scholarly journals A Case of CCDC6-RET Fusion Mutation in Adult Acute Lymphoblastic Leukemia (ALL), a Known Activating Mutation Unreported in ALL

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5136-5136
Author(s):  
Mateo Mejia Saldarriaga ◽  
Amir Steinberg ◽  
Adam F. Binder
Keyword(s):  
Bone Marrow ◽  
Tyrosine Kinase ◽  
Clinical Response ◽  
Cellular Proliferation ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Tyrosine Kinase Receptors ◽  
Promising Alternative ◽  
Clinical Scenarios ◽  
Ret Rearrangement

Abstract INTRODUCTION: Understanding the oncogenesis and the role of the different mutations observed are of crucial importance. They not only have prognostic importance, but also are evolving therapeutic targets. The first targeted mutation in ALL was the BCR-ABL gene(Thomas et al. 2004; Kim et al. 2015), which not only improved overall response and survival in "fit" patients, but also allowed for a promising alternative treatment strategy for elderly or frail patients(Delannoy et al. 2006). More recently, a subgroup of patient were noticed to share the same gene expression profile as Ph-positive, but lack the BCR-ABL translocation. These patients have several different genomic alteration, and are possibly targeted using different TKI's(Den Boer et al. 2009). CASE REPORT A 55 year-old female patient with past medical history of type 2 diabetes and hypothyroidism presented to the ED with 2 weeks of right flank pain radiating to her right groin. Her initial vital signs were stable, and she was afebrile. Her physical exam was remarkable for petechiae with no adenopathy or organomegaly. The patient's initial blood work was remarkable for pancytopenia with normal, coagulation studies, uric acid, renal and liver function except for elevated Alkaline phosphatase (481 U/L) Lactate dehydrogenase (17216 U/L). A bone marrow flow cytometry revealed a population of blasts that represented 77% of the sample. The blast were positive for CD9, CD19, CD10 (variable), CD20, cCD22, CD34, CD45, cCD79a, HLA-DR, and cTdT. These findings were compatible with B-lymphoblastic leukemia. Cytogenetics display abnormal 71-74, del(22) with high hypertriploidy. FoundatioOneHeme® was sent as part of her diagnostic work up, and revealed CCDC6-RET Fusion, FLT3 D835H and D835V, CDKN2A/B loss, DNMT3A Truncation in intron 14, MLL3 S793* and TP53 K132R as genomic alterations. The patient achieved complete response after 1 cycle of R-HyperCVAD and finished induction with this regimen. Afterwards, patient underwent myeloablative allogenic bone marrow transplant from. The patient developed Graft versus host disease 4 months after transplant that required steroids and Ruxolitinib. To date, 413 days since transplant, she continues to be in remission. DISCUSSION Activating mutations involving tyrosine kinase receptors and other molecules involved in intracellular signal amplifications/transduction are known to be important drivers of oncogenesis. Moreover, the presence of the same mutation across different neoplasm, often represents an important pharmacological target. CCDC6-RET and other common fusion partners leads to homodimerization causing ligand independent activation(Truebestein and Leonard 2018). RET autophosphorylation lead to activation of several intracellular transduction cascades involved in cellular proliferation, including MAPK, AKT, PKC, JAK-STAT, PKA and PI3K(Santoro and Carlomagno 2013). RET fusion genes were first seen in patients with PTC, and then identified in subset of patient with NSCLC(Wang et al. 2012). These mutations are considered to have a considerable role in oncogenesis as they are usually mutually exclusive with other alterations such as BRAF and RAS in PTC(Xu et al. 2017; Mitsutake et al. 2006; Kimura et al. 2003), a hypothesis that is supported by clinical response to inhibition of such tyrosine kinase receptors. Several multikinase inhibitors with RET inhibition are available and have been approved for several different indications. Several TKI commercially available have been used in trials of NSCLC and other clinical settings and represent a potential option. RET TKI has showed worse clinical response when compared to EGFR, BRAF and ALK inhibition, possibly due to off target effect leading to poor compliance or less active RET inhhibition. Newer RET specific molecules currently being developed and evaluated in different clinical scenarios(Drilon et al. 2017). To our knowledge, there is no data regarding CCDC6-RET targeting in hematological malignancies. CONCLUSION This is the first report of CCDC6-RET rearrangement in ALL to date. The isolation of CCDC6-RET mutation raise the possibility of directed RET inhibition with selective or multitarget TKI in this disease. Comprehensive assessment of RET rearrangement and point mutations should be conducted in ALL in order to identify the prevalence and clinical implications of this new finding. Disclosures No relevant conflicts of interest to declare.

