ◽

Duncan R Hewett ◽

Krzysztof M Mrozik ◽

Vasilios Panagopoulos ◽

Monika Engelhardt ◽

...

Keyword(s):

Flow Cytometry ◽

Disease Progression ◽

Mononuclear Cells ◽

Plasma Cells ◽

Primary Tumour ◽

Nsg Mice ◽

Rpmi 8226 ◽

Retention Signal

Introduction:Multiple myeloma (MM) disease progression is dependent on the ability of the MM plasma cells (PC) to leave the bone marrow (BM), re-enter the peripheral blood (PB) and disseminate to other BM sites. Previous studies show that expression of CXCL12 by BM stromal cells is crucial for MM PC retention within the BM. However, the mechanisms which overcome this retention signal enabling MM PC egress and dissemination via the PB are poorly understood. Previous studies in haematopoietic progenitor cells have demonstrated that CCL3 overcomes the CXCL12 retention signal to drive mobilisation to the PB (Lord et al. Blood 1995). Here, we examined the role of the CCL3 chemokine receptor CCR1 in driving MM PC dissemination. Methods and results: Initially, we assessed the expression of CCR1 protein on CD138+CD38++CD45loCD19- PC from 28 MM, 8 MGUS and 2 SMM patients by flow cytometry. Results show CCR1 expression is significantly increased in newly diagnosed MM compared with premalignant MGUS and SMM patients (p=0.03; CCR1 MFI mean±SEM, MGUS: 53.0±33.6; SMM: 37.6±8.9 MM: 250.9±71.6). Furthermore, CCR1 expression on PB MM PC positively correlated with PB MM PC numbers (p=0.03; n=11 patients). To identify mechanistically how CCR1 may promote dissemination, the effect of CCL3 on the response to CXCL12 in human myeloma cell lines (HMCL) was assessed in vitro. The migration of RPMI-8226 and OPM2 cells was induced by CCL3 or CXCL12 chemoattractant in a transwell assay. Notably, pre-treatment of RPMI-8226 or OPM2 with CCL3 abrogated migration towards CXCL12 and blocked F-actin remodelling in response to CXCL12 in vitro. These findings suggest that CCL3 can desensitise cells to exogenous CXCL12, providing a potential mechanism facilitating loss of the CXCL12 retention signal. To confirm whether CCR1 is required for driving MM PC dissemination, homozygous CCR1 knockout (KO) cells were generated using a lentiviral CRISPR/Cas9 system in OPM2 cells. CCR1-KO OPM2 cells were confirmed to have no detectable CCR1 expression by flow cytometry and could no longer migrate towards CCL3 in vitro. Empty vector (EV) or CCR1-KO OPM2 MM PC were injected into the tibia of immune-compromised NOD-scidgamma (NSG) mice. After 4 weeks, primary tumour within the injected tibia and disseminated tumour in the PB and the contralateral tibia and femur was assessed by flow cytometry. We found that mice bearing CCR1-KO cells have a 45.5% decrease in primary tumour growth (p=0.008; % GFP+ of total mononuclear cells, EV: 77.2±17.2; CCR1-KO: 42.1±24.4), a 97.8% reduction in PB MM PC (p<0.0001; EV: 1.39±0.7; CCR1-KO: 0.03±0.046) anda 99.9% reduction in BM tumour dissemination (p<0.0001; EV: 49.5±17; CCR1-KO: 0.019±0.013), compared with controls. In a supportive study, CCR1 was expressed in the murine MM cell line 5TGM1 using lentiviral transduction. 5TGM1-CCR1 cells were confirmed to express CCR1 by qPCR and were able to migrate towards CCL3 in vitro. 5TGM1-CCR1 or EV cells were injected into the tibiae of C57BL/KaLwRij mice and allowed to initiate systemic MM disease for 3.5 weeks. Importantly, while 55% of control mice exhibited disseminated tumours, this increased to 92% with CCR1 expression (p<0.0001; n=12/group). These data suggest that CCR1 expression on MM PC may play an important role in MM PC dissemination. To determine whether therapeutic inhibition of CCR1 prevents dissemination, the effect of a small molecule CCR1 inhibitor, CCR1i, was assessed in vivo. OPM2 EV or RPMI-8226 cells were injected into the tibia of NSG mice and, after 3 days, mice were treated with CCR1i (15mg/kg) or vehicle twice daily by oral gavage for 25 days. OPM2-inoculated CCR1i-treated mice had 66.1% lower PB MM PC (p<0.0001; % GFP+ of total mononuclear cells, vehicle: 23.9±7.2; CCR1i: 8.1±3.8) and a 22.1% reduction in BM dissemination (p=0.0002; vehicle: 78.1±4.8;CCR1i: 60.8±7.1) compared with controls. Similarly, CCR1i treatment reduced BM dissemination by 59.6% in RPMI-8226 bearing mice (p<0.0001; % GFP+ of total mononuclear cells, vehicle: 0.86±0.15; CCR1i: 0.26±0.05). This suggests that CCR1 inhibition can slow tumour dissemination in vivo. Conclusion:This study identified CCR1 as a novel driver of MM PC dissemination in vivo, at least in part by overcoming the CXCL12 retention signal. Importantly, this study demonstrated for the first time that targeting CCR1 can be a viable therapeutic strategy to limit dissemination and potentially slow disease progression. Disclosures Croucher: Trovagene: Employment.

