scholarly journals A CRISPR/Cas9 Library Screen in Patients' Leukemia Cells In Vivo

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3945-3945
Author(s):  
Ehsan Bahrami ◽  
Martin Becker ◽  
Anna-Katharina Wirth ◽  
Jan Phillip Schmid ◽  
Tobias Herold ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Lymphoblastic Leukemia ◽  
Drop Out ◽  
Leukemia Cells ◽  
Functional Genomic ◽  
Cell Homing ◽  
Nsg Mice ◽  
Crispr Screen

Background: Functional genomic screens elegantly increase our understanding of biology of leukemias. So far, CRISPR/Cas9 screens are widely performed in cell lines and in genetically engineered mouse models, in vitro and in vivo; here, we extended their use to patient-derived leukemia cells in vivo. Methods Serially transplantable patient-derived xenograft (PDX) models were generated from children and adults with acute lymphoblastic leukemia (ALL). Cas9 was stably expressed in PDX ALL cells using a split form of Cas9 assembled by inteins, facilitating lentiviral-mediated gene delivery. Customized sgRNA library was generated using golden gate cloning, at 5 sgRNAs per target gene. The sgRNA vector additionally expressed a fluorochrome marker and a tag, for sequential magnetic-activated cell sorting (MACS) and flow cytometry (FACS) enrichment of sgRNA transduced PDX cells. Highly enriched Cas9/sgRNA double transgenic cells were transplanted into NSG mice and animals sacrificed after different periods of time. Cells were re-isolated from bone marrow, purified and subjected to PCR-based amplification of sgRNA library followed by next generation sequencing. Differential sgRNA distributions were analysed using a MAGeCK pipeline. Results We aimed to establish a comprehensive CRISPR screen pipeline allowing functional genomic screens in patients' acute leukemia cells. We investigated surface molecules required for cell homing and growth in mice, using a distinct customized sgRNA library. Quality controls of the sgRNA plasmid pool as well as transgenic PDX input samples verified standard distribution of all sgRNAs. As knockout was required at the time point of transplantation, conditions for prolonged culture of PDX ALL cells in vitro were optimized. Before injection into NSG mice, transduced PDX ALL cells were successfully enriched to above 95% using MACS and FACS. Over time in vivo, deep sequencing of re-isolated PDX cells revealed unchanged distribution of control sgRNAs, but strong loss of sgRNAs targeting CXCR4 and ITGB1, suggesting that CXCR4 and ITGB1 might be required for PDX ALL cell homing and engraftment. To validate the findings of drop-out CRISPR screen, we analyzed single sgRNAs targeting CXCR4 and ITGB1 in PDX cells. Competitive in vivo assays monitored by recombinant fluorochrome markers showed that the cells with CXCR4 or ITGB1 knockout had a significant disadvantage in vivo with respect to homing and growth in mice, compared to the control population. Taken together, we established a comprehensive workflow for CRISPR screen in PDX model of ALL in vivo. Our data identify and validate that CXCR4 and ITGB1 are required for homing and growth of PDX ALL cells in mice. Conclusion We show that CRISPR/Cas9 functional genetic screens are feasible in PDX acute leukemia models in vivo and report the first such screen, as far as to our knowledge. Extending CRISPR/Cas9 screens to patients' cells will greatly facilitate our understanding of individual leukemia biology and therapeutic targets in the future. Disclosures Becker: AVA Lifescience GmbH: Consultancy.

The Effects of Anti-High Molecular Weight-Melanoma Associated Antigen (HMW-MAA) Monoclonal Antibodies (mAb) Against 11q23 Positive Acute Leukemia Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4550-4550
Author(s):  
Allison S. Drake ◽  
Brady T. Michael ◽  
Xin Hui Wang ◽  
Sheila J.N. Sait ◽  
Justin C. Earp ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Lymphoblastic Leukemia ◽  
Scid Mice ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
High Dose ◽  
Tumor Inhibition ◽  
Tyrosine Kinase 2

