Development Of Chronic Myelogenous Leukemia Research Articles

Co-Diagnosis of Sickle Cell and Chronic Myelogenous Leukemia: Exploratory Analysis from a Nationally Representative Database

Background: Chronic myelogenous leukemia (CML) developing in patients with sickle cell disease (SCD) has been reported infrequently in case studies to date (1). Prevalent thought relates its infrequency to the shortened life expectancy in the SCD population. However, with advancements in medical care, longevity of these patients has been increasing. Resultingly, risk factors for CML related to their disease and its treatments may accumulate, including chronic inflammation, heightened cell turnover, iron overload, immunomodulation from frequent transfusions, blood and marrow transplantation, and hydroxyurea use (2,3). Most specifically, the elevated and continuous inflammatory cell stimulation due to vaso-occlusion may enhance changes in granulocytic cell differentiation, increasing the risk for CML (3). By leveraging a nationwide database, we aim to describe characteristics associated with the development of CML in SCD patients. Methods: Hospitalizations for SCD and CML were identified using ICD-10 coding from the 2020 National Inpatient Sample (NIS). Those hospitalizations with a co-diagnosis of SCD and CML were included in this study, and further evaluated for demographics, comorbidities, transfusion status, length of stay, mortality, and charges during admission. The NIS is the largest all-payer inpatient database in the US and approximates a 20% stratified sample of inpatient discharges from >5000 hospitals across 48 states (98% of U.S. population). Sampling weights were applied to generate nationally representative estimates. Results: In the 2020 NIS sample upon weighting, there were 60 hospitalizations with a co-diagnosis of SCD and CML. The vast majority of these patients presented with active CML (92%) and in sickle cell crisis (83%). Overall, half of the sample comprised those patients who were between the ages of 29.5-49 years old, with a median age of 40.5. Fifty percent (n=30) of the hospitalizations were female; 92% (n=55) were African American; 58% were hospitalized in the Southern region; 25% in the West, and 17% in the Northeastern United States. The majority of hospitalizations (92%) occurred in teaching hospitals and 83% were in large-bed size hospitals. Common comorbidities were similar to that expected in the general SCD population, including history of transient ischemic attack (42%), history of pulmonary embolism/venous thromboembolism (42%), chronic kidney disease (33%), anemia (33%), and pulmonary hypertension (33%). Seventeen percent of patients had a transfusion during the hospitalization (n=15). The mean length of stay among these hospitalizations was 4.25 days. There were no deaths during these hospitalizations. Mean charges during hospitalization were $53,973. Conclusion: Previously observed in rare case studies, here we present an exploratory analysis from a national inpatient database demonstrating a population of 60 adult patients with SCD and a co-diagnosis of CML. Specifically, we observe concomitant development of CML in the SCD patient population correlated with a younger age (median age 40.5) than expected compared to traditional CML patients (median age 64). There is some evidence to suggest that chronic inflammation associated with sickle cell with extended life expectancies and treatment burden may be associated with myeloid cancers. These studies will need to be followed up with prospective analyses to investigate a potential increased risk of CML in the SCD population. Literature Cited: 1. Chen L et al., Chronic myelogenous leukemia in sickle cell anemia. Arch Pathol Lab Med. 2005;129(3):423-424. doi:10.5858/2005-129-423-CMLISC 2. Brunson A et al., Increased risk of leukemia among sickle cell disease patients in California. Blood. 2017;130(13):1597-1599. doi:10.1182/blood-2017-05-78323. 3. Stankovic et al., Chronic myeloid leukaemia and sickle cell disease: could imatinib prevent vaso-occlusive crisis?. Br J Haematol. 2011;155(2):271-272. doi:10.1111/j.1365-2141.2011.08670.x

Persistence of Drug-Resistant Leukemic Stem Cells and Impaired NK Cell Immunity in CML Patients Depend on MIR300 Antiproliferative and PP2A-Activating Functions

<h3></h3> Persistence of drug-resistant quiescent leukemic stem cells (LSC) and impaired natural killer (NK) cell immune response account for relapse of chronic myelogenous leukemia (CML). Inactivation of protein phosphatase 2A (PP2A) is essential for CML-quiescent LSC survival and NK cell antitumor activity. Here we show that <i>MIR300</i> has antiproliferative and PP2A-activating functions that are dose dependently differentially induced by CCND2/CDK6 and SET inhibition, respectively. <i>MIR300</i> is upregulated in CML LSCs and NK cells by bone marrow microenvironment (BMM) signals to induce quiescence and impair immune response, respectively. Conversely, BCR-ABL1 downregulates <i>MIR300</i> in CML progenitors to prevent growth arrest and PP2A-mediated apoptosis. Quiescent LSCs escape apoptosis by upregulating <i>TUG1</i> long noncoding RNA that uncouples and limits <i>MIR300</i> function to cytostasis. Genetic and pharmacologic <i>MIR300</i> modulation and/or PP2A-activating drug treatment restore NK cell activity, inhibit BMM-induced growth arrest, and selectively trigger LSC apoptosis <i>in vitro</i> and in patient-derived xenografts; hence, the importance of <i>MIR300</i> and PP2A activity for CML development and therapy. <h3>Significance:</h3> Tumor-naïve microenvironment–induced <i>MIR300</i> is the only tumor suppressor miRNA that induces CML LSC quiescence while inhibiting NK cell antitumor immune response, and CML LSC/progenitor cell apoptosis through its anti-proliferative and PP2A-activating functions, respectively. Thus, the importance of <i>MIR300</i> and PP2A-activating drugs for formation/survival and eradication of drug-resistant CML LSCs, respectively. <i>See related commentary by Broxmeyer, p. 13</i>. <i>This article is highlighted in the In This Issue feature, p. 5</i>

Myeloproliferative Neoplasms with Subsequent Development of Chronic Myelogenous Leukemia: A Case Series

Myeloproliferative Neoplasms with Subsequent Development of Chronic Myelogenous Leukemia: A Case Series

Persistence of Drug-Resistant Leukemic Stem Cells and Impaired NK Cell Immunity in CML Patients Depend on MIR300 Antiproliferative and PP2A-Activating Functions.