Disease Propagating Blasts in Standard and High Risk Acute Lymphoblastic Leukemia Are Frequent and of Diverse Immunophenotype.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1421-1421 ◽  
Author(s):  
Klaus Rehe ◽  
Kerrie Wilson ◽  
Hesta McNeill ◽  
Martin Schrappe ◽  
Julie Irving ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
High Risk ◽  
Cancer Stem Cells ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Color Flow ◽  
Nsg Mice ◽  
Limiting Dilution

Abstract Abstract 1421 Poster Board I-444 Conflicting results in the field of cancer stem cells have reignited debate regarding the frequency and identity of cells with the ability to self renew and to propagate the complete phenotype of the malignancy. Initially it was suggested by different studies that cancer stem cells represent only a small minority of the malignant population and that the immunophenotypes of these cells resemble a rather immature type in the cell hierarchy. More recent data from our own and other groups have challenged these findings by demonstrating that cells at different maturity levels within the leukemic hierarchy have cancer stem cell abilities and that the frequency of the leukemia maintaining cell is higher than previously thought (Cancer Cell 2008, 14(1), p47-58). We use an in vivo NOD/scid IL2Rγnull (NSG) mouse intra-femoral transplant model to determine the clonogenicity of sorted candidate leukemic stem cell populations, characterized by specific immunophenotypes. We selected the surface markers CD10 and CD20, in order to differentiate between rather immature and more mature cells. Furthermore we carried out limiting dilution experiments on sorted (CD20) and unsorted leukemic blasts to investigate the frequency of the proposed leukemic stem cells. Flow sorted ALL blasts of CD19+CD20low and CD19+CD20high as well as of CD19+CD10low and CD19+CD10high immunophenotype were transplanted into NSG mice. Sorts were performed on primary patient material and on leukemic blasts that had been harvested following prior passage in mice. Different subtypes of ALL were included (high risk: BCR/ABL (t9;22) positive (patients L4967, L4951, L49101, L8849, L2510), high hyperdiploid/MRD positive high risk (L754, L835), intermediate risk: high WBC/MRD negative (L736, L784), age >10 years (L803)). CD20 sorts were performed on primary patient material (L4951, L49101, L754, L835 and L776), on secondary samples harvested from engrafted primary mice (L4967, L4951, L2510, L736 and L754) and on tertiary samples harvested from engrafted secondary mice (L4967 and L736). In total 151 mice were transplanted, with 122 showing engraftment in consecutive bone marrow punctures or in bone marrow harvests. CD10 sorts were performed on primary patient material (L784 and L49101) and on secondary samples harvested from engrafted primary mice (L4951, L8849, L2510 and L803) with 31 out of 52 mice transplanted with sorted material showing engraftment as seen with CD20 sorted cells. Blasts of all selected immunophenotypes were able to engraft the leukemia in unconditioned NSG mice as determined by 5 color flow cytometry. In particular, sorted cells of both fractions were able to reconstitute the complete phenotype of the leukemia. Harvested cells from engrafted mice could then be re-sorted into high and low antigen expressing fractions and successfully re-engrafted on secondary and tertiary mice. Cell purities of transplanted cells were usually higher than 90% (range 67-100%). The ability of all populations to serially engraft mice demonstrates long-term self-renewal capacity. Two additional patients were used in the limiting dilution assays (high WBC/t(4;11) high risk (L826); low WBC/MRD negative low risk (L792)) and experiments were performed on primary unsorted and secondary sorted material. Cell numbers necessary for ALL engraftment differed between individual leukemias but as little as 100 cells proved to be sufficient in one unsorted and in both the CD19+CD20low and CD19+CD20high fractions (Table 1). Mice transplanted with 10 cells only are still under observation. Table 1 Patient Transplant Population Cell dose Mice engrafted/transplanted L4951 Secondary CD20 high 500 3/3 CD20 low 3/3 CD20 high 100 3/3 CD20 low 3/3 L2510 Secondary CD20 high 3,000 2/4 CD20 low 4/4 CD20 high 300 0/4 CD20 low 1/4 L49101 Primary Unsorted 500 3/4 100 0/4 L792 Primary Unsorted 1,000 5/5 100 1/5 L826 Primary Unsorted 1,000 3/4 100 0/4 In conclusion we present strong evidence that leukemia-propagating cells are much more prevalent than previously thought and that blasts of diverse immunophenotype are able to serially reconstitute the complete leukemia in immune-deficient mice. Disclosures: No relevant conflicts of interest to declare.

Transforming and Tumorogenic Activity of JAK2 by Fusion to BCR: Molecular Mechanisms of Action of a Novel BCR-JAK2 Tyrosin-Kinase.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4683-4683
Author(s):  
Álvaro Cuesta-Domínguez ◽  
Mara Ortega ◽  
Cristina Ormazabal ◽  
Matilde Santos-Roncero ◽  
Marta Galán-Díez ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Nude Mice ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Chimeric Protein ◽  
Tyrosine Kinase Domain ◽  
Molecular Response