Abstract Acute leukemia with 11q23 aberrations is associated with especially poor outcome to standard treatment. HMW-MAA is a membrane bound proteoglycan that has been targeted in the treatment of malignant melanoma. It is widely distributed on 11q23-positive leukemic cells and is not expressed on normal hematopoietic cells. Finally, HMW-MAA is believed to function by signaling through the focal adhesion kinase (Fak)/proline-rich tyrosine kinase 2 (Pyk2) pathways and by supporting angiogenesis via its expression on pericytes. We hypothesized that inhibition of HMW-MAA with mAb directed against specific HMW-MAA determinants would effectively limit the growth of 11q23-positive acute leukemia cells in vitro and in vivo, by affecting Fak/Pyk2 pathways. We examined an 11q23-positive acute myeloid leukemia (AML) cell line (ML-2) and leukemia cells from two patients (one AML, one acute lymphoblastic leukemia) with 11q23 aberrations. Our results demonstrate that ML-2 cells expressed only the VT68.2 and 225.28 determinants, while primary acute leukemia cells expressed VT68.2, 225.28, and 763.74 determinants. Treatment of ML-2 cells with mAb against VT68.2 and 225.28 in vitro did not affect overall proliferation but downregulated phosphorylated (p) Pyk2 expression as determined by Western blotting. Further, anti-HMW-MAA mAb treatment enhanced the anti-proliferative effect of cytarabine (y=0.7875) in vitro. Based on these results, we then evaluated the dose-response relationships and toxicities of anti-HMW-MAA mAb alone and in combination with cytarabine in subcutaneous ML-2 xenografts grown in SCID mice. A trend towards tumor inhibition was noted following high dose (1 mg/kg 2x/week) anti-225 mAb therapy but was not seen at lower (0.25 and 0.5 mg/kg) anti-225 mAb doses. Mice demonstrated no side effects or weight loss as a result of anti-225 therapy. No downregulation of pPyk2 in tumor cells was observed following anti-225 therapy at any dose. Furthermore, combination anti-225.28 HMW-MAA mAb and cytarabine treatment (at increasing cytarabine doses) did not result in improved tumor inhibition as compared to cytarabine alone (p>0.05). We then evaluated the effects of HMW-MAA anti-225.28 and anti-763.74 mAb alone or together for treatment of NOD/SCID mice engrafted with primary 11q23-positive leukemia patient cells. Combination anti-HMW-MAA mAb therapy resulted in no prolongation in survival of engrafted mice as compared to control treated animals. We were able to confirm the presence of high levels of circulating HMW-MAA mAb in the serum of animals 4 days following mAb injection. In addition, explanted ML-2 cells harvested following xenograft growth still maintained expression of the HMW-MAA determinants. We conclude that naïve anti-HMW-MAA mAb neither caused a statistically significant inhibitory effect nor enhanced the effect of cytarabine on 11q23-positive acute leukemia cells in vivo. Future studies using radiolabelled or toxin-conjugated antibodies may enhance the anti-tumor effects of targeted HMW-MAA immunotherapy for 11q23-positive acute leukemia.

The Regulation of β-arrestin1 in Leukemia Initiating Cells of Children B-Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3538-3538
Author(s):  
Lin Zou ◽  
Shan Liu ◽  
Yi Shu ◽  
Ru Qin ◽  
Kang Li ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Mrna Expression ◽  
Promoter Region ◽  
Lymphoblastic Leukemia ◽  
The Self ◽  
Mrna Expression Level ◽  
Self Renewal ◽  
Nsg Mice

