R-DHAP Is Effective in Fludarabine-Refractory CLL, Possibly Via Upregulation of Pro-Apoptotic P73.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3449-3449
Author(s):  
Sanne H. Tonino ◽  
Michel Van Gelder ◽  
Eric Eldering ◽  
Marinus H. J. van Oers ◽  
Arnon P. Kater
Keyword(s):  
Conflicts Of Interest ◽  
Residual Disease ◽  
Tumor Lysis Syndrome ◽  
Remission Induction ◽  
Refractory Disease ◽  
P53 Family ◽  
Bulky Disease ◽  
P53 Response

Abstract Abstract 3449 Poster Board III-337 Allogeneic stem cell transplantation (alloSCT) has the potential to cure high risk CLL patients including those with fludarabine-refractory disease with or without a deletion of 17p. Treatment-related mortality (TRM) of this procedure is significantly reduced by the use of reduced-intensity conditioning regimens (RIC). Risk factors for relapse after alloSCT are refractory or bulky disease. In an ongoing prospective Dutch/ Belgian HOVON trial we study the efficacy of remission-induction with the R-DHAP regimen (rituximab, cytarabine, cisplatinum and dexamethasone) prior to alloSCT in CLL patients with fludarabine refractory disease. During the initiation phase ten fludarabine refractory patients have been treated according to this protocol. The table shows their disease characteristics and response to R-DHAP. patient age FISH bulky disease1 preceding therapies No. of R-DHAP cycles Response 1 36 del 17p + Chl, CVP, FCR, Alem, R-CHOP 1 SD 2 65 normal - Chl, CVP, F 3 PR 3 71 normal + Chl, F, FC, FCR 1 PR 4 51 normal + Chl, F, FCR 4 SD 5 47 del 11q + FCR, Alem 3 PR 6 53 ND - Chl, F, Alem 3 PR 7 54 ND + Chl, F, FCR, R-CHOP 4 PR 8 65 ND + Chl, CVP, F, CHOP, Alem 3 PR 9 57 del 17p + R-CVP, R-CHOP 4 PR 10 50 del 11q + Chl, FCR, Alem 3 CR 1 lymph nodes > 5 cm Eight of ten patients had a response: one CR and seven PR, whereas two had SD. In the patient who achieved a CR, four-color flowcytometry confirmed the absence of minimal residual disease (MRD). One of the two patients with del 17p and both patients with del 11q had a response. Six of the eight patients with bulky disease responded. One patient developed tumor lysis syndrome. One patient, who had been pretreated with alemtuzumab (no 8), developed an opportunistic infection (Aspergillus pneumonia). Most responding patients subsequently underwent alloSCT. The observed response rate in these heavily pre-treated and chemo-refractory patients is remarkable. As chemorefractory disease is highly associated with a dysfunctional p53 response, we hypothesized that effects of this regimen could be independent of p53-function. Therefore the molecular basis of apoptosis induction was studied more in depth in one patient with del 17p and proven p53 dysfunction. Within weeks after the first cycle of R-DHAP peripheral blood lymphocyte counts decreased from 78 × 109/L to 3.3 × 109/L. Simultaneous analysis of RNA-expression levels of 30 apoptosis regulating genes by RT-MLPA in samples taken before and 24 and 48 hours after start of R-DHAP revealed significant upregulation of the pro-apoptotic BH3-only molecule Puma. Recent data indicate that Puma is not only a p53 response gene, but can also be regulated by the p53-family member p73. Indeed, detectable p73 protein levels were found already after 24 hours of treatment in vivo. At the functional level, we found that the pretreatment fludarabine resistance in vitro was abrogated after 48 hours of treatment in vivo. Our data indicate that the R-DHAP regimen has activity in fludarabine-refractory CLL patients, even in those with cytogenetic changes affecting the p53-response. Furthermore, in our preliminary in vitro studies we could show upregulation of the pro-apoptotic protein p73 and concomitant abrogation of fludarabine-resistance. This pathway should be further explored and may provide means to overcome drug-resistance in CLL. Disclosures No relevant conflicts of interest to declare.