Persistence of drug-resistant quiescent leukemic stem cells (LSC) and impaired natural killer (NK) cell immune response account for relapse of chronic myelogenous leukemia (CML). Inactivation of protein phosphatase 2A (PP2A) is essential for CML-quiescent LSC survival and NK cell antitumor activity. Here we show that MIR300 has antiproliferative and PP2A-activating functions that are dose dependently differentially induced by CCND2/CDK6 and SET inhibition, respectively. MIR300 is upregulated in CML LSCs and NK cells by bone marrow microenvironment (BMM) signals to induce quiescence and impair immune response, respectively. Conversely, BCR-ABL1 downregulates MIR300 in CML progenitors to prevent growth arrest and PP2A-mediated apoptosis. Quiescent LSCs escape apoptosis by upregulating TUG1 long noncoding RNA that uncouples and limits MIR300 function to cytostasis. Genetic and pharmacologic MIR300 modulation and/or PP2A-activating drug treatment restore NK cell activity, inhibit BMM-induced growth arrest, and selectively trigger LSC apoptosis in vitro and in patient-derived xenografts; hence, the importance of MIR300 and PP2A activity for CML development and therapy.

Chronic myeloid leukemia stem cells require cell-autonomous pleiotrophin signaling.

Tyrosine kinase inhibitors (TKIs) induce molecular remission in the majority of patients with chronic myelogenous leukemia (CML), but the persistence of CML stem cells hinders cure and necessitates indefinite TKI therapy. We report that CML stem cells upregulate the expression of pleiotrophin (PTN) and require cell-autonomous PTN signaling for CML pathogenesis in BCR/ABL+ mice. Constitutive PTN deletion substantially reduced the numbers of CML stem cells capable of initiating CML in vivo. Hematopoietic cell-specific deletion of PTN suppressed CML development in BCR/ABL+ mice, suggesting that cell-autonomous PTN signaling was necessary for CML disease evolution. Mechanistically, PTN promoted CML stem cell survival and TKI resistance via induction of Jun and the unfolded protein response. Human CML cells were also dependent on cell-autonomous PTN signaling, and anti-PTN antibody suppressed human CML colony formation and CML repopulation in vivo. Our results suggest that targeted inhibition of PTN has therapeutic potential to eradicate CML stem cells.

PARP Inhibition Sensitize BCR-ABL1 Positive Cel

Introduction: BCR-ABL1 play a key role in the development of chronic myelogenous leukemia and a part of Ph1 positive acute lymphoblastic leukemia (ALL). BCR-ABL1 functions as a tyrosine kinase. Whereas, BCR-ABL1 induces genomic instability by downregulation of BRCA1. An innate error of BRCA1, a molecule involved in the homologous recombination repair pathway, causes hereditary breast and ovarian cancer. PARP inhibitor (PARPi) induces synthetic lethality in BRCA defective cell. Therefore, PARP inhibitor is expected to induce efficient cell death with BCR-ABL1 positive cell. In addition, in some previous reports, reduction of PARP1 activity leads to the upregulation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway and BCR-ABL1 tyrosine kinase activates PI3K/AKT pathway. These findings suggest activation of the PI3K/AKT pathway leading to PARPi resistance in BCR-ABL1 positive leukemic cells. Here, we demonstrate that PARP inhibition attenuates BCR-ABL1 mediated leukemogenesis and aberration of factors associated with PARP inhibitor resistance induces cell death to fully transformed leukemic cells. Method: Bone marrow-derived mononuclear cells (MNC) from wild type mice and BCR-ABL1 transgenic (Tg) mice were exposed to PARPi in vivo, and cell death was analyzed Annexin-V positivity. PARPi sensitivity to BCR-ABL1 expressed cell was also investigated in vivo bone marrow transplantation model using mouse hematopoietic stem cell (HCS) infected with BCR-ABL1 expressing retrovirus. To evaluate more precisely the results obtained in vitro and in vivo transplantation model, the genetical approach was also performed. The Parp1 knockout (KO) mice were crossed with BCR-ABL1 Tg mice. Then, Leukemia development and subsequent mouse death were observed. In vitro, HR activity was examined using DR-GFP assay. Genomic instability was investigated using the breakage-fusion-bridge (BFB) generation.Maintenance of HSC as a progenitor of the leukemic cell was analyzed by repopulation activity using colony assay. The growth-inhibitory effect was assessed using BCR-ABL positive cell lines with PARPi and PI3K inhibitor. Results: BCR-ABL1 Tg mice derived MNC showed more hypersensitivity to PARPi. Mouse HCS was infected with BCR-ABL1 expressing retrovirus and transplanted lethally Olaparib or vehicle was administrated intraperitoneal injection one day after transplantation. BCR-ABL1 mediated leukemic death was observed 1 month after transplantation in sham-treated mouse, whereas, Olaparib treated mouse did not develop BCR-ABL1 mediated leukemia. Parp1 KO BCR-ABL1 Tg mice attenuated leukemia development and extended their survival compared with BCR-ABL1 Tg mice. In vitro experiment revealed HR activity was down-regulated by BCR-ABL1 expression in DR-GFP assay. The number of BFB generation was increased in BCR-ABL1 Tg with Parp1 KO background. The colony-forming activity of BCR-ABL1 positive HSC was totally abolished by PARP inhibition after 3 times serial replating, whereas sham-treated HSC retained repopulation activity. However, the effect of PARPi on BCR-ABL positive leukemic cell lines was controversial. Therefore, leukemic cell lines were treated with the PARPi and inhibitors toward the molecules associated with PARPi resistance. As a result, a combination of PARPi with PI3K inhibitor effectively induce cell death in PARPi resistant BCR-ABL1 positive leukemic cell lines. Conclusion and discussion: Tyrosine kinase inhibitor (TKI) is the gold standard of the therapeutic option of BCR-ABL1 positive leukemia. However, TKI monotherapy is not sufficient for complete eradication of leukemic cells. It is highly expected that molecules effectively induce cell death to leukemic cells combined with TKI. PARPi would be one of these candidates. However, PARPi could not induces efficient death in all of the cancer cells that carry the mutation of molecules associated with the HR defect. Comprehensive genetic analysis to reveal PARPi resistance is important for HRR defective cancer cells. Combination therapy of PARPi and inhibitorstoward the molecules associated with PARPi resistance would be a good therapeutic option for Ph1 positive leukemia. Disclosures No relevant conflicts of interest to declare.

