Crisis Chronic Myelogenous Leukemia Research Articles

Deficiency of the E3 Ligase Hoip Impairs Adult Hematopoiesis and Myeloid Leukemia

Ubiquitination is a post-translational modification that plays important roles in the regulation of various cellular processes. The linear ubiquitin chain assembly complex (LUBAC) is the only E3 ubiquitin ligase, which can specifically generate linear ubiquitin chains. LUBAC is composed of three subunits: catalytic Hoip (Rnf31), Hoil-1l (RBCK1), and Sharpin. LUBAC regulates canonical NF-κB signaling pathway and apoptosis. Several studies have shown that LUBAC is required for fetal hematopoiesis and tumorigenesis in several solid cancers, but the role of Hoip, which is the catalytic component of the LUBAC, in adult hematopoiesis and myeloid leukemia is unclear.To address whether Hoip is required for adult hematopoiesis, we used tamoxifen induced Hoip deleted mice (Hoip fl/fl; Rosa26-CreERT). Deletion of Hoip reduced the numbers of almost all cell fractions in bone marrow and peripheral blood, except for long-term hematopoietic stem cell (LT-HSC) fraction (CD34 - Flk2 - Lineage - c-Kit + Sca-1 +: LSK). Deletion of Hoip treated with tamoxifen markedly impaired colony formation of LT-HSCs in vitro. Among competitive transplantation assay using bone marrow cells from Hoip fl/fl; Rosa26-CreERT mice, deletion of Hoip treated with tamoxifen following stable reconstitution 8 weeks after transplantation led to a significant reduction of Hoip-deficient chimerism compared to control in vivo (Figure A). Deletion of Hoip induced apoptosis in bone marrow cells and increased the frequency of CD34 - LSK cells in S/G2/M phase of the cell cycle. Collectively, these data indicated that Hoip is required for adult hematopoiesis.To evaluate the consequences of Hoip deletion in myeloid leukemia propagation and maintenance, we utilized aggressive murine myeloid leukemia models driven by retroviral transduction of oncogenes: MLL-AF9 and NRAS G12V-driven acute myeloid leukemia (AML) and BCR-ABL and NUP98-HOXA9-driven blast crisis of chronic myelogenous leukemia (CML-BC) models. Deletion of Hoip in established leukemia cells treated with tamoxifen markedly impaired colony formation of leukemia cells in vitro and led to a significantly longer survival with reduced disease burden in bone marrow and spleen in vivo in both of leukemic models (Figure B,C). Deletion of Hoip also induced apoptosis in leukemia stem cells (LSCs: c-Kit + Lineage - cells) (Figure D) and increased the frequency of LSCs in S-phase of the cell cycle. Furthermore, we evaluated the contribution of HOIP to human myeloid leukemia. Knockdown of HOIP by small hairpin RNA using lentivirus in several myeloid leukemia cell lines (THP-1, SKM-1, and K562) and primary patient-derived AML cells exhibited reduced numbers of colony forming cells in vitro, and increased apoptosis. LUBAC inhibitor gliotoxin impaired the growth of murine and human leukemia cells. These results indicate that Hoip is essential for propagation and maintenance in murine and human myeloid leukemia.Because LUBAC regulates canonical NF-κB signaling pathway, we evaluated protein levels of p65 phosphorylation on Ser536 (p-p65) and IκBα in leukemia cells by flow cytometric analysis. Reduction in p-p65 and inhibition of IκBα degradation were observed in Hoip-deficient leukemia cells (Figure E), indicating that Hoip plays an important role in regulation of NF-κB signaling in myeloid leukemia.Since Hoip is the central component of LUBAC and the only catalytically active subunit of E3 ubiquitin ligase, we examined the importance of each domain of Hoip for myeloid leukemia propagation using Hoip mutant isoforms. Loss of colony-forming ability of Hoip-deficient AML cells could not be rescued by retroviral transduction of the Hoip mutants with deletion of UBA domain (which is required for interaction with the other LUBAC subunits) and with deletion of RBR domain and with mutation in C879A (which lack ligase activity) (Figure F), indicating that LUBAC ligase activity and interaction with the other LUBAC subunits are critical for leukemia propagation.Thus, our data demonstrated that Hoip is essential for adult hematopoiesis and myeloid leukemia. Given that patients with myeloid leukemia have shown increased activity of NF-κB signaling, inhibition of LUBAC ligase activity could serve as a promising strategy for treating myeloid leukemia. [Display omitted] DisclosuresJimbo: Japan Society for the Promotion of Science: Research Funding. Ito: Japan Society for the Promotion of Science: Research Funding; Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding. Iwama: Nissan Chemical Corporation: Research Funding. Nannya: Otsuka Pharmaceutical Co., Ltd.: Consultancy, Speakers Bureau; Astellas: Speakers Bureau. Konuma: Japan Society for the Promotion of Science: Research Funding; The Japanese Society of Hematology: Research Funding; SGH Foundation: Research Funding; Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding.

