Inhibitors of Chemoresistance Pathways in Combination with Ara-C to Overcome Multidrug Resistance in AML. A Mini Review

Acute myeloid leukemia (AML), the most common type of leukemia in older adults, is a heterogeneous disease that originates from the clonal expansion of undifferentiated hematopoietic progenitor cells. These cells present a remarkable variety of genes and proteins with altered expression and function. Despite significant advances in understanding the molecular panorama of AML and the development of therapies that target mutations, survival has not improved significantly, and the therapy standard is still based on highly toxic chemotherapy, which includes cytarabine (Ara-C) and allogeneic hematopoietic cell transplantation. Approximately 60% of AML patients respond favorably to these treatments and go into complete remission; however, most eventually relapse, develop refractory disease or chemoresistance, and do not survive for more than five years. Therefore, drug resistance that initially occurs in leukemic cells (primary resistance) or that develops during or after treatment (acquired resistance) has become the main obstacle to AML treatment. In this work, the main molecules responsible for generating chemoresistance to Ara-C in AML are discussed, as well as some of the newer strategies to overcome it, such as the inclusion of molecules that can induce synergistic cytotoxicity with Ara-C (MNKI-8e, emodin, metformin and niclosamide), subtoxic concentrations of chemotherapy (PD0332991), and potently antineoplastic treatments that do not damage nonmalignant cells (heteronemin or hydroxyurea + azidothymidine).

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Development of BCR-ABL1 Transgenic Zebrafish Model Reproducing Chronic Myeloid Leukemia (CML) Like-Disease and Providing a New Insight into CML Mechanisms

Cells ◽

10.3390/cells10020445 ◽

2021 ◽

Vol 10 (2) ◽

pp. 445

Author(s):

Daniela Zizioli ◽

Simona Bernardi ◽

Marco Varinelli ◽

Mirko Farina ◽

Luca Mignani ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Fusion Gene ◽

Philadelphia Chromosome ◽

Leukemic Cells ◽

Transgenic Zebrafish ◽

Hematopoietic Tissue ◽

Zebrafish Model ◽

Altered Expression

Zebrafish has proven to be a versatile and reliable experimental in vivo tool to study human hematopoiesis and model hematological malignancies. Transgenic technologies enable the generation of specific leukemia types by the expression of human oncogenes under specific promoters. Using this technology, a variety of myeloid and lymphoid malignancies zebrafish models have been described. Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasia characterized by the BCR-ABL1 fusion gene, derived from the t (9;22) translocation causing the Philadelphia Chromosome (Ph). The BCR-ABL1 protein is a constitutively activated tyrosine kinas inducing the leukemogenesis and resulting in an accumulation of immature leukemic cells into bone marrow and peripheral blood. To model Ph+ CML, a transgenic zebrafish line expressing the human BCR-ABL1 was generated by the Gal4/UAS system, and then crossed with the hsp70-Gal4 transgenic line. The new line named (BCR-ABL1pUAS:CFP/hsp70-Gal4), presented altered expression of hematopoietic markers during embryonic development compared to controls and transgenic larvae showed proliferating hematopoietic cells in the caudal hematopoietic tissue (CHT). The present transgenic zebrafish would be a robust CML model and a high-throughput drug screening tool.

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Clonal expansion and myeloid leukemia progression modeled by multiplex gene editing of murine hematopoietic progenitor cells

Experimental Hematology ◽

10.1016/j.exphem.2018.04.009 ◽

2018 ◽

Vol 64 ◽

pp. 33-44.e5 ◽

Cited By ~ 5

Author(s):

Xiangguo Shi ◽

Ayumi Kitano ◽

Yajian Jiang ◽

Victor Luu ◽

Kevin A. Hoegenauer ◽

...