Spred1 Insufficiency in the Hematopoietic and/or Vascular Compartments of the Bone Marrow (BM) Niche Promotes Aggressive Leukemogenesis in Chronic Myelogenous Leukemia (CML)

Spred1, a member of the Sprouty family of proteins and a negative regulator of RAS-MAPK signaling, is highly expressed in normal hematopoietic stem cells (HSCs) where it negatively regulates self-renewal activity. Lack of Spred1 function has been associated with aberrant hematopoiesis (Tadokoro, 2018). Spred1 knocked-out (KO) mice fed with high-fat diet develop a myeloproliferative phenotype (Tadokoro, 2018), and lower SPRED1 expression in acute myeloid leukemia associates with a poor outcome (Li, 2015; Olsson, 2014; Pasmant, 2015), suggesting a potential role of this gene as a tumor suppressor in myeloid malignancies. In CML, however, the role of Spred1 has not been fully dissected. Thus, we generated Spred1 KO CML (i.e., Spred1-/-SCLtTA/BCR-ABL) mice by crossing Spred1 KO (a gift from Dr. Yoshimura, Japan) with inducible SCLtTA/BCR-ABL CML mice. Spred1 KO mice showed increased cell cycling of BM long-term HSCs (LTHSCs; Lin-Sca-1+c-kit+Flt3-CD150+CD48-; G0: 62% vs 76%), and increased white blood cell (WBC) counts [14 vs 5.9 k/ul at 12 weeks (w) old, n=15 per group, p&lt;0.0001], as compared to wt mice. Upon B/A induction by tetracycline withdrawal, Spred1-/-SCLtTA/BCR-ABL mice had higher WBC (102.5 vs 12 k/ul at 4 w, n=15 per group, p&lt;0.0001), more pronounced splenomegaly (spleen weight: 0.28g vs 0.19g, n=4 per group, p=0.06) and a significantly shorter survival (median: 39 vs 83 days, n=23 per group, p&lt;0.0001) than Spred1 wt CML mice. In Spred1-/-SCLtTA/BCR-ABL mice, we observed a more rapid expansion of circulating mature myeloid cells (CD11b+Gr-1+ cells: 63% vs 25%, n=8 per group, p&lt;0.01) and a deeper decrease of BM LTHSCs (1,385 vs 2,164 per femur, n=5 per group, p&lt;0.01) and increase of spleen LTHSCs (27330 vs 18546, n=5 per group, p&lt;0.01) at 4 w after B/A induction compared with Spred1 wt CML mice. Further, we found a higher fraction of Spred-/-SCLtTA/BCR-ABL mice (33% vs 10%) developed lymph node enlargement, with infiltration with pro-B lymphoblastic cells (B220+CD43+CD19+IgM−) compared with Spred1 wt CML mice. Altogether these features suggested that Spred1 insufficiency accelerates CML development and evolution to more aggressive phases of the disease. Since upregulation of Spred1 reportedly disrupts vascular integrity (Fish, 2008; Wang 2008), a finding that we have also confirmed in the BM niche, in order to evaluate separately the leukemogenic effect of Spred1 expression on different compartments of the BM niche, we generated the following conditional Spred1 KO strains: Spred1flox(f)/fMxl-cre+ (Spred1 KO in HSCs, hereafter called Spred1HSCΔ/Δ), Spred1f/fTie2-cre+ [Spred1 KO in endothelial cells (ECs), hereafter called Spred1ECΔ/Δ], Spred1HSCΔ/ΔSCLtTA/BCR-ABL and Spred1ECΔ/ΔSCLtTA/BCR-ABL by crossing SCLtTA/BCR-ABL with the above Spred1 KO mice. LTHSCs from Spred1HSCΔ/ΔSCLtTA/BCR-ABL mice showed an increase in cell cycling, RAS/MAPK/ERK activity and Bcl-2 levels, and higher engraftment in recipient mice (blood: 9.7% vs 26.5% at 6w, 14.8% vs 42% at 8w, 14.7% vs 48% at 12w, n=10 per group, p&lt;0.01), compared to Spred1 wt CML LTHSCs. Spred1HSCΔ/ΔSCLtTA/BCR-ABL mice (n=15) showed enhanced leukemia progression (WBC: 19 vs 12 k/ul, p=0.004; CD11b+Gr-1+ in blood: 36% vs 25%, p=0.04 at 4 w after B/A induction) and a significantly shorter survival (median: 49.5 vs 83 days, p=0.01) compared to Spred1 wt CML mice (n=20). However, the disease in these mice appeared to be overall less aggressive than global Spred1 KO CML (i.e., Spred1-/-SCLtTA/BCR-ABL) mice (WBC: 19 vs 102 k/ul; CD11b+Gr-1+ in blood: 36 vs 63%; Survival: 49.5 vs 39 days), suggesting that Spred1 depletion in other non-hematopoietic cell compartments may also be important for leukemogenesis. In fact, Spred1ECΔ/ΔSCLtTA/BCR-ABL mice (n=8) showed enhanced leukemia progression (WBC: 26 vs 9.8 k/ul at 4 w after B/A induction, p=0.02), a trend for a reduced survival (median: 56 vs 83 days, p=0.09), and increased arteriolar vascularization, compared to Spred1 wt CML mice (n=20). Mechanistic studies on how endothelial Spred1 insufficiency co-participates in leukemogenesis are ongoing. Altogether our results support a role of Spred1 insufficiency in distinct BM niche compartments to produce a more aggressive CML phenotype, likely through different, but complementary mechanisms. Spred1 may therefore emerge as a novel target for advanced CML. Disclosures No relevant conflicts of interest to declare.