Adaptation of a Xenograft AML Model to Evaluate Chemotherapeutic Efficacy In Vivo

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3304-3304 ◽  
Author(s):  
Mark Wunderlich ◽  
Fu-Sheng Chou ◽  
Mahesh Shrestha ◽  
Benjamin Mizukawa ◽  
James C. Mulloy
Keyword(s):  
Blood Cell ◽  
Treatment Period ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Synergistic Effects ◽  
Day Treatment ◽  
Chemotherapeutic Agents ◽  
Mouse Strains

Abstract Abstract 3304 Although significant progress has been made in the treatment of leukemia, relapse continues to be a major problem, particularly in acute myeloid leukemia (AML). The prognosis for relapsed leukemia is poor, indicating an area for potential improvements. However, animal models to study the response of human AML to chemotherapeutics and subsequent relapse are lacking. Recently we developed an improved NOD/SCID mouse with IL2RG knockout and transgenic expression of myelo-supportive cytokines SCF, GM-CSF, and IL-3 (the NSGS mouse). This mouse is remarkable in its ability to accept human AML grafts more efficiently than all other available strains. When coupled with in vitro derived AML cells, the NSGS mouse allows for a more predictable AML model with shorter latency and smaller range of death than in other mouse strains, including NSG mice. Importantly, very low numbers of cells reliably generate fatal AML in roughly 40 days, even in non-irradiated NSGS mice, allowing for rapid experimental conclusions and reduced toxicity. With the benefits of these unique tools, we sought to develop a model system to evaluate the efficacy of chemotherapeutic agents on human AML cells in vivo. Engrafted mice received a chemotherapy regimen over a 5-day treatment period consisting of a daily dose of cytarabine with simultaneous injection of doxorubicin during the first three days. Treated mice experienced striking weight loss during the treatment period with a nadir at days 8–10 post-treatment. Mice recovered body weight within 3 weeks. Serial complete blood counts indicated a rapid transient drop in total white blood cell and neutrophil counts and a delayed transient drop in red blood cell and platelet numbers, reminiscent of the effects observed in patients undergoing chemotherapy. The drugs successfully targeted the cells of the bone marrow, as evidenced by a profound loss of cellularity in treated mice relative to controls. When mice harboring N-Ras(G12D) positive AML cells were treated at early time points post-transplant, a significant reduction of tumor burden was observed in the BM and PB, with the grafts of treated mice essentially undetectable for weeks after treatment cessation. Nevertheless, treated mice inevitably succumbed to disease, although with a significantly prolonged latency compared to mock treated mice. However, when AML cells containing the FLT3-ITD mutation were used, a shift in disease latency was not reproducibly seen. This data correlates well with patient data showing that FLT3-ITD mutant AML has a worse prognosis than AML samples with N-Ras mutations. Importantly, the reappearance of AML within weeks of treatment affords the opportunity to model drug resistance and relapse, as well as the potential synergistic effects of experimental compounds used in combination with traditional chemotherapy. Additionally, the period following treatment may allow for studies of minimal residual disease as well as the testing of potential maintenance therapies. Finally, this approach permits a detailed analysis of the critical few cancer stem cells that remain after induction therapy with the goal of identifying novel compounds capable of targeting these cells. Disclosures: No relevant conflicts of interest to declare.

Investigating the Expression of the MRD Marker CD58 on Leukaemia Initiating Cells in Childhood Acute Lymphoblastic Leukaemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1887-1887 ◽  
Author(s):  
Charlotte Victoria Cox ◽  
Paraskevi Diamanti ◽  
Allison Blair
Keyword(s):  
Acute Lymphoblastic Leukaemia ◽  
Lymphoblastic Leukaemia ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Model Systems ◽  
Childhood All ◽  
Therapy Assessment ◽  
Childhood Acute Lymphoblastic Leukaemia