Keyword(s):

Progenitor Cells ◽

Myeloid Leukemia ◽

Gene Editing ◽

Clonal Expansion ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor

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Evidence for CD34+ Hematopoietic Progenitor Cell Involvement in Acute Myeloid Leukemia with NPM1 Gene Mutation: Implications for the Cell of Origin

Blood ◽

10.1182/blood.v112.11.307.307 ◽

2008 ◽

Vol 112 (11) ◽

pp. 307-307 ◽

Cited By ~ 1

Author(s):

Maria Paola Martelli ◽

Valentina Pettirossi ◽

Elisabetta Bonifacio ◽

Federica Mezzasoma ◽

Nicla Manes ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Myeloid Leukemia ◽

Hox Genes ◽

Leukemic Cells ◽

Mutant Protein ◽

Cell Of Origin ◽

Hematopoietic Progenitor ◽

Cd34 Cells ◽

Acute Myeloid

Abstract Acute myeloid leukemia expressing mutated NPM1 gene and cytoplasmic nucleophosmin (NPMc+ AML) [Falini B et al, NEJM2005;352:254–266] is a new entity of WHO classification that shows distinctive biological and clinical features, including a unique molecular signature characterized by downregulation of CD34 and upregulation of most HOX genes [Falini B et al, Blood2007;109:874–885]. Involvement of HOX genes in the maintenance of the stem-cell phenotype strongly suggest that AML with mutated NPM1 originates from a multipotent hematopoietic progenitor (HSC). This view is also supported by immunohistological findings showing that AML with mutated NPM1 frequently displays multilineage involvement [Pasqualucci L et al, Blood2006;108:4146–4155]. On the other hand, the frequent negativity of NPMc+ AML for the HSC-associated antigen CD34 raises the question of whether the mutation event occurs in a CD34-negative HSC (these cells have been identified in mice) or whether a minimal pool of CD34-positive NPM1-mutated leukemic cells does exist. Currently, the hierarchical level of stem cell involvement in NPMc+ AML is unknown. To address this issue, we purified CD34+ cells from NPMc+ AML patients and detected NPM1 mutant protein in the sorted population by Western blot with anti-NPM mutant specific antibodies [Martelli MP et al, Leukemia 2008] (Figure 1A). We investigated 6 NPMc+ AML patients presenting at diagnosis with 0.12%, 0.14%, 0.38%, 5%, 22%, and 28% of CD34+ cells in the peripheral blood. In all cases, CD34+ fractions (purity >90%) harboured NPM1 mutant protein, indicating they belong to the leukemic clone (Figure 1B). The percentage of most undifferentiated CD34+/CD38− cells in the CD34+ fractions ranged from 5 to 97%. Notably, in at least one case, all CD34+ NPM1-mutated leukemic cells were CD38−negative. Moreover in all cases, CD34+ NPM1-mutated leukemic cells appeared to express CD123 (IL-3 receptor), considered a marker of the leukemic stem cell and target of potential therapy. Double staining of bone marrow biopsies with anti-CD34 and anti-NPM antibodies revealed that the rare CD34+ cells expressed NPM1 aberrantly in the cytoplasm. Inoculation of CD34+ NPM1-mutated AML cells into sublethally irradiated NOD/SCID mice resulted into leukemia engrafment in various body sites, especially bone marrow, spleen, lung and liver. Preliminary results showed that CD34+ leukemic cells reacquired the same leukemic phenotype as the original patient’s, including CD34-negativity of the leukemic bulk in spite of any lack of differentiation. This finding suggests that NPM1 mutant protein may be involved in downregulation of CD34 antigen, while keeping a gene expression profile typical of the hematopoietic stem cell. These findings suggest the CD34+ fraction contains the SCID-leukemia initiating cells (SL-IC) and point to CD34+/CD38− HSC as the cell of origin of AML with mutated NPM1. Figure Figure

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Retrospective Application of European LeukemiaNet Provisional Criteria for Second-Generation TKI Chronic Myeloid Leukemia

Blood ◽

10.1182/blood.v116.21.2270.2270 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2270-2270

Author(s):

Massimo Breccia ◽

Roberto Latagliata ◽

Fabio Stagno ◽

Antonella Gozzini ◽

Elisabetta Abruzzese ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Second Generation ◽