New Heteroleptic Ruthenium(II) Complexes with Sulfamethoxypyridazine and Diimines as Potential Antitumor Agents.

Two new complexes of Ru(II) with mixed ligands were prepared: [Ru(bpy)2smp](PF6) (1) and [Ru(phen)2smp](PF6) (2), in which smp = sulfamethoxypyridazine; bpy = 2,2′-bipyridine; phen = 1,10-phenanthroline. The complexes have been characterized by elemental and conductivity analyses; infrared, NMR, and electrospray ionization mass spectroscopies; and X-ray diffraction of single crystal. Structural analyses reveal a distorted octahedral geometry around Ru(II) that is bound to two bpy (in 1) or two phen (in 2) via their two heterocyclic nitrogens and to two nitrogen atoms from sulfamethoxypyridazine—one of the methoxypyridazine ring and the sulfonamidic nitrogen, which is deprotonated. Both complexes inhibit the growth of chronic myelogenous leukemia cells. The interaction of the complexes with bovine serum albumin and DNA is described. DNA footprinting using an oligonucleotide as substrate showed the complexes’ preference for thymine base rich sites. It is worth notifying that the complexes interact with the Src homology SH3 domain of the Abl tyrosine kinase protein. Abl protein is involved in signal transduction and implicated in the development of chronic myelogenous leukemia. Nuclear magnetic resonance (NMR) studies of the interaction of complex 2 with the Abl-SH3 domain showed that the most affected residues were T79, G97, W99, and Y115.

DUXAP10 regulates proliferation and apoptosis of chronic myeloid leukemia via PTEN pathway.

To investigate the role of DUXAP10 in chronic myelogenous leukemia (CML) and its underlying mechanism. We detected DUXAP10 expression in 82 CML patients, 12 normal controls, and CML cell line by qRT-PCR (quantitative real-time polymerase chain reaction). After transfection of si-DUXAP10 or si-PTEN in CML cell lines (K652, KG-1), we detected proliferation, cell cycle, and apoptosis by CCK-8 (cell counting kit-8), colony formation assay, and flow cytometry, respectively. Finally, protein expressions of p21, CDK2, Bcl-2, Bax, and PTEN were detected by Western blot. DUXAP10 was upregulated in CML tissues and cells, which was gradually increased in the chronic phase (CP), acceleration phase (AP), and blast phase (BP) of CML. Knockdown of DUXAP10 in K652 and KG-1 cells can remarkably inhibit cell proliferation, promote cycle arrest and apoptosis. Western blot and flow cytometry results demonstrated that DUXAP10 can reduce apoptosis by inhibiting PTEN expression. Overexpressed DUXAP10 accelerates the development and progression of CML by promoting cell proliferation, reducing cell cycle arrest and apoptosis via inhibiting PTEN expression.

Inhibition of ER stress-related IRE1α/CREB/NLRP1 pathway promotes the apoptosis of human chronic myelogenous leukemia cell

Endoplasmic reticulum (ER) stress is induced in chronic myelogenous leukemia (CML) cells. As an important sensor of ER stress, inositol-requiring protein-1α (IRE1α) promotes the survival of acute myeloid leukemia. NLRP1 inflammasome activation promotes metastatic melanoma growth and that IRE1α can increase NLRP1 inflammasome gene expression. This study aimed to investigate the role and molecular mechanism of IRE1α in CML cell growth. We found that overexpression of IRE1α or NLRP1 significantly promoted the proliferation and decreased the apoptosis of CML cells, whereas downregulation of these two genes showed the opposite effects. 4-phenylbutyric acid (4-PBA), an ER stress inhibitor, reduced the expression of IRE1α and NLRP1. IRE1α elevated NLRP1 expression via cAMP responsive element binding protein (CREB) phosphorylation. NLRP1 inflammasome was activated in CML cells and its activation partly reversed ER stress inhibitor-induced cell apoptosis. Furthermore, inhibition of IRE1α/NLRP1 pathway sensitized CML cells to imatinib-mediated apoptosis. Additionally, IRE1α expression was elevated and NLRP1 inflammasome was activated in primary cells from CML patients. Downregulation of IRE1α or NLRP1 suppressed the proliferation and elevated the apoptosis of primary CML cells. Collectively, this study demonstrated that the IRE1α/CREB/NLRP1 pathway contributes to the progression of CML and the development of imatinib resistance. Hence, targeting ER stress-related IRE1α expression or NLRP1 inflammasome activation may block CML development.

Interactions of ruthenium(II) compounds with sulfasalazine and N,N′-heterocyclic ligands with proteins

Two complexes of Ru(II) possessing the general formula [Ru(ssz)2L](PF6) were prepared, in which ssz is sulfasalazine and L is either 2,2′-bipyridine (1) or 1,10-phenanthroline (2). The complexes were characterized by elemental and conductivity analyses, UV–Vis, vibrational, NMR and electrospray ionization mass spectroscopies. The geometry around ruthenium(II) is distorted octahedral. The metal ion is bound to the carboxylate of ssz in a bidentate way and to two heterocyclic nitrogens of bpy or phen. Spectrofluorimetric measurements indicate that complexes bind to BSA with affinity constants ranging from 104 to 105M−1. Both compounds are significantly cytotoxic in a chronic myelogenous leukemia cell line in a concentration dependent manner. UV-light exposure for 5min increases the cytotoxicity of 1 by three times and that of 2 by ten times. In addition, upon UV light irradiation, the complexes are able to damage the bovine serum albumin molecule, at concentrations that do not affect BSA in dark conditions. It is worth to note that the complexes also bind to Abl-SH3 protein, which is responsible for the development of chronic myelogenous leukemia. Interactions with proteins seem to be significant for the cytotoxic and photocytotoxic activity of these complexes.