Abstract Background Leukemia is the most common malignant tumor in children under 15 years old. The main subtype of children leukemia is acute lymphoblastic leukemia (ALL), and B-lineage ALL (B-ALL) accounts for approximately 70%. The leukemia-initiating cells (LICs) are cancer stem cells with long-term repopulating potential and propagation ability, to maintain the leukemia cell phenotype, and possess leukemia-initiating activity. However, the regulation of LICs for the leukemia progression is poorly understood. The multifunctional scaffold proteins β-arrestins are proven to mediate H4 acetylation and gene expression. And β-arrestin2 is found to regulate the initiation and progression of chronic myeloid leukemia (CML). However, the role of β-arrestin1 in B-ALL is still unknown. Our preliminary data showed that both the high expression of β-arrestin1 and high proportion of CD34+CD38- cells are positively correlated with risk stratification and poor prognosis of childhood B-ALL. And β-arrestin1 binds with EZH2 to increase BCR/ABL H4 acetylation and thus promotes CML cell progression in vitro and in vivo. The aim of study is to investigate the essential function of β-arrestin1 in LICs from B-ALL. Materials and Methods The bone marrow (BM) and periphery blood (PB) of children B-ALL patients were collected, isolated and identified LICs by Magnetic-activated cell sorting (MACS) and flow cytometry. The total RNA and protein were purified for gene and protein expression by real-time RT-PCR and Western blot. The leukemia cells (LICs, Raji, and Reh) of β-arrestin1 depletion were constructed by transient or stable screening si-β-arrestin1 (siβ1) lentivirus vector. The serial cell colony formation and NSG mice survival analysis was measured the LICs self-renewal ability. The CCK8 and MTS assays were used to detect the cell proliferation, and annexin V-FITC and PI staining for cell apoptosis. The DNA methylation of gene promoter region was detected by methylation-specific PCR and the methltransferase activity by ELISA. The telomere length was indicated by Southern blot and FISH, and telomerase activity by TRAP. Electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter assay were applied to explain gene transcription. Student’s t test and Log-Rank test were used in the corresponding statistical significance and P<0.05 were considered significant. All the statistical analysis was performed using the GraphPad Prism (Version 5.0) software packages and SPSS 17.0. Results The expression of β-arrestin1 was elevated in LICs from B-ALL patients, and the high level of β-arrestin1 was negatively correlated with the survival of these patients. Further study showed that the loss of β-arrestin1 in B-ALL LICs attenuates their self-renewal capacity and promotes their senescence in vitro and in vivo. The mRNA expression level of β-arrestin1 is negatively correlated with that of PTEN in LICs. Moreover, the DNA methylation of the PTEN promoter region, the activity and the expression of DNMTs were enhanced in the LICs. The inhibition of DNMT1 activity impaired the self-renewal and increased the expression of PTEN of LICs. In addition, depletion of β-arrestin1 significantly decreased DNMT1 activity and PTEN methylation, and consistently increased PTEN expression in LICs. For B-ALL cell senescence, the mRNA expression level of β-arrestin1 is negatively related with the length of telomere, positively related with the activity of telomerase and the mRNA expression of hTERT in B-ALL LICs and engrafted NSG mice. Moreover, the weakened effect of β-arrestin1 on telomere, telomerase and the gene of hTERT were observed by injected the inhibitor of telomerase in leukemic mice. In addition, depletion of β-arrestin1 significantly decreased the binding of SP1 to the promoter of hTERT and thus reduced the transcription of hTERT in B-ALL Raji and Reh cells. Furthermore, β-arrestin1 interacted with P300 to bind with SP1 in the -104bp to -113bp of hTERT core promoter region in B-ALL cells. Conclusions β-arrestin1 could regulate the self-renewal and senescence of LICs from B-ALL, by partially mediating DNMT1 activity and hTERT transcription respectively, indicating that β-arrestin1 is a potential therapeutic target for B-ALL. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
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.

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

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.

scholarly journals Human Cytokine-Induced Memory-like NK Cells Exhibit in Vivo Anti-Leukemia Activity in Xenografted NSG Mice and in Patients with Acute Myeloid Leukemia (AML)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 101-101
Author(s):  
Rizwan Romee ◽  
Rosario Maximillian ◽  
Melissa M Berrien-Elliott ◽  
Julia A Wagner ◽  
Brea A Jewell ◽  
...  
Keyword(s):  
Nk Cells ◽  
Myeloid Leukemia ◽  
Low Dose ◽  
Nk Cell ◽  
Leukemia Cells ◽  
Cell Frequency ◽  
Nsg Mice ◽  
Post Infusion