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Activation of Notch Signaling Mediates Ex Vivo Expansion of Multilineage, Engrafting Murine Hematopoietic Progenitors From Embryonic Sources.

Blood ◽

10.1182/blood.v120.21.2321.2321 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2321-2321

Author(s):

Brandon K Hadland ◽

Barbara Varnum-Finney ◽

Irwin D. Bernstein

Keyword(s):

Notch Signaling ◽

Ex Vivo ◽

Fetal Liver ◽

Signal Pathways ◽

Mouse Bone Marrow ◽

Human Cord Blood ◽

Hematopoietic Stem ◽

Notch Activation

Abstract Abstract 2321 An important goal in the application of pluripotent stem cells (PSC) for therapeutic purposes is the derivation of hematopoietic stem and progenitor cells (HSPC) capable of efficient engraftment in vivo. Fundamental to achieving this goal is improved understanding of key signal pathways required to establish, maintain and expand HSPCs from embryonic sources. Ex vivo activation of Notch signaling in mouse bone marrow and human cord blood-derived HSC can facilitate expansion of rapidly engrafting multilineage progenitors, which has recently been translated for therapeutic purposes. In contrast, similar expansion of engrafting progenitors has not been successful from PSC. This prompted us to evaluate whether embryonic-derived HSPC have capacity to respond to ligand-induced Notch signaling ex vivo, and whether Notch activation could promote expansion of engrafting progenitors from these embryonic sources. We have examined the effects of ex vivo activation of Notch receptors by immobilized, exogenous Notch ligands on highly enriched populations of embryonic HSC and HSC precursors (pre-HSC) at various developmental stages. We find that activation of Notch by the ligand Delta1 within HSC/pre-HSC isolated from embryonic aorta-gonad-mesonephros (AGM) promotes expansion of progenitors with erythromyeloid colony forming potential and T/B-lymphoid potential in vitro, with concurrent expression of surface phenotypes resembling fetal liver-stage HSC. Furthermore, Notch activation in embryonic HSPC also mediates expansion of progenitors with rapidly engrafting myeloid and lymphoid capacity in irradiated mouse models. Our results demonstrate that embryonic stage HSPC have capacity to expand in response to Notch activation, and thus further studies comparing AGM- and PSC-derived hematopoietic precursors are needed to elucidate differences that may account for failure to expand HSPC from PSC. Disclosures: Bernstein: Seattle Genetics, Inc.: Consultancy.

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Insights Into Thrombopoiesis From Infused Human Megakaryocytes Into Mice

Blood ◽

10.1182/blood.v122.21.1165.1165 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1165-1165

Author(s):

Yuhuan Wang ◽

Vincent M. Hayes ◽

Prasuna Paluru ◽

Stella T. Chou ◽

Deborah L. French ◽

...