Bone Marrow Stromal Cell Remodeling Is a Common Feature of Diverse Fibrotic Myeloproliferative Neoplasm Models

Myeloproliferative neoplasms (MPN) are blood cancers initiated by driver mutations that transform hematopoietic stem cells. MPN exhibit gross pathologic bone marrow (BM) stromal remodeling, including damaging myelofibrotic change that leads to dependence on extramedullary hematopoiesis and more severe clinical diseases. Therapies targeting fibrotic change would have broad appeal in the treatment of these diseases. We previously demonstrated a critical role for malignant myeloid cells in remodeling endosteal mesenchymal stromal cells (MSC) into myelofibrotic osteoblast-lineage cells (OBC) in a model of chronic myelogenous leukemia (CML) driven by BCR/ABL (Schepers et al., Cell Stem Cell, 2013). In a separate study in a fibrotic MPN model driven by Jak2V617F, neuropathy and nestin-positive MSC cell death were found critical to disease progression but their involvement in myelofibrosis was not investigated (Arranz et al. Nature. 2014). Our goal is to characterize the type of BM stromal remodeling occurring in non-CML MPN models driven by various mutations and representing a spectrum of disease severity and fibrosis. This includes a minimally fibrotic transgenic Jak2V617F alone model (Jak2V617F model, Xing et al., Blood, 2008) and more advanced fibrotic models driven by MPLW515L expression (MPLW515L model, Pikman et al., PLoS Med, 2006) or combined transgenic Jak2V617F expression with conditional deletion of the polycomb gene EZH2 (Jak2V617F/EZH2-/- model, Sashida et al., JEM, 2016). We found common blood and BM hematopoietic changes in all three models, including thrombocytosis and expansion of myeloid-biased multipotent progenitor BM cells and confirmed the degree of fibrosis using picrosirius red staining of bone sections. Both MPLW515L and Jak2V617F/EZH2-/- heavily fibrotic models demonstrated inhibition of total endosteal MSC, OBC and endothelial cell (EC) numbers during disease development - in most cohorts a greater than 50% decrease in absolute stromal cell numbers was found. In addition, we observed that whole BM cells from Jak2V617F/EZH2-/- mice contained a significantly lower number of totalfibroblast colony forming cells (CFU-F). In co-culture experiments designed to measure direct MSC remodeling induced by malignant cells, both MPLW515L and Jak2V617F/EZH2-/- BM cells inhibited healthy endosteal MSC colony formation over time. In contrast, we found no inhibition of stromal cell numbers or co-culture MSC growth in the minimal fibrotic Jak2V617F model. In initial experiments measuring rare central marrow perivascular MSC, we found reduced LepR+ MSC (Ding et al., Nature, 2012) in both MPLW515L and Jak2V617F/EZH2-/- long bone sections using immunofluorescence. Our results show that fibrotic development in non-CML MPN inhibits stromal cell numbers and function likely via direct effects of malignant hematopoietic cells. This is in contrast to fibrotic CML development where myelofibrotic endosteal stromal cells are expanded. This difference could be partly explained by the type and localization of fibrosis in these various models. The CML model has focal endosteal collagen-I fibrosis which is heavily reliant on osteoblast remodeling, while the MPLW515L and Jak2V617F/EZH2-/- models have more diffuse reticulin central marrow fibrosis which may be produced through a process of stromal cell senescence or differentiation. Overall, this study underscores that a “one size fits all“ approach to understanding myelofibrosis is insufficient. To tease out these differences, we are examining qualitative and quantitative changes in additional central marrow MSC populations, including PDGFR+, Sca-1+ and Gli-1+ MSC, during MPN development as well as assaying the molecular mediators of stromal remodeling. Our long-term goal is to identify therapies that can restore a more normal BM stroma and support healthy hematopoiesis in MPN. DisclosuresNo relevant conflicts of interest to declare.

Genetic polymorphisms associated with increased risk of developing chronic myelogenous leukemia.

Little is known about inherited factors associated with the risk of developing chronic myelogenous leukemia (CML). We used a dedicated DNA chip containing 16 561 single nucleotide polymorphisms (SNPs) covering 1 916 candidate genes to analyze 437 CML patients and 1 144 healthy control individuals. Single SNP association analysis identified 139 SNPs that passed multiple comparisons (1% false discovery rate). The HDAC9, AVEN, SEMA3C, IKBKB, GSTA3, RIPK1 and FGF2 genes were each represented by three SNPs, the PSM family by four SNPs and the SLC15A1 gene by six. Haplotype analysis showed that certain combinations of rare alleles of these genes increased the risk of developing CML by more than two or three-fold. A classification tree model identified five SNPs belonging to the genes PSMB10, TNFRSF10D, PSMB2, PPARD and CYP26B1, which were associated with CML predisposition. A CML-risk-allele score was created using these five SNPs. This score was accurate for discriminating CML status (AUC: 0.61, 95%CI: 0.58-0.64). Interestingly, the score was associated with age at diagnosis and the average number of risk alleles was significantly higher in younger patients. The risk-allele score showed the same distribution in the general population (HapMap CEU samples) as in our control individuals and was associated with differential gene expression patterns of two genes (VAPA and TDRKH). In conclusion, we describe haplotypes and a genetic score that are significantly associated with a predisposition to develop CML. The SNPs identified will also serve to drive fundamental research on the putative role of these genes in CML development.

Modification of Gene Expression, Proliferation, and Function of OP9 Stroma Cells by Bcr-Abl-Expressing Leukemia Cells.