Abstract Abstract 1887 Further improvements in outcome for childhood acute lymphoblastic leukaemia (ALL) will require a better understanding of the underlying biology of this disease and the fundamental mechanisms of drug resistance. The discoveries that a few populations can initiate leukemia in mouse models and that new populations of leukaemia initiating cells (LIC) can be detected following an initial round of transplantation in these models raises important questions about the biology of the leukaemias. If several cell populations have LIC properties, what are the relationships of these populations to each other and which populations are most important to target with therapy? It will also be important to determine whether there is any correlation in the biological properties of LIC identified in the model systems with the response of the patients to therapy. Assessment of minimal residual disease (MRD) levels provides a sensitive measurement of early treatment response and permits detection of the in vivo selected drug resistant population. CD58 (leucocyte function-associated antigen 3; LFA-3) is a useful marker in MRD tracking of B cell precursor (BCP) ALL. CD58 is over expressed in these cases permitting discrimination of leukaemia blasts from normal B cells. In this study we investigated whether CD58 is expressed on LIC populations in childhood ALL. Expression of CD58 and CD34 was assessed in a cohort of 12 diagnostic samples with mixed prognoses and compared to levels detected in 11 normal bone marrow (NBM) samples. Levels of CD58 were significantly higher in the ALL cases (57.4±37.7%) than on NBM cells (21.1±12.2%; p=0.007). Likewise, the CD34+/CD58+ population was larger in ALL cases than in normal cells (22.2±34.7% and 0.25±0.25%, respectively; p=0.05). Cells from eight of the 12 patients, were sorted on the basis of expression or lack of expression of these markers and the functional ability of the sorted subpopulations was assessed in vitro and in vivo. On sorting, the majority of cells were CD34−/CD58− (43.7±39.2%), 20.7±30.7% were CD34−/CD58+, 19±14.3% were CD34+/CD58+ and the CD34+/CD58− population accounted for 16.6±35.3%. Unsorted cells and all 4 sorted populations were set up in long-term culture to assess proliferative capability and the in vivo propagating potential was assessed in NSG mice. All 4 sorted subpopulations proliferated over the 6 week period but the highest levels of expansion were observed in the cultures of CD34+/CD58+ (6–420 fold) and CD34+/CD58− (3–24 fold) cells. Cytogenetic analyses confirmed that leukaemia cells were maintained in the culture system. Results from the in vivo analyses on 5 cases to date indicate that all 4 subpopulations contain LIC. In these cases, higher levels of engraftment were observed with CD34+/CD58+ (up to 20%) and with CD34−/CD58− subpopulations (6.1-98%). Serial transplantation studies will determine whether there are differences in the repopulating and self-renewal abilities of these LIC. These findings suggest that using CD58 alone or in combination with CD34 would be insufficient to track disease progression in ALL. Incorporating additional markers that are commonly used in MRD panels will provide valuable information on LIC populations and facilitate development of improved disease monitoring. Disclosures: No relevant conflicts of interest to declare.

Assessing CD97 and CD99 As Markers of Leukaemia Initiating Cells in Paediatric ALL

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1882-1882 ◽  
Author(s):  
Charlotte Victoria Cox ◽  
Paraskevi Diamanti ◽  
Allison Blair
Keyword(s):  
Functional Capacity ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Survival Rates ◽  
In Vivo Studies ◽  
Nsg Mice ◽  
Early Treatment Response ◽  
Current Therapies

Abstract Abstract 1882 Overall survival rates in paediatric acute lymphoblastic leukaemia (ALL) have dramatically improved but around 20% do not respond to current therapies and subsequently relapse. Leukaemia initiating cells (LIC) are the topic of much investigation, as these cells can self-renew and may have the potential to cause relapse. It has been shown that multiple subpopulations of ALL cells have the ability to initiate the disease in immune deficient mouse models. Therefore, treatment should be targeted at all cells with this capacity, if the disease is to be eradicated. Minimal residual disease (MRD) detection is an invaluable tracking tool to assess early treatment response and recent studies have highlighted potential markers that may improve the sensitivity of MRD detection by flow cytometry. CD97 and CD99 are two markers which were over expressed in paediatric ALL. Incorporating these markers into investigations of LIC may allow discrimination of leukaemia cells from normal haemopoietic stem cells (HSC). In this study we evaluated the expression of CD34 in combination with CD97 in B cell precursor (BCP) ALL cases and CD99 in T-ALL cases and subsequently assessed the functional capacity of the sorted subpopulations in vitro and in vivo. Ten ALL samples (6 B-ALL & 4 T-ALL) with a median age 7 years (range 2–15 years) were studied. One B-ALL case and 3 T-ALL cases were considered high risk by molecular assessment of MRD at day 28 of treatment. Flow cytometric analyses of the ALL samples and 8 normal haemopoietic cell samples demonstrated that both CD97 and CD99 were over expressed in ALL patients (78.9±14.8% & 76.4±32.8%, respectively) when compared to normal haemopoietic cells (14.1±25.4%; p=0.001, 47.1±10%; p=0.03, respectively). Cells were sorted for expression/lack of expression of these markers and proliferation of the sorted cells was assessed in suspension culture over a 6 week period. In the B-ALL patients the CD34+/CD97+ subpopulation represented the bulk of leukaemia cells (65.2±32.1%), the CD34−/CD97+ the smallest fraction (3.3±2.4%) with the CD34+/CD97− and CD34−/CD97− subpopulations representing 21.1±31.5% and 10.5±5.8% of cells, respectively. When the functional capacity of these subpopulations was assessed in vitro greatest expansion was observed in cells derived from CD34+/CD97− subpopulation (2–173 fold) from 9.4×103 at initiation up to 1.5×106 cells at week 6. Expansion was also observed, to a lesser extent in the CD34−/CD97− subpopulation (3.4–28 fold) from 8×103 up to 1.4×106 cells. No expansion was observed in cultures of CD34+/CD97+ and CD34−/CD97− subpopulations but cells were maintained throughout the culture period. These sorted subpopulations were also inoculated into NOD/LtSz-SCID IL-2Rγc null (NSG) mice to evaluate repopulating capacity. To date, engraftment has been achieved with 3 subpopulations; CD34+/CD97+ (3–28.8% CD45+), CD34+/CD97− (0.5–25.5% CD45+) and CD34−/CD97+ (23.8% CD45+) cells. When the functional capacity of T-ALL cases was assessed the CD34+/CD99+ subpopulation represented the bulk of cells at sorting (51.87±47.2%), the CD34+/CD99- subpopulation was the smallest (0.9±0.8%) and the CD34−/CD99+ and CD34−/CD99− subpopulations represented 32.1±38.9% and 27.2±33.4% of cells, respectively. Greatest expansion was observed in cultures of CD34+/CD99- cells (4.6–1798 fold) from 7.5×103 up to 2.6×106 cells at week 6. The other 3 subpopulations expanded to a lesser extent (1.3–216 fold) from 5×103 up to 1.8×106 cells. When the functional capacity of these cells was assessed in NSG mice, engraftment was achieved in all subpopulations; CD34+/CD99+ (87–90.5% CD45+), CD34+/CD99− (1.5–84.9% CD45+), CD34−/CD99+ (31.3–98.6% CD45+) and CD34−/CD99− (3–92.9% CD45+). In some cases, cells recovered from BM of NSG inoculated with CD99− cells had high expression of CD99, typical of the patient samples at diagnosis, indicating that the inoculated CD99− cells had differentiated in vivo. Studies are ongoing to assess the self-renewal capacity of these subpopulations by serial transplantation. The findings to date indicate that targeting CD97 and CD99, either alone or in combination with CD34 would not eliminate all cells with the capacity to initiate and maintain B-ALL and T-ALL, respectively. Further developments in therapy may require targeting leukaemogenic pathways, rather than only cell surface markers to improve survival outcome in paediatric ALL. Disclosures: No relevant conflicts of interest to declare.