Keyword(s):

Half Life ◽

Size Range ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Human Donor ◽

Nsg Mice ◽

Infusion Study ◽

Recipient Animal ◽

Post Infusion

Abstract Thrombopoiesis is the process by which megakaryocytes (Megs) release platelets (Plts), but issues remain as to the detailed in vivo mechanisms underlying this process. We now report new insights into this process by studying infused human Megs into immunocompromized NOD/SCID, gamma-interferon deleted (NSG) mice. Prior in situ microscopy has suggested that Megs release varied-size cytoplasmic fragments up to whole Megs in size into the medullary vascular space. Other studies have suggested that at least a portion of thrombopoiesis occurs by Megs lodged in the lungs. We previously infused ex vivo-generated murine Megs into mice and found that these Megs become entrapped in the animals’ lungs, and in <1.5 hrs, release functional Plts (termed here “Meg-Plts”) that have a similar half-life as infused mouse donor-derived Plts (termed here “Donor-Plts”). To better understand the biology of thrombopoiesis, we have infused ex vivo-generated human Megs into NSG mice. These studies replicated many of the observations seen with infused murine Megs: Human Megs were entrapped in the lungs with delayed release of human Meg-Plts, and these Meg-Plts had the same half-life as infused human Donor-Plts. Human Plts differ from murine Plts in size so this parameter was analyzed following infusion of human Megs using forward cell scatter analysis. We noted that 10 mins post-infusion, the Meg-Plt size range was wide and displayed a non-bell-shaped distribution. This distribution was in contrast to the tight bell-shaped curves seen for the endogenous murine Plts and for infused human Donor-Plts. However, by 3 hrs post-human Meg infusion - at the time of peak Meg-Plt counts - the human Meg-Plts now displayed an identical bell-shaped distribution curve as infused human Donor-Plt. The smaller, human Meg-Plts had disappeared. The size and distribution of these Meg-Plts then remained near identical to Donor-Plts for the remaining portion of the 48 hr post-infusion study. However, after impairing macrophage clearance in NSG recipient mice with clodronate-ladened liposome infusion, the small Meg-Plts did not disappear and were present at 48 hrs. Using thiazole orange (TO) to stain platelets for RNA content, we noted that ∼70% of all Meg-Plts were initially TO+ compared to the steady-state of ∼10% for mouse endogenous platelets. This high TO+ state decreased to near 10% by 24 hrs post-infusion. Up to ∼6 hrs, all of the large Meg-Plts were TO+, while the smaller-sized Meg-Plts were predominantly TO-. Unless the mice were treated with clodronate-ladened liposomes, these TO-, small Meg-Plts disappeared before 6 hrs. In conclusion, these data support that ex vivo-generated human Megs release physiologic platelets in the pulmonary vascular bed of NSG mice with the same size range/distribution and survival as infused human Donor-Plts. Mean Meg-Plt size depends on the species of origin of the infused Megs rather than on the species of the recipient animal. We did not detect large Meg cytoplasmic fragments that underwent further size reduction although our technique may not be capable of detecting small numbers of such fragments or the small size changes that would accompany platelet maturation from preplatelets. Our data also suggest that Megs generated in culture release a wide size range of non-physiologic Plt-like particles that when infused are cleared rapidly by macrophages. Disclosures: No relevant conflicts of interest to declare.

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A Whole Genome In Vivo Crispr Screen in Primary ALL Predicts Leukaemic Relapse

Blood ◽

10.1182/blood.v126.23.2619.2619 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2619-2619

Author(s):

Katherine Dormon ◽

Elda S Latif ◽

Matthew Bashton ◽

Deepali Pal ◽

Matthew Selby ◽

...

Keyword(s):