Abstract Abstract 4683 Chromosomal translocations in human tumors frequently produce fusion genes whose chimeric protein products play an essential role in oncogenesis. Recent reports have found a BCR-JAK2 fusion gene in cases of chronic or acute myeloid leukemia, but the protein had not been characterized. We describe a BCR-JAK2 fusion gene by fluorescence in situ hybridization and RT-PCR amplification from bone marrow at diagnosis of a patient with acute lymphoblastic leukemia. After induction therapy, real time PCR showed persistent molecular response correlating with hematological remission maintained up to present. BCR-JAK2 is a 110 KDa chimeric protein containing the BCR oligomerization domain fused to the JAK2 tyrosine-kinase domain. In vitro analysis showed that BCR-JAK2 was constitutively phosphorylated and was located to the cytoplasm. BCR-JAK2 transformed the IL-3-dependent murine hematopoietic cell line Ba/F3 into IL-3 independent growth and induced STAT5b phosphorylation and translocation into the cell nuclei. The treatment with a JAK2 inhibitor abrogated BCR-JAK2 and STAT5b phosphorylation, leading to apoptosis of transformed Ba/F3 cells. To test whether BCR-JAK2 has tumorogenic ability in vivo, we performed experiments with nude mice, in which we injected subcutaneously cells transduced with the control vector and cells expressing BCR-JAK2. Notably, we only obtained tumors in the flank injected with BCR-JAK2 expressing cells, thus confirming the tumorogenic activity of the BCR-JAK2 fusion protein. We conclude that BCR-JAK2 is a new tyrosine-kinase that induces proliferation and cell survival, which can be abrogated by JAK2 inhibitors. In vitro studies demonstrate that BCR-JAK2 displays transforming activity. Moreover, the nude mice model reveals its ability to cause tumors. Disclosures: No relevant conflicts of interest to declare.

Association of PML Expression with Chemotherapy-Resistance In Multiple Myeloma Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2959-2959
Author(s):  
Daisuke Ohgiya ◽  
Makoto Onizuka ◽  
Hiromichi Matsushita ◽  
Naoya Nakamura ◽  
Hiroshi Kawada ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cellular Proliferation ◽  
Conflicts Of Interest ◽  
Alkylating Agents ◽  
Chemotherapy Resistance ◽  
Nuclear Body ◽  
Rank Test ◽  
Significant Difference ◽  
The Impact

Abstract Abstract 2959 Background: Although several novel agents have improved the prognosis of patients with multiple myeloma (MM), it still remains an incurable disease because of the difficulty to eradicate MM cells by current therapeutic approaches. Recent studies have revealed that a subset of malignant cells, cancer stem cells, contribute to chemotherapy-resistance in cancer treatment. Promyelocytic leukemia gene product (PML), known as a tumor suppressor through a variety of cellular functions in a nuclear macromolecular structure called the PML nuclear body, has been reported to be responsible for the chemotherapy-resistance by regulating cell cycle in chronic myeloid leukemia. We therefore investigated the impact of PML expression on the cellular proliferation status of MM cells and patients' prognoses. Materials/Methods: Bone marrow clot sections from 48 patients with newly diagnosed MM from Jan 1998 to Dec 2009 before any therapy at diagnosis were obtained, and analyzed, according to appropriate procedure approved by IRB at the Tokai University School of Medicine (Kanagawa, Japan) with written informed consent. They were doubly-stained with a combination of anti-PML/anti-CD138 and anti-Ki67/anti-CD138. For evaluation of the relation between PML status and cellular proliferation, the positive rates of PML and Ki67 in CD138 positive cells were compared. For investigation of the impact of PML expression on the prognosis of MM, the patients were divided into 3 groups, according to the PML positive rates in the CD138 positive cells: negative/low (less than 25 percentile: 12 cases), intermediate (from 25 to 75 percentile: 24 cases) and high (more than 75 percentile: 12 cases). Their overall survivals were compared using log-rank test. Furthermore, the PML positive rates between before and after treatments were compared using paired t-test. Results: The median observation period of 48 cases was 915 days. The median age of the patients was 62.5 (38-76) at diagnosis. All the patients were underwent combination chemotherapies containing alkylating agents as initial therapies. Two and nine patients were underwent allogeneic and autologous stem cell transplantation during the clinical courses, respectively. The numbers of patients of international staging system (ISS) stage I, II and III were 17, 14 and 17 cases. The PML positive rates in each case ranged from 0% to 83.8%. They were not correlated with ISS stages (Spearman r = 0.083) and the Ki67 positive rates (Spearman r = -0.13). The PML positive rates in the negative/low, intermediate and the high groups were less than 22.1%, from 22.1 to 56.6% and more than 56.6%, respectively. No significant difference in overall survival was observed among the 3 groups (p>0.05). However, there were significant differences in two year survival rate when the 3 groups were compared (100%, 85.2% and 54.7%; p=0.015) (Fig. 1). In 13 patients whose bone marrow clot sections were sequentially collected, the PML positive rates after treatments were significantly higher than those at diagnosis (p=0.0042) (Fig. 2). Especially, PML positive rates in all the 3 patients from the negative/low group were progressively increased (0.3 to 82.6%, 14.1 to 100%, 19.0 to 37.5%), and 2 of them died due to disease progression. On the other hand, 2 patients whose PML positive rates decreased after treatment were alive more than 5 years without therapies. Conclusion: Our data indicated that the level of the PML expression at diagnosis was a possible prognostic factor for early course of the disease (2 years after diagnosis). Chemotherapies might induce PML expression in MM cells or select PML positive MM cells. These findings suggest that PML expression presumably reflect chemotherapy-resistance in MM cells. The molecular mechanism of the association is now under investigation. Disclosures: No relevant conflicts of interest to declare.