Recurrent and isolated central nervous system blast crisis in chronic myelogenous leukemia

Recurrent and isolated central nervous system blast crisis in chronic myelogenous leukemia

Haploidentical Hematopoietic Stem Cell Transplantation with Post-Transplantation Cyclophosphamide in Children with High-Risk Leukemia Using a Reduced-Intensity Conditioning Regimen and Peripheral Blood as the Stem Cell Source

The use of haploidentical donor hematopoietic stem cell transplantation with post-transplantation cyclophosphamide (Haplo-PTCy) in children is increasing; however, it is still not clear which preparative regimen is best in this setting. We present the long-term results of 42 patients age <18 years with high-risk leukemia who underwent this procedure using a reduced-intensity conditioning regimen (RIC) and peripheral blood as the stem cell source. Twenty-six patients had acute lymphoblastic leukemia, 13 had acute myelogenous leukemia, 2 had juvenile myelomonocytic leukemia, and 1 had blast crisis of chronic myelogenous leukemia. One-third of the patients were in first remission, 50% were in second remission, 14% were in third remission, and 3% had refractory disease. Neutrophil recovery occurred in 100% of the 40 patients alive at day +30, and transplantation-related mortality at 1 year was 14%. The incidence of acute graft-versus-disease (GVHD) grade III-IV was 17%, and the cumulative incidence of moderate to severe chronic GVHD at 1 year was 29%. The median duration of follow-up for surviving patients was 45 months; overall survival and event-free survival at 36 months were 56% and 46%, respectively. Long-term results of this series show that the use of an RIC regimen with peripheral blood stem cells as the cell source, in children with high-risk leukemia who underwent haplo-PTCy has tolerable toxicity, universal engraftment, and good survival rates.

Immunoglobulin Superfamily Member 8 Is Indispensable for Myeloid Leukemia Via Wnt/β-Catenin Signaling Pathway

Immunoglobulin superfamily member 8 (IGSF8, also known as EWI-2, PGRL, and CD316), is a cell surface protein containing 4 immunoglobulin domains. IGSF8 directly binds to the tetraspanin molecules, CD9 and CD81, and modulates cell adhesion, migration, and growth. Previous studies demonstrated that IGSF8 was associated with prognosis and metastasis in several solid tumors. However, the role of IGSF8 in normal hematopoiesis and myeloid leukemia is still unclear. First, we examined the expression levels of Igsf8 in various hematopoietic fraction of wild-type murine bone marrow cells, and found that Igsf8 is expressed in all hematopoietic lineages. To investigate hematopoietic functions of Igsf8, we generated hematopoietic cells specific Igsf8 deleted mice (Igsf8fl/fl; Vav-Cre) and tamoxifen induced Igsf8 deleted mice (Igsf8fl/fl; Rosa26-CreERT). Igsf8fl/fl, Vav-Cre (denoted as Igsf8-/-) mice represented normal maturation. Deletion of Igsf8 did not significantly affect adult hematopoiesis in peripheral blood and bone marrow. Igsf8-/- long-term hematopoietic stem cells (LT-HSCs: CD34- Flk2- c-Kit+ Sca-1+ Lineage- cells) reduced colony forming ability in vitro, and serial competitive transplantation assay showed comparable donor chimerism by 3 months, but led to decrease Igsf8-/- donor chimerism at 4 months and those after second transplantation in vivo. These results suggest that Igsf8 does not affect the adult hematopoiesis, but it can affect their proliferative and reconstitutive capacity of HSCs. To investigate the effects of Igsf8 on myeloid leukemia, we generated MLL-AF9 and NRASG12V-driven acute myelogenous leukemia (AML), or BCR-ABL and NUP98-HOXA9-driven blast crisis of chronic myelogenous leukemia (CML-BC) mice models. Igsf8-/- led to a dramatic reduction in the number of leukemic colonies formed in vitro (Figure 1A). Igsf8-/- leukemia mice showed significantly longer survival in vivo (Figure 1B). This effect was also observed by eliminating Igsf8 expression after leukemia establishment using conditionally deletion. Igsf8-/- AML cells showed decreased S phase fraction. Igsf8-/- leukemia stem cells (LSCs: c-Kit+ Lineage- cells) triggered an increment of the apoptosis, which contribute to significantly lower proportion of LSCs in spleen of Igsf8-/- leukemic mice. Given that Igsf8-/- did not affect homing ability of leukemia cells, these results indicate that Igsf8 is required for propagation of myeloid leukemia and maintenance of LSC. To understand the Igsf8-mediated regulation of myeloid leukemia, we conducted RNA sequencing analysis of LT-HSCs, and LSCs of AML and CML-BC. Gene set enrichment analysis exhibited increase apoptosis related genes and decrease Wnt/β-catenin related genes in Igsf8-/- leukemic cells, but not in LT-HSCs (Figure 1C). Increment of pro-apoptosis genes, and decrement of anti-apoptosis genes and Wnt/β-catenin target genes in Igsf8-/- AML stem cells were validated in quantitative polymerase chain reaction analysis. Further, expression levels of β-catenin protein in Igsf8-/- leukemic cells were significantly lower compared to Igsf8+/+ leukemic cells, but not in normal hematopoietic stem and progenitor cells (Figure 1D). These results suggest that Igsf8 might be critical for myeloid leukemia maintenance via Wnt/β-catenin signaling pathway. Then, we investigated the effects of IGSF8 on human myeloid leukemia. We confirmed IGSF8 expression in several human myeloid leukemia cell line and primary patient-derived leukemia cells. Knockdown of IGSF8 by small hairpin RNA in myeloid leukemia cell lines (THP-1, MV4-11, SKM-1, and K562) and primary patient-derived AML cells exhibited reduced numbers of colony forming cells in vitro. Knockdown of IGSF8 also caused decrease expression of β-CATENIN in AML cell lines. These results indicate that IGSF8 is also required for propagation of human myeloid leukemia cells. Taken together, our present study reveals that Igsf8 is indispensable for myeloid leukemia, but not adult hematopoiesis, suggesting that IGSF8 inhibition should be considered for targeting myeloid leukemia. Disclosures Jimbo: Japan Society for the Promotion of Science: Research Funding. Konuma:SGH Foundation: Research Funding; The Japanese Society of Hematology: Research Funding; Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding. Ito:Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding.