Molecular iodine synergized and sensitized neuroblastoma cells to the antineoplastic effect of ATRA

2020 ◽  
Vol 27 (12) ◽  
pp. 699-710
Author(s):  
Irasema Mendieta ◽  
Gabriel Rodríguez-Gómez ◽  
Bertha Rueda-Zarazúa ◽  
Julia Rodríguez-Castelán ◽  
Winniberg Álvarez-León ◽  
...  
Keyword(s):  
Residual Disease ◽  
Neuroblastoma Cells ◽  
Survivin Expression ◽  
Body Weight Loss ◽  
Immunohistochemical Analysis ◽  
Molecular Iodine ◽  
Tyrosine Kinase Receptor ◽  
Adjuvant Effect

Neuroblastoma (NB) is the most common solid childhood tumor, and all-trans retinoic acid (ATRA) is used as a treatment to decrease minimal residual disease. Molecular iodine (I2) induces differentiation and/or apoptosis in several neoplastic cells through activation of PPARγ nuclear receptors. Here, we analyzed whether the coadministration of I2 and ATRA increases the efficacy of NB treatment. ATRA-sensitive (SH-SY5Y), partially-sensitive (SK-N-BE(2)), and non-sensitive (SK-N-AS) NB cells were used to analyze the effect of I2 and ATRA in vitro and in xenografts (Foxn1 nu/nu mice), exploring actions on cellular viability, differentiation, and molecular responses. In the SH-SY5Y cells, 200 μM I2 caused a 100-fold (0.01 µM) reduction in the antiproliferative dose of ATRA and promoted neurite extension and neural marker expression (tyrosine hydroxylase (TH) and tyrosine kinase receptor alpha (Trk-A)). In SK-N-AS, the I2 supplement sensitized these cells to 0.1 μM ATRA, increasing the ATRA-receptor (RARα) and PPARγ expression, and decreasing the Survivin expression. The I2 supplement increased the mitochondrial membrane potential in SK-N-AS suggesting the participation of mitochondrial-mediated mechanisms involved in the sensibilization to ATRA. In vivo, oral I2 supplementation (0.025%) synergized the antitumor effect of ATRA (1.5 mg/kg BW) and prevented side effects (body weight loss and diarrhea episodes). The immunohistochemical analysis showed that I2 supplementation decreased the intratumoral vasculature (CD34). We suggest that the I2 + ATRA combination should be studied in preclinical and clinical trials to evaluate its potential adjuvant effect in addition to conventional treatments.

scholarly journals Integrin α6 mediates the drug resistance of acute lymphoblastic B-cell leukemia

