Mouse Model Of Acute Promyelocytic Leukemia Research Articles

Tetra-arsenic tetra-sulfide enhances NK-92MI mediated cellular immunotherapy in all-trans retinoic acid-resistant acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is liable to induce disseminated intravascular coagulation and has a high early mortality. Although the combination of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) has significantly improved the complete remission rate, there are still some patients developed drug resistance. Growing evidence suggests that natural killer (NK) cell-mediated immunotherapy as a new treatment can help slow the progression of hematological malignancies. Previous studies also indicated that some tumors exhibited excellent responsiveness to NK cells in vitro. However, many clinical trial results showed that the anti-tumor effect of NK cells infusion alone was not ideal, which may be related to the inactivation of infiltrating NK cells caused by strong immunosuppression in tumor microenvironment, but the specific mechanism remains to be further explored. In the present study, we demonstrated that low doses of tetra-arsenic tetra-sulfide (As4S4) not only enhanced the in vitro killing of NK-92MI against ATRA-resistant APL cells, but also strengthened the growth inhibition of xenografted tumors in APL mouse model. Mechanistically, As4S4 altered the expression of natural killer group 2 member D ligands (NKG2DLs) and cytokines in APL cells, and PD-1 in NK-92MI cells. In addition, database retrieval results further revealed the relationship between the differentially regulated molecules of As4S4 and immune infiltration and its impact on prognosis. In conclusion, our findings confirmed the potential of As4S4 as an adjuvant for NK-92MI in the treatment of ATRA-resistant APL.

CD44–fibrinogen binding promotes bleeding in acute promyelocytic leukemia by in situ fibrin(ogen) deposition

AbstractEarly hemorrhagic death is still the main obstacle for the successful treatment of acute promyelocytic leukemia (APL). However, the mechanisms underlying hemostatic perturbations in APL have not been fully elucidated. Here, we report that CD44 on the membrane of APL blasts and NB4 cells ligated bound fibrinogen, resulting in in situ deposition of fibrin and abnormal fibrin distribution. Clots formed by leukemic cells in response to CD44 and fibrinogen interaction exhibited low permeability and resistance to fibrinolysis. Using flow cytometry and confocal microscopy, we found that CD44 was also involved in platelet and leukemic cell adhesion. CD44 bound activated platelets but not resting platelets through interaction with P-selectin. APL cell-coated fibrinogen-activated platelets directly induce enhanced procoagulant activity of platelets. In vivo studies revealed that CD44 knockdown shortened bleeding time, increased the level of fibrinogen, and elevated the number of platelets by approximately twofold in an APL mouse model. Moreover, CD44 expression on leukemic cells in an APL mouse model was not only associated with bleeding complications but was also related to the wound-healing process and the survival time of APL mice. Collectively, our results suggest that CD44 may be a potential intervention target for preventing bleeding complications in APL.

Metabolic adaptation drives arsenic trioxide resistance in acute promyelocytic leukemia

AbstractAcquired genetic mutations can confer resistance to arsenic trioxide (ATO) in the treatment of acute promyelocytic leukemia (APL). However, such resistance-conferring mutations are rare and do not explain most disease recurrence seen in the clinic. We have generated stable ATO-resistant promyelocytic cell lines that are less sensitive to all-trans retinoic acid (ATRA) and the combination of ATO and ATRA compared with the sensitive cell line. Characterization of these resistant cell lines that were generated in-house showed significant differences in immunophenotype, drug transporter expression, anti-apoptotic protein dependence, and promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA) mutation. Gene expression profiling revealed prominent dysregulation of the cellular metabolic pathways in these ATO-resistant APL cell lines. Glycolytic inhibition by 2-deoxyglucose (2-DG) was sufficient and comparable to the standard of care (ATO) in targeting the sensitive APL cell line. 2-DG was also effective in the in vivo transplantable APL mouse model; however, it did not affect the ATO-resistant cell lines. In contrast, the resistant cell lines were significantly affected by compounds targeting mitochondrial respiration when combined with ATO, irrespective of the ATO resistance-conferring genetic mutations or the pattern of their anti-apoptotic protein dependency. Our data demonstrate that combining mitocans with ATO can overcome ATO resistance. We also show that this combination has potential for treating non-M3 acute myeloid leukemia (AML) and relapsed APL. The translation of this approach in the clinic needs to be explored further.