Embryonic Program Activated during Blast Crisis of Chronic Myelogenous Leukemia (CML) Implicates a TCF7L2 and MYC Cooperative Chromatin Binding.

Chronic myeloid leukemia (CML) is characterized by an inherent genetic instability, which contributes to the progression of the disease towards an accelerated phase (AP) and blast crisis (BC). Several cytogenetic and genomic alterations have been reported in the progression towards BC, but the precise molecular mechanisms of this event are undetermined. Transcription Factor 7 like 2 (TFC7L2) is a member of the TCF family of proteins that are known to activate WNT target genes such as Cyclin D1. TCF7L2 has been shown to be overexpressed in acute myeloid leukemia (AML) and represents a druggable target. We report here that TCF7L2 transcription factor expression was found to be correlated to blast cell numbers during the progression of the disease. In these cells, TCF7L2 CHIP-sequencing highlighted distal cis active enhancer, such as elements in SMAD3, ATF5, and PRMT1 genomic regions and a proximal active transcriptional program of 144 genes. The analysis of CHIP-sequencing of MYC revealed a significant overlapping of TCF7L2 epigenetic program with MYC. The β-catenin activator lithium chloride and the MYC-MAX dimerization inhibitor 10058-F4 significantly modified the expression of three epigenetic targets in the BC cell line K562. These results suggest for the first time the cooperative role of TCF7L2 and MYC during CML-BC and they strengthen previous data showing a possible involvement of embryonic genes in this process.

The Embryonic Program Activated during Blast Crisis of Chronic Myelogenous Leukemia (CML) Implicates a TCF7L2 and MYC Cooperative Chromatin Binding and Represents a Druggable Target