Abstract Natural killer (NK) cells mediate anti-AML responses and previously published clinical trials of adoptive allogeneic NK cell therapy provide proof-of-principle that NK cells may eliminate leukemia cells in patients. However, complete remissions occur in 30-50% of patients with active AML and are typically of limited duration. Thus, improvements are needed for this promising cellular immunotherapy strategy. Following paradigm-shifting studies in mice, it was established that human NK cells exhibit an innate 'memory-like' responses following a brief, combined pre-activation with IL-12, -15, and -18 (Romee R et. al., Blood, 2012). These long-lived memory-like NK cells have an enhanced ability to produce IFN-g in response to restimulation with cytokines or activating receptor ligation, even following extensive proliferation. We hypothesized that memory-like NK cells exhibit enhanced responses to myeloid leukemia. Compared to control NK cells from the same donor, IL-12/15/18-induced memory-like NK cells produced significantly increased IFN-g upon co-culture with primary AML blasts in vitro (P<0.001), following 7 days of rest in low dose IL-15 vitro. In addition, memory-like NK cells had increased granzyme B expression (P<0.01), and enhanced killing of K562 leukemia targets in vitro (P<0.05). Utilizing an in vivo xenograft model of human NK cells in NSG mice (Leong J et. al., BBMT, 2014), IL-12/15/18-induced memory-like NK cells that differentiated in NSG mice for 7 days exhibited increased IFN-g responses after ex vivo re-stimulation with K562 leukemia, confirming their memory-like functionality (P<0.05). To test in vivo responses to human leukemia in this model, luciferase-expressing K562 cells were engrafted into NSG mice (1x106/mouse, IV), and on day 3, groups of mice were injected with IL-12/15/18-pre-activated or control NK cells from the same donor (4x106/mouse). Mice treated with a single dose of memory-like NK cells exhibited significantly improved in vivo leukemia control measured by whole mouse bioluminescent imaging (P=0.03), as well as overall survival (P<0.05), compared to mice treated with control or no NK cells. Based on these pre-clinical findings, we initiated a first-in-human clinical trial of HLA-haploidentical IL-12/15/18-induced memory-like NK cells in patients with AML (NCT01898793). Relapsed/refractory (rel/ref) AML patients receive lymphodepleting non-myeloablative flu/cy conditioning, infusion of a single dose of CD56+CD3- memory-like donor NK cells, followed by two weeks of low dose rhIL-2. Three patients were treated at dose level 1 (0.5x106 cells/kg) and two patients treated at dose level 2 (1.0x106/kg) with no DLTs observed, and accrual continues. Correlative analyses utilizing donor-specific HLA mAbs allow tracking of donor memory-like NK cell frequency and function following adoptive transfer. Donor memory-like NK cells were detectable in the PB and BM of all tested patients with informative HLA (4/5), peak in frequency at 7-8 days post-infusion, and contract after 14-21 days as expected following recipient T cell recovery (Figure). Memory-like NK cells exhibit significantly increased Ki67%+ as a marker of proliferation at day 7 [97.8+1.0% (donor) vs. 21.6+5.5% (recipient), mean+SEM, P<0.001]. Moreover, functional analyses of NK cells at days 7-8 post-infusion reveal increased numbers of donor IFN-g+ NK cells following restimulation with K562 leukemia cells in the same blood [1009+590 (donor) vs. 8+3 (recipient) IFN-g+ NK cells] or BM [686+423 (donor) vs. 4+2 (recipient) IFN-g+ NK cells] samples. Two of four evaluable patients treated with memory-like NK cells had leukemia free BM and PB at days 14 post-therapy, which correlated with BM NK cell frequency and IFN-g production (Figure). CIML007 had rel/ref AML with 48% BM blasts pre-therapy, and had no evidence of leukemia on day 14, 28, and 100 BM biopsies, and has an ongoing complete remission more than 100 days after this therapy. CIML009 had 80% BM blasts pre-therapy, and had no evidence of leukemia on day 14 BM biopsy post-infusion. Thus, human IL-12/15/18-induced memory-like NK cells expand and have enhanced anti-AML function following adoptive transfer in patients, thereby constituting a promising translational innovation for immunotherapy of AML. Figure 1. Figure 1. Disclosures Fehniger: Celgene: Research Funding.

scholarly journals Incorporating Hemoglobin Levels to Map Leukostasis Risk in Acute Leukemia Using Microvasculature-on-Chip Technologies

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 9-10
Author(s):  
Jamie Oakley ◽  
Evelyn K. Williams ◽  
Christina Caruso ◽  
Yumiko Sakurai ◽  
Reginald Tran ◽  
...  
Keyword(s):  
T Cell ◽  
Acute Leukemia ◽  
Blood Viscosity ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Cell Counts ◽  
On Chip ◽  
Wbc Count