Conflicts Of Interest ◽

Acquired Resistance ◽

Chronic Phase ◽

Primary Resistance ◽

Free Survival ◽

Optimal Response ◽

Monitoring Response

Abstract Abstract 2270 An update of the European LeukemiaNet criteria for monitoring response of chronic myeloid leukemia patients was recently published and provisional criteria to evaluate patients during second generation TKI therapy after resistance to imatinib were proposed. In our study we retrospectively tested these criteria in a large series of CML patients resistant to imatinib further treated with second generation TKIs with the aim to analyze the outcome of suboptimal response and failure patients compared to those with optimal response and to validate the provisional criteria for monitoring response. One hundred twenty-seven CML patients resistant to imatinib were collected from 6 different Italian hematologic centers. There were 66 males and 61 females, median age 54 years (range 25–80). Twenty-seven patients were in late chronic phase after IFN resistance. Ninety-seven patients received second-generation TKI after acquired resistance, whereas 30 patients had primary resistance. We found that at different time points (3, 6 and 12 months), patients classified as failure showed significantly worse 2-year overall survival (OS), progression-free survival (PFS) and event-free survival (EFS) than sub-optimal and optimal response patients. At 3 months, “failure” patients, had an OS of 83% compared to 86% of sub-optimal and 97% of optimal response patients (p=0.001); PFS was 77% for failure patients compared to 92% and 99% for sub-optimal and optimal response patients, respectively (p=0.001), whereas EFS was 41% for failure vs 59% for sub-optimal (p=0.001) and 85% and optimal response patients, respectively (sub-optimal vs optimal p<0.001). At 6 months, OS was 82%, 88% and 99% for failure, sub-optimal and optimal response patients (p=0.05), respectively; PFS was 82% for failure compared to 94% and 99% for sub-optimal and optimal response patients, respectively (p=0.001); EFS was 47% vs 69% for failure and sub-optimal response (p=0.001) and 86% for optimal response patients (sub-optimal vs optimal, p<0.001). At 12 months again OS was 84% for failure patients compared to 95% and 99% for sub-optimal and optimal response patients (p=0.04); PFS was 86%, 95% and 99% for failure, sub-optimal and optimal response patients, respectively (p=0.001) and EFS was 48% for failure, 67% for sub-optimal response patients (p=0.002) and 89% for optimal response patients (sub-optimal vs optimal, p<0.001). We found that ELN provisional criteria identified at any times worse EFS for sub-optimal response patients, similar to that of failure patients, and failure criteria at 3 months identified patients who had worse PFS and EFS. ELN provisional criteria for second-generation TKIs treated patients appear to clearly predict outcome and can be useful to identify patients at high risk of progression. Disclosures: No relevant conflicts of interest to declare.

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Induction of NK Cell Reactivity Against Myeloid Leukemia By a Novel Fc-Optimized CD133 Antibody

Blood ◽

10.1182/blood.v126.23.3793.3793 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3793-3793 ◽

Cited By ~ 3

Author(s):

Kathrin Rothfelder ◽

Samuel Koerner ◽

Maya Andre ◽

Julia Leibold ◽

Philaretos Kousis ◽

...

Keyword(s):