Establishment of a Novel CNS Infiltrated Xenograft Model through Engraftment of Patient-Derived Acute Lymphoblastic Leukemic Cells Into NOD/SCID/γc Null Mouse

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3248-3248
Author(s):  
Itaru Kato ◽  
Akira Niwa ◽  
Megumu Saito ◽  
Hisanori Fujino ◽  
Satoshi Saida ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Hematologic Malignancy ◽  
Lymphoblastic Leukemia ◽  
Xenograft Model ◽  
Leukemic Cells ◽  
Null Mouse ◽  
Immunodeficient Mice ◽  
Mouse Xenograft Model ◽  
Nog Mice

Abstract Abstract 3248 Background and Purpose: Acute lymphoblastic leukemia (ALL) is the most common type of childhood hematologic malignancy. Although improvements in treatment regimen have raised the 5-year survival rate as high as 80% for pediatric ALL patients, a minority of patients with various risk factors, including central nervous system (CNS) infiltration continue to have poor prognosis. Recently, bone marrow (BM) microenvironments which support leukemic stem cells have become noticed as an important element which can influence treatment response and relapse of the disease. Although leukemic cells appear to be completely eradicated through treatment, they are thought to survive within bone marrow and/or extramedullary microenvironments, such as CNS, causing disease recurrence. However, little is known about the CNS microenvironment for leukemic cells because of the lack of appropriate animal model. Even though several investigators have tried to establish a CNS infiltrated model of leukemia, major limitation with these studies are the use of leukemic cell lines and the preconditioning of recipient mice, which did not represent CNS leukemia observed in patients. Here we report the establishment of a novel xenograft model for primary human ALL using NOD/SCID/γc null (NOG) mouse. Without irradiation, this model recapitulates CNS as well as extramedullary leukemic infiltration (hereby referred to as the h-leukemic NOG model). Result: Primary bone marrow samples were collected from 9 children with ALL at the time of diagnosis with informed consent. The leukemic cells (1×106cells) were injected into the tail veins of non-irradiated 8- to 10-week old NOG mice. Primary samples from 8 out of 9 patients were successfully engrafted. Engrafted leukemic cells could be serially transplanted into secondary, tertiary and quaternary recipients. Morphological and FACS analyses revealed as high as 95% BM chimerism and showed that blast phenotypes were conserved through serial transplantations. Of note, extramedullary organs including the CNS, liver, spleen, and kidneys showed the leukemic invasion consistent with those of the donor ALL patients. Liver pathology in the h-leukemic NOG model is identical to that seen in the ALL patients. We also showed the existence of a functional niche in the liver mediated by SDF-1/CXCR4 axis. In terms of the CNS involvement, we observed the progressive infiltration of leukemic cells into the Virchow-Robin space that is consistent with the pathology of human ALL patients. Using this model, we examined the mechanism of dissemination and harboring of leukemic cells in the CNS niche. Discussion: NOG mice model for engraftment of human leukemic cells provides useful insights into the biology of ALL and allows us to answer various questions concerning the mechanism of extramedullary invasion and expansion. We have reported that NOG mice have significantly better human hematopoietic cell engraftment in the BM and extramedullary organs than other immunodeficient mice (Hiramatsu H. Blood. 2003), and is capable of supporting the growth of human neoplastic cells (Kato M. Nature. 2009). Here we report that this non-preconditioned mouse xenograft model reproduces leukemic extramedullary involvement, including the CNS, in sustaining leukemic cells. This approach provides a more sophisticated and physiological model suitable for the evaluation of molecular interactions between patient leukemic cells and host niche. Our h-leukemic NOG model will provide a powerful tool to analyze the CNS niche that harbors leukemia initiating cells. Moreover, this model would be a useful platform for developing novel anti-leukemic therapies that target CNS extramedullary niche. Disclosures: No relevant conflicts of interest to declare.

Effects of G-CSF On Acute Lymphoblastic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2564-2564
Author(s):  
Jordan Basnett ◽  
Adam Cisterne ◽  
Kenneth F Bradstock ◽  
Linda J Bendall
Keyword(s):  
Bone Marrow ◽  
Disease Progression ◽  
Microarray Analysis ◽  
Environmental Changes ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Hematopoietic Stem ◽  
Childhood All ◽  
Extracellular Matrix Components ◽  
The Impact