Drug Resistance ◽

Conflicts Of Interest ◽

Gene Set Enrichment Analysis ◽

Whole Genome ◽

Competitive Situation ◽

Nsg Mice ◽

A Genome ◽

Crispr Screen

Abstract Although paediatric acute lymphoblastic leukaemia (ALL) has a favourable prognosis, a number of cases will invariably relapse. One of the major problems associated with relapse is drug resistance, in particular to glucocorticoids, the mainstay of ALL treatment. Examining the underlying mechanisms is complicated by clonal heterogeneity within a patient and the potential impact of the leukaemic niche. To address mechanisms of drug resistance in a patient-relevant setting, we performed a genome-wide in vivo CRISPR screen in primary ALL material. To that end, we took advantage of primografted material from patient L707, who initially presented with a Dexamethasone (DEX) sensitive t(17;19) ALL, but relapsed 5 months after initial diagnosis. We transduced DEX sensitive presentation cells with the full genome GeCKOv2 CRISPR library, before transplantation into immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Mice were subsequently treated with DEX by oral gavage (15mg/kg for 5 weeks, 10mg/kg thereafter). DNA from several engrafted sites in the mouse was extracted and PCR amplified before being sequenced on the Illumina HiSeq2500. Changes in pool complexity were analysed using MaGEcK software to determine which sgRNAs were significantly enriched or depleted. By far the most significantly enriched sgRNAs were those targeting NR3C1, the gene encoding the glucocorticoid receptor. In addition, two of the top five significantly depleted sgRNAs targeted the Plexins, PLXNA1 and PLXND1. Whilst PLXNA1 is expressed at low levels, PLXND1 is highly expressed and has been linked to dexamethasone resistance. Notably, the matched relapsed material from L707 was highly DEX resistant both in tissue culture and when transplanted into NSG mice. SNP 6.0 analysis revealed a 5q deletion in the relapse, spanning 5 genes including NR3C1. Whole genome sequencing showed this was comprised of 2 deletions both targeting NR3C1, with different breakpoints for each allele. The differential gene expression between the L707 presentation and relapse established that NR3C1 was the most significant of all the genes lost at relapse, based on gene set enrichment analysis (GSEA). This contrasts with many ALL cases, where one of the downstream effectors of apoptosis is lost as opposed to NR3C1. Growth of the relapse material in vivo and in vitro was slower than the presentation in a competitive situation, but with DEX treatment the relapse phenotype began to emerge with a small percentage of cells showing a heterozygous deletion of NR3C1. These combined data strongly suggest that the NR3C1 deletion is the main driver of DEX resistance in the L707 relapse. Moreover, it proves that our in vivo CRISPR screen predicted the leukaemic relapse. These results confirm NR3C1 deletion as a driver in glucocorticoid resistance and demonstrate the power of in vivo CRISPR screens to predict mechanisms of gain of drug resistance and subsequent relapse. The parallels that can be drawn between the relapse and the CRISPR screen are striking, giving the indication that the progression from presentation to relapse may follow the same path in a patient derived xenograft setting as it did in the patient. Disclosures No relevant conflicts of interest to declare.

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CD138 Regulates Myeloma Cell Survival, Proliferation, BM Engraftment and Tumor Organization

Blood ◽

10.1182/blood.v126.23.2974.2974 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2974-2974

Author(s):

David R Fooksman ◽

Amitabha Mazumder ◽

Mark McCarron

Keyword(s):

Plasma Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Early Stage ◽

Serum Starvation ◽

High Dose ◽

Rapid Reduction ◽

Myeloma Cells

Abstract Multiple myeloma is the 2nd most common blood cancer in adults with a median survival time of 5 years despite high-dose chemotherapy and bone marrow transplantation interventions. Syndecan-1 or CD138, is a heparan-sulfate coated glycoprotein, which is highly expressed on the surface of plasma cells and myeloma cells, important for adhesion and accumulating survival signals. Expression of CD138 is heterogeneous in myeloma tumors, in vivo and in vitro leading some to speculate it may distinguish stem-like subpopulations. While this role is highly disputed, we investigated the effect of CD138 expression on tumor pathology in vivo. To characterize CD138neg and CD138high subpopulations, we used GFP+ Vk*myc myeloma model from Leif Bergsagel, which develops myeloma tumors in BM and spleen of C57Bl/6 mice. We found CD138high populations were more proliferative in vivo based on EdU incorporation experiments. We transferred equal numbers of sorted subpopulations into hosts and found that CD138high cells generated larger tumors in the BM than CD138neg cells after 12 weeks. Analysis of these tumor-bearing mice revealed that all tumors contained both subpopulations, indicating that these two subsets are hierarchically equivalent. We find that in mice with small tumors, the majority of cells (80% or more) are CD138high cells, while in large tumors, the level drops (to 30-50% of tumor) with higher composition of CD138neg cells. We also find lower CD138 levels on myeloma cells found in the blood compared to BM. Using intravital two-photon time-lapse imaging in the tibial BM, we find that tumor cells from smaller, early stage tumors are physically arrested within the BM parenchyma, while in larger, more advanced tumors, myeloma cells are more motile and active. CD138neg cells were more apoptotic based on ex vivo Annexin V staining following serum starvation. Interestingly, serum starvation led to rapid reduction in CD138 surface expression. Taken together, we propose a model where CD138 expression regulates localization and survival in the BM niche, but is downregulated from the plasma membrane when tumor size outgrows the necessary resources, allowing myeloma cells to migrate and metastasize to distant new locations. Disclosures No relevant conflicts of interest to declare.