Nk Cells ◽

Progenitor Cells ◽

Myeloid Leukemia ◽

Nk Cell ◽

Conflicts Of Interest ◽

Residual Disease ◽

Ex Vivo ◽

Leukemic Cells ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor

Abstract NK cells largely contribute to the success of monoclonal antibody (mAb) application in cancer due to their ability to mediate antibody-dependent cellular cytotoxicity (ADCC), a feature considered critical for therapeutic success. Up to now, no immunotherapeutic antibodies are available for the treatment of myeloid leukemias. Recently, we reported on the development of mAb targeting CD133, which is expressed on a wide variety of tumor cells (Koerner et al., Blood 2014 124:2309). Here we extend our analyses and provide further data on the preclinical characterization of an Fc-engineered CD133 mAb for the treatment of myeloid leukemia. Compared to two other anti-human CD133 mAb (clones AC133 and W6B3), which both bound to primary AML and CML cells in 15/25 and 7/10 cases, respectively, clone 293C3 recognized the leukemic cells in 22/25 AML cases and 7/10 CML cases. Based on these results, clone 293C3 was chosen to generate chimeric mAb with either a wildtype Fc part (293C3-WT) or a variant containing amino acid exchanges (S239D/I332E) to enhance affinity to the activating Fc receptor CD16 on NK cells (293C3-SDIE). Treatment with 293C3-SDIE resulted in significantly enhanced activation, degranulation and lysis of primary CD133-positive AML cells by NK cells in allogeneic and autologous experimental ex vivo settings as compared to its wildtype counterpart. Considering the expression of CD133 on healthy hematopoietic progenitor cells, we further performed colony forming unit assays with healthy bone marrow (BM) cells. In line with the observed lower expression levels of CD133 on healthy compared to malignant hematopoietic cells no relevant toxicity of 293C3-SDIE at the level of committed hematopoietic progenitor cells was observed. Moreover, 293C3-SDIE did not induce lysis of of healthy BM cells by allogeneic or autologous NK cells. In a NOD.Cg-Prkdcscid IL2rgtmWjl/Sz (NSG) xenotransplantation model, induction of ADCC by treatment with 293C3-SDIE resulted in the elimination of patient AML cells by NK cells from a matched human donor. Thus, 293C3-SDIE constitutes an attractive immunotherapeutic compound, in particular for the elimination of minimal residual disease in CD133 bearing leukemia in the context of allogenic SCT. Disclosures No relevant conflicts of interest to declare.

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DNAM-1/CD226 is functionally expressed on acute myeloid leukemia (AML) cells and is associated with favorable prognosis

Scientific Reports ◽

10.1038/s41598-021-97400-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Anna Chashchina ◽

Melanie Märklin ◽

Clemens Hinterleitner ◽

Helmut R. Salih ◽

Jonas S. Heitmann ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Leukemic Cells ◽

Allogenic Stem Cell Transplantation ◽

Favorable Prognosis ◽

Tnf Α ◽

And Function ◽

Prognostic Maker ◽

Acute Myeloid

AbstractDNAM-1 is reportedly expressed on cytotoxic T and NK cells and, upon interaction with its ligands CD112 and CD155, plays an important role in tumor immunosurveillance. It has also been reported to be functionally expressed by myeloid cells, but expression and function on malignant cells of the myeloid lineage have not been studied so far. Here we analyzed expression of DNAM-1 in leukemic cells of acute myeloid leukemia (AML) patients. We found substantial levels of DNAM-1 to be expressed on leukemic blasts in 48 of 62 (> 75%) patients. Interaction of DNAM-1 with its ligands CD112 and CD155 induced release of the immunomodulatory cytokines IL-6, IL-8 IL-10 and TNF-α by AML cells and DNAM-1 expression correlated with a more differentiated phenotype. Multivariate analysis did not show any association of DNAM-1 positivity with established risk factors, but expression was significantly associated with clinical disease course: patients with high DNAM-1 surface levels had significantly longer progression-free and overall survival compared to DNAM-1low patients, independently whether patients had undergone allogenic stem cell transplantation or not. Together, our findings unravel a functional role of DNAM-1 in AML pathophysiology and identify DNAM-1 as a potential novel prognostic maker in AML.

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Leukemia stemness and co-occurring mutations drive resistance to IDH inhibitors in acute myeloid leukemia

10.1101/2020.10.27.357111 ◽

2020 ◽

Author(s):

Feng Wang ◽

Kiyomi Morita ◽

Courtney D DiNardo ◽

Ken Furudate ◽

Tomoyuki Tanaka ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Cytosine Methylation ◽

Acquired Resistance ◽

Primary Resistance ◽

Clinical Resistance ◽

Main Driver ◽

Clinical Responses ◽

Mutant Idh1 ◽

Acute Myeloid

AbstractAllosteric inhibitors of mutant IDH1 or IDH2 induce terminal differentiation of the mutant leukemic blasts and provide durable clinical responses in approximately 40% of acute myeloid leukemia (AML) patients with the mutations. However, primary resistance and acquired resistance to the drugs are major clinical issues. To understand the molecular underpinnings of clinical resistance to IDH inhibitors (IDHi), we performed multipronged genomic analyses (DNA sequencing, RNA sequencing and cytosine methylation profiling) in longitudinally collected specimens from 68 IDH1- or IDH2-mutant AML patients treated with the inhibitors. The analysis revealed that leukemia stemness is a major driver of primary resistance to IDHi, whereas selection of mutations in RUNX1/CEBPA or RAS-RTK pathway genes was the main driver of acquired resistance to IDHi, along with BCOR, homologous IDH gene, and TET2. These data suggest that targeting stemness and certain high-risk co-occurring mutations may overcome resistance to IDHi in AML.