Expression of the Bcr-Abl fusion gene in hematopoietic progenitor cells (HPCs) results in the development of chronic myelogenous leukemia (CML), for which hematopoietic microenvironment plays an important role. We investigated the specific effects of an HPC line transduced with Bcr-Abl, KOBA, on BM-derived OP9 stroma cells. DNA microarray analysis revealed that OP9 cells co-cultured with KOBA cells (OP9/L) show diverse changes in the gene expression. OP9/L cells showed significant down-regulation of Cdkn genes and up-regulation of Icam1, leading to the increased proliferation capacity of OP9 cells and enhanced transmigration of leukemia cells through them. The effects were attributed to direct Notch activation of OP9 cells by KOBA cells. OP9/L cells also showed a markedly altered cytokine gene expression pattern, including a robust increase in a variety of proinflammatory genes and a decrease in hematopoietic cytokines such as Cxcl12, Scf, and Angpt1. Consequently, OP9/L cells promoted the proliferation of KOBA cells more efficiently than parental OP9 cells, whereas the activity supporting normal myelopoiesis was attenuated. In mice bearing KOBA leukemia, the characteristic genetic changes observed in OP9/L cells were reflected differentially in the endothelial cells (ECs) and mesenchymal stroma cells (MCs) of the BM. The ECs were markedly increased with Notch-target gene activation and decreased Cdkn expression, whereas the MCs showed a marked increase in proinflammatory gene expression and a profound decrease in hematopoietic genes. Human CML cell lines also induced essentially similar genetic changes in OP9 cells. Our results suggest that CML cells remodel the hematopoietic microenvironment by changing the gene expression patterns differentially in ECs and MCs of BM.

The -137G/C Polymorphism in Interleukin-18 Gene Promoter Contributes to Chronic Myeloid Leukemia Risk in Turkish Patients

Interleukin-18 (IL-18) is a cytokine which belongs to the interleukin-1 superfamily and secreted by various immune and non-immune cells. Evidence has shown that IL-18 has both anticancer and procancer effects. The aim of this study was to evaluate the relationship between -137G/C (rs187238) IL-18 gene polymorphism and susceptibility to chronic myelogenous leukemia(CML) in Turkish patients. The frequencies of polymorphism -137G/C were studied in thirty chronic myelogenous and thirty healthy individuals. The genotyping was performed by polymerase chain reaction and DNA sequencing analysis. Significant associations were detected between the IL-18 rs187238 (G/C) polymorphism and CML patients. A higher prevalence of the C allele was found in CML cases with respect to controls (p<0.05). The GC heterozygote and CC homozygote genotypes are associated with the risk of CML when compared with controls (OR, 1.9023; 95% CI, 0.6171-5.8636 for GC genotype and OR, 10.3585; 95% CI, 0.5326-201.4622 for CC genotype). These results indicate that individuals that are either heterozygote (GC) or homozygote for CC genotype are associated with the development of chronic myelogenous leukemia in Turkish population. Further large-scale studies combined with haplotype and expression analysis are required to validate the current findings. DisclosuresNo relevant conflicts of interest to declare.

Eradication of Chronic Myelogenous Leukemia By Inactivation of the Polycomb Group Protein EZH2

Chronic myelogenous leukemia (CML) is a stem-cell driven malignancy caused by BCL/ABL, the oncoprotein derived from the Philadelphia chromosome 9/22 translocation. Although tyrosine kinase inhibitors (TKIs) are highly effective in managing chronic phase CML, challenges remain, including acquisition of TKI resistance, toxicities of long-term TKI administration and third-generation TKIs, and persistence of leukemia initiating cells (LICs). Reliable curative chemotherapy remains elusive. Alternative approaches to definitive treatment of CML would enhance current management.We report here that the Polycomb group protein EZH2, a catalytic subunit of Polycomb repressive complex 2 (PRC2), is a potential target for curative chemotherapy for CML. EZH2 is overexpressed in LICs, compared to normal hematopoietic stem cells (HSCs), and is sustained by BCR/ABL signaling, as TKIs suppress EZH2 expression in CML cells. Removal of EZH2 in an engineered mouse model prevents CML initiation and development. Moreover, Ezh2 is also required for maintenance of established CML. In a tamoxifen-inducible EZH2 deletion mouse model, conditional inactivation of EZH2 eradicates existing disease induced by wild-type BCR/ABL or the gatekeeper T315I mutant, and leads to long-term survival. Importantly, EZH2 inactivation also eliminates phenotypic and functional LICs. Relevance of the genetic studies in mice is supported by shRNA knockdown and EZH2-inhibitor treatment of human CML cell lines and primary samples, which leads to cell cycle inhibition, apoptosis, and reduced colony formation. Effects of EZH2 loss in CML are context-specific, as EZH2 is dispensable for HSCs, in part due to decreased EZH1 expression in CML LICs compared to normal HSCs. EZH1 expression decreases even further in LICs upon the loss of EZH2. Thus, loss of EZH2 in CML LICs creates a scenario resembling complete inactivation of PRC2, which is essential for the maintenance of HSCs.EZH2-dependence of CML LICs raises prospects for curative chemotherapy based on addition of EZH2 inhibitors to conventional TKI administration. DisclosuresNo relevant conflicts of interest to declare.