The Role of Kdm6a in Malignant Hematopoiesis

Our preliminary studies identified recurrent, somatic mutations in Kdm6a in a mouse model of acute promyelocytic leukemia (APL). Putative inactivating mutations of Kdm6a (primarily small deletions) occurred in approximately 50% of mouse APL samples, making these the most common acquired progression events in this model. We found somatic mutations of KDM6A in 4 of 200 acute myeloid leukemia (AML) samples. However, in a cohort of 42 patients with APL, we identified no somatic alterations in KDM6A . To better understand the role of Kdm6a in malignant hematopoiesis, we intercrossed our Kdm6a conditional knockout (KO) x Vav1 - Cre mice with a “knock-in” mouse model of APL (Ctsg-PML-RARA mice). To determine whether Kdm6a inactivation cooperates with PML - RARA, we performed a tumor watch that included all genotypes of Kdm6a conditional KO female and male triple cross mice. After 18 months of observation, we found that the latency of APL was significantly decreased in all three cohorts of Kdm6a conditional KO x Vav1 - Cre x Ctsg-PML-RARA mice (Figure 1). This finding was most pronounced in female homozygous Kdm6a KO triple cross mice, which had a median survival of 161 days versus 365 days in the control cohort (P < 0.01 by log-rank test). The Kdm6a -null mice developed the typical phenotype of mouse APL, including splenomegaly, myeloproliferation and leukemic cells that coexpressed Gr-1 and Kit. The triple cross APL cells remained sensitive to all- trans retinoic acid (ATRA) and arsenic trioxide ex vivo . Compared with 56% penetrance in Ctsg-PML-RARA mice, female homozygous and male hemizygous Kdm6a KO x Vav1 - Cre x Ctsg-PML-RARA mice developed APL with 100% penetrance. We fully characterized pre-leukemic hematopoiesis in young (6 to 8-weeks-old) Kdm6a conditional KO x Vav1 - Cre x Ctsg-PML-RARA mice (compared to appropriate littermate controls) to define the underlying mechanism(s) by which Kdm6a inactivation contributes to leukemogenesis. Like our double cross Kdm6a conditional KO x Vav1 - Cre mice, the loss of Kdm6a expression in young female homozygous triple cross mice caused mild thrombocytopenia and splenomegaly. These young mice had neutrophilia and lymphopenia, which the female Kdm6a -null double cross mice only acquired with age. We observed a trend for increased numbers of colonies in a myeloid serial replating assay that was also specific to this cohort. Next, we demonstrated that this cohort had significant decreases in the Kit+Lin-Sca-1+ (KLS) cell, short-term hematopoietic stem cell (ST-HSC), and multipotent progenitor (MPP) hematopoietic stem and precursor compartments. This recapitulated the findings from the young, double cross Kdm6a -null female mice but was more pronounced. We found a concomitant increase in granulocyte-macrophage progenitors (GMPs) and decrease in megakaryocyte-erythroid progenitors (MEPs) that was also specific for the female Kdm6a -null triple cross cohort. Finally, we performed gene expression profiling via exon arrays on flow-sorted SLAM cells from aged (1-year-old) female Kdm6a -null double cross mice and aged controls. Unsupervised hierarchical clustering revealed a clear distinction between cohorts. We identified 1520 differentially expressed genes by an ANOVA analysis (fold change > 2, P < 0.05) between the Kdm6a -null versus WT aged SLAM cells. The list of differentially expressed genes included Pml (downregulated in Kdm6a -null SLAM cells with fold change of 2.35) and Flt3 (upregulated in Kdm6a -null SLAM cells with fold change of 2.32). Expression changes of these two genes may be associated with the mechanism of how inactivation of Kdm6a cooperates with PML-RARA in our mouse model. Pml haploinsuffiency has been show to modestly cooperate with PML-RARA in mouse models of APL, and FLT3 mutations are the most common secondary mutations in APL. In an effort to further elucidate the mechanism by which loss of Kdm6a contributes to malignant hematopoiesis, we have completed RNA-Seq, ATAC-Seq, anti-histone H3 (monomethyl K4) ChIP-Seq, and anti-histone H3 (trimethyl K27) ChIP-Seq studies on mouse APL samples derived from Ctsg-PML-RARA mice that developed spontaneous leukemias (n = 3 Kdm6a -WT samples versus n = 3 Kdm6a -mutant samples). Analysis of this data set is now underway. In sum, our data have demonstrated a functional role of Kdm6a in malignant hematopoiesis and have improved our understanding of its subsequent connection to leukemogenesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Stromal-Cells Downregulate MiR-23a-5p Levels in Myeloid Leukemic Cells to Activate Protective-Autophagy Against Chemotherapeutic Agents

Role of stromal micro-environment in drug resistance has been extensively reported for several cancers. We have demonstrated earlier that there is significant microenvironment mediated drug resistance (EMDR) to arsenic trioxide (ATO) in acute promyelocytic leukemia (APL) and that this was predominantly driven by up-regulation of the NF-kB pathway in the malignant cell (Ganesan S et al. Leukemia 2016). In our current study we have probed the molecular mechanism of EM-DR in further detail. Towards this we evaluated the potential role played by miRNA in EMDR to ATO in APL. Using NGS based small RNA sequencing we identified hsa-miR-23a-5p to be significantly downregulated (adjusted P value <0.01; figure 1a) in NB4 cells upon co-culture with HS-5 stromal cells. We noted that this miRNA is negatively regulated by NF-kB pathway and inversely correlated with NF-kB status in relapsed APL patients (n=8; matched samples). These results were consistent with our earlier reported observations that NF-kB pathway is dysregulated and enhances drug resistance to ATO. Further, we observed that low expression of miR-23a-5p observed in the relapsed patient's samples showed a trend towards an increased association with subsequent second relapse.We also observed miR-23a-5p mimics were able to restore the sensitivity of NB4 cells to ATO in the presence of stromal cells (figure 1b), while NB4/GFP-MAD cells (NF-kB inhibited cell line showing high expression of miR-23a-5p ) were not protected from ATO on stromal co-culture. Looking for the targets of miR-23a-5p , TargetScan analysis data demonstrated a clustering of targets involving toll like receptor genes (TLR) and autophagy pathway genes (TLRs - TLR2, TLR3, TLR4 and TLR5 and autophagy pathway genes - AMBRA1, ATG13, ATG9 and ATG2 ). Previously reported data clearly demonstrated the ability of TLR genes to activate autophagy (Delgado MA. et al. EMBO J 2008). Based on this data we evaluated the role of autophagy in EM-DR. We noted that there was an upregulation of autophagy pathway in leukemic cells upon co-culture with stroma (where miR-23a-5p was downregulated). Re-introducing miR-23a-5p in the leukemic cells targeted autophagy pathway genes which was validated through western blot (figure 1c). We also observed that inhibiting autophagy by chemical inhibitors (bafilomycin A1 and hydroxychloroquine) along with ATO was able to overcome EM-DR to ATO (figure 1d). The in-vivo efficacy of combining ATO along with hydroxychloroquine was further validated in an APL mouse model where we were able to demonstrate a significant decrease in tumor burden on day20 when compared to controls and significantly prolonged the survival of APL mice treated with the combination in comparison to either drug alone.Similar results were obtained with AML cells, where the stromal cells conferred a protective effect over U937 cells against cytarabine (AraC) and daunorubicin (Dnr). The downregulation of miR-23a-5p was also observed in U937 cells upon co-culture. We also observed that re-introducing miR-23a-5p in these cells restored sensitivity to AraC and Dnr. Our results thus illustrates a complex molecular cross-talk between stromal cells and leukemic cells in protecting against the cytotoxic effects of various chemotherapeutic agents in myeloid leukemia. This data along with that reported earlier by us illustrates multiple levels of regulation of the NF-kB pathway and resistance to chemotherapeutic drugs by stromal cell co-culture. Our work thus suggests that miR-23a-5p mimics or autophagy-inhibitors could potentially be effective adjuvants along with chemotherapy/ATO in the treatment of myeloid malignancies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Expression of the Tumor Suppressor WT1 Is Induced By PML-Rara in Acute Promyelocytic Leukemia. Â