Abstract Abstract 2564 G-CSF is commonly used to treat chemotherapy-induced neutropenia and for the mobilization of hematopoietic stem cells for transplantation in patients with leukemia. Administration of G-CSF has profound effects on the bone marrow microenvironment including the cleavage of molecules required for the maintenance of lymphopoiesis, including CXCL12 and VLA-4. We have recently reported that G-CSF results in the dramatic suppression of B-lymphopoiesis. This, together with previous reports by ourselves, and others, showing that disruption of CXCL12 or VLA-4 slow the progression of B-lineage ALL lead us to consider that G-CSF may similarly antagonize the progression of ALL. To explore this possibility, we examined the impact of G-CSF administration on six human ALL xenografts using a NOD/SCID mouse model. Mice were engrafted without radiation and G-CSF commenced when 1% of the bone marrow consisted of ALL cells. G-CSF was administered twice daily for 10 days, at which time all animals were culled and leukemia assessed in the blood, bone marrow and spleens. Surprisingly G-CSF was found to increase disease progression in two of xenografts investigated (1345 and 0398, referred to as G-CSF responsive xenografts hereafter), while the remainder demonstrated a small reduction in leukemia, with one showing a statistical significant decrease. No evidence for a direct mitogenic effect of G-CSF could be demonstrated in any of the xenografts using exogenous G-CSF in vitro cultures in the presence or absence of human or murine stromal support. Consistent with these findings, and previous reports, little to no G-CSF receptor was detected by flow cytometry or microarray analysis of xenografts. Microarray analysis of the xenografts revealed significant differences in gene expression between the G-CSF responsive xenografts and the remainder of the samples. A total of 83 genes were expressed at a higher level and 127 genes at a lower level in the G-CSF responsive xenografts. The more highly expressed genes included cell cycle regulators (eg cyclin A1), adhesion molecules (eg ALCAM), extracellular matrix components and surface receptors. Perhaps the most interesting was the exclusive expression of the acetylcholine receptor (cholinergic receptor, nicotinic, beta 4, nAChRb4) in the G-CSF responsive cases. Analysis of a large public dataset of childhood ALL samples revealed significantly higher expression of this gene in ALL samples with rearranged MLL (p<0.03). However, small numbers of cases in all ALL subgroups had greater than an 2 fold higher expression compared to normal B cell progenitors. The role of nAChR in the response of ALL cells to micro-environmental changes induced by G-CSF remains to be determined, however, nAChR has known roles in cell proliferation and inhibition of apoptosis. Furthermore G-CSF is known to induce acetylcholine production in other tissues. In summary, G-CSF inhibited leukemia progression in the majority of patient xenografts, however, in a subset of samples G-CSF accelerated disease progression. Clinically, G-CSF administration to ALL patients has not been associated with any major adverse outcomes. However our data suggest that a small subset of patients may experience accelerated disease. Identification of features associated with adverse responses to G-CSF will permit the identification of patients for whom G-CSF may present a risk for increased disease progression. Disclosures: No relevant conflicts of interest to declare.

Identification of Non-Cardiotoxic hERG1 Blockers to Overcome Chemoresistance in Acute Lymphoblastic Leukemias

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1506-1506
Author(s):  
Marika Masselli ◽  
Serena Pillozzi ◽  
Massimo D'Amico ◽  
Luca Gasparoli ◽  
Olivia Crociani ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Map Kinases ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Significant Proportion ◽  
Leukemic Cells ◽  
K Channel

Abstract Abstract 1506 Although cure rates for children with acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, have markedly improved over the last two decades, chemotherapy resistance remains a major obstacle to successful treatment in a significant proportion of patients (Pui CH et al. N Engl J Med., 360:2730–2741, 2009). Increasing evidence indicates that bone marrow mesenchymal cells (MSCs) contribute to generate drug resistance in leukemic cells (Konopleva M et al., Leukemia, 16:1713–1724, 2002). We contributed to this topic, describing a novel mechanism through which MSCs protect leukemic cells from chemotherapy (Pillozzi S. et al., Blood, 117:902–914, 2011.). This protection depends on the formation of a macromolecular membrane complex, on the plasma membrane of leukemic cells, the major players being i) the human ether-a-gò-gò-related gene 1 (hERG1) K+ channel, ii) the β1integrin subunit and iii) the SDF-1α receptor CXCR4. In leukemic blasts, the formation of this protein complex activates both the ERK 1/2 MAP kinases and the PI3K/Akt signalling pathways triggering antiapoptotic effects. hERG1 exerts a pivotal role in the complex, as clearly indicated by the effect of hERG1 inhibitors to abrogate MSCs protection against chemotherapeutic drugs. Indeed, E4031, a class III antiarrhythmic that specifically blocks hERG1, enhances the cytotoxicity of drugs commonly used to treat leukemia, both in vitro and in vivo. The latter was tested in a human ALL mouse model, consisting of NOD/SCID mice injected with REH cells, which are relatively resistant to corticosteroids. Mice were treated for 2 weeks with dexamethasone, E4031, or both. Treatment with dexamethasone and E4031 in combination nearly abolished bone marrow engraftment while producing marked apoptosis, and strongly reducing the proportion of leukemic cells in peripheral blood and leukemia infiltration of extramedullary sites. These effects were significantly superior to those obtained by treatment with either dexamethasone alone or E4031 alone. This model corroborated the idea that hERG1 blockers significantly increase the rate of leukemic cell apoptosis in bone marrow and reduced leukemic infiltration of peripheral organs. From a therapeutic viewpoint, to develop a pharmacological strategy based on hERG1 targeting we must consider to circumvent the side effects exerted by hERG1 blockers. Indeed, hERG1 blockers are known to retard the cardiac repolarization, thus lengthening the electrocardiographic QT interval, an effect that in some cases leads to life threatening ventricular arrhythmias (torsades de points). On the whole, it is mandatory to design and test non-cardiotoxic hERG1 blockers as a new strategy to overcome chemoresistance in ALL. On these bases, we tested compounds with potent anti-hERG1 effects, besides E4031, but devoid of cardiotoxicity (e.g. non-torsadogenic hERG1 blockers). Such compounds comprise erythromycin, sertindole and CD160130 (a newly developed drug by BlackSwanPharma GmbH, Leipzig, Germany). We found that such compounds exert a strong anti-leukemic activity both in vitro and in vivo, in the ALL mouse model described above. This is the first study describing the chemotherapeutic effects of non-torsadogenic hERG1 blockers in mouse models of human ALL. This work was supported by grants from the Associazione Genitori contro le Leucemie e Tumori Infantili Noi per Voi, Associazione Italiana per la Ricerca sul Cancro (AIRC) and Istituto Toscano Tumori. Disclosures: No relevant conflicts of interest to declare.