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Altered Multidrug Resistance Phenotype Caused by Anthracycline Analogues and Cytosine Arabinoside in Myeloid Leukemia

Blood ◽

10.1182/blood.v93.12.4086.412k43_4086_4095 ◽

1999 ◽

Vol 93 (12) ◽

pp. 4086-4095 ◽

Cited By ~ 1

Author(s):

Xiu F. Hu ◽

Alison Slater ◽

Phillip Kantharidis ◽

Danny Rischin ◽

Surender Juneja ◽

...

Keyword(s):

Cytosine Arabinoside ◽

Myeloid Leukemia ◽

Ex Vivo ◽

Flow Cytometric Analysis ◽

Leukemic Cells ◽

Short Exposure ◽

Blast Cells ◽

Cytotoxic Stress ◽

And Function

The expression of P-glycoprotein (Pgp) is often increased in acute myeloid leukemia (AML). However, little is known of the regulation of Pgp expression by cytotoxics in AML. We examined whether Pgp expression and function in leukemic blasts was altered after a short exposure to cytotoxics. Blasts were isolated from 19 patients with AML (15 patients) or chronic myeloid leukemia in blastic transformation (BT-CML, 4 patients). Pgp expression and function were analyzed by flow cytometric analysis of MRK 16 binding and Rhodamine 123 retention, respectively. At equitoxic concentrations, ex vivo exposure for 16 hours to the anthracyclines epirubicin (EPI), daunomycin (DAU), idarubicin (IDA), or MX2 or the nucleoside analogue cytosine arabinoside (AraC) differentially upregulated MDR1/Pgp expression in Pgp-negative and Pgp-positive blast cells. In Pgp-negative blasts, all four anthracyclines and AraC significantly increased Pgp expression (P = .01) and Pgp function (P = .03). In contrast, MX2, DAU, and AraC were the most potent in inducing Pgp expression and function in Pgp positive blasts (P < .05). A good correlation between increased Pgp expression and function was observed in Pgp-negative (r = .90, P = .0001) and Pgp-positive blasts (r = .77,P = .0002). This increase in Pgp expression and function was inhibited by the addition of 1 μmol/L PSC 833 to blast cells at the time of their exposure to these cytotoxics. In 1 patient with AML, an increase in Pgp levels was observed in vivo at 4 and 16 hours after the administration of standard chemotherapy with DAU/AraC. Upregulation of Pgp expression was also demonstrated ex vivo in blasts harvested from this patient before the commencement of treatment. In 3 other cases (1 patient with AML and 2 with BT-CML) in which blasts were Pgp negative at the time of initial clinical presentation, serial samples at 1 to 5 months after chemotherapy showed the presence of Pgp-positive blasts. All 3 patients had refractory disease. Interestingly, in all 3 cases, upregulation of Pgp by cytotoxics was demonstrated ex vivo in blasts harvested at the time of presentation. These data suggest that upregulation of the MDR1 gene may represent a normal response of leukemic cells to cytotoxic stress and may contribute to clinical drug resistance.

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Altered Multidrug Resistance Phenotype Caused by Anthracycline Analogues and Cytosine Arabinoside in Myeloid Leukemia

Blood ◽

10.1182/blood.v93.12.4086 ◽

1999 ◽

Vol 93 (12) ◽

pp. 4086-4095 ◽

Cited By ~ 43

Author(s):

Xiu F. Hu ◽

Alison Slater ◽

Phillip Kantharidis ◽

Danny Rischin ◽

Surender Juneja ◽

...