Inactivating mutations in Wilms Tumor 1 (WT1) are found in 5-10% of cases of acute myeloid leukemia (AML). Paradoxically, wild type WT1 is expressed highly in most cases of AML compared to normal hematopoietic progenitors, and high levels of WT1 have been associated with poor outcomes. Therefore, it is not yet clear whether loss-of-function WT1 mutations-or paradoxically, wild type WT1 overexpression-both promote disease progression in AML. We performed a genome-wide screen for mutations in acute promyelocytic leukemia (APL), a morphologic subtype of AML characterized by the PML-RARA fusion. We identified WT1 mutations in 11/42 patients, suggesting that WT1 mutations may cooperate specifically with PML-RARA in APL. Further, expression analysis demonstrated a high level of wild type WT1 expression in non-mutated cases. To test the effect of Wt1 inactivation in a mouse model of APL, we bred mice carrying a floxed Wt1 allele (give Baylor reference) to transgenic mice expressing tamoxifen-inducible Cre recombinase (ERT2-Cre). Tamoxifen treatment leads to deletion of the C-terminal zinc finger domains of Wt1, mimicking some of the mutations that are commonly found in AML/APL cases. These mice were crossed with mice expressing the PML-RARA fusion cDNA in early myeloid progenitors with Cathepsin G (Ctsg) regulatory sequences (Ctsg-PML-RARA). Bone marrow cells from Ctsg-PML-RARA x Wt1fl/fl x ERT2-Cre mice were transplanted into irradiated recipients. Tamoxifen-treated mice (i.e. Wt1 deficient) developed APL with a significantly shortened latency compared to untreated controls (198 days vs. endpoint not reached, N=8-10 each group, p=.0001), suggesting that Wt1 loss-of-function mutations can cooperate with PML-RARA to drive APL. Next, to test the effect of wild type Wt1 overexpression on hematopoietic cells, bone marrow cells from wild type or Ctsg-PML-RARA mice was transduced with retroviruses expressing full length Wt1 cDNA or an empty vector control, and subjected to serial replating. As previously reported, untransduced bone marrow cells from Ctsg-PML-RARA mice were able to form colonies in methylcellulose week after week, while cells overexpressing wild type Wt1 were selected against during serial replating, as demonstrated by a gradual reduction in GFP percentage and Wt1 expression, as measured by western blotting. Interestingly, in contrast to human AML/APL, Wt1 expression could not be detected in a panel of murine tumors with initiating mutations in DNMT3A, PML-RARA, or MLL-AF9 . Taken together, these findings suggest that Wt1 expression suppresses APL development in mice.To reconcile these findings with the high level of WT1 expression in human AML/APL, we speculated that in human progenitor cells, WT1 expression may be induced by leukemia-associated oncogenes (like PML-RARA) as a physiologic, inhibitory response to oncogenic stress. In this case, subsequent inactivating mutations in WT1 would provide an additional growth advantage. Given the observed interspecies difference between WT1/Wt1 expression levels in mouse and human leukemia cells, we used human umbilical cord blood-derived CD34+ cells transduced with retroviruses expressing PML-RARA to model early events in human APL development. CD34+ cells expressing PML-RARA share some characteristics of fully transformed APL cells, including enhanced proliferation in liquid culture, and reduced expression of the myeloid maturation markers CD11b and CD14. WT1 mRNA expression is low in freshly isolated CD34+ cells, but rapidly increases in cells expressing PML-RARA over two weeks in culture (10.5-fold increase compared to empty-vector transduced controls, N=3 separate experiments, p<0.05). Western blotting confirmed robust induction of WT1 protein in transformed cells compared to empty-vector transduced controls, suggesting that WT1 expression is a normal consequence of PML-RARA activation in human hematopoietic progenitor cells. In summary, these findings support a cooperating role for WT1 mutations in APL development, and a potential mechanism to explain the high level of wild type WT1 expression observed in APL cells. Further experiments will be required to determine the downstream mechanisms by which WT1 antagonizes APL development, and whether these observations are generalizable to other AML subtypes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Oncogenic K-ras cooperates with PML-RARα to induce an acute promyelocytic leukemia-like disease

Most patients with acute promyelocytic leukemia (APL) express PML-RAR alpha, the fusion product of t(15;17)(q22;q11.2). Transgenic mice expressing PML-RAR alpha develop APL with long latency, low penetrance, and acquired cytogenetic abnormalities. Based on observations that 4% to 10% of APL patients harbor oncogenic ras mutations, we coexpressed oncogenic K-ras from its endogenous promoter with PML-RAR alpha to generate a short-latency, highly penetrant mouse model of APL. The APL disease was characterized by splenomegaly, leukocytosis, extramedullary hematopoiesis (EMH) in spleen and liver with an increased proportion of immature myeloperoxidase-expressing myeloid forms; transplantability to secondary recipients; and lack of cytogenetic abnormalities. Bone marrow cells showed enhanced self-renewal in vitro. This model establishes a role for oncogenic ras in leukemia pathogenesis and thus validates the oncogenic RAS signaling pathway as a potential target for therapeutic inhibition in leukemia patients. This mouse model should be useful for investigating signaling pathways that promote self-renewal in APL and for testing the in vivo efficacy of RAS signaling pathway inhibitors in conjunction with other targeted therapies such as ATRA (all trans retinoic acid) and arsenic trioxide.