Successful Engrafment After HLA Identical Granulocyte Transfusion As Support Therapy in a Septic Shock Patient with Acute Lymphoblastic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4507-4507
Author(s):  
Roberto Ovilla ◽  
Claudia Barrera-Carmona ◽  
Nicolas Guzman-Bouilloud ◽  
Elizabeth Buganza-Torio ◽  
Rosa Jimenez-Alvarado ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Septic Shock ◽  
Acute Lymphoblastic Leukemia ◽  
Bone Marrow Transplant ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Immunosuppressive Treatment ◽  
Marrow Transplant ◽  
Granulocyte Transfusion ◽  
Shock State

Abstract Abstract 4507 A 53 years old male started in February 2010 with ecchymosis, petechiae and spontaneous gum bleeding. He was diagnosed with acute lymphoblastic leukemia, and was started on HYPER-CVAD, in combination with anti-tumoral lysis syndrome measures and antimicrobial, antiviral y antifungal prophylaxis. After finishing HYPER-CVAD phase A, he developed severe myelosuppression even with the use of G-CSF, 72 hours later he presented with abdominal cramping, fever up to 38.2°C and hypotension, with a high clinical suspicion of neutropenic colitis; his mean arterial pressure average was 45 mmHg, he was started on intravenous colloids, dobutamine and norepinephrine drips. Because of severe myelosuppression, septic shock and myocardial depression, a granulocyte transfusion without previous mobilization was performed with an identical sibling donor with concomitant use of granulocytic colony stimulation factor. Severe myelosuppression was maintained during 7 days, however shock state was reversed some hours after performing granulocyte transfusion, without infectious signs, 36 hours later a mononuclear cell infusion was performed from the same donor, with previous 2 days G-CSF mobilization. A gradual increase in leukocyte number appeared, with 400, 900 and finally 1900. A new bone marrow aspiration was performed, where hematopoietic recovery was confirmed from his sister's cells with confirmation by karyotype and microsatellites. He was then considered bone marrow grafted HLA compatible. On April 2010 he presented with acute diarrheic syndrome secondary to a CMV infection, he was started on ganciclovir and intravenous immunoglobulin. On May he presented an Aspergillus pneumonia that was treated both clinically with antifungal therapy and surgically with thoracoscopy to remove the fungi lesion. On October 2010 he started with graft versus host manifestations on the skin and in the liver. He started immunosuppressive treatment with Prednisone and Sirolimus. On November he developed anogenital herpes zoster infection. After this complication, every GVHD manifestation ceded. Now, 28 months post-bone marrow transplant he presents full remission with no evidence of leukemia. Granulocyte transfusion is an uncommon technique. Preferably it should be done with and identical donor. In this case report, the justification of the procedure was to be able to achieve a remission from a potentially irreversible septic shock; this was successfully done, in an unexpected clinical scenario with severe life threatening, and in a casual manner, the patient achieved a successfully bone marrow transplant, and now 30 months post-granulocyte infusion the patient is free from any evidence of disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Krüppel-like Factor 4 (KLF4) Suppresses T-Cell Acute Lymphoblastic Leukemia By Inhibiting Expression of MAP2K7 and Expansion of Leukemia Initiating Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3569-3569
Author(s):  
Ye Shen ◽  
Chun Shik Park ◽  
Koramit Suppipat ◽  
Takeshi Yamada ◽  
Toni-Ann Mistretta ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Brdu Incorporation ◽  
Jnk Pathway