Hyperleukocytosis, most commonly defined as a white blood cell (WBC) count &gt; 100,000/μL, is an oncologic emergency in acute leukemia that can lead to leukostasis, which occurs when leukemia cells obstruct the microvasculature resulting in significant morbidity and mortality from neurologic (CNS hemorrhage, thrombosis) or pulmonary (respiratory distress, hypoxia) symptoms. The underlying mechanisms are poorly understood but are thought to be related to increased blood viscosity, secondary to high WBC count, leukemia cell aggregation, and the abnormal mechanical properties, size, and cell-cell interactions of leukemia cells. Leukapheresis is a commonly used therapy for rapid cytoreduction in symptomatic patients, but the procedure is not without risks. No existing methods reliably predict leukostasis or guide treatment including the commonly used WBC count, which only loosely correlates with leukostasis and does not accurately describe the blood viscosity at the microvascular level. Importantly, while hematocrit/hemoglobin levels (Hgb) are known to be major contributors to blood viscosity, they have not been systematically assessed in leukostasis risk, and Hgb often decreases as leukemic cell counts rise, complicating the issue. Incorporating Hgb levels may better predict leukostasis and assist physicians balancing the risk of hyperleukocytosis compared to the interventions themselves. To that end, we investigated how the differing presentations of acute leukemia lead to microvessel occlusion, thereby affecting effective blood viscosity at the microvascular level using "microvasculature-on-a-chip" devices that mimic the microvascular geometry (Figure 1) developed by our laboratory. This physiologically relevant microvascular model allows for in vitro investigation as in vivo studies are nearly impossible due to difficulty in visualizing and manipulating the animal microvasculature and cell counts. The devices were microfabricated using polydimethylsiloxane (PDMS). Acute T-cell lymphoblastic (Jurkat) and acute monocytic (THP-1) cell lines were maintained via standard cell culture conditions. Red cells from healthy donors were isolated and mixed with leukemia cells to achieve target Hgb and WBC levels. Various physiologic leukemia "mixtures" were then perfused under physiologic microcirculatory flow conditions through the microvascular device and microchannels occlusion was tracked via videomicroscopy (Figure 2). With T-cell leukemia, Hgb levels affected the risk of "in vitro leukostasis." Specifically, with severe anemia and WBC count less than the hyperleukocytosis range, time to microchannel occlusion was longer, and was more dependent on Hgb rather than WBC count. However, in cases with severe anemia and WBC counts &gt; 100k/μL, WBC count exhibited a stronger effect on occlusion with little dependence on Hgb (Figure 3). At Hgb &gt; 8g/dL, microchannel occlusion was dependent on WBC count regardless of hyperleukocytosis or not. In contrast, our data to date shows that with myeloid leukemia, in vitro leukostasis is not associated with Hgb levels, and is consistent with how myeloid leukemias in vivo cause leukostasis symptoms at lower WBC counts than lymphoid leukemias, not only due to size but also adhesive interactions. These data suggest when determining risk for leukostasis, WBC count should not be the sole determinant. Here we show Hgb levels affect microvascular blood viscosity and propensity for microvascular occlusion, but it appears to have a greater impact with T-cell leukemias versus myeloid leukemias (Figure 4). These studies indicate Hgb is an important clinical parameter for leukostasis risk in acute leukemia and will help inform guidelines for leukapheresis and even phlebotomy, a much simpler and safer procedure, to mitigate hyperviscosity in acute leukemia. These results can also impact decisions regarding the need for red blood cell transfusions, which iatrogenically increase blood viscosity. Studies incorporating patient myeloid and lymphoid leukemia cells and microvasculature-on-chip devices integrating live endothelium to assess leukemia cell adhesion are ongoing. Figure Disclosures Lam: Sanguina, Inc: Current equity holder in private company.

Live Cell Imaging Captures Immune Cell-Mediated Killing of Precursor-B Acute Lymphoblastic Leukemia Cells Targeted with Anti-CD19 (Medi-551) Antibodies: Support for Macrophage and NK Cell In Vivo Activity

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1522-1522
Author(s):  
Ksenia Matlawska-Wasowska ◽  
Dennis Cook ◽  
Samuel R. Stevens ◽  
Elizabeth K. Ward ◽  
Ronald Herbst ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Nk Cells ◽  
Lymphoblastic Leukemia ◽  
Live Cell ◽  
Fluorescent Imaging ◽  
Leukemia Cells ◽  
Strong Activity ◽  
Effector Cells