Arsenic Trioxide Enhances the NK Cell Cytotoxicity Against Acute Promyelocytic Leukemia While Simultaneously Inhibiting Its Bio-Genesis

Natural killer cells (NK) contribute significantly to eradication of cancer cells, and there is increased interest in strategies to enhance it’s efficacy. Therapeutic agents used in the treatment of cancer can impact the immune system in a quantitative and qualitative manner. In this study, we evaluated the impact of arsenic trioxide (ATO) used in the management of acute promyelocytic leukemia (APL) on NK cell reconstitution and function. In patients with APL treated with single agent ATO, there was a significant delay in the reconstitution of circulating NK cells to reach median normal levels from the time of diagnosis (655 days for NK cells vs 145 and 265 days for T cells and B cells, respectively). In vitro experiments demonstrated that ATO significantly reduced the CD34 hematopoietic stem cell (HSC) differentiation to NK cells. Additional experimental data demonstrate that CD34+ sorted cells when exposed to ATO lead to a significant decrease in the expression of IKZF2, ETS1, and TOX transcription factors involved in NK cell differentiation and maturation. In contrast, exposure of NK cells and leukemic cells to low doses of ATO modulates NK cell receptors and malignant cell ligand profile in a direction that enhances NK cell mediated cytolytic activity. We have demonstrated that NK cytolytic activity toward NB4 cell line when exposed to ATO was significantly higher when compared with controls. We also validated this beneficial effect in a mouse model of APL were the median survival with ATO alone and ATO + NK was 44 days (range: 33–46) vs 54 days (range: 52–75). In conclusion, ATO has a differential quantitative and qualitative effect on NK cell activity. This information can potentially be exploited in the management of leukemia.

Abstract B33: Synergistic effect of gefitinib and arsenic trioxide in acute promyelocytic leukemia

Abstract Gefitinib and erlotinib are well-known epidermal growth factor receptor (EGFR) inhibitors approved by the Food and Drug Administration for the treatment of non-small cell lung cancer (NSCLC). Interestingly, patients with synchronous NSCLC and acute myeloid leukemia (AML) treated with erlotinib presented regression of both neoplasms, although AML myeloblasts were shown to lack expression of EGFR. In addition, preclinical studies have shown that gefitinib alone or combined with arsenic trioxide (ATO) or all-trans retinoic acid (ATRA) was able to induce differentiation in the EGFR-negative cell lines of acute promyelocytic leukemia (APL), which is a subtype of AML characterized by the t(15;17)/PML/RARA rearrangement. Our previous study demonstrated that EGFR gene expression levels were associated with prognostic outcomes of APL patients treated according to the International Consortium on APL protocol. However, the EGFR levels were not assessed at protein level. Two phase 3 trials reported that ATRA and ATO therapy was associated with excellent outcomes in APL. The two drugs induced the degradation of the PML/RARA oncoprotein through different mechanisms, with ATRA acting through the proteasome pathway, and ATO functioning through the PML-transformation related protein 53 (Trp53) axis. The purpose of this study was to investigate whether gefitinib or erlotinib has synergistic effects with ATO or ATRA in the treatment of APL. To this end, the APL cell lines NB4 and NB4-R2 were treated with gefitinib (C22H24ClFN4O; Selleck Chemicals, #S7786) and erlotinib (C22H23N3O4; Selleck-Chemicals, #S1025) alone or combined with ATO (As2O3; Sigma-Aldrich, #202673). The stock solutions of gefitinib and erlotinib were prepared in DMSO while ATO was dissolved in 1M NaOH solution. Then each drug was diluted with RPMI medium to the desired final concentration. The median effective dose (ED50) for gefitinib was calculated to be 20.97 and 27.06 µM for NB4 and NB4-R2, respectively. ATO was a potent inducer of apoptosis with an ED50 of 2.27 µM for NB4 and 1.73 µM for NB4-R2 cells. ED50 of ATRA (C20H28O2; Sigma-Aldrich, #R2625) was not calculated because ATRA is a potent differentiation inducer but did not induce cell death of APL blasts. Due to the high value of ED50 for NB4 (78.97 µM) and NB4-R2 (111.36 µM) after 24 h of erlotinib treatment, the analysis of this drug was discontinued. The interaction between gefitinib and ATO demonstrated a moderate synergism for NB4 and NB4-R2 with a combination index values of 0.88 and 0.83, respectively. These in vitro results encouraged us to investigate the in vivo effects of gefitinib alone or in combination with ATO and/or ATRA. To this second end, we developed a syngeneic transplant murine model using leukemic cells obtained from transgenic mice hCG-PML/RARA, which develop a form of leukemia that closely recapitulates the human disease. The engraftment of leukemic cells from hCG-PML-RARα transgenic mice transplanted to wild-type littermate recipients was evaluated by morphology and flow cytometry analysis of the peripheral blood (PB), bone marrow (BM), and spleen. Recipient mice were euthanized and evaluated for signs of engraftment 14 (n=3) and 20 (n=3) days after transplantation. Of note, the morphologic analysis revealed &amp;gt;20% of blasts among nucleated cells in the BM samples. The flow cytometric analysis showed the presence of CD11b+ CD117+ cells in PB (median 10.50, range 8.81-12.20), BM (median 18.30, range 17.80-18.80), and spleen (median 16.45, range 16.00-16.90). These results demonstrate that this rapid and robust murine model is useful to assess the efficacy of EGFR inhibitors in APL. Taken together, our preliminary in vitro results suggest that gefitinib and ATO may have potential application for the APL treatment and that this current syngeneic transplant mouse model of APL is suitable to test this hypothesis. Citation Format: Luciana Yamamoto Almeida, Cleide Lúcia Araújo Silva, Isabel Weinhaüser, Larissa Ananias Cândido, Priscila Santos Scheucher, Camila Cristina Oliveira Menezes Bonaldo, Bárbara Amélia Aparecida Santana, Ana Sílvia Gouvêa Lima Yamada, Eduardo Magalhães Rego. Synergistic effect of gefitinib and arsenic trioxide in acute promyelocytic leukemia [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B33.