Keyword(s):

Cytosine Arabinoside ◽

Myeloid Leukemia ◽

Ex Vivo ◽

Flow Cytometric Analysis ◽

Leukemic Cells ◽

Short Exposure ◽

Blast Cells ◽

Cytotoxic Stress ◽

And Function

Abstract The expression of P-glycoprotein (Pgp) is often increased in acute myeloid leukemia (AML). However, little is known of the regulation of Pgp expression by cytotoxics in AML. We examined whether Pgp expression and function in leukemic blasts was altered after a short exposure to cytotoxics. Blasts were isolated from 19 patients with AML (15 patients) or chronic myeloid leukemia in blastic transformation (BT-CML, 4 patients). Pgp expression and function were analyzed by flow cytometric analysis of MRK 16 binding and Rhodamine 123 retention, respectively. At equitoxic concentrations, ex vivo exposure for 16 hours to the anthracyclines epirubicin (EPI), daunomycin (DAU), idarubicin (IDA), or MX2 or the nucleoside analogue cytosine arabinoside (AraC) differentially upregulated MDR1/Pgp expression in Pgp-negative and Pgp-positive blast cells. In Pgp-negative blasts, all four anthracyclines and AraC significantly increased Pgp expression (P = .01) and Pgp function (P = .03). In contrast, MX2, DAU, and AraC were the most potent in inducing Pgp expression and function in Pgp positive blasts (P < .05). A good correlation between increased Pgp expression and function was observed in Pgp-negative (r = .90, P = .0001) and Pgp-positive blasts (r = .77,P = .0002). This increase in Pgp expression and function was inhibited by the addition of 1 μmol/L PSC 833 to blast cells at the time of their exposure to these cytotoxics. In 1 patient with AML, an increase in Pgp levels was observed in vivo at 4 and 16 hours after the administration of standard chemotherapy with DAU/AraC. Upregulation of Pgp expression was also demonstrated ex vivo in blasts harvested from this patient before the commencement of treatment. In 3 other cases (1 patient with AML and 2 with BT-CML) in which blasts were Pgp negative at the time of initial clinical presentation, serial samples at 1 to 5 months after chemotherapy showed the presence of Pgp-positive blasts. All 3 patients had refractory disease. Interestingly, in all 3 cases, upregulation of Pgp by cytotoxics was demonstrated ex vivo in blasts harvested at the time of presentation. These data suggest that upregulation of the MDR1 gene may represent a normal response of leukemic cells to cytotoxic stress and may contribute to clinical drug resistance.

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Leukemia stemness and co-occurring mutations drive resistance to IDH inhibitors in acute myeloid leukemia

Nature Communications ◽

10.1038/s41467-021-22874-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Feng Wang ◽

Kiyomi Morita ◽

Courtney D. DiNardo ◽

Ken Furudate ◽

Tomoyuki Tanaka ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Cytosine Methylation ◽

Acquired Resistance ◽

Primary Resistance ◽

Clinical Resistance ◽

Main Driver ◽

Clinical Responses ◽

Mutant Idh1 ◽

Acute Myeloid

AbstractAllosteric inhibitors of mutant IDH1 or IDH2 induce terminal differentiation of the mutant leukemic blasts and provide durable clinical responses in approximately 40% of acute myeloid leukemia (AML) patients with the mutations. However, primary resistance and acquired resistance to the drugs are major clinical issues. To understand the molecular underpinnings of clinical resistance to IDH inhibitors (IDHi), we perform multipronged genomic analyses (DNA sequencing, RNA sequencing and cytosine methylation profiling) in longitudinally collected specimens from 60 IDH1- or IDH2-mutant AML patients treated with the inhibitors. The analysis reveals that leukemia stemness is a major driver of primary resistance to IDHi, whereas selection of mutations in RUNX1/CEBPA or RAS-RTK pathway genes is the main driver of acquired resistance to IDHi, along with BCOR, homologous IDH gene, and TET2. These data suggest that targeting stemness and certain high-risk co-occurring mutations may overcome resistance to IDHi in AML.

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