Abstract Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy in children. Although risk-adaptive therapy, CNS-directed chemotherapy and supportive care have improved the survival of ALL patients, disease relapse is still the leading cause of cancer-related death in children. Therefore, new drugs or novel multi-drug combinations are needed as frontline treatments for high-risk patients and as salvage agents for relapsed disease. T-cell ALL (T-ALL) is a subset of ALL that exhibits activating mutations of NOTCH1 in more than 50% of the patients. However, the use of gamma-secretase inhibitors to reduce NOTCH1 activity has not been successful in patients due to limited response and toxicity. Therefore, identification of genetic factors that cooperate with T-ALL leukemogenesis is needed for the development of alternative therapies. KLF4 is a transcription factor that functions as a tumor suppressor or an oncogene depending on cellular context. Our data showed significant reduction of KLF4 transcripts in lymphoblasts from T-ALL patients compared to blood and bone marrow cells from healthy individuals. In consistent with reduced KLF4 levels, these patients exhibit hyper-methylation of CpG islands located between nt -811 and +1190 relative to KLF4 transcription start site. From these findings we hypothesized that KLF4 has tumor suppressor function in T-ALL leukemogenesis. To test our hypothesis, we transduced 5-FU treated bone marrow (BM) cells from control (Klf4fl/fl), Klf4 null (Klf4fl/fl; Vav-iCre) and Klf4 heterozygous (Klf4fl/+; Vav-iCre) mice with retrovirus carrying a NOTCH1 activating mutant (L1601P-ΔP) and then transplanted these BM cells into irradiated recipient mice. In contrast to controls, mice transplanted with transduced Klf4-null BM cells developed T-ALL with significantly higher penetrance (Klf4 null 76.5% v.s. control 21.3%) and shorter latency (Klf4 null 93 days v.s. control 130 days). Interestingly, Klf4 heterozygous group shows similar survival kinetics as Klf4 null group, suggesting that Klf4 haploinsufficiency is enough to accelerate onset of leukemia. To investigate the effect of Klf4 deletion in established leukemia cells, we transplanted NOTCH1 L1601P-ΔP transduced BM cells from Klf4fl/fl; CreER+ mice to induce leukemia. Post-transplantation deletion of the Klf4 gene by tamoxifen administration was able to accelerate T-ALL development compared to mice injected with vehicle. On the cellular level, loss of KLF4 led to increased proliferation of leukemia cells as assessed by in vivo BrdU incorporation, which correlated with decreased levels of p21 protein. Limited dilution transplantation of primary leukemia cells into secondary recipients showed a 9-fold increase of leukemia initiating cells (LIC) frequency in Klf4null leukemia cells compared to controls, suggesting that KLF4 controls expansion of LIC in T-ALL. To elucidate molecular mechanism underlying KLF4 regulation in T-ALL cells, we performed microarray and ChIP-Seq in control and Klf4 null CD4+CD8+ leukemia cells. Combined analyses revealed 202 genes as KLF4 direct targets, of which 11 genes are also deregulated in human T-ALL cells by comparing with published microarray datasets. One of the top upregulated genes is Map2k7, which encodes a kinase upstream of the JNK pathway. Immunoblots in leukemia cells confirmed increased expression of MAP2K7 protein and enhanced phosphorylation of its downstream targets JNK and ATF2. To further investigate the role of JNK pathway in T-ALL, we tested JNK inhibitor SP600125 in human T-ALL cell lines (KOPTK1, DND41, CCRF-CEM, MOLT3). Interestingly, SP600125 showed dose-dependent cytotoxicity in all human T-ALL cell lines tested regardless of their NOTCH1 status. Overall our results showed for the first time that KLF4 functions as a tumor suppressor in T-ALL by regulating proliferation of leukemia cells and frequency of LIC. Additional study elucidated that KLF4 suppresses the JNK pathway via direct transcriptional regulation of MAP2K7. Moreover, the vulnerability of human T-ALL cell lines to JNK inhibition provides a novel target for future therapy in T-ALL patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Low Epression of MiR-18a Distinguishes Pediatric and Adult Acute Lymphoblastic Leukemia from Each Other

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4991-4991
Author(s):  
Neda Mosakhani ◽  
Mohamed El Missiry ◽  
Emmi Vakkila ◽  
Päivi Heikkilä ◽  
Sakari Knuutila ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Immune Cell ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Tissue Samples ◽  
Inflammatory Microenvironment ◽  
Qrt Pcr ◽  
Response To Chemotherapy ◽  
Pediatric All