Blood ◽  
2020 ◽  
Vol 136 (2) ◽  
pp. 210-223 ◽  
Author(s):  
Eun Ji Gang ◽  
Hye Na Kim ◽  
Yao-Te Hsieh ◽  
Yongsheng Ruan ◽  
Heather A. Ogana ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Kinase Inhibitor ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Underlying Mechanism ◽  
Conditional Knockout

Abstract Resistance to multimodal chemotherapy continues to limit the prognosis of acute lymphoblastic leukemia (ALL). This occurs in part through a process called adhesion-mediated drug resistance, which depends on ALL cell adhesion to the stroma through adhesion molecules, including integrins. Integrin α6 has been implicated in minimal residual disease in ALL and in the migration of ALL cells to the central nervous system. However, it has not been evaluated in the context of chemotherapeutic resistance. Here, we show that the anti-human α6-blocking Ab P5G10 induces apoptosis in primary ALL cells in vitro and sensitizes primary ALL cells to chemotherapy or tyrosine kinase inhibition in vitro and in vivo. We further analyzed the underlying mechanism of α6-associated apoptosis using a conditional knockout model of α6 in murine BCR-ABL1+ B-cell ALL cells and showed that α6-deficient ALL cells underwent apoptosis. In vivo deletion of α6 in combination with tyrosine kinase inhibitor (TKI) treatment was more effective in eradicating ALL than treatment with a TKI (nilotinib) alone. Proteomic analysis revealed that α6 deletion in murine ALL was associated with changes in Src signaling, including the upregulation of phosphorylated Lyn (pTyr507) and Fyn (pTyr530). Thus, our data support α6 as a novel therapeutic target for ALL.

scholarly journals An approach for the analysis of relapse and marrow reconstitution after autologous marrow transplantation using retrovirus-mediated gene transfer

Blood ◽  
1992 ◽  
Vol 79 (10) ◽  
pp. 2694-2700 ◽  
Author(s):  
DR Rill ◽  
RC Moen ◽  
M Buschle ◽  
C Bartholomew ◽  
NK Foreman ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Marrow Transplantation ◽  
Residual Disease ◽  
Marker Gene ◽  
Autologous Bone Marrow Transplantation ◽  
Autologous Bone Marrow ◽  
Disease Recurrence ◽  
Malignant Cells

Abstract Autologous bone marrow transplantation (ABMT) is widely used as treatment for malignant disease. Although the major cause of treatment failure is relapse, it is unknown if this arises entirely because of residual disease in the patient or whether contaminating cells in the rescuing marrow contribute. Attempts to purge marrow of its putative residual malignant cells may delay hematopoietic reconstitution and are of uncertain efficacy. We now describe how retrovirus-mediated gene transfer may be used to elucidate the source of relapse after ABMT for acute myeloid leukemia and to evaluate the efficacy of purging. Clonogenic myeloid leukemic blast cells in patient marrow can be transduced with the NeoR gene-containing helper-free retrovirus, LNL6, with an efficacy of 0% to 23.5% (mean, 10.5%). Transduced colonies grow in selective media and the presence of the marker gene can be confirmed in individual malignant colonies by polymerase chain reaction. If such malignant cells remain in harvested “remission” marrow, they will therefore be marked after exposure to LNL6. Detection of the marker gene in the malignant cells present at any later relapse would be firm evidence that residual disease contributed to disease recurrence, and would permit rapid subsequent evaluation of purging techniques. The technique also marks normal marrow progenitors from patients with acute myeloblastic leukemia. These colony-forming cells can be detected in long-term marrow cultures at a frequency of 1% to 18% for up to 10 weeks after exposure to the vector. Animal models and analysis of probability tables both suggest that these levels of marking in vitro are sufficient to provide information about the mechanisms of relapse and the biology of marrow regeneration in vivo. These preclinical data form part of the basis for current clinical studies of gene transfer into marrow before ABMT.

scholarly journals The Molecular Basis of Long First Remissions in Normal Karyotype AML Patients

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3827-3827
Author(s):  
Francesca Ferraro ◽  
Christopher A Miller ◽  
Amy Abdalla ◽  
Nichole Helton ◽  
Nathan Salomonis ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Somatic Mutations ◽  
Conflicts Of Interest ◽  
Normal Karyotype ◽  
High Dose ◽  
Intermediate Risk ◽  
The Mean ◽  
Cebpa Mutations