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In vivo monitoring of JAK/STAT and PI3K/mTOR signal transduction inhibition in pediatric CRLF2-rearranged acute lymphoblastic leukemia (ALL).

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.9506 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 9506-9506

Author(s):

Sarah Kathleen Tasian ◽

Shannon L. Maude ◽

Junior Hall ◽

Tiffaney Vincent ◽

Charles Grenfell Mullighan ◽

...

Keyword(s):

Signal Transduction ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Nsg Mice ◽

Signal Transduction Inhibitors ◽

Background Therapy ◽

Pediatric All ◽

Therapeutic Responses

9506 Background: Therapy intensification for children with B-precursor ALL with high-risk genetic lesions has improved relapse-free survival. CRLF2 rearrangements and JAK2 and IL7RA mutations occur in 10-15% of adult and pediatric ALL patients, most of whom relapse. We and others identified aberrant kinase signatures and perturbed JAK/STAT and PI3K/mTOR signal transduction via in vitro studies of CRLF2-rearranged (CRLF2r) ALLs, suggesting the therapeutic relevance of signal transduction inhibitors (STIs). Our creation of CRLF2r ALL xenograft models has enabled rapid preclinical testing of STIs and measurement of in vivo target inhibition. We hypothesized that inhibition of JAK/STAT and PI3K/mTOR phosphosignaling correlates with therapeutic responses in these models. Methods: NOD/SCID/γc-null (NSG) mice well-engrafted with pediatric ALL samples were treated with the JAK inhibitor ruxolitinib, the mTOR inhibitor sirolimus, or vehicle for 72 hours (for signaling response) or 4 weeks (for therapeutic response). Splenocytes were briefly stimulated ex vivo with thymic stromal lymphopoietin (ligand for CRLF2) and stained with human-specific surface and intracellular phosphoantibodies for multi-parameter phosphoflow cytometry analysis. Results: Ruxolitinib-induced inhibition of phospho (p)-JAK2 and pSTAT5 was most pronounced in non-CRLF2r ALLs with novel JAK2-activating BCR-JAK2 and IL7RA/LNK mutations. Sirolimus potently inhibited pS6 and other PI3K/mTOR pathway phosphoproteins in the CRLF2r r ALLs. PSTAT5 and pS6 inhibition correlated with longer-term ruxolitinib- and sirolimus-induced decreases in ALL cell burden, demonstrating therapeutic responses to STIs. Conclusions: Ruxolitinib inhibited JAK/STAT phosphosignaling and markedly decreased leukemic burden in the JAK2-activating BCR-JAK2 and IL7RA/LNK mutant ALL xenografts. Sirolimus potently inhibited PI3K/mTOR (as well as some JAK/STAT) phosphosignaling and had greater therapeutic efficacy than ruxolitinib in the CRLF2r ALLs. The safety of ruxolitinib and of temsirolimus with cytotoxic chemotherapy are currently being established in Children’s Oncology Group Phase I trials.

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The Influence of Methylsulfonylmethane on Inflammation-Associated Cytokine Release before and following Strenuous Exercise

Journal of Sports Medicine ◽

10.1155/2016/7498359 ◽

2016 ◽

Vol 2016 ◽

pp. 1-9 ◽

Cited By ~ 8

Author(s):

Mariè van der Merwe ◽

Richard J. Bloomer

Keyword(s):