Abstract Abstract 1522 Precursor-B acute lymphoblastic leukemia (pre-B ALL) is the most common malignancy in children and can be cured in a majority of patients. However, cure remains elusive in approximately 20% of patients for reasons that are not well understood. Importantly, survivors commonly develop morbidities that result from dose-intensified treatment with cytotoxic drugs. Here, we investigate the tumoricidal effects of a novel humanized anti-CD19 monoclonal antibody (Medi-551). The a-fucosylated form of this antibody has increased affinity to human FcgammaRIII (CD16) receptor, present on the surface of NK cells and macrophages, mediating antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Medi-551/CD19 complexes internalize slowly and thus remain accessible for effector cells for prolonged periods. We evaluated in vitro ADCC and ADCP activities of primary human NK cells and macrophages (effector cells) against four pre-B ALL cell lines (697, Nalm 6, MHH-Call 3, RS 4;11), as well as freshly isolated patient blasts. We report results of live cell fluorescent imaging studies, characterizing the formation of immunological synapses between Medi 551-bound target leukemia cells and effector cells, as well as the kinetics of both NK-mediated killing and macrophage phagocytosis. The number of the CD19 receptors present on the cell surface is shown to be a factor in effector-mediated killing of Medi-551 targeted leukemia cells. Further, genetic polymorphisms in FcgammaRIII (158 F/V, V/V or F/F) affected in vitro ADCC and ADCP activities with FcgammaRIII 158 V homo- or heterozygotes showing the strongest activity. We also evaluated the efficacy of Medi-551 in a human pre-B ALL murine xenograft model. SCID mice were engrafted with 697 pre-B ALL cells and received either vehicle alone or Medi-551 (3 mg/kg; twice weekly for a total of 5 doses); treatment was started at day 5 after engraftment. Medi-551 treatment markedly lowered disease burden in blood, liver and bone marrow. The lack of cure is consistent with impaired roles for NK cells in this model, since murine NK cells lack FcgammaRIV. Experiments are in progress to improve the model through adoptive transfer of human NK cells. Taken together, the in vitro and in vivo data show that Medi-551 has strong activity against pre-B ALL and support a move forward to early phase trials in this disease. Disclosures: No relevant conflicts of interest to declare.

The Poison Oligonucleotide F10 Is Efficiently Taken Up By, and Highly Effective Against, Acute Lymphoblastic Leukemia While Sparing Normal Hematopoietic Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2674-2674
Author(s):  
Timothy Pardee ◽  
Jamie Jennings-Gee ◽  
Kristin Stadelman ◽  
David L. Caudell ◽  
William Gmeiner
Keyword(s):  
Board Of Directors ◽  
Median Survival ◽  
Survival Benefit ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Advisory Committees ◽  
Previously Treated ◽  
Human Hscs

Abstract The development of imatinib to target the BCR-ABL kinase in chronic phase (CP) CML has changed the natural history of the disease. This success generated much enthusiasm for the approach in the more genetically complex acute lymphoblastic leukemia (ALL). Unfortunately, the durable responses seen in CP CML are not reproduced in BCR-ABL positive ALL. Indeed, all previous attempts to target a single oncogenic pathway in acute leukemias have resulted in transient responses with frequent relapse. An alternative approach is to use agents that target “final common pathways”, i.e. processes that must be accomplished to produce additional leukemia cells regardless of driving mutations. In this paradigm, agents are not judged by differential expression of a target, but by the degree of differential uptake. One pathway to exploit is the known increased uptake of oligonucleotides by ALL cells. F10 is a poison deoxy-oligonucleotide that is a 10mer of the thymidylate synthase (TS) inhibitory 5-fluorouracil (5-FU) metabolite, 5-fluoro-2’-deoxyuridine-5’-O-monophosphate. We sought to determine the uptake and activity of F10 against preclinical ALL models. Using fluorescently labeled F10, we determined that uptake by the human ALL cell lines DG75 and SUP-B15 was rapid and had a profound temperature dependence consistent with an active process. Both cell lines demonstrated increased uptake compared to normal, lineage- depleted marrow cells from C57Bl/6 mice. Using a syngeneic, BCR-ABL-expressing murine ALL model, we confirmed rapid uptake of F10 in vivo. Furthermore, preliminary experiments suggested normal human HSCs have decreased uptake compared to primary patient samples. Consistent with this decreased uptake, F10 treatment did not alter the ability of human HSCs to engraft in immunodeficient mice. F10 treatment resulted in robust induction of apoptosis that could not be equaled by 100 fold more 5-FU. IC50 values for F10 against B6 ALL, Jurkat, DG75, Molt-4, CCRF-CEM, and SUP-B15 were in the picomolar to nanomolar range, with an average value of 2.15 nM (range 0.12 to 5.4 nM). For comparison we also tested 5-FU, doxorubicin, and cytarabine. In all cases, F10 was the most potent agent; over 1000 times more potent than 5-FU. In vivo, F10 treatment was associated with a significant increase in survival in a BCR-ABL driven, syngeneic ALL mouse model (p= 0.0001 by log rank test). Survival was dose-dependent, with median survival extended by 3, 9 or 15 days for 4, 6, or 9 doses, respectively. F10 also protected mice from leukemia-induced weight loss during and for several days after treatment. To confirm the in vivo activity of F10, we treated a separately derived syngeneic model expressing the T315I variant of BCR-ABL. As in the previous model, F10 significantly prolonged survival (p=0.0013). To further extend these results and confirm F10's activity against human ALL cells, we treated DG75, Jurkat, and SUP-B15 xenograft models. As in the murine models, F10 caused regression of disease with several animals cured and resulted in a significant survival benefit (p=0.0112). As the uptake and mechanism of action of F10 is unique we sought to determine the in vivo efficacy of F10 on ALL previously treated with cytarabine. When mice injected with previously treated ALL cells were treated with cytarabine a median survival benefit of only one day (11 vs 12 days) was observed. In contrast F10 treatment resulted in a median survival of 31 days, similar to the survival seen in previously untreated ALL. F10 exposure results in trapped topoisomerase I (Topo1) cleavage complexes in vitro. To determine if this occurs in vivo, we treated leukemic C57Bl/6 or nude mice harboring ALL xenografts with F10. After 24 hours, leukemia cells were harvested and assayed for trapped Topo1 complexes. F10 treatment resulted in detectable trapped Topo1 complexes in both the xenograft and syngeneic models. In summary, F10 exhibited remarkable activity against human and murine ALL cells in vitro and in vivo by inducing apoptosis and trapping Topo1 complexes. These data demonstrate agents that target “final common pathways” but with differential uptake can be safe and effective, even against genetically complex and aggressive leukemias. Disclosures: Pardee: Salzburg Therapeutics: Membership on an entity’s Board of Directors or advisory committees. Gmeiner:Salzburg Therapeutics: Membership on an entity’s Board of Directors or advisory committees.