Lineage-specific function of Engrailed-2 in the progression of chronic myelogenous leukemia to T-cell blast crisis

In hematopoietic malignancies, oncogenic alterations interfere with cellular differentiation and lead to tumoral development. Identification of the proteins regulating differentiation is essential to understand how they are altered in malignancies. Chronic myelogenous leukemia (CML) is a biphasic disease initiated by an alteration taking place in hematopoietic stem cells. CML progresses to a blast crisis (BC) due to a secondary differentiation block in any of the hematopoietic lineages. However, the molecular mechanisms of CML evolution to T-cell BC remain unclear. Here, we have profiled the changes in DNA methylation patterns in human samples from BC-CML, in order to identify genes whose expression is epigenetically silenced during progression to T-cell lineage-specific BC. We have found that the CpG-island of the ENGRAILED-2 (EN2) gene becomes methylated in this progression. Afterwards, we demonstrate that En2 is expressed during T-cell development in mice and humans. Finally, we further show that genetic deletion of En2 in a CML transgenic mouse model induces a T-cell lineage BC that recapitulates human disease. These results identify En2 as a new regulator of T-cell differentiation whose disruption induces a malignant T-cell fate in CML progression, and validate the strategy used to identify new developmental regulators of hematopoiesis.

MTHFR genetic polymorphisms may contribute to the risk of chronic myelogenous leukemia in adults: a meta-analysis of 12 genetic association studies

Chronic myelogenous leukemia (CML) is a complex disease with a genetic basis. The genetic association studies (GASs) that have investigated the association between adult CML and 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms have produced contradictory and inconclusive results. The aim of this meta-analysis is to provide a relatively comprehensive assessment of the association of these polymorphisms with adult CML risk. A literature search for eligible GAS published before September 15, 2013 was conducted in PubMed, Embase, Web of Science, Cochrane Library, and China National Knowledge Infrastructure (CNKI) databases. Pooled odds ratios (ORs) with their corresponding 95% confidence intervals (95% CIs) were used to evaluate the strength of the association under a fixed or random effect model according to heterogeneity test results. All analyses were performed using the Stata software, version 12.0. Twelve case-control studies were included in this meta-analysis with a total of 932 CML patients and 3,465 healthy controls. For MTHFR C677T (dbSNP: rs1801133, C>T), though the pooled ORs were not significant in the overall population, all the ORs greater than 1 suggested an increased risk of CML for carriers of the risk allele. However, stratified analysis based on genotyping method revealed a significant association in the PCR-restriction fragment length polymorphism (RFLP) subgroup, possibly as a result of heterogeneity. For MTHFR A1298C (dbSNP: rs1801131, A>C), the combined results showed that carriers of the C allele may be associated with a decreased risk of adult CML. Stratified analysis showed that the magnitude of this effect was especially significant among Asians, indicating ethnicity differences in adult CML susceptibility. This meta-analysis shows that the C allele of MTHFR A1298C may be associated with a decreased risk in adult CML, especially among Asians, while MTHFR C677T may not be associated with adult CML risk. However, the development of adult CML may be the result of gene-gene and gene-environment interactions, which should be considered in future individual GAS and subsequent meta-analyses.

Heterogeneity Of Leukemic Stem Cell Capacity Of BCR-ABL+ Long-Term Hematopoietic Stem cells In CML Is Associated With Variability In MPL Expression

In chronic myelogenous leukemia (CML), in vivo long-term repopulating and leukemia stem cell (LSC) capacity is restricted to a small population of BCR-ABL+ long-term hematopoietic stem cells (LTHSC). Using an inducible transgenic SCL-tTA/BCR-ABL mouse model of CML, we have shown that leukemic cells with long-term repopulating and leukemia-initiating capacity have the Lin-Sca-1+Kit+Flt3-CD150+CD48- phenotype, also characteristic of normal LTHSC. Limiting dilution transplantation studies show that frequency of cells with LTHSC phenotype with long-term engraftment capacity (1:6) is considerably higher than those with leukemia-initiating capacity (1:80) suggesting that only some LTHSC may have LSC capacity (Cancer Cell 21:577, 2012). To further evaluate the basis for heterogeneity in LSC potential of BCR-ABL+ LTHSC, SCL-tTA/BCR-ABL mice were crossed with GFP expressing mice to allow tracking of donor cells, and a cohort of mice were transplanted with limiting numbers of GFP+LTHSC (200 per mouse) and followed for engraftment of GFP+ cells and development of CML (WBC>10,000/ul). Only 11 of 20 mice developed CML, whereas 9 mice showed long term engraftment without development of CML. GFP+ LTHSC selected from primary recipients were transplanted into secondary recipients (200 per mouse). Seven of 17 mice receiving cells from mice with CML also developed CML after the second transplant, whereas none of the mice receiving cells from non-CML mice developed CML, suggesting the distinction between leukemogenic versus non-leukemogenic LTHSC was maintained after transplantation. LTHSC isolated from primary recipients were also analyzed for expression of several HSC-regulatory genes by multiplex Q-PCR using the Fluidigm system. On hierarchical clustering, LTHSC from mice developing CML clustered separately from LTHSC from mice without CML. Amongst cell surface expressed genes, expression of the thrombopoietin (TPO) receptor MPL (p=0.006) and CD47 (p=0.006) was significantly increased in LTHSC from mice developing CML. We did not see significant differences in BCR-ABL expression in LTHSC from mice with or without CML. We further analyzed the relationship of MPL expression with CML LTHSC function. CML LTHSC (n=6) expressing high levels of MPL (MPLhi, top 10% based on MPL expression) showed significantly increased cell growth (p<0.0001) and CFC potential (p=0.0007) when cultured with TPO (10ng/ml) compared to LTHSC expressing low levels of MPL (MPLlo, lowest 10% based on MPL expression), as well as significantly increased cell growth (p=0.005) and CFC (p=0.03) compared to normal MPLhi LTHSC. Following transplantation, MPLhi LTHSC (200 per mouse) generated significantly higher short-term (4 wks, p=0.008) and long-term (16 wks, p=0.003) engraftment of donor cells compared to MPLlo LTHSC. Seven of 16 mice receiving MPLhi LTHSC developed CML compared to only 1 out of 17 mice receiving MPLlo LTHSC. We next evaluated heterogeneity of MPL expression in LTHSC (CD34+CD38-CD90+ cells) from CML patients and normal subjects. As was seen in murine studies, human CML MPLhi LTHSC cultured with TPO (10ng/ml) showed increased cell growth (p<0.0001) and CFC frequency (p=0.02) compared to CML MPLlo LTHSC, and significantly increased cell growth (p<0.0001) and CFC generation (p=0.02) compared to normal MPLhi LTHSC. Both baseline and TPO stimulated p-Stat3/5 levels were significantly higher in human CML MPLhi LTHSC compared with MPLlo LTHSC (p<0.0001), and in CML compared to normal MPLhi LTHSC. Interestingly p-Stat5 response peaked at 1 hour in CML LTHSC compared to 20 minutes in normal LTHSC, further indicating alterations in MPL signaling in CML LTHSC. Transplantation of CML MPLhi LTHSC (3x104 cells/mouse) into NSG mice resulted in higher engraftment of human myeloid cells in BM at both 4 and 16 weeks (p<0.05) compared with MPLlo LTHSC. Normal MPLhi LTHSC also showed higher engraftment in NSG mice at 4 and 16 weeks compared with MPLlo cells. Our studies indicate that CML LTHSC represent a heterogeneous population with varying LSC capacity. Heterogeneity in LSC capacity is associated with variability in expression of MPL. Higher levels of MPL expression in CML LTHSC are associated with significantly increased Stat3/5 signaling, in vitro and in vivo growth, and LSC capacity. These results identify MPL as a key regulator of LSC potential of BCR-ABL+ LTHSC and a potential target for LSC-directed therapeutics. Disclosures:No relevant conflicts of interest to declare.