HDAC Inhibitors Lead to Significant Survival Benefit in a Novel Fusion Protein TBLR1-Rarα Induced Murine Promyelocytic Leukemia Model

Introduction: TBLR1-RARα is the tenth fusion gene of acute promyelocytic leukemia (APL) first identified in a rare case of APL with t(3;17)(q26;q21) chromosomal translocation in our previous study. The characteristics of its basic structure and functions had been clarified in our previous study. In this study, we successfully established a novel TBLR1-RARα leukemia mouse model (TR mouse) which fully recapitulated the most relevant features of human APLs. The therapeutic effects of retinoic acid (ATRA), arsenic trioxide (As2O3), cytarabine (Ara-C) and histone deacetylase inhibitors (HDACi) on TR mice were examined. The differentially expressed genes (DEGs) between TR mice and normal mice were compared to explore the possible mechanisms and better therapeutic targets for this kind of APL.Methods: pMSCV-TBLR1-RARα-Flag-IRES-GFP (MSCV-TR) and pMSCV-IRES-GFP (vehicle) retroviral plasmids were constructed and transfected 293T packaging cells to produce retroviruses. Lin- cells from C57BL/6 mice bone marrow were purified and infected with MSCV-TR and vehicle retroviral supernatant. For in vitro assay, the GFP+ lin- cells sorted and incubated with or without different concentrations of ATRA were analyzed for the differentiation and proliferation capacity by cell morphology, myeloid markers (CD11b and GR-1) and colony formation assay. For the in vivo experiment, GFP+ lin- cells transfected with indicated retroviral vectors were injected intravenously to lethally irradiated C57BL/6 mice to establish an APL mouse model. Cell surface markers were analyzed by flow cytometry. In treatment assays, GFP+ spleen cells from TR leukemia mice were injected intravenously into recipient mice. The mice were randomly separated into groups and received different treatment with ATRA, As2O3, As2O3 in combination with ATRA, Ara-C, Ara-C in combination with ATRA, chidamide and NL101, respectively. The percentage of GFP+ cells in peripheral blood and body weight were measured dynamically. The survival time of every group was recorded and compared. RNA-seq assay was used to identify DEGs between TR mice and normal mice.Results: In vitro assays indicated that TBLR1-RARα could either block the differentiation of HSCs at a relatively early stage or enhanced the clonogenic potential of cells. The TBLR1-RARα leukemia mouse model was successfully established. During the ten-month observational period, 3 out of 15 mice transplanted with TBLR1-RARα expressing cells developed an APL-like disease. Development of leukemia was not observed in any of the mice in control group. All the leukemia mice had a body weight loss as well as splenomegaly and hepatomegaly. The phenotype analysis revealed that the progenitor markers Sca-1, CD34 and C-kit were positive, the myeloid lineage markers Gr-1 and CD11b were also positive, erythroid lineage marker Ter119 was weekly positive, but the lymphatic lineage marker B220, CD3,CD4 and CD8 were all negative. TR mice treated with 1.5-2.5 mg/kg ATRA alone or together with 2.0 mg/kg As2O3 didn’t survive longer than that of control group, although in vitro differentiation experiment showed that the leukemia cells were sensitive to ATRA. Leukemic mice receiving Ara-C treatment had a much longer survive time. Surprisingly, HDAC inhibitors (12.5 and 25 mg/kg chidamide and 30 mg/kg NL-101) could significantly prolong the survival time of TR mice. Thousands of DEGs had been identified between TR mice and wild type mice, which were widely involved in multiple pathways and participated in various biological functions.Conclusion: The TBLR1-RARα leukemia mouse model was successfully established for the first time, and its main characteristics were clarified. Although the leukemia cells were sensitive to ATRA in vitro, TR mice didn’t benefit from ATRA or As2O3 treatment in vivo. Besides Ara-C, HDAC inhibitors, such as chidamide and NL-101 exhibited potency therapeutic values for TR mice, which provided a new strategy for this kind of refractory APL. What’ more, lots of genes that might be related with the process of leukemogenesis and new therapeutic targets for TR leukemia were identified. This model would serve as a versatile tool to study the mechanisms of leukemogenesis and help to design better strategies for APLs in further studies. DisclosuresNo relevant conflicts of interest to declare.

PML nuclear body disruption impairs DNA double-strand break sensing and repair in APL

Proteins involved in DNA double-strand break (DSB) repair localize within the promyelocytic leukemia nuclear bodies (PML-NBs), whose disruption is at the root of the acute promyelocytic leukemia (APL) pathogenesis. All-trans-retinoic acid (RA) treatment induces PML-RARα degradation, restores PML-NB functions, and causes terminal cell differentiation of APL blasts. However, the precise role of the APL-associated PML-RARα oncoprotein and PML-NB integrity in the DSB response in APL leukemogenesis and tumor suppression is still lacking. Primary leukemia blasts isolated from APL patients showed high phosphorylation levels of H2AX (γ-H2AX), an initial DSBs sensor. By addressing the consequences of ionizing radiation (IR)-induced DSB response in primary APL blasts and RA-responsive and -resistant myeloid cell lines carrying endogenous or ectopically expressed PML-RARα, before and after treatment with RA, we found that the disruption of PML-NBs is associated with delayed DSB response, as revealed by the impaired kinetic of disappearance of γ-H2AX and 53BP1 foci and activation of ATM and of its substrates H2AX, NBN, and CHK2. The disruption of PML-NB integrity by PML-RARα also affects the IR-induced DSB response in a preleukemic mouse model of APL in vivo. We propose the oncoprotein-dependent PML-NB disruption and DDR impairment as relevant early events in APL tumorigenesis.