Chronic myeloid leukemia (CML) is characterized by an inherent genetic instability, which contributes to the progression of the disease towards accelerated and blast crisis (BC). The occurrence of the latter has been hampered by the use of tyrosine kinase inhibitors (TKI), which changed this natural progression, but BC still occurs in patients resistant to TKI. Several cytogenetic (major and minor routes) and genomic (TP53 mutation, p16/INK4A deletions, DNA repair abnormalities such as BRCA1, DNA-PKcs, hnRNP metabolism) events have been reported in the progression towards BC. Previous data have also suggested the involvement of embryonic stem cell program activated in BC cells such as Lin28A. In this work, we have taken advantage of the previously reported gene profiling of BC in a large cohort of patients (Radich et al. 2006) and found a correlation between blast numbers and the involvement of the Transcription Factor 7 like 2 (TCF7L2) in BC. TFC7L2 is a member of the TCF family of proteins that are known to activate WNT target genes such as Cyclin D1. TCF7L2 has been shown to be overexpressed in acute myeloid leukemia (AML) and represents a druggable target (Saenz et al Leukemia 2019). The involvement of TCF7L2 in CML-BC and its interaction with the epigenetic regulators has not been studied so far. The gene correlation study that we have performed using the blast numbers and the expression of TCF7L2 in CD34+ CML cells was found to be highly significant (Pearson test, r = 0.56, p-value=5.2e-4) (Fig.1A). TCF7L2 promoter was classed as active in K562 with ChromHMM Functional genomic analysis. K562 epigenetics peaks of TCF7L2 CHIP-seq were found principally mapped in proximal promoters (39% of the peaks, -3000pb upstream Transcription Starting Sites (TSS), Fig. 1B) and 183 unique peaks matched with promoter of 144 unique genes found to be correlated to the blast number in blood of the CML patients during BC (Fig 1C). This TCF7L2-dependent BC program was characterized to be active because promoters were also found positive for H3K27Ac and negative for H3K27Me3 histone marks, and functionally enriched with binding sites for MYC/MAX interactions (p=1.15e-6). The analysis of CHIP-sequencing of MYC revealed a significant overlapping of TCF7L2 epigenetic program with MYC (fold enrichment: 20.81, p &lt; 2.2e-16). Surprisingly, the TCF7L2 program was found independent of RUNX1 and GATA2 transcriptional program. To determine these potential interactions, we have designed experiments in K562 cell line using the b-catenin activator Lithium Chloride (LiCL2) and the Myc/Max dimerization inhibitor 10058-F4. K562 cells were cultured in the presence of LiCL2 (10mM &amp; 24hours) and the compound 10058-F4 (64µM &amp; 48hours) and the expression of three epigenetic targets was analyzed by Q-RT-PCR in the presence of DMSO controls. The three targets chosen were protein arginine N-methyltransferase (PRMT1), the ATPase/Helicase RUVBL1 and the WD-repeat containing protein WDR77. As expected, after culture with LiCL2, the expression of PRMT1 was increased x 6.3 fold (p=8.49e-13) , that of RUVBL1 by x 1.66 Fold (p=1.67e-6) and that of WDR77 by x 2 fold (p=4.97e) (Fig.1D). On the contrary, the culture of K562 cells in the presence of MYC/MAX inhibitor 10058-F4, decreased the expression of 3 targets as compared to DMSO controls (x 1.6 fold for PRMT1, p=6.52e-5; x2 fold reduction for RUVBL1, p-value=2.71e-5; and x 1.4 fold for WDR77, p =0.0000643). These results show for the first time a cooperative role of TCF7L2 and MYC during blast crisis of CML and provide mechanistic insights into the interactions for the role of MYC in CML blast crisis. In addition they strengthen previous data showing a possible embryonic footprint in the blast development over the hematopoietic differentiation program during progression of the disease and provide a rationale for the pharmacological targeting of BC by the use of MYC/MAX inhibitors such as 10058-F4. Experiments are underway to evaluate the role of these factors and the MYC/MAX inhibitors in primary CML samples. Reference : Radich JP, Dai H, Mao M, Oehler V, Schelter J, Druker B, Sawyers C, Shah N, Stock W, Willman CL, Friend S, Lindsey PS.(2006) :Gene Expression Changes Associated with Progression and Response in Chronic Myeloid Leukemia. Proceedings of the National Academy of Sciences of the United States of America 103 (8): 2794-99. Disclosures Turhan: Incyte: Consultancy, Honoraria; novartis: Honoraria, Research Funding.

Allogeneic Cord Blood Regulatory T Cells Can Prevent Graft Vs. Host Disease and Preserve Graft Vs Leukemia Effect: Update on Phase I/II Clinical Trial