Bone Marrow Osteoblast Ablation Enhances Short-Term Hematopoietic Stem Cells Without Altering Long-Term Hematopoietic Stem Cell Populations and Accelerates Leukemia Development

Hematopoietic stem cells (HSCs) within the bone marrow (BM) microenvironment reside in close proximity to endosteal osteoblasts (OBs). Although OBs have been considered to provide a HSC niche, other studies suggest that perivascular mesenchymal cells or endothelial cells may be the primary HSC niches, and the specific role of OBs in regulation of HSCs requires further clarification. Moreover, the role of OBs in regulating leukemic stem cells (LSC) is even less well studied. To address these questions, we used a conditional OB ablation mouse model (Col2.3Δtk) in which a truncated version of the herpes simplex virus thymidine kinase (Δtk) is expressed under an OB-specific promoter. In these mice, daily intraperitoneal (IP) administration of ganciclovir (GCV) leads to production of a toxic DNA base analogue in OBs, resulting in their death. We crossed Col2.3Δtk mice with Col2.3GFP mice that specifically express GFP in OBs to facilitate assessment of OB ablation. We confirmed that 4 weeks of GCV administration resulted in ablation of endosteal OBs in this model using both immunofluorescence microscopy and flow cytometry analysis. OB ablation was associated with reduced BM cellularity (Δtk+ 3.7e7±3.0e6, Δtk- 4.8e7±3.8e6 per 4 lower extremity bones, p=0.04), but did not alter spleen (SP) cellularity (Δtk+ 5.1e7±5.3e6, Δtk- 6.3e7±7.4e6 cells per SP, p=0.19). OB ablation was also associated with significantly increased numbers of cells with long-term HSC (LTHSC) phenotype (Lin-Kit+Sca-1+Flt3-CD150+CD48-) in both the BM (Δtk+ 6490±1315, Δtk- 4236±922 per 4 lower extremity bones; p=0.03) and SP (Δtk+ 980±473, Δtk- 96±40 per SP; p=0.04). Significant increases in common myeloid progenitor (CMP) (Δtk+ 145114±43608, Δtk- 82200±26754; p=0.002) and granulocyte/monocyte progenitor (GMP) (Δtk+ 51411±17349, Δtk- 20206±9279, p=0.003, p=0.02) numbers were seen in SP of OB-ablated mice, whereas significant alterations in other hematopoietic populations in BM, SP or PB were not seen. We performed limiting-dilution competitive repopulation assays to determine the functional LTHSC potential of BM cells from OB-ablated and control mice. OB-ablated mice demonstrated a higher frequency of short-term repopulating cells compared to LTHSCs from non-ablated mice (5 weeks: Δtk+ 1 in 4,941; Δtk- 1 in 17,351 BM cells) but similar long-term engraftment (15 weeks: Δtk+ 1 in 22,853; Δtk- 1 in 23,137 BM cells). Transplantation of BM cells from primary transplant recipients into secondary recipients demonstrated similar long-term engraftment potential after second transplant. These results suggest that despite increased numbers of phenotypic LTHSCs in OB-ablated mice, the long-term repopulating and self-renewing capacity of BM cells remains unchanged in OB-ablated mice, but on the other hand there is an increase in functional short-term repopulating capacity. Next, to examine the role of OBs in regulation of Chronic Myelogenous Leukemia (CML) stem cells, we crossed the Col2.3GFPΔtk mice with an inducible transgenic BCR-ABL mouse model of CML (ScltTA-BCR/ABL). In these mice withdrawal of tetracycline results in induction of BCR-ABL expression in HSCs and development of a CML-like myeloproliferative disorder. GCV administration to achieve OB ablation was initiated one week prior to BCR-ABL induction by tetracycline withdrawal, and was continued for the duration of the experiment. CML development was monitored by checking blood counts every 2 weeks after induction and mice were followed for survival. We observed significantly accelerated development of CML in OB-ablated versus non-ablated mice, with 50% of the OB-ablated mice dying within 47 days of CML induction, whereas >50% of the non-ablated mice survived to day 73 (p=0.017). Collectively, these studies suggest that BM OBs are not essential for maintenance of long-term repopulating and self-renewing HSCs, but regulate the expansion of short-term HSCs in the BM. Our studies also indicate an important and previously unrecognized role for OBs in regulating the leukemogenicity of CML LSCs. Disclosures:No relevant conflicts of interest to declare.