Synergistic Leukemia Eradication by Combined Treatment with Retinoic Acid and HIF Inhibition by EZN-2208 (PEG-SN38) in Preclinical Models of PML-RARα and PLZF-RARα-Driven Leukemia.

Retinoic acid-arsenic trioxide (ATRA-ATO) combination therapy is the current standard of care for patients with acute promyelocytic leukemia (APL) carrying the oncogenic fusion protein PML-RARα. Despite the high cure rates obtained with this drug combination, resistance to arsenic is recently emerging. Moreover, patients with APL carrying the PLZF-RARα fusion protein are partially resistant to ATRA treatment. Hypoxia-inducible factor-1α (HIF-1α) activation has been recently reported in APL, and EZN-2208 (PEG-SN38) is a compound with HIF-1α inhibitory function currently tested in clinical trials. This study investigates the effect of EZN-2208 in different preclinical APL models, either alone or in combination with ATRA. Efficacy of EZN-2208 in APL was measured in vitro by assessing expression of HIF-1α target genes, cell migration, clonogenicity, and differentiation, vis a vis the cytotoxic and cytostatic effects of this compound. In vivo, EZN-2208 was used in mouse models of APL driven by PML-RARα or PLZF-RARα, either alone or in combination with ATRA. Treatment of APL cell lines with noncytotoxic doses of EZN-2208 causes dose-dependent downregulation of HIF-1α bona fide target genes and affects cell migration and clonogenicity in methylcellulose. In vivo, EZN-2208 impairs leukemia progression and prolongs mice survival in APL mouse models. More importantly, when used in combination with ATRA, EZN-2208 synergizes in debulking leukemia and eradicating leukemia-initiating cells. Our preclinical data suggest that the combination ATRA-EZN-2208 may be tested to treat patients with APL who develop resistance to ATO or patients carrying the PLZF-RARα fusion protein.

Halofuginone inhibits phosphorylation of SMAD-2 reducing angiogenesis and leukemia burden in an acute promyelocytic leukemia mouse model.

BackgroundHalofuginone (HF) is a low-molecular-weight alkaloid that has been demonstrated to interfere with Metalloproteinase-2 (MMP-2) and Tumor Growth Factor-β (TGF-β) function and, to present antiangiogenic, antiproliferative and proapoptotic properties in several solid tumor models. Based on the fact that high levels of Vascular Endothelial Growth Factor (VEGF) and increased angiogenesis have been described in acute myeloid leukemia and associated with disease progression, we studied the in vivo effects of HF using an Acute Promyelocytic Leukemia (APL) mouse model.MethodsNOD/SCID mice were transplanted with leukemic cells from hCG-PML/RARA transgenic mice (TM) and treated with HF 150 μg/kg/day for 21 days. The leukemic infiltration and the percentage of VEGF+ cells were evaluated by morphology and flow cytometry. The effect of HF on the gene expression of several pro- and antiangiogenic factors, phosphorylation of SMAD2 and VEGF secretion was assessed in vitro using NB4 and HUVEC cells.ResultsHF treatment resulted in hematological remission with decreased accumulation of immature cell and lower amounts of VEGF in BM of leukemic mice. In vitro, HF modulated gene expression of several pro- and antiangiogenic factors, reduced VEGF secretion and phosphorylation of SMAD2, blocking TGF-β-signaling.ConclusionTaken together, our results demonstrate that HF inhibits SMAD2 signaling and reduces leukemia growth and angiogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0181-2) contains supplementary material, which is available to authorized users.

Disruption of the PHRF1 Tumor Suppressor Network by PML-RARα Drives Acute Promyelocytic Leukemia Pathogenesis

PHRF1 functions as an essential component of the TGF-β tumor suppressor pathway by triggering degradation of the homeodomain repressor factor TGIF. This leads to redistribution of cPML into the cytoplasm, where it coordinates phosphorylation and activation of Smad2 by the TGF-β receptor. In acute promyelocytic leukemia (APL), acquisition of PML-RARα is known to impede critical aspects of TGF-β signaling, including myeloid differentiation. Although these defects are thought to rely on suppression of cPML activity, the mechanisms underlying this phenomenon remain enigmatic. Here, we find that an abnormal function of PML-RARα is to interfere with TGIF breakdown, presumably by competing with PHRF1 for binding to TGIF, culminating in cPML sequestration and inactivation. Enforcing PHRF1 activity is sufficient to restore TGF-β cytostatic signaling in human blasts and suppress APL formation in a mouse model of APL, providing proof-of-concept data that suppression of PHRF1 activity by PML-RARα represents a critical determinant in APL pathogenesis.