Proteomic, Gene Expression, and Micro-RNA Analysis Of Bone Marrow Mesenchymal Stromal Cells In Acute Myeloid Leukemia Identifies Pro-Inflammatory, Pro-Survival Signatures In Vitro and In Vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3685-3685
Author(s):  
Michael Andreeff ◽  
Rui-yu Wang ◽  
Richard E. Davis ◽  
Rodrigo Jacamo ◽  
Peter P. Ruvolo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Micro Rna ◽  
Leukemia Cells ◽  
Rna Analysis ◽  
Nsg Mice ◽  
Acute Myeloid

Abstract The bone marrow microenvironment (BME) in acute myeloid leukemia (AML) generates resistance signals that protect AML cells/stem cells from chemotherapy. The mechanisms how the BME might support leukemia cell survival are unclear but elucidation of this process could prove useful for therapy design. Here we report new insights specific to stroma functionality in AML. A series of novel experimental approaches were developed including : 1) nanostring micro-RNA and proteomic analysis using reverse-phase protein arrays (RPPAs) of MSC derived from AML patients and normal donors; 2) genome-wide RNA analysis of FACS-sorted MSCs using Illumina arrays of genetically-defined human and murine AML cell lines/primary AML samples after co-culture with normal MSC in vitro; 3) in vivo interaction between genetically-defined murine AML and stromal cells in syngenic C57BL/6J mice, followed by harvesting and FACS-isolation of specific MSC after leukemia engraftment; 4) use of genetically-modified human MSC in vivo in our ectopic humanized bone marrow model in NSG mice (Blood 2012 : 119,4971), followed by bioluminescence growth and homing analysis of human leukemia cells. This model allows the study of in vivo effects of altered MSC on human AML development. 1) Proteomic and transcriptomic analysis of primary MSC from AML patients (n = 106) and normal MSC (n = 71) by RPPA using validated mAbs to 150 proteins and phospho-proteins demonstrated major differences by hierarchical clustering analysis: GSKA, STAT1, PDK1, PP2A, CDKN1A, CDK4, and STAT5AB were significantly over-expressed in AML- vs. normal MSC, while STMN1, SIRT1, SMAD1, SMAD4, HSP90 and EIF2S1 were under-expressed. Differences were observed between MSC from newly diagnosed vs. relapsed AML-MSC. Nanostring analysis of 38 AML-MSC and 24 normal MSC identified differential expression of numerous miRs, a select group of which has been validated so far by qRT-PCR. AML MSC express reduced levels of let-7g, let-7c, miR 21 and miR93, and elevated levels of miR410 compared to normal MSC. Pathways were identified in MSC that might contribute to leukemia survival. 2) Analysis OCI-AML3 cells co-cultured with normal -MSC revealed upregulation of a variety of genes in MSC encoding cytokines and chemokines and gene set enrichment analysis (GSEA) identified activation of NFkB in MSC as a potential cause of these changes. When the ectopic humanized bone marrow model system in NSG mice was used, suppression of NFkB in MSC resulted in a ∼ 50% reduction of AML burden. When murine MSC cultured with wt p53 MLL/ENL-Luc-FLT3-ITD cells were compared to isogenic cells with deleted p53, striking differences were seen in the MSC transcriptome: 429 differentially expressed genes were identified that distinguished co-cultures with p53wt and p53-/- cells, suggesting that AML cells may communicate signals to their microenvironment in a p53-dependent manner. GSEA identified NFkB and HIF-1a as targets, data were confirmed independently, and HIF-1a knockout MSC were found to be inhospitable for AML in the ectopic in vivo model. 3) These syngenic cells were introduced into B57BL/6J mice and MSC were isolated after leukemia engraftment: 147 genes were consistently upregulated and 236 genes downregulated in MSC by their interactions with AML in vivo. Upregulated genes included CTGF, CXCL12, genes related to complement (C4A, C4B, Serpin G1), and IGFBP5, an inhibitor of osteoblast differentiation. Identification of CXCL12 was intriguing as Link's group recently reported the critical role of CXCL12 produced by early MSC in normal hematopoiesis (Nature 2013 : 495,227). Both AML-ETO and MLL-ENL leukemias caused upregulation of CTGF, metalloproteinases, adhesion molecules, and NFkB-related genes in vivo. IPA analysis showed responses in BM-MSC associated with inflammation, cellular movement, cell-cell signaling, cellular growth and proliferation and immune cell trafficking. Conclusion AML cells induce changes in MSC, in short term co-cultures in vitro, or in syngenic systems in vivo, that are consistent with pro-survival, anti-apoptotic, and growth-stimulatory signals that mimic inflammatory responses. Large-scale analysis of primary AML-derived MSC confirms and extends this data. Results facilitate the development of therapeutic strategies to render the BM microenvironment inhospitable to leukemia cells but supportive of normal hematopoiesis. Disclosures: Lowe: Blueprint Medicines: Consultancy; Constellation Pharmaceuticals: Consultancy; Mirimus Inc.: Consultancy.