Previously, we presented the results of a phase I trial of using cord blood (CB) derived regulatory T cells (Tregs) at a dose of 1x106 cells/kg in the prevention of graft vs. host disease (GVHD) in five patients undergoing allogeneic stem cell transplant (SCT). We now present an update of those patients and also the outcome of a single patient treated with CB Tregs at higher dose of 1x107 cells/kg. At the last follow up of 3.5 years, 5 of 6 patients are alive, in complete remission, without GVHD and off immune-suppression (table 1). One patient died of head injury at day 45 post-transplant without GVHD. None of the patients relapsed in spite of having high risk disease including Flt3+ acute myeloid leukemia (AML) (pt#1); refractory mycosis fungoides/ sezary syndrome (pt#2); relapsed refractory multiple myeloma including two autologous SCT (pt#3); myeloid sarcoma (pt#4); AML with complex cytogenetics (pt#5). Specifically, the sixth patient (treated at CB Tregs cell dose of 1x107 cells/kg) had a diagnosis of lymphoid blast crisis of chronic myelogenous leukemia (CML) and underwent allogeneic peripheral blood (PB) matched unrelated donor (MUD) SCT in second chronic phase with the conditioning regimen of fludarabine (40 mg/m2/d on day -5 to -2) and melphalan (140mg/m2 on day -2) and received CB Tregs at a dose of 1x107 cells/kg on day -1. The CB Tregs were manufactured at the MD Anderson GMP facility. Tregs were isolated from 4 out of 6 HLA matched CB unit with a post-thaw total nucleated cell (TNC) count of 1295 x 106 cells with 90% recovery. Enrichment of CD25+ Treg cells was accomplished with directly conjugated anti-CD25 magnetic microbeads and the LS column (Miltenyl) based selection. After selection, the total no. of isolated CB Tregs was 17.6 x 106 cells. These cells were ex-vivo expanded in culture for a total of 14 days in the continued presence of CD3/28 microbeads and interleukin-2. On the day of harvest, a total of 1359 x 106 Treg cells were generated with 96% viability. Based on patient's weight of 67 kg, a total of 67x107 CB Treg cells were infused. The expanded CB Tregs were also evaluated for functionality by using an in vitro suppression assay, where the stimulated CD4+ Tcon cells stained with CellTrace Violet and Tregs were added into a 96-well plate at a 1:1 and 1:2 ratio and incubated for three days at 37oC. As shown in figure 1A, the clinically manufactured CB Tregs were functional and exerted 98% suppression of the proliferating Tcon cells. Subsequently, the patient received PB MUD graft on day 0. The graft had a TNC count of 1900 x106 cells where the CD3+ T cells were at a dose of 400 x106 cells/kg resulting in a Treg: Tcon ratio of 1:40, a significantly lower number of Tregs compared to other published clinical trials using Treg prophylaxis for GVHD. There was no infusion reaction related to the CB Treg infusion, the patient engrafted on day+12 with 100% donor chimerism on day +30. The patient did not develop any grade II-IV GVHD and was off immune suppression by +6 months. At the time of last follow up of 2 years and 4 months, the patient remains alive, in complete remission and without GVHD. We compared the inflammatory cytokine profile as well as GVHD biomarkers for pt #6 to the rest of the five patients who received the lower dose of CB Tregs at 1x106 cells/kg (with up to 400 times Tcon cells in the graft), where 4 patients had developed acute GVHD. The inflammatory biomarkers of IL-6 (fig 1B) and IL-8 (fig 1C) for pt#6 were low to undetectable compared to the other patients. Similarly, the GVHD biomarkers of ST2 (fig 1D); MIG/ CXCL9 (fig 1E) and follistatin (fig 1F) were also low to undetectable in pt#6 compared to the rest. At the time of last follow up, all patients had resolved acute GVHD and were off-immune suppression and in complete remission. Based on these data, we conclude that CB Treg dose of 1x107 cells/kg may be able to prevent GVHD without alleviating the graft vs leukemia effect. Disclosures Popat: Jazz: Consultancy; Incyte: Research Funding; Bayer: Research Funding. Nieto:Affimed: Consultancy; Astra-Zeneca: Research Funding; Novartis: Research Funding; Affimed: Research Funding. Ciurea:Kiadis Pharma: Membership on an entity's Board of Directors or advisory committees, Other: stock holder; MolMed: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Research Funding. Qazilbash:Amgen: Consultancy, Other: Advisory Board; Genzyme: Other: Speaker; Bioclinical: Consultancy; Autolus: Consultancy. Champlin:Actinium: Consultancy; Johnson and Johnson: Consultancy; Sanofi-Genzyme: Research Funding. Parmar:Cellenkos Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Blinatumumab Induces Responses in Extramedulary B-Cell Acute Lymphoid Leukemia (B-ALL) and Lymphoid Blast Crisis Chronic Myelogenous Leukemia (CML), and Rarely Results in CD19 Negative Relapse

Blinatumumab Induces Responses in Extramedulary B-Cell Acute Lymphoid Leukemia (B-ALL) and Lymphoid Blast Crisis Chronic Myelogenous Leukemia (CML), and Rarely Results in CD19 Negative Relapse

Myeloid sarcoma associated with blast crisis in chronic myelogenous leukemia – Case Report

1. Burns A. Observation of surgical anatomy, in Head and Neck. London, England, Royce; 1811. Google Scholar

Extramedullary T-lymphoblastic blast crisis in chronic myelogenous leukemia: A case report of successful diagnosis and treatment.

Extramedullary T-lymphoblastic blast crisis of chronic myelogenous leukemia (CML) is uncommon and the prognosis is poor. It was usually misdiagnosed as the co-existence of T-lymphoblastic lymphoma (T-LBL) and CML. In the present study, we report a patient with CML, who developed extramedullary T-lymphoblastic blast crisis and was successfully treated with human leukocyte antigen (HLA)-mismatched stem cell transplantation. The patient was a 44-year-old man who presented with lymphadenectasis and leucocytosis prior to diagnosis. The bone marrow smear, biopsy and fluorescence in situ hybridization (FISH) of Breakpoint Cluster Region/ Abelson murine leukaemia (BCR/ABL) supported the diagnosis of CML in the chronic phase, while the immunohistochemistry of lymph nodes supported the diagnosis of T-LBL. The FISH test for BCR/ABL in lymph node blast cells was performed and the result was positive; therefore, the patient was diagnosed with extramedullary T-lymphoblastic blast crisis of CML. After several courses of combined chemotherapy, the patient was treated with HLA-mismatched stem cell transplantation and obtained continuous remission for 51 months until the present (September 2013).

A case of pediatric ALL with t(16;21)(p11.2;q22) and FUS-ERG rearrangement.

A case of pediatric ALL with t(16;21)(p11.2;q22) and FUS-ERG rearrangement.