Whole Genome Bisulfite Sequencing of Purified Mouse Promyelocytes Reveals Differentially Methylated Regions in Cells Expressing PML-Rara

Acute promyelocytic leukemia (APL) is an AML subtype that is characterized by aberrant expansion of immature myeloid progenitors and precursors that are arrested at the promyelocyte stage. Almost all APL cases are characterized by the t(15;17)(q22;q11.2) translocation that creates the PML-RARA fusion oncogene. Human APL cells are known to have a canonical expression signature and a specific methylation phenotype that is unique to this form of AML. Our laboratory previously created a mouse model of APL by expressing a human PML-RARA cDNA from the mouse Cathepsin G (Ctsg) locus (Ctsg-PML-RARA), which activates human PML- RARA expression in early myeloid progenitor cells, with peak expression in promyelocytes. After a long latent period (6-12 months), ~60% of these mice develop a clonal, APL-like myeloid malignancy. The long latent period is probably due to the requirement for cooperating mutations that synergize with PML-RARA to accelerate the disease.Human APL samples have a unique gene expression signature that distinguishes them from all other subtypes of AML. We evaluated RNA-Seq data derived from Poly A+ enriched cDNAs obtained from purified promyelocytes derived from 3 young (6 week old) WT and 3 Ctsg-PML-RARA mice. We identified 779 annotated genes that are significantly dysregulated in murine promyelocytes expressing PML-RARA with a log2 fold change >= 2 and P<0.05. Some of these genes included Spib/Pu.1, Pou2af1, Jak2, Runx1, and many others. We also identified a set of 24,018 RNAs in promyelocytes that were defined as novel transcripts. This set contains 7,413 lncRNAs with an FPKM value of >= 2. Differential expression analysis yielded 56 dysregulated lncRNA regions in PML-RARA expressing promyelocytes.To explore the association between gene dysregulation and DNA methylation in promyelocytes, we carried out whole-genome bisulfite sequencing using DNA derived from the purified promyelocytes of a 6 week old Ctsg-PML-RARA mouse, and a WT littermate. We generated a total of approximately 800 million sequencing reads, of which 78% mapped uniquely to the reference genome (mm9); we were able to map ~19 million CpGs with at least 10x coverage. Differential methylation analysis performed on ~4.5 million 1 Kb windows spanning the entire genome identified 17,633 differentially methylated regions with a mean difference of >= 25% and a q-value of < 0.01, the vast majority of which (17,264, 98%) were hypomethylated in the Ctsg-PML-RARA promyelocytes. These windows overlap several known genes, including Runx1, Jak2, Dnmt3a, Gata2, and the Hoxa and Hoxb gene clusters. Using more strict criteria (> 50% mean methylation difference), we identified 87 differentially methylated regions of at least 2 Kb in size. Of these 87 distinct regions, 74 (85%) were hypomethylated in PML-RARA promyelocytes, and 13 were hypermethylated; examples of both as shown in Figure 1. These data strongly suggest that PML-RARA has at least two distinct mechanisms by which it can modify DNA methylation. In regions where CpGs are hypomethylated, PML-RARA may be blocking the normal methylation of CpGs by the de novo DNA methyltransferases Dnmt3a and/or Dnmt3b. In contrast, PML-RARA may be directing de novo methyltransferases to act on the hypermethylated regions. Regardless, these data, when coupled with comprehensive chromatin accessibility mapping and complete RNA sequencing data, should provide new insights into the mechanisms used by PML-RARA to alter gene expression and initiate APL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Activation of a promyelocytic leukemia–tumor protein 53 axis underlies acute promyelocytic leukemia cure

Acute promyelocytic leukemia (APL) is driven by the promyelocytic leukemia (PML)-retinoic acid receptor-α (PML-RARA) fusion protein, which interferes with nuclear receptor signaling and PML nuclear body (NB) assembly. APL is the only malignancy definitively cured by targeted therapies: retinoic acid (RA) and/or arsenic trioxide, which both trigger PML-RARA degradation through nonoverlapping pathways. Yet, the cellular and molecular determinants of treatment efficacy remain disputed. We demonstrate that a functional Pml-transformation-related protein 53 (Trp53) axis is required to eradicate leukemia-initiating cells in a mouse model of APL. Upon RA-induced PML-RARA degradation, normal Pml elicits NB reformation and induces a Trp53 response exhibiting features of senescence but not apoptosis, ultimately abrogating APL-initiating activity. Apart from triggering PML-RARA degradation, arsenic trioxide also targets normal PML to enhance NB reformation, which may explain its clinical potency, alone or with RA. This Pml-Trp53 checkpoint initiated by therapy-triggered NB restoration is specific for PML-RARA-driven APL, but not the RA-resistant promyelocytic leukemia zinc finger (PLZF)-RARA variant. Yet, as NB biogenesis is druggable, it could be therapeutically exploited in non-APL malignancies.

Notch signaling in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is initiated by the PML-RARA fusion oncogene and has a characteristic expression profile that includes high levels of the Notch ligand JAG1. In this study, we used a series of bioinformatic, in vitro, and in vivo assays to assess the role of Notch signaling in human APL samples, and in a PML-RARA knockin mouse model of APL (Ctsg-PML-RARA). We identified a Notch expression signature in both human primary APL cells and in Kit+Lin−Sca1+ (KLS) cells from pre-leukemic Ctsg-PML-RARA mice. Both genetic and pharmacologic inhibition of Notch signaling abrogated the enhanced self-renewal seen in hematopoietic stem/progenitor cells (HSPCs) from pre-leukemic Ctsg-PML-RARA mice, but had no influence on cells from age-matched wildtype mice. In addition, 6 of 9 murine APL tumors tested displayed diminished growth in vitro when Notch signaling was inhibited pharmacologically. Finally, we found that genetic inhibition of Notch signaling with a dominant negative MAML protein reduced APL growth in vivo in a subset of tumors. These findings expand the role of Notch signaling in hematopoietic diseases, and further define the mechanistic events important for PML-RARA-mediated leukemogenesis.