Phase I clinical and pharmacokinetic evaluation of high-dose mitoxantrone in combination with cytarabine in patients with acute leukemia.

1993 ◽  
Vol 11 (10) ◽  
pp. 2002-2009 ◽  
Author(s):  
E J Feldman ◽  
D S Alberts ◽  
Z Arlin ◽  
T Ahmed ◽  
A Mittelman ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Phase I ◽  
Intravenous Infusion ◽  
High Performance ◽  
Lymphoblastic Leukemia ◽  
Myelogenous Leukemia ◽  
High Dose ◽  
Advanced Phase

PURPOSE To determine the maximally tolerated dose of mitoxantrone in combination with cytarabine in patients with acute leukemia and advanced phases of chronic myelogenous leukemia (CML), and to assess the pharmacokinetics of high-dose mitoxantrone in this patient population. PATIENTS AND METHODS In a phase I study, 68 patients with acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), and accelerated- and blastic-phase CML received induction therapy consisting of cytarabine 3 g/m2 by infusion over 3 hours daily for 5 days, with escalating doses of mitoxantrone 40 to 80 mg/m2 over 1 to 2 days by intravenous infusion over 15 minutes. Mitoxantrone pharmacokinetics were evaluated by high-performance liquid chromatography (HPLC) in 15 patients given a single dose of mitoxantrone ranging from 40 to 80 mg/m2 in combination with cytarabine. RESULTS Severe, but reversible hyperbilirubinemia (> three times normal) was considered the dose-limiting toxicity, and was observed in 25% of all patients and in 35% of those who received 70 to 80 mg/m2 of mitoxantrone. Other extramedullary toxicity, including cardiac dysfunction, was mild. Myelosuppression was universal and the median time to complete remission (CR) was 28 days (range, 19 to 77). The CR rate for previously untreated and relapsed patients with AML was 85% (17 of 20) and 38% (seven of 18), respectively. Eighty-three percent (15 of 18) of patients with ALL achieved a CR, including all patients with previously untreated disease. Eight of 12 patients with advanced-phase CML achieved a CR. No significant changes in mean mitoxantrone plasma elimination rates (ie, terminal plasma half-life and total-body clearance rate) occurred as the mitoxantrone dose doubled, indicating linear pharmacokinetics. CONCLUSIONS The recommended phase II dose of mitoxantrone is 80 mg/m2 administered over 15 minutes as a single intravenous infusion in combination with cytarabine 3 g/m2/d for 5 days. At this dose, high concentrations of mitoxantrone are achievable in vivo to levels that have been shown to be extremely cytotoxic in vitro.

Sign in / Sign up

Export Citation Format

Share Document