Hes1 promotes blast crisis in chronic myelogenous leukemia through MMP-9 upregulation in leukemic cells

AbstractHigh levels of HES1 expression are frequently found in BCR-ABL+ chronic myelogenous leukemia in blast crisis (CML-BC). In mouse bone marrow transplantation (BMT) models, co-expression of BCR-ABL and Hes1 induces CML-BC–like disease; however, the underlying mechanism remained elusive. Here, based on gene expression analysis, we show that MMP-9 is upregulated by Hes1 in common myeloid progenitors (CMPs). Analysis of promoter activity demonstrated that Hes1 upregulated MMP-9 by activating NF-κB. Analysis of 20 samples from CML-BC patients showed that MMP-9 was highly expressed in three, with two exhibiting high levels of HES1 expression. Interestingly, MMP-9 deficiency impaired the cobblestone area–forming ability of CMPs expressing BCR-ABL and Hes1 that were in conjunction with a stromal cell layer. In addition, CMPs expressing BCR-ABL and Hes1 secreted MMP-9, promoting the release of soluble Kit-ligand (sKitL) from stromal cells, thereby enhancing proliferation of the leukemic cells. In accordance, mice transplanted with CMPs expressing BCR-ABL and Hes1 exhibited high levels of sKitL as well as MMP-9 in the serum. Importantly, MMP-9 deficiency impaired the development of CML-BC–like disease induced by BCR-ABL and Hes1 in mouse BMT models. The present results suggest that Hes1 promotes the development of CML-BC, partly through MMP-9 upregulation in leukemic cells.

Prognostic Significance of the Lymphoblastic Leukemia-Derived Sequence 1 (LYL1) GeneExpression in Egyptian Patients with AcuteMyeloid Leukemia.

Objective: Aberrant activation of transcription factor genes is the most frequent target of genetic alteration in lymphoid malignancies. The lymphoblastic leukemia-derived sequence 1 (LYL1) gene, which encodes a basic helix-loop helix, was first identified with human T-cell acute leukemia. Recent studies suggest its involvement in myeloid malignancies. We aimed to study the expression percent of oncogene LYL1 in primary and secondary high-risk myeloid leukemia and the impact on prognostic significance in those patients. Materials and Methods: Using quantitative real-time polymerase chain reaction for detection of LYL1 oncogenes, our study was carried out on 39 myeloid leukemia patients including de novo cases, myelodysplastic syndrome (MDS) with transformation, and chronic myelogenous leukemia (CML) in accelerated and blast crisis, in addition to 10 healthy individuals as the reference control. Results: LYL1 expression was increased at least 2 times compared to the controls. The highest expression of this transcription factor was observed in the MDS cases transformed to acute leukemia at 7.3±3.1, p=0.0011. LYL1 expression was found in 68.2%, 75%, and 77.8% of cases of acute myeloid leukemia, CML crisis, and MDS, respectively. Significant correlation of LYL1 overexpression with some subtypes of French-American-British classification was found. There was, for the first time, significant correlation between the blood count at diagnosis and LYL1 expression (p=0.023, 0.002, and 0.031 for white blood cells, hemoglobin, and platelets, respectively). The rate of complete remission was lower with very high levels of LYL1 expression and the risk of relapse increased with higher levels of LYL1 expression, suggesting an unfavorable prognosis for cases with enhanced expression.Conclusion: Overexpression of LYL1 is highly associated with acute myeloid leukemia and shows more expression in MDS with unfavorable prognosis in response to induction chemotherapy. These observations could signal a promising tool for a therapeutic target to basic helix–loop helix protein related to transcription factors, which may improve patient outcome in acute myeloid leukemia, MDS, and CML in blast crisis.

Differential analysis of BM cell morphology, immunophenotypic, cytogenetic characters and prognosis between myeloblastic and lymphoblastic crisis of CML

This study was purposed to investigate the difference of morphology, immunophenotype, cytogenetic features and prognosis between myeloid blast crisis and lymphoid blast crisis of chronic myelogenous leukemia (CML). A total of 31 patients with CML in blastic crisis in Department of Hematology, the First Affiliated Hospital of Xi'an Jiaotong University school of Medicine from 2009 January to 2014 January were enrolled in this study. Out of 31 CML patients, 24 cases were patients with myeloid blast crisis and other 7 cases were patients with lymphoblastic crisis. The clinical data, blast cell percentage in peripheral blood and bone marrow, eosinophil and basophil percentage, immunophenotype, cytogenetic characteristics and prognosis were analyzed. The results indicated that there was no significant difference of blastic cell percentage in peripheral blood and bone marrow of CML with myeloid blast crisis, and the eosinophil and basophil cells could be easily detected. The ratio of blastic cells in BM was higher than that in PB in lymphoid blastic crisis of CML, eosinophil and basophil cells were rare. 7 cases of CML with lymphoid blastic crisis were B ALL with CD10, CD19, CD34, HLA-DR expression, and 2 cases with CD13 and CD33 expression. The lymphoid score was in all CML patients with lymphoid blastic crisis was greater than or equal to 1.5;and 2 patients with CD13 and CD33 expression, and with 1 myeloid score.24 cases of myeloid blastic crisis of CML patients mainly expressed CD33, CD13, CD38, CD34, CD11b and HLA-DR, and their myeloid score greater than or equal to 2, among them the lymphoid scores of 2 patients were 0.5 and 1 score, respectively. All the 31 patients showed 100% Ph(+) chromosome, among them 3 cases also showed other new chromosome aberrations. There was no significant difference of overall survival rate between lymphoid and myeloid blastic crisis of CML, but the overall survival rate of patients treated with tyrosine kinase inhibitor (TKI ) was higher than that in the patients without TKI treatment. It is concluded that eosinophil and basophil cells in peripheral blood of lymphoid blastic crisis were less than that of CML patients with myeloid blastic crisis. Lymphoid blastic crisis of CML patients occurred mostly in B ALL cases with expression of CD10 and CD19. Patients with myeloid blastic crisis of CML mainly expressed CD33, CD13, CD38, CD34, CD11b and HLA-DR, and could be accompanied by other lineage antigen expression, but the score was less than 2. New chromosome aberration is easily observed in myeloid blastic crisis of CML. There is no significant difference of overall survival rate of between CML patients with lymphoid and myeloid blastic crisis, but the overall survival rate of patients treated with TKI is higher than the patients without TKI treatment.