Currently, it is not clear why some patients with acute myeloid leukemia (AML) can be "cured" with chemotherapy alone; are they living with small amounts of disease that is held in check by immunologic (or other) mechanisms, or is their disease really eradicated? The percentage of cytogenetically normal AML patients who have long (>5 years) first remissions (LFRs) after chemotherapy alone is low (about 9.1% in patients <60 years and 1.6% in >60 years1). For this reason, most intermediate risk patients are offered allogeneic transplantation to decrease their risk for relapse. To better understand mechanisms of chemotherapy sensitivity in AML, we performed an analysis of the mutation landscape and persistence, using samples from 8 normal karyotype LFR patients (without CEBPA mutations) who received standard "7+3" induction and high dose cytarabine consolidation as their only therapy. The mean age at diagnosis was 43.5 years, and the mean follow up in first remission is 7.6 years; none of these patients has relapsed to date. For each case, we performed enhanced exome sequencing at diagnosis (235x coverage of the entire exome, and ~1008x coverage of recurrently mutated AML genes). Each case had at least one documented AML driver mutation, with a median of 29 somatic mutations in the exome space. We created probes for 225 mutations (mean 28 per case), and performed error-corrected sequencing (Haloplex) for all available remission samples. The mean depth of Haloplex coverage was 1607x, and each sample had at least one AML-specific mutation assayed, with a sensitivity of 1 cell in 1,750 (0.06%). 7/8 patients demonstrated complete clearance of all mutations in all remission samples tested, which was confirmed with digital droplet PCR for 5 cases, with a sensitivity of detection of 1 cell in 100,000. In one case, we detected a persistent ancestral clone harboring DNMT3AR882H, which can be associated with long first remissions for some patients2. Strikingly, the founding clone in all 8 cases had one or more somatic mutations in genes known to drive cell proliferation (e.g. MYC, FLT3, NRAS, PTPN11, Figure 1 top panel). These are usually subclonal mutations that occur late during leukemic progression, suggesting that the presence of a "proliferative hit" in the founding clone might be important for chemotherapy clearance of all the AML cells in a given patient. To support this hypothesis, we analyzed the mutational clearance of 82 AML cases with paired diagnosis and day 30 post-chemotherapy bone marrow samples. We observed that, whether present in the founding clone or in subclones, mutations in MYC, CEBPA, FLT3, NRAS, and PTPN11 cleared after induction chemotherapy in all samples, while other mutations were often persistent at day 30 (e.g. DNMT3A, IDH1, IDH2, NPM1, TET2; Figure 1 bottom panel). Compared to other published sequencing studies of AML, MYC and NRAS mutations were significantly enriched in this small cohort (MYC p= 0.002, and NRAS p= 0.034), with MYC enrichment being particularly striking (37.5% versus 1.8%). All MYC mutations were canonical single base substitutions occurring in the highly conserved MYC Box 2 domain at the N-terminus of MYC (p.P74Q or p.T73N). Overexpression of MYCP74Q in murine hematopoietic progenitors prolonged MYC half life (89 min vs. 44 min for wild type), and enhanced cytarabine sensitivity at all concentrations tested (range 10-1000 nM, p=0.0003), both in vitro and in a MYC-driven leukemia model in vivo. MYC expression measured with flow cytometry in the blasts of the LFR samples was significantly higher (p=0.045) compared to unfavorable risk (complex karyotype) or other intermediate risk categories, but similar to good risk AML (biallelic CEBPA mutations, core binding factor fusion-associated AML, and AML with isolated NPMc), suggesting that activation of the MYC pathway may represent a shared feature of chemosensitive patients. Taken together, these data suggest that some intermediate patients who are effectively "cured" with chemotherapy alone may not have persistent subclinical disease, nor retained ancestral clones that could potentially contribute to relapse. Importantly, these patients often have mutations driving cell proliferation in the founding clone, indicating that the presence of specific mutations in all malignant cells may be critical for complete AML cell clearance with chemotherapy. 1. Blood Adv. 2018 Jul 10; 2(13): 1645-1650 2. N Engl J Med 2018; 378:1189-1199 Disclosures No relevant conflicts of interest to declare.

scholarly journals Inhibition of Plasmin Generation in Plasma By Heparin, Low Molecular Weight Heparin and a Covalent Antithrombin-Heparin Complex (ATH)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4217-4217
Author(s):  
Gabriela Chang ◽  
Helen M. Atkinson ◽  
Leslie R. Berry ◽  
Anthony K.C. Chan
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Low Molecular Weight Heparin ◽  
Conflicts Of Interest ◽  
Area Under The Curve ◽  
Low Molecular Weight ◽  
Plasminogen Activator Inhibitor 1 ◽  
Significant Difference