Abstract In several adult solid cancers the presence or absence of an inflammatory microenvironment has turned out to be an important prognostic factor. Acute lymphoblastic leukemia (ALL) is seen in both adults and children but the response to chemotherapy and survival is significantly worse in adults than children. Therefore, we wanted to study whether the expression of immune system associated molecular markers would be different in adult and pediatric ALL patients at the time of diagnosis. IDO and FOXP3 were studied from paraffin embedded tissue samples by immunohistochemistry in 12 pediatric and 10 adult bone marrow samples. Inflammation associated miRNA analysis were performed in 19 adult and 79 pediatric ALL patients and involved miR-10, miR-15, miR-16, miR-17-92 cluster, miR-33, miR-146a, miR-150, miR-155, miR-181a, miR-222, miR-223, and miR-339. miRNAs were first analysed by Agilent's miRNA microarray and thereafter validated by qRT-PCR. miRNAs not expressed in at least 75% of one group of samples were excluded. Significance (p <0.05; q<0.1) of differential expression was estimated by t-test for those miRNAs with at least a 2.0 fold change. Sufficient RNA for qRT-PCR was available for 42 pediatric and 19 adult patients. The adult and pediatric ALL patients had quantitatively and qualitatively similar expression of IDO and FOXP3 in leukemic bone marrow samples (p=0.26 and 0.74, respectively). Out of studied miRNAs only miR-18a differed significantly in microarray analysis between adult and pediatric ALL being lower in children (FC -3.74; p 0,0037). Results were confirmed by qRT-PCR (upregulated in adults, FC 3.71, p 0.003161). The other members of the miR-17-92 cluster did not differ significantly. We conclude that pediatric and adult ALL patients have remarkably similar pattern of immune cell associated markers in bone marrow at diagnosis. This is in line with recent evidence that the outcome of the adult ALL patients can be significantly improved if treated with pediatric protocols. However, the low expression of miR-18a in pediatric ALL is interesting and demands further studies. Disclosures No relevant conflicts of interest to declare.

Distinct Transforming and Leukemogenic Activities of Tel-Arg and Tel-Abl Oncogenes in Mice: Possible Negative Role for the Transforming Activity in C-Terminus of Arg

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2845-2845
Author(s):  
Keiko Okuda ◽  
Nari Harakawa ◽  
Richard A VanEtten ◽  
Nihal Patel ◽  
Naochika Domae ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Leukemia ◽  
Tyrosine Kinase ◽  
Intracellular Signaling ◽  
Lymphoblastic Leukemia ◽  
Kinase Domain ◽  
Actin Binding ◽  
Mouse Bone Marrow ◽  
C Terminus ◽  
Transforming Activity

Abstract ARG (ABL2) is a member of ABL tyrosine kinase gene family that is highly homologous to c-ABL (ABL1) in overall domain structure (SH3-SH2-SH1) and amino acid sequence. ARG has recently been implicated in the pathogenesis of human acute leukemia though t(1;12) translocations that fuse a transcription factor gene, ETV6/TEL, to ARG (Iijima et al., Blood2000;95:2126). The resulting TEL-ARG fusion tyrosine kinase is similar in structure to the TEL-ABL fusion found in some acute leukemia and atypical CML patients, and, like TEL-ABL, can transform Ba/F3 cells and fibroblasts in vitro and activate a similar set of intracellular signaling pathways (Iijima et al., Oncogene2002;21:4374). To assess the leukemogenic activity of TEL-ARG, we co-expressed TEL-ARG with GFP in mouse bone marrow using a retroviral bone marrow transduction/transplantation strategy. Whereas TEL-ABL induces rapidly fatal myelolproliferative disease (MPD) in recipient mice (Million et al., Blood2000;96:664), recipients of TEL-ARG-transduced BM did not develop overt MPD, but succumbed instead to long-latency (30–45 weeks) T-cell acute lymphoblastic leukemia/lymphoma characterized by modest leukocytosis and a malignant pleural effusion composed of Thy-1+B220- tumor cells. To study the molecular basis of the marked difference in the leukemogenic activity of TEL-ARG and TEL-ABL, we produced TEL-ARG mutants that swapped the kinase domain or C-terminus of ARG with the corresponding domain in ABL. The mutants were introduced into Ba/F3 cells by retroviral transduction and transduced cells selected for equal expression of the fusion protein by flow sorting for populations with equivalent intensity of GFP fluorescence. All chimeric proteins were expressed and showed equivalent levels of auto-phosphorylation by western blot analysis of the sorted cells. However, the quantitative transforming activity of TEL-ARG in Ba/F3 cells, measured by the number of days required to achieve measurable cell growth following IL-3 deprivation, was significantly lower than for TEL-ABL (25 ± 4.3 days for TEL-ARG vs. 1 to 2 days for TEL-ABL). A TEL-ARG mutant containing the ABL kinase domain was similar to TEL-ARG in this assay, but replacing the ARG C-terminal domain with that of ABL increased Ba/F3 transformation to levels equivalent to TEL-ABL. To further dissect the functional domains that are responsible for this effect, a new series of mutants containing internal deletions in ARG C-terminus of TEL-ARG [aa.826–976 (Delta Box1), aa.977–1213 (Delta Box2), aa 1214–1316 (Delta Box3), aa.1317–1465 (Delta Box4)] were generated. Regarding to their function, only Box2 and Box4 were reported as F- or F,G- actin binding domain. From a preliminary study using the cell lines which express each of the mutants, Delta Box 1 clones obtained an accelerated proliferation which compared with that of Tel-Abl, suggesting that there is a disadvantage for cell proliferation by ARG c-terminus. These results indicate that distinct bio-phenotype associated with Abl family tyrosine kinase is the most likely regulated by their c-terminus and the c-terminus of Arg contains functional subdomain that impairs growth signal induced by Abl family tyrosine kinase.

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