Whole Blood ◽

Mononuclear Cells ◽

Ex Vivo ◽

Knee Extension ◽

Strenuous Exercise ◽

Peripheral Blood Mononuclear ◽

Physically Active ◽

Inflammatory Molecules

Background. Inflammation is associated with strenuous exercise and methylsulfonylmethane (MSM) has been shown to have anti-inflammatory properties.Methods. Physically active men were supplemented with either placebo or MSM (3 grams per day) for 28 days before performing 100 repetitions of eccentric knee extension exercise.Ex vivoandin vitrotesting consisted of evaluating cytokine production in blood (whole blood and isolated peripheral blood mononuclear cells (PBMCs)) exposed to lipopolysaccharide (LPS), before and through 72 hours after exercise, whilein vivotesting included the evaluation of cytokines before and through 72 hours after exercise.Results. LPS stimulation of whole blood after MSM supplementation resulted in decreased induction of IL-1β, with no effect on IL-6, TNF-α, or IL-8. After exercise, there was a reduced response to LPS in the placebo, but MSM resulted in robust release of IL-6 and TNF-α. A small decrease in resting levels of proinflammatory cytokines was noted with MSM, while an acute postexercise increase in IL-10 was observed with MSM.Conclusion. Strenuous exercise causes a robust inflammatory reaction that precludes the cells from efficiently responding to additional stimuli. MSM appears to dampen the release of inflammatory molecules in response to exercise, resulting in a less incendiary environment, allowing cells to still have the capacity to mount an appropriate response to an additional stimulus after exercise.

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Enhancing Functional Platelet Release In Vivo from in Vitro-Grown Megakaryocytes Using the Protein Kinase Inhibitor SU6656

Blood ◽

10.1182/blood.v128.22.1029.1029 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1029-1029

Author(s):

Danuta Jadwiga Jarocha ◽

Karen K Vo ◽

Randolph B Lyde ◽

Vincent M Hayes ◽

Mortimer Poncz

Keyword(s):

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Specific Inhibitor ◽

Annexin V ◽

Rock Inhibitor ◽

Medicine Research ◽

Nsg Mice ◽

Platelet Release

Abstract The clinical demand for platelet transfusions is increasing, threatening the ability to obtain sufficient healthy donors to provide these platelets. Advances in regenerative medicine research have opened the possibility of generating sufficient in vitro-grown megakaryocytes and consequent platelets to supply a portion of the clinical platelet transfusion demand. We have shown that infusing megakaryocytes for obtaining released, functional platelets is a viable alternative strategy than trying to release platelets in vitro. However, for both approaches, in vitro-cultured megakaryocytes have lower ploidy and release fewer platelets than likely occurs in vivo by primary cells. SU6656 inhibitor, a Src kinase inhibitor, has been shown to influence ploidization in several megakaryocyte-like line with purported increase in proplatelets release. However, in our hands, other agents - such as the ROCK inhibitor Y27632 - while increasing polyploidization markedly, inhibited platelet release per infused megakaryocyte in vivo. We grew megakaryocytes from CD34+ cells for 12 days with or without SU6656 (2.5 µM) supplementation during the last 4 days. We found that the SU6656 inhibitor only increased the number of CD34+-derived megakaryocytes by ~15% at the end of the 12 day growth, but more markedly increase the percent of large megakaryocytes measured by FSC parameter in flow cytometry evaluation from 28 up to 41% and percent of high granular megakaryocytes from 27 to 45%. These changes were accompanied with a shift in average ploidy from 4.9 to 6.9 (p<0.0003, N=6). Notably, SU6656-treated megakaryocytes released ~4-fold more platelets per infused megakaryocytes in immunocompromized NSG mice than untreated similarly in vitro-grown megakaryocytes. By 24 hrs, there were 6.5-fold platelets from the infused SU6656-treated megakaryocytes than control untreated (p<0.037, N=6). Released platelets from the drug-treated and untreated megakaryocytes had similar levels of percent thiazole orange positivity as an indication that they were young platelets. Importantly, baseline annexin V, CD62p and PAC1 binding prior to agonist exposure were also similarly and increased to the same extent after thrombin (1U/ml) stimulation. Additionally, incorporation into a growing cremaster laser injury-induced thrombus in vivo was similar further indicating retained function by the platelets released from the drug-treated megakaryocytes. A number of strategies such as modifying the level of transcription factors have been proposed to increase the size, ploidy or proplatelets release from in vitro-grown megakaryocytes. In none of these cases have these released platelets in vivo biology been examined and demonstrated to replicate high release number per megakaryocyte and retained functionality. We show that terminal exposure of in vitro-grown megakaryocytes to the non-specific inhibitor SU6656 significantly increases in vivo yield while leaving in vivo half-life and functionality intact. The exact pathway affected by SU6656 that leads to these results is now being pursued. Disclosures No relevant conflicts of interest to declare.

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