Abstract Introduction: Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) are widely used anticoagulants for thrombosis treatment. However, these anticoagulants have limitations such as increased bleeding, variable dose response, required frequent monitoring, and, in the case of LMWH, inability to inhibit thrombin. This has led to the development of a covalent complex of antithrombin and heparin (ATH), which has been shown to overcome many of these shortcomings. ATH has faster rates of inhibition of many coagulation factors, is able to inhibit clot-bound thrombin, and is a more effective inhibitor of both venous and arterial thrombosis in animal models. Moreover, in a rabbit thrombosis model, ATH has been shown to decrease clot mass and fibrin accretion, while the contrary was observed for UFH. From these observations, it was suggested that ATH may enhance fibrin breakdown and thus led to investigations into the effects of UFH and ATH on fibrinolysis. In vitro studies have shown that UFH enhances antithrombin inhibition of plasmin. In addition, ATH displays a slightly greater inhibition of plasmin generation and activity. Such studies were conducted in purified systems, in the absence of other plasmin inhibitors naturally present in plasma. Therefore, the aim of the present study was to compare the effects of UFH, LMWH, and ATH on plasmin generation in plasma. Methods: At 37°C tissue plasminogen activator (tPA) and soluble fibrin fragments (fib) were added to normal adult pooled platelet poor plasma supplemented with 0.35, 0.7, 1.4, or 2.1 U anti-Xa/ml UFH, LMWH, or ATH, to initiate plasmin generation (8.93nM tPA and 300µg/ml fib). At various time points, subsamples were mixed with excess plasminogen activator inhibitor 1 (PAI-1) (55.12nM) to stop further plasmin generation. The plasmin concentration at each time point was determined using a plasmin-specific chromogenic substrate and a standard curve produced from purified plasmin. Results: Comparisons of mean area under the curve (AUC) for plasmin generation displayed a significant decrease in plasmin generation in the presence of all three anticoagulants at all doses tested (p<0.05). Comparing the anticoagulants at similar doses, plasmin generation was significantly decreased in the presence of ATH (15384.66±1930.23nM/min) compared to LMWH (23892.28±3090.54nM/min) at 0.7 U/ml (p<0.05). At a dose of 1.4 U/ml, there was significantly less plasmin generated, over time, in the presence of UFH (20089.49±3022.1623nM/min) and ATH (19273.86±1805.7323nM/min) when compared to LMWH (24743.18±1265.1023nM/min) (p<0.05). There was no significant difference in plasmin inhibition between UFH and ATH at any of the doses tested. Conclusion: The present study supports previous findings that UFH and ATH can facilitate antithrombin inhibition of plasmin. It is also observed that LMWH catalyzes the inhibition of plasmin by antithrombin but possibly to a lesser extent. These findings suggest that ATH has a similar inhibitory effect on plasmin generation and activity in plasma compared to UFH, despite its overall superior anticoagulant properties. Therefore, previous in vivo observations displaying decrease in clot mass with administration of ATH was due to its enhanced anticoagulant abilities and not fibrinolysis enhancement. These findings add to our understanding of ATH mechanisms of action and aid in its development for clinical use. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Novel Type V CRISPR System with Potential for Genome Editing in the Liver

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1862-1862
Author(s):  
Gregory J. Cost ◽  
Morayma Temoche-Diaz ◽  
Janet Mei ◽  
Cristina N. Butterfield ◽  
Christopher T. Brown ◽  
...  
Keyword(s):  
Amino Acid ◽  
Genome Editing ◽  
Mammalian Cells ◽  
Conflicts Of Interest ◽  
Attractive Alternative ◽  
Vitro Assay ◽  
Crispr System ◽  
Type V

Abstract RNA guided CRISPR genome editing systems can make specific changes to the genomes of mammalian cells and have the potential to treat a range of diseases including those that can be addressed by editing hepatocytes. Attempts to edit the liver in vivo have relied almost exclusively on the Cas9 nucleases derived from the bacteria S treptococcus pyogenes or Staphylococcus aureus to which humans are commonly exposed. Pre-existing immunity to both these proteins has been reported in humans which raises concerns about their in vivo application. In silico analysis of a large metagenomics database followed by testing in mammalian cells in culture identified MG29-1, a novel CRISPR system which is a member of the Type V family but exhibits only 41 % amino acid identity to Francisella tularensis Cas12a/cpf1. MG29-1 is a 1280 amino acid RNA programmable nuclease that utilizes a single guide RNA comprised of a 22 nucleotide (nt) constant region and a 20 to 25 nt spacer, recognizes the PAM KTTN (predicted frequency 1 in 16 bp) and generates staggered cuts. MG29-1 was derived from a sample taken from a hydrothermal vent and it is therefore unlikely that humans will have developed pre-existing immunity to this protein. A screen for sgRNA targeting serum albumin in the mouse liver cell line Hepa1-6 identified 6 guides that generated more than 80% INDELS. The MG29-1 system was optimized for in vivo delivery by screening chemical modifications to the guide that improve stability in mammalian cell lysates while retaining or improving editing activity. Two lead guide chemistries were evaluated in mice using MG29-1 mRNA and sgRNA packaged in lipid nanoparticles (LNP). Three days after a single IV administration on-target editing was evaluated in the liver by Sanger sequencing. The sgRNA that was the most stable in the in vitro assay generated INDELS that ranged from 20 to 25% while a sgRNA with lower in vitro stability failed to generate detectable INDELs. The short sgRNA and small protein size compared to spCas9 makes MG29-1 an attractive alternative to spCas9 for in vivo editing applications. Evaluation of the potential of MG29-1 to perform gene knockouts and gene additions via non-homologous end joining is ongoing. Disclosures No relevant conflicts of interest to declare.