ATM-dependent DNA damage-response pathway as a determinant in chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) begins with an indolent chronic phase, and subsequently progresses to an accelerated or blastic phase. Although several genes are known to be involved in the progression to blastic phase, molecular mechanisms for the evolution toward blast crisis have not been fully identified. Oncogenic stimuli enforce cell proliferation, which requires DNA replication. Unscheduled DNA replication enforced by oncogenic stimuli leads to double strand breaks on DNA. We found the DNA damage-response pathway is activated in bone marrow of chronic-phase CML patients possibly due to an enforced proliferation signal by BCR-ABL expression. Since ataxia telangiectasia mutated (ATM) is a central player of the DNA damage-response pathway, we studied whether loss of this pathway accelerates blast crisis. We crossed Atm-knockout mice with BCR-ABL transgenic mice to test this hypothesis. Interestingly, the loss of one of the Atm alleles was shown to be enough for the acceleration of the blast crisis, which is supported by the finding of increased genomic instability as assayed by breakage–fusion–bridge (BFB) cycle formation. In light of these findings, the DNA damage-response pathway plays a vital role for determination of susceptibility to blast crisis in CML.

Extramedullary blast crisis of chronic myelogenous leukemia as an initial presentation

Extramedullary blast crisis of chronic myelogenous leukemia (CML) is defined as the development of extramedullary disease caused by the infiltration of blasts regardless of proliferation of blasts in the bone marrow. The onset of extramedullary blast crisis in the newly diagnosed patients is known to be extremely rare. Here, we present a case of extramedullary blast crisis of CML as an initial presentation in a 17-year-old female presenting with pain in the left femur tumor. This case was treated successfully with dasatinib and allogeneic hematopoietic stem cell transplantation, with achievement of long-term remission.

A Case with Coexistence of Major and Minor BCR/ABL Fusion Transcript at Lymphoblastic Crisis of Chronic Myelogenous Leukemia in Patients with Major BCR/ ABL Positivity during Chronic Phase

A Case with Coexistence of Major and Minor BCR/ABL Fusion Transcript at Lymphoblastic Crisis of Chronic Myelogenous Leukemia in Patients with Major BCR/ ABL Positivity during Chronic Phase

BCR-ABL1+T-Lymphoblastic Leukemia/Lymphoma Is a Rare Type of Chronic Myelogenous Leukemia Blast Crisis and Has a Poor Prognosis

BCR-ABL1 + - lymphoblastic leukemia/lymphoma (T-LBLL) is rare and may be difficult to distinguish from the also very rare cases of T-lineage blast crisis of chronic myelogenous leukemia (CML). We report 3 cases of BCR-ABL1 + T-LBLL. In all 3 cases, T lineage was confirmed with an extensive immunophenotyping panel. The first patient was a 64-year-old woman with …

Expression of dominant-negative Ikaros isoforms and associated genetic alterations in Chinese adult patients with leukemia

Dominant-negative (DN) Ikaros isoforms, having important roles in pathogenesis of leukemia, are mainly studied in pediatric patients, but little is known about Chinese adult patients. We examined 339 Chinese adult patients with leukemia and demonstrated the different findings between our results and those in several previous studies showing that DN isoforms overexpressed in Philadelphia chromosome positive acute lymphoblastic leukemia (Ph(+)ALL) and lymphoid/mixed blast crisis of chronic myelogenous leukemia. We confirmed that deletion of IKZF1 gene exons 4-7 is responsible for the generation of Ikaros 6 (Ik6). Moreover, we observed that expression of DN isoforms was dynamically consistent with BCR-ABL1 transcript levels, associated with higher incidence of relapse within 3months or poor response to induction chemotherapy in Ph(+)ALL, correlated with high white blood cell, blast cells, CD34 positive cells, and delayed achieving complete hematological remission in ALL patients. In conclusion, this study provides a rationale for the integration of aberrant Ikaros isoforms, notably Ik6 and Ik10, in the evaluation of adult ALL, particularly in Ph(+)ALL patients.