8-CPT-cAMP/all- trans retinoic acid targets t (11;17) acute promyelocytic leukemia through enhanced cell differentiation and PLZF/RARα degradation

The refractoriness of acute promyelocytic leukemia (APL) with t(11;17)(q23;q21) to all-trans retinoic acid (ATRA)-based therapy concerns clinicians and intrigues basic researchers. By using a murine leukemic model carrying both promyelocytic leukemia zinc finger/retinoic acid receptor-α (PLZF/RARα) and RARα/PLZF fusion genes, we discovered that 8-chlorophenylthio adenosine-3', 5'-cyclic monophosphate (8-CPT-cAMP) enhances cellular differentiation and improves gene trans-activation by ATRA in leukemic blasts. Mechanistically, in combination with ATRA, 8-CPT-cAMP activates PKA, causing phosphorylation of PLZF/RARα at Ser765 and resulting in increased dissociation of the silencing mediator for retinoic acid and thyroid hormone receptors/nuclear receptor corepressor from PLZF/RARα. This process results in changes of local chromatin and transcriptional reactivation of the retinoic acid pathway in leukemic cells. Meanwhile, 8-CPT-cAMP also potentiated ATRA-induced degradation of PLZF/RARα through its Ser765 phosphorylation. In vivo treatment of the t(11;17) APL mouse model demonstrated that 8-CPT-cAMP could significantly improve the therapeutic effect of ATRA by targeting a leukemia-initiating cell activity. This combined therapy, which induces enhanced differentiation and oncoprotein degradation, may benefit t(11;17) APL patients.

Lithium Treatment Potentiates Both in Vitro and in Vivo Retinoic Acid Efficacy in APL.

AbstractAbstract 2614We previously demonstrated that although retinoic acid (RA) has targeted efficacy in Acute Promyelocytic Leukemia (APL), heterogeneity exists leading to the appearance of un-targeted clones at the time of relapse. Characterization of these clones is not yet fully unraveled though we and others have previously highlighted the roles of RARα mutations, pharmacogenomics or APL miRNome. We recently identified that the ERK1/2 pathway synergized with RA to restore the transcriptional activity of RA in resistant APL cells, thus restoring RA induced differentiation (Cassinat et al. Mol Cell Biol 2011). These results suggest that targeting interconnected signaling pathways could optimize differentiation therapy efficacy. To this effect, we studied known signaling pathway activators or inhibitors that could potentiate with RA and identified Lithium chloride (LiCl).Treatment of the ATRA sensitive-APL NB4 cell line with LiCl (25mM) decreases proliferation and increases apoptosis (25% and 40% of Annexin V-positive cells at day 1 and 2 respectively) with evidence of caspase 3 cleavage at day 2. Because NB4 cells fully differentiated with RA alone we were unable to observe any synergy when combined with LiCl. Treatment of the RA-resistant APL UF-1 cell line with RA or LiCl alone does not induce differentiation. Combination of RA+LiCl restores differentiation after 3 days of culture (65% CD11b positive and 55% NBT test positive cells). Similar results were obtained with different GSK3 inhibitors, suggesting that the LiCL effects were in part linked to its well characterized GSK3 inhibitory activity. Interestingly, we noted that LiCl treatment induces rapid phosphorylation of ERK1/2 and pretreatment with the MEK/ERK1/2 inhibitor UO126 fully abolished the differentiation induced by the RA+LiCl combination. The combination restores in UF-1 the expression of RA target genes (such as RARα2) to the same levels obtained in NB4 cells treated by RA alone. The level of luciferase activity of an RA responsive element reporter gene was increased with the RA+LiCl combination compared to RA alone. Both target gene expression and luciferase activiy were abolished after inhibition of the MEK/ERK1/2 pathway. Thus, increase in differentiation of UF-1 cells by RA+LiCl is linked to increased RA transcriptional activation. Similar studies in fresh APL patient cells confirmed both the increase in differentiation and level of RA target gene expression and their inhibition by UO126. Finally, to translate these findings in vivo, we used the APL-transplantable mouse model. Plasma lithium levels in treated mice were measured between 0.6 and 1.05 mmol/l, levels reached in humans. When LiCl was combined with RA we repeatedly observed a pronounced survival advantage compared to mice treated by RA alone as evaluated by Kaplan Meier analysis.In this work we demonstrate that LiCl, a well tolerated agent in humans, has the potential, when combined with RA, to restore RA induced transcriptional activation and differentiation in RA resistant APL cells. Furthermore, this combination also increases RA efficacy in an in vivo APL mouse model. Disclosures:Off Label Use: Lithium is a mood modulator administered for bipolar disorders.

Expression and Function of PML-RARA in the Hematopoietic Progenitor Cells of Ctsg-PML-RARA Mice

Because PML-RARA-induced acute promyelocytic leukemia (APL) is a morphologically differentiated leukemia, many groups have speculated about whether its leukemic cell of origin is a committed myeloid precursor (e.g. a promyelocyte) versus an hematopoietic stem/progenitor cell (HSPC). We originally targeted PML-RARA expression with CTSG regulatory elements, based on the early observation that this gene was maximally expressed in cells with promyelocyte morphology. Here, we show that both Ctsg, and PML-RARA targeted to the Ctsg locus (in Ctsg-PML-RARA mice), are expressed in the purified KLS cells of these mice (KLS = Kit+Lin−Sca+, which are highly enriched for HSPCs), and this expression results in biological effects in multi-lineage competitive repopulation assays. Further, we demonstrate the transcriptional consequences of PML-RARA expression in Ctsg-PML-RARA mice in early myeloid development in other myeloid progenitor compartments [common myeloid progenitors (CMPs) and granulocyte/monocyte progenitors (GMPs)], which have a distinct gene expression signature compared to wild-type (WT) mice. Although PML-RARA is indeed expressed at high levels in the promyelocytes of Ctsg-PML-RARA mice and alters the transcriptional signature of these cells, it does not induce their self-renewal. In sum, these results demonstrate that in the Ctsg-PML-RARA mouse model of APL, PML-RARA is expressed in and affects the function of multipotent progenitor cells. Finally, since PML/Pml is normally expressed in the HSPCs of both humans and mice, and since some human APL samples contain TCR rearrangements and express T lineage genes, we suggest that the very early hematopoietic expression of PML-RARA in this mouse model may closely mimic the physiologic expression pattern of PML-RARA in human APL patients.