scholarly journals Novel CD19/CD22 Bicistronic Chimeric Antigen Receptors Outperform Single or Bivalent Cars in Eradicating CD19+CD22+, CD19-, and CD22- Pre-B Leukemia

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 810-810 ◽  
Author(s):  
Haiying Qin ◽  
Sang M Nguyen ◽  
Sneha Ramakrishna ◽  
Samiksha Tarun ◽  
Lila Yang ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Single Chain ◽  
Dual Targeting ◽  
Single Antigen ◽  
Therapeutic Function

Abstract Treatment of pre-B cell acute lymphoblastic leukemia (ALL) using chimeric antigen receptor expressing T cells (CART) targeting CD19 have demonstrated impressive clinical results in children and young adults with up to 70-90% complete remission rate in multiple clinical trials. However, about 30% of patients relapse due to loss of the targeted epitope on CD19 or CART failure. Our CD22-targeted CAR trial has generated promising results in relapsed/refractory ALL, including CD19 antigen negative ALL, but relapse associated with decreased CD22 site density has occurred. Thus, developing strategies to prevent relapses due to changes in antigen expression have the potential to increase the likelihood of durable remissions. In addition, dual targeting of both CD19 and CD22 on pre-B ALL may be synergistic compared to targeting a single antigen, a potential approach to improve efficacy in patients with heterogeneous expression of CD19 and CD22 on leukemic blasts. We describe the systematic development and comparison of the structure and therapeutic function of three different types (over 15 different constructs) of novel CARs targeting both CD19 and CD22: (1) Bivalent Tandem CAR, (2) Bivalent Loop CAR, and (3) Bicistronic CAR. These dual CARs were assembled using CD19- and CD22-binding single chain fragment variable (scFv) regions derived from clinically validated single antigen targeted CARs. They are structurally different in design: both tandem and loop CARs have the CD19 and CD22 scFv covalently linked in the same CAR in different orders, whereas, bicistronic CARs have 2 complete CAR constructs connected with a cleavable linker. The surface expression on the transduced T cell of the CD19/CD22 dual CARs was detected with CD22 Fc and anti-idiotype of CD19 and compared to single CD19 or CD22 CARs. Activities of dual CARs to either CD19 or CD22 were evaluated in vitro with cytotoxicity assays or killing assays against K562 cells expressing either CD19 or CD22 or both antigens and also tested against a leukemia CD19+/CD22+ cell line, NALM6, and NALM6 with CRISPER/CAS9 knockout of CD19 or CD22 or both antigens. Therapeutic function of the top candidates of the dual CARs was then validated in vivo against these NALM6 leukemia lines. Some of these dual CARs were also further tested against patient-derived xenografts. Finally, we tested the dual targeting CARs in an artificial relapse model in which mice were co-injected with a mix of CD19 knockout and CD22 knockout NALM6 leukemia lines. From these studies, we established that the order of the scFv, size of the linker, type of leader sequence, and co-stimulatory domain in the CAR constructs all impact the efficacy of the dual targeting CARs. Tandem, Loop, and Bicistronic CARs all demonstrate some levels of in vitro and in vivo activities, but the bicistronic CAR was most effective at clearing leukemia and preventing relapse. In the CD19+/CD22+ NALM6 model, bicistronic CAR treated mice remain disease free while CD19 CAR or CD22 CAR treated mice already died or relapsed on day 27. In the relapse model, as expected, CD19 or CD22 single CAR T cell treatment resulted in progression of the corresponding antigen-negative NALM6. Treatment with dual targeted bicistronic CARs resulted in clearance of both CD19 and CD22 negative ALL with durable remission. In summary, we described novel CD19/CD22 dual targeting CARs with robust pre-clinical activity against pre-B cell ALL, and validated this approach in the prevention of resistance to single-antigen targeted CARs in preclinical models. Disclosures No relevant conflicts of interest to declare.

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