Nuclear Export (Karyopherin) Inhibitors: A Novel Therapeutic Strategy for Treating Blast Crisis Chronic Myelogenous Leukemia (CML) and Philadelphia-Positive (Ph+) Acute Lymphoblastic Leukemia (ALL) Through Interference with hnRNP Nucleocytoplasmic Shuttling and Rescue of Protein Phosphatase 2A (PP2A) Tumor Suppressor Activity,

Abstract 3758Despite the remarkable response in chronic phase CML, tyrosine kinase inhibitor (TKI)-based therapies do not induce long-term response in myeloid or lymphoid blast crisis CML (CML-BC) and Ph+ ALL, and are unable to kill quiescent Ph+ hematopoietic stem cells. We reported that CML disease progression is characterized by a BCR-ABL1 dose- and kinase-dependent increase in the expression/activity of nucleocytoplasmic-shuttling of heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, E2 and K, which are essential post-transcriptional and translational modulators of critical regulators of cell proliferation, survival and differentiation of CD34+ CML-BC and/or CD34+/CD19+ Ph+ ALL progenitors including SET/PP2A, E2F3, Bcl-xL, C/EBPa, miR-328, and c-Myc. The karyopherin (a class of proteins involved in regulating nuclear import/export) chromosome region maintenance 1 (CRM1) controls the nuclear export of several hnRNPs, and because growth/survival of CML-BC and Ph+ ALL progenitors requires the aberrant cytoplasmic activity of hnRNPs A1, E2 and/or K, we have explored the therapeutic potential of CRM1 inhibitors in p210 and p190 BCR-ABL1+ cell line models of CML-BC and Ph+ ALL, respectively.Thus, the cytokine-dependent myeloid 32Dcl3 and lymphoid BaF3, and the derived cytokine-independent 32D-p210BCR-ABL1 and Baf3-p190BCR-ABL1 mouse progenitors were exposed for 48 hr to different concentrations (0–10 μM) of the CRM1 selective & potent inhibitors of nuclear export (SINE) KPT-185 and KPT-207. MTT viability assays (n=3) revealed that KPT-185 and KPT-207 decreased viability ∼80% in 32D-p210BCR-ABL1 cells (KPT-185≥207) at concentrations ranging from 150–350 nM. Similar results were obtained in Baf3-p190BCR-ABL1 cells as the EC50 was 300nM for KPT-185 and KPT-207. The KPT-SINE not only induced killing, but also affected cytokine-independent growth of BCR-ABL1+ cells: proliferation was inhibited 89% and 81% by KPT-185 and KPT-207, respectively. Notably, growth and survival of non-transformed 32Dcl3 and BaF3 cells was not affected (70–100% viable cells) at concentrations (150–350 nM) of KPT-185 and KPT-207 that impair survival of BCR-ABL1+ cells.Mechanistically, we found that KPT-SINE CRM1 inhibitors altered the nuclear/cytoplasmic ratio of hnRNPs important for BCR-ABL1 leukemogenesis. Indeed, in KPT-207-treated (1 μM; 48h) BCR-ABL1+ cells hnRNP A1, E2 and K accumulated in the cytoplasm. Likewise, KPT-185 treatment (1 μM; 48h) resulted in hnRNP A1 being sequestered in the nucleus whereas hnRNP E2 distribution was not altered and, unexpectedly, levels of hnRNP K expression were markedly decreased in both subcellular compartments.Interestingly, treatment of BCR-ABL1+ cells with KPT-185 and KPT-207 (1 μM; 48h) resulted in 75% and 50% suppression of BCR-ABL1 expression and kinase activity, respectively. Furthermore, KPT-207 also reduced Myc expression in 32D-p210BCR-ABL1 cells; this is consistent with the potential interference of KPT-207 with the proliferation/survival signals triggered by the BCR-ABL1/MAPK/hnRNP K/Myc pathway in CML-BC progenitors.Because both KPT-185 and KPT-207 significantly alter hnRNP A1 localization in addition to impairing BCR-ABL1 expression/activity, and suppression of PP2A tumor suppressor activity is essential for both p210 and p190 BCR-ABL1-driven leukemogenesis, it is highly plausible that KPT-207 and KPT-185 negatively regulate the BCR-ABL1-dependent and hnRNP A1-mediated induction of the PP2A inhibitor SET thereby rescuing PP2A phosphatase activity, which in turn decreases BCR-ABL1 expression and kinase activity, and triggers in vitro and in vivo apoptosis of primary CML-BC and Ph+ ALL progenitors. Indeed, treatment with KPT-207 and KPT-185 (250 nM; 48h) restored PP2A activity in 32D-p210BCR-ABL1 cells to levels similar to those detected in non-transformed 32Dcl3 cells.Although further investigation of KPT-207 and KPT-185 mechanism of action and assessment of their biologic/therapeutic effects in CML-BC and Ph+ ALL mouse models and primary leukemic and normal progenitors is currently ongoing, it is safe to conclude that selective nuclear export (SINE CRM1) inhibitors represent potentially powerful therapeutic tools that, if used alone or in combination with TKIs, might lead to sustained complete molecular remission in CML-BC and Ph+ ALL patients. Disclosures:Shacham:Karyopharm: Equity Ownership. Kauffman:Karyopharm: Equity Ownership.